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Appropriate expression of growth-regulatory genes is essential to ensure normal animal development and to prevent diseases like cancer . Gene regulation at the levels of transcription and translational initiation mediated by the Hippo and Insulin signaling pathways and by the TORC1 complex , respectively , has been well documented . Whether translational control mediated by RNA-binding proteins contributes to the regulation of cellular growth is less clear . Here , we identify Lingerer ( Lig ) , an UBA domain-containing protein , as growth suppressor that associates with the RNA-binding proteins Fragile X mental retardation protein 1 ( FMR1 ) and Caprin ( Capr ) and directly interacts with and regulates the RNA-binding protein Rasputin ( Rin ) in Drosophila melanogaster . lig mutant organs overgrow due to increased proliferation , and a reporter for the JAK/STAT signaling pathway is upregulated in a lig mutant situation . rin , Capr or FMR1 in combination as double mutants , but not the respective single mutants , display lig like phenotypes , implicating a redundant function of Rin , Capr and FMR1 in growth control in epithelial tissues . Thus , Lig regulates cell proliferation during development in concert with Rin , Capr and FMR1 .
Understanding how cells and organs control their growth is a major endeavor in developmental biology . In Drosophila melanogaster and in mammalian systems , genetic studies have revealed a tight regulation mainly at two different layers . Whereas the Hippo and the Insulin receptor signal transduction pathways alter the transcription of growth-regulatory genes via the co-transcriptional factor Yorkie and the transcription factor FoxO , respectively , TORC1 controls translational initiation via 4EBP and S6K [1] . However , increasing evidence indicates that RNA-binding proteins like Fragile X mental retardation 1 protein ( FMR1 ) , mammalian cytoplasmic activation/proliferation associated protein ( Caprin ) and mammalian Ras-GTPase activating protein SH3 domain binding protein ( G3BP ) regulate growth and growth factors at the translational level [2]–[5] . In humans , loss of FMR1 , a protein with one RGG RNA-binding and two KH domains , causes the most common form of inherited mental retardation , the Fragile X syndrome ( FXS ) . Analysis of FMR1 function in the model organisms mouse and Drosophila implicated FMR1 in cell proliferation , cell differentiation and apoptosis in reproductive organs and neuronal tissue via translational regulation of growth-regulatory proteins . For example , FMR1 knockout mice display increased proliferation of adult progenitor/stem cells in two-month-old mice , probably caused by increased protein levels of CDK4 , Cyclin D1 , and GSK3β as a result of missing translational regulation [2] . In Drosophila , FMR1 maintains germline stem cells in ovaries using the miRNA bantam [6] , and brains of FMR1 mutants display increased neuroblast proliferation rates with altered Cyclin E levels [7] . Recently , it was demonstrated that FMR1 associates with the RNA-binding protein Caprin in mice [8] and flies [9] to cooperate in binding to the same mRNA targets ( at least in flies [9] ) . In humans , Caprin-1 and Caprin-2 comprise the homologous region-1 ( HR1 ) and the homologous region-2 ( HR2 ) , which contain RGG motifs . Caprin levels have been correlated with proliferation , e . g . in human T- or B-lymphocytes [10] and the chicken lymphocyte line DT40 [11] . In contrast , inhibition of cell proliferation has been observed e . g . by overexpression of GFP-Caprin-1 in NIH-3T3 cells [10] . Caprin interacts with another RNA-binding protein , G3BP , and binds to growth-associated mRNAs , such as c-myc and cyclin D2 [4] . Drosophila Caprin ( Capr ) , which shares the HR1 domain and three RGG motifs but lacks the HR2 domain , cooperates with FMR1 to regulate the cell cycle via the repression of the CycB and Frühstart mRNAs at the mid-blastula transition in embryos [9] . G3BP consists of an NTF2-like domain and RNA-binding domains ( RRM and RGG ) . It has been implicated in translational control and mRNA decay of growth factors in mammalian model systems . For example , in quiescent Chinese hamster fibroblasts , human G3BP has been reported to bind to the c-myc 3′ UTR and to mediate myc mRNA decay [12] , [13] . Furthermore , in a FilaminC-RasGAP-dependent manner , G3BP regulates two RNA polymerase II kinases , Cdk7 and Cdk9 , at the mRNA level to control growth of cardiac myocytes [3] . However , in Drosophila , it is not known whether FMR1 , Capr and Rasputin ( Rin ) , the fly ortholog of G3BP , regulate cellular growth in epithelial tissues . In this study , we identify the UBA domain-containing protein Lingerer ( Lig ) as a novel interaction partner of FMR1 , Rin and Capr in flies and present genetic , biochemical and cell biological evidence that a complex of Lig with RNA-binding proteins restricts proliferation in growing tissues . Furthermore , we demonstrate that JAK/STAT signaling is activated in lig mutant cells .
In a tissue-specific genetic screen for suppressors of tissue growth [14] , we recovered a complementation group consisting of three EMS-induced recessive lethal alleles based on increased eye and head size ( Figure 1B and 1D ) . By subsequent mapping in combination with complementation tests , rescue experiments and sequencing , we identified lig as the gene responsible for the growth phenotype . lig encodes an conserved ubiquitin-associated ( UBA ) domain-containing protein . All three lig alleles , when placed over ligPP1 , a recessive lethal null allele [15] , or over the deficiency Df ( 2R ) Exel7094 uncovering the lig locus , resulted in lethality in an early pupal stage , forming long and slender pupae ( Figure 1F and Figure S1A and S1B ) as described for lig null mutants [15] . Both the lethality and the clonal overgrowth phenotype were rescued with one copy of a lig genomic rescue construct ( Glig ) ( data not shown and Figure 1C , 1D and 1Z ) but not with a genomic rescue construct containing a frameshift mutation in exon 10 ( GligFS ) ( data not shown and Figure 1Z ) . Sequence analysis of the lig protein-coding sequence of the EMS-induced alleles revealed small deletions ( lig1 , lig2 ) that result in premature stop codons and a point mutation ( lig3 ) , respectively ( Figure 1Z ) . We conclude that all three lig alleles represent null alleles . To determine whether the lig overgrowth phenotype is due to increased cell number or enlarged cell size , we analyzed tangential sections of mosaic compound eyes composed of lig mutant clones and wild-type sister clones surrounded by heterozygous cells . In lig mutant ommatidia , all cell types were normally differentiated and structured and without cell size defects ( Figure 1G ) , suggesting that the overgrowth phenotype is caused by more cells rather than larger cells . Analysis of adult lig mutant eyes revealed a variable ommatidia number . In most cases , the ommatidia number was increased as expected ( Figure 1B and 1D ) , but in some cases , the ommatidia number was equal or even lower than the number in control eyes ( Figure 1D ) . The ommatidia size was not altered in the lig mutant eyes ( Figure S1C ) . The increased or reduced ommatidia number of lig mutant eyes was completely rescued to a control situation by the presence of the Glig transgene ( Figure 1C and 1D ) , thus excluding a second-site mutation as the reason for the variability of the phenotype . Cellular growth is tightly linked to environmental factors like nutrient availability . The variability of the lig mutant eye phenotype might thus depend on food conditions . Indeed , animals raised on food with reduced yeast content ( 25% yeast and 40% yeast , respectively ) were delayed and displayed eyes with a constant increase in ommatidia number ( Figure 1I , 1N , S1E and S1H ) . In contrast , animals grown under normal food conditions ( 100% yeast ) displayed a high variability ( Figure 1K and 1N ) , and this effect was even more pronounced in flies from larvae that developed on food with increased yeast content ( 400% ) ( Figure 1M , 1M' and 1N ) . The diet-dependent phenotype of lig mutant eyes may be explained by varying amino acid levels or by altered developmental timing . To test the former possibility , larvae were grown on 40% yeast-containing food supplemented with the milk protein Casein to 100% protein content . However , this food condition did not increase the variability in lig mutant eyes ( Figure S1G and S1H ) , excluding altered total amino acid levels as the reason for the variable lig phenotype . To investigate the latter possibility , we made use of a Minute mutation to reduce the developmental speed under normal food conditions and to generate eyes largely mutant for lig with the eyFLP/FRT system . In a second experiment , we induced the developing delay by raising the flies at 18°C . Interestingly , the ommatidia number of lig mutant eyes was stably increased only in the Minute experiment ( Figure S1J and S1K ) but variable at 18°C ( Figure S1L ) . However , lig mutant eyes of flies raised on 25% yeast-containing food at 18°C produced a stable overgrowth phenotype ( Figure S1M ) , excluding a temperature sensitivity of lig mutant cells . These results suggest that the diet-dependent phenotype of lig mutant eyes is not dependent on amino acid levels or developmental delay but is probably influenced indirectly by an unknown diet-sensitive process . To investigate whether the variable phenotype is induced by increased apoptosis in lig mutant eyes , we overexpressed the Drosophila inhibitor of apoptosis ( DIAP1 ) or baculovirus caspase inhibitor p35 in lig mutant eyes to block apoptosis . Indeed , lig mutant eyes overexpressing DIAP1 displayed an increased ommatidia number ( Figure 1R and 1S ) in comparison to the control ( Figure 1O and 1S ) . Flies with lig mutant eyes overexpressing p35 were dying as pharate adult except for a few escapers that displayed massively overgrown eye structures ( Figure 1U ) . These results are consistent with published data that DIAP1 overexpression leads to reduced apoptosis rates without developmental consequences [16] , [17] , whereas p35 overexpression abolishes virtually all apoptosis but causes an aberrant morphology probably due to “undead cells” that activate compensatory proliferation ( reviewed in [17] ) . We conclude that lig mutant cells are sensitive to apoptosis . To test whether Lig acts as a general growth regulator , we generated two independent RNAi lines against lig to downregulate lig specifically in different developing tissues ( Figure 1Z ) . The functionality of both RNAi lines was established by ubiquitous expression ( using da-Gal4 as driver ) resulting in pupal lethality like lig mutants ( Figure S1O and S1P ) and by compartment-specific reduction of Lig protein levels in the developing eye using the DE-Gal4 driver line ( Figure S1R' and S1S' ) . Expression of lig RNAi in developing eyes increased the ommatidia number ( Figure S1U , S1V and S1W ) without effecting cell size ( Figure S1X ) under normal food conditions , similar to the lig mutant situation . Consistently , Lig reduction in developing wings by means of RNAi induced overgrowth ( Figure 1W , 1X and 1Y ) , identifying Lig as a general growth regulator . We next tested the effects of lig overexpression in the developing eye under different food conditions using the Gal4/UAS system . To this end , we generated two UAS-lig transgenic lines: UAS-lig is based on the wild-type coding sequence ( based on the release 5 . 45 of the Drosophila genome ) , and UAS-ligR185C/UTR encodes a protein version with an amino acid exchange ( published as wild type in [15] ) , including parts of the 5′ and 3′ UTRs of lig . Overexpression of the transgenes in the proliferating cells of the developing eye led to smaller adult eyes with fewer ommatidia ( Figure 2B , 2C , 2E , 2F , 2H , 2I and 2J ) , and similar effects were obtained for UAS-ligR185C ( Figure S2B and S2C ) , suggesting that the amino acid exchange R185C represents a polymorphism . Whereas the overexpression induced by UAS-lig mildly reduced the ommatidia number independently of the diet ( Figure 2B , 2E , 2H and 2J ) , UAS-ligR185C/UTR strongly decreased the eye size in a diet-dependent manner ( Figure 2C , 2F , 2I and 2J ) . The ligR185C/UTR overexpression eye phenotype was partially rescued in flies grown on 25% yeast-containing food ( Figure 2C , compare to 2F and 2I , 2J ) . Furthermore , lig overexpression in the developing wing led to strong reduction of the adult wing size ( Figure S2G and S2H ) . The ommatidia number of an adult eye depends on the survival and division rate of the cells during eye development . To investigate whether lig overexpression results in inappropriate apoptosis of proliferating cells , we analyzed lig overexpressing clones in the wing and eye imaginal discs of third instar larvae . Indeed , lig overexpressing cells were positive for the apoptosis marker Cleaved Caspase-3 in eye ( Figure S2E ) and wing imaginal discs ( Figure 2L ) , suggesting that an excess of Lig induces programmed cell death . Note that the effect was stronger in wing imaginal discs in comparison to the eye imaginal disc . Consistently , the reduced eye phenotype induced by ligR185C/UTR ( Figure 2N ) was partially rescued by co-overexpression of DIAP1 ( Figure 2P and 2S ) . In addition , the small eye phenotype was also ameliorated by expression of CycE ( Figure 2R and 2S ) . The suppression was further increased by co-overexpression of DIAP1 and CycE ( Figure 2U and 2V ) . These results suggest that the overexpression phenotype of lig is caused by increased apoptosis and reduced cell division . To elucidate the function of Lig , we attempted to identify binding partners of Lig using affinity purification coupled with mass-spectrometry ( AP-MS ) . In this experiment , HA epitope-tagged Lig interacted with Rin , FMR1 and DART1 , a functional Arginine methyl transferase , in Drosophila cultured cells ( Table S1 ) . A complex including Lig , Rin , FMR1 , Capr , and Orb ( oo18 RNA binding ) , the Drosophila cytoplasmic polyadenylation element binding ( CPEB ) protein , has been identified by co-immunoprecipitation ( CoIP ) in ovarian extracts using Orb as bait [18] . To confirm the interactions observed in the AP-MS experiment , we performed co-localization experiments with overexpressed epitope-tagged proteins in cultured Drosophila cells . Lig , FMR1 and Rin localized in punctae in the cytoplasm and were not observed in the nucleus ( Figure S3A , S3B and S3C ) . Co-overexpression of Lig , FMR1 and Rin ( pairwise and all three together ) ( Figure 3A''' , 3B''' and 3D''' ) or Lig and Capr revealed co-localization in cytoplasmic punctae ( 3C''' ) . In contrast , no co-localization was observed between Lig and DART1 ( Figure S3F''' ) . To test whether the endogenous proteins of Lig , Capr , FMR1 and Rin co-localize in cultured Drosophila cells , we transfected the cells with a Cherry-tagged Rin genomic rescue transgene ( GrinCherry ) and performed antibody stainings to visualize Lig , FMR1 and Capr . Rin-Cherry was homogeneously distributed in the cytoplasm ( Figure S3H ) . In some cases , we observed discrete punctae in the cytoplasm suitable for co-localization studies . Indeed , Lig , FMR1 and Capr co-localized with these punctae ( Figure S3I''' , S3J''' and S3K''' ) . However , when we analyzed Lig and Capr localization in cultured Drosophila cells by antibody staining , Lig and Capr co-localized only within bigger dots in few cells ( S3L''' ) . FMR1 interacts with the RISC complex [19] and co-localizes with a P-body marker in cultured Drosophila cells [20] . The co-localization of Lig and FMR1 suggested that Lig also localizes to P-bodies . Therefore , we tested whether Lig punctae overlap with the P-body markers DCP1 and Ago1 [21] using co-overexpression and antibody staining , respectively . Indeed , RFP-Lig and GFP-DCP1 co-localized in cultured Drosophila cells ( Figure 3E''' ) , and GFP-Lig punctae were positive for Ago1 antibody staining ( Figure 3F''' ) . Note that Ago1 was evenly distributed in small punctae in the cytoplasm of untransfected cells ( Figure S3G ) but accumulated in GFP-Lig dots of transfected cells . We conclude that Lig localizes to P-bodies in cultured Drosophila cells . Based on the localization experiments , we focused on the interaction between Lig , FMR1 , Rin and Capr . To test for direct interactions , we performed a yeast two-hybrid ( Y2H ) assay . Lig , FMR1 , Rin , and Capr were N-terminally fused to the activation domain ( AD ) or to the DNA-binding domain ( DBD ) of Gal4 , respectively , and tested for autoactivity ( Figure S4A ) . We used plates lacking adenine ( ADE ) to test for strong interactions and plates lacking histidine ( HIS ) for weak interactions . Lig interacted with Rin but not with FMR1 or Capr in the Y2H assay ( Figure 4A , and data not shown ) , identifying Rin as a direct interaction partner of Lig . The interaction between Lig and Rin was only visible when Lig and Rin were tagged with the AD and the DBD , respectively . To identify the interaction domain in Rin , we generated three Rin protein fragments: Rin1–175 consisting of the NTF2-like domain and the acid-rich region , Rin129–492 containing the acid-rich region and six PxxP motifs , and Rin445–690 containing the RNA recognition motif ( RRM ) and Arginine-Glycine rich region ( RGG ) ( Figure 4B and data not shown ) . In the Y2H assay , the fragment encompassing the NTF2-like domain interacted with Lig ( Figure 4A ) . Proteins with NTF2-like domains like NTF2 , TAP15/p15 and Importinβ have been shown to bind to FxFG , FG and GLFG repeats [22]–[24] . Recently , the structure of the Rin NTF2-like domain was resolved but binding sites for the FG motifs are not conserved [25] . However , analysis of Lig , which consists of a predicted UBA domain at the N-terminus and four conserved regions ( CR2-4 ) [15] ( Figure 4B ) , revealed two FGs in close proximity within the CR3 that could serve as a binding site for the NTF2-like domain of Rin . Indeed , when we mutated the FG repeat to a Leucine-Alanine ( LA ) repeat in Lig , the interaction between Rin and Lig was completely abolished ( Figure 4A and 4B ) . Thus , Rin is a direct interaction partner of Lig , and the interaction occurs via the NTF2-like domain of Rin and the FG repeat of Lig . The physical interaction of Lig with the RNA-binding domain-containing proteins Rin , FMR1 and Capr suggested that Lig is involved in an RNA-regulatory network and regulates growth via Rin , FMR1 and Capr . To investigate this possibility , we first focused on Rin and FMR1 that we identified as binding partners in the AP-MS experiments . No growth phenotypes in Drosophila epithelial tissues have been reported for rin and FMR1 mutants so far . To analyze a putative growth function of FMR1 and Rin , we used the FMR1 null mutant alleles FMR1D113M and FMR1D50M and the rin null mutant allele rin2 , respectively . Flies homozygous for the FMR1 alleles or the rin2 allele are viable and do not display obvious growth phenotypes . Note that rin2 contains a 13 kbp deletion removing the complete coding sequence of rin as well as the Rbp4 and Hrb87F loci ( Figure 5S ) . Hence , we attempted to identify additional rin alleles to exclude secondary effects of Rbp4 and Hrb87F . We wondered whether the P-elements P{GawB}rinNP3248 and P{GawB}rinNP5420 , inserted in the 5′ UTR of rin , are rin alleles and tested them with a Cherry-tagged Rin genomic rescue transgene ( GrinCherry ) . In the course of the rin rescue experiments , we identified a Rin dosage-dependent regulation of GrinCherry . Whereas Rin-Cherry was upregulated in rin mutant clones , Rin-Cherry was slightly downregulated in the sister clone , suggesting a tight regulation of rin to achieve wild-type levels of the gene product ( Figure 5S , S5A' and S5A'' ) . Indeed , cells homozygous for either of the P-elements upregulate RinCherry , verifying both P-elements as rin alleles ( Figure S5B' , S5B'' , S5C' and S5C'' ) . Both P-elements placed over rin2 were viable without phenotypic alterations ( data not shown ) . In the eyFLP/FRT experiment , rin2 but not the rin P-elements or FMR1 mutant eyes showed an increase in ommatidia number ( Figure 5B–5D , S5E–S5G ) under normal food conditions ( 100% yeast ) . In contrast to the lig mutant phenotype , we never observed a variability of the ommatidia number in FMR1 or rin mutant eyes under normal food conditions . Thus , the single mutant phenotypes of FMR1 and rin did not display growth phenotypes similar to the effects caused by lig . In a next step , we tested for functional redundancy using a double mutant situation of rin and FMR1 since FMR1 and Rin are dispensable for viability and are both RNA-binding proteins that co-localize in cultured Drosophila cells . Most rin2 , FMR1D113M homozygous larvae died at an early stage but few escapers that reached the early pupal stage formed long , slender pupae ( Figure 5F ) , reminiscent of the lig null mutant phenotype . Consistently , P{GawB}rinNP3248 , FMR1D50M or P{GawB}rinNP5420 , FMR1D50M over rin2 , FMR1D113M also resulted in long slender pupae ( Figure S5K and S5Q ) . Note that pupae with the P-element P{GawB}rinNP3248 reached a late pupal stage , and pupae with the P-element P{GawB}rinNP5420 developed into adult flies that were dying soon after eclosion . In both combinations , the pupal phenotype and the lethality were rescued by the presence of the GrinCherry transgene ( Figure S5M , S5S and data not shown ) . The two P-elements are therefore likely to represent hypomorphic alleles of rin . We then tested for a redundant function of FMR1 and Rin in growth control by using the eyFLP/FRT system to generate FMR1 , rin double mutant eyes under different food conditions . rin2 , FMR1D113M double mutant eyes consisted of more ommatidia under normal food conditions ( Figure 5H and 5K ) . The double mutant phenotype was rescued to a rin2 and FMR1D113M like phenotype by the presence of a FMR1 genomic rescue transgene ( P{Fmr1 . 14} ) ( Figure 5I and 5K ) and a genomic rin rescue transgene ( Grin ) ( Figure S5T ) , respectively , suggesting a complete rescue for FMR1 and Rin function . However , by the presence of GrinCherry the double mutant eyes were not completely rescued to a FMR1D113M mutant situation ( Figure 5J and 5K ) , suggesting that the C-terminal tag impairs Rin activity . Like in lig mutants , FMR1 rin double mutant eyes were stabilized at reduced food conditions ( 25% yeast ) ( Figure 5M and 5P ) but variable at rich food ( 400% yeast ) ( Figure 5O , 5O' and 5P ) . Furthermore , overexpression of p35 in FMR1 rin double mutant eyes resulted in pharate adults except for some escapers displaying massively overgrown eyes ( Figure 5R ) . Taken together , FMR1 rin double mutant eyes , but not the single mutants , displayed a lig like phenotype , suggesting a functional relationship between lig , FMR1 and rin . Recently , Capr2 null mutants were described to be viable without morphological alterations , and Capr and FMR1 cooperatively regulate the cell cycle at the mid-blastula transition [9] . We wondered whether Capr acts redundantly with FMR1 and Rin in growth control in epithelial tissues . To characterize the Capr growth phenotype , we generated mutant eyes during development using the eyFLP/FRT technique or by downregulation of Capr via RNAi . Note that we used a Minute mutation instead of a cell lethal mutation on the FRT80 chromosome . CaprRNAi overexpression in clones resulted in a strong reduction of Capr protein ( Figure S6A' ) , proving the functionality of the RNAi line . Both Capr2 null mutant eyes and eyes with downregulated Capr displayed slightly reduced eye size in comparison to the controls ( Figure 6B , 6C , 6E and 6L ) . In contrast , downregulation of Capr in FMR1 or rin mutant eyes resulted in overgrown eyes due to more ommatidia ( Figure 6G , 6I and 6L ) . Furthermore , downregulation of Capr in FMR1 rin double mutant eyes resulted in late pupal lethality . Analysis of eyes and heads revealed strongly overgrown structures in pharate adults ( Figure 6K ) , suggesting that FMR1 , Rin and Capr act synergistically in growth regulation . The similarity of the lig and the FMR1 , rin or Capr phenotypes in combination of double mutants prompted us to genetically test whether Lig regulates growth via FMR1 , Rin and Capr . We downregulated lig via RNAi in FMR1 , rin or Capr mutant eyes induced by the eyFLP/FRT system . Note that lig RNAi eyes did not consist of more ommatida under reduced food conditions ( Figure 7B and 7H ) in comparison to flies raised under normal conditions ( Figure S1W ) . Reduced Lig levels in FMR1 or rin mutant eyes increased the eye size due to more ommatidia ( Figure 7D , 7F and 7H ) . Flies with Capr mutant eyes and reduced lig were dying as pharate adults with increased and disturbed eye structures ( Figure 7J ) . We conclude that Lig cooperates with FMR1 , Rin and Capr in growth control . The fact that the single mutants of FMR1 , rin or Capr have no or minor effects on growth regulation , whereas the double mutants have similar effects like lig mutants , suggests that Lig modulates FMR1 , Rin and Capr function in concert . Next we checked the localization and protein levels of FMR1 , Capr and Rin in lig mutant clones induced by the hsFLP/FRT system in developing eyes . Whereas FMR1 showed no localization or abundance alterations ( Figure 7K'' ) and Capr only a slight upregulation ( Figure 7L'' ) in lig mutant cells , Rin-Cherry levels were reduced in lig mutant clones ( Figure 7M'' ) , indicating that Lig mainly regulates Rin levels . Vice versa , Rin-Cherry levels were upregulated in lig overexpressing clones in eye imaginal discs ( Figure S7A' and S7A'' ) . Recently , Rin has been identified as substrate for ubiquitination in the central nervous system [26] . To test whether Lig regulates Rin at the protein level , we induced lig null mutant clones in eye imaginal discs expressing a HA-tagged Rin under the control of an UAS promoter . In this situation , Lig was not able to regulate Rin ( Figure S7B' and S7B'' ) , excluding Lig as stabilizer of the Rin protein . We then investigated whether Lig regulates rin at the transcriptional and/or translational level . Lig overexpression in S2 cells was able to increase Rin-Cherry expressed by GrinCherry ( Figure 7N ) . To generate a translational reporter , we placed the 5′ and 3′ UTRs of rin mRNA upstream and downstream of a Cherry-coding region under control of the ubi promoter . The transcriptional reporter expressed the Cherry-coding sequence and the 3′UTR of rin under control of the rin promoter . Co-expression of lig with the transcriptional reporter construct , but not with the translational reporter , was able to increase Cherry levels , suggesting that Lig impacts on rin transcription ( Figure 7N ) . We demonstrated that Lig regulates cell proliferation in concert with the mRNA binding proteins FMR1 , Rin and Capr . To investigate which growth signaling pathway is altered , we tested readouts for various signaling pathways in lig mutant clones in wing and eye imaginal discs . FMR1 binds to the miRNA bantam to control the fate of germline stem cells [6] . bantam miRNA is a known target of the Hippo signaling pathway [27] , [28] and inhibits the pro-apoptotic gene hid [29] . If Lig regulates the Hippo pathway and/or bantam miRNA , we would expect an upregulation of a minimal Hippo response element ( DIAP4 . 3-GFP ) and downregulation of the bantam sensor . In both experiments we did not observe any alteration of the reporter signal ( Figure S8A'' , S8B'' ) . Consistently , overexpression of lig did not upregulate the bantam sensor ( S8C'' ) . Furthermore , FMR1 was reported to regulate cbl mRNA , a negative regulator of the EGFR , to control germline cell proliferation in ovaries [30] . However , a transcriptional reporter for pointed expression , a target of the EGFR pathway , was not changed in lig mutant clones in eye imaginal discs ( Figure S8D'' ) . Recently , increased Insulin signaling has been observed in FMR1 mutant brains using pAkt as readout [31] . In lig mutant clones in eye imaginal discs , we observed neither an increase of pAkt nor a recruitment of pAkt to the membrane , a sign for active Insulin signaling ( Figure S8E'' ) . The Rin ortholog G3BP is involved in human c-myc mRNA decay by an intrinsic endonuclease activity [12] , [13] . However , we did not detect any alterations of Myc levels in lig mutant clones ( Figure S8F'' ) . Recently , it was demonstrated that G3BP is involved in Wnt/β-catenin signaling by binding and regulation of β-catenin mRNA [5] . To test an involvement of Lig via Rin in Wnt signaling , we stained imaginal discs harboring lig mutant clones for Distal-less ( Dll ) and Senseless ( Sens ) , two target genes of the Wnt signaling pathway in Drosophila . We did not observe any alterations of the Dll expression pattern in wing imaginal discs ( Figure S8G'' ) or of the Sens expression patterns in wing ( Figure S8H'' ) and eye imaginal discs ( Figure S8I'' ) , arguing against an involvement of Lig in Wnt signaling . We also tested Hedgehog , Notch and JAK/STAT signaling . Whereas Ptc and Cut patterns , targets of the Hedgehog and Notch signaling pathway , respectively , were not altered in lig mutant clones ( Figure S8J'' and S8K'' ) , a JAK/STAT reporter ( 10xSTAT92E-GFP ) was upregulated in lig mutant clones . GFP expression from the 10xSTAT92E-GFP reporter was autonomously increased in lig mutant clones in the posterior region of eye discs ( Figure 8A'' ) , in antenna discs ( Figure 8B'' ) and in the pleura and hinge regions of wing discs ( Figure 8C'' ) of early third instar larvae . Consistent with our findings , Lig was identified as negative regulator of JAK/STAT signaling in an RNAi based screen in cultured Drosophila Kc cells [32] . To determine whether Lig has an effect on STAT92E protein levels , we analyzed STAT92E expression in lig mutant clones in eye imaginal discs . We did not observe any alteration of STAT92E levels in the posterior region but an upregulation of STAT92E in the anterior region of the eye imaginal disc ( Figure S8L'' ) . Thus , based on the autonomous effects on the 10xSTAT92E-GFP reporter and on STAT92E levels , Lig regulates intracellular components of the JAK/STAT signaling pathway rather than the ligands .
We have identified Lig as a new growth suppressor in eye and wing epithelial tissues . Whereas eyes mutant for lig consist of more ommatidia without cell size defects , eyes overexpressing lig have a reduced cell number due to increased apoptosis and reduced cell cycle progression . lig mutant eyes are sensitive to apoptosis ( resulting in a variable phenotype under normal food conditions ) but are able to cope with the overgrowth situation when the flies develop under suboptimal growth conditions . Similarly , the reduced eye phenotype of lig overexpressing eyes was partially rescued under suboptimal growth conditions or by expression of DIAP1 , suggesting that the starvation response impacts on the apoptosis rates in imaginal discs . However , we cannot exclude other indirect effects that might be triggered by starvation . In addition to our findings , lig mutants have previously been characterized for their behavioral phenotype in the copulation process [15] and their putative role in neuronal tissues [33] . Lig is conserved from flies to humans , the human orthologs being ubiquitin associated protein 2 ( UBAP2 ) and ubiquitin associated protein 2 like ( UBAP2L ) . UBAP2 has been identified in a Y2H screen as a direct interaction partner of the zona pellucida 3 ( ZP3 ) protein that is involved in sperm binding and acrosomal exocytosis [34] . UBAP2L has been reported to accumulate at ubiquitin-rich aggregates upon proteasome inhibition in human neuroblastoma tissue culture cells , suggesting that the UBA domain is functional [35] . It is currently unknown whether the Lig orthologs are involved in growth regulation , and no interaction partners have been identified except for ZP3 . Several lines of evidence indicate that Lig interacts with FMR1 , Capr and Rin , and via these interactions functions to regulate growth: ( i ) Lig associated with FMR1 and Rin in an AP-MS experiment , ( ii ) Lig co-localized with FMR1 , Capr and Rin , ( iii ) Lig directly interacted with Rin in a Y2H experiment , ( iv ) Lig transcriptionally regulated Rin levels , and ( v ) FMR1 , Capr or rin in combination of double mutants behaved like lig null mutants and ( vi ) lig downregulation in FMR1 , Capr or rin mutant eyes synergistically increased the eye size ( Figure 8D ) . The interaction between Lig and Rin , Capr and FMR1 , three RNA-binding proteins , and the co-localization with P-body components suggests that Lig regulates the translation and/or stability of specific mRNAs of growth-regulatory genes via FMR1 , Capr and Rin function . Indeed , the Drosophila FMR1 and orthologs of Rin are involved in translational regulation of growth-regulatory genes in certain tissues . For example , FMR1 binds bantam miRNA , an inhibitor of the pro-apoptotic gene hid [29] , and regulates cbl , which encodes a component of the EGFR signaling pathway , in germline stem cells [30] . However , bantam miRNA is not regulated by FMR1 in epithelial cells [36] , and Lig was unable to regulate a bantam miRNA reporter . Furthermore , the expression of a pointed transcriptional reporter was unchanged in lig mutant clones , suggesting that cbl regulation by FMR1 is specific to the germline or has only subtle effects in the developing eye . The Rin ortholog G3BP controls myc [12] , [13] , CyclinD2 [4] , cdk7 and cdk9 mRNA [3] . However , it is not known whether this function is conserved for Rin , and we did not observe any alterations of Myc protein levels in lig mutant clones . It will be important to identify mRNAs that are regulated by FMR1 , Capr and Rin in epithelial tissues during development , and to determine whether Lig mediates specificity for certain mRNAs . To identify the signaling pathway that is regulated by Lig , we used readouts for the Hippo , EGFR , Insulin , Hedgehog , Wnt and JAK/STAT signaling pathways . We observed no alterations of all analyzed pathways except for the highly conserved JAK/STAT signaling pathway . The pathway is composed of four modules: the ligands , Upd , Upd2 and Upd3 , the receptor Domeless ( Dome ) , the receptor-associated Janus kinase ( JAK ) Hopscotch ( Hop ) , and the signal transducer and activator of transcription ( STAT ) STAT92E ( reviewed in [37] ) . The involvement of Lig in the JAK/STAT signaling pathway leads to a number of assumptions and questions in the context of our findings . First , the autonomous effect of Lig on the 10xSTAT92E-GFP reporter suggests that Lig regulates the intracellular components ( Dome , Hop or STAT92E ) or modifiers thereof rather than expression of the ligands , which would result in non-autonomous effects . Second , the physical and genetic interactions of Lig with the mRNA binding proteins FMR1 , Capr and Rin raises the question whether Lig directly impacts on the JAK/STAT pathway or whether it modulates the JAK/STAT signaling via FMR1 , Capr and Rin . So far , we cannot exclude either option . However , it was recently demonstrated that upd and STAT92E mRNAs are targets for posttranscriptional regulation via the miRNA pathway [38] , [39] . It will be interesting to determine whether FMR1 , Rin or Capr are involved in this process in the case of STAT92E . Our data provide evidence that FMR1 , Capr and Rin function in a redundant manner in epithelial tissues in growth control , suggesting that they regulate either overlapping sets of mRNAs or different mRNAs encoding proteins with redundant functions . Examples for the former have been described for FMR1 , Capr and G3BP , the human ortholog of Rin . In Drosophila , FMR1 cooperates with Capr , and both proteins bind to the same mRNAs frs and CycB [9] . Similarly , G3BP forms a complex with human Caprin and both interact with myc and CycD mRNAs [4] . Both examples suggest a redundant regulation of these targets . There is no direct evidence for the latter possibility . However , G3BP associates with and translationally regulates tau mRNA in neuronal cells [40] , [41] . In Drosophila , FMR1 negatively regulates futsch mRNA [42] , and the futsch mutant phenotype is suppressed by overexpression of Tau [43] , suggesting a redundant function of Tau and Futsch . Lig impacts on Rin and slightly on Capr but not on FMR1 levels . However , only FMR1 , Capr or rin mutants in combination as double mutants resulted in a lig like phenotype , suggesting that the activity of FMR1 and Capr is altered ( probably at the posttranslational level ) in a lig mutant situation . Our AP-MS experiments also revealed DART1 as a physical binding partner of Lig . Arginine methyl transferases are able to methylate RGG motifs and thereby modulate the binding capability to mRNAs [44] , [45] . Interestingly , FMR1 contains a conserved RGG domain that can be methylated in Drosophila and humans . In humans , protein methyl transferase 1 ( PRMT1 ) , the ortholog of DART1 , mediates the arginine methylation of FMR1 to alter its binding affinity to mRNAs [46] ( Figure 8D ) . Furthermore , G3BP1 , the mouse ortholog of Rin , contains an RGG domain that is methylated by PRMT1 after stimulation of the Wnt signaling pathway to modulate the binding to β-Catenin mRNA [5] . The RGG domain of Rin is weakly conserved and lacks the RGG motifs . It is thus unclear whether Rin can be methylated in the truncated arginine-glycine rich region . Like FMR1 and G3BP , Caprin contains RGG domains , and it was identified as binding partner of PRMT8 , which is closely related to PRMT1 at the sequence level [47] . Further experiments are required to resolve whether Lig is involved in a DART1-mediated methylation of FMR1 and Rin under certain conditions , or whether Lig alters the activity of FMR1 and Capr by another mechanism . Lig , FMR1 , Rin and Capr have been identified as interactors of Orb in Co-IP experiments [18] , suggesting a complex formation of these proteins . Complex formation has been reported for G3BP and Caprin in human cell lines [4] and for Capr and FMR1 in Drosophila [9] and mouse neurons [8] so far ( Figure 8D ) . We were able to demonstrate that Rin , Capr and FMR1 have a redundant function in the eye , and that they localize in the same subcellular structure in cultured Drosophila cells . This raises the question whether the three RNA-binding proteins Capr , Rin and FMR1 are functionally related only in the eye . Systematic analyses of the phenotypes of double mutant combinations will reveal the tissues in which these RNA-binding proteins exert redundant and non-redundant functions . Furthermore , it will be interesting to determine whether Rin and Capr contribute to phenotypes associated with the FXS .
EMS-induced lig mutant alleles were recovered in an unbiased eyFLP/FRT cell lethal screen [14] . lig1 harbors a small deletion of 5 bp ( nucleotides 3959163–3959167 ) and an insertion of an adenine at position 3959174 . lig2 includes a small deletion of 17 bp ( nucleotides 3958424–3958440 ) . The nucleotide positions are based on the release 5 . 45 of the Drosophila genome . lig3 contains a point mutation changing W155 into a stop codon . The following FMR1 , rin and Capr alleles and transgenes were used: ligPP1 [15] , Df ( 2R ) Exel7094 ( BDSC no . 7859 ) , Glig [61B3] [15] , GligFS [86Fb] , FMR1D113M ( BDSC no 6929 , [42] ) , FMR1D50M ( BDSC no 6930 , [42] ) , rin2 ( BDSC no 9303 , [48] ) , P{GawB}rinNP3248 ( DGRC no 104425 ) , P{GawB}rinNP5420 ( DGRC no 113726 ) , Capr2 [9] , UAS-CaprRNAi ( VDRC 110272 ) . The alleles FMR1D113M and FMR1D50M , rin2 , P{GawB}rinNP3248 , P{GawB}rinNP5420 were recombined onto FRT82 . The presence of FMR1D113M and FMR1D50M as well as of rin2 deletions was verified by PCR using the primer pairs FMR1_F , FMR1_R and Rin_F , Rin_R , respectively . Sequencing of the PCR product generated with the primer pair Rin_F , Rin_R revealed the break points of the rin2 deficiency at positions 9473220 and 9486306 . The eyFLP/FRT-cell lethal recombination system [49] or eyFLP/FRT M was used to generate mutant heads . To express UAS transgenes in clones in eye and wing imaginal discs , the Actin-Flp out-Gal4 technique was used [50] . Clones were induced in second instar larvae ( heat shock for 10 min at 37°C , 48 hours before dissection ) , and the imaginal discs were dissected from third instar larvae . Negatively marked mutant clones were generated with the hsFLP/FRT-ubiGFP system . Clones were induced in first instar larvae ( heat shock for 15 min at 37°C , 72 hours before dissection ) , and the eye imaginal discs were dissected from third instar larvae . Additional fly strains used in this study were: nubbin-Gal4 [51] , da-Gal4 ( BDSC ) , DE-Gal4 [52] , ey-Gal4 ( insertion on 2nd chromosome ) [53] , UAS-CycE [54] , EP-Diap1 ( BDSC ) , P{Fmr1 . 14} [55] , UAS-p35 ( BDSC ) , DIAP1-GFP4 . 3 [56] , 10xSTAT92E-GFP [57] , MIR33 bantam sensor ( gift from Stephen Cohen ) , pnt-lacZ ( P{lacW}pntS0998 , former stock collection of Szeged , No . 121625 ) . Genetic experiments were conducted at 25°C . Food with 100% yeast consists of 7 . 5 g sugar , 5 . 5 g corn , 1 g flour , 0 . 8 g Agar , 1 . 5 ml Nipagin/Nipasol and 10 g fresh yeast filled up to 100 ml with tap water . For fly food with 25% or 40% yeast , the yeast amount was reduced to 2 . 5 g and 4 g yeast , respectively . 3 . 3 g Casein was used to substitute 40% yeast-containing food to 100% amino acid-containing food . For fly food with 400% yeast , the yeast amount was 40 g fresh yeast . 10 ml of food was filled into vials with a diameter of 29 mm . For experiments with different food conditions , 100–150 embryos of each cross were collected from apple agar plates and distributed to individual vials . To assess the ommatidia number , flies were exposed to dimethyl ether for 7–10 min before taking scanning electron micrographs with a JEOL 6360 VP microscope . The ommatidia number was counted using a semi-automated ommatidia counter software ( Ommatidia counter , version 0 . 3 , programmed by Vasco Medici , SciTrackS GmbH ) . Pictures from pupae and adult wings were taken with a Keyence VHX-1000 microscope . Tangential eye sections of adult eyes were done as previously described [58] . The Glig was subcloned from pCaSpeR-Glig [15] into the gattb vector using the restriction sites XhoI and XbaI . The frameshift in the GligFS construct was obtained as a spontaneous mutation during the subcloning . For the lig RNAi lines , the regions I ( 308 bp ) and II ( 252 bp ) were amplified with the primer pairs Lig_RNAi_FB , Lig_RNAi_RB and Lig_RNAi_FC , Lig_RNAi_RC , respectively , using pENTR-lig as template . The fragments were first digested with EcoRI and then self-ligated . The resulting inverted repeats were cloned into a modified gattb vector , attB-genxpMF3 . attB-genxpMF3 was generated by cloning a fragment of the pMF3 vector containing the promoter , restriction sites for subcloning of the hairpin and the polyA signal , into the gattb vector using the restriction sites NotI and BamHI . The ligR185C/UTR sequence was subcloned from pUAST-ligR185C/UTR ( gift from Yamamoto lab ) into the pUAST attb vector using the restriction site EcoRI . The lig coding region sequence was amplified from pUAST-ligR185C and cloned into the pENTR vector . Site-directed mutagenesis was used to obtain the lig coding region without the C553T substitution that causes the amino acid exchange R185C . Analysis of UAS-ligR185C revealed similar phenotypes as observed for UAS-lig ( Figure S2B and S2C ) , suggesting that the amino acid exchange R185C represents a polymorphism . pENTR-ligFG-LA was generated by site-directed mutagenesis with the primers LigF_LA and LigR_LA using pENTR lig as template . The coding sequence of rin was cloned into pENTR . LR reaction was used to subclone the coding sequences from pENTR-lig and pENTR-rin into the Gateway vectors pUAST-W-attb and pUAST-HW-attb . The gattB-Grin and gattB-GrinCherry vectors were cloned in two and three steps , respectively . A fragment of 7 . 2 kbp from the P[acman] BAC 13D12 [59] was subcloned into a modified gattb vector using BamHI and AgeI restrictions sites . In the second step , a PCR-amplified fragment of 4 . 6 kbp ( using the primer pair Rin_FA , gRin_R ) was subcloned into the gattb vector containing the 7 . 2 kbp Grin fragment using the restriction sites AgeI and NotI , resulting in the construct gattB-Grin . A cherry coding sequence including a stop codon was fused to the third exon of rin without stop codon and to the 3′ UTR of rin by fusion PCR . Transgenic flies were generated with the site-specific phiC31 integration system using vas-φC31-zh2A; ZH-attP-44F , vas-φC31-zh2A; ZH-attP-51D and vas-φC31-zh2A; ZH-attP-86Fb embryos [60] . S2 cells were cultured and transfected according to standard protocols . The coding sequences of FMR1 , Capr and DART1 were cloned into pENTR . LR reactions were performed to subclone the coding sequences from pENTR-GFP , pENTR-FMR1 , pENTR-Capr , pENTR-DART1 , pENTR-rin , pENTR-lig , pENTR-ligR185C into the Gateway vectors pMHW , pAGW , pARW and pAFW . GFP-DCP1 was used as a P-body marker [61] . For the rin translational reporter construct , the two parts of the 5′ UTR of rin were amplified with the primer pairs EcoRI_Rin_F , Rin_RA and Rin_FB , NotI_Rin_R , respectively , from genomic DNA of y w flies , fused by fusion PCR and subcloned into the gattb vector containing an ubi promoter using the restriction sites EcoRI and NotI . The coding sequence of cherry fused to the 3′ UTR of rin was amplified with the primer pair NotI_Cherry_F , XbaI_Rin_R from the template gattB-RinCherry and subcloned into the gattb-ubi-5′ UTR rin vector using the restriction sites NotI and XbaI . For the rin transcriptional reporter , the ubi-5′ UTR of rin of the translational reporter was replaced with the rin promoter that was amplified with the primer pair gattB_F , Rin_RG from the template gattB-GrinCherry . Western blots were performed according to standard protocols . AP-MS analysis was done as described in [62] . RinF , 5′-CACCATGGTCATGGATGCGACCC-3′; RinR , 5′-GCGACGTCCGTAGTTGCC-3′; FMR1_FB , 5′-CACCATGGAAGATCTCCTCGTG-3′; FMR1_RB , 5′-GGACGTGCCATTGACCAG-3′; Lig_RNAi_I_F , 5′-GAGAATTCCAGCAGCAGACGACGCCTATCA-3′; Lig_RNAi_I_R , 5′-CATCTAGATTCGAGGGTGGTGGCAGCTT-3′; Lig_RNAi_II_F , 5′-GAGAATTCCCACAAATACCGGCAGCAAACA-3′; Lig_RNAi_II_R , 5′-CATCTAGAGTTAAACGGGGGCGGAGTGC-3′; LigF_LA , 5′-GGACGTGCAGTTaGcCGCTCTGGACTTaGcCACGGACGATGG-3′; LigR_LA , 5′- CCATCGTCCGTGgCtAAGTCCAGAGCGgCtAACTGCACGTCC-3′; EcoRI_Rin_F , 5′-TAGAATTCATCATTCACACACCAACACACG-3′; Rin_RA , 5′-CCTAGACGACTGTGTAGCTTTTTTAAGCGATATTTTTCCTCG-3′; Rin_FB , 5′-CGCTTAAAAAAGCTACACAGTCGTCTAGGACTTTTGC-3′; NotI_Rin_R , 5′-ATCGCGGCCGCAGCTGGCGTTTGATTCTTCCTC-3′; NotI_Cherry_F , 5′-TAGCGGCCGCGATGGTGAGCAAGGGCGAGGAGG-3′; XbaI_Rin_R , 5′-ATTCTAGAGTTGCTTGACTTAGTTTGGTTTACG-3′; gattB_F , 5′-GAAAATGCTTGGATTTCACTGG-3′; Rin_FA , 5′-GGTGGCACCACAGCTCAT-3′; gRin_R , 5′-TAGCGGCCGCCAGGCGATTCCGTTCAAGATATTTAATAAATAATAAAG-3′ . S2 cells or eye imaginal discs were fixed in 4% PFA at RT for 20 min and blocked with 2% NDS in 0 . 3% PBT or 1% BSA in 0 . 3% PBT ( only for rabbit α-Cleaved Caspase-3 antibody ) . The following primary and secondary antibodies were used: mouse α-Ago1 ( 1∶300; [63] ) , rabbit α-Cleaved Caspase-3 ( 1∶300 , Cell signaling , Catalog no . 9661 ) , mouse α-Lig-N ( 1∶300 ) [15] , mouse α-FMR1 clone 6A15 ( 1∶300 ) [64] , rabbit α-Capr ( 1∶1000 ) [9] , mouse α-FLAG ( Sigma , F1804 ) , mouse α-HA ( Convance , MMS-101R ) , mouse α-GFP ( Roche , 11814460001 ) , mouse α-mCherry ( Abcam , ab125096 ) , rabbit α-pAkt ( Ser 473 ) ( 1∶300 , Cell signaling , 9277S ) , rabbit α-Myc ( 1∶5000 ) [65] , mouse α-Dll ( Ian Duncan; gift from K . Basler ) , guinea pig α-Sens ( GP55 , 1∶800 , H . Bellen , Baylor College of Medicine , Houston; gift from K . Basler ) , mouse α-Ptc ( 1∶100 , DSHB ) , mouse α-Cut 2B10 ( 1∶100 , DSHB ) , rabbit α-STAT92E ( 1∶1000 ) [66] , goat α-rabbit Cy3 ( GE Healthcare , PA43004 ) , goat α-mouse Cy3 ( GE Healthcare PA43002 ) , α-mouse Cy5 ( GE Healthcare , PA45002 ) , α-mouse HRP ( Jackson ImmunoResearch , 115-035-003 ) . Pictures were taken using a Leica SPE or SP2 confocal laser scanning microscope . Yeast two-hybrid analysis was carried out using Invitrogen's ProQuest Two-Hybrid System with Gateway Technology according to the manufacturer's instructions . Full- length cDNAs and the cDNA fragments of lig , FMR1 , Capr , and rin , and lig256–1333 , ligFG-LA , rin1–175 , rin129–492 and rin445–689 , respectively , were cloned into the Gal4 DNA-binding domain vector pDEST 32 as well as into the Gal4 activation domain vector pDEST 22 . Plasmids were transformed into yeast strain AH109 and plated on SD-Leu-Trp-Ade and SD-Leu-Trp-His ( supplemented with 2 mM 3-AT ) , respectively . | Animal growth is orchestrated by controlled expression of growth-regulatory factors . This regulation is achieved at different molecular levels like transcription , translation initiation , and translational regulation . Whereas transcriptional control and translation initiation of growth components have been well studied , the role of translational control in this process is less well understood . Here , we describe Lingerer ( Lig ) , an UBA domain-containing protein , as a new growth suppressor that associates with the three RNA-binding proteins Fragile X mental retardation protein 1 ( FMR1 ) , Rasputin ( Rin ) and Caprin ( Capr ) . Drosophila FMR1 , Rin and Capr orthologs are known translational regulators . In lig mutants and in FMR1 , Capr and rin in combination as double mutants , organ size is increased due to excess proliferation . These data unveil a growth-regulatory function of Lig , and a redundant function of the RNA-binding proteins FMR1 , Capr and Rin . Our findings demonstrate the involvement of mRNA-binding proteins in epithelial growth control and may also contribute to a better molecular understanding of the Fragile X mental retardation syndrome . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"genetics",
"developmental",
"biology",
"biology"
] | 2013 | The RNA-binding Proteins FMR1, Rasputin and Caprin Act Together with the UBA Protein Lingerer to Restrict Tissue Growth in Drosophila melanogaster |
Familial Dysautonomia ( FD ) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues . Here we generated a conditional knockout ( CKO ) mouse in which exon 20 of IKBKAP is deleted in the nervous system . The CKO FD mice exhibit developmental delays , sensory abnormalities , and less organized dorsal root ganglia ( DRGs ) with attenuated axons compared to wild-type mice . Furthermore , the CKO FD DRGs show elevated HDAC6 levels , reduced acetylated α-tubulin , unstable microtubules , and impairment of axonal retrograde transport of nerve growth factor ( NGF ) . These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms . Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin . Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 ( HDAC6 ) levels and thus increasing acetylation of α-tubulin levels . Thus , we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration .
Familial Dysautonomia ( FD ) is an autosomal recessive congenital neuropathy that occurs almost exclusively in the Ashkenazi Jewish population with a remarkably high carrier frequency ranging from 1 in 18 ( in those of Polish descent ) to 1 in 32 [1] . Individuals with FD suffer from a variety of symptoms including vomiting crises , pneumonia , ataxia , difficulty swallowing , gastrointestinal and cardiovascular dysfunction , and short life spans [2–6] . Previous work discovered that the underlying genetic cause of FD is a point mutation in the IKBKAP gene , which encodes the IκB kinase complex-associated protein ( IKAP ) [7 , 8] . A transition from T to C at position 6 of the 5’ splice site of IKBKAP intron 20 [8] alters the splicing pattern of the IKBKAP gene in a tissue-specific manner: There is a shift from constitutive inclusion of exon 20 to alternative splicing in all tissues , and in the nerve tissues this exon is predominantly skipped [9] . As a result of the exon 20 skipping , a premature stop codon is generated . No truncated protein has been detected in tissues of FD patients [8 , 10 , 11]; however , there is a considerable reduction in full-length IKAP protein expression in the nervous systems of FD patients [8 , 11] . FD patients exhibit abnormal development and progressive depletion of unmyelinated sensory and autonomic neurons [12–16] . Although the central neuropathology in FD is poorly defined , recent MRI studies indicate that FD patients have abnormal proportions of white matter , decreased optic radiation , and cerebellar microstructural alterations compared to healthy volunteers [17] . The lack of IKAP also results in reduced size and numbers of dorsal-root ganglion ( DRG ) and sympathetic ganglion ( SG ) neurons [13 , 18–20] . DRGs are highly polarized cells that depend on active intracellular transport mechanisms in order to survive and properly function . Postsynaptic targets release neurotrophins like nerve growth factor ( NGF ) that move in a retrograde fashion along the axon to the soma to evoke changes in gene expression [21 , 22] . Although alterations in this axonal transport process is linked to many neurodegenerative diseases and may be involved in FD [19 , 23 , 24] , the molecular mechanism that underlies the alterations in transport is unknown . IKAP has been studied extensively , and findings point to an unexpected diversity of IKAP actions . Early findings indicated that IKAP ( also known as ELP1 ) is a subunit of the Elongator complex , important for RNA polymerase II transcription elongation in the nucleus and for histone acetylation [25–29] . As IKAP co-localizes and purifies with cytoplasmic proteins [30–32] , it has been suggested that IKAP functions in tRNA modification [33–35] , exocytosis [36] , cell adhesion and migration , microtubule organization [20 , 32 , 37 , 38] , p53 activation [39] , and c-Jun N-terminal kinase ( JNK ) signaling pathway regulation [19 , 23 , 31] . Recent studies focused on IKAP function in neurons suggest that IKAP influences oligodendrocyte differentiation and myelin formation [40 , 41] , is crucial for vascular and peripheral neural development during embryogenesis [20 , 42 , 43] , regulates NGF signaling , and distributes target innervations [19 , 20] . Deletion of IKAP in migrating neural crest further documented a key role for IKAP in DRG progenitors for correct timing of neurogenesis and survival of TrkA+ nociceptors and thermoreceptors [44] . These findings demonstrate that IKAP plays an essential role during neuronal development . FD patients exhibit progressive DRG neurodegeneration , but the underlying molecular mechanism by which IKAP deficiency result in this degeneration has still not been established . Here we evaluated how IKAP mediates neurodegeneration in FD in vivo using a conditional knockout ( CKO ) mouse model in which exon 20 of IKBKAP is conditionally deleted in the brain and DRGs . Tyrp2-Cre [45] mice were mated with IKBKAPFDloxP/FDloxP mice in which IKBKAP exon 20 flanked by loxP sites ( Fig 1 ) . In the resulting offspring ( Tyrp2-Cre;IKBKAPFDloxP/FDloxP termed the CKOTyrp2 FD mice ) IKBKAP exon 20 is deleted at an early differentiation stage of DRGs in the neural crest [45 , 46] , a step at which IKAP expression was previously shown to be essential [43] . This deletion of IKAP was sufficient to generate the main FD symptoms in these mice including developmental delay , gastrointestinal dysfunction , motor discoordination problems , and reduced thermal perception . Importantly , these CKOTyrp2 mice are viable and therefore enabled us to investigate the roles of IKAP in postmitotic neurons during postnatal stages . In CKOTyrp2 FD mice , DRGs are grossly reduced in size relative to DRGs in control mice and overall the neuronal network formation is compromised . Our analysis of mutant DRGs revealed that IKAP deficiency resulted in less effective NGF axonal transport and suggests that IKAP is required for microtubule stabilization through effects on levels of HDAC6 and acetylated α-tubulin . We further evaluated the ability of a potential therapy to alter neuronal maintenance . Previous FD drug discoveries have mainly attempted to elevate IKAP levels [47–50] . Phosphatidylserine ( PS ) , a food supplement with no reported side effects , elevates IKBKAP transcription in cells generated from FD patients [10 , 51] , in a mouse model for FD [52] , and in preliminary results of clinical trials in FD patients [53] . Mechanistically , PS releases cells generated from FD patients from cell cycle arrest [10] . Also , treatment with PS upregulates IKBKAP transcription by activation of the MAPK/ERK pathway , which activates the transcription factors CREB and ELK1 that bind to the IKBKAP promoter region . This in turn enhances cell mobility [51] . Chronic administration of PS to normal adult rats promotes cell survival as shown by a significant increase in BrdU-positive proliferating cells [54] , a reduction in pro-inflammatory signals [55] , and inactivation of JNK and p38 signals after lipopolysaccharide treatment [56] . Here , using cells cultured from CKOTyrp2 FD and wild-type mice , we examined how PS treatment influences neuronal maintenance . We observed that PS treatment stabilized microtubules by downregulating HDAC6 levels , elevating acetylated α-tubulin levels , and improving NGF axonal transport of DRG neurons . Therefore , PS treatment will likely enhance neuronal survival in FD patients and has potential for treatment of patients with other neurodegenerative disorders that share similar molecular pathways .
Total IKAP knockout in mice results in embryonic lethality between E10 to E12 ( S1A Fig ) [42 , 57] . In FD patients , the mutation in the IKBKAP gene primarily affects the nervous system [9] resulting in the abnormal fetal development and impaired postnatal maintenance of DRG neurons ( S1A Fig ) [13 , 18] . Thus , our goal was to establish a viable FD mouse model in which exon 20 is removed from the IKBKAP gene in the nervous system , mainly in DRGs and to study the role of IKAP in postmitotic neurons . We employed the Tyrp2-Cre line , previously reported to be active in pigmented cells of the eye , embryonic forebrain , and DRG [45] . Cre activation occurs on day E12 . 5 , after DRG differentiation [45 , 46] . We established the CKOTyrp2 FD by mating the humanized IKBKAP knock-in mouse [52] with a Tyrp2-Cre expressing mouse ( Fig 1A and S1B Fig ) . The CKOTyrp2 FD offspring were viable with no significant difference in survival compared to control littermates . Allele inheritance was according to Mendelian ratios and was verified using gDNA PCR ( S1B Fig ) . We analyzed the CKOTyrp2 FD offspring for Cre expression and Cre-mediated recombination of IKBKAP exon 20 using whole-mount immunostaining with the anti-Cre antibody ( Fig 1B–1E ) , verified deletion of exon 20 by PCR of the genomic DNA ( Fig 1F ) , and analyzed IKAP protein levels by western blot ( Fig 1G ) . Control and CKOTyrp2 FD mice had similar levels of IKAP in most tissues , such as the lung ( Fig 1G ) , but in CKOTyrp2 FD mice the levels of IKAP were negligible in the DRG ( 96% reduction compared to controls ) and were reduced by more than 50% in the cerebellum and forebrain ( Fig 1G ) . We evaluated the CKOTyrp2 FD mice for symptoms characteristic of FD . The CKOTyrp2 FD mice had notable developmental delays . CKOTyrp2 FD newborns were significantly smaller than control littermates ( Fig 1H , left panel ) . At 2 months of age , CKOTyrp2 FD mice had 20% lower body weight than control siblings ( Fig 1H , middle panel; mean ± SEM , 21 . 8±1 . 28 g vs . 28 . 5±0 . 7 g , respectively , n = 40 per group , ***p<0 . 001 ) and CKOTyrp2 FD brains weighed 25% less than brains of controls ( Fig 1H , right panel; mean ± SEM , 0 . 37±0 . 02 g vs . 0 . 47±0 . 02 g , respectively , n = 10 per group , **p<0 . 01 ) . These lower weights might result from problems in nursing and/or digestion , both characteristics of FD patients . Moreover , the CKOTyrp2 FD mice had brownish and enlarged , swollen intestines ( Fig 1I ) , which implies gastrointestinal dysmotility [58] as seen in FD patients [6] . Tail hanging evaluations revealed that 3-month-old CKOTyrp2 FD mice show limb-clasping and abnormal posturing behavior when suspended by the tail , whereas we did not observe this in age-matched littermates ( Fig 1J ) . In CKOTyrp2 FD mice , the gap between two hindpaws when the mice were lifted was significantly shorter than that of control littermates ( Fig 1J , n = 5 , ***p<0 . 001 ) . These behaviors are indicative of motor discoordination problems in CKOTyrp2 FD mice [59] . Since FD patients exhibit inappropriate thermal and pain perception , we also performed hot plate analgesia assays as described previously [60] . At 3 months of age , CKOTyrp2 FD mice exhibited less perception of sensation compared to age-matched controls ( Fig 1K; mean ± SEM , 6 . 8±0 . 6 sec . vs . 5 . 2±0 . 4 , n = 20 per group , *p<0 . 05 ) . We next examined whether the lack of IKAP alters expression levels of several genes known to be affected in FD patients and other FD mouse models [24 , 30 , 40 , 41 , 61] . RNA was extracted from the brains and DRGs of 3-month-old CKOTyrp2 FD mice and control littermates . In the CKOTyrp2 FD mouse brains and DRGs we observed lower levels of expression of FD-associated genes compared to controls ( Fig 1L ) . The exception was NGF; levels were not significantly different between CKOTyrp2 FD mice and controls ( Fig 1L ) Taken together , these results demonstrate that the CKOTyrp2 FD mice have reduced IKAP levels in the brain and DRGs and exhibit many of the symptoms of FD . The FD mutation leads to reduced DRG size and number [13 , 18–20 , 62] . We first utilized CKOTyrp2 FD mice to analyze how the loss of IKBKAP disrupts DRG development and survival . IKAP was detected by immunohistochemistry and mouse anti-Isl-1 and anti-Brn3A antibodies were used as DRG markers ( Fig 2A–2J ) . All analyses were performed at E13 . 5 , a day after Cre activation [45 , 46] . In accordance with previous studies [43 , 44] , IKAP was expressed in the DRG at E13 . 5 in control mice ( Fig 2E ) ; in contrast , very little IKAP was detected in the DRGs of CKOTyrp2 FD mice at E13 . 5 ( Fig 2J ) . DRGs were smaller in the CKOTyrp2 FD mice compared to controls ( Fig 2A–2J ) , and quantification of the number of cells in the DRG revealed massive cell loss during the embryonic development in the CKOTyrp2 FD mice ( Fig 2K ) . There was a 45% reduction in the average number of cells in the DRGs ( *p<0 . 05 ) , a 67% reduction in DRG area ( in μm3 , ***p<0 . 001 ) , and a 63% reduction in the Isl-1/Drq5 ratio in CKOTyrp2 FD mice compared to control littermates ( ***p<0 . 001 ) . To determine whether IKAP depletion affects particular subpopulation of nociceptors and thermoreceptors neurons in the DRGs , we performed immunostaining using TrkA antibody . Our analysis did not reveal a difference in numbers of TrkA+ neurons in the CKOTyrp2 FD embryos compared to control DRGs ( S2 Fig ) . We further evaluated DRG degeneration in the CKOTyrp2 FD mice by analyzing DRG axonal projections in CKOTyrp2 FD and control mice . Whole-mount neurofilament immunostaining was performed using an antibody to Tuj-1 in forelimbs of E13 . 5 control and CKOTyrp2 FD mice to reveal the peripheral neuronal network innervation pattern . Tuj-1 staining revealed that nearly all peripheral projections were thinner in mutant forelimbs than in controls ( Fig 2L–2Q ) and that axon patterns were abnormal in CKOTyrp2 FD mice . The Tuj-1 staining was 11% less intense in CKOTyrp2 FD mice compared to that in control littermates ( Fig 2R; mean ROI intensity ± SEM , 51 . 03±2 . 73 vs . 57 . 7±2 . 3; n = 5 per group , *p<0 . 05 ) . These data are in agreement with analyses of induced pluripotent stem cells from FD patients , which revealed defects in the neurogenic propensity to form Tuj-1 sensory neurons [47] . We evaluated axonal projection guidance by measuring the average neurite length and by counting the numbers of branches . On average , neurites were 15% longer in CKOTyrp2 FD forelimbs than in controls ( Fig 2R; mean neurite length normalized to forelimb size ± SEM , 8 . 4±0 . 38 vs . 7 . 2±0 . 31 nm3; *p<0 . 05 ) , and there were 21% more branches in CKOTyrp2 FD neurites than in controls ( Fig 2R; mean of total branches/neurite length ± SEM , 1 . 59±0 . 14 vs . 1 . 31±0 . 08 μm3; *p<0 . 05 ) . Proper functioning of the nervous system depends on the intricate array of connections that are formed during development [63] . Data from previous studies suggest that IKAP regulates peripheral nerve regeneration and that abnormal innervation may underlie the FD neurodegeneration [19 , 20] . To examine this hypothesis , we analyzed IKAP distribution in DRG cultures generated from the CKOTyrp2 FD and control mice . The DRGs cultured from the CKOTyrp2 FD mice had significantly more neurite outgrowth than did control DRGs ( Fig 2S ) , suggesting that the lack of IKAP results in a stress response similar to a conditioning lesion [64] and/or alterations in axonal transport process [65] . Analysis of calcein staining revealed that neurite processes were longer ( Fig 2T; mean neurite length ± SEM , 182 . 7±8 . 1 vs . 145±6 . 2 μm , ***p<0 . 001 ) and had excessive branching ( Fig 2T; mean ± SEM of number of branches per cell , 13 . 7±0 . 76 vs . 10 . 4±0 . 48; *p<0 . 05 ) in CKOTyrp2 FD DRGs compared to control cultures . These findings and the Tuj-1 immunostaining results indicate that downregulation of IKAP increases neurite outgrowth and neuronal branching . Neurotrophic factors such as nerve growth factor ( NGF ) and adhesion molecules like NCAM can regulate axon growth . Interestingly , NCAM and NGF expression levels are correlated with IKAP levels [19 , 24 , 32 , 44] . The levels of NCAM mRNA were notably lower in the extracts of DRGs from CKOTyrp2 FD compared to control mice; however , NGF levels were not significantly different ( Fig 1L ) . We speculated though that subcellular localization of NGF might be altered in CKOTyrp2 FD mice , as neuronal survival , growth , and wiring depend on NGF localization and transport along the axon [66 , 67] . To examine how lack of IKAP affects NGF trafficking along the DRGs , DRG E13 . 5 explants were grown in microfluidic chambers . Labeled NGF was added to the distal side of the chamber , and live cell imaging was used to track the retrograde transport of NGF signaling endosomes as we did previously [68 , 69] ( Fig 3A–3C ) . Analyses revealed 50% decreases in the average instantaneous velocity ( mean ± SEM , 0 . 52±0 . 05 vs . 1 . 03±0 . 04 μm/sec , respectively , ***p<0 . 001 ) and in the average speed ( 0 . 05±0 . 05 vs . 1 . 03±0 . 04 μm/sec , respectively , ***p<0 . 001 ) of NGF transport in CKOTyrp2 FD compared to control DRGs ( Fig 3D ) . Thus , CKOTyrp2 FD DRGs have fewer signaling endosomes per axon and impaired NGF transport compared to DRGs cultured from control littermates . Alterations in the axonal transport process are accompanied by a decrease in acetylation of α-tubulin in many neurodegenerative diseases [66 , 70–72] . Acetylated α-tubulin and intracellular transport are vital to neuronal function and survival [67 , 73] and for microtubule stabilization [74 , 75] . Microtubules form the backbone of axons and are configured by the polymerization of α- and β-tubulin dimers . Previous studies have reported contradictory findings on acetylation of α-tubulin in FD [20 , 23 , 30 , 37 , 38 , 40 , 44 , 76] . To evaluate whether the axonal transport defects we detected in CKOTyrp2 FD DRGs are due to the effect of IKAP on acetylation of α-tubulin , we used immunoblot assays to quantify acetylated α-tubulin in FD CKOTyrp2 mouse DRGs and brains , in fibroblasts derived from FD patients , and in HEK 293nt cells in which IKAP levels were reduced by stable expression of a shRNA targeted to the IKBKAP mRNA ( shIKAP ) ( Fig 4A–4D; *p<0 . 05 ) . We observed a 53% decrease in acetylated α-tubulin in DRGs of CKOTyrp2 FD mice relative to controls ( Fig 4A; *p<0 . 05 ) , and a 49% decrease in the forebrain of CKOTyrp2 FD mice relative to controls ( Fig 4B; *p<0 . 05 ) . In fibroblasts derived from FD patients compared to those from normal controls , there was a 25% reduction in acetylated α-tubulin ( Fig 4C; *p<0 . 05 ) . HEK 293nt cells deficient in IKAP had 14% less acetylated α-tubulin than control cells ( Fig 4D; *p<0 . 05 ) . Data from these experiments indicated that depletion of exon 20 of IKBKAP resulted in a more significant decreased in acetylated α-tubulin levels in neuronal tissues than in non-neuronal cells . These differences in the acetylated α-tubulin levels are likely due to the morphology of neurons , especially the DRGs , which are highly polarized cells with very long axons , made of tubulin subunits . The less dramatic differences in levels of acetylated α-tubulin in fibroblasts and HEK 293nt cells that are deficient in IKAP compared to controls illustrates why alterations in acetylated α-tubulin levels are not necessarily detected in FD patients and might explain the neuronal specificity of the FD phenotype . Reductions in acetylated α-tubulin were previously reported to be accompanied by increased HDAC6 expression [77] . HDAC6 is the major α-tubulin deacetylase , and its expression is abnormally high in patients with neurodegenerative diseases , including Parkinson’s , amyotrophic lateral sclerosis , and Alzheimer’s , and with cancer and pathological autoimmune responses [78] . In each of our models of IKAP deficiency , we observed increased HDAC6 levels relative to controls ( Fig 4A–4D ) . IKAP was previously suggested to impact histone acetylation as part of the Elongator complex [26–29] , and levels of mRNA encoding the catalytic histone acetyltransferase subunit of Elongator protein 3 ( ELP3 ) were significantly downregulated in CKOTyrp2 FD mice compared to control littermates ( Fig 1L ) . We therefore examined the effect of IKAP depletion on acetylation of histone H3K9 . Interestingly , we observed higher H3K9ac levels in CKOTyrp2 FD DRGs than in control DRGs ( Fig 4E; *p<0 . 05 ) , and the opposite in neurons from the forebrain ( Fig 4F; ***p<0 . 001 ) . These results are in line with the previous finding that H3K9 acetylation is elevated in DRGs but not in the central nervous system during neuronal damage [79 , 80] . Thus , IKAP has indirect and tissue-specific functions in neuronal development and function . To further explore the nature of the link between IKAP , microtubules , and histones , we examined fractionated nuclear and cytoplasmic lysates and performed co-immunoprecipitation assay for endogenous IKAP . Since the IKAP effect on acetylated α-tubulin was similar in different cell types ( Fig 4A–4D ) , these experiments were performed in HEK 293nt cells . α-tubulin was used as a marker for cytoplasmic lysate fraction and histone H3 as a nuclear marker . In these extracts , no cross-contamination of fractions was observed , and IKAP was restricted to the cytoplasmic fraction ( Fig 4G ) . DNA was fragmented by limited sonication as described [81] , and anti-IKAP antibody was used to precipitate endogenous IKAP . We observed interactions between IKAP and α-tubulin , JNK , and HDAC6 but no enrichment for histone H3 or ERK ( Fig 4H ) . A reciprocal immunoprecipitation assay using anti-HDAC6 also precipitated α-tubulin and IKAP ( S3 Fig ) . Thus , IKAP is found in a cytoplasmic protein complex together with α-tubulin , JNK , and HDAC6 . PS is a phospholipid that is normally located on the cytoplasmic face of the lipid layer . It is exposed on the outer leaflet of the plasma membrane during apoptosis where it recognized by PS receptors on scavenger macrophages [82] . Though its protective mechanism is not understood , PS administration promotes cell survival [54–56] and was found to elevate IKAP levels in cell line derived from FD patients [10 , 51] , in a mouse model for FD [52] and in preliminary results of clinical trials [53] . Protection may reflect the ability of PS to stimulate IKAP transcription by activating the MAPK/ERK pathway or conceivably exogenous PS may relieve apoptotic stress on neurons and other cell types by saturating macrophage PS receptors [51] . We assume that the beneficial effects of PS result from an increase in IKAP levels in treated cells . Therefore , we first evaluated the effect of PS on levels of acetylated α–tubulin in HEK 293nt cells . Treatment of cells with 200 μg/ml PS elevated acetylated α-tubulin levels by 50% after 24 h ( Fig 5A; *p<0 . 05 ) . We then examined the impact of PS administration on HEK 293nt cells that stably express the shIKAP . In these cells depleted of IKAP , PS treatment resulted in a 44% increase in acetylated α-tubulin levels ( Fig 5B ) . PS also significantly downregulated HDAC6 levels in the shIKAP cells compared to vehicle only treated cells ( Fig 5B ) . This indicates that PS can acts as HDAC6 inhibitor . We next evaluated PS treatment on fibroblasts extracted from FD patients . An increase in acetylated α-tubulin and IKAP levels were also observed as a result of PS administration ( S4A Fig ) . Trichostatin A ( TSA ) , a general HDAC inhibitor [83 , 84] was used as a positive control and had an effect similar to that of treatment with PS on IKAP and acetylated α-tubulin levels ( S4A Fig ) . As another approach to characterize the role of IKAP and the ability of PS to stabilize tubulin , we performed a wound-healing assay . Wound healing is central in a variety of pathologic and physiologic processes including cell growth , differentiation , and cell-cell communication and is a process typically associated with the ability of cells to project tubulin branches . IKAP-depleted cells were previously shown to have impaired migration in wound healing assays [32 , 37] . The shIKAP 293nt HEK cells were grown to confluence in complete media with or without PS , a wound was made using 96-well Wound Maker , and cells were imaged as described [51] . Images and measurements of the wound size as a function of time indicated that in cells that are IKAP deficient migration was enhanced by treatment with either PS or TSA ( S4B Fig ) . These findings suggest that PS works in a similar manner to an HDAC inhibitors and improves the FD phenotype . We next analyzed the ability of PS treatment to improve axonal transport of the CKOTyrp2 FD mouse . No significant effects were detected on FD symptoms ( S5 Fig ) . In this model , exon 20 of the IKBKAP gene is conditionally deleted . This means that PS treatment cannot increase levels of full-length IKAP in these mice . In contrast , in heterozygotes ( CKO/+; in which only one of the IKBKAP alleles has a conditionally deleted exon 20 ) and in wild-type mice , it is possible for PS treatment to elevate IKAP levels from the normal allele . We therefore examined the effect of PS administration on axonal transport in DRGs taken from wild-type ( S6A–S6E Fig ) and CKO/+ mice ( Fig 5C–5G ) . DRG explants were grown in microfluidic chambers and labeled NGF was added to the distal side . Analysis of the retrograde transport of NGF signaling endosomes along the axon , 24 hours after PS addition indicated enhancement of trafficking compared to vehicle-treated controls both in CKO/+ explants ( Fig 5C–5G; ***p<0 . 001 ) and wild-type explants ( S6A–S6E Fig ) . The average instantaneous velocity of NGF transport after PS administration was significantly higher than in vehicle only treated CKO/+ controls ( mean ± SEM , 1 . 4±0 . 07 vs . 1 . 03±0 . 07 μm/sec , respectively , ***p<0 . 001 ) as was the average speed ( 1 . 31±0 . 06 vs . 0 . 97±0 . 06 , μm/sec , respectively , ***p<0 . 001 ) . Moreover , the mean squared displacements and displacement as a function of time indicated that PS treatment resulted in more rapid transport of NGF across the axons ( Fig 5E and 5F and S6C and S6D Fig ) . No significant differences were found between CKO/+ and normal mice ( S7 Fig ) . These experiments show that PS increases levels of IKAP in mice with at least one normal IKBKAP allele , resulting in amelioration of damaged NGF axonal transport and an increase in transport efficiency . Finally , we examined the ability of PS to improve DRG culture outgrowth . DRGs cultured from the CKOTyrp2 FD mice and treated with PS had significantly less neurite outgrowth than did vehicle only controls ( Fig 6A and 6B ) . Analysis of calcein staining revealed that neurite processes were shorter in CKOTyrp2 FD DRGs treated with PS ( Fig 6B; mean neurite length ± SEM , 204 . 3±6 . 3 vs . 240 . 9 . ±6 . 5 μm , ***p<0 . 001 ) and had less branching per cell ( Fig 6B; mean ± SEM 14 . 9±0 . 5 vs . 17 . 1±0 . 5; **p<0 . 01 ) compared to vehicle-treated control cultures . As PS cannot increase IKAP levels in these mice , its affect is most likely independent of IKAP and may involve HDAC6 inhibition and microtubule stability . Indeed , PS had an affect on α-tubulin dynamics in nocodazole-treated fibroblasts derived from FD patients compare to normal controls . Nocodazole treatment collapses the microtubule network as reveled by α-tubulin staining ( S8 Fig ) . Cells were treated with 1 . 0 μM nocodazole and either 200 μg/ml PS or vehicle . Fibroblasts derived from FD patients and from normal controls showed greater resistance to nocodazole treatment and less microtubule depolymerisation when the nocodazole treatment was combined with PS treatment compared to cells treated with nocodazole and vehicle ( S8 Fig ) .
IKAP is a well-studied protein , yet its role in neuronal survival remains controversial . Although it has been known for decades that a mutation that results in mis-splicing of the gene that encodes IKAP causes FD , the molecular mechanism that leads to the progressive neurodegeneration observed in FD patients has been unclear . There have been previous attempts to generate a mouse model for FD . Knock-in mice in which the human IKBKAP locus with the mutation observed in FD patients replaces the reciprocal mouse genomic sequences are fully viable and do not show any FD symptoms other than splicing of IKBKAP in a tissue-specific manner [10 , 52 , 85] . IKBKAP total knockout results in embryonic lethality between E10 and E12 [42 , 57] . The conditional knockout of IKBKAP using a Cre expression system under control of the Wnt-1 promoter resulted in mice that died a few days after birth [20 , 44] . Recently a combined heterozygote model in which one IKBKAP allele is knocked-in with the FD mutation and the second is knocked-out resulted in a mouse that recapitulates many phenotypic features of FD and recreates the same tissue-specific mis-splicing defect seen in FD patients [62] . As IKAP depletion occurs early in embryogenesis in these mice , the roles of IKAP in DRG development and early differentiation cannot be separated from its role in neurodegeneration . Thus , in order to bypass the IKBKAP-/- and Wnt-1 lethality and study the effect of IKAP on neurodegeneration , we established a conditional knockout transgenic mouse line using Tyrp-2 promoter . This conditional knockout results in loss of IKAP at E12 . 5 , after DRG maturation . These mice are viable and mimic the human FD phenotype . These CKOTyrp2 FD mice enabled us to study the role of IKAP in neuronal maintenance in vivo and to establish the mechanism by which PS improves symptoms of neurodegeneration . We first assessed the function of IKAP in DRG development and survival in vivo . First , our data indicate that depletion of IKAP in the brain results in DRGs with grossly reduced size , aberrant peripheral innervations , and excessive axonal growth when compared to controls . Second , DRGs from CKOTyrp2 FD mice exhibit longer neurites and more branches than DRGs derived from normal littermates . These findings are similar to those reported based on analyses of chick and mouse models of FD that showed that IKAP is crucial for peripheral neuron target innervations and NGF signaling [19 , 20 , 42 , 43] . Third , DRG from CKOTyrp2 FD mice had less acetylated α-tubulin than control littermates and impaired NGF axonal transport . Reductions in the acetylated form of α-tubulin were also observed in SGs from these mice . Our data are in agreement with a previous report by Gardiner et al . that demonstrated that retrograde transport was impaired in FD patients [23] . That IKAP is involved in axonal transport was suggested by the finding that exogenously expressed IKAP C-terminal domain co-purifies with the heavy chain of the motor protein dynein [32] and that the absence of IKAP disrupts co-localization of dynein with phosphorylated JNK along axons [19] and causes alterations in expression of several genes important in axonal transport [24] . DRGs are highly polarized cells with very long axons . The structural integrity of these axons is dependent on efficient intracellular transport of cargoes such as neurotrophic factors manufactured in their distant targets cells . Thus , DRGs may be more vulnerable to alterations in axonal transport process than are other neuronal cells types . In DRGs cultured from IKAP-deficient CKOTyrp2 FD mice , NGF transport was impaired relative to control DRGs that express normal levels of IKAP . The unstable neuronal networks formed in IKAP-deficient conditions may underlie the massive DRG neurodegeneration progression observed in FD patients . There is a well-established link between α-tubulin acetylation , protein trafficking , microtubule stability , and neurodegeneration [70–72 , 74 , 75] . Reduced acetylation of α-tubulin correlates with axonal transport defects in Alzheimer’s and Huntington’s disease patients [66] and treatment with an HDAC inhibitor was shown to rescue vesicular transport in a Huntington’s disease model [86] . Moreover , in several FD studies abnormally low levels of α-tubulin acetylation have been reported [38 , 76] . In DRGs and forebrain neurons from our CKOTyrp2 FD mice , we observed significantly increased HDAC6 levels and significantly decreased acetylated α-tubulin levels compared to control littermates . How IKAP levels affect HDAC6 expression remains unclear , although we demonstrated that endogenous IKAP co-purified with HDAC6 and α-tubulin , suggesting that a protein-protein complex might ensure that levels are balanced . Another possibility is that the decrease in acetylated α-tubulin itself stimulates the neuroprotective role of HDAC6 , as a previous study revealed a robust and selective increase in HDAC6 expression following neuronal injury [87] . Thus , the co-purification observed might be due to binding of IKAP and HDAC6 to the same substrate . Furthermore , HDAC6 elevation may underlie the excessive axonal growth since HDAC6 overexpression was shown to increase neurite length [88] . Although IKAP has been suggested to serve as a scaffold protein of the Elongator complex that is responsible for chromatin remodeling during transcription [26 , 37 , 89] , in other studies IKAP has been shown to be restricted to the cytoplasm [31] . One of the functions of the Elongator complex is acetylation of histone H3 [90] , particularly at K9 [37 , 90] . Indeed , we observed that the levels of ELP3 , a component of the Elongator complex , were significantly downregulated upon IKAP depletion . However , our findings suggest that the effect of IKAP on histone acetylation must be indirect . We did not detect IKAP in nuclear fractions , IKAP did not precipitate histone H3 , and the impact on H3K9ac of IKAP depletion depended on the tissue . Mass spectrometry analysis of proteins that precipitated with the C-terminal domain of IKAP identified primarily cytoplasmic proteins [32] , and exogenous IKAP precipitated and partly co-localized with α-tubulin [38] . We assume that changes in H3K9ac levels probably mediate chromatin remodeling during neuronal degeneration in FD; thus , IKAP has an indirect impact on histone H3K9 acetylation . Phosphorylated JNK was previously suggested to mediate the interaction between IKAP and α-tubulin [19 , 23 , 31] , and in our experiments , JNK was precipitated with the anti-IKAP antibody . Recently it was demonstrated that IKBKAP mRNA levels are downregulated in FD patients during crisis [91] , which emphasizes that treatments that elevate transcription of IKBKAP should be an effective therapy for FD . PS was previously found to stimulate the IKBKAP transcription [51] and to elevate IKAP levels in vitro and in vivo [10 , 52] . Here we explored the effects of PS treatment on DRG explants from mice that have only one functional IKBKAP allele and wild-type mice and found that PS treatment enhances NGF retrograde axonal transport , and improves axonal outgrowth . We also identified PS downregulates HDAC6 levels and elevate acetylated α-tubulin levels . We assume that PS treatment increased levels of acetylated α-tubulin levels through its indirect inhibitory effects on HDAC6 as well as activation of Elongator complex and the ELP3 acetyltransferase activities ( Fig 7 ) . We showed that in several assays PS treatment is similar to treatment with the well-characterized HDAC inhibitor TSA . Both PS and an HDAC inhibitor were demonstrated to benefit patients with depression and mood disorder [92] and enhance memory formation [93 , 94] . The responses to treatment with broad-spectrum HDAC inhibitors are heterogeneous , protective in some cases and detrimental in others [95] . Recently PS treatment has been reported to improve memory in Alzheimer’s patients and to reduce hippocampal inflammation and free radical production in a rat model of Alzheimer’s disease [96] . The fact that PS has been used clinically with no serious side effects and promotes neuron survival and axonal transport suggest that PS might be relevant as treatment for other neurodegenerative diseases that share similar pathologic and molecular mechanisms . Additional clinical studies are warranted .
All animal work and all procedures followed guidelines according to the NRC—guide for care and use of laboratory animals . The study was approved by the Institutional Animal Care and Use Committee ( IACUC ) of Tel Aviv University with the approval numbers: M-12-047 ( previous ) and M-15-015 ( current ) , and measures were taken to minimize pain and discomfort . Antibodies used for immunofluorescent staining were as follows: Brn3a ( Chemicon , a gift from the Miguel Weil lab , Tel Aviv University ) , IKAP ( Anaspec or Santa Cruz ) , DRQ5 ( Cell Signaling ) , Isl-1 ( DSHB ) and TrkA ( Almone ) . Antibodies used for whole-mount staining were anti-Cre ( Abcam ) and neuronal class III -tubulin ( anti-Tuj-1; Covance ) which were kind gifts from the Keren Avraham lab ( Tel Aviv University ) and the Avraham Yaron lab ( Weizmann Institute ) . Secondary antibodies were donkey anti-rabbit conjugated to Alexa-594 , donkey anti-mouse Alexa-488 , and donkey anti-goat Alexa-647 ( all from Invitrogen and used 1∶1000 ) . DRG cultures were stained using Hoechst dye and calcein ( Thermo Scientific ) . For immunoblotting , the primary antibodies used were: anti-IKAP ( Anaspec ) , anti-acetylated α-tubulin ( Sigma ) , anti-HDAC6 ( Abcam ) , anti-Hsc70 ( Santa Cruz Biotechnology ) , anti-GAPDH ( GenScript ) , anti-α-tubulin ( Abcam ) , anti-histone H3K9 ( Abcam ) , anti-histone H3 ( Abcam ) , anti-histone H4 ( Millipore ) , anti-JNK ( Santa Cruz Biotechnology ) , anti-pJNK ( Santa Cruz Biotechnology ) , anti-ERK ( Cell Signaling ) , anti-pERK ( Santa Cruz Biotechnology ) . Secondary antibodies donkey anti-rabbit IgG HRP ( Abcam ) , donkey anti-goat IgG ( Abcam ) , or goat anti-mouse IgG ( Jackson ) were used as appropriate . InCog , a lipid composition containing PS-omega 3 , DHA-enriched , referred to here as PS , was dissolved in organic solvent medium chain triglycerides ( MCT ) . Both PS and MCT were obtained from Enzymotec . In all treatments PS was used at 200 μg/ml and was compared to treatment with solvent as a control . The mouse lines employed in this study , Tyrp2-Cre[45] and IKBKAPFDloxP/FDloxP[52] , have been previously described . These lines were used to establish Tyrp2-Cre;IKBKAPFDloxP/FDloxP somatic mutants . In all experiments IKBKAPFDloxP/FDloxP littermates were used as controls . All animal work and all procedures were conducted according to national and international guidelines , and procedures were approved by the Tel Aviv University review board and measures were taken to minimize pain and discomfort . Genotypes were determined by PCR analysis of genomic DNA from tail slips using the High Pure PCR Template Preparation Kit ( Roche ) or KAPA mouse genotyping ( Biosystems ) according to the manufacturer’s instructions . To distinguish IKBKAP wild-type , heterozygous , and homozygous mice , we used primers 5’-GATAGCTAGTCTGTGTTGTAATG-3’ and 5’-CCCTCGTGTGCCCTCAGTG-3’ . Wild-type and homozygous IKBKAPFDloxP/FDloxP mice have genomic fragments of 1140 and 1392 bp , respectively , whereas heterozygous IKBKAPFDloxP/+ mice display both fragments . Cre was detected using 5’-CCGCAGAACCTGAAGATGTTC-3’ and 5’-TCATCAGCTACACCAGAGACG-3’ . Heterozygous Cre mice display a 500-bp fragment . CKOTyrp2 FD and control mice were evaluated for their nociceptive threshold to radiant heat using the hot plate ( Ugo Basile ) paw withdrawal test , as previously described[60] . Briefly , a 40-cm high Plexiglas cylinder was suspended over the 55°C hot plate to give a latency of about 10 seconds for control mice . Withdrawal latency was defined as time between placement on the hot plate and time of withdrawal and licking of hindpaw . Each animal was tested twice , separated by a 30-min rest interval . Student's t-test was used to calculate significance of differences between CKOTyrp2 FD mice and controls . The four-limb tail hanging hindpaw gap evaluations were conducted using Image J . RNA was extracted from CKOTyrp2 FD and control mice using TRI reagent ( Sigma ) and reverse-transcribed using the SuperScript III First Strand kit ( Invitrogen ) with an oligo ( dT ) reverse primer . A qPCR analysis of mRNA expression from mouse brain or DRGs tissue samples were conducted using KAPA SYBR fast qPCR master mix ( Kapa Biosystems ) in a StepOne plus thermocycler PCR machine ( Applied Biosystems ) according to the manufacturer’s instructions . Expression levels of PPIA and GAPDH genes were used as endogenous controls . Brain samples were assayed in triplicate and DRGs were assayed in pulls from four mice . Primers are listed in the supplementary material ( S1 Table ) . Immunofluorescence analysis was performed on 10-μm paraffin or cryo frozen sections as previously described [97] . Stains were analyzed using Image J . For each genotype , we evaluated three to six mice . For each DRG , we counted four to six slices . All fluorescent images were obtained using Super-Resolution Microscope Leica TCS STED or Zeiss confocal microscopy LSM 510 . Whole-mount samples were prepared as follows: at E13 . 5 embryos were dissected and immediately fixed in 4% paraformaldehyde for 6 h 4°C . Embryos were washed and dehydrated through a series of methanol , Dent’s bleach ( methanol:DMSO:hydrogen peroxide ) and in a descending series of methanol concentrations . Embryos were blocked and stained with primary antibody followed by second antibodies overnight at 4°C . Embryos were washed in PBS and incubated in the diluted secondary antibody in blocking solution and then rinsed in a glycerol solution . Embryos were flattened on the basal side in Lab-Tek coverglass sealed mounted chambers ( Nunc ) for observation . For whole-mount statistical analysis , we counted eight to ten neuritis of five forelimbs stained for each group . Adult DRG cultures were cultured as described [98] . Neurons were dissociated by incubation with 100 U of papain followed by 1 mg/ml collagenase-II and 1 . 2 mg/ml dispase . Then DRGs were triturated in HBSS , 10 mM Glucose , and 5 mM HEPES ( pH 7 . 35 ) . Cells were recovered through percoll , plated on laminin , and grown in F12 medium for 48 hr . Neurite outgrowth assays were monitored on a Revolution XD automated imaging system ( Andor ) using calcein AM ( C3100MP ) and Hoechst dye . For rescue experiments , DRG neurons were treated with either PS or an equivalent amount of vehicle for 12 h in DRG medium without NGF to exclude outgrowth effects by NGF . The system automatically collected 10 images per well . Automated analyses of the results were performed using WIS-Neuromath software developed at the Weizmann Institute ( www . weizmann . ac . il/vet/IC/software/wis-neuromath ) [99] . For DRG culture statistical analysis , we filmed on average 70 fields of four different experiments for each group . Human FD fibroblast cells were obtained from the appendices of FD patients and immortalized using telomerase activation . HEK 293nt cells and human FD and normal fibroblast cell lines were cultured in Dulbecco’s modified Eagle’s medium , supplemented with 4 . 5 g/ml glucose , 2 mM L-glutamine , 100 U/ml penicillin , 0 . 1 mg/ml streptomycin , and 10% fetal calf serum . Stable cell lines were prepared using shRNA ( Gencopedia ) according to manufacturer’s instructions . Cells were grown in a 10-cm culture dish , under standard conditions , at 37°C with 5% CO2 . All cell culture materials were purchased from Biological Industries . Total proteins were extracted from the cells using a hypotonic lysis buffer ( 50 mM Tris-HCl , pH 7 . 5 , 1% NP40 , 150 mM NaCl , 0 . 1% SDS , 0 . 5% deoxycholic acid , 1 mM EDTA ) containing protease inhibitor and phosphatase inhibitor cocktails I and II ( Sigma ) . After 20 min centrifugation at 14 , 000 g at 4°C , the supernatant was collected and protein concentrations were measured using BioRad Protein Assay . Cytoplasmic and nuclear fractions were obtained using sucrose gradients as described [81] . Proteins were separated in an 8% SDS-PAGE and then electroblotted onto a Protran nitrocellulose transfer membrane ( Schleicher & Schuell ) . Immunoblots were incubated with primary and secondary antibodies and visualized by enhanced chemiluminescence ( SuperSignal West Pico chemiluminescent substrate; Thermo Scientific ) and exposure to X-ray film . Data was from three separate experiments and was quantified using Image J . Error bars represent SEM . Cells were washed with PBS and crosslinked with 0 . 1% formaldehyde , and total lysates were purified and then re-suspended in immunoprecipitation ( IP ) buffer ( 50 mM HEPES [pH 7 . 6] , 500 mM LiCl , 1 mM EDTA , 0 . 7% DOC , 1% NP-40 , 0 . 1% SDS , 13 CPI ) . Cells were sonicated and incubated 12 h at 4°C with antibody conjugated to protein A Dynabeads ( Invitrogen ) . Samples were washed with IP buffer . Proteins were eluted and subjected to western blot analysis . Samples were reversed crosslinked for 12 h at 55°C , and protein were eluted from the beads by adding 100 ml PBS and 20 ml SDS sample buffer ( 272 mM Tris-HCl [pH 6 . 8] , 30% glycerol , 12% SDS , 20% β-mercaptoethanol , 0 . 01% bromophenol blue ) and incubating in a thermo-shaker for 15 min at 75°C with vigorous shaking . The supernatant was moved to a new tube and boiled for 5 min at 100°C . DRGs were dissected from E13 . 5 mice , followed by separation from meninges and spinal cord . Microfluidic chambers were prepared as described [68 , 69] . A single DRG of up to 1 mm was placed in each explant well , were allowed to adhere for 1 hour after which NGF rich media were added . On DIV 3 cells were starved for 2 hours with NGF-deprived medium , followed by addition of Quantum-Dot labeled NGF to the distal axon compartment . After 30 min incubation in 37°C , retrograde transport along the axon was imaged at 37°C and CO2 controlled environment , using Nikon Eclipse Ti microscope equipped with Yokogawa CSU X-1 spinning disc confocal . Time-lapse image analyses were carried out using Fiji and MATLAB as described [68 , 69] . Cells were seeded in an ImageLock 96-well plate at a density of 27 , 000 cells/well and cultured until confluent . Wounds were inflicted across the cell layer using WoundMaker™ . Cells were washed with PBS and supplemented with fresh medium . Selected wells were incubated with PS ( final concentration 200 μg/ml ) , TSA ( 1 . 5 mM ) or solvent . Migration was monitored using an IncuCyte Zoom microscope ( Essen Bioscience ) , with image acquisition every 60 min for 48 h . The IncuCyte Zoom image analysis software was used to quantify wound closure . Cells were grown on 13-mm glass cover slips . Cells were treated with 1 . 0 μM nocodazole together with 200 μg/ml PS or vehicle . After 24 h , the fibroblasts were fixed for immunofluorescence study . All data were examined using two-tailed Student's t-test . The P-values and number of independent biological replicates ( n ) are indicated in the figure legends and results . | We create a novel FD mouse model , in which exon 20 of IKBKAP was deleted in the nervous system , to study the role of IKAP in the neurodegeneration process . The lack of IKBKAP exon 20 impaired retrograde nerve growth factor ( NGF ) transport and axonal outgrowth . Reduction of IKAP levels resulted in elevated HDAC6 levels and thus reduced acetylated α-tubulin levels . Phosphatidylserine down-regulated HDAC6 levels , furthermore phosphatidylserine treatment facilitated axonal transport and stabilized microtubules . In brief: Naftelberg et al . identify the molecular pathway leading to neurodegeneration using a mouse model of familial dysautonomia and suggest that phosphatidylserine acts as an HDAC6 inhibitor to improve neurologic function . | [
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] | 2016 | Phosphatidylserine Ameliorates Neurodegenerative Symptoms and Enhances Axonal Transport in a Mouse Model of Familial Dysautonomia |
Despite their importance as vectors of human and livestock diseases , relatively little is known about innate antiviral immune pathways in mosquitoes and other insects . Previous work has shown that Culex Vago ( CxVago ) , which is induced and secreted from West Nile virus ( WNV ) -infected mosquito cells , acts as a functional homolog of interferon , by activating Jak-STAT pathway and limiting virus replication in neighbouring cells . Here we describe the Dicer-2-dependent pathway leading to WNV-induced CxVago activation . Using a luciferase reporter assay , we show that a NF-κB-like binding site in CxVago promoter region is conserved in mosquito species and is responsible for induction of CxVago expression following WNV infection . Using dsRNA-based gene knockdown , we show that the NF-κB ortholog , Rel2 , plays significant role in the signaling pathway that activates CxVago in mosquito cells in vitro and in vivo . Using similar approaches , we also show that TRAF , but not TRAF-3 , is involved in activation of Rel2 after viral infection . Overall the study shows that a conserved signaling pathway , which is similar to mammalian interferon activation pathway , is responsible for the induction and antiviral activity of CxVago .
Hematophagous insects ( mosquitoes , sand flies and midges ) serve as vectors of many important viral diseases of humans and livestock . Arthropod-borne viruses are endemic or seasonally epidemic through most regions of the world , accounting for inestimable numbers of human infections , significant rates of mortality , and major impacts on livestock production and trade [1] , [2] . Indeed it has been estimated that over 25% of all emerging viral diseases globally are vector-borne [3] with factors such as global warming , increased population densities and ease of world travel driving the spread into new geographical areas [4] , [5] . Despite their importance in the transmission of viral disease , knowledge of the processes of infection and immunity in insects ( and other invertebrates ) is relatively poor . Insects are known to lack key components of vertebrate immune system , such as interferons , antibodies , lymphocytes and other elements of innate and adaptive immunity [6] . RNA interference ( RNAi ) has been shown to be a major aspect of the insect antiviral response to a wide range of both RNA viruses and DNA viruses [7] , [8] . This involves the detection and degradation of viral double-stranded ( ds ) RNA by Dicer-2 , generating short interfering ( si ) RNAs ( siRNA ) . These viral-siRNAs are subsequently incorporated into the RNA-induced silencing complex ( RISC ) and used to guide the targeted degradation of viral RNAs [9] . There is also evidence that some evolutionarily conserved immune signaling pathways , such as Toll receptor [10] , Imd [11] and Jak-STAT [12] , which direct the induction of antimicrobial peptides in Drosophila , are also involved in the antiviral response . The Toll pathway was initially identified using a Drosophila mutant screen to be involved in the antiviral response [13] , and subsequently was shown to have a role in limiting dengue virus infection in Aedes aegypti mosquitoes [10] . In contrast , using a Sindbis virus replicon system , it has been reported that Drosophila mutants deficient in Toll pathway transcription factors had no effect on viral replication , but that replication was enhanced in mutants deficient in the elements of the Imd pathway [11] . The Jak-STAT pathway was also initially found to be important in antiviral signalling in Drosophila [12] and later has been shown to be play significant role in immunity to dengue virus in mosquitoes [14] . Recently , it has also been shown that Dicer-2 , which is central to the RNAi response , also mediates signaling that leads to induction of Vago , a secreted peptide that also has a role in antiviral immunity in Drosophila and mosquitoes [15] , [16] . Through a mechanism that is independent of the RNAi pathway , it has been shown in mosquito cells that dsRNA-induced , Dicer-2-mediated secretion of Vago inhibits West Nile virus ( WNV ) infection by activating Jak-STAT pathway in the neighboring cells [15] . This suggests that , although structurally unrelated , Culex Vago may have a function similar to mammalian interferons [15] . In vertebrates , viral infection leads to recognition of viral dsRNA by DExD/H-box helicases like RIG-I or MDA-5 [17] . These viral sensors interact with mitochondrial antiviral signaling ( MAVS ) protein via CARD domain [18] , leading to activation of TNF receptor-associated factors ( TRAF ) -3/6 [19] . This is followed by activation and nuclear localization of interferon regulatory factors ( IRFs ) -3/7 and/or nuclear factor-kappa B ( NF-κB ) . NF-κB activation involves phosphorylation and degradation of inhibitory kappa B ( IκB ) which releases NF-κB to translocate into the nucleus [20] . The IRFs and NF-κB by bind to the promoter region of the gene and induce interferon beta via TRAF3 and TRAF6 respectively [21]–[23] . In mosquitoes and other invertebrates viral dsRNA is recognized by Dicer-2 , which contains an amino-terminal DExD/H-box helicase domain similar to RIG-I , ultimately leading to induction of Vago [15] , [16] . However , the intermediate proteins involved in this activation have not been identified . In this report , we investigate the Vago activation pathway following flavivirus infection of mosquito cells in vitro and in vivo . We show that induction of Vago by West Nile virus ( WNV ) or dengue virus ( DENV ) is mediated by Dicer-2-dependent activation of an ortholog of TRAF , which in turn activates the NF-κB ortholog , Rel2 . We also demonstrate that Vago activation is dependent on a conserved NF-κB binding site in the upstream promoter region , suggesting that flavivirus-induced Vago activation occurs by an evolutionarily conserved mechanism .
Hsu ( Culex quinquefasciatus ) and RML-12 ( Aedes albopictus ) cells were maintained at 28°C in Leibovitz's L-15 medium ( Gibco #11415 ) containing 10% tryptose phosphate broth solution , 15% heat-inactivated fetal bovine serum , and 1% penicillin-streptomycin solution . West Nile virus ( NY99 and Kunjin strains ) and dengue-2 virus ( NGC strain ) were used for the study . C6/36 ( Aedes albopictus ) cells were maintained in RPMI media at 28°C were used to propagate the virus . Vero cells maintained in EMEM at 37°C were used for plaque assays . Luciferase reporter constructs were prepared by cloning the 2007 bp region upstream of the initiation codon of the Culex Vago gene . For the core promoter , the region from −10 to +20 bp ( in relation to the transcription start site ) was used . PCR was performed on Culex genomic DNA to amplify the region ( DS231818; region 720994–723000 ) , and using restriction digests to clone it upstream of Renilla luciferase reporter gene . A similar strategy was used to clone sequential deletions of the promoter region in the same vector . Mutations were introduced into the Rel binding site sequence ( CTT→AGG ) using QuickChange Site-Directed Mutagenesis kit ( Stratagene ) according to the manufacturer's protocol . The plasmid was transfected into Hsu cells using Cellfectin ( Invitrogen ) in serum-free medium along with plasmid containing firefly luciferase reporter gene driven by the insect cell promoter OpIE2 . The luciferase reporter assay was performed on cell lysates using Dual-Luciferase Reporter Assay System ( Promega ) according to the manufacturer's instructions . The experiments were conducted at least 3 times , each in triplicates . The controls were set arbitrarily at 1 , and fold-increase over control was plotted as mean ± SD . Total RNA was extracted from cells using the Qiagen RNA extraction kit according to the manufacturer's protocol . Reverse transcription was performed by random hexamers using the First Strand Synthesis kit ( Invitrogen ) . Real-time RT-qPCR was performed using gene-specific primers ( Supplementary Table S2 ) . As an internal control , real-time RT-qPCR was also performed using the housekeeping gene , RpL32 . The control was set arbitrarily at 1 and fold-increase over control was calculated by the ΔΔCt method . The experiments were conducted at least 3 times , each in triplicates . The results were plotted in graph format as mean ± SD . Cells were lysed in RIPA lysis buffer containing protease inhibitor . The cell lysates were collected by centrifuging at 16 , 000×g for 10 min at 4°C . Protein samples ( 10 µg ) were loaded onto polyacrylamide gels ( 4–12% ) . After electrophoresis and transfer to nitrocellulose membranes , proteins were blotted using anti-Vago [15] or anti-V5 ( Invitrogen ) antibody followed by anti-rabbit or anti-mouse secondary antibody , respectively . After adding substrate , the membrane was exposed to film to detect protein levels . Anti-beta-actin antibody was used in immunoblots as a loading control . Plaque assays were performed as previously described [15] . In brief , the supernatant media from the cells infected with WNV ( 10-fold dilutions ) were added onto confluent Vero cell monolayers in 6-well plates . After 1 h incubation at 37°C , the cells were overlaid with medium containing agar . Plaques formed within 72 hpi were counted and the results were plotted graphically . The experiments were conducted at least twice , each with duplicates . Gene-specific dsRNA ( ∼400 nt ) were prepared using the MEGAscript RNAi kit according to the manufacturer's protocol . dsRNAs were transfected into Hsu cells using Cellfectin according to a previously described protocol [15] . dsRNA against green fluorescent protein ( GFP ) was used as a specificity control . A colony of Culex pipiens f . quinquefasciatus mosquitoes was maintained at 25°C and 65% humidity . Three to five day-old female mosquitoes ( n = 60 ) were injected intrathoracically with 0 . 069 µl of dsRNAs against GFP , TRAF or Rel2 . WNV ( Kunjin strain; 1 . 13×10∧7 pfu/ml titer ) or 199 medium ( as control ) was injected similarly 48 h later and the mosquitoes were incubated at 25°C and 65% humidity in an environmental cabinet ( Thermoline Scientific , Smithfield , Australia ) with a wet cotton pad ( 10% sucrose solution ) provided daily as a food source . At 48 hpi , surviving females were collected for analysis . For each group , 2 females were homogenized using a disposable mortar-pestle and RNA extracted using the RNeasy kit ( Qiagen ) was used for real-time RT-qPCR as described above . The process was repeated again for creating duplicate samples . Standard error of the mean ( sem ) was calculated and data analyzed using the non-paired Student's t-test for single mean comparisons .
Culex mosquitoes are known vectors of WNV and Japanese encephalitis virus , whilst Aedes mosquitoes are vectors of DENV and yellow fever virus . To determine whether Vago activation is conserved among flaviviruses and their vector species , Culex quinquefasciatus ( Hsu ) and Aedes albopictus ( RML12 ) cell lines were infected with WNV and DENV , respectively , at a multiplicity of infection ( MOI ) of 5 , and total RNA and protein lysates were collected at 48 h post-infection ( hpi ) . Real-time RT-qPCR using primers specific for Culex ( Cx ) or Aedes ( Ae ) Vago mRNA indicated a 3–4 fold induction ( Fig . 1A ) . Western blotting performed on protein lysates using anti-Vago antibody also showed a significant increase in Vago expression at 48 hpi ( Fig . 1B ) . The results demonstrate that flavivirus-induced Vago activation is a conserved function in mosquitoes . To identify the promoter region responsible for Vago activation , an initial analysis was conducted using PROMO software [24] , which predicted multiple transcription factor binding regions in the 5′ region ∼2 kb upstream of CxVago gene ( using Culex quinquefasciatus genome sequence , GI: 145648993 ) ( Table S1 ) . The 5′ regions ∼2 kb upstream from the transcription start site of Vago orthologs in Culex quinquefasciatus , Aedes aegypti and Anopheles gambiae mosquitoes were then aligned and conserved transcription factor binding sites were identified using phylogenetic footprinting ( Footprinter2 . 0 ) [25] . The results indicated that NF-κB ( Rel ) ( CACCTTCCCC ) and GATA-1 ( TTATCTTT ) transcription factor binding sites are conserved in the Vago promoter region of the three mosquito species ( Table S1 ) . To validate the in-silico data identifying the promoter region responsible for Vago induction , a plasmid was constructed by inserting the ∼2 kb 5′ region upstream of the CxVago gene ( −1 to −2007 ) in front of a luciferase reporter gene . Hsu cells transfected with this reporter plasmid were infected with WNV and luciferase activity in cell lysates was measured 24 hpi . The results indicated a significant ( ∼4-fold ) increase in luciferase activity compared with uninfected control cells ( Fig . 2A ) . Similarly , two fragments of the ∼2 kb promoter region ( −1 to −989 and −990 to −2007 ) were cloned upstream of the luciferase reporter and transfected Hsu cells were infected with WNV . The results indicated a similar increase in luciferase activity in cells transfected with plasmid containing the −1 and −989 bp fragment ( Fig . 2A ) , indicating this region is sufficient and essential for induction of Vago . As a control , Hsu cells transfected with a plasmid containing the whole promoter region ( Vago-Luciferase ) were treated with Vago-containing media , to determine whether Vago was acting in autocrine or paracrine fashion . The results showed no increase in luciferase activity indicating Vago is unable to activate its own promoter region ( Fig . S3 ) . The assay was then repeated using the luciferase reporter plasmid containing smaller fragments of the Vago promoter region of various lengths . The results indicated that a 30 bp region ( −250 to −280 ) containing the predicted NF-κB binding site was sufficient to induce increased luciferase activity following WNV infection ( Fig . 2B ) . To confirm that the NF-κB binding site is responsible for Vago induction , site-specific mutations ( CTT to AGG ) were introduced to disrupt the sequence ( see Materials and Methods ) . WNV infection of Hsu cells transfected with the luciferase reporter plasmid containing mutated NF-κB binding site showed significantly less induction of luciferase activity compared with wild-type NF-κB binding site control ( Fig . 2C ) . These results confirm that NF-κB binding site in the Vago promoter region is essential for its induction following WNV infection . Rel2 ( XP_001862276 ) is the ortholog of human NF-κB ( p105 subunit ) in the Culex genome , with about 30% amino acid identity ( Figure S1 ) . Rel2 has previously been shown to be involved in the Imd pathway in mosquitoes [26] , [27] . Experiments were performed to confirm the significance of Rel2 in Dicer-2-mediated induction of Vago . A long dsRNA was prepared to knockdown expression of Rel2 in Culex cells . Initially , Rel2 dsRNA and the luciferase reporter plasmid containing the −1 to −2007 Vago promoter region were co-transfected into Hsu cells . Cells co-transfected with GFP dsRNA were used as silencing control . The cells were then infected with WNV 24 h post-transfection and luciferase activity was assayed at 24 hpi . The results indicated a significant induction of luciferase activity in WNV-infected control cells treated with GFP dsRNA , but significantly less induction in WNV-infected cells treated with Rel2 dsRNA ( Fig . 3A ) . The results show some induction of luciferase activity after WNV infection in cells transfected with Rel2 dsRNA . This may be due to inefficient knockdown of Rel2 or reduced transfection efficiency when cells are transfected with both Rel2 dsRNA and luciferase plasmids . We also cannot rule out other factors responsible for Vago regulation . Next , to determine whether Vago induction requires activation of Rel2 , Hsu cells were transfected with Rel2 dsRNA and then infected with WNV 24 h post-transfection . Control cells were transfected with GFP dsRNA . Total RNA and supernatant media were collected at 48 hpi . A real-time RT-qPCR assay on the total RNA using Rel2-specific primers showed that treatment with Rel2 dsRNA caused a significant decrease in Rel2 mRNA , indicating knockdown of the target ( Fig . 3B ) . A real-time RT-qPCR using Vago-specific primers indicated that there was more than 3-fold induction of Vago mRNA after WNV infection but less than 2-fold increase in Vago mRNA in cells in which Rel2 was knocked-down , which although statistically significant , was much lower than in control cells ( dsRNA GFP ) infected with WNV ( Fig . 3B ) . A western blot using Vago antibody also showed that Rel2 knockdown reduced Vago protein expression level after WNV infection ( Figure S4 ) . This suggested that Rel2 is required for Vago induction . A real-time qPCR assay was also performed using primers specific for WNV non-structural protein 1 ( NS1 ) gene . The results indicated that viral RNA expression levels were >3 fold higher in the Rel2 knockdown cells than in control cells treated with GFP dsRNA ( Fig . 3C ) . A plaque assay performed on the supernatant media showed significantly higher viral titers ( >1 log ) in Rel2-knockdown cells compared with control cells ( Fig . 3D ) . The results indicate that Rel2 is required for induction of Vago and plays a significant role in antiviral immunity in mosquito cells . WNV infection in mammalian cells leads to activation of interferon regulatory factors ( IRFs ) and NF-κB via TRAF-3 and TRAF-6 , respectively . Experiments were conducted to determine whether an equivalent pathway exists in Culex cells . The Culex quinquefasciatus genome encodes of two TRAF proteins , XP_001858793 ( TRAF-3 ) and XP_001846690 ( TRAF ) , with more than 30% amino acid similarity with human TRAF-3 and TRAF-6 , respectively ( Figure S2 ) . Experiments were initially performed to confirm the significance of TRAFs in Dicer-2-mediated induction of Vago . Long dsRNAs were prepared to knockdown expression of each TRAF in Culex cells . Initially , TRAF3 or TRAF dsRNAs and the luciferase reporter plasmid containing the −1 to −2007 Vago promoter region were co-transfected into Hsu cells . Cells co-transfected with GFP dsRNA were used as silencing control . The cells were then infected with WNV 24 h post-transfection and luciferase activity was assayed at 24 hpi . The results indicated a significant induction of luciferase activity in WNV-infected control cells treated with GFP dsRNA , but significantly less induction in WNV-infected cells treated with TRAF dsRNA ( Fig . S5 ) . Hsu cells were then transfected with TRAF dsRNAs or GFP dsRNA ( control ) and infected with WNV at 24 h post-transfection . Total RNA and the supernatant media were collected at 48 hpi . Real-time RT-qPCR results indicated that TRAF-3 and TRAF dsRNA transfection led to significant silencing of the respective TRAF mRNA in cells without affecting expression of the other TRAF , indicating specificity and efficiency of gene silencing ( Fig . 4A ) . A real-time RT-qPCR assay using Culex Vago primers showed that , compared to control cells , there was no significant WNV-induced increase in Vago mRNA in cells in which TRAF was silenced , suggesting that TRAF was required for Vago induction ( Fig . 4C ) . However , Vago was upregulated in WNV-infected cells treated with TRAF-3 dsRNA , indicating TRAF-3 plays no role in Vago induction . A real-time RT-qPCR assay using WNV NS1 primers indicated that viral RNA levels were significantly higher ( >3 fold ) following TRAF knockdown compared to infected control cells treated with GFP dsRNA , whilst there was no significant difference in WNV RNA levels between infected cells with TRAF-3 dsRNA and control ( GFP dsRNA ) cells ( Fig . 4B ) . Plaque assays performed on the supernatant media showed similar results with significantly higher viral titers ( >1 log ) in cells silenced for TRAF compared with control and TRAF-3 silenced cells ( Fig . 4D ) . These results indicate that TRAF , but not TRAF-3 , is essential for induction of Vago and in-turn plays a significant role in antiviral immunity in mosquito cells . Based on these results , it is possible to conclude that Culex Vago is activated by two different pathways , one involving TRAF and other involving Rel2 . Therefore , experiments were conducted to determine whether both TRAF and Rel2 are part of the same Vago induction pathway downstream of Dicer-2 . The Culex Rel2 gene was cloned into plasmid vector containing the insect cell promoter OpIE2 and a V5 tag ( pIZ-V5 ) . Hsu cells were then transfected with TRAF dsRNA ( or GFP dsRNA as control ) and the pIZ-Rel2-V5 plasmid . Cells were infected with WNV at 24 h post-transfection and cell lysates were collected at 48 hpi . Western blotting performed using anti-V5 antibody detected Rel2-V5 at ∼110 kDa ( Fig . 5A ) . Cells infected with WNV showed cleavage of Rel2-V5 as evidenced by a band at ∼50 kDa , representing the carboxy-terminal inhibitory domain of the protein ( IkB ) tagged with V5 . This indicated cleavage of Rel2 which is a prerequisite for nuclear localization of amino-terminal portion of Rel2 which acts as the activated transcription factor . Cells treated with TRAF dsRNA showed no evidence of cleavage of the ∼110 kDa Rel2-V5 band . This indicates that TRAF is required for WNV-induced activation of Rel2 in Culex cells . A similar experiment performed using Dicer-2 dsRNA indicated that Dicer-2 knockdown inhibited WNV-induced Rel2-V5 cleavage , compared with control cells ( Fig . 5B ) . The results suggest that both Dicer-2 and TRAF are required for Rel2 activation . A mosquito infection model was used to validate results obtained in vitro . Adult 3–5 day old female mosquitoes ( Culex pipiens quinquefasciatus ) were microinjected with dsRNA against GFP , Rel2 or TRAF to knockdown specific genes . The mosquitoes were microinjected 48 h later with WNV ( Kunjin strain ) or cell culture medium ( controls ) and total RNA was collected at 48 hpi from a pool of 4 mosquitoes in each group . CxVago expression , as assayed by real-time RT-qPCR , was induced 3–4 fold in mosquitoes microinjected with GFP dsRNA and Kunjin virus . The results also showed that CxVago expression was not significantly induced in Kunjin challenged mosquitoes microinjected with Rel2 dsRNA or TRAF dsRNA , indicating involvement of these genes in WNV-induced Vago activation ( Fig . 5C ) . The real-time RT-PCR results also showed specific knockdown of Rel2 and TRAF after dsRNA treatment , indicating efficient knockdown of the target genes ( Fig . 5C ) . Changes in viral RNA were also measured by real-time RT-qPCR using WNV NS1 primers . The results indicated a statistically significant higher ( >2fold ) load of WNV RNA in the Rel2 and TRAF knockdown mosquitoes ( Fig . 5C ) .
Mosquito antiviral immunity is a potentially important but poorly characterized aspect of arbovirus transmission . Mosquito immune response pathways could have a significant role in determining vector competence and in allowing efficient infection in the absence of significant pathology which is an intrinsic characteristic of the arbovirus transmission cycle in insects . The RNAi pathway is now known to comprise a major portion of mosquito defense against invading viruses [28]–[30] . Critically , this involves Dicer-2 , an evolutionarily conserved protein containing a carboxy-terminal endoribonuclease domain which cleaves the viral RNA to form viral interfering RNAs . Recent research has also shown that the N-terminal helicase domain of Dicer-2 is similar to DExD/H-box helicase domain found in the mammalian viral RNA sensors RIG-I and MDA5 [16] . In response to viral infections , these mammalian proteins are involved in the induction of interferons which are subsequently secreted , stimulating antiviral activity in other cells . Our previous research has shown that Culex Vago is a functional homolog of mammalian interferons [15] . Culex Vago is induced by WNV infection and , like interferons , is secreted and activates Jak-STAT pathway in the neighboring cells , leading to induction of an antiviral response . However , neither the pathway linking Dicer-2 to Vago induction nor the antiviral effectors induced by Vago had previously been characterized . The results presented here demonstrate that Vago induction occurs in response to flavivirus ( WNV and DENV ) infection in cells from a range of mosquito vector species and , using a WNV-Culex mosquito model , we reveal that this occurs through a novel immune signaling pathway . We show that detection of WNV infection by Dicer-2 leads to activation of TRAF , which in turn triggers cleavage of Rel2 , allowing translocation of the N-terminal activation domain to the nucleus and binding to the NF-κB site in promoter region upstream of the Vago gene . As shown previously , induction and secretion of Vago then stimulates an antiviral response via the Jak-STAT pathway [15] . We also ruled out the possibility that Vago is activated by secretion of Vago from neighbouring cells ( Figure S4 ) . The Vago induction pathway is similar to the RIG-I/TRAF-6/NF-κB-mediated interferon activation pathway , which is activated in response to viral infections in mammalian cells . Interestingly , the mammalian pathway also involves the mitochondrial protein , MAVS , as well as interferon regulatory factors ( IRFs ) , each of which have no apparent orthologs in the mosquito genome . Mammalian RIG-I contains a CARD domain which interacts with a CARD domain in MAVS [18] . This leads to activation of TRAF-3 which induces interferons in IRF-dependent pathway . Stimulation of MAVS also leads to activation of TRAF-6 which induces interferons in NF-κB-dependent fashion [31] . Unlike mammalian RIG-I , Culex Dicer-2 ( and other Dicer-2 orthologs ) lacks a CARD domain and seems to only involve TRAF and NF-κB dependent pathway . The mechanism of Dicer-2 interaction with the downstream proteins remains to be seen . The pathway may also involve unique and still unidentified proteins that link TRAF to Dicer-2 . Experiments to identify these intermediates are currently underway . Using mutant flies and promoter deletion experiments , Deddouche et al [16] reported that induction of drosophila Vago was not mediated by members of NF-κB family of transcription factors . It is possible that regulation of Vago in mosquitoes is different than that in drosophila . Interestingly , Rel2 is also activated by the Imd pathway in response to gram-negative bacterial infection in insects [32] . Peptidoglycans on gram-negative bacteria are recognized by PGRP receptor in insects , which leads to mutlimerization of the receptor . This receptor activation leads to recruitment of adaptor proteins , including Imd [33] , which in turn leads to a cascading reaction involving kinases ( TAK1 and IKK ) which phospholyrate Rel2 . Rel2 consists of an N-terminal nuclear factor containing domain ( Rel-68 ) and an inhibitory C-terminal domain ( Rel-49 ) which anchors Rel2 in the cytoplasm . Rel2 is then cleaved by the caspase , DREDD , releasing the N-terminal domain Rel-68 . This translocates to the nucleus where it is able to activate transcription of genes encoding antimicrobial peptides [34] . In this report , we demonstrated that WNV-induced cleavage of Rel2 occurs via a distinct Dicer-2 and TRAF dependent pathway . Overall , this report identifies a mechanism of activation of CxVago , an interferon functional homolog in mosquitoes . This Dicer-2-dependent pathway is similar to the process of activation of mammalian interferon and involves orthologs of TRAF and Rel2 ( NF-κB ) . Although some of the intricacies of this pathway are yet to be established , the identification of this pathway opens up a novel avenue of mosquito antiviral response regulation . Furthermore , as Dicer-2 is an evolutionarily conserved protein with a canonical domain structure , it raises interesting questions about the evolutionary origins of innate antiviral immunity in vertebrates . | Viruses like West Nile , dengue and Japanese encephalitis are responsible for large number of human and livestock diseases worldwide . These viruses , transmitted by female mosquitoes via saliva during blood-feeding , elicit an immune response in these mosquitoes . The details of this immune response are still being investigated . Dicer2 , which has previously been shown to be involved in RNAi mediated antiviral activity in mosquitoes , contains helicase domain , which leads to activation of antiviral protein , Vago . Vago is functionally similar to mammalian interferon and after secretion activates Jak-STAT pathway in neighboring cells leading to antiviral effect . Here we demonstrate that sensing of viral RNA by Dicer2 leads to activation of TNF receptor-associated factor ( TRAF ) , which in turn leads to cleavage and release of amino-terminal domain of Rel2 , NF-κB ortholog . Rel2 binds to a conserved NF-κB binding site on Vago promoter region leading to its induction . This proposed mechanism of Vago activation is similar to mammalian interferon activation after viral infection . The identification of this novel and evolutionarily conserved pathway downstream of Dicer2 provides new insight into the immune signalling in mosquitoes and other invertebrates . | [
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] | 2014 | Dicer-2-Dependent Activation of Culex Vago Occurs via the TRAF-Rel2 Signaling Pathway |
The M segment of the 2009 pandemic influenza A virus ( IAV ) has been implicated in its emergence into human populations . To elucidate the genetic contributions of the M segment to host adaptation , and the underlying mechanisms , we examined a panel of isogenic viruses that carry avian- or human-derived M segments . Avian , but not human , M segments restricted viral growth and transmission in mammalian model systems , and the restricted growth correlated with increased expression of M2 relative to M1 . M2 overexpression was associated with intracellular accumulation of autophagosomes , which was alleviated by interference of the viral proton channel activity by amantadine treatment . As M1 and M2 are expressed from the M mRNA through alternative splicing , we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication , irrespective of amino acid composition of M1 or M2 . Moreover , in spite of efficient replication , virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently . We conclude that ( i ) determinants of transmission reside in the IAV M2 protein , and that ( ii ) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis .
Influenza A virus ( IAV ) epidemics and pandemics result in widespread and often severe disease [1–3] , as well as considerable societal economic costs [4] . IAV lineages endemic in humans are ultimately derived from those circulating in wild waterfowl . Sporadic transmission of IAV from avian reservoirs to humans occurs mainly through domesticated intermediate hosts such as chickens and pigs [5] . For example , infection of humans with H7N9 and H5N1 subtype IAVs occurs through direct exposure to infected poultry . Although they often cause severe disease , these zoonotic cases have not led to sustained onward transmission to date and have therefore not caused a pandemic . Despite abundant circulation of IAVs at the animal-human interface , pandemics occur only rarely owing to the host specificity of IAV infection . As with all viruses , IAV must exploit host cell functions and overcome antiviral barriers to execute its life cycle . Viral replication in a new host species therefore requires adaptation to re-establish virus-host interactions broken by species-specific features of host cellular processes . IAV can undergo such adaptation through reassortment of intact gene segments between avian IAVs and those already adapted to mammals or through more incremental changes brought about by polymerase error [6] . With the goal of anticipating and mitigating IAV emergence in humans , it is essential to understand the species-specific barriers to infection and the mechanisms by which IAV evolution allows these impediments to be overcome . Changes to viral receptor specificity and polymerase function necessary to overcome host species barriers have been well documented [7–11] , but it is clear that additional features of avian IAVs restrict their growth in mammalian systems [12–14] . Seminal studies , performed in the 1970s , indicated a potential role for the M segment . Avian IAVs were shown to express low levels of M1 protein during abortive infection of mammalian cells , but not avian cells [15–17] , and the limited quantity of viral particles formed during such abortive infections correlated with the amount of M1 protein expressed . Significantly , these studies were conducted prior to the identification of a second gene product expressed from a spliced form of the M segment mRNA: the M2 protein [18 , 19] . Subsequent studies demonstrated that low M1 expression during abortive avian IAV infection in mammalian cells was accompanied by increased M2 mRNA and protein expression , relative to productive infection in avian cells , leading to new hypotheses regarding the mechanisms leading to abortive outcomes [20–22] . Additional evidence for a role of the M segment in host range comes from the in silico identification of positive selection acting on the M segment of the Eurasian avian-like swine lineage , following transfer of this IAV from birds to swine [23] . Moreover , others and we have shown that the M segment of the 2009 pandemic H1N1 ( pH1N1 ) strain carries determinants of transmissibility , suggesting that adaptation of this segment to humans contributed to the emergence of the pH1N1 lineage [24–28] . The mechanistic basis for this contribution; the specific roles , if any , of M1 and M2 proteins to host range; and whether the M segment plays a broader role in IAV host adaptation , remain unclear . It is now recognized that the M segment of IAV encodes the M1 matrix and the M2 proton channel proteins via alternative splicing ( S1A Fig ) [29 , 30] . M1 forms a structural layer under the viral envelope and is a major determinant of virion morphology [24 , 26 , 31 , 32] . Upon endocytosis of virus particles into cells , low pH and high [K+] trigger a conformational change in M1 required for release of viral ribonucleoprotein ( vRNP ) complexes into the cytoplasm [33] . M1 is also critical for the transport of newly replicated vRNPs from the nucleus to the cytoplasm [34–37] . At late stages of infection , cytoplasmic M1 is thought to recruit vRNP complexes to the budding site [38–40] . M2 is a transmembrane protein that tetramerizes to form a proton channel . Within the envelope of incoming virions , this channel allows diffusion of protons into the virion core , thereby triggering the release of vRNPs from M1 , as well as facilitating the intracellular transport of HA protein [41–44] . M2 also plays an important role in the final stages of virion morphogenesis , mediating ESCRT-independent membrane scission [45] . Importantly for the findings reported herein , multiple groups have reported that M2 blocks the fusion of autophagosomes with lysosomes [46–48] . Under normal conditions , this fusion event allows degradation and recycling of organelles and protein aggregates contained within autophagosomes [49] , and is thus important for cellular homeostasis . M2-mediated interference with autolysosome formation therefore stalls autophagic flux and leads to the accumulation of autophagosomes within the cell [46–48] . Many elegant molecular virology studies have revealed that viral strategies for regulating gene expression are central to orchestration of the viral life cycle [50–53] . Here , we report that regulation of M2 expression of IAV is an important determinant of host range . In particular , our data show that the expression of M1 and M2 from alternatively spliced M segment mRNAs is dysregulated when an avian IAV replicates in mammalian cells , and that the resultant overexpression of the M2 proton channel attenuates viral growth by disrupting the cellular autophagy pathway . The observed block in autophagy is consistent with that reported previously for lab-adapted strains of IAV [46–48] . Importantly , however , we see that lab-adapted IAVs express M2 at high levels , comparable to those seen for avian IAV M segments in mammalian cells . In contrast , IAVs carrying M segments well adapted to mammalian systems express relatively little M2 and do not trigger the accumulation of autophagosomes to the same extent as avian-origin and lab-adapted strains . These findings suggest that IAV-induced stalling of autophagic turnover is a result of aberrantly high M2 expression upon infection of mammalian cells with avian IAVs , and which is overcome in human-adapted influenza A lineages . Critically , the mechanism leading to this host-restricted phenotype occurs at the level of gene expression and is driven by host-specific differences in viral nucleic acid sequence , not amino acid changes . This mechanism constitutes a novel paradigm in RNA virus host adaptation , and reveals a new species barrier for IAV , which may be highly relevant for the emergence of avian IAVs into humans .
To assess the contribution of the M segment to host adaptation , we generated a panel of IAVs possessing human- or avian-derived M segments in the A/Puerto Rico/8/34 ( H1N1 ) [PR8] background ( Table 1; Fig 1A; S1B , S1C and S1D Fig ) . The M segments used were selected with the aims of representing circulating human IAVs and capturing the breadth of diversity of M segment sequences present in wild waterfowl . In addition to three wild-type avian M segments , we included in our panel a mutant of the dk/Alb/76 M segment that carries a uniquely identified mutation in M2 ( S89 ) . Reversion of this unique amino acid residue to glycine yields an M2 protein matching the consensus amino acid sequence of IAVs in wild bird reservoirs . The human-adapted M segment analyzed most extensively herein is that of the pandemic A/NL/602/09 ( H1N1 ) strain [NL09] . The relative fitness of PR8-based viruses carrying M segments from the human-adapted NL09 virus or from avian IAVs was evaluated in three avian substrates: embryonated chicken eggs ( ECE ) , chicken origin DF-1 cells , and quail origin QT-6 cells . When grown from low multiplicities of infection ( MOI ) in either ECE or DF-1 cell substrates , all viruses exhibited similar kinetics and reached comparable titers ( Fig 1B and 1C ) . Also , under high MOI growth conditions in DF-1 and QT-6 cells , the growth phenotypes of the PR8 NL09 M and PR8 avian M viruses were similar , although the NL09 M-containing virus trended towards increased growth relative to the avian M-containing viruses in DF-1 cells ( Fig 1D and 1E ) . Nonetheless , at no time did the NL09 M-encoding virus grow to significantly higher titers than the avian M-encoding viruses in any avian host substrate . In contrast , in mammalian cells , human-adapted IAV M segments were found to support improved growth relative to avian-adapted M segments . Viral growth was monitored from low and high MOI in human A549 cells and canine MDCK cells ( Fig 2 ) , and additionally from high MOI in human 293T cells ( S2A Fig ) . In each case , PR8-based viruses carrying the NL09 M segment grew with more rapid kinetics and to higher titers than isogenic viruses carrying avian M segments , although the differences in growth from low MOI did not reach statistical significance . To extend our findings to an independent human lineage of IAV , we evaluated the growth from high MOI in A549 cells of PR8 Pan99 M and PR8 Beth15 M viruses , each of which have an M segment derived from the human seasonal H3N2 lineage ( S1C and S1D Fig ) . We found that both of these human-adapted M segments supported significantly faster kinetics and higher magnitude of growth than the dkAlb76 89G M segment ( S2B Fig ) . Thus , results from multiple different avian and mammalian culture systems reveal defects in the kinetics and magnitude of viral growth associated with avian IAV M segments specifically in mammalian host cells and provide evidence of the acquisition of adaptive changes in the M segment of pH1N1 and H3N2 human influenza A lineages . To evaluate the impact of M segment host adaptation on viral phenotypes in an intact host , we evaluated the PR8 NL09 M virus and the four PR8-based viruses carrying avian M segments in a guinea pig model [54 , 55] . A low inoculation dose of 10 PFU per animal was used to maximize the sensitivity of the system . Each inoculated animal was co-caged with a naïve guinea pig at 24 h post-inoculation and viral infectivity , magnitude and kinetics of growth , and transmission were monitored by collecting nasal lavage samples every other day . Among those animals that were productively infected , total virus sampled in nasal washes over the course of infection was evaluated by calculating the area under the shedding curves shown in Fig 3A . Area under the curve values did not differ significantly among the four viruses with avian M segments , but were significantly higher for PR8 NL09 M virus ( Fig 3A ) . As shown in Fig 3B , fewer guinea pigs were productively infected with 10 PFU when the inoculating virus carried an avian M segment . Differences in the kinetics of viral growth were determined by assessing changes in growth over time using repeated measures ANOVA ( S3 Fig ) . Each virus encoding an avian host-derived M segment replicated with significantly slower kinetics than the PR8 NL09 M virus . The efficiency of transmission was assessed by calculating the proportion of contact animals that contracted infection , considering only cages in which donor guinea pigs were productively infected through intranasal inoculation . While transmission occurred in 7/9 instances for PR8 NL09 M virus , a significantly lower proportion of animals ( 1/20; p<0 . 0001 ) were infected with a virus carrying an avian M segment transmitted from her cage mate ( Fig 3B ) . Thus , data from a mammalian animal model reveal defects in infectivity , growth and transmission associated with avian IAV M segments . Our results suggest the acquisition of adaptive changes in the M segment of pH1N1 human IAV since its introduction from the avian reservoir into mammals . To investigate the mechanism underlying the host-restricted phenotypes conferred by avian-adapted M segments , the steady state levels of the M1 and M2 proteins were evaluated in human A549 and avian DF-1 cells infected at high MOI . Western immunoblotting with the monoclonal antibody E10 , which recognizes the shared N-terminus of M1 and M2 , was used to monitor levels of both proteins concurrently . In the avian cell line , the PR8 NL09 M virus expressed lower levels of M2 than M1 ( Fig 4A ) . When M1 and M2 levels were normalized to those of vinculin and the relative levels of M1 and M2 calculated , the NL09 M2 protein comprised <10% of the total M segment-derived protein expression . Very similar results were seen for four PR8-based viruses carrying avian M segments grown in DF-1 cells . In each case , M2 comprised <20% of the total M segment-derived protein , and in the case of three avian M segment-containing viruses ( PR8 dkAlb76 M; PR8 dkAlb76 M 89G; and PR8 rheaNC93 M ) , the levels of M1 and M2 proteins were not significantly different from those expressed by the PR8 NL09 M virus ( Fig 4B , 4C and 4D ) . In A549 cells , the PR8 NL09 M virus expressed M2 at ~20% of the total M protein , similar to the expression levels observed in infected DF-1 cells ( Fig 5A ) . In contrast , avian M segments yielded markedly higher levels of the M2 protein than were observed for the same viruses grown in avian cells . Here , M2 levels were approximately equal to those of M1 ( Fig 5B , 5C and 5D ) . Moreover , when compared to the NL09 M segment in human cells , levels of M2 protein were significantly higher for avian M segments ( P<0 . 0001 ) ( Fig 5C and 5D ) . Thus , compared to matched virus-host pairings in both human and avian systems , M2 was markedly overexpressed when avian-adapted M segments were introduced into human cells . The M segment vRNA is transcribed by the viral polymerase to give rise to a co-linear mRNA ( mRNA7 ) , which can be spliced by cellular splicing factors to yield mRNA10 or mRNA11 [18 , 56] . The M1 protein is translated from the unspliced mRNA7 , while M2 is expressed from the spliced mRNA10 . To date , no polypeptide corresponding to the short mRNA11 ORF has been identified . Aberrantly high levels of IAV mRNA10 in mammalian cells have been previously reported for the avian IAV , fowl plague virus [20 , 21] . We therefore hypothesized that the high expression levels of M2 protein , observed for avian-adapted M segments in human cells , was due to increased splicing of mRNA7 to yield excessive mRNA10 . We used a RT primer extension assay to quantify the levels of each M segment-derived mRNA species in DF-1 and A549 cells infected with either PR8 NL09 M virus or one of four viruses carrying an avian-adapted M segment . As expected , based on the levels of the encoded proteins , mRNA10 was of low abundance compared to mRNA7 for all viruses in DF-1 cells ( Fig 6A ) . Quantification of band intensities revealed that , in DF-1 cells infected with PR8 NL09 M virus , mRNA7 comprised ~75% , while mRNA10 comprised ~5% of the total M segment-derived mRNA ( Fig 6B ) . For three avian M viruses ( PR8 dkAlb76 M; PR8 dkAlb76 M 89G; and PR8 rheaNC93 M ) , relative levels of mRNA7 were ~70–75% and were not significantly different from those of PR8 NL09 M virus ( Fig 6B ) . For all viruses , mRNA10 was expressed at low levels ( 10–40% of total segment 7 mRNA ) , and in the case of two avian M segment-containing viruses ( PR8 dkAlb76 M; PR8 dkAlb76 M 89G ) , the levels of mRNA10 were not significantly different from those expressed by the PR8 NL09 M virus ( Fig 6C ) . mRNA11 was expressed in each infection at approximately 20% of the total M segment-derived mRNA , and the relative level did not differ significantly with any virus ( Fig 6D ) . Overall , the relative levels of each of the three mRNA species were similar when synthesized from avian- or human-origin encoded M segments in DF-1 cells , correlating with Western immunoblot data on M segment protein expression in these cells . In A549 cells , the PR8 NL09 M virus showed relatively high abundance of mRNA7 and low abundance of mRNA10 , as was seen in DF-1 cells ( Fig 7A ) . By contrast , for viruses carrying avian-adapted M segments , mRNA10 levels exceeded those of mRNA7 in A549 cells ( Fig 7B and 7C ) . Both decreases in mRNA10 levels and increases in mRNA7 levels contributed to the altered relative abundance . Again , no significant differences in relative mRNA11 levels among the viruses were noted ( Fig 7D ) suggesting that changes in splicing to produce mRNA11 do not contribute to changes in M segment mRNA and protein expression observed in A549 cells . Overall , our data point to increased splicing to produce mRNA10 as the underlying mechanism leading to heightened M2 expression from avian M segments in A549 cells , and suggest that cis-acting signals present on the M segment are responsible for driving the disparate M2 expression levels . To gain initial insight into the phenotypic consequences of M2 overexpression , we examined the subcellular localization of M2 in A549 cells infected with PR8 NL09 M virus or one of four PR8-based viruses with avian M segments . At 8 hours-post-infection ( hpi ) in PR8 NL09 M virus-infected cells , M2 showed mainly dispersed cytoplasmic staining , along with weak cytoplasmic membrane localization and some perinuclear accumulation . By contrast , in cells infected with viruses that encoded any of the four avian origin M segments , we noted more abundant M2 protein staining localized at the plasma membrane , and in large puncta near the cell nucleus ( Fig 8A ) . Similar data were obtained from virus-infected 293T cells at 8 hpi ( Fig 8B ) and from both human cell lines at 12 hpi ( S6 Fig ) . This staining pattern , along with data in previously published studies [46–48] , prompted us to investigate whether highly expressed M2 protein was interacting with the cellular autophagy pathway . An interaction between the IAV M2 protein and the cellular autophagy pathway has been reported previously [46–48] . To test whether M2 expression from avian vs . human M segments has differential effects on autophagy , we first evaluated the level of LC3B lipidation in PR8 NL09 M- and PR8 avian M-infected cells . Upon activation of the autophagy pathway , LC3B is modified through the addition of phosphotidylethanolamine [57 , 58] . This lipidated form , called LC3B II , remains associated with the membranes of maturing and mature autophagosomes . Thus , increased levels of LC3B II in the cell are indicative of autophagosome formation; however , under normal conditions LC3B I and II are cycled through an autophagic flux which involves autophagosome maturation and degradation via fusion with lysosomes [59] . A block in this fusion event can therefore lead to accumulation of autophagosomes in the cytoplasm and , consequently , accumulation of LC3B II in the cell . Western immunoblot analysis of LC3B revealed decreased LC3B I and increased LC3B II protein levels in virus-infected cells compared to mock , providing evidence of autophagy activation and/or block during IAV infection of either avian or human cells ( Fig 4A , 4E and 4F; 5A , 5E and 5F ) . In avian DF-1 cells , levels of LC3B I were slightly decreased and levels of LC3B II were slightly increased over mock , but LC3B I and LC3B II levels were generally comparable among human M- and avian M-containing viruses ( Fig 4E and 4F ) . Importantly , human A549 cells infected with PR8 avian M-containing viruses consistently showed lower levels of LC3B I , and higher levels of LC3B II than A549 cells infected with PR8 NL09 M as well as other human M-containing viruses ( Fig 5E and 5F ) . Additionally , the same phenotypes were obtained from the panel of viruses upon infection of human origin 293T cells ( S4A–S4F Fig ) , or canine origin MDCK cells ( S4G–S4L Fig ) . Taken together , these data reveal a correlation between levels of M2 expression and LC3B lipidation ( accumulation of LC3B II ) , suggesting that high levels of M2 present in PR8 avian M-infected cells may be inducing an over-activation of autophagy , or a block in the turnover of autophagosomes . To confirm that M2 protein was co-localizing with LC3-positive autophagic vesicles in mammalian cells , we monitored sub-cellular localization of an overexpressed LC3B-GFP fusion protein in infected cells . Human 293T cells were primarily used for this purpose as they transduce well with GFP-LC3 . Under normal conditions , LC3B-GFP can be seen throughout the cell with a diffuse distribution ( Fig 9 , row 1: mock infected cells ) . Treatment with chloroquine , an inhibitor of autophagy that decreases autophagosome-lysosome fusion , results in the concentration of LC3B-GFP into punctate cytoplasmic structures ( Fig 9 , row 7: CQ-treated cells ) . This relocalization is indicative of the accumulation of autophagosomes [60] . In LC3B-GFP expressing 293T cells infected with PR8 NL09 M virus , we observed little to no increase in GFP-positive cytoplasmic puncta ( Fig 9 , row 2: NL09 M infected cells ) . In cells infected with PR8 avian M-encoding viruses , by contrast , extensive accumulation was apparent , with both the size and number of intracellular vesicles increased relative to PR8 NL09 M infection ( Fig 9 , rows 3–6: Avian M infected cells ) . In addition to these cytoplasmic puncta , viruses with avian M segments were found to redirect a small proportion of LC3B-GFP to the plasma membrane . Notably , the M2 protein co-localized with LC3B-GFP in PR8 avian M-infected cells , both in cytoplasmic puncta and at the plasma membrane . Similar phenotypes were observed in virus-infected A549 cells , at 8 hpi ( S7 Fig ) . Overall , these results suggest that expression of avian M2 protein at high levels interferes with the turnover of autophagosomes and with the localization of LC3B-GFP . In addition , the results reveal a reduction in the tight perinuclear localization of M2 upon LC3B overexpression . As an additional test designed to differentiate autophagy induction from autophagy block in cells infected with IAV carrying avian M segments , we used chloroquine treatment followed by Western immunoblotting for LC3B I and LC3B II . We hypothesized that , if autophagic flux is blocked in cells infected with PR8 avian M viruses , then treatment with chloroquine would not lead to a further increase in LC3B II levels . Conversely , if virus infection of these cells induces autophagy , then blocking their turnover with chloroquine would result in heightened LC3B II over mock-infected , chloroquine-treated cells . As shown in S8A and S8C Fig , we observed a significant difference in the activation of LC3B II following infection with human- or avian M-encoding viruses . This difference in LC3B II activation is lost in the presence of chloroquine . Additionally , as we observed no change in LC3B II levels upon treatment of virus-infected cells with chloroquine , relative to chloroquine-treated , mock-infected cells , we conclude that the high levels of IAV M2 protein expressed from avian M segments , but not human M segments , precipitate a block in the autophagy pathway such that chloroquine treatment cannot bring about further accumulation of autophagosomes . To evaluate the contribution of IAV M2 ion channel activity to the observed stalling of autophagy in PR8 avian M virus-infected cells , we inhibited channel activity with amantadine [61] . Amantadine was added at 1 hpi and the level of LC3B lipidation was assessed at 8 hpi in PR8 NL09 M and PR8 avian M virus-infected A549 cells . The timing of amantadine addition was designed to avoid disruption of the early function of M2 in delivery of the viral genome to the cytoplasm , but allow inhibition of M2 activity in autophagosomes . As noted above ( Fig 5; S4 Fig ) , Western immunoblot analysis of LC3B revealed decreased LC3B I and increased LC3B II upon infection of A549 , 293T or MDCK cells , with avian or human M-encoding IAV , with the effect being stronger for viruses possessing avian M segments . Interestingly , addition of amantadine reversed the intracellular accumulation of LC3B II in both human and avian M-infected A549 cells ( Fig 10 ) . Quantitation of M segment-encoded proteins by Western immunoblot analysis showed no difference in the expression levels of M1 or M2 protein upon treatment with amantadine ( Fig 10A , 10B , 10C and 10D ) , but revealed a statistically significant increase in LC3B I ( Fig 10E ) and decrease in LC3B II ( Fig 10F ) to levels comparable to mock-infected cells . To ensure that the amantadine was working to prevent LC3B II accumulation through inhibition of M2 ion channel activity , we assessed the impact of amantadine treatment on the inhibition of phagosome-lysosome fusion by chloroquine . Amantadine had no impact on LC3B II accumulation in the absence of M2 protein ( Fig 10G–10I ) , indicating that the mechanism of amantadine block of autophagosome accumulation was specifically mediated through interference with M2 function . As a complementary approach to monitor the effects of amantadine , we again used overexpressed LC3B-GFP fusion protein in infected 293T cells at 12 hpi . Using confocal microscopy , we tracked LC3B-GFP localization in the presence and absence of the ion channel blocker . In line with the Western immunoblot analyses obtained in A549 cells , we saw that amantadine reduced the accumulation of LC3B-GFP within cytoplasmic puncta and at the plasma membrane of infected cells ( Fig 11 ) . Of note , amantadine treatment did not alter levels or localization of M2 protein and , moreover , had no detectable effect on LC3B-GFP appearance in chloroquine-treated cells ( Fig 11 ) . Overall , our results indicate that the intracellular ion channel activity of IAV M2 protein is necessary to mediate the observed block in autophagy , and that addition of amantadine can reverse the autophagic phenotypes induced by both human and avian M-encoding IAVs . To test for a causal link between the observed block in autophagy and the restriction of growth seen for PR8 avian M-encoding viruses in mammalian cells , we monitored the effect on viral growth of relieving the autophagy block with amantadine treatment . Specifically , we evaluated single-cycle growth in A549 cells using PR8 NL09 M and PR8 dkAlb76 M 89G viruses . Amantadine was again added at 1 hpi to avoid disruption of the earliest steps of the viral life cycle . While amantadine had no effect on growth of the PR8 NL09 M virus ( P = 0 . 271 ) , a modest but consistent increase in the titer of PR8 dkAlb76 M 89G virus was noted throughout the time-course ( P = 0 . 0007 ) ( Fig 12 ) . This result indicates that M2 ion channel-induced disruption of vesicular homeostasis contributes to the host restriction conferred by avian IAV M segments . To determine whether the observed effects of avian-origin M segments on the viral life cycle and the autophagy pathway are attributable to avian-adapted amino acid sequences or rather to the overexpression of the viral proton channel , we generated a series of chimeric M segments in which the coding changes found in the NL09 M segment were transferred to an avian-adapted RNA background , and vice versa . We introduced all 16 coding changes into M1 and M2 simultaneously , or introduced only the nine or seven changes into M1 or M2 , respectively . The genotypes of this panel of six chimeric viruses are outlined in Table 2 and S9 Fig . Western immunoblot analysis of M1 and M2 proteins expressed from these six chimeric M segments in infected A549 cells is shown in Fig 13A . Among the viruses with the NL09 M nucleic acid background , the PR8 NL M 16mut and PR8 NL M 9mut viruses both showed increased levels of M2 relative to the PR8 NL09 M virus control , indicating that all or some of the coding changes introduced into the M1 ORF impact regulation of M2 gene expression . The PR8 NL M 7mut virus showed similar M1 and M2 expression levels as the PR8 NL09 M control , however . This virus therefore expresses the avian consensus M2 protein at low levels . All viruses with the nucleic acid background of the avian M segment showed relatively high levels of M2 , similar to those seen for the PR8 dkAlb76 M 89G virus control . Thus , in the avian nucleic acid background , we were able to fully separate M2 expression level from M2 and M1 amino acid sequence . Single-cycle growth analysis of the chimeric viruses in A549 cells revealed a strong correlation between M2 expression level and viral yields ( Fig 14A , 14B and 14C ) . Notably , all viruses with the dkAlb76 M 89G-derived nucleic acid background , which express high levels of M2 protein , grew to approximately 10-fold lower titers than the PR8 NL09 M virus , despite expression of NL09 M1 and/or M2 proteins . In addition , the PR8 NL M 7mut virus , which expresses the human M1 and avian M2 proteins at levels typical of human-adapted strains , showed equivalent growth to that seen with the PR8 NL09 M virus , indicating that amino acid changes in M2 are not required for efficient replication in human cells . Notably , the PR8 Av M 9mut virus , which encodes identical M1 and M2 proteins as PR8 NL M 7mut virus ( S9 Fig ) , exhibits approximately 10-fold reduced viral replication relative to PR8 NL M 7mut virus ( P = 0 . 0003 ) ( Fig 14B and 14C ) . This result confirms that overexpression of M2 protein is a dominant negative trait that is deleterious to virus growth . Examination of LC3B lipidation by Western immunoblot analysis also revealed a clear correlation between M2 expression levels and those of LC3B II within infected cells ( Fig 13A ) . Importantly , overexpression of either human or avian M2 during infection boosted LC3B II levels ( compare lanes 2 , 3 , 5 , 6 , 7 and 8 in Fig 13A ) , while expression of avian M2 at levels comparable to those of PR8 NL09 M virus gave low LC3B II levels ( compare lanes 1 and 4 in Fig 13A ) . Confocal images of 293T cells transduced with LC3B-GFP and infected with the chimeric viruses were consistent with these findings . As seen for wild-type avian M segments , high M2 expression again led to the accumulation of LC3B-GFP in perinuclear puncta and at the plasma membrane ( S10 Fig ) . Thus , as with viral growth , the accumulation of autophagosomes within infected cells appears to be triggered by high levels of M2 , regardless of amino acid sequence . To verify that the levels of M1 and M2 protein exhibited by the chimeric M segment-encoding viruses are modulated at the level of mRNA expression , we conducted RT primer extension assays . These data confirmed that differences in the levels of segment 7 mRNA expression ( S11 Fig ) mirrored the observed differences in M1 and M2 protein expression levels . Overall , these data support the premise that splicing signals are at least partly encoded in non-synonymous nucleotide changes residing in the M1 ORF . To evaluate whether correcting expression of the avian M2 protein to levels seen for viruses with human-adapted M segments yielded a well-adapted phenotype in a mammalian animal model , we evaluated growth and transmission of PR8 NL09 M 7mut virus in guinea pigs . Titration of nasal wash samples revealed that the PR8 NL09 M 7mut virus ( which expresses avian M2 at low levels ) had a similar growth phenotype to that of PR8 NL09 M virus: growth kinetics and area under the curve values were not significantly different ( Fig 15; S12 Fig ) . In contrast , analysis of nasal wash samples collected from contact animals indicated that only one of eight inoculated guinea pigs transmitted the PR8 NL09 M 7mut virus to her cage mate . This level of transmission is significantly decreased relative to the PR8 NL09 M virus ( P = 0 . 0339 ) ( Fig 3B ) . Taken together , these results indicate that , when expressed at levels typical for a human-adapted M2 protein , an avian M2 supports efficient replication but inhibits transmission in a mammalian host . The M2-induced block in autophagic flux reported previously was based on multiple IAVs that were originally isolated from human hosts but passaged extensively in laboratory substrates [46–48] . To assess whether these prior data were consistent with our own , we compared M2 expression levels exhibited by one of the lab-adapted strains used , PR8 . Our results show that the relative expression levels of M1 and M2 in PR8-infected 293T cells are comparable to those seen in cells infected with avian M-encoding viruses ( S13 Fig ) . These results suggest that prior reports of M2-induced block in autophagy reflect a feature of mal-adaptation to the host cell .
Our data indicate that dysregulation of gene expression from the IAV M segment contributes to the limited replicative capacity of avian IAVs in mammalian hosts . When transcribed within mammalian cells , avian IAV M segments gave rise to abundant mRNA10 and correspondingly high levels of the encoded M2 proton channel . Comparison to matched virus-host systems indicated that this M2 expression profile was an aberrant feature of avian IAV replication in mammalian cells . When present at high levels , M2 had marked effects on the infected cell , blocking the turnover of autophagic vesicles and redirecting LC3B II , the activated form of a critical autophagy mediator , to the plasma membrane . These effects were found to rely on the proton channel activity of M2 and occurred when either the avian or human M2 protein was overexpressed in infected cells . Importantly , the reduction in viral growth conferred by avian M segments in mammalian systems could be attributed at least in part to disruption of vesicular homeostasis and was fully attributed to the altered expression levels of M1 and M2 . Thus , our data identify the regulation of viral gene expression as a novel host-dependent feature of the IAV lifecycle that contributes to the host range restriction of this virus ( Fig 16 ) . Quantification of IAV encoded mRNAs within infected cells indicated that overexpression of M2 from avian IAV M segments stems from over-production of the corresponding mRNA . M2 is encoded by the mRNA10 transcript , which is generated through splicing of the M1 message , mRNA7 [18 , 56] . Thus , regulation of M2 gene expression is intimately linked to the cellular splicing machinery . Our data indicate that this is a finely balanced interaction between IAV and its host cell , which is susceptible to disruption upon transfer to new species . In this regard , it is notable that the M segment splice donor and acceptor sites are identical between the human and avian IAVs examined . M segment sequences outside of these canonical splice sites have , however , been shown to modulate splicing efficiency [62–65] . Interestingly , mRNA7 association with mammalian SF2/ASF [64] , and with mammalian hnRNPK and NS1-binding protein [65] , have been shown to alter the efficiency of splicing . Nucleotide differences in these binding regions exist between human- and avian-adapted M segments . An interesting area of further investigation will be to test whether there are differences in the binding of human and avian mRNA7 molecules to mammalian SF2/ASF , hnRNPK , and NS1-binding proteins . The gene segments of IAVs circulating in humans are ultimately derived from the avian IAV gene pool . Thus , the contrasting phenotypes conferred by avian- and human-adapted M segments in mammalian cells suggest that a resetting of M segment gene expression to reduce M2 levels has been positively selected following emergence of avian IAVs into mammalian populations . Indeed , our data reveal that this adaptation has occurred in at least two independent incidences: i ) within the Eurasian avian-like swine lineage M , that emerged from birds in the late 1970s and contributed the M segment to the pH1N1 virus prior to 2009; and ii ) within the human seasonal lineage derived from the 1918 pandemic , which contributed its M segment to the H3N2 lineage represented by Pan99 virus . The timing of the adaptive changes is unclear in each case and cannot be inferred from our data . Importantly , M2 expression in human IAV mimics that of avian IAV in avian cells . Thus , restriction of splicing efficiency to achieve low M2 levels appears to be an important feature of IAV host adaptation . Our data furthermore suggest that the negative impact of an autophagy block on viral growth constitutes an important pressure driving this selection . To date , most publications related to autophagy in IAV-infected cells have used the PR8 or WSN lab-adapted strains , the X31 variant of PR8 , or avian IAVs [46 , 47 , 66 , 67] . We found that PR8 virus expresses levels of M2 in mammalian cells that are comparable with avian IAVs . This phenotype may be a result of adaptation to growth in chicken eggs . Owing to the use of viruses carrying lab-adapted M segments , the current literature suggests that IAV infection routinely induces a block in autophagy [46 , 47 , 66 , 67] . We also see evidence of such a block when using viruses that express high levels of M2 , but we propose that this phenotype is a symptom of poor adaptation to the mammalian host cell . Analysis of viruses carrying human M segments in mammalian cells suggests that low levels of M2 and activation , but not blocking , of autophagy are the norm in IAV-infected cells . While the previously reported block in autophagy induced by IAV has generally been interpreted as a viral defense against an antiviral mechanism , a pro-viral role for autophagy is not without precedent . In Dengue virus infection , autophagy has been reported to support viral replication . Specifically , autophagic turnover of lipid droplets in infected cells is thought to provide energy needed for viral growth and to supply phospholipids important during virion assembly [68–70] . For IAV , the availability of activated LC3B protein was reported to be important for viral morphogenesis [46] . Specifically , M2 was implicated in recruitment of LC3B II to the plasma membrane , and disruption of this recruitment was found to affect budding and reduce stability of released virions [46] . We also noted strong redistribution of LC3B-GFP to the plasma membrane during infection with viruses that express high levels of M2 . This relocalization occurred to a lesser extent in a well-matched IAV-host system ( NL09 M in human cells ) , and may play a functionally important role in this context . A role for LC3B II in morphogenesis is consistent with the activation of autophagy having a pro-viral effect . Conversely , our data indicate that a block in autophagic turnover reduces viral growth , potentially owing to disruption of vesicular trafficking that is normally exploited by the virus [71–73] , or due to disruption of cellular metabolic functions on which the virus relies . By inhibiting the proton channel activity of the avian M2 with amantadine , we found that a functional channel is needed to bring about the observed effects on autophagy . Indeed , M2 has been shown previously to block the fusion of autophagosomes with lysosomes in a proton channel-dependent fashion [66] . Surprisingly , however , amantadine treatment was also found to reduce LC3B II accumulation in PR8 NL09 M-infected cells . The pH1N1 M2 protein carries the S31N mutation , which confers resistance to amantadine . Our data suggest that this resistance is not effective at a relatively high concentration of 200 μM amantadine , at least in the context of intracellular viral infection . The mutagenesis of the M segment that we carried out with the goal of separating the effects of M2 expression level from M2 amino acid sequence revealed an important constraint on M1 coding capacity . Namely , the human / avian amino acid polymorphisms introduced into the M1 ORF were found to impact regulation of M2 gene expression . Some or all of these non-synonymous changes to M1 likely modulate the efficiency of mRNA7 splicing . With this new insight in mind , a fresh look at viral phenotypes previously attributed to M1 amino acid variations is warranted . Effects of M1 amino acid sequence on virion morphology [25 , 31 , 32 , 74 , 75] , for example , may in fact be directly or indirectly mediated through changes in intracellular M2 expression level . Notably , while an avian M2 protein was sufficient to support robust viral replication in mammalian systems when expressed at low levels , this sufficiency did not extend to transmission . Virus expressing the avian M2 from a human M segment transmitted poorly , indicating that the IAV M2 carries viral determinants of transmission and that these determinants are not well conserved between avian and human strains . This novel observation also supports the notion that high replicative capacity of a virus in a given host species is not necessarily sufficient for transmission between individuals . In this context , it will be interesting to evaluate the impact of avian M2 amino acid signatures on virion stability in the environment . In summary , our data suggest that adaptive change in the M segment is needed to maintain low expression of M2 in mammalian cells and that , in the absence of such adaptive changes , excess M2 limits viral growth by blocking the autophagy pathway . These findings establish regulation of viral gene expression as a feature of the virus-host interface critical for IAV host range determination and therefore emergence into novel host populations .
The Institutional Animal Care and Use Committee ( IACUC ) of Emory University approved the study protocol under approval number PROTO201700595 . Madin-Darby Canine Kidney ( MDCK ) cells ( a kind gift of Peter Palese , Icahn School of Medicine at Mount Sinai ) were maintained in minimal essential medium ( Gibco ) supplemented with 10% fetal bovine serum ( FBS ) and penicillin-streptomycin . A549 and 293T cells were obtained from the ATCC and maintained in Dulbecco’s minimal essential medium ( DMEM; Gibco ) supplemented with 10% FBS and penicillin-streptomycin . DF-1 cells were obtained from ATCC and maintained in DMEM plus 5% FBS and penicillin-streptomycin . QT-6 cells were obtained from ATCC and maintained in F-12K medium plus 5% FBS , 10% tryptose phosphate and penicillin-streptomycin . All cells were incubated at 37°C with 5% CO2 . All viruses used in this work were generated using reverse genetics techniques [76] . The rescue system for PR8 was a gift of Peter Palese ( Icahn School of Medicine at Mount Sinai ) . The rescue system for A/Panama/2007/99 ( H3N2 ) [Pan/99] virus was initially described in [7]; and that for A/Netherlands/602/2009 ( H1N1 ) [NL09] virus was a gift of Ron Fouchier ( Erasmus Medical Center ) [77] . The NL09 virus was propagated in MDCK cells for two passages . All other virus stocks were generated in 10–11 day old embryonated chicken eggs . Non-synonymous nucleotide changes differentiating NL09 and avian consensus M segments were introduced in a reciprocal fashion into i ) M1 and M2 to generate 16 mut viruses , ii ) M1-only to generate 9 mut viruses , and iii ) M2-only to generate 7 mut viruses . The modified viral cDNAs were synthesized by Genewiz and subcloned into the pDZ reverse genetic vector [78] . Alignment of the FASTA sequences downloaded from Genbank ( or GISAID ) databases was carried out using DNASTAR Lasergene 13 MegAlign software running the Clustal W phylogenetic alignment algorithm . To obtain a consensus nucleotide sequence and to assess the degree of variability in the M segment of H1N1 subtype avian IAVs , we searched the Genbank nucleotide database for full-length M segment sequences derived from avian H1N1 subtype IAV collected between 1976 and 2008 from any temperate Northern hemisphere location . We obtained 262 sequences , which included sequences with partial 3’ and 5’ UTRs , and manually curated the sequences to ( i ) remove swine and human lineage derived H1N1 M segments , retaining 247 sequences , and ( ii ) trim UTR sequences from the retained segments . Alignment of the sequences by Clustal W provided a working consensus sequence and revealed up to 7% variability at the nucleotide level among the selected avian host-derived sequences . To assess the degree of amino acid variability in the M segment of avian IAV of any subtype , we searched Genbank for full-length M1 or M2 protein sequences derived from avian H1Nx to H16Nx subtype IAV , collected between 1970 and 2000 , from any geographical location . These dates were chosen to avoid selecting sequences representing isolates originating from the repeated H5 subtype HPAI incursions into poultry populations which dominate the Genbank database from the early 2000s onwards , and which would likely bias the subsequent analysis . For each hemagglutinin subtype ( H1Nx to H16Nx ) , we aligned the available sequences to generate individual consensus M1 and M2 amino acid sequences ( S1 Table ) , and aligned those consensus sequences to the avian H1N1 subtype consensus M1 and M2 sequences , in order to assess amino acid diversity . The matrix proteins of each subtype , with the exception of H9Nx , were 100% conserved . The H9Nx subtype matrix protein shared 97 . 6% identity to the avian H1N1 sequence . The M2 proteins of each subtype were 100% conserved , excepting H9Nx , H13Nx and H16Nx subtypes , which shared 97 . 2% , 94 . 8% and 93 . 8% identity respectively , to the avian H1N1 consensus sequence . Thus , the amino acid composition of M1 and M2 proteins are highly conserved within wild waterfowl , particularly within isolates that circulate among dabbling ducks and geese . In these host species , both M1 and M2 proteins are at , or close to , 100% identity at the consensus amino acid level . To identify a consensus nucleotide sequence for the 2009 pH1N1 IAV , and compare it to the avian consensus sequence , we searched Genbank for full-length M segments derived from pH1N1 subtype IAV isolated in North America , collected between the 7th June and the 20th June 2009 , which was shortly after emergence of the pH1N1 lineage into the human population . 189 individual nucleotide sequences were obtained , curated , and aligned , to provide a working consensus sequence for the pH1N1 M segment . The pH1N1 M segments possessed >2% variability at the nucleotide level . The A/NL/602/09 ( H1N1 ) ( NL09 ) M segment is a 100% match to the consensus sequence of the 2009 pH1N1 lineage at the nucleotide level , and the M segment from this strain was adopted for use throughout the current study . Interestingly , comparison of the NL09 M segment to the avian consensus sequence revealed 8 . 3% difference at the nucleotide level , and 9 and 7 amino acid changes in M1 and M2 proteins , respectively ( Table 2 ) . These data suggest that pH1N1 M segment possesses a nucleotide sequence that is highly divergent from M segments obtained from viruses circulating within the wildfowl reservoir , and which may encode host adaptive changes . To assess growth , subconfluent cell monolayers in 6-well dishes were inoculated at an MOI of 5 PFU/cell ( for single-cycle growth assays ) or 0 . 01 PFU/cell ( for multi-cycle growth assays ) in a 200 μl volume . Following incubation for 1 h at 37°C , inocula were removed , cells washed 3x with PBS and 2 ml viral growth medium ( MEM ( for MDCK cells ) , DMEM ( for 293T , and DF1 cells ) , or F-12K ( for A549 , and QT-6 cells ) medium plus 3% bovine serum albumin , penicillin streptomycin , and TPCK-treated trypsin ) was added . Infected cells were incubated at 37°C for the remainder of the time-course . At the indicated time points , 120 μl medium was sampled from each dish and 120 μl fresh medium was added to maintain a 2 ml volume . Samples were stored at -80°C and later titered by plaque assay on MDCK cells . Each infection was performed in triplicate and at least three biological replicate infections were carried out on different days . Data are plotted as mean with SD and analyzed by repeated measures ANOVA . Female , Hartley strain , guinea pigs weighing 300–350 g were obtained from Charles River Laboratories . Prior to inoculation or nasal lavage , animals were sedated with a mixture of ketamine ( 30 mg/kg ) and xylazine ( 4 mg/kg ) . Virus used for inoculation was diluted in PBS to allow intranasal inoculation of guinea pigs with 1x101 to 1x103 PFU in a 300 μl volume . Nasal wash samples were collected as described previously [54] , with PBS as the collection fluid . Animals were housed in a Caron 6040 environmental chamber set to 10°C and 20% RH throughout the seven-day period and lids were left off of the cages during this time to ensure environmental control within the cages [79] . A549 or DF-1 cells in 6-well plates were infected with the indicated IAVs and cells were lysed with 2X Laemmli sample buffer ( Bio-rad ) plus 2% beta-mercaptoethanol at 8 hpi . Samples were boiled at 95°C for 10 min and resolved on 4–20% gradient SDS-PAGE gels , followed by transfer to nitrocellulose membranes ( Bio-rad ) , and incubation of membranes with 5% non-fat dry milk/TBST blocking buffer . Antibodies used for immunoblotting were: anti-vinculin monoclonal antibody ( catalog no . V9131; Sigma-Aldrich ) at 1:5000 dilution , IAV nucleoprotein monoclonal antibody ( HT-103; catalog no . EMS010 , Kerafast ) at 1:1000 dilution , IAV matrix protein monoclonal antibody ( E10; catalog no . EMS009 , Kerafast ) at 1:1000 dilution , and LC3B polyclonal antibody ( Thermo Fisher Scientific ) at 1:1000 dilution . Bands were quantified using ImageLab software ( Bio-rad ) after normalization to vinculin . At least three biological replicate viral infections were carried out , with two technical replicates of the immunoblotting for each infection . Data shown with error bars representing SD and statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparisons in GraphPad Prism software . A549 or DF-1 cells in 6-well plates were infected with the specified IAVs and total RNA was extracted 8 hpi using a RNeasy Kit ( Qiagen ) . Total RNA was eluted in RNase-free water and stored at -80 ºC until needed . Radiolabeling of primers: 1 μmol of oligo DNA primer was incubated with 10 μCuries of [γ-32P]ATP and 10 U of T4 PNK ( New England Biolabs ) in 70 mM Tris-HCl ( pH 7 . 6 ) containing 10 mM MgCl2 and 5 mM DTT . Radiolabeling reactions were carried out at 37 ºC for 1 h and diluted to 30 μL with RNase-free water after termination of the reaction . Radiolabeled primers were stored at –20 ºC until needed . 500 ng of total RNA was incubated with 0 . 45 μL unlabeled 5S rRNA primer , 0 . 05 μL radiolabeled 5S rRNA primer , 0 . 25 μL radiolabeled IAV segment 7 vRNA primer , and 0 . 25 μL radiolabeled IAV segment 7 mRNA primer in a final volume of 5 μL . Primers were annealed to RNA by sequential incubations at 95 ºC for 3 min and ice for 3 min . Reactions were pre-warmed at 50 ºC for 5 min . Transcription was initiated by the addition of 50 mM Tris-HCl ( pH 8 . 3 ) buffer containing 75 mM KCl , 3 mM MgCL2 , 10 mM DTT , 2mM dNTP , and 50 units of SuperScript III Reverse Transcriptase ( Thermo Fisher Scientific ) . Reactions were incubated at 50 ºC for 1 h and terminated by the addition of Gel Loading Buffer II ( Thermo Fisher Scientific ) and incubation at 95 ºC for 10 min . Reactions were resolved on denaturing sequencing polyacrylamide gels ( 7M urea , 6% acrylamide ) . Gels were subsequently dried and exposed to a phosphor storage screen overnight . The intensity of bands was analyzed using a Typhoon Trio Imager ( GE Healthcare ) and quantified with ImageQuant software ( GE Healthcare ) after normalization to 5S rRNA . At least three biological replicate viral infections were carried out , with two technical replicates of the RT primer extension assay for each infection . Data are shown with error bars representing SD and statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparisons in GraphPad Prism software . To allow tracking of LC3B II and viral protein , cells grown on collagen-coated coverslips were transduced with lentivirus expressing LC3B-GFP ( BacMamm 2 . 0; catalog no . P36235 , ThermoFisher ) , as follows . 18h post-transduction , 293T or A549 cells were infected with the indicated IAVs at an MOI of 5 PFU/cell and incubated at 37°C . At 8h or 12 h post-IAV infection , cells were washed once with PBS and fixed with 4% paraformaldehyde . Following permeabilization with 0 . 1% Triton X-100 , cells were incubated overnight at 4°C with anti-M2 monoclonal antibody ( E10; catalog no . EMS009 , Kerafast ) . Three washes were performed prior to addition of goat anti-mouse Alexa Fluor-647 secondary antibody ( catalog no . A21241 , Life Technologies ) and incubation for 2 h at room temperature . Antibody dilutions and washes were performed with PBS plus 1% Tween-20 . Cells were treated with DAPI , and coverslips were mounted on slides using Vectashield ( Vector Labs ) mounting medium . Images were obtained with on a Nikon FV1000 confocal microscope at the Emory Integrated Cellular Imaging core facility . Where indicated , chloroquine ( Invitrogen ) was added to cell culture medium at a final concentration of 60 or 120 μM . Amantadine HCl ( catalog no . 1018505 , Sigma ) was added to cell culture medium at a final concentration of 200 μM . Both drugs were added to infected cells 1 h after IAV inoculation . Statistical analyses were performed in GraphPad Prism . Multi-cycle and single-cycle growth in cell culture was assessed in three independent experiments , with three technical sample replicates per experiment . Overall , statistical significance was determined using repeated measures , two-way , multiple comparisons ANOVA on log-transformed mean values , with Bonferroni correction applied to account for comparison of a limited number of means , as well as by assessing pairwise comparisons of viruses at each time point , except for single-cycle growth in cell culture in the presence of amantadine , which was assessed in four independent experiments , with three technical sample replicates per experiment . Overall statistical significance was determined using repeated measures , two-way , multiple comparisons ANOVA on log-transformed values , with Sidak’s correction applied , as well as by assessing pairwise comparisons of viruses at each time point . Growth of each virus in the guinea pig model was assessed in three independent experiments with four guinea pigs per experiment . For transmission analysis , each guinea pig was treated as an independent biological sample . Statistical significance in the magnitude of replication was determined by comparing AUC of growth curves using two-tailed Student’s t-test . Statistical significance in transmission efficiency was determined using unpaired two-tailed Student’s t-test . Significance of differences in the kinetics of replication was determined by assessing the interaction of time and virus using repeated measures , two-way ANOVA on mean log-transformed values , with Bonferroni correction applied to account for comparison of a limited number of means . For experiments presented throughout the manuscript , unless specific values are provided , P values are represented as follows: *< 0 . 05; **<0 . 01; ***< 0 . 001; ****< 0 . 0001 . | Influenza A virus ( IAV ) pandemics arise when a virus adapted to a non-human host overcomes species barriers to successfully infect humans and sustain human-to-human transmission . To gauge the adaptive potential and therefore pandemic risk posed by a particular IAV , it is critical to understand the mechanisms underlying viral adaptation to human hosts . Here , we focused on the role of one of IAV’s eight gene segments , the M segment , in host adaptation . Comparing the growth of IAVs with avian- and human-derived M segments in avian and mammalian systems revealed that the avian M segment restricts viral growth specifically in mammalian cells . We show that the mechanism underlying this host range phenotype is a dysregulation of viral gene expression when the avian IAV M segment is transcribed in mammalian cells . In particular , excess production of the M2 protein results in viral interference with cellular functions on which the virus relies . Our results therefore reveal that the use of cellular machinery to control viral gene expression leaves the virus vulnerable to over- or under-production of critical viral gene products in a new host species . | [
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] | 2019 | Dysregulation of M segment gene expression contributes to influenza A virus host restriction |
A variety of cardiovascular , neurological , and neoplastic conditions have been associated with oxidative stress , i . e . , conditions under which levels of reactive oxygen species ( ROS ) are elevated over significant periods . Nuclear factor erythroid 2-related factor ( Nrf2 ) regulates the transcription of several gene products involved in the protective response to oxidative stress . The transcriptional regulatory and signaling relationships linking gene products involved in the response to oxidative stress are , currently , only partially resolved . Microarray data constitute RNA abundance measures representing gene expression patterns . In some cases , these patterns can identify the molecular interactions of gene products . They can be , in effect , proxies for protein–protein and protein–DNA interactions . Traditional techniques used for clustering coregulated genes on high-throughput gene arrays are rarely capable of distinguishing between direct transcriptional regulatory interactions and indirect ones . In this study , newly developed information-theoretic algorithms that employ the concept of mutual information were used: the Algorithm for the Reconstruction of Accurate Cellular Networks ( ARACNE ) , and Context Likelihood of Relatedness ( CLR ) . These algorithms captured dependencies in the gene expression profiles of the mouse lung , allowing the regulatory effect of Nrf2 in response to oxidative stress to be determined more precisely . In addition , a characterization of promoter sequences of Nrf2 regulatory targets was conducted using a Support Vector Machine classification algorithm to corroborate ARACNE and CLR predictions . Inferred networks were analyzed , compared , and integrated using the Collective Analysis of Biological Interaction Networks ( CABIN ) plug-in of Cytoscape . Using the two network inference algorithms and one machine learning algorithm , a number of both previously known and novel targets of Nrf2 transcriptional activation were identified . Genes predicted as novel Nrf2 targets include Atf1 , Srxn1 , Prnp , Sod2 , Als2 , Nfkbib , and Ppp1r15b . Furthermore , microarray and quantitative RT-PCR experiments following cigarette-smoke-induced oxidative stress in Nrf2+/+ and Nrf2−/− mouse lung affirmed many of the predictions made . Several new potential feed-forward regulatory loops involving Nrf2 , Nqo1 , Srxn1 , Prdx1 , Als2 , Atf1 , Sod1 , and Park7 were predicted . This work shows the promise of network inference algorithms operating on high-throughput gene expression data in identifying transcriptional regulatory and other signaling relationships implicated in mammalian disease .
Sustained elevated levels of reactive oxygen species ( ROS ) have been associated with the etiology of a vast range of pathological conditions . These include a variety of neurodegenerative diseases , cardiovascular diseases , cancer , diabetes mellitus , rheumatoid arthritis , and obstructive sleep apnea [1] . ROSs are highly reactive molecules . They include the superoxide anion , the hydroxyl radical , and hydrogen peroxide . ROSs are a natural by-product of oxygen metabolism . However , ROS levels can dramatically increase during times of environmental stress , causing injury and damage by attacking DNA , protein and lipid , thereby leading to oxidative stress . A number of redox-regulated gene products serve to protect cells from such ROS damage . The antioxidant response element ( ARE ) , a cis-acting DNA element , is known to be activated by oxidative stress and to be responsible for the transcriptional regulation of several redox-regulated gene products [2] . The principal transcription factor that binds to the ARE is Nuclear factor erythroid 2-related factor ( Nrf2 ) [3] . Nrf2 is a basic leucine zipper ( bZIP ) transcription factor that translocates to the nucleus following liberation under oxidative stress conditions from its cytosolic inhibitor Keap1 [4] . In the nucleus , Nrf2 forms dimers with the proteins Maf , Jun , Fos , ATF4 and/or CBP , and regulates transcription by binding to the ARE upstream of a number of target genes [4]–[7] . Established Nrf2-regulated genes include Cu/Zn superoxide dismutase , catalase , thioredoxin , thioredoxin reductase , glutathione reductase , glutathione peroxidase and ferritin ( L ) [3] . All of these genes are involved in the response to oxidative stress . There are several other genes also known to be involved in the response to oxidative stress [1] . The transcriptional regulatory relationships at the mRNA level , and the signaling relationships at the protein level linking these genes and their products are only partially resolved . To find direct regulatory targets of Nrf2 , we use two algorithms that can infer such regulatory links from gene expression data: Context Likelihood of Relatedness ( CLR ) [8] and the Algorithm for the Reconstruction of Accurate Cellular Networks ( ARACNE ) [9]–[11] . These algorithms were applied to the analysis of the mouse lung gene expression datasets to infer regulatory connections between oxidative stress genes . Both of these algorithms use the concept of mutual information ( MI ) from information theory [12] . The pair-wise MI scores calculated are derived from correlations in the patterns of expression of the two genes involved . We also annotate and perform further analysis of the putative target set thus identified . Data derived from the promoter regions of known Nrf2 targets were used to train LibSVM , a machine learning support vector machine classification algorithm [13] . LibSVM was then used to confirm the predictions derived from gene expression data via a separate analysis of upstream DNA sequences of the predicted target genes . We also identify signaling partners of a key Nrf2 target , NAD ( P ) H:quinine oxidoreductase 1 ( Nqo1 ) , shedding light on previously unidentified interactions , many of which are supported by independent microarray and quantitative RT-PCR experiments . . These results demonstrate the promise of network inference algorithms in identifying transcriptional regulatory and other signaling relationships implicated in mammalian disease .
Use of the two network inference algorithms , ARACNE and CLR , on the gene expression data , as well as use of the LibSVM algorithm on sequence data , yielded a number of outcomes where the same regulatory edge was predicted by all three algorithms ( Table 1 ) . ARACNE and CLR use the MI metric on the expression data to identify direct dependencies . LibSVM , trained with sequence data from upstream regions of known Nrf2-regulated genes ( positive examples ) and empirically determined Nrf2-independent genes ( negative examples ) , was used to predict transcription targets from the test set of putative Nrf2-regulated target genes previously identified by ARACNE and CLR . Figure 1 depicts findings of the CLR algorithm when applied to mouse lung microarray data , with a focus on interactions involving Nrf2 determined by using ( see Methods ) the Collective Analysis of Biological Interaction Networks ( CABIN ) software [14] . A z-score cutoff of 2 . 0 on the CLR score set yielded eighteen edges above the cutoff between the probe sets representing the Nfe2l2 gene that produces Nrf2 and the probe sets for other genes in the combined dataset . In other words , the set of gene states for Nfe212 contained enough information on the states of 18 other genes ( probe sets ) to lift their pairwise score two standard deviations or higher above the average CLR score among all genes in the set . Given that Nfe2l2 and other genes are represented by more than one probe set , these eighteen edges yield connections from Nfe2l2 to twelve other genes . Figure 2 is a depiction of the dependencies obtained using the sets of microarrays and the ARACNE algorithm . A high significance threshold for MI values was used , with a p-value of 1e-7 . Post-processing of the inferred edges to remove indirect regulatory relationships was done using a DPI tolerance of 0 . 15 . For a more focused view , interactions involving Nrf2 were selected . Cutoffs for both the ARANCE and CLR algorithms were empirically determined . The cutoffs were pushed as high as possible to exclude false regulatory connections while still retrieving at least a moderate size set of interactions to explore and validate with quantitative RT-PCR , LibSVM , and literature search . In this sense , our work is classic exploratory analysis . All the Nrf2 target genes found using the CLR algorithm were also selected under the ARACNE algorithm under the cutoff values as stated above , and with the parameter settings as given in Methods . However , ARACNE also found additional putative Nrf2 targets . This finding is , however , not an indication that the dependencies identified only by ARACNE are untrustworthy . As summarized in Table 2 , all of the direct dependencies predicted only by ARACNE are backed by the force of biochemical evidence . These observations underscore the power of these inference algorithms ( given large enough datasets ) as potential guides in the search for regulatory and signaling connections in biological networks . Seeking further evidence at the sequence level for direct , DNA-binding regulation between Nrf2 and the potential sets of target genes produced by the ARACNE and CLR runs , we used LibSVM , our selected algorithm for supervised machine learning . The training set in the classification of target gene , non-target gene consisted of features of upstream DNA promoter regions of known Nrf2 transcriptional regulation targets and empirically-determined non-target Nrf2-independent genes ( Text S1 ) . Using the LibSVM nu-SVC classifier at cost = 1 , ν = 0 . 36 and γ = 2−13 , a true positive rate of 0 . 7 or better was obtained under two cross-validation conditions for the genes in the training set . Furthermore , the precision , recall , and area under the ROC curves were 0 . 7 or better ( Table 3 ) . The LibSVM predictions generated on a test set obtained from the dependencies identified by CLR and ARACNE ( Figures 1 and 2 ) posit that Atf1 , Nqo1 , Nfkbib , Prdx1 , Srxn1 , Prnp , Sod2 , Ppp1r15b , Als2 , Idh1 , and Nrf2 ( Nfe2l2 ) are transcriptionally Nrf2-regulated . Of these , Nqo1 , Prdx1 , and Nrf2 are established targets of Nrf2 transcriptional regulation [3] . Tables 1 , 2 , and 4 summarize our results . Table 1 shows all gene targets of possible direct Nrf2 regulation reported by either ARACNE , CLR , or LibSVM , for a total of 21 genes . Table 2 presents what was previously known about these putative target genes , based on our literature search . Array experiments involving wild type ( WT ) and Nrf2 knockout ( NO ) mouse lungs were then conducted to verify the regulatory role of Nrf2 on the expression of the genes identified . The mice were exposed to either air or cigarette smoke ( CS ) . CS-induced elevations of glutathione ( GSH ) and Thiobarbiturate reactive substances ( TBARS ) levels depicted in Figure 3 illustrate the capacity of CS to induce oxidative stress . GSH levels rise in response to oxidative stress , as a protective measure [1] . In the absence of Nrf2 , the CS-induced rise in GSH levels is abolished ( Figure 3A ) . This suggests a requirement for Nrf2 for the rise in GSH levels , and underscores the protective role of Nrf2 . Increases in TBARS indicate increased decomposition of lipid peroxidation products and signal the presence of oxidative stress [15] . However in the absence of Nrf2 , the CS-induced rise in lipid peroxidation as indexed by elevated TBARS levels is enhanced ( Figure 3B ) . This emphasizes a protective role of Nrf2 against CS-induced lipid peroxidation . Thus , microarray data generated from CS-exposed mouse lungs can elucidate the regulation of gene expression in response to oxidative stress . In Figure 4 , microarray data for a cross-section of three stated Nrf2 targets are summarized . Nqo1 and Sod1 have previously been identified as transcription regulatory targets of Nrf2 [3] . Als2 is a novel target arising out of the computational analysis being reported here . We performed a set of measurements showing upregulation of all three genes only in the presence of the Nrf2 gene ( wild type; no knockout ) and CS-induced oxidative stress ( Figure 4 ) . This is additional evidence for a regulatory role for Nrf2 in the expression of Nqo1 , Sod1 and Als2 . Furthermore , quantitative RT-PCR experiments were conducted on a gene set found to be differentially expressed in these CS exposure microarrays as well as identified by ARACNE or CLR as Nrf2 targets . The results affirm the regulatory role of Nrf2 for many of the gene targets predicted by our combined analysis of microarray and sequence data ( Figure 5 ) . Nqo1 , Sod1 , Ercc6 , Prdx6 , Als2 , Txnrd2 , Park7 , Srxn1 , and Epas1 all undergo enhanced upregulation after CS exposure only in the presence of the Nrf2 gene . Thus , we have good evidence from quantitative RT-PCR that Nrf2 positively regulates the expression of these genes . In the case of Sod2 , Ppp1r15b and Fos , CS-induced upregulation is modestly enhanced in the absence of Nrf2 . It is inferred that Nrf2 exerts a negative regulatory influence on the expression of these genes .
Several of the genes that were identified in our work as potential targets of Nrf2 transcriptional regulation in the mouse lung have been implicated in certain neurodegenerative disorders . Of the set of 46 genes of interest ( see Methods ) , six are annotated in the Kyoto Encyclopedia of Genes and Genomes ( KEGG ) [18] with the “Neurodegenerative Disorders” classification . ( Table S2 lists Gene Ontology ( GO ) [19] and KEGG annotation on these 46 genes . ) These six are Crebbp , Apoe , Als2 , Sod1 , Park7 , and Prnp . Five of these six , all except Apoe , were placed by our analysis in the list of 21 potential targets of direct Nrf2 regulation shown in Table 1 . In addition , Nfe2l2 is annotated in KEGG as associated with prion disease . Using the LibSVM algorithm , we found that Nfe2l2 regulates itself ( see Conclusions ) and therefore is included in our set of 21 targets . Thus , of the seven genes marked in KEGG as associated with neurodegenerative disorders , six appear in our set of Nrf2 targets , roughly a two-fold enrichment of what we would expect to see by chance ( 7×21/46 = 3 . 2 genes ) . Death of motor neurons induced by an Amyotrophic Lateral Sclerosis ( ALS ) -linked Sod1 mutant is prevented by the Als2 gene product , alsin [20] . Alsin acts as a guanine nucleotide exchange factor for Rac1 and Rab5 , both GTPases [21] , [22] . A number of protein function-altering Als2 mutations have been identified as causing ALS [23] . Human diseases associated with S-glutathionylation , a common post-translation modification , include PD , diabetes , hyperlipidemia , Friedreich's ataxia , renal cell carcinoma and HIV/AIDS [24] , [25] . The oxidoreductase , Srxn1 , plays a role in signaling by catalyzing reduction following S-glutathionylation [26] . Srxn1 is involved in reversing NO-induced protein glutathionylation; Srxn1 protein deglutathionylation results in the restoration of phosphatase activity of non-receptor-type protein tyrosine phosphatase [25] . Srxn1 also catalyzes the reduction of cysteine sulfinic acids [27] . Park7 , also known as DJ-1 , has been linked to a number of Parkinson's Disease ( PD ) pathways [28] . When oxidized , Park7 acts as a chaperone protein that prevents the characteristic aggregation of certain proteins in PD [29] . Indeed , oxidized forms of this protein accumulate in the brains of some PD and Alzheimer's disease patients [30] . Its functional integrity is so important that up to 1% of PD cases are associated with Park7 mutations [31] . The four traditional classes of prion diseases ( Creutzfeldt–Jakob disease , kuru , fatal familial insomnia , and Gerstmann–Straussler–Scheinker syndrome ) all involve mutations of Prnp and multiple abnormal conformations of its protein product Prp [32] . This set of neurodegenerative diseases has become intensely epidemiologically interesting following the transmission of bovine spongiform encephalopathy to humans and the apparent concomitant emergence of the variant Creutzfeldt–Jakob disease [33] . The ARACNE , CLR , and LibSVM runs in these studies all indicate a regulatory role of Nrf2 on the expression of the Prnp gene in the mouse lung ( Tables 1 and 2 ) . Using the two algorithms ( ARACNE and CLR ) , we establish direct statistical dependencies between the expressions of genes such as Sod1 , Als2 , Srxn1 , and Park7 , and the expression of Nfe2l2 ( the Nrf2 gene ) in the mouse lung . The LibSVM studies affirm that in the case of Als2 and Srxn1 , the direct statistical dependencies indicate transcriptional regulation by Nrf2 . Furthermore , our quantitative RT-PCR experiments show that CS-induced oxidative stress of the mouse lung increases the mRNA expression of several of these genes , and that these increases require the presence of Nrf2 . Experimental evidence ( Figures 4 and 5 ) confirms , for instance , that Als2 is indeed a novel Nrf2 target . Cigarette smoke ( CS ) exposure induces oxidative stress ( Figure 3A and 3B ) and acts as an inducer of Nrf2-mediated transcription . In wild type mice ( but not Nrf2 knockout mice ) , increases in Sod1 , Nqo1 , and Als2 mRNA expressions are observed after CS exposure . These data point to a transcriptional regulatory role for Nrf2 on these genes in the mouse lung . Nqo1 , also known as DT-diaphorase or NAD ( P ) H:quinone oxidoreductase , was found to be a target of direct regulation by Nrf2 under both the CLR algorithm runs ( Figure 1 ) and the ARACNE runs ( Figure 2 ) . In addition , the LibSVM prediction that Nqo1 is trans- criptionally Nrf2-regulated has biochemical proof [34] . Thus , Nqo1 was predicted to be a direct target of Nrf2 by all three methods , and has been confirmed in the literature as such . We therefore considered it suitable for expanding our study of the oxidative stress response beyond Nrf2 . All direct dependencies ( all edges ) involving Nqo1 as determined by the ARACNE runs are shown in Figures 6 and 7 . Although the current literature does not capture all the relationships being identified here , the edges represent a number of plausible regulatory or functional relationships , involving Nqo1 . For instance , there is no previous finding of the direct relationship between Gpx3 ( glutathione peroxidase 3 ) and Nqo1 . However , Gpx3 is distributed in the same fashion as Nqo1 and Sod1 ( Cu/Zn superoxide dismutase ) [35] , [36] . The relationship between Nqo1 and certain other connected nodes , such as Sod1 , have been identified [37] . Both CLR and ARACNE use the concept of mutual information ( MI ) . Why not use Euclidean distance or Pearson correlation for pair-wise calculations , as is done in standard microarray-based gene clustering ? Why use MI ? Unlike Euclidean distance and Pearson correlation , MI does not assume that the relationship between the genes is linear . A major advantage of this information theoretic calculation is its nonparametric nature , and the entropy calculations performed in calculating the MI value do not require any assumptions about the distribution of variables . MI provides a general measurement for dependencies in the data: negative as well as positive , nonlinear as well as linear [38] , [39] . The higher the MI score between two genes , the greater the information we derive on the states of the first gene from the pattern of states in the other , and the greater the likelihood that one of the genes is directly regulating the other . While both ARACNE and CLR are mutual information based algorithms , and while both were applied here to the same microarray datasets , we believe that there is a legitimate reason to conclude that a regulatory connection found by both algorithms is of higher probability of being a true regulatory relationship than if only one of the two algorithms scored such a connection highly . ARACNE and CLR impose a superstructure on the basic MI calculation that differs in important ways . ARACNE also post-processes the results in a different manner . Also , the binning ( discretization ) methods used are different—which can be highly important . Therefore when a gene-to-gene relationship is scored highly by both algorithms , the algorithms have arrived at that conclusion using different calculations . An analogy can be made here to the Oak Ridge National Laboratory GRAIL gene finder tool which uses several algorithms—operating on the same sequence data—and combines their results for improved gene calling . For our resource-limited , time-limited exploratory analysis , we focused on the inferred regulatory connections we believed had the highest probability of proving to be biologically valid and the most robust , that is , on the connections inferred by ARACNE and CLR together . As with the standard clustering metrics , MI calculations are symmetric , yielding identical scores from gene A to gene B and from gene B to gene A . Therefore the directionality ( which of the two genes regulates the other ) cannot be inferred from the MI score alone . More information is needed: is one gene known or suspected to be a transcription factor ? Does one of the two appear to connect ( regulate , as a “hub” ) many other putative targets ? Does one gene connect ( have a high MI score ) to two or more putative target genes in the same operon ? Additional information must be sought , with the regulatory edge in question looked at in the wider context of the entire inferred network . Each edge connecting the nodes in Figures 1 and 2 is subject to one of at least two interpretations . First , we can interpret the edge as a direct dependency between the expression of a transcription factor producing gene and a target non-transcription factor gene , that is , as an indicator of direct transcriptional regulation of the target by the transcription factor via DNA binding of the transcription factor . For instance , in Figure 2 the edge between Nrf2 ( gene Nfe2l2 ) and Sod1 depicts the fact that the expression of Cu/Zn superoxide dismutase ( Sod1 ) is transcriptionally regulated by Nrf2 [40] . Second , if such an edge is one of two or more connections going into a common target , the source gene for one of those edges may be producing a protein necessary for the action of the primary transcription factor also connecting to the common target . For example , this would hold true for Nfe2l2 ( Nrf2 ) and Park7 ( Parkinson disease ( autosomal recessive , early onset ) genes as joint regulators of a common target gene . Park7 has no direct effect on Nfe2l2 mRNA levels . However , it does stabilize the Nrf2 protein produced by Nfe2l2 , and is required for the transcriptional activity of Nrf2 [41] , and thus , through binding to Nrf2 ( rather than directly to DNA near the target ) , Park7 also regulates each of Nrf2's direct targets , with such regulation being reflected in the correlation between Park7 expression levels and that of the target of Nrf2 . In Figure 2 , the edge between Nfe2l2 and Park 7 shows that Nrf2 also exerts regulatory control on the mRNA expression of Park7 itself . Microarray and quantitative RT-PCR data generated from Nrf2 knockout mice ( Figures 4 and 5 ) show CS-induced enhanced mRNA expression of Nqo1 , Sod1 , Ercc6 , Prdx6 , Als2 , Txnrd2 , Park7 , Srxn1 , and Epas1 in wild-type but not Nrf2-knockout mice . In the knockout mice , where Nrf2 is absent , mRNA expression for these genes is dramatically decreased in response to CS ( state of Nrf2 knockout CS-exposed “NOCS” in Figures 4 and 5 ) as compared to wild type with Nrf2 present and active . Thus we infer that Nrf2 is required for the CS-induced increase in Park7 mRNA expression . This assertion holds also for Nqo1 , Sod1 , Ercc6 , Prdx6 , Als2 , Txnrd2 , Srxn1 , and Epas1 , as can be seen in the figures . As noted above , some of the genes reported ( Park 7 , Jun , and Crebbp ) have been investigated and have been found to work with Nrf2 , though they have not previously been identified as genes directly activated by Nrf2 . They remain possible targets of Nrf2 regulation , with a possible fit into the category of feed-forward loops discussed below . Indeed we show that in the absence of Nrf2 , CS elicits a suppression of Park7 and Jun mRNA expression ( see state “NOCS” in Figure 5 ) . Thus the significant mutual information content reported by ARACNE and CLR between each of these genes and Nfe2l2 has biological significance . Edges between the genes producing two transcription factors are subject to similar interpretations as outlined above . In the first case , one of the two transcription factors is a transcriptional regulator of the gene producing the other . In the second case , such an edge can be an indication that the two transcription factors act as coregulators of the expression of other genes , with both proteins working closely together for properly modulated expression of the gene target ( s ) , causing a very tight correlation in their gene expression patterns . These are not mutually exclusive categories . For example , Nrf2 and the transcription factor Atf1 can jointly regulate the target gene ferritin H , and , as our data indicate , Nrf2 can also be a transcriptional activator of Atf1 . In fact , this is common triangular regulatory motif , called a feed-forward loop [42] , between three genes in a transcriptional regulatory network . Such connected subsets of three genes can often form what are known as feed-forward loop ( FFL ) transcriptional regulatory network motifs . These FFL motifs appear in hundreds of gene systems . In this context , gene Nfe212 ( Nrf2 ) would be one of the three genes in an FFL subgraph , having an edge to Nqo1 as an activating regulator of that gene . The direction of the edges from Nqo1 to X , and from X to Nfe2l2 remain to be determined , as well as type of regulation for those two edges–activation or repression . Other examples of possible feed-forward loops are as follows: ( 1 ) The gene product Jun ( whose gene is shown in Figure 2 as connected to the Nrf2-producing Nfe212 gene by a high ARACNE score ) , is part of the activator protein 1 ( AP1 ) transcription factor and is known to serve as a coregulator with Nrf2 in some promoter regions [6] . ( 2 ) The Fos gene product was found to be connected to Nrf2 by high scores using both the CLR algorithm ( Figure 1 ) and the ARACNE algorithm ( Figure 2 ) . Fos can be a component of AP1 and has been shown to negatively regulate ARE-mediated transcription regulation [43] . ( 3 ) Another example is activating transcription factor 1 ( Atf1 ) , shown connected to Nrf2 in both Figures 1 and 2 . Also , there is a recent report by Iwasaki et al . [44] indicating Atf1 is a transcriptional repressor at an anti-oxidant response element , thus modulating target response to Nrf2 , which is the principal transcriptional activator of the antioxidant response element . The regulatory network shown in Figure 6 has inherent within it a number of three-party relations of the kind characterized in Figure 8 , where the edge between Nrf2 and Nqo1 represents transcriptional regulation by Nrf2 . However the edge between Nrf2 and gene X in Figure 8 ( with X representing any of the following: Sod1 , Srxn1 , Txnrd2 , Prdx1 , Prdx2 , Prdx6 , Atf1 , Park7 , or Als2 ) and the edge between X and Nqo1 represent a number of possible transcriptional regulatory relationships , with one gene serving as a final target , and the other two genes functioning as activators or repressors of mRNA expression . Uri Alon [45] has classified the possible feed-forward loops within such a three-node , three-edge relationship into eight types . In the specific case of Nrf2-Nqo1-Sod1 , the transcriptional regulatory influence of Nrf2 on both Nqo1 and Sod1 has been established . Hence we have activation edges from gene Nfe212 ( Nrf2 ) to both Nqo1 and Sod1 . The remaining , less characterized edge represents the Nqo1–Sod1 regulatory relationship . Does Nqo1 directly regulate Sod1—or vice versa ? Watanabe et al . [37] report that inhibition of Nqo1 in lung epithelial ( A549-S ) cells results in inhibition of H2O2 generation by quinones . Exogenous Sod1 also inhibits H2O2 generation by low levels of quinones . Thus inhibition of Nqo1 has the same effect as raising the level of Sod1 . Based on this , we infer that Nqo1 exerts an inhibitory effect on Sod1 . Hence , if Nqo1 is increased , Sod1 should be repressed , and H2O2 generation will not be inhibited . And , therefore , if Nqo1 is inhibited , then H2O2 generation will be inhibited , agreeing with experimental observation . This inferred relationship , resolving the character of the remaining edge , is illustrated in Figure 9 . The subgraph shows an connection from Nqo1 to Sod1 , with Nqo1 acting as an inhibitor . This matches the type 1 incoherent feed-forward loop , which is one of the two most frequent occurring of the eight types of FFLs . ( The other common type is the type 1 coherent feed-forward loop , where all three edges represent transcriptional activation . ) Four possible Nrf2-Nqo1-X feed-forward loops are shown in Figure 10 . The third gene , gene “X” , can be any one of Srxn1 , Prdx1 , Atf1 , or Als2 . All four putative loops have two defined edges , both of which represent transcriptional activation by Nrf2 . However , the third edge , corresponding to a direct regulatory relationship between Nqo1 and gene X , remains to be established . All of these genes are involved in the response to oxidative stress , however . For example , Als2 knockout mice are more susceptible to oxidative stress , and Als2 protects against oxidative stress [46] , [47] . On the basis of the results of our computational analysis , we believe that additional work to confirm direct regulatory relationships between Nqo1 and Srxn1 , Prdx1 , Atf1 , or Als2 would be warranted . As explained in the Data Sources sub-section under Methods , data samples , all from mouse lung , were run on two platforms: the Affymetrix GeneChip Mouse Genome 430 2 . 0 array and the Affymetrix Mouse Expression Set 430 ( MOE430A ) . The latter is a subset of the former . However , for each of our network inference runs data from only a single platform was used , not both . While this limited the number of data points on each gene to something less than if we had combined the two platforms , we thus avoided the problem of comparing gene expression across platforms . The remaining task was that of combining data from multiple laboratories that employ the same microarray platform . ( Table S1 lists the data sources . ) We performed RMA analysis using the affy package in BioConductor , as stated in Data Sources section of Methods . We acknowledge that noise will be introduced when combining array sets from different sources and that this could be a confounding factor . However , we stress that we were functioning in the framework of relatively low-cost , relatively simple exploratory analysis , mining the growing collection of public microarray datasets for identification of candidate regulatory relationships to be later confirmed via LibSVM , quantitative RT-PCR , and literature search . And , hopefully , we are serving as an example of what can be done , with relatively modest cost , in analysis of such datasets , with our work having general application for other researchers analyzing transcriptional regulatory networks . Our working assumption was that multiple-source introduced noise/bias , while hiding regulatory edges whose correlations in gene expression could not rise above such noise , would not prevent at least some true regulatory connections—the strongest ones—from being found by the CLR and ARACNE algorithms . We believe that our assumption bore fruit . In the set of 21 genes reported out by one or more of our three algorithms ( CLR , ARACNE , and LibSVM ) with empirically determined high confidence thresholds , shown in Table 1 , four have been verified in the literature as Nrf2 activation targets: Nqo1 , Prdx1 , Sod1 , and Nfe2l2 itself ( postive autoregulation ) . Two of these four , Nqo1 and Prdx1 , were reported by all three algorithms . Sod1 was reported out by ARACNE , and Nfe2l2 ( Nrf2 ) was reported out by LibSVM ( such autoregulation being undetectable by the other methods ) . Ten more possible gene targets were found by one or more of the algorithms where the literature shows that the product of the gene interacts with Nrf2 as coactivators or coregulators ( Table 2 ) . These ten genes remain as possible Nrf2 targets in the context of the formation of feed-forward loops . Lastly , seven of the 21 were reported out by all three algorithms , but with no literature evidence linking them to Nrf2 ( Table 4 ) . Hence this is the first report of these seven genes ( Als2 , Atf1 , Nfkbib , Ppp1r15b , Prnp , Sod2 , and Srxn1 ) as being strong candidates for direct targets of Nrf2 activation in the mouse lung . Separate RT-PCR experiments indicate that Nrf2 positively regulates the expression of the Nqo1 , Sod1 , Ercc6 , Prdx6 , Als2 , Txnrd2 , Park7 , Srxn1 , and Epas1 genes in the mouse lung ( Figure 5 ) . In addition , these experiments indicate that Nrf2 may negatively regulate the expression of the Sod2 , Ppp1r15b , and Fos genes . Thus these pieces of experimental evidence affirm several inferences made using the CLR , ARACNE , and LibSVM algorithms . We believe our work shows the usefulness of network inference algorithms such as CLR and ARACNE on the growing body of microarray data . Using such algorithms and datasets , exploratory analysis is now possible that can usefully guide laboratory work with a relatively modest effort . Finally , in addition to identifying putative targets of Nrf2 , we extended our analysis of the network downstream of Nrf2 by identifying probable feed-forward loops involving Nqo1 , one of the Nrf2 regulatory targets . We believe further extension of our analysis downstream of Nrf2 is possible , and hope to continue work in this area .
Total RNA was extracted using RNAeasy kit from Qiagen according to the manufacturer's instructions , and 2 µg of total RNA was used for cDNA synthesis . Quantitative PCR analyses were performed by using assay on demand probe sets commercially available from Applied Biosystems . Assays were performed by using the ABI 7000 Taqman system ( Applied Biosystems ) . GAPDH was used for normalization . The cycle threshold ( CT ) value indicates the number of PCR cycles that are necessary for the detection of a fluorescence signal exceeding a fixed threshold . The fold change ( FC ) was calculated by using the following formulas: ΔCT = CT ( GAPDH ) −CT ( target gene ) and , in which ΔCT1 represents the highest CT value among all the samples and ΔCT2 represents the value of a particular sample . Results are expressed as mean values of relative fold changes ( RFC ) for n = 3 with WT Air as the baseline . Total glutathione was determined using a modified Tietze method by measuring reduction of 5 , 5′-dithiobis-2-nitrobenzoic acid in a GSR-couple assay [48] . Thiobarbituric acid reactive substances ( TBARS ) as a measure lipid peroxidation was assessed by the method of Ohkawa et al . [15] . Support Vector Machines ( SVMs ) are a set of supervised machine learning techniques that lie in the family of generalized linear classifiers . They employ a training set , with the SVM classification results scored against the known data classification values , and with the SVM parameters iteratively refined against that metric [50] . SVMs are trained to separate the given binary labeled training data with a hyperplane that is maximally distant from them . After training , the SVM is used to classify new data . SVMs are relatively new , but have already been used extensively in bioinformatics due to their robust performance in classification on sparse and noisy datasets . For our analysis , we used our ( trained ) SVM to identify genes belonging to the set of gene targets directly regulated by Nrf2 . The binary labeled training data was the set of upstream promoter regions from a set of gene targets known to be directly regulated by Nrf2 , combined with the set of promoter regions from a set of genes known not to be directly controlled by Nrf2 . The binary classification to be learned was: target/not target . Thus , the object was to train the SVM to detect those genes that are candidates for targets of direct regulation by Nrf2 , based on the classification the SVM makes from its analysis of the base composition in the upstream promoter region of the candidate . The SVM implementation we used is the LibSVM from Chang and Chih-Jen [13] . Publicly available mouse lung microarray data from seven disparate laboratories were employed , as well as data from the Biswal lab . In all , 260 Affymetrix CEL files from two platforms , Affymetrix GeneChip Mouse Genome 430 2 . 0 array and the Affymetrix Mouse Expression Set 430 ( MOE430A ) , were collected . Of these , 224 arrays were obtained from the publicly available Gene Expression Omnibus Datasets ( Table S1 ) . These mouse lung arrays represent a variety of perturbations of the lung protein-protein interaction network , including gene knockout and ligand treatment . From an R command line ( http://cran . r-project . org/ ) , the affy package of BioConductor ( http://www . bioconductor . org/ ) was used to perform Robust Multi-array Average ( RMA ) analyses on the datasets [51] . The process consisted of the microarray data being normalized and log-transformed , following background correction , according to the method of Irizarry et al . [51] . The RMA analyses were performed on four subsets of the array samples gathered: From the tables generated , data for probe sets representing genes classified under “response to oxidative stress” from the Gene Ontology [19] were then selected . The contents of this class of thirty six genes identified under the GO identifier GO:0006979 are: Aass , Als2 , Apoe , Cat , Cln8 , Ctsb , Cygb , Epas1 , Ercc2 , Ercc6 , Gab1 , Gclm , Gpx1 , Gpx3 , Hif1a , Idh1 , Mtf1 , Nme5 , Nqo1 , Nudt15 , Oxsr1 , Park7 , Ppp1r15b , Prdx1 , Prdx2 , Prdx6 , Prnp , Psmb5 , Sod1 , Sod2 , Srxn1 , Tcf1 , Txnip , Txnrd2 , Xpa , and Ucp3 . In addition , the following relevant possible transcription regulators were added: Nfe2l2 , Ap1gbp1 , Atf1 , Creb1 , Crebbp , Fos , Hsf1 , Jun , Rela , and Nfkbib . The selection was facilitated by a parser we wrote in Lisp [53] for this purpose . All further analyses were confined to these oxidative stress response gene subsets , using four different methods to find direct regulatory targets of Nrf2 . The CLR and ARACNE algorithms were used to examine gene expression patterns in the subsets , and to establish direct dependencies between the expressions of the specified genes and transcription factors such as Nrf2 . The LibSVM utility in the Weka workbench [54] , [55] was used to independently identify , using separate sequence-level data , transcriptional regulatory targets of Nrf2 among the putative Nrf2 target genes returned by the CLR and ARACNE algorithms . This identification was based on a comparison of the promoter regions of the genes to those of known Nrf2 targets . For our fourth analysis method , we matched results from the first three algorithms against Nrf2 gene targets in networks generated using automated literature searches by way of the Agilent Literature Search plug-in [56] of the Cytoscape network visualization platform [57] . We used an implementation of the CLR algorithm within the Software Environment for BIological Network Inference ( SEBINI ) workbench [58] , [59] . The CLR binning parameters were set to use 10 bins , with a spline degree of 3 . The CLR values were converted to z-scores within the SEBINI platform , and a z-score cut-off of 2 . 0 was then employed to select the highest scoring potential regulatory edges . The putative regulatory edges were outputted from SEBINI in Cytoscape Simple Interaction Format ( SIF ) and viewed and analyzed in Cytoscape and CABIN ( as was done with the ARACNE output ) . The p-value for establishing that the mutual information between gene pairs was significant enough to report out was set at 10−7 . The percentage of MI estimates considered as sampling error ( the DPI tolerance ) was set at 0 . 15 . A parser was written in Lisp to convert the outputs into the SIF file format . Each set of edges was thus represented as a network within Cytoscape and CABIN for further analysis . Interactions involving the transcription factor Nrf2 were selected out and entered into Cytoscape and CABIN as smaller-sized networks , for simpler visualization of our Nrf2-based analysis . As detailed in Table S3 , a set of 26 known Nrf2 targets [3] were used for the generation of the true positive part of the LibSVM training set . A set of 23 genes determined to be not Nrf2-regulated [60] formed the true negative part of the training set . The LibSVM Support Vector Machine implementation in the Weka workbench was used for these studies [54] , [55] The results ( Table 3 ) were obtained with normalized data on the nu-SVC classifier , the Radial Basis Function: exp ( −γ*|u−v|2 ) kernel type , with ν = 0 . 36 , γ = 2−13 , cost = 1 . and training set size = 49 . Details on the structure of the LibSVM datasets used are described in Text S1 . Promoter sequences consisting of 1 , 000 nucleotides upstream to 100 nucleotides downstream for each gene were obtained from the Gene Sorter ( http://www . genome . ucsc . edu ) . For each promoter sequence , a vector of size 308 , with elements characterizing features of the sequence , was generated using Common Lisp code . The elements of the vector included a Boolean value indicating whether or not the Antioxidant Response Element ( ARE ) to which Nrf2 binds to activate gene transcription was present . The vector also included numbers characterizing the base pairs stretching between the ARE and the Transcription Start Site ( TSS ) , the ARE and the TFIID bind site , the ARE and the Maf bind site , the ARE and the ATF4 bind site , the ARE and the cAMP Response Element ( CRE ) , and the ARE and the TPA Response Element ( TRE ) . For these characterizations , the three kinds of features used were Composition , Transition and Distribution . Composition is a reference to the proportions of nucleotide base types contributing to the promoter sequence make up . Transitions represent the frequency with which specific nucleotide base types are followed or preceded , within the sequence , by other nucleotide base types . Distribution is a statement concerning the dissemination of specific nucleotide base types within portions of the sequence ( or the entire sequence ) . The data generated was formatted for use within the Weka Workbench software toolkit of machine learning packages in Java [54] . We used the Agilent literature search tool to conduct literature searches [56] . This tool is available as a plug-in for the open source network visualization and analysis tool Cytoscape . It is used to create an inferred network based on published scientific literature for the proteins of interest . The Agilent Literature Search tool takes a protein list and searches for abstracts in several text engines . These search engines include those of the U . S . Patent Office and the National Center for Biotechnology Information ( PubMed ) . The tool parses the search engine output to extract interactions and displays the resulting protein-protein interaction network as a graph within Cytoscape . Literature based evidence is a well recognized way of corroborating interactions detected by other computational prediction methods . Networks found via ARACNE , CLR , and LibSVM were compared to networks identified via this method in order to identify previously identified interactors with Nrf2 and separate out novel Nrf2 targets . As indicated under “Data Sources” above , four sets of RMA-analyzed microarray data constituted the source of four networks for each of the algorithms used . These networks were inputs into the CABIN tool [14] , which is also available as a plug-in for Cytoscape . CABIN was thus used to analyze , compare and merge the inferred networks obtained using ARACNE , CLR , LibSVM and Agilent literature search . CABIN provides the ability to assign weights or confidence to an inferred network , choosing cutoffs by applying dynamic filters . It also provides multiple viewers depicting different abstractions of the data . In this study , the multiple coordinated viewers within CABIN fostered comparison of inferred networks obtained using the algorithms ARACNE , CLR and LibSVM . These networks were further corroborated by combining literature based evidence obtained using the Agilent Literature search tool . Such combined network analysis within CABIN is demonstrated in the screen snapshot shown in Figure 11 . Microarray experiments were conducted with CD-1 Nrf2 wild type ( WT ) and Nrf2 knockout ( NO ) mice exposed to either five continuous hours of cigarette smoke ( CS ) or twenty four hours of air . For the purpose of such studies , approximately 5 hours of continuous CS exposure is about equivalent to one day of cigarette smoking [16] . In the CS-exposed group , there was immediate sacrifice and lung collection after cessation of smoke exposure . In the other group , age-matched air-exposed mice were killed with immediate lung collection following sacrifice . Total RNA was isolated using the Qiagen protocol ( Qiagen Inc . ) . The cDNA was synthesized and Affymetrix microarray ( Mouse genome 430A 2 . 0 array ) was conducted as previously shown [16] . Scanned output files were analyzed by using Affymetrix GeneChip Operating Software version 1 . 3 and were independently normalized to an average intensity of 500 . Further analyses were done as described previously [52] . In addition , the Mann-Whitney pairwise comparison test was performed to rank the results by concordance as an indication of the significance ( P≤0 . 05 ) of each identified change in gene expression . The results for Sod1 , Nqo1 and Als2 indicating mean ( three replicates; n = 3 ) mRNA expression data from the microarrays are shown in Figure 4 . ( WT air exposed ( WTAir ) , WT CS-exposed ( WTCS ) , Nrf2 knockout air-exposed ( NOAir ) and Nrf2 knockout CS-exposed ( NOCS ) ) . | A variety of conditions including certain cancers and heart diseases , diabetes mellitus , and rheumatoid arthritis have been associated with the generation of high levels of highly reactive molecular species under conditions known as “oxidative stress . ” A number of protein molecules have been identified as participants in an elaborate response to oxidative stress . Sustained elevated generation of reactive species can overwhelm this response and lead to disease conditions . In these studies , we make use of data generated from over 250 studies ( microarrays ) in which messenger RNA levels of the gene precursors of mouse lung proteins have been examined collectively . We have made use of computational approaches to help identify the key regulatory relationships among the proteins that respond to oxidative stress . Nrf2 , a protein known as a master regulator of oxidative stress response , was a principal focus of our studies . Among the novel regulatory targets of Nrf2 we identified is Als2 , a protein involved in amyotrophic lateral sclerosis ( Lou Gehrig's disease ) . We also identify important candidate three-party regulatory relationships , one of which involves the recently discovered Srxn1 , an antioxidant protein that reverses S-glutathionylation , a common posttranslational modification associated with diseases such as Parkinson's disease , diabetes , hyperlipidemia , Friedreich's ataxia , renal cell carcinoma , and HIV/AIDS . These studies demonstrate the utility of network inference algorithms and affirm that Nrf2 has a direct regulatory role over the expression of other genes responding to oxidative stress . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [
"computational",
"biology/signaling",
"networks",
"computational",
"biology/transcriptional",
"regulation",
"computational",
"biology/systems",
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] | 2008 | Network Inference Algorithms Elucidate Nrf2 Regulation of Mouse Lung Oxidative Stress |
Mutational robustness quantifies the effect of random mutations on fitness . When mutational robustness is high , most mutations do not change fitness or have only a minor effect on it . From the point of view of fitness landscapes , robust genotypes form neutral networks of almost equal fitness . Using deterministic population models it has been shown that selection favors genotypes inside such networks , which results in increased mutational robustness . Here we demonstrate that this effect is massively enhanced by recombination . Our results are based on a detailed analysis of mesa-shaped fitness landscapes , where we derive precise expressions for the dependence of the robustness on the landscape parameters for recombining and non-recombining populations . In addition , we carry out numerical simulations on different types of random holey landscapes as well as on an empirical fitness landscape . We show that the mutational robustness of a genotype generally correlates with its recombination weight , a new measure that quantifies the likelihood for the genotype to arise from recombination . We argue that the favorable effect of recombination on mutational robustness is a highly universal feature that may have played an important role in the emergence and maintenance of mechanisms of genetic exchange .
The reshuffling of genetic material by recombination is a ubiquitous part of the evolutionary process across the entire range of organismal complexity . Starting with viruses as the simplest evolving entities , recombination occurs largely at random during the coinfection of a cell by more than one virus strain [1] . For bacteria the mechanisms involved in recombination are already more elaborate and present themselves in the form of transformation , transduction and conjugation [2 , 3] . In eukaryotic organisms , sexual reproduction is a nearly universal feature , and recombination is often a necessary condition for the creation of offspring . Although its prevalence in nature is undeniable , the evolution and maintenance of sex is surprising since compared to an asexual population , only half of a sexual population is able to bear offspring and additionally a suitable partner needs to be found [4 , 5] . Whereas the resulting two-fold cost of sex applies only to organisms with differentiated sexes [6] , the fact that genetic reshuffling may break up favorable genetic combinations or introduce harmful variants into the genome poses a problem also to recombining microbes that reproduce asexually [7 , 8] . Since this dilemma was noticed early on in the development of evolutionary theory , many attempts have been undertaken to identify evolutionary benefits of sex and recombination based on general population genetic principles [9–19] . In this article we approach the evolutionary role of recombination from the perspective of fitness landscapes . The fitness landscape is a mapping from genotype to fitness , which encodes the epistatic interactions between mutations and provides a succinct representation of the possible evolutionary trajectories [20] . Previous computational studies addressing the effect of recombination on populations evolving in epistatic fitness landscapes have revealed a rather complex picture , where evolutionary adaptation can be impeded or facilitated depending on , e . g . , the structure of the landscape , the rate of recombination or the time frame of observation [21–26] . Here we focus specifically on the possible benefit of recombination that derives from its ability to enhance the mutational robustness of the population . A living system is said to be robust if it is able to maintain its function in the presence of perturbations [27–31] . In the case of mutational robustness these perturbations are genetic , and the robustness of a genotype is quantified by the number of mutations that it can tolerate without an appreciable change in fitness . Robust genotypes that are connected by mutations therefore form plateaux in the fitness landscape that are commonly referred to as neutral networks [32–35] . Mutational robustness is known to be abundant at various levels of biological organization , but its origins are not well understood . In particular , it is not clear if mutational robustness should be viewed as an evolutionary adaptation , or rather reflects the intrinsic structural constraints of living systems . Arguments in favor of an adaptive origin of robustness were presented by van Nimwegen et al . [32] and by Bornberg-Bauer and Chan [33] , who showed that selection tends to concentrate populations in regions of a neutral network where robustness is higher than average . Whereas this result is widely appreciated , the role of recombination for the evolution of robustness has received much less attention . An early contribution that can be mentioned in this context is due to Boerlijst et al . [36] , who discuss the error threshold in a viral quasi-species model with recombination and point out in a side note that “in sequence space recombination is always inwards pointing . ” This observation was picked up by Wilke and Adami [37] in a review on the evolution of mutational robustness , where they conjecture that the enhancement of robustness by selection should be further amplified by recombination , because “recombination alone always creates sequences that are within the boundaries of the current mutant cloud . ” At about the same time , de Visser et al . discussed a mechanism based on the spreading of robustness modifier alleles in recombining populations [27] ( see also [38] ) . In fact indications of a positive effect of recombination on robustness had been reported earlier in computational studies of the evolution of RNA secondary structure [39] and 2D lattice proteins [40] in the presence and absence of recombination . In these systems the native folding structure of a given sequence is determined by its global free energy minimum . Due to the restricted number of attainable folds , most structures are degenerate in the sense that many sequences fold into the same structure . These sequences form neutral networks in sequence space . Xia and Levitt [40] consider two scenarios , in which the evolution of the lattice proteins is dominated by mutation and by recombination , respectively . The results show that in the latter case the concentration of thermodynamically stable protein sequences is enhanced , which is qualitatively explained by the fact that recombination tends to focus the sequences near the center of their respective neutral network . Therefore most often a single mutation does not change the folding structure . More recently , Azevedo et al . [41] used a model of gene regulatory networks to investigate the origin of negative epistasis , which is a requirement for the advantage of recombination according to the mutational deterministic hypothesis [13] . In this study a gene network is encoded by a matrix of interaction coefficients . It is defined to be viable if its dynamics converges to a stable expression pattern and non-viable otherwise . Thus the underlying fitness landscape is again neutral . Based on their simulation results the authors argue that recombination of interaction matrices reduces the recombinational load , which in turn leads to an increase of mutational robustness and induces negative epistasis as a byproduct . In effect , then , recombination selects for conditions that favor its own maintenance . Other studies along similar lines have been reviewed in [42] . Taken together they suggest that the positive effect of recombination on robustness may be largely independent of the precise structure of the space of genotypes or the genotype-phenotype map . Indeed , a related scenario has also been described in the context of computational evolution of linear genetic programs [43] . Finally , in a numerical study that is similar to ours in spirit , Szöllősi and Derényi considered the effect of recombination on the mutational robustness of populations evolving on different types of neutral fitness landscapes [44] . Using neutral networks that were either generated at random or based on RNA secondary structure , they found that recombination generally enhances mutational robustness by a significant amount . Moreover , they showed that this observation holds not only for infinite populations but also for finite populations , as long as these are sufficiently polymorphic . The goal of this article is to explain these scattered observations in a systematic and quantitative way . For this purpose we begin by a detailed examination of the simplest conceivable setting consisting of a haploid two-locus model with three viable and one lethal genotype [35] . We derive explicit expressions for the robustness as a function of the rates of mutation and recombination that demonstrate the basic phenomenon and guide the exploration of more complex situations . The two-locus results are then generalized to mesa landscapes with L diallelic loci , where genotypes carrying up to k mutations are viable and of equal fitness [45–48] . Using a communal recombination scheme and previous results for multilocus mutation-selection models , we arrive at precise asymptotic results for the mutational robustness for large L and small mutation rates . Subsequently two types of random holey landscape models are considered , including a novel class of sea-cliff landscapes in which the fraction of viable genotypes depends on the distance to a reference sequence . For the isotropic percolation landscape analytic upper and lower bounds on the robustness are derived . As a first step towards a unified explanation for the effect of recombination on mutational robustness we introduce the concept of the recombination weight , which is a measure for the likelihood of a genotype to arise from a recombination event . In analogy to the classic fitness landscape concept in the context of selection [20] , the recombination weight allows one to identify genotypes that are favored by recombination without referring to any specific evolutionary dynamics . We show that recombination weight correlates with mutational robustness for the landscape structures used in this work , thus providing a mechanistic basis for the enhancement of robustness by recombination . Finally , using an empirical fitness landscape as an example , we quantify the competition between selection and recombination as a function of recombination rate . Throughout we describe the evolutionary dynamics by a deterministic , discrete time model that will be introduced in the next section .
We consider a haploid genome with L loci and the corresponding genotype is represented by a sequence σ = ( σ1 , σ2 , … , σL ) of length L . The index i labels genetic loci and each locus carries an allele specified by σi . Here we rely on binary sequences , which means that there are only two different alleles σi ∈ {0 , 1} . This can be either seen as a simplification in the sense that only two alleles are assumed to exist , or in the sense that the genome consisting of all zeros describes the wild type , and the 1’s in the sequence display mutations for which no further distinctions are made . The resulting genotype space is a hypercube of dimension L , where the 2L genotypes represent vertices , and two genotypes that differ at a single locus and are mutually reachable by a point mutation are connected by an edge . A metric is introduced by the Hamming distance d ( σ , κ ) =∑i ( 1−δσiκi ) , ( 1 ) which measures the number of point mutations that separate two genotypes σ and κ . Here and in the following the Kronecker symbol is defined as δxy = 1 if x = y and δxy = 0 otherwise . The genotype σ¯ at maximal distance d ( σ , σ¯ ) =L from a given genotype σ is called its antipodal , and can be defined by σ¯i=1−σi . Finally , in order to generate a fitness landscape , a ( Wrightian ) fitness value wσ is assigned to each genotype . The forces that drive evolution are selection , mutation and recombination . To model the dynamics we use a deterministic , discrete-time model with non-overlapping generations , which can be viewed as an infinite population limit of the Wright-Fisher model . Demographic stochasticity or genetic drift is thus neglected . Numerical simulations of evolution on neutral networks have shown that the infinite population dynamics is already observable for moderate population sizes , which justifies this approximation [32 , 44] . We will return to this point in the Discussion . Once the frequency fσ ( t ) of a genotype σ at generation t is given , the frequency at the next generation is determined in three steps representing selection , mutation , and recombination . After the selection step , the frequency qσ ( t ) is given as qσ ( t ) =wσw¯ ( t ) fσ ( t ) , ( 2 ) where w¯≡∑σwσfσ ( t ) is the mean population fitness at generation t . After the mutation step , the frequency pσ ( t ) is given as pσ ( t ) =∑κUσκqκ ( t ) , ( 3 ) where Uσκ is the probability that an individual with genotype κ mutates to genotype σ in one generation . Here , we assume that alleles at each locus mutate independently , and the mutation probability μ is the same in both directions ( 0 → 1 and 1 → 0 ) and across loci . This leads to the symmetric mutation matrix Uσκ= ( 1−μ ) L−d ( σ , κ ) μd ( σ , κ ) . ( 4 ) In order to incorporate recombination we have to consider the probability that two parents with respective genotypes κ and τ beget a progeny with genotype σ by recombination . This is represented by the following equation: fσ ( t+1 ) =∑κτRσ|κτpκ ( t ) pτ ( t ) . ( 5 ) Descriptively speaking , two genotypes ( κ and τ ) are taken to recombine with a probability that is equal to their frequency in the population ( after selection and mutation ) . The probability for the offspring genotype σ is then given by Rσ|κτ . These probabilities depend of course on the parent genotypes κ and τ but also on the recombination scheme . Here we consider a uniform and a one-point crossover scheme; see Fig 1 for a graphical representation . These two represent extremes in a spectrum of possible recombination schemes . Nevertheless we will show that both lead to qualitatively similar results in the regimes of interest . In the case of uniform crossover the recombination probabilities are given by Rσ|κτ=r2L ( ∏iL ( δσiκi+δσiτi ) ) +1−r2 ( δσκ+δστ ) ( 6 ) and in the case of one point crossover the probabilities can be written as Rσ|κτ=r2 ( L−1 ) ∑n=1L−1[ ( ∏m=1nδσmκm ) ( ∏m=n+1Lδσmτm ) + ( ∏m=1nδσmτm ) ( ∏m=n+1Lδσmκm ) ]+1−r2 ( δσκ+δστ ) . ( 7 ) In both equations a variable r ∈ [0 , 1] appears which describes the recombination rate . For r = 0 no recombination occurs and fσ ( t + 1 ) is the same as pσ ( t ) . For r = 1 recombination is a necessary condition for the creation of offspring ( obligate recombination ) . But also intermediate values of r can be chosen as they occur in nature , e . g . , for bacteria and viruses . In the following we are mostly interested in the equilibrium frequency distribution fσ* of a population , which is determined by the stationarity condition fσ ( t+1 ) =fσ ( t ) =fσ* ( 8 ) for all genotypes σ . From the point of view of fitness landscapes the occurrence of mutational robustness implies that fitness values of neighboring genotypes are degenerate , giving rise to neutral networks in genotype space [29 , 32–35] . In order to model this situation we use two-level landscapes that only differentiate between genotypes that are viable ( wσ = 1 ) or lethal ( wσ = 0 ) . Any selective advantage between viable genotypes is assumed to be negligible . The mutational robustness of a population can then be measured by the average fraction of viable point mutations in an individual , which depends on the population distribution in genotype space [32–34] . It increases if the population mainly adapts to genotypes for which most point mutations are viable . Therefore we define mutational robustness m as the average fraction of viable point mutations of a population , m≡∑σ∈Vmσfσ*withmσ≡nσL . ( 9 ) Here the sum is over the set V of all viable genotypes and nσ is the number of viable point mutations of genotype σ . We will refer to mσ as the mutational robustness of the genotype . The expression is normalized by the total number of loci L , since in an optimal setting the entire population has L viable genotypic neighbors and mσ = 1 for all σ ∈ V . The value of m is thus constrained to be in the range [0 , 1] . We weight the genotypes by their stationary frequencies fσ* , since we want to determine the mutational robustness of populations that are in equilibrium with their environment . In order to elucidate the interplay of recombination and mutational robustness it will prove helpful to introduce a representation of how recombination can transfer genotypes into each other . The number of distinct genotypes that two recombining genotypes are able to create depends on their Hamming distance . In particular , the recombination of two identical genotypes does not create any novelty , whereas a genotype and its antipodal are able to generate all possible genotypes through uniform crossover . Here we introduce a measure which expresses how many pairs of viable genotypes are able to recombine to a specific genotype . It is complementary to the mutational robustness , in the sense that instead of counting the viable mutation neighbors of a genotype , the size of its recombinational neighborhood of viable recombination pairs is determined . The recombinational neighborhood depends on the recombination scheme and the distribution of viable genotypes in the genotype space . For a given recombination scheme the probability for a genotype σ to be the outcome of recombination of two genotypes κ , τ is given by the recombination tensor Rσ|κτ . The recombination weight λσ is therefore obtained by summing the recombination tensor over all ordered pairs of viable genotypes , λσ=12L∑κ∈V , τ∈VRσ|κτ . ( 10 ) It can be seen from ( 5 ) that λσ = 1 when all genotypes are viable , and hence the normalization by 2L ensures that the recombination weight lies in the range [0 , 1] . Under this normalization , the recombination weights sum to ∑σ λσ = |V|2/2L , where |V| stands for the number of viable genotypes . In the following the genotype maximizing λσ will be referred to as the recombination center of the landscape . Since neutral landscapes only differentiate between viable ( unit fitness ) and lethal ( zero fitness ) genotypes , the recombination weight ( 10 ) can alternatively be written as a sum over all ordered pairs of genotypes whereby the recombination tensor is multiplied by the pair’s respective fitness , λσ=12L∑κ , τRσ|κτwκwτ . ( 11 ) In this way the concept naturally generalizes to arbitrary fitness landscapes . In the absence of recombination ( r = 0 ) the recombination weight ( 11 ) of a genotype is simply proportional to its fitness , λσ=w˜wσ , where w˜=2−L∑σwσ is the unweighted average fitness . Within our recombination schemes , the recombination tensor depends linearly on r and , by definition , so does the recombination weight . Accordingly , for general r the recombination weight interpolates linearly between the limiting values at r = 0 and r = 1 . Since λσ for r = 0 is known , the remaining task will be to find λσ for r = 1 .
The simplest fitness landscape to study the mutational robustness of a population would be the haploid two-locus model in which all but one genotype are viable [35]; see Fig 2 for a graphical representation of the model . In this setting the population gains mutational robustness if the frequency of the genotype ( 0 , 0 ) for which both point mutations are viable increases relative to the genotypes ( 0 , 1 ) and ( 1 , 0 ) . This model has been analyzed previously using a unidirectional mutation scheme where reversions 1 → 0 are suppressed [58 , 59] . As a consequence , selection cannot contribute to mutational robustness because the genotype ( 0 , 0 ) goes extinct in the absence of recombination . Here we consider the case of bidirectional , symmetric mutations in which both selection and recombination contribute to robustness . A comparison of the two mutation schemes is provided in S1 Appendix . We proceed to solve the equilibrium condition Eq ( 8 ) . Since the equilibrium genotype frequencies f01* and f10* are the same due to the symmetry of the landscape and the mutation scheme , the recombination step at stationarity reads f00*=p00−ρ ( p00p11−p10p01 ) ⇔f0=p0−ρD , f10/01*=p10/01+ρ ( p00p11−p10p01 ) ⇔f1=p1+2ρD , f11*=p11−ρ ( p00p11−p10p01 ) ⇔f2=p2−ρD , ( 12 ) where pσ is the ( equilibrium ) frequency of genotype σ after the mutation step , fi and pi are the corresponding lumped frequencies [60] of all genotypes with i 1’s , and D≡p00p11−p10p01=p0p2−p12/4 is the linkage disequilibrium after the mutation step . Notice that the one-point and uniform crossover schemes give the same equation form except that the parameter ρ is given by ρ = r in the case of one-point crossover and ρ = r/2 for uniform crossover . However , we would like to emphasize that this is a mere coincidence of the two-locus model which disappears as soon as L is larger than 2 . The lumped frequencies qi of all genotypes with i 1’s after the selection step are given by q0=f01−f2 , q1=f11−f2 , q2=0 . ( 13 ) Applying the mutation step we obtain p0=q0 ( 1−μ ) 2+μ ( 1−μ ) q1=μ ( 1−μ ) + ( 1−μ ) ( 1−2μ ) q0 , p1=q1[ ( 1−μ ) 2+μ2]+2μ ( 1−μ ) q0=1−2μ+2μ2− ( 1−2μ ) 2q0 , p2=μ ( 1−μ ) q1+μ2q0=μ ( 1−μ ) −μ ( 1−2μ ) q0 , D=p0p2−p12/4=−14 ( 1−2μ ) 2 ( 1−q0 ) 2 , ( 14 ) where we have used the normalization q0 + q1 = 1 to express the right hand sides in terms of q0 . Putting everything together , the problem is reduced to solving the following third order polynomial equation for q0 , 0=q0 ( 1−f2 ) −f0=q0 ( 1−p2+ρD ) −p0+ρD=ρ4 ( 1−2μ ) 2 ( 1−q0 ) 2 ( 1+q0 ) +μ[1−2q0−q02−μ ( 1−2q0 ) ( 1+q0 ) ] , ( 15 ) from which we can in principle find exact analytic expressions for fσ* . However , it is difficult to extract useful information from the exact solution . In the following we will therefore provide approximate solutions . If we neglect recombination ( ρ = 0 ) , we obtain the following equilibrium genotype frequency distribution: f00* ( ρ=0 ) =1−μ28−16μ+9μ2−12 ( 2−5μ+3μ2 ) ≈ ( 2−1 ) + ( 52−22 ) μ+O ( μ2 ) , f01/10* ( ρ=0 ) =14 ( 4−9μ+6μ2 ) −1−2μ48−16μ+9μ2≈ ( 1−12 ) + ( 32−94 ) μ+O ( μ2 ) , f11* ( ρ=0 ) =μ2 ( 4−3μ−8−16μ+9μ2 ) ≈ ( 2−2 ) μ+O ( μ2 ) . ( 16 ) When ρ = 1 , which corresponds to the one-point crossover scheme with r = 1 , linkage equilibrium ( f00 f11 = f10 f01 ) is restored after one generation [55] . Accordingly , we can treat each locus independently and get rather simple expressions for fσ* as f00* ( ρ=1 ) =14 ( 2+μ−μ2+4μ ) 2≈1−2μ+2μ+O ( μ3/2 ) , f01/10* ( ρ=1 ) =14 ( 2+μ−μ2+4μ ) ( μ2+4μ−μ ) ≈μ−3μ2+O ( μ3/2 ) , f11* ( ρ=1 ) =14 ( μ2+4μ−μ ) 2≈μ−O ( μ3/2 ) . ( 17 ) We depict the equilibrium solutions for the above two cases in Fig 3 . Now , the mutational robustness m=12 ( 2f00*+f10*+f01* ) =f00*+f10*=f0+12f1 ( 18 ) for the above two cases is obtained as m ( μ , ρ=0 ) =14 ( μ+8−16μ+9μ2 ) ≈12− ( 12−14 ) μ+O ( μ2 ) , ( 19 ) m ( μ , ρ=1 ) =12 ( 2+μ−μ2+4μ ) ≈1−μ+μ2+O ( μ3/2 ) , ( 20 ) which is depicted in Fig 4 . These results encapsulate in a simple form the main topic of this paper . Selection alone ( ρ = 0 ) leads to a moderate increase of robustness from the baseline value m=12 corresponding to a random distribution over genotypes , which is attained at μ=12 , to m=12 for μ → 0 . In contrast , for recombining populations ( ρ = 1 ) robustness is massively enhanced at small mutation rates due to the strong frequency increase of the most robust genotype ( 0 , 0 ) and reaches the maximal value m = 1 at μ = 0 . The underlying mechanism is analogous to Kondrashov’s deterministic mutation hypothesis , which posits that recombination makes selection against deleterious mutations more effective when these interact synergistically [13] . In the present case recombination increases the frequency of the double mutant genotype ( 1 , 1 ) , which is subsequently purged by selection , and thereby effectively drives the frequency of the allele 1 at both loci to zero . The enhancement of the frequency of the genotype ( 0 , 0 ) by recombination is also reflected in the recombination weights , which take on the values λ00=34+ρ4 , λ01=λ10=34−ρ4 , λ11=ρ4 . ( 21 ) Thus the genotype ( 0 , 0 ) is the recombination center of the two-locus landscape . Next we investigate how mutational robustness varies with μ for intermediate recombination rates , assuming that μ is small . As can be seen from Eq ( 15 ) , the asymptotic behavior of the solution for small ρ and μ depends on which of the two parameters is smaller . We first consider the case ρ ≪ μ ≪ 1 . Defining l = ρ/ ( 4μ ) ≪ 1 , Eq ( 15 ) is approximated by 0=l ( 1−q0 ) 2 ( 1+q0 ) +1−2q0−q02−μ ( 1−2q0 ) ( 1+q0 ) , ( 22 ) where we kept terms up to O ( μ ) , since we have not determined whether l is smaller than μ or not . Since q0=2−1 is the solution of Eq ( 22 ) for l = μ = 0 , we set q0=2−1+al+bμ and solve the equation to leading order , which gives q0≈2−1+ ( 3−22 ) l− ( 32−2 ) μ . ( 23 ) The mutational robustness then follows as m=f0+f12=12+p0−p22=12+ ( 1−2μ ) q02≈12+3−222l− ( 12−14 ) μ , ( 24 ) which is consistent with our previous result for ρ = 0; see Eq ( 19 ) . We note that in this regime it is sufficient for the recombination rate to be of order O ( μ2 ) to compensate the negative effect of mutations on mutational robustness , as the two effects cancel when ρ = ρc with ρc=2 ( 5+42 ) μ2≈21 . 3×μ2 . ( 25 ) In the regime ρ ≫ μ , Eq ( 15 ) is approximated as ( 1−4μ ) ( 1−q0 ) 2 ( 1+q0 ) +s ( 1−2q0−q02 ) =0 , ( 26 ) with s = 4μ/ρ . Again we have kept terms up to O ( μ ) because μ and s are of the same order if ρ = O ( 1 ) . Since the solution of Eq ( 26 ) for μ = s = 0 is q0 = 1 , we set q0 = 1 − α with α ≪ 1 . Inserting this into Eq ( 26 ) , we get α≈s . Since α ≫ μ , q0=1−s is the approximate solution to leading order . Hence m=12+ ( 1−2μ ) q02≈1−s4=1−μρ , ( 27 ) which is again consistent with our previous result for ρ = 1 in Eq ( 20 ) . The square root dependence on μ/ρ derives from the corresponding behavior of the genotype frequency f00* and has been noticed previously in the model with unidirectional mutations [58 , 59] . For arbitrary ρ and μ , we have to use the full Eq ( 15 ) . Fig 5 illustrates the behaviour of mutational robustness as a function of the recombination rate for different mutation rates and both recombination schemes . For small μ , a low rate of recombination suffices to bring the robustness close to its maximal value m = 1 . More precisely , according to Eq ( 27 ) , a robustness m > 1 − ϵ is reached for recombination rates ρ > μ/ϵ2 . To summarize , we have seen that analytic results for the two-locus model are easily attainable . For multi-locus models it is much more challenging to derive analytical results , particularly in the presence of recombination . By way of contrast the dynamics induced only by mutation and selection are easier to understand: While mutations increase the genotype diversity in the population , fitter ones grow in frequency through selection , which reduces diversity . Although one might expect that recombination would increase diversity , a number of studies have shown that recombination is more likely to impede the divergence of populations . Recombining populations tend to cluster on single genotypes or in a limited region of a genotype space and furthermore the waiting times for peak shifts in multipeaked fitness landscapes diverge at a critical recombination rate [22 , 26 , 54–56 , 61] . The results for the two-locus model presented above are consistent with this behaviour , as the genotype heterogeneity of the population decreases with increasing recombination rate ( S1 Fig ) . In the following we will investigate how the focusing effect of recombination enhances the mutational robustness of the population in three different multi-locus models . In the mesa landscape it is assumed that up to a certain number k of mutations all genotypes are functional and have unit fitness , whereas genotypes with more than k mutations are lethal and have fitness zero [48] . Hence the fitness landscape is defined as wσ={1 , ifdσ≤k , 0 , otherwise , ( 28 ) where dσ is the Hamming distance to the wild-type sequence ( 0 , 0 , … , 0 ) or , equivalently , the number of loci with allele 1 . We will refer to k as the mesa width or as the critical Hamming distance . Such a scenario can for example be observed in the evolution of regulatory motifs , where the fitness depends on the binding affinity of the regulatory proteins and dσ corresponds to the number of mismatches compared to the original binding motif [45 , 47] . The two-locus model discussed in the preceding section corresponds to the mesa landscape with critical Hamming distance k = 1 and sequence length L = 2 . Here we ask to what extent the behavior observed for the two-locus model generalizes to longer sequences and variable k . Numerical simulations suggest that the strong increase of mutational robustness with recombination rate indeed persists in the general setting , and the particular recombination scheme seems to have only a minor influence; see Fig 6 . Whereas an analytical treatment for general L , k and intermediate recombination rates appears to be out of reach , accurate approximations are available in the limiting case of strong recombination or of no recombination , assuming mutation rate is small . The full derivations for both cases can be found in S1 Appendix . In the following we summarize the main results . In the percolation landscape genotypes are randomly chosen to be viable ( wσ = 1 ) with probability p and lethal ( wσ = 0 ) with probability 1 − p . An interesting property of the percolation model is the emergence of two different landscape regimes [49 , 63–65] . Above the percolation threshold pc , viable genotypes connected by single mutational steps form a cluster that extends over the whole landscape , whereas below pc only isolated small clusters appear . Since the percolation threshold depends inversely on the sequence length , pc≈1L , for large L a small fraction of viable genotypes suffices to create large neutral networks . This allows a population to evolve to distant genotypes without going through lethal regions , and correspondingly the percolation model is often used to study speciation [35 , 49] . A network representation of the percolation model is shown in Fig 8 . The algorithm used to generate this visual representation is explained in S1 Appendix . Fig 9 shows three exemplary stationary genotype frequency distributions on the landscape depicted in Fig 8 . In the absence of recombination the equilibrium frequency distribution is unique , but in the presence of recombination the non-linearity of the dynamics implies that multiple stationary states may exist [54 , 55 , 61] . Fig 9 displays two stationary distributions for r = 1 which are accessed from different initial conditions . It is visually apparent that the recombining populations are concentrated on a small number of highly connected genotypes , leading to a significant increase of mutational robustness . To quantify this effect , the average mutational robustness m¯ is calculated as a function of the recombination rate according to the following numerical protocol: A percolation landscape for given L and p is generated and the initial population is distributed uniformly among all genotypes . The population is evolved in the absence of recombination ( r = 0 ) until the unique equilibrium frequency distribution is reached , for which the mutational robustness m is calculated . Next the recombination rate is increased by predefined increments . After increasing r , the population is again evolved using the stationary state obtained before the increment of r as the initial condition , until it reaches a new stationary state for which the mutational robustness is measured . When the recombination rate has reached r = 1 , a new percolation landscape is generated and the process starts all over again . This is done for an adjustable number of runs over which the average is taken . The results of such a computation are shown in Fig 10 . Similar to the mesa landscapes , a strong increase of mutational robustness is observed already for small rates of recombination , and the effect is largely independent of the recombination scheme . However , in contrast to the mesa landscape the robustness does not reach its maximal value m = 1 for r = 1 and small μ . This reflects the fact that maximally connected genotypes with mσ = 1 are very rare at this particular value of p . For the purpose of comparison we also determined the average mutational robustness m¯0 of a uniform population distribution for the percolation model . Conditioned on the number v = |V| of viable genotypes and assuming that v ≥ 1 , we have m0 ( v , L ) = n ( v , L ) /L , where n ( v , L ) is the average number of viable neighbors of a viable genotype . The latter is given by the expression n ( L , v ) = ( v−1 ) L2L−1 , ( 38 ) since for a given viable genotype there are v − 1 remaining genotypes , each of which has the probability L/ ( 2L − 1 ) to be a neighboring one . Taking into account that the number of viable genotypes is binomially distributed with parameter p and that the empty hypercube ( v = 0 ) should yield m0 = 0 we obtain m¯0=∑v=12L ( v−1 ) 2L−1 ( 2Lv ) pv ( 1−p ) 2L−v=2Lp−1+ ( 1−p ) 2L2L−1 , ( 39 ) which simplifies to m¯0=p when 2Lp ≫ 1 . Note that the condition 2Lp ≫ 1 is naturally satisfied beyond the percolation threshold . Fig 11 illustrates that the dynamics induced by mutation and selection already increase mutational robustness compared to m¯0 and that the addition of recombination even further increases mutational robustness for all values of p . The figure also displays the expected maximum number of viable neighbors of any genotype in the landscape , m¯max , which provides an upper bound on the robustness . The fact that the numerically determined robustness remains below this bound for all p shows that the ability of recombination to locate the most connected genotype is limited . In S1 Appendix it is shown that limL→∞m¯max=1 for p>12 . As outlined above , the algorithm used to generate Figs 10 and 11 computes the mutational robustness of a particular stationary frequency distribution of the recombining population which is smoothly connected to the unique non-recombining stationary state . Although one expects this state to be representative in the sense of being reachable from many initial conditions , for large enough r there can be multiple stationary states that will generally display different robustness ( see Fig 9 ) . To illustrate this point , S7 Fig shows the results of a simulation of the percolation model where all stationary states were identified using localized initial conditions , and the mutational robustness was computed separately for each state . Whereas on average the mutational robustness is always enhanced by recombination , there are rare instances when recombination reduces the robustness compared to the non-recombining case . This may happen , for example , if recombination traps the population on a small island of viable genotypes [22 , 26 , 55 , 56] . In this section we introduce a novel class of fitness-landscape models ( to be called sea-cliff landscapes ) that interpolates between the mesa and percolation landscapes . Similar to the mesa landscape , the fitness values of the sea-cliff model are determined by the distance to a reference genotype κ* . The model differs from the mesa landscape in that it is not assumed that all genotypes have zero fitness beyond a certain number of mutations . Instead , the likelihood for a mutation to be lethal ( to “fall off the cliff” ) is taken to increase with the Hamming distance from the reference genotype . This is mathematically realized by a Heaviside step function θ ( x ) that contains an uncorrelated random contribution ησ and the distance measure d ( σ , κ* ) , wσ=θ[ησ−d ( σ , κ* ) ]={1 , ifησ>d ( σ , κ* ) , 0 , ifησ<d ( σ , κ* ) . ( 40 ) This construction is similar in spirit to the definition of the Rough-Mount-Fuji model [66 , 67] . The average shape of the landscape can be tuned by the mean c and the standard deviation s of the distribution of the random variables ησ , which we assume to be Gaussian in the following . The average fitness at distance d from the reference sequence is then given by w¯ ( d ) =Prob ( wσ=1 ) =12[1−erf ( d−cs2 ) ] , ( 41 ) where erf ( x ) is the error function . Note that the mesa landscape is reproduced if we take the limit s → 0 for fixed c in the range k < c < k + 1 and the percolation landscape is reproduced if we take a joint limit s , |c| → ∞ with c/s fixed . To fix c and s we introduce two distances d< and and d> such that w¯ ( d< ) =0 . 99 and w¯ ( d> ) =0 . 01 , which leads to the relations c=12 ( d<+d> ) ands≈0 . 215 ( d>−d< ) . ( 42 ) The model can be generalized to include several predefined reference sequences , w ( σ ) =θ{∑κ*θ[ησ , κ*−d ( σ , κ* ) ]} , ( 43 ) which allows to create a genotype space with several highly connected clusters . Depending on the Hamming distance between the reference sequences and the variables c and s , clusters can be isolated or connected by viable mutations . Fig 12 shows stationary states in the absence and presence of recombination for two different sea-cliff landscapes with one and two reference genotypes , respectively . Similar to the other landscape models , mutational robustness increases strongly with recombination , due to a population concentration within a neutral cluster . In the presence of two reference genotypes the recombining population should be concentrated within a single cluster . Otherwise lethal genotypes would be predominantly created through recombination of genotypes on different clusters . This observation can also be interpreted in the context of speciation due to genetic incompatibilities [49 , 61] . Without recombination genotypes on both clusters have a nonvanishing frequency , but still the larger cluster is more populated . In contrast to the percolation landscape , robustness reaches a value close to unity for large r , because highly connected genotypes are abundant close to the reference sequence ( S8 Fig ) . Comparing Figs 6 and 10 and S8 Fig , the dependence of mutational robustness on the recombination rate is seen to be strikingly similar . Despite the very different landscape topographies , in all cases a small amount of recombination gives rise to a massive increase in robustness compared to the non-recombining baseline . For the mesa landscape this effect can be plausibly attributed to the focusing property of recombination , which counteracts the entropic spreading towards the fitness brink and localizes the population inside the plateau of viable genotypes . In the case of the holey landscapes , however , it is not evident that focusing the population towards the center of its genotypic range will on average increase robustness , since viable and lethal genotypes are randomly interspersed . To establish the relation between recombination and mutational robustness on the level of individual genotypes , in Fig 13 we plot the recombination weight of each genotype against its robustness mσ . A clear positive correlation between the two quantities is observed both for percolation and sea-cliff landscapes . Additionally we differentiate between viable and lethal genotypes . In the percolation landscape viable genotypes are uniformly distributed in the genotype space , which implies that lethal and viable genotypes have on average the same number of viable point-mutations . Nevertheless the recombination weight of viable genotypes is larger . The fitness of a genotype influences its own recombination weight , because the genotype itself is a possible parental genotype in the recombination event . In non-neutral fitness landscapes the redistribution of the population through recombination competes with selection responding to fitness differences , and the generalized definition ( 11 ) of the recombination weight captures this interplay . To exemplify the relation between recombination weight and mutational robustness in this broader context , we use an empirical fitness landscape for the filamentary fungus Aspergillus niger originally obtained in [68] . In a nutshell , two strains of A . niger ( N411 and N890 ) were fused to a diploid which is unstable and creates two haploids by random chromosome arrangement . Both strains are isogenic to each other , except that N890 has 8 marker mutations on different chromosomes , which were induced by low UV-radiation . Through this process 28 = 256 haploid segregants can theoretically be created of which 186 were isolated in the experiment . As a result of a statistical analysis it was concluded that the missing 70 haploids have zero fitness [69] . In order to illustrate the fitness landscape , a network representation is employed where genotypes are arranged in a plane according to their fitness and their Hamming distance to the wild type , which in this case is the genotype of maximal fitness . In Fig 14A and 14B node sizes are adjusted to the recombination weights and mutational robustness of genotypes , respectively , in order to display the distribution of these quantities . In accordance with the analyses for neutral fitness landscapes , a clear correlation between the recombination weights and mutational robustness is shown in Fig 14C . Since fitness values are not binary we further consider the correlation between the recombination weights and fitness values ( Fig 14D ) . The recombination center is one of the maximally robust genotypes with mσ = 1 , but it is not the fittest within this group . The wild type has maximal fitness but , by comparison , lower robustness ( mσ = 7/8 ) . Fig 15 highlights how the recombination weights change as a function of the recombination rate and how this affects the stationary state of a population . For small recombination rates the recombination weight of each genotype mainly depends on its own fitness , and therefore the wild type coincides with the recombination center . With increasing recombination rate the connectivity of the surrounding genotype network becomes more important and the recombination center switches to a genotype at Hamming distance d = 2 . In contrast to the numerical protocol described previously , in the simulations used to generate Fig 15D–15F the population is reset to a uniform distribution before the recombination rate is increased . Otherwise the population would continue to adapt to the wild type , which has the highest fitness and from which it cannot escape because of peak trapping [22 , 26] . Starting from an initially uniform distribution the population will adapt to one of three possible final genotypes which depend on the recombination rate . For small and large recombination rates the most abundant genotype coincides with the recombination center ( Fig 15D and 15F ) , whereas for intermediate recombination rates the population chooses another genotype that is also located at Hamming distance d = 2 but has higher fitness ( Fig 15E ) . The recombination center ultimately dominates the population , not only because it is maximally connected ( mσ = 1 ) , but also because the genotypes that it is connected to have high fitness . In this sense the sequence of transitions in the most abundant genotype that occur with increasing recombination rate is akin to the scenario described previously in non-recombining populations as the “survival of the flattest” [48 , 70] . Along this sequence mutational robustness increases monotonically whereas the average fitness of the population actually declines ( S9 Fig ) .
Despite a century of research into the evolutionary bases of recombination , a general mechanism explaining the ubiquity of genetic exchange throughout the domains of life has not been found [17 , 18] . Even within the idealized scenario of a population evolving in a fixed environment , whether or not recombination speeds up adaptation and leads to higher fitness levels depends in a complicated way on the structure of the fitness landscape and the parameters of the evolutionary dynamics [21–26] . The most important finding of the present work is that , by comparison , the effect of recombination on mutational robustness is much simpler and highly universal . Irrespective of the number of loci , the structure of the fitness landscape or the recombination scheme , recombination leads to a significant increase of robustness that is usually much stronger than the previously identified effect of selection [32–34] . This suggests that the evolution of recombination may be closely linked to the evolution of robustness , and that similar selective benefits are involved in the two cases . Although the relation of robustness to evolutionary fitness is subtle and not fully understood [27] , it has been convincingly argued that robustness enhances evolvability and hence becomes adaptive in changing environments [29 , 31 , 71 , 72] . A common perspective on recombination , robustness and evolvability can help to develop novel hypotheses about the evolutionary origins of these phenomena that can be tested in future computational or empirical studies . On a quantitative level , we have shown that robustness generally depends on the ratio of recombination to mutation rates , and that the robustness-enhancing effect saturates when r ≫ μ . This observation highlights the importance of r/μ as an evolutionary parameter . Interestingly , even in bacteria and archaea , which have traditionally been regarded as essentially non-recombining , the majority of species displays values of r/μ that are significantly larger than one [73–75] . Similarly , a recent study of the evolution of Siphoviridae phages revealed a ratio of recombination events to mutational substitutions of about 24 [76] . In eukaryotes this ratio is expected to be considerably higher [40] . This indicates that most organisms maintain a rate of recombination that is sufficient to reap its evolutionary benefits in terms of increased robustness . In order to clarify the mechanism through which recombination enhances robustness , we have introduced the concept of the recombination weight , which is a measure for the likelihood of a genotype to arise from the recombination of two viable parental genotypes . The recombination weight defines a “recombination landscape” over the space of genotypes which is similar in spirit to , but distinct from , previous mathematical approaches to conceptualizing the way in which recombining populations navigate a fitness landscape [77] . It is complementary to the more commonly used notion of a recombination load , which refers to the likelihood for a viable genotype to recombine to a lethal one [41 , 42] . In many cases the maximum of the recombination weight correctly predicts the most populated genotype in a recombining population at low mutation rate . Moreover , the concept generalizes to non-neutral landscapes and thus permits to address situations where selection and recombination compete . Provided recombination weight is correlated with mutational robustness for the individual genotypes , this explains the positive effect of recombination on the population-level robustness . Whether or not such a correlation exists will generally depend on the structure of the fitness landscapes . For simple neutral landscapes such as the mesa landscape it is an immediate consequence of the focusing property of recombination , but for more complex neutral networks the relationship between the two quantities is nontrivial and needs to be studied on a case-by-case basis . Although a positive correlation was observed numerically both for the holey landscapes and the empirical landscape considered in this work , it is not difficult to construct landscapes where the genotypes with high recombination weight are not highly robust . As a simple but instructive example , in S10 Fig we show results for an ‘atoll’ landscape where a ring of viable genotypes surrounds a central hole of lethals . Throughout this work the effects of genetic drift have been neglected . We expect that our results will be applicable to finite populations as long as the population is sufficiently diverse rather than being monomorphic . This requires the population-wide mutation rate NμL to be much larger than unity [32 , 44] . If NμL ≪ 1 the population is almost always monomorphic and recombination has no effect . In this regime the population explores the fitness landscape as a random walker and the observed mutational robustness is the uniform robustness m0 . In S11 Fig we present the results of finite population simulations on a mesa landscape , which show a sharp transition from the random walk regime to the behavior predicted by the deterministic theory when NμL ∼ 1 . Future work should be directed towards extending the present investigation to more realistic genotype-phenotype maps arising , for example , from the secondary structures of biopolymers such as RNA or proteins [39 , 40 , 44] , or from simple genetic , metabolic or logical networks [29 , 41 , 43 , 78] . There is ample evidence from numerical studies that a favorable effect of recombination on mutational robustness is present also in these more complex systems , but a detailed analysis of the underlying mechanism has not been carried out . This would entail , in particular , the generalization to genotype spaces composed of sequences carrying more than two alleles per site . We expect that at least part of the analysis for the mesa landscapes carries over to this setting , and in fact some results for the non-recombining case have already been obtained [48] . More importantly , the role of the topology of the corresponding neutral networks in shaping the correlation between recombination weight and robustness needs to be explored systematically . Research along these lines will help to corroborate the relationship between recombination and robustness that we have sketched , and to further elucidate the origins of these two pervasive features of biological evolution . | Two long-standing and seemingly unrelated puzzles in evolutionary biology concern the ubiquity of sexual reproduction and the robustness of organisms against genetic perturbations . Using a theoretical approach based on the concept of a fitness landscape , in this article we argue that the two phenomena may in fact be closely related . In our setting the hereditary information of an organism is encoded in its genotype , which determines it to be either viable or non-viable , and robustness is defined as the fraction of mutations that maintain viability . Previous work has demonstrated that the purging of non-viable genotypes from the population by natural selection leads to a moderate increase in robustness . Here we show that genetic recombination acting in combination with selection massively enhances this effect , an observation that is largely independent of how genotypes are connected by mutations . This suggests that the increase of robustness may be a major driver underlying the evolution of sexual recombination and other forms of genetic exchange throughout the living world . | [
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] | 2019 | Recombination and mutational robustness in neutral fitness landscapes |
The resolution of chromosomes during anaphase is a key step in mitosis . Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements , conditions linked to cancer development . Inactivation of topoisomerase II , condensin , or separase leads to gross chromosome nondisjunction . However , the fate of cells when one or a few chromosomes fail to separate has not been determined . Here , we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm ( cXIIr ) , which allows the characterisation of the cellular fate of the progeny . Surprisingly , we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array . Consequently , one end of the broken chromatid finishes up in each of the new daughter cells , generating a novel type of one-ended double-strand break . Importantly , both daughter cells enter a new cycle and the damage is not detected until the next G2 , when cells arrest in a Rad9-dependent manner . Cytologically , we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11 , indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway . Finally , we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction . We found that aberrant forms of the chromosome were recovered , especially when RAD52 was deleted . Our results demonstrate that , in yeast cells , the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction .
Chromosomes lagging or bridging during anaphase are believed to be one of the main sporadic causes of cytokinesis failure , which leads to tetraploid cells with multicentrosomes , a hallmark of early tumourigenesis [1] , [2] . Conversely , if these anaphase bridges break apart , chromosomes could enter the so-called breakage-fusion-bridge cycle [3]–[5] , which has been related to oncogene amplification and intratumour heterogeneity [6]–[8] . Carcinogens such as cigarette smoke , dysfunction of key cancer genes , bacterial toxins , and , paradoxically , many antitumour chemotherapeutic treatments ( e . g . topoisomerase inhibitors ) are known to cause anaphase bridges [9]–[12] . Chromosomes bridge in anaphase because they have either more than one centromere or problems in resolving the sister chromatids . Most of our knowledge on the biology of sister chromatid resolution comes from studies in yeast . In Saccharomyces cerevisiae , as in the rest of eukaryotes , sister chromatids are kept together after replication by both the cohesin complex and DNA-DNA topological entanglement arising from DNA metabolism ( i . e . , catenations ) [13] . During anaphase onset , cohesion is lost through the regulated cleavage of cohesin by separase [14] , and catenations are removed by the combined actions of condensin [15] and type 2 topoisomerase ( Top2 ) [16] . Yeast mutants for any of these players show knotted nuclear masses in anaphase with trailing distal chromosome regions which cannot be resolved in otherwise bipolarly attached centromeres [15]–[20] . Despite these anaphase problems , all these mutants often perform cytokinesis , leading to a “cut” phenotype characterized by aneuploid daughter cells carrying broken chromosomes [14] , [17] , [21] . Not surprisingly , many daughter cells are not able to enter a new cell cycle after cytokinesis [14]–[16] . This has precluded use of those mutants as tools to follow up the short-term consequences in the progeny of anaphase bridges formed by unresolved sister chromatids . The last genomic region to get resolved in yeast is the ribosomal DNA array ( rDNA ) [19] , [22]–[24] . Importantly , resolution at this locus depends on a third player , besides condensin and Top2: the late mitotic phosphatase Cdc14 [22]–[27] . This is because Cdc14 inactivates transcription by RNA polymerase I in late anaphase , which allows the loading of condensin to the rDNA , its condensation and further resolution with the help of Top2 [22] , [23] , [27]–[31] . Other findings also suggest that these Cdc14 actions could serve to finish up replication within this locus [32] , [33] . When Cdc14 is inactivated by means of thermosensitive conditional alleles such as cdc14-1 , the anaphase segregation problem is much milder than that observed for the other aforementioned mutants . Indeed , cdc14-1 cells get arrested in telophase with the bulk of the nuclear masses segregated yet the rDNA bridging between mother and daughter cells [23] , [24] . In a previous report we demonstrated that re-activation of the thermosensitive protein Cdc14-1 restores its cell cycle functions and is enough to exit mitosis [28] . Nevertheless , a portion of cells do this in spite of failing , in the end , to segregate the rDNA . Because little is known about the behaviour and fate of cells that commit to a new cell cycle once they have failed to resolve sister chromatids , we decided to address these questions taking advantage of this cdc14-1 re-activation phenotype . Herein , we show that cdc14-1 release leads to severing of the rDNA anaphase bridge and a new Rad9-dependent G2/M arrest . We followed the DNA damage response ( DDR ) in these cells and observed that they elicit a Rad52 long-lasting response that is independent of Mre11 . We further discuss how our system provides a model for the study of DNA double strand breaks ( DSB ) where the ends finish up in different compartments ( i . e . , “one-ended” ) .
Since the pioneering works by Hartwell and collaborators on yeast cell cycle control , it is known that conditional mutants for two essential genes , CDC14 and CDC15 , give a telophase block with mostly binucleated dumbbell cells [34] . Nevertheless , we also now know that at least some cdc14 mutants have problems in the resolution and segregation of chromosomes during anaphase [22]–[24] , [33] . As for the rDNA-bearing chromosome XII right arm ( cXIIr ) , the telophase block elicited by the cdc14-1 allele prevents sister chromatid resolution , and therefore segregation , of regions that extend from somewhere within the large rDNA locus to the end of that chromosome arm [23] . Consequently , cXIIr forms an anaphase bridge between the connected daughter nuclei ( Figure 1 for a scheme , “a” phenotype ) . In a previous work , we surprisingly found that , after reactivating the thermosensitive Cdc14-1 protein , cells were able to resume the cell cycle in spite of often failing to complete the resolution and segregation of such distal regions [28] . In general , around 50% of the cells coming out of a cdc14-1 block do not change their missegregation pattern , whereas the other 50% fully complete segregation of cXIIr ( Figure 1 , “a” & “b” phenotypes respectively ) . We began our study by closely monitoring the cell cycle that follows cdc14-1 release in a strain where the cXIIr telomere is labelled ( tetO:1061 ) . We further included a side-by-side isogenic cdc15-2 strain as a control , after confirming that the cXIIr is fully segregated in its telophase block ( Figure S1 ) . In the same experiment we monitored: ( i ) the budding pattern after the release ( Figure 2A ) ; ( ii ) the morphological changes of the nuclei and the overall resolution and segregation of the cXIIr telomere ( Figure 2B and 2C ) ; ( iii ) the changes in DNA content by flow cytometry ( i . e . , bulk replication ) ( Figure 2D ) ; and ( iv ) chromosome behaviour in a pulsed-field gel electrophoresis ( PFGE ) ( i . e . , individual chromosome replication and integrity ) ( Figure 2E ) . For the first two , we looked under the microscope and counted individual cells . The telophase release led to rebudding of the initial dumbbell mitotic cell for both mutants ( Figure 2A ) . Virtually all cells were able to resume the cell cycle in a synchronous way as indicated by the drop of the dumbbell category ( in red ) to values below 10% . Around 120 minutes after the release , most cells had rebudded again . Since most daughter cells remain together for a while after the release , this rebudding gave “threesomes” ( i . e . , single-rebudded or three cell bodies ) and “foursomes” ( i . e . , double-rebudded or four cell bodies ) . Foursomes ( in blue ) remained the most abundant category from 120 minutes onwards ( ∼90% in the cdc14-1 release , and ∼50% in the cdc15-2 release ) . The lesser amount of foursomes in the cdc15-2 release occurred because daughter cells from this mutant were eventually able to separate from each other , whereas daughters for cdc14-1 remained tightly together even long after becoming foursomes ( see below ) . When we followed the release for longer periods we noticed that the budding pattern of the cdc15-2 release became complex and tended to be oscillatory . By contrast , cdc14-1 was much simpler and many cells stalled as the foursome category throughout ( data not shown and Figure S2 ) . A critical difference between the observed threesomes and foursomes for cdc15-2 and cdc14-1 became evident when we looked at the nuclei by DAPI . Thus , cdc15-2 foursomes had 4 nuclei ( i . e . , both daughters have entered and completed another nuclear division round ) in ∼70% of the cases at minute 180 . By contrast , the cdc14-1 release had less than 10% of the foursomes in this situation at that time ( Figure 2B and Figure S2 ) . When we looked at the segregation pattern of the cXIIr telomere ( tetO:1061 ) in these strains , we found that cdc15-2 always segregated it faithfully in the two cell division that took place ( 94 . 0±0 . 1% [mean ± SEM , n = 3] of foursomes with four nuclei had one tetO in each nucleus ) ( Figure 2C for a representative micrograph ) . In the case of cdc14-1 , 63 . 6±1 . 7% ( mean ± SEM , n = 3 ) of foursomes with 2 nuclei had already missegregated cXIIr as expected [28] , and no more cell divisions proceeded in that period ( Figure 2C ) . We repeated this block-and-release experiment in different yeast strains and backgrounds and found that the main conclusion was conserved ( i . e . , long-lasting arrest of many cdc14-1 cells as foursomes in a pre-anaphase stage ) . However , we observed slight differences in terms of synchrony after the release , time of rebudding , and number of daughter cells able to separate from each other . For instance , in the W303 background , cdc15-2 cells got released earlier and the synchrony was much better throughout ( Figure S2 ) . We further explored the spindle apparatus ( spindle itself using Tub1-GFP and spindle pole bodies with Tub4-CFP ) in the cdc14-1 foursomes and observed that each of the two nuclei contained duplicated spindle pole bodies and a metaphase-like spindle ( Figure S3 ) . This shows that the single nucleus observed for each daughter cell in the cdc14-1 foursome represents a genuine pre-anaphase arrest and not a highly tangled anaphase . The fact that cdc14-1 cells stalled as binucleated foursomes after the telophase release indicated that cells got arrested somewhere between S phase ( whose beginning coincides with the rebudding event ) and anaphase . We next narrowed the window of this arrest to G2/M by demonstrating that cells completed DNA replication after the cdc14-1 release . This was possible because , at the time we took samples for microscopy in the above-mentioned experiment , we also took samples for following DNA replication in the cell population by flow cytometry and PFGE ( Figure 2D and 2E ) . When we performed flow cytometry analysis , we observed a duplication of the DNA amount in cells coming from a cdc14-1 release ( Figure 2D ) . Since these cells ended up as foursomes , replication could be clearly assessed by simply observing how cells transited from a 2 N to a 4 N peak . In the case of cdc15-2 , the assessment was a little more difficult since the release gave rise to a complex mixture of single cells ( both unbudded and budded ) and rebudded cells still connected through the cell wall ( threesomes and foursomes ) . However , the three major peaks for DNA content visible during this release accounted well for the observed amounts of each cell type ( Figure 2D , left panel ) , and indicate that these cells also replicated their DNA . An important conclusion we reached from these data is that replication started and finished at the same time for both mutants , at least for the bulk of their DNA . When we performed PFGE for those samples , we further confirmed that chromosome replication is mostly completed for all chromosomes after the cdc14-1 release . We ascertained this using the fact that yeast chromosomes cannot enter a PFGE while being replicated [35] . Thus , we observed that a new replication round for all chromosomes started at around minute 90 in both mutants and that most chromosomes re-entered the gel ∼60 minutes later ( Figure 2E , upper panel; and S4 ) . This individual chromosome replication behaviour fits well with the bulk replication seen by flow cytometry in Figure 2D . Although chromosome XII also started replication after the cdc14-1 release , the recovery of the whole band was incomplete . In fact , we observed just by ethidium bromide staining that chromosome XII became fainter than any other chromosome after the cdc14-1 release ( Figure 2E , upper panels; and S4 ) . This did not happen during the cdc15-2 release . Importantly , when we performed a southern blot with a probe against the rDNA we could see that other shorter bands appeared ( Figure 2E , lower panels , double- and triple-asterisks ) . These new bands were visible after the new round of replication was completed , but they were also visible if we prevented replication after the cdc14-1 release by blocking daughter cells in G1 . Again , this G1 block also led to a 50% drop of chromosome XII band intensity in the ethidium bromide staining; and this drop was specific to the cdc14-1 release ( Figure S4 ) . Besides , a smear above the band for the entire chromosome was also seen during the cdc14-1 release , especially after chromosome replication ( Figure 2E , lower panels , single-asterisk ) . Although we do not know what this smear might be , we speculate that it could account for chromosome XII with replication or recombination intermediates . Interestingly , the cell population in this cdc14-1 strain may have up to three rDNA sizes ( Figure 2E , lower panel , cXII arrow ) ; which would indicate that the rDNA array is more unstable in this mutant . Because the tetO:1061 can still be segregated in ∼50% of the cells coming from a cdc14-1 release [28] , we next decided to specifically assess whether these cells eventually bypass the arrest as foursomes . For that purpose we filmed any re-budding beyond that point and compared it to the previous cXIIr segregation outcome . We followed up 22 dumbbell cdc14-1 cells as they transited out of the telophase arrest on minimal medium agarose patches . Twelve out of these 22 cells ended up missegregating the tetO ( 54 . 5% ) . As expected , all daughter cells rebudded again , although it took around an hour longer than when we performed the release in liquid cultures ( half-life of the dumbbell phenotype was ∼135 minutes for agarose patches versus ∼75 minutes for cells in culture ) . Importantly , no foursomes that originally missegregated the cXIIr had rebudded a third time by 6 hours after the telophase release ( n = 12 ) ; whereas 80% of foursomes had done so when cXIIr segregation had been correct ( n = 10 ) . This difference is statistically very significant ( p<0 . 001 , Fisher's exact test on the 2×2 contingency table ) . At the telophase block , cdc14-1 strains have daughter-to-be cells still connected through the bud neck as cytokinesis has not yet been completed [36] . A key question to understand the observed G2/M block is to address the fate of the cXIIr anaphase bridge after the release; importantly , whether or not cdc14-1 cells complete cytokinesis and hence sever the bridge . We addressed this question two ways . First we looked at karyokinesis microscopically ( i . e . , nuclear fission ) in a strain where the distal part of the rDNA is tagged ( tetO:487 ) and the nuclear TetR-YFP is overexpressed . In this strain we can see both the cXIIr bridge and the nucleoplasm . When Z stacks of microscope pictures were taken at the cdc14-1 block , the tetO:487 was seldom segregated and a clear nucleoplasm bridge was visible across the bud neck ( Figure 3A , 0′ picture; Figure S5 , hollow pointers ) . The nucleoplasm bridge ( soluble TetR-YFP , do not mistake nucleoplasm bridge for anaphase bridge ) was also observed for all cells blocked with the cdc15-2 allele . This suggests that karyokinesis has not yet taken place in both telophase blocks . Noticeably , the nucleoplasm bridge had bulges in the cdc14-1 block ( Figure S5 , filled pointers ) , yet was a thin and straight line in the cdc15-2 block . We tested whether this bulge accommodates the unresolved rDNA and distal regions of the cXIIr by using another yeast strain that also carries the nucleolar marker Net1 fused to CFP . We found that the nucleolus colocalized with the bulge in more than 95% of the cells ( Figure S5B ) . After the cdc14-1 telophase release , the nucleoplasm bridge eventually disappeared in dumbbell cells and was never visible in daughter cells that had already rebudded ( Figure 3A , dumbbells [D] and foursomes [F] at minute 90 ) . A similar behaviour was seen for cdc15-2 release ( data not shown ) . Importantly , no cdc14-1 foursomes had a nucleoplasm bridge , even if they had previously missegregated cXIIr . A conclusive proof that karyokinesis took place before the daughter cells became foursomes was obtained when cells were arrested in G1 right after the cdc14-1 release . Thus , the nucleoplasm bridge was never visible after the release in cells treated with alpha-factor , irrespective of the cXIIr segregation status ( Figure 3A , photo α-F at minute 90 ) . Although rDNA-missegregating cells ended up performing karyokinesis , it was somehow striking we did not observe a delay in the cell cycle during the cdc14-1 release . Such delay is expected since a checkpoint has been described to sense the presence of anaphase bridges in yeast ( i . e . , NoCut checkpoint ) [37] , [38] . We took advantage of having the aforementioned strain to look at the nucleoplasm and the cXIIr bridges simultaneously and check whether the maintenance of the cXIIr bridge after the release correlated with a delay in the karyokinesis ( Figure 3B ) . We performed a time course of the cdc14-1 release and followed daughter cells ( as dumbbells ) throughout the new G1 . We observed that the nucleoplasm bridge ( again do not mistake for the cXIIr bridge ) took around 20 minutes longer to be severed in those cells that finally failed to segregate the cXIIr ( Figure 3B , half-life for the nucleoplasm bridge was ∼30 minutes for cells with segregated cXIIr versus ∼50 minutes for missegregated cXIIr ) . We believe that this 20 minute delay in karyokinesis may account for the NoCut checkpoint . In any case , the time of disappearance of the nucleoplasm bridge ( i . e . , karyokinesis ) was short and the NoCut checkpoint did not preclude cells with the cXIIr bridge from finally completing karyokinesis . In order to confirm that the fate of the cXIIr bridge is to be severed after the cdc14-1 release , we also looked at cytokinesis indirectly . We employed an assay based on the fact that formaldehyde-fixed cells that have not completed cytokinesis are resistant to separation by cell wall digesting enzymes ( i . e . , zymolyase ) [39] . At the cdc14-1 block , when most cells were dumbbells , zymolyase treatment was not able to separate the daughters , although the bud neck that connects them became very thin ( Figure 3C , left-corner photos ) . By contrast , foursomes seen two hours after the release could be split in two ( Figure 3C , main lower photo ) . The drop of foursomes after zymolyase treatment was high ( from ∼70% to ∼3% ) . In another set of experiments , we also counted cell number after zymolyase treatment under different chemical conditions to inhibit either cytokinesis or S-phase . To inhibit cytokinesis , we added the F-actin inhibitor Latrunculin A ( LatA ) [40] . Since its action against cytokinesis is optimal if cells are incubated before they reach telophase but after they have budded , we employed an initial arrest in G2/M [40] . Then we let them transit from the G2/M arrest to the telophase arrest . To inhibit the new S-phase , we blocked cells in G1 with alpha-factor , added at the telophase release . As expected , overall cell number doubled two hours after the release relative to the telophase arrest in a culture without LatA ( Figure 3D ) . Importantly , this separation could be partially prevented by incubating the cdc14-1 cells with LatA ( cdc14-1 release with versus without LatA gave a p = 0 . 036 , Student's T test ) . Moreover , we also demonstrated that cytokinesis occurred before the daughters entered the new S-phase . Thus , alpha-factor prevented dumbbells from becoming foursomes after the release , but it did not circumvent cell separation after zymolyase treatment ( Figure 3D ) . Again , this separation was prevented when LatA was added during the G2/M to telophase transition ( cdc14-1 release to alpha-factor with versus without LatA gave a p = 0 . 028 , Student's T test ) . On the whole , we can conclude from this set of experiments about chromosome XII integrity ( Figure 2E ) and karyo/cytokinesis ( Figure 3 ) that cells physically separate from each other irrespective of the presence of the cXIIr bridge . The logical consequence of this should be the generation of at least a DSB near or within the rDNA . The observed karyokinesis , cytokinesis and cXIIr breakage , followed by the arrest in G2/M in the new cell cycle , likely implies that a DSB-mediated DNA damage checkpoint is activated after the cdc14-1 release . A critical component of this checkpoint is Rad9 . Mutants for this protein allow cells with DSBs to enter a new segregation round [41] . Hence , we decided to check whether our observed pre-anaphase arrest could be overcome by deleting RAD9 . We did this in our cdc14-1 TUB1-GFP strain to follow spindle morphology as well as nuclear division after the release ( Figure 4A ) . In contrast to the single mutant cdc14-1 TUB1-GFP , the double mutant cdc14-1 rad9Δ TUB1-GFP could enter anaphase by 3 hours after the release , becoming foursomes with more than two nuclei masses ( i . e . , at least one of the daughter cells entered a new anaphase ) ( Figure 4A , upper panels ) . Accordingly , when we looked at spindle morphology using Tub1-GFP , we observed a transition from metaphase-like spindles to other patterns in the cdc14-1 rad9Δ double mutant ( mainly G1-like Tub1 dot signals ) ( Figure 4A , lower panels ) . Besides this , we tested the responsiveness of foursomes to the G1-specific pheromone alpha-factor . We reasoned that if cells were able to progress beyond the G2/M arrest , they would become responsive to the pheromone and change their morphology accordingly ( i . e . , acquire the shmoo phenotype ) . Thus , we treated cdc14-1 cells with the pheromone , not at the time of the release as in other experiments above , but after they became foursomes ( 2 hours after the release ) . Then , we left them in alpha-factor for another 3 hours . We first noticed that some rad9Δ backgrounds , like the one that carries the TUB1-GFP , were able to split the foursomes after this 5 hours incubation time . Therefore , we used one of the W303 backgrounds that kept the foursome category in these conditions . Importantly , all cells in most cdc14-1 rad9Δ foursomes were responsive to the pheromone ( Figure 4B ) . Also , these foursomes had four segregated nuclei ( Figure 4C ) . Interestingly , cells in the cdc14-1 RAD9 foursome distributed in three peaks: one with no responsive cells , one with all four cells responsive , and a third subgroup with just two cells responding to the pheromone ( Figure 4B ) . Within this subgroup , the two responsive cells were always a partner and each has a nucleus ( Figure 4C ) . In order to determine where these two cells come from , we repeated this assay with the cdc14-1 strain that carries the labelled cXIIr telomere . We found that in 92% of foursomes ( n = 39 ) both responsive cells had a tetO , whereas there was no tetO in either of the two non-responsive cells . This means that: ( i ) this subgroup came from cdc14-1 cells where missegregation occurred in the first place , and ( ii ) the cell that retained the intact chromosome XII plus the broken cXIIr was able to eventually pass the G2/M arrest . Therefore , we concluded that cells coming from a cdc14-1 release activated the Rad9 checkpoint to prevent daughter cells from entering anaphase , and that this G2/M arrest persists for a long time in the daughter cell that lost an intact copy of chromosome XII ( i . e . , DC1 ) . The Rad9-dependent cell cycle arrest means that daughter cells sense the DSB ( s ) . Therefore , they must accordingly trigger a DNA damage response ( DDR ) . At this point , we started looking at proteins that cytologically mark this DDR by appearance in nuclear foci . Rad52 is a key mediator in the DDR that comprises the preferred homologous recombination ( HR ) pathway for repair [42] . This pathway is central in the DDRs that occur throughout S-phase and well into mitosis [43] . We reasoned that , because daughter cells reached and completed S-phase on schedule after a cdc14-1 release ( Figure 2 ) and then get arrested in G2/M in a Rad9-dependent manner ( Figure 2 and Figure 4 ) , Rad52 should be involved in the DDR . Importantly , Rad52-YFP forms widely studied nuclear foci after induced DNA damage [43] . Thus , we looked at Rad52 foci in our telophase block-and-release experiments . We indeed observed foci after a cdc14-1 release for a subset of cells ( Figure 5 ) . Rad52 foci number and intensity were clearly superior in the cdc14-1 release relative to a side-by-side experiment with the cdc15-2 strain ( Figure 5A ) . Importantly , there was no difference between the strains when growing asynchronously at 25°C ( only ∼5% of budded cells had foci ) . Foci were observed at the telophase block for neither cdc14-1 nor cdc15-2 , further indicating that DNA damage has not yet taken place at this stage . Foci started around 90 minutes after the release and always after rebudding ( Figure 5B for a typical time-lapse movie ) . Furthermore , Rad52 foci were rather dynamic at the beginning of the new S-phase and , within the subgroup that , at some point , had Rad52-YFP foci , tended to end up as either just one major focus in the foursomes ( in one of its two nuclei ) or 2 foci , one located in each nucleus ( Figure 5A and 5B ) . Remarkably , the percentage of foursomes acquiring at least one long-lasting single Rad52-YFP focus during the release was ∼50% ( Figure 5A ) . We noticed that this percentage of cells was equivalent to that of rDNA/cXIIr missegregation [28] . We therefore hypothesised that cells with Rad52 foci may represent those that had failed in rDNA segregation . We addressed this important question in two ways . First we made use of a second mutation that worsens rDNA segregation after a cdc14-1 release ( i . e . , deletion of FOB1 gene ) [28] ( Figure S6 ) . Second , we double labelled two cdc14-1 strains ( one in the S288C background and the other one in W303 ) with a tag for the rDNA and a tag for the Rad52 protein . For the S288C background , we employed our strain with the tetO:487 and added a RAD52-RedStar2 allele ( Figure S7 ) . As for W303 , we employed a previously described strain that bears both Rad52-YFP and a tag inserted within the rDNA ( tetOs/TetR-mRFP system ) [44] and that we made cdc14-1 ( Figure 5C ) . By using the cdc14-1 fob1Δ double mutant , we could correlate worsening of the rDNA segregation with a higher frequency of foursomes carrying at least one bright Rad52 focus ( Figure S6; p<0 . 0001 , Pearson's chi-square test ) . On the other hand , double labelling of Rad52 and the rDNA further and strongly confirmed that Rad52 foci are more frequent in cells that missegregated the rDNA ( Figure 5C and Figure S7; p<0 . 0001 , Pearson's chi-square test ) . Moreover , these strains allowed us to determine that the first and strongest Rad52 focus appeared in the daughter cell that does not carry the tetOs ( i . e . , cell DC1 ) . Thus , 75% of these foci were located in that cell versus only 8% of Rad52 single foci seen in the daughter cell that carries the tetOs ( the remaining 17% of foursomes had one Rad52 focus in each daughter cell ) . We believe that this is an important result since the genetic material that each daughter carries in the anaphase bridge is different as stated above ( Figure 1 , “a” phenotype ) . Hence , “DC1” cell ( the one without the tetOs ) bears just one broken copy of the resolved part of chromosome XII ( from left telomere to somewhere within the rDNA ) , whereas “DC2” cell carries one entire sister chromatid plus the unresolved part of the other one ( from somewhere within the rDNA to the right telomere ) . The fact that Rad52 foci are stronger and long-lasting in DC1 might indicate that this cell struggles to repair the DSB , while DC2 might end up repairing its broken end . This is in agreement with what we observed when deleted RAD9 ( Figure 4B ) . Finally , it is worth mentioning that , for those Rad52 foci visible in the tetO-carrying nuclei , the fluorescent dots were almost always in close proximity ( Figure 5D ) . However , these Rad52 foci did not localize within the nucleolus when we used a nucleolar marker ( Figure S8 ) . This is not surprising though as broken rDNA sequences are transported out of the nucleolus towards nuclear Rad52 factories [44] . Overall , these observations fit well with the prediction of a DDR occurring preferentially in those daughter cells that failed in rDNA segregation during the preceding division . We expected the DSB to occur shortly after the release into G1 , when karyo- and cytokinesis took place ( Figure 3 ) and chromosome XII appeared partly broken ( Figure 2E ) . We were intrigued by the fact that cells did not , however , delay G1 ( Figure 2 ) . A possible explanation for this anomaly would be that the DSB is clean ( i . e . , with little associated single-stranded DNA [ssDNA] at the edges ) . This is the type of DSB generated by inducible endonucleases like HO as opposed to DSBs obtained after ionizing irradiation , which are rich in ssDNA ( i . e . , ragged ends ) [45] , [46] . It has been shown that clean ends are poorly resected in G1 , forming little ssDNA , whereas ragged DSBs can already bind ssDNA-binding proteins such as the RPA complex . The formation of ssDNA and the binding of the RPA complex to it are key steps in checkpoint activation [47] , [48] . Because of this , we also included the RPA complex as a reporter to study the DDR that follows the cdc14-1 release . YFP-tagged Rfa1 ( one of the complex subunits ) also forms foci under the presence of DSBs [49] . Crucially , Rfa1 can form foci in G1 provided that the DSB takes place at this stage and , as just mentioned , the break is ragged [46] , [49] , [50] . Thus , we observed Rfa1 foci for a subset of cells coming from a cdc14-1 release ( Figure 6A and 6B ) . Rfa1 foci were as dynamic as those of Rad52 and also tended to end up as a major focus , one per nucleus in the foursome at the most ( Figure 6C ) . Moreover , it was noticeable that Rfa1-YFP eventually gave very intense foci ( Figure 6B ) . Nevertheless , all foci began to appear around 60′–90′ after the release ( Figure 6A ) , and always after rebudding ( Figure 6C ) . Accordingly , foci were not observed in cdc14-1 cells transiting from telophase to a G1 arrest with alpha-factor ( Figure 6B ) . This likely means that the new type of DSB generated by the severing of the cXIIr bridge has little associated ssDNA ( i . e . , the DSB is clean ) and therefore it is not recognized by the RPA complex in G1 . In the canonical model for DSB recognition and repair by HR , RPA and Rad52 are downstream players to the MRX complex ( Mre11-Rad50-Xrs2 ) [49] . This complex is supposed to recognize each DSB end , bring them together and help in the first stages of end processing to allow template searching for HR . The expected “one-ended” nature of the DSB , a consequence of the anaphase bridge severing ( i . e . , the two ends cannot be brought together ) , prompted us to further study this important component in the DSB signalling and repair . We made use of Mre11-YFP as a reporter of the DSB-specific MRX complex . Unlike Rad52 and Rfa1 , Mre11 foci have been observed in all cell cycle stages ( including G1 ) and for all types of “two-ended” DSBs generated [46] , [49] . We were not able to observe Mre11 foci for cdc14-1 throughout the release ( less than 1% of cells at any one time point in 10 minutes intervals , data not shown ) . Nevertheless , Mre11 was fully functional in cdc14-1 cells growing at the permissive temperature since it forms foci when DSBs were chemically generated ( Figure S9 ) . We further ruled out any role of the MRX complex in the observed Rad52-dependent response after the release by looking at Rad52 foci in a cdc14-1 mre11Δ double mutant . Indeed , we saw Rad52 foci at a number and intensity comparable to that of a cdc14-1 MRE11 strain in foursomes taken 2 and 4 hours after the telophase release ( Figure 7A and 7B ) . Because mre11Δ gave some background of Rad52 foci at the cdc14-1 arrest ( Figure 7A , time 0′ ) , we filmed cells during the release and observed that about 75% of cells with Rad52 foci at the arrest never entered a new cell cycle ( data not shown ) . Therefore , almost all foci measured in the foursomes likely came from cells without foci at the previous arrest . We thus concluded that Rad52 foci in cdc14-1 foursomes were independent of Mre11 . Although the cXIIr is a hotspot for missegregation in cdc14 mutants , it seems not to be the only genomic region affected . Thus , at least one telomere of a chromosome other than XII appeared missegregated at the telophase block in previous works that made used of another thermosensitive allele ( i . e . , cdc14-3 ) [22] , [33] . Therefore , we decided to address whether telomeres other than cXIIr were also missegregated during the cdc14-1 release . We looked at four different telomeres located in two chromosomes ( V & XIV ) [51] . Chromosome V has the right telomere labelled with the lacO/LacI-CFP system , whereas its left telomere ( V-L ) is labelled with the tetO/TetR-YFP system . On the other hand , chromosome XIV has the right telomere ( XIV-R ) labelled with the tetO/TetR-YFP system and its left telomere labelled with the lacO/LacI-CFP system . It is important to note that telomere V-L was the one used in the above-mentioned cdc14-3 studies . When we carried out the cdc14-1 telophase block at 37°C we often failed to detect the CFP signal , so we focused on missegregation after the release ( CFP signal recovered after the temperature drop ) . Since the cdc15-2 release gave only few binucleated foursomes ( Figure 2 and Figure S2 ) , and in order to avoid a possible bias , we compared the cdc14-1 release to the cdc15-2 telophase block . To preserve the CFP signal , we arrested cdc15-2 cells at 34°C ( at least for the YFP-labelled telomeres there was no difference between 34°C and 37°C in terms of segregation , data not shown ) . We observed that missegregation in cdc14-1 binucleated foursomes was low for all four telomeres and comparable to that observed at the cdc15-2 block ( Table 1 ) . From these data , and from the pattern of chromosome integrity shown in Figure S4 , we can conclude that in a cdc14-1 release many chromosomes are expected to be fully segregated . Thus , the anaphase bridge severed after the cdc14-1 release must be relatively enriched with cXIIr fragments . In a previous paper , we demonstrated that less than 1% of daughter cells can survive passage through multiple mitoses ( >25 ) without Cdc14 ( regulated overexpression of the cyclin-dependent kinase inhibitor Sic1 through GAL-SIC1 was employed to overcome Cdc14 roles in the Mitotic Exit Network ) [28] . In that work , we found that all survivors had dramatically shortened the rDNA locus ( although other chromosome rearrangements were not obvious from the PFGE analysis ) . We also showed that the small survival capability depended on Rad52 ( i . e . , HR is needed to repair the DNA damage and survive ) . This prompted us to study whether our cdc14-1 block-and-release approach , where only one cell cycle is compromised , leads to similar results . Because Rad52 also seems to play an important role after the transient Cdc14 inactivation ( Figure 5 , Figures S6 and S7 ) , we also tested whether RAD52 was essential in this system by including a double mutant cdc14-1 rad52Δ . Thus , we performed the block-and-release experiment for both cdc14-1 and cdc14-1 rad52Δ and plated the foursomes to obtain isolated colonies after 3–5 days ( Figure 8A ) . We also plated cells right before the block-and-release experiment , while growing asynchronously at 25°C . At the time of plating , we counted cells with a haemocytometer to determine overall viability . Surprisingly , we did not see a great loss of viability after the transient Cdc14 inactivation ( Figure 8A and table underneath ) . As for the single cdc14-1 mutant , this loss was around 25% at the most , whereas double mutant cdc14-1 rad52Δ showed no drop in viability at all . This demonstrates that at least one of the daughter cells of the foursome often survives and gives raise to a colony . Taking into account that 50% of foursomes missegregated the cXIIr ( equivalent values of missegregation were seen during a cdc14-1 rad52Δ release: 48% ) , the observed percentage of viable cells might indicate that 50% and 100% of the daughter cells that carry an intact cXII ( i . e . , DC2 ) must survive in cdc14-1 and cdc14-1 rad52Δ respectively . Other results we showed above already pointed towards this possibility . For instance , DC2 was often able to pass the G2/M block after a while ( Figure 4B ) . Besides , Rad52 foci seem to eventually disappear in that cell . All these data indicate that DC2 might sometimes repair the damage and carry on dividing until it forms a colony . Importantly , we did notice that around one third of those colonies grew much more slowly in cdc14-1 ( Figure 8A , “s” colonies ) . These slow-growing colonies were also observed when cdc14-1 cells where plated while normally growing at the permissive temperature . However , there was a three-fold increase in their number when plated after the transient Cdc14 inactivation ( Figure 8A and table underneath ) . Strikingly , deletion of RAD52 prevented these very slow-growing colonies from appearing , although most colonies grew ∼30% more slowly after the cdc14-1 release ( Figure 8A and table underneath ) . The different effects of both the block-and-release experiment and the presence of Rad52 on the colony size of survivors prompted us to analyse the state of chromosome XII in the different outcomes ( Figure 8B ) . Thus , we grew several colonies at the permissive temperature and carried out PFGE . We found that chromosome XII was shorter in the cdc14-1 rad52Δ strain we used ( Figure 8B ) than in its parental cdc14-1 RAD52 strain . Interestingly , chromosome XII was highly unstable in the cdc14-1 rad52Δ survivors , whereas it remained more constant in cdc14-1 RAD52 , even in the slow-growing survivors ( Figure 8B ) . One of the cdc14-1 RAD52 survivors ( #b4 ) could have duplicated chromosome XII as suggested by the presence of two rDNA-containing bands . From this set of experiments we conclude that many foursomes where cXIIr missegregation occurred can still carry on dividing for many generations ( DC2 likely seeds these survivors ) . In addition to this , chromosome XII rearrangements and a reduced fitness are frequent outcomes of transient inactivation of Cdc14 for one cell cycle .
The major manifestation of entering anaphase without completing sister chromatid resolution is the appearance of anaphase bridges . Herein , we have introduced a new model to study the short-term consequences of these bridges based on the primary phenotype observed for the cdc14-1 mutant of Saccharomyces cerevisiae [23] , [24] , [28] . From a technical point of view this model presents several key advantages that facilitate cell biology studies on anaphase bridges: ( i ) non-resolution specificity for few genomic regions ( e . g . , cXIIr , see below ) ; ( ii ) cell mixtures of segregated and missegregated cXIIr in the same population and experiment [28] ( Figure 2 ) ; ( iii ) synchrony of the cells exiting mitosis ( Figure 2 ) ; ( iv ) capability to monitor and cross-compare both daughter cells as they remain together after a cdc14-1 release ( Figure 2 and Figure 3 ) ; and ( v ) availability of a proper parallel control that mostly behaves like cdc14-1 but does segregate the cXIIr ( i . e . , cdc15-2 conditional allele ) [22] ( Figure 2 , Figures S1 and S4 ) . Because Cdc14 controls condensin and Top2 in anaphase and directs their activities to the rDNA [22] , [27] , [28] , [30] , [31] , the overall expectation of our system is that the cdc14-1 anaphase bridge is like those of condensin and top2 mutants , however mainly restricted to a single chromosome arm ( i . e . , cXIIr ) . Therefore , it is interesting to compare our results to those previously reported for condensin and top2 mutants . We also include here mutants for cohesin removal due to their similarities . All these mutants form anaphase bridges comprised of trailing and distally unresolved sister chromatids as we depict in Figure 1 and Figure 9 ( just for chromosome XII in those figures [i . e . , cdc14-1] , more chromosome arms are like cXIIr for these other mutants ) . This pattern of non-resolution likely arises from the spindle forces being able to slide cohesin and catenations away from bipolarly attached centromeres . Importantly , these mutants differ in the extent of non-resolution along the chromosome arms and the number of affected chromosomes , cohesin-removal mutants having the strongest phenotype and cdc14-1 the mildest [14] , [19] , [20] , [23] . Accordingly , a common outcome in cells where cohesin or catenation removal have been impaired is the appearance of an anaphase where the nuclear mass cannot be split in two . For instance , condensin or top2 conditional mutants show rod-like nuclei in anaphase [15] , [16] . The same outcome is seen in mutants where cohesin cleavage is inhibited in anaphase ( e . g . , separase mutants or non-cleavable forms of cohesin ) [14] . Importantly , this unresolved nucleus does not abort cytokinesis , which eventually takes place leading to a “cut” phenotype in all cases . This phenotype is characterized by aneuploid daughter cells carrying broken chromosomes [14] , [16] , [32] . Another common feature of those daughter cells is that many are unable to resume the cell cycle , likely because of the massive chromosome breakage observed . The results we present in this work indicate that the anaphase bridge in cdc14-1 and its fate is somewhat different . First , the cdc14-1 block does not lead to a rod-like nucleus in anaphase , rather it is able to split the two DNA masses , which end up in each daughter cell [23]–[25] , [34] . Likely , this is the consequence of most chromosome arms being able to segregate at the block . It is important to point out that we have assessed four telomeres of two other chromosomes ( V and XIV ) and found little missegregation in cdc14-1 foursomes relative to a cdc15-2 block ( Table 1 ) . Moreover , the drop of band intensity in the PFGE was only seen for chromosome XII in the cdc14-1 release ( Figure 2E and Figure S4 ) . Taking into account that , even in top2 and condensin mutants , small and medium-sized chromosomes segregate despite the rod-like nuclear phenotype [19] , [21] , we believe that the anaphase bridge in cdc14-1 mutants must comprise few chromosome arms; and that those severed by cytokinesis after the cdc14-1 release are fewer than for the other mutants . This in turn would explain why both daughter cells reach G2/M ( Figure 2 and Figure S2 ) . If more than four chromosome arms were severed , we would expect a G1 delay [45] , which we did not observe . On the other hand , in all mutants but cdc14-1 , cytokinesis is followed by cell separation . This makes difficult to follow up and cross-compare both daughter cells , a key advantage we show for cdc14-1 . It is unclear why cdc14-1 daughter cells are unable to separate from each other . However , this phenotype can be also seen when overcoming the cdc14-1 block by overexpressing Sic1 [28] , [52]; and it was then shown that cytokinesis was completed [52] . Our data also suggest that cytokinesis is completed after cdc14-1 re-activation ( Figure 3 ) . Thus , a possible role of Cdc14 in cell septation could be responsible for this phenotype ( Figure 2 ) . A role that could actually be extended to the mitotic exit network as cdc15-2 also has a partial defect in cell separation . Finally , the anaphase bridges formed in cohesin and some top2 mutants have been employed to define a checkpoint that delays cytokinesis ( i . e . , NoCut checkpoint ) [37] . The actual length of such delay has been difficult to measure . In our case , the cdc14-1 anaphase bridge gave a short delay of about 20 minutes when comparing karyokinesis relative to the maintenance of the cXIIr bridge after the release ( Figure 3B ) , and likely accounts for the NoCut checkpoint . Nevertheless , this delay was difficult to see by other means . For instance , it was observed neither relative to cdc15-2 ( at least in the S288C background ) nor as a biphasic drop of dumbbell cells in cdc14-1 ( i . e . , cells able to segregate the cXIIr versus those with the cXIIr bridge ) ( Figure 2A and Figure S2 ) . Besides , the dynamics of the spindle disassembly after the release were quick for dumbbell cells ( within the first hour , Figure S3A , left panel ) . It may be possible that a greater number of chromosomes in the anaphase bridge obtained by other mutants may trigger a stronger checkpoint signal . As stated above , the cdc14-1 anaphase bridge is supposed to be similar to condensin and top2 mutants , yet restricted to cXIIr . Notably , there are other situations where we can predict anaphase bridges of a different nature . For instance , anaphase bridges formed by partly replicated chromatids which nevertheless enter anaphase . For instance , sic1 and smc5/6 mutants behave this way [53] , [54] . As with the difference between top2 and cdc14-1 , sic1 and smc5/6 also differ in the actual number of chromosomes in the bridge , cXIIr being enriched in mutants for the Smc5/6 complex [44] , [53] , [54] . Despite the cytological similarities of the anaphase bridges between top2 and sic1 , and between cdc14-1 and smc5/6 , the bridge appeared broken in anaphase before completing cytokinesis in sic1 and smc5/6 mutants and a DDR can be also observed in that cell cycle stage [53] , [54] . These findings highlight a key difference between the anaphase bridges formed by tangled sister chromatids and those where replication is incomplete: breakage before cytokinesis occurs in the latter , perhaps due to more fragile DNA in the unreplicated material . Finally , it is interesting to point out that other cdc14 mutants might enter anaphase with unreplicated DNA as well [33] . However , the behaviour of our cdc14-1 bridge is much closer to top2 and condensin than to what is observed in sic1 and smc5/6 . This is , the chromosome can enter a PFGE in the cdc14-1 block ( Figure 2E ) and no DDR is observed at the block ( Figure 5A and Figure 6A ) . Another distinct anaphase bridge is that accomplished by the use of conditional dicentric chromosomes [55]–[57] . Like our cdc14-1 model , this approach has multiple technical advantages such as: ( i ) an anaphase bridge formed by a single chromosome; and ( ii ) cells with and without the bridge in the same population and experiment ( ∼50% chance of having two centromeres within a single sister chromatid attached to opposing SPBs ) . Nevertheless , the physical nature of the bridge is rather different . In the dicentric model , the bridge is formed by the sister chromatids being in an anti-parallel conformation . Moreover , sisters are supposed to be completely resolved from each other . In cdc14-1 , the bridge is often formed by just one sister , the other one being out of the cytokinetic plane ( Figure 1 and Figure 9 ) [23] . Thus , the expected DSBs and the broken genetic material in daughter cells are different when a dicentric chromosome is used . In relation to this system , it is interesting to note that the conditional dicentric chromosome triggers a Rad9-dependent mid-anaphase checkpoint ( characterized by short spindles ) that we did not see ( data not shown ) [24] , [56] . Regardless , this and other dicentric models , like top2/condensin/cdc14-1 mutants , do not seem to break the anaphase bridge until cytokinesis takes place [57] , [58] . A key conclusion of this work is that at least one DSB near or within the rDNA is produced after the release from the cdc14-1 block ( Figure 2 , Figure 3 , Figure 4 ) . Unlike DSBs generated by endonucleases , radiation or any other means within a single nucleus [49] , DSBs generated during anaphase bridge severing cause the ends of the broken DNA molecule ( s ) to migrate to opposing compartments which cannot be brought together anymore ( i . e . , nuclei of daughter cells ) . In the first scenario , the two ends of the DSB can be physically tied again and repaired by either non-homologous end joining ( NHEJ ) or HR . However , in the second case , the severing of the DNA molecule during nuclear division leads to a DSB where only one end can be found in each daughter nucleus ( i . e . , a “one-ended” DSB ) . Importantly , the state of each daughter cell is actually different with regard to chromosome XII dose ( see Figure 1 and Figure 9 for schemes ) . While one cell ( i . e . , “DC2” ) would have an entire chromosome XII plus the broken distal region of the right arm of the same chromosome ( from the DSB to the telomere ) , the other one ( i . e . , “DC1” ) would retain a fragment of a single chromosome XII ( from the left telomere to the DSB , including its centromere ) . It is difficult to envisage how each DSB end might be repaired . For instance , break-induced replication , de novo telomere addition , chromosome translocation and/or elimination of the broken sisters might well be possible . Confounding matters , if the DSB takes place within the rDNA , which may happen often according to our data , cells can find a template for HR in another copy of the array . This latter situation can lead to an uncertain outcome ( e . g . , extrachromosomal circles ? ) . Whichever way daughter cells face the problem , our results provide several interesting observations: i ) the DSB ( s ) does not trigger a strong DDR in the new G1 ( Figure 2 and Figure 6 ) ; ii ) the MRX complex ( i . e . , Mre11 ) has no role in DSB ( s ) processing ( Figure 7 and Figure S9 ) ; iii ) the Rad9 checkpoint protein , the RPA complex and Rad52 are part of the mechanism to deal with these DSBs as soon as both daughter cells reach S-phase ( Figure 4 , Figure 5 , Figure 6 , Figure S6 , and Figure S7 ) ; iv ) the activity of these key proteins is long lasting and cumulative , especially in the daughter cell that only carries a fragmented cXIIr copy ( i . e . , DC1 ) ( Figure 4 , Figure 5 , Figure 6 ) ; and v ) DC2 often survives and might get rid of the broken distal fragment of cXIIr in order to do so ( without using it as a template for HR , see below and Figure 8 ) . In relation to the absence of both a G1 arrest and Rfa1 foci in the new G1 , our results indicate that the DSB generated after cdc14-1 release is similar to that generated by endonucleases ( i . e . , a “clean” DSB ) as opposed to those generated by ionizing radiation ( i . e . , “ragged” DSBs ) [45] , [46] , [49] . Also , it indicates that the number of DSBs should be below four or five ( i . e . , few chromosome arms are part of the anaphase bridge ) [45] . Besides this , it is interesting that the processing of these DSBs is independent of Mre11 ( Figure 7 ) . Perhaps the MRX complex is not needed because the one-ended nature of each DSB means that there is no need to join both broken ends . Perhaps other molecular players are required in this context . In any case , others have previously reported that cells deficient in Mre11 and other MRX components can still generate a strong DDR and repair DSB through HR [59] . An alternative explanation for the Rfa1/Rad52 foci would be related to a sort of de novo damage generated as a consequence of DNA replication through an unrepaired or faultily-repaired chromosome XII . Future works are also needed in this direction . As for the long-term consequences of this type of one-ended DSB , it is interesting that we observed that the DC2 cell can eventually recover in many cases ( Figure 4 and Figure 8 ) . Most surprising was the fact that viability got better when RAD52 was deleted . We interpret this as indicating that checkpoint adaptation followed by loss of the acentric fragment might be the main pathway that allows DC2 to progress . Accordingly , surviving cdc14-1 rad52Δ foursomes grew ∼30% more slowly ( they actually took around one extra day to be visible on plates ) relative to the same cells plated before the block-and-release experiment ( Figure 8A ) . Indeed , the presence of Rad52 might compromise the chances of cdc14-1 DC2 cells surviving with a good fitness ( Figure 8A ) . Contrary to expectations , though , PFGE of survivors showed that chromosome XII was more unstable in cdc14-1 rad52Δ . Also , the slow-growing colonies of cdc14-1 RAD52 did not show visible abnormal chromosome patterns . Despite our having checked only a few surviving colonies , it is interesting that none show evidence of chromosome XII rearrangements that involve translocations , although one might have duplicated the chromosome . Thus , we concluded that the DC2 cell very often survives and that it might repair the broken cXIIr in two ways; one which is dependent on Rad52 ( e . g . , through break induced replication , a likely event at least in the survivor with two chromosome XIIs ) , and a second Rad52-independent manner that somehow makes more likely changes in chromosome XII size . As far as we know , this is the first time that an analysis of the DNA damage generated by cytokinetic severing of a single chromosome is conducted in yeast . A recent paper has just described the DDR after cytokinesis severs lagging chromosomes in human cells [60] . Many conclusions from that paper agree with those we observed in our work , although the system is clearly distinct ( i . e . , more than one chromosome is affected , both sister chromatids are severed , etc ) . In these human cells , DSBs arise after cytokinesis and are often repaired by NHEJ in G1 , leading to aberrant chromosomes . The difference in the mechanism of repair in our yeast system is nevertheless expected , since yeast basically rely on HR acting through S and G2 rather than NHEJ in G1 [42] . Another key difference between both systems is the ploidy of the dividing cells . Human cells are diploids and may repair broken sisters using homologous chromosomes as templates . Our yeast strains were all haploids . It would be interesting to study whether cdc14-1 diploids also missegregate cXIIr and whether the DDR is different from what we describe here for haploids . Future work will be carried out to this aim . In this study we have assessed the fate of cells that have an anaphase bridge formed by the right arm of chromosome XII ( Figure 9 for a model and summary ) . We show how cells can go through a new G1 , although they sever the bridge as they complete cytokinesis , and reach G2/M where they get arrested in a Rad9-dependent manner . We also show that the expected DNA damage response comprised RPA and Rad52 , but is independent of Mre11 . All these data shed light on how one-ended DSBs generated by a “cut” phenotype may be processed in eukaryotic cells . This work provides the first systematic study of the cell responses to a previous failure in sister chromatid resolution .
All yeast strains used in this work are listed in Table 2 . Strains with the tetOs along chromosome XII right arm and those with tags for chromosome XIV telomeres were S288C background . Those with Rad52-YFP , Rfa1-YFP , Mre11-YFP , GFP-Tub1 and Tub4-CFP tags , and those with tags for chromosome V telomeres were W303 . C-terminal tagging with GFP variants , gene deletions and allele replacements were performed using PCR methods [61] , [62] . All strains were grown overnight at 25°C in YPD media . For telophase block-and-release experiments , asynchronous cultures were first adjusted to OD660/ml = 0 . 2 , incubated at 37°C for 3 h in air orbital incubators and then shifted back to 25°C . To arrest cells in G1 in the cell cycle that follows the telophase release , cells were treated with alpha-factor ( 50 ng/ml ) for 2 hours after the 25°C shift ( all tested strains were bar1Δ ) . Flow cytometry analysis was carried out as described [54] in a BD FACScalibur machine , adjusting the peaks for 1 N and 2 N with an asynchronous culture at 25°C before reading the samples . PFGE to see all yeast chromosomes was performed using a CHEF DR-III system ( Bio-Rad ) in a 0 . 8% agarose gel in 0 . 5× TBE buffer and run at 12°C for 20 h at 6 V/cm with an initial switching time of 80 seconds , a final of 150 seconds , and an angle of 120° . PFGE to assess the size of chromosome XII was performed at 3 V/cm for 68 h with 300 and 900 seconds of initial and final switching time respectively . Ethidium bromide was used to visualize the chromosome bands in the gel . Band quantifications were performed with ImageJ software ( NIH ) . Chromosome XII band ( s ) was identified by Southern blot using a Digoxigenin-labelled probe ( Roche ) against the NTS2 region within the rDNA . Fluorescent proteins and chromosome tags were analysed by wide-field fluorescence microscopy . Series of z-focal plane images ( 10–20 planes , 0 . 15–0 . 3 µm depth ) were collected on a Leica DMI6000 , using a 63×/1 . 30 immersion objective and an ultrasensitive DFC 350 digital camera , and processed with the AF6000 software ( Leica ) . Scale bars in micrographs depict 5 µm . For nuclear morphology studies , DNA was stained using DAPI at 4 µg/ml final concentration after short cell treatment with 1% Triton X-100 . Time-lapse movies were filmed without Triton/DAPI treatment on minimal medium agarose patches . Imaging was done at room temperature . Nucleoplasm pictures using nuclear-tagged TetR-YFP was also done without Triton/DAPI treatment . Rad52 foci recognition was performed either manually or using the CellProfiler software [63] . For the latter , whole images were normalized following the procedure: most intense focus in the first photo taken was set to 1 , least intense pixel of the background was set to 0 . A lower threshold of 0 . 1 was set for foci recognition . Cytokinesis was monitored as previously described [39] with minor modifications . Briefly , aliquots of cells were fixed directly in the growth media by the addition of formaldehyde to 5% final concentration . After incubation at 25°C for 1 h with gentle rocking , fixed cells were washed twice with PBS and then once with 1 M sorbitol in 50 mM KPO4 , pH 7 . 5 . Cells were incubated with 0 . 2 mg/ml zymolyase 20T ( Zymo Research ) in the above sorbitol buffer containing 2 mM DTT for 20 minutes at 37°C . After zymolyase treatment , cell numbers were counted on a haemocytometer . Special chemical treatments in these assays ( i . e . , nocodazole , alpha-factor and latrunculin A ) were performed as follows: The arrest in G2/M was carried with 15 µg/ml of nocodazole at 25°C for 2 . 5 hours . Latrunculin A ( 100 µM ) was added right at the G2/M release and alpha-factor ( 50 ng/ml ) was added right at the telophase release . Release from the G2/M arrest was accomplished by washing away the nocodazole . Incubation for 1 . 5 hours at 37°C was used to block cells in telophase after a G2/M release . For the telophase release , cultures were shifted back to 25°C . Samples for this cytokinesis assay were taken at the telophase block and two hours after the telophase release . We used a cdc14-1 strain of the W303 background in these assays because it gave better synchrony , especially during the double block-and-release experiments ( first at G2/M and then at telophase ) . Error bars in graphs represent the standard error of the mean ( SEM ) unless stated otherwise . The number of experiments is indicated in the corresponding figure legend or table . Statistic inference for cross-comparison of categorical variables distributions were performed by the Fisher's exact test when a 2×2 contingency table could be built ( e . g . , segregation vs . missegregation ) . For other categorical variables with more than two possible outcomes ( e . g . , number of Rad52 foci ) , the Pearson's chi-square test was employed . Individual comparisons between means of independent experiments were performed by the Student's T test . All tests were two-tailed . | When cells divide they must segregate copies of their chromosomes to each of their daughters . A particular harmful situation arises when those copies are glued to each other ( i . e . , nondisjunction ) at the moment of division . Previously , it has been possible to genetically favour this scenario , yet it has been difficult to limit the extent of nondisjunction to a single chromosome . We have developed and studied a yeast model where we control nondisjunction of one of its sixteen chromosomes . We show that dividing cells manage to complete nuclear and cell fission and therefore break that chromosome . We further show that new daughter cells then trigger a DNA damage response , yet only after they initiate a new round of replication . Remarkably , an uncommon repair strategy seems to be used to deal with this damage , which involves part of the homologous recombination machinery ( i . e . , RPA complex and Rad52 ) but lacks its primary sensor Mre11 . Importantly though , both daughter cells arrest their cell cycle in G2 to prevent further damage from occurring . After a while , the cell that still carries an entire copy of the chromosome often survives , leading to aberrant forms of the chromosome in the progeny . | [
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] | 2012 | Nondisjunction of a Single Chromosome Leads to Breakage and Activation of DNA Damage Checkpoint in G2 |
Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology . Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data . This approach is called reverse engineering . It has been successfully applied to many gene networks in various biological systems . However , to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge . Here , we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster . A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data . We have developed a simplified data processing pipeline that considerably increases the throughput of the method , but results in data of reduced accuracy compared to those previously used for gap gene network inference . We demonstrate that we can infer the correct network structure using our reduced data set , and investigate minimal data requirements for successful reverse engineering . Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data , while it is less important to precisely measure expression levels . Based on this , we define minimal data requirements for gap gene network inference . Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort . This enables more widespread use of the method in different developmental contexts and organisms . Such systematic application of data-driven models to real-world networks has enormous potential . Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to discover whether there are rules or regularities governing development and evolution of complex multi-cellular organisms .
Elucidating the regulatory structure and dynamics of gene networks is a major objective in biology . The inference of regulatory networks from gene expression data is known as reverse engineering [1]–[7] . It is being widely and successfully applied , from microbes to animals ( see , for example , [8]–[16] ) . Many reverse engineering studies aim to determine regulatory structure from large-scale perturbation- or time-series data based on microarray or transcriptome-sequencing technology ( reviewed in [5] , [17] ) . This approach has two significant limitations: first , spatial information on gene expression is lost , since homogenised tissue samples or disaggregated cells are studied . And second , most resulting models are of a static and probabilistic nature , which cannot be used to investigate network dynamics ( for example [18]–[20] ) . If dynamical models are used , they are often linear ( for example [21]–[23] ) . Network inference using complex non-linear dynamical models is deemed a considerable technical challenge [6] , [17] , [24] . However , there are many important biological questions that absolutely require consideration of non-linear and spatial aspects of a system . Here we discuss such a case , and show that reverse engineering can be used for its study with a reasonable amount of experimental and computational effort . Our research focuses on how developmental gene regulatory networks produce spatial patterns in multi-cellular organisms , and how these patterns evolve through changes in the underlying structure of the network [25] , [26] . In this context , reverse engineering is implemented by fitting non-linear systems of differential equations to quantitative , spatial gene expression data ( reviewed in [7] ) . There are many network modelling formalisms [27]–[29] , and a number of powerful global non-linear optimisation methods [7] , [30] , [31] , which are suitable for this task . So far , only a small number of developmental systems have been reverse-engineered using dynamical models ( see , for example , [32]–[34] ) . One of those is the ( trunk ) gap gene network of the vinegar fly Drosophila melanogaster ( reviewed in [35] ) . This regulatory network consists of four genes—hunchback ( hb ) , Krüppel ( Kr ) , giant ( gt ) and knirps ( kni ) —which all encode transcription factors . Gap genes are involved in establishing the segmented body plan of the animal . They are active during a very early period of Drosophila development , called the blastoderm stage , which occurs before the onset of gastrulation . At this stage , the embryo consists of a multi-nucleate syncytium allowing transcription factors to diffuse through the tissue . Gap genes are expressed in broad , overlapping domains along the embryo's major , or antero-posterior ( A–P ) axis . They are regulated by long-range gradients of transcription factors encoded by maternal co-ordinate genes bicoid ( bcd ) , hunchback ( hb ) , and caudal ( cad ) , and are repressed by the terminal gap genes tailless ( tll ) and huckebein ( hkb ) in the pole regions of the embryo . Maternal co-ordinate and gap genes form the first two tiers of the segmentation gene hierarchy in Drosophila . Together they regulate pair-rule and segment-polarity genes , the latter forming a molecular pre-pattern that leads to morphological segmentation at later stages of development ( see , [36] , [37] , for review ) . The particular reverse-engineering approach we use to investigate the gap gene network is called the gene circuit method [1] , [38] , [39] ( Figure 1 ) . It uses mathematical models called gene circuits that represent the basic properties of the embryo and the transcriptional regulatory interactions underlying the network . Gene circuits are described in detail in Materials and Methods . Here we provide a brief overview of the model , which consists of a row of dividing nuclei ( Figure 1 , top left ) each harbouring an identical version of the regulatory network . There are three processes that occur within and between nuclei: ( 1 ) regulated gene product synthesis , ( 2 ) Fickian gene product diffusion , and ( 3 ) linear gene product decay ( Figure 1 , top middle ) . Regulatory interactions that direct synthesis are represented by a genetic interconnectivity matrix: each regulatory weight in this matrix can represent activation , repression , or no interaction depending on whether it is positive , negative or ( close to ) zero ( Figure 1 , bottom panel , right ) . The interconnectivity matrix can also be displayed as a network diagram ( Figure 1 , bottom panel , left ) . Note that the values of regulatory weights are not set a priori . Instead , they are estimated by fitting the model to quantitative gene expression data . Those model solutions that fit the data well are analysed to characterize the regulatory structure and dynamics of the network ( Figure 1 , bottom ) . In this way , gene circuits act as tools to extract regulatory information from quantitative data . Previous reverse-engineering studies of the gap gene network were based on quantitative expression data obtained by visualising the distribution of gap gene mRNA [40] , or protein products [41]–[43] using fluorescent whole-mount in situ hybridisation , or antibody staining ( immunofluorescence ) respectively . Stained embryos were imaged using confocal laser-scanning microscopy , and the resulting expression profiles were quantified using a processing pipeline that includes image segmentation to identify nuclei , time classification , removal of non-specific background staining , data registration to remove embryo-to-embryo variability , and data integration ( reviewed in [44] ) . It took years of effort by several researchers to establish the protein data set [41] , [43] . mRNA data , on the other hand , were acquired by one of the authors of this paper in considerably less time [40] . However , these mRNA data remain incomplete in that they only cover a subset of gap genes ( Kr , kni , and gt ) during the earliest stages of expression . These previous reverse-engineering studies have yielded many new insights into gap gene regulation , which would have been difficult to obtain by experimental approaches alone . An early pioneering study predicted a co-operative effect between maternal factors Bcd and Hb on the regulation of gap gene expression [45] . Later efforts uncovered a mechanism for the dynamic anterior shift of gap domains over time [46] , [47] , removed ambiguities in the published experimental evidence [48] , [49] , identified core mechanisms for gap gene regulation [48] , [50] , and explained the robustness of the system against variable levels of maternal inputs [47] , [51] . Taken together , these studies clearly demonstrate the utility and feasibility of the approach: over the past two decades , reverse engineering has contributed significantly to our understanding of gap gene regulation . It would be extremely interesting to apply the gene circuit method to other developmental systems . In our view , reverse engineering has tremendous potential for the study of gene regulatory networks in development and evolution . For instance , gene circuits could be used to reconstruct homologous developmental regulatory networks across a range of species , to compare their regulatory structure and dynamical behaviour [26] . This could be used to map which regulatory changes in a network correspond to which changes in gene expression during evolution . Alternatively , it would also be highly interesting to compare network structures and dynamics between different developmental processes . Despite this potential , the application of dynamic , non-linear reverse-engineering approaches beyond gap genes in Drosophila has been very limited . The main reason for this , we suspect , is the following: collection of high-quality data sets—such as the spatio-temporal profiling of gap genes described above—is costly both in terms of time and resources . It is clearly the bottleneck of the approach . Protocols based on immunofluorescence require antibodies , which are difficult and expensive to obtain . Confocal microscopy is time-consuming and laborious , since a large number of embryo images need to be scanned . Moreover , while protocols for data acquisition and quantification work efficiently in Drosophila , their application to less well-established experimental models is not trivial . In particular , it is often difficult to adapt fluorescent staining protocols to non-model species . Thus , in order to make the gene circuit method more widely applicable—and hence useful for the study of developmental gene regulatory networks—it is imperative that we simplify the method . We address an important question which applies to reverse-engineering approaches in general: how much , and what kind of data are required to successfully infer a gene regulatory network ? Answering this question in the context of the gap genes will allow us to minimise the cost of data acquisition and processing . This , in turn , will decrease the barrier for applying reverse-engineering methodology to other developmental systems , many of which are similar in kind and complexity to the gap gene network . The quality of a gene circuit model depends directly on the quality of the data it was fit to . What matters most in this regard is the timing and position of expression domain boundaries with respect to each other . The relative level of expression in each domain is less crucial . For instance , early gap gene circuit models did not capture the formation of the abdominal kni domain correctly ( see Figure 2 in [45] ) . This was due to the incorrect relative position of this domain in the data resulting in a large gap between it and the posterior hb domain ( see Figure 1 , ibid . ) . This defect is no longer present in more recent models based on data with the abdominal kni domain positioned accurately while still only measuring relative levels of protein concentration [46] , [48]–[50] . In this study , we present a simplified reverse-engineering protocol and apply it to a new , quantitative data set of gap gene mRNA expression in Drosophila . We demonstrate how mRNA expression data derived from a colorimetric ( enzymatic ) protocol for in situ hybridisation can be used to infer the regulatory structure and dynamics of the gap gene network . We compare our results with those obtained in previous studies based on protein expression data , and show that they predict equivalent regulatory mechanisms that are consistent with experimental evidence . In addition , we show that our simplified data set can be reduced even further while still yielding correct predictions . In this way , we define a set of minimal requirements for the successful inference of gap gene regulatory network structure and dynamics . These minimal requirements suggest that the adapted gene circuit method can be applied to a variety of developmental systems with a reasonable amount of effort . Such wider application of reverse-engineering methods will enable us to carry out systematic and comparative analyses of developmental gene regulatory networks .
cDNA clones were ordered from the Drosophila Genomics Resource Center ( DGRC; dgrc . cgb . indiana . edu ) and used to make riboprobes labelled with DIG and/or FITC . Wild-type blastoderm-stage Drosophila embryos were collected after 4 hrs of egg laying and stained with a colorimetric in situ hybridisation protocol adapted from [52] and [53] . In brief , fixed and dehydrated embryos were re-hydrated by washing 1x in PBT/methanol ( embryos are allowed to sink before the solution is removed ) , 2x in PBT , and 1×5 min in PBT . Embryos were incubated in PBT containing 0 . 179U proteinase K for 1 hr on ice , then washed 2x in ice-cold PBT . Embryos were post-fixed for 25 min with 5% formaldehyde in PBT with mild shaking , then washed 1x followed by 2×5 min in PBT . Embryos were pre-hybridised by washing 1×10 min in equal volumes PBT and hybridisation buffer ( 50% formamide , 5xSSC , 5 µg/ml yeast tRNA , 100 mg/ml salmon-sperm DNA , 50 µg/ml heparin , 0 . 1% tween-20 in DEPC-treated water ) , 1×2 min in hybridisation buffer , and 1×1 hr in hybridisation buffer at 56°C . Hybridisation was carried out overnight: 0 . 5–1 ng/µl of probe ( s ) were added after heating at 80°C in a small amount of hybridisation buffer for 3 minutes . Post-hybridisation , the embryos were washed 1×15 min and 2×30 min in hybridisation buffer , 1×15 min in equal volumes of PBT and hybridisation buffer , and 4×15 min in PBT . Blocking steps were carried out using 5% heat-treated goat serum in PBT for 30 min , followed by incubation with anti-DIG or anti-FITC antibodies conjugated with alkaline phosphatase ( Roche ) at 1∶2000 in 5% heat-treated goat serum in PBT for 1 hr . Unbound antibody was removed with washes of 3x followed by washes of 4×15 min in PBT . To prepare for staining , embryos were washed 2×5 min in AP buffer ( 100 mM NaCl , 50 mM MgCl , 100 mM Tris pH 9 . 5 , 0 . 1% tween-20 ) . Staining was carried out in the dark by the addition of AP buffer containing 0 . 1 mg/ml NBT and 0 . 05 mg/ml BCIP . Staining was stopped with 3x washes in PBT . For single staining ( one probe ) , embryos were washed a further 3×10 min then counter-stained ( see below ) . For double staining ( two probes ) , alkaline phosphatase was inactivated by washing 1×1 min , then 1×10 min in glycine buffer ( 0 . 1 M glycine 0 . 1% tween pH 2 ) followed by 3×10 min in PBT . Blocking , antibody incubation and washing steps were carried out as described above . To prepare for staining , embryos were washed 2×5 min in Fast Red buffer ( 100 mM Tris pH 8 . 2 , 0 . 1% tween-20 ) . Staining was carried out in the dark by the addition of Fast Red solution ( 1 Fast Red tablet ( Roche ) dissolved in 2 ml Fast Red buffer ) . Staining was stopped with 3×1 min followed by 3×10 min washes in PBT . Nuclei were counter-stained by a 10-min incubation in PBT containing 0 . 3 µM DAPI , followed by washes of 3x followed by 3×10 min in PBT . Embryos were cleared through a series into 70% glycerol:PBT , of which 30 µl were mounted per slide . All washes were done on a nutator , except for those in proteinase K . An overview of image acquisition and processing steps is shown in Figure 2 . For each embryo , four images were acquired using a compound , wide-field , fluorescence microscope: ( A ) a bright-field image ( Figure 2A ) , ( B ) a fluorescent image of the DAPI nuclear counter-stain ( Figure 2B ) , ( C ) a differential interference contrast ( DIC ) image ( Figure 2C ) , and ( D ) a DIC image of membrane morphology on the dorsal side of the embryo ( Figure 2D ) . Images A–C were acquired using a 10x objective , image D using a 40x objective . Images A and B are focused on the surface , images C and D on the sagittal plane of the embryo . All images were taken at 8-bit accuracy , thus setting the range per RGB channel to [0 , 255] . Only laterally oriented embryos were selected for processing . Gene expression patterns were extracted from embryo images as follows . Binary masks covering the whole embryo are calculated using a sequence of image segmentation steps on the DIC image ( Figure 2C ) . Intermediate steps are shown in Figure 2E/F: 1 . the RGB image is converted to gray-scale , 2 . gamma correction is applied to increase contrast , 3 . the image is inverted , 4 . Sobel edge detection is carried out , 5 . dilation operations are applied to the resulting binary image , and 6 . holes are filled [54] , [55] . This results in a number of contiguous binary blobs in the mask image . All blobs touching the image border are removed , and only the largest blob is retained . Finally , a smooth whole-embryo mask is created by applying a Gaussian filter to the remaining blob . This mask and all raw images ( A–C ) were rotated and cropped as described in [56] such that the embryo's major , or antero-posterior ( A–P ) axis is horizontal . If necessary , embryo images were flipped manually to a canonical orientation such that anterior is to the left , and dorsal is up ( Figure 2G–J ) . To extract gene expression profiles from an embryo , a smooth cubic spline was generated with five equidistant knots through the main branch of the skeleton of the embryo mask ( Figure 2K; [55] , [57] ) . Along the spline , we extract average RGB-values over a band of 10% mask height ( 5% above and below the spline curve ) , resulting in raw profiles for each RGB channel of the bright-field image ( Figure 2H ) along the A–P axis ( Figure 2K , L ) . Values for FastRed or NBT/BCIP staining were then calculated as follows: FastRed = green − red , and NBT/BCIP = red , where ‘red’ and ‘green’ refer to inverted RGB colour channels extracted from the bright-field image . Inspecting 1D-graphs of the resulting profiles ( Figure 2M ) , boundaries for gene expression domains were extracted manually for each embryo: each boundary was labelled with a unique identification number ( see Supplementary Material ) , and two points ( x0 , y0 ) and ( x2 , y2 ) were determined that indicate the beginning and end of the boundary , where staining levels approach background and maximum levels respectively . A middle , third control point ( x1 , y1 ) was automatically calculated from the other two points by taking the average for x , and locating the corresponding expression level y . Hence represent relative position along the A–P axis ( in percent , where 0% is the anterior pole ) , and represent the relative intensity level of the staining . Points x0 , 2 and y0 , 2 were used as anchor points for cubic splines with fixed zero-derivatives at their end knots . Finally , splines were normalised such that the expression level at the starting point was 0 , and the expression level at the end point was 1 ( Figure 2N ) . Integrated time-series of gene expression were prepared as follows . Embryos were staged into separate cleavage cycles ( defined as the period between mitotic divisions n-1 and n; e . g . cycle 13 spans the time between mitoses 12 and 13 ) based on nuclear density and number of nuclei in images showing DAPI nuclear counter-stains ( Figure 2B; time classification for early embryos was described previously in [40] ) . C14A was further subdivided into eight equally spaced time classes ( T1–8 ) based on membrane morphology from high-resolution DIC images ( Figure 2D; time classification for late embryos as described in [44] ) . Expression domain boundaries were grouped by gene , stage and boundary identification number ( see above ) . Average boundary positions were determined by calculating separately the median start and end points for each group , which were then used for fitting a median-boundary spline as described for individual boundaries above . Finally , we combined different domain boundaries for each gene at each time class into an integrated , normalised expression profile along the A–P axis ( Figure 2O ) . Spatial registration of domains was performed by checking the integrated expression profiles against double stained embryos ( i . e . embryos stained for two gap genes , for instance hb and Kr ) to verify the relative spatial order of gap gene expression domains ( data not shown ) . The following post-processing steps had to be applied to our data to make them suitable for model fitting and comparison ( see Results , and Figure S1 of the online Supporting Information ) : ( 1 ) We collected our normalised , integrated mRNA expression data into 50 ( C13 ) or 100 ( C14A ) bins to reflect the approximate number of nuclei along the A–P axis [46] , [48] , [49] . ( 2 ) We scaled the intensity of our expression data linearly along the A–P axis from 50% A–P position ( mid-embryo; ×1 . 0 ) to both termini ( poles of the embryo; ×0 . 5 ) to reflect the higher intensity of central versus more terminal gap domains [43] . For Drosophila expression data , this is a reasonable assumption , but not an essential requirement . Omitting this post-processing step resulted in qualitatively equivalent results ( data not shown ) . ( 3 ) We also scaled our expression data along the time axis by a second-degree spline with a peak of expression during early C14A , to capture the gradual accumulation ( during C12 , C13 and early C14A ) and degradation ( during late C14A ) of gap gene mRNA ( [40] , [43] , and our unpublished data ) . Normalised boundaries were scaled to 0 . 1 at the onset of expression ( early C13 , t = 0 . 0 min ) , 1 . 0 at around T5 ( t = 48 . 0 min ) , and 0 . 7 at gastrulation time ( t = 71 . 1 min ) , which is the final time point . ( 4 ) We multiplied our mRNA expression data by a constant factor of 200 . This makes the scale of both mRNA and protein data match as closely as possible , and therefore facilitates comparison to models obtained with Drosophila protein data [46]–[51] . The posterior Kr domain , which arises in late C14A , was removed from the data used for model fitting to avoid modelling artefacts . This domain is known to be under regulatory control of the terminal gap genes with additional inputs from the Forkhead ( Fkh ) transcription factor ( not included in this study ) , and it does not participate in segment determination [58] . Fitting models using a weighted least squares ( WLS ) protocol ( see below ) requires a weight for each data point indicating its associated variation . As our mRNA expression data do not provide such information , weights were created from normalised , integrated mRNA expression data according to the formula: with being the normalised staining intensity and v the corresponding weight . This proportionality of variation with expression level reflects the fact that gap domains ( showing high levels of expression ) show more variation than those regions of the embryo in which a gene is not expressed [40] , [43] . Gene circuits used for model fitting require external inputs based on data for maternal gradients ( Bcd and Cad ) as well as terminal gap genes ( Tll and Hkb ) . Depending on the scenario we wanted to test ( see Results ) , expression profiles for these inputs were taken from previously published quantitative protein data [43] , [49] , or were approximated as follows . Bcd: a time-independent ( i . e . constant ) anterior gradient was created by fitting an exponential curve to all available Bcd data across time and space . Cad: an artificial posterior morphogen gradient was created using 2D thin-plate splines [59] based on the following minimal set of features of the Cad protein gradient: ( 1 ) the gradient should be complementary to the anterior ( Bcd ) gradient; ( 2 ) as development progresses , there should be increasing repression in the abdominal region ( ∼50–80% A–P position ) ; and ( 3 ) a posterior stripe should develop from time class 6 in C14A onwards at around 80% A–P position . Tll/Hkb: we replaced protein expression data with mRNA expression data for some of our reverse-engineering runs ( see Results ) . Due to the relative constancy of tll and hkb expression patterns over time [43] , [49] , we created time-invariant expression profiles for these genes by averaging boundary positions across all cleavage cycles and time classes . We use gene circuits for model fitting ( reverse engineering ) as described in [1] , [38] , [39] , [46]–[51] . In brief , a gene circuit is a hybrid dynamical model incorporating discrete mitotic divisions of nuclei , as well as continuous gene regulatory dynamics within each nucleus . Each cleavage cycle consists of interphase , mitosis and division . During interphase , the change in gene product concentration ( representing mRNA or protein , depending on the simulation ) for gene a in nucleus i over time t is governed by the following of ordinary differential equation ( ODE ) ( 1 ) The three terms on the right-hand side of the equation define regulated gene product synthesis , diffusion and decay respectively . is the maximum synthesis rate; the diffusion rate ( dependent on the distance between nuclei , which halves at every cleavage division: n defines the number of previous divisions ) ; is the decay rate for the product of gene a . The sigmoid regulation-expression function captures the basic regulatory dynamics , and is defined as follows: ( 2 ) where ( 3 ) with the set of trunk gap genes defined as , and the set of external inputs as . Matrices W and E define the interactions between , respectively , the trunk gap genes themselves , and between the external inputs and trunk gap genes . The elements of these matrices , wba and ema , are called regulatory weights . These weights define the effect of b on a ( or m to a ) which can be ( 1 ) positive ( activating gene product synthesis ) , ( 2 ) negative ( inhibiting synthesis ) , or ( 3 ) ( close to ) zero ( no regulatory interaction ) . ha is a threshold parameter representing uniformly distributed maternal factors . During mitosis , gene product synthesis is set to zero . After mitosis follows division , which is instantaneous . At division , gene product concentrations are copied equally to both daughter nuclei . Finally , diffusion is implemented with no-flux boundary conditions . Our models cover the trunk region of the embryo , from 35 to 87% A–P position . This region is somewhat reduced compared to 35 to 92% for earlier protein models [46]–[51] , but covers the same set of gap domains , due to the slightly more anterior position of mRNA vs . protein domains . This results in gene circuits consisting of systems of 108 ODEs at C13 , and 212 ODEs at C14A . Gap gene circuits were solved numerically from the beginning of C13 ( t = 0 min ) when gap proteins reach detectable levels , to the onset of gastrulation and the end of C14A ( t = 71 . 100 min ) . We use the same division schedule as in [46] , [48] , [49]: mitosis occurs from 16 . 0 min to 21 . 0 min . At the end of mitosis , nuclear division takes place . Initial conditions for gap genes were calculated by interpolation between data points at C12 ( t = −6 . 200 min ) and C13 ( t = 10 . 550 min ) using the same temporal scaling scheme as described in the previous section . Initial conditions of the external inputs were taken from [49] . Time classes in C14A correspond to the following time points ( in minutes ) : T1 , t = 24 . 225; T2 , t = 30 . 475; T3 , t = 36 . 725; T4 , t = 42 . 975; T5 , t = 49 . 225; T6 , t = 55 . 475; T7 , t = 61 . 725; T8 , t = 67 . 975 ( see Figure 2 in [48] ) . We follow a reverse engineering protocol as described in [46] , [48] , [49] . To estimate the values for parameters W , E , R , h , D and λ of the gene circuit model we performed global optimisation by means of parallel Lam Simulated Annealing ( pLSA ) [60]–[62] on the Mare Nostrum supercomputer at the Barcelona Computing Center ( BSC; http://www . bsc . es ) . Per optimisation run we used 50 processor cores for an average duration of about 7 hours . Simulated Annealing requires that candidate solutions have an associated cost ( or energy ) function that is minimised during the optimisation . We adapt the cost function from [49] as follows: ( 4 ) with T the set of time points ( C13 , C14A: T1–8 ) at which expression data is available , Nc ( n ) the number of nuclei after n divisions ( 50 at C13 , 100 at C14A ) , representing positive weights associated with each data point , and referring to the expression level of gene product a at nucleus I and time t as derived from the experiments . If weights are all set to 1 . 0 , the ‘cost’ equation represents a fit by ordinary least squares ( OLS ) , which is the cost function previously used with gene circuit models [46]–[51] , [63] . For fitting by weighted least squares ( WLS ) , we use variable weights , which are inversely related to the level of expression ( calculated as described in ‘Additional Data Processing for Model Fitting and Comparison’ ) . This penalises ectopic gap gene expression , and improves frequency and quality of good fitting solutions as reported in [49] . Based on previous studies using gap gene circuit models , we fix certain parameters without negatively affecting the quality of the fits [46] , [48] , [49] . In the gene interaction matrix E we fix interactions of Hkb to zero , with the exception of Hkb→hb . Furthermore , we take ha = −2 . 5 , for all gap genes , and for respectively [49] . To report the goodness of a fit , we use the root mean square ( RMS ) , defined by ( 5 ) with the total number of data points in our data set . Since the RMS is independent of weights and the number of nuclei ( data points ) in the model , it allows us to compare WLS and OLS , as well as mRNA- and protein-based solutions . Solutions were selected for further analysis by several tests . Firstly , gene circuits were tested for numerical stability with respect to the solver ( also known as solver sensitivity ) and with respect to minute changes in parameter values ( or the ‘brittleness’ of a solution ) . Subsequently , gene circuits were checked for visible gene expression patterning defects by means of visual inspection ( see Text S1 for a categorisation of commonly encountered defects ) . Statistical analysis of parameter estimates was performed as described in [49] , [64] . Here we only provide a short description of the calculation of parameter confidence intervals . Reverse engineering results in a vector of estimates . Once the parameter vector is found , a posteriori identifiability analysis [65]–[67] reveals how reliable the obtained estimate is . The ellipsoidal region around in which the true parameter vector lies with a certain probability ( we set ) is defined by ( 6 ) where ( 7 ) and and the number of parameters and data points , respectively . is the upper part of Fisher's distribution with and degrees of freedom . J is the Jacobian matrix of size defined as , where is the vector of weighted discrepancies between model output and data . From equation 6 one can derive dependent and independent confidence intervals for each parameter estimate . These are , respectively , ( 8 ) and ( 9 ) Here and are obtained from the singular value decomposition of . It is well known that in the presence of strong correlations between parameters , the dependent confidence intervals underestimate the confidence region while the independent confidence intervals overestimate it . For detailed explanations of these statistical quantities and their derivations we refer the reader to [49] , [64] , and references therein . Image processing and extraction of expression domain boundaries were performed using a custom-made processing pipeline with a graphical user interface developed in Java ( using the ImageJ API; http://rsbweb . nih . gov/ij ) . Intermediate processing steps and domain boundary positions were stored in a MySQL database , with a web interface ( SuperFly ) developed by the CRG Bioinformatics Core Facility . SuperFly is available online at: http://superfly . crg . es . We used scripts written in Python , Perl and R for the preparation of integrated data sets , for the generation of artificial external inputs , and for analysis of gene circuit models . Code for numerical solution and optimisation of gene circuits by pLSA [39] , [45]–[49] , [51] , [62] is implemented in C , using the GNU Scientific Library ( GSL , www . gnu . org/software/gsl ) , the Sundials ODE Solver Library [68] , and the Open MPI message-passing interface ( www . open-mpi . org ) . For numerical integration of ODEs , we use an implicit variable-order , adaptive-stepsize , multi-step method; a band-direct solver calculates the set of equations that is generated at each integration step [68] .
Over the last two decades , the potential of reverse engineering has been demonstrated by a pioneering case study—led by John Reinitz and colleagues—where gene circuits have been used to characterise and analyse the gap gene network in Drosophila melanogaster [40] , [45]–[51] . Despite this , the gene circuit method has not yet been applied more widely . One reason for this is that it took many years to establish the required quantitative data set of spatial gap protein expression patterns [41]–[43] . We have developed a simplified protocol for data acquisition and processing , which allows us to create a quantitative data set of spatial gene expression patterns in a time span of months rather than years . Instead of using protein expression data , we have quantified mRNA expression patterns by colorimetric ( enzymatic ) in situ hybridisation , imaged using a wide-field , compound fluorescence microscope . The resulting data set is of reduced quality compared to the original protein data . Here , we address the question whether it can still be used to reconstruct the regulatory structure and dynamics of the gap gene system in a manner which is consistent with previous efforts based on modelling , as well as genetic and molecular approaches to study gap gene regulation . Before we present our modelling results , we provide a quantitative characterisation of our mRNA data , and compare them to the gap gene protein expression data described in [43] . Table 1 shows the number of embryos on which our mRNA data are based . Figure 3 illustrates the quality and resolution of embryo images underlying the two data sets . It shows time series of mRNA expression patterns for the trunk gap genes hb , gt , Kr , and kni produced with a colorimetric ( enzymatic ) in situ hybridisation protocol ( columns 1–4 on the left ) , in comparison to protein expression data for Gt , Kni and the pair-rule protein Even-skipped ( Eve ) from the FlyEx database ( http://urchin . spbcas . ru/flyex; [69] , [70]; column 5 to the right ) . We used images such as the ones shown in columns 1–4 of Figure 3 to quantify the position of gap gene domain boundaries across space and time . This was done by applying the image-processing pipeline as described in Materials and Methods . In brief , we used texture-based image segmentation to create whole-embryo masks , which were used to rotate and crop embryo images . The developmental stage of each embryo was determined based on numbers of nuclei and membrane morphology as previously described [44] ( see also Materials and Methods ) . We then extracted raw profiles of gene expression within a 10% strip along the embryo's lateral midline , and we determined the position of expression domain boundaries . The results of this analysis are shown in Table 2 and Figure 4 . A detailed description of gap gene mRNA patterns can be found in Text S2 . As mentioned in the Introduction , we are mainly interested in the dynamics of gap domain boundary positions across space and time . Figure 4 compares those dynamics between mRNA and protein data . It is evident that mRNA expression patterns resemble those of proteins closely . The relative position of all domains with respect to each other is preserved at all time points . Anterior shifts in domain positions over time also mirror each other between mRNA and protein data: while mRNA domains are always more anterior than protein domains at equivalent stages ( as reported in [46] , Figure 3 ) , the extent of the domain shifts is similar between the two data sets ( see Table S1 ) . Finally , domain sizes are similar as well ( see Table S2 ) , although protein domains of the gap genes are slightly larger than those of their transcripts . However , there are also notable differences between mRNA and protein data . First , all mRNA patterns arise earlier than those of their corresponding proteins: mRNA expression of all gap genes is initiated before C13 ( [40] , and references therein ) , and gap mRNA domains are well established at early cycle 13 . In contrast , protein levels have only just begun to be detectable , and increase rapidly , during that stage [43] . This is due to the delay caused by mRNA processing , nuclear export , and translation . Similarly , there is an evident lag between shifting positions of gap domains ( see also Figure 3 in [46] ) , again indicating a significant delay between the dynamics of mRNA and protein patterns . In summary , while overall expression dynamics are similar between mRNA and protein , both the timing of expression and the absolute positions of gap domain boundaries differ between the two data sets . The main aim of this study is to show that the gene circuit method—originally developed for protein expression data—can be adapted to work successfully with expression profiles derived from mRNA . To achieve this , gene circuit models were fit to mRNA expression patterns derived from our data set of boundary positions for hb , gt , Kr , and kni . External inputs to the model—regulatory contributions from maternal gradients Bcd and Cad , as well as terminal gap genes tll and hkb , which are not themselves regulated by trunk gap genes—were calculated based on protein data as described in Materials and Methods . Our models run from early cleavage cycle 13 ( C13 ) , when gap proteins start to accumulate , to the end of C14A , when gastrulation starts . They span the trunk region of the embryo from 35 to 87% A–P position . This region is located slightly more anteriorly and is somewhat smaller than that used previously for protein models ( 35–92% A–P position [46] , [48]–[50] ) . However , it covers the equivalent set of gap gene expression patterns , since mRNA domains are located more anteriorly and are slightly less wide than their corresponding protein domains ( see Figure 4 , and Tables 2 and S2 ) . Fitting gap genes to the original range of 35–92% A–P position yielded equivalent results to those reported below ( data not shown ) . The quality of a gene circuit depends crucially on the relative position and dynamics of gap domain boundaries , while levels of expression are of secondary importance ( see Introduction ) . However , there are two problems with the loss of information on gap gene product concentrations due to the way our data were processed . The first is that , after data normalisation , boundaries in our data set appear suddenly , without gradual build-up of gene product levels ( see , for example , Figure 2O ) . This is clearly unrealistic , and leads to problems with the numerical stability of our gene circuit models , since very short time scales ( and hence , very high production rates ) will be favoured by the model-fitting procedure ( data not shown ) . We have addressed this problem by re-scaling the data over time using a second-degree spline with a peak of expression at early C14A , capturing the gradual accumulation of mRNA during C13 and early C14A , as well as its degradation during late C14A ( [40] , [43] , and our unpublished data ) . The second problem concerns the relative timing and intensity of expression in different gap gene domains . While central gap domains arise early , and show intense staining levels by C14A , more terminal ones arise later , and show less intense staining [43] . For instance , the anterior mRNA domain of hb arises at C9 or C10 from the maternal Hb gradient , while the posterior hb domain only appears at C13 , with much lower intensity of expression [71]–[74] . We have addressed this problem by re-scaling the data along the A–P axis , using a scaling factor of 1 . 0 in the middle of the embryo ( at 50% A–P position ) that linearly decreases to 0 . 5 at the poles ( 0 and 100% A–P position ) . Note that this scaling step is justified by our prior knowledge of the gap gene system , but is not strictly required to generate the results we present below ( see Discussion ) . Finally , data were multiplied by a factor of 200 , and collected into 50 bins ( at C13 ) and 100 bins ( at C14A ) along the A–P axis to facilitate comparison with models based on protein data ( see Materials and Methods and Figure S1 for details ) . We used both ordinary ( OLS ) and weighted least-squares ( WLS ) fits to our full mRNA data set ( see Materials and Methods ) . On one hand , the WLS method has the advantage of penalising ectopic expression outside the observed gap domains , and has been shown to be more effective than OLS for protein-based circuits [49] . On the other , it requires us to calculate the variance of each data point in our data set , which is not an obvious task given our data-processing methods . For this reason , we had to estimate weight values for the WLS cost function based on the observation that variance is higher in regions with high levels of gene expression than in those that show low , or now expression at all ( see Materials and Methods for details ) . We performed 150 fitting runs each with OLS and WLS cost functions respectively . Because of potential artefacts caused by overfitting , one cannot simply select those circuits with the lowest residual scores for further analysis . Instead , we inspected all solutions visually to detect obvious patterning defects ( observed defects are described in Text S1 ) . Only solutions without defects were selected . Using the WLS cost function resulted in a much higher fraction of runs with good fits to the mRNA expression data compared to OLS . For OLS solutions , only 10/150 ( 6 . 7% ) were suitable for further analysis ( Figure S2 ) . Their residual errors—as measured by the RMS score ( defined in Materials and Methods ) —range from 16 . 6 to 20 . 9 , with a median value of 17 . 453 . For WLS solutions , 52/150 ( 34 . 7% ) could be retained ( Figure 5A ) . As is usual for WLS fitting [49] , they show slightly higher RMS scores—between 20 . 0 and 21 . 7 , with a median value of 21 . 052 . Overall , the selected gene circuits are extremely similar for both OLS and WLS ( compare Figure 5A to Figure S3 ) . Both show correct relative timing and positioning of gap domains , and reproduce the positional shifts of posterior domains towards the anterior . However , there are slight inaccuracies concerning the appearance and placement of domain boundaries . The formation of all gap domains , but particularly the posterior hb domain , is slightly delayed ( Figure 5A , T1 ) , and several boundaries are offset by 1–3 nuclei at specific points in time compared to the data ( see , for example , the anterior boundary of kni at T1 , and its posterior boundary at T3 in Figure 5A ) . These slight defects result in patterns that reproduce the data less faithfully than those obtained by WLS fits to protein data ( Figure 5B; models from [49] ) . This is reflected in the corresponding RMS scores: 10 . 5 for protein data versus 16 . 6 , the best score from our mRNA fits . In addition , our mRNA-based circuits show increased variability in model output between solutions compared to protein-based models ( Figure 5 , Figure S3 ) . Apart from these minor differences , however , there is significant agreement between all three sets of gene circuits . This similarity in expression patterns is reflected in the parameter values of our models . Distributions of estimated parameter values for regulatory weights of WLS-mRNA and WLS-protein solutions are shown as scatter plots in Figure 6A . Corresponding genetic interconnectivity matrices are compared in Figure 6B . It is clear from inspecting these matrices that a large majority of interactions are qualitatively the same in mRNA- and protein-based circuits ( although increased variability in model output is reflected in increased variability of estimated parameter values for mRNA circuits ) . Repression corresponds to repression , and activation to activation , for a majority of circuits in both cases . Only six gap-gap cross-interactions are predicted to be different ( emphasised by black frames in Figure 6B , and discussed in detail in Box 1 ) , while all external inputs ( bcd , cad , tll and hkb ) interact with gap genes in the same manner for both data sets . In summary , none of the differences between mRNA- and protein-based circuits shown in Figure 6B are inconsistent with regulatory mechanisms for gap gene regulation postulated previously [46] , [48]–[50] ( see Box 1 ) . The network recovered from both data sets is essentially equivalent . Our models—just as those obtained with protein data—predict that gap genes are regulated by ( 1 ) broad activation by maternal gradients , ( 2 ) auto-activation terms , which are non-essential , but serve to maintain sharply defined domain boundaries [50] , ( 3 ) strong mutual repression between non-overlapping gap genes ( hb and kni , Kr and gt; we call this mechanism ‘alternating cushions’ for reasons explained in [35] ) , ( 4 ) weak repression between overlapping gap domains showing posterior dominance which leads to dynamic anterior shifts in domain position , and ( 5 ) additional repression of gap genes at the posterior pole by terminal gap genes tll and hkb ( Figure 6D; see [35] for review ) . Sets of parameter estimates based on reduced-quality mRNA data show increased variability compared to protein-based circuits—both in terms of the distribution of parameter values ( Figure 6A ) , and the regulatory categories they fall into ( Figure 6B ) . Nevertheless , we have shown that we can recover consistent regulatory mechanisms from these estimates , if we consider the consensus network structure , that is , those regulatory categories into which a majority of the estimated parameter values fall into . In this section , we examine if our parameter estimates are also determinable in the statistical sense defined by Ashyraliyev et al . [49] , [64] . This is also known as practical parameter determinability analysis . It is achieved by assuming that our fitting problem has a single optimal solution , which we call the ‘true’ solution . We can then calculate confidence intervals around each one of our parameter estimates . These intervals determine a range of parameter values , which include—with a given probability of 95%—the true solution to the problem . A parameter is determinable , if its confidence interval lies entirely within one of our three regulatory categories: repression ( parameter value less than −0 . 005 ) , no interaction ( between −0 . 005 and 0 . 005 ) , or activation ( greater than 0 . 005 ) . It is weakly determinable if its interval intersects two of these categories , but excludes the third . There are two different ways to calculate these confidence intervals: dependent intervals tend to underestimate the extent of the confidence region , while independent intervals have the tendency to overestimate it ( see Materials and Methods for details ) . As in previous studies [49] , [64] , we use independent intervals to estimate the determinability of a parameter . Under specific conditions , gap gene circuits fit to protein data can yield parameter estimates , which are very well determined ( Table 3 , top row ) . Specifically , estimates from WLS fits are much more determinable than those obtained by OLS , if parameter values for diffusion rates ( D ) , threshold parameters ( h ) , and certain regulatory weights ( the effect of Hkb on gt , Kr , and kni ) are fixed during optimisation [49] . In contrast , determinability of parameter estimates is poor in both OLS and WLS fits to mRNA data ( Table 3 , rows 5 and 6 ) . This loss of determinability could be due to three reasons: ( 1 ) the use of mRNA instead of protein data; ( 2 ) our data processing method ( and approximation of variances for WLS ) ; and ( 3 ) the smaller embryo region covered by mRNA-based circuits . To distinguish between these three possibilities , we performed several series of optimisation runs . First , we used the original protein data set with approximated variances that only depend on expression level ( as described for our mRNA data in Materials and Methods ) . This yielded a level of parameter determinability , which was somewhat worse , but still comparable to the original protein data set ( Table 3 , row 2 ) . We then approximated expression boundaries in the integrated protein data set by our spline-based method , to emulate domain and boundary shapes equivalent to the mRNA-based data . Again , this yielded decreased but still reasonable parameter determinability ( Table 3 , row 3 ) . Based on this , we conclude that neither the use of approximated weights , nor the use of approximated boundaries can account for all the loss of determinability observed in mRNA-based circuits . Finally , we performed fits to mRNA data on an extended region of 58 nuclei , equivalent to the region used for protein-based gene circuits . As for other mRNA-based solutions , determinability was poor , significantly decreased compared to any of the protein fits ( Table 3 , row 4 ) . Taken together , the above evidence indicates that the most relevant factor for the loss of statistical determinability is the use of mRNA- instead of protein-based expression data . In the previous sections , we have established that it is possible to reverse-engineer a developmental gene regulatory network from mRNA expression profiles . Even though these mRNA-based networks lack statistical determinability , the observation that we consistently recover a qualitatively equivalent network generates a certain confidence in the methodology . Next , we wanted to define the minimal requirements for gap gene circuits in terms of experimental data . As mentioned in the Introduction , the most time-consuming and hence limiting step involved in this method is the acquisition and processing of the quantitative experimental data required for model fitting . Thus , we would like to reduce and simplify our data as much as possible . To this end , we have performed a series of model fitting runs with reduced data sets ( either by decreasing the number of boundaries , or the number of time classes ) , or with artificially generated data for external inputs ( to simulate the case where quantitative protein data on maternal gradients and terminal gap genes would be unavailable ) . Table 4 presents an overview of the distinct data sets used for model fitting , while the analysis of the resulting gene circuits is summarised in Figure 7 ( based on interconnectivity matrices shown in Text S3 ) . Our results show that a smaller fraction of solutions obtained by fitting to reduced data sets are usable for analysis . This applies to both reduction of the number of boundaries and time points present in a data set ( Figure 7 , 2nd column ) . In contrast , the optimum and median RMS scores of these fits do not show any clear trend , although RMS scores slightly increase as boundaries are removed ( Figure 7 , 4th column ) . Using artificial external inputs did not significantly affect the number of usable solutions or the RMS score . We conclude that our approach is surprisingly robust to the reduction in quality of our data set . Only in one case—reduction of the number of boundaries to 20%—did we completely fail to get any solutions suitable for further analysis . While a good fit to the data is important , it is even more crucial that gene circuits represent consistent gene network structures and regulatory mechanisms . For this reason , we examined whether models fit to reduced or artificial data incorporate the five basic mechanisms of gap gene expression described in the previous section . First , we looked at mutual repression of non-overlapping gap genes ( alternating cushions ) . This mechanism is recovered extremely robustly: it is present in all selected solutions ( Figure 7 , blue column ) . In agreement with previous analyses [50] , we conclude that it lies at the core of gap gene regulation . With respect to auto-activation , we observe that all mRNA-based circuits behaved similarly , with the exception of the gene circuits from fits with artificial maternal gradients ( Table 4 , and Figure 7 , green columns ) . In general , hb , gt and kni were auto-activating , while Kr was not ( or not consistently , at least ) . In addition fits with artificial maternal gradients show reduced presence of gt auto-activation . The variability in these results can be explained by the fact that auto-activation is not essential for positioning gap domains in gene circuit models [50] . To analyse the presence or absence of the domain shift mechanism [46] , we examined net effects of repression between overlapping gap domains as described in the previous section ( Figure 7 , red columns; see also Figure 6C ) . We observed that two domain interactions were consistently present in our models: the net repression of Kr by Kni , and that of kni by Gt . However , net repression of gt by Hb was only recovered in about 10–50% of solutions . In those circuits that do not show this effect , Gt represses hb in the anterior to reproduce the peak of hb expression in the middle of the embryo . We have shown elsewhere that this is a modelling artefact [48] . Repression of hb in the posterior is overcome in these circuits by strong activation of hb by Tll ( data not shown ) , an interaction which is likely to be indirect , and therefore not supported by experimental evidence [75] . Unlike the interactions described above , mutual repression between the overlapping domains of hb and Kr does not contribute to gap domain shifts [46]–[48] . If we consider the net effect of these reciprocal interactions , hb is repressed by Kr in the majority of cases ( Figure 7 , orange column ) . In contrast , most protein-based circuits show net repression of Kr by Hb . We explain this as follows: the border between hb and Kr is maintained at the same A–P position over time ( Figure 4; see also [43] , [46] ) . There are several regulatory mechanisms that can accomplish this . Protein-based circuits exhibit auto-activation on both sides plus a minimal net repressive effect of Hb on Kr , while mRNA-based circuits favour auto-activation of hb only , plus stabilisation of the hb/Kr border by a slight net repression of Kr on hb ( Figure 7 , green and orange columns ) . The experimental evidence on which of these alternative mechanisms applies in the embryo remains inconclusive: while the potential presence of Kr auto-activation [76] favours the first , the absence of any defect in the posterior hb boundary in Kr mutants supports the second mechanism [77] , [78] . Next , we looked at activation of gap genes by the maternal factors Bcd and Cad . This mechanism is recovered very robustly ( Figure 7 , purple column ) , in agreement with previous studies [46] , [48]–[50] . Note that the summary bar graphs of Figure 7 ( purple column ) omit the effect of Cad on hb and Kr , as these interactions are an artifactual aspect of the model ( [48] , and references therein ) . Finally , the terminal gap genes Tll and Hkb repress gap genes at the posterior pole of the embryo . In general , we recover this mechanism robustly ( Figure 7 , brown column ) . However , if Tll and Hkb protein gradients are substituted by mRNA profiles ( see Table 4 ) , about 20% of the solutions fail to show repression of Tll on gt . In these circuits , repression of gt by Hb is sufficient for the retraction of its posterior domain from the pole ( data not shown ) . Interestingly , recovery of this interaction is rescued in those fits in which both maternal and terminal external inputs were replaced by ‘artificial’ patterns ( see Figure 7 ) . In summary , we have managed to recover surprisingly correct and accurate gap gene regulatory mechanisms even with data sets of severely reduced coverage and/or quality . This does not imply that we can reconstitute specific gene networks with arbitrary data . On the contrary , successful network inference requires very specific conditions for the data used in model fitting . We will revisit this important point in the Discussion .
Does the fact that we still recover the correct gap gene network using mRNA data imply that our method lacks specificity ? Would it infer the same network with any kind of data ? The following evidence demonstrates that the answer to these questions is a clear no , and suggests minimal conditions for the expression data that must be met for inference to be specific and consistent . First of all , gap gene circuit models fail to correctly predict gap gene expression in the head region of the embryo ( anterior of 35% A–P position ) [45] , [48] . In this region , additional regulators ( the head gap genes which are not included in our models; see [35] for review ) are required for correct regulation and expression . This implies that the model-fitting procedure is specific: it fails when relevant regulatory factors or mechanisms are missing . In contrast , we have mentioned earlier that accurate measurements of absolute expression levels do not seem to be crucial for correct network inference ( see Introduction ) . Our results suggest that we can recover the structure of the gap gene network even if relative levels are only approximated in the data . This confirms that the most important expression feature for network inference is the dynamic positioning of expression boundaries . This is further corroborated by the following: early attempts at reverse-engineering the gap gene system using gene circuits exhibited various patterning defects and showed inconsistent network structures between independent model fits [45] . In contrast , we are able to infer correct network structure using data sets with spatial and temporal resolution similar to that used in [45] ( Figure 7 ) . Moreover , our data sets approximate gap protein domain positions with mRNA data ( Figure 4 ) . This suggests that the failure of inference in the earlier study was not caused by a lack of accuracy or resolution , but rather by qualitative errors regarding the relative placement of domains in the data used for model fitting . As mentioned in the Introduction , the abdominal kni domain was placed too far anterior in those data , leading to an exaggerated overlap with the central Kr domain , and an artefactual gap between the domains of kni and hb in the posterior of the embryo ( see Figure 1 in [45] ) . Similar problems affected an early study of the eve gene: in the data used for fitting , the position of the fifth expression stripe was shifted compared to the domains of its gap gene regulators , which led to inaccurate prediction of the regulatory mechanism underlying its expression [39] . What differs between these early attempts and more recent reverse-engineering studies is that both protein- [43] and mRNA-based data used in the latter capture the relative arrangement and timing of gap domains correctly . Both data sets show qualitatively equivalent expression patterns ( see Results , Figures 3 and 4 ) . The order in which gap domains are arranged along the A–P axis , as well as the order in which they appear relative to one another are the same for mRNA and protein data . Evidently , this temporal and spatial order is determined by the specific regulatory structure of the network ( given suitable kinetic parameters ) . This , in turn , allows us to robustly recover the specific regulatory structure of the gap gene network as long as our data capture their particular kind of spatio-temporal dynamics . Obviously , there are limits to the amount of inaccuracy the data can contain with regard to absolute positions and variability of the patterns . This is shown by the fact that we were unable to recover any usable gap gene circuits with data sets reduced to 20% of the original number of measured domain boundaries ( Figure 7 ) . These data sets only contain about 1–3 embryos per boundary per time class , while the smallest successful data set ( 40% data ) has about 1–7 embryos on average . In general , the number of embryos that need to be processed will depend on the natural variability of the patterns and the quality of the experimental protocols: noisy expression data require larger sample sizes . Similarly , temporal resolution will depend on the time scale of patterning dynamics . In our case , only 3 time classes ( each about 25 min apart ) were sufficient to recover a correct regulatory network . This is probably due to the fact that gap domains shift and develop smoothly over time ( see Figure 4 ) . Systems with more abrupt or uneven changes in expression patterns will require a higher temporal resolution . Our investigation of minimal data requirements for model fitting is of an empirical nature . It should be corroborated and extended in the future by more systematic and rigorous approaches based on methods for optimal experimental design ( OED , reviewed in [79] ) . OED uses algorithms for global optimisation and calculation of confidence intervals similar to those used here to predict which measurements in a data set are most relevant to accurately estimate parameter values ( see , for example , [80] , [81] ) . This could be used for a more rational design of data sets used for reverse-engineering by guiding the choice of observables and time points that are most informative to infer network structure and dynamics . However , applying OED to real-world , complex , non-linear systems remains challenging , and has only be achieved in exceptional cases ( see [82] ) . Therefore , it is beyond the aim and scope of our current study . In summary , our results demonstrate that the amount of data required for reverse engineering is much lower than previously thought . The necessary data sets can be acquired and processed by a single researcher within a few months , without the need for expensive equipment . The precise number of embryos to be processed , and the required temporal resolution need to be adapted according to the features of the system under study , the main condition being that the data capture the relative timing and spatial arrangement of expression domains correctly . The results reported in this study—together with earlier evidence from protein-based circuits [49]—indicate that weighted least squares ( WLS ) fits perform much better than ordinary least squares ( OLS ) . In principle , however , WLS fits require the accurate measurement of variances in expression levels to calculate the weights for the sum of squares . This is not possible in our current data quantification framework . Moreover , in general it increases the amount of work required for data acquisition and processing considerably . First , methods that measure the relative level of expression accurately are technically more challenging and require more work than those presented here ( see previous section ) . And second , enough individual expression patterns need to be quantified for the measured variance to be reliable . We avoid this complication by using approximated weights for WLS , which are simply proportional to the expression level of a gene . This assumption is mainly based on methodological reasoning ( although it is also supported by experimental evidence on protein expression patterns [40] , [43] ) . It is crucial to avoid small ectopic expression domains in non-expressing regions that can exert significant regulatory effects if interaction weights are sufficiently large . Several such ectopic domains have been observed in fits using OLS ( our data and [47] , [49] , [51] ) . WLS fits effectively suppress these modelling artefacts , as long as the penalty for ectopic expression imposed by small variances in non-expressing regions are sufficiently high . This suggests that our choice of approximated variances is justified for practical reasons , since it emphasises the importance of boundary positions during the fitting process . Although we do recover the same network from our mRNA-based models as that predicted by protein-based circuits , there is much increased variability in the estimated parameter values ( Figure 6A , B ) . This is also reflected in the loss of parameter determinability we observe in our results ( Table 3 ) . In practice , both of these phenomena imply that our mRNA-based models do not predict one , but rather a small set of possible network structures , while protein-based models predicted a specific , single network ( see Figure 6B ) . This is not surprising , since the quality of a gene circuit model reflects the quality of the data it was fit to . But of course it is a problem , although , we would argue , not a fundamental one . It can easily be addressed by combining the reverse-engineering approach with experimental ( genetic and molecular ) verification . Let us illustrate this with an example: there are several alternative network variants that occur in our circuits . They are all minor in that they differ from each other in one or two interactions at most . One of these variants occurs in models in which the posterior domain of hb arises due to strong and direct activation by Tll , rather than the absence of repression by Gt , its immediate anterior neighbour ( see Results ) . This alternative mechanism is a plausible explanation for the observed expression dynamics . However , it is not compatible with experimental evidence , which enabled us to classify it as an artefact of the model . Note that both the experimental evidence ( reviewed in [35] ) and predictions based on gene circuits [48] , [49] contain such ambiguities . Fortunately , these unresolved ( and potentially not resolvable ) cases have only a limited overlap , since genetic and reverse-engineering approaches are complementary to each other: one inferring regulatory interactions from mutants , the other from wild-type expression patterns [48] . In other words , interactions which are not clearly supported by experimental evidence are mostly unambiguous in our gene circuits , while others that are ambiguous in our models ( as , for example , the repression of hb by Gt ) , are clearly supported ( or excluded ) based on experimental evidence . But what about systems which have been less well studied than Drosophila ? In such systems , we do not have a comprehensive experimental literature to compare our results to . Instead , model predictions will have to be tested using genetic approaches such as mutant analysis , over-expression assays , or gene knock-down by RNA interference ( RNAi ) . Again , the reverse-engineering method is most powerful when used in conjunction with complementary experimental approaches . By minimising the amount of quantitative data required for reverse-engineering a developmental gene regulatory network , we have removed a major bottleneck for applying the method more widely . Still , this method is unlikely to be scalable to systems that are orders of magnitude larger than the one studied here . Microscopy and image acquisition remain labour-intensive , and our quantification pipeline still requires a series of manual interventions , such as positioning the splines that are fit to expression boundaries , or time classification of embryos . It remains a major challenge to fully automate these steps . Therefore , the effort required to quantify hundreds or thousands of spatial gene expression patterns is still considerable , even if robust and fast methods are used ( see , for example , [83] , [84] ) . Moreover , global non-linear optimisation is computationally intensive , and may not yield unique solutions in large regulatory systems . On the other hand , many pattern-forming networks are similar in complexity and nature to the gap gene system . One example in Drosophila is the dorso-ventral patterning system in the early embryo [85]–[87] . Other developmental networks occur in the context of cellularised tissues and often involve more than just one spatial dimension . It is straightforward to extend the gene circuit method to such systems . Complicating factors , such as post-transcriptional regulation , cell-to-cell signalling , or tissue movements and growth , can readily be accommodated in the gene circuit modelling formalism [38] . There are several examples of tractable cellular patterning systems in Drosophila: mesoderm and heart development [88] , [89] , morphogen-based patterning in wing imaginal discs [87] , [90] , or the thoracic bristle patterning system [91] , [92] . Examples beyond Drosophila include vulval induction in the roundworm Caenorhabditis elegans ( see , for example , [93] ) , or the dorso-ventral patterning system of the vertebrate neural tube [94] . Generalisation of the method is further facilitated by the fact that the approximations we have used are based on straightforward assumptions that do not require any in-depth understanding of the system under study . Our approximation of relative gene expression levels assumes smooth increase and decrease of gene product levels . The scaling of expression levels from the centre to the terminal ends of the embryo is a reasonable assumption in the case of Drosophila , but may be omitted in any other system as it is not strictly required for inferring the correct network structure . Approximated variances simply assume low levels of variability in non-expressing regions . Artificial external inputs were based on qualitative inspection of maternal gradients and expression of terminal gap genes . All of these assumptions can be applied to systems other than the gap gene network . The only potentially problematic assumption concerns the use of mRNA expression profiles . Obviously , one should have some qualitative evidence that mRNA patterns indeed resemble protein profiles before relying on such data . Application of reverse engineering to a large number of developmental systems in different organisms would allow us to investigate the dynamics of pattern-forming gene networks in a quantitative and systematic manner [26] . Our simplification of the method indicates that this is achievable within a reasonable amount of effort . The potential benefits of such a research programme are significant . Only through quantitative investigation of specific , experimentally accessible , gene networks will we be able to better understand the principles that govern development and pattern formation . A particularly interesting application of the gene circuit method is the comparative analysis of homologous gene networks across different species [26] . Such a comparative analysis allows us to identify conserved and divergent regulatory interactions in an evolving network . Moreover , changes in regulatory mechanisms can be mapped—rigorously and unambiguously—to observable differences in gene expression between species . We are currently performing such an analysis between gap genes in Drosophila and other dipteran species . It will allow us , for the first time , to study the evolution of a real developmental gene regulatory network in a detailed and quantitative manner . This is a crucial step towards a more general investigation into the causal relation between evolution at the genotypic and the phenotypic level . | To better understand multi-cellular organisms we need a better and more systematic understanding of the complex regulatory networks that govern their development and evolution . However , this problem is far from trivial . Regulatory networks involve many factors interacting in a non-linear manner , which makes it difficult to study them without the help of computers . Here , we investigate a computational method , reverse engineering , which allows us to reconstitute real-world regulatory networks in silico . As a case study , we investigate the gap gene network involved in determining the position of body segments during early development of Drosophila . We visualise spatial gap gene expression patterns using in situ hybridisation and microscopy . The resulting embryo images are quantified to measure the position of expression domain boundaries . We then use computational models as tools to extract regulatory information from the data . We investigate what kind , and how much data are required for successful network inference . Our results reveal that much less effort is required for reverse-engineering networks than previously thought . This opens the possibility of investigating a large number of developmental networks using this approach , which in turn will lead to a more general understanding of the rules and principles underlying development in animals and plants . | [
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] | 2012 | Efficient Reverse-Engineering of a Developmental Gene Regulatory Network |
Flowering time relies on the integration of intrinsic developmental cues and environmental signals . FLC and its downstream target FT are key players in the floral transition in Arabidopsis . Here , we characterized the expression pattern and function of JMJ18 , a novel JmjC domain-containing histone H3K4 demethylase gene in Arabidopsis . JMJ18 was dominantly expressed in companion cells; its temporal expression pattern was negatively and positively correlated with that of FLC and FT , respectively , during vegetative development . Mutations in JMJ18 resulted in a weak late-flowering phenotype , while JMJ18 overexpressors exhibited an obvious early-flowering phenotype . JMJ18 displayed demethylase activity toward H3K4me3 and H3K4me2 , and bound FLC chromatin directly . The levels of H3K4me3 and H3K4me2 in chromatins of FLC clade genes and the expression of FLC clade genes were reduced , whereas FT expression was induced and the protein expression of FT increased in JMJ18 overexpressor lines . The early-flowering phenotype caused by the overexpression of JMJ18 was mainly dependent on the functional FT . Our findings suggest that the companion cell–dominant and developmentally regulated JMJ18 binds directly to the FLC locus , reducing the level of H3K4 methylation in FLC chromatin and repressing the expression of FLC , thereby promoting the expression of FT in companion cells to stimulate flowering .
DNA is packaged as chromatin in eukaryotic cells . Nucleosomes , which consist of an octamer of four histones wrapped around 146 base pairs of DNA , are the fundamental unit of chromatin [1] . The flexible N-terminal tails of histones , which protrude from the nucleosome core particle , are subject to several types of covalent modification , including acetylation , methylation , phosphorylation , and ubiquitylation [2] . Each of these modifications is reversible and is required for the dynamic regulation of gene expression [3] . In vivo , the lysine residues in histones display three distinct methylation states: mono- ( me1 ) , di- ( me2 ) , and tri-methylated ( me3 ) . These differences in methylation are important for the recognition of chromatin by chromatin modulators and for the recruitment of other modulators and regulators [4] , 5 , 6 , 7 , 8 . In addition , lysine methylation at different sites within the histone protein plays distinct roles in gene activation and repression [2] , [9] . For example , the methylation of H3K4 and H3K36 is correlated with gene activation , while transcriptional repression has been demonstrated in genomic regions with increased levels of H3K9 and H3K27 methylation [3] , [10] , [11] , [12] . Various developmental processes are regulated by histone methylation in animals and plants . For example , H3K27 methylation mediated by the PRC2 complex is required for embryonic development and stem cell identity in mammals and controls most steps in the development of Arabidopsis [9] , [13] , [14] , [15] . The methylation state of histone proteins is determined by the balance between methylation and demethylation , which is mediated by histone methyltransferases and demethylases , respectively [16] , [17] , [18] . However , the reversibility of histone methylation in vivo is the latest to be discovered compared to other covalent forms of histone modification . The amine oxidase LSD1 was the first histone demethylase found to demethylate H3K4me2 and H3K4me1 through an FAD-dependent oxidation reaction [18] . LSD1 demethylase family proteins are unable to remove methyl groups from tri-methylated lysines , suggesting the presence of other histone demethylases in eukaryotic cells [18] . More recently , a family of JmjC domain-containing proteins was characterized as histone demethylases which were able to reduce any one of the three histone lysine methylation states at several specific sites in yeast and animals [17] , [19] , [20] , [21] . These histone demethylases are involved in many biological processes in animals , including spermatogenesis , HOX gene regulation , and germ cell development [19] , [22] , [23] . FLD and LDLs are the homologs of human LSD1 in Arabidopsis . They promote the floral transition by constitutively inhibiting the expression of FLC [24] , [25] , [26] . The Arabidopsis genome contains 21 JmjC family proteins [27]; however , only five of them have been characterized , and they have been found to be involved in RNA silencing , DNA methylation , flowering time control , circadian clock regulation , BR signaling and shoot regeneration in vitro [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] . Flowering at the appropriate time is the most important factor in achieving reproductive success . To produce the next generation , plants rely on intricate signaling pathways involving a variety of intrinsic factors , including developmental stage and age , and environmental cues such as photoperiod , temperature , and light quantity [37] , [38] , [39] . Thus , distinct environmental conditions modulate endogenous gene expression to ensure flowering at the correct time [12] , [40] . FLOWERING LOCUS C ( FLC ) encodes a MADS-box transcription factor and floral repressor that regulates flowering time in a dosage-dependent manner by integrating the vernalization , autonomous , PAF1 complex , and H2B ubiquitination pathways [11] , [41] , [42] , [43] , [44] , [45] . FLC and the functional locus FRIGIDA ( FRI ) act together to produce winter-annual Arabidopsis accessions , which must be exposed to cold temperature for several weeks to repress FLC expression and promote flowering in the following spring [46] , [47] . As endogenous factors , autonomous pathway genes consecutively repress FLC expression [48] , [49] . In addition , the FLC antisense transcript affects the expression of sense transcript , thereby influencing flowering time in Arabidopsis [50] , [51] , [52] . FLC expression is predominant in the shoot apical meristem ( SAM ) ; however , it is also expressed in vascular tissue in young leaves and the root tip [44] , [53] , [54] . FLC expressed in the SAM and leaf vascular tissue contributes to the control of flowering time in Arabidopsis [55] . During vernalization , VIN3 binds FLC chromatin , thereby repressing its expression in the SAM [53] . The components of the autonomous pathway repress , while those of the PAF1 complex activate , FLC expression in the SAM [11] , [37] , [43] . Thus , although the regulation of FLC expression in the SAM has been extensively studied , little is known about how FLC expression is regulated in leaf vascular tissue . Histone modification plays crucial roles in the regulation of FLC expression in Arabidopsis . In FLC chromatin , H3K4 hyper-tri-methylation and acetylation are associated with the activation of gene expression [43] , [56] , [57] , [58] . The methylation of H3K27 and H3K9 in FLC chromatin leads to the repression of FLC expression and is required for maintenance of the repression of FLC expression in plants growing in the following spring after vernalization [12] , [53] , [59] . In addition , H2B monoubiquitination is required for the maintenance of high levels of H3K4me3 and H3K36me2 [11] , [45] . A loss of H2B monoubiquitination decreases the level of H3K4me3 and H3K36me2 , leading to the repression of FLC transcription and early flowering [11] , [45] . FLOWERING LOCUS T ( FT ) , which is a component of the photoperiod pathway , coordinates signals from the vernalization , autonomous , PAF1 complex , and photoperiod pathways to promote flowering in response to increase in day length [60] , [61] , [62] , [63] . FT expression is restricted to leaf phloem companion cells [62] . FT travels from leaves to the SAM , where it interacts with the bZIP transcription factor FD to stimulate floral meristem initiation [64] , [65] , [66] , [67] , [68] . As a mobile and systemic signal , FT integrates photoperiod- and FLC-dependent pathways to control flowering time by regulating the expression of floral identity genes . It was shown previously that the companion cells specifically expressed FLC represses FT expression in leaf companion cells and delays the expression of its cofactor FD in the SAM [55] . Thus , FLC systemically blocks the function of FT to repress flowering . However , the regulation of FLC expression in companion cells for the control of floral development has not been characterized . As mentioned above , several JmjC domain-containing proteins have been reported to be involved in flowering time control [28] , [29] , . Among them , JMJ14 is a member of JARID family with H3K4 demethylase activity , and is involved in flowering time control through the repression of floral integrators [28] , [29] , [33] , [69] . It is not yet clear whether those floral integrator genes are the direct targets of JMJ14 [28] , [29] , [69] . ELF6 acts as a repressor in photoperiod pathway [31] , and its closest homolog REF6 acts as a FLC repressor in flowering time regulation [31] . Recent results suggest that REF6 is a H3K27 demethylase; thus , FLC is not likely to be the direct target of REF6 [70] . No JmjC domain-containing histone demethylases that target FLC locus have yet been found . In this paper , we describe the expression pattern and function of JMJ18 , a novel JmjC domain-containing histone H3K4 demethylase in Arabidopsis . JMJ18 was predominant expressed in phloem companion cells , and its level of expression increased during vegetative development . JMJ18 was found to promote the floral transition in Arabidopsis by binding FLC chromatin and demethylating H3K4 methylation , leading to the repression of FLC and enhanced expression of the downstream flowering activator FT in companion cells .
JMJ18 ( At1g30810 ) belongs to the evolutionarily conserved JARID1 family . Previous studies have shown that JARID1 family proteins demethylate H3K4 methylations in yeast , animal and Arabidosis [28] , [29] , [33] , [69] , [71] , [72] , [73] . To determine whether JMJ18 exhibits histone demethylase activity , we expressed recombinant His-tagged JMJ18 and purified the recombinant protein from insect cells ( Figure S1A and S1B ) . A MALDI-TOF mass spectrometric analysis-based demethylation assay was used to detect the histone demethylation activity of JMJ18 . Purified His-JMJ18 was incubated with a variety of histone peptides representing the tri- and di-methylated states of the four lysine residues in histone H3 . As shown in Figure 1A , JMJ18 converted H3K4me3 to H3K4me2 , but did not alter the H3K4me2 methylation state or the methylation status of any other lysine residue in the peptides ( summarized in Figure S1C ) . To investigate the demethylase activity of JMJ18 further , we purified two truncated versions of JMJ18 , JMJ18Δ141–144 and JMJ18Δ141–158 , in which the partial linker sequence between JmjN and JmjC was deleted ( Figure 1B ) , and examined the histone demethylase activity of each using the assay described above . Similar to full-length JMJ18 , the truncated proteins were H3K4-specific demethylases ( Figure 1B and Figure S1C ) . Interestingly , both truncated forms of JMJ18 exhibited H3K4me3 and H3K4me2 demethylase activity , and they were able to convert H3K4me3 to H3K4me2 and H3K4me1 in the presence of H3K4me3 peptide ( Figure 1B and Figure S1C ) . This suggests that the linker between JmjN and JmjC blocks the enzyme's demethylase activity toward H3K4me2 in vitro . However , they were unable to demethylate H3K4me2 to H3K3me1 if H3K4me2 peptide was used as the substrate ( Figure 1B and Figure S1C ) . We further observed that the absence of α-ketoglutarate ( α-KG ) or presence of EDTA completely abolished the enzyme's demethylase activity ( Figure 1C ) , while a lack of Fe ( II ) strongly inhibited the enzyme's activity ( Figure 1C ) . Taken together , our results suggest that JMJ18 functions as an H3K4-specific demethylase in vitro , and that its activity is dependent on α-KG and Fe ( II ) . To assess the biological function of JMJ18 , we obtained three T-DNA insertion lines from the ABRC [74]: jmj18-1 ( SALK_073442 ) , jmj18-2 ( GABI_649D05 ) , and jmj18-3 ( SAIL_861_F02 ) ( Figure 2A ) . Reverse transcription ( RT ) -PCR analysis revealed a lack of full-length JMJ18 mRNA in jmj18-1 and jmj18-2; however , a partial transcript was detected in both lines ( Figure 2B ) . In the third line , the T-DNA was inserted into exon 4 ( Figure 2A ) , and full-length mRNA was detected at a reduced level compared to that in wild-type plants , and the transcript was confirmed by sequencing , suggesting the existence of a knock-down allele ( Figure 2B ) . All three alleles exhibited a weak but reproducible late-flowering phenotype under long- ( LD ) and short-day ( SD ) conditions ( Figure 2C , 2D and Table 1 ) . This late flowering phenotype in jmj18-1 was complemented by JMJ18:JMJ18-GFP transformation ( Figure S2 ) . Knock-down lines for JMJ18 expression by using both double strand RNA interference ( RNAi ) and artificial micro-RNA ( amiR ) were generated , and these lines also exhibit weak late-flowering phenotype under LD conditions ( Figure S3 and Table S1 ) . To address the molecular mechanism underlying this phenotype , we examined the expression of three important floral regulators , FLC , CONSTANS ( CO ) , and FT , by quantitative real-time PCR ( qRT-PCR ) . No obvious change in expression compared to wild-type was detected at various vegetative developmental stages , but statistical significant for the expression of FLC and FT between wild-type and jmj18 mutants if any at later vegetative developmental stages ( Figure 2E–2G and Figure S4 ) . This suggests that the changes in gene expression are mild , which is consistent with the mild late-flowering phenotype in the jmj18 mutants and knock-down lines . To gain a deeper understanding of the function of JMJ18 in Arabidopsis , we examined its expression pattern using wild-type Arabidopsis plants carrying a JMJ18 promoter-fused GUS reporter construct . The whole intergenic region between JMJ18 and its upstream gene was first selected to be the promoter of JMJ18 ( Figure S5A ) . GUS expression was detected in vascular tissue collected from the cotyledons , young leaves , and roots of seedlings ( Figure 3A and 3B ) . Similarly , GUS was detected in the vascular tissue of adult leaves and flowers ( Figure 3C and 3D ) . Similar expression pattern was obtained if the length of promoter was increased from 1 . 4 to 1 . 7 kb ( Figure S5A–S5D ) . Next , JMJ18 expression was examined in detail by microscopy . Longitudinal section analysis showed that JMJ18:GUS was expressed in both the shoot and root phloem , as well as in protophloem , but not in the SAM in seedlings ( Figure 3E and 3F ) . Further , JMJ18 expression was restricted to the cell files of protophloem in the root tip ( Figure 3H and Figure S5E–S5J ) and phloem companion cells in mature roots ( Figure 3F and 3G ) . These results suggest that JMJ18 is predominant expressed in companion cells . The phloem cell-dominant expression of JMJ18 was further confirmed in transgenic plants expressing GFP-tagged JMJ18 under the control of the JMJ18 promoter ( Figure 3I–3K ) . JMJ18-GFP was expressed in the protophloem in root tip and phloem in other tissues ( Figure 3I–3K and Figure S6A–S6C ) . We also found that JMJ18-GFP was localized to the nucleus ( Figure 3I and Figure S6D ) , which is consistent with its function as a histone demethylase . To further verify the expression pattern of JMJ18 , the companion cell-specific SUC2 promoter [75] , [76] was used to drive JMJ18-GFP expression in wild-type plants . The expression pattern of SUC2:JMJ18-GFP was similar to that of JMJ18:JMJ18-GFP ( Figure 3K and 3L ) . In addition , we generated JMJ18:JMJ18-RFP and SUC2:JMJ18-GFP double-transgenic plants and found that JMJ18-RFP and JMJ18-GFP were colocalized in the nuclei of companion cells ( Figure 3M–3P ) . It was also observed that JMJ18-GUS was also detected in pollen if the anther was stained longer time ( Figure S5K–S5N ) . These results demonstrate that JMJ18 is expressed predominant in phloem companion cells , similar to the floral integrator FT in vegetative developmental stage [62] . To determine the developmental expression profiles of JMJ18 , wild-type seedlings grown under LD conditions were harvested at dusk on days 6 , 9 , 12 , and 15 for RNA isolation . The plants did not form floral meristems; day 15 was the last day before floral meristem formation based on our microscopic observations . JMJ18 was expressed at a low level in young seedlings; however , its expression progressively increased during vegetative development from day 6 to day 15 ( Figure 4A ) . Thus , the expression of JMJ18 is developmentally regulated . FT expression was also examined using the same batch of materials . Consistent with previous data [60] , [61] , FT expression was weak in young seedlings; however , it increased steadily as the plants matured ( Figure 4A ) . There was a significant positive correlation between the expression patterns of JMJ18 and FT during vegetative development , with a correlation coefficient of 0 . 986 ( Figure 4A ) . To further investigate the function of JMJ18 , we overexpressed JMJ18 using its endogenous promoter ( JMJ18 promoter ) , the companion cell-specific SUC2 promoter , and the constitutively expressed CaMV35S promoter of which is expressed in and outside of the companion cells in wild-type plants . All three types of overexpression lines exhibited earlier flowering compared to wild-type under inductive LD and non-inductive SD conditions ( Figure 4B , 4C and Table 2 ) . Overall , the SUC2:JMJ18-GFP transgenic lines flowered earlier than the JMJ18:JMJ18-GFP transgenic plants; however , the 35S:JMJ18-GFP transgenic lines flowered before either of the other two ( Figure 4B , 4C and Table 2 ) . This suggests that the overexpression phenotype was JMJ18 dose-dependent . To verify this prediction , total protein was extracted from eleven-day-old transgenic plants harvested at dusk , and the abundance of JMJ18 was assessed by Western blotting using anti-JMJ18 antibodies generated against recombinant JMJ18 in rabbits . Consistent with the early-flowering phenotype described above , the expression of JMJ18 in the transgenic plants was as follows ( in order from lowest to highest ) : JMJ18:JMJ18-GFP , SUC2:JMJ18-GFP , and 35S:JMJ18-GFP ( Figure 4E and Figure S7 ) . Taking our flowering time and JMJ18 expression data together , the overexpression of JMJ18 promotes flowering in a JMJ18 dose-dependent manner . The strong positive correlation between the JMJ18 and FT spatio-temporal expression patterns ( Figure 3 , Figure 4A , and Figure S5 ) suggests that JMJ18 regulates FT expression in companion cells to control floral development . To investigate this possibility , total RNA was isolated from the same batch of materials used to examine JMJ18 expression . FT expression was increased to varying degrees in all three types of overexpression lines , and the degree of increase in each line was correlated with the abundance of JMJ18 and flowering time ( Figure 4D , 4E and Table 2 ) . Similar results were obtained for the expression of TSF , a homolog of FT , in JMJ18 overexpression lines ( Figure S8 ) . It has been shown that FT is one of the most important components of florigen , and its abundance determines flowering behavior [66] , [67] , [68] . Thus , we next examined the abundance of FT in JMJ18 overexpression lines by Western blotting using anti-FT antibodies generated against recombinant FT in rabbits . The FT level detected in each line was consistent with the mRNA level and abundance of JMJ18 ( Figure 4D and 4E ) . These results indicate that JMJ18 induces FT and its homologs such as TSF transcription and enhances their accumulation to accelerate the vegetative-to-reproductive transition . To test whether FT function is genetically necessary for facilitation of the vegetative-to-reproductive transition by JMJ18 , ft-11 was crossed with the SUC2:JMJ18-GFP line . The mutation in FT dramatically suppressed the early-flowering phenotype of the SUC2:JMJ18-GFP plants ( Figure 5A–5C ) , suggesting that FT is required for the function of JMJ18 in flowering control . In the photoperiod pathway , CO is responsible for FT induction , and FT expression is obviously delayed in a CO mutant background even under inductive LD conditions [60] , [61] . FD is an SAM-specific transcription factor that interacts with FT in the SAM [64] . Both FT and FD are required for floral meristem formation [64] , [65] . The mutation of CO or FD also abolished the SUC2:JMJ18-GFP early-flowering phenotype , such as SUC2:JMJ18-GFP co-11 or SUC2:JMJ18-GFP fd-5 , flowered at a similar time to co-11 or fd-5 ( Figure 5A–5C ) . In addition , we found that overexpression of JMJ18 in mutant background , such as SUC2:JMJ18-GFP ft-11 , SUC2:JMJ18-GFP co-11 and SUC2:JMJ18-GFP fd-5 , flowered slightly earlier than the single mutants ft-11 , co-11 , and fd-5 , in terms of both flowering time and rosette leaf number ( Figure 5A–5C ) . These genetic data demonstrate that promotion of the floral transition by JMJ18 mainly depends on functional FT . JMJ18 has four conserved domains: the JmjN , JmjC , zinc-finger , and FY-rich domains ( Figure 2A ) . Due to the obvious early-flowering phenotype of JMJ18 overexpressors , we overexpressed truncated JMJ18 ( Figure 6A and Figure S9A ) in wild-type plants to determine the domain ( s ) in JMJ18 necessary for its function in planta . The early-flowering phenotype of 35S:JMJ18 ( containing the entire JMJ18 CDS ) was echoed by that of 35S:JMJ18-GFP ( Figure 6B and 6C ) , suggesting that blocking of the free of C-terminus of JMJ18 by GFP does not affect its function . 35S:NCZ ( containing the JmjN , JmjC , and zinc-finger domains but lacking the FY-rich domain ) transgenic plants exhibited a shorter life cycle than wild-type plants; however , the effect was weaker than that observed in 35S:JMJ18 plants ( Figure 6B and 6C ) . Regardless , no early-flowering phenotype was observed in those plants overexpressing truncated JMJ18 with a deletion in both the zinc-finger and FY-rich domains ( 35S:NC ) ( Figure 6B and 6C ) . Northern blot analysis was used to determine whether these truncated transcripts were expressed in the transgenic plants . As shown in Figure 6D , all of the truncated transcripts were expressed . The transgenic plants overexpressing truncated JMJ18 with a deletion in both the JmjN and JmjC domains displayed similar flowering time compared to wild-type plants ( Figure S9 ) . Thus , these data suggest that the JmjN , JmjC , and zinc-finger domains are required for the function of JMJ18 , while the FY-rich domain is not fully necessary for its function but affects its activity . The 35S:T ( encoding a truncated protein identical to that in jmj18-1 and containing the JmjN , JmjC , and zinc-finger domains ) transgenic plants also displayed an obviously earlier floral transition compared to wild-type plants ( Figure 6A–6D ) , which indicates that jmj18-1 and jmj18-2 are weak alleles . And the result is consistent with their weak early-flowering phenotype ( Figure 2C , 2D and Table 1 ) . H3K4 methylation is associated with gene activation; thus , H3K4 demethylases should work as gene repressors , rather than as activators . Therefore , FT is not likely to be the direct target of JMJ18 . The target of JMJ18 could be an upstream repressor of FT . If this is the case , the floral repressor FLC is a good candidate . To examine this possibility , the effect of JMJ18 overexpression on FLC expression was examined in JMJ18 overexpressor plants . FLC expression was significantly repressed in all JMJ18 overexpressor lines examined , and the degree of repression was positively correlated with the abundance of JMJ18 , but negatively with the level of FT ( Figure 4E and Figure 7A ) , the other members of FLC clade genes ( MAF1 to MAF5 ) were also repressed to different extent in JMJ18 overexpressors ( Figure 7C ) ; in comparison , there was no obvious change in the expression of CO , an upstream activator of FT ( Figure 7B ) . To analyze the temporal expression pattern of FLC and its relationship to that of JMJ18 during vegetative development , we measured FLC expression in plants grown under LD conditions on days 6 , 9 , 12 , and 15 as described above . FLC expression was strong at the seedling stage , then decreased during vegetative development , reaching its lowest level before floral meristem formation on day 15 ( Figure 7D ) . There was a strong negative correlation between the developmental expression patterns of JMJ18 and FLC during vegetative development ( r = −0 . 950 ) . Thus , we propose that JMJ18 is a repressor of FLC . We generated jmj18-1 flc-3 to test whether JMJ18 interacts genetically with FLC . The FLC mutation mainly blocks the late-flowering phenotype of jmj18-1 ( Figure 7E , 7G and Table S2 ) . Thus , the mutation in FLC did not enhance the early-flowering phenotype of the JMJ18 overexpressors . In addition , overexpression of JMJ18 in flc , such as SUC2:JMJ18-GFP flc-3 , flowered earlier than flc-3 and at almost the same time as SUC2:JMJ18-GFP ) ( Figure 7F , 7G and Table S2 ) , indicating that endogenous JMJ18 enhances flowering mainly by repressing FLC in wild-type plants , while overexpressing JMJ18 recognized FLC and MAFs as targets . Taken together , those results indicate that JMJ18 and FLC belong to the same genetic pathway in flowering time control , and JMJ18 functions in upstream of FLC . Our previous result suggested that JMJ18 functions as an H3K4-specific demethylase in vitro ( Figure 1 and Figure S1 ) . To verify the histone demethylase activity of JMJ18 in vivo , nucleoproteins were extracted from jmj18 , our JMJ18 overexpressor lines , and wild-type plants . There was no obvious change in the levels of H3K4me3 , H3K4me2 , and H3K4me1 among the wild-type , jmj18 , and JMJ18 overexpressor plants on a global scale ( Figure 8A and 8B ) . A slight decrease in H3K4me3 was detected in the 35S:JMJ18-GFP plants ( Figure 8B ) , but no obvious difference was detected between wild-type and the other JMJ18 overexpressors ( Figure 8B ) . Thus , endogenous JMJ18 may function as a gene-specific H3K4 demethylase in vivo , while overexpressed JMJ18 can target many other loci to demethylate H3K4me3 once it is expressed in many cell types . To verify whether JMJ18 demethylates FLC chromatin , chromatin immunoprecipitation ( ChIP ) was done to detect the level of H3K4 methylation across the entire FLC chromatin region . In wild-type plants , the regions around the transcription start site ( regions B4 , B5 , and B6 ) had higher levels of H3K4me3 than the other regions ( Figure 9A and 9B ) , which is consistent with previous data [11] , [43] . However , the H3K4me3 level was obviously decreased in SUC2:JMJ18-GFP plants across the entire FLC chromatin region compared to that in wild-type plants ( Figure 9B ) . However , the H3K4me3 levels at the house keeping gene ACTIN , which contains high level of H3K4me3 , and transposon elements AtMu1 and AtSN1 , which are lack of H3K4me3 , were not obviously changed in SUC2:JMJ18-GFP plants compared to wild-type plants ( Figure S10 ) . The similar results were obtained by using normalizing H3K4me3 level to total H3 and to the input ( Figure S11 ) . The level of H3K4me2 in FLC chromatin was also decreased in SUC2:JMJ18-GFP overexpressor plants compared to wild-type plants ( Figure 9C ) . To analyze whether JMJ18 regulates the expression of MAFs through the control of the H3K4 methylation state at their loci , the H3K4me3 and me2 modification levels were detected in SUC2:JMJ18-GFP and wild-type plants . The levels of H3K4me3 at the chromatins of five MAFs were obviously decreased . While , the levels of H3K4me2 were slightly decreased at MAF4 , MAF5 but not MAF1 , MAF2 and MAF3 ( Figure 9D , 9E ) . These results indicate that JMJ18 demethylates H3K4me3 and H3K4me2 within the FLC and MAFs loci in vivo . JMJ18 controls FLC expression by modulating the H3K4 methylation level at FLC chromatin , suggesting that JMJ18 associates with the FLC locus and mediates the methylation of FLC chromatin directly . To investigate this possibility , ChIP was used to detect the binding of JMJ18 to the FLC locus . Due to the increased level of nonspecific binding of GFP to Arabidopsis chromatin under our experimental conditions , we used SUC2:JMJ18-myc transgenic plants instead of SUC2:JMJ18-GFP plants in our assay . The SUC2:JMJ18-myc transgenic plants exhibited similar phenotypes to the SUC2:JMJ18-GFP plants , including an early-flowering phenotype , altered FLC and FT expression , and reduced H3K4me3 and H3K4me2 levels across FLC chromatin ( Figure S12 ) . A c-myc-specific antibody was used to precipitate chromatin from wild-type and SUC2:JMJ18-myc plants . Compared to the wild-type plants , the FLC chromatin of the transgenic plants was significantly enriched with JMJ18 protein , with varying levels of occupancy across the entire FLC chromatin region , while , there was no obvious binding of JMJ18 to the chromatin of ACTIN , which was a negative control , compared with its binding to FLC chromatin ( Figure 9F ) . Regions B5 and B6 exhibited the highest binding ability ( Figure 9F ) , whereas the H3K4 methylation level was higher than in the other regions ( Figure 9B and 9C ) , demonstrating that JMJ18 associates with FLC chromatin and that the binding of JMJ18 to FLC is necessary for the dynamic of H3K4 methylation in FLC chromatin . FRI is the major determinant of ecotype differences in flowering time in Arabidopsis [46] , [47] . To examine the influence of JMJ18 on flowering time promotion , JMJ18 was overexpressed in FRI plants by the introduction of FRI into JMJ18:JMJ18-GFP , SUC2:JMJ18-GFP , and 35S:JMJ18-GFP transgenic lines via crossing , respectively . The overexpression of JMJ18 significantly suppressed the late-flowering phenotype of FRI in a JMJ18 dose-dependent manner ( Figure 10A and Table 3 ) . FRI represses the floral transition by activating FLC expression [46] , [77] . To determine whether JMJ18 suppresses the FRI late-flowering phenotype by decreasing FLC expression , FLC transcription in FRI wild-type and JMJ18 overexpressors was measured . FLC expression was decreased in the transgenic lines compared to that in the FRI background , with a more pronounced effect in leaves than in complete seedlings except for 35S:JMJ18-GFP ( Figure 10B and 10C ) , which is consistent with the expression pattern of FLC and JMJ18 in our transgenic lines . This is because FLC was expressed in both the SAM and companion cells , with predominant expression in the SAM , while both the JMJ18 and SUC2 promoters were specifically expressed in companion cells . Thus , this result supports the notions that JMJ18 promotes flowering in FRI by repressing FLC expression , and that FLC expressed in both the SAM and companion cells contributes to flowering time control . Consistent with our previous results ( Figure 4D and Figure S12D ) and early-flowering phenotype ( Figure 10A and Table 3 ) , the expression of FT in these transgenic lines was obviously activated ( Figure 10D ) , indicating that JMJ18 represses FLC expression and induces FT expression in an FRI background as well .
Several JARID1 family proteins have been characterized as H3K4 demethylases in various organisms [9] , [28] , [29] , [33] , [69] , [71] , [72] , [73] . There are 6 members of JARID1 proteins in Arabidopsis , JMJ14 ( at4g20400 ) , JMJ15 ( at2g34880 ) , JMJ16 ( at1g08620 ) , JMJ17 ( at1g63490 ) , JMJ18 ( at1g30810 ) , and JMJ19 ( at2g38950 ) . To examine whether Arabidopsis JMJ18 is a real histone demethylase , we characterized its histone demethylase activity in vitro and in vivo . We examined the histone demethylase activity of JMJ18 in vitro by purifying recombinant JMJ18 from insect cells and analyzing it by MALDI-TOF mass spectrometry . In that analysis , JMJ18 exhibited histone H3K4-specific demethylase activity ( Figure 1A , 1B , and Figure S1 ) , which was dependent on Fe ( II ) and α-KG ( Figure 1C ) . Additionally , JMJ18 was able to demethylate histone H3K4me3 to H3K4me2 and H3K4me1 ( Figure 1A , 1B , and Figures S1C ) . This observation was confirmed in vivo using FLC and MAFs chromatins ( Figure 9B–9E , Figures S11A–11B and S12E–S12F ) , but not in a genome-wide analysis of histone H3 in jmj18 mutants or in overexpressing lines driven by JMJ18- or SUC2- promoters ( Figure 8 ) , and the H3K4me3 modification was not obviously changed at ACTIN , AtMu1 and AtSN1 loci neither ( Figures S10 and S11C ) , suggesting that JMJ18 may function as a gene-specific H3K4 demethylase in Arabidopsis . However , it is obvious that global H3K4me3 in 35S:JMJ18-GFP is reduced ( Figure 8B ) , indicating that JMJ18 can target many other loci to demethylate H3K4me3 but not H3K4me2 once it is expressed in many cell types . Thus , the target specificity of JMJ18 can be fulfilled by gene specific targeting mechanisms or expression restriction or both . It was observed that the reduction in H3K4me2 is less than that H3K4me3 in the chromatins FLC and MAFs by JMJ18 overexpression ( Figure 9B–9E and –S12F ) , and full-length JMJ18 lacks demethylase activity towards H3K4me2 to H3K4me1 ( Figure 1A and Figure S1C ) , indicating that JMJ18 favors H3K4me3 to H3K4me2 demethylation . Our results also indicate that JARID1 family proteins are conserved histone H3K4 demethylases from human to plants . Interestingly , full-length JMJ18 only demethylated H3K4me3 to H3K4me2 ( Figure 1A ) . However , truncated JMJ18 lacking the partial linker between the JmjN and JmjC domains exhibited demethylase activity toward both H3K4me3 and H3K4me2 ( Figure 1B and Figure S1C ) . Taken together ( Figure 9B–9E and Figure S12E–S12F ) , our results suggest that the linker between the JmjN and JmjC domains in JMJ18 blocks its H3K4me2 demethylase activity in vitro . This linker could not block the H3K4me2 demethylase activity of JMJ18 in vivo by functioning as a protein-interaction domain; however , this requires further study . At any rate , this observation raises an interesting question as to the structural function of the linker sequence in JMJ18 itself and within the JMJ18 complex . Cell- or tissue-specific expression is a significant part of enabling a gene to achieve its function in plant cell fate determination and development [64] , [65] , [78] . Since the transgenic plants carrying JMJ18:JMJ18-GFP transformation could complement the late-flowering phenotype of jmj18-1 ( Figure S2 ) , it was possible to characterize the expression pattern of JMJ18 in Arabidopsis based on JMJ18 promoter-reporter expression ( Figure 3 , Figures S5 and S6 ) . We found that JMJ18:GUS and JMJ18:GFP were expressed in the vascular tissue of cotyledons , young leaves , adult leaves , hypocotyl and flowers ( Figure 3A , 3C , 3D; Figures S5 and S6 ) , not expressed in the SAM where FLC was expressed dominantly ( Figure 3E ) . In the roots of the transgenic plants , signals for GUS and GFP were predominantly observed in protophloem at the root tip , and in phloem in other regions of the root ( Figure 3B , 3F–3K; Figures S5 and S6D ) . In addition , different length of promoter-driven reporter exhibits similar expression pattern ( Figure 3 , Figures S5 and S6 ) , indicating that JMJ18 is predominantly expressed in the phloem of vascular tissue during vegetative development . Recently , Hong et al . ( 2009 ) reported that the expression of JMJ18 ( Atjmj8 in their study ) is expressed broader in root and leaf [79] . We constructed a same version of JMJ18 promoter-GUS fusion as Hong et al ( 2009 ) and transformed it into the wild-type plants . We observed that all 21 independent lines transformed with 1 . 7 kb length promoter-GUS we examined exhibits similar expression pattern , and the two versions of promoter ( 1 . 4 and 1 . 7 kb in length ) exhibit very similar expression patterns ( Figure 3 , Figures S5 and S6 ) . One possibility for the discrepancy between the two studies is due to the over-staining of GUS of tissues in study of Hong et al . ( 2009 ) [79] . Phloem is composed of sieve elements and companion cells . An analysis of the cell-specific expression pattern of JMJ18:GFP in phloem indicated that JMJ18 is predominantly expressed in phloem companion cells ( Figure 3I , 3K , and Figure S5 ) . This result was confirmed by examining the cellular localization of tagged JMJ18 in the phloem using Arabidopsis coexpressing JMJ18:JMJ18-RFP and SUC2:JMJ18-GFP . SUC2 is a companion cell-specific gene in Arabidopsis [76] , [80] . JMJ18-RFP and -GFP were colocalized in the same cell in the roots of the transgenic plants ( Figure 3M–3P ) , indicating that JMJ18 is predominantly expressed in companion cells . Thus , our results reveal that JMJ18 is a companion cell-dominant histone H3K4 demethylase in Arabidopsis . In addition , the expression of JMJ18 increased during vegetative development , reaching its high level before the formation of the floral meristem ( Figure 4A ) . This suggests that JMJ18 encodes a companion cell-dominant , developmentally-regulated histone demethylase in Arabidopsis . To determine the role of JMJ18 in Arabidopsis development , we characterized three jmj18 T-DNA insertion mutants . All three mutants displayed a late-flowering phenotype under LD and SD conditions ( Figure 2C , 2D and Table 1 ) . The knock-down lines for JMJ18 using both RNAi and amiR also exhibit delayed flowering ( Figure S3 and Table S1 ) . The late-flowering phenotype in jmj18 is complemented by transforming JMJ18:JMJ18-GFP to jmj18 ( Figure S2 ) . However , the flowering phenotype was weak , and the changes in the expression of flowering marker genes in jmj18 were very mild ( Figure 2E–2G , Figures S3 and S4 ) . To determine the reason for the weak phenotype of jmj18 , we further characterized the three jmj18 alleles . We found that the T-DNA was inserted at the 3′-end of JMJ18 , behind the JmjN , JmjC , and zinc-finger domains , in both jmj18-1 and jmj18-2 ( Figure 2A ) . Partial transcripts of JMJ18 were detected in jmj18-1 and jmj18-2 ( Figure 2B ) . In addition , the truncated JMJ18 in jmj18-1 and jmj18-2 was functional in planta ( Figure 6B and 6C ) . For the third allele , although the T-DNA was inserted in the fourth exon of JMJ18 , full-length JMJ18 mRNA was detected at reduced levels in jmj18-3 ( Figure 2B ) . These results indicate that the three jmj18 alleles were weak mutants . Unfortunately , no other allele for jmj18 is currently available . However , the increase in JMJ18 activity caused by the overexpression of JMJ18 significantly enhanced flowering in Arabidopsis ( Figure 4B , 4C; Figure 6B , 6C; Figure 10A; Table 2 and Table 3; and ) . This observation was true for all JMJ18 overexpressors driven by the constitutive CaMV35S promoter , companion cell-specific SUC2 promoter , and endogenous JMJ18 promoter ( Figure 4B , 4C; Figure 6B , 6C; Figure 10A; Table 2 and Table 3; and Figure S12A , S12B ) . The above data suggest that JMJ18 is a flowering time regulator in Arabidopsis . FLC is a central repressor of flowering in Arabidopsis that works in part through the repression of a companion cell-specific flowering activator , FT [55] . FLC is expressed in both the SAM and companion cells , and the expressed FLC in both tissues contributes to flowering time control in Arabidopsis [55] . The regulation of FLC expression in the SAM has been extensively studied [11] , [24] , [43] , [53]; however , little is known about the regulation of FLC expression in companion cells . In the present work , we found that JMJ18 was predominantly expressed in companion cells in vegetative tissue ( Figure 3 , Figures S5 and S6 ) . In addition , we confirmed that the expression pattern of JMJ18 was strongly negatively correlated with that of FLC , but strongly positively correlated with that of FT , during vegetative development in Arabidopsis ( Figure 4A and Figure 7D ) . These results suggest that JMJ18 works as an endogenous developmental signal to regulate the expression of FLC and FT in the control of flowering time . We further found that JMJ18 binds FLC chromatin ( Figure 9F ) , and decreases the levels of H3K4me3 and H3K4m2 in FLC chromatin ( Figure 9B , 9C; Figures S11A and S12E–S12F ) . In addition , an increase in JMJ18 abundance obviously decreased the mRNA levels of FLC and MAFs ( Figure 4E; Figure 7A , 7C , 7D; Figure 10B , 10C; and Figure S12C ) , and obviously increased the expression of FT at both the mRNA and protein levels ( Figure 4D , 4E; Figure 10D; Figure S12D ) . Furthermore , the promotion of flowering time by JMJ18 was mainly dependent on functional FT ( Figure 5A–5C ) , but was not enhanced by mutation in FLC ( Figure 7F , 7G , and Table 2 ) . These results indicate that JMJ18 works as an endogenous , companion cell-dominant developmental signal that directly represses FLC expression and indirectly induces FT expression in companion cells to control flowering time during vegetative development in Arabidopsis . It was noted that overexpressed JMJ18-GFP in companion cells reduced both levels of H3K4me3 and H3K4me2 on FLC , but only H3K4me3 on MAFs ( Figure 9B–9E and Figure S12E–S12F ) . The difference of the decrease in the levels of H3K4 methylation between FLC and MAFs in JMJ18 overexpression line may suggest that JMJ18 is more important for the regulation of FLC than MAFs in Arabidopsis . In Arabidopsis , the flowering transition is a complicated process . It needs to integrate the environmental cues and internal signals; in addition , these signals will be sensed and regulated in different tissues or organs in the plant [81] , [82] . There are two main tissues involved in the flowering control: the vascular tissue in the leaf and the SAM [37] , [83] . The character of the apical meristem is determined not only by the processes occurring in apical meristem , but also by the signals transmitted from vascular tissue . Thus , different flowering regulators should be functional and regulated in distinct tissues or steps , integrated in the SAM for the precise control of flowering time . As flowering regulators , the JmjC domain-containing demethylases are functional in both vascular tissue and SAM , and target different genes . Since REF6 is characterized as a H3K27 demethylase and represses FLC in both SAM and leaves , FLC can not be the direct target of REF6 [31] , [70] . However , its homolog ELF6 , an H3K4 demethylase , is expressed in leaves and targets FT to repress flowering [28] . Another H3K4 demethylase , JMJ14 , is restricted in leaves to repress flowering by decreasing the expression of the floral integrators including FT , SOC1 , LFY and AP1 [28] , [29] , [69] . In this report , we demonstrated that JMJ18 is a H3K4 demethylase and specially expressed in the vascular tissue ( Figure 1 , Figure 3 , Figure 9; Figures S5 , S6 , S11 , and S12E–S12F ) . The expression of JMJ18 is developmental-regulated ( Figure 4A ) . JMJ18 directly represses the expression of FLC and releases the expression of FT in vascular tissue ( Figure 4 , Figure 5 , Figure 7 , Figure 9 , Figure 10 , and Figure S12 ) , then the released FT is transmitted from the vascular tissue to the SAM to stimulating flowering . These results suggest that plants may have evolved to the point where they use a family of proteins with different expression patterns which target various genes to integrate environmental cues and internal signals to precisely control flowering time . Thus , this study provides novel insight into the regulation of FLC expression in companion cells and the epigenetic control of the floral transition in Arabidopsis .
The Arabidopsis plants used in our experiments were of the Columbia-0 ecotype except for FRI . The seeds were first sterilized with 2 . 25% bleach , then washed three times with water , stratified for three days at 4°C , and then put on Murashige and Skoog ( MS ) medium ( Sigma-Aldrich ) containing 1% sucrose and 0 . 3% phytagel ( Sigma-Aldrich ) . Following ten days of growth under LD ( 16 h of light , 22°C/8 h of dark , 18°C ) or SD ( 8 h of light , 22°C /16 h of dark , 18°C ) conditions in a growth chamber ( Percival CU36L5 ) under a cool white fluorescent light ( 160 µmolm−2 s−1 ) , the plants were transplanted to soil and grown in a growth room under LD ( 16 h of light , 22°C/8 h of dark , 18°C ) or SD ( 8 h of light , 22°C/6 h of dark , 18°C ) conditions at 50% relative humidity . jmj18-1 ( SALK_073442 ) , jmj18-2 ( GABI-649D05 ) , jmj18-3 ( SAIL-861-F02 ) , ft-11 ( GABI-290E08 ) , and co-11 ( SAIL_24_H04 ) were obtained from the ABRC . flc-3 , fd-5 , and FRI-Col were described previously [44] , [64] , [84] . All mutations were confirmed by PCR and sequencing . To construct JMJ18:GUS , 1 , 361 and 1667-bp genomic sequence upstream of the JMJ18 ATG were PCR-amplified and fused with GUS . The primers used for the two versions of promoter were forward for 1 , 361 bp 5′-CTGCAGACAAGACAGAAGAAGCAGGAAAC-3′ , 1667 bp 5′-CTGCAGTGCGAGTTCTTCTTTCAATGTG-3′ and reverse for both , 5′-TCTAGAATGAATTGAAAAATCAATACTACTCAA-3′ . The SUC2 promoter was cloned as described previously [76] , [80] using the primers SUC2 promoter forward 5′-CTGCAGAAAATCTGGTTTCATATTAATTTCA-3′ , and reverse 5′-TCTAGAATTTGACAAACCAAGAAAGTAAGA-3′ . We used the following primers to amplify the full-length JMJ18 CDS: JMJ18 forward 5′-TCTAGAATGGAAAATCCTCCATTAGAATC-3′ , and reverse , 5′-GGATCCCCATCAAATCTACTCCGAAAAGTT-3′ . GFP , RFP , or 6×c-myc was fused to the C-terminus of the JMJ18 CDS to produce tagged JMJ18 . To construct 35S:NC , 35S:NCZ , 35S:T , 35S:JMJ18 and 35S:ZY , the following primers were used to amplify the truncated or full-length JMJ18 CDS: JMJ18 forward 5′-TCTAGAATGGAAAATCCTCCATTAGAATC-3′; JMJ18 NC reverse 5′-GAGCTCTTAATATAGTTCCACAGCGTTCTGTC-3′; JMJ18 NCZ reverse GAGCTCTTAACCTTCCTTTAACTTCTTCTCCTC-3′; JMJ18 T reverse , 5′-GAGCTCTTAAACTATAGAGGGTGAGAGGAAACC-3′; JMJ ZY forward , 5′-TCTAGAATGCAGAACGCTGTGGAACTATATAG-3′ and JMJ18 CDS reverse 5′-GAGCTCTTACATCAAATCTACTCCGAAAAGTT-3′ . For plant transformation , the constructs were transformed into Agrobacterium tumefaciens strain GV3101 . Plants were transformed using the floral dip method [85] . Transformants were selected on MS medium containing kanamycin or hygromycin . Single insertion lines were selected based on the segregation of antibiotic resistance . Full-length and truncated ( JMJ18Δ420–432 bp and JMJ18Δ420–474 bp ) versions of JMJ18 were generated by PCR amplification from Arabidopsis cDNA and cloned into the pFastBac expression vector ( Invitrogen ) . Baculoviruses expressing the various proteins were generated by transfecting the appropriate vector into High5 insect cells . The proteins were purified using Ni-NTA affinity resin ( Qiagen ) . Select fractions were concentrated and purified by size-exclusion chromatography . The histone demethylase assay and MALDI-TOF mass spectrometry were performed as described previously [21] . About 2 µg of purified full-length or truncated recombinant JMJ18 were incubated with 10 µM peptides at 37°C for 3 h in histone demethylase reaction buffer ( 50 mM Tris-HCl [pH 7 . 5] , 150 mM NaCl , 50 µM [NH4]2Fe[SO4]2 , 2 mM ascorbic acid , and 1 mM α-KG ) . The reaction was stopped by freezing , and the mixture was diluted for mass spectrometry . The peptides used were: H3K4me3 ARTK ( me3 ) QTARKS , H3K4me2 ARTK ( me2 ) QTARKS , H3K36me3 ATGGVK ( me3 ) KPHRYR , H3K36me2 ATGGVK ( me2 ) KPHRYR , H3K9me3 ARTKQTARK ( me3 ) STGGKAPRAQ , H3K9me2 ARTKQTARK ( me2 ) STGGKAY , H3K27me3 ATKAARK ( me3 ) SAPATG , and H3K27me2 ATKAARK ( me2 ) SAPATG . GUS staining was performed as described previously [86] , [87] . Twenty-four independent JMJ18:GUS T2 lines were used for histochemical analysis , with at least ten individual plants observed for each line . Pictures were taken using a stereomicroscope ( Leica MZ16 ) or light microscope ( Zeiss Imager M1 ) . For the observation of GFP fluorescence , roots were cut from seedlings grown on MS medium under LD conditions for seven days . For JMJ18:JMJ18-GFP , 20 independent lines were observed , with at least ten individual plants analyzed per line . Images were collected using a Zeiss LSM 510 Meta confocal laser scanning microscope as described previously [80] . JMJ18:JMJ18-RFP was constructed to pCAMBIA2300 and introduced into SUC2:JMJ18-GFP ( in pCAMBIA1300 ) transgenic plants . The double transformants were selected on MS medium containing kanamycin and hygromycin . Transgenic lines expressing JMJ18-GFP and -RFP were selected and photographed using a Zeiss LSM 510 Meta confocal laser scanning microscope . Total RNA was isolated from the plant materials indicated in the text using RNAiso plus ( Takara ) and treated with RNA-free DNase ( Promega ) to remove all remaining DNA . Three micrograms of total RNA were used to synthesize first-strand cDNA with a reverse transcription kit ( Fermentas ) . The cDNAs were diluted to 60 µl with sterilized water . One microliter of diluted cDNA was used for real-time PCR amplification with SYBR Premix Ex Taq ( Takara ) . Three biological replicates were performed to calculate the mRNA abundance . Standard deviations were calculated from the replicates . The primers used were listed in Table S3 . Plant tissue ( 1 . 5 g ) was collected after growth under LD conditions for eleven days . ChIP was performed as described previously with three biological replicates [88] , [89] . The results are shown as absolute enrichment compared to the input or to total H3 . The antibodies used were: anti-H3K4me3 ( Upstate 04-745 ) , -H3K4me2 ( Upstate 07-030 ) , and -c-myc ( Sigma-Aldrich M4439 ) . The primers used to measure the amount of DNA from the ChIP products were listed in Table S4 . Purified recombinant JMJ18 from insect cells and FT from E . coli were used as antigens to immunize rabbits . Each rabbit was immunized once every fourteen days with 1 mg of protein . After four successive immunizations , the anti-serum was examined by ELISA and Western blotting using total protein from the mutant and overexpressor lines . For immunoblotting , Arabidopsis seedlings were ground to a powder in liquid nitrogen then homogenized in extraction buffer ( 10 mM Tris-HCl [pH 7 . 5] , 150 mM NaCl , 10 mM MgCl2 , 1% Nonidet P-40 , complete protease inhibitor [Roche] , and 1 mM phenylmethylsulfonyl fluoride ) . The extracts were then centrifuged , the pellet removed , and the supernatant boiled in 6× SDS sample buffer . The proteins in the samples were separated by 8% SDS-PAGE , transferred to polyvinylidene difluoride membranes ( Millipore ) , and detected using different antibodies . The antibody used for Tubulin detection was anti-α-Tubulin ( Sigma-Aldrich T5168 ) . To knock-down the expression of JMJ18 , a double strand RNA and artificial microRNA interference approaches were used . For a double strand RNA RNAi , a unique 616 bp JMJ18 sequence was amplified by PCR . The gene-specific primers were: forward 5′-GGGGTACCTCTAGAGGTGAAGGCTCTTTGGGAACTC-3′ , reverse 5′-CGGGATCCGAGCTCCTCGACCGATACACCAAGGTTC-3′ . The self-complementary hairpin RNA was constructed to pTCK303 as described by Wesley et al . ( 2001 ) [90] . Three primer pairs were designed through the web ( http://wmd2 . weigelworld . org/cgi-bin/mirnatools . pl ? page=1 ) for artificial microRNA interference assay . The primers are: amiR-s , 5′-GATGAGTCTTTAAAATGCAGGGCTCTCTCTTTTGTATTCC-3′ , amiR-a , 5′-GAGCCCTGCATTTTAAAGACTCATCAAAGAGAATCAATGA-3′ , amiR*s , 5′-GAGCACTGCATTTTATAGACTCTTCACAGGTCGTGATATG-3′ , amiR*a , 5′-GAAGAGTCTATAAAATGCAGTGCTCTACATATATATTCCT-3′ . The constructs for amiRNAi were established by following the protocols from Schwab et al . ( Schwab et al . , 2006 ) . The resulting three constructs were delivered to wild-type through agrobacterium–mediated transformation to generate JMJ18 amiRNA interference lines . Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: ACTIN ( At5g09810 ) , CO ( At5g15840 ) , FLC ( At5g10140 ) , FT ( At1g65480 ) , FRI ( At4g00650 ) , JMJ18 ( At1g30810 ) , MAF1 ( At1g77080 ) , MAF2 ( At5g65050 ) , MAF3 ( At5g65060 ) , MAF4 ( At5g65070 ) , MAF5 ( At5g65080 ) , SUC2 ( At1g22710 ) and TSF ( At4g20370 ) . | Flowering is an important developmental transition during plant life cycle and the key process for production of the next generation . Flowering time is controlled by both intrinsic developmental and environmental signals . FLC and its target FT work as repressor and activator , respectively , to regulate flowering time in Arabidopsis; thus the regulation of FLC and FT expression is the key for the control of floral transition . Epigenetic modifications are critical for transcription regulation . Here , we show that a novel JmjC domain-containing histone H3K4 demethylase , JMJ18 , is a key regulator for the expression of FLC and FT in companion cells and flowering time . JMJ18 is dominantly expressed in vascular tissue; its temporal expression pattern was developmentally regulated , and negatively and positively correlated with FLC and FT , respectively . JMJ18 mutation leads to weak late-flowering , while JMJ18 overexpressor exhibited an obvious early-flowering phenotype . JMJ18 binds to chromatin of FLC , represses its expression , and releases expression of FT in companion cells . Our results suggest that JMJ18 is a developmentally regulated companion cell–dominantly expressed signal to control flowering time by binding to FLC—reducing level of H3K4 methylation in FLC and repressing expression of FLC—thereby promoting expression of FT in companion cells during vegetative development in Arabidopsis . | [
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] | 2012 | A Companion Cell–Dominant and Developmentally Regulated H3K4 Demethylase Controls Flowering Time in Arabidopsis via the Repression of FLC Expression |
Determining the fundamental architectural design of complex nervous systems will lead to significant medical and technological advances . Yet it remains unclear how nervous systems evolved highly efficient networks with near optimal sharing of pathways that yet produce multiple distinct behaviors to reach the organism’s goals . To determine this , the nematode roundworm Caenorhabditis elegans is an attractive model system . Progress has been made in delineating the behavioral circuits of the C . elegans , however , many details are unclear , including the specific functions of every neuron and synapse , as well as the extent the behavioral circuits are separate and parallel versus integrative and serial . Network analysis provides a normative approach to help specify the network design . We investigated the vulnerability of the Caenorhabditis elegans connectome by performing computational experiments that ( a ) “attacked” 279 individual neurons and 2 , 990 weighted synaptic connections ( composed of 6 , 393 chemical synapses and 890 electrical junctions ) and ( b ) quantified the effects of each removal on global network properties that influence information processing . The analysis identified 12 critical neurons and 29 critical synapses for establishing fundamental network properties . These critical constituents were found to be control elements—i . e . , those with the most influence over multiple underlying pathways . Additionally , the critical synapses formed into circuit-level pathways . These emergent pathways provide evidence for ( a ) the importance of backward locomotion , avoidance behavior , and social feeding behavior to the organism; ( b ) the potential roles of specific neurons whose functions have been unclear; and ( c ) both parallel and serial design elements in the connectome—i . e . , specific evidence for a mixed architectural design .
Understanding the fundamental architectural design of nervous systems will provide insights into nervous system function and how mental illness arises from its dysfunction . The design derives from a trade-off between physical wiring costs and functional complexity [1–4] . Nervous systems mediate behavior to achieve the organism’s goals , and thus the network must be composed of behavioral circuits ( e . g . , to forage , to avoid danger , to mate ) . The punitive nature of wiring costs , however , demands highly efficient solutions , producing short and shared pathways whenever possible . How then do nervous systems solve the problem of having distinct behavioral circuits , on the one hand , and near maximized sharing of pathways and information , on the other ? To determine this , it is useful to examine a nervous system that is both well specified and highly tractable , making the nematode roundworm Caenorhabditis elegans an attractive model system , especially since its complete connectome is available for analysis . Considerable progress has been made in delineating behavioral circuits [5–9] of the C . elegans including , for example , mechanosensation [10–12] , chemosensation [13] , feeding [14 , 15] , exploration [14 , 15] , egg laying [16–18] , mating [7 , 19–22] , and tap withdrawal [23 , 24] ( i . e . , avoidance of a vibrating “tap” ) . These circuits appear to follow a general pattern of sensory neurons ( S ) to 1–3 layers of interneurons ( I ) , with the final interneuron layer being command interneurons ( i . e . , direct control of motor neurons ) , to one or more motor neurons ( M ) that directly control muscle activity ( i . e . , S → I → M pattern ) [7 , 14 , 25] . Some subcircuits have also been characterized , for example , for both forward and backward locomotion , and more generally for foraging behavior [6 , 14] . Nonetheless , many details remain unclear , not only with respect to the specific functions of every neuron and synapse , but also the extent to which the behavioral circuits are largely separate and parallel versus having a more integrative and serial design ( i . e . , input → central processing → output ) [7 , 14] . These issues are especially exacerbated in the C . elegans connectome because it is highly interconnected , with evidence that behavioral functions such as navigation entail activity across a large fraction of the nervous system [26] . Graph theoretic analysis of neuronal networks provides a normative approach to help quantitatively specify the network structure and dynamics—in which neurons are network nodes , and synapses ( whether chemical or gap junctions ) are connections or edges between the nodes . Important network properties of the connectome have been established , including a layout that is nearly optimized to minimize wiring costs [1 , 3 , 4 , 8 , 27] . The near optimal wiring suggests strong selection pressure on efficiency , and thus , near optimally efficient information processing: e . g . , path sharing whenever possible . And characteristics have been identified that reflect evolved complexity in light of this constraint , especially small-worldness ( i . e . , high clustering for functional specialization , together with significant interconnections across the network for efficient information integration and transfer ) , and nonrandom distributions of highly connected neurons as well as those that support high volume traffic [4 , 7 , 8 , 27–30] . Larger information processing structures in the network have also been identified , such as a set of highly connected interneurons that form hubs and “rich clubs” that integrate information to drive locomotion [27 , 31] . Modularity analyses have also attempted to delineate the main functional modules based on high interconnectivity within modules versus lower across them [8 , 27 , 28] . Although specific results across studies differ based on the modularity algorithm used , these studies demonstrate that identified modules align with known behavioral circuits , providing some evidence for separable behavioral circuits . At the same time , the results also show elements of an integrative and serial design ( i . e . , to the extent modules align with input , central , and output processing layers ) . Taken together , current findings may suggest a fundamentally mixed architectural design . An important next step , then , is to build on these findings by unequivocally identifying specific critical substructures of the connectome , which will in turn help clarify the architectural design principles . To this end , we take an evolutionary and developmental ( evo-devo ) perspective and seek to identify the most critical elements of this information processing system , i . e . , those whose addition provided the most ‘bang for the buck’ for information propagation , integration and processing [32–34] . To determine this , in the current study we conducted a computational network-vulnerability analysis that systematically removed network components and calculated the effects of each loss on key network properties , with the view that functional loss upon removal reveals what their addition enables . We also take the perspective that critical components in real-world nervous systems may not always depend on local individual properties independent of context , such that significance may emerge from this context . The vulnerability analysis also enables an assessment of these contextual effects [35 , 36] . Using this approach , we tested all neurons and synapses to assure no misassignments: i . e . , some considered critical when not ( e . g . , highly connected but redundant ) , or those that may appear not , but in fact are , from a more global network perspective .
We analyzed the most recent published connectome of the C . elegans hermaphrodite as a combined ( i . e . , chemical synapses and gap junctions ) , directed , and weighted network ( see Methods and S1 Text ) [5 , 15 , 29] . The S1 Text contains an examination of the general network properties of the intact network , including circular wiring diagrams , comparisons of the three neuron classes ( sensory , interneurons , and motor ) on different network properties , and robustness and information propagation analyses . We then sought to identify the most critical neurons in the C . elegans connectome . To do this , we asked which ones had the greatest impact on network function when they are lost . Thus , we conducted a vulnerability analysis , attacking each of the 279 neurons in the connectome and calculating the change in global network properties when the neuron is removed ( see Methods for analysis details and explanation of notation used; S1 File ) [35 , 36] . Three fundamental properties that determine efficient information processing in a network are ( 1 ) specialized processing in the form of local clusters , ( 2 ) global information integration and coordination via short path lengths between nodes , and ( 3 ) the control of information flow by providing a route that is shared by multiple pathways . These three properties were assessed by calculating the network vulnerability , V , ( i . e . , the relative change in value after elimination of the network component ) with respect to the clustering coefficient ( VC ) , global efficiency ( VE ) , which assesses the network’s average path length , and the average betweenness centrality ( VB ) , which represents the average of number of shortest paths that travel through each network component ( i . e . , neurons here , synapses below ) ( detailed in the Methods and S1 Text ) . To determine the critical neurons , we reasoned that the most influential ones should yield network vulnerability scores after deletion that are clear outliers from the rest of the population , reflecting extreme functional loss in the network with respect to the specific fundamental network property . The most conservative criterion used in the Towlson et al . [27] study to identify the rich club was selecting those that were 3SD or more above the average , which we adopted here ( Fig 1; S2 File ) . This stricter criterion enabled the identification of the most critical neurons , it provided a standard value often used in identifying outliers ( 3SD ) , and it continued the convention established by Towlson et al . [27] , enabling a direct comparison of findings across studies . Thus , we defined a neuron as critical for a given network property when a deletion of the neuron produced a vulnerability over three SDs above the mean vulnerability value of all 279 neuronal attacks . Our analysis identified 2 , 5 , and 8 critical neurons for VC , VE , and VB , respectively , yielding 12 ( i . e . , 4 . 3% ) critical neurons in all ( Table 1; S1 and S2 Files ) . Of the 12 critical neurons , 9 are interneurons , 2 are motor neurons , and 1 is sensory , reflecting the general importance of interneurons in information processing in the C . elegans connectome , as has been reported previously [27] . Moreover , of the 7 interneurons with known function , 5 are considered command interneurons that control locomotion [27] . To obtain further insight into the critical neurons , we examined their development times ( Fig 2A; Table 1 ) [27] . All critical neurons for both the VC ( 2 interneurons ) and VE ( 5 interneurons ) analyses developed early . For VB , 5 interneurons and one motor neuron ( DA01 ) developed early , whereas 2 neurons developed late ( after hatching ) : one sensory ( AQR ) and one motor ( VD01 ) . Taken together , all interneurons and 1 motor neuron ( DA01 ) developed early , whereas , one sensory and 1 motor neuron developed late . Thus , as found by Towlson et al . [27] , we also found that all of the critical interneurons formed in the early development phase before twitching ( 470 min after fertilization; first visible motor activity ) , presumably attesting to their importance in establishing the connectome topology . In addition , we note that the early developing motor neuron ( DA01 ) is less peripheral than the late developing one with respect to functional connectivity ( VD01 ) in the fully formed adult connectome [6] . Thus , our results also suggest a general ‘inside-out’ developmental pattern , with the most peripheral neurons tending to develop last , presumably requiring environmental experience after hatching [27 , 37] . Examining the specific results for VC , E , B , the AVA neurons stand out , being critical for multiple network properties ( Table 1; S1 File ) . AVAL and AVAR were critical for both VC and VE , meaning that they most affect network clustering and efficiency . AVAL was additionally critical for VB , although this measure ( i . e . , VB ( i ) ) actually decreased when AVAL was removed ( without taking the absolute value ) ( S1 File ) . This decrease indicates that a main pathway segment was removed with AVAL , leaving longer alternative routes ( resulting in increased B ( i ) overall , XB ( i ) ) . Thus , the results show that the AVA neurons ( and AVAL in particular ) are dominant ones in the connectome , critically influencing network clustering , interconnectivity , and information flow . Given AVA’s known role in backward movement control , these results suggest that backward movement likely plays a key role in locomotor maneuverability in potentially multiple biological functions; and to the extent backward movement in response to environmental stimuli might be construed as avoidance behavior , the results might also highlight the significance of avoidance behavior in general . We discuss possible biological function further below . For VE , 5 neurons were found to have the greatest effect on global efficiency: besides the two AVA neurons , DVA ( I ) and PVCL/R ( I ) ( i . e . , both left and right ) were also identified ( Table 1; S2 File ) . All five critical neurons for VE are interneurons , which would indeed be those expected to have significant influence on global efficiency ( yet only these 5 were found to be critical ) . Since all have been implicated in either forward locomotion for avoiding aversive stimuli or backward locomotion , it suggests that the most influential nodes on global efficiency in the connectome perform locomotion control , and again , especially with respect to backward locomotion and avoidance behavior . For VB , 8 neurons were found to have the greatest effect on betweenness centrality: 1 sensory ( AQR ) , 5 interneurons ( AVAL , AVER , AVHL , DVC , PVPR ) , and 2 motor ( DA01 , VD01 ) ( Table 1; S1 File ) . Thus , with respect to network information propagation , all neuronal types ( S , I , and M ) , related to potentially multiple biological functions are implicated , which we examine further in the synapse analysis . During this study , AVHL and DVC were conspicuous critical neurons whose biological function had been unknown ( there has since been new evidence for DVC involvement in locomotion ) [38] . We also note that due to the integrative nature of the C . elegans connectome , it is possible that many neurons ( especially interneurons ) participate in multiple functions . We therefore conducted an analysis based on known biological function of neighbors to help determine the possible functions of AVHL and DVC ( S1 Text; S8 Fig ) . Indeed , our analysis suggests multiple possible functions for both neurons , most notably ventral cord pioneering , chemotaxis , and locomotion for AVHL , and locomotion and ventral cord pioneering for DVC . We discuss possible biological functions for these neurons further below . We also note that although ventral cord pioneering is important for axon guidance during neuronal development of the C . elegans connectome , if these neurons remain integrated in the connectome in the fully developed adult , they likely participate in functions other than ventral cord pioneering . This consideration would then point to chemotaxis and locomotion for AVHL , and locomotion for DVC in the adult neuronal network . We next attempted to better understand the general characteristics of the critical neurons . For example , were they the most highly connected ? Degree does appear important , with 6 critical neurons being in the top 10 for degree ( these 6 being members of the rich club [27] ) . However , we also found 6 other critical neurons , and none were among the next 18 for degree , with all 12 critical neurons being only in the top 113 ( of 279 total ) ( Tables D and E in S1 Text ) . To further examine the properties of the critical neurons , we first compared all critical neurons to the noncritical ones on multiple network factors using the Mann-Whitney U test ( Fig 2B ) . The critical neurons have higher degree ( D ) , strength ( Str ) , average weight ( AW ) , nodal efficiency ( E ( i ) ) , and nodal betweenness centrality ( B ( i ) ) values than the noncritical neurons . Only the nodal clustering coefficient ( C ( i ) ) showed no differences between the critical and noncritical groups . Thus , as a group , the critical neurons were more connected , more strongly connected , more closely connected to others in the network ( i . e . , the shortest average path lengths ) , and generally positioned as important control centers regarding the number of shortest pathways passing through them and thus the number of pathways influenced by them . These results support others that have shown the significance of nodal network properties such as degree and betweenness centrality in the connectome [7 , 8 , 27–29] . Overall , multiple factors distinguish the critical neurons , but it is important to clarify their impact on each vulnerability measure VC , E , B individually . One might assume that each vulnerability measure would be most affected by its corresponding nodal property: e . g . , individual neuron values for clustering coefficient ( C ( i ) ) determining global VC . However , this is not what we found—in fact in no case was the same local nodal property the leading factor influencing global network vulnerability . To evaluate the relationship of global VC , E , B to nodal network properties of the intact network , we examined correlations of D , Str , AW , C ( i ) , E ( i ) , B ( i ) to VC , E , B without taking the absolute values ( Table F in S1 Text ) . The S1 Text contains a detailed discussion of the results . In sum , neurons most critical for network clustering ( VC ) send dual ( or more ) projections to other neurons that project to each other , producing the largest clusters . Those most critical for global efficiency ( VE ) generally have the most influence—or control—in the network , regarding the number of pathways sharing them and thus the number influenced by them . Thus , the critical VE neurons do not necessarily have high individual efficiency values , but they do normally have high individual betweenness values . And those critical for network betweenness ( VB ) also appeared to be those with most control in the network , but the effect is more contextual—it depends on the other possible available routes when the node is lost ( detours ) , the loss in nodal betweenness centrality of the attacked node , and the changes generated in others due to the loss of the attacked connections . Most critical neurons for VB also tended to be connected to other nodes with high nodal betweenness centrality , creating a linked control structure . The previous analysis of critical neurons determined the significance of each neuron’s constellation of connections to the network . An individual synapse analysis provides a finer grain analysis of each constituent element [39 , 40] . We therefore next conducted a vulnerability edge analysis by attacking each one of the 2 , 990 synaptic connections ( S3 File ) . Because the criterion used to identify critical neurons ( 3SD or above ) proved too liberal for the synapses ( potentially labeling too many as most critical and obscuring differences among them ) , we used the following procedure . First , for each list of VC , VE , and VB values ( Fig 3A–3C ) , we transformed the values to the distance from the mean in SD integer units ( i . e . , 1SD , 2SD , etc . ) , and ordered this list from highest to lowest . Second , we created a histogram distribution for each ordered list and identified the first major change in the slope ( see Fig 3D ) . Third , we compared the SD values of the first change points for VC , VE , and VB and used the most conservative value ( the highest SD distance ) as the criterion for all three vulnerability measures . From this procedure , 6SD was identified from the VE values and used as the criterion to identify the critical synapses ( Fig 3A–3D ) . The analysis identified 7 , 13 , and 17 synapses as critical for VC , VE , and VB , respectively , implicating 29 ( i . e . , ~1% ) critical synapses ( Table 2; Fig 3 ) . 28 of the 29 synapses are excitatory , suggesting that they promote information transmission . The one exception , VD01 ( M ) → DVC ( I ) , is inhibitory , which we note further below . In addition , 26 of the 29 pairs ( 89 . 7% ) contain at least one interneuron , 14 ( 48 . 3% ) contain at least one motor neuron , and 5 ( 17 . 2% ) contained one sensory neuron . Thus , the critical synapses also appear to relate predominately to interneurons , but also included motor and sensory neurons . Since inherently costly long-range connections should be minimized , one might expect an overrepresentation of these among the most critical synapses , as they would provide critical waypoints for the dispersed nervous system . Indeed , we did find a bias for long-range connections as calculated by the direct Euclidean distance of soma positions between two neurons: 16 of the 29 ( 55 . 2% ) , compared to roughly 10% in the entire connectome ( Fig 3E ) [1 , 3 , 4] . These connections were similar for all vulnerability results: for VC , 4/7 ( 57% ) ; VE , 6/13 ( 46% ) ; VB , 9/17 ( 53% ) . Finally , regarding connectome laterality , we did find asymmetries between the left and right neuronal pairs , in which certain neuron types were critical only for the left or right ( e . g . , neurons AVER , AVHL , PVPR ) . Overall , however , we found that there is no clear overall laterality bias in the critical neurons ( 4 left , 5 right ) or synapses ( source node: 10 left , 12 right; sink node: 6 left , 8 right ) . The left/right differences that we did find , however , warrant future examination of laterality in the C . elegans connectome [41] . We next examined the vulnerability edge results for each network property VC , E , B more closely ( Table 2 ) . For VC , of 7 total , 2 were AVA connections to each other ( i . e . , AVAL ( I ) ←→ AVAR ( I ) ) , 4 were AVAL/R ( i . e . , both left and right ) to motor neurons DA07 ( M ) and AS08 ( M ) , and 1 was motor to motor ( VA08 ( M ) → DD04 ( M ) ) . With respect to biological function , AVA , DAn , and VAn have well-established involvement in backward locomotor control , and DDn and ASn in the coordination of locomotion ( between forward and backward ) . Thus , the results show that the most dominant synapses that drive coordinated local processing occurs with locomotor control , and in particular , backward locomotion . The analysis of VB identifies 17 critical synapses with the largest influence on betweenness centrality: 15/17 ( 88% ) contain at least one interneuron in the pair , 6/17 ( 35% ) at least one motor neuron , and 3/17 ( 18% ) have one sensory neuron , with 7 of the 17 ( 41% ) links being interneuron to interneuron ( I → I ) , and the remaining 10 being some combination of sensory , interneurons , and motor neurons . To better understand the significance of these critical segments in the network for overall betweenness centrality , we examined their distribution . If these segments were independent , they should be fairly evenly distributed throughout the connectome . If , however , there were an interdependency among them , some pattern may emerge . In fact , we found that the synapses formed three separate pathways ( see blue lines in Fig 3E ) . To see these more clearly we collapsed left and right neuron pairs ( e . g . , AVAL or AVAR as AVA ) . Justification for the left/right collapsing is taken from the biological work on the C . elegans , which normally shows symmetric function for neuron pairs [3 , 6] . As shown in Fig 4A , the first AVA-based critical pathway begins with RIB ( I ) → AVE ( I ) → AVA ( I ) and has two routes after this . Route ( a ) ( from RIB ( I ) to VD01 ( M ) ) is in fact a significant segment of the well-established circuit for backward locomotion . Although the role of route ( b ) ( RIB to PVC ) has been suggested to be secondary coordination of backward with forward locomotion , the VB analysis nonetheless suggests that this is an important pathway for locomotor control . As shown in Fig 4B , the second PVP-based critical pathway begins with AQR ( S ) ←→ PVP ( I ) and has two routes after this . For route ( a ) , the results suggest a critical sensory ( AQR ) to motor ( VD01 ) circuit . To date , AQR has been implicated in aerotaxis ( O2 and CO2 ) , regulating social feeding ( i . e . , behavior when individuals aggregate at bacterial patches ) and bordering behavior ( i . e . , aggregation in densest part of bacterial patch ) , and suppressing innate immunity [5 , 42–44] . Since evidence shows that aerotaxis also regulates social feeding [43] , we summarize these functions as the regulation of ( 1 ) social feeding [42 , 43] and ( 2 ) internal immunity responses [44] . The motor neuron VD01 is implicated in locomotion [5 , 6 , 15] . Given that route ( a ) ends in locomotor behavior , the results suggest that the identified circuit is involved in social feeding . Moreover , the results thus predict involvement of both PVP and DVC in this behavioral circuit , whose functional roles in the fully developed adult network to date remain unclear ( although there is evidence for PVP involvement in ventral cord pioneering during development; and there is new evidence for DVC involvement in locomotion ) [38 , 45–47] . More specifically , if we use the general behavioral circuit structure as a guide , S --> I1 --> I2 --> M , it provides further suggestive evidence for the functions of PVP ( I1: sensorimotor integration ) and DVC ( I2: information integration , motor control ) [7 , 14] . The behavioral function of route ( b ) ( AQR ( S ) ←→ PVP ( I ) → AVH ( I ) ) is less clear . However , since route ( a ) implicates PVP in social feeding behavior , this may also implicate route ( b ) in the same , suggesting a possible functional role for AVH , whose function currently remains unknown . The more local analysis of the potential biological functions of AVH and DVC based on the functions of their neighbors ( reported in the S1 Text ) also appears to further corroborate these findings by implicating both AVHL and DVC in locomotor control ( among other possible functions , which is also to be expected ) . Finally , the VB analysis also identified the RMD ( M ) → OLL ( S ) link as a critical pathway segment ( Fig 4C ) . OLL has been implicated in aversive stimulus sensation and RMD in avoidance responses of the nose/head . Interestingly , the motor to sensory link as an important pathway highlights the loop structure in the C . elegans circuitry , with the motor to sensory link closing the loop [7] . This loop structure component suggests that multiple motor signals likely converge on sensory processing , suggesting significant action-influenced perception . Indeed , given the bidirectional links in the two main pathways between motor and interneurons , interneurons among themselves , and interneurons and sensory neurons , the VB results suggest that these are important recurrent connections in the connectome circuitry , and further suggest that recurrent feedback loops for central processing ( middle layers ) and action influences on perception are important general principles of nervous system function [7] . The VE synapse analysis identified 13 critical links with the greatest effect on global efficiency , with all nodes being interneurons or motor neurons , and all connections containing at least one interneuron and over ½ at least 1 motor neuron: 6 I → I , 4 I → M , 3 M → I ( see red lines in Fig 3E ) . The interneuron-to-interneuron links are expected to be critical information processing structures in the network , and the others show the significance of motor control . More specifically , two critical pathways were identified , which were again AVA- and PVP-based ( Fig 5A and 5B ) . Thus , the VE results show that components of both the AVA- and PVP-based pathways are important control segments that have the greatest influence on global network efficiency . Combining the results for all critical neurons , synapses and vulnerabilities , they appear to converge on three main pathways . These pathways are most clearly defined by the VB results ( Fig 4A ) , which identify the synapses with the largest effect on network betweenness centrality , the VE results then highlight the components with the largest effect on global efficiency , and the VC results show where the most critical clustering occurs . First , there is an AVA-based pathway , beginning with RIB ( I ) → AVE ( I ) , and ending with either motor neurons or PVC . This pathway strongly matches the backward locomotion control circuit , attesting to the significance of backward locomotion in the C . elegans nervous system . Second , there is a PVP-based pathway . This pathway is composed of sensory ( AQR ) , interneurons ( PVP , DVC , AVH ) , and a motor neuron ( VD01 ) . The sensory to motor structure reveals a complete behavioral circuit , and implicates social feeding behavior . The third critical pathway segment is RMD ( M ) → OLL ( S ) that again identifies avoidance control as important—in this case , aversive stimulus avoidance with respect to nose/head . The motor to sensory directional link also highlights the importance of the loop structure in the C . elegans circuitry [7] . To better understand the nature of the critical synapses , we examined the two main local properties of edges: strength ( Str ) ( i . e . , the weight of the given synapse ) and edge betweenness centrality ( EBC , a betweenness measure for edges; see S1 Text ) . We first compared the critical and noncritical synapses on Str and EBC using the Mann-Whitney U test ( Fig 4C ) , and the critical synapses indeed showed significantly higher Str and EBC than the noncritical synapses . Thus , like the critical neurons , the critical synapses have stronger connections and were those with the most shortest paths running through them—i . e . , with the highest volume traffic . As stated previously , constituents with such high volume traffic can be considered as control structures by virtue of their influence over the multiple pathways passing through them . To clarify how Str and EBC related to each vulnerability measure VC , E , B , we next examined the correlations ( without absolute value for VC , E , B ) , and all were statistically significant ( Table F in S1 Text ) . The S1 Text contains a detailed discussion of the results . In sum , like neurons , the synapses most critical for betweenness centrality generally have the highest control themselves , and also tend to link control nodes together . For global efficiency , also like neurons , they generally are those with the highest control in the network , regarding the number of pathways sharing them . There were , however , five exceptions for global efficiency and betweenness centrality ( detailed in the S1 Text ) , and they suggest that , like betweenness centrality , global efficiency can also be critically affected by context . Finally , for clustering ( VC ) , critical synapses appeared to participate in shared projections to neighbors , producing clusters .
Function derives from form , and with unrelenting natural selection , nervous systems must achieve complexity as efficiently as possible . Network analysis provides a normative approach to analyze the solution , and vulnerability analysis helps provide a more experimental and less assumption-laden analysis of individual component contributions to information processing . In this study , we tested the effects of attacks on every neuron and synaptic connection in the C . elegans connectome to identify and characterize the most critical constituents of the network . To our knowledge , this is the first study to analyze network robustness for individual node and edge attacks on an entire nervous system . We identified 12 neurons and 29 synapses critical for clustering , information integration and propagation . Although one might have expected the clustering , efficiency , and betweenness values for individual neurons and synapses to be the most important factors determining these same properties at the network level , control structures—i . e . , those that influence multiple others—prove the most important . From an evolutionary-development ( evo-devo ) perspective , the additions of higher-order control structures can produce the largest effects on information processing , providing the most ‘bang for the buck’ . Thus , to most affect clustering , new neurons should project paired synapses to neighbors . To most affect global efficiency , new neurons or synapses should be placed in central positions that shorten the largest number of prior existing pathways . These more centralized neurons not only contribute to overall efficiency and information integration , they also become traffic centers , and thereby have greater influence ( and thus control ) in the connectome . Finally , to most affect network traffic flow ( i . e . , global betweenness ) , new neurons or synapses should provide new pathways that decrease a larger number of path lengths and should also normally link to other control structures , leading to the formation of chains of control units ( i . e . , node1 → node2 → node3 ) . Not only were there critical control structures at the cellular level ( neurons and synapses ) , the synapses organized into larger control structures at a higher circuit level—into full or partial circuits . Thus , the vulnerability analysis seemed to uncover fractal-like control in the network [48] . Yet these network control systems must translate to circuits that accomplish the organism’s biological goals , and there was in fact a close correspondence between the identified critical pathways and biological function . The AVA-based pathway is a large component of the backward locomotor circuitry . Identifying this circuitry as a control pathway with respect to its relative influence in the network suggests that there is high volume use of this circuitry . This in turn suggests that backward locomotion likely has multiple trigger stimuli and is utilized by multiple biological functions that require maneuverability . Indeed , given the apparent need for the worm to reverse prior to changing directions ( e . g . , for omega turns ) , the finding may highlight the significance of backward movement for navigation more generally . At the same time , to the extent that backward movement reflects the avoidance of particular trigger stimuli , it might also point to the importance of avoidance behavior . For the PVP-based pathway , given the sensory to motor structure , as well as the AQR neuron being implicated in social feeding and the VD01 neuron in locomotor control , our analysis appears to implicate a social-feeding circuit as a second major critical pathway in the connectome . Moreover , the pathway result implicates PVP , DVC , and AVH involvement in social feeding—neurons whose complete functional roles in information processing in the adult network remain unclear . The third pathway segment has also been implicated in avoidance behavior . Taken together , our results suggest that the network topology is particularly organized around backward movement , avoidance behavior and social feeding , suggesting a primary significance of these functions to the organism . Indeed , backward movement is likely required for maneuverability in multiple behavioral functions . Thus , the C . elegans appears to have evolved under strong selection pressures for maneuverability , to avoid undesirable and harmful circumstances and to negotiate social interactions . The significance of social behavior would highlight the fundamental importance of behavioral strategies in animals to overcome inevitable competition for resources . With respect to architectural design , the AVA-based pathway has three general components: a second-layer interneuron ( RIB ) , command neurons ( AVE , AVA , PVC ) , and motor neurons . Thus , it is a subcircuit for information integration , sensorimotor integration , motor control , and motor performance . Moreover , beginning with a second layer interneuron ( RIB ) , it suggests that multiple prior circuit segments may use this critical pathway to produce backward locomotion: for example , for multiple avoidance stimuli to tap into this ( backward locomotion ) circuit segment . This finding is comparable to Gray et al . ’s suggestion of a “common substrate” foraging subcircuit [14] . Such subcircuit structures lend support for a serial-based architectural design structure ( i . e . , input → central processing → output ) . At the same time , our second PVP-based pathway contained a sensory to motor structure that suggests a more complete behavioral circuit , and itself provides evidence for separable behavioral circuits , and thus a parallel design structure [7] . Taken together , our findings point to a mixed or hybrid architectural design . Modularity studies of the C . elegans have appeared to find evidence for this mixture—with modules aligning both with known biological function to some degree ( supporting parallelism ) , as well as with the input-output serial design structure to some degree ( e . g . , sensory and first-layer interneurons dominating one module , second-layer interneurons another , and command interneurons and motor neurons another ) [8 , 28] . Our study highlights and extends these findings by identifying specific neurons , synapses and pathways using a vulnerability approach that implicates specific circuit structures as having critical roles in the connectome . Our findings may also extend those by others who have found significant neural activity across a large extent of the nervous system during activities such as locomotion [26] . Even when such global processing occurs , there is likely a hierarchy of importance or influence among the network components , which can be characterized . There are multiple avenues for further investigations . For example , future studies should explore other taxa to help determine the evolutionary trajectory of nervous systems by identifying shared and diverging properties with C . elegans . They could also induce multiple perturbations on the network , such as multiple simultaneous attacks , to help clarify how network components interact to affect network processing properties [31 , 49] . In addition , because our findings derive from theoretical analyses , they require empirical verification , especially to test the hypotheses generated from our study: most notably , the functional roles of PVP ( sensorimotor integration ) , DVC ( information integration , motor control ) , and AVH ( information integration , motor control ) in social feeding behavior ( as well as the other possible roles of DVC and AVH derived in the S1 Text ) . The nematode C . elegans is a valuable model system to identify potentially fundamental design elements of nervous systems . Further specification of these principles in the C . elegans and other nervous systems , including that of humans , will lead to the ultimate goals of a true understanding of nervous system design , and the subsequent medical and technological advances that follow .
We analyzed the published neuronal wiring data of the nematode C . elegans hermaphrodite that has information on 279 neurons ( pharyngeal and unconnected neurons excluded ) with 6 , 393 chemical synapses and 890 electrical junctions [5 , 15 , 29] . We examined the directed and weighted full network , generated by combining the gap junction and chemical synapse networks . The weight of a connection between two neurons was defined as the number of gap junctions and chemical synapse contacts between them . Thus , the total number of directed connections , which we call “synapses” , between all neurons were represented as 2 , 990 synaptic connections with weights . We have also followed the typical naming conventions used for the connectome . Many neurons have symmetric left , right pairs , which are denoted as “L” and “R” . Thus , for example , the so-called AVA neurons have both AVAL and AVAR counterparts , sometimes written as AVAL/R . Motor neurons are organized into classes and numbered , such that “DA01” , for example , is the first motor neuron of class “DAn” , where n represents the number . We denote a synapse with “→” , such as AVAL ( I ) → AVAR ( I ) , and we put neuron type , sensory ( S ) , interneuron ( I ) , or motor ( M ) , in parenthesis when useful . Finally , to save space we sometimes write VC , VE , and VB as VC , E , B . We defined an attack on a neuron in the C . elegans connectome as a deletion of all connections of a target neuron ( also called node ) , but the target neuron was still in the adjacency matrix with zero degree . Furthermore , we also defined an attack on a synapse as a deletion of the target connection ( also called edge ) . Therefore , 279 neuronal attacked networks and 2 , 990 synaptic attacked networks were constructed ( detailed in the S1 Text Methods ) . All attacked networks had a corresponding 279 by 279 adjacency matrix that contained the direction and weight edge information , representing the synaptic connections between neurons . We also examined the connectome’s topological structure with respect to robustness and information propagation potential . These analyses included a characterization of ( a ) leaf nodes ( i . e . , ones that have only one connection ) in the intact network , ( b ) isolation from single neuronal attacks based on network fraction ( i . e . , the extent individual neurons or subnetworks are isolated ) , and ( c ) reachability ( whether each neuron pair in the connectome has a connected path between them ) both in the intact connectome and after neuronal deletions ( detailed in the S1 Text ) . We quantified vulnerability , V , to determine the effect of the loss of a neuron or synapse on global information processing , as measured by three key graph-theoretic structural properties [50]: the clustering coefficient ( VC ) , global efficiency ( VE ) , and the average betweenness centrality of the network ( VB ) . The vulnerability of a network to a particular loss might also be considered the degree of lethality or redundancy , or the functionality enabled by a component’s addition [35 , 36] . Vulnerability with respect to each network property was defined as the relative change in value after elimination of the neuron or synapse , namely Vx ( i ) =|X−X ( i ) |X , ( 1 ) where X is the value of the network property x in the original intact network , and X ( i ) is the value of the property after the attack of neuron i . For each synapse i → j , ( i , j ) was used in place of ( i ) . | One of the most important scientific aims is to uncover the functional design principles of nervous systems . To reach this aim , it is useful to examine a complex nervous system that is both well specified and highly tractable , making the nematode roundworm Caenorhabditis elegans an attractive model system , especially since it is the only complete connectome currently available for analysis . In this computational study , we tested the effects of individual attacks on every neuron and synaptic connection in the C . elegans connectome to identify and characterize the most critical constituents of the network by quantifying the changes in key network properties of the connectome that influence information processing . Our analysis identified 12 neurons and 29 synapses critical to clustering , information integration and propagation . These critical constituents formed circuit-level structures that control network processing in the C . elegans connectome . We believe our study provides a significant advance in the understanding of the network topology of the C . elegans connectome , and provides insights into the fundamental architectural design of complex nervous systems . | [
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] | 2016 | Vulnerability-Based Critical Neurons, Synapses, and Pathways in the Caenorhabditis elegans Connectome |
Rice flowering is controlled by changes in the photoperiod that promote the transition to the reproductive phase as days become shorter . Natural genetic variation for flowering time has been largely documented and has been instrumental to define the genetics of the photoperiodic pathway , as well as providing valuable material for artificial selection of varieties better adapted to local environments . We mined genetic variation in a collection of rice varieties highly adapted to European regions and isolated distinct variants of the long day repressor HEADING DATE 1 ( Hd1 ) that perturb its expression or protein function . Specific variants allowed us to define novel features of the photoperiodic flowering pathway . We demonstrate that a histone fold domain scaffold formed by GRAIN YIELD , PLANT HEIGHT AND HEADING DATE 8 ( Ghd8 ) and several NF-YC subunits can accommodate distinct proteins , including Hd1 and PSEUDO RESPONSE REGULATOR 37 ( PRR37 ) , and that the resulting OsNF-Y complex containing Hd1 can bind a specific sequence in the promoter of HEADING DATE 3A ( Hd3a ) . Artificial selection has locally favored an Hd1 variant unable to assemble in such heterotrimeric complex . The causal polymorphism was defined as a single conserved lysine in the CCT domain of the Hd1 protein . Our results indicate how genetic variation can be stratified and explored at multiple levels , and how its description can contribute to the molecular understanding of basic developmental processes .
Rice ( Oryza sativa L . ) was originally domesticated ~8000 years ago in tropical Asia . Archeological remains and genome re-sequencing indicated southern China as the region of first cultivation , despite the debate regarding the domestication dynamics is still open [1–3] . Although of tropical origin , rice is currently cultivated across a broad latitudinal range [4 , 5] . Expansion to temperate areas required selection of varieties better adapted to local environmental conditions . Tolerance to low temperatures and day length-insensitive flowering have been crucial adaptive traits under selection . Flowering ( or heading date ) is the result of an intricate series of pathways that mediate between environmental inputs and the production of molecules inducing flowering , known as florigens . Photoperiod is the major environmental cue that rice plants utilize to measure seasonal time [6] . Rice , as well as other important cereals including sorghum and maize , is a short day ( SD ) plant in which flowering is induced as the duration of the light phase during the day does not exceed a critical threshold . Under such inductive conditions , expression of HEADING DATE 3a ( Hd3a ) and RICE FLOWERING LOCUS T1 ( RFT1 ) , florigenic genes highly similar to Arabidopsis FLOWERING LOCUS T ( FT ) , is induced in the vascular tissue of leaves [7–11] . Expression of the florigens is triggered by EARLY HEADING DATE 1 ( Ehd1 ) encoding a B-type response regulator protein central to the photoperiodic flowering network [12] . The Ehd1 gene is unique to rice and its transcription is strongly controlled at diurnal and seasonal levels [12 , 13] . Mapping of QTLs identified several major regulators of Ehd1 expression that , upon cloning , were shown to encode transcription factors belonging to distinct protein groups [14–20] . In particular , GRAIN YIELD , PLANT HEIGHT AND HEADING DATE 7 ( Ghd7 ) and PSEUDO RESPONSE REGULATOR 37 ( PRR37 ) encode CCT ( CONSTANS , CONSTANS-like , TOC1 ) domain proteins , whereas Ghd8 encodes the NF-YB11 subunit of the NF-Y transcription factor complex [14–16]: all of them encode strong floral repressors . Artificial selection of rice varieties adapted to grow in Europe or Asia has taken advantage of loss-of-function alleles at such loci , because de-repression of Ehd1 expression results in up-regulation of the florigens and subsequent flowering also under non-inductive day lengths [14–16 , 21 , 22] . Sensitivity to day length can be compromised to the extent that pyramiding of specific mutations completely abolishes it [23 , 24] . A major repressor of Ehd1 transcription is encoded by HEADING DATE 1 ( Hd1 ) , a zinc-finger CCT-domain transcription factor , homologous to CONSTANS of Arabidopsis [10 , 23 , 25 , 26] . As opposed to CO that promotes flowering under inductive long days ( LD ) , Hd1 performs a dual function , because under LD conditions it delays flowering , whereas under SD conditions it promotes it by inducing expression of the florigens [10 , 27] . Similarly to floral repressor genes already mentioned , extensive allelic variation has been described at the Hd1 locus that includes a plethora of loss-of-function alleles associated to varieties adapted to a broad latitudinal range [21 , 23 , 28–31] . However , the dual molecular function of Hd1 and the modes of repression of florigenic loci under LD are poorly understood . Although extensive genetic variation for flowering time traits has been described , more allelic variants must exist within local germplasm collections , because described genetic diversity appears insufficient to fully account for reduced sensitivity to day length of all varieties . Additionally , most efforts have focused on the identification of polymorphisms creating clear loss-of-function mutations , such as frame shifts or premature stops codons . Such first level of investigation provides an important but limited description of standing variation and additional levels have to be explored . Comparisons between rice and Arabidopsis can help to derive and test hypotheses concerning protein function , albeit these two species diverged ~150M years ago and the extent of conservation of the Hd1-Hd3a/CO-FT modules is debated [32 , 33] . In Arabidopsis , CO can interact with several AtNF-YB and AtNF-YC subunits [34 , 35] . Induction of FT expression and flowering mediated by CO requires some of these subunits , because nf-yb2 nf-yb3 or nf-yc3 nf-yc4 nf-yc9 mutants fail to induce FT expression and flower late under LD [35 , 36] . Additionally , the early flowering phenotype of plants overexpressing CO from a strong promoter is limited by the double nf-yb2 nf-yb3 or triple nf-yc3 nf-yc4 nf-yc9 mutations [35 , 37] . The CO protein can directly bind the proximal promoter of FT in vivo [38] or CO Response Elements ( CORE ) in vitro [37] . Whether the recruitment of CO to the FT promoter is enhanced by the interaction with NF-YB and NF-YC subunits upon formation of a CO-containing NF-Y heterotrimeric complex is currently unknown . It is similarly unclear if rice relies on a trimeric NF-Y system to regulate expression of the florigens and flowering . In this study , we used varieties flowering at higher latitudes to identify novel polymorphisms at loci relevant for photoperiodic adaptation . Two novel and common Hd1 alleles were found , both sufficient to create a non-functional variant . Taking advantage of such genetic tools , we hypothesized and demonstrated the formation of a NF-Y heterotrimeric complex containing Hd1 , capable of binding to a conserved response element in the Hd3a promoter . Genetic variation at Hd1 can impinge on trimer formation and the floral transition . Our results suggest how multiple layers of variation can be stratified at the same locus and independently exploited during artificial selection . Additionally , they show how genetic diversity can provide unique molecular variants to understand specific developmental processes at the molecular level .
Artificial selection of loss-of-function mutations in floral repressor genes , including Hd1 , Ghd7 , Ghd8 and PRR37 has been an effective strategy to expand rice cultivation to higher latitudes in both Asia and Europe [4 , 23 , 28 , 39 , 40] . However , the genetic determinants that allowed expansion have not been fully determined for all varieties and additional major regulators or novel haplotypes not previously described are likely to be present in several accessions . A genetic approach was used to identify candidate genes conferring reduced sensitivity to day length . A segregating population was obtained by crossing Nipponbare ( NB ) with Erythroceros Hokkaido ( EH ) , a temperate japonica variety from Poland [29] . The genome of NB harbors functional Hd1 , Ghd7 , Ghd8 and PRR37 that confer sensitivity to day length ( Photoperiod Sensitivity Index , PSI = 0 . 69 ) , whereas EH is insensitive to the photoperiod ( PSI = 0 . 16 ) and flowers very early regardless of external light conditions ( Fig 1 ) . Flowering of the resulting F2 individuals was scored under LD conditions ( 16L/8D ) and followed a normal distribution ( Fig 1A ) . The EH parental line was examined with molecular markers designed on known mutant alleles of floral repressors , revealing the presence of homozygous ghd7-0a and prr37-2a loss-of-function genes [14 , 16 , 23 , 40] . Consistently with this finding , the earliest flowering F2 plants co-segregated with these mutant alleles ( S1 Fig ) . The Hd1 locus of EH ( Hd1EH ) was sequenced , including the coding region ( CDS ) , introns and untranslated 5’ and 3’ regions and was found to be identical to haplotype Hd1-VII [23] . The Hd1EH allele apparently encodes for a functional protein , based on the absence of indels creating frame shifts or premature stop codons in the CDS . However , it co-segregated with very early heading plants , effectively behaving as a non-functional LD repressor or as constitutive activator of flowering ( Fig 1A ) . To distinguish the effects of ghd7 , prr37 and Hd1EH on flowering , F3 plants bearing single or multiple mutations were selected and heading dates were scored under natural long days ( NLD ) in Milan ( 45 . 47°N ) ( S2 Fig ) . Under NLD , the Hd1EH allele strongly promoted flowering and pyramiding of prr37 further accelerated it , indicating additive effects . Combinations of prr37 and ghd7 produced the shortest cycle length ( S2 Fig ) . To assess the differences between Hd1NB and Hd1EH we selected F3 lines carrying wild type Ghd7 and PRR37 alleles and scored heading dates under increasing photoperiods ( Fig 1B ) . Phenotypic differences between photoperiodic treatments were mild in lines harboring the Hd1EH allele compared to lines harboring the Hd1NB allele , confirming that genotypes containing Hd1EH have reduced sensitivity to day length . To understand the functional effects of Hd1EH on flowering , the mRNA levels of downstream targets of Hd1 were quantified under LD conditions in selected F3 genotypes harboring the Hd1EH or Hd1NB alleles . The Hd1EH allele caused precocious transcription of the florigens , and particularly that of RFT1 , compared to plants carrying the Hd1NB allele ( Fig 2C and 2D ) . Expression of Ehd1 was also elevated in plants bearing the Hd1EH allele ( Fig 2B ) . As Hd1 represses Ehd1 under LD conditions [23] , increased transcription of Ehd1 could be explained by reduced functionality or expression of Hd1 . Interestingly , expression of Hd1 from plants harboring the Hd1EH allele was almost undetectable during the entire time course under LD ( Fig 2A ) , a feature not previously described for any Hd1 allele . Transcript abundance of Hd1 shows a diurnal rhythm and to assess if low levels of Hd1EH were caused by the time of sampling , diurnal time courses were collected and gene expression quantified during a 24h cycle under LD . Transcriptional levels of Hd1 were strongly reduced in EH during the entire diurnal cycle , de-repressing Ehd1 and promoting expression of the florigens ( S3 Fig ) . Low levels of Hd1 mRNA were detected also under SD , indicating that gene expression was not affected by the photoperiod ( S3 Fig ) . These data indicate that Hd1EH is never expressed and it can be considered a loss-of-function allele . Silencing of Hd1 is therefore an effective strategy to promote heading and indicates the existence of a tunable layer of variation creating phenotypic diversity . Regulatory elements in the Hd1 promoter could be responsible for variation of its transcription . To test this hypothesis , fourteen varieties were chosen from the European Rice Core Collection ( ERCC ) that harbored functional alleles of Ghd7 , Ghd8 and PRR37 and three distinct alleles at the Hd1 locus , including Hd1NB , Hd1EH and Hd1 from Volano ( Hd1Vol ) , a widely cultivated , high-yielding variety from Italy [41] . About 1 . 2 Kb of DNA upstream of the ATG was sequenced in this panel and all varieties carrying the Hd1EH alleles were found to contain a sequence of ~4 . 4Kb at position -166bp , annotated as mobile element ( GenBank accession AB300057 . 1 ) ( Fig 3A ) . Whether this DNA sequence has features of a transposable or retrotransposable element is unclear . However , some sequences in the mobile element are expressed ( Genbank accession AK101779 . 1 ) . An additional copy of this same element is present in the rice genome , on chromosome 6 . Hd1 was expressed in all varieties except those bearing the mobile element ( Fig 3A ) . Using diagnostic primers , 242 varieties belonging to the ERCC were screened and 92 ( 38% ) were identified that contained the mobile element ( S1 Table ) . The same screen performed on a world panel , including 77 varieties belonging to all Oryza genetic groups , identified only two bearing the mobile element , suggesting that the non-expressed allele is more widely distributed among European varieties ( S2 Table ) . Among all varieties of the ERCC bearing functional copies of Hd1 , Ghd7 , Ghd8 and PRR37 , as defined by Gómez-Ariza et al . ( 102 accessions in total ) , two groups were distinguished based on the presence or absence of the mobile element ( S1 Table ) . Expression of Hd1 was quantified in 4-week-old seedlings grown under LD . Transcript levels were undetectable in varieties bearing the promoter insertion , whereas expression of Ghd7 and PRR37 was similar between the two groups ( Fig 3B–3D ) . To clear the effects of the genetic background , we cloned the Hd1 promoters of NB and EH , and used them to drive expression of the ß-glucuronidase gene ( GUS ) upon transformation into NB and EH calli . The pHd1NB>>GUS vector was active in both the NB and EH calli , whereas the pHd1EH>>GUS could not produce expression spots in EH and NB calli , indicating that failure to express the reporter was caused by cis-acting elements in pHd1EH ( Fig 3E ) . Taken together , these data indicate that insertion of a mobile element in the Hd1 promoter prevents its expression . This is sufficient to reduce sensitivity to day length in several varieties and adapt them to European regions . These results also show how a transposable element has been instrumental to human selection to spread cultivation of a major cereal at higher latitudes , and add to the prominent roles that transposons have played during domestication and later diversification of crops [42–45] . Of the 102 varieties belonging to the ERCC and categorized as having functional copies of LD floral repressors [23] , 64 silence expression of Hd1 through a mobile element inserted in its regulatory regions . From the remaining pool that expresses Hd1 , Volano ( Vol ) was selected and crossed with NB to produce a recombinant population suitable for QTL mapping . An F2 segregating progeny comprising 138 individuals was grown under controlled LD ( 16L/8D ) and heading dates were scored ( Fig 4A ) . A normal distribution for days to heading was observed with several plants flowering very late and showing transgressive segregation . The DNA of twenty individuals from the earliest and latest flowering plants was bulked separately and a QTL-Seq approach was applied to identify the loci responsible for heading date variation [46] . A strong peak in the ΔSNP score was detected with high statistical significance on chromosome 6 , representing the major locus controlling flowering in this cross ( Fig 4B ) . The QTL corresponded to the position of the Hd1 locus ( Fig 4B ) . The normal distribution for days to heading suggested the existence of additional genes . Two QTLs were identified on chromosome 1 and 10 in which the NB allele promoted and delayed flowering , respectively . However , their statistical significance was lower compared to the QTL on chromosome 6 , possibly due to low sequencing coverage and/or dominance effects of the QTLs ( S4 Fig ) [46] . The coding sequence of Hd1Vol includes two in-frame insertions , several non-synonymous and conservative substitutions , and the deletion of a Lysine in the CCT domain , when compared to Hd1NB ( S5 Fig ) . Therefore , it is not interrupted by deletions or frame shifts and based on genetic evidences , it could be hypothesized that impaired function is caused by abolished mRNA expression , altered protein activity or mislocalization . Quantification of mRNA showed that Hd1 transcripts abundance was reduced , but not abolished in Volano when compared to NB [23] , and among the flowering repressors , also Ghd7 and PRR37 transcripts showed reduced diurnal cycling amplitude ( S6 Fig ) . We transiently expressed the Hd1NB-GFP and Hd1Vol-GFP proteins in tobacco leaves under an inducible promoter and observed that both protein variants accumulated after induction and were targeted to the nucleus ( S7 Fig ) . These data indicate that Hd1Vol is expressed , cycles normally and is targeted correctly . We therefore tested whether the polymorphisms in Hd1Vol prevent interactions with relevant partners binding to target genes . The AtNF-YB2/3 and AtNF-YC3/4/9 proteins are necessary for FT expression and flowering of Arabidopsis [34–36] . NF-YB and NF-YC form a histone fold domain ( HFD ) scaffold that accommodates NF-YA , the sequence-specific subunit of the CCAAT-binding trimer NF-Y [47–49] . Binding of AtNF-Y to the CCAAT box in the distal promoter of FT controls its expression and HFDs were shown to interact with CO [37 , 50 , 51] . In rice the corresponding components are encoded by Hd1 , Ghd8 ( OsNF-YB11 ) and NF-YC subunits . Whether a NF-Y trimeric complex formed in rice and artificial selection of polymorphisms in single components affected features of the complex , including protein-protein interactions or DNA binding properties , has never been addressed . Assembly of a trimeric complex by distinct NF-Y components was therefore tested using yeast-two and three-hybrid assays . First , transcripts of the seven NF-YC genes encoded in the rice genome were quantified , to select for those expressed in leaves where the complex is likely formed . Transcripts of NF-YC3 and NF-YC5 were not expressed under LD conditions ( S8 Fig ) ; the remaining genes were all expressed at similar levels , except NF-YC2 , whose diurnal oscillations were wider both under LD and SD ( S8 Fig ) . The NF-YC1 , NF-YC2 and NF-YC7 proteins were then expressed in yeast together with Ghd8 . Heterodimeric interactions were observed for all combinations , as well as Ghd8 homodimerization ( S3 Table ) . No direct interactions were detected between Hd1NB or Hd1Vol and NF-YC1 , NF-YC7 or Ghd8 . Therefore , we could not reproduce recent data indicating interaction between Hd1 and Ghd8 , possibly because different cultivars were used [52] . As both BD:NF-YC2 and BD:Hd1 fusion proteins could autoactivate the yeast reporters , their interaction could not be determined . The NF-YB and NF-YC subunits form the histone fold domain scaffold that accommodates the third subunit of the trimeric complex . Using yeast-three-hybrid assays , a strong interaction was observed between Hd1NB/NF-YC1/Ghd8 and Hd1NB/NF-YC7/Ghd8 . However , Hd1Vol could not interact with the NF-YC1/Ghd8 or NF-YC7/Ghd8 heterodimers ( Table 1 ) , indicating that some polymorphisms in Hd1Vol prevent the formation of the heterotrimer . OsPRR37 is a major LD repressor whose CCT-domain shows homology to the CCT of Hd1 and structural homology to NF-YA [16 , 40 , 53] . To address the combinatorial properties of the rice NF-Y complex , OsPRR37 was used in a yeast-three-hybrid assay together with NF-YC subunits and Ghd8 . Growth of yeast on selective media indicated that the OsPRR37 protein could interact with the NF-YC1/Ghd8 and NF-YC7/Ghd8 heterodimers ( Table 1 ) . Taken together , these data indicate that ( i ) a trimeric complex can be assembled between Hd1 , Ghd8 and distinct NF-YC subunits , among which at least NF-YC1 and NF-YC7 interact strongly within the trimer , ( ii ) genetic variation creates protein variants unable to interact with the HFD and ( iii ) OsPRR37 ( and possibly other PRR proteins ) can replace Hd1 in the heterotrimer . The formation of a NF-Y trimer shown above and the resemblance of the CCT domain to the NF-YA domain required for CCAAT-binding [53] , suggest that the CCT of Hd1 could impart sequence-specificity to the trimer , as well as being sufficient for heterotrimerization [34 , 50] . We assessed the DNA-binding properties of the complex by electrophoretic mobility shift assays ( EMSA ) . We produced the HFDs of OsNF-YC7 and Ghd8 , and the CCT domains of Hd1NB and Hd1Vol in E . coli . Note that CCT-Hd1NB and CCT-Hd1Vol differ only for a lysine , missing in Vol and lying in the first part of the CCT domain . This region , based on structural homology with the NF-YA A1 helix [53 , 54] , is involved in protein-protein interactions with HFD proteins and highly conserved among CO-like proteins in monocots and dicots ( S5 Fig ) . The DNA probe was selected within the Hd3a proximal promoter region , based on the presence and conservation of a CO Response Element 2 ( CORE2 ) located at -169bp [37 , 51] . Fig 4C shows that a shifted band was detected when CCT-Hd1NB/Ghd8/OsNF-YC7 were incubated together , reconstituting a trimeric complex . The band shift was not observed in the presence of CCT-Hd1Vol or when Ghd8/OsNF-YC7 was missing ( Fig 4C ) . To check for specificity , we challenged the complex with an unlabeled oligonucleotide identical to the DNA probe , or containing mutations in the CORE2 site ( Fig 4D ) . The wild type oligonucleotide , but not the mutated one , competed binding efficiently . Overall , these data corroborate the yeast analysis indicating that Hd1NB forms a trimer with the OsNF-Y HFDs , and confirm that Hd1Vol , defective in HFDs association , is unable to bind a CORE2 element in DNA-binding assays . Finally , we checked the distribution of the Hd1ΔK337 polymorphism in the ERCC and found a total of 38 varieties sharing this mutation ( Fig 4E ) . Notably , 33 of these belonged to the subset of 102 accessions mentioned above ( S1 Table ) . Thus , the large majority of expressed variants of Hd1 have been likely selected because they compromise the repressor function of the complex .
The genetic architecture of the rice photoperiodic pathway heavily relies on floral repressor genes encoding transcription factors . These have been the first components to be isolated by genetic mapping in the flowering regulatory network and include Hd1 , Ghd7 , Ghd8 and PRR37 [14 , 16 , 25 , 55–57] . The position of such genes in the network initially suggested the existence of separate regulatory branches having partly unrelated effects . The Ghd7 and Ghd8 mutants have been isolated as independent regulators of Ehd1 [15 , 55] . Mutations in PRR37 have been initially believed to repress flowering by limiting Hd3a expression but not that of Ehd1 or RFT1 [16] . A later study indicated that PRR37 acts upstream of both florigens by controlling Ehd1 expression [40] . Until recently , the pathways centered on Hd1 and Ehd1 have been considered independent and acting in parallel , but recent data established a connection between Hd1 activity and Ehd1 expression , demonstrating Hd1 to be an upstream repressor of Ehd1 under LD [23] . The data presented in this study indicate that Hd1 , Ghd8 and PRR37 proteins do not act independently but rather assemble into a higher-order NF-Y protein complex that constitutes the molecular core of the photoperiodic pathway ( Fig 5A ) . The recent demonstration of a molecular interaction between Hd1 and Ghd7 proteins at the Ehd1 promoter , despite not directly implicating a heterotrimeric complex , further corroborates this interpretation [58] . Finally , binding of the heterotrimer to an element present in the Hd3a promoter suggests the existence of multiple targets for the OsNF-Y complex within the flowering network . The NF-Y complex is a sequence-specific heterotrimeric transcription factor formed by histone-like subunits and common to eukaryotes [59] . However , whereas in animals and fungi each component of the complex is encoded by a single gene , plant genomes have largely amplified the number of subunits and in species such as rice or Arabidopsis hundreds of combinations of the NF-YA , B and C subunits are possible , that fine tune the spatio-temporal regulation of gene expression while enormously expanding the range of regulated processes [48 , 60–62] . In rice , the major NF-YB subunit involved in flowering time regulation is Ghd8 ( OsNF-YB11 ) , however , other components , including OsNF-YB10 and OsNF-YB8 share high sequence similarity with Ghd8 and might have a role as regulators of flowering in rice [53] . This view is corroborated by the fact that late flowering of Arabidopsis nf-yb2 nf-yb3 double mutants is rescued by expression of OsNF-YB10 or OsNF-YB8 , suggesting an effect on flowering time control , at least in a heterologous system [63] . Additionally , overexpression of OsNF-YB7 and OsNF-YB9 delays flowering in rice under LD conditions [64] . Despite their sequence being only weakly related to that of Ghd8 , they might compete with Ghd8 in the complex that includes it , or form an alternative one . Therefore , although Ghd8 is a prominent regulator , the existence of other NF-YB subunits regulating flowering in the Hd1/PRR37 containing complexes cannot be ruled out and deserves further attention . Based on yeast interactions and DNA-protein binding assays we showed that OsNF-YC1 and OsNF-YC7 can interact with Ghd8 and CCT-domain proteins including , at least , Hd1 and PRR37 . Trimer formation with OsNF-YC2 and OsNF-YC4 could not be formally demonstrated because of auto-activation of yeast reporters . However , dimeric interactions between OsNF-YC2 and OsNF-YB8 , OsNF-YB10 and OsNF-YB11 have been reported [65] . Most importantly , genetic evidences support a role for OsNF-YC2 in the control of flowering in rice [65] . Transcriptional silencing of OsNF-YC2 by RNA-interference ( RNAi ) resulted in accelerated flowering under LD conditions , whereas its overexpression under the maize ubiquitin promoter strongly delayed flowering [65] . A milder effect on LD repression of flowering has been reported for OsNF-YC4 , whereas OsNF-YC6 seems to have no role in flowering time control [65] . Taken together , these data indicate that in rice cells multiple HFD scaffolds can form and possibly bind to NF-YA or CCT domain proteins to control heading . Demonstration of a heterotrimeric interaction between HFD dimers and PRR37 indicates for the first time that the HFD scaffold can bind proteins different from NF-YA or CO and CO-related proteins , all of which share a structurally similar , albeit not identical , CCT domain [34] . These findings further expand the combinatorial properties of the complex and might suggest a competitive mode of assembly , whereby Hd1 or other related proteins , including PRR-like or Ghd7-like factors , dynamically replace each other while interacting with the HFDs . Such model has been previously proposed for the CO2 and VRN2 proteins that were shown to compete with each other for binding to NF-YA , B or C components of wheat [47] , but could be much more diversified among plant species as more CCT interactors become implicated in trimer formation . Additional combinations could be provided by direct interactions between CCT domain proteins . Recently , a direct interaction between Hd1 and Ghd7 was reported , and the Ghd7 protein was shown to bind the Ehd1 promoter [58] . Whether Hd1 or other NF-Y subunits are required for Ghd7 binding to DNA is still unclear . Also , how the dynamical assembly of proteins around Hd1 is regulated is unknown . The NFYB/C dimers and Ghd7 could compete with each other for interacting with Hd1 , similarly to CO2 and VRN2 wheat proteins , either at specific times of the day or season . Alternatively , Hd1 could be the scaffold on which both HFD proteins and Ghd7 interact , forming a large and unique LD repressor complex . Since DNA specificity is determined by NF-YA or CCT domain proteins , a further layer of variation is provided by the sequences bound by such components , possibly being the CCAAT box [50] , CORE elements ( [37 , 51] and R . M . , N . G . personal communication ) or morning elements [66] . Finally , a crucial issue to address is when or in which cells an Hd1-containing complex is predominant over a PRR-containing complex to regulate expression of Ehd1 , Hd3a or other genes , and how the dynamics of assembly and activity of alternative complexes are regulated at diurnal or seasonal levels . Not secondary to this question is the fact that since PRR proteins are central components of plants circadian clocks [67] , the rhythm of expression of several genes other than those involved in flowering time control , might be dependent upon specific higher-order NF-Y complexes . Tissue-specific and temporal patterns of expression of NF-Y genes could help distinguishing between complexes possibly involved in regulation of photoperiodic responses ( that take place in the vascular tissue of leaves or in cells of the shoot apex ) from those involved in circadian clock functions ( that take place in most cell types ) [35 , 61] . Based on recent findings and on the results presented in this study , it is worth reconsidering the comparison between the photoperiodic regulatory networks of rice and Arabidopsis . Day length responses in rice are not controlled by distinct pathways but by a unique one , whose regulatory elements converge on Ehd1 [12] . Homologs of Ehd1 have not been identified in Arabidopsis or other dicot species , but they are present in the genomes of monocots , thus encoding a function not shared by all plants , and that likely evolved after the split between monocots and dicots about 150M years ago [68 , 69] . The gene works as an upstream transcriptional activator of Hd3a and RFT1 and promotes flowering under SD also in the absence of a functional Hd1 [12 , 70] . However , its repression under LD is mediated by genes whose homologs are present in Arabidopsis , and function in the regulation of flowering time also in dicot species . Similarly to Arabidopsis , the OsGI , Hd1 , Hd3a/RFT1 genetic cascade is present in rice as well . The origin of the CO function and the conservation of the CO-FT module across Angiosperms has been challenged [32] . Simon et al . , proposed that a CO function has evolved in the Brassicaceae family only after the most recent genome duplication that occurred within the family and that is not shared by its sister family [33] . Evolution of such function created a flexible switch to trigger flowering under LD . According to this interpretation , the Hd1 function might have evolved by convergent evolution . Consistent with a distinct origin ( and distinct environmental pressures of tropical vs temperate areas ) , it is to be considered that a major function of Hd1 is to repress flowering under LD , and this function seems prominent compared to its function as SD flowering activator . These functions are not shared by Arabidopsis CO and the repressive activity in particular is directed to Ehd1 [23 , 58] . Therefore , the Ehd1 function seems to have been added to , or co-evolved with an existing network containing homologs that are shared with Arabidopsis , and that Ehd1 became central to the photoperiodic pathway of rice , as well as a hub gathering signals also from other environmental cues [71] . The DNA binding assays performed with heterotrimeric complexes indicates that the Hd1-containing NF-Y complex has the capacity to bind a CORE2 element from the Hd3a promoter . Similar assays have demonstrated that CO can bind the FT promoter and that CORE sequences are necessary for binding [37] . Thus , protein-DNA interactions between Hd1-Hd3a and CO-FT suggest the existence of similar regulatory modules in rice and Arabidopsis . However , since the CO function evolved only recently in the Brassicaceae and the Hd1 function evolved by convergent evolution , the existence of such modules and their similar arrangement is striking [33] . This might be indicative of their robustness at the core of the photoperiodic pathway . The Hd1 protein could bind also to the Ehd1 proximal promoter , as shown by chromatin immunoprecipitation assays , although no CORE sites have been identified in such region [58] . These data point to a three-node coherent feed forward loop of regulation under LD , directly linking Hd1 to Ehd1 and Hd3a ( Fig 5B and 5C ) . This mechanism might have evolved because the presence of an Ehd1 floral inductive function unlinked from an Hd1 repressive function could have resulted in the induction of Hd3a/RFT1 expression also under LD . However , with both Hd3a and Ehd1 under direct control of Hd1 , this problem would be overcome and long photoperiods would prevent flowering by limiting all floral activators . The role of RFT1 in such feed forward loop remains to be addressed . However , searches for CORE elements resulted in the identification of additional sites in both Hd3a and RFT1 loci . Follow up studies in vivo will test if these can be effective binding sites for NF-Y repressor complexes .
The Japanese rice variety Nipponbare ( NB , Hd1 Ghd7 PRR37 Ghd8 hd6 ) was crossed with Erythroceros Hokkaido ( EH , Hd1EH ghd7 prr37 Ghd8 Hd6 ) to produce a recombinant F2 population comprising 215 individuals . Depending on the genotype , F2 individuals were selected and heading dates were determined using F3 plants under different photoperiodic conditions . Volano ( Vol , Hd1Vol Ghd7 PRR37 Ghd8 ) , a high-yielding variety from Italy , was crossed with NB and heading dates of 138 F2 individuals were scored . The core collection comprising 242 varieties cultivated in Europe has been already described in[23] . Details of the accessions and genotypes of Hd1 , Ghd7 , PRR37 and Ghd8 are available in S1 Table . Plants were grown under controlled conditions in Conviron PGR15 chambers or greenhouses under LD ( 16h light ) or SD ( 10h light ) photoperiodic regimes . Day and night temperatures were 28°C and 24°C , respectively . Humidity was set at 70% during the day and ~90% during the night . Field experiments were performed at the Botanical Garden Città Studi , Milan ( 45 . 47°N ) . Seeds were sown in a cold greenhouse on Apr 11 , 2014 and transplanted in an irrigated field on May 17 , 2014 . Heading dates were scored from ~30 plants/genotype . The photoperiod sensitivity index ( PSI ) was calculated as in [23] . Genomic DNA was prepared from leaves using a modified CTAB and chloroform:isoamyl alcohol method [72] . Genotyping of the NBxEH F2 population was performed using markers for the prr37-2a and ghd7-0 alleles [23] , whereas distribution of the Hd1NB and Hd1EH alleles was determined using primers listed in S4 Table . Genomic DNA was amplified using LA Taq from TaKaRa in Buffer I , according to manufacturer’s indications . For each PCR reaction , DNA was initially incubated five minutes at 95°C , followed by 40 cycles of amplification ( 95°C 30 seconds 58°C 30 seconds and 72°C 1 minute ) . The same PCR profile was applied to all PCR reactions , extending or shortening the extension time depending on the expected fragment size . By using the same PCR conditions , the European rice varieties were screened for the presence of the 4 . 4Kb mobile element in the Hd1 promoter , using forward 5’-promoter-anchored and reverse 3’-promoter-anchored primers in combination with primers designed within the mobile element . Additionally , a pair designed around the insertion site that could amplify only in the absence of the mobile element was used ( S4 Table ) . Sequencing reactions were prepared and analyzed according to [23] . Total RNA was extracted using the TRI Reagent ( Sigma Aldrich ) from the distal part of young leaves collected from at least three independent plants . Genomic DNA was digested using TURBO DNAse ( Life Technologies ) and the RNA was precipitated with sodium acetate and ethanol and resuspended in water . After quantification of total RNA , 1μg was retrotranscribed with SuperScriptII Reverse Transcriptase ( Invitrogen ) and oligo-dT according to manufacturers’ instructions . The cDNA product was diluted 10 fold with sterile water . Transcripts were quantified in a Realplex2 ( Eppendorf ) . Reactions were carried out using 3μl of cDNA as template , 5μl of 2X Maxima SYBR Green qPCR Master Mix ( Thermo Scientific ) and 0 . 2μl of each primer ( final concentration 10μM ) and ddH2O to a final volume of 10μL . A list of primers used for mRNA quantification is available in S4 Table . In particular , primers used to detect Hd1 expression are located in the 3’UTR region , that was sequenced and found to be identical between Hd1EH and Hd1NB , excluding the possibility that the primers used could not detect one of the two allelic variants . The coding sequence of Hd1NB was fused at the C-terminus with mCherry and that of Hd1Vol was fused with GFP in pABind vectors [73] . Expression of the fusion proteins was under a β-estradiol inducible promoter . Tobacco leaves were infiltrated with Agrobacterium cultures containing the plasmids . A 20μM β-estradiol solution was sprayed on leaves 3–12 hours before observation of epidermal cells using a confocal microscope . The coding sequences of genes used in yeast two- and three-hybrid assays were amplified from cDNA prepared from mature leaves using primers listed in S4 Table . The full length clone of Ghd8 was synthesized by GENEWIZ Inc . ( South Plainfield , NJ ) whereas OsPRR37 and NF-YC4 clones were obtained from the Rice Genome Resource Center ( http://www . rgrc . dna . affrc . go . jp/index . html . en ) . All genes were cloned in pDONR207 ( Life Technologies ) . Each entry clone was recombined with pGADT7 and pGBKT7 ( Clontech ) , to obtain AD- and BD-fusion proteins . The AH109 and Y187 strains were used in yeast transformation as described in the Clontech manual for the Matchmaker Gold yeast-two-hybrid system . Transformed cultures were selected on YSD media lacking leucine ( Leu ) , tryptophan ( Trp ) or adenine ( Ade ) for pGADT7 , pGBKT7 and pTFT1 , respectively . Protein-protein interactions were assessed by streaking colonies on YSD media lacking Leu , Trp and Histidine ( His ) for Y2H experiments and on media lacking Leu , Trp , Ade and His for Y3H experiments . The strength of the interactions was evaluated by streaking colonies on increasing amounts of 3-aminotriazole ( 3AT ) . Yeast growth was verified after 6 days at 30°C . Each experiment has been repeated at least 3 times using independent clones . The QTL-Seq approach has been previously described [46] . Briefly , DNA was prepared using the C-TAB method to extract genomic DNA individually from the twenty earliest and twenty latest flowering plants , within a total population of 138 F2s . DNA was quantified and two DNA pools of early and late flowering plants were produced using 1μg of genomic DNA per each plant . The whole genome was re-sequenced using Illumina HiSeq 2500 with chemistry v4 at Eurofins ( Germany ) , producing 125bp paired ends reads . Whole-genome resequencing yielded 18896 and 24961Mbp , with an approximate coverage of 39 and 52 fold for the early and late flowering pools , respectively . Filtered short reads were aligned to the NB reference genome and SNP indexes were calculated for the early and late heading bulks . SNP values of less than 0 . 3 in both samples were removed and a ΔSNP-index was determined and plotted on the chromosome maps . Finally , using a sliding window analysis candidate QTLs were visualized . The cDNAs encoding the HFD of Ghd8 and OsNF-YC7 were synthesized by Eurofins Genomics and subcloned in pmcnCS EATCH using NdeI/BamHI restriction sites ( S5 Fig ) . Only Ghd8 was tagged with 6x-His[74] . The CCT domains of Hd1NB and Hd1Vol were synthesized by Eurofins Genomics . The resulting proteins were tagged with 6x-His at the C-terminus and subcloned in pmcnEATCH . Soluble HFD heterodimers were produced in E . coli by co-expression in BL21 ( DE3 ) strains by IPTG induction , and purified using the HisSelect resin ( SIGMA ) . The CCT domains of Hd1NB and Hd1Vol were purified separately . Proteins were eluted in Buffer A ( 10mM Tris pH8 . 0 , 400mM NaCl , 2mM MgCl2 ) , containing 250mM imidazole . Purified proteins were dialyzed against Buffer B ( 10mM Tris-Cl pH8 . 0 , 400mM NaCl , 2mM DTT , 10% glycerol ) . The protocol for EMSA was adapted from [48 , 50] . CCT-Hd1/Ghd8/OsNF-YC7 heterotrimeric complex assembly and CORE2 DNA-binding was tested with Cy5-labeled oligos ( Fig 4D ) . Ghd8/OsNF-YC7 dimers ( 60nM ) and CCT-Hd1NB or CCT-Hd1Vol ( 360nM ) were mixed in a final volume of 16μl with Cy5-5’-labeled CORE2 probe in a reaction buffer ( 20nM ds-oligo , 12mM Tris-HCl pH8 . 0 , 50mM KCl , 62 . 5mM NaCl , 0 . 5mM EDTA , 5mM MgCl2 , 2 . 5mM DTT , 0 . 2mg/ml BSA , 5% glycerol , 100ng polydA-dT ) . To test the specificity of the binding , identical reactions were prepared with increasingly higher concentrations of unlabeled or mutated oligonucleotide competitors or TE buffer , as indicated in Fig 4C . Reactions were incubated at 30°C for 30min , and subsequently resolved by polyacrylamide gel electrophoresis . Fluorescence signals were detected using a Chemidoc MP system ( Bio-Rad ) with ImageLab software . The promoter regions of NB ( 580bp ) and EH ( 4996bp including the mobile element ) were amplified with primers suitable for Gateway cloning and recombined into pDONR207 vectors ( Life Technologies ) . Positive clones were confirmed by sequencing and recombined into pBGWFS7 , to drive expression of a GFP-GUS reporter gene , using LR clonase ( Life Technologies ) . Embryogenic calli of NB and EH were derived from scutella of mature seeds . For callus induction , seeds were dehusked , sterilized and placed on basal NB-medium plates ( pH5 . 8 ) , supplemented with 3mg/L 2 , 4D , 0 . 25mg/L cytochine and 30g/L glucose , for three weeks in the dark and 28°C . Proliferating embryogenic calli were subcultured on fresh medium for another three weeks before biolistic transformation . Four hours before the bombardment and 16 hours after , calli were transferred on NB osmotic medium containing 34 . 6g/L of both mannitol and sorbitol , and they were kept in the dark at 28°C . After the osmotic treatment , calli were placed on basal MS medium supplemented with 3mg/L 2 , 4 D for two days before GUS staining . Non-bombarded calli and calli bombarded with gold microcarries only were used as negative controls . The Biolistic PDS/1000 helium system ( BioRad , USA ) was used with the following parameters: ruptor disc pressure , 1100psi; macrocarrier to stop screening distance , 9cm; vacuum pressure , 28 inches of mercury ( inHg ) ; gold microparticle size , 1μm . For macrocarrier preparation , 4μg of plasmids were precipitated with 25μl of CaCl2 ( 2 . 5M ) , 10μl of spermidine ( 0 , 1M ) and 25μl of gold particles ( 60mg/ml ) . The DNA bound to gold microcarriers were washed and re-suspended in ethanol . 10μl of gold microcarrier were then spotted on each macrocarrier for biolistic transformation . Biolistic transformation was repeated twice . After bombardment , calli were incubated in the dark at 28°C for at least two days . The viable calli were then incubated in 90% cold acetone at -20°C for 20’ , washed twice with NaPO4 buffer and finally transferred into the GUS histochemical reagent containing 1mM K4Fe ( CN ) 6 , 1mM K3Fe ( CN ) 6 , 0 , 1M NaPO4 , 10mM EDTA , 0 , 1x Triton , 2mM X-Gluc . Samples were vacuum infiltrated for 20’ and then incubated for 24h at 37°C . After staining , samples were cleared with 70% ethanol . | Many plant species flower in response to changes in day length and can be categorized depending on their requirements for long or short days . Rice has tropical origins and normally flowers in response to shortening days . However , artificial selection operated by ancient farmers or modern breeders adapted rice cultivation to several environments , including those typical of temperate regions characterized by long days during the cropping season . Modifications of the genetic network controlling flowering that are causal to such expansion have been the subject of extensive studies , but the full complement of genes that regulate it and the molecular bases of their activity remains unknown . We took advantage of germplasm cultivated in Europe—and highly adapted to flower under long days–to isolate widespread variants of the HEADING DATE 1 ( Hd1 ) gene that limits flowering in temperate areas , and showed that such variants are non-functional and unable to prevent long day flowering . We identified the DNA changes causing the gene to be non-functional and used such mutant alleles as tools to demonstrate that Hd1 can bind a specific DNA sequence in the promoter of a florigenic rice gene . Mining genetic diversity becomes thus instrumental to define the molecular properties of regulatory pathways . | [
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"organisms"
] | 2017 | Transcriptional and Post-transcriptional Mechanisms Limit Heading Date 1 (Hd1) Function to Adapt Rice to High Latitudes |
In excess of % of human cancer incidents have a viral cofactor . Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered . Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts . However , technical problems such as low abundances of viral transcripts in large volumes of sequencing data , viral sequence divergence , and homology between viral and human factors significantly confound identification of tumor viruses . We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors . We apply the approach to conducting the first systematic search for viruses in neuroblastoma , the most common cancer in infancy . The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor . However , a viral etiology of neuroblastoma is currently contested . We mapped transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses . Analysis of controls , comparisons with related methods , and statistical estimates demonstrate the high sensitivity of our approach . Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses . Likewise , de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens . While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data , in principle , this is rendered less likely by the high sensitivity of our approach and the number of biological replicates analyzed . Therefore , our results suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely .
To date , pathogenic agents are known to be causally related to 20% of human cancer cases [1] and significantly affect the global health burden of this disease [2] . The majority of these agents comprise oncogenic viruses such as human papilloma virus ( HPV ) , Epstein-Barr virus ( EBV ) , hepatitis B virus ( HBV ) , and hepatitis C virus ( HCV ) [3] . Characterizing the oncogenic potential of viral pathogens has important consequences for prevention , diagnosis , and treatment of malignant neoplasms [4] , [5] . Tumor viruses in particular have received renewed attention in the context of recent global efforts to characterize the etiology of cancer [6] , [7] . Consequently , viral cofactors for several idiopathic cancers are currently investigated [8] and epidemiological indicators suggest that additional human tumor viruses remain to be discovered [9] . Neuroblastoma is a heterogeneous embryonal tumor [10] , [11] that is accountable for 15% of deaths caused by malignant conditions in children [12] . The disease is associated with an exceptionally low median age of presentation of months [13] and is often diagnosed in utero . Metastatic neuroblastoma has two biologically divergent subtypes . Stage 4S is characterized by an age of presentation between in utero and months , metastases confined to liver , skin , lymph nodes and bone marrow , and its ability to regress spontaneously [14] , [15] . In contrast , stage 4 tumors are presented at any age , demonstrate high infiltration rates in bone marrow and bone , and are most often progressive [10] , [16] . While genes related to neuronal differentiation have been described to be upregulated in stage 4S in comparison to stage 4 neuroblastoma , thereby indicating distinct levels of neuronal differentiation [17] , little is currently known about the differences between molecular etiologies of stage 4 and stage 4S neuroblastoma . The variation of clinical outcomes between neuroblastoma subtypes indicates distinct genetic and environmental factors affecting the development of this malignancy . Interestingly , the early onset of the disease overlaps with periods of high susceptibility to viral infections and is reminiscent of acute lymphoblastic leukemia – another pediatric tumor with uncertain etiology for which an infective cofactor has long been suspected [18] . Furthermore , epidemiological studies have associated reduced neuroblastoma risk with immunologic indicators such as previous childhood infections , day care attendance , and breast feeding [19] , [20] that are suggestive of an infective cofactor [21] . While transforming polyomaviruses such as JCV and BKV were previously identified within neuroblastoma samples and other pediatric embryonal tumors [22]–[24] , newer studies seem to render these associations inconclusive [25] . Therefore , the role of pathogenic cofactors of neuroblastoma oncogenesis remains unresolved . In general , the search for suspected viral cofactors of idiopathic diseases requires systematic screening of human tissues for viral biomarkers such as virus-derived nucleotide sequences . Unfortunately , viruses are of polyphyletic origin and thus lack common universal marker genes as they are frequently exploited in metagenomics studies targeting cellular microorganisms . Consequently , it is not currently possible to specifically PCR-amplify viral nucleotide sequences within a given tissue without prior information about the infective agent being sought [26] . As a result , several systematic assays for pathogen detection have been developed that do not rely on targeted PCR-amplification of viral factors [27] and were employed to identify Kaposi's sarcoma-associated herpes virus ( KSHV ) as a human tumor virus [28] . These systematic approaches were recently supplemented by sensitive deep sequencing technologies [27] . These technologies were recently applied to exclude several cancer-virus associations based on negative evidence [29] , [30] and aided in the identification of MCPyV , a human polyomavirus , as a cofactor of Merkel cell carcinoma [31] . Deep sequencing technologies have enabled detection of both known and novel viruses with unprecedented sensitivity [32] . However , the large numbers of sequence fragments ( “reads” ) generated by these methods necessitate data reduction approaches for filtering and condensing the list of putative viral transcripts . Two such approaches are currently represented in the literature: digital transcript subtraction that discards human sequence homologs from the sequence data and considers the remaining transcripts as potential viral signatures [30] , [31] , [33]–[39] , and de-novo sequence assembly that aims to reconstruct whole viral genomes from overlapping reads [40]–[43] . Recently , variants of these of two approaches have been implemented in several computational pipelines such as PathSeq [44] , RINS [45] , and CaPSID [46] . Identification of tumor viruses in particular poses several important challenges to existing computational pipelines . Confounding factors such as loss of viral genetic material from progressed tumors as well as limited replication competence or latent replication strategies often result in low or selective transcription of tumor viruses [5] . In addition , viral oncogenes homologous to human factors and chimeric transcripts originating from proviral insertion sites may share significant sequence similarity with human transcripts [47] , thus making unequivocal identification of viral factors difficult . Last , high rates of viral sequence divergence from ( dsDNA viruses ) up to ( ssRNA viruses ) substitutions per site and year [48] , [49] hinder recognition of known viruses based on known reference sequences . We have developed Virana , a novel computational approach specifically tailored to detecting low-abundance transcripts that diverge from known viral reference sequences or share significant sequence homology with human factors . In particular , our method maps sequence reads to a combined reference database comprising the human genome and all known viral reference sequences . The approach is configured to allow for high mismatch rates and mappings to multiple reference sequences ( ‘multimaps’ ) . By using this combined and sensitive mapping strategy , our approach is especially well suited for detecting human-viral chimeric transcripts and viruses diverging from known references . In contrast to existing subtractive approaches for viral transcript discovery , our method abstains from discarding reads homologous to the human genome from further analysis . Instead , Virana exploits multimaps to assign sequence reads to a homologous context comprising human reference transcripts and viral reference genomes . These homologous regions retain the full , unfiltered information contained in the raw sequence data while also being amenable to further analyses by multiple sequence alignments , human-viral phylogenies , and orthogonal taxonomic annotations , thus greatly aiding in the interpretation of the results . We applied our novel approach on an overall number of deep sequencing transcriptomes of stage 4 and stage 4S metastatic neuroblastoma in order to identify putative viral cofactors associated with this idiopathic disease .
Primary neuroblastoma samples from stage 4 ( progressive ) patients ( ) and stage 4S ( regressive ) patients ( were obtained prior to treatment from the central neuroblastoma tumor bank at the University Hospital of Cologne , Germany . None of the tumors harbored amplification of the MYCN proto-oncogene as determined by two independent laboratories for each case by fluorescence in situ hybridisation ( FISH ) and Southern blot [50] . Only neuroblastoma samples with a tumor cell content of above % as assessed by a pathologist were selected for deep sequencing . Integrity of RNA was evaluated using the Bioanalyzer ( Agilent Technologies ) and only samples with an RNA integrity number of at least were considered for further processing . Quality of all neuroblastoma samples and related deep sequencing data was additionally confirmed by an orthogonal computational analysis focusing on human gene expression in the context of differential splicing [51] . All patients were enrolled in the German Neuroblastoma trials with informed consent . In order to validate our approach we additionally employed a positive control panel consisting of tumors with known viral cofactors . An EBV-positive B-cell-lymphoma ( BCL ) was received from the Pediatric Oncology and Hematology Department of the Hannover Medical School . Deep-sequencing reads obtained from full transcriptome libraries of two HPV18-positive HeLa samples ( HeLa ) and a HPV16-positive primary cervical squamous cell carcinoma ( ceSCC ) were downloaded from the Short Read Archive ( SRA ) and preprocessed as specified in the original publication [30] . Transcriptome data of a HBV-positive hepatocellular carcinoma ( HCC ) HKCI-5 cell line with confirmed HBV integration events was downloaded from the SRA based on information in the original publication [52] . A negative control panel consisting of a normal brain transcriptome generated as part of the Illumina BodyMap 2 . 0 project was obtained from the SRA at run accession number ERR030882 . mRNA libraries of the EBV-positive B-cell lymphoma and neuroblastomas were prepared following the Illumina RNA Sample Preparation Kit and Guide ( Part # Rev . A ) . For each sample , g high-quality total RNA was processed for mRNA purification , chemical fragmentation , first strand synthesis , second strand synthesis , end repair , ′-end adenylation , adapter ligation , and PCR amplification . Validated libraries underwent gel size selection and final paired-end sequencing with an effective read length of bp on the Illumina Genome Analyzer IIx following Illumina standard protocols . Additionally , libraries for two of the neuroblastoma samples were generated using the same protocols and sequenced with an effective paired-end read length of bp on a Illumina HiSeq . All libraries had insert size distributions approximating bp , bp as later confirmed by read mapping . The data were filtered according to signal purity by the Illumina Realtime Analysis ( RTA ) software . In this study we employ simulated sequencing data from three viral genomes that are homologous to human factors . Reads originating from the ABL1-homologue of the Abelson murine leukemia virus ( A-MuLV , GI:9626953 , positions ) , from the the gag region of HERVK22I ( obtained from Repbase [53] , positions ) , and from Bo17 , a GCNT3-homolog of the bovine herpesvirus 4 ( BoHV-4 , GI:13095578 , positions ) were generated in silico by dwgsim , a read simulator based on wgsim [54] . In addition , we produced simulated chimeric transcripts by fusing each of the aforementioned sequence regions to the human TP53 gene , a known proto-oncogene ( UCSC build hg19 , GRCh37 , chr17 , positions 7572926–7579569 ) . These artificial fusion transcripts were generated using Fusim [55] based on TP53 exon models obtained from the UCSC refGene database [56] . Fusion transcripts were then used as templates for generating simulated data sets with dwgsim . In all cases , dwgsim was applied using the default empirical error model . Paired-end read lengths and insert size distributions were chosen according to the neuroblastoma sequencing data ( see above ) . Additional simulated sequencing data generated by a related publication were analyzed as described in Section “Estimation of read mapping sensitivity” . Sample panels containing neuroblastoma transcriptomes sequenced at bp and bp effective read lengths are denoted as NB1 and NB2 , respectively . While the NB1 panel contains seven transcriptomes of neuroblastoma stages 4 and 4S each , the NB2 panel contains one sample of stages 4 and 4S each ( see Table 1 ) . Positive control panels of human cancer transcriptomes with known viral cofactors ( BCL , HeLa , ceSCC , and HCC ) are denoted as POS . The negative control panel consisting of a normal human brain transcriptome is denoted as NEG . The current assembly of the human reference genome ( UCSC build hg19 , GRCh37 ) as well as corresponding refGene splice-site annotations were obtained from UCSC . Splice variant annotations and cDNA sequences for the human genome were downloaded from Ensembl [57] . A set of all available complete viral reference genomes and their taxonomic lineages were obtained from NCBI via the E-utilities web service [58] and the database query: “Viruses[Organism] AND srcdb_refseq[PROP] NOT cellular organisms [ORGN]” . In addition , we obtained consensus reference sequences for all human endogenous retroviruses ( HERV-K/HML-2 ) represented in Repbase ( Primate HERV , HERVK11DI , HERVK11I , HERVK13I , HERVK22I , HERVK3I , HERVK9I , HERVKC4 ) ) [53] . All reference genomes were combined into a single human-viral reference database for Virana . Since RINS and CaPSID cannot use such a combined database , human and viral reference sequences were collected within two separate databases for these approaches . Paired-end reads from the neuroblastoma panels and positive control panels were quality-controlled with an in-house sequence analysis framework in order to identify sample contamination , adapter contamination , and batch effects . After quality control , the sequence data consisted of Gbp ( NB1 ) , Gbp ( NB2 ) , Gbp ( POS ) , and Gbp ( NEG ) of sequence reads , respectively ( see Table 1 ) . All data were mapped against a combined human-viral reference database with the splicing-aware and gapped read mapper STAR [59] in paired-end mode . While Virana considers the read mapper to be a replaceable component , in principle , we decided to employ STAR due to its mapping speed , high sensitivity settings , and its consideration of putative chimeric transcripts . We configured the mapper for high sensitivity by following recommendations of the author of STAR ( personal communication ) . In particular , we set the rate of acceptable mismatches to times the length of each read and the seedSearchStartLmax and winAnchorMultimapNmax parameters to and , respectively . The minimum length of chimeric segments ( chimSegmentMin ) was reduced to in order to detect fusion transcripts at short read lengths . Known splice sites from splice annotations of the human reference genome as well as canonical splice sites were considered in the mapping . For each read , multiple mapping locations with alignment score distances of up to ranks relative to the best score were permitted ( ‘multimaps’ ) . Read alignments were stored in standardized BAM files . STAR supports detection of chimeric transcripts by reporting discordant read pairs whose ends map to different chromosomes . These discordant read pairs were employed in further analyses as detailed in the next section . In order to identify putative new viral transcripts , read pairs with at least one unmapped read end were extracted from BAM files by the Samtools suite [54] and assembled into longer contigs by the de-novo transcriptome assemblers Trinity [60] and Oases [61] using default parameters . Oases was configured for using different k-mer values in order to facilitate reconstruction of low-abundance viral transcripts . Contigs of length less than bp were considered to be spurious assemblies and excluded from further processing . Virana supports detection of human-viral chimeric transcripts in two different manners . First , the read mapper employed in our study is able to partially align reads that contain a human-viral chimeric breakpoint to multiple reference sequences . Consequently , these partially aligned reads can be detected by Virana within the generic analysis of homologous regions ( see below ) . The second , more sensitive approach to detecting chimeric transcripts is based on paired-end read information . Since the STAR mapper assigns reads to a combined reference database comprising both human and viral reference sequences , ends of paired-end reads whose inserts span the breakpoint of a chimeric transcript will be aligned to different reference sequences . These discordant read pairs are reported by STAR during read mapping ( see above ) and can further be filtered by mismatch score or sequence complexity in order to yield a high-confidence list of chimeric transcripts . A distinguishing feature of Virana is its ability to automatically reconstruct the homologous context of reads that map to both viral and human reference sequences . This homologous context is constructed in four steps: Consensus sequences can be further processed by phylogenetic analyses . For generating phylogenies , Virana employs the software PhyML [64] following the maximum likelihood approach and using default parameters recommended by the HIV sequence database ( http://hiv . lanl . gov , GTR model of nucleotide substitution , transition/transversion ratio: 4 , gamma shape parameter: 1 , number of substation rate categories: 4 , approximate Likelihood Ratio Test ( aLRT ) using SH-like supports where applicable ) . We note that the topology of the phylogenetic trees constructed in this manner is stable with regard to the model choice; while more complex model parameters may yield better likelihoods in some instances , these differences do not influence interpretation of our results . In this study , we additionally compare consensus sequences of aligned HOGs as well as de-novo assembled sequence contigs to nucleotide ( NCBI NT ) and protein ( NCBI NR ) reference archives in order to assign transcripts to a taxonomic origin . To this end , we employ several BLAST [58] search strategies ( BLASTN , BLASTX , and TBLASTX ) with sensitive word sizes ( , , and , respectively ) . TBLASTX bypasses synonymous mutations during similarity search and is particularly suited for detecting functionally conserved homologs . This approach is therefore recommended for discovering remote similarities [65] and is widely used in environmental metagenomics [66] . A permissive E-value threshold of is used for all comparisons in order to reduce the possibility of missing true viral hits . For each query transcript and search strategy , the three highest-scoring reference sequences are extracted from the BLAST results . Subsequently , descriptions , taxonomic information , and available gene annotations for high-scoring reference hits are pooled and query transcripts are assigned a putative viral , human , or ambiguous origin based on the pooled information . In order to limit the search space of the computationally intensive TBLASTX procedure , we constrain the allowed taxonomic origin of reference sequences to only viral ( NCBI taxon ID ) or human ( NCBI taxon ID ) hits while excluding artificial sequences ( NCBI taxon ID ) using the NCBI database query “ ( ( ( txid10239 [ORGN] ) OR ( txid9606 [ORGN] ) OR ( human [ORGN] ) ) NOT ( txid81077 [ORGN] ) ) ” . We quantify the ability of our novel method Virana and the related methods RINS [45] and CaPSID [46] at detecting diverged viral transcripts among human sequence data by employing a recently published validation data set [46] . This data set consists of a negative control background set of reads simulated from the human reference genome that is spiked with four sets of reads simulated from viral reference genomes . Nucleotide positions within reads of each of the four viral spike-in data sets are mutated randomly independently and uniformly with a set-specific probability before being merged with the background data set . The set of viral reference sequences represents different viral families that infect plants ( Cherry green ring mottle virus , Cestrum yellow leaf curling virus , Elm mottle virus , East African cassava mosaic virus ) , birds ( Gallid herpesvirus 1 ) , insects ( Cotesia congregata bracovirus ) , bacteria ( Guinea pig Chlamydia phage ) , amphibians ( Frog adenovirus 1 ) , and mammals ( Rat coronavirus Parker , Banna virus ) . All five data sets ( non-spiked human negative control and four human-viral spike-in sets ) are analyzed by Virana , RINS , and CaPSID using identical reference sequences as described in Section “Reference genomes” . Sensitivity ( fraction of correctly identified viral reads among all viral reads ) and specificity ( fraction of falsely identified human reads among all human reads ) of viral read detection are determined for each method and data set . Analyses are performed with either default parameters ( Virana ) , parameters published in the original validation data set ( CaPSID ) , or settings adapted by us in order to maximize sensitivity ( RINS: minimal contig length decreased to , read lengths and insert size distributions according to input data ) . Since all methods map to the same complete viral reference set , reads from a particular viral genome of the validation data set may be distributed across several closely related reference genomes , all of which may be considered valid mappings . For this reason , we added post-processing steps to CaPSID and RINS and performed this validation on the level of viral taxonomic families rather than on the level of single viral species . We note , however , that results of all tested methods including Virana retain information on single viral species throughout the analysis . In particular , sensitivity and specificity of the methods change only minimally if data is analyzed on the single species level . Analysis of the human-viral homologous regions and chimeric transcripts based on simulated read data ( see Section “Simulated sequencing data” ) was conducted by configuring CaPSID , RINS , and Virana analogous to the previous section . For the validation of fusion transcript detection , the number of true positives is set to the number of all reads originating from the human-viral fusion transcript . Since all detection methods in this validation are configured to only report reads mapping to the viral part of the fusion transcript , sensitivity estimates are scaled down equally for all methods in this particular validation . Analysis of discordant read ends in order to detect the origins of chimeric transcripts was performed as described before ( see Section “Detection of chimeric transcripts” ) . Expanding on related work [34] , [35] , we quantify the theoretical sensitivity of Virana by estimating the number of viral transcripts per cell that are required for achieving a certain minimal sequencing coverage at a probability of at least 95% . Based on human genome annotations obtained from UCSC , we determined an average length of human coding sequences ( CDS ) of bp . By conservatively assuming that an idealized cell contains mRNAs [34] of average length fragmented at bp as a result of library preparation , an expected number of cDNA fragments are generated per cell . For a given viral transcript of length and a viral transcript abundance per cell , we expect a number of viral transcript fragments . Assuming a theoretical , unbiased sequencing process , the probability of sequencing a viral transcript fragment among the overall transcript fragments is . Given a single-end read length of , a number reads are required to achieve a sequence coverage of that viral transcript . The probability of observing at least reads during sequencing with a sequencing depth is specified by the cumulative binomial distribution function with parameters , and . Due to numerical instabilities of computing the cumulative binomial distribution for large values , we exploit the Central Limit Theorem and estimate by the Camp-Paulson normal approximation to the binomial distribution . This approach has a negligible approximation error of , where [67] . Our approach further depends on successfully reconstructed homologous regions , each requiring an empirically determined minimum number of transcripts separated by no more than base pairs . Although the probability of a homologous region being successfully constructed from viral transcripts at a given sequence coverage can be derived analytically for a special case [68] , this solution neither considers edge effects occurring for small transcripts nor takes into account the distribution of insert sizes of paired-end reads . We therefore approach the problem empirically by in silico simulation of paired-end reads that are assigned randomly independently and uniformly to transcripts of different lengths and at varying coverages . This simulation process addresses the aforementioned confounding factors by considering transcript boundaries and sampling insert sizes from a normal distribution parametrized according to neuroblastoma sequence data employed in this study ( see Section “Library preparation and sequencing” ) . An mean estimator for and its standard error were derived by averaging the success rates of homologous region constructions across simulations for each transcript length , read length , region linkage , and read coverage . All sequence data generated in this study are publicly available in the European Nucleotide Archive ( ENA ) at study accession number PRJEB4441 . Software implementations of our method and all validation procedures are available at http://mpi-inf . mpg . de/∼sven/virana .
In order to compare Virana and the two subtractive approaches CaPSID and RINS in a controlled environment we rely on a previously published simulated data set consisting of a negative control data set free of viral reads , here denoted as background set . The background set is used to construct four additional validation data sets spiked with viral reads at increasing rates of sequence divergence ( 0% , 5% , 10% , 25% , see Materials and Methods ) . Performance is quantified in terms of sensitivity and specificity ( see Materials and Methods ) . Applying all three viral detection methods on the validation data sets reveals comparatively high rates of correctly detected viral reads for CaPSID and RINS at low sequence divergences between 0% and 5% . Specifically , the two subtractive methods achieve fold higher sensitivities compared to Virana ( sensitivities of versus for subtractive approaches and Virana , respectively , see Figure 2 ) . In contrast , Virana substantially surpasses subtractive approaches at higher rates of viral sequence divergence ( 10–25% ) , offering comparatively stable sensitivities between -fold and -fold higher than Capsid and RINS , respectively ( sensitivities of versus for subtractive approaches and Virana , respectively , see Figure 2 , left panel ) . Notably , while subtractive approaches fail to identify 20–90% of viruses in settings of high sequence divergence , Virana is the only approach able to reliably detect the full set of viruses in all validation scenarios ( see Figure 2 , right panel ) . As a result of Virana's ability to detect human-viral transcript homologs , reads originating from several human endogenous retroviruses ( HERVs ) that are part of the human reference genome but technically also belong to the viral family Retroviridae are detected in validation data at all levels of sequence divergence . Since the detected HERV reads originate from the human rather than from the viral part of the validation data , these reads classified as false positive ( FP ) hits for the purpose of this validation . As a result of this artifact , Virana exhibits a slightly lowered specificity compared to subtractive approaches ( 0 . 99985 versus 1 . 0 for Virana and CaPSID/RINS , respectively ) . However , we note that HERV reads are correctly classified by Virana during homologous region construction and by optional BLAST-based taxonomic annotation . These reads can therefore be safely and automatically ignored in subsequent analyses if HERV expression is of no interest to the researcher . In spite of the involved construction process of homologous regions , Virana is fastest among the three viral detection approaches , requiring only about half an hour per sample analyzed . In contrast , RINS and CaPSID require two to times longer per sample , respectively ( see Figure 3 ) . Interestingly , the majority of time spend by CaPSID is lost on subtraction , indicating that this step is a limiting factor of subtractive approaches . We note than reported times are based on analyses using a single compute core . Since all evaluated methods benefit from multithreading , dedicating additional compute cores to the analysis allows for further reduction in processing time . Having established Virana's ability to detect reads sampled at comparatively high coverage from viral genomes with low or no human-viral sequence similarity , we next test the sensitivity of the viral detection methods in a more challenging scenario involving gene regions of animal viruses that have close human homologs and are sampled at low sequencing coverages . Three such human-viral homologs are used in the analysis: V-ABL of the acutely transforming retrovirus A-MuLV , Bo17 of herpesvirus BoHV-4 ( a model virus for oncogenic gammaherpesviruses such as EBV and KSHV and implied in several animal cancers [69] ) and gag of HERV-K ( HML2 ) 22I , a class of human endogenous retroviruses associated with some forms of breast cancer [70] ) . Validation is based on simulated sequencing data and split into two scenarios ( see Materials and Methods for details ) . Within the first scenario , simulated sequencing reads are sampled directly from human-viral homologs while in the second scenario reads are generated from artificial fusion transcripts that each involve one of the three homologs fused to the human TP53 proto-oncogene . The resulting human-viral fusion transcripts mimic transcriptional signals indicating retroviral integration or homologous recombination of viral DNA next to a human gene which may result in activation of the latter by insertional mutagenesis . We apply the viral detection methods Virana , CaPSID , and RINS on these two validation data sets in order to evaluate sensitivity at detecting viral genes that are similar to human factors either due to natural sequence homology or due to gene fusions . Performance is quantified by detection sensitivity , specificity , as well as by the absolute number of reads correctly detected . While all methods performed at a perfect specificity of , only Virana detects viral transcripts at all coverages and with two to three-fold higher sensitivities compared to competing methods ( Figure 4 ) . In particular , sequence reads originating from endogenous retroviruses were almost always subtracted from the analysis by RINS and CaPSID . In addition , RINS seemed to be confounded by low sequencing coverage , a fact most probably resulting from its heavy reliance on de-novo transcript assembly . Subsequent analysis of discordantly mapped read pairs by Virana ( see Materials and Methods ) correctly identified the TP53 gene as fusion partner of both V-ABL and Bo17 , indicating that detection of human-viral chimeras is reliable even at low twofold coverage . Due to the repeat nature of the HERV-K sequence in the human genome and the resulting re-occurrence of HERV-K homologs at multiple loci in the human reference it was not possible to unambiguously identify the fusion partner of the HERV-K gag gene . Due to a variety of factors ( see Discussion ) human tumor viruses often replicate at very low levels within the infected cell . Determining the required sequencing depth for detecting viral transcripts present at specific cellular abundances is therefore crucial for planning transcriptome experiments designed to identify tumor viruses . Based on statistical arguments and average mRNA sizes ( see Materials and Methods ) , we inferred the minimal abundances of viral transcripts required in an average cell required for detection depending ( 1 ) on the length of the transcript being sought and ( 2 ) on the sequencing depth employed in the experiment . Here we report results for an average viral cDNA-transcript ( bp ) , an average viral transcript region analyzed in the validation of human-viral homologs ( Bo17 and vABL , bp , see previous section ) , an average length human CDS ( bp ) , and the genome size of a small tumor virus ( A-MuLV , bp ) . Based on these estimates and given an average sequencing depth as employed in the NB1 analysis panel , Virana requires a minimum twofold sequence coverage of an average viral cDNA transcript in order to detect the transcript within a homologous region with % probability ( Figure 5 , upper left quadrant , dashed blue vertical line ) . This sequence coverage is produced with % probability if at least one viral transcript is present per cell , on average ( Figure 6 , upper left quadrant , dashed blue vertical line ) . The number of viral transcripts per cell required for detection is inversely related to transcript length and sequencing depth , in principle: at a transcript length corresponding to a small viral genome ( bp ) and a per-sample sequencing depth of % of the sequencing depth generated in the NB1 panel , a transcript coverage of and at least viral transcripts per cell are required for reliable detection ( Figure 6 , upper right panel , dotted black vertical line ) . In order to evaluate Virana on experimental data we conducted an analysis of several positive and negative control samples with a cumulative size of Gbp . The negative control sequencing data originates from a normal brain transcriptome that is suitable as a control for neuroblastoma data . Positive controls span a range of cancer transcriptomes that are associated with several viral cofactors such as a hepatocellular carcinoma ( HCC ) cell line with proviral integration of Hepatitis B virus , a cervical squamous cell carcinoma ( ceSCC ) and two HeLa cell line samples with associated human papillomavirus ( HPV ) , and a Ebstein-Barr virus ( EBV ) positive B-cell lymphoma ( BCL ) . As displayed in Figure 7 ( upper part ) , analysis of the brain negative control sample demonstrates that viral transcription is ubiquitous even in normal ( non-cancerous ) samples . Specifically , several bacteriophages of the taxonomic families Microvirodae , Myoviridae , Podoviridae , and Siphovoridae indicate sample contamination with bacteria as well as technical spike-ins ( http://res . illumina . com/documents/products/technotes/technote_phixcontrolv3 . pdf ) . Remarkably , the Coliphage phi-X174 genome of the family Microvirodae could be fully assembled by Virana's homologous region construction , yielding a single fragment of 99% sequence identity and 100% coverage compared to the phi-x174 reference genome . In addition , several retroviral and flaviviral hits at low abundances of reads per million reads mapped ( RPMM ) highlight human factors such as HERV-Ks ( endogenous retroviruses ) as well as human proto-oncogenes SRC/ABL and DNAJC14/RP11 that have close homologs in the viral families Retroviridae and Flaviviridae , respectively . The taxonomic ambiguity of these regions is automatically identified during Virana's homologous region construction and confirmed by optional BLAST-based annotation compared to NCBI nt and nr databases ( as indicated by thinner bars in Figure 7 ) . Analysis of positive control samples resulted in homologous regions ( HORs ) spanning five viral families ( see Figure 7 , lower part ) . Viral cofactors associated with each of the cancer samples are correctly recovered at a high dynamic range of read abundances between RPMM ( HCC with integrated HBV provirus ) and RPMM ( HeLa cell line associated with HPV18 ) . In addition , several viral fragments were successfully reconstructed within HORs of the positive control samples , such as a bp long EBV segment containing latency-associated factors EBNA 3b , 3c , and 4a ( 80% sequence identity with the wild type genome ) as well as a bp long HBV fragment containing the oncogenic HBV-X gene ( 98% sequence identity compared with Hepatitis B virus isolate HK1476 ) . Similar to results on the negative control brain sample , several HORs with lower abundances assigned to the taxonomic families Retroviridae and Flaviviridae represent human-viral sequence homologies that are automatically flagged to be of ambiguous taxonomic status by Virana . Interestingly , the HCC sample was also investigated in recent work focusing on detecting viral integration events [52] . In this recent study , the authors confirmed one integration event by Sanger sequencing while alluding to two additional events still awaiting experimental validation . By analyzing discordantly mapped read ends , Virana could correctly identify all three HBV fusion events involving human genes TRRAP ( 11 read pairs ) , ZNF48 ( 11 read pairs ) , and PLB1 ( 6 read pairs ) as part of the primary mapping procedure . Deep-sequencing of neuroblastoma samples on two sequencing platforms yielded Gbp ( NB1 ) and Gbp ( NB2 ) of mapped read pairs ( including multimaps ) , respectively ( see Table 2 ) . While samples were sequenced independently and marked with unique identifiers to allow for sample tracking at each step of the analysis , reads from each sample panel and each tumor stage ( 4 or 4S ) were pooled for analysis . Processing the pooled sample panels with Virana resulted in homologous regions representing four viral families ( see Figure 8 ) . All HORs were associated with low relative read abundances of RPMM compared to confirmed viral signatures of experimental positive controls ( RPMM , see Figure 7 ) . Several homologous regions assigned to bacteriophage viral families Baculoviridae and Myoviridae are attributable to sample contamination . Reads assigned to viral families Retroviridae and Flaviviridae were determined to originate from either endogenous elements ( HERVs ) or from human proto-oncogenes that have close homologs in pestiviruses and acutely transforming retroviruses . HORs associated with these viral families were automatically assigned human or ambiguous taxonomic origin by Virana , as indicated by narrower bars in Figure 8 . We undertook manual investigation of homologous relationships within each ambiguous HOR by analyzing multiple sequence alignments and phylogenetic trees of the respective regions . These analyses revealed unambiguous clusterings of neuroblastoma sequence reads near human or endogenous factors in all cases ( see Figure 9 for an example phylogeny ) . No significant differences in viral expression signatures between neuroblastoma 4 and 4S stages could be detected except for HERV-K endogenous retroviruses which display higher abundances in stage 4S ( NB1: 56 RPMM , NB2: 28 RPMM ) than in stage 4 ( NB1: 41 RPMM , NB2: 15 RPMM ) neuroblastomas . All reads assigned to homologous regions were further analyzed for evidence of chimeric transcription ( see Materials and Methods ) . While several read pairs with putative chimeric mappings could be identified , all viral chimeric read ends were clustered within low-complexity regions of the viral genomes . Analyses revealed that these putative chimeric mappings represent sequencing errors and low-complexity templates that non-specifically attracted reads of similarly low sequence complexity . No cluster of chimeric reads located at a specifically viral genome location and representing a human-viral breakpoint could be identified . In order to identify transcripts of novel viruses that do not map to known references , we generated de-novo transcriptome assemblies of all unmapped reads . We applied the two de Bruijn graph based assembly methods Oases[61] and Trinity[60] that demonstrated best-in-class performance in recent evaluations [71] on sequencing data of the NB2 panel . This sequencing data is especially amenable to assembly due to its long read length ( see Table 1 ) . Assembly resulted in and reconstructed neuroblastoma 4S contigs for Oases , and Trinity , respectively ( see Figure 10 ) . Assembly of the neuroblastoma 4 sample yielded and contigs from the same methods . Results of Oases and Trinity assemblies are comparable in terms of contig length . All contigs were subjected to taxonomic annotation using high-sensitivity TBLASTX annotation based on human and viral content of the NCBI nt and nr databases ( see Materials and Methods ) . Overall , contigs ( of contigs of any specific assembly ) were identified to be of putative viral origin . contigs were assigned to bacteriophage references and excluded from further analysis . Based on searches against the full NCBI nr and nt databases followed by manual inspection , all remaining contigs were determined to display higher similarities to bacterial or human sequences than to any viral reference .
Neuroblastoma is a pediatric tumor of the sympathetic nervous system that represents the most common form of cancer in infancy . It is characterized by a striking diversity in biology and clinical behaviour of its subtypes . This heterogeneity as well as supporting epidemiological findings are highly suggestive of infectious cofactors involved in genesis and maintenance of the disease [19] , [20] . While several studies utilizing technologies with lower sensitivity compared to our approach have identified human polyomaviruses in neuroblastoma and pediatric embryonal tumors [22]–[24] , newer investigations seem to render these associations inconclusive [25] . However , viral commensals of the families polyomaviridae and adenoviridae are indeed suspected to acquire rare transforming properties as a consequence of viral latency or defective replication [72] and to encode oncogenes [73] , [74] whose carcinogenic potential in human is currently investigated [8] , [75] . We undertook the first systematic search for known and unknown viruses in transcriptomes of metastatic neuroblastoma by analyzing deep sequencing RNA-Seq data of metastatic neuroblastomas from two tumor stages as well as positive and negative experimental controls . Several high-throughput methods for detecting viral sequence reads among human RNA-Seq data have been developed . Among these methods , PathSeq , CaPSID and RINS are most prominent due to their design as reusable computational pipelines . In this study we selected CaPSID and RINS due to their high performance and public availability and compared their detection performance with that of our novel method Virana . Both CaPSID and RINS follow a subtractive approach , e . g . they separately map input data to viral and human reference sequences and subtract viral read mappings that are similar to the human genome from the analysis . While CaPSID is conceptualised as a generalised framework that supports the subtraction process by means of a database and a web server , RINS features an integrated pipeline that splits input reads into shorter fragments in order to increase mapping sensitivity , followed by transcriptome assembly of putative viral reads into full length transcripts . Both RNA and DNA viruses may share considerable sequence homology to human factors due to reasons such as lateral gene transfer , oncogene capture , ancestral endogenization , or insertional mutagenesis leading to chimeric transcripts [47] . Such homologous transcripts may display human-viral sequence similarities of 86% ( Bovine Herpes virus ) and up to 92% ( acutely transforming retroviruses ) . Subtractive approaches silently discard these transcript from the analysis due to their similarity to the human reference genome . In contrast , our novel method Virana follows a radically different approach . Instead of separate mapping to viral and human reference database followed by digital subtraction , Virana undertakes a particularly sensitive read mapping to a combined set of human and viral references . By allowing for multimaps , this mapping strategy facilitates discovery of viral transcripts regardless of their similarity to human factors . Apart from being conceptually simpler by relying on only one mapping step and discarding the subtraction procedure that is both possibly erroneous and computationally costly , this approach empowers the mapper to make informed decisions about relative alignment quality by weighing different human and viral reference positions against each other . As a direct consequence of this increased mapping quality , paired-end reads can be mapped across human and viral references , allowing for detection of human-viral chimeric transcription and proviral integration events . We quantitatively validated Virana's approach both in settings involving simulated reads as well as in real-world scenarios involving experimental positive and negative controls . In these validations , Virana displays significantly higher detection sensitivities than competing approaches especially at high rates of viral sequence divergence exceeding % that are common for tumor viruses [76]–[78] . As a consequence , Virana was the only method able to detect all viral families independent of sequence divergence in the validation data set . In spite of the additional processing undertaken by our method , Virana features between and two and three times faster execution speeds compared to related methods . Interestingly , viral reads analyzed in the sequence divergence validation originate from a broad array of viral species , only two of which infect mammalian hosts and none of which display significant human-viral sequence homology . As a consequence , this validation favors subtractive approaches by reducing the danger of erroneous subtraction of viral reads that are similar to the human genome . In addition , the sequence divergence validation contained reads sampled at high coverage . However , transcripts of tumor viruses are often expressed at only low cellular abundances and are thus expected to have low sequence coverage . We therefore next validated the ability of viral detection approaches to detect viral transcripts homologous to human factors at varying levels of sequence coverage . Virana , by virtue of not relying on digital subtraction , demonstrated superior sensitivity at this validation both in settings of natural sequence homology as well as in cases of human-viral chimeric transcription . Specifically , Virana was the only method able to detect evidence for all viruses even at low twofold coverages . We observed that both RINS and CaPSID discarded a substantial amount of human-viral homologous transcripts due to their high similarity to the human reference genome , a fact that explains the lower performance of these methods in this validation scenario . Analysis of positive and negative experimental controls further reveals that Virana is able to detect viral transcripts associated with four types of cancer at a high dynamic range of relative abundances . While Virana displays a slightly reduced specificity in simulated and experimental evaluations , these false positive hits are limited to only two viral families ( Flaviviridae and Retroviridae ) that display high sequence similarity to human factors . These hits are additionally annotated with an ambiguous taxonomic origin by Virana . In addition , Virana provides extensive support for investigating such ambiguous viral hits by analyzing the homologous context of putative viral reads in a context of multiple sequence alignments and phylogenies . In principle , several biological confounding factors may hinder detection of viral transcripts by any sequence-based method . Low concentration and extratumoral location of viral producer cells [8] or selection of growth-autonomous cells in progressed tumors [79] can significantly dilute the number of viral transcripts in a sample . Additionally , known tumor viruses such as high-risk HPV strains , EBV , and MCPyV selectively transcribe their genome during viral latency ( HPV: E6/7 [80] , [81] , EBV: EBNA1/2 [82]–[84] , MCPyV: large T antigen [31] , [85] ) , thus generating only low abundances of tens ( MCPyV [31] ) to hundreds ( KSHV [86] , EBV [87] ) of transcripts per cell . Last , transcription of human oncogenic factors modulated by viral [88] or endogenous [89] , [90] retroviral promoters as well as ‘hit-and-run’ mechanisms of viral oncogenesis that imply loss of viral material [91] , [92] may predispose cells to transformation without requiring maintenance of viral transcripts . Our approach aims to counteract these confounding factors by two strategies: first by sequencing neuroblastoma transcriptomes at comparatively high depth in order to detect rare transcripts and second by using several biological replicates at different tumor stages , thus reducing the probability of total loss of viral material from all analyzed samples . Based on statistical estimations concerning Virana's homologous region construction process and the sequencing depth of our experimental data , we can conclude that our approach requires minimal abundances of only two average-length viral transcripts per cell even under adverse conditions such as high viral divergence or extensive human-viral sequence homology . While representing a theoretical sensitivity that may be altered by sequencing biases [93] , these copy numbers compare very favorably with related estimates reporting minimal abundances of one to several complete viral genomes per cell [27] , . After applying Virana to several positive control panels of human cancers with known viral cofactors and accurately reconstructing large fragments of viruses that are causally related to the respective tumors , we analyzed neuroblastoma transcriptomes at high sequencing depth and using two different sequencing platforms . Analyses of neuroblastoma transcriptomes resulted in the detection of putative viral transcripts with high local sequence similarity to several viral families . However , automatic taxonomic annotation as well as detailed manual inspection of homologous regions pertaining to these families revealed the human or bacteriophage origin of all transcripts . While we could find differences in the abundance of HERV-K transcripts between neuroblastoma stages 4 and 4S , the causative role of HERV transcription with regard to oncogenesis is currently unclear [94] and , as to our knowledge , only tentative associations with specific cancers have been made as to date [70] . Apart from these tentative differences in HERV-K abundances , no quantitative difference between neuroblastoma stages 4 and 4S could be identified with regard to viral transcription . In conclusion , our observations provide negative evidence regarding the contested question of putative viral cofactors of metastatic neuroblastoma by suggesting that viruses are unlikely to be frequent cofactors in the maintenance of metastatic neuroblastoma . | Many human cancers are caused by infections with tumor viruses and identification of these pathogens is considered a critical contribution to cancer prevention . Deep sequencing enables us to systematically investigate viral nucleotide signatures in order to either verify or exclude the existence of viruses in idiopathic human cancers . We have developed Virana , a novel computational approach for identifying tumor viruses in human cancers that is applicable to a wide variety of tumors and viruses . Virana firstly addresses several important biological confounding factors that may hinder successful detection of these pathogens . We applied our approach in the first systematic search for cancer-causing viruses in metastatic neuroblastoma , the most common form of cancer in infancy . Although the heterogeneous clinical progression of this disease as well as epidemiological and virological findings are suggestive of a pathogenic cofactor , the viral etiology of neuroblastoma is currently contested . We conducted an analysis of experimental controls , comparisons with related approaches , as well as statistical analyses in order to validate our method . In spite of the high sensitivity of our approach , analyses of neuroblastoma transcriptomes did not provide evidence for the existence of any known or unknown human viruses . Our results therefore suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely . | [
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] | [] | 2013 | Sensitive Detection of Viral Transcripts in Human Tumor Transcriptomes |
Proteases perform numerous vital functions in flatworms , many of which are likely to be conserved throughout the phylum Platyhelminthes . Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation . The flatworm Schmidtea mediterranea , or planaria , is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference ( RNAi ) . In this study , we were interested in deconvoluting the digestive protease system that exists in the planarian gut . To do this , we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S . mediterranea . Using a panel of fluorescent substrates , we show that this treatment produces a sharp increase in proteolytic activity . These enzymes have broad yet diverse substrate specificity profiles . Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases . However , treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion . Specific RNAi knockdown of the cathepsin B-like cysteine protease ( SmedCB ) reduced protein degradation in vivo . Immunohistochemistry and whole-mount in situ hybridization ( WISH ) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen . Finally , we show that the knockdown of SmedCB reduces the speed of tissue regeneration . Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms , potentially uncovering drug targets in parasites .
The family Platyhelminthes contains an estimated 25 , 000 species , including the free-living planaria Schmidtea mediterranea . This freshwater flatworm has been an experimental model for over a century due to its regenerative capacity . Worms can fully regenerate missing body tissues from fragments as small as <1/200th of their original size [1] . This is due to the large population of stem cells , or neoblasts , that make up approximately 30% of all adult tissues in the worm [2] . The S . mediterranea genome has been sequenced and these worms are highly amenable to the use of RNAi , making them an ideal flatworm for study [3 , 4] . While S . mediterranea is commonly used as a model for regeneration , it has recently been proposed as a model for parasitic flatworms , much like the free-living Caenorhabditis elegans is used as a model for other parasitic nematodes [5] . Over half of the known members of Platyhelminthes are human or veterinary parasites , including worms of the genus Schistosoma , the causative agents of the disease schistosomiasis . Schistosomiasis affects more than 200 million people worldwide and is the second most common parasitic disease behind malaria [6] . However , these and other parasitic worms are difficult to study due to their complex , multi-host life cycles and their resistance to biochemical tools such as RNA interference ( RNAi ) . S . mediterranea and the parasite S . mansoni share several similarities in their reproductive systems , protonephridia , and stem cell populations [7] . Both are triploblastic , bilaterally symmetric metazoans and contain nervous , digestive , and secretory systems [8] . In addition , these organisms share over 85% of their predicted proteome , including several families of proteases [9] . For digestion , S . mansoni and S . mediterranea have a blind-end , bifurcated gut that distributes digested food throughout the body [10] . Adult female S . mansoni worms will utilize several distinct proteases to rapidly digest red blood cells at a rate of 500 per minute [11] . While vertebrates rely on serine proteases from the trypsin family ( clan PA ) for protein digestion , invertebrate helminths generally use cysteine proteases from the clan CA ( papain-like proteases ) and aspartic proteases from the clan AA . Cysteine and aspartyl proteases are also key to digestion in other invertebrates like nematodes and arthropods . It appears that the preference for digestive serine proteases occurred during the evolution of arthropods or mollusks [12 , 13 , 14] . Three cysteine cathepsin proteases perform the majority of digestive function in helminths: cathepsins B , C , and L . These clan CA proteases are found in many flatworm parasites , including the trematodes Fasciola hepatica , Clonorchis sinesis , and Opisthorchis viverrini [11 , 15] . Immunohistochemistry suggests that cathepsins B , C , and L are associated strongly with the gastrodermis , vitellaria , and tegument [15] . These proteases work in concert to degrade hemoglobin and albumin in the acidic helminth gut [16] . Inhibition of cysteine proteases has been shown to kill parasites both in vitro and in vivo , suggesting that these proteases are important for worm viability [17 , 16] . Previous work has shown that inhibition of schistosome cysteine cathepsin protease activity in infected mice has led to a reduced worm and egg burden and an improvement in organ pathology [18] . Schistosoma flatworms can be induced to regurgitate their gut contents and the cysteine proteases cathepsin B , C , and L , as well as an aspartyl protease ( cathepsin D ) and an asparaginyl endopeptidase ( legumain ) , have been identified [19 , 10] . Cathepsin B1 , or SmCB1 , represents the most abundant cysteine peptidase activity measurable in both adult schistosomes and gastrointestinal content ( GIC ) extracts . Along with degrading host hemoglobin and albumin , SmCB1 has been shown to degrade several immunoglobulins in vitro , suggesting possible roles in immune evasion [20] . Furthermore , SmCB1 has been suggested as a drug target , a potential vaccine target , and a serodiagnositc marker [21 , 22] . Although schistosomula larvae with cathepsin B reduced by RNAi were still viable , these worms showed a significant decrease in growth compared to control groups [23] . This suggests that SmCB1 plays an important role in digestion such that a lack of activity has a negative effect on the acquisition of nutrients for growth . Very little is known about the function of proteolytic enzymes in planaria and if conservation in enzymatic function exists between parasitic and free-living flatworms . Given the important role played by S . mansoni gut proteases in digestion , we investigated the role of these enzymes in the gut of S . mediterranea . Using proteomics and a global protease substrate profiling method , referred to as multiplex substrate profiling by mass spectrometry ( MSP-MS ) , we identified and detected active proteases in S . mediterranea gastrointestinal contents . Using RNAi and specific protease inhibitors , we probed the function of several protease families in vivo to determine the roles of these enzymes in protein digestion . We further examined a cathepsin B-like cysteine protease and its localization in S . mediterranea . We hypothesized that planarians utilize cysteine proteases for digestion and that they perform similar roles in free-living flatworms as in parasitic helminths .
A clonal line [4] of diploid , asexual Schmidtea mediterranea [24] was used in all experiments . Worms were maintained as described previously at room temperature ( 20–22°C ) in 1x Montjuïc salts ( worm water ) , pH 7 . 2 , and were fed every two weeks with organic beef liver [25] . Unless otherwise stated , animals were starved for two weeks prior to use in experiments . Worms were starved for one week and washed several times in 1x Montjuïc salts before the addition of 3% EtOH for one hour to induce regurgitation . Treatment with low-percentage ethanol does not lead to long-term damage of worms [26] . Control samples were treated with water . Worm regurgitant was collected and filtered using a 50mm Filter Unit ( Nalgene ) , then concentrated 50-fold using at an Amicon Ultra 10K MWCO Centrifugal Filter at 8 , 000 x g and 4°C . The concentrated vomit was stored at -80°C . Protein identification in S . mediterranea regurgitant was performed using peptide sequencing by mass spectrometry . 10μg total protein was digested with trypsin , extracted , desalted using C18 zip-tips ( Rainin ) , and lyophilized . Liquid chromatography-tandem mass spectrometry was performed as previously described [27] . Algorithms in the BLAST2Go program ( v2 . 7 . 2 ) were used to search for proteins with shared sequence features to proteins in S . mediterranea vomit . Searches were conducted against the SwissProt or NCBInr databases using the National Center for Biotechnology Information Server ( July 27 , 2015 , blast . ncbi . nlm . nih . gov/Blast . cgi ) and the top ranking hits reported in S1 Table . Protease activity in regurgitant from three replicate tanks were compared using a mixture of 7 internally quenched fluorescent substrates available from CPC Scientific , Sunnyvale , California ( Table 1 ) . These substrates were chosen based on their diverse sequence composition to enable detection of multiple protease classes . Worm regurgitant was diluted 7 . 5-fold in assay buffer containing 2 . 5 μM of each substrate , 100 mM NaCl , 2 mM DTT , 0 . 01% Tween-20 and 20 mM Citrate-Phosphate buffer at pH 3 . 5 , 5 . 5 or 7 . 5 . For inhibitor assays , the regurgitant from three replicate tanks were combined at equal volume and incubated with 10 μM Pepstatin-A , 10 μM E-64 , 1 mM 1 , 10-Phenanthroline , 0 . 5 mM AEBSF or 1% DMSO . Assays were run for 1 hour at room temperature in black round bottom microplates using an excitation wavelength of 330 nm and emission wavelength of 400 nm . Activity was reported as change in fluorescent units per second . The MSP-MS assay was performed as previously described with minor modifications [28] . Worm regurgitant ( 500ng/mL ) was assayed with an equal molar mixture of 124 tetradecapeptides ( 500nM each ) in a total reaction volume of 300μL . Assays were performed at pH 3 . 5 , 5 . 5 , or 7 . 5 and incubated for 15 , 60 , 240 , or 1200 minutes before quenching with concentrated formic acid to a final pH of 2 . 5 . Samples were desalted with C18 zip-tips ( Rainin ) and analyzed by LC-MS/MS sequencing . Mass spectrometry and data analysis were performed as described previously [29] . To compare substrate specificity , iceLogo software ( http://iomics . ugent . be/icelogoserver/ ) [30] was used to generate the specificity signature for amino acids at ±4 positions adjacent to the identified cleavage site . Sequences of Schistosoma proteases were obtained via BLAST and used to manually identify homologs found using the Schmidtea mediterranea database ( SmedDb; http://planaria . neuro . utah . edu ) [4] . Hits were checked via BLAST; reciprocal best hits were scored as putative homologs . The major cathepsin B homolog was isolated from planaria lysate ( ~50 planarians in lysis buffer; 100mM Tris pH 7 . 5 , 200mM NaCl , 1% NP-40 , 0 . 1% SDS , 1XTBS ) . Lysate was separated on a MonoQ 10/100 GL column ( GE Healthcare Life Sciences ) and assayed for protease activity with Z-RR-AMC ( BACHEM ) . Fractions of cathepsin B activity were pooled and separated on a Superdex 200 10/300 GL column ( GE Healthcare Life Sciences ) which had been tested with a Gel Filtration Calibration Kit LMW ( GE Healthcare ) to determine at what volume the cathepsin B homolog would elute . The resulting samples with proteolytic activity were run on a 10% SDS-PAGE gel , silver stained , and cut into bands for analysis on PE-Biosystems Voyager Elite STR MALDI-TOF . The band corresponding to the most proteolytic activity and correct size was determined to be the cathepsin B homolog identified by SmedDb . To measure protease activity of worm lysates , planarians were ground with mortar and pestle for thirty seconds and incubated with lysis buffer ( 100mM Tris pH 7 . 5 , 200mM NaCl , 1% NP-40 , 0 . 1% SDS , 1XTBS ) for one hour on ice with occasional vortexing . Samples were spun in a microcentrifuge at maximum speed , 4°C for twenty minutes . The supernatant was saved and activity was measured by adding assay mix ( 5mMDTT , 50mM sodium citrate , pH5 . 5 ) containing 50μM fluorescent peptide , either Z-FR-AMC or Z-RR-AMC ( BACHEM ) . Fluorescence was measured ( excitation 360nm , absorbance 460nm ) using a FlexStation fluorometer ( Molecular Devices ) and SoftMax Pro 4 . 8 software . Both kinetic and endpoint assays were used . Chemical inhibitors of cathepsin B , K11777 ( UCSF , CDIPD ) and CA-074 ( Sigma ) , were added at 50μM concentration two hours prior to addition of fluorescent peptides for select experiments . Protease activity was also measured with activity-based probes DCG-04 and BMV109 , both gifts from Matthew Bogyo [31 , 32] . Probes were added to 1μM final volume in samples containing 5mM DTT , pH 5 . 5 for 1 hour at 37°C before imaging Cy5 levels via Typhoon Trio ( GE Healthcare Life Sciences ) . In vitro double-stranded RNA ( dsRNA ) was synthesized from a PCR template of cathepsin B using T3 and T7 polymerases ( Promega ) . The dsRNA was injected as described [33] . Worms were injected with three 33 . 2nL pulses of dsRNA ( 100ng total ) once a day over three consecutive days . To determine the efficiency of knockdown , quantitative real-time PCR ( qPCR ) was performed using an Mx3005P QPCR System ( Agilent Technologies ) and LightCycler SYBR Green Master I ( Roche ) . mRNA levels of cathepsin genes were compared to a standard internal control clone , GAPDH ( Accession number: AY067285 . 1 ) to normalize RNA starting material . The efficiency and specificity of the primers was tested via serial dilutions of primers and cDNA template according to the protocol [34] . Primers used: GAPDH: 5’-AGCTCCATTGGCGAAAGTTA-3’ , 5’- CTTTTGCTGCACCAGTTGAA -3’; CB1: 5’-CTAGATTCCAAACCGTTTCGGACA-3’ , 5’-CAAGCTGCCAAAGAAAAGTTCAGG-3’; CL1: 5’-CAAGGCTATCCAGCAAATGG-3’ , 5’-GAATCAACGCATTTGCAAT-3’; CD: 5’-GGCGAAATCACAATTGGAAC-3’ , 5’-ATTTTCCATTCGATACGGCA-3’ . Planaria were fed pureed liver with tetramethylrhodamine conjugated bovine serum albumin ( RhBSA , Molecular Probes ) . Worms were starved one week prior to feeding; RNAi treated worms were fed one week after injections were completed . Unless otherwise stated , worms treated with chemical inhibitors ( K11777 , pepstatin ) received drug treatment for one hour prior to feeding . Worms were fed in the dark for one hour before being transferred into worm water ( with or without appropriate drug ) without food for one hour . Live worms were placed on slides on ice for a few minutes to stop worm movement before imaging . Images were taken using an Axiovert 40 CFL microscope and Axiovision Rel . 4 . 8 . 2 software ( Zeiss ) . Fluorescence was quantified using ImageJ . Live worms were imaged again after 24 or 48 hours . Measurements were evaluated by paired t-tests; differences were considered significant with P<0 . 05 . The catalytic region of S . mediterranea cathepsin B ( based on the reference sequence mk4 . 000308 . 13 . 01 ) was cloned into pET28a and expressed in Escherichia coli . Protein was purified under reducing conditions ( 8M urea ) on Ni-NTA Agarose ( Qiagen ) and dialyzed before mouse immunization . Sera was pooled and purified with NAb Protein G Spin Columns ( Pierce ) . Three peptide regions of cathepsin B were selected and synthesized by New England Peptide , and combined to immunize rabbits by Covance . Antibodies were purified with NAb Protein G Spin Columns ( Pierce ) . DNA template for in vitro transcription of anti-sense RNA probes was amplified from a PCR sequence containing the full length SmedCB1 gene . Probes were synthesized in an in vitro transcription with DIG-12-UTP ( Roche ) and precipitated with lithium chloride and ethanol according to the Roche suggested protocol . Probes were then resuspended and stored; animals between 2–5mm were processed for WISH as previously described [35] with modifications [36] . All animals were treated with 1:100 dilution of SmedCB probe . Animals were killed by treatment with 5% N-Acetyl L-Cysteine for five minutes at room temperature , fixed in 4% formaldehyde , and dehydrated in 50% MeOH . Planarians were embedded in paraffin , sliced , and treated with antibodies described above . For immunofluorescence , anti-rabbit and anti-mouse secondary antibodies with Alexa Fluor 488nm ( LifeTechnonolgies ) were used at 1:100 . All imaging was performed using AxioImage . M1 , Axiovision Rel . 4 . 8 . 2 software ( Zeiss ) . Worms for electron microscopy were fixed and processed using the same procedures as the immunofluorescence . Worms were treated with the anti-zymogen antibody described above and an anti-rabbit 10nm gold preadsorbed secondary antibody ( AbCam ) . To measure the effect of cathepsin B RNAi on regeneration , planarians were amputated one full day after injections were complete . Worms treated with cathepsin B inhibitors were also amputated pre- and post-pharyngeally and immediately placed in worm water containing the appropriate drug . Unless otherwise indicated , images for size quantitation were taken immediately after amputation ( Day 0 ) and again on days 5 , 8 , 11 , and 14 . Worms saved for RNA extraction for qPCR were also frozen at -80°C on these days . Live images were taken using an AxioZoom . V16 microscope and Axiovision Rel . 4 . 8 . 2 software ( Zeiss ) . Phenotypes were scored by measuring body areas with ImageJ . Each planaria amputated fragment ( head , pharynx , and tail ) was measured for change in total area and blastema size . Changes in area were evaluated with paired t-tests; differences were considered significant with P<0 . 05 .
A preliminary BLAST search using sequences from previously identified S . mansoni proteases against the S . mediterranea database ( http://smedgd . neuro . utah . edu ) revealed that several protease families are conserved between both parasitic and free-living flatworms ( Table 2 ) . Among the conserved protease families were aspartyl and cysteine cathepsin proteases , including cathepsin B , D , L1 , and L2/L3 , all of which are involved in blood feeding and found in the gut of in Schistosoma worms . The aspartyl proteases cathepsin D had two putative homologs , while the cysteine protease cathepsin L1 and L2/L3 each had several putative homologs . Unlike S . mansoni , only one gene corresponding to cathepsin B was found in S . mediterranea . Interestingly , two well-characterized S . mansoni proteases were absent in the S . mediterranea genome . These enzymes are the serine protease , cercarial elastase , which is involved in skin invasion , and the gut-associated asparaginyl endopeptidase called legumain [10] . To identify and characterize intestinal tract proteases in S . mediterranea , worms were placed in 3% ethanol to induce regurgitation ( S1 Video ) . These worms were starved for a week to avoid contamination by proteases from the S . mediterranea food source . Protease activity in the worm regurgitant was assessed using a mixture of internally quenched fluorescent substrates . These substrates consist of 7-mer to 10-mer peptide sequences flanked by 7-methoxycoumarin on the amino terminus and dinitrophenol conjugated to lysine on the carboxyl terminus . Protease activity from replicate S . mediterranea tanks was assayed in pH 3 . 5 , 5 . 5 and 7 . 5 buffer . Activity was highest at pH 3 . 5 and lowest at pH 7 . 5 . No activity was detected in the water from worms that were not exposed to ethanol ( Fig 1A ) . The increase in protease activity due to ethanol treatment proved that we had successfully enriched for the gut proteases of S . mediterranea . The regurgitant from the replicate tanks was pooled and the substrate specificity profile was uncovered using an unbiased substrate profiling assay consisting of 124 physiochemically diverse peptides of 14 residues each [28] . Cleavage of these peptides was detected by LC-MS/MS sequencing after incubation of regurgitant with the peptide mixture at pH 3 . 5 , 5 . 5 and 7 . 5 . An aliquot of each reaction was removed after 15 , 60 , 240 , and 1200 minutes . Proteases active at pH 3 . 5 and 5 . 5 cleaved at more sites than the proteases that were active in the pH 7 . 5 , indicating a broader substrate specificity ( Fig 1B ) . Proteases active in the pH 3 . 5 buffer had a preference for all hydrophobic residues except Val and Pro at most positions between P3 and P4ʹ ( Fig 1C ) . In the pH 5 . 5 buffer , hydrophobic residues were also found at high frequency in most sub-sites , except for P2ʹ , which prefers Arg . In addition , Tyr and Arg are well tolerated at P1 and P1ʹ . Unlike the cleavage sites detected in the pH 3 . 5 and 5 . 5 assays , protease activity at pH 7 . 5 was dominated by exo-peptidases . After 15 minutes incubation , 30 out of 36 cleavage sites detected occurred at the amino or carboxyl termini of the 14-mer peptides . After 60 minutes incubation , 68 out of the 94 cleavage sites had occurred at the termini and the overall substrate preference was for arginine at P1 and norleucine at P1ʹ . Taken together , these data indicate that multiple proteases are present in the gut of S . mediterranea that are capable of cleaving diverse set of peptide bonds over a broad pH range . In order to better understand the proteases responsible for cleaving the peptide substrates , we incubated S . mediterranea regurgitant with the class specific protease inhibitors pepstatin-A , E-64 , 1 , 10-phenanthroline , and AEBSF . These compounds are standard inhibitors of aspartyl , cysteine , metallo- , and serine proteases , respectively . Under all conditions tested , AEBSF failed to inhibit activity , indicating that there were no active serine proteases present in the regurgitate ( Fig 1D ) . At each pH , 1 , 10-phenanthroline reduced activity by more than 50% , while pepstatin and E-64 inhibited protease activity at pH 3 . 5 and pH 5 . 5 only . To identify the specific proteins responsible for the aspartyl- , cysteine- and metallo-protease activity in the S . mediterranea regurgitant , we performed a proteomic analysis on the regurgitant proteins . Proteins were digested with trypsin , and the resulting peptides were sequenced with liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) . These peptides were compared to a database of putative proteins from the asexual S . mediterranea strain ( http://smedgd . stowers . org/downloads/ ) . Peptides were ranked based on their intensity sums . We identified a total of 122 proteins , including 7 proteases ( S1 Table ) . Based on peptide count , the two metalloproteases , astacin-2 and astacin-5 appear to be the most abundant proteases in the regurgitant . Recombinant astacin from the parasitic nematode , Teladorsagia circumcincta was expressed in E . coli and found to be sensitive to 1 , 10-phenanthroline [37] . Therefore , it is likely that astacin-2 and -5 are the source of the 1 , 10-phenanthroline-sensitive activity in S . mediterranea regurgitant . We also found that one regurgitant sample contained three other metalloproteases: an M12 protease , astacin 1 , and two M10 matrix metalloproteinases , mmp1 and mmp2 . Although our inhibition studies determined that there was one or more pepstatin-sensitive aspartyl protease in the gut , we were unable to identify this protease by mass spectrometry; it is possible that aspartyl proteases are in lower abundance than metallo- or cysteine proteases . However , we were able to detect two clan CA cysteine proteases; a cathepsin B-like and a cathepsin L1 protease that are likely to be the E-64 sensitive enzymes in the pH 5 . 5 assay . As noted above , cysteine , aspartyl , and metalloproteases are all active in the regurgitant of S . mediterranea and are all likely to play important yet distinct roles in protein digestion . To investigate the multi-enzyme model of digestion , we utilized a chemical knockdown strategy using protease inhibitors to monitor the effect of these protease classes . Starved worms were fed beef liver coated with rhodamine-labeled bovine serum albumin ( Rh-BSA ) , which has been used previously to assay digestive proteases in S . mansoni [10] . Fluorescence generated by cleavage of quenched rhodamine-BSA molecules is proportional to the activity of digestive enzymes; lower fluorescence indicates a decrease in digestion ( Fig 2A ) . Measurements are represented as fluorescence over worm area to correct for differences in worm size . We first examined the role of cysteine proteases , specifically the two identified in the worm regurgitant: cathepsin B and cathepsin L . While E-64 is the standard pan-cysteine protease inhibitor in vitro , its poor cell-permeability limits its effects in vivo [38] . However , the vinyl sulfone K11777 is a potent chemical inhibitor of both cysteine proteases cathepsin B and L , is cell-permeable , and has been shown to reduce the parasite burden of schistosomes in mice [39 , 18] . Chemical inhibition of cysteine cathepsin proteases by K11777 strongly reduced digestion in the planaria gut . Worms pretreated with K11777 were fed RhBSA and fluorescence was imaged as a measurement of proteolytic activity . K11777 treated worms exhibited a 38% reduction in fluorescence compared to untreated controls ( Fig 2B and 2E ) . This decrease in signal persisted over time , and twenty-four hours post feeding treated worms had a 42% decrease in signal . As one or more pepstatin-sensitive aspartic proteases are present in the S . mediterranea regurgitant , we used this inhibitor to determine the role of these enzymes in protein digestion . Worms treated with pepstatin resulted in a 16% reduction in RhBSA activity immediately after feeding compared to the untreated worms . After 24 hours , only a 6% decrease in activity was evident . However , co-treatment with pepstatin and K11777 had a dramatic effect on digestion . Treated worms were on average 46% and 37% less bright than vehicle treated worms after one and twenty-four hours , respectively ( Fig 2C and 2F ) . The representative image in Fig 2C shows that very little fluorescence , indicative of albumin degradation , was observed in worms where both cysteine and aspartic proteases were inhibited . Due to the presence of metalloproteases in the worm regurgitant as documented by biochemical assays and the proteome analysis , 1 , 10-phenathroline was used to examine the effect of reducing the metalloprotease activity . However , 1 , 10-phenanthroline was highly toxic to the worms and therefore two alternative metalloprotease inhibitors , bestatin and EDTA , were used for the in vivo digestion studies . Representative images in Fig 2D show that no decrease in fluorescence due to albumin degradation was detected; therefore , digestion of rhodamine-labeled albumin was unaffected by metalloprotease inhibitors ( Fig 2D and 2F ) . The reduction in protein digestion in S . mediterranea that occurs in the presence of a combined treatment of a cysteine and aspartyl protease inhibitor confirms that these proteases are the major digestive enzymes in the worm gut . Although we could not confirm the exact aspartyl protease present , we predicted that this enzyme was a cathepsin D-like protein found in the genome of planaria . Primer pairs were designed against the mRNA sequence of cathepsin D and the two cysteine proteases , cathepsin B and L . We first used RT-PCR to quantify the levels of cysteine and aspartyl proteases over time following starvation . Worms starved over a three-week period showed a 50–65% reduction in cathepsin D levels and a 65–75% reduction in cathepsin B mRNA levels while cathepsin L levels were reduced by only 20% ( Fig 3A ) . These data suggest that cathepsin B and D expression is regulated by food intake while cathepsin L expression is less affected by changes in feeding conditions . To examine the specific role ( s ) played by SmedCB , we used RNAi to assess whether loss of function was detrimental to worm viability and survival . Worms were first starved for two weeks before injection with ~100ng SmedCB dsRNA for three consecutive days . This protocol resulted in 80% reduction in SmedCB mRNA levels after fourteen days when compared to untreated worms ( Fig 4A ) . This RNAi knockdown was specific to SmedCB and did not significantly impact mRNA levels of related proteases cathepsin L or cathepsin D ( Fig 3B ) . Using the standard cathepsin B fluorescent substrate , z-Arg-Arg-AMC [40] , protease activity was reduced in worm lysates by 81% fourteen days after SmedCB mRNA knock-down . ( Fig 4B ) . Activity using this substrate was confirmed to be derived from cathepsin B because CA-074 , a highly selective cathepsin B inhibitor , reduced total activity by 71% ( Fig 5 ) . In addition , protein hydrolysis using rhodamine-labeled albumin was reduced by 20% for 48 hours after feeding ( Fig 4C ) . Taken together , these data confirm that SmedCB specifically plays a central role in protein digestion in S . mediterranea , much like cathepsin B1 does in Schistosoma mansoni . Schistosoma mansoni expresses two isoforms of cathepsin B; SmCB1 is found in the gut and performs a digestive function , while SmCB2 is localized to the tegument where it serves an unknown purpose . We hypothesized that SmedCB would also be found in the gut of S . mediterranea , as indicated by its presence in the worm vomit , and would perform a role in digestion . In order to determine the localization of SmedCB in S . mediterranea , we first needed to confirm that this protein was detectable in a whole worm lysate . Using the activity based probe DCG-04 [31] , which specifically targets cysteine proteases , we observed a biotinylated band on an SDS-PAGE gel at 27 kDa . Mass spectrometry sequencing of the excised band confirmed that its identity as SmedCB ( S2 Table ) . Knowing that SmedCB was present in whole worm lysate and not just in the regurgitation , we developed antibodies that could detect procathepsin B and mature cathepsin B . Peptides encoding a region of the propeptide , as well as the catalytic domain , were synthesized for antibody development ( Fig 6A ) . Both antibodies specifically labeled SmedCB in the planaria lysate ( Fig 6B ) . Immunohistochemistry using the zymogen SmedCB antibody labeled cells surrounding the intestinal lumen ( Fig 6C and 6D ) . Localization of the catalytic region of SmedCB , found in both the full-length zymogen as well as the catalytically activated protein , was performed using an antibody generated against recombinant protein expressed in Escherichia coli . Immunohistochemistry showed that mouse antisera against the catalytic domain of SmedCB also labeled cellular vesicles surrounding the intestinal lumen ( Fig 6E and 6F ) . Length of starvation did not affect localization of SmedCB ( Fig 6G ) . Previous histological analysis of planaria had identified two types of intestinal cells: “phagocytes” that absorb food for intracellular digestion , and secretory “goblet cells” that release digestive enzymes into the intestinal lumen [41 , 42 , 43] . Electron microscopy revealed that the proSmedCB localizes to vesicular structures within these intestinal cells ( Fig 6H and 6I ) but not neighboring cells , like lipid droplets ( Fig 6J ) . SmedCB localization was also confirmed via whole-mount in situ hybridization ( WISH ) to identify SmedCB RNA expression . The WISH protocol previously established [35] , and modified [36] , was used to maximize signal sensitivity . Planaria starved for four or eight days prior to sample preparation showed similar expression patterns despite the difference in worm feeding ( Fig 7A and 7B ) . SmedCB is highly expressed throughout the branched intestine of the worm , confirming the labeling seen with immunohistochemistry . Furthermore , there appear to be punctae of SmedCB labeling in the mesenchyme , especially in the head where the gut signal is less pronounced . In regenerating worm fragments , labeling of the growing branched intestine as well as the punctae seen in intact worms is observed , although there are several differences in SmedCB expression . Planaria were cut into three segments: heads , tails , and pharynxes . Amputations were performed three , five , and seven days prior to treatment to assess the changes in SmedCB expression during regeneration . Head segments exhibit concentrated labeling near the blastema , the region of newly regenerated body tissue , throughout the time course . ( Fig 7C ) . Regenerating pharynxes show some increased signal near the blastema , but this is only seen on worms that have been recently amputated . Worms that have healed over five and seven days no longer have an intense level of SmedCB near the blastema sites ( Fig 7D ) . The blastema labeling in tail segments is observed at three and five days after amputation , but SmedCB appears to be localized to the same area as intact worms after a week of regeneration: the branched intestine ( Fig 7E ) . Because of the interest in the regenerative ability of S . mediterranea , we also examined whether SmedCB might be required for regeneration as well as digestion . Another protease , transmembrane matrix-metalloproteinase A ( Smed mt-mmpA ) , has been shown to modulate cell migration and delay new tissue growth [44] . Cathepsin B may modulate tissue growth as well given its localization to the regenerating blastema during in situ hybridization . We first tested whether SmedCB activity changed after feeding and amputation by incubating worm lysate with the activity-based probe DCG-04 . This probe contains an electrophilic “warhead” , which becomes covalently attached to the nucleophilic residue of cysteine proteases . Specificity of the probe for various protease targets is achieved via a linker region between the warhead and a biotin tag , which is used to visualize protease labeling [32] . Following digestion , an increase in SmedCB labeling was observed for 2–6 days before returning to baseline during worm starvation , suggesting that cathepsin B was active when food was present , but inactive once digestion was finished ( Fig 8A ) . Interestingly , a similar trend was observed when worms were amputated following digestion ( Fig 8B ) . S . mediterranea worms take 7–8 days to fully regenerate; high SmedCB activity during this period suggests a possible use for cathepsin B during worm growth and regeneration . SmedCB RNAi treated worms did not show major defects in regeneration , although regenerating worm segments did grow less than untreated worms ( Fig 8C ) . This was quantified through the relative size increase of regenerating worms . Relative size increase was measured comparing the change between day 14 and day 0 worms divided by initial size to normalize for any variation in the size of selected animals . This trend of decreased growth in RNAi worms was consistent over two and three weeks , but it was not statistically significant . Therefore , despite the change in active protease levels observed in regenerating worm lysates , SmedCB alone does not appear to play an essential role in growth rates of S . mediterranea during regeneration . We next examined whether inhibition of cathepsin B and L in tandem had any effect on regeneration and found that in contrast to RNAi of SmedCB alone , chemical inhibition of both proteases greatly reduced growth of treated worm fragments ( Fig 8D ) . This effect was dose dependent . In general , head fragments have fewer neoblasts than pharynxes and tails [45] . Therefore , even vehicle treated heads do not grow much larger than their original size after amputation . Untreated heads had a relative size increase of only 0 . 045 , as compared to 1 . 0 for pharynxes and 0 . 93 for tails . This means that while pharynxes and tails increased their size by almost 100% over two weeks of regeneration , heads tended to remain the same size . K11777 treated worms had significant reductions in growth and , in the case of heads , increased shrinking . Heads treated with 40μM K11777 were almost 20% smaller than when they were amputated . Pharynx and tail relative size increase stood at 0 . 72 and 0 . 41 , respectively . Compared to vehicle treated worms , all three regions of worms had a significantly lower relative size increase when treated with K11777 . These data suggest that while knockdown of SmedCB alone is not enough to hinder to regeneration , chemical inhibition of SmedCB and cathepsin L proteases inhibits planaria ability to grow following amputation .
Proteases perform many vital functions in both free-living and parasitic flatworms . For free-living flatworms like Schmidtea mediterranea , effective protein digestion is essential to growth and reproduction . Maturity of asexual planarians depends on size , which directly correlates with feeding; the more an animal is fed , the more it will divide and reproduce [46] . The major digestive enzymes in the family Platyhelminthes are cysteine proteases , in contrast to vertebrates , which predominantly use serine proteases . Several proteases often function as a network for protein digestion . The parasitic worm Schistosoma mansoni uses a proteolytic cascade of cysteine and aspartyl proteases to degrade host albumin and hemoglobin into amino acids . S . mansoni relies on the cysteine protease cathepsin B for digestion of albumin , although cathepsin L , legumain , and the aspartic protease cathepsin D are involved in later steps of albumin processing [10] . In contrast to the role of cathepsins B and L in albumin degradation , cathepsin D plays the primary role in hemoglobin digestion by schistosomes [47] . Interestingly , while homologs of cathepsin B , L , and D were found in free-living Schmidtea worms , no homolog for legumain was identified ( Table 2 ) . It is therefore presumed that legumain performs a “parasitic” function , although its precise role in the host-parasite relationship is unknown [48] . Legumain has been implicated in the digestion of host hemoglobin in flatworm parasites [10 , 49] as well as other blood-feeding ecto-parasites , like ticks [50] . We were able to induce regurgitation in S . mediterranea to examine the major proteases present and active in the flatworm gut lumen . We used peptide substrates and class specific protease inhibitors to determine that aspartyl , cysteine , and metalloproteases were present and active in the worm regurgitant . Treatment of live worms with a combination of aspartyl and a cysteine protease inhibitor reduced protein degradation in the gut by 46% . While the physiological pH of the S . mediterranea gut is unknown , S . mansoni regurgitant has been estimated to be pH 6 . 0–6 . 8 [20] while the C . elegans gut pH varies from pH 3 . 6 to pH 6 . 0 depending on specific location [51] . Previous work found that optimal degradation of albumin and hemoglobin by S . mansoni proteases , occurred at pH 4 . 0 and not pH 6 . 0 [10] . Pepsin-type aspartyl proteases generally have little or no activity above pH 5 . 5 and therefore the pH of S . mediterranea is more acidic than pH 5 . 5 since pepstatin has such a profound effect on albumin degradation . In support of this , the number of cleavages sites generated by proteases active at pH 3 . 5 and 5 . 5 were considerably greater than at pH 7 . 5 . In fact , only metalloproteases with exo-peptidase activity were detected a neutral pH and these enzymes are likely to play a role in generating single amino acids from peptide termini in the later stages of degradation . Taken together , we hypothesize that protein degradation is initiated at pH 5 . 5 or lower by a combination of aspartyl and cysteine proteases and these peptides are further processed by exo-peptidases in a region of the gut where the pH is closer to neutral . Feeding assays with S . mediterranea confirmed the importance of cysteine and aspartyl proteases in vivo . Knockdown of cathepsin B activity through RNAi significantly decreased protein digestion in worms . This mirrors the function of SmCB1 in S . mansoni . Concurrent inhibition of SmedCB and cathepsin L activity using the inhibitor K11777 resulted in a further decrease in digestive ability . This implies some redundancy in the activities of cathepsin B and L . When SmedCB is specifically knocked down with RNAi , cathepsin L can compensate for some of the loss in digestive ability . However , when both proteases were chemically inhibited , digestion of rhodamine-labeled albumin was profoundly decreased . Aspartyl proteases also act in the process of digestion in S . mediterranea . Although no aspartyl proteases were detected in the worm regurgitant by mass spectrometry , it is possible that cathepsin D is not secreted directly into the intestinal lumen . Previous work in S . japonicum found that cathepsin D localized in digestive vacuolar compartments lining the gastrodermis , where it aids in the breakdown of host hemoglobin [52 , 47] . The primary site of action of cathepsin D in these worms is thought to be in the gastrodermal lysosome or endosome . Blocking the action of aspartyl proteases with pepstatin resulted in a significant decrease in digestion in live S . mediterranea , and concurrent inhibition of cysteine and aspartyl proteases saw an almost complete loss of digestive ability . This suggests that initial digestion might take place in the intestinal lumen via the action of cysteine proteases and the remaining fragments are taken up into cells where further degradation by aspartyl proteases occurs . Inhibition of both cysteine and aspartyl proteases showed a dramatic decrease in digestive ability , suggesting that both of these classes of proteases are critical for proper digestion in the worm gut . Inhibition of metalloproteases had no effect on digestive ability , suggesting that the metalloproteases detected in the worm regurgitant are not involved directly in the breakdown of food . SmedCB RNA expression was visible throughout the branched intestine . This suggests that SmedCB transcription occurs throughout the gut epithelium followed by packaging of the translated protease in adjacent intestinal cells . This is confirmed by antibody localization of translated protein in secretory vesicles of intestinal cells . This suggests that SmedCB performs a similar role to SmCB1 in S . mansoni and that vesicles are used to store full-length SmedCB before cleavage of the zymogen form and secretion into the intestinal lumen . In regenerating worms , SmedCB is expressed in the newly formed gut as well as near the blastema during early stages of growth . It is unclear whether this expression is due solely to the formation of the gut near the blastema , or if SmedCB plays other important roles during tissue remodeling , requiring its increased presence and activity at the site of regeneration . While SmedCB alone does not impact the ability of S . mediterranea worms to regenerate , inhibiting the activity of both cathepsin B and L did result in a significant decrease in growth rate . During regeneration , old structures are broken down through both autophagy and apoptosis [53] . Apoptosis occurs in two waves: an initial localized response near the wound site and followed by a systemic response . Even preexisting tissues undergo apoptosis in order to maintain their correct proportions within the worm [54] . Perhaps cysteine proteases are involved in tissue remodeling and turnover during regeneration and their absence slows the rate of this process . The lack of severe morphological effects during regeneration in cathepsin B and L inhibited worms suggests that while these proteases may play a small role in growth , they are not entirely essential to worm survival . We have exploited the ease of use of Schmidtea to confirm the conservation of a major cysteine protease in free-living and parasitic flatworms . We have found that not only is cathepsin B involved in digestion in free-living worms , but it also plays a role in growth and regeneration . Schistosome parasites also have neoblast-like stem cells , so there may be other aspects of worm growth or life cycle alterations that are conserved between planarians and Schistosoma [7] . | Certain parasitic flatworms pose serious threats to human health . Understanding more about the biology of flatworms can lead to new and better drug targets . However , due to the complex life cycles of parasitic flatworms , it has been suggested that a non-parasitic flatworm , Schmidtea mediterranea , be used to study flatworm biology . This worm can be used as a model to study processes that all flatworms have in common; these findings can be applied back to parasitic flatworms . Digestion occurs in all worms and relies on the activity of protease enzymes to break down protein . The authors were able to induce regurgitation in S . mediterranea and study the contents of the worm gut . These worms have several types of proteases present , although it seems that only two types , called cysteine and aspartyl proteases , are important in digestion in live worms . Furthermore , one specific cysteine protease , cathepsin B , is responsible for the bulk of the digestive capabilities in S . mediterranea . Cysteine proteases also appear to be involved in the ability of S . mediterranea to regenerate damaged body tissues . Understanding protease activity in S . mediterranea can provide insight as to the roles of these enzymes in medically relevant parasitic flatworms . | [
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] | 2016 | Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria |
In Europe , the elimination of wildlife rabies using oral rabies vaccination [ORV] of foxes for more than 30 years has been a success story . Since a comprehensive review on the scope of the different oral rabies vaccine baits distributed across Europe has not been available yet , we evaluated the use of different vaccine baits over the entire period of ORV [1978–2014] . Our findings provide valuable insights into the complexity of ORV programs in terms of vaccine related issues . More than 10 oral vaccines against rabies were used over the past four decades . Depending on many factors , the extent to which oral rabies virus vaccines were used varied considerably resulting in huge differences in the number of vaccine doses disseminated in ORV campaigns as well as in large spatial and temporal overlaps . Although vaccine virus strains derived from the SAD rabies virus isolate were the most widely used , the success of ORV campaigns in Europe cannot be assigned to a single oral rabies virus vaccine alone . Rather , the successful elimination of fox rabies is the result of an interaction of different key components of ORV campaigns , i . e . vaccine strain , vaccine bait and strategy of distribution .
Elimination of infectious diseases from wildlife populations is a major challenge . Thus , until a few decades ago , elimination of wildlife-mediated rabies from Europe was considered wishful thinking . This paralleled the results of early conventional control measures aimed at stopping the expansion of fox rabies in Europe [1 , 2] . Almost all attempts to reduce the fox population below a threshold , where the chain of infection would be interrupted and the epidemic would fade out , using intensive culling , poisoning , and trapping failed [3] . Moreover , scientists , veterinary and public health authorities came to understand that focusing on reduction of the fox population evidently was counterproductive as it disrupted the social and spatial organization of foxes , thereby increasing contact rates and disease incidence [3 , 4] . The development of attenuated oral rabies virus vaccines in the second half of the 20th century provided a unique opportunity to target elimination of the infection circulating in European wildlife [5 , 6] . Concerted inter-sectorial applied research activities in a few European countries such as Switzerland , Germany and France pioneered the basic techniques for a suitable strategy for oral rabies vaccination ( ORV ) of foxes . These included the type of baits , timing of vaccination campaigns , bait delivery densities , adequate modes of bait distribution , and duration and monitoring of ORV campaigns [2 , 7] . What started as a small but ground-breaking field trial in Switzerland in 1978 [8] soon became the most widely-used model for controlling and eliminating a zoonosis in its wildlife reservoir host . Triggered by promising results of first field trials in the 1980s and accelerated by a strong political commitment of European governments and the co-financing policy of the European Union ( EU ) for member states and neighboring non-EU countries [9] , many rabies affected European countries implemented long-term ORV programs [10] . However , it was not until further scientific and technical achievements , e . g . the development of manufacturing methods for baits and the application of computer supported automatic aerial distribution equipment for baits were made that large-scale vaccination campaigns became possible [2 , 11] . Despite perceived challenges and individual setbacks at national or international levels [10 , 12] , the current rabies situation provides an impressive testimony of the efficiency and future potential of wildlife vaccination [2 , 13] . Within the past 30 years the overall rabies incidence in Europe decreased by approximately 80% compared to the peak year 1984 during which 24 , 315 rabies cases were reported ( www . who-rabies-bulletin . org ) . Furthermore , the disease was completely eliminated from Western and Central Europe [2 , 13 , 14] . The proportion of landscape area ever affected by rabies and an index capturing the size and overlap of successive ORV campaigns were identified as factors having statistically significant effects on the number of campaigns required to control and eliminate rabies [13] . Highly potent and safe oral rabies vaccines have been the key for the elimination of wildlife rabies in Europe and several regions of North America [10 , 15] . All rabies vaccines used for oral immunization of wildlife in Europe are based on live replication-competent vaccine viruses [15] . The first generation vaccines were attenuated rabies viruses developed by conventional in vivo and/or in vitro serial passaging of virulent field virus isolates resulting in e . g . SAD Bern , SAD B19 , SAD P5/88 , Vnukovo-32 , or RV-97 [16–19] . The second generation was developed by selection of monoclonal antibody escape mutants , e . g . SAD VA1 , SAG1 , or SAG2 [20–23] . Later , site-directed mutagenesis [reverse genetics] led to the development of a third generation of oral rabies vaccines , e . g . ERA G333 [24] . Finally , a recombinant vaccinia virus expressing the rabies virus glycoprotein from the ERA strain [V-RG] [25 , 26] has been used . The progenitor of almost all attenuated rabies virus-based vaccines currently in use in Europe is the vaccine virus strain SAD Bern [27] , a derivative of the original SAD ( Street Alabama Dufferin ) virus isolated from a rabid dog in 1935 in the US [28] . The limited available information on the use of those vaccines in ORV programs in Europe [7 , 29 , 30] resulted in speculations whether the efficiency of ORV programs in Europe can be attributed to a single vaccine only or whether certain vaccines are more efficient than others under field conditions . However , a comprehensive review of the scope of oral rabies virus vaccines distributed across Europe had not been available . We therefore evaluated the spatio-temporal use of different oral rabies virus vaccines in individual European ORV programs over the past 37 years .
The study region encompassed all countries in Europe that implemented ORV programs on their territories between 1978 and 2014 ( Fig 1 ) . The standard ORV approach applied typically comprised of ( i ) performance of ORV campaigns twice a year ( spring and autumn ) , ( ii ) an average bait density of 20–25 baits/km2 , ( iii ) aerial and manual ( mainly at the beginning of ORV , later complementary ) distribution of vaccine baits , and ( iv ) a flight line distance of 500 to 2000 m in case of aerial distribution of baits [13] . In a few exceptions ORV campaigns were only conducted once a year ( Italy , 1984 ) or additional campaigns were conducted either in summer or in winter ( France , Germany 2005 , Italy 2009 ) or at short intervals ( Germany , 2005; Italy , 2010 ) [31 , 32] . As part of the terms of reference as a WHO Collaborating Centre for Rabies Surveillance and Research , data related to ORV programs of individual European countries for the past 37 years were collected from three different sources including ( i ) information provided by veterinary or other competent authorities ( list of contributors to the WHO Rabies Bulletin Europe , http://www . who-rabies-bulletin . org ) upon request , ( ii ) presentations of members states given at meetings the Standing Committee on the Food Chain and Animal Health—Section: "Animal health and animal welfare" ( SCFCAH ) [http://ec . europa . eu/food/committees/regulatory/scfcah/animal_health/index_en . htm] , and ( iii ) official websites of competent authorities of European countries concerned . For each ORV campaign ( spring , summer , autumn , winter or other ) carried out during the observation period data comprised information on the size of vaccination areas , the timing of vaccination campaigns , bait density , mode of bait distribution , and oral rabies vaccine strains used . The size and location of individual vaccination areas was either requested as shape files or , if not available , as scanned maps from publications or presentations [13] . For countries that implemented ORV programs during the review period we established a GIS database with individual campaign based datasets using ArcGIS software [Esri Inc . , version 10 . 2 . , Redlands , California , USA] . Maps of vaccination areas that were not available as shape files were digitalized , converted into the GIS databases as previously described [33] , and subsequently sent to competent authorities or rabies experts of the respective European countries for validation and revision , if necessary . Information on the oral rabies vaccines used was assigned to any individual vaccination area or sub-region ( national units of territories ) therein . Data of the entire observation period were subsequently stratified , compiled and displayed in maps . Using ArcGIS analysis tools we calculated the size of the area exclusively vaccinated in Europe with a single oral rabies vaccine or with multiple vaccines by considering vaccine combinations . For each oral rabies vaccine used during the observation period we also calculated the area in which it was used . The total number of vaccine baits for each individual vaccine distributed in Europe was computed based on the size of the cumulative vaccine-specific vaccination areas and an assumed average bait density of 20 baits/km2 .
During the past 37 years , 30 European countries implemented ORV programs . In autumn 1978 , Switzerland conducted the first European ORV campaign ever; the last country to join efforts to eliminate fox-mediated rabies was Albania in 2014 . In seven countries , ORV programs were discontinued and re-initiated either due to re-infection , establishment of a vaccination belt ( cordon sanitaire ) as a result of unfavorable rabies situation in neighboring regions , or budget constraints . During the review period 10 countries successfully eliminated fox and raccoon dog-mediated rabies from their territory using ORV ( Fig 1 ) . Since 1978 , the total area ever vaccinated encompassed 2 . 5 million km2 , within which a total of ten different attenuated rabies virus-based vaccine strains and one recombinant vaccine were used [Fig 2] . In the early days of ORV in Europe ( 1978–1984 ) baits consisted of chicken heads each containing a sachet made of plastic and aluminum foil with 1 . 8 ml SAD vaccine . Since the development of novel manufactured baits in 1983 , later almost all vaccine baits in Europe ( SAD P5/88 , SAD B19 , SAD Berne , V-RG , SAG-1 , SAG-2 , SAD VA1 ) have used fish meal as attractant , while the carrier substance differed between the baits ( paraffin , polymer , coconut fat ) . Bait casings usually contained 150 mg of tetracycline as a biomarker . About 1 . 52 million km2 ( 60 . 8% ) of the total area comprising different regions in 25 countries were exclusively vaccinated with a single representative of the available 11 vaccines strains . In contrast , in 702 , 482 km2 ( 28 . 1% ) and 278 , 415 km2 ( 11 . 1% ) of the area , two and three , or more than three vaccines were distributed , respectively , over the complete time span of ORV in Europe ( Tables 1–3 ) . When the size of vaccination areas covered in all individual vaccination campaigns in respective countries between 1978 and 2014 ( Fig 1 ) was summarized , the cumulative vaccination area encompassed 33 . 25 million km2 . Assuming an average bait density of 20 baits/km2 , the estimated total number of vaccine baits distributed between 1978 and 2014 amounts to 665 million ( Fig 3 ) . The spatio-temporal use of the different vaccine strains varied considerably . The temporal use of the different vaccine strains is depicted in Fig 4 . With a time span of 35 and 32 years of application and an area of 1 . 96 and 1 . 12 million km2 covered with at least a single campaign by SAD Bern and SAD B19 , respectively , these two vaccines were the most widely used throughout Europe . V-RG , SAD P5/88 and SAG2 had been used to a much more limited extent . Although having been authorized for more than 20 years , the area ever vaccinated with vaccine baits of those three vaccine strains comprised 0 . 54 , 0 . 31 and 0 . 21 million km2 , respectively ( Fig 5A–5D ) . In contrast , the remaining vaccine strains were only used at a minor scale during a shorter period of time ( <11 years ) ( Figs 3 and 6A and 6B ) with a limited number of baits distributed in the field .
We show that the extent to which oral rabies virus vaccines were used in Europe varies considerably in space and time . Next to the type of vaccine virus strains used , there is significant difference in the number of vaccine doses distributed in ORV campaigns as well as tremendous spatial and temporal overlap . Although SAD derived vaccine virus strains are the most widely used , the success of ORV campaigns in Europe cannot be attributed to a single oral rabies virus vaccine or a specific group of vaccines ( e . g . first vs . second generation vaccines or modified live vs . recombinant vaccines ) . Result are likely due to the interaction of different key components , programs and strategies , including adequate distribution of efficacious vaccine-baits that led to the elimination of fox rabies from vast areas of Europe [13] . | Oral rabies vaccination [ORV] is the pre-eminent example of successful vaccination of wildlife populations which has resulted in virtual elimination of fox-mediated rabies from large parts of Western and Central Europe . This achievement is unprecedented in history . Attractive species-specific baits , efficient vaccination strategy , and highly potent and safe oral rabies vaccines have been instrumental for the success . Numerous oral rabies vaccines for wildlife have been developed over the past four decades . However , information on the use of those vaccines in operational ORV programs in Europe that exists has until now not been fully publicly accessible . Here , we provide the first comprehensive review of the spatio-temporal use of oral rabies vaccines over the entire period of ORV [1978–2014] . Although ‘first generation’ vaccine virus strains derived from the SAD rabies isolate are the most widely used , the results do not support significant differences in efficacy of these strains under field conditions nor can success of ORV be attributed solely to one vaccine type . In contrast , vaccination strategy is of major importance . Our analyses could help in the development of adequate rabies control strategies by documenting the importance of vaccination strategy , in which oral rabies vaccines are only one contributing component among other parameters . | [
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] | [] | 2015 | Spatio-temporal Use of Oral Rabies Vaccines in Fox Rabies Elimination Programmes in Europe |
Diet profoundly affects metabolism and incidences of age-related diseases . Animals adapt their physiology to different food-types , modulating complex life-history traits like aging . The molecular mechanisms linking adaptive capacity to diet with aging are less known . We identify FLR-4 kinase as a novel modulator of aging in C . elegans , depending on bacterial diet . FLR-4 functions to prevent differential activation of the p38MAPK pathway in response to diverse food-types , thereby maintaining normal life span . In a kinase-dead flr-4 mutant , E . coli HT115 ( K12 strain ) , but not the standard diet OP50 ( B strain ) , is able to activate p38MAPK , elevate expression of cytoprotective genes through the nuclear hormone receptor NHR-8 and enhance life span . Interestingly , flr-4 and dietary restriction utilize similar pathways for longevity assurance , suggesting cross-talks between cellular modules that respond to diet quality and quantity . Together , our study discovers a new C . elegans gene-diet pair that controls the plasticity of aging .
Animals dwell in a complex ecosystem where they interact with a host of other organisms; some of them may alter their life history traits . For example , the nematode Caenorhabditis elegans is found in decaying moist vegetation that is co-inhabited by different types of bacteria that they feed on . So , the worms are often exposed to various pathogenic bacteria that they either avoid or use a conserved innate immunity pathway to counter . The worms are also presented with a range of bacteria of different nutritional values that they choose between [1–3] . They encounter E . coli¸ Bacillus and Comamonas etc . that are known to modulate development , reproduction , fat storage and life span [2 , 4–7] . In the laboratory , worms are mostly maintained on E . coli OP50 but are often exposed to the RNAseIII-deficient HT115 during RNAi experiments . The two strains differ considerably , particularly in terms of carbohydrate content , with the OP50 strain considered as a low quality diet that induces less satiety and promotes higher fat storage [3 , 6–9] . Since the rate of aging is greatly influenced by dietary composition , worms seem to have evolved intricate adaptive strategies to maintain normal aging [10 , 11] . As a result , although the two diets differentially affect metabolism , the worms are able to ensure relatively normal life span when fed either bacteria [7] . How C . elegans sense different diet to alter metabolism and life history traits , including complex traits like aging , is an emerging area of research . These studies are being facilitated by the discovery of gene-diet pairs where the function of a gene becomes discernible only on a particular diet [12] . Previous work has shown that sensory neurons may process signals that differentiate between different food-types and regulate life span in flies and worms [10 , 13 , 14] . In C . elegans , the neuromedin U receptor-like gene , nmur-1 mutant as well as osm-3 ( kinesin motor protein required for cilia formation ) mutant has extended life span on OP50 but not on HT115 [10 , 14] . These life spans were found to be dependent on the FOXO transcription factor , DAF-16 [14] . On the other hand , the proline metabolism gene alh-6 works in the muscle to preserve mitochondrial structure and functional homeostasis in response to OP50 [11] . This requires a functional NMUR-1 receptor signalling [11] . Interestingly and intuitively , it would appear that the intestine may also contribute to this phenomenon as it gets to sample different food that the worms ingest . However , the role of intestine in food-type-dependent life span regulation is not as well-characterized . Here we show that a serine-threonine kinase gene , flr-4 regulates food-type-dependent life span by functioning both in the neurons and the intestine . We find that when flr-4 is knocked down by RNAi or its function disrupted by a P223S missense mutation in its kinase domain , life span is dramatically increased . The life span of the kinase-dead mutant flr-4 ( n2259 ) is increased only when the mutant is fed HT115 and not OP50 . We show that knocking down flr-4 leads to increased cytoprotective xenobiotic detoxification pathway ( XDP ) gene expression , through the nuclear hormone receptor NHR-8 , that plays a causal role in its increased life span . Interestingly , this elevated gene expression as well as the increased life span is dependent on the conserved p38 MAPK signalling . In flr-4 ( n2259 ) , OP50 is unable to strongly activate the p38 MAPK while HT115 leads to increased phosphorylation of the MAPK . Consequently , in the mutant , HT115 is able to increase the levels of the XDP genes while OP50 does not . Finally , we demonstrate that FLR-4 uses a pathway similar to DR to ensure longevity , dependent on FOXA/PHA-4 but independent of FOXO/DAF-16 . Together , our study establishes flr-4 as a new longevity gene that controls adaptive capacity of C . elegans towards bacterial diet by preventing differential activation of XDP genes through p38MAPK pathway , dependent on food-type .
Flr-4 was originally identified in a screen for genes that regulate fluoride resistance and the mutants exhibit temperature-sensitive defecation defects [15] . However , at 20 oC they do not have defects in defecation [15] . We initially became interested in the flr-4 gene as it has 26% homology and 40% identity to drl-1 [16] , a gene we have recently characterized to be involved in Dietary Restriction ( DR ) . Knocking down flr-4 using a cDNA RNAi construct increased life span dramatically ( average life span increase 40–60% , Fig 1A , S1 and S2 Tables ) . Similar life span extension was observed in absence of FUDR , a DNA synthesis inhibitor used to arrest confounding effects of progeny population during life span analysis ( S1A Fig ) . The increased life span was also associated with better health as evident from lower lipofuscin pigment accumulation ( S2A Fig ) , lesser muscular atrophy with age ( S2B Fig ) and consequently , better motility ( S2C Fig ) . These worms were smaller in size ( S2D Fig ) but did not have any major defects in developmental rates ( S3 Fig ) . However , the increase in life span was not associated with enhanced tolerance towards heat stress ( S4B Fig ) ; UV stress tolerance was only increased 13–15% compared to 40–60% increase in life span ( S4A Fig ) . The increased life span of flr-4 knock down was also not dependent on the heat shock transcription factor hsf-1 ( S4C and S4D Fig ) . Thus , flr-4 seems to decouple longevity from stress resistance and is a novel longevity modulator . Next , we asked whether an flr-4 mutant has attributes similar to the flr-4 RNAi . We used the flr-4 ( n2259 ) allele that has a P223S missense mutation in the activation loop of the protein kinase domain and shows increased fluoride resistance [15] . This allele has similar developmental rates as wild-type ( S5 Fig ) and shows no dauer arrest [15] . We found that flr-4 ( n2259 ) increased life span that was not further increased when grown on flr-4 RNAi ( Fig 1B , S1 and S2 Tables ) . The life span of the mutant was also not affected by the presence of FUDR ( S1B Fig ) . This suggests that flr-4 ( n2259 ) behaves as a null allele to regulate life span . C . elegans is known to respond differentially to bacterial diet to modulate life history traits , including life span [5–7 , 14 , 17] . Interestingly , we found that the life span extension in flr-4 ( n2259 ) is dependent on the food-type . When the mutant was grown on the E . coli HT115 ( a K12 strain ) , life span was dramatically extended ( Fig 1C , S1 and S2 Tables ) . In contrast , when grown on E . coli OP50 ( a B strain ) , no extension of life span was observed ( Fig 1C ) . We checked for differences in pumping under these conditions and found no change ( S2E Fig ) . Also , ingestion of RFP-labelled beads was similar in WT and flr-4 ( n2259 ) ( S2F Fig ) , suggesting that the differences in life span are not due to altered feeding behaviour . Furthermore , the developmental rate was similar in both the strains when grown on the two E . coli strains ( S5 Fig ) . Remarkably , knocking down flr-4 using an OP50-based RNAi system [18] increased life span similar to that of the HT115-based system ( Fig 1D ) , suggesting that the food-type-dependent life span extension may be attributed to the kinase function of FLR-4 . This conclusion is further supported by the observation that rescuing flr-4 ( n2259 ) with a kinase-proficient wild-type transgene suppresses the increased life span on HT115 ( S16 Fig ) . Together , FLR-4 kinase suppresses pro-longevity cues in a food-type dependent manner . Next , we asked where FLR-4 functions to regulate longevity . We constructed a flr-4p::gfp transgenic line and found that the flr-4 promoter drove expression of gfp in the intestine and a few neurons ( Fig 2A ) . We used a tissue-specific RNAi system to determine the tissue where flr-4 functions . We found that flr-4 knockdown specifically in the intestine or the neurons was sufficient for life span extension; no extension in life span was observed when the gene is solely knocked down in muscle or hypodermis ( Fig 2B–2E , S1 and S2 Tables ) . Together , flr-4 functions in the intestine and neurons to negatively regulate life span of the worms . In C . elegans , longevity genes need to be knocked down at temporally distinct points in development to increase life span . For example , the mitochondrial electron transport gene cco-1 or the MEKK-3-like kinase drl-1 needs to be knocked down early in development to increase life span while insulin-like signalling pathway functions in adulthood to exhibit the beneficial longevity effects [16 , 19 , 20] . We initiated flr-4 RNAi at different stages of development of the worms and found that knocking down at L1 or L2 produced maximum life span extension; knocking down at L3 or later had diminished or no effect ( S6A–S6E Fig ) . Together , flr-4 functions in the intestine and neurons , during larval development to regulate adult life span . Since FLR-4 is required to suppress the effect of food-type on longevity , we asked what signalling cascade may be mediating this effect . The p38 MAPK pathway is a central signalling mediator required for mounting the innate immune response when worms are challenged with pathogens [21–25] . The worm p38 homolog PMK-1 is activated by its upstream MAPKKK NSY-1 and MAPKK SEK-1 [26 , 27] . The TIR domain adaptor protein TIR-1 , an ortholog of human SARM [28 , 29] and UNC-43 , a Ca2+/calmodulin-dependent protein kinase II ( CaMKII ) [30] lie further upstream of these serine-threonine kinases . While TIR-1 works upstream of the p38 MAPK pathway to regulate innate immunity genes [28 , 29] , both TIR-1 and UNC-43 functions in the neurons to control neuronal cell fate and asymmetric patterns of odorant receptor expression [30 , 31] . We grew wild-type , pmk-1 ( km25 ) , sek-1 ( km4 ) , nsy-1 ( ag3 ) , nsy-1 ( ok593 ) , tir-1 ( tm3036 ) and unc-43 ( e403 ) on control or flr-4 RNAi and performed life span analysis . We found that increased life span on flr-4 RNAi is suppressed when any of the kinases in the p38 MAPK pathway or tir-1 or unc-43 is mutated ( Fig 3A , S1 and S2 Tables ) . Interestingly , pmk-3 is not required for the life span extension , showing specificity of the process ( S1 and S2 Tables ) . Similar suppression of life span was observed when flr-4 ( n2259 ) was grown on sek-1 RNAi ( S7A Fig ) . We also created a flr-4 ( n2259 ) :sek-1 ( km4 ) double mutant and found that the life span was suppressed ( S7B Fig ) . Further , in order to determine biochemically whether knocking down flr-4 activates the p38 MAPK , we performed western blot analysis using a phospo-PMK-1-specific antibody ( Figs 3B and S14 ) . We found that in WT worms when flr-4 is knocked down , PMK-1 phosphorylation increases in a sek-1-dependent manner . Further , TIR-1 or UNC-43 seems to be working in the same linear pathway as flr-4; in the tir-1 ( 3036 ) and unc-43 ( e408 ) , flr-4 knockdown failed to increase phosphorylation of PMK-1 ( S7C Fig ) . Together , the FLR-4 longevity signals are mediated by the p38 MAPK pathway . In order to understand how flr-4 knockdown increases life span , we performed transcriptomic analysis of wild-type worms grown on control or flr-4 RNAi . We found that 1957 genes were upregulated ( > 2 folds , P ≤ 0 . 05 ) while 538 genes were down-regulated . We determined the biological functions of these genes using DAVID [32] and show that they are significantly enriched for genes involved in the Xenobiotic Detoxification Pathway ( XDP ) ( Figs 4A and S8A ) . All these cytoprotective XDP genes are expressed in the intestine of the worms ( www . wormbase . org ) . Using quantitative reverse transcriptase PCR ( qRT-PCR ) , we verified 13 genes that were upregulated in our RNA-seq experiment ( Figs 4B and S8B ) . We also used a transgenic worm expressing gfp driven by the cyp-35B1 promoter and show that the expression of GFP is enhanced in the hind-gut region , when the worms were grown on flr-4 RNAi ( S8C Fig ) . Next , in order to determine whether p38 MAPK pathway has any role in the regulation of these cytoprotective genes , we performed qRT-PCR analysis of these genes after knocking down flr-4 in sek-1 ( km4 ) . Interestingly , majority of the XDP genes that were upregulated in the wild-type failed to do so in sek-1 ( km4 ) ( Figs 4C and S8D ) . Since the levels of some of the genes do not fall back to basal level , it is possible that other signalling pathways or transcriptional regulators are also involved . These experiments suggested that p38 MAPK pathway regulates XDP genes downstream of flr-4 . In order to determine whether XDP genes are indeed required for life span extension brought about by flr-4 knockdown , we used a mutant of nhr-8 , a transcription factor required for XDP gene expression [16 , 33] . First , we knocked down flr-4 by RNAi in nhr-8 ( ok186 ) and found that it failed to increase life span to the same extent as in wild-type ( Fig 4D ) . We also knocked down nhr-8 using RNAi in flr-4 ( n2259 ) , and found that the life span of the mutant is significantly suppressed ( S8E Fig ) . Finally , we show that in nhr-8 ( ok186 ) , the XDP genes fail to upregulate to the same extent as in WT , when flr-4 is knocked down ( S8F Fig ) . Thus , increased expression of XDP genes is required for life span extension by flr-4 knockdown . Next , we asked whether NHR-8 functions downstream of p38 MAPK pathway to regulate XDP genes . For this , we decoupled p38 MAPK from flr-4 and activated it by knocking down the phosphatase VHP-1 using RNAi [34 , 35] . Knocking down vhp-1 led to upregulation of cyp-35B1 , as measured by increased GFP expression in the cyp-35B1p::gfp transgenic line . This enhancement was suppressed in the cyp-35B1p::gfp;nhr-8 ( ok1853 ) worms , showing that NHR-8 functions downstream of p38MAPK ( Fig 4E ) . QRT-PCR analysis showed that three other XDP genes that are upregulated on vhp-1 knockdown are dependent of NHR-8 ( Fig 4F ) . Interestingly , knocking down vhp-1 was not sufficient to increase life span of WT ( S8G Fig ) , suggesting that additional downstream events may have to be coactivated in order to get life span benefits similar to flr-4 knockdown . Since flr-4 ( n2259 ) shows differential response to OP50 and HT115 to extend life span , we suspected that this may be due to the ability of a diet to upregulate a specific set of genes . So , we performed transcriptomic analysis of wild-type and the mutant on the two bacterial diets . We found that the XDP genes are upregulated only when flr-4 ( n2259 ) was grown on HT115 but not when grown on OP50 ( Fig 5A ) . We verified several of these genes using qRT-PCR and found them to be upregulated only on HT115 ( Figs 5B and S9A ) . Additionally , we used the flr-4 ( n2259 ) ;cyp-35B1p::gfp strain to show that the expression of GFP is induced only when the worms are grown on HT115 ( Fig 5C ) . Together , this shows that flr-4 mutant worms mount a specific p38-dependent transcriptional response when fed HT115 that provide cytoprotective benefits leading to enhanced life span . Next , we asked whether only HT115 can differentially activate the p38 MAPK pathway . For this , we performed western blot analysis with WT and flr-4 ( n2259 ) grown on HT115 or OP50 . Interestingly , we found that flr-4 ( n2259 ) grown on HT115 showed enhanced phosphorylation of PMK-1 compared to wild-type ( Figs 5D , S9B and S15 ) . However , the levels of phosphorylation were unchanged in WT maintained on the two bacteria . Thus , FLR-4 prevents differential activation of p38 MAPK dependent on diet , maintaining adaptive capacity in C . elegans . Since flr-4 mutant worms responded differentially to diet , we evaluated the interaction of the gene with two nutrient sensing pathways . First , using RNAi we knocked down flr-4 in the IIS pathway mutant daf-2 ( e1370 ) and found that the life span of the mutant is further extended , suggesting independent mechanisms ( Fig 6A , S1 and S2 Tables ) . On the other hand , the life span of eat-2 ( ad1116 ) is not further extended; in fact , the life span was suppressed by 10–14% ( Fig 6B ) . However , flr-4 knockdown does not affect pharyngeal pumping of eat-2 ( ad1116 ) , similar to wild-type ( S13 Fig ) , showing that the lack of additive effect on life span with eat-2 ( ad1116 ) is not mechanical . In case of another genetic mimic of DR [16] , the extended life span of drl-1 RNAi worms was also not extended further by flr-4 mutation ( S10A Fig ) . This suggested that flr-4 uses cellular signalling pathways utilized by DR , but not the IIS pathway to ensure longevity . This was further supported by the fact that life span of flr-4 RNAi , as in eat-2 mutants and on drl-1 knockdown , was dependent only on the FOXA transcription factor PHA-4 , and not the FOXO factor DAF-16 that is required by IIS pathway mutants ( Figs 6C , 6D and S10B ) . In the pha-4 ( zu225 ) , life span extension by flr-4 knockdown was completely abrogated . Interestingly , the life span of the flr-4 ( n2259 ) or flr-4 RNAi worms was independent of the NRF2 ortholog , SKN-1 , a common output of insulin-like signalling and DR [36 , 37] ( S10C and S10D Fig ) ; skn-1 abrogation by mutation or RNAi affects the life span of WT and flr-4 knockdown worms to similar extent . Further , similar to DR [16 , 38] , the flr-4 RNAi did not further extend the already long life span of germline defective mutants ( S11 Fig ) . Like many long lived mutants , flr-4 mutants have delayed reproductive span and lower brood size compared to WT , mainly on HT115 ( S12 Fig ) . This may be due to more resource allocation towards somatic maintenance during DR [39] and may be caused by lower germ cell proliferation as seen in case of insulin-IGF-1 signalling pathway mutants [40] . The fact that FLR-4 utilizes the DR machinery for longevity assurance is also consistent with its role in ensuring adaptive capacity to diet .
A complex interaction between genes and diet determines the rate of aging and predisposes an individual to age-related diseases . The term gene-diet pair is used when the consequences of mutating a gene is visible only on a specific diet [12] . Surprisingly , only a few gene-diet pair have been identified that regulates aging , mainly through studies in C . elegans [12] . In this report , we identify a novel gene-diet pair and show that the adaptive capacity to different food-type is modulated by the protein kinase FLR-4 . This protein prevents differential activation of the p38 MAPK pathway dependent on the food-type and consequently , the expression of cytoprotective genes by transcription factor NHR-8 ( Fig 6E ) . Interestingly , this pathway overlaps with the DR pathway , suggesting a cross-talk between cellular signalling that senses food quality and quantity to regulate life history traits like aging . FLR-4 is a serine-threonine protein kinase similar to mammalian Cyclin-dependent protein kinase 3 ( 31% identity , 50% similarity , E value 8e-22 ) . It was initially identified in a screen for genes involved in fluoride tolerance [41] , but was subsequently shown to have defects in ultradian rhythm in the intestine that controls defecation [15] . In this study , we elucidate a novel function for FLR-4 in the intestine and neurons that is independent of its role in defection . The temperature-sensitive kinase dead mutant flr-4 ( n2259 ) has normal defecation cycle at 20 oC [15] , a temperature at which most of our assays were performed . In view of the central role that this kinase plays in controlling multiple important phenotypes , future research needs to be directed towards finding its immediate cellular targets . This is particularly important as we found that the food-type-dependence is specific to the kinase dead mutant; RNAi knockdown using an OP50-based system also increased life span . This suggests that the kinase-dead mutant may not be able to phosphorylate a substrate ( s ) that is required to maintain life span homeostasis on the different bacterial diets . Considering the importance of gene-diet pairs in aging and disease , our understanding of the mechanisms of adaptive capacity to food-type is still in its infancy . Previous studies have identified a few genes that play a role in this process . Notably among them are the RICTOR ortholog rict-1 [6] , neuromedin U receptor ortholog nmur-1 [14] and mitochondrial 1-pyrroline-5-carboxylate dehydrogenase ( P5CDH ) alh-6 [11] . The rict-1 mutants have phenotypes similar to flr-4 mutant worms such that they have shorter life span on OP50 while exhibiting life span extension on HT115 [6] . However , in contrast to the flr-4 , rict-1 regulates feeding behaviour when an animal encounters diets of different qualities . The rict-1 mutants have different pumping rates and exhibit avoidance behaviour on food of diverse quality [6 , 12] . The alh-6 mutants , on the other hand , show accelerated aging when fed OP50 while retaining normal rates of aging on HT115 [11] . Interestingly , the nmur-1 mutants have longer life span on OP50 but not on HT115 [14] . Thus , these gene-diet pairs seem to control diverse aspects of an animal’s response to different food . In future , it will be interesting to study the interaction of these genes with flr-4 , considering the fact that opposing phenotypes controlled by these genes may indicate homeostatic control of life span in response to different diet . As the above mutants differ in their response to different diet , they may also activate diverse signalling cascades . This is apparent from the fact that rict-1 and nmur-1 interact differentially with the downstream components of the insulin signalling pathway [6 , 14 , 42] . While the life span extension of rict-1 knockdown is dependent on NRF2 ortholog skn-1 , nmur-1 mutants require the FOXO transcription factor DAF-16 . We found that the flr-4 mutants require the FOXA transcription factor PHA-4 for life span extension and is independent of DAF-16; its transcriptional response may thus be different from other gene-diet pairs . Since both alh-6 and flr-4 may signal through a pathway used by the eat-2 model of DR , it will be interesting to study the transcription factor requirements of the former . Comparative gene expression profiles of these mutants on different diet will help us understand the complex gene expression modalities controlled by these gene-diet pairs . Gustatory and olfactory neurons that perceive chemical signals have previously been shown to affect life span [10 , 14 , 43] . Here we show that flr-4 knockdown in the neurons can also increase life span . In fact , the life span extension by flr-4 RNAi requires the CAMKII ortholog UNC-43 and SARM ortholog TIR-1 that is known to act in the neurons to determine cell fate . On the other hand , intestine-specific knockdown of flr-4 also increases life span . Although it appears intuitive to suggest that the gut may play an important role in sensing different diet that is ingested , the role of the intestine in food-type-dependent life span extension is less known . Interestingly , TORC2 that also regulates food-type-dependent life span , requires SKN-1/NRF2 in the intestine to regulate life span [42] . However , FLR-4 life span is independent on skn-1 , indicating to extensive insulation as well as cross-talks among these pathways . Future research needs to be directed to understand the partitioning of the p38 MAPK pathway in the neurons and intestine as well as their cross-talk to regulate flr-4-mediated life span . Animals in the wild , unlike those in laboratories , are exposed to a wide variety of food that they have adapted to . Being able to utilize a wide range of diet is evolutionarily advantageous as the animals can survive when their optimal diet is depleted . Giant panda that depend mainly on bamboo for nutrition is facing extinction due to loss of habitat ( wwf . panda . org ) [44] . Since diet influences the rate of aging , the animals have evolved intricate mechanisms to maintain homeostasis . In addition to the quality of diet , the quantity of food regulates the plasticity of aging . As a result , DR is able to delay aging and increase life span across the animal kingdom [45 , 46] . In our study as well as in that of alh-6 [11] , we observe genetic overlap with DR , suggesting that organisms have evolved cellular modules that evaluate both quality and quantity of diet to regulate life span . We show that FLR-4 signals through the p38 MAPK pathway to regulate the expression of cytoprotective genes dependent on diet . In C . elegans , this pathway has been extensively characterized for its role in mediating innate immune response towards pathogenic bacteria as well as in mounting an oxidative stress response [21 , 23 , 47–49] . On the other hand , the XDP genes have been shown to provide cytoprotective effects leading to enhanced life span in multiple models of longevity [16 , 50–52] . The fact that FLR-4 would signal differences of diet through the p38 MAPK seems quite intuitive for an organism that feeds on bacteria and uses this same pathway to differentially activate immune genes on encountering pathogens . But in case of flr-4 knock down , immune effector genes are not upregulated , showing specificity of this module . However , in mammals , the p38 MAPK has important role in regulating metabolism in liver and adipocytes during fasting , mediated by glucagon and insulin [53] . It will be interesting to study the role of p38 MAPK pathway in gene-diet interaction networks in mammals . How flr-4 mutants sense the differences in bacterial food remains to be answered . One possibility is that the mutants become sensitive to the presence or absence of a metabolite secreted by the bacteria and mount the specific response , whereas the WT worms are able to maintain homeostasis . Detailed metabolomics study will be able to reveal the exact nature of the molecule . One interesting observation is that genes that are upregulated in flr-4 ( n2259 ) , grown specifically on HT115 , are enriched in amino acid metabolism . Previous studies have shown that methionine metabolism greatly influences life span , metabolism , and stress resistance [54–56] . Vitamin B12 acts an important cofactor in methionine as well as propionic acid metabolism , maintaining optimal level of Homocysteine and propionic acid , thereby preventing toxicity [54] . It is possible that the molecule may be vitamin B12 , as seen in case of Comamonas [17 , 57 , 58] . In line with this idea , we found that the levels of the metabolic sensor acdh-1 is much suppressed in HT115-fed flr-4 ( n2259 ) in our RNA-seq data , similar to the effect of vitamin B12 treatment . However , flr-4 mutants do not have any significant defect in development or fat storage . In future , why flr-4 mutants become sensitive to a metabolite or whether the two bacteria differ in production of soluble metabolites needs to be addressed . We also need to understand why the RNAi knockdown of the gene do not induce food-type-dependent life span response . Together , our study discovers a new gene-diet pair that controls the plasticity of aging in C . elegans and reveals a complete signal transduction cascade involved in this process .
C . elegans strains used in this study were obtained from the Caenorhabditis Genetics Center and maintained on NGM agar plates at 20°C , unless otherwise stated , on Escherichia coli OP50 lawns . All RNAi experiments were initiated with synchronized L1 worms . Strains used in the study are: N2 Bristol as wild-type , flr-4 ( n2259 ) X , rde-1 ( ne219 ) V , rde-1 ( ne219 ) V;kzIs9 , rde-1 ( ne219 ) V;kzIs20 , rde-1 ( ne213 ) V;kbIs7 , sid-1 ( pk3321 ) V , sid-1 ( pk3321 ) V;uIs69 V , ccIs4251 [pSAK2 ( myo-3::NGFP-LacZ ) ] , tir-1 ( tm3036 ) III , unc-43 ( e408 ) IV , nsy-1 ( ag3 ) II , nsy-1 ( ok593 ) II , sek-1 ( km4 ) X , pmk-1 ( km25 ) IV , pmk-3 ( ok169 ) IV , flr-4 ( n2259 ) X; sek-1 ( km4 ) X , nhr-8 ( ok186 ) IV , daf-2 ( e1370 ) III , bvIs5 [cyp-35B1p::GFP + gcy-7p::GFP] referred to as cyp-35B1p::gfp in this manuscript , nhr-8 ( ok186 ) IV;bvIs5 , flr-4 ( n2259 ) X;bvIs5 , rrf-3 ( pk1426 ) II;eat-2 ( ad1116 ) II , rrf-3 ( pk1426 ) II , daf-16 ( mgdf50 ) I , smg-1 ( cc546 ) , smg-1 ( cc546 ) I;pha-4 ( zu225 ) V , skn-1 ( zu169 ) IV/nT1[unc- ? ( n754 ) let- ? ] ( IV;V ) , glp-1 ( e2141 ) III , gld-1 ( op236 ) I , glp-4 ( bn2ts ) I , hsf-1 ( sy441 ) . Gravid adult worms , initially grown on E . coli OP50 , were bleached and the eggs were L1 synchronized in M9 buffer for 16 hours before placing them on the respective RNAi plates ( say ‘X’ gene RNAi ) . Once worms reached L4 stage , they were transferred to intermediate RNAi plates ( seeded with the same ‘X’ gene RNAi ) for 12 hours . After that , the worms were picked onto fresh ‘X’ gene RNAi plates overlaid with 5-fluorodeoxyuridine ( FUDR , final concentration 0 . 1 mg/ml of media ) . For life span analysis on plates without FUDR , worms were transferred to fresh plates on alternate days till the end of the reproductive span . Life span scoring was initiated at day 7 of adulthood and continued every alternate day . For statistical analyses of survival , OASIS software ( http://sbi . postech . ac . kr/oasis ) was used and P-values were calculated by using a log rank ( Mantel-Cox method ) test . For temporal requirement experiments , L1 synchronized worms were placed on control RNAi plates . Worms from the plates were transferred to flr-4 RNAi plates at L2 , L3 , L4 or YA stages . FUDR was overlaid on the plates 12 hrs after the worms reached L4 . For life span on different bacterial feed , L1 synchronized worms were placed on HT115 and OP50-seeded plates and the lifespan was initiated as mentioned above . All life span analysis referred in the main text is provided in the S1 Table . Two independent biological replicates are provided in S2 Table . A total of 20 worms each from control or flr-4 RNAi plates were transferred to an unseeded plate on day 2 , 5 and 10 of adulthood . Each worm was gently prodded on the tail with a platinum wire and total number of body bends per 30 seconds was counted . A body bend was scored every time the area behind the pharynx reached a maximum bend in the opposite direction from the last bend counted . The ccIs4251 [pSAK2 ( myo-3::NGFP-LacZ ) ] worms were grown on control or flr-4 RNAi plates . On day 2 , 5 and 10 of adulthood , the worms were paralyzed on 2% agarose pads in the presence of 20 mM sodium azide . Photographs of the worms were captured at 630X magnification using an AxioImager M2 microscope ( Carl Zeiss , Germany ) fitted with Axiocam MRm [Excitation 488nm and Absorbance at 520nm] . For each RNAi , at least 10 nuclei of 10 worms each were photographed . Morphology of each muscle nuclei was scored as ‘intact’ , ‘moderately damaged’ or ‘severely damaged’ . A nucleus was scored as ‘intact’ if it had intact membrane with no degradation , ‘moderately damaged’ when the nuclear membrane appeared to disintegrate but the nucleoplasm displayed no or very little dark patches and ‘severely damaged’ when the nuclei had increased nucleolar size , dark patches in the nucleoplasm , distorted appearance and membrane disintegration . To determine lipofuscin autofluorescence , 20 worms each grown on control or flr-4 RNAi were anesthetized in 20mM Sodium Azide and mounted on 2% agarose pads on day 1 , 5 and 10 of adulthood . The worms were visualized under microscope using FITC filter and images were captured using a constant exposure time ( 1 . 2 sec ) . Twenty five L4 worms , grown on respective bacterial feed , were picked and placed onto NGM plates seeded with 250:1 ( vol:vol ) of bacteria and Fluoresbrites Multifluorescent microspheres/RFP beads ( 0 . 2 μm diameter , Polyscience Inc . , USA ) . After 10 minutes , worms were collected and washed twice with 1X M9 buffer to remove any bead attached to the body surface . Worms were finally re-suspended in 30 μl of 1X M9 buffer and transferred to a freshly prepared 2% agarose pad slides . The images of worms were taken using AxioImager M2 microscope ( Carl Zeiss , Germany ) . Quantification was performed using NIH ImageJ software . An one minute video of Day 1 adult worms was taken using Axiocam MRm camera attached to M205FA microscope ( Leica , Germany ) . The video was slowed down and pharyngeal pumping was counted for that 10 second period . Worms were imaged one day after they reached L4 using Axiocam MRm camera attached to an AxioImager M2 microscope ( Carl Zeiss , Germany ) . Area of the worms was quantified using NIH ImageJ software . flr-4 RNAi: The full length cDNA sequence of flr-4 was amplified using primers listed in S3 Table and cloned into pL4440 RNAi vector . A transcriptional fusion of the flr-4 promoter and a green fluorescent protein ( GFP ) gene was constructed in pPD95 . 75 . The 3 . 5 kb promoter region upstream of start codon of F09B12 . 6 was amplified using primers listed in S3 Table and HiFidelity PCR system ( Kapa Biosystems , USA ) and cloned into pPD95 . 75 using BamHI and KpnI restriction sites . The recombinant plasmid was injected at a concentration of 5ng/μl into the syncytial gonad of wild-type worms along with 100ng/μl pRF4 ( rol-6 ) co-injection marker using a Microinjection setup consisting of Nikon TiS inverted microscope fitted with Eppendorf Femtojet Express and Transferman NK2 . Transformants were selected based on the rolling phenotype as well as the presence of GFP expression . Fluorescence images of transgenic worms were captured under AxioImager M2 microscope ( Carl Zeiss , Germany ) fitted with Axiocam MRm at 40X magnification [Excitation 488nm and Absorbance at 520nm] . The full length cDNA sequence of flr-4 was amplified using primers listed in S3 Table . The gfp sequence of Pflr-4::gfp plasmid was then excised using KpnI and EcoRI and replaced with the amplified flr-4 cDNA sequence , generating the Pflr-4::flr-4 cDNA construct . The Pflr-4::flr-4 cDNA construct and pRF4 were co-injected in the germline of flr-4 ( n2259 ) ( concentrations: 150 ng/μL pRF4 and 5 ng/μL Pflr-4::flr-4 cDNA ) . Wild-type and flr-4 ( n2259 ) roller lines were generated by injecting 150 ng/μL pRF4 . Lines were maintained by picking rollers . Synchronized L1 worms grown on OP50 or RNAi plates were collected at Day 1 of adulthood in M9 buffer and washed thrice using M9 buffer . Then , Trizol was added to about 4 times the volume of the worm pellet and the worms lysed using two freeze thaw cycles , followed by vigorous vortexing . RNA was purified by phenol:chloroform:isoamylalcohol extraction and isopropanol precipitation . For quantitative Reverse Transcriptase PCR ( qRT-PCR ) experiments , the concentrations of the RNA were determined using NanoDrop 2000 ( Thermo Scientific , USA ) and the quality of the ribosomal 28 S and 18 S on denaturing agarose gel was used for evaluation of RNA integrity . For transcriptomic analysis , the quality was evaluated using Bioanalyzer ( Agilent , USA ) and only RNA with RIN number above 9 was used for RNA-seq . About 2 . 5 μg of RNA was converted to cDNA using Superscript III Reverse Transcriptase enzyme and poly-T primers ( Invitrogen , USA ) . QRT-PCR analysis was performed using the DyNAmo Flash SYBR Green mastermix ( Thermo Scientific , USA ) and Realplex PCR system ( Eppendorf , USA ) to determine the relative gene expression levels . Statistical analysis was performed using GraphPad 7 . 0 . All the primers used are listed in S3 Table . RNA-Sequencing ( RNA-seq ) libraries of WT grown on Control RNAi or flr-4 RNAi , and WT or flr-4 ( n2259 ) grown on HT115 or OP50 at Day 1 adulthood were prepared as recommended by the Illumina TruSeq RNA Sample Preparation kit using Low-Throughput ( LT ) Protocol ( Illumina , Inc . , USA ) . Sequencing of libraries was performed using Illumina GAIIX for 78 cycles including 6 additional cycles for index read . Sequence reads were aligned using CLC Genomics Workbench 6 . 5 . 1 with default setting against C . elegans genome assembly ( WS231 ) . Unpaired group comparisons , based on RPKM ( Reads per Kilobase per Million mapped reads ) , were chosen as expression values for comparing the samples . A fold change ±2 . 0 and P value ≤0 . 05 ( Kal's Z test ) were used to filter the differentially expressed genes . GO‐term enrichment analysis was performed using the DAVID Bioinformatics Database [32] . The sequencing data is available as BioProject ID: PRJNA362992 . Synchronized L1 worms , grown on OP50 or HT115-seeded plates , were collected at Day 1 of adulthood in 1xM9 buffer and washed thrice using the same buffer . The pellet was freeze-thawed 3 times in a protein extraction buffer ( 20 mM Hepes buffer pH 7 . 9 , 25% glycerol , 0 . 42 mM NaCl , 1 . 5 mM MgCl2 hexahydrate , 0 . 2 mM EDTA dihydrate , 0 . 5 mM DTT ) in presence of a protease inhibitor cocktail ( Sigma , USA ) , sonicated in a waterbath-based sonicator ( Diagenode , USA ) and centrifuged at 10 , 000 rpm for 10 mins . The protein concentration in the supernatant was estimated by using Bradford reagent ( BioRad , USA ) . About 30 μg of protein was separated on a 12% SDS-PAGE and transferred to Nitrocellulose membrane . The membranes were blocked for 1 hour in 5% non-fat milk and 5% BSA dissolved in 1X TBST ( TBS with 0 . 1% Tween 20 ) and probed with anti-PMK-1 antibody ( 1:2 , 000 dilution in blocking buffer; Cell Signaling Technology , USA ) or anti-phospho-PMK-1 antibody ( 1:2 , 000 dilution in blocking buffer; Cell Signaling Technology , USA ) , incubated overnight at 4°C . Next day , the membranes were washed thrice with 1X TBST and further incubated with 1:10 , 000-diluted secondary antibody ( anti-rabbit conjugated to HRP , Cell Signaling Technology , USA ) for 1 hr at room temperature . The blots were then washed 4–5 times with 1X TBST , each wash lasting 10 min . The blots were developed using enhanced chemiluminiscent substrate ( Millipore , USA ) . For the quantification of PMK-1 activity , the band intensities of pPMK-1 and total PMK-1 were quantified using ImageJ software ( National Institutes of Health , Bethesda , MD; http://rsb . info . nih . gov/ij/ ) and divided with the intensity of the beta-actin bands . The value thus acquired for pPMK-1 was then divided by that of total PMK-1 and represented as percentage . The immunoblots of four independent experiments were quantified . Well-fed young adult worms from control RNAi-seeded plates were collected and washed thrice in M9 buffer . The worm pellet was then divided into two halves; to one half 120 μl of 1X M9 buffer was added while to the other half 120 μl of 1X M9 buffer containing 20mM sodium arsenite was added . After incubation at 20°C for 20 minutes , the worms were washed thrice with M9 buffer . The worm pellet was then processed for protein isolation and western blotting using the above-mentioned method . Worms were grown on respective RNAi from L1 onwards . For each RNAi , four 60 mm unseeded NGM plates with approximately 25 L4 worms per plate were irradiated using a 254 nm UV bulb at 10Jm-2min-1 in a CL-1000 UV Crosslinker ( Ultra-Violet Products Limited ) , followed by transfer to the respective RNAi-seeded NGM . All UV-resistance assays were performed at 20 oC . Survival to stress was scored every 24 hrs post UV exposure . The worms were grown on RNAi plate as above . For each RNAi , three 60 mm NGM plates with approximately 40 L4 worms per plate were incubated at 35 oC . Animal survival was scored every 60 min . Wild-type or flr-4 ( n2259 ) mutant worms were grown on two different E . coli feed , OP50 or HT115 till late L4 stage . Five worms were picked onto fresh plates ( OP50 or HT115 seeded ) and allowed to lay eggs for 24 hours . Three such plates were used for each assay so that ‘n’ was 15 per experiment . The worms were then transferred to fresh plates every day and the eggs/L1s on previous day’s plate were counted . Worms that crawled off the plates or ruptured before the fertile period ended were discarded . Eggs that produced viable progeny were considered as total L1s and the un-hatched eggs were considered as dead eggs . Pool of total L1s and dead eggs are defined as brood size . Data is presented as brood size ± SEM . For calculating reproductive span , total number of L1s is expressed per worm per day . Data is shown as viable progenies plotted against number of days , with SEM at each time point . Gravid adult worms , initially grown on E . coli OP50 , were bleached and the eggs were L1 synchronized in M9 buffer for 16 hours before placing them on seeded NGM plates . The worms were then scored every 12 hours till 60th hour for their development stage . The study was performed with approval from the Institutional Biosafety committee . Only invertebrate nematodes were used for the study . | For animals living in the wild , being able to utilize a wide range of diet is evolutionarily advantageous as they can survive even when their optimal diet is depleted . Since diet is known to influence the rate of aging , animals seem to have evolved intricate mechanisms to maintain homeostasis and normal life span , but the molecular mechanisms are less understood . Using a small nematode , C . elegans as a model , we show that the adaptive capacity to different diet is maintained by a kinase gene . When this gene is mutated , worms start living longer on one strain of bacterial diet but not on the other . We identify the molecular cascade required for this food-type-dependent longevity . We show that this cascade of events significantly overlaps with the pathway that determine food quantity-dependent life span enhancement . Our study thus elucidates a part of the molecular monitoring system that regulates longevity dependent on the available quality and quantity of diet . | [
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] | 2018 | A novel gene-diet pair modulates C. elegans aging |
Here we formulate a mechanistic mathematical model to describe the growth dynamics of P . aeruginosa in the presence of the β-lactam antibiotic meropenem . The model is mechanistic in the sense that carrying capacity is taken into account through the dynamics of nutrient availability rather than via logistic growth . In accordance with our experimental results we incorporate a sub-population of cells , differing in morphology from the normal bacillary shape of P . aeruginosa bacteria , which we assume have immunity from direct antibiotic action . By fitting this model to experimental data we obtain parameter values that give insight into the growth of a bacterial population that includes different cell morphologies . The analysis of two parameters sets , that produce different long term behaviour , allows us to manipulate the system theoretically in order to explore the advantages of a shape transition that may potentially be a mechanism that allows P . aeruginosa to withstand antibiotic effects . Our results suggest that inhibition of this shape transition may be detrimental to bacterial growth and thus suggest that the transition may be a defensive mechanism implemented by bacterial machinery . In addition to this we provide strong theoretical evidence for the potential therapeutic strategy of using antimicrobial peptides ( AMPs ) in combination with meropenem . This proposed combination therapy exploits the shape transition as AMPs induce cell lysis by forming pores in the cytoplasmic membrane , which becomes exposed in the spherical cells .
Antimicrobial resistance ( AMR ) is now acknowledged as an urgent global health threat and the severity of the situation was highlighted by the World Health Organization 2014 report that discusses the increasing incidence of resistance-induced health problems in every region of the world [1] . A “post-antibiotic” era is described , where even a simple infection can become fatal as current drug strategies fail to ameliorate previously manageable infections . It is imperative that we try to gain a deeper understanding of currently used drug treatments and specifically the mechanism of action of a drug and the consequential response of a bacterial population . Elucidating the mechanistic interactions between bacteria and antibiotic increases our understanding of how pathogens react in response to antimicrobials and the concurrent impact on the selective pressure that can influence the emergence of resistance . A popular strategy used to investigate mechanisms of action is the examination of the morphology of treated bacteria . This is a relatively simple experimental procedure that can be used as an initial preliminary step in an investigation or to provide further evidence to support a suspected mechanistic interaction . Investigation into bacterial response has shown that many bacteria undergo changes in their morphology as a result of antibacterial action . Morphological changes such as filamentation ( cell elongation ) , localised swelling and bulge formation can often be attributed to specific antibiotic mechanisms of action [2] . For example , antibiotic agents that alter lateral cell wall synthesis by disrupting the peptidoglycan-synthesizing enzymes can often cause cells to decrease in length , producing ovoid cells [3 , 4] . Observations like this can be the result of multiple mechanistic interactions between the antibiotic and the bacteria and it can also be difficult to differentiate between changes in morphology . Many of these structural changes may occur to varying extents depending on factors such as the antibiotic concentration , incubation conditions and how long the bacteria is exposed to the agent [2 , 5] . Although this can result in structural heterogeneity within bacterial populations , any observations of changes in cellular morphology can still act as an indication of the occurrence of a certain mechanism of action . One bacterial species that shows significant changes to its morphology in response to antimicrobials is Pseudomonas aeruginosa , a Gram-negative pathogenic bacteria that also shows notable levels of resistance to many antibiotics . This extremely versatile , opportunistic pathogen is able to acquire nutrients from a wide range of organic matter , meaning that it can easily infect damaged tissues in animals and humans . P . aeruginosa is an example of a nosocomial pathogen , a characteristic that arises from its ability to survive in moist environments and on hospital instruments such as catheters . Infections are often found in airways , urinary tracts and in burns and wounds and can often be asymptomatic until a biofilm forms . This can overwhelm the immune system and cause bacteraemia , pneumonia and sepsis , and can ultimately lead to death; this makes P . aeruginosa especially threatening to those who are immunocompromised , including in particular patients with cystic fibrosis [6] . There are several antibiotics that still have activity against P . aeruginosa including some carbapenems , a class of β-lactams , the most widely used group of antibiotics . Identified by possessing a β-lactam ring , this class of antibiotics inhibits cell wall synthesis by binding to penicillin binding proteins ( PBPs ) . Inhibition of cell wall synthesis results in structural changes to the bacteria and ultimately this leads to lysis . Some strains of P . aeruginosa display resistance to β-lactam antibiotics , for example through the production of β-lactamases and the upregulation of efflux pumps . Additionally , there is evidence that some genetically susceptible strains are able to tolerate the presence of many β-lactams , including meropenem , for periods of time . Meropenem , a carbapenem that has been shown to have greater antibacterial action against P . aeruginosa compared to other carbapenems , induces several morphological changes in this bacteria including filamentation and spheroplast formation [7] . Its enhanced antibacterial activity and the varying resultant responses in morphology are often attributed to its affinity for both PBP2 and PBP3 enzymes , which lead to inhibition of peptidoglycan synthesis at different regions of the bacterial wall . The work of Monahan et al . [8] investigates the tolerance of P . aeruginosa to β-lactam antibiotics , including meropenem , and suggests that the ability of the bacteria to withstand high concentrations of β-lactams can be attributed to a rapid transition in cell morphology . This population-wide change , activated by antibiotic exposure , is described as a survival strategy that results in the bacteria evading antibiotic effects . The experimental results show that antibiotic exposure induces a transition in morphology that produces a subpopulation of spherical cells that possess a defective cell wall . These spherical cells are thought to be able to survive antibiotic effects since β-lactam drugs target the ability of the bacteria to synthesise its cell wall . It was first thought that the cell wall deficient spherical cells would lyse and not be viable due to the delicate structure of the cell , however , it was found that not only did the whole population of cells transition from rod to spherical-shaped within 24 hours , but also 84% of these cells were viable and able to transition back to rod-shaped cells in the absence of the meropenem [8] . This population of spherical shaped cells can therefore evade the effects of the antibiotic and lay dormant until the antibiotic is no longer a threat , at which point they possess the ability to transition back into rod-shaped cells and resume proliferation . Whether this large scale transition is an observation of the mechanistic interactions between the drug and the bacteria or a mechanism used as a defensive strategy controlled by the bacteria , the viability of these cells and the possibility of a reverse transition could explain some of the persistent characteristics of P . aeruginosa . Persistence is described as a result of phenotypic heterogeneity and this phenomenon occurs due to the formation of a subpopulation of persister cells with potentially different growth and survival rates . These cells , often developing due to environmental stresses , lay dormant until more favourable conditions occur; at which point , the possibility of reversion results in persistent infections that do not necessarily rely on resistance genes . The varying characteristics of the subpopulations we see when meropenem is added to P . aeruginosa form an analogous environment . The task of providing new treatment strategies and exploring the interactions between drugs and bacteria has traditionally fallen to the pharmaceutical industry but with the waning effectiveness of classic antibiotic strategies , it is imperative that we can harness ideas and techniques from other disciplines in order to explore how we can control antibiotic resistance . Mathematical modelling and in particular the study of population dynamics , is increasingly being used to study the changes in growth of bacterial populations , the effects of antibiotics and the emergence of resistance . Here we develop a system of differential equations in order to describe the bacterial growth of P . aeruginosa in the presence of the antibiotic meropenem with the inclusion of a morphological transition that is activated by the presence of the antibiotic . Whether this transition is a result of direct antibiotic action or attributable to an intrinsic survival mechanism , we assume that these cell wall deficient spherical cells evade antibacterial effects , as evidenced by the data in [8] and our own data . The model is formulated to to be entirely mechanistic with growth depending explicitly on nutrient availability . Using in vitro data of growth curves of P . aeruginosa in the presence of varying concentrations of the antibiotic meropenem , we obtain parameter estimates for the model that enable it to reproduce experimentally observed behaviour . Analysis of the results to changes in these parameters allows us to explore the effects of the shape transition on bacterial susceptibility to meropenem . We note that our investigation will focus solely on a meropenem-susceptible strain of P . aeruginosa; our concern is not with the mechanisms of resistance that are often attributed to P . aeruginosa , such as the production of β-lactamases or the upregulation of efflux pumps . Rather , we wish to investigate a shape transition that permits meropenem-susceptible strains of P . aeruginosa to tolerate the presence of antibiotics .
Single colonies of the P . aeruginosa strain PA1008 , a meropenem susceptible isolate derived from a hospital burn unit [9] , were inoculated into LB broth and grown for around 16 hours at 37°C , shaking at 200 rpm . Single colonies of the P . aeruginosa strain PA1008 were inoculated into 5 ml of LB broth and grown overnight at 37°C , shaking at 200 rpm . The following day , the cultures were diluted to an OD600 of 0 . 4 , which would be diluted to 0 . 2 following the addition of the antibiotic . Dilutions of meropenem were made using LB broth to give final concentrations . 50μl of bacteria culture was added to the appropriate wells of a sterile , 96 well , flat bottom microtitre plate , followed by 50μl of the appropriate meropenem dilution . Media controls were then added to the plate for each meropenem concentration , consisting of 50μl of each meropenem dilution plus 50μl of LB broth . The OD600 of each well was taken using a FLUOstar Omega plate reader every 30 minutes for 24 hours . Between OD readings the plate was shaken for 27 minutes . The temperature was set to 37°C for the duration of the assay . In order to successfully parametrise the model of Eqs 2–6 we have used two sets of data , which we will refer to as data set 1 and data set 2 . The first data set is used as parametrisation data; this consists of three biological replicates , each with three technical repeats . The meropenem concentrations used for this data set were 0μg ml−1 , 2μg ml−1 , 4μg ml−1 , 10μg ml−1 , 20μg ml−1 , 40μg ml−1 and 200μg ml−1 . For each concentration we display the mean of the nine resulting growth curves . The error bars are formulated by calculating the standard deviation of the means of the three biological replicates . Data set 2 is also referred to as the test data and includes one biological replicate with three technical repeats; the meropenem concentrations used for data set 2 were 0μg ml−1 , 0 . 5μg ml−1 , 1μg ml−1 , 5μg ml−1 , 50μg ml−1 and 100μg ml−1 . For each concentration , we calculate the mean value of the three resulting growth curves at each time point and the standard deviation error bars for this data set represent the standard deviation over the three technical repeats . The parameter estimates were obtained using the MATLAB function fminsearch , which uses the Nelder-Mead algorithm as described in [10] . The fminsearch function fits parameters that minimise a specified objective function given some initial estimates . We employ the following objective function , R = 1 n ( | | ( H + S 2 ) - y | | 2 | | y | | 2 ) . ( 1 ) The vectors H and S are the solutions for rod-shaped and spherical cells respectively at the time points used in the experiments . The vector y consists of the OD values obtained in the experiment and n is the number of data values . This equation evaluates the mean relative error of the difference between the data and the solution to the system at each time point . We use the relative error in our objective function as unlike the absolute error the relative error should not be skewed by large or anomalous data points . We note here that the contributions of rod and spherical shaped cells to the OD differ in the objective function . OD is a measurement of the absorbency of a material; it measures how much light can pass through a sample and therefore reflects the total number of cells present in the inoculum . Rod and spherical cells will contribute differently to the OD measurement as they differ in size and absorbency . Therefore , rather than attempt to scale OD to obtain a total cell number , we work in OD and based on our biological insight we have assumed that spherical cells contribute half the absorbance of rod cells to the OD measurements . When using the MATLAB function fminsearch to parametrise we also apply a constraint that restricts parameter values to non-negative values . To do this we apply a penalty to the objective function that increases its value significantly if a parameter is less than zero; we simply multiply the objective function value by 1000 ( i . e . we make the penalised objective function much larger than we anticipate the relative error using the fitted parameters to be ) . Deterministic ODE models are commonly used to model bacterial growth and generally use mass action kinetics to assign rates of biological processes to the species involved . Bacterial growth can be described using a variety of growth curves outlined in [11] , with arguably the most commonly used being the logistic growth curve , proposed by Verhulst in 1845 . Logistic growth describes growth at a rate that decreases , and ultimately ceases , as the population size reaches a saturating carrying capacity [12–15] . This incorporates competition for resources by making the rate of growth dependent on the relation between the current population and the saturating population constant . This means that it does not explicitly take into account the nutrient available to the bacteria for growth; this can be incorporated using a density dependent rate , such as Monod kinetics [16] . Using Monod kinetics to model bacterial growth provides a more mechanistic approach that offers more meaningful constants as opposed to the phenomenological nature of the logistic equation . Antimicrobial action can be incorporated using well established concepts from pharmacokinetics and pharmacodynamics . Antibiotic degradation is most commonly modelled via first order kinetics [17–19] and antibiotic effects are often modelled using a density dependent term that will saturate as a consequence of limited binding receptors . Other notable examples of mathematical modelling in bacterial growth and antibiotic action can be seen in [12 , 20–29] . Previous models that include subpopulations of persister cells have been used to investigate the growth dynamics of bacteria and the effects of dosing regimens on the persister population , for example [30–32] . In [30] , a simple mathematical model is formulated using quantitive measurements to describe persistence of single cells of Escherichia coli . The results highlight the need to consider persistent subpopulations and suggest their potential as treatment targets . A general model for persister formation is developed in [31] and is used to investigate the effectiveness of a periodic dosing regime and explore the impact the regime has on the persister population . The investigation in [32] follows on from [31] , yet this model is extended by assuming that persister formation occurs due to the production of toxins within the bacteria . The model we will develop will incorporate the specific shape transition witnessed in P . aeruginosa population dynamics to explore the persistent nature of the cell wall deficient spherical cells . The assumptions made for the model are based upon the results in [8] , which we have reproduced experimentally , with our results displayed in Fig 1 . The images indicate changes to cell morphology shortly after exposure to the antibiotic; spherical shaped cells can be seen after only one hour . After 22 hours we see rod-shaped cells that are in the process of dividing ( Fig 1 ( b ) ) and this indicates that the cells can revert back from the spherical form and continue proliferation . Additionally , the fluorescence microscopy results support the results of [8]; most of the cells stained green , indicating that these spherical forms were viable . In accordance with these findings , our compartmental model follows the interactions described in Fig 2 . We introduce variables ( defined in Table 1 ) for rod-shaped bacteria ( H ) and spherical-shaped bacteria ( S ) . Here , we assume that the rod-shaped cell population consists of all those cells that are susceptible to the antibiotic , this may include cells that have shown signs of localised swelling or filamentation . In this model we will refer to this population as rod-shaped whereas the spherical cell population is made up of cells that have transitioned into this form due to antibiotic exposure . Experimental data has shown that the spherical cells are susceptible to lysis due to internal turgor pressure and that this causes the cells to expand before they lyse . For this model we will contain all spherical cells , regardless of their size , in the same population . In addition to these variables we define the antibiotic concentration to be A and the proportion of nutrient remaining in the system , N . Including a nutrient dependent growth term , instead of the commonly used logistic growth term , will allow us to formulate a mechanistic model . Fig 2 also includes a population of dead cells , however we do not include this variable in the model or results . Any lysed cells will quickly disappear from the culture , we can therefore assume that the contribution made to the optical density by the lysed cells can be considered negligible . This assumption is supported by Fig 1; with only a minority of cells in the image fluorescing red ( indicating a dead cell ) we can assume that the majority of dead cells have completely lysed and disappeared . We assign parameters for the rates of growth , death and transition as defined in Table 2 . Nutrient dependent growth will occur at the rate r and proliferation will concurrently incur a decrease in nutrient that occurs at the rate r ˜ . For this model we assume that only rod-shaped cells can proliferate . This assumption is based on existing literature , experimentally characterising the proliferation of usually rod-shaped bacteria that have undergone conversion to spherical cells as a result of shedding the cell wall [33] . The experiments demonstrated that although proliferation of spherical cells was possible , it was only possible under specific osmoprotective conditions and even then proceeds in only a very small subset of spherical cells , with a doubling time 10-fold higher than that of the rod-shaped cells . Conventional proliferation becomes impossible due to the compromised integrity of the spherical cell wall; bacterial proliferation is heavily dependent on correct positioning of the cell wall and without this it is unlikely that a cell will successfully divide . Although P . aeruginosa was not included in these experiments , it was suggested that similar proliferation occurs across all bacteria; due to the high doubling time we will assume that spherical growth is negligible . Meropenem is bactericidal hence its action is modelled via a death term distinct from natural death [34] . This additional term differs from some models that absorb the antibiotic effects into the model by means of a reduction in bacterial growth , yet this approach would be more suited to bacteriostatic antibiotics [17 , 25] . The antimicrobial effects on the rod-shaped population is modelled using a density dependent term to incorporate a saturating effect . Attempts at using a linear term for this resulted in unsuccessful parameter fits ( results omitted ) and a saturating term was found to be more suitable in describing the effects of the antibiotic; this is in keeping with previous models of antibiotic treated bacteria [14 , 17 , 35] . We assume that the meropenem only inhibits rod-shaped cell survival as experimental results indicate that transitioning to a spherical form mediates tolerance to lethal β-lactam antibiotic concentrations for multiple bacterial species [8 , 33] . We assign ρ to be the maximum death rate of rod-shaped cells due to antibiotic and A50 to be the antibiotic concentration needed for half maximal killing . We assume that the meropenem molecules will enter the cell and bind to the PBPs in order to inhibit cell wall synthesis . In this model we assume that this binding is irreversible due to mode of action of β-lactams . The antibiotic molecules covalently modify PBPs upon binding [36] and therefore when a bacterium lyses , any bound antibiotic is unlikely to be released back into the extracellular domain . The irreversibility of the antibiotic results in antibiotic decay and we will take this to happen at a rate , ρ ˜ . Investigation into this parameter has shown that relaxing this parameter and setting ρ ˜ = 0 μg ml−1 OD−1 min−1 does not affect the results significantly ( results omitted ) yet we include this assumption as it has an impact on long-term model predictions of bacterial growth . In addition to this , we take antibiotic decay to follow first order kinetics at a rate α . In concordance with [8] , we assume that spherical cells evade the effects of the antibiotic . The primary objective of this model is to investigate whether we can describe the population dynamics of P . aeruginosa in the presence of meropenem by including the morphological transition witnessed in experimental data . We model this transition from rod-shaped cells to spherical-shaped cells with a saturating term , similar to that used for the bactericidal effects of the antibiotic on the rod-shaped cell population . Following exposure to the meropenem the rod-shaped cells transition to a spherical form at a maximum rate γ and T50 defines the concentration for which we get the half maximal transition rate . Following [8 , 33] we include the process of reversion within our model: antibiotic is removed from samples in order to experimentally show that spherical cells can transition back to the bacillary form , implying that this occurs independently from the presence of the antibiotic . Previous models of persister populations have assumed that reversion does not occur when a constant dose of antibiotic is administered [31 , 32] . However , in our model , antibiotic may degrade out of the system and even when present the spherical cells may not be directly exposed to antibiotic at all times . Reversion will be independent from the antibiotic concentration and we define δ to be the rate at which this occurs . Supported by the microscopy results in Fig 1 , this reverse transition also lends weight to the assumption that antibiotic decays as it suggests that the pressure on the bacteria from the antibiotic has decreased . Finally , following our own fluorescence microscopy data , we assume that both types of cell will lyse naturally . In our microscopy results we witnessed lysed ( red ) rod-shaped cells even in the absence of antibiotic , indicating that some level of natural death must occur . Subsequently , we assume that spherical cells also lyse naturally due to the increased fragility of the cell structure . Lysed cells degraded sufficiently quickly that we may assume they do not contribute to the OD settings in our experimental set-up . The spherical cells lyse at a rate ψ , whilst rod-shaped bacteria lyse at a rate ϕ . Due to the availability of nutrient and general fitness of the rod-shaped cells we would expect ϕ to be small and due to the compromised integrity of the cell wall in spherical cells we expect ϕ < ψ . Following mass action kinetics we produce the following model: d H d t = r N H - ( γ A T 50 + A ) H + δ S - ( ρ A A 50 + A ) H - ϕ H , ( 2 ) d S d t = ( γ A T 50 + A ) H - δ S - ψ S , ( 3 ) d A d t = - α A - ( ρ ˜ A A 50 + A ) H , ( 4 ) d N d t = - r ˜ N H , ( 5 ) with initial conditions H ( 0 ) = H 0 , S ( 0 ) = 0 , A ( 0 ) = A 0 , N ( 0 ) = 1 . ( 6 ) As N is defined to be the proportion of initial nutrient remaining instead of a concentration we can specify N ( 0 ) = 1 . All variables and parameters are defined in Tables 1 and 2 respectively .
Our primary interest in formulating this model is to investigate the proposed morphological change observed in our experimental data of P . aeruginosa . This shape transition has been associated with the ability of the bacteria to withstand high levels of antibiotic and using suitable parameters we can reproduce the bacterial regrowth witnessed in our experimental data . We can now manipulate the system by changing parameters associated with the transition mechanism to investigate the impact the shape transition has on bacterial susceptibility and produce predictions that could facilitate treatment design . Fig 10 ( a ) –10 ( f ) display numerical simulations used for the analysis in this section . We note here that the results using Θ1 are qualitatively the same regardless of the initial antibiotic concentration and these are also similar to the results when using Θ2 with initial antibiotic doses of >2μg ml−1 . The results for Θ2 differ for higher ( >2μg ml−1 ) and lower ( 2μg ml−1 ) antibiotic concentrations and therefore we only include simulations using Θ2 for brevity ( i . e . the Θ1 results are equivalent to the Θ2 results at 10μg ml−1 . We have chosen to display simulations using the initial antibiotic dose of A0 = 2μg ml−1 as the results in this case differ from the other concentrations and A0 = 10μg ml−1 as this case represents a clinically relevant antibiotic concentration .
Following the conclusions made by Monahan et al . and supported by our own data , we have formulated a model that describes the growth dynamics of P . aeruginosa in the presence of meropenem , with the inclusion of a sub-population of cells with an incomplete cell wall that results in an altered shape and size . We have made assumptions regarding the inducement of this morphological shift and by successfully fitting it to experimental data we obtained two parameter sets that successfully fit the data yet result in qualitatively different predictions of the long-term bacterial dynamics and different predictions when we investigate the values of some of the model parameters . We can attempt to address the ambiguity surrounding the cause of structural changes to the bacterial population using this model . By assuming that the change in bacterial morphology occurs due to the presence of antibiotic exposure , whether the transition is an intrinsic internal response of the bacteria or a result of antibiotic action , the model formulated allows for both of these possibilities . Research suggests that the formation of spheroplasts , with a depleted peptidoglycan layer and unstable osmotic properties , may be the final structural change witnessed before lysis , implying that the transition may be due to antibiotic effects and not an intrinsic mechanism . Although this hypothesis may contradict the findings in [8] , the model is still suitably formulated under this assumption . The death rate of the bacillary cells due to antibiotic can be seen to represent those cells that are eliminated by the antibiotic before any significant changes in morphotype occur . Those that do display changes in their ultrastructure undergo transition into the spherical population and are then subject to a higher death rate; however this mechanism is not explicitly dependent on antibiotic . Another way of looking at this would be to suggest that if the shift in population structure is solely the result of peptidoglycan inhibition , induced by the antibiotic , then once a bacterium has converted into a spherical form the bactericidal damage has already been induced and lysis is no longer depend on antibiotic presence . Similarly , allowing for a reverse transition would suggest that the damage made by the antibiotic is reversible . Much of our results , however , support the hypothesis proposed by Monahan et al . [8] that this transition is in fact a purposeful evasion mechanism , induced by antibiotic exposure , yet ultimately implemented by a responsive cellular mechanism . Under the assumption that spherical cells are immune to antibiotic effects , and by using suitable parameters , we have shown that the transition may lead to the recovery of population levels in the absence of meropenem , which we have seen in both our microscopy results and growth curves using low antibiotic concentrations . Although these results are restricted by limited data , the repercussions of this theory could give insight into the persistent characteristics of P . aeruginosa and explain its ability to sometimes withstand high levels of carpabenems . This could be seen as a characteristic of resistance that is displayed intrinsically without any reliance on the inheritance of resistance genes or acquisition of specific mutations that warrant resistance to the antibiotic . Owing to this , we must consider the possible impact of the shape transition in the wider context of antibiotic resistance; if intrinsic in nature then the use of this mechanism could work hand-in-hand with other resistance mechanisms . Additionally , possessing a mechanism that causes this kind of morphological shift can be linked with persistence and the threat of recurrent infection that comes along with phenotypic persistence . By formulating a mechanistic model we have obtained parameter values that are not only potentially measurable but are also more meaningful and directly relate to the amount of nutrient in the experiment . The model can successfully describe the overall trends witnessed in the growth curves and although the model does not capture the growth dynamics over the first few hours of the experiment perfectly , the impact of this on the applications of this model is arguable as our interests lie with the long term predictions of population growth , which are accurately predicted . The error over this region increases for the higher antibiotic doses yet we note these are much higher than the recommended clinical dose of 10−20μg ml−1 , therefore any discrepancies obtained for these concentrations should have no clinical relevance . We also take note of the large error bars for the initial condition: the high level of variance between the measurements taken at this time point means it could be difficult to achieve a fit of high confidence in this region regardless . Furthermore , having parametrised the model from total bacterial load ( OD measurements ) the model is then able to correctly predict the underlying dynamics of the individual rod- and spherical-shaped populations ( calculated from the microscopy data ) . Manipulation of the parameters obtained allows us to investigate the impact of inhibiting the transition mechanism . Our results suggest that inhibiting the spherical transition would be detrimental to bacterial populations in most cases with inhibition leading to faster depletion and lower OD levels over a long time scale . An exception to this is when we use parameter set Θ2 and initial antibiotic concentration of 2μg ml−1; in this case , inhibition is only detrimental to growth in the short term , whereas long term predictions are the same regardless of the level of inhibition . In most cases ( including all antibiotic concentrations for Θ1 ) the results imply that inhibition of the spherical transition would be desirable in treating a P . aeruginosa infection and thus support the hypothesis that this may be an intrinsic defence strategy of the bacteria . When the transition to spheres was inhibited , the simulations also displayed a characteristic of higher OD levels over the first few hours than with no inhibition; if these results were to be directly translated to an in vivo infection , this could imply a higher level of bacterial virulence . However , in order to formulate biological conclusions based on these results we must consider the virulent properties of the different cell types we include in the model . A higher OD value does not automatically signify a higher level of virulence since we cannot assume that the virulence of spherical cells is half that of the rod-shaped cells , an assumption we make for the OD . Virulence in P . aeruginosa is to a large extent based on the Type III secretion system and secreted toxins; both of which depend on outer membrane components for successful secretion . It is probable that the spherical cells are less virulent than the rod-shaped cells due to their compromised outer membrane . If this assumption is correct then although inhibition of the shape transition predicts higher antibiotic action over the total time , a higher initial bacterial load would not be a desirable effect of a treatment when these are predominantly rod-shaped cells . Increased virulence could be threatening to a patient’s health and this would be especially dangerous when treating immunocompromised patients . It is also worth noting here that if the morphological changes witnessed are a result of antibiotic action then inhibiting this mechanism could be counter-productive to the application of meropenem . Envisaging a drug that could inhibit the bacterium from shedding the cell membrane , thus keeping it intact , this could work directly against the mechanism of action of the antibiotic . An alternative strategy would be to target the reverse reaction and our results indicate that this approach could be beneficial , depending on the virulent properties of the spherical cells . By inhibiting this reaction we predict a higher proportion of spherical cells and this enables successful suppression of a lower proportion of remaining rod-shaped cells without exhausting the antibiotic supply . Following this phase of rod-shaped cell death , the model predicts that for sufficient inhibition , the bacteria attains similar spherical-only population levels regardless of the parameter set used or initial antibiotic concentration . Though in many cases the estimated population level is higher than we would predict using the default parameter values , this would not be an undesirable outcome under the assumption that spherical cells are not as virulent as rod-shaped cells due to the compromised cell membrane . Owing to the high proportion of spherical cells we predict when inhibiting the spherical to rod transition , we could consider the combination therapy of meropenem and AMPs with a drug that would inhibit the reverse transition . Simulations suggest that if we sufficiently inhibit the reverse transition rate then any increase in spherical cell death would result in faster killing of the bacterial population and extended antibiotic presence regardless of the parameter set used or initial antibiotic concentration ( results omitted for brevity ) . Here we define sufficient inhibition of the reverse transition to be when we choose a value for δ that predicts population levels comprised of mostly spherical cells . If we do not attempt to inhibit the morphological transition and instead pursue the application of a drug that increases the death rate of spherical cells alone , we predict lower OD levels . This supports the results detailed in [8] that promote the use of AMPs as a supplementary agent in combination therapy with the meropenem and empiric studies have also found that β-lactam and AMPs act synergistically to inhibit growth [41] . This could be a very suitable strategy for treating a P . aeruginosa infection , however further investigation into the resultant impact this may have on the emergence of resistance would be needed . The use of AMPs would impose a direct fitness cost on the spherical cells and if the transition occurs as a result of an intrinsic mechanism then this could impact selection for resistant phenotypes . However , due to the already-depleted cell wall and lowered natural survival rate of the spherical cells , it is uncertain whether imposing a further fitness cost on the bacteria would impact the possible emergence of resistance . The same evaluation would need to be made if a strategy targeting inhibition of either the forward or reverse transition was pursued; any target that results in lowered fitness could influence the selection for resistant phenotypes . Additionally , if we were to inhibit the rod to spherical shape transition , resistance would imply a bacterium not transitioning to a spherical form and this would imply that it would maintain a rod-shape morphology and remain susceptible to the antibiotic . Using an extended model it is possible to simulate the system with multiple strains ( i . e . meropenem-resistant and -susceptible strains , similar to [14] ) of the bacteria and we are currently investigating whether or not a resistant strain would flourish in this environment . We have formulated a model to predict the growth dynamics of P . aeruginosa witnessed in vitro . In order to make conclusions in a clinical setting we would have to translate the model to an in vivo model and extend it to include antibiotic dosing instead of the single antibiotic application used in our experiments . We would also need to consider how nutrient availability would differ in vivo . Nutrient availability in vivo is likely to be lower than in the nutrient rich growth media used in experiments , but it may be replenished over time . Numerical simulations ( omitted ) indicate that if nutrient-abundance is maintained for longer , the bacteria may withstand higher concentrations of antibiotic . In conclusion , by formulating a model based on biological mechanisms , including a morphological transition , into a mathematical model for population growth , we have successfully obtained parameters values that describe the rates of the mechanisms involved . Crucially , extending this model will allow for a better prediction of how these potential therapies may impact the emergence and development of drug resistance and parameter analyses could hint at strategies to combat the threat of resistance . Our analysis suggests that inhibition of the morphological transition could be a suitable target for a treatment strategy . | Antimicrobial resistance is an urgent global health threat and it is critical that we formulate alternative treatment strategies to combat bacterial infections . To do this we must understand how bacteria respond to currently used antibiotics . Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients , a top cause of hospital-acquired infections in the UK and is currently listed as a critical priority in a list of antibiotic-resistant bacteria produced by the World Health Organisation . P . aeruginosa can change shape in the presence of certain antibiotics that work by targeting cell wall synthesis . The bacteria make the reversible transition from the native rod shape to a fragile spherical shape by shedding the cell wall and in doing so they evade the effects of the antibiotic . We formulate a system of equations that describes the growth of the bacteria including the shape transition we witness when we add antibiotic . Fitting this model to experimental data , we obtain parameter values that we then vary to make predictions on how inhibiting the shape transition or increasing the death rate of spherical cells would affect the overall bacterial growth . These predictions can support suitable combination therapies and hint towards alternative treatment strategies . | [
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] | 2018 | Mathematical modelling of the antibiotic-induced morphological transition of Pseudomonas aeruginosa |
Hepatitis B virus ( HBV ) replicates its 3 kb DNA genome through capsid-internal reverse transcription , initiated by assembly of 120 core protein ( HBc ) dimers around a complex of viral pregenomic ( pg ) RNA and polymerase . Following synthesis of relaxed circular ( RC ) DNA capsids can be enveloped and secreted as stable virions . Upon infection of a new cell , however , the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular ( ccc ) DNA . HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain ( CTD ) with intrinsically strong non-specific RNA binding activity . Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events . However , neither the relevant enzymes nor their target sites in HBc are firmly established . Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc . Combining Phos-tag gel electrophoresis , mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 ( SRPK1 ) ; fewer sites were phosphorylated by PKA and PKC . Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility , without major structure changes in the capsid shell . The bulk of capsids from human hepatoma cells was similarly highly , yet non-identically , phosphorylated as by SRPK1 . While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex . The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target .
Chronic infection with hepatitis B virus ( HBV ) puts more than 250 million people at a greatly increased risk to develop terminal liver disease [1] . HBV , the prototypic hepadnavirus , is a small enveloped virus that replicates its 3 kb DNA genome through capsid-internal reverse transcription of a pregenomic ( pg ) RNA ( reviewed in [2 , 3] ) . The virion envelope consists of a lipid bilayer into which the small ( S ) , middle ( M; PreS2/S ) and large ( L; PreS1/PreS2/S ) surface proteins are embedded [4–6] . Binding of L to the HBV receptor sodium taurocholate cotransporting polypeptide ( NTCP ) is essential for infection ( reviewed in [7] ) ; in addition , L contributes a "matrix domain" that interacts with the capsid for virion morphogenesis ( reviewed in [8] ) . The icosahedral HBV capsid ( core particle ) is composed of 120 dimers ( triangulation number T = 4 ) of a single core protein ( HBc ) species of 183–185 amino acids ( aa ) in length; a minor capsid class ( T = 3 ) comprises 90 HBc dimers . The HBc monomer encompasses an N terminal assembly domain [9] , linked through residues 141–149 [10] to an arginine-rich C terminal domain ( CTD; Fig 1A ) . The CTD is crucial for specific co-encapsidation of a complex of pgRNA and viral polymerase ( P protein ) during replication but it can also mediate non-sequence-specific packaging of RNA ( Fig 1B ) , e . g . when HBc is expressed in E . coli [9 , 11–13] . Structural analyses of such capsid-like particles ( CLPs ) , mostly from CTD-less variants like HBc149 [9] , revealed five α-helices [14–16] . Helices α3 and α4 form an antiparallel hairpin; for dimerization , two such hairpins associate into four-helix bundles which protrude as spikes from the capsid surface ( Fig 1B ) . Helix α5 plus the downstream sequence to position 140 harbor the major inter-dimer contacts . Little is known about the structure of the CTD; in current CLP structures the visible sequence commonly ends within the linker [10 , 14–19] . Regarding CTD disposition most data support a luminal localization [20–23] . However , a permanent internal disposition as well as a static nucleic binding capacity are incompatible with the full set of HBc functions in the viral life-cycle ( Fig 1C ) . Beyond pgRNA/P protein encapsidation these include CTD-mediated reverse transcription [24] into single-stranded ( ss ) minus-DNA and then partly double-stranded ( ds ) relaxed circular ( RC ) DNA [24–26]; capsid envelopment for secretion of virions [8]; and , upon infection of a new cell , transport of the viral genome to the nuclear pore so as to release the RC-DNA into the nucleoplasm [27 , 28] for conversion into covalently closed circular ( ccc ) DNA [29 , 30] . Nuclear transport requires binding of CTD-encoded nuclear localization signals ( NLSs; [31] ) to cytosolic transport receptors [32–35] such that at least one CTD per capsid must become exposed [18 , 27 , 32 , 36 , 37] . Hence the capsid must safely stow ss and also ds nucleic acid with twice as many negative charges and then orderly let go of it . A likely mechanism underlying these CTD dynamics is transient phosphorylation , early-on hinted at by a capsid-associated protein kinase activity [38 , 39] . However , neither this kinase nor other potentially HBc-relevant kinases have unambiguously been identified . Proposed candidates include Ca2+ and/or lipid-activated protein kinase C ( PKC; [36 , 40] ) ; cyclic AMP dependent protein kinase A ( PKA; [41 , 42] ) ; serine/arginine-rich protein kinase 1 and 2 ( SRPK1 , SRPK2; [43] ) ; cyclin-dependent kinase 2 ( CDK2; [44] ) ; and polo-like kinase 1 ( PLK1; [45] ) . Major analytical challenges are the presence in human cells of >500 protein kinases plus >200 phosphatases [46]; the repetitve nature of the HBc CTD sequence ( Fig 1A ) ; the limited specificity of pharmacological kinase inhibitors; and especially the lack of assays that feasibly distinguish non-phosphorylated and differently phosphorylated HBc species . As a surrogate , various studies assessed the impact of serine and/or threonine ( S/T ) replacements in HBc by alanine ( A ) to prevent phosphorylation , and by aspartic acid ( D ) or glutamic acid ( E ) to mimic phosphorylation [47–50] . Collectively these data suggest that phosphorylation is necessary early and some dephosphorylation late during replication . However , genetic mimics cannot model the dynamics of phosphorylation . Phospho-HBc-specific antibodies [51 , 52] are valuable but they interrogate only few of the many options for phosphorylation site occupancy . Correlation with specific replication states is further convoluted by the variety of capsid forms , including nonenveloped capsids [53] and the recently found enveloped genome-less capsids ( "empty virions"; [52] ) which seem to by far outnumber infectious virions ( reviewed in [54] ) . The most direct evidence for the dynamics in core protein phosphorylation comes from duck HBV ( DHBV ) . Intracellular DHBV core protein ( DHBc ) showed up to four distinct phosphorylation-dependent bands in normal SDS-PAGE [55] whereas virion-derived DHBc displayed a single , non-phosphorylated band , as confirmed by mass spectrometry ( MS ) [56] . The presence in virions of mostly mature viral ds DNA [57] , similarly observed for HBV [58] , supported the "maturation signal" hypothesis [57] whereby capsid-internal genome maturation , perhaps sensed by the CTDs and/or their phosphorylation status , exposes interaction sites for L protein on the capsid surface ( Fig 1D ) . The finding that also empty though not ss nucleic acid containing capsids can be enveloped prompted a revised model whereby RNA or ssDNA actively inhibit envelopment [52 , 59] . A cryoEM comparison between recombinant RNA containing capsids and serum virus-derived , supposedly dsDNA bearing capsids had indeed revealed subtle differences [17] . However , at the time empty virions in serum [52 , 60] were not known; hence potential envelopment-relevant structural differences and their correlation with HBc phosphorylation remain open ( Fig 1C , i-iii ) . Some natural HBc mutations such as F97L [61–63] promote secretion of immature , ssDNA containing virions . Such capsid proteins might by default adopt an envelopment-proficient conformation ( Fig 1D ) . Yet , while affecting in vitro assembly [64] no differences in stability or morphology between wild-type and F97L HBc particles were detectable [63 , 65] . However , a potential role of phosphorylation could not be assessed at the time . In sum a large body of data supports correlations between HBc phosphorylation , genome maturation and capsid structure , including CTD disposition and envelopment-competence; however , fundamental information for disentangling these interdependencies is lacking . In order to generate such information we here established the efficient recombinant production of distinct phospho-HBc species and showed the feasibility of Phos-tag SDS-PAGE [66] for their differentiation . Using MS and mutagenesis we identified seven hydroxy amino acid residues in the CTD as target sites for SRPK1 [34 , 43] while PKA and PKC modified fewer , yet to be mapped sites . Full occupancy of the SRPK1 sites massively reduced HBc183 CLP RNA content while abrogation of one phospho-site restored substantial RNA binding . Full phosphorylation also caused differences in CTD accessibility to trypsin , regardless of the F97L mutation . Importantly , in human hepatoma cells the bulk of HBc appeared as highly phosphorylated as SRPK1-coexpressed HBc . These data pave the way for a comprehensive characterization of the substrate properties of hepadnaviral core proteins for individual kinases and phosphatases . Virologically they suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few dephosphorylation events could enable specific packaging of the pgRNA/P protein complex .
Eukaryote-like S/T protein kinases are rare in eubacteria [67] and apparently absent from E . coli laboratory strains . Hence co-expression of HBc with a mammalian kinase should allow the background-free evaluation of its action on HBc . In a first step we created T7 promoter-based vectors carrying an E . coli codon usage adapted HBc gene ( HBc183opt ) to boost expression of full-length HBc183 ( Fig 1A ) . CLP yields were indeed around three-fold higher ( S1 Fig ) compared to the older non-optimized HBc gene [68] in the same pET28a2 vector [69 , 70] , and they increased further using pRSF-Duet derived vectors ( see below ) . For the coexpression approach the recombinant kinase must be enzymatically active and target sites in HBc must not be sequestered by CLP formation . We thus chose human SRPK1 as the first candidate HBc kinase . Beyond its very high affinity for the HBc CTD [34 , 43 , 71] we had already shown HBc phosphorylating activity of the full-length enzyme in bacteria [11]; here we used an N terminally His6-tagged variant , NHisSRPK1ΔNS1 , in which an internal deletion boosts soluble expression in bacteria [72] . To minimize experimental variations seen when HBc and kinase were expressed from two separate plasmids [11] we devised a single-vector dual expression system; it features a Tet repressor / anhydrotetracycline ( AHT ) inducible Tet promoter cassette plus an isopropyl β-D-1-thiogalactopyranoside ( IPTG ) regulatable T7 promoter cassette ( S2 Fig ) . Functionality was shown by inducer-specific expression of eGFP from one and mCherry from the other cassette ( S2 Fig ) . HBc183opt in the T7 cassette was expressed at up to five-fold higher levels than from the pET28a2 vector ( S3 Fig ) . Of note , expression from the Tet cassette was affected by the specific ORF sequence . For instance , the basal level of NHisSRPK1ΔNS1 expression without AHT was still about one third as high as with inducer ( S3A Fig ) ; hence in subsequent SRPK1 coexpression experiments AHT was omitted . Either way , in line with its high affinity for HBc [34 , 43] most of the SRPK1 cosedimented with the CLPs , both from wild-type and F97L HBc ( S3 Fig ) ; separation was achieved by immobilized metal ion chromatography ( IMAC ) under semi-denaturing conditions ( S4 Fig ) . Altogether , the system provided easy access to milligram amounts of CLPs per 100 ml of induced bacterial culture . Low level co-expression in E . coli of full-length SRPK1 with HBc183 had generated a mixture of HBc species containing from one to several phosphoryl groups [11]; this prompted the follow-up use of a triple S>E variant , HBc183-EEE ( S155E_S162E_S170E ) as a homogeneous genetic mimic of such partially phosphorylated HBc183 [34 , 35] . HBc183-EEE CLPs contained ~25% less RNA than wild-type HBc183 CLPs [73] . To evaluate the impact on CLP RNA content of the much higher NHisSRPK1ΔNS1 expression in our new system we separated the CLPs by NAGE , then stained packaged RNA by ethidium bromide ( EB ) and the protein shell by Coomassie Brillant Blue ( CB ) [9 , 74]; other stains , e . g . Sybr Green 2 ( SG2 ) for RNA and Sypro Ruby ( SR ) for protein [75] , could be used instead ( see below ) . The intensity of RNA vs . protein stain per band is proportional to the CLP´s RNA content . Fig 2A shows such an analysis for HBc183_F97L expressed in the absence ( -SRPK ) vs . presence of SRPK1 . Despite much weaker EB staining the +SRPK1 samples showed even stronger CB staining; in either case abundant CLPs were visible in negative staining EM; wild-type HBc183 CLPs gave comparable results . To appreciate the extent of reduction in RNA content caused by NHisSRPK1ΔNS1 coexpression we used a reference set of CLPs from C terminally truncated HBc proteins , starting with the CTD-less variant HBc140 . SG2 and SR allowed semiquantitative assessment of the relative fluorescence intensities of each band ( Fig 2B ) using a laser scanner ( Typhoon FL7000 , GE Healthcare ) . The ratio of RNA: protein fluorescence for non-phosphorylated HBc183 CLPs was set to 100% , to which the ratios for the other CLPs were normalized . Accordingly , SRPK1-coexpressed HBc183 CLPs contained nominally 27% as much RNA as the HBc183 reference CLPs , very similar to CTD-less HBc140 CLPs . For the other variants , the ratios increased with increasing CTD length , in accord with the electrostatic "charge balance" hypothesis [13 , 26 , 76] . An estimate of absolute CLP RNA content as molar ratio of nucleotides ( N ) per core protein ( P ) subunit ( N/P ) was obtained from UV-VIS spectra by deconvoluting the superimposed RNA and protein absorbances [73]; spectra were recorded using gradient-enriched CLPs incubated with 1/100 the amount of ( w/w ) of RNase A and subsequently dialyzed to remove non-packaged RNA . For HBc183 CLPs we obtained ( N/P ) values ± standard deviation ( SD ) of 16 . 4±2 . 2 ( n = 8 ) ; for a T = 4 CLP this corresponds to an RNA content equivalent to ~4 , 000 nt , similar to other reports [12 , 73] . Much in contrast , SRPK1-coexpressed HBc183 CLPs gave a ratio of only 2 . 3±0 . 5 ( n = 8 ) , not significantly different from CTD-less HBc140 ( 2 . 9±1 . 3; n = 4 ) and HBc149 CLPs ( 1 . 8±1 . 5; n = 4 ) . Intermediate RNA contents were found for HBc161 , 163 , 166 and 173 CLPs , as shown in Fig 2C in comparison with the NAGE/SG2:SR procedure . While the apparent overestimation of low RNA contents by the latter method could later be reduced by several improvements ( Materials and Methods ) , both data sets corroborated that SRPK1 coexpression reduced RNA content as strongly as deletion of the entire CTD . This implied a phosphorylation extent sufficient to shift assembly to an RNA-independent , protein-protein interaction driven mechanism [37 , 77] , perhaps by neutralizing most of the positive CTD charges [13] . The Phos-tag chelator coordinated with two Me2+ ions binds to phosphoryl groups , including in proteins [78] . In SDS-PAGE gels containing copolymerized Phos-tag acrylamide and loaded with Mn2+ or Zn2+ phospho-proteins migrate more slowly than their non-phosphorylated forms [66] . Hence this technique appeared attractive to detect HBc phosphorylation . After optimization we routinely used home-made gels containing 75 μM Phos-tag acrylamide loaded with 150 μM Mn2+ . As shown in Fig 3 , in normal SDS-PAGE both HBc183 and HBc183_F97L migrated to the same ~21 kDa position , regardless of SRPK1 coexpression . In Phos-tag SDS-PAGE , however , exclusively the kinase-coexpressed proteins exerted a drastically reduced mobility . Band ladders produced in in vitro SRPK1 phosphorylation and lambda phosphatase dephosphorylation reactions with a non-assembling GFP-CTD fusion protein suggested phosphorylation at five or more positions . Analogous coexpression of HBc183 with the catalytic domains of PKA ( Genbank NP_032880 . 1 ) and PKC theta ( Genbank NP_001269573 . 1 ) , chosen owing to their reportedly functional expression in E . coli [79 , 80] , caused a less pronounced retardation ( see below ) . The number of phosphoryl groups introduced into HBc183 by SRPK1 was analyzed by matrix-assisted laser desorption/ionization ( MALDI ) time-of-flight ( TOF ) MS . The major MH+ peaks in the relevant 21 kDa region of the spectra had mass/charge ( m/z ) values of 21 , 103 for mono-expressed HBc183 and 21 , 664 for SRPK1-coexpressed HBc183 . These values match most closely to the calculated masses of 21 , 116 Da for completely unmodified HBc183 , and to 21 , 676 Da for a seven-fold phosphorylated HBc183 ( S5 Fig; each phosphoryl group contributes ~80 Da ) . Notably , for SRPK1-coexpressed HBc140 and HBc149 the main peaks still corresponded to the non-phosphorylated proteins; hence the assembly domain and the linker sequence lack SRPK1 target sites . For more accurate mass determination we devised a method ( S1 protocol ) to isolate just the CTD peptide from a tobacco etch virus ( TEV ) protease cleavable NHisGFP-TEV-CTD fusion protein ( Fig 4A ) , expressed with or without kinase . Cleavage reduces the relevant masses to ~5 , 000 Da , with a synthetic N-acetylated CTD peptide serving as reference ( Fig 4B ) . Methods development included replacement of the C terminal Cys residue by Ala ( C183A in full-length HBc numbering ) . In brief , the NHisGFP-TEV-CTD protein was enriched by Ni2+ IMAC under semi-denaturing conditions , then incubated with His-tagged TEV protease [81] to release the CTD peptide; all His-tagged components ( uncleaved GFP fusion protein , the GFP-containing cleavage product and TEV protease ) were removed by another round of IMAC . The clipped-off CTD peptide in the flow-through was concentrated and further enriched by ultrafiltration . An SDS-PAGE analysis of the final products obtained upon coexpression with SRPK1ΔNS1 or the PKA catalytic domain ( PKAcd ) or without kinase , alongside the synthetic CTD peptide , is shown in Fig 4C . Importantly , MS of the SRPK1 sample ( Fig 4D ) showed one major peak with excellent agreement to a seven-fold but not six- or eight-fold phosphorylated CTD peptide . Hence there are seven SRPK1 target sites in the HBc sequence 146–183 . The m/z values for the synthetic and the non-phosphorylated fusion protein-derived CTD peptide were <1 Da off the calculated masses ( S6 Fig ) . The PKA-coexpressed sample showed two main peaks , matching four and five phosphoryl groups , respectively; for full-length HBc183 as substrate the products from PKA and PKC coexpression carried only three phosphoryl groups ( S7 Fig ) , possibly due to assembly-mediated target site sequestration . To identify the seven SRPK1 sites in HBc we individually mutated the seven S residues plus one T residue in the CTD ( S155 , T160 , S162 , S168 , S170 , S172 , S178 and S181 ) to A , coexpressed the mutant proteins with SRPK1 and monitored phosphorylation by MS ( Fig 5A ) and by immunoblotting after Phos-tag SDS-PAGE . For all mutants but one MS revealed a best match to the six-fold phosphorylated forms , i . e . loss of one phosphorylation site . The exception was mutant S181A which still matched best to a seven-fold phosphorylated species . Hence all hydroxy amino acids in the HBc CTD including T160 yet except S181 are substrates for SRPK1 . Double and triple S/T>A mutants confirmed the data as the number of lost phosphorylation sites always corresponded to the number of S/T replacements by A ( or by G or R ) , except when S181 was mutated ( S7 Fig ) . In line with their at least six-fold phosphorylation all single S/T variants were similarly strongly retarded in Phos-tag SDS-PAGE as SRPK1-coexpressed wild-type HBc183 , as shown by immunoblotting ( Fig 5B ) with the assembly domain-directed anti-HBc mAb 1D8 [82] . Notably , though , the correlation between the number of phosphorylation sites and mobility was not strictly linear . The six-fold phosphorylated variants S155A , S176A , and S178A migrated about as slowly as the seven-fold phosphorylated wild-type protein and variant S181A . In contrast , the likewise six-fold phosphorylated variants T160A , S162A and S170 ( marked with asterisks in Fig 5B ) had distinctly higher mobilities . Hence beyond the number of phosphoryl groups also their sequence context contributes to the interaction with the Phos-tag . Knowledge of the S/T>A variants´ phosphorylation status also enabled a more detailed characterization of the epitope requirements of mAb T2212 ( see below ) . We then evaluated the impact of the S/T>A mutations on CLP RNA content by the NAGE based SG2/SR staining assay ( Fig 5C , S8 Fig ) but using a more elaborate protocol ( Material and Methods ) . Owing to the lower background signals the relative RNA contents of HBc140 , HBc149 and SRPK1-coexpressed HBc183 as well as HBc183_S181A CLPs were now estimated to ≤10% of that in unmodified HBc183 CLPs ( Fig 5C ) . The mostly three-fold phosphorylation by PKA and PKC caused only minor reductions ( significant only for PKC ) . Remarkably , preventing phosphorylation at one of the seven SRPK1 sites was sufficient to increase RNA contents to generally ~20–30% of non-phosphorylated HBc183 CLPs; additional phosphorylation site mutations further increased RNA content ( S8 Fig ) . Intriguingly , the single S170A mutation restored RNA content to ~50% of unmodified wild-type HBc183 CLPs , and this difference to the other single variants was significant ( p<0 . 001; n = 6 ) . Hence phosphorylation of S170 may be particularly effective in suppressing RNA binding by the HBc CTD . HBc183 CLPs are sensitive against SDS [9 , 65]; hence titration with SDS might reveal whether high-level phosphorylation and/or the concomitant low RNA content affect CLP stability . To this end we incubated HBc183 CLPs and HBc183_F97L CLPs expressed in the absence vs . presence of SRPK1 with either 1x TAE electrophoresis buffer ( Ø ) , or a native glycerol-based DNA loading buffer ( Ø* ) , or increasing amounts of a 6x DNA loading buffer ( NEB Purple ) containing 0 . 48% SDS ( 0 . 08% at 1x concentration ) . RNA and protein were monitored by EB and CB staining; expectedly , SRPK1-coexpressed CLPs showed only faint EB versus CB signals ( Fig 6 , right panels ) . At 0 . 024% SDS the nonphosphorylated CLPs released some of the RNA ( faster migrating EB smear ) although the major band remained at the original position . At 0 . 048% SDS , the fast RNA smear increased , while the original CLP band disappeared in favor of a slower , less intense band from which EB signals emanated upwards to the cathode; still higher SDS concentrations enhanced these effects . CB staining of the latter bands suggests they may represent complexes of HBc183 proteins with exposed CTDs ( forced to move towards the cathode ) to which some of the initially encapsidated RNA is bound . In accord with previous data [65] no clear difference was detectable for wild-type vs . F97L CLPs . Interestingly the SRPK1-phosphorylated low RNA CLPs also showed a sharp mobility transition between 0 . 024% and 0 . 048% SDS , although the new products migrated a bit slower than those from the nonphosphorylated CLPs . In sum , SDS sensitivity of the HBc CLPs was neither affected by seven-fold phosphorylation of the CTD , nor by RNA content or the F97L mutation . The high arginine content makes the entire HBc CTD an excellent substrate for trypsin; however , protease accessibility is affected by the CLP structure [37 , 65 , 83] . In our hands , trypsin had converted part of the unmodified wild-type HBc183 into a major product with HBc149-like mobility [83] . To reveal potential alterations of this pattern by phosphorylation , RNA content and/or the F97L mutation , we monitored the kinetics of trypsin action on non-phosphorylated vs . phosphorylated CLPs from wild-type and F97L HBc183 protein . First we addressed a potential inhibition of trypsin cleavage by CTD phosphorylation by comparing the trypsin sensitivity of the non-assembling NHis-GFP-TEV-CTD fusion protein ( see Fig 4A ) expressed in the absence vs . presence of SRPK1 . Both proteins were completely cleaved with similar kinetics ( Fig 7A , left panel ) . Under the same conditions , HBc183 in non-phosphorylated CLPs was only partially cleaved within 30 min ( Fig 7A , right panel ) , with about half the molecules remaining intact and the other half displaying HBc149-like mobility; this pattern remained stable over 2 h . Higher temporal resolution ( Fig 7B ) revealed the transitory formation of intermediate mobility bands which had mostly disappeared at 30 min in favor of the stable HBc149-like product ( labeled "4" ) ; thereafter the ratio between presumably intact HBc183 ( labeled "1" ) and product "4" remained constant for at least 4 h . NAGE did not reveal significant alterations in EB vs . CB staining over time ( Fig 7B , lower panels ) . Hence loss of half the CTDs affected neither RNA content nor surface charge , indicating the overall CLP structure remained intact . Comparable results with HBc183_F97L CLPs ( Fig 7C ) ruled out major effects of the F97L mutation . In contrast , SRPK1-coexpressed HBc183 CLPs ( Fig 7D , top panel ) presented two additional discrete , stable products ( "2" and "3" ) of which product 3 eventually reached similar levels as the remaining full-length protein . By comparison with HBc marker proteins ( Fig 7D , lower panel ) products 3 and 2 resulted from cleavage between positions 157–159 and 166–172 , respectively . HBc_F97L CLPs produced a very similar four-band pattern in SDS-PAGE ( Fig 7E , top panel ) ; in NAGE CLP mobility and RNA content again remained unaltered ( Fig 7E , bottom panels ) . While quantitation of the band 1 to 4 proportions could be disturbed by the presence of not fully intact CLPs , formation of bands 2 and 3 exclusively from the phospho-CLPs strongly implies distinct steric constraints for the band 2 and 3 processing sites compared to unmodified CLPs . Scenarios compatible with the data ( complete CTD extrusion vs . looping out of CTD parts ) are shown in S9 Fig . To directly assess the impact of seven-fold CTD phosphorylation and/or low RNA content on capsid structure we analyzed nonphosphorylated vs . SRPK1-phosphorylated HBc183 CLPs by cryoEM ( S10A and S10 Fig ) and calculated image reconstructions ( Fig 8 ) at 7 . 8 Å ( B = 707 Å2 ) and 7 . 9 Å ( B = 979 Å2 ) resolution ( S10C Fig ) . Most striking was a distinct shell of internal density exclusively in the non-phosphorylated particles ( Fig 8B ) which must represent CTDs plus packaged RNA . The relatively high B-factors suggested some structural heterogeneity within each CLP class , hence the gross features in the shells of unmodified vs . phosphorylated particles appeared very similar . However , in defined areas significantly different grey value distributions occurred ( Fig 8C; see S2 Protocol for significance calculations ) . For the SRPK1 coexpressed CLPs they could be best described as reduced order in the N terminal regions which flank the spikes ( Fig 8C bottom vs . top: less red color around the three-fold axes ) , plus an apparent stretching of the spike helices and the inter-dimer contact regions ( Fig 8C bottom vs . top: more blue color within the dimer contours ) . To improve resolution for fitting these differences into atomic models of HBc we vitrified additional aliquots of the same sample preparations which , fortuitously , had been stored for 1 . 5 years at 4°C . Corroborating the high stability of HBc CLPs , NAGE , SDS-PAGE and Phos-tag SDS-PAGE revealed no signs of degradation or loss of phosphorylation . Surprisingly , however , by 3D-classification the majority of these "aged" CLPs , especially the phosphorylated ones , grouped into a distinct class from the bulk of fresh particles ( vitrified within two weeks post preparation ) used above . The aged particle reconstructions had higher resolution and lower B-factors ( 6 . 3 Å and 6 . 6 Å , and 555 Å2 and 466 Å2 , respectively , for non-phosphorylated vs . SRPK1-phosphorylated CLPs ) , in line with increased ordering with time . For the non-phosphorylated CLPs the differences were only minor but for the phospho-HBc particles they were pronounced . As in the comparison with fresh unmodified CLPs ( Fig 8C ) the freshly vitrified phospho-CLPs showed extended spikes and less ordered N termini ( S11A Fig , top ) whereas after ageing they appeared very similar to the non-phosphorylated ones ( S11A Fig , bottom ) . This suggests a conformational maturation process , although not necessarily in the classic sense of gaining envelopment competence . Fitting the HBc149-derived crystal structure 1QGT [16] into these reconstructions accounted for most density in the assembly domains but left unaccounted internal density close to the inner CLP surface , likely related to CTDs plus RNA in the non-phosphorylated CLPs yet only CTDs in the phospho-CLPs . The undisturbed view onto the inner shell surface of the latter particles revealed extensive tube-like structures around the 5-fold symmetry axes ( Fig 8D , bottom vs . top ) . Connecting to the last visible residues in the crystal structure they probably represent residues from the linker plus the CTD . The respective density extended towards the feet of the spikes , with apparently direct contacts to R112 in both fresh and matured CLPs ( S11B Fig ) . Their visibility implies rather stable interactions , perhaps including electrostatic binding between R112 and phosphorylated CTD residues in the phospho-CLPs ( S9C and S9D Fig ) . Similar though less prominent extra density was also seen in the non-phosphorylated CLPs , although their high RNA content makes a clear distinction between nucleic acid and protein residues difficult ( S11B Fig ) . Recent data suggest that most HBc in mammalian cells from replicating as well as non-replicating vectors is phosphorylated [13 , 52] yet the number of phospho-sites per HBc monomer is unknown . We therefore analyzed HBc in intracellular capsids from the stable HBV producing TetOFF HepG2 . 117 line [84] by Phos-tag SDS-PAGE , using our recombinant phospho-HBc183 proteins as markers . To minimize band distortions seen with crude cytoplasmic lysate we first enriched capsids by sedimentation through 10–60% ( w/v ) Nycodenz gradients ( Fig 9A; S12A Fig ) . The presence of HBV-typical replicative DNA intermediates was corroborated by Southern blotting ( Fig 9B ) ; however , less vs . more mature species were barely separated and the presence of empty particles [54] remains to be addressed . Regardless of this , Phos-tag SDS-PAGE showed a comparably strong retardation of the bulk HBc signals as for SRPK1-phosphorylated recombinant HBc183 ( Fig 9C , short exposure ) . Longer exposure revealed several very weak intermediate mobility bands in the HepG2 . 117 samples , similar to those seen for CLPs coexpressed with PKA and PKC , and for non-phosphorylated CLPs; however , >95% of the band intensity was concentrated in the slowest migrating band . Though this would be in line with an SRPK1-like phosphorylation further investigation using the phospho-CTD specific mAb T2212 [51] indicated a non-identical phosphorylation pattern . As reported mAb T2212 did not recognize unmodified recombinant HBc183 , yet it reacted strongly with the PKA- and some of the PKC-phosphorylated HBc species . Interestingly , fully SRPK1-phosphorylated HBc gave almost no signal whereas single alanine replacements of the phospho acceptor sites S162 , S168 and , weakly , S176 and S178 but not S170 restored reactivity ( S12 Fig ) . Hence the T2212 epitope appears to depend on the presence of phosphoryl groups at some positions , including S170 , yet their absence from nearby sites . To assess whether mAb T2212-reactive phospho-HBc species are present in enveloped particles we separated extracellular particles from HepG2 . 117 cells and from a new HBc183-only expressing Huh7 line by NAGE; recombinant HBc183 CLPs served as control ( Fig 9D ) . Immunoblotting revealed a fast migrating band in the human cell-derived samples which corresponded to naked capsids , as indicated by comigration with bacterial HBc183 CLPs; as before , the latter did not react with mAb T2212 but were detected by the anti-assembly domain mAb 312 ( marked by ** ) . Importantly , mAb T2212 revealed a second , slower migrating band exclusively in the HepG2 . 117 samples which also stained with the anti-HBs mAb 9H9 [85] . Hence capsids containing phosphorylated HBc can be enveloped . More detailed conclusions will require separation from the naked capsids and efficient fractionation of enveloped capsids with differing nucleic acid content .
Our MS data confirmed the absence from E . coli BL21 cells of enzymes capable of phosphorylating any residue in HBc183 ( Figs 3 and 4; S5 and S6 Figs ) . Hence all previously reported properties of E . coli derived HBc CLPs relate to the fully unphosphorylated state . Vice versa , co-expressing a eukaryotic kinase then allows to single out how that specific kinase acts on HBc , as shown here for SRPK1 and per proof-of-principle for PKA and PKC . There are still caveats for interpretation , including the concentration and ratio of kinase to HBc substrate in the bacteria , plus the potential sequestration of CTD-embedded target sites in the capsid interior . ATP is present in E . coli at concentrations of ≥1 mM [86] and thus not limiting . However , a low translation rate of a kinase compared to HBc , or poor solubility and/or low affinity for HBc could all affect phosphorylation efficiency . The higher levels of soluble SRPK1ΔNS1 ( S3 and S4 Figs ) likely explain the homogeneous seven-fold phosphorylation found here compared to the mixed phospho-HBc species seen upon coexpressing full-length SRPK1 [11] . An impact of target sequestration on phosphorylation efficiency is supported by the MS data for PKA coexpression which indicated predominantly three-fold phosphorylation of self-assembling HBc183 ( S7 Fig ) , yet four- and five-fold phosphorylation of the non-assembling GFP-CTD protein ( S6 Fig ) . For SRPK1 the seven identified phosphorylation sites likely represent the maximum number; notably they include T160 ( Fig 5 ) although SRPK1 is mainly considered as a serine kinase [72 , 87] . Furthermore , the apparent stability of bacterial phospho-HBc in the absence of Phos-Stop inhibitor suggests the absence of phosphatases able to revert the mammalian-type phosphorylations . A compromised substrate specificity in the E . coli system is unlikely because none of the eleven S and twelve T residues upstream of the CTD showed any indication of phosphorylation when HBc140 or HBc149 were coexpressed with SRPK1 ( S7 Fig ) . In sum , these data advocate a broad applicability of our recombinant phospho-HBc system to reveal the impact of individual kinases ( and with adaptations , of phosphatases ) on HBc . Different from conventional SDS-PAGE Phos-tag SDS-PAGE resolved HBc species containing from none to five or six phosphoryl groups . While higher phosphorylation generally correlated with stronger retardation , discrimination amongst highly phosphorylated species was less clear , also owing to sequence context specific effects . ( Fig 5A and 5B ) . It will be interesting to see whether variants with unusual Phos-tag mobility shifts also display specific biological features . For instance , two of the three single-site mutations causing lower than average retardation , S162A and S170A ( Fig 5C ) , had the strongest negative impact on pgRNA encapsidation [47 , 48 , 88] . Coexpression with SRPK1 of additional HBc183 S/T>A mutants would provide a unique , comprehensive panel of reference proteins for more systematic investigations . Uniform seven-fold phosphorylation by SRPK1 enabled to correlate a defined phospho-status with basic properties of HBc . Most striking was the drastic reduction in CLP RNA content ( Figs 2 , 5 , 6 and 7 ) . This has not been seen with partly phosphorylated HBc or with phosphorylation mimicking S/T>D , E variants . For instance , a variant termed 7E with seven S/T>E replacements in the CTD , expressed in HEK293 cells , reportedly contained still half as much RNA as nonphosphorylated bacterial CLPs [89]; similar results were seen for an all-S>D variant ( S7D ) expressed in E . coli [13] . Likely , the much stronger impairment of RNA binding by seven phosphoryl groups versus seven carboxyl groups is owed to their different chemistries . Seryl- and threonyl-phosphomonoesters are dibasic acids . With pKa values of 1 . 2 and 6 . 5 [90] even the second phosphoryl hydroxy group will be mostly deprotonated at physiological pH; hence each phosphoryl group can contribute nearly two negative charges . Seven-fold CTD phosphorylation would thus suffice to largely neutralize the 15 net positive charges ( Fig 1 ) , minimize the CTD´s general RNA binding capacity , and explain the low RNA content of the resulting CLPs . This interpretation is supported by inverse experiments in eukaryotic settings where complete inhibition of CTD phosphorylation by S/T>A mutations led to the formation of non-specifically RNA-filled rather than empty HBc CLPs [13 , 89] . Practically , low RNA CLPs from SRPK1 coexpression in E . coli of full-length HBc variants bearing heterologous sequences could be of interest for vaccine applications [74] . Seven-fold phosphorylation and low RNA content had little impact on CLP stability against SDS ( Fig 6 ) whereas trypsin treatment revealed specific phosphorylation and/or RNA content-dependent differences; their independence from the F97L mutation suggests that the structural changes causing the premature virion secretion phenotype of the F97L variant are either very subtle or not sufficiently long-lived to be observed [91] , as corroborated in a recent high resolution cryoEM study [92] . Non-phosphorylated CLPs yielded one major HBc149-like product ( "band 4" in Fig 7B and 7C ) , whereas the SRPK1-phosphorylated CLPs gave two additional , stable intermediates ( Fig 7C and 7D; S9 Fig ) . These data are reminiscent of but also distinct from artificially RNA-depleted HBc-EEE vs . WT CLPs [37]; there , a band similar in size to our band 3 also accumulated over time , however , in both wild-type and HBc-EEE CLPst . The more pronounced differences in our experiments may relate to the higher number of modifications per CTD plus the chemical differences outlined above . Two models that could explain formation of distinct trypsin processing products ( S9C and S9D Fig ) would invoke either complete extrusion of some CTDs , or the looping-out of selective trypsin target sites with the very C terminal CTD residues still internally disposed . In the phospho-HBc CLPs this could be mediated by electrostatic interactions between the phosphoryl groups and positively charged side-chains in the CTDs and/or on the inner CLP lining , as proposed for HBc-EEE CLPs [37] . Maintained integrity and RNA content of the trypsin-treated CLPs ( Fig 7B , 7C and 7E ) appear in better accord with the looping-out model . The current cryoEM reconstructions did not resolve this issue but they revealed distinct phosphorylation-dependent differences . The lack in SRPK1-coexpressed HBc183 CLPs of internal RNA density ( Fig 8B ) enabled allocating the remaining extra density to the linker plus CTD residues which formed tube-like structures around the five-folds , in apparent contact with residues lining the capsid lumen , i . e . R112 ( S11B Fig ) . Freshly vitrified phospho-CLPs showed well detectable local differences in their assembly domains compared to unmodified CLPs which largely disappeared upon long-term storage . In particular the very N termini adopted a more ordered conformation , as in both fresh and aged unmodified CLPs . A potential physiological relevance of this slow rearrangement in the phospho-CLPs remains to be determined . Notably , though , replacement of the N-proximal P5 lowered virus secretion [93] and P5 is close in space to residues 95–97 implied in interactions with L protein [94] . The bulk of HBc183 in capsids from human cells was strongly retarded in Phos-tag SDS-PAGE ( Fig 9C , S12B Fig ) , in line with an independent report [13] . Comparison with the recombinant phospho-HBc proteins indicated an SRPK1-like seven-fold phosphorylation status , in line with a physiological role of SRPK1 as a HBc kinase [34 , 43 , 71] . However , Phos-tag retardation alone would be also compatible with a slightly lower or higher phosphorylation extent ( Fig 5 ) . Also , the strong reactivity of mAb T2212 with the human cell-derived but not the recombinant phospho-HBc ( S12 Fig ) indicates at least one phosphorylation-dependent difference . If SRPK1 was indeed the main HBc kinase , ( a ) compatible phosphorylation pattern ( s ) could arise from one or few selective dephosphorylation events . Alternatively , however , the combined action of other kinases ( and phosphatases ) could lead to a similar pattern . Notably , mAb T2212 detected phosphorylated HBc in enveloped particles ( Fig 9 ) , as proposed for empty virions [52] . Further exploration using our new tools , with more efficient separation of the various HBV particles [54] , will help to accurately ascribe specific phosphorylation patterns to individual particle types . At any rate does the high phosphorylation level of most HBc183 in human cells imply a similar suppression of general RNA binding capacity as in recombinant SRPK1 phosphorylated HBc183 . As outlined in Fig 10 , in favor of viral replication this would counteract encapsidation of irrelevant RNAs [13 , 89] yet also impair pgRNA packaging , giving empty capsids and empty virions . This dilemma could plausibly be resolved by partial dephosphorylation , possibly at just one site ( Fig 5C ) . Most effective would be a phosphatase activity associated with the pgRNA—P protein complex , providing spatio-temporal control of the dephosphorylation event ( s ) ; HBc dephosphorylation coincident with pgRNA packaging has indeed very recently been suggested [95] . HBc dimers joining the nascent capsid , but not bulk HBc , could then as well become partly dephosphorylated , enabling stable packaging of the entire 3 . 5 kb pgRNA . With progressing dsDNA synthesis the co-packaged phosphatase activity could gradually release more CTD phosphoryl groups until this electrostatic buffer is emptied [13 , 26 , 75] . One might further speculate that timely envelopment blocks the supply of dNTPs into the capsid before the ( + ) -DNA gap is completely filled , and that this represents the most stable state for a DNA containing nucleocapsid ready to leave the cell as virion . Conversely , after infection of a new cell continued DNA synthesis plus re-phosphorylation might create an excess of negative charge and destabilize the capsid in preparation for uncoating . More work will be needed to substantiate this model , but the concept as such lets previous statements on the importance of HBc phosphorylation for HBV replication appear oversimplified . Rather than directly promoting pgRNA encapsidation HBc phosphorylation seems to act indirectly by blocking competing interactions with non-specific RNA . In turn , with high-level HBc phosphorylation as default , dephoshorylation becomes as important for viral replication as phosphorylation . Hence production of replication-competent HBV nucleocapsids appears to depend on an intricately balanced level of HBc phosphorylation . The excess of empty over genome-containing HB virions [52 , 60] indicates that proper execution of this program is delicate; hence even small perturbations might have severe consequences , inviting therapeutic exploitation , e . g . by kinase [96] or phosphatase inhibitors . However , such strategies will require a deeper understanding of the involved host enzymes and the substrate properties of HBc for which our study provides valuable new tools . On the basic side , these tools can feasibly be adapted to other viruses , including but not limited to relatives of HBV [97] . Preliminary data indicate , for instance , that the core protein of DHBV , a virus capable of replicating in human cells [98] , is also highly phosphorylated ( at eight yet to be mapped sites ) by human SRPK1 in the recombinant coexpression system whereas the core protein of the fish Cichlid nackedna virus ( CNDV; [97] ) is not . Compatibility with the cellular kinases and phosphatases may thus well be a determinant of viral host and tissue tropism .
HBc expression vector pET28a2-HBc183 [69] carries a T7 promoter controlled synthetic HBc gene [68] for a genotype D ( GenBank: V01460 . 1 ) core protein . The HBc183opt ORF used here encodes the same aa sequence but was adapted to E . coli codon usage ( GeneOptimizer software; ThermoFisher/GeneArt ) , increasing CLP yields ~3-fold ( S1 Fig ) . The new pRSF expression vectors are based on pRSFDuet-1 ( Novagen ) which harbors two T7 promoter cassettes and the lacI repressor gene for IPTG inducible coexpression of two ORFs . For HBc mono-expression the HBc183opt ORF and its derivatives were usually inserted after the 5´ T7 promoter , with simultaneous deletion of the 3´ T7 promoter cassette ( plasmids pRSF_T7-HBcNNNopt; NNN specifies the last HBc aa present ) . For coexpression , the upstream T7 promoter was replaced by a Tet promoter , and in addition a gene for the Tet repressor was inserted into the plasmid ( S2A Fig ) for separate inducibility by anhydrotetracycline ( AHT ) . Kinase ORFs were inserted under Tet promoter control , HBc ORFs under T7 promoter control ( plasmids pRSF_Tet-X_T7-HBcNNNopt , where X denotes the ORF under Tet promoter control ) . Employed kinase ORFs encoded an N-terminally His6-tagged version of SRPK1ΔNS1 [72]; the catalytic subunit alpha isoform 1 ( aa 1–351 ) of PKA ( NP_032880 . 1 ) ; and the C-terminal catalytic domain ( aa 397–740 ) of human PKC theta isoform X1 ( XP_005252553 . 1 ) . The latter two ORFs had previously been expressed in bacteria [79 , 80] and here were obtained as E . coli expression optimized DNA strings ( ThermoFisher/GeneArt ) . In the non-assembling CTD control constructs the HBc aa 1–145 part was replaced by the ORF for N-terminally His6-tagged eGFP [99] followed by a Gly2 linker and a recognition site for TEV protease ( see Fig 4A ) . Cloning was done by conventional restriction-based methods or using the Q5 mutagenesis kit ( NEB ) . All constructs were verified by Sanger sequencing . Expression of HBc CLPs followed previously described procedures [99 , 100] , as detailed in reference [101] . In brief , E . coli BL21*CP served as expression host . T7 promoter and Tet promoter driven expression from the pRSF plasmids were induced by IPTG ( 1 mM final concentration ) and/or 0 . 2 μg/ml AHT , respectively; cultures ( usually 200 ml ) were then shaken for 12–16 h at 20–25°C . Cell lysis included treatment with lysozyme , Triton X-100 and benzonase ( Merck-Millipore ) in the presence of protease-inhibitor cocktail ( Roche ) with subsequent sonication; in kinase coexpressions , Phos-Stop phosphatase inhibitor ( Roche ) was included . Cleared cell lysates were subjected to sedimentation ( TST41 . 14 rotor; 41 , 000 rpm for 2 h at 20°C ) through 10%-60% sucrose step gradients in TN150 buffer ( 25 mM Tris/HCl , 150 mM NaCl , pH 7 . 4 ) . For longer term storage at -80°C , peak fractions were dialysed into storage buffer ( 50 mM Tris/HCl pH 7 . 5 , 5 mM EDTA , 5% ( w/v ) sucrose , 2 mM DTT ) . Isolated CTD peptides from TEV-cleavable GFP fusions were obtained as detailed in S1 Protocol . NAGE was performed in 1% agarose gels in 1x TAE buffer ( 40 mM Tris , 20 mM acetic acid , 1 mM EDTA ) as previously described [9 , 23 , 99] . For routine detection of nucleic acids gels contained 0 . 5 μg/ml ethidium bromide ( EB ) ; subsequent protein staining was done using Coomassie Brilliant Blue R250 ( CB ) followed by extensive destaining in fixing buffer ( 50% MeOH , 7% acetic acid , v/v in H2O ) . Alternatively , gels run without EB were stained for RNA using 1x Sybr Green 2 ( SG2 ) RNA stain in TAE buffer ( from 10 , 000x in DMSO; FMC Bioproducts ) , followed by protein staining with ready-to-use Sypro Ruby ( SR ) Protein Gel Stain ( BioRad ) . Fluorescence signals were recorded using a laser Scanner ( Typhoon FLA 7000; GE Healthcare ) set at excitation 473 nm / filter Y520 nm ( SG2 ) and 473 nm / filter O580 nm ( SR ) . Signals were quantified using ImageQuant software . Higher quantitation accuracy was achieved by several adapations of the protocol , as described next . Critical issues for robust signal quantitation included weak SG2 staining ( especially for low RNA content CLPs ) , concentration-dependent variations in SR signal intensities , SG2 to SR signal carry-over during Laser scanning , and high background staining . Implemented countermeasures were to use gels no thicker than 5–6 mm; loading similar protein amounts of different CLPs and possibly two different amounts of the same CLPs; and including on each gel a well-characterized wild-type CLP preparation as standard to account for variations in staining intensity . SG2 staining over background was improved by doubling the dye concentration ( 1:5 , 000 dilution ) , a 1 h staining period , plus ≥3 washes with TAE buffer prior to SG2 laser scanning . For improved SR staining gels were washed twice in fixing buffer , after which the SG2 staining was no longer detectable; fixation also prevented band broadening by diffusion during the subsequent overnight incubation in SR protein gel stain . After two additional washes in distilled water SR fluorescence was recorded as described above . For each gel , the ratio of SG2: SR fluorescence intensity in the wild-type HBc183 standard was set to 100% to which the respective intensity ratios from the test CLPs were normalized . Applying this procedure reduced the calculated relative RNA content of CTD-less and SRPK1-phosphorylated CLPs from >25% of that of unmodified HBc183 CLPs ( Fig 2C ) to ≤10% ( Fig 5C , S8 Fig ) . RNA content was calculated from CLP sample absorbances at 260 , 280 , 340 and 360 nm [73] . For high purity CLP preparations , e . g . HBc183 CLPs from two sequential sucrose gradients [101] , ten-fold dilutions in H2O were measured in 1 cm path length cuvettes in an Ultrospec 7000 instrument ( GE Healthcare ) . For single gradient CLP preparations which may contain free RNA [101] aliquots from the respective fractions ( routinely containing 0 . 5–4 mg/ml of HBc protein ) were incubated with 1/100 ( w/w ) of RNase A for 30 min at room temperature , then dialyzed ( 30 kDa MWCO ) against TN 150 buffer . UV/VIS absorbances of the resulting solutions were then monitored without further dilution in a QIAxpert instrument operating with 0 . 1 cm path length ( 2 μl volume ) microcuvettes . Molar nucleotide per core protein monomer ( N/P ) ratios were calculated as described [73] . Mn2+-Phos-tag SDS-PAGE was performed by adding Phos-tag acrylamide AAL-107 ( Wako Pure Chemical Corporation ) plus MnCl2 to the acrylamide solutions for conventional Lämmli SDS-PAGE resolving gels , as recommended by the manufacturer . In our hands , the best separation of differently phosphorylated HBc183 species was obtained using 15% acrylamide gels supplemented with 75 μM Phos-tag acrylamide and 150 μM MnCl2 . Immunoblotting after NAGE or conventional SDS-PAGE was performed as previously described [69 , 102] . Low transfer efficiency to polyvinylidene difluoride ( PVDF ) membrane after Phos-tag SDS-PAGE gels was improved by sequentially soaking the gels for 10 min each in transfer buffer A ( 39 mM glycine , 48 mM Tris , 3 . 75% ( w/v ) SDS , 20% ( v/v ) MeOH ) containing 10 mM EDTA , 1 mM EDTA , and no EDTA , plus the use of a wet blot rather than a semi-dry transfer system . HBc specific antibodies employed were the anti-assembly domain mouse mAbs 312 [103] and 1D8 [82] , both recognizing a linear epitope exposed on intact CLPs and SDS-denatured HBc protein; the capsid-specific mAbs 275 [104] and 3120 [105] , obtained from Tokyo Future Style , Inc . ( catalogue no . : 2AHC22 ) ; and the anti-phospho-CTD mAb T2212 [51] , also from Tokyo Future Style ( catalogue no . : 2AHC23 ) . HBsAg on NAGE blots was detected by human mAb 9H9 [85] which recognizes a conformational epitope in S . Bound mAbs were visualized using horse raddish peroxidase ( PO ) , either as direct mAb conjugate or via appropriate secondary antibody-PO conjugates , and chemiluminescent substrates . About 5 μg of sucrose gradient enriched HBc CLPs were incubated for 30 min with increasing amounts of a 6x DNA loading buffer containing 0 . 48% SDS ( NEB Purple ) to yield final SDS concentrations from 0 . 024% to 0 . 24%; samples incubated in a non-denaturing 6x loading buffer served as control . Reaction products were monitored by NAGE in 1% gels; EB fluorescence signals were recorded using a laser scanner ( excitation 532 nm / filter O580 nm ) . Proteins were subsequently stained by CB . For partial trypsin digests the respective HBc CLPs were incubated with 1/100 ( w/w ) the amount of sequencing grade trypsin ( Promega ) in TN50 buffer ( 25 mM Tris/HCl , 50 mM NaCl , pH 7 . 5 ) at 30°C . At various time points aliquots were withdrawn and digestion was stopped by adding 4- ( 2-aminoethyl ) -benzenesulfonyl fluoride hydrochloride ( AEBSF; Applichem ) to a final concentration of 0 . 2 mM . Formation of cleavage products and intactness of particles were analyzed by SDS-PAGE and NAGE . MS analyses were performed at the Institut de Biologie et Chimie des Protéines , UMR5086 , CNRS/Université Lyon 1 , France on a Sciex Voyager DE-PRO MALDI-TOF instrument , using sucrose gradient enriched CLPs diluted 1:100 ( v/v ) in sinapic acid as matrix . Electron microscopy was carried out in the Edinburgh , UK , cryoEM facility . Detailed procedures for sample preparation , micrograph recording , particle selection , image processing , refinement , difference map calculation , and molecular fitting are given in S2 Protocol . In brief , gradient-enriched CLP samples were vitrifed as described [106] using a manual freezing apparatus with an environmental chamber [107] at room temperature . Before use , grids ( Quantifoil R1 . 3/1 . 2 ) were glow discharged in air for 1 min with a current of 30 μA using a Quorumtec sputter coater . Micrographs were recorded on a FEI Tecnai F20 microscope operated at 200 kV and a 8192 pixels x 8192 pixels CMOS camera ( TVIPS F816 ) . Only particles with round shape and crisp appearance were subsequently used for reconstructions . Resolution and B-factors were calculated with Relion [108] . Fitting was done using Chimera [109] , based on pdb 1QGT [16] . The human hepatoma cell line HepG2 and its TetOFF HBV derivative HepG2 . 117 [84] were cultured as described [84 , 110] . The stable , constitutively HBc183 producing cell line H4-15 was derived from the human hepatoma cell line Huh7 by CRISPR/Cas9-mediated homologous recombination of a HBc expression cassette into the AAVS1 locus ( Peter Zimmermann , MSc thesis; University of Freiburg , 2017 ) . Cytoplasmic HBV capsids were obtained from TetOFF HepG2 . 117 cells cultured for 10 days in the absence of doxycycline [84] . Cytoplasmic lysates were prepared using NP40 lysis buffer ( 50 mM Tris/HCl , 140 mM NaCl , pH 8 , with 0 . 5% ( v/v ) NP40 detergent ) supplemented with Phos-Stop . To enrich capsids , lysates were sedimented through a 10% - 60% ( w/v ) Nycodenz step gradient for 2 h at 4°C in a TST 41 . 14 rotor at 41 , 000 rpm ( 302 , 500 g ) and harvested in 14 fractions . Detection of HBV DNA in capsids and by Southern blotting was performed using 32P labeled HBV DNA probes as described [84 , 111] . Extracellular particles in the culture supernatants of HepG2 . 117 and H4-15 cells were enriched by precipitation with PEG8000 [84 , 112] . Unless indicated otherwise data are expressed as mean ± standard deviation ( SD ) from ≥3 experiments . Comparisons between multiple groups were performed using One-way ANOVA and Tukey´s post test ( Graphpad Prism 5 ) . Differences between means of two paired groups were evaluated using Student's t-test . P-values of p<0 . 05 were regarded as statistically significant . | The liver-pathogenic hepatitis B virus ( HBV ) is a small enveloped DNA virus that replicates through reverse transcription of a pregenomic ( pg ) RNA . This requires specific encapsidation of pgRNA and viral polymerase into a shell of 240 core protein ( HBc ) subunits . Capsid-internal formation of relaxed circular ( RC ) DNA enables the particle to leave the cell as stable virion; yet , when infecting a new cell it must release the RC-DNA for conversion into another , plasmid-like DNA that templates new viral RNAs . This up and down in nucleic acid interactions is presumably regulated by transient phosphorylation of HBc , mainly in its arginine-rich C terminal domain ( CTD ) which displays strong non-sequence-specific RNA binding . However , neither the phosphorylation sites nor the relevant enzymes are well defined . We developed a recombinant system to produce kinase-specific phospho-HBc species , and adapted a feasible gel assay for their separation . By mutagenesis and mass spectrometry we identified seven target sites for a major candidate kinase , SRPK1 , in the CTD . As full SRPK1 phosphorylation thwarted non-specific RNA binding the comparably high phosphorylation of HBc in human cells suggests how specific pgRNA encapsidation might be achieved . Our new tool set will facilitate disentangling the role of HBc phosphorylation in HBV infection and exploiting it as potential therapeutic target . | [
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] | 2018 | Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure |
Herpes simplex virus ( HSV ) types 1 and 2 are highly prevalent human neurotropic pathogens that cause a variety of diseases , including lethal encephalitis . The relationship between HSV and the host immune system is one of the main determinants of the infection outcome . Chemokines play relevant roles in antiviral response and immunopathology , but the modulation of chemokine function by HSV is not well understood . We have addressed the modulation of chemokine function mediated by HSV . By using surface plasmon resonance and crosslinking assays we show that secreted glycoprotein G ( SgG ) from both HSV-1 and HSV-2 binds chemokines with high affinity . Chemokine binding activity was also observed in the supernatant of HSV-2 infected cells and in the plasma membrane of cells infected with HSV-1 wild type but not with a gG deficient HSV-1 mutant . Cell-binding and competition experiments indicate that the interaction takes place through the glycosaminoglycan-binding domain of the chemokine . The functional relevance of the interaction was determined both in vitro , by performing transwell assays , time-lapse microscopy , and signal transduction experiments; and in vivo , using the air pouch model of inflammation . Interestingly , and in contrast to what has been observed for previously described viral chemokine binding proteins , HSV SgGs do not inhibit chemokine function . On the contrary , HSV SgGs enhance chemotaxis both in vitro and in vivo through increasing directionality , potency and receptor signaling . This is the first report , to our knowledge , of a viral chemokine binding protein from a human pathogen that increases chemokine function and points towards a previously undescribed strategy of immune modulation mediated by viruses .
Herpes simplex virus type 1 and 2 ( HSV-1 and HSV-2 , respectively ) and varizella zoster virus ( VZV ) are the three human members of the Alphaherpesvirinae subfamily , which establish latency in the sensory ganglia of the peripheral nervous system . Both HSV-1 and -2 are highly prevalent viruses with values around 90% for HSV-1 and 12–20% for HSV-2 in adult populations of industrialized countries , reaching up to 80% for HSV-2 in developing countries [1] , [2] . Infection by HSV can be either asymptomatic , show mild symptoms in localized tissues or cause severe diseases such as stromal keratitis or herpes simplex encephalitis ( HSE ) , with high mortality and neurologic morbidity [3] . HSV infection of neonates can result in disseminated disease including infection of the central nervous system or involve several organs with mortality reaching 80% [4] . The causes of such different outcomes following HSV infection or reactivation are unknown but involve the interplay between the virus and the immune response . Chemokines are essential elements of the antiviral response . They constitute a family of chemotactic cytokines that orchestrate leukocyte migration to sites of injury or infection [5] . Chemokines also play relevant roles in the developing and mature nervous system [6] . The chemokine network contains more than 45 chemokines and around 20 G-protein coupled receptors ( GPCR ) . There are 4 subfamilies of chemokines classified on C , CC , CXC and CX3C . All chemokines are secreted . CXCL16 and CX3CL1 are also present as membrane-anchored forms . The chemokine network is complex , highly regulated and promiscuous , with some receptors interacting with more than one chemokine and some chemokines binding to more than one receptor . Alterations in the chemokine network are responsible for inflammatory , autoimmune diseases and the establishment of chronic pain [7] , [8] . Binding of chemokine to glycosaminoglycans ( GAGs ) is relevant for chemokine function . GAGs promote chemokine oligomerization , mediate retention of chemokines onto the cell surface allowing chemokine recruitment in tissues , increase their local concentration in the microenvironment surrounding the GPCR , and modulate receptor recognition [9] . Interaction of the chemokine with the GPCR triggers a signal cascade that includes stimulation of mitogen activated protein kinases ( MAPKs ) such as Janus-N-terminal kinase 1 and 2 ( JNK1-2 ) , extracellular signal-regulated kinase 1-2 ( ERK1/2 ) and p38 [10] . The proper function of chemokines is essential to trigger an appropriate and effective antiviral response . An exacerbated immune response , often triggered or maintained by chemokines , may lead to immunopathology . Patients suffering from HSE present higher level of chemokine expression in the cerebrospinal fluid than healthy individuals suggesting a relevant role for chemokines in the pathogenesis of HSE [11] . Both pox- and herpesviruses express proteins that interfere with chemokine function playing relevant roles in viral cycle , immune evasion and pathogenesis [12] . One of the strategies of chemokine interference involves the expression of secreted viral proteins that bind chemokines and inhibit chemokine function [13] . These proteins have been termed viral chemokine binding proteins ( vCKBP ) . They lack amino acid sequence similarities among themselves or with host chemokine receptors , making difficult the detection of such proteins by sequence analysis . We , and others , have previously shown that secreted glycoprotein G ( gG ) from some non-human alphaherpesviruses binds to chemokines and inhibits chemokine function . Examples of such viruses are bovine herpesvirus 5 ( BHV-5 ) , equine herpesvirus 1 and 3 ( EHV-1 and EHV-3 ) [14] , [15] , pseudorabies virus ( PRV ) [16] and infectious laryngotracheitis virus [17] . Chemokine-binding activity was not observed when supernatants of cells infected with the human viruses VZV , HSV-1 and HSV-2 were tested using different radio-iodinated chemokines [14] . In the case of VZV , the gene encoding for gG is not present within its genome . However , both HSV-1 and HSV-2 contain the open reading frame us4 encoding gG . HSV-1 and HSV-2 gG ( gG1 and gG2 , respectively ) are present on the viral particle and on the plasma membrane of infected cells [18]–[20] . gG2 is further processed and an N-terminal fragment is secreted to the medium of the infected cells [19] , [20] . On the contrary , gG1 is not secreted , similarly to the rest of HSV glycoproteins . The functions of HSV-1 and HSV-2 gGs are not well understood . Two reports point to a role of the HSV gGs in the initial steps of entry . HSV-1 gG seems to be important for the infection of polarized epithelial cells [21] . The non-secreted portion of HSV-2 gG binds heparin and the cellular plasma membrane [22] . Deletion or disruption of us4 attenuates HSV-1 in vivo , indicating that gG is a virulence factor , although the mechanism ( s ) beneath such phenotype are unknown [23]–[25] . The main aim of this study was to investigate the modulation of the immune system by HSV . We focused initially on identifying the function of HSV gG and its possible interaction with chemokines . We show here that secreted , soluble HSV gG ( SgG ) binds both CC and CXC chemokines with high affinity through the GAG-binding domain of the chemokine . Moreover , we could detect chemokine-binding activity in the plasma membrane of HSV-1 infected cells and in the supernatant of HSV-2 infected cells . Further experiments indicate that HSV-1 full-length gG and secreted , soluble HSV gG ( SgG ) are responsible for this activity . In complete contrast to all previously described vCKBPs , HSV-1 and HSV-2 SgGs are not inhibitors of chemokine function . Instead , they increase chemokine-mediated cell migration both in vitro and in vivo through a mechanism that involves GPCR signaling and phosphorylation of MAPKs . HSV SgGs increase the potency of the chemokine , and the directionality of cell movement . This constitutes , to our knowledge , the first description of a chemokine binding protein expressed by a human pathogen that potentiates chemokine function . The data presented here suggest the existence of a novel viral mechanism of immune modulation and provide tools to investigate the pathways controlling chemotaxis . Given the relevant roles played by chemokines in both the immune and nervous systems , enhancement of chemokine function by HSV gG may be important for HSV-mediated immunopathogenesis .
To test whether HSV gGs bind chemokines , we expressed soluble , secreted forms of gG1 and gG2 ( SgG1 and SgG2 , respectively ) , lacking the transmembrane and cytoplasmic domains , in insect cells infected with recombinant baculovirus vectors ( Figure 1A; Protocol S1; Text S1 ) . Following infection , SgG1 and SgG2 were purified from the supernatant of Hi-5 insect cell cultures by affinity chromatography and the purity of the preparation was determined by Coomassie staining ( Figure 1B ) . We routinely obtained two separate bands when SgG1 was expressed in insect cells , probably due to different levels of SgG1 glycosylation . A monoclonal antibody raised against gG1 [18] reacted with purified SgG1 but not SgG2 ( Figure 1C , middle panel ) whereas a monoclonal anti-SgG2 [26] recognized SgG2 only ( Figure 1C , right panel ) . The anti-His antibody reacted with both proteins ( Figure 1C , left panel ) . Both purified SgG1 and SgG2 were covalently coupled to BIAcore CM5 chips and tested for chemokine binding by Surface Plasmon Resonance ( SPR ) . A screening with 44 commercially available human ( h ) chemokines ( Protocol S2 ) was performed by injecting each chemokine in a BIAcore X biosensor . Both SgG1 and SgG2 bound with high affinity hCCL18 , hCCL25 , hCCL26 , hCCL28 , hCXCL9 , hCXCL10 , hCXCL11 , hCXCL12α , hCXCL12β , hCXCL13 and hCXCL14 , and SgG2 also bound hCCL22 with high affinity ( Figure 2A and Table 1 ) . As negative controls for chemokine binding we used the cysteine-rich domain ( CRD ) of ectromelia virus cytokine response modifier B ( CrmB ) , previously shown to lack chemokine-binding activity [27] ( not shown ) . The affinity constants of the interactions between SgG1 , SgG2 and the different chemokines were calculated using the SPR technology ( Table 1 ) . Both SgG1 and SgG2 interacted with chemokines with high affinity , in the nanomolar range . The interaction between HSV SgGs and chemokines was also observed by cross-linking assays ( Protocol S3; Text S1 ) using radio-iodinated recombinant hCCL25 , hCXCL10 , and hCXCL12α ( Figure 2B–D ) . As a negative control we employed CrmB-CRD ( Figure 2C ) . Competition assays with [125I]-hCXCL12α and increasing concentrations of cold hCXCL12α showed the specificity of SgG2-chemokine interaction ( Figure 2D ) . We addressed whether chemokine-binding activity was present in the HSV-1 infected cells . To this end we infected BHK-21 cells ( Protocol S4 and S5; Text S1 ) with HSV-1 wt and an HSV-1 virus where expression of gG had been disrupted by the insertion of the β-galactosidase gene [23] and determined binding of [125I]-hCXCL10 to the cells 14 to 16 hours post infection ( h . p . i . ) . We could detect chemokine binding to HSV-1 wt-infected cells ( Figure 3A; Protocol S5 ) . Binding was not observed when the deletion mutant HSV-1ΔgG was used . We also obtained supernatants from mock- or HSV-2 infected Vero cells 36 h . p . i . , and performed a crosslinking assay with [125I]-hCXCL12α . Two bands could be detected in the crosslinking assay ( Figure 3B ) that could correspond to the high mannose 72 kDa precursor and the 34 kDa secreted protein produced during gG2 expression and processing [19] , [20] . Another possibility is that the higher molecular weight band observed corresponds to an SgG2 dimer complexed with chemokine . To function properly , chemokines need to interact with both GAGs and GPCRs . We investigated the chemokine domain involved in the interaction with HSV SgGs using two experimental approaches . First , to address whether HSV SgGs could affect chemokine-receptor interaction , we performed binding assays of [125I]-hCXCL12α and [125I]-hCCL25 with MOLT-4 cells ( Protocol S4 and S6 ) expressing endogenous hCXCR4 ( the receptor for hCXCL12 ) and hCCR9 ( the receptor for hCCL25 ) in the presence of SgG-containing supernatant ( not shown ) . We also performed binding assays of [125I]-hCXCL12α to MonoMac-1 cells expressing endogenous hCXCR4 ( not shown ) . As a positive control , addition of supernatant containing BHV-5 SgG inhibited [125I]-hCXCL12α binding to MOLT-4 cells [14] ( not shown ) . However , similar amounts of SgG1 or SgG2 did not decrease [125I]-hCXCL12α binding to MOLT-4 cells , MonoMac-1 cells or [125I]-hCCL25 binding to MOLT-4 ( not shown ) compared to the mock sample . Thus , SgGs do not inhibit binding of the chemokines to their receptors . Second , to determine the implication of the GAG-binding domain of the chemokine in the interaction with HSV SgGs we utilized the SPR technology . The amount of chemokine binding to SgGs , covalently bound to a BIAcore chip , in the absence of heparin was considered 100% of binding ( Figure 4 ) . Competition experiments showed that increasing concentrations of heparin impaired chemokine binding to both SgG1 and SgG2 in a significant manner ( Figure 4 ) . As a control , each of the different heparin concentrations used were injected independently to confirm that no direct heparin binding to the chip occurred ( not shown ) . In summary , these results indicate that SgG1 and SgG2 interact preferentially with the GAG-binding domain of the chemokine and do not block the binding of chemokines to cell surface specific receptors . We , and others , have previously shown that gG encoded by several non-human alphaherpesviruses inhibits chemotaxis [14]–[17] , [28] . To examine the functional role of the interaction between HSV SgGs and chemokines we performed cell migration experiments . First we addressed whether the chemokine-binding activity observed in the supernatant of HSV-2 infected cells could have any effect on chemotaxis . We incubated CXCL12β with supernatant from mock- or HSV-2-infected cells and performed a chemotactic assay with MonoMac-1 cells ( monocyte-like ) , a cell line that expresses hCXCR4 , the receptor for hCXCL12 . The supernatant from HSV-2-infected cells significantly enhanced chemokine function in a dose dependent manner when compared to the supernatant from mock-infected cells ( Figure 5A ) . To address whether this effect could be due to SgG , we performed chemotactic experiments using several cell lines and recombinant protein . Incubation of SgG1 with hCXCL12β resulted in higher MOLT-4 migration ( Figure 5B ) . A similar result was obtained with SgG2 whereas BHV-5 SgG inhibited hCXCL12β migration ( not shown ) . We then incubated SgG1 and SgG2 with hCXCL13 and tested their effect on mouse B cells ( m300-19 ) stably transfected with hCXCR5 , the receptor for hCXCL13 ( Figure 5C , Protocol S4 ) . Inhibition of migration was observed with the vCKBP M3 , as expected [29] , [30] ( Figure 5C ) . However , SgG1 and SgG2 required the presence of the chemokine and were not able to induce chemotaxis on their own ( Figure 5C ) . The parental m300-19 cells , which do not express hCXCR5 , did not respond to the hCXCL13 stimulus ( not shown ) . To test whether binding to the chemokine was necessary for the enhancing effect , we performed chemotaxis experiments using MonoMac-1 , a cell line expressing hCXCR4 and hCCR2 , the receptor for hCCL2 , a chemokine not bound by HSV SgGs ( Figure 2 and Table 1 ) . The enhancement in chemotaxis mediated by SgGs required SgG-chemokine interaction since SgG2 did not have any effect on the chemotactic properties of hCCL2 ( Figure 5D ) , whereas it was able to potentiate hCXCL12β . A similar result was obtained with SgG1 ( not shown ) . In all cases , the enhancement in chemotaxis was dose dependent and significant . The effect of SgGs on chemotaxis was dependent on G protein activation since addition of pertussis toxin ( PTX ) inhibited both hCXCL12β-mediated cell migration and its enhancement mediated by SgGs ( Figure 5E ) . Finally , we examined the effect of SgG1 and SgG2 on hCXCL12β-mediated cell migration utilizing increasing concentrations of hCXCL12β and a constant molar ratio ( 1∶100 ) between the chemokine and SgG ( Figure 5F ) . The effect of hCXCL12β on in vitro cell migration had the characteristic bell-shaped curve ( not shown ) . As a control we used PRV-SgG , which inhibited chemokine-mediated migration [16] . However , both SgG1 and SgG2 enhanced the potency of hCXCL12 , displacing the chemotactic bell-shaped curve towards lower concentrations of the chemokine . To analyze the impact of HSV SgG on different aspects of chemotaxis in real time we performed time-lapse video microscopy using freshly isolated human monocytes and hCXCL12β . The chemokine , alone or in combination with SgG2 , was released from a micropipette with constant backpressure . Analysis of tracks recorded by time-lapse video microscopy from cell cultures stimulated with CXCL12β ( Video S1 ) or CXCL12β-SgG2 ( Video S2 ) clearly showed that chemotaxis in the presence of the viral protein was enhanced , compared to the migration towards the chemokine alone ( Videos S1 , S2 and Figure 6B ) . SgG2 was not able to trigger migration in the absence of the chemokine ( Video S3 ) . Consistent with our data from transwell assays ( Figure 5 ) , SgG2 greatly enhanced the number of human monocytes that moved towards a given concentration of the chemoattractant ( Figure 6 ) . The cells sensed the chemokine gradient from longer distance to the dispensing pipette than when chemokine was dispensed alone . Chemotactic parameters , i . e . velocity , FMI and distance traveled , were calculated during an initial 10-min period . The velocity of the cell movement and the Forward Migration Index ( FMI ) , i . e . the ratio of the net distance the cell progressed in the forward direction to the total distance the cell traveled , were significantly increased when SgG2 was bound to CXCL12β ( Figure 6C , D ) . Moreover , the cells travelled a longer distance when the chemokine and SgG2 were dispensed together than when the chemokine was dispensed alone ( Figure 6E ) . Similar results were obtained when using CXCL12α ( not shown ) . Transwell experiments performed in parallel with freshly isolated human monocytes confirmed the SgG2-mediated enhancement of CXCL12β chemotaxis observed by video microscopy ( Figure 6F ) . MAPKs are involved in several cellular processes including cell migration [31] . Binding of chemokine to its receptor activates a signaling cascade that involves phosphorylation and , thereby , activation of MAPKs . Incubation of MonoMac-1 cells with low doses of hCXCL12β resulted in low activation of MAPKs ( Figure 7 ) . Pre-incubation of different concentrations of hCXCL12β with a constant molar ratio ( 1∶200 ) of SgG1 enhanced the phosphorylation of ERK ( Figure 7A and B ) . The SgG1-mediated increase in the phosphorylation of JNK1-2 was dose-dependent ( Figure 7C and D ) . Similar results were obtained with SgG2 ( not shown ) . Densitometer analysis of the blots shows a dose-dependent enhancement of MAPK activation in the range of 5 fold for both ERK and JNK at the highest chemokine concentration . These results showed , using a different biological assay , a similar enhancement of chemokine activity mediated by HSV SgGs . Activation of CXCR4 results in the dissociation of GDP from the Gαβγ heterotrimer followed by association of GTP to the Gα subunit . In order to measure the effect of HSV SgG on receptor occupancy we performed a [35S]-GTPγS binding assay . The results show that the incubation of CXCL12β with SgG results in higher levels of [35S]-GTPγS incorporation ( Figure 7E ) . We tested the functional relevance of SgG2-chemokine interaction in vivo using the mouse air pouch model , by performing injections of chemokine alone or in combination with SgG2 . Injection of 0 . 2 µg of mCXCL12α or mCCL28 induced the migration of leukocytes into the air cavity ( Figure 8 ) . The presence of 2 µg SgG2 enhanced CXCL12α-mediated migration ( Figure 8A ) of total leukocytes ( top panel , P<0 . 001 ) , lymphocytes ( middle panel , P<0 . 001 ) and granulocytes ( bottom panel , P<0 . 05 ) . As a control , we used 2 µg recombinant secreted gG from PRV ( PRV-SgG ) , a vCKBP shown to inhibit chemotaxis [16] . PRV-SgG significantly inhibited CXCL12α-mediated chemotaxis of total leukocytes ( top panel , P<0 . 001 ) and granulocytes ( bottom panel , P<0 . 05 ) . CCL28-mediated chemotaxis ( Figure 8B ) of total leukocytes ( top panel ) and lymphocytes ( middle panel ) was significantly increased by SgG2 ( P<0 . 05 ) , whereas the migration of granulocytes ( bottom panel ) was not affected by SgG2 . This could be explained by the specificity of CCL28 in driving T cell chemotaxis . In contrast to the inhibition observed when CXCL12 was used , PRV-SgG did not significantly inhibit CCL28-mediated chemotaxis . This may be due to uncontrolled factors such as the stability of the PRV-SgG-CCL28 complex in vivo or the indirect activation of other chemoattractants that may also induce migration . Injection of SgG2 or PRV-SgG alone , in the absence of chemokine , did not result in differences in leukocyte chemotaxis when compared to PBS injection .
HSV glycoproteins play relevant roles in the viral cycle and pathogenesis , and constitute promising vaccine candidates [32] , [33] . Among all HSV glycoproteins , gG is the least well characterized and its function has not been fully elucidated . A role for HSV gG on virus entry has been suggested . HSV-1 gG seems to be important for the infection of , but not initial binding to , polarized cells through the apical surface [21] . The non-secreted domain of HSV-2 gG could participate in initial interaction of the virion with the cell surface [21] , [22] . A synthetic peptide encompassing amino acids 190–205 from the secreted domain of HSV-2 gG was found to have a proinflammatory role in vitro when bound to the formyl peptide receptor [34] . However , until present , no function has been attributed to the full-length secreted portion of HSV-2 gG . Here , we have investigated the function of secreted forms of gG from HSV-1 and HSV-2 . We show for the first time a chemokine-binding activity both in HSV-1 infected cells and in the supernatant of HSV-2 infected cells . Disruption of the HSV-1 gG expression abrogated chemokine binding suggesting that HSV gG is the protein responsible for the interaction . We could indeed show that both HSV-1 and HSV-2 SgG bind with high affinity , in the nanomolar range , CC and CXC chemokines . This interaction was demonstrated by the use of two different experimental approaches: crosslinking assays and SPR . Finally , and more importantly , we describe the first vCKBP , to our knowledge , with the ability to increase chemotaxis both in vitro and in vivo by enhancing the potency of the chemokine and the directionality of cell migration . HSV SgGs enhancement of chemotaxis required the interaction with the chemokine through the chemokine GAG-binding domain and involved signaling through the GPCR and activation of MAPKs . We confirmed that supernatant containing gG secreted following HSV-2 infection enhances chemokine-mediated migration of leukocytes . Moreover , in preliminary experiments we have found that membrane-anchored gG expressed during HSV-1 replication in cell culture also enhances chemokine activity ( N . M . -M . and A . V . -B . , unpublished data ) . During evolution , viruses have developed strategies to modulate the host immune response . Inhibition of chemokine function through the expression of vCKBP is a common strategy in members of the Poxviridae family [12] , [35] indicating the importance of chemokines in antiviral defense . In the Herpesviridae family , however , there are only three examples of vCKBP reported to date , two of them expressed by animal viruses -gG from alphaherpesviruses and M3 from murine herpesvirus 68- and one expressed by a human pathogen , pUL21 . 5 encoded by human cytomegalovirus [14] , [36] . In addition , interaction of HSV gB with a reduced number of chemokines has been reported [37] . However , this interaction was of low affinity , in the micromolar range [37] compared to the nanomolar range observed for all vCKBP [14] , [16] , [17] , [29] , [30] , [36] . Moreover , gB did not seem to have an effect on chemotaxis [37] . Nearly all previously described vCKBP have been shown to inhibit chemotaxis either in vitro or in vivo . As a general rule , vCKBPs inhibit chemokine function through impairing chemokine-receptor interaction or chemokine presentation by GAGs [38] . For instance , gG from some animal alphaherpesviruses blocks chemokine interaction with its receptor [14] , [28] and with GAGs [14] inhibiting chemotaxis [14] , [16] , [17] . To date , there are no reports of a vCKBP that potentiates chemokine function either in vitro or in vivo . HSV SgG is , therefore , the first vCKBP described , to our knowledge , which enhances chemokine function both in vitro and in vivo . Our studies with SgG1 and SgG2 show that these viral proteins interact with the GAG-binding domain of the chemokines and enhance the chemokine activation of GPCRs . Chemokine-GAG interaction is required for correct chemokine function in vivo [9] . Several reports show that GAG-binding deficient chemokines are functionally impaired in vivo and when in vitro migration and invasion assays are performed [39] , [40] . GAGs also modify chemokine quaternary structure and this seems to be required for chemokine function [39] , [41] . We propose a model in which SgG1 and SgG2 act similarly to the GAGs , maybe by increasing the local chemokine concentration , modifying the chemokine quaternary structure or improving chemokine presentation to the receptor so that signaling is enhanced . This would cause the observed activation of chemokine signaling at lower doses of chemokine when gG is present . This contrasts with the related gGs encoded by non-human herpesviruses , which have been shown to inhibit chemokine-mediated signal transduction and cell migration [14]–[17] . It appears that HSV-1 and HSV-2 have evolved a vCKBP to enhance , rather than to inhibit , chemokine function , and this may represent an advantage to these human herpesviruses . The functional relevance of chemokine enhancement in HSV life cycle and pathogenesis is unknown . The role of alphaherpesvirus gG in vivo is not fully understood . Results presented in several reports indicate that gG from animal alphaherpesviruses is relevant for pathogenesis and immune modulation [15] , [17] . There are currently no data on the role of HSV-2 gG on pathogenesis . Three independent reports show that lack of gG expression in HSV-1 leads to different degrees of virus attenuation [23]–[25] . Thus , lower viral titers were detected in mouse tissues infected through scarification of the ear with an HSV-1 mutant lacking gG [23] . A double us3/us4 deletion mutant ( with us3 encoding a kinase and us4 encoding gG ) was attenuated following intracranial injection [24] . However , the relative contribution of either protein in that animal model could not be defined . Mutation of the us4 gene by the use of transposon Tn5 resulted in a HSV-1 mutant that was less pathogenic , was deficient in its ability to replicate in the mouse central nervous system and caused a delay in encephalitis induction [25] . The mechanisms of attenuation of HSV-1 gG mutant viruses are unknown , but the discovery that HSV-1 gG enhances chemokine function points to a role of HSV gG on deregulation of chemokine function that could explain the lower pathogenicity observed with the mutant viruses . Although there are not yet systematic analyses on the expression of all known chemokines on the tissues relevant for HSV infection , the information obtained by several laboratories supports the relevance of chemokines on HSV infection and pathogenesis . The expression of some chemokines is upregulated upon HSV-1 and HSV-2 infection [42] , [43] leading to leukocyte infiltration , which may be as pathogenic as viral infection [44] . In fact , chemokines are important in HSE pathogenesis in humans [11] . Deficiency in CXCR3 or CCR5 increases susceptibility to genital HSV-2 infection although through different mechanisms [43] , [45] . Interestingly , the lack of CXCR3 does not result in lower leukocyte recruitment . On the contrary , CXCR3−/− mice show an increase in viral titers , infiltrating cells and neuropathology accompanied by a higher level of cytokine and chemokine expression in brain and spinal cord [46] . Differences were observed between CXCL10−/− and CXCR3−/− ( the receptor for CXCL10 ) mice when challenged with ocular HSV-1 infection [47] , [48] . However , CXCR3−/− responded like CXCL9−/− or CXCL10−/− in a genital model of HSV-2 infection [46] . There are also differences in susceptibility depending on the route of infection and the nature of the pathogen employed . The redundancy of the chemokine network may be beneath some of these differences and discrepancies . The chemokines bound by SgG1 and SgG2 are expressed in tissues relevant for HSV infection , replication and spread . Among other cell types , mucosal epithelial cells express CCL25 , CCL28 and CXCL13: ( 1 ) CCL25 expression is upregulated during oral wound healing [49]; ( 2 ) CCL28 is expressed in airway epithelial cells [50]; and ( 3 ) CXCL13 is required for the organization and function of the nasal-associated lymphoid tissue [51] . Human corneal keratinocytes express CXCL9 , CXCL10 and CXCL11 , expression that can be further induced by proinflammatory cytokines [52] . CXCL14 expression in taste-bud cells is remarkably high and secreted to the saliva [53] . Among other tissues , CXCL12 is expressed in nervous tissues where it has been suggested to play a role in leukocyte extravasation [54] . CXCL12 also induces migration of neural progenitors , is required for axonal elongation and pathfinding , is relevant for neurotoxicity and neurotransmission in the adult nervous system and contributes to chronic pain [6] , [8] . Thus , modulation of the activity of chemokines mediated by gG1 and gG2 could occur in tissues infected by HSV and play a role in HSV biology . Enhancement of chemokine function by HSV SgGs could impact at least four different scenarios relevant for HSV spread and pathogenesis . First , enhancement of GPCR signaling could aid in the early steps of infection and in viral replication . In fact , MAPK activation is required for efficient HSV replication [55] . In this scenario gG1 , due to its presence in the viral particle and at the plasma membrane of the infected cells , may play a more relevant role than gG2 , which is processed secreting its chemokine-binding domain . Second , increase in the level of infiltrating leukocytes , or differences in the composition of such infiltrate , could skew the immune response and favor viral replication . The fact that HSV SgGs only bind 11–12 out of 45 human chemokines with high affinity suggests the existence of a selectivity and specificity in the modulation of the immune response . Third , enhancement in the migration of a particular leukocyte population could recruit cells that may be subsequently infected by HSV , enhancing viral load . Fourth , modulation of chemokines present in the nervous system , such as CXCL12 , could play a role in the initial infection of the ganglia , sites of HSV latency , and increase the ability of HSV to persist and cause disease . The impact of HSV gG-chemokine interaction on HSV biology requires further characterization . In summary , this is the first report of a vCKBP that enhances chemokine function and suggests a novel mechanism of immune modulation mediated by a highly relevant and prevalent human pathogen . The findings reported here shall foster further investigations on the role of HSV gG on pathogenesis and immune modulation and will allow the design of novel immunomodulators , antiviral drugs and tools to study chemokine function .
All animal experiments were performed in compliance with Irish Department of Health and Children regulations and approved by the Trinity College Dublin's BioResources ethical review board . Human peripheral blood monocytes were prepared from buffy coats obtained from the local donor bank ( “Servizio Trasfusione , Svizzera Italiana” , CH-6900 Lugano , Switzerland ) , with oral consent from the donors according to Swiss regulations . The use of buffy coats was approved by the institutional review board “Comitato Etico Cantonale , CH-6501 Bellinzona , Switzerland” and the experimental studies were approved by the “Dipartimento della Sanitá e della Socialitá” . The interactions between chemokines and SgGs and their affinity constants were determined by SPR technology using a Biacore X biosensor ( GE Healthcare ) as previously described [16] . Both proteins were dialyzed against acetate buffer ( pH 5 . 0 for SgG1 and pH 5 . 5 for SgG2 ) prior to amine-coupling of the recombinant proteins in CM5 chips . Chemokines that did not bind under kinetic conditions were considered negative and not taken into further consideration for the study . In competition experiments with heparin the chemokine was injected at 100 nM alone or with increasing concentrations of heparin in HBS-EP buffer ( 10 mM Hepes , 150 mM , NaCl , 3 mM EDTA , 0 . 005% ( vol/vol ) surfactant P20 , pH 7 . 4 ) at a flow rate of 10 µl/min , and association and dissociation were monitored . All Biacore sensorgrams were analyzed with the software Biaevaluation 3 . 2 . Bulk refractive index changes were removed by subtracting the reference flow cell responses , and the average response of a blank injection was subtracted from all analyte sensorgrams to remove systematic artifacts . Competition experiments were carried out incubating 0 . 5 pmol of [125I]-hCCL25 or [125I]-hCXCL12α with or without different concentrations of SgGs ( or baculovirus supernatants ) at 4°C in binding medium ( RPMI 1640 containing 1%FBS and 20 mM HEPES pH 7 . 4 ) during 1 h at 4°C . Then , 3×106 MOLT-4 or MonoMac cells were added to the mixture and incubated for further 2 h at 4°C with gentle agitation , subjected to phthalate oil centrifugation , washed twice with PBS , and cell-bound chemokine was determined using a gamma-counter . Chemokines were placed in the lower compartment of 24-well transwell plates ( Costar ) or in 96-well ChemoTx System plates ( Neuro Probe Inc . , MD , USA ) with or without recombinant gGs in RPMI 1640 containing 1% FBS . MOLT-4 , MonoMac-1 , m300-19 and m300-19-hCXCR5 cells were placed on the upper compartment ( 3×105 cells in the 24-well transwell plate and 1 . 25×105 cells in the 96-well ChemoTx System plate , with the exception of m300-19-hCXCR5 where 2 . 5×105 cells were used ) . To test the effect of supernatant from mock- or HSV-2-infected cells in chemotaxis , the cells were infected in the presence of Optimem ( Gibco ) and the supernatants were collected 36 h . p . i . These supernatants were inactivated with psoralen as previously described [56] and concentrated 10 times using a Vivaspin 500 ( Sartorius ) prior to use . Both chambers were separated by a 3 µm ( for MOLT-4 , MonoMac-1 cells and monocytes ) or 5 µm ( for m300-19 and m300-19-hCXCR5 cells ) pore size filter . The plates were incubated at 37°C during 2–4 h and the number of cells in the lower chamber was determined using a flowcytometer ( for 24-well transwell plates ) or by staining the cells with 5 µl of CellTiter 96 aqueous one solution cell proliferation assay ( Promega , WI , USA ) during 2 h at 37°C and measuring absorbance at 492 nm , with the exception of monocytes and m300-19-hCXCR5 which were counted with a light microscopy . When the CellTiter 96 aqueous one solution cell proliferation assay was used , known amounts of cells were incubated with the CellTiter solution to quantify the number of migrated cells . When used , PTX was incubated with MonoMac-1 cells overnight at a concentration of 0 . 1 µg/ml , prior to the chemotaxis experiment . Monocytes were isolated from blood of healthy donors by negative selection using Monocyte Isolation kit II ( MACS Miltenyi Biotec ) . Peripheral blood mononuclear cells ( PBMBs ) were isolated from heparinized blood by Ficoll ( Lymphoprep ) gradient centrifugation . Cells were resuspended in MACs buffer and incubated with FcR blocking reagent at 4°C . Monocytes were purified by negative selection according to the manufacturer's protocol . Time-lapse video microscopy analysis of chemotaxis was performed immediately with a Leica DI6000 microscope stand connected to a SP5 scan head equipped with a temperature controlled chamber ( Cube , LIS , Basel ) . Freshly isolated monocytes were placed in a humidified and CO2-controlled incubator , which was mounted on the microscope stage ( Brick , LIS , Basel ) . Cells were resuspended in D-PBS containing calcium and magnesium ( Invitrogen ) supplemented with 1% FBS , Pen/Strep , 0 . 04 mM sodium pyruvate , 1 mg/ml fatty acid free BSA ( Sigma ) , 1 mg/ml glucose ( Fluka ) . Cells were plated on glass bottom petri-dishes ( MatTek cultureware ) which were coated previously with D-poly-lysine ( 5 µg/ml ) and subsequently overlaid with 3 µg/ml VCAM-1 ( BD Biosciences ) at 4°C overnight . Before plating the cells , coated-dishes were treated with PBS containing FBS and BSA to block non-specific binding . Chemokine was dispensed with a micropipette ( Femtotip II , Eppendorf ) controlled by a micromanipulator ( Eppendorf ) at a constant backpressure of 30 hPa ( Femtojet , Eppendorf ) . Chemokine alone or in combination with SgGs was added to 106 MonoMac-1 cells and incubated during 1 min at 37°C . Cells were lysed in lysis buffer ( 20 mM triethanolamine pH 8 . 0 , 300 mM NaCl , 2 mM EDTA , 20% glycerol , 1% digitonin and proteinase inhibitors ) . The lysate was analyzed by western blotting using anti-phospho-ERK , anti-phospho-P38 ( Cell Signaling Technology ) or anti-phospho-JNK1/2 polyclonal antibodies ( Abcam ) . Blots were scanned and the densities of the bands were analyzed and compared with the Image J 1 . 43 software normalizing the densities obtained from each band from the MAPK blots to their respective loading controls . Age-matched female C57BL/6 mice from Harlan ( Bicester , U . K . ) were housed in a specific pathogen-free facility in individually ventilated and filtered cages under positive pressure . All animal experiments were performed in compliance with Irish Department of Health and Children regulations and approved by the Trinity College Dublin's BioResources ethical review board . Dorsal air pouches were induced in mice as described [57] . In brief , 5 ml of sterile-filtered air was injected subcutaneously into the dorsal skin of mice , with air pouches re-inflated with 3 ml of sterile air 3 days later . The dorsal air pouches of groups of 5–6 mice were injected 2 days later with 0 . 2 µg chemokine alone or in combination with 2 µg SgG . Mice were killed and air pouches were lavaged with PBS 3 h later . The air pouch aspirate was centrifuged and total leukocytes cells were counted . Cells were stained with a panel of mAbs for surface markers for flow cytometric cell characterization as described [58] . mAbs used were from BD Biosciences; PerCP anti-CD4 ( RM4-5 ) , PerCP-Cy5 . 5 anti-CD19 ( 1D3 ) , PerCP anti-CD8a ( 53-6 . 7 ) , PerCP anti-CD11b ( M1/70 ) and eBioscience: PE anti-Ly6G ( RB6/8C5 ) . Cells were defined as lymphocytes ( CD4+CD8+CD19+ ) and Ly6GhiCD11b+ granulocytes ( neutrophils ) . Data were collected on a CyAn ( Beckman Coulter ) and analyzed using FlowJo ( Tree Star ) . Quadrants were drawn using appropriate isotype-controls and data plotted on logarithmic scale density- or dot-plots . Statistical analyses of data were performed with the program GraphPad Prism . The significant value ( P value ) for the parameters measured in all assays was calculated using the student's t-test with the exception of the ones obtained in the air-pouch model experiments which was calculated using the one-way analysis of variance ( ANOVA ) . | Chemokines are chemotactic cytokines that direct the flux of leukocytes to the site of injury and infection , playing a relevant role in the antiviral response . An uncontrolled , unorganized chemokine response is beneath the onset and maintenance of several immunopathologies . During millions of years of evolution , viruses have developed strategies to modulate the host immune system . One of such strategies consists on the secretion of viral proteins that bind to and inhibit the function of chemokines . However , the modulation of the chemokine network mediated by the highly prevalent human pathogen herpes simplex virus ( HSV ) is unknown . We have addressed this issue and show that HSV-1 , causing cold sores and encephalitis and HSV-2 , causing urogenital tract infections , interact with chemokines . We determined that the viral protein responsible for such activity is glycoprotein G ( gG ) . gG binds chemokines with high affinity and , in contrast to all viral chemokine binding proteins described to date that inhibit chemokine function , we found that HSV gG potentiates chemokine function in vitro and in vivo . The implications of such potentiation in HSV viral cycle , pathogenesis and chemokine function are discussed . | [
"Abstract",
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] | 2012 | Enhancement of Chemokine Function as an Immunomodulatory Strategy Employed by Human Herpesviruses |
To study VSV entry and the fate of incoming matrix ( M ) protein during virus uncoating we used recombinant viruses encoding M proteins with a C-terminal tetracysteine tag that could be fluorescently labeled using biarsenical ( Lumio ) compounds . We found that uncoating occurs early in the endocytic pathway and is inhibited by expression of dominant-negative ( DN ) Rab5 , but is not inhibited by DN-Rab7 or DN-Rab11 . Uncoating , as defined by the separation of nucleocapsids from M protein , occurred between 15 and 20 minutes post-entry and did not require microtubules or an intact actin cytoskeleton . Unexpectedly , the bulk of M protein remained associated with endosomal membranes after uncoating and was eventually trafficked to recycling endosomes . Another small , but significant fraction of M distributed to nuclear pore complexes , which was also not dependent on microtubules or polymerized actin . Quantification of fluorescence from high-resolution confocal micrographs indicated that after membrane fusion , M protein diffuses across the endosomal membrane with a concomitant increase in fluorescence from the Lumio label which occurred soon after the release of RNPs into the cytoplasm . These data support a new model for VSV uncoating in which RNPs are released from M which remains bound to the endosomal membrane rather than the dissociation of M protein from RNPs after release of the complex into the cytoplasm following membrane fusion .
The entry of enveloped viruses that utilize the clathrin-dependent endocytic pathway involves attachment of virus to the cell surface and uptake of virions in coated vesicles that are transported to early or late endosomes . When virions reach a compartment in which the lumen has the appropriate pH there is an acid-induced fusion of the endosomal and viral membranes which results in virus uncoating and release of the genome into the cytoplasm [1] , [2] . Vesicular stomatitis virus ( VSV ) , a prototypic enveloped , nonsegmented , negative-strand RNA virus in the Rhabdoviridae family enters host cells through the clathrin- and pH-dependent endocytic pathway [3] , [4] , [5] , [6] . The genome of VSV encodes five major viral proteins: the nucleocapsid protein ( N ) , the phosphoprotein ( P ) , the matrix protein ( M ) , the glycoprotein ( G ) , and the large polymerase protein ( L ) . The viral genome is encapsidated by the N protein and associates with the viral RNA-dependent RNA polymerase ( RdRp ) , which consists of a complex of the L and P proteins . The N-RNA-RdRp collectively forms the ribonucleoprotein ( RNP ) complex . The M protein within virions is associated with RNPs in structures called skeletons [7] , [8] . Recently , cryo-EM imaging of intact VSV particles showed that the RNP skeleton consists of a compact left-handed helix bounded by an outer layer of M protein which anchors the RNP to the viral membrane [9] . Protruding from the virion surface are glycoprotein spikes consisting of G protein trimers . G protein is responsible for attachment of virions to cells and fusion of the endosomal and viral membranes , which results in the transfer of the RNP into the cytoplasm where VSV replication occurs [3] . Early models of VSV uncoating proposed that either directly after or concomitant with membrane fusion , M protein dissociates from RNPs , which results in decondensation of the skeleton [7] , [8] and completes virus uncoating [10] . More recently , it was proposed that VSV initially fuses with vesicles found within multivesicular bodies and that the release of nucleocapsids into the cytoplasm occurs after transport to late endosomes , which requires a back-fusion event [11] , and that Tsg101 controls the endosome-to-cytosol release of nucleocapsids [12] . After uncoating , the decondensed RNP serves as a template for transcription of viral mRNAs by the packaged RNA-dependent RNA polymerase . VSV uncoating , defined as the dissociation of M from RNPs , is an essential step which is required for a productive infection [13] . Without uncoating it is thought that transcription of viral mRNAs will not occur since it has been shown that transcription from RNPs is inhibited by M protein [14] , [15] , [16] . This essential step is also shared by another enveloped negative-strand virus , influenza virus , which requires the release of its matrix protein ( M1 ) for a productive infection [17] , [18] . To better understand the requirements for VSV uncoating , we generated recombinant VSV ( rVSV ) which encoded M proteins that had tetracysteine ( -CCRECC- ) Lumio tags at either the N- or C-terminus , and recovered infectious virus [19] . Similar rVSVs have been described by others [20] and have been used to study VSV entry and assembly . Previously , using rVSV containing M protein labeled with Lumio Green ( rVSV-MLG ) , we found that during entry the interior of VSV virions become acidified and that acidification required G protein [19] . Furthermore , we obtained evidence that virion acidification enhanced M protein release and proposed a model in which G protein-induced acidification facilitates VSV uncoating by causing subtle conformational changes in M protein which results in the dissociation of M from RNPs . In this report we examine the kinetics of VSV uncoating and the fate of virion-associated M protein and RNPs during VSV entry and after uncoating using rVSV-MLG . We show that after fusion of the viral membrane with endosomes , the bulk of M protein remained associated with vesicular structures and eventually colocalized with markers for recycling endosomes . Although most of M remained bound to endosomal membranes , a small , but significant fraction of M protein was released and localized to the nuclear envelope . The delivery of M protein to the nuclear envelope was not dependent on microtubules or an intact actin cytoskeleton . Using confocal microscopy we observed that RNPs entered the cytoplasm and physically separated from M protein between 15 and 20 minutes post-entry and that the release of RNPs into the cytoplasm was also not dependent on microtubules or intact microfilaments . Following RNP release , the membrane-bound M protein diffuses across the endosomal membrane with a concomitant increase in MLG fluorescence . Collectively , these data provide strong evidence that VSV uncoating involves the direct release of RNPs from membrane-associated M protein into the cytoplasm where viral transcription can take place to initiate a productive infection .
Recently , using rVSV containing Lumio-Green labeled M protein ( rVSV-MLG ) , we reported that very soon after endocytosis the interior of VSV virions become acidified resulting in a decrease in input MLG fluorescence [19] . This is followed by a recovery of fluorescence to the original input levels at approximately 10 minutes post-entry , which we proposed is due to exposure of MLG to the neutral pH of the cytoplasm and therefore is a marker for VSV membrane fusion and the initiation of virus uncoating [19] . These results are consistent with models in which the release of VSV RNPs occurs from early endosomes . To further define the intracellular location of VSV uncoating we examined the effect of dominant-negative ( DN ) Rab proteins on rVSV-wt infection . Rab5 , 7 and 11 are GTPases that are required for the movement of endocytosed cargo to early ( Rab5 ) , late ( Rab7 ) , or recycling ( Rab11 ) endosomes [21] , [22] , [23] . Expression of DN-Rab5 prevents fusion of endocytic vesicles with early endosomes , while DN-Rab7 and 11 prevent movement from early to late , or to recycling endosomes , respectively . BHK-21 cells were transfected with plasmids encoding eGFP-tagged DN-Rabs and then the cells were infected with rVSV-wt 18 h post-transfection . The cells were fixed 8 hours later , permeabilized , and stained for VSV nucleocapsid ( N ) protein using an N-specific monoclonal antibody ( mAb-10G4; [24] ) conjugated to Alexa Fluor-568 . As shown in Fig . 1 , most cells expressing DN-Rab7 and DN-Rab11 were infected , but cells expressing DN-Rab5 were not and infection was inhibited by ∼70% . Expression of the wt-Rab proteins had only minimal effects on VSV infection ( Fig . 1B ) . These data support previous reports which showed VSV could infect cells expressing DN-Rab7 , but not cells expressing DN-Rab5 [4] , [5] . Inhibition of VSV infection by DN-Rab5 was highly significant ( p = 0 . 002 ) , but the small inhibition observed with DN-Rab11 was not ( p = 0 . 1549 ) . To ensure our DN-Rab7 construct was functional we examined the effect of the DN-Rabs on influenza A virus infection . As shown previously [5] , DN-Rab5 and 7 inhibited influenza virus infection ( Fig . 1B ) , while DN-Rab11 did not . Collectively , these data indicate that the majority of VSV virions fuse and uncoat after delivery to early endosomes and that VSV infection does not require transfer to late endosomes . To study VSV uncoating and the fate of incoming M protein , rVSV-MLG and transferrin-Texas Red ( Tfn-TR ) were adsorbed to cells for 90 minutes at 4°C to prevent endocytosis and then entry was initiated by replacing the inoculum with media pre-warmed to 37°C . The Tfn-TR was used as a surface ( t-0 ) and endosomal ( post t-0 ) marker for most of the studies described below . To differentiate surface-bound from endocytosed Tfn-TR , the cells were treated with acidic buffer containing an iron chelating agent , which removes transferrin bound to transferrin receptor on the cell surface . Acid washing ( AW ) of cells prior to the addition of warm media and the initiation of endocytosis resulted in the loss of the Tfn-TR signal ( Fig . 2 , t-0 AW ) , but had no effect on bound virus . Five minutes after transferring the cells to 37°C virtually all of the Tfn-TR was endocytosed ( Fig . 2 , t-5 AW ) . Based on colocalization of Tfn-TR and MLG fluorescence , the vast majority of rVSV-MLG was also endocytosed , which is consistent with two recent reports showing that VSV is rapidly internalized [4] , [6] . Because of concerns that acid treatment may induce membrane fusion of surface bound virus , all of the subsequent studies were performed without an acid wash step . To examine the kinetics of virus entry and to follow the trafficking of M protein , rVSV-MLG and Tfn-TR were bound to cells in the cold as described in Fig . 2 , and after the addition of warm media the cells were incubated for the specified times and examined by laser scanning confocal microscopy ( LSCM; Fig . 3 ) . At t-0 , rVSV-MLG and Tfn-TR decorated the cell surface and showed some colocalization . After 5 min ( not shown ) both markers were internalized and most of the rVSV-MLG virions were in transferrin-positive endosomes . Between 10 to 30 minutes , endosomes containing Tfn-TR and MLG moved from the cell periphery towards the cell interior . Notably , the bulk of MLG remained associated with Tfn-TR-positive endosomes until the last time point examined ( t-150 ) . In addition to the endosomal-associated fraction a small amount of MLG began to accumulate on what appeared to be the nuclear envelope beginning 20 minutes post entry ( Fig . 3 , arrowheads ) . Association of MLG with this structure continued to increase up to the last time point examined . At no time did we observe Tfn-TR associated with the nuclear envelope suggesting this was not due to membrane recycling to the endoplasmic reticulum surrounding the nucleus . To determine if trafficking of MLG to the nuclear envelope required membrane fusion and virus uncoating rVSV-MLG and Tfn-TR were bound to cells that had been pretreated with bafilomycin A1 , which inhibits endosome acidification and prevents VSV infection , and examined by LSCM at various times as described for Fig . 3 . At no time did we observe MLG associating with the nuclear envelope in cells treated with bafilomycin A1 ( Fig . 4A ) . To determine if ongoing protein synthesis was required we synchronized fusion of the viral and endosomal membrane using ammonium chloride in the presence of cycloheximide . Ammonium chloride ( NH4Cl ) is a lysosomotropic agent that prevents acidification of endosomes and thereby inhibits fusion and uncoating of endocytosed virions [10] , [25] , but unlike bafilomycin A1 , NH4Cl can be easily washed out of cells allowing rapid re-acidification of endocytic compartments . Following entry in the presence of NH4Cl , the inoculum and NH4Cl were removed , the cells were washed 4 times , and media containing cycloheximide was added . Cells were fixed either immediately or 60 minutes after NH4Cl removal . The cells were then permeabilized and stained with anti-Nup62 antibody , which binds to the nuclear pore complex ( NPC ) on the nuclear envelope , and with an anti-M mAb ( 23H12; [24] ) and examined by LSCM ( Fig . 4B ) . At t-0 , rVSV-MLG particles were in small vesicular structures ( endosomes ) and no M protein was detected at the nuclear envelope ( Fig . 4B , top panels ) . Similar to the results shown in Fig . 3 , at t-60 the bulk of MLG was found in large , mostly perinuclear endosomes and a small but significant amount of MLG was localized to the nuclear envelope ( Fig . 4B , t-60 arrows ) . These data indicate that while most of the incoming ( e . g . virion-associated ) M protein remains associated with Tfn-positive endosomes , a small amount of M trafficks to the NPC after uncoating . As reported by others , the M mAb ( 23H12 ) did not detect M protein bound to nuclear envelope ( Fig . 4B , yellow arrows ) , which explains why earlier studies examining VSV uncoating [10] did not observe delivery of M protein to the NPC . The epitope recognized by the 23H12 M mAb overlaps the region of M that binds to the nuclear shuttling protein RaeI [26] , [27] , suggesting that the fraction of MLG which associates with the nuclear envelope is likely complexed with RaeI . To determine if microtubules or microfilaments are required for the delivery of MLG to the nuclear envelope cells were inoculated with rVSV-MLG in the presence of NH4Cl for 60 minutes and then the cytoskeletal inhibitors cytochalasin D or nocodazole were added for 30 minutes . The NH4Cl was removed by washing the cells with media containing cycloheximide either with or without cytoskeletal inhibitors and incubated for an additional 60 minutes . The cells were then fixed and stained for filamentous actin using Texas Red-phalloidin ( Fig . 5A ) , or for tubulin ( Fig . 5B ) . Either in the absence ( top panels ) or presence of the inhibitors ( bottom panels ) , MLG was found in association with endosomes and at the nuclear envelope ( arrows ) . These results indicate that delivery of MLG to the nuclear envelope does not require an intact actin cytoskeleton or microtubules . The images in Fig . 3 show that the bulk of MLG remains associated with Tfn-positive endosomes for at least 150 min after entry . To determine the identity of these perinuclear structures we used markers that corresponded to lysosomes ( LAMP-1 ) , late endosomes/lysosomes ( Mannose-6-Phosphate receptor , M-6-P-R ) , or recycling endosomes ( Tfn-TR and Rab11 ) . rVSV-MLG was endocytosed in the presence of NH4Cl as described for Fig . 4 , the inoculum and NH4Cl were removed and the cells were incubated for an additional 60 min and then stained using antibodies to the various endosomal markers , or visualized directly for Tfn-TR . Quantification of MLG colocalization with the various markers indicated that most of incoming M protein remains associated with membranes that mature into recycling endosomes ( Fig . 6C and D ) , while only some MLG is delivered to late endosomes ( Fig . 6B ) and lysosomes ( Fig . 6A ) . It is not known if VSV RNPs are released into the cytoplasm , or if they remain associated with membranes after uncoating in vivo . To further define the site of virus uncoating we asked whether we could detect nucleocapsids physically separating from MLG after endocytosis . Cells were inoculated with rVSV-MLG as described in Fig . 3 , except that Tfn-TR was not included , and entry was initiated after transfer to 37°C . Cells were fixed and RNPs were detected by staining for N protein at the times indicated . At t-5 , the majority of RNPs colocalized with MLG ( Fig . 7A and C ) . Beginning approximately 10 minutes post-entry RNPs could be seen separating from MLG-endosomes . The largest change in RNP-MLG colocalization occurred between 15 and 20 minutes; quantification of the fraction of RNPs that colocalized with MLG decreased from approximately 80% at t-5 through t-15 to less than 50% at t-20 ( Fig . 7B ) . We also examined RNP-M separation biochemically using a cell fractionation protocol to determine if the results seen by fluorescence confocal microscopy could be reproduced by an independent method . As shown in Fig . 8A , RNPs redistributed from intact virions at t-0 to a cytoplasmic fraction between 10 and 20 minutes post-entry , similar to the time when RNPs dissociated from MLG in Fig . 7 . Likewise , M protein redistributed to a fraction that was enriched in endosomal markers beginning at 10 minutes and continuing until the end of the experiment ( Fig . 8B ) . Thus , the distribution of RNPs and M protein correlated well when using either confocal microscopy or a cell fractionation assay . To determine if RNP separation required microtubules rVSV-MLG was endocytosed in the presence of NH4Cl for 1 hour and then nocodazole and cycloheximide were added for 30 minutes to depolymerize microtubules and stop protein synthesis . The cells were either fixed immediately ( Fig . 9A ) or were incubated in the presence of both inhibitors , or with nocodazole only , for an additional 2 hours . Quantification of MLG and N protein colocalization showed that at t-0 , 92 . 8% ( +/−3 . 4% , n = 50 cells ) of RNPs colocalized with MLG , whereas by t-120 ( Fig . 9B ) most of the nucleocapsids had separated from M protein as indicated by only 31 . 3% ( +/−8 . 2% , n = 50 cells ) of RNP-MLG colocalization . Similar to that observed in Figs . 3 , 4 and 5 , there was very distinct MLG fluorescence associated with the nuclear envelope at t-120 , but importantly there was no N protein staining of the nuclear membrane . This indicates that M protein localizes to the nuclear envelope alone and not in association with RNPs . As previously reported by others [28] , we also observed that VSV transcription and viral protein synthesis can occur following infection of cells that lack an organized microtubule network ( Fig . 9C ) , as indicated by the extensive N protein staining throughout the cytoplasm which represents new N protein synthesis . These results also confirm that the N protein staining seen in Fig . 9B represents incoming RNPs , and is not from new N protein synthesis . When fusion between the virus and endosomal membranes occurs , an asymmetry in the endosomal membrane is generated by addition of the viral membrane to the endosomal membrane at the site of membrane fusion . Based on the tight association of MLG with the viral membrane it should be possible to visualize the virus-endosome fusion event using MLG as a marker . To examine the spatial distribution of MLG during virus entry and uncoating we analyzed high magnification confocal images of endosomes from an experiment similar to that described in Fig . 3 . For this analysis we used a 100X-1 . 4 N . A . apochromat objective and digitally magnified the image 5× prior to capture using 1024×1024 pixel resolution . Figure 10A shows an example of an image collected 15 minutes post-entry which clearly shows an asymmetric distribution of MLG relative to Tfn-TR within an endosome . To determine if this asymmetry required membrane fusion we produced MLG virus particles lacking G protein ( ΔG-MLG ) . We have shown previously that “bald” ΔG-VSV is able to bind and enter cells , albeit somewhat less efficiently than wt-VSV , and that the virus is non-infectious due to the lack of G protein [29] . In contrast to infectious rVSV-MLG , we found that MLG from ΔG-MLG particles did not acquire an asymmetrical distribution ( Fig . 10B ) , presumably because the particle remained within the lumen of the endosome . As might be expected , we also did not observe MLG from ΔG-MLG particles associating with the nuclear envelope at any time point ( data not shown ) . In addition to observing MLG at the site of membrane fusion , we followed the fate of MLG post-fusion . We found that by 20 to 30 minutes post-entry the asymmetry began to disappear and the size of the MLG-positive membrane area increased , reaching a plateau after approximately 60 minutes ( Fig . 10C and D ) . These data suggest that after binding of virus to cells on ice , virus-endosome fusion occurs between 10 and 15 minutes after warming cells to 37°C . The data also suggest that as M protein is exposed to the cytoplasm following membrane fusion it begins to diffuse across the endosomal membrane where it becomes evenly distributed as the lipids of the viral and endosomal membranes continue to mix . The kinetics of the increase in MLG surface area correlated with our previously published results [19] showing that after an initial low pH-induced decrease in MLG fluorescence , there was a steady increase in fluorescence up to approximately 60 minutes post-entry . Based on the results shown in Fig . 10D , the increase in fluorescence likely represents reduced packing of MLG as it transitions from the ordered helical lattice found within virions [9] , to a more diffuse form found after uncoating has occurred . This change in fluorescence and movement of MLG across the endosomal membrane also correlates temporally with the reduction in colocalization of RNPs with MLG , which represents the release of RNPs into the cytoplasm seen in Fig . 7 .
An important new finding from these studies was that most of the incoming M protein remained associated with an endosomal membrane at both early and at late times after entry . This was seen both in cells that had virus bound to the cell surface in the cold , as well as in cells where virus fusion was synchronized by NH4Cl washout . By quantifying the amount of N protein that had separated from MLG endosomes ( Fig . 7 ) , it appears that greater than 75% of the virus had fused , therefore only a small fraction represents unfused virus . Thus , most of the incoming virus enters cells in a productive manner and the bulk of M protein remains bound to the endosomal membrane . The topology of the viral membrane after fusion would predict that the endosomal-associated M would be exposed to the cytoplasm; therefore the binding of M to the endosomal membrane must be quite strong . As the infection progressed , e . g . 60 to 150 minutes post-entry , M protein was observed in large perinuclear structures , which stained for markers of recycling endosomes . Following uncoating a smaller proportion of M was apparently released and rapidly localized to the nuclear envelope . These observations correlate nicely with an earlier study which showed using a biochemical assay that ∼15% of input M was released into the cytosol after entry [10]; however , in that study the distinct nuclear envelope localization was not observed . Our results support the finding that only a fraction of the incoming M protein is released in a soluble form after uncoating . However , this small amount of M colocalized with Nup62 , suggesting that this soluble fraction binds to nuclear pore complexes ( NPCs ) . These data are consistent with reports that GFP-M fusion proteins can associate with NPCs [27] . Based on our finding that M protein from incoming virus particles also binds to NPCs , we propose that upon VSV entry , a small amount of M protein is released after uncoating and that this fraction associates with NPCs where it may block nucleocytoplasmic transport [30] , thus representing an early effort by the virus to mute the innate immune response even before viral protein synthesis has begun . The mechanism by which M is trafficked to the NPCs is less clear . We observed localization of MLG on the nuclear envelope in the absence of polymerized actin ( Fig . 5A ) or tubulin ( Fig . 5B ) . This was unexpected since early studies indicated that M protein binds to the cytoskeleton [31] , [32] and it was assumed that M trafficking to the nuclear envelope involves microfilaments or microtubules . Alternatively , trafficking of M to the NPC may involve M binding to RaeI . It is known that binding of M to RaeI results in inhibition of mRNA nucleo-cytoplasmic transport and that RaeI binds to microtubules in mitotic spindle complexes [33] , [34] . Therefore , M could bind to RaeI in the cytoplasm and be delivered to the NPCs; however , we observed M trafficking to the nuclear envelope in the presence of nocodazole , suggesting that localization to the NPC likely occurs by a different mechanism . Our results also shed new light on the mechanism of VSV entry and uncoating . There are currently two models proposed for how VSV nucleocapsids are delivered to the cytoplasm . Both models begin with the well-accepted paradigm that VSV particles are endocytosed via the clathrin-dependent pathway and are delivered to early endosomes . In the traditional model of VSV entry , once the pH of the early endosomal lumen becomes acidified to ∼pH 6 . 3 , G protein undergoes conformational changes which induce fusion of the viral envelope with the endosomal membrane and the RNP is exposed to the cytoplasm where primary transcription occurs . This model has gained support through the use of siRNAs to knock-down essential Rab proteins required for maturation of early to late endosomes [5] , [21] and by use of specific inhibitors and TIRF microscopy [4] . A second model was recently proposed in which fusion occurs early , but RNPs are not released immediately and instead are trafficked in multi-vesicular bodies to late endosomes where a “back-fusion” event occurs which releases RNPs into the cytoplasm [11] , [12] . Our data support the traditional model ( at least for the majority of particles ) in which VSV virions fuse early in the endocytic pathway and RNPs are released , with the unexpected observation that a significant pool of M protein remains associated with the endosomal membrane long after RNP release has occurred . The use of fluorescently labeled M and the direct visualization of M distribution by confocal microscopy have allowed us to describe for the first time the spatio-temporal relationship of viral proteins during VSV uncoating ( Fig . 11 ) . The following events are proposed based on these data . Within 2 to 5 minutes , virions are internalized and between 5 and 10 minutes they are exposed to low pH , which can be detected by the loss of MLG fluorescence within the virion interior ( Fig . 11 , step A , [19] ) . It is well established that low pH also causes conformational changes in G protein [35] , [36] which bring about the membrane fusion event [37] , [38] required for VSV infection ( Fig . 11 , step B ) . During entry we found that MLG became asymmetrically distributed and localized to one side of the endosomal membrane , which we propose occurs as the virus membrane merges with the delimiting membrane of the endosome where the RNP is released into the cytoplasm ( Fig . 11 , step C ) . There was a sharp decrease in the colocalization of RNPs with MLG between 15 and 20 minutes post-entry , but since our analysis measured the physical separation of RNPs and MLG , these data suggest that the initial uncoating event would occur prior to this time . We also propose that interactions between the condensed RNP with MLG on the viral membrane maintains MLG in an organized lattice which initially restricted the quantum yield of MLG fluorescence and that after the release of RNPs into the cytoplasm this organization is lost resulting in the diffusion of MLG across the endosomal membrane and the concomitant increase in MLG fluorescence which we observed beginning approximately 15 minutes post-entry ( Fig . 11 , step D ) . As a result of uncoating , some M is released into the cytoplasm and associates with NPCs ( Fig . 11 , step E ) . By generating rVSVs encoding M-Lumio with other viral proteins fused to red , yellow , or cyan fluorescent proteins , or multiple Lumio-tagged viral proteins that are differentially labeled with Lumio-Green and Lumio-Red , it should be possible to gain further insight to the early events in VSV entry and the mechanisms of VSV uncoating .
BHK-21 cells on 100 mm plates at ∼95% confluency were infected with rVSV-ML or G-complemented ΔG-ML for 1 hour as described previously [19] and then at 4 hours post-infection ( hpi ) the cells were washed twice with reduced-serum Opti-MEM I ( Invitrogen ) and replaced with 10 ml of Opti-MEM I containing 200 nM Lumio Green ( Invitrogen ) . The supernatant was collected 18 hpi and labeled virus was concentrated by centrifugation over a 20% sucrose cushion at 38 , 000 rpm in an SW41 swinging bucket rotor ( Beckman ) for 45 minutes . The viral pellet was resuspended on ice in 1 ml of 10% sucrose-TN buffer ( 10 mM Tris , pH 7 . 4;150 mM NaCl ) and the virus was stored at −80°C . Titers were determined by standard plaque assay on BHK-21 cells and protein concentration was determined by a BCA protein assay ( Pierce ) according to the manufacturer's directions . Fluorescence for each virus preparation was determined as described [19] . BHK-21 or MDCK cells were transfected with plasmids ( generous gifts from Dr . Terry Dermody , Vanderbilt University ) expressing either wild-type ( wt ) or dominant-negative ( DN ) Rab proteins using either Lipofectamine ( Invitrogen ) for BHK cells , or Effectene ( Qiagen ) for MDCKs according to the manufacturers instructions . The wt and DN-Rab5 ( S34N; [39] , [40] ) , Rab7 ( N125I; [41] ) , or Rab11 ( S25N; [42] , [43] ) proteins were fused to eGFP in the plasmid p-eGFP ( Clontech ) . Eighteen hours post-transfection the BHK-21 cells were infected with rVSV-wt ( MOI = 10 ) and the MDCK cells were infected with A/Aichi/2/68 ( H3N2 ) influenza ( influenza stocks were a kind gift from Dr . Charles Russell , St . Jude Children's Research Hospital ) and fixed at 8 hr ( VSV ) , or 15 hr ( influenza ) post-infection . The cells were then stained for VSV N protein using monoclonal 10G4 [24] , or a polyclonal influenza NP antibody conjugated to biotin ( generous gift from Dr . Richard Webby , St . Jude Children's Research Hospital ) . Fluorescence micrographs were collected using a Zeiss Axioplan 2 microscope equipped with an HR Axiocam camera and Axiovision software . Percent inhibition was determined from approximately 90 to 150 cells in 10–20 individual fields by counting the total number of Rab-positive ( eGFP-positive ) cells in a field that were also VSV N- or influenza NP-positive using the formula [ ( 1− ( virus-positive cells/Rab-positive cells ) ) *100] . rVSV-MLG infected cells were washed twice with phosphate-buffered saline , pH 7 . 4 ( PBS ) and then fixed for 15 minutes with 3% paraformaldehyde ( PFA ) in PBS at room temperature . The fix solution was removed and the cells washed twice with PBS containing 10 mM glycine and 0 . 05% sodium azide ( PBS-glycine ) , and then permeabilized with 1% Triton X-100 in PBS-glycine at ambient temperature for 1 minute . After permeabilization , the cells were washed twice with PBS-glycine and then stained with the following antibodies as indicated in the figure legends: a ) M protein using monoclonal antibody ( mAb ) 23H12 [24] conjugated to rhodamine , b ) VSV N protein using mAb 10G4 [24] conjugated to rhodamine or after incubation with a secondary goat anti-mouse antibody conjugated to Alexa Fluor-633 , c ) Nup62 using mAb #610497 ( BD Biosciences ) , d ) mannose 6 phosphate receptor ( M-6-P-R ) using mAb #MA1-066 ( Affinity BioReagents ) , e ) LAMP-1 using an anti-LAMP-1 mAb ( Developmental Studies Hybridoma Bank at the University of Iowa ) , f ) EEA1 using a polyclonal antibody ( #2411 , Cell Signaling Technology ) , g ) Rab11 using a polyclonal antibody ( #71-5300 , Zymed Laboratories ) , or h ) microtubules using anti-α tubulin mAb ( #A-11126 , Molecular Probes ) . The unconjugated primary antibodies were detected with goat anti-mouse , or goat anti-rabbit secondary antibody conjugated to rhodamine ( Jackson Research Laboratories ) , or for anti-Nup62 , goat anti-mouse labeled with Zenon IgG2b Alexa Fluor-647 ( #A-21242 Invitrogen; Molecular Probes ) . Phalloidin conjugated to Texas Red-X ( Invitrogen; Molecular Probes ) was used to stain actin . The distribution of the indicated proteins was examined using laser scanning confocal microscopy ( Zeiss LSM 510 and AIM software version 3 . 2 ) with the multi-track setting and 488nm , 543nm , or 633nm laser excitation in 1 micron optical sections . Percent colocalization of M and N protein was accomplished using the Profile function of the Zeiss LSM Physiology software package by counting the number of N protein puncta that colocalized with M in a total of 50 cells from three different experiments . Colocalization of MLG with the endosomal markers was quantified by selecting individual cells ( n = 5 ) and using the colocalization function of the LSM Physiology software . Colocalization of MLG with endosomal markers was determined from a 1 micron optical slice near the center plane of the cell . For colocalization analysis , the threshold settings for both green and red pixels were set at 100 . BHK-21 cells plated onto 35 mm glass bottomed culture dishes ( MatTek Corporation ) were washed twice with ice-cold Opti-MEM I ( Invitrogen ) and then placed at 4°C for 15 minutes . Lumio-labeled virus ( MOI 50 ) and human transferrin conjugated to Texas Red ( 50 µg ) were adsorbed in 0 . 1 ml ice-cold Opti-MEM I for 60 minutes with rocking every 15 minutes on ice . Surface binding was examined by washing the cells 3 times with ice cold PBS . The cells were placed on ice and washed once with ice-cold 100µM desferrioxamine in PBS for 5 minutes to chelate residual iron . The cells were then washed once with ice-cold acid wash buffer ( 100mM sodium acetate , 50mM NaCl , pH 5 . 5 ) for 5 minutes to release transferrin-TR from transferrin receptors on the cell surface . After the acid washing the cells were washed 3 times with PBS and then incubated for 5 minutes in Opti-MEM I at 37°C . For MLG trafficking without acid washing , the ice-cold inoculum was replaced with Opti-MEM I warmed to 37°C and the cells were incubated for the times indicated . MLG and Tfn-TR were imaged in live ( non-fixed ) cells on a Zeiss LSM 510 laser scanning confocal microscopy using a heated stage set to 37°C and an objective heating collar using alternating 488nm and 543nm laser excitation in multi-track mode . The images were collected using identical detector gain and offset settings for each time point . The non-entry time point ( t-0 ) was examined following addition of 2 ml of ice-cold Opti-MEM I on a stage at ambient temperature . To reduce problems of sample photobleaching , separate plates were examined for each time point . The fractionation protocol was essentially that described previously by others [44] , with the following modification for infection with VSV . BHK-21 cells plated in 35 mm dishes were washed twice with ice-cold Opti-MEM I ( Invitrogen ) and then placed at 4°C for 10 minutes . rVSV-wt ( MOI 50 ) was adsorbed in 0 . 3 ml ice-cold Opti-MEM I for 90 minutes with rocking every 15 minutes on ice . The inoculum was removed and cells were washed twice with ice-cold Opti-MEM I and then twice with ice-cold PBS . The cells were then either harvested immediately by incubation with 1 ml ice-cold MES buffer ( 150 mM NaCl with 25 mM MES , pH 6 . 5 ) for approximately 10 minutes ( until the cells could be removed from the plate by pipetting ) , or were incubated for the times indicated in Opti-MEM I at 37°C . After incubation at 37°C the cells were quickly cooled by the addition of ice-cold PBS and then incubation for 10 minutes in ice-cold MES buffer after which the cell suspensions were transferred into a 1 . 5 ml microfuge tube on ice . All subsequent steps were performed on ice and with a microcentrifuge cooled to 4°C . Cells were disrupted using a 1 ml syringe fitted with a 25-gauge needle by forcing the cell suspension rapidly through the needle 20 times . Cell disruption was assessed via trypan blue staining , which showed >95% of the cells were trypan blue permeable after the treatment . The cell suspension was then centrifuged at 1000×g for 10 minutes . The supernatant was transferred to a clean tube on ice and the supernatant fraction was centrifuged again at 1000×g , to remove residual pelletable material . The first 1000×g pellet was kept on ice . The supernatant was transferred to a new tube and then spun at 16 , 000×g for 10 minutes . The pellet from the 16 , 000×g spin ( P16 ) was washed once with ice-cold MES buffer , repelleted and then resuspended in SDS-PAGE sample buffer . The supernatant ( S16 ) was precipitated with 10% trichloroacetic acid ( TCA ) and the pellet resuspended in SDS-PAGE sample buffer . The pellet from the initial 1000×g spin was washed once with ice-cold MES buffer , respun and then the pellet was resuspended in NDG buffer ( 1% Nonidet-40; 0 . 5% deoxycholate; 10% glycerol; 137 mM NaCl and 20 mM Tris , pH 8 . 0 ) . After incubation on ice for 2 minutes the suspension was centrifuged at 16 , 000×g for 10 minutes . The pellet ( NDG pellet ) was washed once in NDG buffer , repelleted and then resuspended in SDS-PAGE sample buffer . The supernatant ( NDG supt ) was TCA precipitated and suspended in SDS-PAGE sample buffer . Fractions were electrophoresed on a 9% acrylamide gel containing SDS , transferred to Immobilon membrane and processed for immunoblot detection using the following antibodies with detection using the Pierce Dura-West Detection Reagent as described by the manufacturer . N and M proteins in the relevant fractions were quantified using Image J after scanning films and importing the images into Photoshop ( Adobe ) as . TIFFs . Antibodies used were a ) polyclonal anti-VSV ( #4006-F; Whitt lab ) , b ) anti-Nup62 ( mAb #610497; BD Biosciences ) , c ) anti-EEA1 ( polyclonal antibody #2411; Cell Signaling Technology ) , d ) anti-Rab11 ( polyclonal antibody #71-5300; Zymed Laboratories ) , e ) anti-α tubulin ( mAb #A-11126; Molecular Probes ) , and f ) anti-mitochondrial membrane protein OxPhos Complex-I 39 kDa ( CI-39; mAb #A21344; Molecular Probes ) . To prevent the acidification of endosomes we used either the proton ATPase inhibitor bafilomycin A1 as described previously [19] , or the lysosomotropic reagent NH4Cl . For NH4Cl treatment used to synchronize fusion of virions with endosomal membranes , BHK-21 cells were washed twice with PBS and then washed twice with PBS containing 100mM NH4Cl . Virus inocula , either with or without 50 µg of transferrin-TR , were adsorbed in serum-free DMEM ( SF-DMEM ) containing 100mM NH4Cl for 60 or 90 minutes . After adsorption the cells were washed with PBS four times to remove the NH4Cl and then either fixed immediately ( t-0 ) with 3% paraformaldehyde in PBS , pH 7 . 4 , or after 60 minutes ( t-60 ) following the addition of 2 ml of SF-DMEM containing 10µg/ml cycloheximide at 37°C . Synchronized fusion assays using the cytoskeletal inhibitors cytochalasin D or nocodazole were performed as described above except virus was adsorbed for 60 minutes in SF-DMEM with 100mM NH4Cl and the medium was replaced with SF-DMEM containing 10µM of the cytoskeletal inhibitor and 100mM NH4Cl for 30 minutes . The NH4Cl was then washed out as described above except that the cytoskeletal inhibitors remained for the time indicated and then the cells were fixed with 3% PFA and prepared for IF . | Vesicular stomatitis virus ( VSV ) is a prototypic enveloped virus that enters cells following endocytosis and a low pH-dependent membrane fusion event between the viral and endosomal membrane . To initiate a productive infection the viral nucleocapsid must dissociate from the matrix ( M ) protein , which underlies the viral membrane , in a process known as uncoating . The requirements for VSV uncoating are poorly understood . Here we used a virus containing fluorescent M protein to follow VSV uncoating in live cells . This analysis resulted in three new findings which provide for the first time a description of matrix and nucleocapsid trafficking during VSV uncoating . We found that most of the M protein remains bound to the endosomal membrane after virus-endosome fusion and that the nucleocapsid is released into the cytoplasm where replication occurs . While most of M remains membrane-bound , a small but detectable fraction is released during uncoating and is trafficked to nuclear pores . This has not been previously observed and may aid in shutting down host responses to infection . Collectively we provide the first spatio-temporal description of VSV uncoating by visualizing the uncoating process in live cells . | [
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] | 2010 | A Spatio-Temporal Analysis of Matrix Protein and Nucleocapsid Trafficking during Vesicular Stomatitis Virus Uncoating |
Mycobacterium tuberculosis is sensitive to nitric oxide generated by inducible nitric oxide synthase ( iNOS ) . Consequently , to ensure its survival in macrophages , M . tuberculosis inhibits iNOS recruitment to its phagosome by an unknown mechanism . Here we report the mechanism underlying this process , whereby mycobacteria affect the scaffolding protein EBP50 , which normally binds to iNOS and links it to the actin cytoskeleton . Phagosomes harboring live mycobacteria showed reduced capacity to retain EBP50 , consistent with lower iNOS recruitment . EBP50 was found on purified phagosomes , and its expression increased upon macrophage activation , paralleling expression changes seen with iNOS . Overexpression of EBP50 increased while EBP50 knockdown decreased iNOS recruitment to phagosomes . Knockdown of EBP50 enhanced mycobacterial survival in activated macrophages . We tested another actin organizer , coronin-1 , implicated in mycobacterium-macrophage interaction for contribution to iNOS exclusion . A knockdown of coronin-1 resulted in increased iNOS recruitment to model latex bead phagosomes but did not increase iNOS recruitment to phagosomes with live mycobacteria and did not affect mycobacterial survival . Our findings are consistent with a model for the block in iNOS association with mycobacterial phagosomes as a mechanism dependent primarly on reduced EBP50 recruitment .
Approximately one-third of the world's population is asymptomatically infected with Mycobacterium tuberculosis . This represents the reservoir leading to 8 million cases of active disease and 2 million deaths annually from tuberculosis . Being an intracellular pathogen , M . tuberculosis is able to infect and replicate in host macrophages by defending against and manipulating macrophage antimicrobial responses [1] . After entry into macrophages via phagocytosis , M . tuberculosis has the capacity to avoid phagosome-lysosome fusion by interfering with phagolysosome biogenesis pathways , a trait that has been well recognized and studied [2–6] . The important questions that have not been fully addressed thus far are whether or not and how M . tuberculosis manipulates other macrophage microbicidal responses aside from phagolysosomal biogenesis , such as the production of reactive oxygen intermediates and reactive nitrogen intermediates ( RNI ) . The production of nitric oxide ( NO . ) and other reactive nitrogen metabolites has been shown to play an important effector role in innate immunity [7] . NO . is a highly reactive and diffusible free radical , soluble in both lipids and water , and capable of reacting with oxygen and reactive oxygen intermediates to form NO2 , NO2− , NO3− , N2O3 , and the highly mycobactericidal ONOO− [8–10] . Inducible nitric oxide synthase ( iNOS ) is the major generator of RNI in immune cells and is a tightly regulated enzyme [11] . It has been demonstrated that iNOS is important in immune control of M . tuberculosis as evidenced by compromised handling of infections in mice lacking the gene for iNOS [12]; iNOS is believed to perform a similar function in human macrophages , although this issue has not been fully resolved [12] . Studies investigating defenses against RNI in M . tuberculosis have demonstrated the importance of several mycobacterial proteins against RNI [13 , 14] , further underscoring the potential importance of iNOS in host protection against M . tuberculosis . Since iNOS products are highly reactive radicals , if NO . is generated at intracellular sites distant from intended targets ( e . g . , away from phagosomes containing bacteria ) , it may be consumed or spent before it can reach the microbe . Thus , the proximity of iNOS to the intracellular targets is likely to play a role . Recently , it was reported that M . tuberculosis inhibits trafficking of iNOS to the mycobacterial phagosome [15] . Here , we investigated the mechanism by which iNOS is normally recruited to phagosomes and the properties of the mycobacterial phagosome responsible for iNOS exclusion . Since trafficking of iNOS to phagosomes in a macrophage depends on a functional actin cytoskeleton [15] , we looked at actin cytoskeleton regulation and iNOS scaffolding molecules interacting directly or indirectly with actin . Ezrin/radixin/moesin ( ERM ) -binding phosphoprotein 50 ( EBP50 ) , also known as Na/H+ exchange regulatory factor 1 , is a scaffolding protein responsible for the anchoring of various cellular proteins to the actin cytoskeleton through its linkage to ERM proteins . A 358-residue protein , with an ERM-binding domain and two PSD-95/Dlg-1 , Drosophila disk large/ZO-1 ( PDZ ) domains , EBP50 has been shown to bind to iNOS through one of its PDZ domains and the SAL motif at the C-terminal end of iNOS [16] . We report that the localization of iNOS to phagosomes is an EBP50-dependent process and that EBP50 , like iNOS , is upregulated in macrophages activated with interferon-gamma ( IFN-γ ) and lipopolysaccharide ( LPS ) . EBP50 is targeted by live mycobacteria to inhibit recruitment of iNOS to the vicinity of their phagosomes . This provides a new understanding of how iNOS is trafficked in macrophages .
Model latex bead phagosomes have been used to demonstrate actin-dependent recruitment of iNOS to the vicinity of phagosomes formed in IFN-γ- and LPS-activated macrophages [15] . This process is disrupted when cells are infected with M . tuberculosis . Since mycobacterial factors affecting intracellular trafficking in infected cells include preformed products such as lipids [6] , we tested whether mycobacterial viability was required for iNOS exclusion . To induce iNOS expression , RAW 264 . 7 macrophages were treated with IFN-γ and LPS for 16 h , infected for 10 min with latex beads , live or heat-killed M . tuberculosis var . bovis BCG labeled with Texas Red under conditions not affecting bacterial viability [4] , and localization of endogenous iNOS relative to the phagosomes analyzed by fluorescence confocal microscopy ( Figure 1A ) . In accordance with previous studies , phagosomes harboring live mycobacteria showed a reduction in iNOS recruitment as compared to latex bead phagosomes ( 32% ± 3% versus 47% ± 4% , p = 0 . 0484 ) ( Figure 1B ) . In contrast , heat- killed mycobacteria were unable to disrupt the localization of iNOS to the phagosome when compared to latex bead phagosomes ( 54% ± 4% versus 47% ± 4% , p = 0 . 2870 ) ( Figure 1B ) . These results indicate that the inhibition of iNOS recruitment to the vicinity of mycobacterial phagosomes is an active mechanism that requires live mycobacteria . In polarized epithelial cells , iNOS localization depends on EBP50 [16] . Here we tested the role of EBP50 on iNOS distribution in macrophages . Latex bead phagosomes were isolated as previously described [15] ( Figure 2A ) and probed for EBP50 by western blotting ( Figure 2A ) . Endogenous EBP50 was detected on latex bead compartments , purified by floatation . This was confirmed using an independent phagosome purification protocol , with phagosomes containing magnetic beads . Along with EBP50 , iNOS was present on purified model magnetic bead phagosomes ( Figure 2B ) . This result indicates that iNOS and EBP50 are both recruited to model latex bead , or magnetic bead phagosomes . Furthermore , EBP50-GFP colocalized with iNOS in macrophages ( Figure 2C ) . A functional role of EBP50 in iNOS recruitment to phagosomes was tested next . Activated macrophages expressing EBP50-GFP were infected with latex beads and analyzed by fluorescence microscopy for localization of endogenous iNOS to model latex bead phagosomes . Latex bead phagosomes in cells expressing EBP50-GFP showed some increase in iNOS recruitment to the latex bead phagosomes when compared to cells expressing control enhanced green fluorescent protein ( 56% ± 11% versus 41% ± 5 . 5% ) , albeit the statistical significance cut-off of <0 . 05 was not reached ( Figure 3A and 3B ) . Nevertheless , this trend suggested that EBP50 might play a role in trafficking of iNOS to phagosomes . To test this , short interfering RNA ( siRNA ) directed against EBP50 was used to knockdown the endogenous EBP50 . Macrophages were transfected with either control ( scramble ) siRNA or EBP50 siRNA , treated with IFN-γ and LPS for 16 h , and then infected with latex beads . EBP50 knockdown expression was confirmed by western blotting ( Figure 3C ) . In cells with EBP50 siRNA , there was a decrease in recruitment of iNOS to the latex bead phagosome as compared to cells transfected with control siRNA ( 30% ± 2% versus 40% ± 2 . 5% p = 0 . 0254 ) ( Figure 3D and 3E ) . This level of decrease was similar to that observed in comparisons between phagosomes harboring live mycobacteria , dead mycobacteria , or latex beads ( Figure 1 ) . These results indicate that EBP50 plays a role in the recruitment of iNOS to latex bead phagosomes . Finally , we tested the involvement of EBP50 in recruitment of iNOS to mycobacterial phagosomes . In cells transfected with EBP50 siRNA , iNOS localization to phagosomes containing dead mycobacteria was reduced by 37% ± 7% compared to cells transfected with control siRNA ( Figure 3F and 3G ) . Overall , these results demonstrate that EBP50 is essential for recruitment of iNOS to model and mycobacterial phagosomes . Expression of iNOS is induced upon macrophage stimulation with IFN-γ and LPS . If EBP50 controls iNOS localization , we hypothesized that EBP50 expression might also be affected . We tested whether EBP50 protein levels changed upon macrophage activation with IFN-γ and LPS . Cells were incubated in media ( control ) or media containing IFN-γ ( 200 U/ml ) and LPS ( 200 ng/ml ) for 16 h , and then protein extracts analyzed for EBP50 content by western blotting . As expected , iNOS was undetectable under resting conditions , but was strongly upregulated upon treatment of IFN-γ and LPS ( Figure 4A ) . EBP50 showed a similar induction pattern , although some EBP50 was detectable even without activation . Upon stimulation , there was a strong increase in EBP50 levels ( Figure 4A and 4C ) . Increase in expression of EBP50 by IFN-γ and LPS parallels that of iNOS , consistent with a model in which EBP50 directs the proper trafficking of iNOS in macrophages . To determine the contribution of different signaling pathways ( LPS and IFN-γ ) in EBP50 induction , macrophages were treated separately with LPS or IFN-γ . EBP50 was upregulated upon treatment with LPS but not with IFN-γ alone; iNOS was also upregulated with LPS and to a lesser extent with IFN-γ alone ( Figure 4B and 4D ) . The expression of EBP50 was further increased when IFN-γ was included along with LPS treatment ( Figure 4B and 4D ) . These data indicate that EBP50 expression is regulated primarily by the LPS signaling pathway , and that , as for iNOS , IFN-γ synergizes with the LPS effect . To investigate whether EBP50 played a role in mycobacterial effects on iNOS distribution , we examined recruitment of EBP50 to the phagosomes containing latex beads , live and dead mycobacteria , applying the previously described 4-D confocal microscopy approach [17] . Macrophages were transfected with EBP50-GFP , treated with IFN-γ and LPS for 16 h , and then allowed to phagocytose latex beads or mycobacteria . The dynamics of EBP50 recruitment to the phagosome was monitored by live confocal microscopy . Latex bead phagosomes rapidly acquired EBP50-GFP upon entry into macrophages ( Figure 5A and 5D , Video S1 ) , followed by its loss at later time points . A rapid recruitment of EBP50 was also observed with dead and live mycobacteria phagosomes ( Figure 5B–5D , Videos S2 , S3 , and S4 , respectively ) . However , live mycobacterial phagosomes lost EBP50 faster than other phagosomes ( Figure 5C and 5D , Videos S3 and S4 ) . These kinetic studies strongly indicate that live mycobacteria diminish the dwell time of EBP50 on phagosomes resulting in its premature desorption relative to phagosomes with dead mycobacteria or latex beads . The time-resolved studies with EBP50 localization relative to phagosomes are indicative of the positive role of EBP50 in iNOS localization . However , the transient association of EBP50 with phagosomes suggests that other factors could play a role in maintaining the differences in steady-state distribution of iNOS relative to phagosomes with live mycobacteria versus dead mycobacteria or latex beads . Hence , we investigated another actin-organizing element , coronin-1 , known to be recruited to phagosomes with live but not dead mycobacteria [18] . Thus , we hypothesized that recruitment of coronin-1 to live mycobacterial phagosomes could affect iNOS distribution , since our earlier studies showed that actin rearrangements are essential for proper positioning of iNOS [19] . To test this hypothesis , coronin-1 expression was knocked down by siRNA and the distribution of iNOS relative to phagosomes examined . Cells were transfected with either control ( scramble ) siRNA or coronin-1 siRNA , treated with IFN-γ and LPS for 16 h , and then allowed to phagocytose latex beads . In cells treated with coronin-1 siRNA , there was a significant increase in the recruitment of iNOS to the latex bead phagosome as compared to cells transfected with control siRNA ( 65 . 2% ± 0 . 8% versus 48 . 0% ± 0 . 9% p < 0 . 0001 ) ( Figure 6A and 6B ) . Coronin-1 knockdown expression was confirmed by western blotting ( Figure 6C ) . Next , we tested whether coronin-1 knockdown could rescue iNOS recruitment to live mycobacteria phagosomes . Cells were transfected with either control ( scramble ) siRNA or coronin-1 siRNA , treated with IFN-γ and LPS for 16 h , and then allowed to phagocytose live mycobacteria . Cells transfected with coronin-1 siRNA showed the same percentage of iNOS localization with live mycobacterial phagosomes relative to cells transfected with control siRNA ( Figure 6D ) . These results show that coronin-1 plays a negative regulatory role in the recruitment of iNOS to model phagosome , but it is not a contributing factor in mycobacteria-induced inhibition of iNOS recruitment to phagosomes . If iNOS recruitment to mycobacterial phagosomes occurs via EBP50 , then a knockdown of EBP50 should increase survival of mycobacteria in macrophages . In order to test this , RAW 264 . 7 murine macrophages were transfected with control ( scramble ) , EBP50 , or coronin-1 siRNA , activated with IFN-γ and LPS for 16 h , and infected with M . bovis BCG . At indicated time points , cells were lysed and plated for colony forming unit ( CFU ) determination . The EBP50 knockdown resulted in a significant increase in mycobacterial survival compared to control siRNA ( Figure 7 ) . However , no difference in BCG survival was seen in macrophages transfected with coronin-1 siRNA ( Figure 7 ) . These results indicate that EBP50 is essential for mycobactericidal activity in LPS- and IFN-γ-treated macrophages .
In this study , we have demonstrated that EBP50 plays a significant role in the localization of iNOS to phagosomes in macrophages . A plausible model emerges with EBP50 linking iNOS and the actin cytoskeleton to phagosomes and explains in part how mycobacteria inhibit iNOS recruitment due to reduced EBP50 dwell time on mycobacterial phagosomes . Our findings , along with previous observations [15] , demonstrate that iNOS is not properly localized relative to the phagosomes harboring live M . tuberculosis , and that this localization of iNOS includes an EBP50-dependent mechanism . M . tuberculosis is an intracellular pathogen that is exceptionally well adapted to ensure its intracellular survival and persistence in macrophages . It has evolved mechanisms to counter multiple and often independently acting bactericidal effectors in phagocytic cells . In this study we have demonstrated that live mycobacteria are able to inhibit localization of iNOS to the phagosome . This finding indicates that the mislocalization of iNOS relative to phagosomes is an active process similar to other phenotypes observed only with live mycobacteria , such as inhibition of phagosome maturation [3 , 4 , 18 , 20] . The mechanism responsible for a decrease in iNOS localization to live mycobacterial phagosomes involves a kinetic change in EBP50 association with mycobacterial phagosomes . Previous observations have demonstrated that the SAL motif at the C-terminal end of iNOS can bind to a PDZ domain of EBP50 in epithelial cells [16] . The SAL motif was further found to be important in the proper apical localization of iNOS and vectorial output of NO in epithelial cells , allowing for maximal efficiency of delivering NO . to its targets while reducing general cellular toxicity . We found that EBP50 copurifies with iNOS on isolated model phagosomes . Furthermore , EBP50-GFP and iNOS colocalize by immunofluorescence in macrophages . Biochemical and morphological colocalization of iNOS and EBP50 is not coincidental , as functional experiments both with EBP50-GFP and endogenous EBP50 knockdowns have internally consistent effects on iNOS localization . Depletion of endogenous EBP50 by siRNA inhibited iNOS localization to both model and dead mycobacteria phagosomes similar to the effects seen with live mycobacteria . Most importantly , a knockdown of EBP50 interferes with mycobactericidal mechanisms in LPS- and IFN-γ-activated macrophages . Other pathogens have been reported to interfere with membrane-cytoskeleton linker proteins such as EBP50 and the ERM family of proteins [21 , 22] . Listeria monocytogenes , another intracellular pathogen , was shown to recruit ERM proteins to form plasma membrane protrusions used for cell-to-cell transmission [22] . These protrusions are dependent on ERM protein interactions with a membrane component and actin , as well as proper phosphorylation of ERM proteins . Another study has identified EBP50 as a molecular target for the Enteropathogenic Escherichia coli effector protein Map [21] . With a motif similar to the PDZ binding motif ( SAL ) of iNOS , Map uses a TRL sequence to bind to the PDZ1 domain of EBP50 . In the case of Map , its binding to EBP50 induces a proteolytic degradation of EBP50 , causing a cleavage of the ezrin-binding domain in the Map-bound EBP50 , rendering the affected EBP50 unable to associate with its ERM binding partners . With effects on pathophysiology of infections [21] , EBP50 modulates the proper localization of its partner ERM proteins [23] in a process that controls its own localization . Taken together , the precedents of pathogen interactions with EBP50 and ERM proteins , the in vitro role of ERM proteins and actin on general phagosome biology [24 , 25] , and the role of EBP50 in iNOS recruitment to phagosomes support the targeting of EBP50 by M . tuberculosis as a process contributing to exclusion of iNOS and mycobacterial survival [21 , 22 , 26] . Previously , actin [25 , 27 , 28] and the ERM proteins ezrin [25] and moesin [25 , 27] have been found on model latex bead phagosomes . These reports and our findings indicate that iNOS is most likely recruited by EBP50 and ERM proteins to the vicinity of the phagosomal membrane . An EBP50-iNOS complex is recruited to the phagosome by EBP50 , possibly in association with ERM proteins ultimately leading to entrapment of iNOS in an actin network polymerized around the phagosome [25 , 27 , 28] . This model of EBP50 playing a role in positioning of iNOS is supported by the observation that endogenous EBP50 levels are increased coordinately with iNOS upon macrophage activation . Macrophage activation-dependent increase in EBP50 levels places this protein into the category of immune response factors . Coronin-1 , which acts as a negative regulator of actin branch polymerization [19 , 29] , still lacks a physiological role explaining its previously reported survival-conferring association with live mycobacterial phagosomes [18] , albeit some effects on Ca2+ have been recently reported [30] . By being recruited to phagosomes and exerting its anti-actin branching effects [19] , coronin-1 may contribute to iNOS localization as demonstrated here in the case of latex bead phagosomes . So why did knockdown of coronin-1 not result in iNOS recruitment to mycobacterial phagosomes ? Recently , Deghmane et al . [31] have shown that IFN-γ activation results in disappearance of coronin-1 from live mycobacteria phagosomes . This work explains why a knockdown of coronin-1 did not rescue iNOS recruitment to phagosome containing live mycobacteria . Alternatively , coronin-1 knockdown may not be sufficient to overcome the effect of mycobacteria on iNOS localization , via EBP50 . In conclusion , we have described a mechanism endowing mycobacteria to defend themselves against iNOS-dependent microbicidal capabilities of macrophages . Pharmacological approaches aimed at counteracting effects of mycobacteria on EBP50 may therefore lead to increased clearance of M . tuberculosis .
The murine macrophage cell line RAW 264 . 7 was maintained in DMEM supplemented with 4 mM L-glutamine and 10% fetal bovine serum ( FBS ) . Mycobacterium tuberculosis var . bovis BCG was grown in Middlebrook 7H9 broth , and BCG expressing GFP was maintained in 7H9 broth containing 25 μg/ml kanamycin . Mycobacterium was heat-killed for 10 min at 90 °C . Mycobacteria ( live and dead ) and latex beads ( 1 . 0 μm and 3 . 0 μm ) were labeled with Texas Red-succinimidyl ester ( 0 . 5 mg/ml ) , or Alexa 568 succinimidyl ester ( 5 μg/ml ) in PBS for 1 h . Labeled mycobacteria and latex beads were washed three times in PBS , homogenized , and opsonized in DMEM supplemented with 10% FBS . Viability of bacterial cultures was determined by comparing the OD600 counts ( where an OD600 of 0 . 1 is equivalent to 1 × 107 cfu/ml ) to those obtained by plating for CFU determination . Percentage of viability was 76 . 05 ± 6 . 891 . EBP50-GFP was from Dr . S . Lambert ( University of Massachusetts Medical School , Massachusetts ) . Transfection of RAW 264 . 7 cells was carried out as previously described [4] . Briefly , 5 × 106 cells were resuspended in nucleoporator buffer ( Amaxa Biosystems ) containing 5 μg of DNA or 1 . 5-μg siRNA , and nucleoporated according to manufacturer's protocol . Cells were then either plated on cover slips or cultured in flasks 24 h prior to bacterial infections or biochemical experiments . Rabbit polyclonal antibody to EBP50 was from Affinity BioReagents . Rabbit polyclonal and mouse monoclonal antibodies against iNOS were from Transduction Laboratories . Mouse monoclonal antibodies against GAPDH and actin were from Abcam . Coronin-1 antibody was from J . Pieters ( University of Basel , Switzerland ) . Murine IFN-γ , LPS , and latex beads were purchased from Sigma-Aldrich . Secondary antibodies conjugated to Alexa 488 and 568 were from Molecular Probes . RAW 264 . 7 macrophages were stimulated with IFN-γ and LPS ( 200 U/ml and 200 ng/ml ) . For immunofluorescence experiments , macrophages were seeded onto cover slips in 12-well tissue culture plates at a density of 3 . 0 × 105 cells per cover slip and exposed to 500 U/ml of IFN-γ and 500 ng/ml of LPS for 16 h prior to infection . For western blotting and purification of latex bead phagosomes , macrophages were incubated with both IFN-γ ( 500 U/ml ) and LPS ( 500 ng/ml ) in their respective flask for 16 h prior to lysis or homogenization . For immunoblotting , cells were washed in PBS and lysed with buffer containing 50 mM Tris HCl ( pH 7 . 4 ) , 150 mM NaCl , 0 . 25 % deoxycholate , 1 mM EDTA , 1% NP-40 , Leupeptin ( 10 μg/ml ) , Pepstatin ( 1 μg/ml ) , E64 ( 1 . 79 μg/ml ) , TLCK ( 10 μg/ml ) , 1 mM activated Na3VO4 , and 1 mM NaF . 50 μg of protein was loaded and separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose . Staining was revealed with SuperSignal West Dura chemiluminescent substrate ( Pierce ) . GAPDH and actin were used as a loading control . Latex bead phagosomal compartments were isolated from latex bead–infected RAW 264 . 7 macrophages as described previously [32] . Magnetic bead phagosomes were prepared as follows: Macrophages were incubated for 1 h at 37 °C , 5% CO2 with magnetic particles ( Polysciences ) ( 1/45 , v/v ) in 150 mM NaCl , 20 mM HEPES ( pH 7 . 4 ) , 6 . 5 mM glucose , 1 mg/ml BSA . After three washes with cold PBS , cells were lysed in homogenization buffer ( HB ) ( 250 mM sucrose , 20 mM Hepes , [pH 7 . 2] , protease inhibitors ) by passing through 22-gauge needles connected to a two-syringe apparatus . Post-nuclear-supernatant ( PNS ) was generated and phagosomes were isolated using a magnetic separator ( Polysciences ) , resuspended , and washed four times with HB . PNS ( 60 μg ) and phagosomes ( 20 μg ) were analyzed by western blot , equal amounts of protein were loaded for untreated and IFN-γ- and LPS-treated cells . RAW 264 . 7 cells grown on cover slips were fixed with 1% paraformaldehyde followed by membrane permeabilization using 0 . 2% Saponin or 0 . 1% Triton X-100 . After appropriate antibody incubations , cover slips were mounted using Permafluor Aqueous mounting medium ( Immunon ) . Collection of 1-μm thick optical sections was performed using an Axiovert 200 M microscope with an Axioscope 63× oil objective and LSM 5 Pascal or LSM 510 META systems ( Carl Zeiss ) . Images were cropped using Adobe Photoshop CS2 . An average of two phagosomes per cell were counted with an average of 135 cells per condition . Live cell confocal microscopy was performed as previously described [4] . For quantification , maximum intensity projections and mean-intensity projections were used for rendering and quantitative analysis as previously described [17] . Relative fluorescence intensity in cytosol ( RFUc ) was substracted from RFU on phagosome ( RFUp ) . Each kinetic was normalized to percentage of maximum RFU ( RFUp − RFUc ) . Mycobacteria were homogenized to remove clumps and centrifuged at 800 rpm for 1 min for synchronization of uptake by transfected RAW 264 . 7 macrophages . Cells were washed three times in PBS and lysed in cold water 1 h and 3 d post-infection . Serial dilutions of cell lysates were plated onto 7H11 plates containing ADC for CFU determination . The results were normalized to 1 h time point and are represented as percent relative to control siRNA . Statistical analysis was done on three independent experiments using Wilcoxon matched pairs test . | Mycobacterium tuberculosis infects one third of the world's population , with the majority of infected individuals being asymptomatic while running a lifetime risk of developing active disease . The key to the success of M . tuberculosis as a recalcitrant human pathogen is its ability to parasitize macrophages and persist in these cells or their derivatives for long periods of time . We still do not have complete knowledge of the full repertoire of M . tuberculosis determinants that allow it to evade bactericidal mechanisms of the macrophage . Here we report the mechanism by which M . tuberculosis eludes effective elimination by nitric oxide , a radical with antimycobacterial properties that is generated by the inducible form of nitric oxide synthase . It was generally assumed that nitric oxide synthase , upon induction by the major anti-tuberculosis cytokine interferon gamma , simply homogeneously fills up the macrophage like a sack and generates nitric oxide throughout the cell . The present study shows that nitric oxide synthase is not randomly distributed in macrophages , and that its positioning in the cell is dictated by interactions with the scaffolding protein EBP50 , shown here to be induced during macrophage activation . Thus , not only do the phagocytic cells increase the amount of nitric oxide synthase , but they also have a system to deliver and keep this enzyme in the vicinity of phagosomes . This is of significance , as nitric oxide is a highly reactive radical , and its generation somewhere else in the cell would lead to it being spent by the time it diffuses to the site of intended action , such as mycobacterium-laden phagosomes . It turns out , as this study shows , that M . tuberculosis interferes with the process of EBP50-guided positioning of the inducible nitric oxide synthase , thus avoiding delivery and accumulation of this enzyme and its noxious products near the phagosome where nitric oxide would have the best chance of inhibiting intracellular mycobacteria . | [
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] | 2007 | Mechanism of Inducible Nitric Oxide Synthase Exclusion from Mycobacterial Phagosomes |
Dengue is an important public health problem in the Philippines . We sought to describe the trends in dengue research in the country . We searched four databases and identified published studies on dengue research in the Philippines during the past 60 years . We reviewed 135 eligible studies , of which 33% were descriptive epidemiologic studies or case series , 16% were entomologic or vector control studies , 12% were studies on dengue virology and serologic response , 10% were socio-behavioral and economics studies , 8% were clinical trials , 7% were on burden of disease , 7% were investigations on markers of disease severity , 5% were on dengue diagnostics , and 2% were modeling studies . During the last decade , dengue research in the Philippines has increased and evolved from simple descriptive studies to those with more complex and diverse designs . We identified several key topics where more research would be useful .
Dengue is a mosquito-borne , acute febrile illness that is an important public health problem in tropical countries . In the early 1950’s , the disease was described in the Philippines as hemorrhagic fever or infectious acute thrombocytopenic purpura [1 , 2] . Dengue continues to cause considerable concern in the country because of its widespread endemicity , the minimal success of vector control strategies , the possibility of severe disease caused by sequential infection by a different serotype , the potential for fatal outcomes and the consequent social and economic burden . The four dengue virus serotypes circulate in the country where the disease is predominantly reported among children [3] . Findings from dengue studies could provide policy-makers with information needed for rational decision-making regarding dengue preventive and control efforts . The focus of dengue research may vary widely . This could include basic laboratory research , the estimation of dengue seroprevalence and incidence; the assessment of risk factors for severe disease; the quantification of its economic burden; the elucidation of local transmission and epidemiology; the development of improved diagnostic tests or the evaluation of interventions . We reviewed published studies on dengue research in the Philippines during the past 60 years . The objective of the review is to better understand the trends in dengue research and the findings from these studies . The results of the review could provide an impression of local capacity and infrastructure for dengue research and help determine important knowledge gaps . These gaps need to be identified since research interest and support for funding can only be achieved if scientists , decision makers and other stakeholders are able to understand developments related to the disease and recognize areas where more information is needed .
The Philippines is an archipelago of 7 , 107 islands and is located in the western Pacific Ocean in Southeastern Asia . The population of the Philippines in 2015 was 100 , 981 , 437 [4] . Philippine health status indicators show that the country lags behind most of Southeast and North Asia in terms of health outcomes [5] . Communicable diseases continue to be major causes of morbidity and mortality in the country . Health care in the Philippines is provided through a mixed public-private system . This systematic review was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analyses ( PRISMA ) guidelines [6] . In June 2018 , we searched articles on PubMed , the Cochrane Library , ScienceDirect and the Health Research and Development Information Network ( HERDIN ) from 1 January 1958 to 31 December 2017 combining MeSH and free-text terms for the following: dengue , “dengue fever” , “hemorrhagic fever” , “dengue hemorrhagic fever” , “dengue shock syndrome” , DF , DHF , DSS and Philippines without any language or age restrictions . The search on HERDIN , an electronic database of health research in the Philippines , was done to ensure that articles from local journals not indexed on international databases are included . The completed PRISMA checklist ( S1 Table ) is shown in the Supporting information . There is no protocol for this systematic review . The articles were compiled in Endnote ( Thomson Reuters , San Francisco , CA , USA ) . Titles and abstracts were screened for eligibility . Published articles on dengue research in the Philippines and on Filipinos that reported objectives , methods and results or descriptive epidemiologic and case reports were included . We excluded unpublished articles , studies that were not focused on dengue or not focused on the Philippines , those reporting aggregated results from various countries or analysis of a global or regional collection of viral isolates and specimens from which findings specific to the Philippines could not be retrieved , those reporting the same data from another publication ( duplicates ) , reviews and updates ( not original research ) , meeting or news reports , program descriptions , commentaries , guidelines on dengue ( prevention , treatment or diagnosis ) and studies on expatriates and non-Filipinos . Towards the goal of assessing the broad picture of dengue research in the Philippines , we included studies that met the basic standard requirements and did not exclude studies based on methodology or risk of bias or selective reporting . The relevant full papers were downloaded and reviewed in detail . Information from each eligible paper was extracted and entered into an Excel spread sheet ( Microsoft Office 2007 , Seattle , WA , USA ) . These included the study title , the year of publication , the journal , the study site primary location , type of study , brief methods and study findings . The summary measures were descriptive . We compared the annual number of Philippine-related dengue publications with other markers . As a measure of economic growth in the country , we assessed the Philippine Gross Domestic Product ( GDP ) per capita ( in current US dollars ) in 1960 ( the earliest year data was available ) and in 2017 [7] . For comparison , we also obtained the annual number of publications worldwide on PubMed combining the terms: dengue , “dengue fever” , “hemorrhagic fever” , “dengue hemorrhagic fever” , “dengue shock syndrome” , DF , DHF , DSS , from 1958 to 2017 , without location , language or age restrictions .
The most common studies during the 1960’s were descriptive and these types of studies continue to be published in recent years . The 44 publications included in this category described demographic , clinical and laboratory findings in Filipino patients with suspected or confirmed dengue in hospital or community settings [8–51] . One study of 100 patients who died of clinically-diagnosed dengue hemorrhagic fever reported necropsy findings of intravascular thrombosis and hemorrhages; dengue virus ( DENV ) was isolated in 32 per cent of the patients [18] . A re-analysis of dengue experimental infection studies in the 1920’s allowed the calculation of an average incubation period for dengue infection of about 6 days [33] . One article described the dengue prevention and response strategies applied after a natural disaster , Typhoon Haiyan that occurred in 2013 [44] while another paper characterized hospital admissions to a tertiary care hospital , including dengue cases , after the typhoon [47] . Five studies assessed the correlation between dengue fever and climate or weather patterns [34 , 35 , 40 , 41 , 51] . Longer-term comparative reporting and analysis of dengue fever from around the country would be useful to assess geographic and temporal epidemiologic patterns , risk factors for severe disease , variations in clinical management and changes in case-fatality rates . These studies help improve our understanding of the dengue vectors , which could be useful in developing effective control strategies . Of the 21 articles in this category [52–72] , six investigated dengue mosquito vector key breeding sites and potential interventions [52 , 56–58 , 60 , 64] , three described the response to or efficiency of vector control measures introduced in communities [54 , 59 , 61] , five assessed the larvicidal activity of various agents against Aedes aegypti [55 , 62 , 65 , 68 , 70] , three explored the characteristics and behavior of Ae . aegypti or Ae . albopictus [63 , 67 , 72] , one quantified vertical transmission of dengue viruses in Ae . aegypti [66] , two described the population and genetic changes of Ae . aegypti populations during the dry and wet seasons [53 , 69] and one investigated the role of different water-holding containers on the development of Ae . aegypti [71] . As newer strategies become available ( e . g . mosquito sterilization and Wolbachia-based approaches ) , it will be important to investigate these vector control methods in the country . In 1960 , an article described how viruses isolated from specimens collected in Manila ( 12 from human sera and 2 from wild-caught mosquitoes ) were adapted to suckling mice and shown to be dengue viruses [73] . This was followed by the publication of 15 studies on virologic and serologic aspects of dengue in the Philippines [74–88] . These included one from 1974 reporting how antibody assessments of sera collected from nine participants of dengue experimental infection studies in the 1920’s showed that DENV 1 and 4 were transmitted in these experiments [75] . Several studies described the isolation of various dengue serotypes circulating in the community [76 , 77 , 79 , 81 , 84] . A paper compared the nucleotide and amino acid sequences of the nonstructural-1 gene of dengue virus serotype 3 isolated in Metro Manila [78] and another described the molecular epidemiology of DENV 2 [82] . Two studies assessed the presence of dengue antibodies among monkeys in the Philippines suggesting possible sylvatic transmission cycles [80 , 86] . In another study , flow cytometric analysis of peripheral blood samples from clinically suspected dengue cases found that B cells are a major replication site for dengue viruses [83] . More recent studies described the continued circulation of a single genotype of DENV 2 in the Philippines [87] and the modulatory effects of compounds on dengue virus infected cells [88] . Continued monitoring of the circulating dengue viruses in the Philippines would help in understanding better the epidemiology of the disease . Together with epidemiologic studies that quantify the incidence and seroprevalence of disease , socio-behavioral and economic research provides information on how dengue impacts affected communities . There were nine dengue socio-behavioral studies [89–93 , 95 , 96 , 98 , 100] . Six assessed dengue-related knowledge and preventive practices in different communities [89 , 90 , 92 , 93 , 96 , 98] . Two were multi-country studies that included the Philippines and used questionnaires and focus group discussions to assess policymakers’ views on dengue and the need for a dengue vaccine [91] and health care providers’ use of dengue clinical guidelines [95] . One documented anecdotal use of a local herb in the treatment of dengue [100] . In light of the recent dengue vaccination controversy in the country , a study on policymakers’ understanding of dengue's complicated pathophysiology and immunologic responses would be useful in addressing unresolved issues and also for considering what would be needed when implementing future dengue control strategies . There were four economics studies [94 , 97 , 99 , 101] . One published in 2008 , prior to the licensure of the first dengue vaccine , used a contingent valuation survey and found a high willingness to pay and household demand for a dengue vaccine [94] . In another study , investigators assessed the economic and disease burden of dengue in 12 Southeast Asian countries [97] . For the Philippines , they calculated the direct cost for each hospitalized and ambulatory dengue case ( in 2010 US dollars ) of $177 and $47 , respectively , plus indirect costs of $36 and $17 , respectively . In a later publication , an annual average of 842 , 867 clinically diagnosed dengue cases in the Philippines was estimated , with direct medical costs ( in 2012 US dollars ) of $345 million ( $3 . 26 per capita ) [99] . The potential cost-effectiveness of a dengue vaccination program was discussed in another paper [101] . It will be useful to estimate the economic benefits of new dengue control methods in the country , as they become available . Of the 11 publications on dengue-related clinical trials , four were on therapeutic interventions [102–105] and seven were on vaccine trials [106–112] . The therapeutic interventions assessed included a hemostatic agent [102] , fluids [103] and immunoglobulin [104 , 105] . Multi-country randomized controlled trials of candidate dengue vaccines included study sites in the Philippines and the seven papers we identified reported on vaccine safety , immunogenicity and efficacy [106–108 , 110–112] , as well as concomitant dengue and MMR vaccination [109] . As newer dengue vaccines and therapeutics become available , it will be important to investigate these interventions in the country . Ten studies assessed the burden of dengue infections [113–122] . A study from 1992 reported an attack rate of 0 . 2 dengue cases per 1 , 000 population for the period of July to December 1990 in Zamboanga city [113] . On a national scale , the annual dengue surveillance data from the Philippines ( included among other countries in the World Health Organization Western Pacific Region ) showed dengue fever notification rates of 1 . 5 per 1 , 000 population in 2010 , 1 . 3 per 1 , 000 population in 2011 and 1 . 9 per 1 , 000 population in 2012 [115 , 116 , 118] . Another paper quantified epidemiologic trends in dengue disease burden in 5 Asian countries , including the Philippines , over a 30-year period using data from DengueNet and the WHO [122] . The estimated dengue incidence and mortality in the Philippines increased by 24% and 29% , respectively , but the authors acknowledged that implementation of more sensitive surveillance methods over the study period may have contributed to a reporting bias . These data provide an overall picture but are based on routine passive notification , often of clinically diagnosed cases , and may be weakened by incomplete reporting and delays . Among the burden of disease articles , incidence of laboratory-confirmed symptomatic dengue infections were estimated in several prospective surveillance studies that actively followed a cohort for acute febrile illness [114 , 117 , 119–121] . Incidence was calculated using the number of new cases arising from the defined cohort as the numerator and the years of observation time contributed by each person in the cohort as the denominator . Table 1 shows the estimated incidence of laboratory-confirmed symptomatic dengue infections from the articles . In the first study , Capeding and co-workers followed 4 , 441 healthy infants; and dengue infection was confirmed by serotype specific reverse transcriptase-polymerase chain reaction ( RT-PCR ) in acute-phase sera and dengue IgM/IgG enzyme linked immunosorbent assay ( ELISA ) in paired acute and convalescent phase sera [114] . The incidence of symptomatic ( clinically apparent ) infant dengue infections was 16 per 1 , 000 person-years ( Table 1 ) , of which hospitalized episodes occurred at 8 per 1 , 000 person-years . Serologic testing of serial blood samples from a subset of 250 infants without reported febrile illnesses in 2007 showed an incidence of clinically-inapparent dengue infections ( defined as a > 4-fold rise in dengue virus 50% plaque-reduction neutralization titers between two time points with a monotypic pattern ) , that was 6-fold higher than that of symptomatic infections at 103 per 1 , 000 person-years ( 95% CI 64–155 ) . Second , in a multi-center study , 300 healthy children 2 to 14 years at two sites in the Philippines were actively followed for febrile illness and dengue was confirmed using a nonstructural protein 1 ( NS1 ) antigen ELISA in acute serum samples and IgM/IgG ELISA in both acute and convalescent samples [117] . The incidence of confirmed symptomatic dengue infections was 34 per 1 , 000 person-years ( Table 1 ) . In the third study , 854 participants 6 months to over 50 years of age underwent active fever surveillance and annual serological assessment [119] . Acute sera were tested by dengue PCR and acute/convalescent samples by dengue IgM/IgG ELISA to identify symptomatic infections while enrolment and 12-month samples were tested by dengue hemagglutination inhibition assay to identify subclinical infections . The incidence of symptomatic dengue infection was 16 per 1 , 000 person-years ( Table 1 ) and clinically inapparent dengue infections occurred at 70 per 1 , 000 person-years ( 95% CI 54–90 ) . Symptomatic dengue rarely occurred in those older than 15 years . Fourth , two articles reported the incidence of virologically-confirmed dengue in the control group of a multi-center phase 3 trial of a dengue vaccine , including 1 , 166 participants 2 to 16 years of age at two Philippine study sites [120 , 121] . The children were followed for acute febrile illness and dengue infection was confirmed by means of both NS 1 antigen and RT-PCR assays . The incidence of symptomatic dengue infection was 66 per 1 , 000 person-years ( Table 1 ) , of which hospitalized episodes occurred at 7 per 1 , 000 person-years ( 95% CI 4–12 ) . In comparison with the national data described above , these incidence data provide a more accurate estimate of the burden of dengue because of the active surveillance in a defined cohort and the laboratory-confirmation of cases . But they are limited by having been conducted at only three sites ( Laguna , Metro Manila and Cebu ) in the country . The wide differences in incidence of laboratory-confirmed symptomatic dengue infections in the studies ( Table 1 ) are due to the different age groups in the cohort and varying time periods ( dengue has seasonal and cyclical epidemic patterns ) but may also reflect variations in the dengue force of infection across the sites . Additionally , differences in fever detection methods and diagnostic confirmatory tests may have contributed to the variation in the incidence estimates . We derived data on dengue seroprevalence in Filipinos from two studies that conducted baseline serologic assessments prior to fever surveillance [119 , 120] . First , among participants over 6 months of age in Cebu City , dengue seroprevalence assessed by hemagglutination inhibition assay increased sharply with age [119] . The proportion of participants with a multitypic dengue serologic profile was 40% in the 6 month to 5-year-old age group compared to 99% in the 31 to 50 year olds . Second , baseline dengue seropositivity prior to vaccination , assessed in 604 Filipino children by plaque-reduction seroneutralization assay , was 78% overall and 58% , 75% , 86% and 93% in the 2–4 , 5–8 , 9–12 and 13–16 year old age group , respectively [120] . Ten studies looked for associations between biomarkers and clinical presentation of dengue disease . Eight studies assessed levels of various immune-related or enzymatic biomarkers [123–127 , 130–132] , while two evaluated the potential role of adiposity [128 , 129] . More research is needed to better understand the host characteristics that contribute to dengue disease severity . There are several methods available for the diagnosis of dengue fever , including virus isolation , detection of viral components ( RNA or antigen ) and serological assays . In the Philippines , RT-PCR is the confirmatory test of choice but RT-PCR is expensive and time consuming , requires technical expertise and high-level laboratory equipment and does not provide immediate results that could be used for patient care . Dengue rapid diagnostic tests are used at the point-of-care but have insufficient sensitivity and specificity . We found seven published studies that assessed various dengue diagnostic tests , including ELISA [133–135 , 138] , fluorogenic real-time RT-PCR [136] and rapid diagnostic tests [137 , 139] . The gold standard used for comparison in these studies was conventional RT-PCR . Definitive diagnosis of dengue is important for the clinical management of patients , disease surveillance and outbreak investigations . A dengue diagnostic assay with sufficient sensitivity and specificity , that is less cumbersome than RT-PCR and with results immediately available for clinical care would be very useful . There were three studies that used modeling techniques to estimate dengue burden and describe disease patterns [140–142] . Using historical epidemiological , environmental , socio-economic and climate data , one study developed prediction models for future dengue incidence in the Philippines [140] . From an analysis of 18 years of dengue surveillance reports in eight countries in Southeast Asia , including the Philippines , investigators found strong patterns of synchronous dengue transmission across the entire region coinciding with elevated temperatures associated with anomalies in Pacific Ocean surface temperatures ( Oceanic Niño index ) [141] . Another study estimated 794 , 255 annual dengue episodes and a disease burden of 535 DALYs per million population in the Philippines extrapolated from passive routinely-collected data compared with results from a prospective community-based cohort study at one site [142] . Modeling studies may be useful in the evaluation of dengue interventions or control studies that become available in the future , especially when field studies are not feasible .
We report on published , dengue research in the Philippines during the past 60 years . During the last decade , there have been an increasing number of dengue studies in the Philippines . From the 1960’s to the 1990’s , the studies were mainly descriptive epidemiologic assessments and case series , but during the recent years , the types of investigations have become more complex and diverse . We believe this reflects advancement in local research capacity and infrastructure . The improvement has coincided with an increase in annual GDP per capita . Globally , there has also been an upsurge in dengue-related publications over the recent decades , probably due to an increasing interest in dengue together with its geographic expansion , more research publications from dengue-endemic countries , the assessment of recently developed strategies against the disease , as well as the proliferation of medical journals . Despite the increase in dengue research in the Philippines , we identified several dengue knowledge gaps . The vast majority were descriptive short-term hospital- or community-based studies . A longer-term comparative assessment of dengue epidemiologic patterns by site and year would be useful to understand the bigger picture of dengue in the country . As newer vector control methods and vaccine and therapeutic interventions become available , it will be important to investigate these strategies in the country . Sociobehavioral , economics and modeling studies related to these future interventions would be important to assess their impact . More studies on basic laboratory research , including continued monitoring of the circulating dengue viruses in the country and dengue serologic response would help to provide a better understanding of dengue epidemiology in the country . The incidence and seroprevalence data are available from a few sites and it is not known whether this is generalizable to other areas of the country . Aside from these important research areas , it is essential that basic dengue information and updated findings be communicated to policymakers , health workers , academics and other stakeholders . Researchers may need to liaison with the media to avoid miscommunication to the general public . This is especially important to avoid issues arising from misunderstanding when new control measures are implemented . Perhaps the recent controversy that surrounded the dengue vaccination program could have been avoided by prior detailed communication and education for more informed decision-making . There are several limitations of this review . First , although we searched four databases ( including a local repository ) , it is possible that some publications were missed . Second , there was some overlap in topics covered by some papers and we selected the main theme covered in the classification and assessment of results . Third , although the majority of the articles ( 117/135 or 87% ) included a Filipino author affiliated with a Philippine institution , foreign collaborators led many of the projects for which much of the laboratory work and data analysis were done outside the Philippines . Although dengue research capacity and infrastructure in the Philippines appears to have significantly increased during the recent decades , we are not able to exactly quantify the improvement . As local investigators gain more experience in developing proposals , obtaining grants and implementing research , we hope that more dengue projects will be lead by Filipino scientists . Fourth , this review on identifying dengue research gaps is just one step towards defining specific questions of interest on dengue in the Philippines . There needs to be a fuller engagement of scientists , policymakers and the public and the development of a continuing method to assess the evolving dengue research needs of the country . The importance of dengue research is justified by the data showing a significant burden of the disease . These studies indicated a symptomatic laboratory-confirmed dengue incidence of 16 to 66 per 1 , 000 person-years ( depending on the age group , the year when the study was done , the intensity of the surveillance method and the diagnostic method ) , while the incidence of hospitalized dengue was estimated at 7 to 8 per 1 , 000 person-years . Furthermore , clinically inapparent or asymptomatic dengue infections occur quite frequently , many folds higher than symptomatic dengue , due to the intense transmission of the virus . The available incidence and seroprevalence data confirm the high endemicity of dengue infections in the country , which results in a heavy socio-economic burden . The epidemiology of dengue varies in different geographical areas around the world . Describing what is happening in the Philippines can provide a template for other dengue-endemic areas . A standardized protocol could be developed from this and other reviews [143] for those who wish to conduct a similar activity in other dengue-endemic countries . Publishing data on the research needed to improve health care delivery is part of the communication that is central and key to successful implementation of public health programs . This is particularly true in the Philippines where dengue vaccination has recently been in the limelight when it was introduced in 2016 and stopped the year after . Initial introduction and subsequent events that resulted in highly controversial issues were partly due to misunderstanding of dengue's complicated pathophysiology and immunologic responses . In conclusion , this review showed that dengue studies in the country have increased in number and evolved from simple to more complicated types of investigations . We identified several important areas for increased research efforts . Studies such as this can help raise awareness on the significance of the disease and the need for better treatment and preventive strategies . | Dengue is a disease caused by four separate but related viruses transmitted by mosquitos . In this systematic review , we aimed to describe dengue research in the Philippines , where the disease is of great concern , to better understand the types of dengue research and the main findings and important gaps . We identified 135 studies that described dengue research in the Philippines during the past 60 years . Our review showed that in the early years , dengue studies were mainly simple descriptive studies and case reports . Recently the types of investigations have become more complex and diverse , reflecting advancement in local research capacity and infrastructure but more research activity would be beneficial in several areas . | [
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] | 2019 | Trends in dengue research in the Philippines: A systematic review |
The polarization of CD4+ T cells into distinct T helper cell lineages is essential for protective immunity against infection , but aberrant T cell polarization can cause autoimmunity . The transcription factor T-bet ( TBX21 ) specifies the Th1 lineage and represses alternative T cell fates . Genome-wide association studies have identified single nucleotide polymorphisms ( SNPs ) that may be causative for autoimmune diseases . The majority of these polymorphisms are located within non-coding distal regulatory elements . It is considered that these genetic variants contribute to disease by altering the binding of regulatory proteins and thus gene expression , but whether these variants alter the binding of lineage-specifying transcription factors has not been determined . Here , we show that SNPs associated with the mucosal inflammatory diseases Crohn’s disease , ulcerative colitis ( UC ) and celiac disease , but not rheumatoid arthritis or psoriasis , are enriched at T-bet binding sites . Furthermore , we identify disease-associated variants that alter T-bet binding in vitro and in vivo . ChIP-seq for T-bet in individuals heterozygous for the celiac disease-associated SNPs rs1465321 and rs2058622 and the IBD-associated SNPs rs1551398 and rs1551399 , reveals decreased binding to the minor disease-associated alleles . Furthermore , we show that rs1465321 is an expression quantitative trait locus ( eQTL ) for the neighboring gene IL18RAP , with decreased T-bet binding associated with decreased expression of this gene . These results suggest that genetic polymorphisms may predispose individuals to mucosal autoimmune disease through alterations in T-bet binding . Other disease-associated variants may similarly act by modulating the binding of lineage-specifying transcription factors in a tissue-selective and disease-specific manner .
The differentiation of naïve CD4+ T cells into distinct T helper cell ( Th ) lineages is essential for adaptive immunity . The original paradigm of interferon-gamma ( IFN-γ ) producing T-helper 1 ( Th1 ) , and type-2 ( Interleukin 4 , 5 , and 13 ) cytokine producing Th2 cells has expanded to include both Interleukin-17 ( IL-17 ) producing Th17 and anti-inflammatory T-regulatory ( Treg ) cells . Th cell differentiation is controlled by a set of master regulatory or lineage-specifying transcription factors , with the T-box family member T-bet necessary and sufficient for Th1 cell differentiation . GATA3 , RORγT and FOXP3 perform parallel roles in Th2 , Th17 and Treg cells , respectively [1] . Importantly , T-bet inhibits alternative lineage fate specification , repressing both the Th17 and Th2 lineages [2–4] . Inappropriate Th cell activation and polarization can lead to autoimmunity . Worldwide , autoimmune and auto-inflammatory diseases are now estimated to affect nearly 10% of the population [5] . The incidence of inflammatory bowel diseases ( IBD ) , including Crohn’s disease and UC , and celiac disease , is rising rapidly , with more than 1 . 4 million people affected in the USA alone [6] . A role for T-bet is particularly apparent in the mucosal immune system and has been linked to IBD and celiac disease [7] . The expression of T-bet is upregulated in lamina propria T cells of patients with Crohn’s and celiac disease and ex vivo culture of biopsies from untreated celiac patients with gliadin increases T-bet expression through STAT1 activation [8 , 9] . In addition to this , it is now apparent that mucosal inflammation is also driven by IL-17 , which is enhanced by IL-23 receptor signals in effector T cells [10] . Loss of T-bet in the innate immune system leads to a transmissible form of ulcerative colitis in the TRUC ( T-bet and Rag deficient Ulcerative Colitis ) model , driven by transcriptional derepression of TNF in colonic mononuclear phagocytes [11–13] . This susceptibility has also been shown to be dependent on IL-17 and mediated via repression of IL-7 receptor expression by T-bet in innate lymphoid cells ( ILCs ) [11] . T-bet has subsequently been shown to play a role in the development of the NKp46+ CCR6- subset of IL-22 expressing ILCs that , in turn , are important for protecting the epithelial barrier during Salmonella enterica infection [14 , 15] . Autoimmune diseases cluster in families , suggesting a large genetic component [16] . Genome-wide association studies ( GWAS ) have identified hundreds of risk loci for autoimmune diseases , including for IBD and celiac disease [16–23] . The majority of autoimmune disease-associated SNPs lie outside of gene coding regions in intergenic or intronic regions [24] . This can make it challenging to understand the molecular basis of how a genetic variant predisposes to disease . Furthermore , the causal variant can be difficult to identify from the large clusters of SNPs in linkage disequilibrium that tend to be identified by GWAS . Thus , efforts have been made to identify SNPs located within regulatory elements marked by open chromatin , histone modifications associated with active enhancers or known/predicted transcription factor binding sites [21 , 24–32] . Some of these variants have been shown to modulate transcription factor binding or epigenetic regulation . Genetic variants that alter DNase I hypersensitivity [27 , 33 , 34] , DNA methylation [35–38] , histone modification [27 , 39–43] , and the binding of transcriptional regulators to DNA [27 , 33 , 34 , 44–51] , have been identified , suggesting potential causal mechanisms . Although previous studies have demonstrated enrichment of transcription factor binding sites at disease-associated polymorphisms , whether specific disease causing variants act to alter the binding of T cell lineage-specifying factors has not been investigated . Having previously mapped T-bet binding across the genome in human Th1 cells [52–54] we used a systematic functional GWAS ( fGWAS ) approach to determine the degree to which disease-associated SNPs were enriched within T-bet binding sites . SNPs were then tested for effects on T-bet binding in vitro using a novel flow cytometric assay and in vivo by allele-specific ChIP-seq . We report here that SNPs associated with mucosal inflammatory diseases are selectively enriched at T-bet binding sites . Furthermore , we show that the celiac disease associated variants of rs1465321 and rs2058622 , and the IBD-associated variants of rs1551398 and rs1551399 , exhibit decreased T-bet binding in vivo . We further demonstrate that the genes associated with these SNPs , IL18RAP and TRIB1 , respectively , are transcriptionally regulated by T-bet and that rs1465321 is an expression quantitative trait locus ( eQTL ) for IL18RAP . Taken together , these data mechanistically link alterations in T-bet binding to disease predisposition .
To identify disease-associated polymorphisms at T-bet binding sites , we compared the locations of GWAS hits listed in the National Human Genome Research Institute ( NHGRI ) catalogue [55] with binding sites for T-bet in primary human Th1 cells [52–54] . As the published trait-associated SNP may not be the most functionally relevant [28] , SNPs in high linkage disequilibrium LD ( r2 >0 . 8 ) were also examined . This returned a list of 926 unique SNPs located at T-bet binding sites ( hereafter referred to as T-bet hit-SNPs; Fig 1A and 1B , S1A Fig and S1 Table ) . In line with previous reports , a minority ( 143 ) of the T-bet hit-SNPs were the putative causal SNP from GWAS data , with the others being in high LD ( total of 621 independent LD blocks ) . Examination of the location of T-bet hit-SNPs in relation to protein-coding genes revealed that the majority ( 63% ) were distal ( >1 kb ) to gene promoters . As expected , H3K27ac and DNaseI hypersensitivity were highly enriched in Th1 cells at T-bet hit-SNPs compared with all disease-associated SNPs , consistent with these being located within active regulatory elements ( Fig 1C ) . As T-bet is only expressed in cells of the immune system , we hypothesised that T-bet hit-SNPs would be primarily associated with autoimmune diseases . To test this , we used fGWAS [56] , a hierarchical model that assesses relative enrichment of GWAS associations within various functional elements . This model splits the genome into large blocks ( larger than regions of linkage disequilibrium ) , assesses whether each block contains a SNP associated with the trait of interest or not and then searches among supplied functional annotations for those that improve the likelihood of predicting the presence of a trait-associated SNP , finally predicting which SNP in the block is most likely causal . To test whether disease-associated SNPs were enriched at T-bet binding sites , we gathered GWAS data for the Th1-associated auto-inflammatory conditions celiac disease , Crohn’s disease , UC , rheumatoid arthritis ( RA ) , psoriasis and , as a negative non-immune control , coronary artery disease ( Fig 2 ) . We compared T-bet binding sites with a number of other relevant functional annotations , including Th1 and Th2 cell DHS [57] , H3K27ac [58] , and sites of histone modification and transcription factor binding in immune cell lines from the ENCODE project [26] and other sources ( S2 Table ) . Notably , we found that SNPs associated with all of the mucosal immune-mediated diseases tested ( Crohn’s disease , UC and celiac disease ) were enriched at T-bet binding sites , with the effect in Crohn’s disease being particularly striking . Enrichment at T-bet binding sites was similar to , or stronger than , DHS and H3K27ac and , in the case of Crohn’s and celiac disease , stronger than any other sets of transcription factor binding sites . As expected , SNPs associated with coronary artery disease were not enriched at T-bet binding sites . Of interest , no enrichment for T-bet binding sites was observed for RA- or psoriasis-associated SNPs , suggesting a specific role for altered T-bet binding in mucosal inflammatory disease . To confirm that T-bet binding is enriched at IBD-associated SNPs , we compared T-bet binding sites to a set of credible SNPs identified at 94 IBD-associated loci [21] . We found that T-bet binding sites were more highly associated with credible SNPs than other SNPs at the same loci ( 93 bound by T-bet , p = 1 . 4x10-5 , Fisher exact test ) . Furthermore , within the set of credible SNPs , the higher the posterior probability for causality , the more likely that the SNP overlapped a T-bet binding site ( p = 6 . 3x10-6 , continuous binomial regression , S1B Fig ) . The association of T-bet binding with causal SNPs is highlighted by the finding that , of the 93 credible SNPs bound by T-bet , 11 are the lead variants for their loci . Three of these ( rs74465132 , rs1887428 and rs61839660 ) have a posterior probability for causality of greater than 95% . These data suggest that the strong association of these SNPs with IBD is related to T-bet binding at these sites . Having identified a set of SNPs overlapping T-bet binding sites , we next asked whether these sequence variants altered T-bet binding . The traditional pull-down technique is time intensive and semi-quantitative . Therefore , we explored whether transcription factor binding could be assayed using a flow cytometric readout . In this technique , which we call OligoFlow , a fluorochrome-labelled antibody for the transcription factor of interest is added to the oligonucleotide-bead / lysate mix , and the Median Fluorescence Intensity ( MFI ) of the beads is assessed by flow cytometry as a quantitative measure of binding efficiency ( Fig 3A ) . To validate this new technique , a positive control oligonucleotide ( Motif+ ) was designed to incorporate the previously identified consensus sequence [54] surrounded by non-specific sequence ( S3 Table ) . A negative control oligo ( Motif- ) incorporated mutations of two key residues within the motif . OligoFlow was conducted with lysate from either the YT human cell line , which constitutively expresses T-bet [59] , or lysate from primary human CD4+ cells polarised under Th1 conditions in culture . The positive and negative control oligonucleotides showed a clear difference in MFI ( Fig 3A ) and thus OligoFlow can successfully discriminate positive and negative transcription factor binding events . We then proceeded to test a subset of our T-bet hit-SNPs that were also associated with H3K27ac or near genes of immunological interest . SNPs that showed differential binding were tested at least five times . Within each experiment , the MFI of each allele was normalised to the MFI of the negative control and significantly altered binding between alleles across all experiments was assessed using a paired t-test . Three T-bet hit-SNPs exhibited significantly different binding to the two alleles; rs1465321 , located within the second intron of IL18R1 , rs1006353 , 22 . 5 kb upstream of MTIF3 , and rs11135484 , within an intron of ERAP2 ( Fig 3B ) . Differential T-bet binding to the two alleles of rs1465321 were confirmed by traditional oligonucleotide pull-down ( S2 Fig ) . All 3 SNPs are [A/G] with A as the minor allele . In each case , allele A is also in LD with alleles associated with for the trait under investigation . rs1465321 is in high LD with multiple SNPs associated with celiac disease , including rs13015714 and rs917997 , identified as the strongest risk alleles for celiac disease in 2q12 . 1 [18 , 60] , with the disease-associated alleles linked to reduced IL18RAP expression [60] . rs1465321 and rs11135484 have also been associated with Crohn’s disease [18 , 22 , 60 , 61] , but not in a more recent study [21] . For rs1465321 and rs1006353 , the minor disease-associated A allele binds T-bet less strongly than the G allele ( Fig 3C–3F ) . In contrast , for rs11135484 , the A allele binds T-bet more strongly than the G allele ( Fig 3G and 3H ) . We conclude that disease-associated genetic variants can alter T-bet binding to DNA in vitro . Motif analysis has often been used to predict transcription factor binding sites affected by genetic variants . We previously derived a consensus T-bet motif from T-bet binding sites in human Th1 cells [54] and repeated this analysis with duplicate T-bet ChIP-seq data ( Fig 4 ) . The three T-bet hit-SNPs that showed altered binding in OligoFlow were then examined for whether they disrupted such a T-bet binding motif . In the case of rs1006353 , the G allele formed part of a T-bet binding motif , whereas the A allele abolished this binding site ( Fig 4B ) . However , neither of the other two SNPs , rs1465321 and rs11135484 , overlapped a predicted T-bet binding motif ( Fig 4B ) . Thus , over-reliance on motif analysis can result in SNPs with the potential to alter transcription factor binding sites being missed and highlights the importance of using experimental validation to confirm binding of the relevant transcription factor . We next sought to confirm that T-bet exhibited differential binding to disease-associated SNPs in vivo . We focused on rs1465321 , because it lies within the IL18R1/IL18RAP gene locus that we have previously identified as a T-bet target [54] and because disease-associated alleles in high LD are associated with reduced IL18RAP expression and disease [60] . Primary naive CD4+ T cells were purified from the peripheral blood of two individuals heterozygous for this SNP and were polarised into the Th1 lineage . We then performed ChIP-seq for T-bet in these cells , as previously described [54] . We aligned the reads for the T-bet ChIP-enriched DNA and input controls to the reference human genome and then counted the number of reads matching the major or minor alleles in the inputs and ChIP samples . In the input DNA samples , there were approximately equal numbers of reads for the two alleles in both individuals . In comparison , the T-bet ChIP reads showed significantly lower enrichment for the minor A allele in both donors ( Fig 5A and 5B ) . There was also a significant allelic imbalance for T-bet binding at the neighbouring SNP rs2058622 , which is in high LD ( r2 = 1 . 0 ) with rs1465321 ( Fig 5A and 5B ) . To determine whether T-bet exhibited allelic imbalanced binding at any other loci , we identified all SNPs that exhibited heterozygosity in both individuals . Of the heterozygous SNPs that overlapped a T-bet binding site , 19 exhibited significant allelic imbalanced binding in both donors after adjustment for multiple hypothesis testing ( Fig 5C , S4 Table ) . These included the IBD-associated SNPs rs1551398 and rs1551399 [21] , situated 86bp apart and downstream of TRIB1 ( Fig 5C , S1 and S3 Figs ) . We conclude that the two alleles of rs1465321 exhibit different levels of T-bet binding in vivo , with the disease associated A allele bound significantly less , and that the credible IBD variants rs1551398 and rs1551399 also influence T-bet binding . Having identified rs1465321 , rs2058622 , rs1551398 and rs1551399 as disease associated SNPs that modulate T-bet binding in vivo , we next determined whether there was a functional relationship between T-bet binding and the genes associated with these SNPs . rs1465321 and rs2058622 are in high LD with SNPs associated with low expression of IL18RAP in celiac disease [60] . The IBD-associated SNPs rs15513998 and rs1551399 are associated with TRIB1 [21] . To determine whether there was a functional relationship between T-bet binding and IL18RAP and TRIB1 expression , we compared gene expression profiles of wild type and T-bet-/- naïve CD4+ T cells polarised in Th1 conditions . As expected , genes known to be positively regulated by T-bet were significantly downregulated in T-bet-/- cells , including Interferon-γ ( Ifng ) and Tim-3 ( Havcr2 ) , while the housekeeping genes Gapdh , Actb and Hprt remained unchanged ( Fig 6A ) . Il18rap was also significantly downregulated in the absence of T-bet , implying a positive regulatory role for T-bet in modulating its expression ( Fig 6A ) . In contrast , Trib1 was significantly upregulated in T-bet-/- cells , implying that T-bet functions to repress this gene . Consistent with a direct role for T-bet in regulating Il18rap and Trib1 expression , multiple T-bet binding sites were located within intronic regions of murine Il18rap and downstream of Trib1 ( S4 Fig ) . Thus , these data support a direct role for T-bet binding in the regulation of IL18RAP and TRIB1 expression . We next explored whether the genotype of rs1465321 could control the expression of nearby genes and how this potential eQTL related to celiac disease susceptibility ( Fig 6B ) . Celiac disease association was based on a case control association study of 12 , 041 celiac disease cases and 12 , 228 controls [23] . Using a gene expression dataset of 1 , 214 samples [62] we found a strong correlation between rs1465321 genotypes and IL18RAP expression level ( p<10−100 , Fig 6C ) . No other gene showed a significant association with rs1465321 . However , this SNP did not display the greatest eQTL association compared with other variants in the region , which could suggest a lack of a causal role . Moreover , using a previously developed methodology [63] , we established that the eQTL and disease association signals in the IL18RAP regions were unlikely to be driven by the same genetic variant ( posterior probability supporting a shared variant < 1% , Fig 6C ) . However , a stepwise regression analysis of the eQTL data shows that after accounting for the primary eQTL signal ( conditional on rs1985329 ) , a second eQTL association was clearly detectable ( p<10−30 ) . This suggested that at least two independent variants , with distinct biological mechanisms , are controlling IL18RAP mRNA expression . Interestingly , this secondary eQTL signal co-localized with the celiac disease risk signal ( Fig 6D , posterior probability supporting a shared variant > 99% ) . Moreover , rs1465321 is one of the most strongly associated genetic variants for this secondary eQTL signal , with the disease-associated A allele , which exhibited reduced T-bet binding , associated with reduced IL18RAP expression . Therefore , our combined fine-mapping disease eQTL data are consistent with rs1465321 affecting IL18RAP expression through altered binding of T-bet .
We have found that IBD and celiac disease-associated SNPs are significantly enriched at T-bet binding sites . Surprisingly , this association is not observed for RA or psoriasis , suggesting it may be specific for mucosal inflammatory disease . Furthermore , we have identified genetic variants that alter T-bet binding to DNA , both in vitro and in vivo , including rs1465321 , which we also identify as an eQTL for IL18RAP and celiac disease . Thus , these data provide a mechanistic explanation for why a single base change at this locus is associated with changes in gene expression and disease risk . Although some studies have identified sequence variants that modulate transcription factor binding , alterations in the binding of Th lineage-specifying factors at disease-associated variants has not previously been identified . Our discovery that SNPs associated with IBD and celiac disease alter T-bet occupancy reveals that genetic variants can have a significant impact on the function of key master regulator transcription factors that govern cell fate . The strong association of T-bet binding sites with mucosal autoimmune/inflammatory diseases suggests that other disease-associated variants also act to alter the binding of this critical immune regulator , with important consequences for T cell polarisation and lineage-specific gene expression . That T-bet binding sites are associated with mucosal autoimmune disease , but not with RA or psoriasis is somewhat surprising , because all of these diseases have been linked to aberrant Th1 responses [2] . However , mucosal disease is more strongly associated with aberrant Th17 responses , which are repressed by T-bet [3 , 4 , 64 , 65] , providing a mechanistic rationale for our findings . We and others have recently shown that T-bet plays a critical and non-redundant role in the function of ILCs [2 , 7 , 11–15] . It is therefore feasible that the association of mucosal autoimmune disease-associated SNPs with T-bet binding sites reflects alterations to T-bet binding in ILCs , which have a key regulatory role at mucosal surfaces . Expanding our fGWAS analysis to other autoimmune conditions will be necessary to fully establish the specificity of the association of T-bet with SNPs associated with mucosal disease . Significantly , we have demonstrated that T-bet binding is enriched at disease-associated SNPs that have high posterior probabilities [21] . This suggests that more T-bet bound variants will be discovered when other IBD loci are subjected to fine-mapping analysis . We further found that the disease-associated alleles of rs1551398 and rs1551399 both reduce T-bet binding in vivo . These SNPs are located upstream of TRIB1 , a gene that is upregulated in the mucosa of both UC and CD patients [66] . Consistent with this , we find that T-bet functions to repress Trib1 expression , suggesting that the disease-associated alleles may increase disease risk by abrogating T-bet-mediated repression of this gene . T-bet also binds at 2 other sites near TRIB1 ( rs28510097 and rs1551400 ) and , together , these 4 SNPs account for 55% posterior probability of association for this locus [21] . We also identified rs1465321 , located within an intron of IL18R1 , to exhibit allele-imbalanced T-bet binding . This SNP is an eQTL for IL18RAP and celiac disease risk , with the minor disease-associated allele linked with reduced T-bet binding and IL18RAP gene expression . IL18RAP and IL18R1 together form the IL-18 receptor . Signaling through this receptor , IL-18 synergizes with IL-12 to induce IFNγ . rs1465321 is in high LD with the lead SNP in this locus for celiac disease [60] . Although our data are consistent with rs1465321 altering IL18RAP expression through altered binding of T-bet , we cannot rule out that variants in strong LD with rs1465321 could also be causal , such as rs2058622 that also exhibits allele-imbalanced T-bet binding . Given that T-bet acts through multiple sites to regulate its target genes [52 , 54 , 67 , 68] , it is likely to be the combined effect of the haplotype that is relevant . ChIP-seq for T-bet in individuals heterozygous for other disease-associated SNPs will likely reveal further examples of genetic variants that modulate T-bet binding . Our finding that there are two independent eQTLs for IL18RAP , and that only one of these is associated with celiac disease ( Fig 6 ) , suggests that the level of IL18RAP expression may not be functionally relevant for disease susceptibility . Alternatively , it is possible that the two independent eQTLs for IL18RAP represent different enhancers that mediate transcriptional activation in different cells or in response to different stimuli , and that IL18RAP expression level is only relevant for celiac disease in one cell type or in response to a particular signal . Attempts to determine the likely effect of non-coding sequence variants have mostly focused on identifying overlapping transcription factor binding motifs or overlapping sites of transcription factor binding , DNase I hypersensitivity or DNA and histone modification . Our analysis of allele-specific T-bet ChIP-seq data shows that genetic variants within transcription factor binding sites do not necessarily alter transcription factor binding . Similarly , genetic variants that do impact transcription factor binding do not necessarily lie within the predicted motif . Thus , confirmation of allele-specific binding events is necessary to confirm that a SNP does indeed impact transcription factor function and provides a mechanistic link between genetic variation and disease risk . We have established the feasibility of using flow cytometry to assay allelic effects on transcription factor binding , and validated this technique through both the traditional pull-down assay and allele-specific ChIP-seq . As flow cytometric methods can be easily automated , this method provides a more rapid means to assay large numbers of allelic variants compared to traditional pull-down methods . Using this OligoFlow method , we identified alterations in T-bet binding at rs11135484 , in high LD with a SNP associated with Crohn’s disease and with rs1006353 , the closest neighbor of which is MTIF3 , associated with body mass index [69] . Interestingly , T-bet has been linked with regulation of insulin sensitivity and visceral adiposity [70] . In summary , we have identified a specific association between T-bet binding sites and mucosal autoimmune disease variants and determined that such genetic variants modulate T-bet binding in cells . This suggests that altered binding of T cell master regulators can predispose individuals to specific autoimmune and inflammatory conditions . This study establishes a scalable method that can be used to explore the impact of genetic variation on the function of other lineage-specifying transcriptional factors . These insights will identify molecular mechanisms that underlie the genetic basis of autoimmune diseases and suggest new therapies for their treatment .
ChIP-seq for T-bet in human Th1 cells was performed previously [53–54] ( GEO accessions: GSE31320 and GSE62486 ) and binding sites were identified from the merged dataset with MACS 1 . 4 ( p<10−7 ) [71] . The positions of T-bet peaks were identified relative to gene transcription start sites annotated in RefSeq . The GWAS catalogue was downloaded from the NHGRI [55] on December 12th 2014 . SNPs were checked against dbSNP and 4 SNPs called ‘suspect’ removed . SNPs that had been merged with other IDs were checked against HapMap3 and the ID given in HapMap3 used in downstream analysis . SNPs not in HapMap3 were removed , giving 13 , 936 autosomal SNPs in the final analysis . Data were analysed using the bioconductor snpMatrix programme ( recently updated to snpStats ) [72 , 73] . SNPs in high LD ( r2> 0 . 8 with a SNP from the GWAS catalogue ) were obtained from HapMap3 [74] , giving a total of 127 , 594 SNPs . These were then overlapped with the T-bet binding sites . To identify the number of independent LD blocks were represented by the 926 T-bet bound SNPs , we used the SNPclip module of LDlink to reduce any SNPs in high LD to a single tag SNP , using a R2 threshold of 0 . 8 and a MAF ( Minimum Allele Frequency ) threshold of 0 . 01 . ChIP-seq data for IgG , H3K27ac and total H3 were taken from GSE62486 . Sequence reads were trimmed to remove low quality bases and to remove adapters and aligned using Bowtie ( default settings ) to hg19 . Peaks of H3K27ac were identified with MACS ( p<10−7 ) [71] . DHS data were obtained from ENCODE ( GEO accession GSM736592 ) [75 , 76] . Average binding profiles were calculated across 4 kb windows centred on hit-SNPs using ngsplot [77] . Data were visualized on the UCSC genome browser by calculating tag density in 10bp windows , normalizing to reads per million total reads and subtracting background ( input for T-bet and H3 for H3K27ac ) , as described [54] . Individuals heterozygous for rs1465321 were identified from the Twins UK cohort at the Guy’s and St Thomas’ NHS Foundation Trust ( GSTT ) Bioresource , where HumanHap610Q Illumina array data is available for all registered participants . The Illlumina calling algorithm [78] was used to assign genotypes from array data . Before imputation , quality controls were applied , with exclusion of all samples with: ( 1 ) call rate <98% , ( 2 ) heterozygosity across all SNPs ≥2 standard deviations from the sample mean; ( 3 ) evidence of non-European ancestry as assessed by PCA comparison with HapMap3 populations; ( 4 ) observed pairwise IBD probabilities suggestive of sample identity errors . We also corrected zygosity based on IBD probabilities . Quality controls were also applied to each individual SNP using the following exclusion criteria: ( 1 ) Hardy-Weinberg p-value <10−6 ( assessed in a set of unrelated samples ) ; ( 2 ) MAF <1% ( assessed in a set of unrelated samples ) ; ( 3 ) SNP call rate <97% ( SNPs with MAF ≥5% ) or < 99% ( for 1% ≤ MAF < 5% ) . Finally all the alleles were aligned to the forward strand of HapMap2 . After completion of both sample and SNP quality controls checks , imputation was performed using the IMPUTE software package ( v2 ) [79] using HapMap2 as a reference panel ( HapMap2 , rel . 22 , combined CEU+YRI+ASN panels ) . Heterozygous SNPs were selected using PLINK ( version 1 . 0 . 7 ) [80] “—recode-rlist” option on the imputed dataset . A final QC stage was applied on all the heterozygous SNPs , excluding all those polymorphisms with an imputation quality score ≤ 0 . 8 . In accordance with the Department of Health’s Research Governance Framework for Health and Social Care , ethical approval for this study was gained from the South London Research Ethics Committee ( Ref:15/LO/0151 ) , and from the Department of Research and Development at GSTT NHS Trust ( Ref:RJ115/N122 ) . Approval was also gained from the GSTT National Institute of Health Research ( NIHR ) Bioresource for recruitment of individuals registered on the Bioresource and heterozygous for rs1465321 . All of the subjects in this study gave written consent . Blood was taken from two individuals heterozygous for the desired SNP . CD4+ T cells were purified from whole blood leukocytes using CD4 microbeads ( Miltenyi Biotec ) and naïve CD4+ T-cells sorted by FACS selection for CD4+ CD45RA+ CD4RO- CD25- CCR7+ cells . Sorted naïve T-cells were activated with anti-CD3/CD28 and polarized under Th1 conditions ( IL2 , IL12 and anti-IL4 ) for 7 days [54] . Cells were then crosslinked and ChIP-seq for T-bet performed with a custom-made polyclonal antibody [54] . Libraries were quantified using the KAPA library quantification kit and sequenced ( 150 bp single-end ) with an Illumina NextSeq . Sequence reads were trimmed and aligned to hg19 as before . Peak regions for both donor 1 and 2 were identified separately using MACS 1 . 4 . Broad shallow peaks were filtered , intersecting peaks identified with Bedtools ( n = 8185 ) , and then narrowed to the central 400 bp . Potential SNP variants within these intersecting peak regions were extracted from dbSNP version 138 ( assembly hg19 , n = 490 , 310 ) . SNP sites for further analysis were determined from the Bowtie aligned bam files as containing >1 reads with both Ref and Alt bases in both ChIP and Input samples from both donors ( n = 9058 ) . This list was then compared to the set of heterozygous SNPs identified by the SNP array analysis ( n = 2621 high confidence heterozygous SNPs ) . Reads surrounding these sites were extracted into R using the Bioconductor Rsamtools and GenomicRanges packages . The reads were split by Ref and Alt alignment for visualization using the GenomicAlignments package . To test whether T-bet exhibited allelic imbalanced binding at rs1465321 and at SNPs in high LD , we used a binomial test . Donor 1 and 2 p-values were combined using the Fisher method . rs1465321 and rs2058622 showed significant allelic imbalance ( p<0 . 01 ) in the T-bet ChIP samples and allelic balance ( p>0 . 1 ) in the Input samples . To identify other heterozygous SNPs that exhibited allelic-imbalanced T-bet binding , we used a binomial test to identify heterozygous SNPs at which significantly more reads were reported for one allele compared to the other in both T-bet ChIP samples ( Benjamini-Hochberg adjusted p<0 . 05 ) but not imbalanced in the Input samples from either donor ( unadjusted p>0 . 4 ) . This produced a list of 19 additional SNPs ( S4 Table ) . T-bet ChIP-seq data from donors 1 and 2 heterozygous for rs1465321 are available at GEO under accession GSE81881 . Data-sets for wild-type and T-bet deficient CD4+ T cells polarised in Th1 and Th2 conditions were obtained from GEO ( GSE38808 ) . Raw reads were aligned to the mm10 build of the murine genome using Subread [81] , and subsequently mapped to RefSeq genes using featureCounts [82] . DESeq2 was used to normalise read counts by size factors , and call differentially regulated genes using an empirical Bayes model and the Wald test followed by Benjamini-Hochberg correction for multiple testing [83] . The presence of T-bet motifs was assessed using FIMO [84] using previously compiled matrices for T-bet binding obtained by ChIP-seq [54] . Sequences for T-bet binding sites were obtained from the hg19 reference genome and SNPs were manually altered to the alternative allele . fGWAS analysis was performed as described in [56] using fGWAS version 0 . 3 . 3 with case control setting . Data were prepared for fGWAS using R and the GenomicRanges package to compute overlap between binding sites and SNPs . Publicly available GWAS data were downloaded from the websites of the relevant consortiums for UC , Crohn’s disease ( http://www . ibdgenetics . org/downloads . html ) , coronary artery disease ( http://www . cardiogramplusc4d . org/downloads/ ) , and rheumatoid arthritis ( http://plaza . umin . ac . jp/~yokada/datasource/software . htm ) . Psoriasis data are from [85] . T-bet binding sites were identified as described above . ENCODE ChIP-seq , FAIRE-seq and DNaseI hypersensitivity datasets were obtained from the ENCODE website in bed format ( http://ftp . ebi . ac . uk/pub/databases/ensembl/encode/integration_data_jan2011 ) . The complete ENCODE datasets combines DNaseI ( 125 annotations ) , FAIRE-seq ( 24 annotations ) , histone marks ( 117 annotations ) and transcription factor binding site datasets ( S2 Table ) . In addition , we included GATA3 binding sites in Th1 and Th2 cells ( GSE31320 ) [54] , FOXP3 binding sites in Tregs [86] , NF-κB binding sites in lymphoblastoid cells [47] ( GSE19486 ) , and H3K27ac [58] and DHS [57] in Th1 and Th2 cells . Celiac disease association was based on a case control association study of 12 , 041 celiac disease cases and 12 , 228 controls [23] . Gene expression data were taken from [62] . eQTL analysis was performed as described [63] . eQTL p-values were obtained by fitting a linear trend test regression between the expression of each gene and all variants 200 kb upstream and downstream from each probe . Posterior computation was performed as described [63] . The regional association plots for the eQTL and Biomarker datasets were created using LocusZoom [87] ( http://csg . sph . umich . edu/locuszoom/ ) . Colocalisation analysis was performed using the R package COLOC [63] based on single variant summary statistics ( log odds ratio , standard error for the log odds ratio for case control and effect size and standard error for effect size for eQTL study , in addition to MAF and physical position for each variant ) and with the default settings provided with the R package . Human CD4+ cells were isolated from buffy coats ( UK National Blood Service , used under REC reference number 10/H0804/65 from SE London Research Ethics Committee 2 ) using RosetteSep human CD4+ T cell enrichment cocktail ( STEMCELL Technologies ) according to manufacturer’s instructions and polarised towards a Th1 phenotype in supplemented RPMI as described in above . Cells were harvested after a total of seven days of culture . YT cells were cultured in RPMI medium ( PAA ) supplemented with 50 units/ml penicillin , 50 μg/ml streptomycin ( Gibco ) , 10 mM HEPES buffer solution ( Fisher Scientific ) , 1 mM sodium pyruvate ( Gibco ) , 1 × minimum essential medium-non essential amino acids ( Gibco ) , 2 mM L-glutamine ( Gibco ) and 10% foetal bovine serum ( PAA ) . All cells were maintained at 37°C in 5% CO2 . Forward and reverse single-stranded oligos ( Integrated DNA Technologies , S3 Table ) for each allele of each SNP were annealed by incubating at 94°C for 5 mins , 65°C for 10 mins , 25°C for 10 mins and 4°C thereafter in annealing buffer ( 50 mM Tris pH 8 , 7 mM MgCl2 and 1 mM DTT ) . For pull-down and western blot , 20μl of streptavidin agarose beads ( Sigma ) were used per sample . For OligoFlow , 50 μl of Sphero streptavidin polystyrene particles ( Spherotech #SVP-100-4 ) were used per sample . Beads were washed twice in PBS and then once in annealing buffer . Beads were then incubated with double-stranded oligonucleotides for 1 hr at 4°C , washed twice in oligo buffer ( 10 mM Tris pH 8 , 100 mM NaCl , 0 . 1 mM EDTA , 1 mM DTT , 5% glycerol , 1 mg/ml BSA Fraction V , 20 μg/ml dI/dC ( Sigma , P4929 ) and Complete protease inhibitor ( Roche ) and finally resuspended in 450 μl oligo buffer . Cells ( 30 million per sample ) were washed twice in PBS and lysed in 1 ml hypotonic buffer ( 20 mM HEPES pH 8 , 10 mM KCl , 1 mM MgCl2 , 0 . 1% Triton X-100 , 5% glycerol , 1 mM DTT and Complete protease inhibitor ) on ice for 5 mins . Lysed cells were pelleted and resuspended in 150 μl hypertonic buffer ( 20 mM HEPES pH 8 , 400 mM NaCl , 1 mM EDTA , 0 . 1% Triton X-100 , 5% glycerol , 1 mM DTT and Complete protease inhibitor ) . Debris was pelleted , 180 μl of supernatant containing nuclear extract added to the beads and incubated on a rotor for 1 hour at 4°C . For western blotting , samples were then washed three times in oligo buffer and resuspended in SDS loading buffer . For OligoFlow , 0 . 25 μg of anti-T-bet Alexa647 antibody ( clone 4B10 , BioLegend ) was added and samples incubated for a further 1 hr at 4°C . Data ( at least 30 , 000 events ) were acquired on a FACSCanto flow cytometer ( BD Biosciences ) . Oligonucleotide pull-down samples were heated in SDS loading buffer before transfer to nitrocellulose membrane . Samples were blocked in 5% milk in TBS-T ( 1 hr , RT ) and incubated with 1:1000 anti-T-bet ( clone eBio4B10 ( eBioscience ) ; 4°C overnight ) . Blots were washed before addition of anti-mouse-HRP ( GE Healthcare ) and visualised with Enhanced Chemiluminescent Substrate ( PerkinElmer ) and exposed to film . | Research to date has identified many genetic variants that are more common in people with a particular disease . However , in conditions that reflect multiple genetic and environmental factors , it is difficult to know with certainty if and why any particular genetic variant is causative and the mechanism that may underlie this . Such variants are often outside of protein-coding exons , instead falling in regions that regulate gene expression . In these cases , the genetic variation may alter transcription factor binding and subsequent gene expression . In this study , we have examined how genetic variation affects T-bet binding to DNA , as a key transcriptional regulatory mechanism in the immune response . An inability to mount this response effectively can result in increased susceptibility to infections or cancer , while a response that is too strong , or wrongly targeted , can result in uncontrolled/chronic inflammatory and autoimmune conditions . We have found that T-bet binding sites are specifically enriched in genetic variants associated with the mucosal autoinflammatory diseases UC , Crohn’s disease and celiac disease . We also identify genetic variants that alter T-bet binding and gene expression . This discovery thus identifies a molecular mechanism through which genetic variants can be associated with increased risk of mucosal autoimmune disease . | [
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] | 2017 | Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease |
The precise identification of Human Leukocyte Antigen class I ( HLA-I ) binding motifs plays a central role in our ability to understand and predict ( neo- ) antigen presentation in infectious diseases and cancer . Here , by exploiting co-occurrence of HLA-I alleles across ten newly generated as well as forty public HLA peptidomics datasets comprising more than 115 , 000 unique peptides , we show that we can rapidly and accurately identify many HLA-I binding motifs and map them to their corresponding alleles without any a priori knowledge of HLA-I binding specificity . Our approach recapitulates and refines known motifs for 43 of the most frequent alleles , uncovers new motifs for 9 alleles that up to now had less than five known ligands and provides a scalable framework to incorporate additional HLA peptidomics studies in the future . The refined motifs improve neo-antigen and cancer testis antigen predictions , indicating that unbiased HLA peptidomics data are ideal for in silico predictions of neo-antigens from tumor exome sequencing data . The new motifs further reveal distant modulation of the binding specificity at P2 for some HLA-I alleles by residues in the HLA-I binding site but outside of the B-pocket and we unravel the underlying mechanisms by protein structure analysis , mutagenesis and in vitro binding assays .
HLA-I molecules play a central role in defence mechanisms against pathogens and immune recognition of cancer cells . Their main functionality is to bind short peptides ( mainly 9- to 12-mers ) coming from degradation products of endogenous or viral proteins . The peptides are cleaved in the proteasome , transported by the transporter associated with antigen processing ( TAP ) complex , loaded onto the HLA-I molecules in the ER and presented at the cell surface [1] . Non-self peptides presented on HLA-I molecules , such as those derived from degradation of viral proteins , mutated proteins ( referred to as neo-antigens ) , and other cancer specific and abnormally expressed proteins can then be recognized by CD8 T cells and elicit cytolytic activity . Neo-antigens have recently emerged as promising targets for development of personalized cancer immunotherapy [2] . Human cells express three HLA-I genes ( HLA-A/B/C ) . These genes are the most polymorphic of the human genome and currently more than 12 , 000 different alleles have been observed [3] . Such a high polymorphism makes it challenging to model the different binding specificities of each allele and predict antigens presented at the cell surface . Information about binding motifs ( mathematically defined here as Position Weight Matrices and graphically represented as sequence logos ) of HLA-I molecules has been mainly obtained from biochemical assays where chemically synthesized peptides are tested in vitro for binding . This in vitro approach is experimentally laborious , time consuming and expensive . Currently , the most frequent HLA-I alleles have thousands of known ligands that provide a detailed description of their binding specificity . Many of these ligands are stored in very important resources such as IEDB [4 , 5] and have been used to train machine learning algorithms for HLA-I ligand predictions [6–11] . As a result , for frequent alleles in Caucasian populations , much is known about their binding specificity . However , the vast majority ( >95% ) of HLA-I alleles still lack documented ligands and despite very valuable algorithmic developments to generalize prediction methods to any allele [12] , it remains more challenging to make accurate predictions for alleles without known ligands . Importantly , although these alleles are only found in a small fraction of the Caucasian population , they are more frequently encountered in other ethnic groups and gaining more understanding of their binding specificity would be desirable for expanding the scope of therapeutic strategies relying on HLA-I ligand predictions . Moreover , many alleles with known motifs are supported by only a few tens of peptides and some of these ligands have been selected based on a priori expectations of the binding specificity rather than unbiased screening of random peptide libraries . Such potentially biased datasets can be sub-optimal for training HLA-I ligand predictors . Mass-spectrometry ( MS ) analysis of HLA-I binding peptides eluted from cell lines or tissue samples is a promising alternative to the use of HLA-I ligand interaction predictions [13] and MS is increasingly used to directly identify viral [14 , 15] or cancer-specific ( neo- ) antigens [13 , 16–20] . For neo-antigen discovery , tumor exome sequencing is first performed . Patient-specific non-synonymous somatic alterations are included in a customized database . MS-MS spectra of HLA ligands eluted from the patient’s tissue samples are searched against this expanded database permitting either the wild-type or the mutated peptide sequences to be identified . Neo-antigens directly identified this way , or alternatively predicted in-silico , may be further validated using targeted mass-spectrometry approaches in which isotopically heavy-labeled synthetic peptides are spiked into the HLA ligands eluted from the patient’s tumor tissue . Identification of co-eluting pairs of heavy ( standard ) and light ( endogenous ) peptides validate the presentation of the neo-antigen in the investigated tissue [19] . However , this technique is only applicable to a small fraction of samples due to the large amount of material that is required for MS analysis ( typically 1cm3 of tissue material , or 1x108 cells in culture ) and the complexity of these experiments . In addition to potentially immunologically relevant ( neo- ) antigens , tens of thousands of endogenous peptides naturally presented on HLA-I molecules are identified in such HLA peptidomics studies , providing a unique opportunity to collect very large numbers of HLA-I ligands that can be used to better understand the binding properties of HLA-I molecules . The challenge in studying HLA-I motifs based on such pooled peptidomics data from unmodified cell lines or tissue samples is to determine the allele on which each peptide was displayed . The most widely used approach is to predict binding affinity of each peptide to each allele present in a sample [21] . Recent studies by ourselves and others have shown that HLA-I motifs can be identified in HLA peptidomics datasets in an unsupervised way by grouping peptides based on their sequence similarity [17 , 22–25] . However , this strategy still relies on previous information about HLA-I binding specificity when associating predicted motifs with HLA-I alleles and is therefore restricted to alleles whose motifs have been already characterized . Here , we describe a computational framework for direct identification and annotation of dozens of HLA-I motifs without any a priori information about HLA-I binding specificity by taking advantage of co-occurrence of HLA-I alleles across both newly generated and publicly available HLA peptidomics datasets . Our approach recapitulates and refines motifs for many common alleles and uncovers new motifs for eight alleles for which , until this study , no ligand had been documented . Importantly , this approach is highly scalable and will enable continuous refinement of motifs for known alleles and determination of novel motifs for uncharacterized alleles as more HLA peptidomics data will be acquired in the future . Training HLA-I ligand predictors based on the refined motifs significantly improves neo-antigen predictions in tumor samples with experimentally determined neo-antigens . Our large collection of HLA-I ligands further allowed us to unravel some of the molecular determinants of HLA-I binding motifs and revealed allosteric modulation of HLA-I binding specificity . To elucidate the underlying molecular mechanisms , we show how a single point mutation ( W97R ) in HLA-B14:02 outside of the B pocket significantly changes the amino acid preferences at P2 in the ligands .
To study the binding properties of HLA-I alleles without relying on a priori assumption on their binding specificity and investigate whether this unbiased approach could improve neo-antigen predictions from exome sequencing data , we reasoned that HLA-I binding motifs might be identified across samples with in-depth and accurate HLA peptidomics data by taking advantage of co-occurrence of HLA-I alleles . To this end , we measured the HLA peptidome eluted from six B cell lines , two in vitro expanded tumor-infiltrating lymphocytes ( TILs ) samples and two leukapheresis samples ( peripheral blood mononuclear cells ) selected based on their high diversity of HLA-I alleles ( see Methods and S1 Dataset ) . By applying a stringent false discovery rate for peptides identification of 1% , we accurately identified 47 , 023 unique peptides displayed on 32 HLA-I molecules . To expand the coverage of HLA-I alleles , we further collected 40 publicly available high-quality HLA peptidomics datasets [17 , 18 , 22 , 23 , 26–28] ( see Methods and S2 Dataset ) . Our final data consists of a total of 50 HLA peptidomics datasets covering 66 different HLA-I alleles ( 18 HLA-A , 32 HLA-B and 16 HLA-C alleles , see Table A in S1 Supporting Information ) . The number of unique HLA-I ligand interactions across all samples reaches 252 , 165 for a total of 119 , 035 unique peptides ( 9- to 14-mers ) , which makes it , to our knowledge , the largest currently available collection of HLA peptidomics datasets both in terms of number of peptides and diversity of HLA-I molecules . Binding motifs in each HLA peptidomics dataset were identified for 9- and 10-mers ( see Fig A in S1 Supporting Information ) using a motif discovery algorithm initially developed for multiple specificity analysis [29 , 30] and recently applied to the analysis of a small dataset of seven HLA peptidomics studies [24] . Importantly , this method does not rely on HLA-I peptide interaction predictions ( see Methods ) . To assign each motif to its allele even in the absence of a priori information about the alleles’ binding specificity , we developed a novel computational strategy illustrated in Fig 1 . In this example , one allele ( HLA-A24:02 ) was shared between all three samples . Remarkably , exactly one identical motif was shared between the three samples . As such , one can predict that this motif corresponds to the shared allele . Similarly , two alleles ( HLA-A01:01 and HLA-C06:02 ) were shared between exactly two samples and here again two motifs were shared among the corresponding samples , and could therefore be annotated to their corresponding alleles . Moreover , if one sample shares all but one allele with another sample , it can be inferred that the motif that is not shared corresponds to the unshared allele ( see example in Fig B in S1 Supporting Information ) , even if some of the shared motifs have not been annotated yet . Finally , if all but one motif had been annotated in a sample to all but one allele , one can infer that the remaining motif corresponds to the remaining allele . These three ideas can then be recursively applied to identify HLA-I motifs across our large collection HLA peptidomics datasets ( see Methods ) . Of note , motifs identified in distinct samples that have some alleles in common show very high similarity ( Fig 1 and Fig B in S1 Supporting Information ) and our new approach builds upon this remarkable inherent reproducibility of in-depth and accurate HLA peptidomics data . We applied our algorithm to the 50 HLA peptidomics datasets considered in this study . In total , motifs could be found for 44 different alleles without relying on any a priori assumption of HLA-I binding specificity ( Fig 2A ) . These include seven alleles ( HLA-B13:02 , HLA-B14:01 , HLA-B15:11 , HLA-B15:18 , HLA-B18:03 , HLA-B39:24 and HLA-C07:04 ) that did not have known ligands in IEDB , and 5 additional ones ( HLA-B38:01 , HLA-B39:06 , HLA-B41:01 , HLA-B56:01 , HLA-C07:01 ) that had less than 50 known ligands . To validate our predictions , we compared the motifs predicted by our fully unsupervised method with known motifs derived from IEDB [4] , when available . Despite some differences ( e . g . HLA-A25:01 motif at P9 ) affecting especially alleles with low number of ligands in IEDB ( Fig C in S1 Supporting Information ) , we observed an overall high similarity confirming the reliability of our predicted motifs ( Fig 2A ) . However , it is important to realize that even small differences in the motifs can have important effects on the performance of predictors that are trained on such data when ranking very large lists of potential epitopes . When comparing with data recently obtained by HLA peptidomics analysis of mono-allelic cell lines [31] , a very high similarity was also observed ( Fig 2B , stars in Fig C in S1 Supporting Information ) , which further validates our computational approach for HLA-I motif identification and annotation from in-depth pooled HLA peptidomics data . As expected from many previous studies , alleles with the same two first digits code showed high similarity in their binding specificity , apart from HLA-B15 alleles which are known to be more diverse [32] . This includes many of the new motifs ( e . g . , HLA-B14:01 vs HLA-B14:02; HLA-B18:03 vs HLA-B18:01; HLA-B39:24 vs HLA-B39:06 ) , which provides further evidences of the accuracy of our predictions for these uncharacterized alleles . For the most frequent HLA-I alleles , including several shown in Fig 2A , a good description of their binding motifs can be already obtained from existing databases [4] . To further expand our collection of HLA-I binding motifs , we used similarity to the binding motifs derived from IEDB ligands to annotate motifs that could not be assigned to their corresponding allele by the fully unsupervised algorithm , following the approach previously introduced by ourselves in ref [24] ( see Methods ) . This enabled us to determine the binding motifs of 8 additional alleles ( Fig 2C ) . Of note , the new motif of HLA-A02:20 was predicted by observing that it was the only motif not annotated in one sample ( RA957 ) and could only be annotated to this allele based on the motifs identified in other samples for all the other alleles ( see Fig A in S1 Supporting Information ) . The final list of motifs for the 52 alleles and detailed comparison with IEDB derived data , when available , is shown in Fig D in S1 Supporting Information . Importantly , for the majority of alleles considered in this study , the motifs are supported by significantly more ligands than what is available in existing databases ( Fig E in S1 Supporting Information ) , and in total our approach enabled us to collect 88’051 unique 9-mer peptide HLA-I interactions for all alleles annotated in this work , compared to the 57’651 interactions available in IEDB for the same set of alleles . 14 out of a total of 195 motifs ( corresponding to 5’703 9-mer peptide-HLA interactions ) could not be annotated by our approach ( see Fig A in S1 Supporting Information ) . To investigate how our approach depends on the number of samples in which a motif is found , we show in Fig F in S1 Supporting Information the distance between our predicted motifs and those derived from mono-allelic cell lines ( Fig F ( a ) in S1 Supporting Information ) or those pooled from all samples ( Fig F ( b ) in S1 Supporting Information ) as a function of the number of samples ( i . e . sub-sampling ) . As expected , higher similarity could be observed by integrating several samples , justifying our idea of collecting as much data as possible from different HLA peptidomics datasets to refine our motifs . But overall , all distances are very small ( D2 < 0 . 03 ) , highlighting the excellent reproducibility of the motifs deconvoluted from HLA peptidomics datasets . To explore the statistical significance of the motifs associated to the same alleles , we followed the approach of ref . [33] and compared the similarity ( both Euclidean distance and BLiC score ) between each pair of motifs ( mi , mj ) annotated to the same allele h ( i = 1 , …Nh , j = 1…Nh , i≠j with Nh the number of motif annotated to allele h ) to the distribution of similarity values between motif mi and all known HLA-I motifs [33] ( see Methods ) . Fig G in S1 Supporting Information shows that more than 99% of the pairs of motifs associated to the same alleles have a statistically significant similarity ( P<0 . 05 ) , confirming the few examples shown in Fig 1 . Exceptions consist mainly of motifs annotated to HLA-C alleles , which are more degenerate and therefore more difficult to deconvolute [24] . We therefore recognize that our motifs are likely less accurate for HLA-C alleles , but emphasize that these alleles are also poorly described in existing databases or literature . We further explored the effect of the threshold T = 0 . 078 on Euclidean distance manually defined in this work ( see Methods ) . As expected lower values of T still results in highly similar motifs annotated to the same alleles , but in fewer alleles to which motifs can be annotated ( Fig H in S1 Supporting Information ) . Reversely , larger values tend to increase the number of alleles with annotated motifs , but at some point ( T>0 . 09 ) more distinct motifs ( P>0 . 05 , based on Euclidean distance ) become associated to the same alleles ( Fig H in S1 Supporting Information ) . The full pipeline was also applied on 10-mers identified by MS across the 50 HLA peptidomics studies and revealed six new motifs for poorly characterized alleles in IEDB ( Fig I in S1 Supporting Information ) . Different technical biases may affect MS data , which could undermine their use for training HLA-I ligand predictors . To investigate this potential issue , we computed amino acid frequencies at non-anchor positions ( P4 to P7 ) in our HLA peptidomics data , excluding alleles displaying anchor residues at these positions ( see Methods and Table B in S1 Supporting Information ) . The reason for focusing on middle positions is that they display low specificity ( especially in 9-mers , see discussion in [24 , 34 , 35] for longer peptides ) and therefore could provide a global view of potential MS biases on amino acid frequencies that is not affected by the constraints of binding to HLA-I molecules . As expected , we observed a good correlation between amino acid frequencies at non-anchor positions in our HLA peptidomics data and in the human proteome ( r = 0 . 85 ) ( Fig 3 and Fig J in S1 Supporting Information ) . The most important difference that could strongly affect predictors trained on such data was found for cysteine , which is prone to post-translational modifications that are typically not included in database searches and was observed at very low frequency in the HLA peptidomics data ( the same observation was recently made in mono-allelic cell lines [31] ) . Moreover , IEDB data clearly indicate that cysteine can be found at non-anchor positions , including for immunogenic epitopes , and therefore the low frequency observed in MS data very likely corresponds to a technical bias . Other amino acids were less under- or over-represented and the differences observed with the human proteome may also reflect some residual specificity at non-anchor positions . Moreover , no clear pattern emerged from these data with respect to amino acid biophysical properties ( e . g . , charge , hydrophobicity , size ) . Overall , our results suggest that HLA peptidomics data do not show strong technical biases , apart from under-representation of cysteine ( see next section for a proposed method on how to compensate this bias ) , and therefore could provide ideal data for training HLA-I peptide interaction predictors , especially for ligands coming from human cells like neo-antigens . To test whether our unique dataset of naturally presented peptides could help predicting HLA-I ligands , including neo-antigens in tumors based on exome sequencing data , we trained a predictor of HLA-I ligands ( referred to as MixMHCpred ) . As MS only includes positive examples and HLA-I ligands in general do not show strong amino acid correlations between different positions ( see discussion in [24] for some exceptions ) , we built Position Weight Matrices ( PWMs ) for each of the 52 alleles . These PWMs were built by pooling together all peptides assigned to each allele across all our HLA peptidomics datasets ( see Methods and Fig 1 ) . We further included MS data from mono-allelic cell lines for 6 rare alleles that were not present in our dataset , resulting in a total of 58 alleles available in our predictor . To correct for the low detection of cysteine observed in HLA peptidomics data we further renormalized our predictions by amino acid frequencies at non-anchor positions ( see Methods and Table B in S1 Supporting Information ) . As a first validation , we attempted to re-predict naturally presented peptides experimentally identified in ten mono-allelic cell lines whose alleles overlapped with our dataset [31] . For this analysis , we did not include data from these mono-allelic cell lines in our training set . To assess our ability to predict naturally presented peptides , we added 99-fold excess of decoy peptides randomly selected from the human proteome to each mono-allelic cell line dataset and measured the fraction of Positives among the top 1% Predictions ( PP1% , i . e . , True Positive Rate among the top 1% ) , which in the case of 99-fold excess of decoy is equivalent to both the Precision and the Recall , since the number of predictions ( top 1% ) is equal to the number of actual positives ( 1% ) . For all but one allele , our algorithm showed higher or equal predictive power compared to standard HLA-I ligand predictors [8 , 12 , 36] ( Fig 4A ) . We further measured the average Area Under the Curve ( AUC ) for these alleles and obtained quite similar values ( 0 . 978 for MixMHCpred , 0 . 976 for NetMHC , 0 . 979 for NetMHCpan and 0 . 977 for NetMHCstabpan ) . However , we emphasize that most random peptides used as negatives are quite distinct from the positives , which can explain the very high AUC values and we suggest that precision values for top 1% of the predictions shown in Fig 4A are more representative of the actual performance of the algorithms . We then collected currently available datasets that included direct identification of neo-antigens displayed on cancer cells as well as exome sequencing data ( Mel5 , Mel8 , Mel15 from [17] and 12T from [20] ) for a total of ten 9- and 10-mers mutated peptides experimentally found to be presented on cancer cells ( see Table 1 ) . This dataset has the unique advantage of not being restricted to peptides selected based on in silico predictions , and is therefore an ideal testing set for benchmarking our predictor . Moreover , as these studies are quite recent , the neo-antigens used here as testing set are not part of the training set of any existing algorithm . In particular , they are not part of the large training set used in this study since we only included wild-type human peptides in our pipeline . We then retrieved all possible 9- and 10-mer peptides that encompassed each missense mutation ( S3 Dataset ) and ranked separately for each patient these potential neo-antigens based on the score of our predictor ( see Methods and Table 1 ) . Remarkably , six of the ten neo-antigens fell among the top 25 predicted peptides , suggesting that by testing as few as 25 mutated peptides per sample , we could identify more than half of the neo-antigens identified by MS ( Table 1 ) . Considering that the total number of potential neo-antigens ( i . e . 9- and 10-mers containing a missense mutation ) can be as large as 25 , 000 for tumors with high mutational load , our predictor trained on naturally presented human HLA-I ligands clearly enabled us to significantly reduce the number of peptides that would need to be experimentally tested to identify neo-antigens from exome sequencing data . We further added two datasets of neo-antigens identified in lung cancer patients ( L011 and L013 ) [37] , although only peptides pre-selected based on binding affinities predicted with existing tools [12] were tested in this study . Here again , our predictor ranked one neo-antigen in the top 25 predicted peptides in both samples ( Table C in S1 Supporting Information ) . When comparing with standard tools that are widely used to narrow-down the list of potential neo-antigens predicted from exome sequencing data [8 , 12 , 36] , our method trained on HLA peptidomics data showed clear improvement ( Table 1 ) with a mean AUC value of 0 . 979 , compared to 0 . 932 for NetMHC [8] , 0 . 942 for NetMHCpan [12] and 0 . 945 for NetMHCstabpan [36] ( Fig 4B ) and increased number of neo-antigens in the top 1% of the predictions ( i . e . , typically what is experimentally tested for immunogenicity ) across all six samples ( Fig 4C ) . This is especially clear for the 12T sample , where the single neo-antigen was very well predicted by our model and poorly predicted by existing tools ( >6’000nM with HLA-B51:01 , see also Table 1 and [20] ) . We still note that , due to the low number of neo-antigens publicly available together with exome sequencing data , performance metrics in Fig 4B and 4C can be sensitive to one neo-antigen being better or less well predicted and we stress that the values shown in Fig 4B and 4C are simply a graphical way of looking at data shown in Table 1 and Table C in S1 Supporting Information . Importantly , even if we did not include in the training of our predictor MS data ( i . e . wild-type peptides ) from the samples in which the neo-antigens were identified , neo-antigens were still more accurately predicted compared to other tools ( see Fig K in S1 Supporting Information ) . This demonstrates that our approach for neo-antigen predictions from the list of somatic mutations identified by exome sequencing of tumors does not require HLA peptidomics data from the same sample where neo-antigens had been identified . Nevertheless , both our predictor and standard prediction tools failed to identify some neo-antigens ( e . g . , KLILWRGLK from NCAPG2 P333L mutation , see Table 1 ) . This suggests that , when enough tumor material is available for HLA peptidomics analyses , direct identification of neo-antigens with MS should still be performed to optimally enrich in true positives the list of ligands to be experimentally tested for immunogenicity [16 , 19] . The number of studies reporting both neo-antigens and exome sequencing results is still limited . To benchmark our algorithm with larger datasets of immunologically relevant tumor antigens , we tested our ability to predict epitopes from cancer testis antigens . We retrieved all epitopes listed in the CT database [38] ( see Methods and Table D in S1 Supporting Information ) . We then assessed how our predictor could prioritize these epitopes from all possible peptides encoded by these cancer testis antigens . Although we cannot exclude that some of these epitopes had been selected for experimental testing after prediction by older versions of HLA-I ligand predictors , we still observed improvement using our predictor trained only on naturally presented HLA-I ligands , both in terms of AUC and fraction of true positives that fall in the top 1% of the predictions ( Fig 4D and 4E ) . This indicates that improvement in prediction accuracy is not restricted to elution data ( see similar results in [24] ) . MS data can contain false positives for many different reasons , such as co-eluting peptide contaminations or errors in the computational identification of peptides from the spectra . Therefore , despite the high quality of HLA peptidomics datasets generated in this study ( <1% FDR ) , we do expect our data to contain some noise . To test the robustness towards contaminations of our motif discovery and annotation pipeline , and our HLA-I ligand predictor , we incorporated 5% of random peptides from the human proteome into all HLA peptidomics datasets considered in this work and rerun the whole motif annotation pipeline and training of the predictor . Remarkably , the accuracy of the predictions was only very modestly affected by this noise and predictions were still better than with other existing tools ( Fig L in S1 Supporting Information ) . To explore the effect of wrong peptide identification , we reprocessed with MaxQuant [39] the three MS samples shown in Fig 1 and chose the second best hit for 1% of the peptides . Overall the motifs predicted by our approach remained almost unchanged ( Fig M in S1 Supporting Information ) . This suggests that our pipeline is robust and indicates that the wealth of unbiased and accurate data provided by MS can compensate the inherent contaminations , when using these data for training HLA-I ligand predictors . An important step in our predictor is the renormalization by amino acid frequencies observed at non-anchor positions , which was designed to correct for biases in MS data . As expected , doing this renormalization step with amino acid frequencies observed in the human proteome ( or no renormalization at all ) results in very low frequency of cysteine-containing peptides among the top predicted ligands . As such , it improves the predictions of MS data ( see especially Fig N ( a ) in S1 Supporting Information ) , but decreases the performance in other datasets ( e . g . , Fig N ( b-c ) in S1 Supporting Information for L011 and L013 ) . These results highlight the importance of carefully considering MS biases when including such data to train predictors in order to avoid over-fitting elution data . We anticipate that additional work may further improve this step , such as inclusion of cysteine modifications in spectral searches [31] or better estimations of the expected baseline amino acid frequencies in HLA peptidomics datasets . One of our novel HLA-I motifs describes the binding specificity of HLA-A02:20 ( Fig 2C ) . HLA-A02 binding motifs have been widely studied . However , HLA-A02:20 motif differs from standard HLA-A02 motifs at P1 , with a clear preference for charged residues ( Fig 5A ) . Interestingly , HLA-A02:20 is among the very few ( <2% ) HLA-A02 alleles that do not have a conserved lysine pointing towards P1 at position 66 ( residue numbering follows 2BNQ X-ray structure hereafter ) . Instead an asparagine is found there ( Fig 5A ) , and this residue is the only difference with the sequence of the very common A02:01 allele . To explore how the absence of lysine at position 66 could explain the observed difference in binding specificity , we collected all HLA-I alleles showing preference for charged amino acids at P1 ( see Fig O in S1 Supporting Information ) . All of them had either asparagine or isoleucine at position 66 . We then explored available crystal structures of HLA-I peptide complexes with charged residues at P1 . HLA-B57:03 was crystalized with such a ligand ( KAFSPEVI ) [40] . Superposing the crystal structure of this complex with the structure of HLA-A02:01 provides a possible mechanism for understanding the change in binding specificity at P1 . In HLA-A02:01 , lysine at position 66 interacts with the hydroxyl group of serine at P1 ( Fig 5A , green sidechains ) . Such a conformation would not be compatible with a longer residue . Reversely , when asparagine was found at position 66 , it did not point towards P1 ( Fig 5A , pink sidechains ) , thereby freeing space for larger sidechains like lysine or arginine at P1 . Overall , our analysis indicates that the presence of asparagine at residue 66 may be responsible for the change in binding specificity between HLA-A02:01 and HLA-A02:20 . More generally , our results suggest that lysine at residue 66 in HLA-I alleles strongly disfavours charged residues at P1 . The new motif identified for HLA-B15:18 ( Fig 2A ) displayed strong preference for histidine at P2 , which is not often observed in HLA-I ligands . To gain insights into the mechanisms underlying this less common binding motif , we surveyed all alleles that show preference for histidine at P2 ( Fig P ( a ) in S1 Supporting Information ) . Sequence and structure analysis showed that all of them have a conserved P2 binding site , commonly referred to as the B pocket ( see Fig 5B ) . However , several HLA-B14 alleles have exactly the same B pocket but show specificity for arginine at P2 ( Fig P ( b ) in S1 Supporting Information ) . Among them , HLA-B14:02 had the highest sequence similarity to HLA-B15:18 , with only 8 different residues in the peptide binding domain , none of them making any contact with arginine at P2 in the crystal structure of HLA-B14:02 ( orange residues in Fig 5C ) . This suggests that the difference in binding specificity at P2 between HLA-B14:02 and HLA-B15:18 is likely explained by allosteric mechanisms . Of particular interest is residue 97 ( W in HLA-B14:02 and R in HLA-B15:18 ) , which is in the HLA-I binding site and contacts the peptide ( mainly P3 to P6 , Fig 5C ) but is more than 7Å away from the arginine sidechain at P2 . This residue is part of a network of aligned aromatic residues ( Y9 , W97 and F116 ) in HLA-B14:02 ( Fig 5C ) compatible with pi-pi interactions . Interestingly , X-ray structures with Arg at position 97 ( e . g . , 4O2C ) show a flip in the orientation of Y9 sidechain , which reduces the size of the B pocket . We therefore hypothesized that mutating residue 97 into arginine in HLA-B14:02 may indirectly modify the binding specificity at P2 and explain the preference for histidine observed in the HLA-B15:18 motif . To test our hypothesis , we generated a construct for HLA-B14:02 wild-type ( wt ) and W97R mutant . We tested several ligands of HLA-B15:18 identified in our HLA peptidomics data with histidine at P2 , which were predicted to show enhanced binding to HLA-B14:02 W97R . As expected , a strong decrease in binding stability was observed between HLA-B14:02 W97R and HLA-B14:02 wt ( Fig 5D ) . Reversely , when testing the same peptides with arginine at P2 , a significant increase in stability was observed between HLA-B14:02 W97R and HLA-B14:02 wt ( Fig 5D ) . For instance , binding of the peptide AHTKPRPAL was fully abolished in HLA-B14:02 wt , but was rescued when changing histidine to arginine at P2 . Although other residues may also play a role in the binding specificity differences between HLA-B14:02 and HLA-B15:18 , all of them are further away from P2 . Overall , our results show that HLA-I binding specificity at P2 can be modulated by amino acids outside of the B pocket , and that residue 97 can act as a switch of the binding specificity at P2 . These binding experiments further confirm the motifs predicted for HLA-B14:01 and HLA-B15:18 alleles .
Despite decades of work to characterize the binding motifs of the most common HLA-I alleles , unbiased peptide screening approaches have not been commonly used in the past . This is mainly because both the N- and the C-terminus of the peptides are engaged in binding to HLA-I molecules , thereby preventing the use of high-throughput techniques for peptide screening like phage display . To address this issue , we developed a novel algorithm to rapidly identify and annotate HLA-I binding motifs in a fully unsupervised way using in-depth and accurate HLA peptidomics data from unmodified cell lines and tissue samples . This enabled us to refine models of binding specificity for many alleles with few ligands in existing databases and characterize the binding properties of eight HLA-I alleles that had no known ligands until this study . Our approach is conceptually similar to existing approaches to deconvolute peptide epitopes by identifying shared peptides between different pools showing T cell reactivity in ELISpot experiments , but had never been applied to motif annotation across HLA peptidomics datasets . Remarkably , our predicted motifs displayed high similarity with known motifs for common alleles , including motifs derived from HLA peptidomics analyses of mono-allelic cell lines [31] ( Fig 2 ) , and the MS-induced technical bias ( mainly low detection of cysteine ) could be compensated by renormalization with expected amino acid frequencies . This suggests that HLA peptidomics data are optimal to train HLA-I ligand interaction predictors , as confirmed by our ability to accurately predict from exome sequencing data several neo-antigens identified in tumor samples . These observations are in line with recent results obtained with predictors trained on HLA peptidomics data from mono-allelic cell lines for 16 human class I alleles [31] and mouse class II alleles [43] . Moreover , similar results seem to be observed when including MS data in the training set of NetMHCpan tools [44] . Although mass spectrometry may miss a fraction of the actually presented and immunogenic neo-antigens , those detected by MS are likely presented at high level on cancer cells . Therefore , accurately predicting such dominant neo-antigens is promising to prioritize targets for cancer immunotherapy . Importantly , the improvement in prediction accuracy we report here comes primarily from refinement of known HLA-I motifs , since the less frequent alleles for which we uncovered new motifs were in general not part of our testing sets . For instance , differences are observed at P9 between the motif of HLA-A25:01 obtained from HLA peptidomics data ( preference for F/W/Y/L ) and from IEDB ligands ( preference for Y/L/M/F ) ( Fig 2A ) . This likely explains why the neo-antigen ETSKQVTRW was poorly predicted by standard tools [8 , 12] ( predicted IC50 > 3 , 000nM ) . Similar observations apply for the neo-antigen DANSFLQSV found in [20] ( predicted IC50 > 6 , 000nM with HLA-B51:01 ) . Although these differences may look relatively small on the logos ( Fig 2A ) , they play an important role when ranking tens of thousands of potential epitopes with HLA-I ligand predictors . Moreover , as our predictor is only trained on naturally presented ligands , it may also capture some features of antigen presentation of endogenous peptides beyond the binding to HLA-I molecules . Along this line , it is interesting to note that a smaller improvement in prediction accuracy had been observed when attempting to predict ligands from the SYFPEITHI database [45] ( including a large fraction of viral peptides ) with HLA peptidomics data [24] . Although the dataset used in this previous study was significantly smaller , this observation suggests that HLA peptidomics data may be especially well suited for training predictors of human endogenous or mutated HLA-I ligands . Overall , our work highlights the importance of carefully determining HLA-I motifs , including for alleles that already have some known ligands , based on unsupervised analysis of naturally presented human HLA-I ligands for neo-antigen discovery . In this work , we did not attempt to optimize the prediction algorithm itself , but rather focused on optimizing the training data and carefully correcting for MS biases , which in our view is more important for improving predictions of HLA-I ligands , since HLA-I ligands do not display strong correlations among the different residue positions . Nevertheless , we cannot exclude that using neural networks or other machine learning tools may further improve the predictions , and we anticipate that our large collection and assignment of unbiased HLA-I ligands may help exploring new amino acid correlation patterns among HLA-I ligands ( see example in [24] ) . Currently our predictor is limited to 9- and 10-mers , which is the most common length of HLA-I ligands and accounts for more than 80% of the HLA peptidome [17] . Although motif identification may work in some cases for 11-mers [24] , the automated motif deconvolution and annotation becomes less accurate , especially for samples with less than 10’000 peptides identified by MS . Therefore , rather than including sub-optimal motifs in our predictor , we focused in this work on 9- and 10-mers . We anticipate that manual curation of HLA-I motifs in pooled HLA peptidomics datasets or the use of mono-allelic cell lines [31] may be more appropriate for training predictors for longer peptides . Importantly , not including 11-mers has no influence on the predictions for 9- and 10-mers , since peptides of different length are treated separately in the current framework ( see ref . [8] for possible algorithmic extensions to include peptides of different length in the training set of HLA-I ligand predictors ) . Our work enabled us to identify motifs for uncharacterized alleles and is to date the predictor entirely trained on naturally presented peptides with the largest allelic coverage , including all frequent HLA-I alleles in the Caucasian population . However , the number of HLA-I alleles for which predictions are available ( 58 in total ) is still smaller than what other tools can do ( especially tools like NetMHCpan [12] that can make predictions for any allele ) . Despite this limitation , we emphasize that our work provides the first scalable framework to integrate HLA peptidomics datasets that will be or are being generated for neo-antigen discovery in cancer immunotherapy and therefore will enable increasing the allele coverage as new studies are published . Moreover , our results suggest that improving existing models describing the binding specificity of relatively common HLA-I alleles may be as important as expanding allele coverage to rare alleles for neo-antigen discovery . All HLA peptidomics datasets used in this work were generated with only 1% FDR and are of high purity . For this reason , and also to prevent including potential biases or removing important data , we decided not to filter our data with existing HLA-I ligand predictors , but we expect some contaminants in our large sets of peptides . Moreover , in a few cases , the motifs for some alleles were not detectable ( see HLA-C12:03 in Fig 1 ) . This suggests that the ( few ) peptides binding to this allele may contaminate the other motifs . This is a known situation when analysing HLA peptidomics data with unsupervised approaches [24 , 25 , 46] . As previously observed , it affects especially HLA-C alleles which are often poorly expressed and whose binding specificities are more redundant [24 , 47] . However , our results show that some level of noise is tolerated for training our predictor and can still lead to improvement over existing tools ( Fig 4 and Fig L in S1 Supporting Information ) . We also stress that no existing HLA-ligand interaction dataset used for training predictors is free of false-positives and for many alleles the number of ligands is significantly lower ( Fig E in S1 Supporting Information ) . In a few cases , the motifs of two alleles could not be split because of the very high binding specificity similarity of these alleles ( e . g . , HLA-C07:01 and HLA-C07:02 in Fig 1 ) . We emphasize that this does not preclude the use of HLA peptidomics data for training HLA-I ligand predictors , since many other samples in our dataset contained only one of these two alleles together with other non-overlapping ones . As such our strategy takes advantage of our large collection of HLA peptidomics datasets to naturally overcome cases where the deconvolution could not be fully achieved in one given sample . Direct identification of neo-antigens with MS shows higher specificity compared to our predictions based on exome sequencing data [16 , 17] , as expected . However , it is important to realize that these experiments are challenging and can be carried out only in a small subset of patients with enough tumor material . Moreover , in many cases , no neo-antigen is found by MS . As such , our work provides a scalable approach to capitalize on large MS data obtained from some patients or cell lines in order to improve predictions of neo-antigens in other patients where MS analysis of the immunopeptidome could not be carried out and only exome sequencing data are available . This work may also find applications in other jawed vertebrate species where reference motifs for MHC-I alleles are poorly described , since the only requirement of our approach is the availability of MHC-I specific antibodies and MHC typing information . Previous computational approaches for neo-antigen predictions have shown that incorporation of gene expression data could improve accuracy [31 , 48] . Considering that our predictor using only exome sequencing information ( i . e . , the list of somatic mutations ) already enabled us to improve predictions in the cancer samples analysed in this work , we anticipate that integrating additional information such as gene expression , when available , may lead to even more accurate predictions of neo-antigens . Results shown in Fig 5 further emphasize the power of in-depth sampling of the HLA-I ligand space to inform us about molecular mechanisms underlying HLA-I binding properties [49] . Considering the rapid expansion of HLA peptidomics experiments performed in cancer immunotherapy research [13 , 17 , 18 , 20 , 27 , 28] , we anticipate that our approach for HLA-I motif identification and annotation will enable similar analyses in the future to uncover other molecular determinants of HLA-I binding specificity . Overall , our work shows for the first time that HLA-I motifs can be reliably identified across in-depth and accurate HLA peptidomics datasets without relying on HLA-I interaction prediction tools or a priori knowledge of HLA-I binding specificity . This unsupervised and scalable approach refines known HLA-I binding motifs and expands our understanding of HLA-I binding specificities to a few additional alleles without documented ligands . As such , this work is a powerful alternative to synthesizing every peptide for in vitro binding assays , or to genetically modifying [31] or transfecting cell lines with soluble HLA-I alleles [34 , 35 , 50] , and may save substantially amount of money and time for HLA-I motif determination . Our results further contribute to our global understanding of HLA-I binding properties and improve neo-antigen predictions from exome sequencing data . This work may therefore facilitate identification of clinically relevant targets for cancer immunotherapy , especially when direct identification of neo-antigens with MS cannot be experimentally done .
Informed consent of the participants was obtained following requirements of the institutional review board ( Ethics Commission , University Hospital of Lausanne ( CHUV ) ) . We carefully selected ten donors expressing a broad range of HLA-I alleles and generated novel HLA peptidomics data ( Table A in S1 Supporting Information ) . EBV-transformed human B-cell lines CD165 , GD149 , PD42 , CM467 , RA957 and MD155 were maintained in RPMI 1640 + GlutaMAX medium ( Gibco , Paisley , UK ) supplemented with 10% FBS ( Gibco ) and 1% Penicillin/Streptomycin Solution ( BioConcept , Allschwil , Switzerland ) . TIL were expanded from two melanoma tumors following established protocols [51 , 52] . Informed consent of the participants was obtained following requirements of the institutional review board ( Ethics Commission , University Hospital of Lausanne ( CHUV ) ) . Briefly , fresh tumor samples were cut in small fragments and placed in 24-well plate containing RPMI CTS grade ( Life Technologies Europe BV , Switzerland ) , 10% Human serum ( Valley Biomedical , USA ) , 0 . 025 M HEPES ( Life Technologies Europe BV , Switzerland ) , 55 μmol/L 2-Mercaptoethanol ( Life Technologies Europe BV , Switzerland ) and supplemented with a high concentration of IL-2 ( Proleukin , 6 , 000 IU/mL , Novartis , Switzerland ) for three to five weeks . Following this initial pre-REP , TIL were then expanded in using a REP approach . To do so , 25 x106 TIL were stimulated with irradiated feeder cells , anti-CD3 ( OKT3 , 30 ng/mL , Miltenyi biotech ) and high dose IL-2 ( 3 , 000 IU/mL ) for 14 days . The final cell product was washed and prepared using a cell harvester ( LoVo , Fresenius Kabi ) . Leukapheresis samples ( Apher1 and 6 ) were obtained from blood donors from the Service régional vaudois de transfusion sanguine , Lausanne . Upon receival of TIL and leukapheresis samples , the cells were washed with PBS on ice , aliquoted and stored as dry pellets at -80°C until use . High resolution 4-digit HLA-I typing was performed at the Laboratory of Diagnostics , Service of Immunology and Allergy , CHUV , Lausanne . W6/32 monoclonal antibodies were purified from the supernatant of HB95 cells grown in CELLLine CL-1000 flasks ( Sigma-Aldrich , Missouri , USA ) using Protein-A Sepharose ( Invitrogen , California , USA ) . We extracted the HLA-I peptidome from 2–5 biological replicates per cell line or patient material . The cell counts ranged from 1 x 108 to 3 x 108 cells per replicate . Lysis was performed with 0 . 25% sodium deoxycholate ( Sigma-Aldrich ) , 0 . 2 mM iodoacetamide ( Sigma-Aldrich ) , 1 mM EDTA , 1:200 Protease Inhibitors Cocktail ( Sigma , Missouri , USA ) , 1 mM Phenylmethylsulfonylfluoride ( Roche , Mannheim , Germany ) , 1% octyl-beta-D glucopyranoside ( Sigma ) in PBS at 4°C for 1 hr . The lysates were cleared by centrifugation with a table-top centrifuge ( Eppendorf Centrifuge 5430R , Schönenbuch , Switzerland ) at 4°C at 14200 rpm for 20 min . Immuno-affinity purification was performed by passing the cleared lysates through Protein-A Sepharose covalently bound to W6-32 antibodies . Affinity columns were then washed with at least 6 column volumes of 150 mM NaCl and 20 mM Tris HCl ( buffer A ) , 6 column volumes of 400 mM NaCl and 20 mM Tris HCl and lastly with another 6 column washes of buffer A . Finally , affinity columns were washed with at least 2 column volumes of 20 mM Tris HCl , pH 8 . HLA-I complexes were eluted by addition of 1% trifluoroacetic acid ( TFA , Merck , Darmstadt , Switzerland ) for each sample . HLA-I complexes with HLA-I peptides were loaded on Sep-Pak tC18 ( Waters , Massachusetts , USA ) cartridges which were pre-washed with 80% acetonitrile ( ACN , Merck ) in 0 . 1% TFA and 0 . 1% TFA only . After loading , cartridges were washed twice with 0 . 1% TFA before separation and elution of HLA-I peptides from the more hydrophobic HLA-I heavy chains with 30% ACN in 0 . 1% TFA . The HLA-I peptides were dried using vacuum centrifugation ( Eppendorf Concentrator Plus , Schönenbuch , Switzerland ) and re-suspended in a final volume of 12 uL 0 . 1% TFA . For MS analysis , we injected 5 uL of these peptides per run . Measurements of HLA-I peptidomics samples were acquired using the nanoflow UHPLC Easy nLC 1200 ( Thermo Fisher Scientific , Germering , Germany ) coupled online to a Q Exactive HF Orbitrap mass spectrometer ( Thermo Fischer Scientific , Bremen , Germany ) or with Dionex Ultimate RSLC3000 nanoLC ( Thermo Fischer Scientific , Sunnyvale , CA ) coupled online to an Orbitrap Fusion Mass Spectrometer ( Thermo Fischer Scientific , San Jose , CA ) , both with a nanoelectrospray ion source . We packed an uncoated PicoTip 8μm tip opening with diameter of 50 cm x 75 um with a ReproSil-Pur C18 1 . 9 μm particles and 120 Å pore size resin ( Dr . Maisch GmbH , Ammerbuch-Entringen , Germany ) re-suspended in Methanol . The analytical column was heated to 50°C using a column oven . Peptides were eluted with a linear gradient of 2–30% buffer B ( 80% ACN and 0 . 1% formic acid ) at a flow rate of 250 nl/min over 90 min . Data was acquired with data-dependent “top10” method , which isolates the ten most intense ions and fragments them by higher-energy collisional dissociation ( HCD ) with a normalized collision energy of 27% and 32% for the Q Exactive HF and Fusion instruments , respectively . For the Q Exactive HF instrument the MS scan range was set to 300 to 1 , 650 m/z with a resolution of 60 , 000 ( 200 m/z ) and a target value of 3e6 ions . The ten most intense ions were sequentially isolated and accumulated to an AGC target value of 1e5 with a maximum injection time of 120 ms and MS/MS resolution was 15 , 000 ( 200 m/z ) . For the Fusion , a resolution of 120 , 000 ( 200 m/z ) and a target value of 4e5 ions were set . The ten most intense ions accumulated to an AGC target value of 1e5 with a maximum injection time of 120 ms and MS/MS resolution was 15 , 000 ( 200 m/z ) . The peptide match option was disabled . Dynamic exclusion of fragmented m/z values from further selection was set for 20 or 30 seconds with the Q Exactive HF and Fusion instruments , respectively . We employed the MaxQuant computational proteomics platform [39] version 1 . 5 . 3 . 2 to search the peak lists against the UniProt databases ( Human 85 , 919 entries , May 2014 ) and a file containing 247 frequently observed contaminants . N-terminal acetylation ( 42 . 010565 Da ) and methionine oxidation ( 15 . 994915 Da ) were set as variable modifications . The second peptide identification option in Andromeda was enabled . A false discovery rate of 0 . 01 was required for peptides and no protein false discovery rate was set . The enzyme specificity was set as unspecific . Possible sequence matches were restricted to 8 to 15 amino acids , a maximum peptides mass of 1 , 500 Da and a maximum charge state of three . The initial allowed mass deviation of the precursor ion was set to 6 ppm and the maximum fragment mass deviation was set to 20 ppm . We enabled the ‘match between runs’ option , which allows matching of identifications across different replicates of the same biological sample in a time window of 0 . 5 min and an initial alignment time window of 20 min . To expand the number of samples and survey an even broader range of HLA-I alleles , we included in this study forty publicly available HLA peptidomics data from seven recent studies [17 , 18 , 22 , 23 , 26–28] . Only samples with HLA-I typing were used . Peptides identified in the recent study [18] in different repeats and under different treatments were pooled together to generate one list of unique peptides per sample . Since the published peptidomics datasets from Pearson et al . [28] were filtered to include only peptides with predicted affinity scores of less or equal to 1250 nM , we re-processed the mass spectrometer raw data using MaxQuant with similar settings as mentioned above except that peptide length was set to 8–25 mers ( S2 Dataset ) . HLA typing information was retrieved from the original publications . In one case ( THP-1 cell lines ) , the typing is controversial [53] . In this work , we used the typing determined by the authors of the HLA peptidomics study where the data came from [23] . The high fraction ( >50% of the peptides ) displaying a clear A24:02 and B35:03 motif based on our unsupervised deconvolution further indicates that these alleles are truly expressed in the sample on which the HLA peptidomics analysis was performed . Known HLA-I ligands were retrieved from IEDB ( mhc_ligand_full . csv file , as of Oct 2016 ) [4] . All ligands annotated as positives with a given HLA-I allele ( i . e . , “Positive-High” , “Positive-Intermediate” , “Positive-Low” and “Positive” ) were used to build the IEDB reference motifs ( Fig 2A and 2C ) . Ligands coming from HLA peptidomics studies analysed in this work were not considered to prevent circularity in the motif comparisons and because the HLA-I alleles to which these peptides bind were not experimentally determined . Position Weight matrices ( PWMs ) representing binding motifs in IEDB and used to compare with motifs derived from our deconvolution of HLA peptidomics datasets were built by computing the frequency of each amino acid at each position and using a random count of 1 for each amino acid at each position . All 16 HLA peptidomics datasets obtained from mono-allelic cell lines were downloaded from [31] . Motifs used in the comparison presented in Fig 2B were built in the same way as for IEDB data . When benchmarking our ability to re-predict such data with , we considered 9-mers from the ten alleles that overlapped our set of deconvoluted HLA peptidomics data and added 99-fold excess of random peptides from the human proteome . The fraction of positives ( i . e . , MS peptides identified in these mono-allelic cell lines ) predicted in the top 1% was used to assess the prediction accuracy with different methods . Peptides with a clear tryptic peptide signature ( i . e . , R/K at the last position for all alleles except HLA-A03:01 and HLA-A31:01 ) were manually removed . An algorithm based on mixture models and initially developed for multiple specificity analysis in peptide ligands [29 , 30] was used to identify binding motifs in each dataset analysed in this work . Briefly , all peptides pooled by mass spectrometry analysis of eluted peptide-HLA-I complexes in a given sample were first split into different groups according to their size ( 9–10 mers ) . All 9- and 10-mers ligands were then modelled using multiple PWMs [24] . The results of such analysis consist of a set of PWMs that describe distinct motifs for each HLA peptidomics datasets ( see Fig A in S1 Supporting Information ) and probabilities ( i . e . , responsibilities ) for each peptide to be associated with each motif . Sequence logos representing HLA-I motifs were generated with the LoLa software ( http://baderlab . org/Software/LOLA ) . The command-line script for the mixture model ( “MixMHCp” ) can be downloaded at: https://github . com/GfellerLab/MixMHCp . The availability of high-quality HLA peptidomics data from several samples with diverse HLA-I alleles suggest that one could infer which motifs correspond to which HLA-I alleles without relying on comparison with known motifs . For instance , if two samples share exactly one HLA-I allele , it is expected that the shared motif will originate from the shared allele ( Fig 1 ) . To exploit this type of patterns of shared HLA-I alleles , we designed the following algorithm: Comparison of motifs was performed using ( squared ) Euclidean distance between the corresponding PWMs: D2=1L∑i=120∑A=1L ( MiA−MiA′ ) 2 , where M and M’ stands for two PWMs ( i . e . , 20 x L matrices ) to be compared and L is the peptide length . A threshold of T = 0 . 078 was used to define similar motifs based on visual inspection of the results . Cases of inconsistencies ( i . e . , distances larger that T ) between motifs mapped to the same allele were automatically eliminated . The final binding motif for each HLA-I allele ( Fig 2 and Fig D in S1 Supporting Information ) was built by combining peptides from each sample that had been associated with the corresponding allele . Other measures of similarity between PWMs [33] did not improve the results ( see for instance comparison between Euclidean distance and Jensen-Shannon divergence in Fig Q in S1 Supporting Information for the example shown in Fig 1 ) and we therefore used the Euclidean distance throughout this study . To further study the statistical significance of the similarity between motifs annotated to the same alleles , we computed the P-value of the similarity for each pair of motifs annotated to the same allele . To this end , we used both the Euclidean distance and the BLiC score with standard Dirichlet priors and hyper-parameters αj ( j = 1…20 ) equal to one , as introduced in ref . [33] . This BLiC score was re-implemented using 20 amino acids ( instead of the 4 nucleotides ) and the background distribution was taken as the expected amino acid frequencies in MS data ( see Table B in S1 Supporting Information ) . For a pair of motifs ( mi , mj ) , empirical P-values were estimated by comparing the similarity between motifs mi and mj to the similarity between motif mi and all known HLA-I motifs ( i . e , 107 in total , taking only alleles with more than 20 unique ligands in IEDB and excluding the allele to which the motif mi had been annotated ) . The distributions of P-values for all pairs of motifs annotated to the same alleles ( i . e . , for each allele h , i = 1 , …Nh , j = 1…Nh , with i≠j and Nh the number of motifs annotated to allele h ) are shown in Fig G in S1 Supporting Information for both the Euclidean distance and the BLiC score [33] , and indicate that for more than 99% of the pairs of motifs annotated to the same alleles , the similarity has a P-value smaller than 0 . 05 . The few pairs with P-values larger than 0 . 05 come mainly from HLA-C allele , as expected since they are less expressed and more degenerate , and therefore more challenging to describe . We further studied the impact of the threshold T and explored several values between 0 . 01 and 0 . 14 . As shown in Fig H in S1 Supporting Information , smaller values of T result in fewer alleles to which motifs can be annotated , as expected . Reversely , for larger thresholds the algorithm seems to behave badly and many more motifs annotated to the same alleles are no longer statistically similar . Data in Fig H in S1 Supporting Information suggest that reasonable results can be obtained for thresholds between 0 . 05 and 0 . 09 , which is compatible with our manual choice of 0 . 078 . In practice , HLA-A and HLA-B alleles tend to be more expressed and therefore give rise to a stronger signal in HLA peptidomics data . We therefore used first our deconvolution method [24] , setting the number of motifs equal to the number of HLA-A and HLA-B alleles and identified HLA-A and HLA-B motifs with the algorithm introduced above ( Step 1 ) . We then ran our deconvolution method [24] without restricting the number of clusters ( Step 2 ) and identified motifs corresponding to HLA-A and HLA-B alleles based on the similarity with those identified in Step 1 . The remaining motifs were then analysed across all samples with the algorithm introduced above . To expand the identification of binding motifs for alleles without known ligands , we used data from IEDB for HLA-I alleles with well-described binding motifs . In practice , for all HLA-I alleles in our samples that had not been mapped to motifs in the fully unsupervised approach and have more than twenty different ligands in IEDB , PWMs were built from IEDB data . These PWMs were used to scan the remaining motifs in each sample that contained the corresponding alleles . Motifs were mapped to HLA-I alleles if exactly one PWM obtained with the mixture model was found to be similar to the IEDB-derived motif ( i . e . , Euclidean distance smaller than T , as before ) . The unsupervised procedure described above was then applied to the remaining motifs to identify new motifs for alleles without ligands in IEDB . To have reliable estimates of the potential technical biases due to MS , amino acid frequencies were computed at non-anchor positions ( P4 to P7 ) for alleles in our HLA peptidomics datasets ( 9-mers ) . Alleles showing some specificity at these positions ( A02:01 , A02:05 , A02:06 , A02:20 , A25:01 , A26:01 , A29:02 , B08:01 , B14:01 , B14:02 , C03:03 , and C07:04 , see Fig D in S1 Supporting Information ) were excluded from this analysis . The average frequencies of amino acids across alleles were then compared against the human proteome using Pearson correlation coefficient ( Fig 3 ) . We also performed the same analysis with HLA-I ligands ( 9-mers ) from IEDB splitting between those obtained by MS and those obtained by other assays ( “non-MS data” ) ( see Fig J in S1 Supporting Information ) . To enable meaningful comparison between these datasets , only alleles present in our HLA peptidomics data , with more than 100 ligands in both IEDB MS and non-MS data were considered in this analysis ( 14 alleles in total , see Fig D in S1 Supporting Information ) . For each HLA-I allele , PWMs were built from all peptides associated to this allele across all samples where the binding motif could be identified , using the highest responsibility values of the mixture model [24] . The frequency of each amino acid was first computed . Pseudocounts were added using the approach described in [54] , based on the BLOSUM62 substitution matrix with parameter β = 200 . The score of a given peptide ( X1 , …XN ) was computed by summing the logarithm of the corresponding PWM entries , including renormalization by expected amino acid frequencies: S=1N∑i=1Nlog ( pXi , iqXi ) . Here qA stands for frequency of amino acid A at non-anchor positions ( Fig 3 and Table B in S1 Supporting Information ) , pA , i stands for the PWM entry corresponding to amino acid A at position i , and N stands for the length of the peptide ( N = 9 , 10 ) . The final score of a peptide was taken as the maximal score across all alleles present in a given sample and a P-value estimate is computed by comparing with distribution of scores obtained from 100 , 000 randomly selected peptides from the human proteome , so as to have similar amino acid frequencies compared to endogenous ligands ( see MixMHCpred1 . 0 results ) . To test our ability to predict neo-antigens , we used four melanoma samples in which ten neo-antigens ( 9- and 10-mers ) have been directly identified with in-depth immunopeptidomics analyses of the tumor samples: Mel5 , Mel8 , Mel15 from [17] and 12T [20] . Missense mutations ( i . e . cancer specific non-synonymous point mutations ) identified by exome sequencing in those four melanoma samples [17] were retrieved and a list of all possible 9- and 10-mer peptides encompassing each mutation was built ( S3 Dataset ) . Multiple transcripts corresponding to the same genes were merged so that each mutated peptide appears only once in the list . The total number of potential neo-antigens in each sample is shown in Table 1 . Predictions for each HLA-I allele of each sample were carried out with the model described above . Peptides were ranked based on the highest score over the different alleles present in their sample . In parallel , affinity predictions with NetMHC ( v4 . 0 ) [8] and NetMHCpan ( v3 . 0 ) [12] and stability predictions with NetMHCstabpan ( v1 . 0 ) [36] were performed for the same peptides and peptides were ranked based on predicted affinity using the highest score ( i . e . , lowest Kd ) over all alleles . Only HLA-A and HLA-B alleles were considered since HLA-C alleles are known to show much lower expression and both our predictor and NetMHC could not be run for some HLA-C alleles in these melanoma patients . Ranking of the neo-antigens compared to all possible peptides containing a missense mutation is shown in Table 1 with either our predictor ( MixMHCpred ) or the other tools mentioned above . Area Under the Curves were also computed ( Fig 4B ) , as well as the fraction of neo-antigens that fell among the top 1% of the predictions ( PP1% , which typically corresponds to what can be experimentally tested ) ( Fig 4C ) , to provide a graphical visualization of the results in Table 1 . The same analysis was applied to study neo-antigens ( 9- and 10-mers ) recently identified in two lung cancer patients [37] ( L011: FAFQEYDSF , GTSAPRKKK , SVTNEFCLK , RSMRTVYGLF , GPEELGLPM and L013: YSNYYCGLRY , ALQSRLQAL , KVCCCQILL ) and the ranking of these epitopes with different HLA-ligand predictors with respect to the full list of potential epitopes is shown in Table C in S1 Supporting Information ( see also Fig 4B and 4C ) . To assess how much our improved predictions of neo-antigens in the three melanoma samples of [17] depend on HLA peptidomics data generated from these samples , we performed a careful cross-sample validation . For each of the three samples where neo-eptiopes had been identified ( Mel15 , Mel8 , Mel5 ) [17] , we re-run our entire pipeline ( i . e . , annotation of HLA-I motifs across HLA peptidomics datasets + construction of PWMs for each allele ) without the HLA peptidomics data coming from this sample . The PWMs were then used to rank all possible peptides ( 9- and 10-mers ) encompassing each mutation . Overall , the predictions changed very little ( Fig K in S1 Supporting Information ) . All cancer testis antigens with annotated epitopes ( 9- or 10-mers ) and HLA restriction information were retrieved from the CTDatabase [38] for all alleles considered in our predictor ( see Table C in S1 Supporting Information ) . All other possible peptides along these proteins ( 9- or 10-mers ) were used as negatives when benchmarking the predictions on this dataset . HLA-I sequences were retrieved from IMGT database [3] . All protein structures analysed in this work were downloaded from the PDB . Residues forming the P2 binding site in HLA-B14:02 ( PDB: 3BVN [42] ) were determined using a standard cut-off of 5Å from any heavy atoms of arginine at P2 . W97R mutation was introduced into HLA-B14:02 wt by overlap extension PCR and confirmed by DNA sequencing . BL21 ( DE3 ) pLys bacterial cells were used to produce HLA-B14:02 wt and W97R as inclusion bodies . Four peptides with histidine or arginine at P2 ( A[H/R]TKPRPAL , G[H/R]YDRSKSL , A[H/R]FAKSISL , H[H/R]FEKAVTL ) were synthesized at the Peptide Facility ( UNIL , Lausanne ) with free N and C-termini ( 1mg of each peptide , > 80% purity ) . Peptides with histidine at P2 come from our HLA peptidomics data and were assigned to HLA-B15:18 by our mixture model algorithm . Based on our analysis of HLA sequence and structure , peptides with histidine at P2 are predicted to interact with HLA-B14:02 W97R , while peptides with arginine at P2 are predicted to bind better HLA-B14:02 wt . Synthetic peptides were incubated separately with denaturated HLA-B14:01 wt and HLA-B14:01 W97R mutant refolded by dilution in the presence of biotinylated beta-2 microglobulin proteins at temperature T = 4°C for 48 hours . The solution was then incubated at 37°C and samples were retrieved at time t = 0h , 8h , 24h , 48h and t = 72h . The known HLA-B14:02 ligand IRHENRMVL was used for positive controls . Negative controls consist of absence of peptides . koff were determined by fitting exponential curves to the light intensity values obtained by ELISA at different time points . Half-lives were computed as ln ( 2 ) /koff . Values shown in Fig 5D correspond to the average over two replicates . For two peptides showing exceptionally high binding stability , only lower bounds on half-lives could be determined ( dashed lines in Fig 5D ) . | Predicting the differences between cancer and normal cells that are visible to the immune system is of central importance for cancer immunotherapy . Here we introduce a novel computational framework to harness the wealth of data from in-depth HLA peptidomics studies , including ten novel high quality ( <1% FDR ) datasets generated for this work , to improve predictions of peptides displayed on HLA-I molecules . These high-throughput and unbiased data enable us to refine models of HLA-I binding specificity for many alleles ( including some that had no ligand until this study ) and improve predictions of neo-antigens from exome sequencing data in melanoma and lung cancer samples . Moreover , the refined description of HLA-I binding specificity reveals cases of allosteric modulation of HLA-I binding specificity at the second amino acid position ( P2 ) of their ligands by residues that are part of the HLA-I binding site but outside of the B pocket . | [
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] | 2017 | Deciphering HLA-I motifs across HLA peptidomes improves neo-antigen predictions and identifies allostery regulating HLA specificity |
Malaria ( Plasmodium spp . ) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies . The most important human malaria parasite , Plasmodium falciparum , undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut . Here , we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans . Specifically , we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99% . Of those mosquitoes that were infected , we observed a 75–99% reduction in parasite load . In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked . The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan . Thus , activation of Akt signaling reduced the number of infected mosquitoes , the number of malaria parasites per infected mosquito , and the duration of mosquito infectivity .
Malaria is one of the world's most severe public health concerns , killing nearly one million people annually [1] . The disease is caused by infection with parasites of the genus Plasmodium that are transmitted by female anopheline mosquitoes . Shortly after an infective bloodmeal is consumed by the mosquito , motile ookinetes develop and attempt to invade the mosquito midgut . Ookinetes that successfully traverse the midgut epithelium form non-motile oocysts and develop on the midgut for a minimum of 12 days before rupturing and releasing sporozoites capable of invading the salivary glands . Following salivary gland invasion by sporozoites , and within 16 days after ingestion of an infectious bloodmeal , the mosquito becomes infective to humans and remains so for the duration of its life . Midgut invasion by the parasite is highly risky and a majority of the parasites perish before developing into oocysts [2] , [3] . Further , Anopheles stephensi mosquitoes – the leading vector of malaria in India , parts of Asia and the Middle East and the focus of our work – rarely survive more than two weeks in the field [4]–[6] . These observations suggest that only the oldest mosquitoes in a population are capable of transmitting malaria and that even a modest reduction in lifespan could significantly impact parasite transmission . The insulin/insulin-like growth factor 1 signaling ( IIS ) cascade plays a critical role in the regulation of innate immunity and lifespan in a wide range of vertebrate and invertebrate organisms [7] , [8] . IIS is initiated through the binding of insulin-like peptides ( ILPs ) to the insulin receptor , leading to a series of downstream phosphorylation events that include the key signaling protein Akt . Activation of IIS results in translocation of Akt to the cell membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 ( PDK1 ) . Activated Akt then phosphorylates the forkhead transcription factor FOXO1 , preventing it from entering the nucleus and activating transcription of target genes [9] . In model invertebrates , the IIS cascade has been linked to both innate immunity and lifespan regulation . In the nematode Caenorhabditis elegans , disruption of the insulin receptor orthologue daf-2 leads to decreased IIS , extension of lifespan [10] and increased resistance to bacterial infection [11] . In contrast , loss of function mutations in the FOXO1 orthologue daf-16 result in nematodes that are sensitive to infection [11] and short-lived [12] . As in C . elegans , disruption of the IIS can lead to lifespan extension in the fruit fly Drosophila melanogaster [13]–[15] . Recent work has also demonstrated that activation of the Toll cascade , a key pathway in fly immunity , inhibits IIS in the fly [16] . These observations confirm that the connections observed in humans between innate immunity , metabolism and aging are evolutionarily conserved ( reviewed in [17] ) . Lifespan extension due to IIS disruption is tissue-dependant , although the tissues involved can vary within and across genera . In C . elegans [18] and D . melanogaster [15] , the nervous system is a key IIS center . In D . melanogaster , disruption of IIS in the fat body can also lead to lifespan extension [15] . Overexpression of the transcription factor daf-16 in the C . elegans intestine extends lifespan [19] . Our previous work with A . stephensi suggests that the analogous mosquito tissue – the midgut – is also a center of IIS . In particular , we have shown that ingested human insulin can activate IIS in midgut epithelial cells and significantly decrease the lifespan of A . stephensi mosquitoes [20] , implying a direct relationship between exogenous insulin from the mammalian bloodmeal , activation of the midgut IIS , and lifespan . Therefore , we predicted that genetic manipulation of key IIS components in the midgut would offer a unique strategy for disrupting P . falciparum development while simultaneously decreasing the lifespan of the mosquito below the extrinsic incubation period ( EIP ) or the time required for malaria parasite development . We genetically engineered A . stephensi to express an active variant of the mosquito Akt under the control of the midgut-specific carboxypeptidase ( CP ) promoter . As predicted , increased Akt signaling in the midgut significantly reduced malaria parasite development and mosquito lifespan . Both the number of infected mosquitoes and the average number of parasites per mosquito were reduced in transgenic mosquitoes relative to controls . In addition , transgenic mosquitoes had significantly shorter lifespans than non-transgenic siblings reared under identical conditions . These results demonstrate that manipulation of one signaling protein , Akt , in the mosquito midgut can affect both mosquito innate immunity and lifespan .
We generated a transgenic A . stephensi line overexpressing an activated form of Akt under the control of the midgut-specific CP promoter . Activated Akt was generated by a myristoylation sequence encoded at the amino terminus . An HA epitope at the carboxy terminus ( myr-AsteAkt-HA ) facilitated protein identification . The construct was inserted into the pBac[3XP3-DsRedafm] plasmid vector [21] for transformation into the A . stephensi genome ( Fig . 1A ) . We injected approximately 4400 embryos with a mixture of the pBac[3XP3-DsRedafm]CP-myr-AsteAkt-HA donor plasmid and the phsp-pBac helper plasmid , resulting in approximately 176 adult mosquitoes whose progeny were screened for DsRed eye fluorescence ( Fig . 1B ) . We isolated three F1 progeny with stable DsRed eye fluorescence , from which we established a stable line ( Fig . 1C ) . Transgenic mosquitoes were maintained as a heterozygous line by outcrossing the mosquitoes in each generation to non-transgenic colony A . stephensi . A homozygous line was generated after approximately 20 generations of outcrossing and used to verify the viability of homozygous mosquitoes and to test the effect of increased Akt signaling on P . falciparum development in the midgut . We assessed myr-AsteAkt-HA transcript expression levels during mosquito development and found that myr-AsteAkt-HA is primarily expressed in pupae and adult female stages ( Figure S1 , Text S1 ) . In adult females , the myr-AsteAkt-HA transcript and protein were only detected within the midgut of transgenic mosquitoes ( Figs . 1D and 1E ) . No expression was observed in the carcass of transgenic mosquitoes or in the midgut or carcass of non-transgenic mosquitoes . Surprisingly , we observed high levels of transcript ( Fig . 2A ) and protein ( Fig . 2B ) in the midguts of both non-bloodfed and bloodfed transgenic mosquitoes . Transcript expression increased slightly 2–6 h after bloodfeeding and increased dramatically between 24–48 h after the bloodmeal ( Fig . 2A ) . Protein expression increased 2–12 h after the bloodmeal as would be expected for the CP promoter , but was reduced during the latter half of the reproductive cycle ( 24–48 h ) ( Figs . 2B and 2C ) . The myristoylation sequence at the amino terminus was expected to target myr-AsteAkt-HA to the cell membrane to be phosphorylated and activated by PDK1 , eliminating the need for Akt binding to the upstream IIS component phosphoinositide ( 3 , 4 , 5 ) -trisphosphate and endogenous insulin signaling in general . To assess membrane localization of myr-AsteAkt-HA , we performed immunocytochemistry on both midgut sections ( Fig . 3A ) and whole midguts ( Fig . 3B ) of transgenic and non-transgenic mosquitoes using an anti-HA-fluorescein antibody . Strong staining of midgut epithelial cells was observed only in transgenic mosquitoes and no expression was observed in non-transgenic mosquitoes . A majority of the staining in midguts from transgenic mosquitoes was localized to the cell membrane as expected with the myristoylation sequence ( Fig . 3A and 3B – white arrows ) . To confirm this result , we isolated the nuclei , cell membranes , and cytoplasm from midgut epithelia of transgenic mosquitoes and compared transgene protein levels in these fractions . The transgene protein was detected only in the cell membrane fraction at levels similar to those observed in an intact midgut ( Fig . 3C ) . FOXO1 is key transcription factor in the IIS cascade that is directly phosphorylated by Akt . Human insulin induced FOXO1 phosphorylation in the midguts of bloodfed , non-transgenic A . stephensi ( Fig . 4A ) . In CP-myr-AsteAkt-HA-expressing mosquitoes , we also observed a marked increase in midgut FOXO1 phosphorylation relative to non-transgenic sibling mosquitoes even though a bloodmeal was not provided ( Fig . 4B ) . This indicates that myr-AsteAkt-HA is active and capable of phosphorylating downstream IIS effectors . In sum , both human insulin and myr-AsteAkt-HA induced FOXO1 phosphorylation in vivo . Increased Akt activity in the midgut epithelium led to major reductions in both the percentage of mosquitoes infected with P . falciparum and the number of oocysts in the midguts of infected mosquitoes ( Fig . 5 ) . The percentage of mosquitoes with one or more oocysts decreased from an average of 58 . 5% ( 36–86% ) in non-transgenic controls to 10 . 5% ( 2–14% ) in heterozyogous myr-AsteAkt mosquitoes ( Fig . 5A , p<0 . 0001; pooled across replicates ) . Similarly , the intensity of infection was reduced by 95 . 6% from an average of 3 . 9 oocysts/midgut ( 0–45; n = 200 ) in non-transgenic controls to 0 . 18 ( 0–6; n = 200 ) in myr-AsteAkt mosquitoes ( Fig . 5B ) . This rate of inhibition is higher than rates reported for other anti-parasite effector molecules , including SM1 ( 81 . 6% ) , PLA2 ( 87% ) , anti-HAP2 ( 81 . 1% ) , and anti-chitinase ( 91 . 3% ) [22]–[25] . We also assessed the effect of doubling myr-AsteAkt expression by establishing a homozygous transgenic line . P . falciparum infection in the homozygous line was completely blocked , with no viable oocysts observed in any of the midguts ( Fig . 6; n = 90 ) . In contrast , 62% of control mosquitoes had at least one oocyst , with an average of 6 . 6 parasites per midgut ( 0–76; n = 150 ) . A recent study demonstrated that the combination of two effector molecules , defensin A and cercropin A , was capable of completely blocking the development of the avian malaria parasite Plasmodium gallinaceum in Aedes aegypti [26] . However , our data constitute the first example of a single effector molecule in a transgenic mosquito completely blocking invasion by the human malaria parasite . We hypothesized that increased activation of IIS due to expression of myr-AsteAkt-HA in the midgut would alter the lifespan of sugarfed and bloodfed mosquitoes relative to non-transgenic controls . In contrast to bloodfeeding , mosquitoes provided with sugar only do not enter a reproductive cycle or produce eggs . In replicated assays , sugarfed transgenic mosquitoes lived an average of 18 . 85 ( 17 . 16–20 . 29 ) days compared to 23 . 02 ( 22 . 17–24 . 04 ) days for non-transgenic control siblings , a decrease of nearly 20% ( Fig . 7A; p<0 . 0001 to p = 0 . 0001 ) . This same trend was observed in transgenic mosquitoes provided with weekly bloodmeals and given the opportunity to produce a weekly clutch of eggs . Bloodfed transgenic mosquitoes survived an average of 17 . 44 ( 16 . 54–19 . 24 ) days compared with 21 . 32 ( 17 . 94–25 . 75 ) days for non-transgenic control siblings , a reduction of more than 18% ( Fig . 7B; p = 0 . 0058 to 0 . 0482 ) . An important measure for malaria control is the percent of the population that survives long enough to transmit the malaria parasite . If one assumes that ( 1 ) a female mosquito finds a mate on the first day after adult eclosion , ( 2 ) she acquires an infective bloodmeal on the second day , and ( 3 ) the parasite develops and invades the salivary glands 14 days after taking the infected bloodmeal , then that mosquito must survive a minimum of 16 days to successfully transmit P . falciparum ( blue areas Figs . 7A and 7B ) . Under our conditions , an average of 59% of the non-transgenic mosquitoes given weekly bloodmeals were still alive at day 16 compared to 44% of the myr-AsteAkt transgenic mosquitoes . Comparing the area under the lifespan curves of the transgenic and non-transgenic siblings after 16 days , we observed a 53% reduction in sugarfed mosquitoes and a 48% reduction in bloodfed mosquitoes . This indicates that the population of competent malaria vectors can be reduced by half with a modest 20% reduction in lifespan . Fitness costs due to the generation of the transgenic line or the transgene itself were likely minimal due to transgene insertion into non-coding sequence ( Figure S2 , Text S1 ) and repeated out-crossing to non-transgenic mosquitoes . In addition , lifespan studies were performed using sibling transgenic and non-transgenic mosquitoes to minimize genetic differences and were performed with sibling mosquitoes reared as larvae in the same pans of water and separated as pupae to minimize environmental differences . Insulin signaling regulates reproduction in a wide range of organisms . In insects , including mosquitoes , IIS has been shown to regulate steroidogenesis in the ovaries and vitellogenesis in the fat body [27] , [28] . Although IIS in the midgut has not previously been implicated in the regulation of reproduction , we examined whether any differences in egg production between the transgenic and non-transgenic siblings could be detected . For the five replicates in which zero egg counts were recorded , there were no significant differences between transgenic and non-transgenic females in the number of eggs laid ( Table 1 ) . Among those that laid eggs , only one replicate of the six indicated a significant difference between non-transgenic and transgenic females . The remaining replicates indicated no difference between genotypes ( Table 1 ) . There was no difference between genotypes in whether or not females laid eggs . To ensure that differences in egg production were not due to the size of the blood meal , the amount of blood ingested was also compared between transgenic and non-transgenic females . For all five replicates , there was no significant difference between genotypes in the amount of blood ingested ( Figure S3A and S3B , Text S1 ) . In addition , no obvious differences were observed between transgenic and non-transgenic sibling mosquitoes in the amount of undigested BSA remaining at 24 h after blood ingestion ( Figure S3C , Text S1 ) .
Mosquitoes require a bloodmeal to initiate a reproductive cycle and produce eggs . Within this bloodmeal are insulin , insulin-like growth factor 1 , and various other factors that circulate in the blood of the human host . Our previous work demonstrated that some of these factors , including human insulin and human TGF-β1 , activate mitogen-activated protein ( MAP ) kinase and phosphoinositide-3 kinase ( PI3K ) signaling cascades in the mosquito midgut [20] , [29] . Here , we used transgenesis to overexpress a key component of the IIS cascade , Akt , in the A . stephensi midgut to induce signaling independent of exogenous insulin . We observed significant reductions in both the prevalence and intensity of P . falciparum infections in transgenic mosquitoes following the consumption of an infective bloodmeal . We also observed a reduction in lifespan consistent with that observed in insulin-fed A . stephensi [20] , indicating that the mosquito midgut plays a central role in regulating lifespan . Myristoylated Akt localized to the midgut epithelial cell membrane in transgenic A . stephensi ( Fig . 3 ) where it was activated to subsequently phosphorylate the downstream effector protein FOXO1 ( Fig . 4C ) . This parallels FOXO1 phosphorylation in the midguts of mosquitoes fed bloodmeals containing insulin ( Fig 4A ) . Taken together , these results suggest that the mechanisms of parasite and lifespan reduction observed in CP-myr-AsteAkt-HA transgenic mosquitoes are dependent on the activation of the PI3K/Akt/FOXO arm of the IIS cascade . It is noteworthy that Akt has been defined as “a critical signaling node within all cells of higher eukaryotes and one of the most important and versatile protein kinases at the core of human physiology and disease [30] . ” Akt has more than 100 experimentally verified substrates and broad crosstalk between a variety of biologically important signal transduction pathways . Thus , the mechanisms through which tissue-specific Akt overexpression regulates innate immunity and lifespan are likely to be complex [30] . A carboxypeptidase promoter drives the myr-AsteAkt-HA transgene , so we expected expression to rise shortly after a bloodmeal was consumed and to be midgut-specific . Expression of myr-AsteAkt-HA was indeed specific to the midgut ( Fig . 1D and E ) , but the timing of expression was unexpected since both transcript and protein were observed even in the absence of a bloodmeal ( Fig . 2 ) . As expected for a gene regulated by a carboxypeptidase promoter , however , protein expression increased following ingestion of the bloodmeal . Leaky transgene expression has been observed with this promoter , resulting in expression prior to bloodfeeding [31] or late in the reproductive cycle [32] . The process of generating a transgenic mosquito strain could also explain the unexpected expression patterns . For example , the transgene may have inserted near an enhancer DNA sequence , resulting in greater gene and protein expression [33] . Although this pattern of myr-AsteAkt-HA expression was unexpected , it was ultimately advantageous because increased insulin signaling is maintained for the apparent duration of adult female life and does not depend on consumption of a bloodmeal for activity . Thus , the anti-parasite activity and lifespan effects of myr-AsteAkt-HA will occur regardless of the timing and quantity of bloodmeals that are consumed by a transgenic mosquito . Increased insulin signaling in the mosquito midgut , whether through ingestion of exogenous insulin [20] or overexpression of active IIS proteins such as Akt , can significantly reduce mosquito lifespan and inhibit P . falciparum development . Importantly , we observed that increased AsteAkt expression in the homozygous line increased parasite resistance to the point that oocyst formation on the midgut was completely blocked . Although it will likely be necessary to deploy heterozygous mosquitoes for any future transmission blocking strategy , our data suggest that an increase in myr-AsteAkt expression , possibly through manipulation of the promoter or transgene insertion site , could yield heterozygous mosquitoes that are resistant to P . falcipaurm infection . Lifespan reduction can also impact malaria parasite prevalence based on the combined effects of a relatively short natural lifespan of A . stephensi [4]–[6] and a relatively lengthy parasite development time . In particular , models of vector competence routinely demonstrate that the daily probability of survival is the single most important factor in determining how effectively a mosquito transmits a pathogen [34] . All else being equal , even modest reductions in lifespan will have significant effects on disease transmission . In summary , we have developed a novel mechanism to reduce the transmission of the human malaria parasite P . falciparum . This approach is based on the manipulation of two key physiological parameters , lifespan and innate immunity , through activation of a single signaling protein , Akt . Increased Akt activity significantly reduced infection prevalence in the mosquito host at the same time that it reduced the infective period of the mosquito lifespan . A multi-component approach to transgenesis focused on manipulation of the IIS cascade could be combined with overexpression of additional anti-parasite effectors to effectively block parasite transmission , reduce lifespan , and increase fecundity . Perhaps more importantly , a multi-component approach could prevent the escape of adaptive parasite variants , providing a powerful new tool for malaria control .
Anopheles stephensi mosquitoes were maintained at 28°C , 75% RH , on a 16∶8 light∶dark photoperiod . Larval mosquitoes were fed cat food pellets ( Purina ) . Adult mosquitoes were fed ad libitum on a 10% dextrose solution . Porcine blood ( UA Meat Science facility ) supplemented with sodium citrate ( 0 . 38% ) and warmed to 37°C was used for colony maintenance and bloodfeeding experiments . For feeding experiments , engorged females were separated from unfed and partially fed mosquitoes and maintained on 10% dextrose until needed . Females used for post-oviposition studies were transferred 48 h post-blood meal to a new container and allowed to oviposit on moistened filter papers overnight . The A . gambiae carboxypeptidase ( CP ) promoter was kindly provided by Dr . Luciano Moreira [32] . The 5′ promoter was amplified with primers designed to remove the signal peptide , start methionine and Kozak consensus sequence , and to add XhoI and NotI restriction digest sites . The modified 5′ CP promoter was ligated into the phsp-pBac shuttle plasmid using XhoI and NotI sites [21] . The SV40 3′ UTR was ligated into the EcoRI site of phsp-pBac . A Kozak consensus sequence ( CCAACCATGG ) and Src myristoylation sequence ( MGSSKSKPKDPSQR ) were added to the 5′ end of AsteAkt , while an HA epitope ( YPYDVPDYA ) was added to the 3′ end . This construct was inserted it into the phsp-pBac shuttle containing the CP promoter and SV40 3′ UTR . Finally , the CP-myr-AsteAkt-HA-SV40 construct was ligated into the pBac[3XP3-DsRedafm] construct to generate the pBac[3XP3-DsRedafm]CP-myr-AsteAkt-HA plasmid for injection into A . stephensi embryos . Donor ( 500 ng/µl ) and helper ( 200 ng/µl ) plasmids were injected into newly oviposited embryos , which were then reared to adulthood and screened for transgene insertion as described by Lobo et al [35] . Lifespan and reproduction experiments were initiated only after five generations of outcrossing the transgenic line to a non-transgenic lab strain . Crosses between heterozygous transgenic and non-transgenic mosquitoes produced a 50/50 ratio of transgenic to non-transgenic siblings . Midguts and carcasses ( whole body without midgut ) were collected from ten transgenic females prior to bloodfeeding and at 2 , 6 , 12 , 24 , 48 , and 72 h PBM . Total RNA was extracted using RNeasy kit ( Qiagen ) , treated with DNase 1 ( Fermentas ) and cDNA was synthesized using High Capacity cDNA ReverseTranscription Kit ( Applied Biosystems ) with random hexamer primers . Quantitative real-time PCR ( qRT-PCR ) was performed using Maxima SYBP Green/ROX qPCR master mix ( Fermentas ) and an ABI 7300 real-time PCR system . Myr-AsteAKT-HA-specific primers ( forward: 5′-TTACCGGTGAAAGTGTGGAGCTGA-3′; reverse: 5′-AAGCGTAATCTGGCACATCGTATGG-3′; efficiency - 98% ) were used to detect myr-AsteAKT-HA in midguts and carcasses . Myr-AsteAKT-HA expression was normalized to ribosomal protein S7 expression . qRT-PCR reactions were performed in triplicate and the experiment was replicated twice with separate cohorts of mosquitoes . Immunoblots were performed with one midgut equivalent of protein as previously described [36] . Myr-AsteAkt-HA protein levels were detected using an anti-HA antibody ( 1∶20 , 000 dilution; Roche ) . RT-PCR and immunoblot assays were replicated three times with separate cohorts of mosquitoes . A total of 100 3- to 5-day-old female A . stephensi mosquitoes were fed artificial bloodmeals supplemented with 1 . 7×10−3 µmol of human insulin or an equivalent volume of insulin buffer as described in Kang et al [20] . Immunoblot analyses of protein phosphorylation from 60 midguts per treatment group were conducted as previously described [20] . Midgut samples were probed with anti-phospho FOXO1A/FOXO3A antibody ( 1∶1000 dilution , Millipore ) or an anti-GADPH antibody ( 1∶10 , 000 dilution , Abcam ) to assess protein loading . In the CP-myr-AsteAkt transgenic mosquitoes midguts were subjected to immunoblot analysis as described above , and were probed with anti-phospho-FOXO1A antibody ( 1∶10 , 000 dilution; Millipore ) . Five midgut equivalents of protein were used per lane . Blots were stripped and re-probed with an anti-GADPH antibody ( 1∶40 , 000 dilution , CST ) to assess protein loading . For whole mount immunocytochemistry studies , midguts were dissected from 10 transgenic and 10 non-transgenic mosquitoes in 1× Aedes saline ( 125 mM NaCl , 5mM KCl , 1 . 85 mM CaCl2 , pH 6 . 5 ) and opened into a midgut sheet . Immunocytochemistry was performed as described by Riehle and Brown [37] , except that an anti-HA antibody conjugated to fluorescein ( 1∶1000 , Roche ) was used without a secondary antibody . All samples were imaged at identical settings to facilitate comparison . Experiments were replicated a minimum of three times with separate cohorts of mosquitoes . For immunocytochemistry using paraffin embedded sections , midguts were dissected from 10 transgenic and 10 non-transgenic mosquitoes in 1× Aedes saline and 10× Complete protease inhibitors ( Roche ) . Midguts were immediately transferred to 4% paraformaldehyde in PBS for 2 h at RT and then stored in 70% EtOH at 4°C until embedded . The midguts were embedded in paraffin at the University of Arizona histology center and cut to obtain 4 . 5–5 µM sections . The paraffin was removed by two xylene washes of 10 min and the samples were hydrated through a series of solutions of decreasing ethanol concentration ( 100 , 95 , 70 , 50 and 30% ) . The tissues were washed in PBS with 0 . 1% Tween 20 ( PBS-T ) and then blocked in a solution of 2% BSA/PBS-T for 2 h at RT . The slides were incubated overnight in a humid chamber with a 1∶500 dilution of the anti-HA antibody conjugated to fluorescein . The tissues were washed 3× in PBS-T for 15 min at RT and observed under a Nikon Eclipse E600 fluorescent microscope . Images were acquired using a SPOT camera system ( Diagnostic Instruments Inc ) at identical settings for all fluorescent images . To verify the subcellular localization of myr-AsteAkt-HA , we prepared midgut cell membranes , nuclei and cytoplasm from midguts from transgenic and non-transgenic A . stephensi as described by Brown et al [38] . The three sub-cellular fractions were subjected to immunoblot analysis using the anti-HA antibody as described above with replicated samples from three separate cohorts of mosquitoes . Cultures of P . falciparum NF54 were initiated at 1% parasitemia in 10% heat-inactivated human serum , and 6% washed human RBCs in RPMI 1640 with HEPES ( Gibco ) and hypoxanthine . Stage V gametocytes were evident by day 15 and exflagellation was evaluated on the day prior to and the day of mosquito feeding . For our assays , 5-day old female transgenic and non-transgenic A . stephensi were fed on mature gametocyte culture diluted with human erythrocytes and heat-inactivated serum . On day 10 , midguts from fully gravid females were dissected in PBS and stained with 1% mercurochrome/PBS to visualize P . falciparum oocysts . Oocysts were counted for each midgut and mean oocysts per midgut ( infection intensity ) and percentages of infected mosquitoes ( infection prevalence; infection = at least one oocyst ) were calculated from all dissected mosquitoes . Transgenic mosquitoes heterozygous for the CP-myr-AsteAkt-HA construct were mated with non-transgenic mosquitoes to generate 50% transgenic and 50% non-transgenic mosquitoes . The resulting larvae were reared together under identical conditions and separated based on DsRed fluorescence in the eyes of pupae under a fluorescent stereomicroscope . Female mosquitoes were separated into four treatment groups: transgenic bloodfed , transgenic sugarfed , non-transgenic bloodfed , and non-transgenic sugarfed . Bloodfed mosquitoes were given weekly bloodmeals throughout their entire adult life in addition to 10% dextrose ad libitum , while sugarfed mosquitoes were only provided 10% dextrose ad libitum . Daily mortality for each treatment was recorded and dead mosquitoes were removed until all mosquitoes had perished . These experiments were replicated twice . A third experiment was conducted using approximately 500 mosquitoes per treatment to verify the initial results . Transgenic CP-myr-AsteAkt-HA females and their non-transgenic siblings were mated with colony males shortly after emergence . At 5–7 days post-emergence , females were starved overnight and then fed a blood meal . Fully engorged females were placed into individual cages and provided with an oviposition site and 10% dextrose ad libitum . Oviposition sites were removed 72 h after bloodfeeding and the numbers of eggs were counted . The experiment was repeated six times with separate cohorts of mosquitoes . In the first experiment , data were recorded only for those mosquitoes that laid eggs . In subsequent replicates , the number of individuals that did not lay eggs was recorded . For each replicate , the non-normally distributed egg counts were first analyzed using a Wilcoxon test to determine if there was a significant difference between transgenic and non-transgenic females . Parasite prevalence and oocyst numbers were analyzed to determine whether transgenic mosquitoes were more resistant than their nontransgenic siblings . The data were analyzed in two ways , first by determining whether genotype was an important predictor of resistance within replicates and also pooled across replicates . This allowed us to infer , in part , why replicates within the same experiment differed . In contrast , for the pooled data sets , we included replicate as a random effect to control for inter-replicate variation without explicitly estimating their mean values . Parasite prevalence data were analyzed to determine whether infection status ( infected or not ) depended on genotype . The data were analyzed for each replicate separately using a logistic regression with genotype as a fixed effect . Data for all replicates were then combined and analyzed using a generalized linear mixed model with replicate and genotype included as a random and fixed effect , respectively , in the model . Significant differences were detected using a Wald χ2 statistic . Oocyst counts were square-root transformed to correct for overdispersion prior to using a generalized linear mixed model analysis . Data were first analyzed for each replicate separately to test for the fixed effect of genotype . The data were then combined across replicates and analyzed using replicate as a random effect and genotype as a fixed effect . Significant differences were detected using Wald's F statistic . Analysis of survival curves was conducted using the Kaplan Meier method [39] and significant differences were detected using the Wilcoxon test as previously described [20] . | For malaria transmission to occur , a mosquito must ingest and harbor the parasites for approximately two weeks while the parasites complete multiple developmental stages . Until development is complete and the malaria parasites invade the mosquito salivary glands , transmission to another host cannot occur . Upon completion of parasite development , transmission is possible with every subsequent bite . In this study we demonstrate that tissue-specific overexpression of a single activated protein kinase that is essential to insulin signaling in the mosquito can dramatically reduce parasite development . This kinase – Akt – has been described as a critical cell signaling node that regulates a range of physiological processes . In addition to the impact on parasite development , increased Akt signaling also reduced the average mosquito lifespan relative to controls , thereby limiting the window of opportunity for successful parasite transmission . Thus , we demonstrate that genetic manipulation of one key signaling protein directly reduces parasite development in the insect vector as well as the duration of mosquito infectivity . | [
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] | 2010 | Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes |
The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells . In addition to age and family history , other significant risk factors for glaucoma include elevated intraocular pressure ( IOP ) and myopia . The complexity of glaucoma has made it difficult to model in animals , but also challenging to identify responsible genes . We have used zebrafish to identify a genetically complex , recessive mutant that shows risk factors for glaucoma including adult onset severe myopia , elevated IOP , and progressive retinal ganglion cell pathology . Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 ( lrp2 ) underlie the mutant phenotype . Lrp2 , previously named Megalin , functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog , Bone morphogenic protein 4 , retinol-binding protein , vitamin D-binding protein , and apolipoprotein E , among others . Detailed phenotype analyses indicated that as lrp2 mutant fish age , many individuals—but not all—develop high IOP and severe myopia with obviously enlarged eye globes . This results in retinal stretch and prolonged stress to retinal ganglion cells , which ultimately show signs of pathogenesis . Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease .
The multi-factorial nature of many ocular diseases poses a major challenge in understanding their molecular etiology and in engineering animal models to study mechanisms of pathology . Macular degeneration , myopia , and glaucoma are examples of prevalent and disruptive complex ocular diseases . While characterization of complement factor genes has provided insight into most cases of macular degeneration [1] , no major genetic pathway has been found to underlie myopia or glaucoma . Myopia is the most common human ocular disorder worldwide and is caused by abnormal growth of the eye resulting in refractive error [2] , [3] . Myopia also increases risk for other visual impairing diseases including glaucoma [4] . The glaucomas are a heterogeneous group of progressive blinding disorders that result from damage to retinal ganglion cells and their axons [5] . Important risk factors for glaucoma include elevated intraocular pressure ( IOP ) , age , family history , and myopia [6] . Although traditional human genetic analysis has been limited in identifying causative genes for complex disorders , mutational screens in animals can provide insights into disease etiology . Recently , progress has been made on establishing the zebrafish model to study phenotypes associated with glaucoma . From a forward-genetic perspective , zebrafish offer a major advantage in studying complex disease , in that large pedigrees can be efficiently generated with moderate space and time requirements . Through a mutational screen for adult ocular defects , we identified a complex mutant , bugeye , that manifests multiple adult-onset phenotypes associated with glaucoma including enlarged eyes with myopia , elevated IOP , and damage to retinal ganglion cells . Using linkage analysis we discovered non-sense mutations in low density lipoprotein receptor-related protein 2 ( lrp2 ) for bugeye , as well as within a non-complementing allele . Lrp2 is a large transmembrane protein of the LDL-receptor related protein ( Lrp ) family [7] . Lrp2 participates in receptor-mediated endocytosis and has a host of identified ligands including signaling molecules like Sonic hedgehog and Bone morphogenetic protein 4 , vitamin and hormone binding proteins , apolipoproteins , among others [8] . Lrp2 is expressed on cells of the renal proximal tubule , choroid plexus , developing neural tube , intestine , thyroid , and inner ear . Within the eye , Lrp2 is expressed on retinal pigment epithelial cells as well as ciliary epithelial cells [7]–[9] . In humans , mutations in LRP2 result in Donnai-Barrow syndrome [10] , a rare disease characterized by a spectrum of phenotypes including agenesis of the corpus collosum , diaphragmatic hernia , sensonurial deafness , hypertelorism , buphthalmia ( enlarged eye globes ) and high myopia [11] , [12] . As the eyes of bugeye zebrafish are also highly myopic , Lrp2 may be critical in regulating emmetropic eye growth across species . The strong association of myopia with glaucoma [13] makes bugeye an attractive model to study the genetic and molecular pathways involved in these ocular diseases .
The bugeye zebrafish mutant was identified in a three-generation forward-genetic screen for adult ocular abnormalities . Mutants were easily identified by 6 months as their eyes were visibly enlarged ( Figure 1A , 1C ) . Interestingly , the degree of eye enlargement often varied between the two eyes of a single fish ( Figure S1A–S1G ) . Occasionally the phenotype presented only in one eye , and the other eye remained normal in size ( Figure S1B ) . To address whether ocular enlargement in mutants might represent a retinoblastoma phenotype , we analyzed eyes by histology . Instead of obvious cellular overgrowth we found that the retina was notably thinner in all layers ( Figure 1B , 1D ) . As buphthalmia is often associated with elevated IOP , we used servo-null electrophysiology to measure the eye pressures in mutants and wild-type siblings [14] . Compared to wild-type fish , bugeye mutants consistently showed elevated IOPs ( Figure 1I ) . In addition , the rare fish that presented the phenotype in a unilateral manner had normal pressure in the wild-type sized eye and elevated pressure in the enlarged eye ( Figure S1H ) . IOP is maintained by the balance of aqueous humor production and drainage . Like mammals , aqueous humor in zebrafish is produced in the ciliary epithelium and drained at the iridocorneal angle . However , unlike mammals where drainage occurs circumferentially throughout the angle region , aqueous outflow for zebrafish is facilitated through a discrete ventrally localized canalicular network [15] . Histology did not reveal obvious disorganization in either the dorsal ciliary epithelium ( Figure 1E , 1G ) or in the ventral canalicular outflow network ( Figure 1F , 1H ) . However , the ciliary epithelium occasionally appeared mildly hypertrophied ( Figure 1G , arrow ) and the angle region of mutants was more prone than wild-type specimens to separation between the iris and corneal tissues during histological preparation ( Figure 1H , asterisk ) . Additional characterization of these regions at the time of phenotype onset confirmed these observations ( Figure S2 ) . The original bugeye mutants presented in the third generation of a three-generation screen , suggesting the mutation was recessive . However , only 3 fish out of a family of 28 showed the phenotype and therefore the penetrance was lower than predicted for a simple recessive mutation ( ∼9% vs . 25% predicted ) . Moreover , incrossing 2 of those original mutant fish resulted in 25 progeny that showed large eyes and 18 that never developed the phenotype . Again , if the mutation was a simple recessive mutation , incrossing should have resulted in all progeny showing the phenotype . To better characterize inheritance and establish recombinant mapping panels to genetically position the mutant locus , we set up a series of test-crosses . Table 1 summarizes the results of incross , outcross and backcross matings over multiple generations and genetic backgrounds ( Table 1 ) . The data indicate that the bugeye phenotype is most likely caused by a single recessive mutation , but like many multi-factorial complex diseases , the penetrance was modified by common wild-type backgrounds and/or by non-genetic factors . To map the mutant locus , progeny from single pair backcross matings were used for whole-genome linkage analysis . Co-segregation for markers on chromosome 9 and the mutant phenotype was found ( Figure 2A ) . Informatively , no other linkage in the genome was noted , consistent with the single recessive causative mutation hypothesis . Public databases revealed that the lrp2 gene was within the critical recombinant interval . Given the similarity of the bugeye phenotype to those caused by LRP2 mutations in humans , we sequenced this candidate gene . Analysis of lrp2 cDNA from bugeyemw1 mutants revealed a T to A conversion that changes a cysteine to a stop codon at predicted amino acid position 23 ( C23X ) ( Figure 2B , 2C ) . Through an independent genetic screen we identified a second large eye mutant that like the bugeyemw1 allele , presented in adulthood and showed reduced penetrance . Intercrosses between this mutant ( allele p5bnc ) and bugeyemw1 were non-complementing and suggested that lrp2 may also be affected in the p5bnc mutant . Indeed , sequencing of p5bnc cDNA revealed a separate non-sense mutation , also very early in the coding region of lrp2 ( bugeyep5bnc , Q413X ) ( Figure 2B , 2C ) . To test whether somatic reversion or alternate splicing around the non-sense mutations might underlie the reduced penetrance or variability often observed between the left and right eyes , we sequenced ocular cDNA in affected and unaffected eyes . However , we did not find evidence of mosaicism or alternate splicing surrounding the mutations , suggesting the penetrance and phenotype variability is influenced by other genes , epigenetics , and/or unpredictable changes in physiology which affects the phenotypes . In mammalian eyes , the multi-ligand receptor Lrp2 is known to be expressed in the developing and adult retinal pigment epithelium ( RPE ) and ciliary epithelium . We therefore analyzed Lrp2 expression in wild-type , bugeye mutant larvae treated with phenyl-thio-urea ( PTU ) , which blocks pigmentation and allows visualization of potential RPE immunoreactivity . As predicted , strong immunoreactivity was found in wild-type RPE and ciliary epithelium . Other regions of expression noted in wild-type fish included forebrain ventricles , regions of the inner ear , proximal pronephros , and gut epithelium ( data not shown ) . All Lrp2 immunoreactivity was completely absent in mutant larvae for both bugeye alleles ( Figure 2D , 2E and data not shown ) . We next developed genotyping protocols for both mutant alleles and confirmed that large-eyed fish never showed wild-type lrp2 genotypes ( Figure 2F , 2G ) . We also used this assay to test whether the reduced penetrance of the ocular phenotype could be explained by increased larval lethality of lrp2 mutants . However , we found that all genotypes were represented in Mendelian ratios in the adult progeny of either heterozygous or backcross pairwise matings , despite the fact that some homozygous mutants never developed the enlarged eye phenotype . Cumulatively , these data indicate that lrp2 mutations are responsible for the large-eyed phenotype in bugeye and that the reduced penetrance and variability in eye enlargement are due to either common ( yet unknown ) genetic background differences and/or non-genetic factors such as physiological modifiers of the mutation . Having established the causative gene for bugeye , we next investigated the onset of the ocular phenotype and quantified the pathology . To characterize the development of enlarged eyes in bugeye/lrp2 mutants we performed longitudinal studies tracking wild-type and mutant fish from 1-12 months . The zebrafish eye reaches its final adult anatomy by approximately 1 month of age [16] . Because overall growth rates can vary between equally aged fish — even within the same tank — we used the ratio of eye size to body length ( E:B ) to determine the relative size of the eye . This ratio remained constant in wild-type fish , allowing comparison of relative eye size between individuals regardless of the overall growth of the fish . Although this ratio remained flat as wild-type fish grew , the E:B ratio increased over time for most lrp2 mutants ( Figure 3A ) . Despite individual variability , the average body length growth rates between wild-type and lrp2 mutant fish were indistinguishable ( Figure S3 ) . For the E:B ratio , no wild-type fish had a value greater than 0 . 05 ( most fell between 0 . 02 and 0 . 04 ) , and mutants with visibly enlarged eyes had an E:B ratio ≥0 . 07 . The onset of large eyes was variable both within shared tanks of siblings and between generations , but a statistically significant difference between mutant and wild-type fish was consistently found at 2 months ( Figure 3A and data not shown ) . In general , lrp2 mutant eyes become visibly enlarged in adults between 2–6 months and eye growth often plateaus between 8–12 months . Histological cross-sections of lrp2 mutant eyes revealed that the region with the greatest increase in size was the depth of the vitreous chamber ( Figure 3B ) . This suggests that in large-eyed mutants , the retina lies behind the point at which the lens focuses light and the eyes are therefore myopic . We calculated the relative refractive error ( RRE ) , an estimate for the degree of myopia , for lrp2 mutant eyes relative to wild-type eyes at 1 and 2 months using retina and lens radius measurements collected from histological sections . Using the RRE equation , a myopic eye has a negative value and a hyperopic eye is positive ( Figure 3C; Methods ) . We found that lrp2 mutant eyes are slightly myopic at 1 month , but become significantly more myopic by 2 months ( Figure 3D ) . Histology suggested retinal cell density was affected in lrp2 mutant eyes ( Figure 4 ) . At 1 month , before eyes of mutant fish were visibly enlarged , there was a small reduction in retinal cell density as compared to wild-type fish ( Figure 4A , 4B ) . By 2 months , when the onset of large eyes had occurred in some mutants but not in others , there was a significant difference in cell density in all layers of the retina ( Figure 4C , 4D ) . As expected , at 6 months when relative eye size was greater overall , there was a further decrease in cell density ( Figure 4E , 4F ) . When considering retinal cell density for each layer as a function of relative eye size ( as measured by the ratio of the retinal cross-section length to body length ) , we found that for mutants , the relation between neuron density and relative eye size decreased in a linear manner ( Figure 5A ) . The same was true when considering just the absolute size of eye ( as measured by retinal cross-section length , Figure 5B ) . Interestingly , there was an increase in photoreceptor density in larger eyes for wild-type fish ( Figure 5B ) . When considering cell density for wild-type and mutant eyes of the same absolute size , but of different ages in order to match size , density was still reduced in lrp2 mutant fish ( Figure 5C ) . For this comparison we evaluated retinal cell density of 6-month old wild-type fish and 2-month old lrp2 mutant fish , each that had retinal lengths that fell between 2–3 mm . Importantly , there was no significant change in cell density for the retinal ganglion cells layer between 2–6 months in wild-type fish . For the inner nuclear and photoreceptor layers , there was a small , but significant change ( ANOVA , p<0 . 001 ) , where the cellular densities increased with age . Together , these data suggest that the reduced neuron density seen in lrp2 mutant retinas is not simply due to an acceleration of normal ocular growth . Despite the reduced cell density in mutant eyes , total retinal cell number was estimated to be greater than wild-type , owing to the much larger eye size overall . We estimated total retinal cell numbers by considering the retina area as that of the surface area for half a sphere and extrapolated total cell numbers using density data . These calculations showed that mutant eyes with E:B ratios >0 . 07 had significantly increased numbers of total neurons . More directly , analysis of DNA content , which is proportional to total cell number , confirmed that large-eyed mutant fish ( EB ratio >0 . 07 ) had more cells , even though retinal cell density was much lower ( data not shown ) . The altered retinal cell density in lrp2 mutants could be due to either insufficient cell generation to match scleral growth and remodeling , or through increased cell death . To address these possibilities we analyzed by immunofluorescence the number of proliferating cells within the ciliary margin zone ( using Minichromosome maintenance homolog 5 , Mcm5 antibodies ) and the number of apoptotic cells across the retina ( using activated-Caspase3 antibodies ) . Mcm5 is required for DNA replication and is expressed throughout the cell cycle in all proliferating cells , but the protein is rapidly lost in post-mitotic cells . Proteolytic cleavage of Caspase3 , recognized by the activated-Caspase3 antibody , is one of the last steps in the apoptosis cascade and marks cells committed to die in a number of contexts , including glaucoma . At 1 month , proliferation in both wild-type and lrp2 mutant retinas was primarily confined to the ciliary marginal zone , a stem cell niche where ongoing proliferation from multipotent elongated neuroepithelial cells is known to occur in fish [17] ( Figure 6A–6C ) . For each genotype , occasional Mcm5-positive cells were also located in the inner nuclear layer , which have previously been shown to be rod progenitor cells in teleost fish [18]–[20] . At 2 months , cell counts indicated a reduction in Mcm5-positive cells per CMZ niche in bugeye fish , suggesting maintenance of stem cells was inadequate to match eye globe growth ( Figure 6D–6F ) . Consistent with this observation , a role for Lrp2 in maintaining neuronal stem cells of the adult mouse forebrain has been recently described [21] . Similar to analysis of proliferation , cryosections of wild-type and lrp2 mutant retinas were used to investigate cell death . However , very few dying cells were noted in sections of retina from either condition . Similar results were obtained using the TUNEL assay to characterize dying cells . We therefore used activated-Caspase3 immunoreactivity on control and lrp2 mutant flat-mounted retinas to observe all neurons from individual samples . Even by flat-mount analysis , there was little apoptosis up to 6 months of age ( Figure 6G ) , although at these times bugeye mutants showed trends towards increased numbers of activated-Caspase3-positive cells . By 12 months , apoptosis in bugeye retinas was significantly elevated . We also noted that activated-Caspase3 immunoreactivity from all ages was restricted to the retinal ganglion cell layer ( Figure 6H ) . It is possible , however , that some cells , including those outside of the ganglion cell layer die by Caspase3- and TUNEL-independent mechanisms . Overall , these data indicate that initially , as lrp2 mutant eyes expand , proliferation is not sufficient to maintain proper cell density and later , perhaps following mechanical stress imposed by retinal stretch , retinal ganglion cells begin to die . In the following studies we evaluated the onset of retinal ganglion cell stress and pathology . Relative expression levels of twelve genes known to be up-regulated in animal models of retinal ganglion cell injury was surveyed by quantitative RT-PCR . This panel of markers included three transcripts expressed in microglia ( aif1l , [22] , [23]; apoeb , [24] , [25]; arg1 , [26] , [27] ) , one expressed in Müller glia and astrocytes ( gfap , [28] , [29] ) , and eight expressed in retinal ganglion cells ( atf3 , [30] , [31]; c1q , [32]–[34]; c-jun , [31] , [35] , [36]; gap43 , [37] , [38]; klf6a , [31] , [39]; socs3a and socs3b , [31] , [40] , [41]; thy1 , [42] , [43] ) . Analysis was conducted on cDNA isolated from pooled 1-month-old retinas , a time just prior to when mutant eyes were measurably enlarged . We chose this early time-point to avoid measuring changes that might simply reflect significant alterations in cell proportions and density . With this assay , we found induction primarily of transcripts associated with retinal ganglion cells , but not for the glia-associated genes ( Figure 7A ) . To investigate whether the markers of retinal ganglion cell stress correlated with optic nerve pathology , we first compared sagittal sections of wild-type and lrp2 mutant optic nerve heads from 6-month-old fish by light microscopy . We then analyzed cross-sections of wild-type and lrp2 mutant optic nerves , just posterior to the optic nerve head from 7- and 12-month-old fish by transmission electron microscopy ( TEM ) . In zebrafish , like other teleost fish as well as some rodents , the optic nerve head is comprised of an astroglial lamina without obvious elastin-collagen rich laminar plates as observed in primates [44]–[46] . In addition , as the optic nerve exits the fish eye , it is initially unmyelinated , like that in humans and most mammals [47]–[50] . Histology of the optic nerve head did not reveal excavation or cupping in lrp2 mutants , but did indicate mutant nerves were larger , consistent with increased total numbers of retinal ganglion cells in the large-eyed fish ( Figure 7B ) . Optic nerve cross-sections for TEM were collected distal to the exit point from the eye within the myelinated region of the optic nerve , which is adjacent to the site of axonal injury in glaucoma [51]–[53] . Nerve damage was scored as 1 ) degenerating axons , as noted by electron-dense appearance , 2 ) axons having an unraveled myelinated sheath , or 3 ) space left behind by a shrunken and degenerating axon . At both ages , examples for each type of pathology were found in wild-type and lrp2 mutant optic nerves ( Figure 7C ) . Surprisingly , when total counts were normalized to area ( mm2 ) there were no differences between genotypes or ages ( Figure 7D ) . Because the ultrastructural signature of degenerating axons following a crush injury is relatively short-lived in the optic nerve tract of teleost fish as compared to mammals [54] , we utilized a genetic tool to label damaged and regenerating axons over a longer period of time [55] . We crossed Tg ( 3 . 6Frgap43:GFP ) mil1 transgenic fish with lrp2 homozygous mutants and then used the resulting progeny to backcross with non-transgenic lrp2 mutant fish . This breeding scheme resulted in families with equal proportions of lrp2 heterozygous and homozygous mutant fish carrying single insertions of the 3 . 6Frgap43:GFP transgene . This transgene contains 3 . 6 kb of regulatory sequence ( 5′ flanking region and first intron ) from theTakifugu rubripes gap43 locus driving GFP . Importantly , in these transgenic fish , GFP is expressed in axons following injury [55] . For our analysis , we compared large-eyed lrp2 homozygous mutant fish ( >0 . 07 E:B ratio ) to normal-eyed heterozygous siblings ( Figure 8K–8T ) . In all large-eyed mutant fish we observed strong activation of GFP in a sub-set of retinal ganglion cells . In the majority of mutant retinas examined ( 6 of 6 at 6 months , Figure 8P–8T; and 10 of 12 at 12 months , data not shown ) , there was a characteristic axon ‘wandering’ and ‘circling’ around the optic nerve head . This axon phenotype , where GFP-positive axons approached the optic nerve head in a disorganized and circuitous fashion , was never observed in retinas from age matched lrp2 heterozygotes ( Figure 8K–8O ) or from 12-month wild-type fish that carried the 3 . 6Frgap43:GFP transgene ( data not shown ) . The transgene was activated with variability at 2 months in both wild-type or lrp2 mutant fish ( Figure 8A–8J ) , but the wandering axon phenotype was only rarely observed in mutants at this early timepoint . Weak expression of the transgene was noted in the nerve fiber layer of non-mutant retinas , consistent with the ongoing neurogenesis of zebrafish . In addition , older wild-type fish occasionally showed stronger GFP-positive axons , suggesting sporadic age-related degeneration . In wild-type eyes , all of the low-GFP expressing axons , as well as the occasional high-GFP expressing axons , exited the eye directly without wandering or circling the optic nerve head like those of mutants . To address whether the chronic stress conditions of lrp2 mutants differ from acute injury , we performed optic nerve crushes on adult gap43:GFP fish . At 6 days post-crush there was significant up-regulation of GFP across the retina ( Figure S4 ) . By 5 weeks post-crush , when axons had regrown [56] , there was only an occasional wandering axon . Most samples following nerve crush , however , showed accurate and direct axon targeting through the optic nerve head . By 11 weeks post-crush , there was significant reduction in transgene activation and no axons showed wandering or circling at the optic nerve head like age-matched lrp2 mutants . This comparison highlights the differences between the chronic stresses caused by the lrp2 mutation versus the acute , crush injury model , in which the genetic model results in changes at the optic nerve head that are not evident in the post-nerve head crush paradigm .
A major challenge with research on either myopia or glaucoma is identifying genetic lesions that impact the diseases . Recently , genome wide association studies for both diseases revealed non-coding associated changes , but the predicted effects on phenotypes were small and the actual gene products affected by the intergenic alterations have yet to be identified [57]–[59] . In addition to defining genetic susceptibilities for glaucoma , there is a need to understand and model how other risk factors like age , raised IOP , and myopia itself affect the onset , severity , and progression of neuropathology . In our studies we identified non-sense mutations in zebrafish lrp2 that lead to phenotypes that are known risk factors for glaucoma . These phenotypes included increased IOP , enlarged eye globes with significant refractive errors , decreased retinal neuron density , activation of retinal ganglion cell stress genes , and distinct axon pathology at the optic nerve head . The zebrafish lrp2 mutants have similar heritable phenotypes to the black moor goldfish [60] , [61] and the RCS;rdy- rat [62] . It will be interesting to see if lrp2 or pathway genes are affected in either of those models . Similarly , it is possible that alterations to genes that control pathways affected by loss of Lrp2 might influence myopia or forms of glaucoma . To date , however , polymorphisms in Lrp2 have only been linked with urate and cholesterol levels in serum [63] , [64] and the molecular and cellular pathways affected by loss of Lrp2 that impact the ocular phenotypes remain uncharacterized . In general , Lrp2 functions in regulation and homeostasis of multiple bioactive molecules including vitamins , hormones , nutrients , and growth factors through localized tissue delivery or reuptake by epithelia . In knowing the affected gene , the zebrafish mutants hold promise in shedding light on how de-regulated signaling and homeostasis affect phenotypes such as elevated IOP or excessive eye growth . While it is tempting to speculate that the excessive eye growth in lrp2 mutants is due to the elevated IOP , our studies do not rule out the possibility that these two phenotypes are distinct . In fact , the only two Donnai-Barrow patients who have had their IOPs reported ( each with non-sense mutations in LRP2 ) , showed values in the normal range [65] . Despite normal IOPs , the eyes of the two young siblings were enlarged and showed high myopia . Furthermore , as an endocytic receptor found on the RPE , Lrp2 is an interesting candidate as a direct regulator of emmetropization [66] . Potentially , Lrp2 mediates the availability or transport of signaling molecules from the retina to affect remodeling within the sclera . In this context Lrp2 might be key in facilitating the matching of visual input with axial length of the eye . Nonetheless , relationships between eye pressure and size are established and the elevated IOP in zebrafish lrp2 mutants is likely to be at least contributory to the observed buphthalmia . Consistent with this possibility , in the few mutant fish where the eye phenotype presented in a unilateral manner , IOPs were normal in unaffected eyes , yet elevated in enlarged ones . Indeed , expression of Lrp2 on the ciliary epithelium suggests a direct role in IOP regulation , particularly considering the function of Lrp2 at other sites of fluid regulation . For example in mice , Lrp2 has been shown to regulate glomerular filtration in the proximal tubule of the kidney and in the choroid plexus the receptor modulates homeostasis of cerebrospinal fluid [67]–[71] . A significant characteristic of lrp2 mutant fish is the strong relationship between abnormal eye globe growth , retinal thinning , and activation of retinal ganglion cell stress markers . In this context , lrp2 mutants have value as a genetic model for studying the effects of protracted mechanical stress on retinal ganglion cells , their axons , and the associated glia . As this phenotype relates to glaucoma , it was surprising that mutant fish did not show significantly elevated optic nerve pathology with TEM analysis . It is possible that the stresses induced by lrp2 mutations simply do not reach a threshold to cause ultrastructural pathology . Alternatively , low-grade stress may actually “pre-condition” and promote protective mechanisms in the mutant neurons [72] , [73] . However , the lack of a difference in ultrastructure pathology between mutant and wild-type siblings could also be explained by the surprisingly high number of pathological events noted in the wild-type fish . This perhaps relates to the regenerative capacity of teleosts [56] , [74] and a relaxation of selective pressure to maintain nerve health with normal aging . Through evolution , fish may have lost highly-robust nerve protective mechanisms against age-related stresses , and instead rely on ongoing growth and regeneration to maintain vision , perhaps accounting for the unexpected pathology scored in wild-type optic nerves . In addition , because a higher proportion of the ganglion cell axons in lrp2 mutant fish are in fact younger than those of wild-type siblings ( due to the excessive ongoing generation of neurons in their eyes ) , many of the optic nerve profiles might be expected to in fact look healthier in a relative manner . The modest death of retinal ganglion cells in lrp2 mutants was less surprising . First , extended retinal ganglion cell soma survival , despite axonal damage and dysfunction , is known for the DBA/2 mouse glaucoma model . DBA/2 mice show a pigment dispersion-related glaucoma with elevated IOP [75] , [76] . In young DBA/2 mice , axons at the nerve head often show focal insults with many having dystrophic features [51] . In many aged animals , axons are clearly degenerative [51] . Most retinal ganglion cells , however , survive for extended periods of time and their disconnected proximal ( intra-retinal ) axons take on reactive and stressed characteristics [51] , [77] , [78] . Second , the resilient nature of retinal ganglion cells in teleosts has been well characterized . In fact for goldfish , experimental axotomy or optic nerve crush results in less than 10% death of retinal ganglion cells [79] , and in zebrafish only 20% of the lesioned neurons are reported to die [80] . In contrast , optic nerve axotomy in mammals results in apoptosis of nearly all retinal ganglion cells [81]–[83] . The regrowth of axons in teleosts occurs over a course of weeks and results in correct axon pathfinding and appropriate tectal innervation [84] , [85] . In contrast , in lrp2 mutants , retinal ganglion cells appear to be under prolonged mechanical stress from the stretching and growth of the eye globe . This was evident from the changes in retinal density with eye enlargement and the activation of retinal ganglion cell stress markers . Of interest , axon regrowth through the optic nerve was affected in lrp2 mutants . The wandering and circling phenotype of the gap43:GFP axons in large-eyed mutants is reminiscent of the EphB3-dependent ‘reactive plasticity’ following optic nerve injury in mice [86] , [87] . Regardless , of why bugeye/lrp2 mutants do not show dramatic retinal ganglion cell death , this fact emphasizes that while these fish model initiating risk factors for glaucoma , they do not model the end stages of the disease . Lrp2 mutations in humans and mice are often lethal , but always developmentally relevant , particularly within the nervous system [71] , [88] , [89] . Our analyses of both bugeye alleles indicate Lrp2 is dispensable for survival in zebrafish . Furthermore , we did not detect morphological phenotypes in mutant embryos , similar to the observations following oligonucleotide knock-down of zebrafish lrp2 [67] . The total lack of lethality in zebrafish lrp2 mutants may be due to species differences in respiration , as mice mutants often die from respiratory failure at birth . Alternatively , there may be compensation from other Lrp family members in zebrafish . Compensation from Lrp family members may also explain the lack of obvious developmental defects . More detailed studies of the zebrafish mutant embryos and larvae are warranted to assess whether subtle defects exist . In summary , we have identified mutations in lrp2 that cause adult-onset ocular pathogenesis in zebrafish . While mutants appear normal during larval stages of development , as young adults they develop enlarged eyes with elevated IOP . Over time , retinal cell density becomes significantly reduced due to insufficient proliferation of marginal zone stem cells and increased neuronal cell death . Markers of retinal ganglion cell stress become elevated and damaged and/or regenerating axons at the optic nerve head show a characteristic wandering and circling phenotype . These fish will be valuable for future studies on the signaling and cellular mechanism of myopia and other risk factors for glaucoma .
Wild-type and mutant zebrafish ( Dano rerio ) were maintained at 28°C with a 14 on/10 off light cycle and were feed a standard diet [90] . All animal husbandry and experiments were approved and conducted in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin . bugeye; lrp2mw1 ( this study ) bugeye; lrp2p5bnc ( this study ) Tg ( 3 . 6Frgap43:GFP ) mil1 [55] lrp2 , HM_754616 aif1l , NM_198870 apoeb , NM_131098 arg1 , XM_001922563 gfap , NM_131373 atf3 , NM_200964 c1q , NM_001005976 c-jun , NM_199987 gap43 , NM_131341 klf6a , NM_201461 socs3a , NM_199950 socs3b , NM_213304 thy1 , NM_198065 Servo-null electrophysiology was used to measure IOPs as described previously [14] . Mapping panels of 6 month adult mutant fish ( obviously enlarged eyes ) were collected from backcross pedigrees . Bulked segregant analysis , using pooled samples of mutant genomic DNA and individual parental DNA , was conducted with simple sequence repeat ( SSR ) markers to establish linkage to Chromosome 9 . For higher-resolution mapping , sequencing of parental genomic DNA in regions associated with the closest linked microsatellite markers was done to find additional SSRs . These new SSRs were then used to refine the critical interval by analyzing single mutant fish . PCR was performed on DNA isolated using the Puregene kit ( Qiagen , Germantown , MD ) from tailfin-clips , using primers designed to amplify the allele specific mutations in lrp2: bug mw1 F: CGTTATTTTCTGTCTAGGTTCAGGTTA , bug mw1 R: GAAAAGAAAAGATTGATACATACGG bug p5bnc F: GTGTGTTTTCTGAAAACTGTCAAGC , bug p5bnc R: CTTTGCAGCTGGTAATGAAAATCCACACCAACAGCGGCTCCTCTGTCCTA . Underlined letter in primer denotes mutant nucleotide , bolded letter denotes a single nucleotide change in the primer to generate a novel restriction site for each allele ( bug mw1: MseI; bug p5bnc AvrII ) . Fish were anesthetized with 0 . 05% Tricaine and body lengths were measured in side-view from the tip of the head to the end of the trunk ( before the caudal fin ) . To measure eye size , anesthetized fish were imaged at a fixed magnification from a dorsal perspective using a Nikon CoolPix995 camera attached to a Leica MZFLIII microscope . These images were imported into Metamorph software ( Universal Imaging Corp , Philadelphia , PA ) , and the area of each eye from the dorsal view was traced using the Region Measurements function . Lens radius ( L ) was measured from histological cross sections; retina radius ( R ) was back-calculated by assuming the retina to be a semi-circle , measuring the length of the retina , and taking that measurement as half the circumference of a circle ( so R = length of retina/π ) . Sections with minimal distortion from processing were used and no attempts to correct for distortions were made . A focal length ( F ) of 2 . 32 x L for the lens was used as in studies with goldfish [91] . RRE was calculated as 1- ( R/F ) . By this calculation , all wild type fish were predicted to be slightly hyperopic ( RRE >1 ) , likely due to fixation artifact . To adjust this , the ratio of ( R/F ) was multiplied by a constant factor for both genotypes at each age ( 1 month , 1 . 15; 2 months , 1 . 18 ) , so that on average , the wild type fish were emmetropic ( RRE = 0 ) . Heads were removed from terminally anesthetized fish and fixed overnight in gluteraldehyde/paraformaldehyde at 4°C , washed three times in PBS , and dehydrated in increasing ethanol solutions ( 50% , 70% , 80% , 90% , 95% , 100% , 100% , 100% ) for 10 minutes each , all at RT . The heads were then infiltrated with propylene oxide for 15 minutes twice , then a 1∶1 mix propylene oxide:epon for 2 hours at RT . An additional equal volume of epon was added to the samples and these were incubated overnight with culture tube caps off so that the propylene oxide would evaporate . Heads were bisected when necessary to fit in block-molds , embedded in epon , and baked for at least 24 hours at 65°C . Semi-thin sections were cut on a Leica RM2255 microtome and stained with 1% Toluidine , 1% Borax . For each eye , 5 non-consecutive sections were imaged from the central retina ( sections with the largest lens diameter ) with a 40X objective on a Nikon E600FN microscope with a Photometrics CoolSnap camera attached . Each image was printed and the nuclei in each layer of the retina were counted . The average of the 5 sections was calculated and represented 1 data point . For sample condition , between 6–12 eyes were scored in this manner . Zebrafish embryos or isolated eyes were fixed overnight at 4°C in 4% PFA ( pH 7 . 4 , in PBS ) , washed three times for 10 minutes in PBS , then infiltrated with increasing concentrations of sucrose ( 15% , 30% ) for 2 hours each at 4°C , followed by overnight incubation in HistoPrep freezing media ( Fischer Scientific , Pittsburgh , PA ) . Cryoprotected embryos were embedded in HistoPrep and flash frozen , sectioned at 10–12 µM and collected on Supercharge Plus slides ( Fischer Scientific ) . Cryosections were allowed to dry on the slide for 1hr at RT , and the edge of the slide was traced with a PAP pen . Slides were rinsed briefly with PBTD ( PBS +1% DMSO +1% Tween-20 ) to rehydrate the tissue , and then incubated in block ( 5% donkey serum in PBTD ) for 2 hours at RT . Primary antibody was diluted in block ( Sheep-anti-Lrp2 1∶1000 , gift from Dr . Thomas Willnow ( Max Delbruck Center , Berlin , Germany ) ) and incubated on slides overnight at 4°C . Antibody was removed and slides were washed three times with PBTD rinses , and secondary antibody diluted in block ( Cy3-Donkey anti-Sheep 1∶250 , Jackson ImmunoResearch , Westgrove , PA ) was incubated at RT for 1 . 5 hours . Secondary antibody was removed with three washes of PBTD , and slides were mounted in 1∶1 PBS to glycerol with 0 . 1% Hoechst nuclear stain ( cryosections ) . Images were collected using a Nikon C1 confocal microscope . The same procedure was followed for dissected whole adult retinas prior to flat-mount analysis , using anti-cleaved caspase-3 primary antibody ( 1∶500 , Cell Signaling Technology , Danvers , MA ) and DyLight 488 secondary ( 1∶1000 , Jackson ImmunoResearch ) . 1 month fish measuring between 10–12mm were anesthetized in Tricaine , and both eyes were removed and placed immediately in TRIzol ( Invitrogen ) . Each sample was a pool of 3 pairs of eyes ( 6 eyes per sample ) , and 4 samples were used for each genotype . RNA was isolated following the Invitrogen protocol . Reverse-transcription PCR was carried out following the protocol for SuperScript III First Strand Synthesis ( Invitrogen ) . Gene specific primers were used as follows to amplify the genes of interest: Aif1l ( F: CAACATGGACTTACAAGGCG , R: TCCTCTTCGTCTCTGTACTTCTG ) ; ApoEb ( F: GTGCAAAACATCAAGGGCTC , R: GGGTCATCTGGGTTTGGAG ) ; Arg1 ( F: TGGGCATCAAAACCTTCTCC , R: AAACTCAGATGGATCGGCTTC ) ; Atf3 ( F: AGCCTGCATGAACACTGAG , R: TTTTCCTTCGGTCGTTCTCC ) ; C1q ( F: CTCTGCTGACACCTGTCCTG , R: GGTGGTCCTTTCAGACCAAA ) ; c-Jun ( F: ACGTGGGACTTCTCAAACTG , R: TCTTGGGACACAGAAACTGG ) ; Gap43 ( F: GAAGGCAATGCACAGAAAGAG , R: TGCTGGTTTGGATTCCTCAG ) ; Gfap ( F: AAGCTCTGCAAGACGAGATC , R: GCTTAGACACATCCAGATCCAC ) ; Klf6 ( F: CACTTAAAAGCACATCAGCGG , R: GAAGTGTCGGGTTAGCTCATC ) ; Socs3a ( F: CATTCAACAAAAGAGACTCATAGGC , R: TGTGGGTTATCATGGCGATAC ) ; Socs3b ( F: CCCAAGATTGAGTCGGATAACG , R: ACCAACACAAAGCCCAGAG ) ; Thy-1 ( F: CCGGTGTCAATCATTCAAACTG , R: CAGTGGGAAAGTGAGGAAGG ) . Initially , PCR products were amplified with Accuprime Taq HighFidelity ( Invitrogen ) , and sequenced to verify specificity . Real-time analysis was performed on a Bio-Rad iCycler using iQ SYBR Green SuperMix ( Bio-Rad ) . 3-step PCR with a 57°C annealing temperature was used for all primer sets except Arg1 , Atf3 , and Thy1 , which used a 2-step PCR with a 54°C annealing temperature to eliminate a non-specific product . All samples were run in triplicate , and fold change was calculated using the ΔΔCt method , with Ef1α as the housekeeping gene for all primer sets . Heads were removed from terminally anesthetized fish . In a Petri dish filled with buffer , the optic nerves were dissected from the heads first by removing the skin , skeleton , and connective tissue , leaving the eyes and attached nerves and tectum intact . The tectum was cut from the nerves , leaving the nerves intertwined at the chiasm . The nerves were separated by gently pulling on the eye globes with forceps , and making a cut with an 8 mm Spring Scissors ( Fine Science Tools ) when necessary . Dissected nerves with attached eyes were then incubated overnight at 4°C in gluteraldehyde/paraformaldehyde fixative . Heads were washed three times in 0 . 1M PO4 buffer , and then most of the eye globe removed by using the 8 mm scissors to make a circumferential cut around the optic nerve head , leaving a small portion of the posterior eye attached to the dissected nerve . The nerves were post-fixed in gluteraldehyde/paraformaldehyde for 1 hr at room temperature , washed 3X in 0 . 1M PO4 buffer , fixed in 1% buffered Osmuium for 1 hr on ice , and washed 3X with ice cold water . The following steps were all done at room temperature: nerves were dehydrated in an increasing series of MeOH ( 30% , 50% , 70% , 95% , 100% , 100% , 100% ) , then infiltrated with acetonitrile , 2X for 15minutes each , followed by 2 hours in a 1:1 mix of acetonitrile and EM Epon , and finally incubated in 100% EM Epon overnight , embedded in molds , and baked for at least 24 hours at 65°C . The blocks were trimmed to between 100–200 microns past the optic nerve head on a Leica RM2255 microtome , and ultra-thin sections were cut and plated on a grid , and imaged using a Hitachi H600 transmission electron microscope . The entire nerve cross-section was canvassed at 8000X , and 10–16 representative images were collected from each nerve at this magnification . Quantitative assessment of nerve pathology was conducted in a double-blinded manner in which both the TEM microscopist and the individual scoring pathology for the samples was unaware of the sample genotype . Eyes were dissected from terminally anesthetized adult fish and fixed overnight at 4°C in 4% PFA ( pH 7 . 4 , in PBS ) , then washed three times in PBS . In a Petri dish filled with PBS , a circumferential cut was made at front of the eye with a scalpel , near the border of the anterior and posterior segments . The anterior segment was discarded , followed by removal of the sclera from the posterior segment . The remaining retina with RPE was post-fixed 1-2 hrs with 4% PFA ( pH 7 . 4 , in PBS ) , washed in PBS , and then laid flat on a slide by making incisions through the retina so that it would lay flat . Whole retinas were mounted on the slides with 20 µl of Vectashield Mounting Medium ( Vector Labs , Burlingame , CA ) , and coverslipped . For retinas used for anti-activated-caspase-3 immunofluorescence , antibody incubations were done after removal of the anterior segment and sclera , but prior to flat-mount analysis . | Complex genetic inheritance , including variable penetrance and severity , underlies many common eye diseases . In this study , we present analysis of a zebrafish mutant , bugeye , which shows complex inheritance of multiple ocular phenotypes that are known risk factors for glaucoma , including high myopia , elevated intraocular pressure , and up-regulation of stress-response genes in retinal ganglion cells . Molecular genetic analysis revealed that mutations in low density lipoprotein receptor-related protein 2 ( lrp2 ) underlie the mutant phenotypes . Lrp2 is a large transmembrane protein expressed in epithelia of the eye . It facilitates transport and clearance of multiple secreted bioactive factors through receptor-mediated endocytosis . Glaucoma , a progressive blinding disorder , usually presents in adulthood and is characterized by optic nerve damage followed by ganglion cell death . In bugeye/lrp2 mutants , ganglion cell death was significantly elevated , but surprisingly moderate , and therefore they do not model this endpoint of glaucoma . As such , bugeye/lrp2 mutants should be considered valuable as a genetic model ( A ) for buphthalmia , myopia , and regulated eye growth; ( B ) for identifying genes and pathways that modify the observed ocular phenotypes; and ( C ) for studying the initiation of retinal ganglion cell pathology in the context of high myopia and elevated intraocular pressure . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"genetics",
"and",
"genomics/disease",
"models",
"neurological",
"disorders/neuro-ophthalmology",
"and",
"neuro-otology",
"ophthalmology/glaucoma",
"cell",
"biology/cell",
"signaling"
] | 2011 | Mutations in Zebrafish lrp2 Result in Adult-Onset Ocular Pathogenesis That Models Myopia and Other Risk Factors for Glaucoma |
Cationic antimicrobial peptides ( CAMPs ) serve as the first line of defense of the innate immune system against invading microbial pathogens . Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids ( TAs ) with D-alanine , but the exact mechanism of resistance is not fully understood . Here , we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus ( GBS ) with a series of CAMPs having different properties . The data reveal that: ( i ) D-alanylation of lipoteichoic acids ( LTAs ) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; ( ii ) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and ( iii ) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density , as seen by Transmission Electron Microscopy , and reduces the penetration of CAMPs through the cell wall . Furthermore , Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant . We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall , rather than by reducing the electrostatic interactions of the peptide with the cell surface . Overall , our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance .
The innate immune system of almost all living organisms produce cationic antimicrobial peptides ( CAMPs ) to protect against bacterial invaders . CAMPs are gene-encoded peptides that differ in their primary amino acid sequences [1] , [2] but most native CAMPs share a well-defined α-helix or β-strand secondary structures and display a net positive charge of mainly +2 to +9 [3] , [4] . In contrast to conventional antibiotics that interact with specific targets , many CAMPs bind and perturb the bacterial membrane . Nevertheless , throughout evolution certain gram-positive bacteria have evolved sophisticated regulatory mechanisms to modify their surface properties in order to overcome killing by CAMPs . These are typified by two-component systems ( TCSs ) that sense and respond to environmental CAMPs [5]–[8] . The cell wall of gram-positive bacteria is a complex network composed mainly of peptidoglycan ( PGN ) and teichoic acids ( TAs ) , both of which are essential for maintaining the structural integrity and shape of the bacterial cell . Teichoic acids are negatively charged poly-glycerophosphate ( Gro-P ) chains that can be either covalently linked to PGN ( i . e . wall teichoic acids or WTAs ) or anchored to the cytoplasmic membrane ( i . e . lipoteichoic acids or LTAs ) [9] . WTAs and LTAs are assembled via different pathways . The anionic property of teichoic acids confers a global negative charge , which is thought to contribute to the preferential accumulation of CAMPs on the bacterial cell surface [10] . Subsequently , these peptides traverse the PGN barrier , reach the anionic phospholipid of the cytoplasmic membrane , and perturb it via several mechanisms , depending on the peptide used [11]–[13] . In gram-positive bacteria , resistance to CAMPs is mainly due to an increase of the positive surface charge through increase in D-alanylation of teichoic acids ( TAs ) mediated by the dlt operon gene products and/or incorporation of L-lysine into phosphatidylglycerol , the major membrane lipid , mediated by the mprF gene product [10] . Deletion of the dlt operon leads to the complete absence of D-alanyl esters of TAs in Staphylococcus aureus ( S . aureus ) [14] , Group A Streptococcus [15] , Streptococcus pneumoniae [16] , Enterococcus faecalis [17] , and Group B Streptococcus ( GBS ) [14] , which results in an increased susceptibility to various cationic antimicrobial agents . It has been demonstrated that lowering the anionicity of the cell wall causes a reduction in the electrostatic attraction between S . aureus and the cationic antibiotics gallidermin [14] and vancomycin [18] . Therefore , reduced electrostatic attraction represents one of the mechanisms by which D-alanylation of TAs can mediate CAMP resistance . However , the molecular mechanism by which D-alanylation of TAs mediates resistance has not been fully described . GBS , also known as Streptococcus agalactiae , is a leading cause of invasive infections ( pneumonia , septicaemia , and meningitis ) in the neonate , and a serious cause of mortality or morbidity in adults with underlying diseases [19] , [20] . Interestingly , this bacterium alike most if not all streptococci apparently lack the genes coding the essential enzymes for glycerophosphate WTA synthesis that are present in bacilli , enteroccci , lactobacilli , listeria , and staphylococci [21] and D-alanylation of LTAs appears to be the main mechanism of resistance to CAMPs . In order to better understand how modification of LTAs charge contributes to the resistance of GBS to CAMPs , we investigated its interaction with selected natural and de-novo designed CAMPs having different biophysical properties . For that purpose , we utilized a multidisciplinary approach combining microbiological and biophysical methods to highlight differences between various steps of the interaction of CAMPs with intact WT GBS and its isogenic dltA mutant . Our results suggest that incorporation of D-alanine into LTAs induce CAMPs resistance in GBS by modifying the rigidity and permeability of the cell wall rather than by affecting the electrostatic-driven binding of CAMPs to bacterial surface .
The investigated GBS strains were previously characterized revealing that 20 . 8% of the glycerophosphate residues of the LTAs of the WT strain NEM316 were substituted with D-alanyl esters whereas insertional inactivation of dltA caused complete absence of D-alanine [21] . The dltA mutant was highly susceptible to colistin compared to the WT strain and resistance was recovered at an intermediate level in a complemented strain where D-alanine incorporation was partially restored to 12 . 8% , confirming that there is a positive correlation between D-alanine content and resistance [22] . Here , we investigated whether there is a correlation between resistance and specific peptide property ( net charge , hydrophobicity , charge density and length ) . For that purpose , a group of nine peptides having different properties was investigated . The antibacterial activity of the selected CAMPs was determined against WT GBS and its isogenic dltA strain . The list includes two natural linear CAMPs ( magainin 2 and the human cathelicidin LL37 ) , two natural cyclic CAMPs ( polymyxin B and colistin ) , four well characterized short de-novo designed peptides , and a lipopeptide composed of lysine and leucine repeats ( K9L6 , K6L9 , 5D-K6L9 , K5L7 and C8-K5L7 ) ( Table 1 ) . Magainin 2 and LL37 are α-helical peptides with a net charge of +4 and +7 , respectively [12] . K6L9 and 5D-K6L9 share the same 15 aa sequence , have a net charge of +7 , but differ in their secondary structure . While K6L9 is 90% α-helical in PE/PG membrane bilayers and in lipopolysaccharide ( LPS ) multi-bilayers , the incorporation of D-amino acids reduced its α-helix content to 40% . This modification has been shown to improve its antibacterial activity against various gram-negative and gram-positive bacteria [23] , [24] . The peptide K9L6 is highly cationic ( +10 ) but exhibits poor antibacterial activity . Similar activity was found for K5L7 , but coupling of a short fatty acid ( C8-K5L7 ) strongly improved its antimicrobial activity [25] . The antimicrobial activities of all the peptides against GBS NEM316 WT strain and relevant isogenic mutants are summarized in Table 2 . The data reveal that , irrespective of their net positive charge ranging from +4 to +7 , all natural peptides ( magainin 2 , LL37 , colistin and polymyxin B ) are significantly more active against the dltA mutant ( more than a 2-fold dilution ) compared to the WT . In contrast , the activities of the designed peptides against the WT and dltA mutant strains are similar regardless of their charge ( ranging from +5 to +10 ) . We therefore try to hierarchize the biophysical properties that dictate the activity of the tested linear peptides by analyzing the minimal inhibitory concentration ( MIC ) ratios between the WT and dltA mutant ( Figure 1 ) . Interestingly , in contrast to what is expected , increased activity against the mutant does not correlate with peptides net charge . For example , LL37 is more active on the mutant compared to the WT . In contrast , the similarly charged ( +7 ) K6L9 or 5D-K6L9 peptides display the same activity on both strains . In addition , magainin 2 is also 4-fold more active on the dltA mutant compared to the WT although its net charge is only +4 ( Figure 1A ) . Morever , despite its +10 net charge , K9L6 displays weak activity on both strains . This could be due to its low hydrophobicity resulting in reduced ability to disrupt the membrane . However , there is no obvious correlation between hydrophobicity and antibacterial activity . For example , the peptides LL37 , magainin 2 , K6L9 , and the lipopeptide C8-K5L7 have similar high hydrophobicity , but only the first two are more active on the dltA mutant ( Figure 1B ) . Interestingly , the MIC ratios obtained with linear cationic peptides ( +4 to +10 ) , display a significant positive correlation with the peptide length ( R2 = 0 . 705 ) but a significant negative correlation ( R2 = 0 . 797 ) with the charge density ( Figure 1C and 1D , respectively ) . The data suggest that if two peptides of different sizes possess an identical cationic charge , the activity of the longer peptide is more impaired by LTA D-alanylation . Note that in comparison to the α-helical LL37 and magainin 2 , the cyclic peptides polymixin B and colistin have high charge density but are 8-fold more active against the dltA mutant . This is probably attributed to different mechanisms of actions which involve specific binding to lipid II rather than membrane perforation [4] , [26] , [27] . Since CAMPs are thought to interact with cell surface components prior to their incorporation into the membrane , we investigated whether other surface modifications beside LTAs D-alanylation could affect peptide-cell wall interaction . To address this question , peptides were tested for their activities against isogenic mutants that were unable to ( i ) express the transmembrane protein MprF ( Gbs2090 ) thought to incorporate L-lysine into membrane phosphatidylglycerol , ( ii ) synthesize membrane-bound lipoproteins ( lgt , lsp ) or the capsular polysaccharide ( cpsD ) , or ( iii ) anchor LPXTG proteins to peptidoglycan ( srtA ) . Our results show no significant difference in the MICs of the WT compared to these mutant strains ( Table 2 ) , suggesting that the corresponding GBS surface components do not significantly contribute to CAMPs resistance under our experimental conditions and do not affect peptide-wall interactions . Thus , in GBS , resistance to CAMPs is mainly due to D-alanylation of LTAs . Many CAMPs exhibit their antibacterial activity through direct interaction with and subsequent perforation of the membrane . To determine whether altered susceptibility to CAMPs correlates with changes in membrane permeability , we measured the entrance of the cationic dye SYTOX green into the WT and dltA strains . SYTOX green cannot enter intact bacteria unless the membrane is disrupted by external compounds . Upon penetration into bacteria and binding to intracellular nucleic acids , its signal increases drastically . A dose-dependent enhancement of signal intensity was found following exposure to selected CAMPs . In agreement with the MIC assay , only magainin 2 and LL37 induced significantly stronger signal with the dltA mutant , compared to the parental strain ( Figure 2A and 2B ) . Expectedly , the highly potent 5D-K6L9 was similarly active on both strains ( Figure 2C ) whereas the weakly potent K5L7 induced an equally low signal ( Figure 2D ) . These results confirm that D-alanylation of LTAs protects the membrane from perturbation by CAMPs . It is commonly admitted that esterification of WTAs and LTAs with D-alanyl esters reduces the surface cationic binding capacity , thereby leading to a reduced electrostatic binding of CAMPs and to an increased resistance [28] . GBS is devoid of WTAs but possess a branched rhamnose-rich carbohydrate similarly anchored to the peptidoglycan ( the so-called group B antigen ) [29]–[31] . This polysaccharidic sructure also displays an anionic character conferred by its high phosphate content which can mask differences in surface charge due to D-alanylation of LTAs . We therefore evaluated differences between the surface charge of GBS WT and dltA mutant strains by measuring their ability to bind the cationic protein cytochrome C [14] . To confirm the role of DltA in charge modification , an isogenic complemented dltA ( dltA comp . ) strain was also tested . The data reveal a 3-fold increased binding of cytochrome C to the dltA mutant compared to the WT strain and that complementation restored cytochrome C binding to the WT level ( Figure 3A ) . A similar trend was found in S . aureus and Group A Streptococcus [14] , [15] . The binding of CAMPs to the bacteria was then tested under high ( 160 mM NaCl ) and low ( 16 mM NaCl ) ionic strength conditions . The data reveal a direct correlation between the charge of the peptides and their binding capacity: LL37 ( +6 ) >K5L7 ( +5 ) >Magainin 2 ( +3 ) in both conditions ( Figure 3B ) . In these experiments , the peptides labeled with NBD at their N-terminus possess the net charge of the unlabeled peptides minus one , while their antimicrobial activity was unchanged ( data not shown ) . As expected , a marked reduction in binding was observed under high ionic strength buffer due to electrostatic masking of charges . Surprisingly , despite the huge difference in surface charge between the WT and the dltA mutant under low ionic environment , there was no significant increase in the binding of the tested peptides to the dltA strain . A similar result was found also for the peptides displaying no increased activity toward the mutant strain ( Figure S1 ) . Resistance to CAMPs could result from a thickened cell wall as demonstrated for S . aureus [32] . To address this possibility , we accurately measured the GBS cell wall thickness from high resolution transmission electron microscopy ( TEM ) images by using freeze substitution method . The morphology of the GBS cell wall is typical of that reported for other streptococci [33] , being composed of two compact laminae: an intensely electron opaque inner layer that lies the cytoplasmic membrane and a less electron opaque outer layer ( Figure 4 ) . Our measurements reveal no difference between the average thickness of the WT ( 31 . 9±6 . 7 nm ) and that of the dltA mutant ( 32 . 8±6 . 1 nm ) . However , the inner region of the WT cell wall was more heavily stained with metal ions ( lead citrate and uranyl acetate ) , as compared to the dltA mutant strain ( Figure 4 ) . These two observations were confirmed using a dltA complemented strain which had similar morphology as the WT strain . The heavy staining density cannot be attributed to increase in metal binding since the WT has less anionic charge then the dltA strain . An alternative explanation is that higher amounts of metals are trapped in the cell wall matrix of the WT and dltA complemented strain [22] . Therefore , we hypothesize that D-alanylation of LTAs increases the packing density of the GBS cell wall but does not modify its thickness . We used AFM to investigate the influence of D-alanylation of LTAs on the mechanical properties of the cell wall . This method enables to measure nanoscale changes in bacterial surface with a minimal interference with cell integrity . When bacteria were grown without CAMPs , WT and its isogenic dltA strain did not exhibit any apparent difference in surface morphology , as imaged in an amplitude mode ( Figure 5A and 5B ) and in topography mode ( Figure 5E and 5F ) . This observation is supported by calculation of the distribution of surface heights in WT and dltA mutant for which the root mean square ( RMS ) of the height distribution ( 1 . 14±0 . 32 nm and 1 . 22±0 . 21 nm , respectively ) are similar ( Figure 6A ) . Treatment with LL37 has significantly altered the morphology of WT and dltA mutant with a more pronounced effect on the mutant strain ( Figure 5C and 5D in amplitude images and 5G and 5H in topography images ) . This results in an increase in the average RMS of the height distribution for the dltA mutant ( 2 . 28±0 . 24 ) compared to the WT strain ( 1 . 8±0 . 19 ) ( Figure 6A ) . We also characterized the surface rigidity of non-treated WT , dltA mutant and dltA complemented strains by PeakForce QNM , which is based on a force-volume approach , and determination of the average value of DMT-modulus [34] , [35] . The data reveal that the rigidity of cell wall of the WT strain is more than 20-fold higher than that of the dltA mutant ( 177 . 37±19 . 54 MPa and 7 . 87±0 . 66 MPa , respectively ) . Complementation with a functional dltA gene restored the rigidity to a value of 150 . 50±19 . 10 ( Figure 6B ) . The value calculated for the GBS WT strain is in the range of those estimated for non-fixed air dried Escherichia coli and S . aureus [36] and live , agarose encapsulated E . coli ( 50–150 MPa ) , Bacillus subtilis ( 100–200 MPa ) and Pseudomonas aeruginosa ( 100–200 MPa ) [37] . We further investigated whether the increased cell wall density resulting from D-alanylation of LTAs can reduce CAMPs penetration through the cell wall barrier by following the interaction of fluorescently labeled CAMPs with intact bacteria . Earlier studies demonstrated that CAMPs can aggregate upon their interaction with cell wall lipopolysaccharides [11] , [23] , [24] , [38] . Here , we investigated if the peptides display different aggregation states when bound to the WT or the dltA mutant strains . This was done by monitoring changes in the signal intensity of rhodamine-labeled peptides following their interaction with the bacterial surface under under high ( 160 mM NaCl ) and low ( 16 mM NaCl ) ionic strength conditions . Addition of bacteria to the rhodamine-labeled peptides induced self-quenching of fluorescence for all tested peptides . However , no significant difference in aggregation was found in the WT compared to the dltA strain ( Figure 7A and Table 3 ) . Similar trend of aggregation was obtained when commercial LTAs isolated from S . aureus were used instead of intact bacteria which demonstrated that the change in aggregation state is due to the interaction of CAMPs with membrane-bound LTAs of GBS cells ( Figure S2A ) . We further examined the ability of the CAMPs to traverse the cell wall barrier and interact with the phospholipid membrane using NBD labeled peptides . The fluorescence emission of NBD , unlike that of rhodamine , is highly enhanced in a lipidic environment . Therefore , the signal output of NBD is strongly affected by its proximity to the membrane . We first monitored the interaction of NBD-labeled peptides with liposomes composed of cardiolipin and phosphatidylglycerol ( CL∶PG ) , anionic lipids composing membranes of streptococci [39] but lacking all cell wall components including peptidoglycan and LTAs . Upon addition of liposomes to the NBD-labeled peptides , the signal increased rapidly due to the localization of the peptide in the hydrophobic environment of the membrane ( Figure S2B ) . In comparison , addition of bacteria to either NBD-LL37 or NBD-magainin 2 resulted in a slower rate of signal increase ( Figure 7B ) , suggesting that the cell wall delays the peptides from reaching the phospholipid membrane . With intact GBS cells , the signal increase due to peptide penetration at low ionic strength environment was higher for the dltA mutant in comparison to the WT strain , whereas only a slight differences was observed under high ionic strength for magainin 2 but not for LL37 ( Table 3 ) . In contrast , the inactive NBD-K5L7 increased its signal by only 1 . 8% in 16 mM NaCl and displayed a slight reduction in signal in 160 mM solution , suggesting that most of the peptide remained trapped in the cell wall and did not reach the membrane . Together , our findings suggests that electrostatic interactions between LTAs are major contributors to the ability of the bacteria to block penetration of the CAMPs .
The purpose of this study was to investigate the mechanism by which D-alanylation of LTAs confers resistance to CAMPs in GBS , a species chosen as a model of gram-positive bacterium devoid of WTAs . Our data will be discussed in line with the three following major findings: ( i ) D-alanylation of LTAs modulates the resistance towards only a subset of CAMPs whose activity is in a relation to their length and charge density; ( ii ) CAMPs resistance due to increased surface cationic charge is not due to decreased amounts of bound peptides to bacteria; and ( iii ) D-alanylation of LTAs reduces the penetration of CAMPs through the cell wall by increasing its density , most likely by altering the conformation of LTAs . ( i ) Several studies have shown that inactivation of the dltABCD genes in different gram-positive species , including GBS , result in an increased susceptibility to various natural CAMPs [14] , [22] , [40] . Here , our data revealed a similar trend when the GBS dltA mutant was exposed to natural CAMPs but not to de-novo designed linear peptides ( Table 2 ) . Increased activity against the dltA mutant strain did not correlate with peptides net charge or hydrophobicity . However , reasonable correlations were found with peptides length ( R2 = 0 . 705 ) and charge density ( R2 = 0 . 797 ) ( Figure 1 ) . Although the main mechanism of bacterial killing of the linear peptides used in this study is membrane pertubation , deviations from the rules may be attributed to differences in the specific mode of action of each peptide . Consistently , the fact that the length and charge density of a peptide correlates with the WT/dltA MIC ratio , whereas its net charge does not influence its bactericidal activity , suggests the existence of at least one alternative mechanism of intrinsic resistance besides electrostatic repulsion . The data on the efflux of SYTOX green into the non-growing cells due to treatment with CAMPs ( Figure 2 ) confirmed that incorporation of D-alanyl resisdues into LTAs protect the membrane integrity by reducing the capability of the CAMPs to disrupt it . This observation was not due to peptide interaction with cell wall components such as the capsular polysaccharide and surface proteins . In addition , we provide evidence that under our conditions the transmembrane MprF protein thought to aminoacylate the membrane phospholipids did not significantly contribute to resistance towards the CAMPs used in this study ( Table 2 ) . ( ii ) The activities of cationic peptides , such as nisin and gallidermin , against GBS and S . aureus decrease as the bacterial electropositive surface charge increases through incorporation of D-alanyl residues in LTAs [14] , [22] . Note that these antibiotics target the cell wall through electrostatic interactions and dock specifically on the cell wall precursor lipid II to inhibit peptidoglycan synthesis [4] , [26] , [27] . Therefore , the activity of these molecules is expected to be highly affected by surface charge modification . A similar mechanism of electrostatic repulsion was demonstrated for the activity of the staphylococcal mprF gene product which attenuates membrane charge [41] . However , a recent study by Kilelee et al . showed that physiological concentrations of lysyl-PG enhances the resistance of model membranes to the CAMP 6W-RP-1 due to a decrease in lipid clustering rather than by a mechanism of electrostatic repulsion [42] . Under our experimental settings , the GBS mprF mutant was found as resistant as the parental WT strain to all CAMPs tested in this study and this result is currently under investigation . Our results reveal a direct correlation between the net charge of the peptides and their binding capacity to either WT or dltA mutant strains but no significant difference was observed in the binding of a given peptide to either strains ( Figure 3 and Figure S1 ) . On the other hand , the cytochrome C protein binds in higher amounts to the dltA mutant compared to the WT strain which indicate that there is a significant difference between their overall surface charge . Therefore , the binding of the tested CAMPs , unlike that of cytochrome C , seems less affected by changes in GBS cell wall charge . These results suggest that charge repulsion is not the major mechanism by which D-alanylation of LTAs promotes CAMPs resistance , as shown in S . aureus for the human group IIA phospholipase A2 [43] . ( iii ) The secondary structure of LTAs highly depend on the presence of environmental cations . Under low ionic-strength conditions , the anionic LTA chains repel each other and therefore exist in an extended conformation as it was also proposed for LPS molecules of gram-negative bacteria [44] . In high ionic-strength solutions , it has been shown that the electrostatic screening of the poly ( Gro-P ) moiety of LTA by sodium ions induced random-coil conformation which in turn increased the packing density of the matrix [45] . In this model , incorporation of D-alanyl residues should mask the anionic phosphates of the LTAs to reduce repulsion between neighboring molecules [46] , [47] . We postulated that this structural modification could affect the mechanical resistance of the cell wall to CAMPs . Indeed , TEM analysis showed that D-alanylation of LTAs increase the density of the cell wall ( Figure 4 ) . Moreover , AFM analysis revealed that the surface rigidity of WT GBS strain is more than 20-fold higher than that of the dltA mutant and that gene complementation restores rigidity to values similar to the WT strain ( Figure 6B ) . As a likely consequence , the WT strain displays reduced alterations of its surface topography following treatment with LL37 ( Figure 5 ) and attenuated penetration of fluorescently labeled CAMPs through the cell wall barrier ( Figure 7 and Table 3 ) . Our results show that high NaCl concentration reduces the penetration of CAMPs through the cell wall of the dltA strain to restore the WT behaviour ( Table 3 ) . Therefore , electrostatic screening of cell wall charge can be achieved either through D-alanylation of LTAs or by electrostatic interaction with metal cations . This delicate interplay between environmental cations and incorporation of D-alanyl residues , which could be regulated at the transcriptional level [20] , [42] , allow the bacteria to change the mechanical properties of its cell wall to resist CAMPs attack . In conclusion , our work constitue a detailed investigation of the mechanism by which D-alanylation of LTAs can mediate resistance to CAMPs in GBS . Based on our observations , we suggest that D-alanylation of LTAs increase the packing of the cell wall of gram-positive bacteria to reduce the effective peptide concentration over the membrane . Our findings uncover a novel protective role of the cell wall against CAMPs and therefore constitute an advance in our understanding of bacterial defense mechanisms against these molecules .
Rink amide MBHA resin and 9-fluorenylmethoxycarbonyl ( Fmoc ) amino acids were purchased from Calibochem-Novabiochem AG ( Switzerland ) . Other reagents used for peptide synthesis include N , N-diisopropylethylamine ( DIEA , Sigma- Aldrich ) , dimethylformamide , dicheloromethane , and piperidine ( Biolab , Israel ) . 4-chloro-7-nitrobenz-2-oxa-1 , 3-diazole fluoride ( NBD-F ) , rhodamine-N-hydroxysuccinimide ( Rho-N ) and SYTOX green were purchased from Molecular Probes ( Junction City , OR , USA ) . LTAs from Staphylococcus aureus ( L2515 ) and Cytochrome C were purchased from Sigma-Aldrich ( Rehovot , Israel ) . Cardiolipin and phosphatidylglycerol were purchased from Avanti Polar Lipids ( Alabaster , Alabama ) . The procedure conducted as described previously [23] . Briefly , peptides were synthesized using the Fmoc solid phase method on Rink amide resin ( 0 . 68 meq/mg ) . For fluorescent labeling of the N-terminus of the peptides , resin-bound peptides were treated with NBD or rhodamine dissolved in dimethyl formamide ( DMF ) , washed thoroughly with DMF and then with methylene chloride , dried and then cleaved . The peptides were purified ( greater than 98% homogeneity ) by reverse phase high performance liquid chromatography ( RP-HPLC ) on a C4 column using a linear gradient of 30–70% acetonitrile in 0 . 1% trifluoroacetic acid ( TFA ) for 40 minutes . The peptides were subjected to amino acid and mass spectrometry analysis to confirm their composition . The bacterial strains used in this study and their main characteristics are listed in Table S1 . The MICs of each antimicrobial peptide were tested in Todd-Hewitt broth ( THB ) buffered with 100 mM HEPES in 96-well polypropylene microplates ( Costar , Cambridge , MA ) by a dilution method . Bacteria ( 2×106 CFU ) were added in triplicates to wells containing increasing concentrations of the antimicrobial peptides . Plates were incubated at 37°C with shaking overnight and then read ( OD600 nm ) using microplate reader ( Labsystems Multiskanat ) for bacterial growth . The MICs90 was considered to be the peptide concentration that inhibited 90% growth . Bacteria were washed twice , resuspended in sodium phosphate buffer ( SPB , 20 mM , pH 7 . 4 ) , and incubated with 1 µM SYTOX green for 20 min in the dark with agitation [48] . Bacteria were added to a 96 well black plate ( Nunc , Denmark ) containing increasing concentrations of CAMPs and the increase in fluorescence , due to penetration and binding of the dye to intracellular DNA , was immediately monitored ( excitation of 485 nm and emission of 520 nm ) . Results are mean values ±SD of three independent experiments , performed in triplicates . Bacteria were harvested and washed twice with PBS ( 16 mM , pH 7 . 2 ) . Cells from 200 µl aliquots ( OD600 nm adjusted to 4 ) were incubated ( shaking in dark ) with 0 . 5 mg/ml Cytochrome C . For peptide binding , 200 µl bacterial aliquots ( OD600 nm adjusted to 0 . 75 ) were incubated with NBD labeled peptides ( 1 µM ) . Samples were centrifuged ( 14 , 000 g , 3 min ) and 100 µl of the supernatant were collected and assayed photometrically ( Cytochrome C , 530 nm ) or fluorescently ( NBD excitation at 467 nm and emission at 530 nm ) . In order to dissolve any peptide aggregates that may quench the fluorescent signal , 50 µl of 6 M guanidine hydrochloride were added to the NBD-peptide wells . Data is presented as a percentage of the maximum signal ( peptide only ) ± SD of three independent experiments , carried out in triplicate . Real time tracking of the changes in the fluorescent signal of rhodamine or NBD labeled peptides was performed using an SLM-Aminco Bowman series 2-luminescence spectrophotometer ( SLM-Aminco , Rochester , NY , USA ) at room temperature . Typical spectral bandwidths were 5 nm for excitation and 5 nm for emission . The ability of the peptides to traverse the cell wall and interact with the membrane was evaluated using 1 µM peptide , a concentration that did not disrupt membrane integrity in the SYTOX green experiments . The peptides were placed in a 5×5-mm quartz cuvette with constant magnetic stirring . Following signal stabilization , bacterial suspension ( 10 µl , OD600 nm adjusted to 4 ) or liposomes suspension ( 100 µM lipid final concentration ) composed of cardiolipid/PG ( 1∶1 M/M ) [49] was added to the cuvette and the change in signal was monitored over time . AFM imaging was as described previously [50] . Prior to the measurements , bacterial culture was washed , resuspended in PBS and adjusted to OD600 nm of 0 . 5 . Bacteria were immobilized on freshly cleaved MICA coated with poly-L-lysine ( 0 . 01 mg/ml ) , washed to remove unattached bacteria , and gently fixated with 1 . 5% glutaraldehyde for 10 min . Samples were washed again with DDW to remove glutaraldehyde traces and left to dry overnight at room temperature . Images of bacteria were acquired with MultiMode AFM ( Bruker , Santa Barbara , CA ) equipped with the Nanoscope V controller and a small scanner . Images were recorded in air , at room temperature ( 22–24°C ) , in PeakForce QNM ( quantitative nanomechanical mapping ) mode using silicon nitride WSlevers ( ORC8-PS-W , Olympus ) with a nominal spring constant of 0 . 76 N/m . PeakForce QNM AFM imaging mode yields quantitative nanomechanical mapping of material properties , including DMT modulus and adhesion . In the same time sample topography is imaged with high resolution ( 1024 pixels ) and minimized sample distortion due to the fine adjustment of the force applied to the sample surface . The applied force was adjusted around 1 nanonewton and the scan rate was set to 1 Hz . Imaging was carried out at different scales to verify the consistency and robustness of the evaluated structures . Numerical data presented is the mean value ( ±SD ) of the root-mean-square ( RMS ) of surface roughness or DMT modulus of four 150×150 nm samples , taken from a 700×700 nm field ( n≥8 cells for each treatment ) . Cells were centrifuged and the pellet was loaded on aluminum discs with depth of 100 µm ( Engineering Office M . Wohlwend GmbH , Switzerland ) and covered with a flat disc . The sandwiched sample was frozen in a HPM010 high-pressure freezing machine ( Bal-Tec , Liechtenstein ) . Cells were subsequently freeze-substituted in a AFS2 freeze substitution device ( Leica Microsystems , Austria ) in anhydrous acetone containing 2% glutaraldehyde and 0 . 2% tannic acid osmium tetroxide for 3 days at −90°c and then warmed up to −30°c over 24 hours . Samples were washed three times with acetone , incubated for one hour at room temperature with 2% osmium tetroxide , washed three times with acetone and infiltrated for 5–7 days at room temperature in a series of increasing concentration of Epon in acetone . After polymerization at 60°c , 60–80 nm sections were stained with uranyl acetate and lead citrate and examined in a Tecnai T12 electron microscope ( FEI , Holland ) operating at 120 kV , utilizing a 2k by 2k ES500W Erlangshen CCD camera ( Gatan , UK ) . Results were analyzed using a single factor ANOVA test . Values of p<0 . 05 were considered statistically significant . | Cationic antimicrobial peptides ( CAMPs ) represent important evolutionarily conserved elements of innate immunity and their killing mechanism involves bacterial cell wall permeation . As a result , gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids ( TAs ) following incorporation of D-alanyl residues to neutralize their surface charge , a reaction catalyzed by the dlt operon gene product . Here , we demonstrate that this electrochemical modification changes the barrier properties of Group B Streptococcus cell wall and inactivation of the dlt operon activity results in CAMP sensitivity . However , despite the major increase in the surface charge of the mutant , no increased electrostatic binding of CAMPs is observed . Rather , D-alanine incorporation protects the bacterial membrane by reducing the penetration of CAMPs through the cell wall . Accordingly , a dlt mutant was more susceptible to perforation by CAMPs and its cell wall nanostructure was significantly altered . Overall , we demonstrate a novel protective role of the cell wall against CAMPs which should enable bacterial invaders to survive upon host's colonization . | [
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] | 2012 | D-Alanylation of Lipoteichoic Acids Confers Resistance to Cationic Peptides in Group B Streptococcus by Increasing the Cell Wall Density |
Amphotericin B provides improved therapy for visceral leishmaniasis ( VL ) caused by Leishmania donovani , with single dose liposomal-encapsulated Ambisome providing the best cure rates . The VL elimination program aims to reduce the incidence rate in the Indian subcontinent to <1/10 , 000 population/year . Ability to predict which asymptomatic individuals ( e . g . anti-leishmanial IgG and/or Leishmania-specific modified Quantiferon positive ) will progress to clinical VL would help in monitoring disease outbreaks . Here we examined whole blood transcriptional profiles associated with asymptomatic infection , active disease , and in treated cases . Two independent microarray experiments were performed , with analysis focussed primarily on differentially expressed genes ( DEGs ) concordant across both experiments . No DEGs were identified for IgG or Quantiferon positive asymptomatic groups compared to negative healthy endemic controls . We therefore concentrated on comparing concordant DEGs from active cases with all healthy controls , and in examining differences in the transcriptome following different regimens of drug treatment . In these comparisons 6 major themes emerged: ( i ) expression of genes and enrichment of gene sets associated with erythrocyte function in active cases; ( ii ) strong evidence for enrichment of gene sets involved in cell cycle in comparing active cases with healthy controls; ( iii ) identification of IFNG encoding interferon-γ as the major hub gene in concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; ( iv ) enrichment for interleukin signalling ( IL-1/3/4/6/7/8 ) and a prominent role for CXCL10/9/11 and chemokine signalling pathways in comparing active cases with treated cases; ( v ) the novel identification of Aryl Hydrocarbon Receptor signalling as a significant canonical pathway when comparing active cases with healthy controls or with treated cases; and ( vi ) global expression profiling support for more effective cure at day 30 post-treatment with a single dose of liposomal encapsulated amphotericin B compared to multi-dose non-liposomal amphotericin B treatment over 30 days . ( 296 words; 300 words allowed ) .
Visceral leishmaniasis ( VL ) , also known as kala-azar , is a potentially fatal disease caused by obligate intracellular parasites of the Leishmania donovani complex . VL is a serious public health problem in indigenous and rural populations in India , causing high morbidity and mortality , as well as major costs to both local and national health budgets . The estimated annual global incidence of VL is 200 , 000 to 400 , 000 , with up to 50 , 000 deaths annually occurring principally in India , Bangladesh , Sudan , South Sudan , Ethiopia and Brazil [1] . In India , improvements in drug therapy have been afforded through the introduction of amphotericin B treatment , with single dose liposomal encapsulated Ambisome providing the best cure rates and now being used as the preferred treatment regime in the VL elimination program [2] . However , with the potential development of drug resistance to each new therapeutic approach [3] , there remains a continuing need for improved and more accurate methods of early diagnosis , as well as ability to monitor responses to treatment and to predict disease outcome . These objectives are also important in relation to the World Health Organization-supported VL elimination initiative in the Indian subcontinent , which aims at reducing the incidence rate of VL in the region to below 1 per 10 , 000 population per year by 2020 [4] . Monitoring disease outbreaks in the context of the elimination program will be an important goal , including the ability to determine which individuals displaying asymptomatic disease , as monitored by anti-leishmanial IgG [5 , 6] and/or Leishmania-specific modified Quantiferon responses [7] , will progress to clinical VL disease [6] . In recent years , the use of whole blood transcriptional profiling in humans has provided a better understanding of the host response to infectious disease , leading to the identification of blood signatures and potential biomarkers for use in diagnosis , prognosis and treatment monitoring ( reviewed [8] ) . Pioneering studies using this approach were successful in identifying a neutrophil-driven interferon ( IFN ) -inducible blood transcriptional signature for active tuberculosis that involved both IFN-γ and type I IFN-α/β signalling [9] and was subsequently confirmed in multiple countries world-wide ( reviewed [8] ) . This neutrophil-driven interferon signature was present in active disease but absent in both latent infection and in healthy controls [9] . While an IFN-inducible signature was also identified in patients with the autoimmune disease systemic lupus erythematosus , there were differences in the signatures that also distinguished the two profiles from each other [9] . Viral infections [10] and bacterial infections like melioidosis [11] are also broadly characterised by IFN-inducible gene expression , but whole blood signatures have been identified that are able to discriminate between bacterial and viral infections [10 , 12] , as well as between different viral infections [10] . In HIV , blood transcriptional signatures have been identified that distinguish between rapid compared to slow progression to disease [13] . Blood signatures have also been identified which distinguish between children who acquire dengue virus fever compared to those who develop dengue haemorrhagic fever [14 , 15] . There are also signatures that distinguish between pulmonary and extra-pulmonary tuberculosis [16] , as well as between pulmonary tuberculosis , pulmonary sarcoidosis , pneumonias and lung cancers [17] . A transcriptional signature that can be used to monitor treatment response is also a valuable goal in infectious disease . Again , studies from two cohorts followed longitudinally in South Africa show that the transcriptional signature of active tuberculosis disease rapidly diminishes with successful treatment [18 , 19] . More recently , whole blood transcriptional profiling has been used to study human host responses to protozoan pathogens such as malaria [20 , 21] and Chagas disease caused by Trypanosoma cruzi [22 , 23] . Expression profiling has also been applied in the context of animal models [24–26] and in human studies [15 , 27–29] of the leishmaniases . In particular , whole blood transcriptomics was used to compare expression profiles in patients with active VL caused by L . infantum with asymptomatic infected individuals , patients under remission from VL , and controls [27] . While VL patients exhibited profiles reflecting activation of T cells via MHC Class I signalling and type I interferon , patients in remission showed heterogeneous profiles associated with T cell activation , type I interferon signalling , cell cycle , activation of Notch signalling , and an increased proportion of B cells . Asymptomatics ( as determined by a positive delayed type hypersensitivity response to leishmanial antigen ) and uninfected individuals exhibited similar gene expression profiles . Here we also set out to determine whole blood transcriptional profiles that might distinguish unifected individuals from asymptomatic infection or active disease caused by L . donovani in India , as well as to monitor the changes in transcriptional profiles that accompanied drug treatment . Whilst we were unable to detect a signature that distinguished asymptomatic ( IgG antibody positive [5 , 6] , or modified Quantiferon positive [7] ) individuals from healthy endemic controls who were negative by these two assays , we were able to determine the transcriptional profile of active VL cases , and to demonstrate interesting differences in return to baseline between patients treated with non-liposomal compared to liposomal-encapsulated ( Ambisome ) amphotericin B .
The enrolment of human subjects complies with the principles laid down in the Helsinki declaration . Institutional ethical approval ( reference numbers: Dean/2012-2013/89 ) was obtained from the ethical review board of Banaras Hindu University ( BHU ) , Varanasi , India . Informed written consent was obtained from each participant at the time of enrolment , or from their legal guardian if they were under 18 years old . Only patients who had not previously received treatment and who agreed to participate in the study were enrolled . All clinical treatment and follow-up records were maintained using standardised case report forms on an electronic server . All patient data were analysed anonymously . In this study two independent microarray experiments were performed . For experiment 1 , samples were collected between February and April 2011 . For experiment 2 , samples were collected between April and July 2012 . Samples were collected at the Kala-azar Medical Research Center ( KAMRC ) , Muzaffarpur , Bihar , India , or in nearby field sites for some asymptomatic individuals and endemic controls . Active VL cases were diagnosed by experienced clinicians based on clinical signs , including fever ( >2 weeks ) , splenomegaly , positive serology for recombinant antigen ( r ) -K39 and/or by microscopic demonstration of Leishmania amastigotes in splenic aspirate smears . VL patients were treated according to routine clinical care with either ( a ) experiment 1: 0 . 75 mg/kg non-liposomal amphotericin B daily for 15 days ( N = 3 ) , or on alternate days over 30 days ( N = 7 ) , by infusion ( i . e . 15 doses in all; total dose 11 . 25 mg/kg over 30 days ) ; or ( b ) experiment 2: 10 mg/kg of Ambisome ( liposome-encapsulated amphotericin B ) as a single dose by infusion . Blood samples were collected pre- ( N = 10 experiment 1; N = 11 experiment 2 ) and post- ( day 30; N = 10 experiment 1; N = 11 experiment 2 ) treatment . There were 9 paired pre-/post-treatment samples for experiment 1; 10 for experiment 2 . Healthy control subjects included ( i ) asymptomatic individuals ( N = 2 experiment 1; N = 6 experiment 2 ) who had sustained high anti-leishmanial antibody levels by direct agglutination test ( DAT titer ≥1:25 , 600 ) over two annual surveys prior to blood collection for profiling [6]; ( ii ) asymptomatic individuals ( N = 8 experiment 1; N = 9 experiment 2 ) who were positive by Leishmania-specific modified quantiferon assays [7] over two annual surveys prior to blood collection for profiling; and ( iii ) Serology ( DAT titer ≤1:1600 ) and quantiferon negative healthy endemic controls ( N = 6 experiment 1; N = 10 experiment 2 ) who were negative by both assays over two annual surveys prior to blood collection for profiling . Sample sizes are for post-QC samples used in expression profiling studies ( see below ) . Further clinical and demographic details on participants are provided in S1 Table . The work flow for data analysis is provided in S1 Fig . Whole blood ( 5 mL ) collected by venepuncture was immediately placed into Paxgene tubes ( QIAGEN GmbH , Germany ) and stored at -80°C for later processing for RNA . RNA was extracted using PAXgene Blood RNA kits ( QIAGEN GmbH , Germany ) according to manufacturer’s instructions . RNA integrity and purity were checked using Tape Station 4200 ( Agilent Technologies , USA ) . Samples used for beadchip analysis had RNA integrity ( RIN ) mean±SD values 6 . 75±0 . 67 ( range 5 . 5–7 . 7 ) . Globin mRNA was depleted using GLOBINclear-Human kits ( ThermoFischer Scientific , USA ) . RNA was reverse transcribed and biotin-labelled using the Illumina TotalPrep RNA Amplification kit ( ThermoFischer Scientific , USA ) . The resulting biotinylated cRNA was hybridised to Illumina HT12v4 Expression BeadChips , specifically HumanHT-12_V4_0_R2_15002873_B , containing 47 , 323 genome wide gene probes , and 887 control probes . Samples from different control or clinical groups were distributed evenly across 3 ( experiment 1 ) or 4 ( experiment 2 ) beadchips . All RNA preparation and processing of samples over beadchips was carried out at Sandor Lifesciences Pvt . Ltd . ( Hyderabad , India ) . All data analysis was carried out in R Version 3 . 4 . 3 ( Smooth Sidewalk - https://www . r-project . org/ ) and RStudio ( version 1 . 1 . 383 ) . The Bioconductor package Lumi [30] was used to read in raw expression values and perform quality control . Background correction and quantile normalisation of the data was carried out using the Bioconductor package Limma [31] . Pre-processing of the microarray data and removal of non-expressed ( detection P-value > 0 . 05 in all arrays ) and poor quality probes previously shown to have unreliable annotation [32] provided 21 , 959 and 23 , 466 probe sets which passed QC in experiments 1 and 2 , respectively . Principal components analysis ( PCA ) and unsupervised cluster analysis ( Pearson’s correlation coefficient; hclust = complete ) of normalised data was performed in R . Data was visualised using the R packages ggplot2 ( 3 . 1 . 2 ) [33] and pheatmap ( 1 . 0 . 12 ) [34] . Differential expression analysis using linear modelling and empirical Bayes methods was carried out in the Bioconductor package Limma [31] for comparisons between control and clinical groups , as indicated . The threshold for differential expression was a log2-fold-change ≥1 ( i . e . ≥2-fold ) and/or Benjamini-Hochberg [35] adjusted p-value ( Padj ) ≤0 . 05 , as indicated . Genes achieving these thresholds were taken forward in analyses using the gene set enrichment tool Enrichr [36] , and using Ingenuity Pathway Analysis ( IPA ) ( Ingenuity Systems , www . ingenuity . com ) to identify canonical pathways , upstream regulators , and gene networks . Enrichr [36 , 37] accesses a wide range of open access databases to identify terms ( pathways/processes/disease states ) for which the gene set is enriched . Input to Enrichr comprised lists of DEGs for specific between-group comparisons , as indicated , and did not include expression level data for individual genes . Enrichr uses four scores to report enrichment: a p-value ( reported here as Pnominal ) calculated using Fisher’s exact test; a q-value ( reported here as Padj ) which is the Benjamin-Hochberg adjusted p-value; a rank or z-score of the deviation from the expected rank by the Fisher’s exact test; and a combined score which is a combination of the p-value and z-score calculated by multiplying the two scores using the formula c = ln ( p ) *z . This z-score and the combined score correct for biases in ranking of term lists based solely on Fisher’s exact test [37] and outperform other enrichment methods in benchmarking studies [36] . The Enrichr z-score is not an activation score . IPA uses the Ingenuity Knowledge Base , an extensive database comprising biological pathways and functional annotations derived from the interactions between genes , proteins , complexes , drugs , tissues and disease , to carry out all its analyses . Benjamini-Hochberg correction was applied where applicable and Padj ≤ 0 . 05 was used to filter all results . Canonical pathway predicts known biological pathways that are changing based on the pattern of gene expression . The p-value uses Fisher’s Exact Test and does not consider the directional effect of one molecule on another , or the direction of change of molecules in the dataset . The significance level is the most important metric . The Z-score in IPA canonical pathway analysis is an activation z-score that takes account of known directional effects of one molecule on another or on a process , and the direction of change of molecules in the dataset . However , just because a pathway does not have a good z-score does not make it uninteresting . Upstream Regulator Analysis within IPA was employed to predict if there were any endogenous genes/cytokines/transcription factors which may be responsible for the observed gene expression patterns . If an upstream regulator is identified , an activation Z-score is calculated based on the fold change values of its target genes within the dataset . A Z-score ≥2 suggests that an upstream regulator is activated whereas a Z-score ≤-2 suggests it is inhibited , with active VL cases being the experimental group of baseline comparator . IPA also generates a “Top Tox List” pathway which provides an indication of toxic or pathogenic pathways that could be amenable to therapeutic intervention . Networks were constructed in IPA using the “Connect” option under the “Build” functionality . Genes with no previously documented interactions were removed from the diagram and the functions of each network were inferred from the remaining connected genes in each time-point . Nonparametric Gene Set Enrichment Analysis ( GSEA [38] ) using expression values to rank genes by their differential expression between two phenotypes was also used as an additional tool to ensure that important differences were not missed due to stringency of parametric methods , especially in the comparing the uninfected healthy endemic control groups with healthy antibody positive or healthy quantiferon positive groups . For this analysis we compared our data to the Blood Transcription Module gene list for antibody responses to vaccines ( [39] in addition to the canonical pathway ( CP ) collection of the GSEA-MSigDB C2 curated gene sets ( C2 ) [38] . GSEA was run for 1 , 000 permutations using weighted enrichment statistic and signal-to-noise ranking metric .
Two independent microarray experiments were carried out to compare transcriptional profiles across clinical groups that included active VL cases pre-treatment ( N = 10 experiment 1; N = 11 experiment 2 ) , drug treated VL cases ( N = 10 experiment 1; N = 11 experiment 2 ) , modified quantiferon [7] positive asymptomatic individuals ( N = 8 experiment 1; N = 9 experiment 2 ) , high Leishmania-specific antibody positive ( by DAT ) asymptomatic individuals ( N = 2 experiment 1; N = 6 experiment 2 ) , and endemic healthy controls ( N = 6 experiment 1; N = 10 experiment 2 ) who were both modified quantiferon negative and antibody negative by DAT . PCA of the top 500 most variable probes ( Fig 1 ) across all pairwise comparisons of samples showed that principal component 1 ( PC1 ) accounted for 45% ( experiment 1; Fig 1A and 1B ) and 31% ( experiment 2; Fig 1D and 1E ) of the variation and resolved active cases compared to endemic healthy control and asymptomatic groups . The latter were not well resolved from each other in either experiment . Treated patients sat intermediate between , and overlapping with , both active cases and control/asymptomatic groups in experiment 1 but showed greater overlap with control/asymptomatic groups in experiment 2 ( see below ) . This is particularly apparent when comparing plots of PC1 by PC3 ( Fig 1B and 1E ) . Unsupervised hierarchical cluster analysis also ( Fig 1C and 1F ) provided discrete clusters of active cases compared to control and asymptomatic individuals , with treated cases interspersed with both active cases and control groups and not falling into a single discrete cluster in either experiment . Consistent with the PCA plots ( Fig 1 ) , there were no differentially expressed probes representing genes ( i . e . Benjamini-Hochberg [35] Padj ≤0 . 05 ) when comparing either modified quantiferon positive asymptomatic individuals with endemic healthy controls , or when comparing high antibody titer individuals with endemic healthy controls , in either experiment 1 or experiment 2 . The additional non-parametric analysis carried out using GSEA also failed to identify gene sets enriched in controls compared to high antibody titre or modified quantiferon positive asymptomatic individuals that could be replicated across the two experiments ( S2 and S3 Tables ) . For the analyses presented below , these groups were therefore analysed as one group referred to as “controls” or “healthy controls” in all further differential expression analyses . Differential expression analysis focused on the comparison of ( i ) active VL cases versus controls , ( ii ) treated VL cases versus controls , and ( iii ) active versus treated VL cases . Log2-fold-change in experiment 1 was highly correlated with log2-fold-change in experiment 2 across all probes . Table 1 shows the number of differentially expressed probes representing genes in experiment 1 and experiment 2 for each comparison as well as the number of differentially expressed probes that replicated and were concordant for direction of effect between the two cohorts . At Padj ≤0 . 05 , there are 2 , 584 concordant differentially expressed probes in common when comparing active cases with controls , 37 concordant probes when comparing treated cases with controls , and 221 concordant probes when comparing active and treated cases . At the more stringent threshold of ≥2-fold change there were 439 , 8 , and 42 concordant probes for these comparisons , respectively . Of note , we found a greater number of transcriptional differences between treated cases and controls in experiment 1 compared to experiment 2 ( Table 1; differentially expressed probes are 1132 and 126 , respectively , at Padj ≤0 . 05 ) . One explanation for this could be the different treatment regimen employed in the two cohorts . VL patients of the first experiment were treated with 15 doses of a non-liposomal form of amphotericin B over 30 days . In experiment 2 patients received a single dose of liposomal amphotericin B , which has shown better efficacy for the treatment of VL [40 , 41] . The effect of treatment regimen on whole blood transcriptional profiles is further indicated by the comparison of active and treated cases . In this case , fewer differences in transcriptional regulation are observed between active and treated cases in experiment 1 as opposed to experiment 2 ( Table 1; differentially expressed probes are 654 and 1317 , respectively , at Padj ≤0 . 05 ) , in which patients have received a more efficacious therapy . These findings agree with the PCA results ( Fig 1 ) , in which treated cases of the experiment 1 cohort form a more discrete group between active cases and controls ( Fig 1A and 1B ) whereas treated cases of the experiment 2 cohort are grouped more closely to controls ( Fig 1D and 1E ) . Due to the small number of concordant differentially expressed genes identified for the treated cases versus controls ( Table 1; 37 at Padj ≤0 . 05 , 8 at Padj ≤0 . 05 and ≥2-fold change ) , only the concordant differentially expressed gene sets for active cases versus controls and active cases versus treated cases were used in subsequent pathway and gene set enrichment analyses . S1 and S2 Data provide spreadsheets of the data from experiments 1 and 2 respectively for all concordant DEGs that were significant at Padj<0 . 05 ) . Heatmaps were generated for individual expression levels for probes representing the top 10 concordant genes expressed at a higher ( “induced” ) level ( Fig 2A ) , and the top 10 concordant genes expressed at a lower ( “repressed” ) level ( Fig 2B ) , in active cases compared to controls in experiment 1 . Heatmaps for the same “induced” and “repressed” probes/genes in experiment 2 are presented in Fig 2C and 2D . Of note 8/10 “repressed” genes were also in the top 10 most highly differentially expressed “repressed” genes in experiment 2; all 10 genes achieved ≥2-fold change in both experiments . Amongst these 10 most “repressed” genes were: peptidase inhibitor 3 ( PI3 ) , a known antimicrobial peptide for bacteria and fungi that is upregulated by lipopolysaccharide and cytokines; the C-C chemokine ligand 23 ( CCL23; represented by 2 probes ) which acts as a chemoattractant for resting ( but not active ) T cells , monocytes , and to a lesser extent neutrophils; G-protein-coupled C-C motif chemokine receptor 3 ( CCR3 ) which binds CCL10 ( eotaxin ) , CCL26 ( eotaxin-3 ) , CCL7 ( MCP3 ) , CCL13 ( MCP4 ) and CCL5 ( RANTES ) that likewise act as chemoattractants for eosinophils , monocytes and neutrophils; ALOX15 which is a lipoxygenase known to regulate inflammation and immunity; and the G-protein-coupled prostaglandin D2 receptor 2 ( PTGDR2 alias GPR44 ) that is preferentially expressed in CD4 effector T helper 2 ( Th2 ) cells and mediates pro-inflammatory chemotaxis of eosinophils , basophils and Th2 cells . For the “induced” genes ( Fig 2A and 2C ) , only 2/10 ( the top 2 in both experiments ) were also in the top 10 “induced” genes in experiment 2 , but all achieved ≥2-fold change in both experiments . In addition to type I interferon inducible 27 ( IFI27 ) and complement C1q B chain ( C1QB ) genes , there was a bias amongst the most strongly “induced” genes towards genes involved in erythrocyte function , including: glycophorin B ( GYPB ) , a major sialoglycoprotein of the human erythrocyte membrane; Rh D blood group antigens ( RHD ) ; hemoglobin subunit delta ( HBD ) ; 5'-aminolevulinate synthase 2 ( ALAS2 ) an erythroid-specific enzyme located in the mitochondrion and involved in heme biosynthesis; carbonic anhydrase 1 ( CA1 ) which is found at its highest level in erythrocytes; atypical chemokine receptor 1 ( Duffy blood group ) ( ACHR1 alias DARC ) known for its role as the erythrocyte receptor for Plasmodium vivax and P . knowlesi; and 2 , 3-diphosphoglycerate ( 2 , 3-DPG ) ( BPGM ) found at high concentrations in red blood cells where it binds to and decreases the oxygen affinity of haemoglobin . To gain a more global picture of the impact of differential gene expression , the 391 genes represented by 439 probes that were concordant for differential gene expression ( Padj ≤0 . 05; ≥2-fold change ) between active cases and controls in experiments 1 and 2 were taken forward in Ingenuity Pathway ( IPA ) and gene-set enrichment ( Enrichr ) analyses . IPA network analysis indicated that 254 of these genes are joined in a single network ( Fig 3 ) , with IFNG as the major hub gene ( i . e . with most connections to other genes in the network ) , and other major hub genes including CCNA2 , CXCL10 , SPI1 , SNCA , CHEK1 , MCM2 , AURKB , RARA , CDK1 , CDC20 , and FOXM1 . The top Ingenuity Canonical Pathways for the 391 genes that achieved Padj <0 . 05 and ≥2-fold change ( Table 2 ) were Estrogen-mediated S-phase Entry ( P = 6 . 46x10-5; Padj = 0 . 019; z-score 2 ) , Mitotic Roles of Polo-Like Kinases ( P = 1 . 20x10-4; Padj = 0 . 019; z-score 1 . 89 ) , Aryl Hydrocarbon Receptor ( AHR ) Signalling ( P = 1 . 51x10-4; Padj = 0 . 019; z-score 1 . 89 ) , and Heme Biosynthesis II ( P = 3 . 72x10-4; Padj = 0 . 035 ) . Although not achieving Padj≤0 . 05 , identification of the Th2 pathway ( P = 1 . 22x10-3; Padj = 0 . 074; z-score -0 . 82 ) and Activation of Th1 and Th2 Pathway ( P = 1 . 32x10-3; Padj = 0 . 074 ) as nominally significant canonical pathways is consistent with prior knowledge of immune responses to leishmaniasis . Activation of AHR signalling ( z-score 1 . 89 ) as a top Ingenuity Canonical Pathway is reflective of increasing recognition of the role of AHR signalling in immunity , including the ability of AHR ligands to significantly induce cell secretion of IL-10 and inhibit IL-1β and IL-6 production in dendritic cells , and to promote IL-10 production and suppress IL-17 expression in CD4 ( + ) T cells [42–44] . It is also reflected in the identification of RARA , CCNA2 and CHEK1 genes from the AHR pathway ( Table 2 ) as major hub genes ( Fig 3 ) . AHR signalling was also identified as top in the Ingenuity “Top Tox List” pathway ( P = 4 . 16x10-4 ) indicative of its role as a toxic pathology endpoint that could be amenable to therapeutic intervention . Schematic representation of the core AHR canonical pathway overlaid with concordant gene expression data ( Padj<0 . 05 ) for experiment 1 ( Fig 4 ) for active cases relative to healthy controls shows differential gene expression that includes core players AHR and the AHR nuclear translocator ( ARNT ) in the AHR pathway , as well as for key phase I metabolising enzymes ( CYPB1 , ALDH5A1 , ALD3B1 and ALD3A2 ) . The full pathway , including cross-talk between AHR and other signalling pathways that lead to noncanonical mechanisms of action of AHR and its ligands , overlaid with expression data from experiments 1 ( S2 Fig ) and 2 ( S3 Fig ) , highlight a total of 28 concordant genes that all achieve differential gene expression at Padj<0 . 05 . These demonstrate the interplay between the top IPA-identified canonical pathways , with AHR function influencing cell proliferation and estrogen receptor signalling pathways , while heme derivatives biliverdin and bilirubin are known to act as endogenous ligands for AHR [45 , 46] . Identification of Mitotic Roles of Polo-Like Kinases as a top canonical pathway is indicative of cell proliferative activity that is consistent with CDC20 and CDK1 ( Table 2 ) as major hub genes in the network ( Fig 3 ) , and with identification of the cyclin-dependent kinase inhibitor CDKN1A as the top inhibited upstream regulator ( Activation z-score = -2 . 764; P = 5 . 4x10-26 ) in IPA . Using Enrichr ( S4 Table ) , signalling pathways involved in cell cycle predominated amongst the top pathways using the Reactome 2016 ( “Cell cycle_Homo sapiens” , “Cell Cycle , Mitotic_Homo sapiens” , and multiple other pathways involved in cell cycle ) , WikiPathways 2016 ( “Cell Cycle Homo sapiens” ) , KEGG 2016 ( “Cell cycle_Homo sapiens” ) , and NCI-Nature 2016 ( “Aurora B signalling” , “Aurora A signalling” and the “FOXM1 transcription factor network for Homo sapiens” , all of which play key roles in cell cycle progression ) databases . CDK1 was also identified as the top PPI Hub Protein using Enrichr ( S4 Table ) . Consistent with our top 10 “induced” gene list , other database comparisons using Enrichr ( S4 Table ) identified gene sets associated with erythrocyte function including “erythroid cell” ( Jensen Tissues Table ) , “abnormal erythrocyte morphology” and multiple other erythrocyte-related phenotypes ( MGI Mammalian Phenotype 2017 ) , “CD71+Early Erythroid” ( Human Gene Atlas ) , “congenital haemolytic anaemia” ( Jensen Diseases ) , and “Haemoglobin’s Chaperone pathway” ( BIOCARTA_2016 ) . Heatmaps were generated for individual expression levels for the top 10 concordant genes expressed at a higher level ( Fig 5A ) , and the top 10 concordant genes expressed at a lower level ( Fig 5B ) , in active cases compared to treated cases in experiment 1 . Heatmaps were also generated for the same “induced” and “repressed” probes/genes in experiment 2 ( Fig 5C and 5D ) . In this case , 6/10 and 7/10 top genes from experiment 1 were also in the top 10 most highly differentially expressed genes for “induced” and “repressed” gene sets in experiment 2 , respectively , and all achieved fold-change >2 in both experiments . Amongst the 10 most “repressed” genes in experiments 1 and 2 were 3 genes also observed in the comparison of active cases with healthy controls: peptidase inhibitor 3 ( PI3 ) , as noted above known as an antimicrobial peptide for bacteria and fungi; ALPL which encodes an alkaline phosphatase that plays a role in bone mineralization; and CACNA2D3 which encodes the alpha2delta3 subunit of the voltage-dependent calcium channel complex . Of additional interest in this comparison were “repressed” genes: CHI3L1 which encodes a chitinase-like protein that lacks chitinase activity but is secreted by activated macrophages and neutrophils; EMR3 ( ADGRE3 ) encoding an adhesion G protein-coupled receptor expressed predominantly in cells of the immune system and playing a role in myeloid-myeloid interactions during inflammation; and MMP25 that encodes matrix metallopeptidase 25 which inactivates alpha-1 proteinase inhibitor produced by activated neutrophils during inflammation thereby facilitating transendothelial migration of neutrophils to inflammatory sites . Of interest amongst the top 10 “induced” genes in both experiments were: CXCL10 encoding a chemokine of the CXC subfamily that is a ligand for CXCR3 , binding to which results in stimulation of monocyte , natural killer and T-cell migration; IFNG encoding interferon-γ , well known for its role in macrophage activation for anti-leishmanial activity; and GBP1 that encodes a guanylate binding protein induced by interferon . As there were only 42 concordant genes that achieved ≥2-fold change in expression , a more global picture of the impact of differential gene expression was obtained by performing IPA and Enrichr analyses using the full set of 210 genes represented by 221 probes that were concordant for differential gene expression at Padj ≤0 . 05 . IPA network analysis indicated that 85 of these genes are joined in a single network ( Fig 6 ) , with IFNG as the major hub gene ( i . e . with most connections to other genes in the network ) , and other major hub genes including STAT1 , SPI1 , RARA , NOTCH1 and MAPK3 . The top canonical pathways included pathogenesis of multiple sclerosis ( Table 2 ) , consistent with interconnections between CXCL10/CXCL9/CXCL11 and major hub genes IFNG and STAT1 ( Fig 6 ) , and the Notch signalling pathway . Aryl hydrocarbon receptor signalling was also identified as a canonical pathway in this analysis at a nominal P = 0 . 003 ( Table 2 ) . Enrichment for chemokine signalling and Notch signalling pathways were also supported by analyses undertaken using Enrichr ( Reactome 2016; WikiPathways 2016 , and KEGG 2016 pathways; S5 Table ) . Consistent with this were top LINCS_L1000_Ligand_Perturbations_Up ( S5 Table ) for which perturbations of TNFA , IFNG , IL1 , IFNA , and HGF were all significant at Padj<0 . 01 . These ligand perturbations were all associated with differential expression at CXCL10 , and commonly also at CXCL11 , CXCL9 , and STAT1 . The major cell types associated with the treatment response were CD14+ monocytes and CD33+ myeloid cell populations ( Human Gene Atlas; S5 Table ) . As noted above , we found more differentially expressed probes between treated cases and controls , along with fewer differentially expressed probes between active and treated cases , in experiment 1 compared to experiment 2 ( Table 1 ) . We hypothesize that this is due to more effective treatment using liposome encapsulated amphotericin B in experiment 2 compared to the non-liposomal form of the drug employed during experiment 1 . We therefore examined the genes that were discordant between active cases and treated cases across the two experiments to understand differences in the cure response . In support of the more efficient cure rate in experiment 2 , 7/10 of the top “repressed” genes ( namely: OLIG1 , OLIG2 , PTGDR2 alias GPR44 , CCR3 , CCL23 , ALOX15 , SLC29A1 ) identified as differentially expressed between active cases and treated cases in experiment 2 but not experiment 1 were the same genes that were most repressed in the concordant genes comparing active cases with healthy controls . In comparison , 0/10 of the top “repressed” genes identified as differentially expressed between active cases and treated cases in experiment 1 ( but not experiment 2 ) matched the comparison of concordant genes for active cases and healthy controls . That is , treated cases in experiment 2 were behaving more like healthy controls than were treated cases in experiment 1 . To gain a more global picture of differential gene expression that might inform mechanistic differences in cure rates between the two therapeutic regimes , the 417 genes ( from 443 probes ) that were differentially expressed between active cases and treated cases in experiment 1 but not experiment 2 , and the 988 genes ( from 1096 probes ) that were differentially expressed between active cases and treated cases in experiment 2 but not experiment 1 , were analysed in Enrichr for gene-set enrichment . S6 and S7 Tables present details of the pathways and gene sets that contrast molecular events that characterise the two different treatment groups . These are summarised in Table 3 . For the 988 genes that were differentially expressed between active cases and treated cases in experiment 2 but not in experiment 1 ( S6 Table ) signalling pathways involved in cell cycle predominated amongst the top pathways using the Reactome 2016 ( “Cell cycle_Homo sapiens” ) , Wiki Pathways 2016 ( “Cell Cycle Homo sapiens” ) , KEGG 2016 ( “Cell cycle_Homo sapiens” ) , and NCI-Nature 2016 ( “Aurora B signalling” ) databases . In every case there were multiple other pathways involved in cell cycle that achieved rank z-scores <-1 and Padj ≤0 . 01 . This pattern recapitulates the results obtained in the earlier comparison of concordant genes for active cases and healthy controls ( S4 Table ) , with CDK1 again identified as the top PPI Hub Protein for this gene set ( S7 Table ) . Consistent with an enhanced rate of cure , multiple immune response signalling pathways ( Table 3 and S6 Table ) were also identified in this gene set , including IL-1 , IL-3 , IL-4 , IL-6 , IL-7 and IL-8 signalling pathways ( Reactome 2016 , Wiki Pathways 2016 , and NCI Nature 2016 databases ) , Delta-Notch signalling ( Wiki Pathways database ) , chemokine signalling ( Wiki Pathways 2016 and KEGG 2016 databases ) including specifically IL-8/CXCR2-mediated and CXCR4-mediated signalling ( NCI Nature 2016 database ) , and Fc gamma R-mediated phagocytosis Homo sapiens ( KEGG 2016 database ) . Of note , IL-4 was identified as the most significantly down-regulated perturbed ligand pathway ( LINCS_L1000_Perturbed_Down; rank z-score -1 . 8 , Padj 6 . 36x10-11 ) in this set of genes differentially expressed in active versus treated cases in experiment 2 but not experiment 1 . None of these databases showed significant gene set enrichment when interrogated with the 417 genes identified as differentially expressed between active cases and treated controls in experiment 1 but not in experiment 2 , i . e . they are not present in Table 3 or S7 Table which compare other enriched gene sets showing differences of interest between experiments 1 and 2 . For example , all PPI Hub Proteins identified as significant for the 988 genes that were differentially expressed between active cases and treated cases in experiment 2 but not experiment 1 were related to cell cycle ( S7 Table ) . In contrast , the 5 significant matches to gene sets for PPI Hub Proteins for the 417 genes differentially expressed between active cases and treated cases in experiment 1 but not experiment 2 included the inhibitor of NFκB NFKBIA and the SMAD-signalling pathway gene SMAD9 which transduces signals from members of the TGFβ family . Mutations in NFKBIA are associated with T-cell immunodeficiency [47] . SMAD9 ( aliases SMAD8 , SMAD8A , SMAD8B , SMAD8/9 ) transduces signals following ligation of TGFβ family members known as bone morphogenesis proteins ( BMPs ) to specific BMP ( TGFβ family ) receptors . Enrichr identified enrichment for a gene set matching genes differentially expressed in BMP4-treated cells ( SILAC-Phosphoproteomic Database; P = 5 . 4x10-5 , Padj = 0 . 003 , rank z-score = -1 . 74 ) from the 417 but not the 988 genes ( S7 Table ) . Another difference was enrichment of the “CD71+Early Erythroid” ( Human Gene Atlas ) gene set in the 417 genes , while the 988 genes were enriched for gene sets ( S7 Table Human Gene Atlas database ) associated with B lymphoblasts , CD105+ endothelial , CD33+ myeloid , and CD14+ monocytes but not erythroid cells . Overall these analyses of discordant gene sets between experiments 1 and 2 are consistent with our hypothesis that patients treated with a single dose of liposomal amphotericin B ( experiment 2 ) were at a more advanced stage of cure at day 30 post treatment than patients treated with multi-dose non-liposomal amphotericin B ( experiment 1 ) .
In this study we have analysed whole blood transcriptomic data to further understand the pathogenesis of VL . One original goal of the study was to identify transcriptomic signatures that might differentiate asymptomatic infections from uninfected controls . In our attempt to achieve this we compared both modified quantiferon positive asymptomatic individuals and high antibody positive asymptomatic individuals with healthy endemic controls who were negative for these assays . In the event , we did not find signatures that would be diagnostic for either of these asymptomatic groups compared to negative controls . This was despite longitudinal epidemiological evidence from our study area showing that high antibody individuals are the group at most risk of progressing to clinical VL [6] . However , in that study we observed that high antibody individuals progressed to clinical VL within one year . In our study we selected individuals who had sustained high DAT titres for more than two annual surveys . Hence , we were effectively selecting for a subset of asymptomatic individuals who were resistant to progression to clinical disease . Similarly , there was no significant difference in the odds of progression to clinical disease in individuals who were positive by the modified quantiferon assay [6] , and we found no evidence for a whole blood transcriptional signature to distinguish these individuals from uninfected endemic healthy controls . Our results therefore mirror those of Gardinassi and coworkers [27] who likewise found no significant differences in whole blood transcriptional signatures between asymptomatic individuals infected with L . infantum in Brazil , as determined by positive delayed type hypersensitivity to leishmanial skin-test antigen , and uninfected endemic controls . A more detailed longitudinal study will be required to detect transcriptional signatures early after exposure to L . donovani or L . infantum to identify signatures that may be predictive of progression to disease in asymptomatic individuals positive for antibody or cellular immunity to leishmanial antigens . In India it may be particularly interesting to identify signatures for those high titre DAT antibody individuals who progress to disease within 9 months from those who do not . Our failure to identify signatures to detect asymptomatic infection meant that our attention focussed on understanding disease pathogenesis by comparing whole blood transcriptomes from active cases with all healthy controls , and in examining differences in the transcriptome following different regimens of drug treatment . In these comparisons 6 major themes emerged: ( i ) expression of genes and enrichment of gene sets associated with erythrocyte function in active cases; ( ii ) strong evidence for enrichment of gene sets involved in cell cycle in comparing active cases with healthy controls ( or with more effective cure in experiment 2 ) ; ( iii ) identification of IFNG encoding interferon-γ as the major hub gene in concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; ( iv ) enrichment for interleukin signalling ( IL-1/3/4/6/7/8 ) and a prominent role for CXCL10/9/11 and chemokine signalling pathways in the comparison of active cases with treated cases; ( v ) the novel identification of AHR signalling as a significant IPA canonical pathway identified from concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; and ( vi ) global expression profiling support for more effective cure at day 30 post-treatment with a single dose of liposomal encapsulated amphotericin B compared to multi-dose treatment over 30 days . Interesting in our analysis of top differentially expressed genes and enriched gene sets/pathways between active cases and healthy controls was the predominance of gene sets associated with erythroid cells and function . A recent systematic review [48] found that anaemia has an overall prevalence higher than 90% in VL . Pathogenesis of anaemia based on clinical observations included the presence of anti-erythrocyte antibodies , dysfunction in erythropoiesis , and hemophagocytosis in spleen or bone marrow . Of these , the authors of this review conclude that hemophagocytosis is the most likely cause [48] . The results of our study indicate differential regulation of gene sets associated with abnormal erythrocyte morphology , erythropoiesis , erythrocyte physiology , erythrocyte osmotic lysis , along with decreased haematocrit , spherocytosis and reticulocytosis . The gene sets defining these erythrocyte phenotypes therefore suggest mechanisms other than just hemophagocytosis and could provide important signatures to monitor clinical cure . This is especially relevant given our observation that erythroid related genes were present amongst the discordant genes that were differentially expressed between active cases and cases treated with multi-dose amphotericin B ( experiment 1 ) in which the degree of clinical cure was not as progressed for the same period of treatment with a single dose of liposomal amphotericin B ( experiment 2 ) . Many of the individual cell-cycle and immune-related ( e . g . Notch signalling , interleukin and chemokine signalling ) signalling pathways that were perturbed in active cases relative to cured cases or healthy controls were also observed in the similar study of whole blood expression profiling carried out by Gardinassi and coworkers [27] in relation to VL caused by L . infantum in Brazil . However , a common feature of both the comparison of active cases with healthy controls , and of active cases with treated cases , in our study was the identification of IFNG encoding interferon-γ as the major hub gene . This was not itself surprising since interferon-γ plays a key role in activating macrophages to kill L . donovani parasites [49] . Studies across the leishmaniases have generally supported the notion that type 1 immune responses and the production of interferon-γ are vital for macrophage activation and parasite elimination [50–52] . It was interesting in our study that transcript levels for IFNG were higher in active cases than treated cases , where enhanced interferon-γ responses might have been expected to accompany drug cure . Nonetheless , it concurs with our observations that CD4+ T cells in whole blood from active VL patients and treated patients secrete high levels of interferon-γ following stimulation with crude Leishmania antigen [53 , 54] , the difference being that only active VL cases secreted IL-10 concurrently with interferon-γ [54] . The higher transcript abundance for IFNG in active compared to treated cases in our study suggests return to baseline with treatment in the latter . Gardinassi and coworkers similarly found higher transcript levels for IFNG in active compared to treated cases [27] . Accompanying the central role of IFNG as a hub gene when comparing active cases with treated cases was evidence for perturbation of multiple cytokines , including IFNG , IFNA , IL-1 , IL-6 , and TNF , all of which were supported by differentially expressed gene signatures that generally included CXCL10/11/9 and STAT1 . This CXCL10/11/9 chemokine gene expression signature also accounted for the identification of “pathogenesis of multiple sclerosis” [55] as the top disease-related canonical pathway identified using IPA , consistent with a proinflammatory response contributing to disease pathology in active VL . “Pathogenesis of multiple sclerosis” was also identified as a top canonical pathway in spleen tissue and splenic macrophages from L . donovani infected hamsters [26] , a study in which the authors also noted high interferon-γ expression that was ineffective in directing macrophage activation and parasite killing . STAT1 is a transcription factor activated by ligation of interferon-γ receptors . CXCL10/11/9 are all induced by interferon-γ , all bind to CXCR3 , and between them have multiple roles as chemoattractants for monocytes and macrophages , T cells , NK cells , and dendritic cells , and in promoting T cell adhesion . CXCL10 and CXCL9 were also identified as the most highly “induced” genes in comparing lesion transcript profiles with normal skin of patients with American cutaneous leishmaniasis , consistent with their roles in inflammatory cell recruitment [28] . Cxcl9 , Gbp1 ( encoding the interferon-γ-induced guanylate binding protein GBP1 identified here as one of the top 10 induced genes when comparing active versus treated cases ) , and Ifng were also identified as part of a common signature of 26 genes upregulated in blood , spleen and liver throughout the course of experimental infection with L . donovani in susceptible BALB/c mice , with Cxcl9 and Gbp1 reported as hub genes from a STRING analysis [24] . Given the many studies that have identified the importance of regulatory IL-10 in VL pathogenesis [54 , 56–59] , it was of some interest in our study that IL10 was not identified as a top differentially expressed gene or as a significantly enriched signalling pathway in either comparison of active cases with healthy controls , or of active cases with treated cases . Nor did we observed perturbation of IL10R as has been reported in experimental transcriptional profiling studies of VL [24] . Indeed , downregulated expression of the type 2 cytokine gene IL4 was the strongest response associated with effective cure in liposome-encapsulated amphotericin B treated cases , in line with previous studies showing that IL-4 levels were two-fold higher in VL patients who had failed treatment compared to previously untreated patients , whereas IL-10 levels were comparable in both [58] . One novel observation of our study was identification of AHR signalling as the top canonical pathway when comparing transcriptomes between active cases and healthy controls or treated cases . Through crosstalk between signalling pathways , AHR ligands have been shown to significantly induce IL-10 secretion and inhibit IL-1β and IL-6 production in dendritic cells , and to promote IL-10 production and suppress IL-17 expression in CD4+ T cells [42–44] . IL-17 is a potent activator of neutrophils , both through lineage expansion and through their recruitment by regulating chemokine expression . While IL-17 perturbation was not identified in our whole blood transcriptional profiles associated with human VL , evidence from murine models [60] demonstrate a strong role for IL-17 and neutrophils in parasite clearance from liver and spleen . Duthie and coworkers [59] have shown that both IL-10 and IL-17 cytokines are elevated in the serum of active VL patients , reverting to baseline levels with standard antimonial treatments . AHR activation has also been shown to inhibit inflammation through upregulation of IL-22 [61] , another cytokine that has been shown to be significantly higher in Leishmania antigen stimulated peripheral blood mononuclear cells from active VL cases compared to treated cases [62] . AHR activation during VL may underpin the complex regulation of pro- and anti-inflammatory responses during disease pathogenesis and during response to therapy . Of potential translational importance in our study was the additional identification of AHR signalling pathway at the top of the Ingenuity “Top Tox List” indicative of its role as a toxic pathology endpoint that could be amenable to therapeutic intervention . AHR locates to the cytoplasm in a stable complex that includes HSP90 observed as a differentially regulated gene in our comparison of active cases with healthy controls . Ligand binding occurs in the cytoplasm and triggers AHR translocation to the nucleus where it binds with ARNT to act as a transcription factor . Both AHR and ARNT were differentially expressed between active VL cases and controls in our study . The AHR response was first associated with xenobiotic induction of metabolizing enzymes , such as the induction of cytochrome P450 , family 1 , subfamily A , polypeptide 1 ( Cyp1a1 ) following exposure to the polychlorinated dibenzo-p-dioxin 2 , 3 , 7 , 8-Tetrachlorodibenzo-p-dioxin [63] . Multiple AHR ligands are known to induce a “gene battery” of metabolizing enzymes involved in oxidative stress response , cell cycle and apoptosis [64] , amongst which are CYP1B1 , ALD3B1 , ALD3B and ALDH5A1 that were differentially expressed between active VL cases and healthy controls . Transcriptomic profiling of M . tuberculosis infected macrophages uncovered evidence for the generation of endogenous AHR ligands through induction of enzymes controlling tryptophan catabolism [65] . The generation of endogenous AHR ligands may likewise explain the role of AHR signalling in VL . For example , heme derivatives biliverdin and bilirubin have both been shown to act as endogenous ligands for AHR , as have arachidonic acid metabolites such as prostaglandins and leukotrienes [45 , 46] . The former would be consistent with the strong perturbation of erythrocyte function between active VL cases and controls observed in our study . Importantly , addition of exogenous AHR ligands enhanced M . tuberculosis infection associated AHR transactivation to stimulate expression of AHR target genes , including IL-1β and IL-23 which stimulate T cell subsets to produce IL-22 . This suggests that administration of exogenous ligands could be used as a therapeutic intervention , especially in the knowledge that different exogenous AHR ligands can modulate either regulatory T cell or inflammatory T helper 17 cell differentiation in a ligand-specific fashion to suppress or exacerbate autoimmune disease [66] . One of the potential benefits of gene expression profiling is the identification of gene signatures that could be used in the diagnosis of disease and in the monitoring of treatment efficacy . In this respect it is remarkable that 9 of the top 10 DEGs found to be more highly expressed in active cases compared to healthy controls in our study ( i . e . all except DARC ) were also found to be more highly expressed in active cases compared to healthy controls in the Brazilian whole blood expression profiling study of L . infantum [27] . Similarly , 5 ( PI3 , CCR3 , OLIG1 , CACNA2D3 , ALPL ) of the top 10 DEGs found to be reduced in expression in active cases relative to healthy controls were also found to be reduced in expression in active L . infantum cases . More extensive cross-matching of the gene lists from the two studies identifies larger sets of concordant genes to be used as signatures for VL disease that cross the divides of geography and species and could be tested in other regions endemic for VL disease . In relation to treatment monitoring , 6 ( CXCL10 , ANKRD22 , MT1G , IFNG , GBP1 , SEPT4 ) of the top 10 genes retaining higher expression in cases relative to treated cases were also concordant across the two studies . While there was no concordance for the top 10 genes expressed at reduced levels in active cases compared to treated cases , this could reflect the effect of different treatment protocols ( pentavalent antimony in Brazil versus two forms of Amphotericin B in India ) . As we observed in our comparison of different Amphotericin B treatment strategies , the rate of return to control levels of gene expression differs across treatments . Nevertheless , in our study we observed some concordance ( PI3 , CACNA2D3 , ALPL ) between the genes that were more highly expressed in all treated cases and healthy controls relative to active cases . A signature that combines gene markers of active disease with genes that represent return to healthy baseline would be valuable in the monitoring of treatment efficacy . Overall , our study has made some novel observations in relation to gene signatures that accompany both active VL disease and clinical cure in treated cases that could provide translatable targets for the development of novel or drug repurposed therapeutic interventions . Furthermore , by studying in more detail the discordant gene patterns that accompanied treatment with single dose liposome encapsulated amphotericin B versus multi-dose non-liposomal amphotericin B we were able to define gene signatures that could be used to monitor progress towards clinical cure . | Visceral leishmaniasis ( VL ) , also known as kala-azar , is a potentially fatal disease caused by intracellular parasites of the Leishmania donovani complex . VL is a serious public health problem in rural India , causing high morbidity and mortality , as well as major costs to local and national health budgets . Amphotericin B provides improved therapy for VL with single dose liposomal-encapsulated Ambisome , now affordable through WHO-negotiated price reductions , providing the best cure rates . The VL elimination program aims to reduce the incidence rate in the Indian subcontinent to <1/10 , 000 population/year . By assessing immune responses to parasites in people infected with L . donovani , but with different clinical status , we can determine the requirements for immune cell development and predict which asymptomatic individuals , for example healthy individuals with high anti-leishmanial antibody levels , will progress to clinical VL . This will help in monitoring disease outbreaks . In this study we looked at global gene expression patterns in whole blood to try to understand more about asymptomatic infection , active VL , and the progress to cure in cases treated with single or multi-dose amphotericin B . The signatures of gene expression identified aid in our understanding of disease pathogenesis and provide novel targets for therapeutic intervention in the future . | [
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] | 2019 | Transcriptional blood signatures for active and amphotericin B treated visceral leishmaniasis in India |
Integrating and conjugative elements ( ICEs ) are one of the three principal types of self-transmissible mobile genetic elements in bacteria . ICEs , like plasmids , transfer via conjugation; but unlike plasmids and similar to many phages , these elements integrate into and replicate along with the host chromosome . Members of the SXT/R391 family of ICEs have been isolated from several species of gram-negative bacteria , including Vibrio cholerae , the cause of cholera , where they have been important vectors for disseminating genes conferring resistance to antibiotics . Here we developed a plasmid-based system to capture and isolate SXT/R391 ICEs for sequencing . Comparative analyses of the genomes of 13 SXT/R391 ICEs derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes that serve as a scaffold capable of mobilizing an array of variable DNA . Furthermore , selection pressure to maintain ICE mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions . The variable genes confer diverse element-specific phenotypes , such as resistance to antibiotics . Functional analysis of a set of deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of most of the dispensable core genes are unknown . Several lines of evidence suggest that there has been extensive recombination between SXT/R391 ICEs , resulting in re-assortment of their respective variable gene content . Furthermore , our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of mobile genetic elements .
There are three types of self-transmissible mobile genetic elements: plasmids , bacteriophages and integrative conjugative elements ( ICEs ) . All three classes of elements enable horizontal transmission of genetic information and all have had major impacts on bacterial evolution [1]–[4] . ICEs , ( aka conjugation transposons ) , like plasmids , are transmitted via conjugation; however , unlike plasmids , ICEs integrate into and replicate along with the chromosome . Following integration , ICEs can excise from the chromosome and form circular molecules that are intermediates in ICE transfer . Plasmids and phages have been the subject of more extensive study than ICEs and while there is growing understanding of the molecular aspects of several ICEs [5]–[10] , to date there have been few reports of comparative ICE genomics [11] , [12] and consequently understanding of ICE evolution is only beginning to be unraveled . Diverse ICEs have been identified in a variety of gram-positive and gram–negative organisms [13] . These elements utilize a variety of genes to mediate the core ICE functions of chromosome integration , excision and conjugation . In addition to a core gene set , ICEs routinely contain genes that confer specific phenotypes upon their hosts , such as resistance to antibiotics and heavy metals [14]–[18] , aromatic compound degradation [19] or nitrogen fixation [20] . SXT is an ∼100 Kb ICE that was originally discovered in Vibrio cholerae O139 [16] , the first non-O1 serogroup to cause epidemic cholera [21] . SXT encodes resistances to several antibiotics , including sulfamethoxazole and trimethoprim ( which together are often abbreviated as SXT ) that had previously been useful in the treatment of cholera . Since the emergence of V . cholerae O139 on the Indian subcontinent in 1992 , SXT or a similar ICE has been found in most clinical isolates of V . cholerae , including V . cholerae serogroup O1 , from both Asia and Africa . Other vibrio species besides V . cholerae have also been found to harbor SXT-related ICEs [22] . Furthermore , SXT-like ICEs are not restricted to vibrio species , as such ICEs have been detected in Photobacterium damselae , Shewanella putrefaciens and Providencia alcalifaciens [23]–[25] . Moreover , Hochhut et al [26] found that SXT is genetically and functionally related to the so-called ‘Inc J’ element R391 , which was derived from a South African Providencia rettgeri strain isolated in 1967 [27] . It is now clear that Inc J elements are SXT-related ICEs that were originally misclassified as plasmids . In the laboratory , SXT has a fairly broad host range and can be transmitted between a variety of gram-negative organisms [16] . The SXT/R391 family of ICEs is now known to include more than 30 elements that have been detected in clinical and environmental isolates of several species of γ- proteobacteria from disparate locations around the globe [28] . SXT/R391 ICEs are grouped together as an ICE family because they all encode a nearly identical integrase , Int . Int , a tyrosine recombinase , is considered a defining feature of these elements because it enables their site-specific integration into the 5′ end of prfC , a conserved chromosomal gene that encodes peptide chain release factor 3 [29] . Int mediates recombination between nearly identical element and chromosome sequences , attP and attB respectively [29] . When an SXT/R391 ICE excises from the chromosome , Int , aided by Xis , a recombination directionality factor , mediates the reverse reaction - recombination between the extreme right and left ends ( attR and attL ) of the integrated element - thereby reconstituting attP and attB [6] , [29] . The excised circular SXT form is thought to be the principal substrate for its conjugative transfer . The genes that encode activities required for SXT transfer ( tra genes ) were originally found to be distantly related to certain plasmid tra genes [30]–[32] . The tra genes encode proteins important for processing DNA for transfer , mating pair formation and generating the conjugation machinery . Regulation of SXT excision and transfer is at least in part governed by a pathway that resembles the pathway governing the lytic development of the phage lambda . Agents that damage DNA and induce the bacterial SOS response are thought to stimulate the cleavage and inactivation of SetR , an SXT encoded λ cI-related repressor , which represses expression of setD and setC , transcription activators that promote expression of int and tra genes [5] . The complete nucleotide sequences of SXT ( 99 . 5kb ) and R391 ( 89kb ) were the first SXT/R391 ICE family genomes to be reported [14] , [32] . Comparative [33] and functional genomic analyses [5] , [32] revealed that these 2 ICEs share a set of conserved core genes that mediate their integration/excision ( int and xis ) , conjugative transfer ( various tra genes ) , and regulation ( setR , setCD ) . In addition to the conserved genes , these 2 ICEs contain element specific genes that confer element specific properties such as resistance to antibiotics or heavy metals . Interestingly , many of these genes were found in identical locations in SXT and R391 , leading Beaber et al [33] to propose that there are ‘hotspots’ where SXT/R391 ICEs can acquire new DNA . The genomes of two additional SXT/R391 ICEs , ICEPdaSpa1 , isolated from Photobacterium damselae [23] , and ICESpuPO1 , derived from an environmental isolate of Shewanella putrefaciens [24] are now also known . These two genomes also share most of the conserved set of core genes present in SXT and R391 and contain element specific DNA . Determination of the sequences of SXT/R391 family ICE genomes was a fairly arduous task due to their size and predominantly chromosomal localization . Here , we developed a method to capture and then sequence complete SXT/R391 ICE genomes . In addition , we identified 3 as yet unannotated SXT/R391 ICE genomes in the database of completed bacterial genomes . Comparative analyses of the 13 SXT/R391 genomes now available allowed us to greatly refine our understanding of the organization and conservation of the core genes that are present in all members of this ICE family . Comparative and functional analyses also facilitated our proposal of the minimal functional SXT/R391 ICE genome . Furthermore , this work provides new knowledge of the considerable diversity of genes and potential accessory functions encoded by the variable DNA found in these mobile elements . Finally , this comparative genomics approach has allowed us to garner clues regarding the evolution of this class of mobile elements .
To date , ICE sequencing has been cumbersome because it has typically required construction of chromosome-derived cosmid libraries and screening for sequences that hybridize to ICE probes [23] , [32] . We constructed a vector ( pIceCap ) that enables capture of complete SXT/R391 ICE genomes on a low-copy plasmid to simplify the protocol for ICE sequencing . This plasmid is a derivative of the single-copy modified F plasmid pXX704 [34] , [35] , which contains a minimal set of genes for F replication and segregation but lacks genes enabling conjugation . We modified pXX704 to include an ∼400bp fragment that encompasses the SXT/R391 attachment site ( attB ) and thereby enabled Int-catalyzed site-specific recombination between attB on pIceCap and attP on an excised and transferred ICE to drive ICE capture ( Figure 1 ) . Conjugations between an SXT/R391 ICE-bearing donor strain and an E . coli recipient deleted for prfC ( and thus chromosomal attB ) and harboring pIceCap yielded exconjugants containing the transferred ICE integrated into pIceCap ( Figure 1 ) . We used the ΔprfC recipient to bias integration of the transferred ICE into pIceCap rather than the chromosome . In these experiments , we selected for exconjugants containing the transferred ICE integrated into pIceCap , using an antibiotic marker present on the ICE as well as a marker present in pIceCap . The low copy IceCap::ICE plasmid was then isolated and used as a substrate for shotgun sequencing . We also found that the IceCap::ICE plasmids were transmissible . Thus , in principle this technique should facilitate capture of ICEs that do not harbor genes conferring resistance to antibiotics , by mating out the IceCap::ICE plasmid into a new recipient and selecting for the marker on pIceCap . A list of the 13 SXT/R391 ICEs whose genomes were analyzed and compared in this study is shown in Table 1 . All of the ICEs included in our analyses contain an int gene that was amplifiable using PCR primers for intsxt [29] . They were isolated on 4 continents and from the Pacific Ocean during a span of more than 4 decades . They are derived from 7 different genera of γ-proteobacteria and the ICEs derived from V . cholerae strains are from both clinical and environmental isolates of 3 different V . cholerae serogroups . Five of these ICE genome sequences were determined at the J . Craig Venter Institute ( JCVI ) using the ICE capture system described above ( Table 1 , rows 1–5 ) . In addition , we sequenced ICEVflInd1 , also at the JCVI , by isolating cosmids that encompassed this V . fluvialis derived ICE prior to developing the ICE capture technique ( Table 1 , row 6 ) . Table 1 ( rows 7–10 ) also includes 4 previously unannotated ICE genomes that we found in BLAST searches of the NCBI database of completed but as yet unannotated genomes; 3 of these ICEs are clearly members of SXT/R391 ICE family since they are integrated into their respective host's prfC locus and contain int genes that are predicted to encode Int proteins that are 99% identical to Intsxt . The fourth element , ICEVchBan8 does not encode an Intsxt orthologue; however , this element contains nearly identical homologues of most of the known conserved core SXT/R391 ICE family genes . ICEVchBan8 will be discussed in more detail below but since it does not contain an Intsxt orthologue it is not considered a member of the SXT/R391 family of ICEs and thus not included in our comparative study . Finally , Table 1 also includes the 4 SXT/R391 ICEs that were previously sequenced ( Table 1 , rows 11–14 ) . Despite the diversity of our sources for SXT/R391 ICEs , the genomes of two pairs of ICEs that we analyzed proved to be very similar . SXTMO10 and ICEVchInd4 only differed by 13 SNPs in 7 genes and by the absence from ICEVchInd4 of dfr18 , a gene conferring trimethoprim resistance . These ICEs were derived from V . cholerae O139 strains isolated in India from different cities at different times: SXTMO10 from Chennai in 1992 and ICEVchInd4 from Kolkata in 1997 . The high degree of similarity of these two ICE genomes suggests that ICEs can be fairly stable over time . ICEVchBan9 and ICEVchMoz10 were also extremely similar although ICEVchMoz10 lacks dfrA1 , another allele for trimethoprim resistance . These two ICEs were derived from V . cholerae O1 strains from Bangladesh ( 1994 ) and Mozambique ( 2004 ) respectively . The great similarity of these ICEs suggests that there has been spread of SXT-related ICEs between Asia and Africa in recent times . Studies of CTX prophage genomes have also suggested the spread of V . cholerae strains between these continents [36] . The ICEs listed in Table 1 were initially compared using MAUVE [37] and LAGAN [38] , programs that enable visualization of conserved and variable regions on a global scale . All of the SXT/R391 ICEs we analyzed share a common structure and have sizes ranging from 79 , 733 bp to 108 , 623 bp ( Table 1 and Figure 2 ) . They contain syntenous sets of 52 conserved core genes ( Figure 2A ) that total approximately 47kb and encode proteins with an average of 97% identity to those encoded by SXT . All of the individual ICEs also contain DNA that is relatively specific for individual elements ( Figure 2B ) ; the differences in the sizes of the variable regions accounts for the range in ICE sizes . Five sites within the conserved SXT/R391 ICE structure have variable DNA present in all of the ICEs in Figure 2 . Four of these sites were previously termed ‘hotspots’ for ICE acquisition of new DNA [33] . Due to similarities between SXT and R391 , the fifth hotspot only became apparent through our comparison of the 13 ICEs examined here . Each of these hotspots ( HS1 to HS5 in Figure 2B ) is found in an intergenic region ( see below ) , suggesting that the acquisition of these variable DNA regions has not interrupted core ICE gene functions . In addition , some of the ICEs have variable DNA inserted in additional intergenic locations or in rumB ( labeled I–IV in Figure 2B ) . Previous analyses [32] indicated that the insertion in rumB , did not impair SXT transmissibility . Overall , comparison of these 13 SXT/R391 ICE genomes suggests that: 1 ) these elements consist of the same perfectly syntenous and nearly identical 52 core genes that serve as a scaffold ( see below ) capable of mobilizing a large range of variable DNA; and 2 ) selection pressure to maintain ICE mobility has restricted insertions of variable DNA into sites that do not interrupt core functions . The 52 core genes present in all the SXT/R391 ICEs analyzed include sets of genes that are known to be required for the key ICE functions of integration/excision , conjugative transfer and regulation [32] as well as many genes of unknown function . Most genes of known or putative ( based on homology ) function ( coded by gray shading or hatch marks in Figure 2A ) are clustered with genes that have related functions . For example , int and xis , genes required for integration and excision , are adjacent and setR , and setC/D , the key SXT regulators are near each other at the extreme 3′ end of the elements , although separated by 4 conserved genes of unknown function . Each ICE also has four gene clusters implicated in conjugative DNA processing and transfer ( shown in light gray in Figure 2A ) . Finally , each of the ICEs has a nearly identical origin of transfer ( oriT ) , a cis-acting DNA site that is thought to be nicked to initiate DNA processing events during conjugative transfer [39] , in the same relative location . The conserved core genes include approximately as many genes of unknown function as genes of known function . Some of the genes of unknown function are found either interspersed amongst gene clusters that likely comprise functional modules ( e . g s091 between traD and s043 ) while others are grouped together ( e . g . most genes between traN and traF ) . In several cases , the interspersed genes appear to be part of operons with genes of known function ( e . g . s086-s082 maybe in an operon with setDC ) . In addition to sharing 52 core genes , all of the ICE genomes analyzed contain variable DNA regions , ranging in size from 676 to 29 , 210 bp . Most of the variable DNA sequences are found in 5 intergenic hotspots ( Figure 2B ) . However , some ICEs contain additional variable DNA inserts outside the 5 hotspots . For example , SXT and five other ICEs in Figure 2 have variable DNA segments , corresponding to related ISCR2 elements , disrupting rumB ( Figure 2B , site III ) . ISCR2 elements are IS91-like transposable elements that tend to accumulate antibiotic resistance genes [40] . Interestingly , it is unusual for the contents of the hotspots and other variable regions to be found in only one ICE . Instead , the variable gene content of most of the ICEs shown in Figure 2B is found in more than one ICE . For example , ICESpuPO1 , ICEPalBan1 , and ICEVflInd1 , all have identical contents in hotspot 5 ( lavender genes in hotspot 5 in Figure 2B ) ; however , the contents of the other hotspots in these 3 elements are almost entirely different . Thus , the variable gene content of the SXT/R391 ICEs reveals that these elements are mosaics . The overlapping distribution of variable DNA segments seen in the ICEs in Figure 2B suggests that recombination among this family of mobile elements may be extensive . In addition , in some instances , the variable regions appear subject to additional genetic modifications . For example , ICEPdaSpa1 and ICEVchBan9 contain ICE-specific DNA nested within the shared sequences inserted at hotspot 5 DNA ( the green and pink genes in hotspot 5 in these elements , Figure 2B ) . The variable genes encode a large array of functions and only a few will be discussed here . A complete list of the diverse genes found in the hotspots is found in Table S1 . Although we cannot predict functions for many genes found in the hotspots , since they lack homology to genes of known function , at least a subset of the known genes seem likely to confer an adaptive advantage upon their hosts . Most of the ICE antibiotic resistance genes are found within transposon-like structures ( e . g . , the ISCR2 elements noted above ) but four ICEs contain a dfrA1 cassette , which confers resistance to trimethoprim [25] , in a class IV integron located in hotspot 3 . A disproportionate number of variable genes are likely involved in DNA modification , recombination or repair , as they are predicted to encode diverse putative restriction-modification systems , helicases and endonucleases . Such genes may provide the host with barriers to invasion by foreign DNA including phage infection and/or promote the integrity of the ICE genome during its transfer between hosts . Three ICEs contain genes that encode diguanylate cyclases [41] in hotspot 3 . These enzymes catalyze the formation of cyclic-diguanosine monophosphate ( c-di-GMP ) , a second messenger molecule that regulates biofilm formation , motility and virulence in several organisms including V . cholerae [42] , [43] . Most SXT/R391 ICEs contain mosA and mosT in hotspot 2 . These two genes encode a novel toxin-antitoxin pair that promotes SXT maintenance by killing or severely inhibiting the growth of cells that have lost this element [44] . Not all ICEs in the SXT/R391 family contain mosAT; however , those lacking these genes may encode similar systems to prevent ICE loss . For instance , R391 and ICEVchMex1 contain two genes ( orf2 and orf3 ) encoding a predicted HipA-like toxin and a predicted transcriptional repressor distantly related to the antitoxin HipB . The variable regions found in the 5 hotspots are found exclusively in intergenic regions , punctuating the conserved ICE backbone ( Figure 2 ) . The boundaries between the conserved and variable sequences were mapped on the nucleotide level and compared ( Figure 3A–3E ) . Each hotspot had a distinct boundary . Remarkably , even though the contents of the variable regions markedly differ , with few exceptions the left and right boundaries between conserved and variable DNA for each hotspot was identical among all the ICEs ( Figure 3 ) . For example , the left junctions of the inserts in hotspot 2 immediately follow the stop codon of traA and the right junctions are exactly 79 bp upstream of the start of s054 ( Figure 3B ) , despite the fact that the DNA contents within these borders greatly differ . In hotspot 2 , the right junction appears to begin with a 15 bp sequence that has two variants ( Figure 3B , brown & light brown sequence ) . These sequences may reflect the presence of earlier insertions that have since been partially replaced . A similar pattern was found adjacent to the left boundary of hotspot 4 in several ICEs ( Figure 3D , lines 3–6 ) . Once an insertion is acquired , the number of permissive sites for the addition of new variable DNA likely increases . There are two exceptions to the precise boundaries between variable and conserved DNA . Hotspot 1 and hotspot 3 in ICEVchMex1 and ICEPdaSpa1 , respectively , contain variable DNA that extends beyond the boundary exhibited by all the other ICEs in these locations ( Figure 3A , line 3 , and Figure 3C , line 7 ) . The only boundary that could not be identified was the left border of hotspot 5 , the region containing genes between s026 and traI . As discussed below , s026 is the least conserved core gene and its variability obscured any consensus sequence abutting the variable DNA . Perhaps this border has eroded because s026 is not required for ICE mobility [32] . The relative precision of most boundaries between conserved and variable DNA sequences in all the ICEs analyzed suggests that a particular recombination mechanism , such as bet/exo-mediated recombination , may explain the acquisition of the variable regions . However , at this point , we cannot exclude the possibility that the precise locations for variable DNA insertions simply reflects selection for optimal ICE fitness; i . e . , ICEs can optimally accommodate variable DNA in these locations while preserving their essential functions . Unexpectedly , BLAST analyses revealed that most of the conserved core SXT/R391 genes are also present in IncA/C conjugative plasmids . These multidrug resistance plasmids are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources [45] , [46] . Recently , members of this family of plasmids have also been identified in Yersinia pestis , including from a patient with bubonic plague [47] , and in aquatic γ-proteobacteria [48] , including Vibrio cholerae [49] , [50] . To date , the closest known relatives of the SXT/R391 transfer proteins are found in the IncA/C plasmids . Every predicted SXT transfer protein is encoded by the IncA/C plasmid pIP1202 isolated from Y . pestis [50] and the identities of these predicted protein sequences vary from 34 to 78% ( Figure 4A ) . Furthermore , there is perfect synteny between the four gene clusters encoding the respective conjugative machineries of these two mobile elements ( yellow and orange genes in Figure 4A ) . Despite the extensive similarity of the SXT and IncA/C conjugative transfer systems , these plasmids lack homologues of setR and setD/C as well as int/xis , suggesting that regulation of conjugative transfer differs between these elements . The similarity of IncA/C plasmids and SXT/R391 ICEs is not limited to genes important for conjugal DNA transfer . Ten genes of unknown function ( shown in black in Figure 4A ) , some of which are interspersed within likely tra gene operons and some of which are clustered together between traN and traF , are similar in the two elements . Furthermore , most of these ten genes are in identical locations in the two elements . Both elements also contain homologs of bet and exo ( shown in green in Figure 4A ) ; these are the only known homologs of the λ Red recombination genes found outside of bacteriophages . Together , the similarity of DNA sequences and organization of SXT/R391 ICEs and IncA/C plasmids suggests that these elements have a common ancestor . The fact that the contents of the hotspots in the two classes of elements are entirely distinct suggests that their evolutionary paths diverged prior to acquisition of these variable DNA segments . The conservation of the 52 core genes in all 13 SXT/R391 ICEs analyzed suggested that many or even all of these genes would be required for key ICE functions of excision/integration , conjugative transfer and regulation . The presence of ten ICE core genes of unknown function in IncA/C plasmids ( black genes in Figure 4A ) is also consistent with the hypothesis that these genes might be required for ICE transfer . However , our previous work demonstrated that not all genes recognized here as part of the conserved core gene set are required for SXT transfer . Beaber et al showed that deletion of rumB – s026 ( which includes 5 cores genes ) from SXT had no detectable influence on SXT excision or transfer [32] . Therefore , we systematically deleted all of the core ICE genes whose contributions had not previously been assessed , in order to explore the hypothesis that these genes ( especially those also present in IncA/C plasmids ) would be essential for ICE transfer and to define the minimum functional SXT/R391 gene set . Surprisingly , deletion of most of the ICE core genes of unknown function , including genes with homologues in IncA/C plasmids , did not alter SXT transfer efficiency . Deletion of s002 or s003 , which are located downstream of int in all SXT/R391 ICEs , did not alter the frequency of SXT transfer; similarly , deletion of s082 , s083 , and s084 , core genes of unknown function that are found near the opposite end of SXT/R391 ICEs but not in IncA/C plasmids , also did not influence SXT transfer frequency ( Figure 4B ) . Furthermore , deletion of s091 , which is found between traD and s043 in ICEs and IncA/C plasmids , did not reduce SXT transfer ( Figure 4B ) . In contrast , deletion of s043 , which has weak homology to traJ in the F plasmid ( a gene important in DNA processing ) and is located in a transfer cluster containing traI and traD , abolished transfer ( Figure 4B , Δd ) , suggesting that s043 , here re-named traJ is required for SXT transfer . It is unlikely that the transfer defect of SXTΔtraJ can be explained by polar effects of the deletion on downstream genes , since traJ appears to be the last gene of an operon found immediately upstream of hotspot 1 . Similarly , deletion of s054 , which is found immediately 5′ of traC and is homologous to a disulfide-bond isomerase dsbC , also abolished transfer ( Figure 4B , Δe ) . Interestingly , disulfide bond-isomerases are present in several other conjugative systems [51] . However , it is not clear at this point if the deletion of s054 from SXT accounts for the transfer defect of SXTΔs054 , since we could not restore transfer by complementation . Additionally , Beaber et al found that deletion of s060 through s073 in SXT , which includes 7 genes that are also found in IncA/C plasmids reduced SXT transfer more than 100-fold [32] . We constructed several smaller deletions in this region and found that deletion of s063 , which is also found in pIP1202 , reduced the transfer frequency of SXT by ∼100-fold , nearly the same amount as deleting the entire region ( Figure 4B ) . Complementation analyses revealed that the absence of s063 accounted for the transfer defect of SXTΔs063 ( data not shown ) . Even though SXTΔs063 was still capable of transfer , in our view , the drastic reduction in the transfer frequency of this mutant warrants inclusion of s063 into the minimum functional SXT ICE genome ( shown in Figure 4C ) . Other deletions in this region , including deletions of bet , exo , s067 , s068 and s070 , which have orthologues in IncA/C plasmids , resulted in ≤10-fold reductions in transfer frequency . We therefore did not include these genes in the minimal functional core SXT/R391 genome ( Figure 4C ) . The findings from our experiments testing the transfer frequencies of SXT derivatives harboring core gene deletions ( shown in Figure 4B ) , coupled with our previous work demonstrating the requirements for the predicted SXT tra genes in the element's transfer [32] , suggest a minimal functional SXT/R391 ICE structure as shown in Figure 4C . This minimum element is ∼29 . 7 kb and consists of 25 genes . Genes with related functions , which in some cases encode proteins that likely form large functional complexes ( such as the conjugation apparatus ) , are grouped together in the minimal genome . At the left end of the minimum ICE genomes are xis and int , the integration/excision module of SXT/R391 ICEs . In the minimal ICE genome , the ICE oriT and mobI , which encodes a protein required for SXT transfer [39] , are no longer separated from the other genes ( traIDJ ) that are also thought to play roles in the DNA processing events required for conjugative DNA transfer . The genes required for formation of the conjugation machinery , including the pilus , and mating pair formation and stabilization [32] , [39] are divided between three clusters ( denoted mpf1-3 in Figure 4C ) . Finally , at the right end of the minimal functional genome are the genes that regulate ICE transfer ( setC/D and setR ) . Thus , the minimal functional SXT/R391 ICE is relatively small and organized into 3 discrete functional modules that mediate excision/integration , conjugation , and regulation . Even though deletion of 27 out of 52 SXT/R391 ICE core genes proved to have little or no effect on SXT transfer frequency , and hence these genes were not included in Figure 4C , it is reasonable to presume that these genes encode functions that enhance ICE fitness given their conservation . For example , the presence of highly conserved bet and exo genes in all SXT/R391 ICEs suggests that there has been selection pressure to maintain this ICE-encoded recombination system that promotes ICE diversity by facilitating inter ICE recombination ( G Garriss , MK Waldor , V Burrus , in press ) . A key challenge for future studies will be to determine how core genes of unknown function promote ICE fitness . To identify genes in the SXT/R391 core genome that may be subject to different selection pressures , we compared the percent identity of each ICE's core genes to the corresponding SXT gene ( Figure 5 ) . Most of the ICEs' core genes exhibited 94% to 98% identity on the nucleotide level to SXT's core genes . There was no discernable difference in the degree of conservation of most core genes that were or were not part of the minimal ICE , suggesting that there are equal selective pressures on essential and non-essential genes . However , we identified 8 genes ( s026 , traI , orfZ , s073 , traF , eex , s086 , and setR ) that exhibit significantly different degrees of conservation ( Figure 5 and Figure S1 ) . Three of these showed unusually high conservation , while the other 5 had below average conservation . Two of the highly conserved genes , setR and s086 , are found at the extreme 3′ end of the elements . The conservation of setR may reflect the key role of this gene in controlling SXT gene expression . S086 may also play a role in regulating SXT transfer [52] . The other highly conserved gene , orfZ , is found between bet and exo and has no known function . s026 and s073 are the most divergent of all the genes in the backbone . s026 encodes a hypothetical protein with homologues in many gram negative organisms . Although S026 is predicted to contain a conserved domain , COG2378 , which has a putative role in transcription regulation , this protein is not required for SXT transfer [32] . The significant divergence of s026 along with its lack of essentiality suggests that this gene could become a pseudogene . A similar argument could be made for s073 , which encodes a hypothetical protein that is also not required for ICE transfer . However , this argument does not hold for traI or traF , two genes which are essential for ICE transfer . Although the reasons which account for the different degrees of conservation of these 8 core genes are hard to ascertain at this point , the data in Figure 5 suggests that individual core genes are subject to different evolutionary pressures . We created phylogenetic trees for each core gene based on their respective nucleotide sequences to further explore the evolution of the conserved backbone of SXT/R391 ICEs . Since we found such a high degree of conservation for most of the core genes , the bootstrap values for most of these trees were relatively low . Thus , we concentrated on the most polymorphic genes found in Figure 5 , s026 , s073 , traI , and eex , for phylogenetic analyses . As shown in Figure 6A , the trees for s026 , traI and s073 exhibit 3 distinct branching patterns . The lack of similarity in these phylogenetic trees suggests that either individual core genes have evolved independently or that high degrees of recombination mask their common evolutionary history . The latter hypothesis seems more likely since experimental findings have revealed that SXT/R391 ICEs can co-exist in a host chromosome in tandem [26] and recombination between tandem elements can yield novel hybrid ICEs with considerable frequency [53] ( G Garriss , MK Waldor , V Burrus , in press ) . Also , as noted above , the distributions of variable genes among the ICEs shown in Figure 2 also supports the idea that inter-ICE recombination is commonplace . Unlike most core genes , the trees for traG and eex were similar . In these two trees , the ICEs segregate into two evolutionarily distinct groups ( Figure 6B ) , confirming and extending previous observations that revealed that there are two groups of eex and traG sequences in SXT/R391 ICEs [54] . These two groups correspond to the two functional SXT/R391 ICE exclusion groups . Interactions between traG and eex of the same group mediate ICE exclusion [55] . Thus , the identical 2 clusters of traG and eex sequences observed in their respective trees reveals the co-evolution of the traG/eex functional unit . The two groups of eex sequences can also be observed in Figure 5 where the bifurcating pattern reveals the 2 exclusion groups . This pattern is difficult to discern for traG , perhaps because of the large size of this multi-functional gene . The sequence of ICEVchBan8 , which was derived from a non-O1 , non-O139 V . cholerae strain , is incomplete but it appears to contain 49 out of 52 SXT/R391 core genes . However , since this strain lacks Intsxt it was not included in our comparative analyses above . It is not known if ICEVchBan8 is capable of excision or transmission; however , it contains a P4-like integrase and a putative xis . It is tempting to speculate that the genome of ICEVchBan8 provides an illustration of how acquisition ( presumably via recombination ) of a new integration/excision module may generate a novel ICE family . Comparative analysis of the genomes of the 13 SXT/R391 ICEs studied here has greatly refined our understanding of this group of mobile genetic elements . These elements , which have been isolated from 4 continents and the depths of the Pacific Ocean , all have an identical genetic structure , consisting of the same syntenous set of 52 conserved core genes that are interrupted by clusters of diverse variable genes . All the elements have insertions of variable DNA segments in the same five intergenic hotspots that interrupt the conserved backbone . Furthermore , some of the elements have additional insertions outside the hotspots; however , in all cases the acquisition of variable DNA has not compromised the integrity of the core genes required for ICE mobility . Functional analyses revealed that less than half of the conserved genes are necessary for ICE transmissibility and the contributions of the 27 core genes of unknown function to ICE fitness remains an open question . Finally , several observations presented here suggest that recombination between SXT/R391 ICEs has been a major force in shaping the genomes of this widespread family of mobile elements . Although comparisons of the 13 ICE genomes analyzed here strongly suggest that these mobile elements have undergone extensive recombination during their evolutionary histories , there is a remarkable degree of similarity among the SXT/R391 ICEs . All of these ICEs consist of the same syntenous and nearly identical 52 genes . In contrast , other families of closely related mobile elements , such as lambdoid or T4-like phages for example , exhibit greater diversity [56] , [57] . Since the elements that we sequenced were isolated from several different host species and from diverse locations , the great degree of similarity of the SXT/R391 ICE family does not likely reflect bias in the elements that we sequenced . It is possible that this family of mobile elements is a relatively recent creation of evolution and has yet to undergo significant diversification . To date , relatively few formal comparative genomic analyses of other ICE families have been reported . Mohd-Zain et al [11] identified several diverse ICEs and genomic islands that shared a largely syntenous set of core genes with ICEHin1056 , an ICE originally identified in Haemophilus influenzae . However , even though these elements share a similar genomic organization , they exhibit far greater variability in the sites of insertion of variable DNA and in the degree of conservation in their core genes compared to SXT/R391 ICEs . Thus , although this group of elements appears to share a common ancestor , they seem to have diverged earlier in evolutionary history than the SXT/R391 ICEs . However , when comparative genomic analyses were restricted to ICEHin1056-related ICEs found in only two Haemophilus sp . , Juhas et al found that , like the SXT/R391 family of ICEs , these 7 ICEHin1056-related ICEs share greater than 90% similarity at the DNA level in their nearly syntenous set of core genes [12] . It will be interesting to learn the extent of conservation of genetic structure and DNA sequence in additional ICE families to obtain a wider perspective on ICE evolution . Comparative genomic studies of bacteriophages have led to the idea that the full range of phage sequences are part of common but extremely diverse gene pool [58] , [59] . The SXT/R391 ICE genomes suggest that there may be an even larger network of phylogenetic relationships linking sequences found in all types of mobile genetic elements including phages , plasmids , ICEs and transposons . The genomes of SXT/R391 ICEs appear to be amalgams of genes commonly associated with other types of mobile elements . Many of the ICE core genes are usually associated with phages , such as int , bet , exo and setR , or with plasmids , such as the tra genes . Additionally , the SXT/R391 ICEs and IncA/C plasmids clearly have a common ancestor , as we found that the entire set of SXT/R391 tra genes are also present in IncA/C plasmids . Thus , the genes present in all types of mobile genetic elements appear to contribute to a common gene pool from which novel variants of particular elements ( such as ICEVchBan8 ) or perhaps even novel types of mobile genetic elements can arise .
ICEPalBan1 , ICEVchMex1 , ICEVchInd4 , ICEVchInd5 and ICEVchBan5 were isolated using the plasmid capture system described in Figure 1 . The SXT chromosomal attachment sequence , attB , was introduced into the modified F plasmid pXX704 [34] to create pIceCap . This plasmid was then introduced into a ΔprfC derivative of the TcR E . coli strain CAG18439 . Exconjugants derived from matings between this strain and those harboring the 5 ICEs listed above resulted in strains carrying a pIceCap::ICE plasmid . Once captured , the plasmids were isolated using the Qiagen plasmid midi kit for low-copy plasmids ( Qiagen ) . Isolated pIceCap::ICE plasmids were then sequenced . ICEVflInd genome was determined by sequencing several overlapping cosmids that encompassed this ICE's genome . Briefly , genomic DNA from a Vibrio fluvialis strain carrying ICEVflInd was prepared using the GNome DNA kit ( QBIOgene ) . Sau3A1 restricted genomic DNA was used to create a SuperCos1 ( Stratagene ) -based cosmid library according the manufacture's instructions . The library was subsequently screened for cosmids containing ICE-specific sequences using PCR with primers to conserved core ICE sequences . Four cosmids containing overlapping ICEVflInd sequences were identified and sequenced . The genomes of 6 ICEs were sequenced by the Sanger random shotgun method [60] . Briefly , small insert plasmid libraries ( 2–3 kb ) were constructed by random nebulization and cloning of pIceCap::ICE DNA or of cosmid DNA for ICEVflInd . In the initial random sequencing phase , 8–12 fold sequence coverage was achieved . The sequences of either pIceCap or pSuperCos were subtracted and the remaining sequences were assembled using the Celera Assembler [61] . An initial set of open reading frames ( ORFs ) that likely encode proteins was identified using GLIMMER [62] , and those shorter than 90 base pairs ( bp ) as well as some of those with overlaps eliminated . Nucleotide and amino acid conservation were assessed with the appropriate BLAST algorithms . ICEs were aligned using clustalW with default settings [63] . MAUVE [37] and LAGAN [38] were used to identify core genes in Figure 2 . To map the boundaries of the hotspots , sequence comparisons were made using MAUVE and then manually compared to find boundaries between conserved and variable DNA as shown in Figure 3 . Phylogenetic trees were generated from alignments of nucleotide sequences using the neighbor-joining method as implemented by ClustalX software , version 2 . 011 [64] . The reliability of each tree was subjected to a bootstrap test with 1000 replications . Trees were edited using FigTree 1 . 22 ( http://tree . bio . ed . ac . uk/software/figtree/ ) . CAG81439 harboring SXT was used as the host strain to create the SXT deletion mutants shown in Figure 3; the deletions were constructed using one-step gene inactivation as previously described [44] , [65] . The primers used to create the deletion mutants are available upon request . Matings were conducted as previously described [16] , [44] using deletion mutants and a KnR E . coli recipient , CAG18420 . Exconjugants were selected on LB agar plates containing chloramphenicol , 20µg/ml ( for SXT selection ) and kanamycin , 50 µg/ml . The frequency of exconjugant formation was calculated by dividing the number of exconjugants by the number of donors . | Integrative and conjugative elements ( ICEs ) are a class of mobile genetic elements that are key mediators of horizontal gene flow in bacteria . These elements integrate into the host chromosome , yet are able to excise and transfer via conjugation . Our understanding of ICE evolution is rudimentary . Here , we developed a method to capture ICEs on plasmids , thus facilitating their sequencing . Comparative analyses of the DNA sequences of ICEs from the same family revealed that they have an identical genetic structure consisting of syntenous , highly conserved core genes that are interrupted by clusters of diverse variable genes . Unexpectedly , many genes in the core backbone proved non-essential for ICE transfer . Comparisons of the variable gene content in the ICEs analyzed revealed that these elements are mosaics whose genomes have been shaped by inter–ICE recombination . Finally , our work suggests that ICEs contribute to a larger gene pool that connects all types of mobile elements . | [
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] | 2009 | Comparative ICE Genomics: Insights into the Evolution of the SXT/R391 Family of ICEs |
Lrig proteins are conserved transmembrane proteins that modulate a variety of signaling pathways from worm to humans . In mammals , there are three family members – Lrig1 , Lrig2 , and Lrig3 – that are defined by closely related extracellular domains with a similar arrangement of leucine rich repeats and immunoglobulin domains . However , the intracellular domains show little homology . Lrig1 inhibits EGF signaling through internalization and degradation of ErbB receptors . Although Lrig3 can also bind ErbB receptors in vitro , it is unclear whether Lrig2 and Lrig3 exhibit similar functions to Lrig1 . To gain insights into Lrig gene functions in vivo , we compared the expression and function of the Lrigs in the inner ear , which offers a sensitive system for detecting effects on morphogenesis and function . We find that all three family members are expressed in the inner ear throughout development , with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2 expressed more broadly . Lrig1 and Lrig3 overlap prominently in the developing vestibular apparatus and simultaneous removal of both genes disrupts inner ear morphogenesis . This suggests that these two family members act redundantly in the otic epithelium . In contrast , although Lrig1 and Lrig2 are frequently co-expressed , Lrig1−/−;Lrig2−/− double mutant ears show no enhanced structural abnormalities . At later stages , Lrig1 expression is sustained in non-sensory tissues , whereas Lrig2 levels are enhanced in neurons and sensory epithelia . Consistent with these distinct expression patterns , Lrig1 and Lrig2 mutant mice exhibit different forms of impaired auditory responsiveness . Notably , Lrig1−/−;Lrig2−/− double mutant mice display vestibular deficits and suffer from a more severe auditory defect that is accompanied by a cochlear innervation phenotype not present in single mutants . Thus , Lrig genes appear to act both redundantly and independently , with Lrig2 emerging as the most functionally distinct family member .
Protein-protein interactions are critical for diverse and complex biological functions throughout the animal kingdom , including nervous system development , cell adhesion and signaling , tissue morphogenesis , the immune response and human disease [1]–[4] . This functional diversity is accomplished by superfamilies of proteins harboring combinations of common protein recognition motifs . For instance , the human genome encodes hundreds of proteins with extracellular leucine rich repeats ( LRR ) , a 20–30 amino acid motif that forms a characteristic horseshoe structure for protein-protein interactions [5] , [6] . Similarly , the large immunoglobulin ( Ig ) superfamily of cell adhesion molecules is defined by the presence of Ig domains , which can mediate highly specific homophilic and heterophilic binding [7] , [8] . Despite their abundance , LRR and Ig motifs are rarely found in the same protein , with only several dozen mammalian genes encoding LRR-Ig proteins that fall into twelve gene families [3] , [9] , [10] . Most of these proteins are vertebrate-specific and show discrete expression in the developing nervous system , suggesting that expansion of the LRR-Ig family may have contributed to the increased complexity of the vertebrate nervous system . Consistent with this idea , several LRR-Ig proteins have been shown to control highly specific cell-cell interactions underlying synapse formation and other aspects of nervous system development [2] . The invertebrate-specific Kekkon proteins , on the other hand , modulate signaling by binding to and downregulating EGF receptors [11] , [12] . Within the LRR-Ig family , only the Lrig subfamily contains both invertebrate and vertebrate members [3] , indicating that analysis of this family may provide general insights into the evolution of LRR-Ig proteins . The leucine-rich repeat and immunoglobulin-like domain proteins ( Lrigs ) are single pass transmembrane proteins with extracellular domains containing fifteen LRRs , three Ig-like domains and intracellular domains of varying length [13] . The fly and worm genomes each contain a single Lrig gene . This family is expanded in the vertebrate genome , which encodes for three family members [14]: Lrig1 ( formerly Lig1 ) , Lrig2 , and Lrig3 . The extracellular domains are highly conserved within the family , but the cytoplasmic domains diverge significantly , with no motifs common to flies , worms , or vertebrates . This suggests that Lrig family members may interact with similar binding partners yet ultimately exert distinct downstream effects . Most of what is known about Lrig function has come from analysis of Lrig1 , which is downregulated in several human cancers [15] . Consistent with its proposed role as a tumor suppressor gene , Lrig1 can control the activity of several receptor tyrosine kinases ( rTKs ) with important effects on cell proliferation and survival . For instance , Lrig1 negatively regulates members of the ErbB family of receptors by promoting receptor degradation [16]–[18] . In support of this , Lrig1 regulates EGFR levels in primary human keratinocytes [19] , and loss of Lrig1 results in increased EGF signaling and excess intestinal stem cell proliferation , tumor formation and psoriasis-like hyperplasia in mice [20]–[22] . However , Lrig1 can also inhibit Met and Ret rTK activation [23] , [24] , suggesting that Lrig1 activity extends beyond regulation of EGF signaling . How any Lrig protein functions at the molecular level remains a mystery . Whether Lrig3 shares some properties with Lrig1 remains an open question . As predicted by homology in their extracellular domains , both Lrig1 and Lrig3 can bind to ErbB receptors [25] . However , although downregulation of Lrig3 in human glioma cells caused enhanced EGFR levels [26] , more recent studies indicate that Lrig3 actually opposes Lrig1's effects on EGF signaling [18] . In addition , similar to Lrig1's ability to interact with a variety of receptors , Lrig3 also binds to FGF receptors and regulates FGF and Wnt signaling in Xenopus [27] . Whereas several phenotypes reported in Lrig1 mutant mice have been associated with changes in EGF signaling , loss of Lrig3 leads to a disruption in the three-dimensional structure of the inner ear that is not easily explained by altered ErbB signaling [25] , [28] . Thus , it is not yet clear how the functions identified for Lrig1 and Lrig3 in vitro translate to their actions in vivo . Comparison of Lrig1 and Lrig2 , on the other hand , has suggested key differences . First , reduction of Lrig2 either lowers or has no effect on EGFR levels in vitro [18] , [29] . Consistent with this observation , Lrig2 does not behave like a typical tumor suppressor in humans . For instance , Lrig2 expression can be increased in some human tumors , and a combination of high levels of Lrig2 and low levels of Lrig1 correlates with a poor prognosis for a type of early-stage squamous cell carcinoma [30] . Similarly , overexpression of Lrig2 correlates with invasiveness of pituitary adenoma [31] . In addition , studies of Lrig protein expression in human tumors have revealed fundamental differences in the subcellular distribution of these family members [32] , [33] . Although Lrig2 phenotypes have not yet been described in mice , loss of LRIG2 causes Urofacial Syndrome in humans , which is characterized by abnormal bladder function and altered facial expression , possibly due to abnormal innervation [34] . In order to clarify whether Lrig genes mediate common biological functions in vivo , we have taken a genetic approach in mice . We have focused our analysis on the development and function of the inner ear , an exquisitely complex structure whose perfect form and function is crucial for the senses of hearing and balance [35] , [36] . The spiral-shaped cochlea mediates the sense of hearing . Head position and motion is sensed by movement of fluid within the vestibular system , which consists of three semicircular canals oriented in the three dimensions of space , and a saccule and utricle that detect linear acceleration and gravity . The inner ear contains six sensory epithelia , which contain the sensory hair cells . Vestibular hair cells in the two maculae and three cristae detect motion of the head , whereas auditory hair cells in the organ of Corti respond to specific frequencies of sound . Vestibular and auditory information is transmitted from the inner ear to the brain by primary sensory neurons in the vestibular or spiral ganglia respectively . The inner ear provides an unusually sensitive system for analysis of gene function since small changes in the formation or structure of the inner ear can cause profound functional deficits in hearing and balance . For instance , Lrig3 mutant mice exhibit hyperactivity and run in circles due to truncation of a single semicircular canal [28] . Further , Lrigs have been shown to modulate BMP , FGF , and Wnt signaling pathways , which all play important roles in the morphogenesis and patterning of the inner ear [35] . Thus , analysis of the inner ear provides an ideal opportunity to uncover the in vivo actions of the Lrigs . Here , we analyzed several features of inner ear development and function in single and double Lrig mouse mutants . Our results suggest that Lrig1 and Lrig3 cooperate during morphogenesis . Lrig1 and Lrig2 , on the other hand , control largely distinct aspects of inner ear development and function , yet act redundantly to ensure proper innervation of the cochlea .
To be able to compare and contrast Lrig gene function in the inner ear , it is critical to know when and where each family member is expressed . Any sites of overlap offer an opportunity to examine redundancy , whereas unique sites of expression can be used to reveal the biological significance of individual family members . For instance , Lrig3 is the only family member expressed in the developing lateral semicircular canal and Lrig3 mutant mice circle due to defects in this canal . However , although Lrig3 is also expressed in other regions of the inner ear , Lrig3 mutant mice exhibit normal auditory responses , with no other obvious changes in the structure or function of the inner ear [28] . This raises the possibility that other Lrig genes compensate for the loss of Lrig3 . Therefore , to begin to determine whether these three family members play overlapping functions , we compared their expression patterns in the inner ear , either by in situ hybridization ( Lrig1 ) or by examining the expression of βgeo reporter genes inserted into the Lrig2 ( Figure S1 ) and Lrig3 [28] loci . Given the known role for Lrig3 in canal morphogenesis , we first compared expression patterns at embryonic day 12 . 5 ( E12 . 5 ) , just before the canals begin to acquire their mature morphology . The inner ear develops from the otic vesicle , a simple sphere of epithelium that invaginates from the epidermis overlying the hindbrain beginning around E9 in mouse [35] . Over the next several days , the vestibular apparatus and endolymphatic duct develop from the dorsal half of the otic vesicle , while the cochlea extends ventrally ( Figure 1A ) . Beginning around E12 , the semicircular canals are sculpted from the vertical and lateral pouches . The utricle and saccule develop from an intermediate region called the atrium [37] . In parallel , signaling events establish restricted sensory regions , which ultimately produce hair cells and support cells in the mature sensory epithelia in the canals ( the cristae ) , the utricle and saccule ( the maculae ) , and the cochlea ( the organ of Corti ) . Non-sensory regions in the cochlea go on to form the lateral wall , inner sulcus , and Reissner's membrane . Consistent with previous studies [28] , Lrig1 and Lrig3 showed remarkably restricted yet related patterns of expression at E12 . 5 , overlapping both in the atrium and in the non-sensory domain of the cochlea ( Figure 1B , C ) . In contrast , Lrig2-βgeo activity was evident throughout the early otic epithelium ( Figures 1C and S2 ) . Indeed , Lrig2-βgeo expression appeared nearly ubiquitous at all stages examined , although the levels varied in different tissues ( Figure S2 ) . To determine whether Lrig1 and Lrig2 , like Lrig3 , help determine the three-dimensional structure of the inner ear , we generated and analyzed Lrig1 and Lrig2 mutant mice . Lrig1 mutant mice harbor a gene trap insertion in the third intron of the Lrig1 locus , and Lrig2 mutants contain a gene trap insertion after exon 11 ( Figure S1 ) . These gene trap insertions are predicted to interfere with normal splicing of endogenous transcripts , instead producing transmembrane fusion proteins that are targeted to the lysosome and therefore unlikely to exert any effect [38] . Western blot and immunostaining studies confirmed that Lrig1 and Lrig2 protein levels are severely reduced in each mutant background ( Figures S1 and S3 ) . In contrast to Lrig3 mutants , however , both Lrig1 and Lrig2 single mutant animals exhibited normal inner ear morphologies at E14 . 5 ( Fig . 2B , C , E ) . Given the striking co-expression of Lrig1 and Lrig3 , we wondered whether combined loss of these two family members would provide any evidence for similar functions . Indeed , inner ear development is more severely disrupted in Lrig1−/−;Lrig3−/− double mutant mice than in either single mutant ( Table 1 ) . For example , the utricle and saccule fail to separate ( Figure 2G , arrowhead ) , consistent with the co-expression of Lrig1 and Lrig3 in the embryonic atrium ( Figure 1 ) . In addition , the posterior canal is abnormally small and misshapen ( Figure 2H , I ) . To see whether Lrig1 and Lrig3 also cooperate in the lateral canal , we took advantage of the fact that the lateral canal phenotype is only partially penetrant in Lrig3 mutants maintained on this background , with truncation or thinning observed in only 33% of the animals ( Figure 2D , Table 1 ) . However , loss of either one or two copies of Lrig1 did not strongly enhance this phenotype ( Figure 2D , G , I; Table 1 ) , consistent with the fact that Lrig1 and Lrig3 are not obviously co-expressed in the lateral canal epithelium [28] . The fact that new phenotypes emerge only in sites of Lrig1/Lrig3 co-expression strongly suggests that these two family members act redundantly during inner ear morphogenesis . In contrast , Lrig1 and Lrig2 do not appear to cooperate here , as Lrig1−/−;Lrig2−/− double mutant ears developed normally ( Figure 2F ) despite the extensive co-expression of Lrig1 and Lrig2 at E12 . 5 ( Figure 1B , D ) . To gain a broader view of genetic interactions among Lrig family members , we asked whether either Lrig2 or Lrig3 exert overlapping functions with Lrig1 in other regions of the inner ear . In support of this idea , unlike either single mutant , Lrig1−/−; Lrig3−/− double mutant animals die at or before birth ( Table 2 ) and suffer from an array of morphogenetic phenotypes , including microphthalmia and skeletal malformations ( data not shown ) . Although the presence of new defects suggests that Lrig1 and Lrig3 likely work together in many other tissues , this lethality prevented analysis of any other aspects of inner ear function . In contrast , Lrig1−/− , Lrig2−/− , and Lrig1−/−;Lrig2−/− mutant mice survive past the onset of hearing . We therefore focused the rest of the analysis on Lrig1 and Lrig2 . As previously reported [20] , Lrig1−/− mice frequently die within the first postnatal week when maintained on an inbred background ( Table 2 ) . Lrig2−/− mice were born in normal Mendelian ratios and showed no obvious defects ( Table 2 ) . However , very few Lrig1−/−;Lrig2−/− double mutant animals survived to six weeks of age . A small percentage of Lrig1−/−; Lrig2−/− double mutants survived to adulthood ( Table 2 ) and were noticeably runty during adolescence . More strikingly , half of the double mutant survivors exhibited a mild vestibular defect with circling behavior ( 3 of 6 animals , see video S1 ) . Since neither surviving Lrig1−/− nor Lrig2−/− animals showed any signs of circling , this observation suggests that Lrig1 and Lrig2 may work together in the vestibular system . To investigate this possibility , we performed a more detailed analysis of expression in the vestibular system by double labeling with an anti-Lrig1 antibody and an anti-β-galactosidase antibody to detect Lrig2-βgeo . Lrig2 gene trap heterozygotes were used due to the lack of antibodies that reliably detect Lrig2 protein in tissue . Lrig2-βgeo should serve as an accurate read-out of the pattern of Lrig2 expression , but it should be noted that there may be subtle differences in the stability of the Lrig2-βgeo protein compared to endogenous Lrig2 . However , our observations of Lrig2-βgeo expression match previous reports of Lrig2 transcription [10] , so any discrepancies are likely to be minor . No Lrig1 labeling was detected in Lrig1 mutant tissue , confirming that this antibody detects only this family member ( Figure S3 ) . Consistent with results from in situ hybridization and X-gal staining ( Figures 1 and S2 ) , double labeling at E12 . 5 revealed highly restricted expression of Lrig1 protein in the atrium and cochlea , with broad Lrig2-βgeo expression throughout the otic epithelium and in the surrounding mesenchyme ( Figure 3A , B ) . Within the atrium , Lrig1 was restricted to non-sensory regions , which flank the Sox2-positive sensory patches that eventually give rise to the maculae ( Figure 3C ) . This pattern was maintained after formation of the utricle and saccule , with expression in the transitional epithelium adjacent to the utricular macula and in the extramacular epithelium of the saccule at E16 . 5 ( Figure 3D , F ) , E18 . 5 ( Figure S3 ) and P15 ( Figure 3H–J ) . Lrig2-βgeo , in contrast , was expressed throughout sensory and non-sensory regions of the vestibular organs at E16 . 5 and continuing through the first postnatal week ( Figure 3E , G and S2 ) . However , by P15 , Lrig2-βgeo levels were noticeably enhanced in the utricular and saccular maculae as well as the cristae ( Figure 3E , G and data not shown ) . In contrast , Lrig1 protein was not detected in the vestibular sensory epithelia at any stage . Thus , Lrig1 and Lrig2 are co-expressed in non-sensory regions of the utricle and saccule , but only Lrig2 seems to be present in the sensory epithelia . One prominent site of overlapping expression was the vestibular ganglion , which communicates head position information to the brain . Lrig1 was present at low levels in the neuronal cell bodies , with intense expression in projections to the utricle , saccule , and lateral crista at E16 . 5 ( Figure 3D ) , E18 . 5 ( Figure S3 ) , and P15 ( Figure 3H–J ) . Lrig2-βgeo was also present in the vestibular ganglion at all stages , with enriched expression at P15 ( Figures 3H and S2 , and data not shown ) . The co-expression of Lrig1 and Lrig2 in the vestibular ganglion , particularly at postnatal stages , may explain why Lrig1−/−;Lrig2−/− double mutant animals display occasional circling behavior , since the gross structure of the inner ear is unaffected ( Figure 2F ) and Lrig1 and Lrig2 are not co-expressed in the sensory epithelia at any stage ( Figure 3 ) . Thus , it is possible that Lrig1 and Lrig2 act redundantly in the vestibular ganglion neurons or non-sensory epithelium , though they do not cooperate during the initial formation of the vestibular apparatus . As in the vestibular system , Lrig1 and Lrig2 showed largely distinct patterns of expression in the cochlea , overlapping only in non-sensory regions . Lrig1 protein was restricted to non-sensory regions of the cochlea at all stages , with maintained expression only in Reissner's membrane , a structure that regulates the endolymph environment that is critical for cochlear function ( Figure 4A–C ) [39] . Lrig2-βgeo , on the other hand , appeared ubiquitous in the cochlear epithelium and surrounding mesenchyme at E12 . 5 and E16 . 5 ( Figure 4A′–B′ ) . However , similar to the vestibular system , expression was elevated in sensory and neural tissues postnatally ( Figures 4C′ and S2 ) . Although Lrig1 was not detected in the spiral ganglion neurons or their projections at any stage , expression was apparent in the mesenchyme in the region that the spiral ganglion neuron neurites grow through to reach the cochlear duct ( Figure 4A , and data not shown ) . In summary , although Lrig1 and Lrig2 are at times co-expressed in the vestibular system and cochlea , these two family members show fundamentally different expression patterns , which contrasts with the obvious similarities in the expression of Lrig1 and Lrig3 at all stages examined ( Figure 1 and [28] ) . To assess the relative contributions of Lrig1 and Lrig2 to cochlear function , we tested auditory responsiveness in single and double mutant mice using two complementary assays . First , we recorded Distortion Product Otoacoustic Emissions ( DPOAEs ) , which are generated by the cochlea in response to simultaneous presentation of two slightly dissimilar pure tone frequency stimuli . Production of DPOAEs depends on outer hair cell ( OHC ) function , and DPOAE thresholds will increase if hair cells are missing , damaged , or cannot be properly stimulated due to changes in cochlear mechanics . Second , we recorded Auditory Brainstem Responses ( ABRs ) . ABRs reflect the sum of neuronal activity in response to sound stimulation , starting with the initial activation of spiral ganglion neurons ( wave 1 ) and following with activation in the auditory brainstem ( waves 2–5 ) . Sensitivity is assessed by determining the lowest intensity sound stimulus ( i . e . the threshold ) that is able to generate an ABR response . In addition , the strength of the neuronal response can be evaluated by measuring the latency and amplitude of the first wave . By altering the frequency of the pure tone stimuli , function can be tested along the length of the cochlea , from high frequencies in the base to low frequencies in the apex . Together , these tests offer a sensitive way to identify impairments in the ability of the cochlea to detect and respond to acoustic stimuli . DPOAE and ABR measurements revealed that Lrig1 , but not Lrig2 , is necessary for normal auditory sensitivity . Lrig1 mutants showed significantly elevated DPOAE and ABR thresholds in response to 11 . 3 and 16 kHz stimuli , which typically elicit the lowest threshold responses in control animals ( Figure 5 and Table S2 ) . Whereas control animals reliably detected 16 kHz DPOAE stimuli as quiet as 15 dB , mutants did not respond until the sounds were 45 dB , which is ∼30 times more intense . Thresholds were also elevated in response to lower ( 5 . 6 and 8 kHz ) and higher ( 22 . 6 and 32 kHz ) frequencies , but these differences were not statistically significant since sensitivity is already reduced in these regions of control cochleae ( for example , 57 . 43±2 . 37 dB for wild-type vs . 71 . 86±4 . 5 dB for Lrig1−/− animals presented with a 32 kHz stimulus ) . Lrig2 mutants , on the other hand , responded with the same sensitivity as control littermates . Similarly , Lrig1+/−;Lrig2−/− mutants also demonstrated normal thresholds . However , loss of either one or two copies of Lrig2 from Lrig1 mutants strongly enhanced the effect , such that the outer hair cell response of Lrig1−/−;Lrig2+/− and Lrig1−/−;Lrig2−/− animals only occurred in response to sounds greater than 55 dB across all frequencies ( Table S1 ) . To understand how loss of Lrig2 might exacerbate the Lrig1 phenotype , we looked more closely at the nature of the ABR waveforms in all single and double mutant combinations ( Figure 6 ) . As expected , in Lrig1 mutants the amplitude of the first wave was significantly diminished in response to a range of frequencies and sound intensities ( Figure 6C , D , and Tables S3 and S4 ) . Combined with the increased thresholds , this suggests that the neural response is decreased because the cochlea is not able to detect sounds with sufficient sensitivity . Remarkably , despite the lack of any effect on thresholds , Lrig2 mutants showed a similar response: the amplitude of the first wave was significantly decreased relative to controls at multiple frequencies and across sound intensities ( Figure 6C , D , and Tables S3 and S4 ) . Latencies were also increased ( Figure 6D ) . Thus , whereas Lrig1 is critical for the initial detection of sound , Lrig2 is required for the subsequent neuronal response . Since Lrig2 is uniquely enriched in the spiral ganglion neurons throughout life , these findings suggest that Lrig1 and Lrig2 control distinct aspects of cochlear function . Amplitudes and latencies were even more affected in double mutants , as expected based on the increased thresholds . Although Lrig1−/−;Lrig2−/− double mutants exhibit a fully penetrant auditory response deficit , the cochlea showed no gross malformations either at E19 ( Figure S4A , B ) or in adults ( data not shown ) . The cochlear duct had a normal histological appearance , consistent with the absence of any morphological defect at E14 . 5 ( Figure 2 ) . In addition , immunostaining confirmed the presence of hair cells and neurons in each turn of the cochlea , with spiral ganglion neurites extending to contact hair cells in the organ of Corti ( Figure S4C , D ) . Similarly , in the few double mutant animals that survived past early postnatal stages , there was no obvious change in the number or organization of hair cells and spiral ganglion neurons ( data not shown ) . However , the overall pattern of cochlear innervation was clearly disrupted in double mutants , as revealed by immunolabeling for neurofilament , which labels both afferent and efferent neurites ( Figure 7A–C ) . Whereas control neurites aligned in regularly spaced radial bundles that were clearly separated from each other ( Figure 7A ) , the mutant neurites were noticeably defasciculated and the gaps between the bundles were smaller and present only intermittently ( Figure 7C ) . More strikingly , the inner spiral bundle ( ISB , bracket ) was reduced , indicating a possible change in the innervation of the cochlea by efferent neurons from the hindbrain . In contrast , no obvious changes were apparent in Lrig1 or Lrig2 single mutants ( Figure S4E–G ) . To determine whether Lrig1 and Lrig2 might act redundantly in certain contexts , we looked more closely at the efferent innervation of the cochlea by staining for choline acetyltransferase ( ChAT ) [40] and synaptophysin in single and double mutant animals . Consistent with results from neurofilament-staining , efferent innervation of the cochlea was noticeably sparser in double mutant animals ( n = 4 ) compared to controls ( n = 8 ) ( Figure 7D , F ) . In contrast , cochleae from Lrig1−/− ( n = 2 ) and Lrig1+/−;Lrig2−/− ( n = 4 ) animals were unaffected ( Figure S4G , H ) . Due to the nature of the crosses used to generate sufficient numbers of double mutant animals , Lrig2−/− single mutant animals were not available for analysis of efferent innervation . However , the normal pattern of neurofilament staining ( Figure S4G ) together with the lack of defects in the Lrig1+/−;Lrig2−/− cochlea ( Figure S4H′ ) indicates that Lrig2 is not required on its own and that Lrig1 can fully compensate for reduced Lrig2 activity . On the other hand , cochleae from Lrig1−/−;Lrig2+/− animals ( n = 4 ) exhibited an intermediate phenotype ( Figure 7E ) , which fits with their diminished auditory responsiveness . Taken together , these findings indicate that Lrig1 and Lrig2 exert overlapping functions during cochlear innervation , perhaps uncovering a novel role for Lrig proteins in the nervous system . Moreover , the absence of any obvious morphogenetic or gross cochlear patterning defects argues against the idea that Lrig1 and Lrig2 act redundantly to control any of the major signaling pathways , consistent with their distinct effects in vitro and in cancer .
Here , we used genetic analysis in mice to compare and contrast the effects of Lrig2 and Lrig3 to the founding member of the family , Lrig1 . By analyzing multiple aspects of inner ear development and function , we found that Lrig1 and Lrig3 cooperate to control inner ear morphogenesis , whereas Lrig1 and Lrig2 appear to affect largely distinct aspects of inner ear function . Our results highlight the biological significance of all three Lrig genes in vivo and provide insights into the functional diversity of the LRR-Ig superfamily of proteins . Our findings add to a growing body of work underscoring the similarities between Lrig1 and Lrig3 . At the molecular level , both Lrig1 and Lrig3 can bind multiple members of the EGF receptor family and show a similar subcellular distribution , with expression on the cell surface and in intracellular vesicles [25] , [41] . Moreover , both family members also interact with other rTKs [23] , [24] , [27] , indicating that the Lrig ectodomain does not mediate selective binding . In addition , in vitro studies suggest that both Lrig1 and Lrig3 can act as negative regulators of signaling pathways [16] , [17] , [23] , [24] , [26] , [27] . Our findings suggest that Lrig1 and Lrig3 also exhibit common activities in vivo . For instance , Lrig1 and Lrig3 show strikingly similar patterns of expression within multiple tissues throughout development [10] , [28] . Moreover , Lrig1−/−;Lrig3−/− double mutants exhibit much more dramatic phenotypes than either single mutant . Importantly , new phenotypes emerge at sites of co-expression , such as the developing utricle and saccule . Conversely , the strongest phenotype in the Lrig3 mutant ear is in the lateral canal , which is one of the few sites where Lrig1 and Lrig3 do not overlap . Curiously , although Netrin1 is a key effector of Lrig3 activity in the lateral canal , the atrium develops normally in Netrin1−/− mice ( A . M . N . and L . V . G . , unpublished observation ) , suggesting that Lrig1 and Lrig3 mediate their effects through additional molecules in this region of the inner ear . Consistent with this idea , neither the anterior nor posterior canal was truncated in Lrig1−/−;Lrig3−/− double mutant inner ears , despite the known role of Netrin1 there [42] . One likely explanation is that Lrig1 and Lrig3 modulate a broadly active signaling pathway that controls expression of Netrin1 in the lateral canal , but that other target genes are responsible for effects elsewhere in the inner ear . Indeed , our results suggest that both of these Lrig proteins mediate their effects through key signaling pathways underlying morphogenesis , as Lrig1−/−;Lrig3−/− double mutants die at or before birth with obvious morphogenetic malformations in multiple tissues . A much more detailed analysis of each affected tissue will be needed to pinpoint the pathways involved . Although Lrig1 and Lrig3 appear to cooperate during inner ear morphogenesis , each protein also has its own distinct biological functions . Indeed , the phenotypes already reported in Lrig1 mutant mice indicate that this family member may play a particularly prominent role in EGF signaling and cell proliferation [20]–[22] . Similarly , despite the extensive overlap of Lrig1 and Lrig3 in the ear , loss of Lrig1 is sufficient to cause a significant auditory phenotype , as evidenced by increased DPOAE and ABR thresholds . No auditory phenotypes were detected in Lrig3 mutant mice , in contrast [28] . Moreover , a role for ErbB receptors in inner ear morphogenesis has not been described , and in fact , broad inhibition of ErbB activity has no effect on canal formation in chicks [25] . On the other hand , BMP and FGF signaling is critical for inner ear morphogenesis [35] . Thus , one possibility is that Lrig1 and Lrig3 work together to modulate signaling through BMP or FGF pathways , but that Lrig1 is the dominant regulator of the EGF pathway in vivo . In support of this idea , Lrig1 and Lrig3 can actually exert opposing effects on ErbB receptor levels in vitro , with Lrig1 reinforcing its effects by decreasing Lrig3 levels [18] . Hence , the added loss of Lrig3 might not be expected to exacerbate the effects of Lrig1 on EGF signaling in vivo . Whether Lrig1 and Lrig3 also exert reciprocal effects on the FGF receptor or other putative targets has not yet been examined . An important step towards resolving these apparent differences will be to determine the nature of the pathways affected by both Lrig1 and Lrig3 in vivo . Analysis of Lrig2 indicates that this family member has acquired particularly independent functions . Unlike Lrig1 and Lrig3 , Lrig2 seems to be expressed nearly ubiquitously , although final confirmation awaits the production of reliable anti-Lrig2 antibodies . Such broad expression is not typical for proteins that function in developmental signaling pathways , which tend to show more restricted patterns of expression . Notably , despite the fact that Lrig2 is apparently present in every site of Lrig1 expression , no new morphogenetic phenotypes are uncovered in Lrig1−/−;Lrig2−/− double mutant mice . Thus , Lrig2 is not sufficient to compensate for the combined loss of Lrig1 and Lrig3 , whereas Lrig3 can direct proper inner ear morphogenesis even in the absence of both Lrig1 and Lrig2 . Although final proof will require analysis of triple mutant animals , the contrasting phenotypes seen in each set of double mutants strongly suggest that Lrig2 does not affect the same pathways as Lrig1 or Lrig3 . These genetic results fit with previous reports that Lrig2 behaves differently in vitro and in human tumors [18] , [29]–[31] , [33] . It is also possible that some residual function persists in Lrig2 gene trap mice , thereby obscuring redundant effects . However , this seems unlikely given the sensitivity of the inner ear to even subtle changes in signaling levels , as well as the fact that we were able to detect effects on auditory responsiveness in Lrig2−/− mutants . In addition , loss of just one copy of Lrig2 was sufficient to exacerbate the Lrig1−/− phenotype , as assessed by DPOAE analysis . Although our work provides a useful starting point , analysis of independent Lrig2 alleles may reveal additional functions for this protein in the future . Lrig2 seems to exert distinct effects from Lrig1 and Lrig3 on the basic signaling events that underlie patterning and morphogenesis , with an independent function in neurons . Indeed , although Lrig2 mutant mice are outwardly normal , they do not process sound properly . Specifically , although mutant animals can detect sounds with normal sensitivity , the subsequent neuronal response is attenuated , as evidenced by significantly decreased ABR amplitudes across multiple frequencies . Consistent with this phenotype , Lrig2 is present in spiral ganglion neurons throughout life , with particularly enhanced expression after the onset of hearing . How Lrig2 affects spiral ganglion neuron function remains unclear , though , as there were no obvious defects in the gross innervation of the cochlea in Lrig2 mutant mice . This is not entirely unexpected , as many forms of human deafness are not associated with overt changes in the structure or organization of the cochlea . A role for Lrig2 in the brainstem may exist since Lrig2 mutant mice exhibit auditory brainstem response deficits and LRIG2 is crucial for brainstem mediated bladder control and facial expressions in humans [34] . Although hearing defects have not been reported , our findings suggest that it may be worth investigating whether any patients experience subtle auditory processing defects that might not be detected using standard auditory testing methods . Our results also imply that Lrig1 and Lrig2 may cooperate in limited contexts . Indeed , Lrig1−/−;Lrig2−/− double mutant mice do show enhanced phenotypes relative to the single mutants . For instance , some double mutants show mild circling behavior and hyperactivity that is not seen in either single mutant . More strikingly , acoustic responsiveness is severely impaired in all double mutants , with both DPOAE and ABR thresholds increased across frequencies . The ABR effect may be mostly additive , as Lrig1 and Lrig2 single mutants each exhibit a different kind of auditory defect: Lrig1 is required for the detection of sound and Lrig2 is required for the appropriate neuronal response . The changes in DPOAE thresholds , on the other hand , could be due to redundancy , as loss of even one copy of Lrig2 enhances the Lrig1 mutant phenotype , despite the fact that thresholds are normal in Lrig2 mutants . Since the amplification of the cochlear response by OHC activity is non-linear , DPOAEs offer an unusually sensitive measure of function . Hence , it is possible that the contribution of Lrig2 is too minor to see on its own , but that this small effect is uncovered once OHCs stop responding optimally , as occurs in Lrig1 mutants . Alternatively , Lrig1 and Lrig2 may play similar roles in Reissner's membrane , which is the only apparent site of co-expression in the mature cochlea . Reissner's membrane controls sodium homeostasis in the cochlear endolymph , and changes in the endolymph are known to lead to deafness [36] , [39] . Intriguingly , Lrig1 and Lrig2 are also co-expressed in analogous non-sensory tissues in the utricle and saccule . Hence , a change in endolymph composition could also explain the mild vestibular phenotype uncovered in Lrig1−/−;Lrig2−/− double mutants . An alternative explanation for the enhanced phenotypes is that Lrig1 and Lrig2 cooperate specifically in neuronal populations . In support of this idea , Lrig1 and Lrig2 are co-expressed in the vestibular ganglion and loss of both genes creates a vestibular deficit . More strikingly , efferent innervation is noticeably sparse in double mutant animals , but not obviously altered in either single mutant . The efferent neurons play an important role in modulating OHC responsiveness [43] , so any change in their organization or function could cause the enhanced DPOAE phenotype . In support of this idea , an intermediate efferent phenotype was noted in Lrig1−/−;Lrig2+/− animals , paralleling their abnormal DPOAE responses . Understanding the origin of this defect will be challenging , however , due to the high lethality of Lrig1−/−;Lrig2−/− animals . Nevertheless , taken together with the proposal that the phenotypes seen in Urofacial Syndrome patients are due to abnormal innervation by neurons in the brainstem , these observations suggest that Lrig2 may play a particularly important role in the nervous system , much like the vertebrate-specific LRR-Ig proteins . Lrig1 , on the other hand , may exhibit dual effects , acting like the ancient LRR-Ig proteins to regulate signaling in most contexts , but taking on new functions typical of other LRR-Ig proteins when present in neurons . Our findings underscore the need to delve more deeply into the functions of all three family members in the nervous system . The divergence that occurs within the Lrig gene family may be analogous to the more general diversification in functions for the expanded LRR-Ig superfamily in vertebrates . Analysis of function across species strongly suggests that the original function of Lrig proteins is to bind rTKs and regulate their activity . There are single Lrig orthologs both in worms ( sma-10 ) and flies ( lambik ) . Although nothing is known about lambik function , sma-10 is required for normal regulation of BMP signaling and hence body size in worms [44] . Interestingly , lambik can substitute for sma-10 in vivo . Similarly , Sma-10 binds both invertebrate and vertebrate BMP receptors . However , whereas Lrig1 acts as a negative regulator , sma-10 has a positive effect on BMP signaling . Thus , Lrig proteins from diverse species appear to share the ability to bind to cell-surface receptors , but the consequences of these interactions vary . Similarly , Lrig family members within a single species may have diverged to acquire distinct signaling properties mediated by their intracellular domains . In the case of Lrig1 and Lrig3 , the divergence from ancestral Lrig function is minimal . Lrig2 , however , seems to have gained new and distinct functions . The presence of new behavioral phenotypes in Lrig1−/−;Lrig2−/− double mutant mice suggests that this poorly understood activity may in fact be shared by Lrig1 in some contexts . Although the view of Lrig function in vivo is far from complete , our findings may provide important insights into the origin and activities of vertebrate-specific branches of the LRR-Ig superfamily .
All mice were back-crossed and maintained for more than six generations on the C57BL/6N strain ( Charles River Laboratories ) . The mouse line Lrig1Gt ( GST4169C6 ) contains the VICTR48 gene trap vector ( Lexicon Genetics ) in the Lrig1 locus ( Figure S1 ) and was obtained from the Texas Institute for Genomic Medicine ( TIGM ) at Houston , TX via the Knock Out Mouse Program ( KOMP ) at the University of California , Davis . The RST656 mouse line contains the GTOTMpfs gene trap vector [45] in the Lrig2 locus ( Figure S1 ) . This results in production of a fusion between Lrig2 and βgeo , which mediates neomycin resistance as well as β-galactosidase activity all under the control of the endogenous Lrig2 promoter . Mice were generated by the Mouse Gene Manipulation Facility of Boston Children's Hospital Intellectual and Developmental Disabilities Research Center ( IDDRC ) which is supported by NIHP30-HD18655 . Lrig3 mutant mice contain a deletion of exon 1 and were derived from the Lrig3flox allele which has been previously described [25] . Genotype distribution ( Table 1 ) was assessed for surviving animals at 1 week and 6 weeks of age . For timed pregnancies , embryonic day 0 . 5 ( E0 . 5 ) was defined as noon on the day a copulatory plug was present . All mice were maintained in accordance with institutional and National Institutes of Health ( NIH ) guidelines approved by the Institutional Animal Care and Use Committee ( IACUC ) at Harvard Medical School . Rat polyclonal antiserum to Lrig2 was raised against the intracellular domain of mouse Lrig2 protein expressed in bacteria ( Dana-Farber/Harvard Cancer Center Monoclonal Antibody Core ) . E12 . 5 littermate embryos were lysed in 50 mM Tris ( pH 7 . 4 ) , 150 mM NaCl , 1% Igepal CA 630 ( NP-40 ) , 0 . 5% sodium deoxycholate , 0 . 1% sodium dodecyl sulfate ( SDS ) and 1 mM Pefabloc ( Roche ) . Western blot analysis was performed using standard protocols and a 1∶2000 dilution of anti-Lrig2 serum or 1∶8000 dilution of anti-actin antibody ( Abcam ab8226 ) . Non-radioactive in situ hybridization for Lrig1 was performed on cryosections of mouse E12 . 5 tissue as described [28] . A detailed protocol is available at http://goodrich . med . harvard . edu/resources/resources_protocol . htm . Tissue was fixed for 1 hour in 4% paraformaldehyde ( PFA ) /phosphate buffered saline ( PBS ) , equilibrated in 30% sucrose/PBS at 4°C , and embedded in Neg50 ( Richard-Allan Scientific ) . Cryosections transverse to the ear ( Figure 1A ) were cut and incubated in 1 mg/ml X-Gal ( Sigma-Aldrich ) in X-Gal buffer , post-fixed in 4% PFA/PBS for 1 hour at 4°C , and mounted using Glycerol Gelatin mounting medium ( Sigma-Aldrich ) . E12 . 5 and E16 . 5 mouse heads were collected and fixed for 1 hour at 4°C in 4% PFA/PBS , equilibrated in 30% sucrose/PBS at 4°C , and embedded in Neg50 ( Richard-Allan Scientific ) . E19 heads were hemisected , fixed overnight at 4°C in 4% PFA/PBS and processed the same way . P15 animals were perfused with 4% PFA/PBS , the head hemisected and the brain removed , and the remaining tissue was post-fixed in 4% PFA/PBS for 1 hour at room temperature , decalcified in 0 . 12M EDTA/PBS overnight at room temperature followed by several days at 4°C , and embedded as before . Tissue from 6 week old animals was fixed overnight at 4°C in 4% PFA/PBS and decalcified for 5 to 7 days in 0 . 12M EDTA/PBS at 4°C prior to immunostaining . Cryosections cut transverse to the ear were blocked in PBS+3% bovine serum albumin ( BSA ) and permeabilized in wash solution ( PBS+1% BSA+0 . 1% Triton X-100 ) . Primary antibodies were added in wash solution at the following concentrations: β-galactosidase ( 1∶300 , MP Biomedicals 08559761 ) , Lrig1 ( 1∶75–300 , R&D Systems AF3688 ) , Neurofilament H ( 1∶1000 , Millipore AB5539 ) , and Sox2 ( 1∶500 , Millipore AB5603 ) . Whole cochleae were blocked in PBS with 1% Triton X-100 and 5% normal donkey serum for one hour , followed by a 20 hour incubation at 37°C in primary antibodies diluted in blocking solution at the following concentrations: Choline acetyltransferase ( 1∶200 , Millipore AB144P ) , Neurofilament H ( 1∶1000 ) , and Synaptophysin ( 1∶200 , Synaptic Systems 101011 ) . Alexa-conjugated secondary antibodies were used for signal detection . Tissue was imaged on an Olympus Fluoview FV1000 confocal microscope or a Nikon E800 compound microscope . For wholemount cochleae , the middle turn of the cochlea was imaged . The overall pattern of innervation in each image was scored as either “normal” , “intermediate” , or “abnormal” by three observers blind to genotype . Auditory brainstem recordings ( ABRs ) and distortion product otoacoustic emissions ( DPOAE ) recordings were performed on the right ears ( unless otherwise indicated ) of mice at 6 weeks of age in a soundproof chamber maintained at 32°C . Prior to recordings , mutant mice were observed for circling behavior . Mice were anesthetized with ketamine ( 100 mg/kg ) and xylazine ( 10 mg/kg ) prior to recordings , which were performed as previously described [46] . Littermate control animals were included in each round of recordings . Due to the high lethality rate of Lrig1−/−;Lrig2−/− double mutants , animals used for recordings were generated by Lrig1+/−;RST656+/− intercrosses as well as crosses using Lrig1+/−;RST656−/− or Lrig1−/−;RST656+/− animals . Additionally , recordings were made from both ears of the Lrig1−/−;Lrig2−/− double mutants . Average ABR waveforms were plotted using MATLAB ( MathWorks ) and a script written by Ann E . Hickox in the laboratory of Dr . Charles Liberman ( EPL Laboratories , Massachusetts Eye and Ear Infirmary , Boston , MA ) . E14 . 5 mouse heads were fixed overnight at 4°C with Bodian's Fix , dehydrated in 100% ethanol , and then cleared overnight in methyl salicylate . Heads were hemisected , and White All Purpose Correction Fluid ( Sanford Corporation ) diluted in methyl salicylate was injected into the cochlea with a pulled glass pipette and Hamilton syringe . Filled ears were imaged in methyl salicylate using an Olympus MVX10 microscope to capture image stacks at approximately 30 µm intervals through the ear . Image stacks were processed using Image J software [47] and the Stack Focuser plugin ( author Michael Umorin ) to produce a single image representation ( Figure 3 ) . | The mammalian genome encodes three Lrig family members - Lrig1 , Lrig2 , and Lrig3 . Lrig proteins share a characteristic extracellular domain that can bind to a variety of signaling receptors , but the three family members show little homology in the cytoplasmic domain . Lrig1 is a tumor suppressor gene required for normal EGF signaling . Whether Lrig2 and Lrig3 play similar roles is not known . To address this gap in knowledge , we compared the expression and function of Lrigs in the mouse inner ear , which is responsible for hearing and balance . Even subtle changes in the inner ear cause easily detected deficits in hearing and balance , making it an ideal system for analysis of gene function . We find that Lrigs can act both redundantly and independently in the inner ear , with Lrig1 and Lrig3 cooperating to control morphogenesis and Lrig1 and Lrig2 acting independently to ensure proper cochlear function . However , loss of both Lrig1 and Lrig2 causes a more severe auditory response deficit and additionally causes a vestibular defect , suggesting some overlapping activities . Our findings provide new insights into the in vivo functions for the Lrig genes , which play important roles in vertebrate development and disease . | [
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] | [] | 2013 | In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear |
The ability of DENV2 to display different morphologies ( hence different antigenic properties ) complicates vaccine and therapeutics development . Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to “bumpy” surfaced morphology when the temperature is switched from 29°C at 37°C . Here we identified five envelope ( E ) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies . Several mutations performed on the smooth DENV2 infectious clone destabilized the surface , as observed by cryoEM . Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins . In contrast , three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C , and only at high fever temperature ( 40°C ) did they become “bumpy” . These results imply vaccines should contain particles representing both morphologies . For prophylactic and therapeutic treatments , this study also informs on which types of antibodies should be used at different stages of an infection , i . e . , those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus .
Dengue virus ( DENV ) infects ~400 million people annually around the world especially in the tropical and sub-tropical regions [1 , 2] . It causes diseases ranging from mild dengue fever to severe dengue hemorrhagic fever ( DHF ) and dengue shock syndrome ( DSS ) . DENV is a flavivirus , and is transmitted by the mosquito vectors Aedes aegypti and Aedes albopictus . Other important human pathogens in the flaviviridae family are West Nile ( WNV ) , Yellow fever , Japanese encephalitis and Zika virus . DENV comprises of four serotypes ( DENV1-4 ) . The sequence variability between the four serotypes is about 25 to 40% and between strains within a serotype is ~3%[3 , 4] . The development of vaccines is complicated by the presence of four serotypes . When an individual is infected with one serotype , subsequent infection with another serotype may result in the development of DHF or DSS . This antibody-dependent enhancement ( ADE ) phenomenon is proposed to be due to antibodies elicited in a primary infection against the first serotype subsequently binding to but not neutralizing the other serotype in the second infection . Non-neutralizing or sub-neutralizing concentrations of antibodies binding to the virus may help to concentrate the virus onto the Fcγ receptor on the surface of monocytes and macrophage cells , thereby leading to an enhancement of infection [5] . This suggests that an effective vaccine should simultaneously stimulate equally strong neutralizing antibody responses against all four serotypes . Currently , several vaccine candidates have been tested in clinical trials . Thus far , CYD-TDV is the only licensed dengue vaccine . However , this tetravalent dengue vaccine showed poor efficacy towards DENV2 and moderate efficacy to DENV1 , DENV3 and DENV4 [6 , 7] . Interestingly , we previously observed that the DENV2 mature virus structure can be heterogenous at 37°C , which poses an additional complication when designing vaccines . DENV contains a 11kb positive sense RNA genome encoding three structural proteins and seven non-structural proteins . Of the three structural proteins , the envelope ( E ) protein is the major surface protein on the virus and therefore is the primary antigen that elicits the neutralizing antibody response . The mature DENV virion is composed of 180 copies of E proteins . Previous studies showed that DENV2 changes from a smooth compact to a loose bumpy surface morphology when the temperature is raised from 28°C to 37°C [8 , 9] . On the smooth compact particles , the E proteins are tightly packed together [10]: three E protein dimers lie parallel to each other forming a raft , and thirty such rafts are organized into a herringbone pattern ( S1 Fig ) . There are extensive interactions between the E proteins in the intra-dimer , inter-dimer and inter-raft interfaces . However , in the bumpy particles , the increased temperature loosens the tight interactions between the E proteins breaking all inter-dimer , inter-raft and some intra-dimer interactions [8 , 9] ( S1 Fig ) . This results in the E proteins moving outwards , forming a larger diameter virion particle [8 , 9] . The quaternary arrangement of the E proteins on the smooth and bumpy surface particles are therefore very different from each other . It was shown previously that the highly neutralizing antibodies against the smooth particles mainly bind across surface exposed regions of the E proteins at the inter-dimer interface , locking the E proteins together and preventing the structural reorganization necessary for fusion of the virus with the endosomal membrane [11] . However , in the bumpy particles , the inter-dimer relationship between the E proteins is completely broken; these types of antibodies may therefore not be able to bind and neutralize the virus [8 , 12] . The individual E proteins on the bumpy surfaced particles exhibit higher solvent accessibility than the smooth particles; as a result , antibodies binding to lower-order E protein assemblies such as the dimer or monomer could then be more effective [12–14] . Conversely , antibodies binding to these lower-order E protein assemblies may not bind to the smooth particles well , as the epitope may be partially or completely hidden due to the tight interactions between the E proteins . This may also potentially lead to lower antibody occupancy , increasing the chance of ADE . The different morphologies of mature DENV2 therefore impart very different antigenic properties . Despite the morphological differences , both viruses are infectious [8] and thus may pose an additional complication for vaccine development . Here we sought to answer the following questions: ( 1 ) Which E protein residue ( s ) have an impact on conferring the ability of DENV2 to change its morphology at 37°C , and how does this happen at the molecular level ? ( 2 ) Since the bumpy particles were observed in laboratory adapted DENV2 strains , do they exist in clinical isolates ? ( 3 ) If the clinical isolate does become bumpy , what temperature is required for this structural transition ? Answering these questions should inform on strategies for vaccine and therapeutics development . Here we identified two DENV2 New Guinea C ( NGC ) strains with different passage histories from two different laboratories; one is routinely passaged in BHK-21 , while the other is passaged in C6/36 cells , showing smooth or bumpy surface morphologies at 37°C , respectively . Comparison of these two strains showed five relatively chemically conserved amino acid changes . A series of E protein mutations were performed on the infectious clone of the smooth DENV2 NGC particle . We showed that subtle mutations at several places can make the virus bumpy at 37°C . Examination of four clinical strains showed three with smooth surface structures at 37°C , and all became bumpy at 40°C . We identified two distinct residues that when individually mutated , switched the virus to bumpy surfaced at 37°C; these residues exist at the intra-dimer interface , and molecular dynamics simulations revealed how they destabilized the E protein intra-dimeric interactions at the atomic level .
CryoEM micrographs and 2D-averages of the particles of two DENV2 NGC strains from different labs ( with different passage histories ) ( Fig 1A ) , hereafter named NGC-1 and NGC-2 , showed the surface of NGC-1 particles mostly remains smooth with some broken particles at 37°C , while NGC-2 particles turned bumpy ( S5A Fig , S6 Fig ) . Comparison of their E protein sequences showed five differences at amino acid positions 6 , 71 , 112 , 124 and 402 ( Fig 1B ) . These differences do not involve drastic changes in the electrostatic characteristics of the residues . At positions 6 , 71 and 402 , the differences between NGC-1 and NGC-2 are in the bulkiness of residues while maintaining their charge and polarity: I6M , D71E and I402F . G112S results in an increased in hydrophilicity , whereas conversely , N124I causes an increase in hydrophobicity . In the 3 . 5Å resolution DENV2 cryoEM structure [15] , the side chains of residues 71 and 124 are not involved in E-to-E interactions and are projected towards the outside environment ( Fig 1B ) . Residue 402 is present on the E protein helical stem region and is facing the viral lipid membrane . None of the five residues exist at the E protein inter-dimer or inter-raft interfaces ( Fig 1B ) in the smooth surfaced DENV2 structure[15] . Only residue 6 lies at the E protein intra-dimer interface . To identify the determinant ( s ) that enable the particles to turn bumpy at 37°C , we conducted mutagenesis studies to change all five residues ( positions 6 , 71 , 112 , 124 and 402 ) on the E protein of the smooth surfaced NGC-1 infectious clone to those observed in NGC-2 ( Fig 1C ) . Simultaneous substitution of all five residues ( I6M , D71E , G112S , N124I and I402F ) in an M1 mutant showed that the particles turned bumpy at 37°C ( Fig 1D ) . Since the M1 mutant contains the E protein sequence identical to NGC-2 , but the rest of the structural and non-structural proteins belong to NGC-1 , this suggests that its ability to become bumpy is solely due to determinants on the E protein . M2 , M3 and M4 mutants ( Fig 1C ) were generated and all resulted in more particles becoming bumpy at 37°C ( Fig 1D , S5 Fig , S7 Fig ) . In the M2-4 mutants , residue 124 was not mutated because this residue is facing outwards and not involved in any E-E protein interactions . M2 contains four substitutions ( I6M , D71E , G112S and I402F ) , and showed a bumpy surface ( Fig 1D , S5 Fig , S7 Fig ) indicating that one or a combination of these mutations is important for causing the change in morphology . M3 , which carries three ( D71E , G112S and I402F ) of the four substitutions in M2 , showed a bumpy surface but the particles were more structurally unstable , as more broken particles were observed compared to the other mutants . This suggests that the extra substitution at residue 6 in M2 compared to M3 leads to the stabilization of the bumpy particle . Interestingly the M4 mutant , which contains only one mutation , I6M , also turned the smooth surface virus into bumpy particles . In conclusion , there are redundancies in substitutions in various positions that turn the virus bumpy , and only subtle changes in the chemical characteristics of the residues are required . To test the thermal stability of the DENV2 NGC mutants , we pre-incubated the viruses at different temperatures ( 4°C , 29°C , 37°C and 40°C ) for 30 mins before conducting plaque assay at 29°C using BHK-21 cells . These temperatures were chosen to mimic the physiological temperature in the mosquitoes ( 29°C ) , human host ( 37°C ) and also when humans are experiencing high fever ( 40°C ) . The percentage of plaques , for each incubation temperature of virus , were calculated by comparing to the same virus pre-incubated at 4°C . For the DENV2 NGC strains ( NGC-1 and NGC-2 ) and mutants ( M1-4 ) ( Fig 2A ) , all showed similarly high stability when pre-incubated at 4°C and 29°C . At 40°C , they were all equally unstable . The most obvious differences in stability between NGC-1 compared to the other viruses occurred at 37°C , with the NGC-1 strain significantly more stable than the others . This is correlated with their differences in morphologies between NGC-1 ( smooth ) and the other viruses ( bumpy ) at 37°C , while at 29°C and below , they have smooth surfaces . Separate growth curve experiments were performed ( Fig 2C ) , where NGC-1 , M1 , M3 and M4 mutants ( without pre-incubation at respective temperatures ) were allowed to infect mammalian cells ( HuH-7 , BHK-21 ) at 37°C , and mosquito cells ( C6/36 ) at 29°C . All viruses were infected at the same multiplicity of infection ( MOI ) of 0 . 1 . The growth curves were followed by taking samples at different time points and the amount of virus in the supernatant determined by either plaque or focus-forming assays , and real-time polymerase chain reaction ( RT-PCR ) . 12 hours post infection of virus in HuH-7 and BHK-21 cells ( 37°C ) showed similar quantities of virus by RT-PCR , suggesting that they bind to the cells with equal efficiencies . However , starting at 24 hours post-infection , both plaque and RT-PCR started to show differences in growth rate between the NGC mutants , largely with the smooth surfaced NGC-1 growing slightly slower than the bumpy viruses ( M1 and M4 ) . However , the smooth surfaced NGC-1 virus and the bumpy surfaced M3 virus growth curves largely overlap in the mammalian cell , which may be due to the structural instability of the M3 mutant as observed by cryoEM ( Fig 1D ) . When tested in C6/36 cells where virus is grown at 29°C , all viruses have similar growth curves; this is expected as they all have smooth surfaces at this temperature . Together , the results suggest that morphological differences between mutants at 37°C likely play an important role in affecting their growth . Interestingly , the NGC-1 and M4 strains in mammalian cell lines ( HuH-7 and BHK-21 ) showed the biggest differences in their growth rate , yet they differ by only one residue ( I6M ) . Not considering M3 , as the particles were structurally unstable , the bumpy particles formed by the M1 and M4 mutants seemed to have slightly higher growth rates than that of the smooth surface NGC-1 strain in mammalian cells . In conclusion , the smooth surface particles ( NGC-1 ) seemed to be more thermally stable than the bumpy particles ( NGC-2 and M1-4 ) . In terms of growth rate in mammalian cells , the bumpy particles ( M1 and M4 ) showed slightly faster growth rates than the smooth particle ( NGC-1 ) while in C6/36 cells where all viruses are grown at 29°C , they would have similar smooth surface morphologies and thus could lead to the observed similar growth rate . Previous studies showing DENV2 particles [8 , 9] that can become bumpy at 37°C were mostly observed in laboratory adapted strains: 16681[9] , NGC [8] , and WRAIR strain S16803[8] . We tested the naturally circulated DENV2 strains for their morphological changes at both 37°C and 40°C . Four clinical strains ( 05K4155 , PVP94/07 , PVP103/07 , SL56 ) were used , three were isolated from Singapore and one from Sri Lanka . PVP94/07 and PVP103/07 were both isolated in the same year ( 2007 ) in Singapore suggesting they are from the same outbreak , whereas the rest are from different outbreaks ( Table 1 ) . The viruses were grown in C6/36 cells; the part of the viral genome encoding prM-E of the particles used for cryoEM were sequenced and showed that limited passages ( ~4–7 passages ) did not result in additional mutations compared to the original viruses . Particles of DENV2 PVP94/07 , 05K4155 and SL56 strains exhibited a smooth surface morphology at 37°C whereas PVP103/07 appeared bumpy ( Figs 3A , S5B and S8 ) . This suggests that the clinical strains can adopt both morphologies . When heated to 40ºC mimicking high fever conditions , all the clinical strains became bumpy ( Figs 3A , S5B and S8 ) . Thermal stability tests of the clinical strains at 37°C were done ( Fig 2B ) , and the strains were ranked from the most to the least stable in the following order: the smooth surfaced strains PVP94/07 , 05K4155 , SL56 , and finally the bumpy surfaced PVP103/07 . This shows a similar trend as that observed with the NGC mutants , suggesting that the bumpy particles of clinical strains at 37°C likely cause them to be less stable than the smooth particles . Interestingly , DENV2 PVP94/07 and PVP103/07 which were isolated within the same year are different in morphology: PVP94/07 ( smooth ) and PVP103/07 ( bumpy ) , yet their E protein sequences differ by only one amino acid , at position 262: threonine in PVP94/07 and methionine in PVP103/07 ( B ) . Therefore , one could be a mutant of the other . The T262M substitution between PVP97/07 and PVP103/07 is located at the intra-dimer E protein interface ( Fig 3B ) . Interestingly , the mutation of a single residue , I6M , in the NGC-1 E protein ( Fig 1C ) , which is also located at the intra-dimer interface , also changed the smooth surfaced virus to bumpy . This suggests that destabilization of the E protein dimer may play an important role in controlling the morphology of particles at 37°C . There is only one amino acid difference in the E protein , T262M , between PVP94/07 ( smooth ) and PVP103/07 ( bumpy ) strains . This allows us to study the difference in the neutralization profile of various antibodies to viruses that have different morphologies but minimal amino acid sequence variation in their E protein . We used antibodies that have been shown to be neutralizing and bind to different regions on the E protein . Monoclonal antibodies ( MAb ) 4G2 [16 , 17] and 4 . 8A [18] were shown to bind to E protein domain II . The epitope for 4G2 is thought to be partially hidden on the smooth virus surface [16] . Epitopes bound by MAbs 1A1D-2 [19] , 2D22 [12] and C10 [20] had been previously shown by crystallography or cryoEM . MAb 1A1D-2 bind to domain III and the epitope is partially exposed on the compact smooth virus surface [19] while the MAb 2D22 and C10 recognize epitopes across the E protein dimer ( EDE antibodies ) . MAb C10 when exposed to soluble E proteins can assemble the monomeric E proteins into dimers [20] . We tested their neutralization profiles to DENV2 PVP94/07 ( smooth ) and PVP103/07 ( bumpy ) strains . MAbs 4G2 , 4 . 8A and 1A1D-2 have differing neutralizing capabilities to the different virus morphologies . They neutralized the bumpy surface virus better than the smooth surfaced one ( Fig 4A–4C ) , suggesting that their epitopes are more accessible in the bumpy surface morphology . The EDE antibodies 2D22 and C10 , on the other hand , neutralized both morphologies equally well ( Fig 4D and 4E ) . We characterized the efficiencies of the smooth ( PVP94/07 ) and bumpy ( PVP103/07 ) surfaced DENV2 to attach to cells , and also to fuse with liposomes—steps that mimic the early cell entry process . To determine the cell attachment capabilities of the smooth ( PVP94/07 ) and bumpy ( PVP103/07 ) surfaced DENV2 ( virus structural changes were induced by incubation at 37°C ) , we determined the amount of these viruses bound to 4°C pre-cooled Aedes albopictus C6/36 and human HuH-7 cells by quantitative RT-PCR . Results showed there were slightly less bumpy PVP103/07 particles bound to C6/36 cells compared to the smooth PVP94/07 DENV2 , however , their difference is not statistically significant ( Fig 4F ) . As for HuH-7 cells , we detected significantly larger amounts of bound PVP103/07 compared to PVP94/07 ( Fig 4F ) . We also characterized the ability of the smooth ( PVP94/07 ) and bumpy ( PVP103/07 ) surfaced DENV2 to fuse with liposomes at pH 5 . 0 . We detected higher levels of fusion of PVP103/07 to liposomes than PVP94/07 ( Fig 4G ) . Together the results suggest that the bumpy surfaced virus is better than the smooth surfaced virus particles in attaching to cells and fusing with the endosomal membrane during cell entry . These observations are consistent with the higher growth rate of the bumpy NGC M4 mutant , which has a substitution at amino acid position 6 of the E protein ( also at the E protein dimer interface ) , than the smooth surfaced NGC-1 in HuH-7 cells ( Fig 2C ) . We have shown that the single point mutation I6M in the DENV2 NGC-1 E protein changes the surface of the virus to bumpy at 37°C ( Fig 1 ) . In order to explore the structural basis for this change , we have employed atomically detailed , explicitly solvated molecular dynamics ( MD ) simulations of the NGC-1 ( EI6 ) and M4 ( EM6 ) states using the ~800 residue E protein ectodomain dimers for EI6 and EM6 at 37°C ( see details in Methods ) . The N-terminal loop encompassing residues 1–10 retained its cryoEM determined dimeric conformation in EI6 over the 300 ns simulation trajectory ( S2 Fig ) . The I6 side chain of both E protein protomers within a dimer remained embedded within the interior of this well-defined turn throughout the simulations ( Fig 5A and 5C ) . In contrast , a conformational change was observed in each N-terminal loop of the EM6 protomers within the dimer within <100 ns ( S2 Fig ) . This was due to the presence of the bulkier sulfur atom of the methionine thioether group , which led to its sidechain flipping outwards ( Fig 5B ) , influencing the local secondary structure of the loop ( Fig 5B and 5D ) . In chain A , the M6 substitution led to an extended loop structure , while that in chain B led to the formation of a helical structure ( Fig 5B and 5D ) . The latter is in agreement with the propensity for methionine to favour α-helical conformations [21] . This “local switch” in the I6M mutant also caused spontaneous global changes across the E protein dimer , highlighted by filtering the “noise” from the trajectory using principal component analysis ( PCA ) in order to visualize the largest-amplitude motions , as shown in S1 Movie . Domain I was observed to shift “outwards” with respect to domain III of each chain ( Fig 5 ) , and domain II also then reoriented leading to a change in the relative position of the two antiparallel helical segments at the centre of the dimeric complex . This would be expected to disrupt the symmetry-related contacts on the smooth virus surface , leading to it becoming bumpy at 37°C . Comparison of DENV2 clinical isolate PVP94/07 with PVP103/07 showed the T262M substitution in the E protein resulted in the virus becoming “bumpy” at 37°C ( Fig 3 ) . We employed a similar MD simulation protocol as above to study the dimer stability of both ET262 and EM262 systems . The ET262 showed a stable conformation relative to the cryoEM structure over 300 ns ( S2 Movie ) . The relative orientation of the two E protein chains was well maintained ( Fig 6A ) , while the two antiparallel helical segments encompassing residues 250–265 at the centre of the dimeric complex retained their experimentally defined interface in terms of buried surface ( Fig 8A ) and structure ( Fig 6A and 6C ) throughout the simulation time , with the T262 side chains of opposing chains separated by ~1 . 6 nm ( Fig 6A , 6C and 6E ) . A persistent intra-helical hydrogen bond between the T262 side chain hydroxyl and the G258 backbone carbonyl contributed to the stability of this helical region ( Fig 6F ) . In contrast , EM262 was significantly less stable compared to the ET262 ( S3 Movie ) . The loss of the intra-helical hydrogen bond between M262 and G258 led to the rearrangement into a new interface that was stable over hundreds of nanoseconds ( Fig 6B and 6D ) . This was accompanied by the formation of a hydrophobic interaction between the side chains of the two M262 residues each from an opposing chain , with a final separation of ~0 . 25 nm ( Fig 6E ) , as indicated by the total buried area between the central helical segments ( Fig 8A ) . This resulted in a large-scale conformational change in the E protein dimer , as illustrated via PCA ( Fig 8B and S4 Movie ) . Thus , in both protomers , domain II shifted outwards with respect to domains I and III , while domain III in only one of the protomers ( Fig 6B ) was observed to turn towards domain II of the other protomer ( Fig 6B ) . These changes are expected to disrupt the symmetry-related contacts in the smooth compact virus structure , leading to the virus turning bumpy as observed in our cryoEM micrograph of the respective clinical isolates ( Fig 3 ) .
Changes in DENV2 morphology will influence its recognition by antibodies . Questions on whether this morphological diversity occurs in those circulating in nature , and if primary E protein sequence analysis alone can accurately predict the morphology , are important for vaccine and therapeutics design . To answer the question on whether primary sequence can inform on the morphology of DENV2 , we mutated different individual or sets of multiple residues of the E protein of the bumpy surface DENV NGC ( NGC-2 ) into the infectious clone of the smooth surfaced NGC ( NGC-1 ) virus . The results showed that most mutations—even chemically subtle ones—changed smooth viruses into bumpy surfaced ones , suggesting multiple regions of the virus E protein could destabilize the surface . Since subtle mutations at different positions can have an effect on the DENV2 morphology , it is therefore unlikely that one could predict with confidence , from the primary sequence alone , the morphology of DENV2 . Goo et al . , 2017 [22] , showed yet another mutation , T198F ( from small polar side chain ( T ) to bulky hydrophobic side chain ( F ) ) on the DI-DII hinge , not included in our study , that makes a previously cryptic epitope on DIII more accessible on both WNV and DENV1 for antibody binding . The authors suggested that due to the mutation , the virus likely becomes bumpy , thereby increasing the accessibility of the epitope on DIII for antibody neutralization . However , they did not show the morphology of the virus with cryoEM . The I6M mutation that changes the smooth surface NGC strain into the bumpy form ( M4 mutant ) ( Fig 1 ) , and the T262M difference between clinical isolates ( PVP94/07 and PVP103/07 ) ( Fig 3 ) that results in PVP103/07 having bumpy surface at 37°C , are located at the interface of the E-E protein dimeric interactions . MD simulations showed how these substitutions ( Fig 7 and Fig 8 ) destabilized the E protein dimer and they may then cause larger scale changes in the quaternary arrangements of the E proteins on the smooth virus particle surface . In this study , we were able to observe one laboratory-passaged DENV2 NGC strain from an infectious clone , having a smooth surface morphology . In contrast , other laboratory-adapted strains ( WRAIR strain S16803 , 16681 and our lab DENV2 NGC ) were shown to have a bumpy surface morphology at 37°C . As for the clinical isolates examined , three out of four strains have smooth surface at 37°C . This may suggest that there could be a selective pressure in vivo for smooth surfaced particles . Thermal stability tests of the NGC mutants showed the smooth surfaced virus ( NGC-1 ) is the most thermally stable virus compared to the bumpy surfaced mutants at 37°C ( Fig 2A ) . Previous studies comparing dengue viruses to Zika virus , which has a smooth surface even at 40°C , showed Zika virus retains infectivity after incubation at 40°C while the DENV viruses are comparatively much lower in infectivity at elevated temperatures . This correlation suggests the smooth viral surface imparts thermal stability [23] . However , other than the difference in their surface morphologies , these viruses also have significant sequence differences in other parts of the genome , thereby adding more variability in the interpretation of the data . The latter possibility could be excluded by a previous study using chimeric Zika and DENV , showing that when the viral structural prM-E genes from these viruses were switched , this also switches their thermostability characteristics [24] . Although the smooth NGC-1 particles are more thermally stable ( Fig 2A ) , the growth curve ( without pre-incubating virus at respective temperatures ) ( Fig 2C ) in mammalian cells showed them to have a slightly slower growth rate than the bumpy ( M1 and M4 ) particles at 37°C . This correlates with our attachment and fusion assay results comparing the smooth PVP94/07 and bumpy PVP103/07; the PVP94/07 particles were shown to have a lower extent of attachment to human cells and also reduced fusion to liposomes ( Fig 4F and 4G ) . We speculate that perhaps in the host , the cells for DENV are not readily available , and the viruses may need to travel a distance to find their next target cell and therefore , their thermal stability at 37°C in the absence of host cells becomes a more important factor than the growth rate in infected cells , leading to selection of the smooth particles . In the lab , we generally passage DENV in C6/36 cells at 29°C , and at this temperature , all viruses ( regardless of whether they carry substitutions that lead to a bumpy surface at 37°C ) will have smooth surfaces , and therefore the selection pressure is low . We have shown previously that the DENV2 NGC strain ( NGC-2 ) , when incubated at 37°C , contains four structural classes of particles—one class is the smooth surface particles , while the other four classes are expanded particles that looked bumpy . Since our thermal stability tests showed that the smooth surface particles are more stable , it could be possible that one can select for smooth particles by pre-incubating the NGC-2 at 37°C for 30 mins before infecting BHK-21 cells at 37°C; however , this may require multiple passages . Co-incidentally , DENV2 NGC-1 which has a smooth surface , has been continuously passaged in BHK-21 cells at 37°C . Our studies showed that the in vitro versus the in vivo passaged virus may display largely different morphologies and comparison of their E protein sequences showed some residue differences ( S3 Fig ) . Although they have largely chemically conserved residues , our studies showed even subtle changes can cause major morphological changes . This suggests that the use of lab adapted strains may not be completely representative of the viruses naturally circulating when studying antigenicity . This was also shown in a study by Chaichana et al . [25] in which it was demonstrated that laboratory-adapted DENV2 strain 16681 displayed higher levels of ADE compared to patient-derived DENV2 . Leah et . al . [26] using human sera neutralization cartography have also shown that strains within a serotype can be as antigenically diverse as between DENV serotypes . We therefore suggest that laboratory experiments should use seed virus cultures derived from infectious clones of the clinical isolates displaying different morphologies , so as to ensure translatability of the results to viruses in nature . We detected that limited passaging ( 4–7 passages ) in C6/36 cells of the clinical isolates did not cause any E protein mutations in the virus , however , extended passaging should best be avoided . The capability of antibodies to neutralize viruses may depend on the accessibility of the respective epitope ( s ) , which will vary depending on the virus surface morphology . For example , the E protein fusion loop is only partially exposed on the smooth surface DENV and therefore , antibodies targeting this region can only achieve partial occupancy which may lead to ADE [27 , 28] , whereas on the bumpy particles , this region is much more accessible , allowing antibody neutralization . Antibodies that are highly neutralizing against the smooth surfaced DENV1 [13 , 29] , DENV3 [11] and WNV [30] particles have been shown to bind across E protein dimers , a conformation highly dependent on the preservation of the quaternary structure of the smooth surface virus . These antibodies would not be able to bind to the bumpy particles as the E proteins are arranged differently . Indeed , comparison of the neutralization activities of several antibodies ( that bind to different regions of the E proteins ) to the smooth PVP94/07 and bumpy surfaced PVP103/07 DENV2 clinical isolates , ( Fig 4A–4E ) showed that they can have different potencies against different virus morphologies . We showed here that for the four clinical strains , three have a smooth surface while one is bumpy at 37°C; this suggests that for vaccine development , DENV2 should include both morphologies so that the vaccine may then elicit protective antibody responses to all morphologies . As for antibody therapy , for prophylactic use where the patient body temperature is at 37°C , an antibody mixture that binds efficiently to both smooth and bumpy particles should be used . For therapeutics , where usually the patient is experiencing fever , our cryoEM data shows that all clinical strains at 40°C appear to have bumpy surfaces , suggesting that antibodies that bind efficiently to lower-order assemblies of E proteins ( monomers or dimers ) should be used .
Aedes albopictus clone C6/36 ( American Type Culture Collection ( ATCC ) CRL1660 , USA ) and BHK-21 ( ATCC CCL10 ) cells were cultured in RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum ( FBS ) ( Gibco , Brazil ) and incubated in 5% CO2 at 29°C and 37°C respectively . HuH-7 ( Japanese Collection of Research Bioresources ( JCRB ) Cell Bank JCRB0403 , Japan ) cells were cultured in DMEM medium supplemented with heat-inactivated 10% FBS and incubated in 5% CO2 . DENV2 clinical isolates were from the Early Dengue infection and outcome study ( EDEN ) ( 05K4155 , PVP94/07 and PVP103/07; Singapore ) [31] and Aravinda de Silva ( SL56; Sri Lanka ) . The general procedure for construction of DENV cDNA has been described elsewhere . [32] . Briefly , a single E protein mutation was first introduced into a shuttle vector using a QuikChange III XL site-directed mutagenesis kit ( Stratagene ) . The mutated DNA fragments from the shuttle vectors were sequenced and then swapped into the pACYC-FL-NGC cDNA clone through . For triple or pentadruple mutations ( D71E-G112S-I402F or I6M-D71E-G112S-N124I-I402F ) , multiple rounds of mutagenesis were performed to engineer all mutations into a shutter vector . Each round of mutagenesis introduced one mutation into the shutter vector and the introduced mutation was confirmed by sequencing . The fragments containing the mutations were then engineered into the pACYC-FL-NGC plasmid by using restriction enzymes BsrGI and NheI . All cDNA plasmids were again verified by DNA sequencing . The assay protocols used were described previously [32] . Briefly , RNAs were transcribed from linearized full-length cDNA plasmids using T7 mMessage mMachine Transcription kit ( Thermo Fisher Scientific ) and then electroporated into BHK-21 cells . Viral E protein synthesis in transfected cells was monitored by IFA with DEN-immune mouse 4G2 ( American Type Culture Collection ) and anti-mouse immunoglobulin G conjugated with fluorescein isothiocyanate ( FITC ) as the primary and secondary antibodies , respectively . The culture fluid was harvested on day 4 or 5 post transfection and stored at -80°C for subsequent plaque assays . The method for virus production and purification has been described previously [34] . The clinical isolates used in this study were passaged not more than 7 times . Briefly , all the strains of DENV2 were propagated in Aedes albopictus C6/36 cells at 29°C at 5% CO2 . The infection was carried out at a confluency of 80% and at MOI of 0 . 1 for 2h . This was followed by the replacement of the inoculum with fresh media containing 2% FBS . The viruses were harvested 4 days post-infection and centrifuged to remove cell debris . Precipitation of the virus from the media by 8% w/v polyethylene glycol 8000 in NTE ( 10mM Tris-HCl pH8 , 120mM NaCl and 1mM EDTA ) was carried out overnight at 4°C followed by centrifugation through a 24% w/v sucrose cushion . This was further purified using a 10 to 30% w/v potassium tartrate gradient . The virus band was then extracted , concentrated through a 100k-Da filter and buffer exchanged to NTE buffer . The virus purity was verified using Coomassie Blue-stained SDS-PAGE gel to ensure suitability for cryoEM imaging . The clinical isolate strains were adjusted to pH7 . 4 using 1M Tris-Cl pH7 . 4 at a final concentration of 100mM Tris-HCl . The various strains of DENV2 were aliquoted and incubated at their respective temperature ( 4°C , 37°C or 40°C ) for 30 min followed by ~2h at 4°C . Subsequently , 2 . 2 μL of the virus sample was pipetted to a carbon-coated lacey carbon grid ( Ted Pella ) , blotted with filter paper , snap frozen in liquid ethane using FEI Vitrobot Mark IV and stored in liquid nitrogen . The virus particles were imaged using a Titan Krios cryo-electron microscope equipped with a field emission gun of 300kV . The images were detected using a direct electron detector ( Falcon , FEI ) and collected manually . Images were collected at 47 , 000x magnification with pixel size of 1 . 71 Å . Contrast transfer function parameters were estimated using the Gctf programme[35] . For each image dataset , particle picking was done manually using the e2boxer in EMAN2 [36] and then 2D classification was done on these particles using Relion2 . 1[37] . The preparation of viral genome for sequencing was modified from Christenbury JW et al ( 2010 ) . Briefly , viral RNA was extracted from purified virus using QIAamp Viral RNA Mini Kit in accordance to manufacturer’s protocol . cDNA was synthesized from the viral RNA using SuperScript III First-Strand Synthesis System according to manufacturer’s protocol with primer D2a5B [38] . Subsequently , the cDNA was amplified in a polymerase chain reaction ( PCR ) with high fidelity PfuUltra II Fusion HS DNA polymerase using previously reported primers d2s1C and d2a18 [38] . The PCR products were then run in a 1% agarose gel containing 1X SYBR Safe and visualized . Upon determining the absence of non-specific products , the PCR products were purified using Qiagen MiniElute PCR Purification Kit . Purified PCR products were then quantified using Nanodrop . The purified PCR products were submitted for single pass DNA sequencing using primers listed in S1 Table that covers overlapping regions . Primers used in DNA sequencing were designed from the multiple sequence alignment of DENV2 sequences available in NCBI Nucleotide database . Chromatograms obtained from DNA sequencing were analysed in FinchTV software . The sense sequences were verified against the antisense sequences . The regions that encoded for prM and E protein were extracted and translated using ExPASy translate tool . Sequence alignment was carried out on the translated sequences using Clustal Omega . Each virus strain was diluted to about 500 PFU/ml and split to four aliquots , followed by incubation at 4°C , 29°C , 37°C and 40°C respectively for 30 min . Subsequently , each virus strain at the different temperatures were used to infect BHK-21 seeded in 24-well plates for 1 . 5 hour at 29°C before washed with phosphate-buffered saline ( PBS ) , overlaid with carboxy-methyl cellulose and incubated at 37°C . After 3–5 days , the cells were fixed and stained . Plaque forming units ( PFU ) were then tabulated as percentages to that of the control ( 4°C ) . The neutralization activities of MAb 4G2 , 4 . 8A , 1A1D2 , 2D22 and C10 to clinical isolates PVP94/07 and PVP103/07 were determined by PRNT . Both DENV2 strains PVP94/07 and PVP103/07 were pre-incubated at 37°C for 30 min to induce structural changes before they were exposed to MAbs . Two-fold serial dilutions of each MAb were done and then they were incubated with equal volumes of virus at 37°C for 30 min . 100 μL of these mixtures were layered on BHK-21 cells in 24-well plates and incubated at 37°C for 1 h . The infected cells were washed with PBS , overlaid with carboxyl-methyl cellulose and then further incubated at 37°C for 3–4 days . Cells were fixed and stained . Percentage neutralization was determined from the comparison of the number of plaque forming units ( PFU ) in each antibody dilution to the control without MAbs . PRNT50 is the concentration of the antibody that results in 50% reduction in PFU . HuH-7 ( Japanese Collection of Research Bioresources ( JCRB ) Cell Bank JCRB0403 , Japan ) , BHK-21 and C6/36 cells were seeded into 12-well plates . The next day , the cells were infected with DENV WT and mutants at MOI of 0 . 1 . The MOI determined using plaque assay in BHK-21 was used for the assays on HuH-7 and BHK-21 cells . The MOI determined using focal forming assay ( FFA ) in C6/36 was used for the assays on C6/36 cells . After 1 h incubation , the virus inocula were removed . The cells were washed twice with phosphate buffered saline and cultured with 1 ml of fresh medium per well . The culture medium was collected at indicated time points and stored at -80°C . Virus titers were determined by using RT-PCR and either plaque assays ( samples from HuH-7 and BHK-21 cells ) or FFA ( samples from C6/36 cells ) . C6/36 was seeded in 24 well plate and incubated overnight . Virus samples were 10-fold serially diluted before infecting the cells for 1h at 29°C . The cells were then overlaid with carboxy-methyl cellulose and incubated at 29°C for 4 to 5 days . Thereafter , the overlay was removed and cells were washed with phosphate buffered saline before being fixed with 80% acetone . The cells were then blocked with 5% milk ( Anlene , New Zealand ) then incubated with 4G2 followed by horseradish peroxidase conjugated anti-mouse antibody . The foci were developed using 3 , 3'-diaminobenzidine substrate ( Dako ) . The focus forming unit ( FFU ) were counted and tabulated . DENV2 strains PVP94/07 and PVP103/07 were pre-incubated at 37°C for 30 min to induce structural changes . C6/36 and HuH-7 grown in 24-well plate were pre-cooled at 4°C before incubated with the DENV2 strains PVP94/07 and PVP103/07 at multiplicity of genome containing particles ( MOG ) of 10 , 000 at 4°C for 1 hour . After incubation , the cells were washed 3 times with cold PBS and RNA were extracted using RNeasy Mini Kit ( Qiagen ) . cDNA was synthesized using qScript cDNA supermix . Taqman real-time PCR was performed using a mixture of primers specific to virus and also housekeeping β-actin gene; listed in S2 Table [39–42] . The genome copies of the DENV2 strains were normalized to the housekeeping gene β-actin and the relative fold difference of the genome copies of the strain PVP103/07 to that of PVP94/07 was determined using the following 2-ΔΔCT method [43]: Relativechangeincopynumber=2−[ ( CT , PVP103/07−CT , β−actin ) − ( CT , PVP94/07−CT , β−actin ) ] Membranes of the purified DENV2 strains PVP94/07 and PVP103/07 were labelled with the lipophilic fluorescent probe 1 , 1′-dioctadecyl-3 , 3 , 3′ , 3′-tetramethylindodicarbocyanine , 4-chlorobenzenesulfonate ( DiD ) dye ( Invitrogen ) as described previously[44] . Briefly , virus was incubated at 28°C for 30 min with 1 mM DiD dye dissolved in DMSO . The DiD-labelled virus was filtered through a PD-10 desalting column ( GE Healthcare ) to remove unbound dye . Labelled virus was used within 2 days . Equal volumes of virus and liposomes were mixed on ice and diluted with 20 μl of 2% BSA in NTE . 60 μl of pre-chilled low pH buffer ( 50 mM MES ) was then added . The total volume of the sample is 100 μl . The fluorescence was recorded at excitation and emission wavelengths of 633 nm and 665 nm , respectively , using a Tecan Infinite M200 microplate reader every minute over 30 minutes at 37°C . The maximum amount of lipid mixing was determined by adding 20 μl of 2% of Triton X-100 and measuring the fluorescence . The concentration of virus used in the experiment was standardized based on the maximum amount of lipid mixing prior to the experiment . The extent of fusion reported is calculated by dividing the experimental fluorescence ( at t = 30 mins ) with the fluorescence after the addition of Triton X-100 and normalized to the ratio of the extent of fusion between the two viruses alone at pH 5 . 0 . The initial conformation used for simulations of the dimeric E protein ectodomain were obtained from the cryoEM structure of DENV2 in its mature state ( protein data bank entry: 3J27 [15] , residues 1–395 ) under conditions of neutral pH . For consistency with the experimentally studied strain PVP94/07 ( corresponding to the ET262 simulation system ) , several in silico point mutations ( E47K , Q52H , I61V , D71A , K126E , H149N , I164V , N390S ) were introduced using PyMol ( https://www . pymol . org ) . In addition , for strain PVP103/07 , the additional T262M mutation was introduced ( corresponding to the EM262 simulation system ) . Similarly , for strain NGC-1 ( corresponding to the EI6 simulation system ) , the mutation E47K was introduced , along with the additional I6M mutation for strain NGC-M4 ( corresponding to the EM6 simulation system ) . Each construct was placed in the centre of a cubic box ( 20x20x20 nm3 ) and solvated with approximately 120 , 000 TIP3P[45] water molecules . All simulations were performed using the Amber99SB*-ILDN-Q forcefield . [46] Ionizable residues and termini were treated in their fully charged state , with sodium ions added to neutralize the overall system charge . MD simulations were performed with GROMACS 5 . 0 . 2 . [47] Equations of motion were integrated with the Verlet leapfrog algorithm using a 2 fs time step . Bond lengths were constrained with the LINCS algorithm . [48] The cutoff distance was switched from 0 . 9 to 1 . 2 nm for the short-range neighbour list and van der Waal’s interactions . The Particle Mesh Ewald ( PME ) [49] method was applied for long-range electrostatic interactions with a 1 . 2 nm real space cutoff . The velocity rescale thermostat with an additional stochastic term[50] and Parinello-Rahman[51] barostat were used to maintain the temperature at 37°C and pressure at 1 bar , respectively . Initial velocities were set according to a Maxwell distribution . Periodic boundaries were applied in all directions . Initial configurations were minimized using the steepest descent algorithm , followed by equilibration with position restraints on protein heavy atoms with a force constant of 1 , 000 kJ mol-1 nm-2 in the NVT and subsequently NPT ensembles , for 5 ns and 10 ns , respectively . Production runs for each system were generated for 300 ns in the NPT ensemble , without restraints . Simulations were performed on an in-house Linux cluster of 7 nodes comprised of 2 GPUs ( Nvidia K20 ) and 20 CPUs ( Intel Xeon CPU E5-2680 v2 @ 2 . 8 GHz ) each or the National Supercomputing Centre Singapore ( http://www . nscc . sg ) using 4 nodes comprised of 1 GPU ( Nvidia K40t ) and 24 CPUs ( Intel Xeon CPU-E5-2690 v3 @ 2 . 60 GHz ) each . Secondary structure was analysed using VMD software . [52] Covariance analysis and PCA[53] was performed for all Cα atoms in each 300 ns trajectory . The first principal component accounted for ~50% of the total variance in each case . Hydrogen bonds were measured based on a donor–acceptor distance cut-off of 0 . 35 nm and a hydrogen–donor–acceptor angle cut-off of 30° . All structural analyses were performed using tools within the GROMACS and VMD packages . Data were analysed and graphs were generated using Graphpad Prism 6 software . Paired student t-test was used to determine significance . Sequence alignment was done using Multialn[54] and depicted using ESPript[33] . Structural analysis was carried out using USCF Chimera [55] on cryoEM structures of compact ( PDB accession code 3J27 ) and expanded DENV2 ( PDB accession code 3ZKO ) . | DENV2 particles have been shown to change their morphologies ( compact smooth to loose bumpy surfaced ) when temperature is switched from 28°C to 37°C . We used two DENV2 viruses both belonging to the same strain designation but with a different passage history—one of which exhibited the smooth surfaced morphology while the other was bumpy surfaced , observed by cryoEM . We mutated residues in the E protein of the DENV2 infectious clone that has the smooth surfaced morphology to determine if any could result in a bumpy morphology . Results showed several different mutations could lead to this change . Using molecular dynamics simulations , we showed how these mutations likely destabilize the E protein dimeric interactions . We investigated whether the bumpy morphology also occurs in DENV2 clinical isolates , and showed that these viruses can exhibit both morphologies , indicating that vaccine and therapeutics development should target both virus forms . | [
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] | 2019 | Molecular basis of dengue virus serotype 2 morphological switch from 29°C to 37°C |
Filamentous actin ( F-actin ) and non-muscle myosin II motors drive cell motility and cell shape changes that guide large scale tissue movements during embryonic morphogenesis . To gain a better understanding of the role of actomyosin in vivo , we have developed a two-dimensional ( 2D ) computational model to study emergent phenomena of dynamic unbranched actomyosin arrays in the cell cortex . These phenomena include actomyosin punctuated contractions , or "actin asters" that form within quiescent F-actin networks . Punctuated contractions involve both formation of high intensity aster-like structures and disassembly of those same structures . Our 2D model allows us to explore the kinematics of filament polarity sorting , segregation of motors , and morphology of F-actin arrays that emerge as the model structure and biophysical properties are varied . Our model demonstrates the complex , emergent feedback between filament reorganization and motor transport that generate as well as disassemble actin asters . Since intracellular actomyosin dynamics are thought to be controlled by localization of scaffold proteins that bind F-actin or their myosin motors we also apply our 2D model to recapitulate in vitro studies that have revealed complex patterns of actomyosin that assemble from patterning filaments and motor complexes with microcontact printing . Although we use a minimal representation of filament , motor , and cross-linker biophysics , our model establishes a framework for investigating the role of other actin binding proteins , how they might alter actomyosin dynamics , and makes predictions that can be tested experimentally within live cells as well as within in vitro models .
Dynamic actomyosin networks play a critical role in development by providing motive forces for cell shape change and morphogenesis , and by establishing tissue mechanical properties [1–3] . For instance , actomyosin can form a contractile actin purse-string , a rope-like structure of bundled F-actin spanning multiple cells at the margin of the lateral epidermis that contracts and contributes to dorsal closure in Drosophila [4–6] . Contractile actomyosin networks in the medioapical domain of epithelial cells can also drive cell shape change leading to bending of epithelial sheets during gastrulation in Drosophila [7] . In addition to regulating force or stress production actomyosin is responsible for establishing the mechanical properties of the embryo that resist stress and guide tissue deformation . For instance , actomyosin controls much of the viscoelastic properties of Xenopus during gastrulation and neurulation as dorsal axial tissues converge and extend [8–10] . On the cellular scale , the biomechanical function of actomyosin is a direct target of many signaling pathways that pattern cell identities and behaviors in the embryo . For instance , Wnt-signaling during mediolateral cell intercalation appears to control force production and stiffness by regulating F-actin polymerization and myosin II contractility [11 , 12] . Given the importance of actomyosin networks and their relevance to most , if not all , morphogenetic processes during development , we know a great deal about their composition and molecular-scale processes . By contrast , surprisingly little is known about the mechanisms that coordinate the large-scale spatial and temporal dynamics of actomyosin network assembly and contraction during morphogenesis . Live imaging of fluorescently tagged F-actin and myosin regulatory light chain have revealed that actomyosin networks in the cell cortex are very dynamic , forming transient structures that turn-over or remodel in minutes [13–16] . Unbranched F-actin networks within the cell cortex are both less dense and less organized than F-actin in lamellipodia or circumapical junctions . Time-lapse sequences using fluorescent proteins conjugated to actin-binding domains from moesin or utrophin , or minimal synthetic actin-binding domains ( e . g . life-act ) reveal dynamic heterogeneous arrays of F-actin . Aster-like structures are often observed in these time-lapse sequences . Such asters form and persist for a few minutes before dissipating [11 , 12 , 17–20] ( Fig 1A; S1 Video ) . The central cores of actin asters are enriched with active myosin II which appears to lag the assembly of the aster ( Fig 1B; S2 Video ) . Asters are observed in a variety of morphogenetic events where they are strongly correlated with cell shape change [3 , 21] . Actin asters can be seen on surfaces other than the apical cell cortex . F-actin networks in the basal cortex of the epithelium and basolateral cortex of mesenchymal cells in Xenopus embryos ( Fig 1C ) adopt less dense morphologies than those in the apical cell cortex ( Fig 1D ) . A diverse range of F-actin morphologies , including asters in the apical cortex can be seen within different classes of epithelial cells even at the same stage of development: for instance , highly aligned F-actin in prospective epidermal cells bordering the blastopore lip ( Fig 1E ) [23] , or high frequency actomyosin contractions in the neural epithelium as the neural folds form and neural tube closes ( Fig 1F ) . Complex actomyosin dynamics can also be studied in vitro as reconstituted gels ( e . g . [24 , 25] ) or within microcontact printed arrays [26–28] . One advantage of reconstituted gel models is that their mechanical or rheological properties can be measured directly [29 , 30] as the composition of the gel is changed , e . g . addition of purified actin cross-linking proteins [31] . Such studies reveal how factors that alter the morphology of stable actin networks correlate with changes in material properties . Microcontact printing has been used to create patterns of actin polymerizing protein ( Nucleating Promoting Factor pWA ) , myosin motors ( myosin VI or myosin II ) , or capping protein ( CapZ ) [26–28] . Once actin binding factors or motors are immobilized , or printed onto a glass substrate , purified G-actin and additional factors are added , and the evolution of the network is followed by time-lapse confocal or total internal reflection fluorescence microscopy . Actin dynamics that arise from printed patterns can serve as physical analogs of the native actin cortex , allowing more detailed correlation of cytoskeletal protein-protein interactions with mechanical properties and kinematics of actomyosin networks , and have provided many insights into in vivo actomyosin dynamics . Reconstituted in vitro systems can be viewed as physical analogs for cortical actin in the cell and complement theoretical computational analog models we describe next . Complex dynamics of actomyosin arrays observed in vivo and in vitro have inspired theoretical and computational models seeking to connect the biophysical interactions of F-actin and myosin II motors to macroscopic phenomena [32] . Early biophysical models on the origin of muscle contraction forces were closely coupled to experiments; for instance , Huxley's model connected microscopic structural analysis of striated muscle [33] to dynamics and metabolism of contractility measured experimentally [34] . More recently efforts have sought to explain phenomenological events driven by less ordered arrays of actomyosin typically found in epithelial and mesenchymal cells . Such filament arrays have been the subject of theoretical and computational models investigating how interactions between actin filaments , myosin II motors and their regulators might drive emergence of ordered arrays or allow disordered arrays to generate or transmit force . Continuum models of actomyosin mechanics and bulk dynamics have emerged from studies of active polar polymers ( e . g . [35] ) to explain complex patterns of F-actin seen in cells ( e . g . [36–42] ) . Such models often combine , or lump , specific parameters of actin and myosin biophysical interactions into bulk-stresses or modulus and have succeeded in representing mesoscopic behaviors of actomyosin seen in tissues , cells , and reconstituted actomyosin gels [25] . Live cell imaging of actomyosin dynamics have inspired development of microstructurally realistic computational models . One strategy to simulate complex actomyosin dynamics involves the adoption of agent based computational simulations similar to those used to study microtubule dynamics [43] . These approaches utilize agent-based microscopic models that represent individual actin filaments and proteins such as cross-linkers that interact to modulate filament connectivity . Individual mini-thick filaments of myosin II can be included to drive filament rearrangement ( e . g . [44–51] ) . Each agent , or molecular component can interact with others through defined biochemical and biophysical processes to drive changes in the filament array . Our own microstructural actomyosin models have been motivated by these efforts and by the need to understand how actomyosin asters emerge within embryonic cells and how these structures may be coordinated and generate force during development [52] . Several groups have recently applied similar microscopic computational models to explore the complex morphologies that emerge from actomyosin interactions . Models have been used to understand the complex actin morphologies that emerge from in vitro experiments with recombinant proteins microcontact printed in patterns or confined in specific ring-like geometries [53 , 54] . Other models have been constructed to understand the dual function of actomyosin arrays in both generating and transmitting forces to neighboring cells [50] or the role of actin polymerization and alignment during assembly and constriction of the cytokinetic furrow [42 , 46] . Efforts similar to our own have also sought to understand remodeling of filament arrays by motors [51] , the evolution of stress production and clustering of actin within the 2D cell cortex [55] , and to explore the potential contribution of stress-induced actin filament severing [48] to actomyosin mechanics . Increasing computational requirements for these simulations led us to seek simplifications that would allow us to compare the importance of various protein-protein interactions and association rates that shape actomyosin arrays within the cell cortex and how cells might disassemble actin arrays or spatially control morphologies through localization of actin-binding proteins . To understand how dynamic ordered arrays such as asters might emerge from disordered networks we developed a 2D "search , capture , remodel , and traffic" model that incorporates dynamic aspects of in vivo F-actin and myosin motor interactions . We find this model captures many of the observed behaviors of in vitro model system actomyosin . By including F-actin and myosin motor interactions and their effect on transporting myosin motors we have advanced beyond current computational and in vitro models , including our previous rotational model [47] , to investigate the important biophysical interactions that shape actin asters . To extend microstructural models to study actomyosin networks confined to thin layers such as the embryonic cell cortex , we created a model of a two-dimensional array of actin filaments with myosin motors . Since there is a strong positive correlation between "material" elastic modulus and actin cross-linking in both reconstituted gels [56–60] and the cortical actin [61] we also chose to investigate the role of cross-linkers on filament-motor dynamics by implementing cross-linker agents and testing their effectiveness in shaping asters . Although changes in network morphology can be qualitatively correlated with specific perturbations , in this paper we investigate how actomyosin asters arise and may be shaped by changing conditions in the cell and discuss how programs of development and morphogenesis might control cortical actomyosin dynamics to drive and guide tissue movement .
To illustrate the biophysical processes incorporated in our actomyosin model , we first simulate a sparse network of 50 filaments and 250 motors ( Fig 2A–2E ) . Each filament in our model represents a fixed length actin microfilament . Each motor represents a multiprotein complex of multiple non-muscle myosin II motors composed of heavy and light chains that self-assemble into a myosin filament [62] also referred to as a bipolar myosin II mini thick filament [52] . As motors bind and walk toward the plus- or barbed-end on pairs of filaments we observe polarity sorting and the co-emergence of a dense cluster of motors and filaments that we refer to as an “aster” ( see Materials and Methods for model equations and implementation and S1 Table for model specific parameters; Fig 2F ) . Asters can be recognized by the divergence of their component filaments ( see Materials and Methods for divergence calculations; Fig 2G; divergence indicates aster centers by sites where filament orientations reverse ) . Briefly , we assigned vectors originating from filament plus-ends and summed the orientation for box areas covering the domain ( Vi , j→=[Vxi , j , Vyi , j] ) . We then used a second order derivative approximation to determine the divergence in x ( divXi , j ) and y ( divYi , j ) for each box and summed these to determine the total divergence . As the aster forms , the motor-generated internal-network forces decrease to a steady state value ( Fig 2H ) , and the network morphology reaches a dynamically stable form with filament plus-ends gathered at the aster center . Each simulation begins with a disordered initial distribution of filaments and motors; to compare different conditions we carried out 100 simulations and compared the mean internal-network forces ( Fig 2I ) . The steady state of force over 100 simulations is normally distributed ( 0 . 09 pN ± 36% , S1 Fig ) . The asters produced in our simulations , are formed as plus-ends of filaments pack together in the center with minus ends radiating outward . For the remainder of this study we present single simulations to represent behaviors observed over multiple repetitions from different initial configurations . To understand dynamics of the model under more physiological conditions we increased filament and motor density to 1 , 000 filaments and 5 , 000 motors ( Fig 3A; S3 Video ) . The denser actin network is reorganized by the myosin motors through an intermediate , isotropic ring structure ( over the first 2 . 5 seconds; Fig 3A ) , which continues to contract into a stable aster ( from 7 . 5 to 10 s; Fig 3A ) . To compare dynamic remodeling of model filament-motor arrays with in vivo data collected from time-lapse sequences we generated time-lapse sequences of models mimicking the linear additive fluorescence signal observed in confocal time-lapse sequences . Computed synthetic time-lapse sequences allowed us to analyze aster formation with image-based tools commonly applied to live-cell F-actin dynamics [11 , 17 , 63] . As an illustration , we track the normalized mean intensity of filaments within a fixed circular region of interest ( ROI ) , to observe intensity increases over the first second as the network begins to form a ring structure , but drops after 2 seconds ( Fig 3B ) as the ring forms outside of the ROI . At this time , filaments are "swept" into the ring by motors and depleted in the center . Intensity at the center gradually increases as the ring of filaments and motors contract into the centralized aster from 2 . 5 to 7 . 5 s ( Fig 3A , Fig 3B ) . The normalized mean intensity then maintains a high level reflecting compaction of the ring into the aster together with filament turnover since filaments are randomly depolymerized and relocated randomly to places within the hexagon , including regions outside of the ROI . A kymograph provides another method of quantifying the aster emergence ( Fig 3C , upper panel ) and also reveals movement of filaments into the aster similar to that seen in live-cell time-lapses of F-actin in Xenopus cells [11 , 17] . For comparison , we include a kymograph from a simulation with high filament turnover ( p2 , 5 s-1 ) where asters do not assemble ( Fig 3C , lower panel ) . Asters generated in our simulations are qualitatively similar to punctuated actomyosin contractions observed in vivo during epithelial morphogenetic movements such as Drosophila apical constriction [7] , establishment of anterior-posterior polarity in C . elegans embryos [64 , 65] , and dorsal closure in Drosophila [66] . Internal-network forces and plus-end positions evolve in a pattern qualitatively similar to that observed in the sparse network . The internal network force ( Fig 3D ) , and the filament divergence at the end of the simulation ( Fig 3E and 3F ) approach a steady-state similar to that reached by the sparse network ( Fig 2H ) . Mean motor generated force at steady state in cases lacking aster formation is higher than the case when the aster is formed ( Fig 3D ) . Furthermore , not all filaments are recruited into the aster ( Fig 3E and 3F ) due to the rapid transport and trapping of motors by the centrally concentrated plus-ends of the radial filament array . Polarized filaments in the aster direct all bound motors toward the aster's center ( Fig 3A , myosin location in green ) . Motors are trapped by the aster core; as a motor moves to and falls off the filament plus-end in the core , it diffuses until it binds to another filament , however , at the core it only finds other plus-ends that all direct the motor into the aster center . Our initial simulations demonstrate that aster formation is highly robust and that once assembled , an aster is a dynamically stable structure . In many cases actomyosin contractions observed in cells can be stable ( e . g . asters in Xenopus at sites of bundled extracellular matrix , or in 'nodes' late in gastrulation ) but often contractions are transient [11 , 17] . To fully recapitulate the in vivo dynamics of punctuated actomyosin contractions , asters would need to dissipate after formation . Such pulsatile contractions are not an emergent feature in our model , rather , we suspect transition between a stable aster and its disassembly requires altered rules or conditions that control F-actin and myosin II function . To understand the influence of biophysical properties of F-actin and myosin mini thick filaments on aster formation and stability we carried out a series of simulations varying these parameters . In most cases , simulations reach steady state within 1 , 000 time steps ( 10 s ) ; longer simulations to 3 , 000 ( 30 s ) or 6 , 000 ( 60 s ) time steps were carried out on the remaining cases ( see S2 Table and S1 Text for aster identification methods ) . Simulations were performed with each parameter allowed to vary through their physiological range ( S1 Table ) . In simulations where filament turn-over rates are zero ( e . g . p2 set to 0 ) , the majority of filaments quickly rearrange due to motor interactions , with plus-ends in the center , forming an aster ( Fig 4 and S4 Fig ) . Rapid aster formation traps motors and limits their access and thus their interactions with non-aster filaments . Furthermore , the abundance of unbound motors is reduced outside the aster , limiting the domain contributing filaments to the forming aster . If we reduce the ability of the motors to stay attached to filaments , we slow the recruitment of filaments into the aster . As the turnover rate of filaments increases , asters can no longer form ( p2 , 5 s-1 ) since filaments are removed from nascent asters and are randomly redistributed . Once a filament turns over , any motor attachments are severed . Therefore , when the turnover rate of filaments is high , the time motors spend exerting forces on filaments to organize them into an aster is greatly reduced . Thus , aster formation can be slowed or inhibited by high rates of turnover or by reducing the length of time motors interact with filaments . Changing filament length alters aster formation in a manner similar to alterations observed when filament density or turn-over is changed in vivo . Shorter filaments ( L , 0 . 25 μm ) result in formation of multiple small asters whereas longer filaments ( L , 2 μm ) create single , domain encompassing asters ( Fig 4; S5 Video ) . Asters produced with short filaments are similar to actin networks observed in vivo after treatments with Cytochalasin D or Latrunculin B , which depolymerize actin to produce sparse actin networks [11] . Additionally , small asters have been observed to interact physically when filament length is reduced [67] . The formation of multiple asters can be attributed to reducing filament length as well as reducing the area searched by unbound motors . One of the effects of motors on network remodeling can be understood in the mechanism of motor transport on filaments; bound motors move faster through the domain than by simple diffusion . When filaments are shorter , motors traverse shorter distances along each filament slowing the process of filament polarity sorting and assembly of filaments into asters . Motors cannot link short filaments from two different asters that would otherwise enable the merger of multiple smaller asters formed by larger filaments . Lastly , increased filament length leads to dense actin networks similar to those observed in vivo when F-actin is stabilized with Jasplakinolide [11] . Changing the dynamic viscosity , η , alters the rate that motor forces produce filament translation and rotational movement ( 5 pN s/μm2; the dynamic viscosity of water is 8 . 9x10-4 pN s/μm2 ) . Simulations with a lower viscous cytoplasm ( 0 . 1 pN s/μm2 ) result in more bundling of filaments in the aster ( Fig 4 ) . With the exception of motor contractility , we see few changes in aster formation as we alter parameters that change motor interactions with filaments . Strongly increasing the rate of motor detachment ( p0 , 10 s-1; Fig 5 ) can contribute to the formation of multiple asters as motors exert short bursts of force onto filament pairs . Changing the rate of motor attachment only disrupts aster formation when the rate is low ( p1 , 1 s-1 , Fig 5 ) . Alternatively , motor contractility , or spring stiffness , can alter aster formation; extremely compliant motors , below physiologically measured values , are never able to rearrange filaments into a single , central aster , instead remaining more concentrated at the periphery or assembling multiple asters at the periphery ( k , 0 . 5 or 1 pN/μm , Fig 5 ) . This condition may recapitulate in vivo conditions where the small molecule ROCK inhibitor Y-27632 reduces the incidence of contractions [11] . Conversely , increased motor contractility drives filament networks into dense , single aster structures that are similar to ones observed in vivo after application of the myosin phosphatase inhibitor Calyculin A [11] . Surprisingly , changes to motor velocity did not drive differences in aster formation . When motors move rapidly ( v , 3 μm/s ) , they are unable to spend sufficient time on filament pairs to pull them into the aster before the motors are trapped ( Fig 5 ) . By contrast , the distance motors can search and stretch before detaching ( r ) strongly affected aster formation ( Fig 5 ) . The distance a motor may search as it seeks a binding site on nearby filaments limits the number of filaments that are within range and to a lesser extent , limits the time a motor interacts with filament pairs before detaching ( S6 Fig ) . In summary , actomyosin aster formation was robust to variations in myosin motor detachment rates , increased attachment rates , increased motor stiffness , and changes in motor speed . However , several factors reduced the ability of asters to form or resulted in multiple small asters; these changes include low motor attachment rate , low motor stiffness , lower motor search or stretch thresholds ( r , 0 . 05 μm or 0 . 15 μm ) , high filament turn-over ( p2 , 5 s-1 ) , short filaments ( L , 0 . 25 μm or 0 . 5 μm ) , and high dynamic viscosity ( η , 5 pN s μm-2 or 10 pN s μm-2 ) . The ability to recruit more filaments into an aster increased with increasing motor stretch ( r > 0 . 6 μm ) , or increased filament length ( L > 1 . 5 μm ) . We did not find any conditions that produced oscillating or episodic formation and disassembly of actin asters . To understand the molecular mechanisms that a cell might use to disassemble an aster , we carried out a series of simulations that began with an aster in steady-state , at which point we changed a parameter and observed the dynamics of the aster over time . In particular we wondered whether asters could transit from one stable state into another stable state . Since there are a few parameter ranges that do not generate single asters by the end of our simulations ( t , 1 , 000 time steps or 10 s; Fig 6 ) , we sought to understand what would occur when conditions were changed so we switched parameters from values that would induce an aster to values that had not previously organized an aster . To identify conditions that might destabilize an aster we changed conditions that formed stable asters to conditions that had not assembled de novo asters ( Figs 4 and 5 ) . As we expected , most cases led to aster disassembly , for instance , decreasing the motor attachment rate ( p1 , from 10 to 1 s-1 ) or increasing the filament turn-over rate ( p2 , from 0 . 7 to 5 s-1 ) completely annihilated a stable aster . However , some cases of parameter switching resulted in a small remnant of the initial aster ( Fig 6A ) . Such small stable asters were found after decreasing motor stiffness ( k , from 3 to 1 pN/μm ) , reducing the distance a motor can stretch ( r , from 0 . 3 to 0 . 15 μm ) , and decreasing filament length ( L , from 1 to 0 . 5 μm ) . We note that all of these cases resulted in a marked decrease in the number of filaments in the remnant filament aster ( S7 Fig ) . Interestingly , decreasing filament length did not break the stable aster into multiple , smaller asters; at the start of this simulation most motors are already trapped at the aster core and remain sequestered after the parameter switch . Thus , after filaments are shortened , the initial core of the aster persists and continues to trap motors , preventing them from assembling other asters . We next asked whether we could induce an aster from conditions that had not initially produced a stable aster ( Figs 4 and 5 ) . In almost all cases we found parameter switches could generate single stable asters ( Fig 6B ) . An exception was found when increased filament length ( L , from 0 . 5 to 1 μm; S6 Video ) generated two asters , however , replicate simulations over longer durations revealed only limited cases where asters merged ( S8 Fig , see sims 3 , 4 , and 9 ) . Our model simulations of aster fusion after changing filament length highlights the rapid changes in structure and dynamics of the cytoskeleton that can be attained by fine tuning actin filament length . Our simulations suggest that cells control episodic aster formation , or punctuated contractions by controlling a limited set of biophysical interactions . In some cases , actomyosin network function may be regulated by scaffolding proteins that localize the actomyosin network to specific sites in the cortex [68–70] . To understand how actomyosin morphology might be shaped by protein localization we simulated cases in which filaments or motors are tethered to specific positions ( Fig 7 ) . One such study reconstituted F-actin with Myosin VI and II , and α-actinin and examined the morphology and assembly dynamics of F-actin arrays [26] from patterns of immobilized actomyosin regulators . This study immobilized the actin nucleating protein pWA in a bar pattern on a glass substrate and then added a mixture of G-actin , myosin VI , and ARP2/3 . Long actin filaments formed after two minutes with minus-ends confined to the “bar” and plus or barbed ends extending away . To simulate this experiment , we initially localized 50% of the filament minus ends to a rectangular region ( Fig 7A , S7 Video ) and allowed the remaining filaments and myosin motors to move throughout the entire simulation domain . Motors followed the same rules as our previous simulations; we observed motors reoriented and contracted filaments into an aster distributed over a small subdomain of the bar . Applying our simulation to a more artificial case where filaments were immobilized qualitatively reproduced the actomyosin arrays observed experimentally . Since it is not clear how 3D bulk concentrations in reconstituted systems should be represented by 2D model agents or how fractional ‘activity’ of interacting filaments , motors , and cross-linkers should be represented , we have simulated the experiments using the same domains and number of filaments and motors as our previous examples . Large scale structures up to 1000s of μm across assembled in reconstituted systems involve both early phase of filament nucleation and later phase of filament remodeling . Our simulations are not trying to capture the dynamics of the nucleation phase of the actin filaments , but rather sought to represent the second phase of myosin remodeling actin filaments as might be expected within scaffold-localized subdomains of the cell cortex . Scaffold proteins can also serve to localize myosin motors and may also regulate actomyosin function in the cortex . In another set of in vitro reconstitution studies , myosin VI motors were immobilized to a bar shape on a glass substrate , binding one leg of the motor and leaving the other leg free to bind and move filaments [26] . They observed that the bar of motors pulled a bar of F-actin to the motor location . Our simulation can recreate these same patterns of motor directed filament movements ( Fig 7B , S8 Video ) . To test whether modest levels of immobilized motors could produce the same result , we included free motors in the simulation so that fixed motors accounted for 12 . 5% of the total ( see S2 Text for an explanation of this percentage ) . We observed the free motor network initially contracts filaments into a characteristic isotropic ring , but that tethered motors subsequently remodel the ring into two asters . The tethered motors then pull the asters toward the lower domain , and finally merge the multiple asters into a single aster over the tethered motor domain ( Fig 7B ) . This result suggests that localized myosin can quickly reposition existing filament networks . We also observed tethered motors can initially deplete filaments in adjacent regions , preventing secondary asters from forming in close proximity to the tethered motors . We also considered the possibility that F-actin binding scaffolds ( i . e . anchoring a portion of filaments into a fixed location ) may play a role in formation of the actomyosin arrays ( see S2 Text; S9 Fig ) . In this case emergent asters co-align with the position of fixed filaments ( S9 Fig ) . By contrast , a more complex set of aster structures arises from fixing motors . The divergence maps ( Fig 7 ) show that aster-like structures form over both fixed filament and tethered motor sites but a second aster appears in the space adjacent to the sites of tethered motors . Paradoxically , this dynamically stable aster does not move to the site of fixed motors . The plots of the mean motor force show that fixed filament and tethered motor systems ( Fig 7 ) evolve stably along distinct trajectories . In conclusion , by localizing filaments or motors our simulations can qualitatively recreate actomyosin arrays produced in reconstituted systems where filaments or motors are localized by microcontact printing . Stress fibers , cytokinetic furrows , and adherens junctions all contain parallel arrays of F-actin filaments [71–74] . Yet , the most common structure produced by our simulations is a radial aster . We have not observed stable parallel or anti-parallel arrays or co-aligned filaments; however , we observed many cases where asters form via an intermediate ring-shaped filament array that is composed of co-aligned , anti-parallel filaments . Since the processes that cross-link actin within these transient structures might represent native processes we wondered whether F-actin cross-linkers could stabilize the formation of transiently parallel F-actin arrays . A wide range of actin bundling or cross-linking proteins have been described [75] . Bundling proteins are a family of actin-binding proteins that bridge and hold together two different actin filaments . These cross-linkers all contain calponin homology ( CH ) domains that mediate their attachment to F-actin [76] . Bundling proteins vary in their stability , how closely they bind pairs of filaments , the orientation of actin in the bundle , and the specific sites on F-actin that they recognize . Fimbrin and fascin , for example , bind to a pair of filaments with the same polar direction , at binding sites every 3 . 5 to 5 actin subunits , and keep the F-actin pairs 10 nm apart [75] . In addition , fimbrin and fascin limit myosin motors access to bundled filaments . By contrast , α-actinin , another cross-linker binds and orients filaments and maintains a spacing of 40 nm , which is thought to allow myosin motors to interact and contract the network [74 , 77] . Filamin , another F-actin cross-linker , forms a v-shaped link and binds at the cross-over point between orthogonal filaments . Networks cross-linked by filamin are relatively flexible and can deform [74] . To model fimbrin , fascin , α-actinin and the more general class of CH containing F-actin cross-linking proteins we introduce to our simulations a population of cross-linkers that only bind filament pairs that are co-oriented within a range of angles up to π/8 ( 22 . 5° ) . We simulate cross-linkers by letting the cross-linker search and bind filament pairs in the way we simulate motor binding . Once bound we allowed cross-linkers to stretch to 40 nm , similar to the distance spanned by myosin filaments [78] . As the case with motors , multiple cross-linkers can attach to each filament and once bound to a pair of filaments the cross-linker exerts spring-like forces at the binding site on each filament . While different cross-linkers may differ in their ability to interfere with motor processivity , for simplicity , we assume that motors can pass through any bound cross-linker . Surprisingly , simulations with cross-linkers do not produce significant numbers of parallel filaments , but do slow aster formation ( Fig 8 , S9 Video ) . We next asked whether cross-linkers could alter aster formation and found that only a three-fold excess of cross-linkers to motors ( 3 , 750 , compared 1250 motors ) could inhibit asters from forming ( S10 Fig ) . Since simulations with cross-linkers generate transient ring alignment of filaments rather than parallel stress-fiber-like arrays , we hypothesize that bundling cross-linkers might stabilize parallel arrays through other mechanisms , such as altering filament turn over or inhibiting motor-mediated remodeling . Since parallel filaments do not emerge from the addition of cross-linkers , we asked whether actin cross-linking proteins might stabilize initially parallel filaments . To test this we included cross-linkers within a simulation that started with aligned filaments ( within 22°; Fig 9 ) . By pre-forming parallel filament arrays we expected cross-linkers ( 1 , 250 ) would bind filaments and allow filament arrays to resist reorientation by motors ( 3 , 750 ) . Instead , we found motors were still able to reorient and remodel filament arrays into asters ( Fig 9 ) . In conclusion , the addition of actin cross-linking proteins to our model can slow or prevent the central aster from forming . Tight parallel bundles of filaments as seen in stress fibers , for example , do not emerge for our model even in cases where excess parallel binding cross-linkers are added .
Our mesoscale molecular dynamics model recapitulates native cortical actomyosin dynamics by simulating myosin II motor search , binding to actin filaments , and polarized motor movements that contract and remodel filament arrays into aster-like structures . Asters emerge as filaments undergo "polarity-sorting" [51] and motors are transported to and trapped within aster cores . Our findings confirm previous 2D studies that have simulated aster emergence from simple motor-filament interactions [51 , 55] . We have further confirmed that increasing rates of actin turnover can inhibit aster formation [55] in accordance with predictions from our earlier rotational model [47] and a simplified 1D model ( S3 Text and S11 Fig ) . As the rate of filament turnover increased in the 1D model , the number and alignment of filaments in the contraction and the force generated by the motors increased . Although the geometry and boundary conditions of the 1D models differ significantly from our current model , in 2D we observe asters become less dense as filament turn-over rates increase ( Fig 4; Fig 10 ) and that steady state force exerted by the motors also increases ( S12 Fig ) . Complex patterns of motor transport and activity emerge from our model without an explicit feedback mechanism such as a catch-bond . In the absence of filament turnover , motors quickly align filaments into arrays which in turn sequester motors , removing the motors from active force production . As filament turnover increases the compactness of the aster is reduced and fewer motors are sequestered leading to higher levels of force production ( S13 Fig; S12 Video ) . Disordered , single aster , and multiple aster states of F-actin in the cortex and the transitions between them can be understood in terms of the work-energy principle that shapes the network and the filament turnover processes that destabilize the network ( Fig 10 ) . Filaments and motors form a dissipative system that remodels as a consequence of both elastic work and losses due to viscous dissipation ( Fig 10A ) . ATP hydrolysis by filament bound motors is the ultimate power source responsible for driving filaments to remodel but the dynamic time-scale and morphology of the array that emerges depends on reactive dissipative forces that resist movement of the filaments and transport of motors along those filaments . In our simulations we can track motors states as they bind filaments and segregate from filaments; these evolving changes in motor association with filaments first increases force production that is then dissipated as filaments sort into asters ( S14 Fig ) . While work stored in an array's elastic energy can be dissipated as motor coupled filaments are depolymerized , our calculations suggest this form of dissipation is negligible compared to the energy lost to viscous dissipation ( Fig 10B; S14 Fig ) . Whereas previous studies have focused on force production and transmission through the elastic network we propose new biophysical studies on frictional forces operating in the cortex . Interestingly , moderate rates of actin turnover enhance the rate of frictional forces by maintaining high rates of filament motion . The process of aster formation is commonly associated with cell shape change and force production but our model suggests that by trapping motors in their core asters deplete motors from other regions of the cortex and are indicators of low levels of contractility . The processes that drive both transient F-actin rings and dense plus-end rich aster cores in our simulations could be tested by assays of actin polarity and myosin II motor distribution . A recent simulation using an approach similar to ours has suggested either cross-linkers or branched F-actin networks are required to stabilize actin rings [54] . However , in our simulations ring structures emerge transiently as asters form and do not require that we initiate the simulation with a ring-structure , cross-linkers , or branching factors . A previous , simpler version of our model [79] also produced ring-like arrays of F-actin when boundary conditions were imposed that mimicked stiff surroundings of cell-cell or cell-ECM junctions . One possible reason our results differ from Ennomani et al . [54] is the parameters used to control motor activity . Motors in Ennomani et al . are stiffer than ours ( 100 pN/μm versus 3 pN/μm; Table 2 ) , with a rupture force of 3 . 65 pN compared to our maximum motor exerted force of 0 . 9 pN . Additionally , our simulations involve more motors ( 5 , 000 compared to Ennomani et al . ’s 2 , 000 motors ) that bind F-actin more frequently ( 10 s-1 versus Ennomani et al . ’s 5 s-1 ) and move more quickly ( 1 μm/s versus Ennomani et al . ’s 0 . 3 μm/s ) . Thus , each myosin motor implemented in our model does more work in remodeling filament arrays than those in Ennomani et al . , even though the maximum force exerted by our motors is four-fold lower . Fewer active motors and highly stable filament cross-linkers may be responsible for stabilizing filaments into persistent ring-like arrays . Episodic actomyosin contractions of the cell cortex suggest the need for episodic mechanical or biochemical signaling to drive cycles of aster assembly and disassembly . In vivo cortical actomyosin networks can exhibit pulsatile contractions whereas our 2D models result in a steady state contraction . Rather than capturing the dynamic signaling environment of the cell , our 2D simulations recreate reconstituted actomyosin gels which contract in a similar manner to produce a large stable structure [25 , 31 , 80 , 81] . Asters generated in our simulations are highly stable and require specific changes in the biophysical conditions for disassembly ( Fig 10 ) . By switching model parameters after a steady state had been achieved , we identified several strategies that might disrupt or trigger formation of an aster including: 1 ) changing the motor attachment rate tenfold , 2 ) changing the motor stretch by half , and 3 ) changing the filament length by half . Our model predictions suggest quantitative studies of aster dynamics after mutant myosin II motors , or factors , such as capping factor to regulate the length of F-actin in the cortex , or signaling pathways that modulate these effectors are targeted to the cell cortex . Alternatively , it has been suggested that cycles of contraction and relaxation in the actin cortex might reflect F-actin disassembly as highly compressed F-actin filaments bend and are severed [82] . A recent 2D microstructural model of contracting filament networks demonstrated that tensed networks can drive filament bending that could be sufficient for severing but do so well before recognizable asters form [48] . We did not incorporate mechanisms to sever or depolymerize filaments under tension , however , we note that F-actin within stable asters experience very low forces since most motors are segregated from potentially productive pairs of filaments . Instead , F-actin arrays in our simulations generate highest tension early in aster formation as broadly distributed motors remodel filament arrays . Interestingly , both models confirm findings from our earlier 1D model that ordered arrays generate the lowest levels of force [47] . Thus , feedback through tension-mediated filament disassembly appears unlikely to generate cycles of cortical aster assembly and disassembly . To investigate mechanisms that might stabilize or orient filament arrays we introduced actin cross-linking proteins into our simulations ( Fig 8 ) . In the light of prior modeling studies [51 , 53 , 55 , 83] we were surprised that cross-linkers did not stabilize oriented filaments in our simulations . Instead , we found that filaments cross-linked at a preferred angle of orientation are not sufficiently stable against motor-mediated remodeling or under high rates of filament turnover . Steric locking of filament arrays , allowed in Mak et al . [55] , but not present in our simulations may serve to increase stress and lock motors into rigor states and to stabilize the F-actin array . In vivo cross-linkers may inhibit motor access or drive motors to dissociate from filaments and substantially reduce the long range transport of motors . Our simulations do not account for the ability of some cross-linkers to block motor access [84] , instead our simulated motors can walk past the cross linkers as they do in α-actinin cross-linked filaments . Motor-blocking cross-linkers could additionally lower the dwell time of the motor on a filament and slow or reduce polarity sorting of filaments . To test the role of cross-linking proteins more realistically would require inhibiting motor processivity , varying binding kinetics , cross-linker length , and the ability of cross-linkers to hold filaments at fixed angles to match the biophysical and biochemical behaviors of specific F-actin cross-linking proteins . There is growing adoption of microscopic computational models for studying the complex interactions that shape the cytoskeleton [32 , 85] . Our model of cortical actomyosin dynamics shares many features with recent mesoscale molecular simulations of actomyosin arrays but extends those models with constraints that are unique to the thin cortex of embryonic cells . Mesoscale molecular models are advantageous because they can examine the influence of external mechanical conditions on disordered filament network arrays . Our model is formally similar to other simulations based on Langevin dynamics ( e . g . Cytosim [53 , 54] ) , and like these other models , incorporates experimentally determined parameters that describe biophysical behaviors of actomyosin networks . Differences in model construction , initial conditions , boundary conditions , and implementation of biochemical and biophysical processes make it challenging to directly compare predictions of these models , however , these computational models reveal how actin filament polarity , actin motors , and actin turnover and polymerization drive the emergence of distinctive actin array morphologies . The results of our 2D model qualitatively parallel findings from a simpler 1D rotational model [47] and a 1D linear model ( S3 Text and S11 Fig ) where higher rates of F-actin turn-over affected , but did not totally inhibit formation of a quasi-steady state contractile filament array . Although the geometry and boundary conditions of the 1D models differ significantly from our current model , in 2D we observe asters that become less dense as filament turn-over rates are increased ( Fig 4; Fig 10 ) with increasing levels of steady state force and network work ( S14 Fig ) . Increasing computational requirements for microstructural simulations led us to seek simplifications that would allow us to capture the key elements that shape actomyosin arrays within the cell cortex and aid in understanding how cells might disassemble actin arrays or spatially control morphologies through localization of actin-binding proteins . Our simplified model of actomyosin interactions have provided novel insights into the relative importance of elastic energy storage and viscous dissipation that suggests filament frictional forces may play a key role in transmitting work from actomyosin networks to cell shape and tissue mechanics . Complex patterns of motor transport and activity emerge from our model without imposing an explicit feedback mechanism . In the absence of filament turnover motors quickly align filaments into arrays which in turn sequester motors , removing them from active force production . As filament turnover increases the compactness of the aster is reduced and fewer motors are sequestered leading to higher levels of force production . In addition , aster-formation becomes less likely as filament turnover increases since motors are not able to coordinate large scale remodeling of the filament array . The asters produced by our model resemble those generated by other groups , however we are able to produce asters without explicit feedback mechanisms [86] . Our model implements search , capture , remodel , and traffic processes by analogy with the 'search , capture , pull , and release' model developed to describe the involvement of actomyosin in the cytokinetic furrow of fission yeast [46] . In our model , polarized arrays of filaments transport motors towards the filament plus-ends , and directional alignment of filament arrays serves to transport motors , sequestering them within the cortex . Together , these processes generate a stable aster with filament plus-ends concentrated in a small central region , which depletes motors from regions with high minus-end densities and traps motors in the aster center . Motor trafficking on oriented filament arrays results in motors becoming trapped in filament plus-end “cages” because they are unable to move out of the aster center; motors that escape the cage via diffusion quickly bind new filaments that direct them back to the center of the aster . A key prediction of our model is that filaments within contracted actin asters are polarized with their plus-ends embedded in the aster center . The dynamics of actomyosin arrays simulated here may be analogous to a case of microtubule-kinesin arrays which remodel into “pineapple” morphologies and multiple aster-like arrays with centrally located plus-ends [87] . Similar to microtubule-kinesin arrays , our model predicts that F-actin plus-end localization and myosin trafficking play critical roles in the formation of asters . The polarity of F-actin or locations of myosin II motors within these dynamic contractions have not yet been resolved but actin filament polarity might be revealed in fixed cells since there are no methods to visualize F-actin plus-ends in live cells . Identifying the in vivo polarity of F-actin arrays predicted by our model will require improved experimental techniques . F-actin plus-end trackers , analogous to CLIP-170-GFP used to track microtubule plus-ends [88] , are not currently available for studying F-actin plus-end dynamics . Our model can be extended to include more biophysical and biochemical realism . For instance , simulated filaments have a fixed length and a single parameter controlling turn-over . This has allowed us to test roles of filament density and length independent of turn-over rates . However , in the cell , filament turnover , length , and density would be closely coupled; higher depolymerization rates may generate shorter actin filaments at lower density . Additionally , filaments in our model do not interact physically with one another . Filaments can pass through each other as they rearrange or can be packed at high density without nematic or lateral association effects on their orientation . As a model utilizing Langevin dynamics , our model can be easily extended to include complex programs of polymerization ( e . g . [89] ) , steric interactions ( e . g . [53] ) , or other biochemical or mechanical interactions . Furthermore , our model makes predictions about the roles of myosin stiffness and filament orientation that would be difficult to examine without fine-grained experimental control . As actomyosin models continue to evolve they will be more able to guide and interpret in vivo studies .
Xenopus laevis aquatic frogs used in this study were cared for according to principles and standard operating procedures established by the University of Pittsburgh IACUC protocol #15025409 ( PHS Assurance Number: A3187-01 ) . Planar arrays of actomyosin within the cell cortex are confined to a 0 . 2 μm thick volume beneath the plasma membrane [90] which we simulate with a 2D array of discrete agents representing F-actin filaments ( referred to as filaments ) and non-muscle myosin mini-thick filaments ( referred to as motors ) . To capture the dynamics of these networks we use experimentally determined biophysical parameters or ranges of parameters ( S1 Table ) . Motors connecting two filaments apply forces to those filaments . Forces are then summed and drive filaments to rearrange within a viscous media . Filaments , motors and cross-linker dynamics and movements are carried out within a Monte Carlo framework . In the sections below we describe the model rationale and implementation details . In cells , filamentous actin ( microfilaments or F-actin ) can vary in length from a few G-actin subunits to more than 10 μm . F-actin exhibits a distinct polarity of plus or barbed and minus- ends with distinctive polymerization rates . F-actin can polymerize or depolymerize depending on the concentrations of G-actin and polymerization factors [91] . Filament polarity also directs myosin motor movement to the filament plus-end . These fundamental properties of F-actin are likely shared throughout most living cells [92] . Filaments in our simulations are represented as polarized cylindrical rods with a fixed length of 1 μm . Because the scale of our simulations are small compared to large in vitro actin filament arrays , we have modeled F-actin as rigid and not semi-flexible . Initially our simulation places filaments at random positions ( xi , yi ) and angles ( θi ) within a hexagonal boundary . The filaments then move in response to forces exerted by attached , stretched motor complexes . In the cell , F-actin length can grow and shrink through polymerization and depolymerization ( Fig 2A ) . A fully realized model of F-actin would require tracking filament subunits ( e . g . G-actin ) and their differential addition and removal from the plus and minus-ends of F-actin . However , for simplicity we represent filament polymerization processes in the simulation with a single turn-over rate that removes a randomly chosen filament and adds a new filament in a random location and orientation in one time step . Any motors attached to a filament that turns over detach . Such stochastic events of filament depolymerization ( for rates see S1 Table ) introduce an element of spatial "noise" similar to that implemented in our earlier model [47] . One aspect of filament dynamics is treadmilling where the addition of new G-actin monomers to the plus or barbed end of the existing F-actin is faster than the subtraction of monomer from the minus end causing F-actin to “move” in the direction of the plus or barbed end . In order to determine how fast F-actin would move due to treadmilling , we considered the rate of in vivo treadmilling from Selve and Wegner [93] of 0 . 21 molecules/second , and the typical size of a G-actin monomer of 4–7 nm . The forward velocity of any F-actin due to treadmilling would then be 8 . 4 x 10−4 to 1 . 47 x 10−3 μm/s . Our simulation time step is 0 . 01 s , which would mean a filament would move between 8 . 4 x 10−6 to 1 . 47 x 10−5 μm , which is small so we have assumed no contribution of treadmilling to filament movement in the simulation . Multiple non-muscle myosin heavy chain and regulatory light chains are associated with F-actin as myosin filaments ( also known as mini thick filaments , [94] ) . Myosin filaments take a variety of forms but are generally composed of 15 to 30 myosin motor heads at either end of a bipolar myosin filament ( Lecuit et al , 2011 ) . The stiffness of myosin filaments have been measured and they can generate a range of forces from 240 to 21 , 000 pN [95–97] . In our model , each end of the motor can independently bind to an actin filament and processively walk to the filament plus-end ( Fig 2B ) . If the two ends of a motor move apart on two filaments and separate , they exert a spring-like force on the two filaments at their respective points of attachment ( Fig 2C and 2D ) . For simplicity , simulated motors , representing single mini thick-filaments , have 0 rest length . As they stretch between two filaments they exert equal and oppositely directed spring forces at their attachments on each filament . The maximum force a motor complex can exert is based on the motor’s spring stiffness and its maximal stretch ( S1 Table ) . Motor movement along each filament is implicitly due to ATP-dependent cross bridge cycling of myosin motors within the myosin filament . We do not explicitly represent the biophysics of this cross bridge cycling because the time steps in the simulation are longer than the time scale of cross bridge cycling . Instead , we simulate motor processivity with a constant , plus-end directed motor velocity . We have modeled filament associated motors as non-muscle myosin II mini-thick filaments . A single motor operates as a two headed Hookean spring . The heads on either end of mini-thick filament interact with actin filaments and extend as a spring with a spring stiffness constant of 3 pN/μm . Previous work [98] had performed in vitro assessments of the spring stiffness for individual non-muscle myosin II mini-thick filaments as being around 300 pN/μm , but with force measurements of 1–10 pN . Motors can exist in three states , either free diffusing , attached to a single filament , or attached to two filaments . Motors only exert forces when they are bound to two filaments . Attachment and detachment of a motor to a filament is probabilistic with independent rates . Free diffusing motors may bind to a pair of filaments located within their search radius , and they will attach to two different filaments if more than one filament is within the search radius . This radius is based on the size of a free myosin mini thick filament ( ~300 nm ) . This search length also serves as the maximum stretch a motor is allowed ( Fig 2E ) . If only one filament is near the motor , the motor attaches to that one filament and processively moves toward the filament plus-end . At each time step a motor bound to one filament will seek a second filament within the defined search radius . If a filament is found , the motor binds stochastically according to the attachment rate . Motors bound to one or more filaments detach from a filament once the motor reaches the plus-end of the filament , is stretched past its threshold stretch radius , or is stochastically selected to detach according to the detachment rate . Motors detached from single filaments join either the free diffusing pool or , if remaining attached to another filament , move toward the plus-end of the bound filament . In order to recapitulate actomyosin dynamics within the cell cortex we extended our previous rotational model of filament motor interactions [47] to a two-dimensional domain where additional biophysical modeling allowed free filament movement ( see Table 1 ) . Movements of motors and filaments are advanced according to finite difference scheme . With each time step , we first calculated the forces from all motors acting on filament i . I . e . if a motor j is attached to a filament i , then it has a nonzero length ( len ) , otherwise , motor j has a zero length . The resulting forces for each motor on each filament are then calculated: Fj=[k* ( Aj−aj ) k* ( Bj−bj ) ] ( 2 ) We transform to the parallel and perpendicular coordinate system of the filament using the following rotation matrix: XRi=[cos ( θi ) sin ( θi ) −sin ( θi ) cos ( θi ) ][xiyi]FRj=[cos ( θi ) sin ( θi ) −sin ( θi ) cos ( θi ) ]Fj ( 3 ) We then update the positions of the filament by updating the center of mass based on the parallel and perpendicular translations , and the angle of orientation by the applied torque . First , we determine frictional drag coefficients for the cylinder in each of these directions . To calculate viscous drag , we use the following drag constants , where p is the ratio of the length ( L ) to diameter ( di ) of the cylinder , and γperp = 0 . 84 , γpar = 0 . 114 and γrot = −0 . 662 are constants when p = ∞ [99] . We assume the filaments experience high shear , so the dynamic viscosity , η , is higher than water . Γperp=4πηLlogp+γperpΓpar=2πηLlogp+γparΓrot=13πηL3logp+γrot ( 4 ) Next , we update the positions of the filaments by updating the center of mass based on the parallel and perpendicular translations , and the angle of orientation by the applied torque . XRni ( 1 ) =XRi ( 1 ) +dt*1Γpar∑jFRj ( 1 ) XRni ( 2 ) =XRi ( 2 ) +dt*1Γperp∑jFRj ( 2 ) θni=θi+dt*1Γrot∑jlenj*FRj ( 2 ) ( 5 ) We then transform the updated positions for the filament back into the original coordinate system using the inverse rotation matrix . Simulated actomyosin interactions occur within an open 2D hexagonal domain . The thickness of cortical F-actin in Xenopus embryonic cells [11] and cultured cells [90] is approximately 0 . 2 μm . Cortical actomyosin arrays are essentially planar when compared to the 20 to 40 μm diameter the Xenopus embryonic cells . Unlike approaches utilizing periodic boundary condition , filaments or motors that move out of the hexagonal domain are randomly re-inserted into the domain . Parallel filaments are not attracted to one another and do not interact sterically but instead slide through and past each other . Simulations were implemented in Matlab ( Mathworks , Inc , Natick , MA ) and the plots carried out in ImageJ [100] . For most cases , the simulations were run for 1 , 000 time steps at a step size of 0 . 01s , which translates to approximately 10 seconds ( in vivo contractions in Xenopus typically form in 45 seconds [11] ) . This time was chosen for comparison because aster stability in the standard case had been reached . We confirmed aster stability through inspection of long duration simulations ( S5 Fig , S15 Fig , and S3 Table ) . Aster formation is also accompanied by a reduction in mean motor force ( S2 Fig ) . Mean force decreases as more motors become trapped in the center of the aster and spend less time remodeling or aligning filaments . Model time-lapse sequences simulate fluorescence microscope images by additively increasing intensity of a pixel when more than one filament or motor are present . Single image frames , kymographs , and intensity profiles were generated from simulated time-lapse sequences using ImageJ . Mean intensity profile plots were generated by determining a region of interest , in the case of Fig 3B the ROI was a circle , and then calculating the mean intensity within the ROI over time . To quantify aster structural evolution , we have employed two strategies . First , using image analysis tools previously used to assess F-actin networks in vivo [11 , 17 , 63] , and using divergence of filament orientations to identify how filaments are oriented during aster evolution ( S1 Text ) . The first method segments actin-dense regions and can track the number , duration , and movements of actin-asters . Quantitation with this method shows that large stable asters form and persist in a quasi-stable configuration , and identifies small transient asters that form at the periphery before being drawn into the large central aster ( Fig 3G; S3 Fig ) . Our second strategy to assess filament networks uses exact locations of filaments and motors to calculate the mean motor generated forces and clustering of filament plus-ends over time . Asters were identified by the divergence of filaments in a small grid divided into L/8 size boxes . Our algorithm determined which filament plus-ends were located within the box , and vector orientations for each filament within the box were added to determine a summed orientation for each box ( Vi , j→=[Vxi , j , Vyi , j] ) . We then used a second order derivative approximation to determine the divergence in x ( divXi , j ) and y ( divYi , j ) for each box . The total divergence is the sum of divergence in x and y and was plotted using a pseudocolor scale to highlight where a sharp transition from low divergence to high divergence is spatially located for any time during the simulation . If filaments are randomly distributed the divergence will be 0 , but for an aster formation , the divergence will be negative at the center of the aster and then sharply transition to positive in the area surrounding the center of the aster . Microstructural simulations of cytoskeletal dynamics provide unique insights into the fundamental biophysical and biochemical processes that guide cell shape and morphogenesis [85] . Several papers have been published that use similar modeling approaches to those we present in this paper [50 , 51 , 54 , 55] . Two of these papers [54 , 55] apply Langevin dynamics to microstructural models of F-actin and myosin motors similar to our own . Both models find F-actin contractions , either through motor-based contraction [55] or through defined filament cross-linkers [54] . The mesoscopic structures these two models produce , e . g . actin asters [55] and ring-like structures [54] , are similar to the actin asters and transient ring structures produced in our simulations . The first of these models by Mak , et al . [55] simulates actomyosin networks with actin filaments , myosin motors , and actin cross-linking proteins . By comparison to our minimal representation , the Mak model includes a diverse array of complex interactions including specialized catch-bonds in myosin motors , steric interactions between filaments , and filament treadmilling . In addition , actin binding proteins that cross-link filaments are included and allowed to stabilize the filament array prior to initiation of motor activity . Despite these differences in formulation , both our approach and that by Mak et al . reveal emergence of actomyosin asters that are inhibited by increasing rates of filament turnover . However , implementation differences in motor activity can produce different results as cross-linking proteins are incorporated . In our models , cross-linking proteins slow the emergence of asters but do not completely block their formation unless provided in vast excess . We suspect this difference reflects differences in motor function and differences in the initial conditions . Mak , et al . have chosen to include motile motors that exert forces on actin filaments through an extremely stiff connection between the myosin II tail ( or backbone ) domains as they bind two filaments . Like our model , myosin becomes extended between the pair of bound filaments and force increases . In our model , the motors can dissociate stochastically , dissociate once they reach to plus-end , or once they extend to their threshold . By contrast motors in Mak slow and lock in a rigor state once they reach a threshold stall force ( 5 . 7 pN; Table 2 ) . The maximum stall force is not much different than maximum exerted force of 0 . 9 pN but the subsequent stalling of motor processivity may lead to more stable filament networks and increase the apparent cross-linker density . Furthermore , bringing the network to equilibrium with cross-linkers may further stabilize the network against continued polarity sorting seen in our simulations . The recent paper by Ennomani et al [54] also simulated motors as Hookean springs to investigate the formation and stabilization of F-actin rings similar to those that form during cytokinesis . Their simulations operated on initially stabilized actomyosin rings and revealed that actin filaments must be branched or ordered by F-actin cross-linking proteins before contraction . By contrast , in our model ring-like contractile structures emerged as transient features during aster formation . Rings occur as the initial wave of contraction sweeps filaments inward from the boundary . Rings form as inward movement of filaments is initially faster than polarity sorting but occurs without pre-patterning filament alignment or polarity . We also found that addition of actin cross-linking proteins did not stabilize transient rings and only prevented aster formation only when present in excess . We suspected that our simulations differed in the implementation of motor function , since we found motors were capable of driving polarity sorting in filament arrays even when those arrays are cross-linked . Since Ennomani et al . implemented myosin motors in a manner similar to our implementation , we can directly compare parameters . We note that their motors are stiffer than ours ( 100 pN/μm versus 3 pN/μm; Table 2 ) , with a rupture force , the point where motors would fall off of a filament , of 3 . 65 pN compared to our max motor exerted force of 0 . 9 pN . Additionally , our simulations involve more motors ( 5 , 000 compared to Ennomani et al . ’s 2 , 000 motors ) that bind F-actin more frequently ( 10 s-1 versus Ennomani et al . ’s 5 s-1 ) and move more quickly ( 1 μm/s versus Ennomani et al . ’s 0 . 3 μm/s ) . Each myosin motor implemented in our model carry out more work in remodeling filament arrays than those in Ennomani et al . , even though the maximum force exerted by our motors is four times less . Lastly , there is a seemingly large difference in the viscosity between our model and the other two recent actomyosin models . In part , this difference arises from the difference between effective viscosity and dynamic viscosity . Mak et al . ’s value is closer to that of water and is included in their calculations for extensional , bending , repulsive and thermal forces for the Brownian motion of filaments , motors , and actin binding proteins . Ennomani et al . ’s value is higher and closer to our value ( 0 . 18 pN s/μm2 versus our value of 1 pN s/μm2 ) , but it is not immediately obvious how viscosity in this model influences of filament motion beyond the description that “actin filaments are modeled as elastic fibers surrounded by an immobile viscous fluid” [54] . Viscosity in our model directly regulates the motion of filaments in response to forces exerted by the motors; filament rotation and translation are opposed by viscous drag on a rigid , 1 μm cylindrical rod with a diameter of 8 nm and is based on low Reynolds number hydrodynamics [101] . More detailed models of motor-filament interactions may be more realistic but the increased complexity required to simulate more realistic interactions is not always necessary to demonstrate complex emergent behaviors of actomyosin arrays in the cell cortex . The simplicity of our approach complements existing actomyosin modeling efforts and highlights the strength of microstructural computational methods in exploring the role of F-actin and myosin in shaping the complex mechanics that control cell- and tissue-mechanics and morphogenesis . | Recent genetic and mechanical studies of embryonic development reveal a critical role for intracellular scaffolds in generating the shape of the embryo and constructing internal organs . In this paper we developed computer simulations of these scaffolds , composed of filamentous actin ( F-actin ) , a rod-like protein polymer , and mini-thick filaments , composed of non-muscle myosin II , forming a two headed spring-like complex of motor proteins that can walk on , and remodel F-actin networks . Using simulations of these dynamic interactions , we can carry out virtual experiments where we change the physics and chemistry of F-actin polymers , their associated myosin motors , and cross-linkers and observe the changes in scaffolds that emerge . For example , by modulating the motor stiffness of the myosin motors in our model we can observe the formation or loss of large aster-like structures . Such fine-grained control over the physical properties of motors or filaments within simulations are not currently possible with biological experiments , even where mutant proteins or small molecule inhibitors can be targeted to specific sites on filaments or motors . Our approach reflects a growing adoption of simulation methods to investigate microscopic features that shape actomyosin arrays and the mesoscale effects of molecular scale processes . We expect predictions from these models will drive more refined experimental approaches to expose the many roles of actomyosin in development . | [
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] | 2018 | Emergent mechanics of actomyosin drive punctuated contractions and shape network morphology in the cell cortex |
It has been argued that the limited genetic diversity and reduced allelic heterogeneity observed in isolated founder populations facilitates discovery of loci contributing to both Mendelian and complex disease . A strong founder effect , severe isolation , and substantial inbreeding have dramatically reduced genetic diversity in natives from the island of Kosrae , Federated States of Micronesia , who exhibit a high prevalence of obesity and other metabolic disorders . We hypothesized that genetic drift and possibly natural selection on Kosrae might have increased the frequency of previously rare genetic variants with relatively large effects , making these alleles readily detectable in genome-wide association analysis . However , mapping in large , inbred cohorts introduces analytic challenges , as extensive relatedness between subjects violates the assumptions of independence upon which traditional association test statistics are based . We performed genome-wide association analysis for 15 quantitative traits in 2 , 906 members of the Kosrae population , using novel approaches to manage the extreme relatedness in the sample . As positive controls , we observe association to known loci for plasma cholesterol , triglycerides , and C-reactive protein and to a compelling candidate loci for thyroid stimulating hormone and fasting plasma glucose . We show that our study is well powered to detect common alleles explaining ≥5% phenotypic variance . However , no such large effects were observed with genome-wide significance , arguing that even in such a severely inbred population , common alleles typically have modest effects . Finally , we show that a majority of common variants discovered in Caucasians have indistinguishable effect sizes on Kosrae , despite the major differences in population genetics and environment .
The use of isolated populations has a long history in genetic mapping , with benefits including founder effects , reduced genetic diversity , reduced genetic and environmental heterogeneity , and large , multi-generational pedigrees [1]–[3] . The resulting reduction in allelic heterogeneity has contributed to the success of genetic linkage and positional cloning approaches in isolated populations , particularly for the identification of Mendelian disease mutations [1] . While multiple rare mutations may segregate in an outbred population , founding events and subsequent population bottlenecks may reduce allelic diversity such that a single mutation dominates the allelic spectrum in an isolated population . In addition , previously rare mutant alleles may increase in frequency through genetic drift or natural selection , thus contributing more substantially to trait variation than in outbred populations and increasing the power of genetic mapping studies . Conceivably , the same properties that make isolated populations valuable for Mendelian trait genetics may be exploited for genome-wide association approaches to the study of complex genetic traits [3] . We have been studying the native population of Kosrae , Federated States of Micronesia , under the hypothesis that power to detect mutant alleles might be enhanced by reduced allelic heterogeneity , and that different genes ( and thus biological insights ) might be obtained . Our initial analyses of genotyping data from 30 Kosraen trios and ∼110 , 000 genome-wide SNPs showed that Kosraens exhibit strikingly reduced haplotype diversity and extended LD , likely resulting from a strong founder effect and repeated population bottlenecks [4]–[6] . These features were much more dramatic than in commonly cited “founder” populations such as Finland and Iceland . Our prior analyses , including resequencing on Kosrae , suggested that fixed marker sets such as the Affymetrix SNP genotyping products would provide better coverage for common variants in Kosraens than in any HapMap population [4] . We also previously observed that native Kosraens exhibit elevated rates of obesity and diabetes , as seen in other indigenous populations [7]–[10] . It is likely that many common mechanisms underlie the rising prevalence of obesity and metabolic disease in both Caucasian and native populations . However , given the reduced genetic diversity of isolated populations , the high prevalence of metabolic disease raises the possibilities that population-specific disease loci and fewer disease loci of relatively larger effect segregate in Kosraens . The genetic architecture of isolated populations introduces analytic challenges which confound traditional association tests [11] . Inbreeding and the historical lack of random mating in a small population violate assumptions such as Hardy-Weinberg equilibrium which underlie many association test statistics . Members of isolated populations descend from a small number of founders , thus are related , typically in large families . In addition to “known” relationships , cryptic relatedness further confounds the test statistic , as more distant relationships may be unreported or incorrectly specified during patient interview . We ascertained over 3 , 100 Kosraen adults in three screens spanning a decade and performed genome-wide association studies for 15 quantitative traits in this cohort . To do so , we developed analytic strategies to address the complexities of studying a population in which the majority of subjects are related . Our work includes: extensive validation of the extended Kosrae pedigree; identifying an analytic approach to maximize power; calibrating the association score to correct for relatedness in the cohort; and application of this method to the analysis of 15 quantitative traits . Results from the genome-wide association analyses validate our approach by detecting previously known loci for LDL-C , HDL-C , triglycerides and C-reactive protein . Additionally , our data suggest novel loci contributing to phenotypic variation in thyroid stimulating hormone ( TSH ) and fasting plasma glucose ( FPG ) . While empirical power calculations suggest our study is well-powered to detect common variants of relatively large effect ( ≥5% variance explained ) with genome-wide significance , no such effects were observed in our data with convincing statistical support .
We performed a population-based screen of native Kosraens over three separate visits to the island ( Table 1 ) . The 1994 cohort was described previously [7] , [12] . Self-reported family relationships were recorded for use in constructing pedigrees and blood was collected for DNA extraction and genotyping . A rich phenotypic dataset was collected for a majority of the adult population of the island , including measurements of height , weight , body mass index ( BMI ) , waist circumference , plasma leptin , percent body fat , fasting plasma glucose , blood pressure , plasma lipids ( ApoA1 , HDL-C , ApoB , LDL-C , total cholesterol , triglycerides ) , thyroid stimulating hormone ( TSH ) , and plasma C-reactive protein ( CRP ) . Phenotypic data were carefully reviewed for errors in data entry , unit conversion and spurious measurements , and to verify that measurements of related traits are correlated ( e . g . , r2>0 . 7 between BMI and waist circumference ) . Any values that could not be reconciled were excluded from the analysis . Heritability estimates for each trait are typically within published ranges; mean values , distribution , number of phenotyped individuals , and heritability estimates for each trait can be found in Dataset S1 . A total of 2 , 906 individuals were successfully genotyped using the Affymetrix 500 k mapping assay ( minimum per-chip call rate 95% ) ( Table S1 ) . SNPs were excluded from the analysis for the following reasons: mapping to multiple genomic locations ( n = 3 , 462 ) ; missing >5% data ( n = 43 , 849 ) ; or more than 10 Mendelian errors observed ( n = 5 , 887 ) ( Figure S1 ) . Hardy-Weinberg equilibrium was not used as a quality filter , as it is difficult to assess in our highly related cohort using standard formulae . For the purposes of SNP quality control , allele frequencies were estimated assuming all 2 , 906 genotyped individuals are unrelated . After excluding monomorphic SNPs ( n = 30 , 581 ) , 408 , 775 SNPs passed technical quality filters , including 78 , 862 SNPs of very low frequency in Kosraens ( 0<MAF<0 . 01 ) . We next used data from 2 , 906 individuals genotyped for 400 , 301 polymorphic , autosomal SNPs to validate the Kosrae pedigree . Genetic accuracy of the Kosrae pedigree was assessed using pairwise identity-by-descent ( IBD ) estimates generated in PLINK [13] . For three types of known relationships ( parent-child , full sibling , and half-sibling ) , pairs of genotyped individuals were evaluated to determine whether estimates of the proportion IBD zero , one or two copies were consistent with the relationship reported by the patients and their families . The pedigree was corrected to reflect the true genetic relationship between pairs of individuals whose IBD estimates were inconsistent with self-reported relationships ( Table 2 ) . For example , 2 , 553 parent-child pairs reported by study participants were validated by genetic data , while 141 parent-child pairs were identified using IBD estimates where the relationship was previously unknown , was misreported , or not reported by study participants . In some cases , individuals were added to the pedigree as “placeholders . ” For example , if genetic data indicated that one individual of a reported sibship was actually a maternal half-sibling , an ungenotyped “placeholder” was added to the pedigree as the father of the newly-discovered half-sib . Discrepancies between the genealogical and genetic pedigrees on Kosrae are not unexpected given the inherent inaccuracies of self-reported relationships , and are also consistent with known adoption practices on the island . Changes to the pedigree were made based on data from a related pair in which both individuals were genotyped . However , successive iterations of pedigree validation and correction for fully genotyped , first-degree relatives produced a “ripple effect , ” also improving the accuracy of relationships involving individuals not genotyped with the 500 k assay ( Table 2 ) , and second-degree and other higher-order relationships across the extended pedigree . After extensive comparisons with genetic data , the extended Kosrae pedigree spans eight generations and includes over 4 , 300 individuals ( living or deceased ) , averaging four individuals per sibship ( range 1–12 ) . Nearly all ( n = 2 , 900 ) of the subjects successfully genotyped with the Affymetrix 500 k assay can be joined in a single extended pedigree , with an additional six individuals forming three independent nuclear families . We count 58 consanguineous offspring as well as numerous marriage loops . Nearly 30% of all genotyped individuals have two genotyped parents . Fifty-six individuals appear distantly related or unrelated to any other study participants . Our goal was to develop an analytic framework that accommodates the complex familial relationships in the Kosraen cohort while maximizing power to detect association . We were unable to identify or develop software capable of simultaneously computing over a complex pedigree of 2 , 900 individuals and >330 , 000 SNPs . Thus , our strategy became to break the pedigree into smaller units; a similar approach was recently taken by Przeworski and colleagues in their study of recombination in the Hutterites [14] . Below we also describe the data simulation framework used to perform controlled comparisons between analytic approaches , leading to the selection of an association test . We use empirical power calculations to determine an effective sample size for our highly related cohort and estimate the power of our study across a range of effect sizes . We applied our method for association analyses in a related cohort to the study of 15 quantitative traits in native Kosraens . We broke the extended pedigree to create smaller units that could be feasibly analyzed by existing software packages for large numbers of markers , while maximizing the number of genotyped individuals included in the analysis ( as an initial , rough proxy for power ) and maintaining some degree of information about relatedness between study participants . Alternative methods for breaking the pedigree were systematically explored , as described in the Materials and Methods . We selected sibships-without-parents as the unit of analysis ( Figure 1A ) . The 2 , 848 non-consanguineous genotyped Kosraens were grouped into 586 sibships consisting of two or more individuals who share a mother and father ( Figure 1B ) . Any genotyped parents are considered only in the context of the parents' sibship . Of the individuals not included in a sibship of size ≥2 ( n = 612 ) , a subset was identified in which any two members of the subset were related to the degree of first cousins or less , as determined by genome-wide IBD sharing . In the context of association analyses , this subset of individuals can be considered as sibships of size one , where relatedness between family groups is no more than first cousins . The actual number of individuals included in the association analysis varies with the availability of phenotypic data , as individuals lacking phenotype data were omitted from the analysis of each trait . The extended Kosrae pedigree was thus broken into sibships for analysis of each of 15 quantitative traits . For example , in LDL-C , 560 sibships size ≥2 and 240 sibships of size 1 ( n = 2 , 366 individuals total ) were analyzed for association . For BMI , the analysis was limited to individuals who had reached full adult height ( females age ≥22 and males age ≥24 ) [15] , and comprised 2 , 073 individuals in 467 sibships of size ≥2 and 202 sibships of size 1 . Since the Kosrae cohort spans multiple generations , members of one sibship are frequently parents or cousins of other sibships . Because traditional association tests assume independence between family groups , we anticipated that relatedness between sibships would inflate the association test statistic [16] , [17] . We used simulation to evaluate association tests and the distribution of association statistics , with the goal of selecting an association test that maximized power for our chosen family configuration of sibships-without-parents . We compared two different approaches for association analyses of quantitative traits: a within-family test vs . a combined within- and between-family test . We selected the FBAT software to represent within-family test statistics , with the expectation that it would be robust to population stratification and relatedness between families [18] . The QFAM module in PLINK includes options for within-only ( PLINK/QFAM-Within ) as well as within- and between-family tests ( PLINK/QFAM-Total ) [13] . Both options of PLINK/QFAM use permutation testing to derive empirical p-values; however , we expected the between-family test to exhibit score inflation due to known relatedness between sibships . We used a modified simulation framework to evaluate and compare performance of the association approaches . An effect of known size was spiked into a Kosraen phenotype ( BMI ) and analyzed using observed Kosraen genotypes and family structure . We chose to modify an observed phenotype instead of simulating genotypes in order to preserve the complex familial correlations between genotype and phenotype on Kosrae . We selected BMI as a representative quantitative phenotype for its moderate heritability ( h2 = 0 . 47 on Kosrae ) and near-complete phenotyping in our cohort . Genotype data for 1 , 000 SNPs were randomly drawn from the larger dataset . After omitting rare SNPs ( MAF<0 . 01 ) , 770 SNPs remained . For each simulation , we modified the BMI phenotype to contain an association to a single SNP contributing an additional 1% to the total phenotypic variance . While this constitutes a fairly substantial single locus effect , it constitutes a small influence on the trait as a whole that does not distort the overall heritability and genome-wide relationships between genotype and phenotype . A total of 770 modified phenotypes were generated , each containing an artificial association to a different SNP in addition to the heritable and other variation in the observed BMI phenotype . Across datasets , the randomly-selected SNP associated with the spiked-in effect spanned a range of allele frequencies greater than 0 . 01 . Each dataset was analyzed in parallel using FBAT for a within-family association test , or using PLINK/QFAM for within-only or combined within- and between-family tests . The performance of each method was evaluated by tallying across datasets the rank of the spiked SNP within its respective dataset . The method that consistently assigned a higher rank to the spiked SNP was identified as the more powerful approach for association analyses . While genomic control is used in the actual association tests to control the false positive rate , we note that rank order is not changed by genomic control , and thus we did not employ it at this stage of evaluating methods . Comparison of the within-only vs . combined within- and between-family association test confirmed that greatest power , as measured by the rank order of the true effects , was obtained through the use of a combined within- and between-family association test ( Figure 2 ) . Within-family tests implemented in FBAT and PLINK/QFAM-Within identified the spiked SNP as the best result in 36% and 43% of all spiked datasets , respectively . A combined within- and between-family test as implemented in PLINK/QFAM-Total increased identification of the spike as the best-associated SNP to 68% . PLINK/QFAM-Total also ranked a greater proportion of spiked SNPs in the top 5 results than FBAT ( 78% PLINK/QFAM-Total vs . 65% FBAT ) , indicating that the between-family test adds substantial power to the study . In a full genome scan of ∼340 , 000 markers , these rank thresholds approximately correspond to the top 440 or 2 , 200 results , respectively , for a true effect explaining 1% of the variance . We then examined p-value distributions for the PLINK/QFAM-Within and PLINK/QFAM-Total tests ( data not shown ) . As expected , p-values for the within-family test follow the null distribution while the combined within- and between-family ( QFAM-Total ) test exhibited a systematic deviation from the null . Such inflation of the nominal association score is typical for genotyping artifacts as well as excess known or cryptic relatedness in a between-family test of association , and was anticipated from the known relatedness between sibships . We determined that the major source of score inflation in the combined within- and between-family association test ( QFAM-Total ) was relatedness in the cohort ( Dataset S1 , Figure S2 ) . Conceptually , including closely related family units resembles population stratification , as the allele frequencies ( from IBD ) and phenotypes ( from heritability and shared environment ) are correlated in family members . In all subsequent analyses , we applied genomic control to adjust the association scores for excess relatedness [19] . After calibrating the distribution of test statistics to the null , we again evaluated the within-only vs . within- and between-family association tests using p-values to compare performance and to assess study power . Because the ability to estimate power accurately is poor when power is very low , we sought to improve power to discriminate between performances of the two tests by examining the spiked dataset containing an effect explaining 2% phenotypic variance . Using p-values as the measure of significance , we confirmed that the combined within- and between-family association test implemented in PLINK/QFAM-Total has 24 . 4% power compared to the within-family only test at 15 . 3% power to achieve an arbitrary threshold of p<10−6 ( Figure 3 ) . Based on these preliminary analyses , we selected an analytic strategy as follows . The extended Kosrae pedigree is broken into smaller family units , namely sibships without parents . The remaining individuals are filtered on identity-by-descent estimates to produce a set of individuals related no more closely than first cousins , i . e . sibships of size one . The complete set of all sibships is filtered for each trait to include only individuals who are both successfully genotyped and phenotyped . Sibships are analyzed using a combined within- and between-family association test as implemented in PLINK/QFAM-Total , including permutation testing to correct for within-family correlation between genotype and phenotype . Finally , we compensate for relatedness between family units and any residual stratification by applying genomic control . We used the simulation data above to estimate the effective sample size for the BMI phenotype by direct observation for small effects ( Figure 4A ) and by extrapolation for larger effects ( Figure 4B ) . Power from the 2 , 073 individuals analyzed in Kosraen sibships for BMI is comparable to that obtained from a study of 840 unrelated individuals . The more than two-fold size reduction from the actual cohort composed of sibships to an effective number of unrelated individuals highlights the excess of relatedness among our study participants . Given the effective sample size of our cohort , we then used the Genetic Power Calculator [20] to estimate study power for effects explaining larger proportions of phenotypic variance , as our hypothesis was that such effects might exist on Kosrae ( Figure 4B ) . Our study has ∼87% power to detect effects explaining 5% phenotypic variance at a genome-wide significant threshold of p<5×10−8 , and >95% power to detect such effects at p<10−6 . We concluded that our genome-wide association strategy for quantitative traits on Kosrae is well-powered to identify loci with relatively strong genetic effects , should they exist and are tagged by SNPs on the genotyping array . We applied our strategy for association analyses to the examination of 15 quantitative traits , using measurements from clinical screenings in 1994 , 2001 and 2003 . As anticipated from the known relatedness between sibships , scores were inflated compared to the null distribution . Score inflation ranged from λ = 1 . 10 for fasting plasma glucose to λ = 2 . 05 for HDL-C ( Figure S3 ) . Score inflation was correlated strongly with trait heritability ( r2 = 0 . 42 ) . For reference , Table 3 provides a list of SNPs with p≤10−5 for each trait , including genome-wide significant association ( p≤5×10−8 ) between SNPs on chromosome 11 and triglycerides . Quantile-quantile ( QQ ) plots and the respective genomic control correction factors ( λ ) for select traits are shown in Figure 5; plots for all 15 quantitative traits are presented in Figure S3 . The results from our genome wide scans indicate an excess of association signal over that expected by chance for LDL-C , triglycerides and thyroid stimulating hormone . We observed strong association between SNPs in previously established loci and HDL-C , LDL-C , triglyceride levels , TSH and CRP , supporting the validity of our analytic approach . For HDL-C , we observe association with rs4783962 and rs1800775 near CETP ( p = 1 . 68×10−4 and 1 . 71×10−4 , respectively ) , with the same allele and direction of effect as reported in Caucasian populations [21] , [22] . The best-associated SNP for LDL-C is rs4420638 in the APOE/C1/C4/C2 gene cluster on chromosome 19 ( p = 1 . 89×10−7 ) , a known locus influencing plasma levels of LDL-C and total cholesterol [23] . We also observed association between LDL-C levels and multiple SNPs in and around the gene encoding HMG-CoA reductase ( HMGCR ) , the target for cholesterol-lowering statin drugs [24] , [25] . Studies in Caucasian cohorts recently established this locus as a true association , with genome-wide significant p-values ∼1×10−20 [21] . For TSH , three SNPs ( rs4704397 , rs6885099 , and rs2046045 ) previously identified in Caucasian cohorts are also associated in Kosraens , with p-values ranging from 3×10−4 to 1 . 8×10−4 [26] . For CRP , SNPs at the CRP and HNF1A gene loci show association and the same direction of effect on Kosrae ( p = 2 . 0×10−5 and 3×10−4 , respectively ) as previously observed in a Caucasian cohort , in which the associated SNPs were either directly genotyped or are in perfect correlation ( r2 = 1 in both HapMap CEU and ASN ) [27] . The strongest association for CRP on Kosrae is with rs4420638 near the APOE gene ( p = 1 . 6×10−6; Table 3 ) , another previously known locus [27] , [28] . This SNP is less well-correlated with the most highly-associated SNP reported in the literature ( rs2075650; r2 = 0 . 37 and 0 . 49 in HapMap CEU and ASN , respectively ) [27] . Seven SNPs near APOC3/A5 have genome-wide significant association ( p<5×10−8 ) with triglyceride levels ( p = 1 . 2×10−9 to 8 . 6×10−9 ) , including specific variants not previously implicated in Caucasian cohorts . In Kosrae , the best-associated SNP for triglyceride levels ( rs7396835 , p = 1 . 2×10−9 ) is ∼23 kb downstream of the variant recognized in Caucasians ( rs2266788 ) . Correlation between these two SNPs has not been evaluated in Kosraens , since rs2266788 is neither directly genotyped nor well-covered by other SNPs in our 500 k dataset . However , rs2266788 is uncorrelated in HapMap Asian or Caucasian samples ( r2≤0 . 32 and r2≤0 . 14 , respectively ) with any of the seven SNPs associated with triglycerides in Kosraens . As the causal variant for this locus has not been identified , it remains to be determined whether these seven SNPs tag a causal allele common to both populations or whether independent causal variants segregate in these two ethnic groups . Besides SNPs near APOC3/A5 and triglycerides , no other loci across all 15 traits achieved genome-wide significance ( Table 3 ) . The most compelling evidence for a novel finding is observed in the association results for thyroid stimulating hormone ( TSH ) . Among the top 20 results for TSH , 10 SNPs ( p = 9 . 9×10−7 to 4 . 8×10−6 ) map to chromosome 9 at 97 . 6–97 . 8 Mb , a region encompassing the gene thyroid transcription factor 2 ( TTF-2 ) ( MIM 602617 ) . Missense mutations in TTF-2 have been reported in conjunction with thyroid agenesis and congenital hypothyroidism in humans [29]–[31] . The two best-associated SNPs for TSH , rs755109 and rs10983893 , are located ∼19 kb upstream and ∼77 kb downstream of the TTF-2 coding region , respectively . Analyses conditioning on the best-associated SNP , rs755109 , suggest that a single association signal underlies association between SNPs in this region and plasma levels of TSH in Kosraens ( data not shown ) . In another example of association observed near a strong biological candidate , rs10998046 on chromosome 10 near MAWBP ( MIM 612189 ) is modestly associated with fasting plasma glucose in Kosraens ( p = 1 . 12×10−4 ) . Upregulated expression of this gene has been reported in rat models of insulin resistance [32] . Meta-analysis of these data with publicly available results from the Diabetes Genetics Initiative [33] produces a combined p-value of 2 . 10×10−6 , with 7 other neighboring SNPs producing combined p-values of 2 . 53×10−6 to 5 . 73×10−6 . While extended linkage disequilibrium limits our ability to identify the causal variant in Kosraens or to exclude association to other genes in the associated region , these loci represent promising results for follow-up in other cohorts . Our study was motivated by the hypothesis that the reduction in allelic heterogeneity resulting from the founder effect on Kosrae , combined with drift and natural selection , might produce common variants with relatively large effects segregating through the population . Empirical power calculations demonstrated that effects ≥5% should be readily detected ( 95% power ) at p≤10−6 . Given these power calculations , and the evidence for a series of known associated loci as described above , the consistent lack of strong association across the majority of traits argues strongly that common variants of large effect are unusual on Kosrae ( Table 3 ) , as they are in the larger populations studied to date in GWAS . The best observed p-value for each trait at a novel locus ranged from 1 . 9×10−5 for fasting plasma glucose ( rs10745259 ) to 8 . 4×10−7 for waist circumference ( rs2222328 ) . Only two of fifteen traits , TSH and waist circumference , have strong , novel associations with p≤10−6 . No novel associations were detected in any trait with p<8×10−7 . Interestingly , these data indicate that on Kosrae , common variants in LD with SNPs on the genotyping array are of small effect in this founder population , similar to the genetic architecture observed in Caucasian populations . If the effect sizes for common variants are similar in Kosraens and Europeans , then the lack of strong associations is unsurprising given the modest size of our cohort . We observe modest evidence for SNPs near multiple other loci which have been convincingly replicated in other cohorts [21] , [22] , [27] , [33] , [34] , including HDL-C and LPL ( p = 0 . 025 , rs17411024 ) or LIPC ( p = 4 . 5×10−3 , rs11071386 ) ; triglycerides and GCKR ( p = 0 . 015 , rs780094 ) , and LDL-C and APOB ( 1 . 6×10−3 , rs7575840 ) . These and other common variants of small effect identified in Caucasian populations may also influence trait variation on Kosrae , despite lack of genome-wide statistical significance for association . We examined data for previously associated SNPs not under the null hypothesis of no effect on trait value , but rather under the alternative hypothesis that the effect seen in Europeans was also observed on Kosrae . We compared the effect sizes ( β-coefficients ) and allele frequencies for known loci in Caucasians to those observed in our study . Specifically , we identified 45 established associations for BMI , height , lipids , fasting plasma glucose , TSH and CRP ( Table 4 ) where the best-associated SNP in the literature was directly genotyped in Kosraens or had a strong proxy ( r2≥0 . 95 ) in HapMap Caucasians and Asians [18]–[22] , [34]–[36] . A test of heterogeneity for the magnitude and direction of β-coefficients was performed for 39 SNPs with MAF≥0 . 05 . Six SNPs were omitted from the comparison of effect sizes , as there is little power to estimate the individual effects for SNPs observed at very low frequencies . Of the 39 SNPs examined for effect sizes on Kosrae , only 6 loci had significantly different ( p≤0 . 01 ) effects in Caucasians and Kosraens ( p = 5 . 5×10−42 to 6 . 8×10−4 ) , of which 4 SNPs were associated with height in Caucasians . Over 70% of the loci evaluated ( n = 28 ) had effects which were of indistinguishable magnitude and direction ( p≥0 . 1 ) in the two populations . We next considered whether differences in allele frequency could underlie the lack of association in Kosraens to loci with strong support in European studies . Of 45 established associations ( Table 4 ) , allele frequencies were compared for 36 SNPs directly typed on the Affymetrix array . For each risk allele , we identified SNPs on the Affymetrix array with frequencies in HapMap CEU within 2% of the risk allele frequency in HapMap CEU . For that set of SNPs , we generated a distribution of allele frequency differences between HapMap CEU and Kosrae . To determine whether a risk allele has an unusual difference in frequency between Kosraen and HapMap CEU , we examined the difference in frequency for the risk allele in the context of the complete distribution of allele frequencies . Over 85% of the SNPs evaluated ( n = 32 ) have statistically indistinguishable frequencies in Kosraens and Europeans ( empirical p≥0 . 1 ) while none of the loci examined had significantly different ( empirical p≤0 . 01 ) frequencies in the two populations . Together , these data suggest that Kosraens segregate many of the common variants that have been identified in Caucasian populations , and that effect sizes for a majority of those variants on Kosrae is not detectably different from that observed in Caucasians despite a dramatically different population history and environment . The empirical similarity of these genetic architectures lends support to the concept of combining association studies across populations to take advantage of neutrally arising differences in allele frequency and LD patterns to aid in confirmation and fine mapping of common disease variants .
We describe genome-wide association analyses in a population-based cohort with extensive family structure , and explore the value of genetic studies in a population isolate with high levels of linkage disequilibrium and relative allelic homogeneity [4] . Our goal was to take advantage of the population genetic features of this isolated population while maximizing the power to detect associations . We broke the extended Kosrae pedigree into sibships to create a computationally tractable dataset that uses as many genotyped individuals as possible . Empirical power calculations show that testing for association both within and between sibships gives more power than within-family tests alone . We used permutation testing and genomic control to correct for score inflation . Association to true biological variants was clearly observed for several known lipid loci , including APOE , CETP , HMGCR and APOC3/A5 . Our ability to detect multiple loci with known association indicates that our analytic strategy is adequate to identify true disease loci . Suggestive evidence for association was observed for thyroid stimulating factor ( TSH ) to SNPs in the gene encoding thyroid transcription factor 2 ( TTF-2 ) , a strong biological candidate with no previously known association . Associations near APOC3/A5 for triglycerides and near TTF-2 for TSH also highlight the possibility of island-specific variants or differences in LD between Kosraens and Caucasians that may be useful in identifying causal variants common to both populations . Our study tests the hypothesis that reduced genetic diversity , genetic drift and/or natural selection might have resulted in a class of common alleles with large effects on metabolic phenotypes . Reduced diversity is evident in our study and consistent with our earlier observations [4] , with 20% of the SNPs ( n>109 , 000 ) passing technical quality filters having minor allele frequencies <0 . 01 in Kosraens . Empirical estimates of study power showed that we have 95% power to observe effects explaining ≥5% phenotypic variance at p<10−6 . And yet , no large effects of this sort were detected . This is similar to the pattern observed in other populations , where the majority of common variants have individually modest effects , typically explaining ≤2% of phenotypic variance [21] , [22] , [35]–[37] . Our genome-wide data expand on and confirm previous work suggesting that many genes of small effect influence trait variation in both outbred and founder populations such as Kosrae [38] . While our cohort encompasses ∼65% of the adult population on Kosrae , limited sample size , coupled with substantial relatedness between study participants , reduces the power of our study in comparison to recently published genome-wide association studies and meta-analyses for common diseases . It is interesting , given the widespread and reasonable predictions that gene-by-gene and gene-by-environment effects modulate marginal associations , that a comparison of allele frequencies and the direction/magnitude of effects for loci originally identified in Caucasian cohorts shows that a majority with statistically indistinguishable effects in Kosraens . We also note that the set of biologically relevant variants influencing metabolic traits is unlikely to be wholly identical between Kosraens and Caucasians . For example , heritability of total plasma cholesterol is similar in Kosraens and Caucasians , but the population mean is approximately 20 mg/dL lower in Kosraens . Variants specific to Kosraens may underlie the phenotypic difference between populations; these variants may lie in novel genes or genes previously implicated in disease or trait variation . Identification of such variants in Kosraens and other ethnic groups may shed light on biological pathways and aspects of disease biology that might otherwise be overlooked in purely Caucasian studies . Validation of any novel association results in our study is hampered by the lack of genome-wide scans in cohorts with an ethnic origin and population history similar to Kosrae . The majority of studies to date have been performed in Caucasians . Further work is required to assess in Kosraens the extent of genetic drift and selection under strikingly different environmental pressures . Replication of true “island” variants would likely be difficult or impossible in existing Caucasian cohorts and underscores the need for the inclusion of diverse ethnic groups in genetic studies . It is also worth noting that although extended LD on Kosrae facilitates locus identification through greater genome coverage , it hampers fine-mapping efforts . In the event that novel association results can be validated or replicated in other populations , a comparison of LD patterns between populations will likely be important to identify the causal variant . While future methods will no doubt improve on our analytical approach , we describe approaches which may be useful to others undertaking genetic studies in population isolates . Tools for validating pedigrees with genetic data greatly facilitated the review of millions of pairwise IBD estimates , highlighted inconsistencies in the reported pedigree , and assisted in the identification of previously unknown first-degree relationships . We show that applying a combined within- and between-family test of association to the subunits of a large extended pedigree increases study power . In addition , the simulation framework we describe for empirical power calculations will be useful for evaluating and comparing the performance of other methods for association analyses as they become available . The current analysis assesses the role of common variants influencing phenotype on the island of Kosrae , but does not evaluate the role of rare variants . In fact , the analytic challenges posed by extensive relatedness in this cohort and the previously demonstrated extended LD in the population suggest that Kosraens may be particularly informative for other analytic methods such as homozygosity mapping . Recent , severe population bottlenecks and subsequent rapid expansion have greatly enriched Kosraens for long stretches of homozygosity . These homozygous segments act as proxies for rare recessive variants segregating in the population and are predicted to greatly increase our power to detect such variants . We are currently developing methods for homozygosity mapping in this unique population . We anticipate that homozygosity approaches for the detection of rare , recessive alleles , coupled with direct sequencing studies to characterize variation on Kosrae not captured by existing genotyping platforms , will complement the association studies for common variants presented here . Together , these three approaches will provide a more complete picture of genetic variation in population isolates , and the underlying role of drift and natural selection on the architecture of metabolic traits on Kosrae .
The study was approved by the Institutional Review Boards at all participating institutions , including Rockefeller University ( protocol #JFN-0282-0707 ) , Massachusetts Institute of Technology ( COUHES protocol #0602110607 ) and Massachusetts General Hospital ( protocol #2006-P-000211/6; MGH ) . All patients provided written informed consent ( in English or Kosraen ) for the collection of samples and subsequent analysis . During screenings performed in 1994 , 2001 and 2003 , patients were recruited by public announcements and came to the clinic following an overnight fast . The 1994 screen was described previously [7] , [12] . Briefly , informed consent was obtained from each patient ( forms available in Kosraen ) , along with self-reported information on identity of family members , medical history , current medications , lifestyle , diet , exercise , and ethnicity . Blood was collected from Kosraens in the fasted state and centrifuged . Plasma and buffy coats were frozen and shipped to Rockefeller University for serological assays and DNA extraction , respectively . IRB approval was obtained from all participating institutions . Quantitative trait measurements were log- or square root- transformed to approach normality , adjusted for age and gender where applicable , and converted to Z-scores . An average Z-score was used for patients screened in multiple collection years or monozygotic twins . Individual trait descriptions are available in the Supplemental materials ( Dataset S1 ) . Genotypes were analyzed for association with fifteen quantitative traits: body mass index ( BMI ) , height , weight , waist circumference , plasma leptin , percent body fat , diastolic and systolic blood pressure , fasting plasma glucose , thyroid stimulating hormone , HDL-C , LDL-C , total plasma cholesterol , triglycerides and high-sensitivity plasma C-reactive protein . Data from the Affymetrix 500 k assay were generated at Affymetrix , South San Francisco , CA . Genotypes were called with the BRLMM algorithm . A minimum call rate of 95% was required for each chip ( Table S1 ) . The two chips in the 500 k assay ( enzyme fractions Sty and Nsp ) were matched by genotype concordance and gender concordance between each chip and the clinical data for that sample . Of the ∼3 , 100 subjects ascertained , 2 , 906 individuals were successfully genotyped according to these criteria . Per-SNP quality filters included: mapping to a unique genomic location , minimum per-SNP call rate of 95% , fewer than 10 Mendelian errors , and minor allele frequency ( MAF ) >0 . 408 , 775 SNPs met these criteria . For the purposes of SNP quality control , allele frequencies were estimated assuming all 2 , 906 genotyped individuals were unrelated . Hardy-Weinberg equilibrium was not used as a quality filter , as it cannot be assessed by standard formulae in our highly related cohort . Autosomal SNPs with MAF≥0 . 01 were analyzed for association with each trait , where MAF was calculated using the individuals phenotyped for that trait . Sibling relationships were accounted for according to default procedures in PLINK . The number of SNPs analyzed ranged from 332 , 890 ( TSH ) to 345 , 026 ( Height ) . Study participants provided names and birthdates of their relatives during the patient interview . Information from multiple patient records was cross-referenced and used to reconstruct extended pedigrees . Relationships reported by subjects in the 1994 screen were validated genetically using Mendelian inheritance checks and identity-by-state analyses with microsatellite markers and the pedigree was modified to reflect the genetically accurate relationships [12] , [39] . Subjects screened in 2001 and 2003 were originally incorporated into the pedigree on the basis of genealogical information . SNP genotyping data were subjected to identity-by-descent estimation using PLINK [13] . Thresholds of IBD sharing for parent-child , full-sibling , and half-sibling relationships were empirically determined from the distribution of genome-wide IBD scores for known relationships . We used empirical ratios of total sharing and the proportion of genome shared in 0 , 1 , or 2 copies between two individuals to evaluate whether genetic evidence supported putative relationships reported in the Kosrae pedigree . A complete list of putative first-degree relative pairs ( parent-child , sibling , half-sibling ) was extracted from the pedigree . For each putative first-degree relative pair , we examined genetic evidence supporting or refuting that relationship and corrected relationships in the Kosrae pedigree accordingly . For example , in a set of individuals forming a putative nuclear family , we verified that all combinations of parent-child relationships and sibling relationships met our criteria for genetic relatedness . “Placeholder” individuals were added to the pedigree as necessary to reflect genetic relationships , such as the addition of a “placeholder” father for a newly-discovered maternal half-sibling . The correction of numerous relationship pairs and our ability to detect cryptic relationships enabled consolidation or elimination of over 70 non-genotyped ancestors , resulting in “tightening” of the Kosrae family tree . Thirteen pairs of monozygotic twins and thirty-five duplicate sample pairs were identified by genotype similarity . Sample identity was confirmed from patient records ( name , birth date ) and one subject from each pair was included in the association analyses . We identified 58 offspring from consanguineous matings , where a common ancestor could be identified in the extended Kosrae pedigree; these consanguineous offspring were excluded from association analyses . An additional nine individuals were excluded from the dataset , as they self-reported non-Micronesian ethnicities and could not be connected to the pedigree . Three approaches were considered to break the extended pedigree into smaller units . Founders consist of a filtered subset of sibships-without-parents , such that any genotyped offspring of a founder sibship are removed from the dataset . Founder sibships are drawn primarily from the upper levels of the Kosrae family tree . For example , 582 “founders” in 247 sibships were identified for the BMI phenotype . Sibships without parents consist of two or more individuals known to share both parents . Since the Kosrae cohort spans multiple generations , members of one family group are frequently parents or cousins of other family groups . Information about parents is used to define a sibship; however , any genotyped parents are considered only in the context of their own siblings . For BMI , 1 , 871 individuals were included in 467 sibships of size ≥2 . Nuclear families consist of two genotyped parents and one or more offspring . Where one genotyped parent is available , offspring are included as a sibship without parents and the genotyped parent is included in the context of its own siblings . Where no genotyped parents are available , individuals contribute as members of a sibship without parents . We performed empirical evaluations of power for each association method using simulated datasets . We “spiked” an effect of known size ( explaining an additional 0 . 5% , 1% or 2% variance ) into an existing Kosraen phenotype ( BMI ) and analyzed this modified phenotype with observed Kosraen genotypes , thereby retaining the true genotype-phenotype correlation between related study subjects . A subset of 1 , 000 SNPs across the genome were randomly selected and filtered to retain SNPs with MAF>0 . 01 . The remaining 770 SNPs were analyzed for association with the spiked phenotype . A total of 770 spiked phenotypes were generated , in which each phenotype was altered to reflect association to a different SNP of the random subset . These 770 phenotypic datasets were analyzed for association to the spiked SNP using FBAT and PLINK/QFAM under an additive model [13] , [40] . We used empirical power estimates from the BMI “spiking” experiment and the module for variance components QTL association for sibships in the Genetic Power Calculator [20] to estimate the effective sample size of our cohort , or the number of unrelated individuals required to obtain power equivalent to that provided by the Kosraen sibships . Power calculations were performed assuming no dominance , minor allele frequency of 0 . 2 , and direct genotyping of the causal variant . For BMI , the 2 , 073 individuals included in the association analysis have power equivalent to ∼840 unrelated individuals . Quantitative trait data were analyzed under an additive model using the QFAM module of PLINK [13] . Nominal scores were permuted to obtain an empirical p-value while maintaining familial correlation between genotype and phenotype . The permutation procedure employed by QFAM corrects for relatedness within families . Between-family relatedness is not addressed in QFAM and is the major source of score inflation ( see Dataset S1 , Figure S2 ) . Genomic control was used to correct for score inflation introduced by relatedness between family units ( sibships ) [19] . We account for multiple testing by assuming a threshold of p≤5×10−8 for genome-wide significance , following the work of Pe'er et al ( 2008 ) and Dudbridge and Gusnanto ( 2008 ) [41] , [42] . This approach assumes approximately 106 independent tests across the genome and requires an additional p≤0 . 05 . This significance threshold is likely conservative on Kosrae , where the true number of independent tests is likely to be smaller because of the extended LD , and so alleviates the multiple testing burden . Association results for known , associated loci were drawn from studies in large Caucasian cohorts for multiple traits . For each Caucasian risk allele where the SNP was directly genotyped in Kosraens , we determined its frequency in HapMap CEU . We identified SNPs on the Affymetrix array that have frequencies in HapMap CEU within 2% of the frequency of the risk allele in HapMap CEU . For each SNP in that set , we calculated the difference in allele frequency between HapMap CEU and Kosrae . These values were used to generate an empirical distribution of allele frequency differences . For the risk allele , we calculate the difference in allele frequency between HapMap CEU and Kosrae , and place this difference on the empirical distribution to determine significance . Association results for known , associated loci were drawn from studies in large Caucasian cohorts for multiple traits . Caucasian loci were limited to SNPs directly genotyped in Kosraens , or where a strong proxy was genotyped in Kosraens ( r2≥0 . 95 in HapMap CEU and ASN ) . SNPs with MAF<0 . 05 on Kosrae were omitted from comparison , as power is low to estimate effect sizes accurately for rare SNPs . We assumed the Caucasian β estimates for each of the traits to be a fixed value . A test of heterogeneity for the magnitude and direction of the effect in Caucasians and Kosraens was performed as follows [43]: Where βk and βc are the effect sizes for Kosrae and Caucasian populations , is the standard deviation on the βk , and is distributed like a T . | Isolated populations have contributed to the discovery of loci with simple Mendelian segregation and large effects on disease risk or trait variation . We hypothesized that the use of isolated populations might also facilitate the discovery of common alleles contributing to complex traits with relatively larger effects . However , the use of association analyses to map common loci influencing trait variation in large , inbred cohorts introduces analytic challenges , as extensive relatedness between subjects violates the assumptions of independence upon which traditional association test statistics are based . We developed an analytic strategy to perform genome-wide association studies in an inbred family containing over 2 , 800 individuals from the island of Kosrae , Federated States of Micronesia . No alleles with large effect were observed with strong statistical support in any of the 15 traits examined , suggesting that the contribution of individual common variants to complex trait variation in Kosraens is typically not much greater than that observed in other populations . We show that the effects of many loci previously identified in Caucasian populations are indistinguishable in Caucasians and Kosraens , despite very different population genetics and environmental influences . | [
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] | 2009 | Genome-Wide Association Studies in an Isolated Founder Population from the Pacific Island of Kosrae |
Neurofibromatosis type 1 ( NF1 ) , a genetic disease that affects 1 in 3 , 000 , is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein ( GAP ) for Ras . Among Drosophila melanogaster Nf1 ( dNf1 ) null mutant phenotypes , learning/memory deficits and reduced overall growth resemble human NF1 symptoms . These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3′-5′ cyclic adenosine monophosphate ( cAMP ) dependent Protein Kinase A ( PKA ) pathway , or phenocopied by inhibiting this pathway . However , whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial . To shed light on this issue we screened 486 1st and 2nd chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of the dNf1 pupal size defect , identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference ( RNAi ) knockdown . Validating the screen , identified suppressors include the previously implicated dAlk tyrosine kinase , its activating ligand jelly belly ( jeb ) , two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling . Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor . Providing mechanistic clues , we show that dAlk , jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction ( NMJ ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone ( AKH ) -producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency . Finally , supporting our previous contention that ALK might be a therapeutic target in NF1 , we report that human ALK is expressed in cells that give rise to NF1 tumors and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man .
RASopathies , caused by mutations that activate Ras/ERK signaling , are a group of related disorders with features that include facial dysmorphism , skeletal , skin and cardiac defects , cognitive deficits , reduced growth and an increased cancer risk [1] . Neurofibromatosis type 1 ( NF1; OMIM 162200 ) , caused by loss of a RasGAP , and Noonan syndrome , caused by mutations that alter Ras/ERK pathway proteins SOS1 , KRAS , NRAS , RAF1 , BRAF , CBL , PTPN11 , or SHOC2 , are the most common members of this group , affecting 1 in 3 , 000 , or as many as 1 in 1 , 000 live births , respectively [2] , [3] . The genetics of these disorders provides a strong argument that excess Ras/ERK signaling underlies common RASopathy symptoms , and much effort remains focused on attenuating Ras/ERK signaling as a strategy for therapeutic intervention . However , whether life-long pharmacological inhibition of Ras/ERK signaling is a viable strategy to treat the full range of often non-life-threatening , but nonetheless serious symptoms of these chronic disorders , remains an open question . This motivates our work to better understand the molecular and cellular pathways responsible for NF1 symptom development , in the hope this will identify more specific therapeutic targets . We have been interested in using Drosophila melanogaster as a model to investigate NF1 functions in vivo , following our identification of a conserved dNf1 ortholog predicting a protein that is 60% identical to human neurofibromin over its entire 2802 amino acid length [4] . Like human neurofibromin , the Drosophila protein functions as a GAP for conventional ( dRas1 ) and R-Ras-like ( dRas2 ) GTPases [4] , [5] . This functional conservation made it all the more surprising when both initially identified dNf1 homozygous null mutant phenotypes , a postembryonic growth deficiency and a neuropeptide-elicited NMJ electrophysiological defect , appeared insensitive to genetic manipulations that attenuate Ras signaling strength , but were suppressed by increasing signaling through the cAMP-dependent PKA pathway [4] , [6] . The genetic link between dNf1 and cAMP/PKA led to further studies , which demonstrated that similar to many children with NF1 [7] , and Nf1+/− mice [8] , dNf1−/− flies exhibit specific learning and memory deficits [9] . Biochemical studies with fly brain extracts further revealed that loss of dNf1 is associated with reduced GTP-γS-stimulated but not basal adenylyl cyclase ( AC ) activity [9] , and with defects in both classical and unconventional AC pathways [10] . Arguing that the cAMP related function of NF1 is evolutionary conserved , GTP-γS-stimulated AC activity and cAMP levels were also reduced in E12 . 5 Nf1−/− mouse brain [11] , and defects in cAMP generation appear to explain the unique sensitivity to Nf1 heterozygosity of murine central nervous system neurons [12] . Arguing that NF1 may regulate cAMP signaling at least in part in a cell autonomous manner , reduced cAMP levels and AC activity were also found in NF1 deficient human astrocytes [13] . Thus , while there is little doubt that aberrant AC signaling is an evolutionary conserved NF1 phenotype , we and others have reached conflicting conclusions about the underlying mechanism . Based on Drosophila phenotypic rescue studies with human NF1 transgenes , others reported that neurofibromin has physically separable functions as a negative regulator of Ras and a positive mediator of AC/PKA signaling . This conclusion followed from findings that NF1-GAP activity was not required to rescue dNf1 size [10] or learning [14] phenotypes , whereas a transgene encoding a C-terminal part of human neurofibromin that did not include the GAP catalytic domain did suppress both defects . In obvious conflict , in similar experiments with dNf1 transgenes , we found that neuronal expression of a functional NF1-GAP catalytic segment was necessary and sufficient to suppress the systemic growth defect , and that other protein segments had no effect . Moreover , the dNf1 growth defect was also suppressed by neuronal expression of the Drosophila p120RasGAP ortholog , and although we extended earlier findings by showing that heterozygous loss of dRas1 or dRas2 , or of a comprehensive set of Ras effector proteins did not modify the growth defect , these mutations also did not reduce the elevated phospho-ERK level in the dNf1 central nervous system ( CNS ) . However , some Ras/ERK pathway double mutants did suppress both defects , leading us to conclude that excess neuronal Ras/ERK signaling is the proximal cause of the non-cell-autonomous dNf1 growth defect [5] . Further supporting this notion , recent work implicated the neuronal dAlk tyrosine kinase receptor and its activating ligand jelly belly ( jeb ) as rate-limiting activators of dNf1 regulated Ras/ERK pathways responsible for both systemic growth and olfactory learning defects [15] . The above evidence underlies our hypothesis that loss of dNf1 increases neuronal dAlk/Ras/ERK activity , which in turn causes reduced cAMP/PKA signaling , which may or may not be cell-autonomous . Obviously , identifying additional components of dNf1-regulated growth controlling pathways followed by functional analysis might help to test this hypothesis . Here we report results of a dNf1 growth deficiency modifier screen , which identified components of tyrosine kinase/Ras/ERK and neuropeptide/cAMP/PKA pathways in addition to genes involved in synaptic morphogenesis and functioning . Further analysis showed that the requirement for dNf1 and cAMP/PKA in Drosophila growth regulation involves different tissues , with dNf1 required broadly in larval neurons , and cAMP/PKA signaling specifically in AKH-producing cells and perhaps in other parts of the neuroendocrine ring gland . These results , and the recent discovery of a novel dNf1 synaptic overgrowth phenotype [16] that is also suppressed by several genes identified in our screen , set the stage for further work to more precisely define how loss of dNf1 causes Ras/ERK and other signaling defects , the ultimate consequence of which is reduced systemic growth .
Animals use elaborate hormonal mechanisms to coordinate nutrient availability and feeding with changes in metabolism and overall growth . Since starvation or crowding during the larval phase of the Drosophila life cycle reduces systemic growth [17] , we first examined whether the small size of dNf1 mutants reflected reduced feeding . Arguing against this hypothesis , wild-type and dNf1 larvae ingested similar amounts of dye-stained food throughout their development ( Figure 1A ) . Unlike a pumpless ( ppl ) mutant [18] , dNf1 larvae also showed no tendency to move away from a food source ( Figure 1B ) . Analysis of the expression of the starvation-inducible Pepck and Lip3 genes [18] provided further evidence that loss of dNf1 does not phenocopy starvation ( Figure 1C ) . Mechanisms that control Drosophila growth have been the topic of intense study and much has been learned about how an interplay between insulin-like peptide ( ILP ) controlled growth rate and ecdysone controlled growth duration determines overall growth ( see [19] and [20] for reviews ) . Arguing against an important role for ecdysone or other factors that control the length of the larval growth period , no differences in the expression of canonical ecdysone-regulated genes was found ( results not shown ) and no difference in developmental timing between wild-type and dNf1 mutants was detected ( Figure 1D and S1 ) . Rather , a reduced growth rate throughout larval development results in an approximately 25% weight reduction of dNf1 pupae relative to isogenic controls ( Figure 1E and S1 ) . Drosophila ILPs control systemic growth , metabolism , longevity , and female fecundity [21]–[24] . Among the eight Drosophila ILP genes , Ilp2 , Ilp3 and Ilp5 are co-expressed in bilateral clusters of seven insulin-producing neurosecretory cells ( IPCs ) in the larval brain [21] . Ablation of these cells causes a severe reduction in overall size , which is rescued by inducing the expression of a hsp70-Ilp2 transgene [22] , [23] . However , several results argue against a role for ILPs in the dNf1 growth defect . Firstly , two hypomorphic insulin receptor alleles , InR05545 and InR327 , did not affect dNf1 pupal size ( Figure 1F ) . Secondly , qRT-PCR analysis of RNA extracted from wandering wild-type and dNf1 third instar larvae detected no major differences in the expression of Ilp1 ( not shown ) , Ilp2 , Ilp3 , Ilp5 , Ilp6 and Ilp7 in fed larvae . Among the three IPC expressed ILP genes , the expression of Ilp3 and Ilp5 is reduced in response to starvation [21] . Starved wild-type and dNf1 larvae showed a similar reduction in Ilp5 expression , whereas Ilp3 showed a less pronounced response ( Figure 1G ) . Thirdly , while certain insulin receptor or insulin receptor substrate ( chico ) mutants have an up to 85% increased life span [25] , [26] , the lifespan of dNf1 mutants and isogenic controls was comparable ( Figure 1H ) . We note that others previously reported a reduced life span for the originally identified dNf1 p-element alleles , generated in a different genetic background [27] . Finally , we previously showed that Ilp2-GAL4 driven UAS-dNf1 expression in IPCs did not rescue the dNf1 size defect [5] . Although daily heat shocking of hsp70-ilp2 carrying larvae increased the size of dNf1 pupae , indicating that mutants do not lack the ability to respond to insulin , similar induction of this transgene , as previously noted [21] , also substantially increased the size of wild-type controls ( Figure 1I ) . Thus , reduced insulin signaling does not provide an obvious explanation for the slower dNf1 growth rate , prompting us to perform a screen to identify other genes involved in dNf1-mediated systemic growth control . While most dNf1 defects are poorly suited for use in modifier screens , the postembryonic growth defect is robust and readily quantified during the pupal stage [4] . However , using this phenotype in a screen is complicated by the fact that organismal size is sexually dimorphic ( females are larger than males ) and affected by population density , feeding , environmental factors and genetic background differences . With these confounding factors in mind , we used the crossing schemes outlined in Figure 2 to test collections of isogenic 1st and 2nd chromosome deficiencies for dNf1E2 pupal size modifier effects or synthetic lethal interactions . For each of 139 1st and 347 2nd chromosome deficiencies from the Exelixis [28] , DrosDel [29] or Bloomington Stock Center ( BSC ) collections , we generated Df ( 1 ) /+; Nf1E2/Nf1E2 ( Figure 2A ) or Df ( 2 ) /+; Nf1E2/Nf1E2 ( Figure 2B ) stocks , respectively . Notably , our work identified only few synthetic lethal interactions , and in all cases tested the synthetic lethality has been specific to the chromosome carrying the Nf1E2 allele , and not observed when the same deficiency was tested in Nf1E2/Nf1E1 null trans-heterozygotes [5] . To guard against size differences caused by inadvertent differences in population density or environmental conditions , each deficiency was scored at least twice using an initial rough caliper measurement of pupae attached to the side of culture vials . For each candidate modifying deficiency thus identified , microscopy combined with image analysis was used to determine the precise head-to-tail length of at least 40 pupae , which were then allowed to individually eclose in order to establish their sex . Several controls were next performed to eliminate non-specific modifiers or artifactual results . First , for all suppressors the continued presence of the Nf1E2 nonsense mutation was confirmed by a PCR assay ( Figure S2 ) . Secondly , as a critical specificity control , all modifying deficiencies were analyzed in a wild-type background to eliminate those that affect pupal size irrespective of dNf1 genotype . Further analysis of some of these non-specific modifiers demonstrated that loss of Act57B dominantly increases pupal size , whereas heterozygous loss of the glutamate transporter Eaat1 has the opposite effect . Thirdly , because pupal size is a function of larval growth rate and duration , modifying deficiencies were monitored for obvious changes in developmental timing . Table 1 shows the number of screened chromosome 1 , 2L and 2R deficiencies , the fraction of genes uncovered and the number of dNf1 and wild-type pupal size modifying deficiencies and loci identified . Figure 2C shows the magnitude of the pupal size modification of typical enhancers and suppressors . The number of modifying deficiencies exceeds the number of identified loci , because many modifying deficiencies uncover overlapping genomic segments ( Figure 3 ) . Not unexpectedly , individual modifying deficiencies increase or decrease dNf1 pupal size to different extents ( Figure 4 ) . Some large non-modifying deficiencies identified in our screen completely overlapped with smaller modifying ones . In such cases , stocks were re-ordered and reanalyzed . If these tests replicated the original results , genetic complementation analysis or PCR amplification using transposon and flanking sequence-specific primers was used to confirm the mapping of the deficiencies in question . This procedure identified several mismapped or mislabeled deficiencies , most of which have since been withdrawn by stock centers . Any suspect or recessive modifying deficiency , or any deficiency that uncovers genes with non-specific size phenotypes , such as Minute loci [30] , [31] , were eliminated from further analysis . Table S1 lists these deficiencies and the reason for their exclusion . During work to identify genes responsible for observed effects , we prioritized genes uncovered by suppressing deficiencies over those uncovered by enhancers . We also prioritized modifying loci uncovered by more than one deficiency , strong modifiers over weak ones , and genes uncovered by smaller deficiencies over those uncovered by larger ones , reasoning that effects of smaller deficiencies are more likely due to the loss of single genes . Validating the screen , suppressing Df ( 2R ) Exel7144 uncovers dAlk and partially overlapping suppressing Df ( 2R ) BSC199 and Df ( 2R ) BSC699 each uncover the gene for its activating ligand , jeb , both previously identified as dominant suppressors of dNf1 size , learning , and neuronal ERK over-activation phenotypes [15] . Other uncovered candidate modifiers , such as PKA catalytic and regulatory subunit genes , were tested in crosses with loss-of-function alleles and/or by tissue-specific knockdown using at least two independent UAS-RNAi transgenes , most of which were obtained from the Vienna Drosophila Stock Center ( VDRC ) [32] . For deficiencies that lacked obvious candidate modifiers , we used the UAS-RNAi approach to more broadly screen uncovered genes . Figure S3 shows examples of modifiers identified by this latter approach . Although the nutrient sensing fat body and other tissues outside of the CNS play important roles in Drosophila growth control [33] , [34] , candidate modifiers have only been tested by RNAi knockdown in neurons or glial cells . We focused on these cell types , because neuronal UAS-dNf1 expression sufficed to suppress the growth phenotype [5] . The dNf1 pupal size modifiers identified to date can be classified into three non-exclusive categories , the first of which consists of the previously implicated dAlk/jeb receptor/ligand pair and two not previously implicated other genes involved in Ras-mediated signal transduction . Another expected category includes genes involved in cAMP/PKA signaling , including the previously reported dnc cAMP phosphodiesterase suppressor [35] , and the newly identified PKA catalytic subunit gene , PKA-C1 , which acts as an enhancer . This group also includes the CCKLR-17D1 drosulfakinin receptor , recently implicated as a cAMP-coupled promoter of synaptic growth [36] , which is particularly interesting given the recent identification of a dNf1 larval NMJ overgrowth phenotype [16] . Finally , our screen also identified multiple genes whose roles in dNf1 growth control had not been anticipated and whose functional relevance remains to be established . Several genes in this group are predominantly expressed in brain or have known neuronal functions , including genes coding for the aforementioned CCKLR-17D1 receptor , the synaptic scaffold protein Dap160 , the neuronal RNA binding protein elav , the neuronal Na , K ATPase interacting protein NKAIN [37] , and the larval brain and alimentary channel expressed amino acid transporter NAAT1 [38] . Other genes in this group include CKIIbeta2 , encoding a casein kinase regulatory subunit , the endosomal trafficking proteins deep-orange and carnation , the Notch modifier heparan sulfate 3-O sulfotransferase Hs3st-B [39] , and the ubiquitin E3 ligases HERC2 , which acts as a suppressor , and CUL3 , which has the opposite effect . Table 2 lists deficiencies that modify dNf1 but not wild-type pupal size , limited to those for which the responsible gene has been identified . Table S2 identifies all analyzed deficiencies , indicates which modified dNf1 pupal size ( providing female pupal sizes as a gauge of modification strength ) , which also altered wild-type pupal size , and which deficiencies altered developmental timing . We previously reported that the dAlk receptor tyrosine kinase [40] acts as a rate-limiting activator of neuronal Ras/ERK pathways responsible for dNf1 size and learning defects [15] . Therefore , the fact that the dAlk and jeb genes are uncovered by one and two suppressing deficiencies , respectively ( Table 2 ) , validates our screen . Others recently reported that Jeb/dAlk signaling allows brain growth to be spared at the expense of other tissues in nutrient restricted Drosophila , and identified a glial cell niche around neuroblasts as the source of Jeb under these conditions [41] . To determine whether glial cells also produce Jeb involved in overall growth control under normal conditions , we used glial and neuronal Gal4 drivers to test the effect of tissue-specific jeb and dAlk knockdown . Arguing that neurons are the main source of Jeb involved in systemic growth control under non-starvation conditions , jeb knockdown with the Ras2-Gal4 , C23-Gal4 , and n-syb-Gal4 neuronal drivers [5] increased dNf1E2 pupal size ( Figure 5A ) , whereas the Nrv2-Gal4 , Eaat1-Gal4 and Gli-Gal4 glial drivers had no effect ( data not shown ) . The only glial driver that gave rise to partial rescue was the pan-glial repo-Gal4 line , although this effect was not enhanced by co-expressing UAS-Dcr2 . Control experiments showed that any driver used in these and other experiments had no effect on pupal size in the absence of UAS transgenes or vice-versa , that UAS transgenes had no effect in the absence of Gal4 drivers ( Figure 5A and data not shown ) . Finally , extending previous findings and further confirming a role for jeb as a dominant dNf1 size defect suppressor , the jebweli loss-of-function allele [42] dominantly increased dNf1 pupal size ( Figure 5B ) Previously , heterozygous mutations affecting RAF/MEK/ERK kinase cascade components Draf ( pole hole; phl ) , Dsor1/dMEK , or ERK/rolled ( rl ) , did not modify dNf1 size [5] . In agreement , two phl-uncovering deficiencies , Df ( 1 ) ED6574 and Df ( 1 ) ED11354 , did not score as modifiers ( Table S2 ) . No rl uncovering deficiencies were analyzed , but Df ( 1 ) Exel9049 , which is among the stronger suppressors identified , deletes Dsor1 and only two other genes , the neurogenic gene almondex ( amx ) , and CG17754 , predicting a BTB and Kelch domain protein . Arguing that reduced Ras/ERK signaling upon loss of Dsor1 combined with abnormal neuronal differentiation due to loss of amx may synergistically cause the observed strong effect , Ras2-Gal4 driven UAS-RNAi transgenes targeting either gene , while causing pupal lethality at 25°C , increased dNf1 pupal size at lower temperatures ( Figure 5C ) . Moreover , suppression of the dNf1 pupal size defect was also observed upon individual heterozygous loss of either Dsor1 or amx , although at least with the tested alleles , combined loss of both genes did not have a more pronounced effect ( Figure 5B ) . Previously , we did not observe suppression of the dNf1E2 pupal size defect in crosses with the Dsor1S-1221 allele [5] . A potential explanation may be that Dsor1LH110 is a null mutant [43] , whereas the molecular nature of Dsor1S-1221 is undetermined . Genetic background differences between these Dsor1 alleles are another potential explanation for the discrepant results . Multiple screens aimed at identifying genes involved in Drosophila tyrosine kinase/Ras signaling have been performed [44]–[52] . Among the genes identified , several are uncovered by 1st and 2nd chromosome deficiencies that do not modify dNf1 size . Suppressing Df ( 2R ) BSC161 uncovers 27 genes including connector enhancer of KSR ( cnk ) , a scaffold protein that functions as a bimodal ( both positive and negative ) regulator of RAS/MAPK signaling [53] , [54] . Supporting a role for cnk as a dNf1 modifier , the cnkXE-385 and cnkE-2083 alleles acted as dominant suppressors ( Figure 5B ) , and suppression was also observed upon RNAi-mediated Cnk knockdown using Ras2-Gal4 or P ( GawB ) C23-Gal4 neuronal drivers ( Figure 5C ) . However , Df ( 2R ) BSC154 , which uncovers cnk and only nine other genes , did not score as a modifier ( Table S2 ) . The dNf1 growth defect is suppressed by heat shock-induced expression of a constitutively active murine PKA catalytic subunit transgene , called PKA* [4] , or by loss of the dunce ( dnc ) cAMP phosphodiesterase [35] . Further validating our screen , two dnc uncovering deficiencies and another that removes the region immediately upstream of the dnc coding region , all scored as suppressors ( Table 2 ) . Moreover , the Pka-R2 gene , encoding a cAMP binding regulatory PKA subunit , whose dissociation from the catalytic subunit activates the latter , is uncovered by two additional suppressing deficiencies , whereas a deficiency that uncovers the major Pka-C1 catalytic subunit gene scored as an enhancer ( Table 2 ) . Df ( 1 ) ED7261 , which uncovers the rutabaga ( rut ) adenylyl cyclase , did not score as a modifier ( not shown ) . Confirmation of dnc and Pka-C1 as the genes responsible for the observed effects was obtained in crosses with three dnc and three Pka-C1 loss-of-function alleles ( Table 2 ) . Pka-R2 remains an attractive candidate suppressor , but expression Pka-R2RNAi transgenes in neurons had no effect and its role as a dNf1 modifier remains unconfirmed ( results not shown ) . Recently , the cAMP-coupled CCKLR-17D1 drosulfakinin receptor , but not its closely related CCKLR-17D3 paralog , was identified as a positive regulator of synaptic growth [36] . The CCKLR-17D1 gene is uncovered by three suppressing deficiencies , including Df ( 1 ) Exel9051 , which uncovers only three other genes . The closely linked CCKLR-17D3 paralog is not uncovered by Df ( 1 ) Exel9051 , and while Ras2-Gal4 or P ( GawB ) C23-Gal4 driven neuronal CCKLR-17D1 RNAi expression strongly suppressed the dNf1 pupal size defect , similar suppression of CCKLR-17D3 had no effect ( Figure 6A ) . Beyond CCKLR-17D1 , several dNf1 size modifiers are expressed in brain and/or have neuronal functions . Among these , dynamin-associated protein 160 ( Dap160 ) is an intersectin-related scaffold implicated in synaptic vesicle exocytosis and neuroblast proliferation [55]–[58] . Dap160 is uncovered by suppressing deficiencies Df ( 2L ) Exel6047 and Df ( 2L ) BSC302 , whose region of overlap encompasses ten genes . We note that Df ( 2L ) Exel6047 also uncovers the Drosophila Ret tyrosine kinase gene , the human ortholog of which is the receptor for glial-derived neurotrophic factor . Ret initially appeared an especially attractive candidate suppressor , because activating RET and inactivating NF1 mutations can both lead to human pheochromocytoma [59] , and because Drosophila Ret is expressed in larval brain neurons that resemble neuroendocrine cells [60] . However , among multiple lines of evidence that argue against a role for Ret in the dNf1 growth defect , UAS-dNf1 re-expression directed by a newly generated Ret-Gal4 driver that recapitulates the endogenous larval brain Ret expression pattern ( Figure S4B ) , or RNAi-mediated Ret inhibition , did not modify dNf1 pupal size , nor did expression of a UAS-Ret K805A kinase dead transgene . Moreover , Ret-Gal4 driven expression of UAS-Ret transgenes carrying the activating C695R mutation , which mimics a mutation found in multiple endocrine neoplasia type 2 did not phenocopy the dNf1 reduced growth phenotype , although the same transgene did produce the previously described rough eye phenotype when driven by GMR-Gal4 [60]; Figure S4C] . Further arguing against a role in dNf1 growth control , Ret is uncovered by non-modifying Df ( 2L ) BSC312 . By contrast , Dap160 loss-of-function alleles ( Dap160Δ1 and Dap160Δ2; [56] ) , or Dap160 RNAi expression driven by three neuronal Gal4 drivers , suppressed the dNf1 pupal size defect , identifying it as the responsible modifier ( Figure 6B ) . The gene for the neuronal RNA binding protein elav is uncovered by suppressing Df ( 1 ) Exel6221 and Df ( 1 ) ED6396 whose region of overlap includes just three other genes . Identifying elav as the responsible modifier , elav1 and elavG0031 alleles strongly suppressed ( Figure 6C ) . Rab9 is a modifier uncovered by suppressing deficiency Df ( 2L ) Exel8041 . Neuronal but not glial Rab9RNAi expression increases dNf1 pupal size , and the same result is seen upon neuronal expression of a Rab9 dominant negative [61] mutant ( Figure 6D ) . NAAT1 , coding for a larval gut and brain expressed amino acid transporter with a unique affinity for D-amino acids [38] , is uncovered by suppressing Df ( 1 ) Exel6290 and Df ( 1 ) BSC533 whose region of overlap includes only four other genes . Identifying NAAT1 as the responsible suppressor , three neuronal Gal4 lines driving the expression of three NAAT1 targeting RNAi transgenes suppressed the dNf1 size defect , whereas Repo-Gal4 driven glial expression had no effect ( Figure 7A and Table 2 ) . Mammalian E3 ubiquitin ligase HERC2 controls the ubiquitin-dependent assembly of DNA repair proteins on damaged chromosomes [62] . Drosophila HERC2 is uncovered by suppressing deficiency Df ( 1 ) Exel6254 , which also uncovers the syx16 , coding for syntaxin 16 . No HERC2 alleles exist , but Ras2-Gal4 driven expression of a UAS-HERC2RNAi transgene ( v105374 ) strongly suppressed the dNf1 pupal size defect ( Figure 7A ) , whereas similar knockdown of Syx16 had no statistically significant effect ( not shown ) . The gene for another E3 ligase component , Cul-3 , is uncovered by three enhancing deficiencies , and a Cul-3 loss-of-function allele or Ras2-Gal4 driven expression of a Cul-3 RNAi transgene both enhanced the dNf1 size defect , identifying it as the responsible gene ( Table 2 ) . Suppressing Df ( 1 ) Exel9068 uncovers only four genes , including one encoding the TORC2 complex subunit Rictor . However , systematic Ras2-Gal4 driven RNAi knockdown of Df ( 1 ) Exel9068 uncovered genes identified Hs3st-B , encoding one of two Drosophila heparan sulfate 3-O sulfotransferases , as a potent dNf1 size defect suppressor ( Figure 7A ) , whereas knockdown of Rictor had no effect ( not shown ) . Others previously identified Hs3st-B as a positive regulator of Notch signaling [39] . However , the heparan sulfate proteoglycan substrates of Hs3st-B bind various growth factors and other ligands and have been implicated in a variety of biological processes . Exactly why loss of Hs3st-B suppresses the dNf1 growth defect remains to be determined . Two functionally related dNf1 growth defect suppressors carnation ( car/Vps33A ) and deep-orange ( dor/Vps18 ) , encode subunits of the Class C Vacuolar Protein Sorting ( VPS ) complex , required for the delivery of endosomal vesicles to lysosomes [63]; Figure 7B] . The Vps16A gene encodes a third member of this complex [64] , but whether Vps16A located on the 3rd chromosome also acts as a dNf1 suppressor , or whether pharmacological inhibition of lysosomal degradation affects dNf1 pupal size are questions that remain to be answered . B4/Susi is a coiled-coil protein without obvious orthologs outside of insects . It functions as a negative regulator of Drosophila class I phosphatidylinositol-3 kinase Pi3K92E/Dp110 by binding to its Pi3K21B/dP60 regulatory subunit . Homozygous B4 mutants have an increased body size [65] , which may explain why Ras2-Gal4-driven RNAi-mediated suppression of B4 , uncovered by suppressing deficiency Df ( 2L ) BSC147 , increased dNf1 pupal size ( not shown ) . However , whether B4 is the responsible dominant modifier is doubtful , given that it is also uncovered by Df ( 2L ) BSC692 , a non-modifying deficiency . Moreover , we previously found that heterozygous loss of Pi3K21B , or neuronal expression of a dominant negative Pi3K92E transgene , did not modify dNf1 pupal size [5] . Beyond B4 , dNf1 size modifying deficiencies uncovered no genes involved in the canonical growth regulating pathways mediated by insulin and ecdysone . Indeed , several such genes were uncovered by non-modifying deficiencies . Among these genes , fat body expressed insulin-like growth factor Ilp6 , which regulates larval growth in the post-feeding phase [66] , [67] , is uncovered by two non-modifying deficiencies . A single non-modifying deficiency , Df ( 2L ) BSC206 , uncovers both the chico and pten genes , whose products antagonistically control insulin-stimulated Pi3K92E/Dp110 activity , leading to changes in body , organ , and cell size [68] , [69] . Among subunits of the cell growth regulating mTORC1 complex , raptor is uncovered by three and Tor by one non-modifying deficiency . Among genes implicated in ecdysone signaling , the ecdysone co-receptor ultraspiracle and the ecdysone-induced growth regulating DHR4 nuclear receptor [70] are each uncovered by non-modifying deficiencies , and two such deficiencies uncover Ptth , coding for prothoracicotropic hormone , which provides developmental timing cues by stimulating the production of ecdysone [71] , [72] . These results reinforce our conclusion that the canonical growth regulating pathways involving insulin and ecdysone play no obvious roles in dNf1 growth control . Several results argue that defects in Ras/ERK and cAMP/PKA signaling responsible for the dNf1 growth defect involve non-overlapping cell populations . Firstly , heat shock-induced hsp70-PKA* , or Ras2-Gal4 induced attenuated UAS-PKA* transgene ( see below ) expression rescued the dNf1 pupal size defect , but failed to reduce the elevated larval brain phospho-ERK level ( Figure 8A ) . Moreover , several neuronal RNAi drivers that increase dNf1 pupal size when driving UAS-dNf1 [5] , failed to modify this phenotype when driving dncRNAi transgenes , even in the presence of the UAS-Dcr-2 RNAi enhancer ( Table 3 ) . This prompted us to investigate whether genetic manipulation of cAMP/PKA signaling in cells other than dNf1 requiring neurons was more effective . To manipulate cAMP/PKA signaling tissue-specifically we used three UAS-dncRNAi transgenes . We also generated a series of attenuated UAS-PKA* transgenes using vectors with modified Gal4-inducible promoters harboring just 2 , 3 or 4 Gal4-binding UAS elements ( Figure 8B and C ) . We made the latter transgenes because a UAS-PKA* expression using the five UAS element containing standard UAS-T vector is lethal in combination with most Gal4 drivers [73] . As reported previously [74] , driving UAS-dNf1 ubiquitously with Act5C-Gal4 , or broadly in neurons with elav-Gal4 , Ras2-Gal4 , c23-Gal4 , or 386Y-Gal4 restored dNf1 pupal size , whereas driving the same transgene with more restricted neuronal or non-neuronal drivers had no effect ( Figure 8D and Table 3 ) . By contrast , driving the expression of UAS-dncRNAi or attenuated UAS-PKA* transgenes with the same set of broadly expressed neuronal drivers was ineffective ( Tables 3 and S5 ) . We note that expression of the 2×UAS-PKA* and 3×-UAS-PKA* transgenes was generally well tolerated , whereas the 4×UAS-PKA* and the 5×UAS-PKA* transgenes exhibited increasing levels of lethality ( Tables 3 and S5 ) . Arguing that rescue of the dNf1 growth defect by manipulating cAMP/PKA signaling or dNf1 expression involves different cells , strong pupal size rescue was observed by increasing cAMP/PKA signaling in adipokinetic hormone-producing cells at the base of the neuroendocrine ring gland using the Akh-Gal4 driver ( Figure 8D ) . Rescue was also observed with the Feb36-Gal4 and Aug21-Gal4 ring gland drivers ( Figure 8D ) , which give rise to expression in the corpora allata , the source of juvenile hormone , but not with the P0206-Gal4 or Mai60-Gal4 drivers , which express predominantly in the prothoracic gland ( Table 3 ) . The tissue specificity of all Gal4 drivers used in this and other experiments was verified by microscopic observation of dissected UAS-GFP expressing larvae ( Table S4 and Figures 8E–H and S5 ) . During larval development , significant expansion of the NMJ arbor must occur , reflecting the steady muscle growth that takes place during larval life . As the NMJ grows , additional branches and boutons are added to the initial synaptic arbor that forms during late embryonic stages upon motor axon contact with its target muscle . As a result , at the wandering third instar stage , wild-type NMJs contain a highly stereotyped , segment specific number of synaptic boutons [75] . Recently , it was reported that dNf1 functions presynaptically to constrain NMJ synaptic growth and neurotransmission [16] . In dNf1 null mutant wandering third instar larvae , while the distribution of major presynaptic proteins is unaffected , increased overall size and synaptic bouton number is apparent at multiple NMJs , supporting a specific role for dNf1 in restricting NMJ expansion [16] . Several dNf1 suppressors that emerged in the current screen have also been linked to synapse morphogenesis , including CCKLR-17D1 , which functions as a promoter of NMJ growth [36] . As our screen identified CCKLR-17D1 as a dominant dNf1 size defect suppressor , we wanted to confirm the dNf1 NMJ phenotype and test whether CCKLR-17D1 and other suppressors affected this defect . By quantifying bouton number at the NMJ on muscles 6 and 7 , we confirmed that dNf1 mutants have a significant increase in mean bouton number ( Figure 9A and B ) . In addition , this analysis confirmed previously published phenotypes for dAlk , jeb and CCKLR-17D1 [36] , [76] . Importantly , the dNf1 synaptic overgrowth phenotype is dominantly suppressed by CCKLR-17D1 , dAlk , jeb , and cnk alleles ( Figure 9B ) , arguing that all four genes are epistatic to dNf1 . As a control we analyzed an allele of spitz ( spi ) , which encodes an EGF-like growth factor and is uncovered by suppressing Df ( 2L ) Exel8041 . However , spi shows no genetic interaction with dNf1 , as loss of spi modified neither the pupal size nor the NMJ overgrowth phenotypes ( Figure 9B and data not shown ) . The identification of dAlk as a suppressor of all hitherto analyzed dNf1 defects prompted us to explore whether human ALK represents a therapeutic target in NF1 . Given our hypothesis that NF1 negatively regulates ALK stimulated Ras/ERK signaling , in order to play such a role , ALK and NF1 must be co-expressed in cells that give rise to symptoms . We previously found that dNf1 and dAlk expression overlaps extensively in Drosophila larval and adult CNS [15] , and the expression of orthologs of both genes also overlaps in the murine CNS [77] , [78] . While overlapping CNS expression is compatible with a role for ALK in NF1-associated cognitive dysfunction , a causative role in another hallmark NF1 symptom , peripheral nerve-associated tumors , is less obvious . Among the near universal symptoms on NF1 , benign neurofibromas consist of Schwann cells , perineurial fibroblasts , infiltrating mast cells , and nerve elements , with the Schwann cells sustaining the second NF1 hit [79] . To test whether increased ALK signaling in the absence of NF1 might play a role in the development of neurofibromas , we used reverse transcription/PCR to detect the presence or absence of ALK mRNA in neurofibroma-derived NF1−/− Schwann cells and NF1+/− fibroblasts , using RNAs kindly provided by Drs . Eric Legius and Eline Beert . In these experiments , two different primer sets readily detected ALK mRNA in NF1−/− Schwann cells , but not in NF1+/− fibroblasts derived from the same tumors ( Figure S6 ) . To test whether functional interactions between NF1 and ALK exist in human cells , we used the SK-SY5Y and Kelly neuroblastoma cells , both of which harbor constitutively active F1174L ALK alleles , and both of which are highly sensitive to pharmacological ALK inhibition [80] . Compatible with a role for NF1 as a negative regulator of mitogenic ALK/RAS signals , qRT-PCR verified NF1 knockdown with two shRNA retroviral vectors increased the resistance of both lines to ALK inhibitors NVP-TAE684 and Crizotinib ( Figures 10A , 10C and S7 ) . Compatible with a model in which NF1 negatively regulates ALK/RAS signaling , NF1 knockdown resulted in elevated ERK and AKT activation ( Figures 10B ) . Moreover , expression of activated KRAS , BRAF , or MEK transgenes , but not of other Ras effector transgenes , in SH-SY5Y cells conferred similar resistance to ALK inhibition ( Figure S8 ) .
The work reported here was motivated by the fact that human NF1 is a characteristically variable disease , the severity of which is controlled at least in part by symptom-specific modifier genes [81] . Thus , a genetic analysis in Drosophila might not only reveal molecular pathways controlled by the highly conserved ( 50% identical ) dNf1 protein , but also provide clues to the identity of human modifiers , which by virtue of their rate-limiting roles in symptom development might serve as therapeutic targets . The current work was also motivated by the fact that , for reasons that remain poorly understood , most dNf1 null mutant phenotypes are rescued by increasing , or phenocopied by decreasing , cAMP/PKA signaling . The identification of genetic modifiers of a cAMP/PKA sensitive defect might reveal how loss of dNf1 affects cAMP/PKA signaling , and help to resolve the long-standing controversy as to whether dNf1 affects cAMP/PKA signaling directly , independent of its role as a Ras regulator [10] , [27] , or indirectly , secondary to a Ras signaling defect [5] , [15] . While recognizing that none of the thus far identified dNf1 phenotypes are ideally suited for use in modifier screens , we selected the pupal size defect as the phenotype to analyze in our screen for three main reasons . First , pupariation occurs at the end of the larval growth period , and pupal size is readily assessed by inspecting pupae attached to the side of culture vials , making this phenotype amenable to a large-scale screen . Second , the growth defect is among several cAMP/PKA sensitive dNf1 phenotypes . Finally , reduced growth is also a symptom of human NF1 and other RASopathies [1] , [82] . However , while compelling reasons support the selection of this phenotype , confounding factors include that Drosophila size is a sexually dimorphic phenotype affected by population density , feeding , environmental conditions such as temperature , and genetic background differences . Moreover , while heterozygous dNf1 mutants are marginally smaller than wild-type pupae [5] , the more robust size phenotype ( ∼15% reduction in linear dimensions , ∼25% reduction in weight ) used in our screen is only observed upon homozygous loss of dNf1 . Thus , our screen was not designed to find modifiers that act on the dNf1 protein itself , like the recently identified SPRED proteins [83] . Finally , organism size is a function of growth rate and duration , both of which are regulated by hormonal cascades that involve cross-talk between the larval brain , the neuroendocrine ring gland , the fat body and other tissues [19] , [84] . Thus , a screen for modifiers of dNf1-regulated growth may uncover genes involved in various aspects of systemic growth control . Early attempts to identify dNf1 pupal size modifiers were abandoned when >95% of large X-ray induced 2nd chromosome deficiencies were found to be lethal in a dNf1 background ( Glenn Cowley , Iswar Hariharan and A . B . , unpublished ) , or when a pilot chemical mutagenesis screen found the reliable mapping of identified enhancer or suppressor mutations to be impracticable ( Suzanne Brill , Iswar Hariharan and A . B . , unpublished ) . Both aborted screens informed the current effort , which used precisely defined small deficiencies , isogenic crossing schemes and experimental protocols that guarded against population density differences . In total we analyzed 486 1st and 2nd chromosome deficiencies that together uncover well over 80% of chromosome 1 , 2L and 2R genes ( Table 1 ) . Among the screened deficiencies , 132 ( 27 . 2% ) significantly modified dNf1 pupal size ( p<0 . 01; two-tailed Student's t-test ) . While this is a large number , 20 deficiencies were subsequently eliminated because they also affect wild-type size . Several modifying deficiencies also uncover overlapping genomic segments , further reducing the number of dNf1 modifying loci to 76 . During follow-up studies aimed at identifying responsible genes , we prioritized genes uncovered by suppressing deficiencies over those uncovered by enhancing ones , modifiers uncovered by overlapping deficiencies over those uncovered by single deletions , modifiers uncovered by small deficiencies over those uncovered by larger ones and stronger modifiers over weaker ones . We also limited ourselves to genes that function in the nervous system , based on the consideration that dNf1 re-expression in larval neurons is sufficient to suppress the growth defect [5] . We previously reported that dNf1 growth and learning defects are phenocopied by increasing neuronal Jeb/dAlk/ERK signaling , and suppressed by genetic or pharmacological attenuation of this pathway [15] . Validating our screen , deficiencies that uncover jeb and dAlk were identified as dominant dNf1 size defect suppressors . Others recently reported that Jeb/dAlk signaling allows brain growth to be spared at the expense of other tissues in nutrient restricted Drosophila and identified a glial cell niche around neuroblasts as the source of Jeb under these conditions [41] . However , Jeb involved in systemic growth appears of mainly neuronal origin , as RNAi-mediated jeb knockdown in neurons increased dNf1 pupal size , whereas only one of four tested glial drivers produced partial rescue ( Figure 5A ) . The identification of cAMP/PKA pathway modifiers dnc , PKA-C1 and tentatively PKA-R2 further validates our screen . Arguing that increased PKA activity doesn't suppress dNf1 defects by attenuating Ras/Raf/MEK/ERK signaling , hsp70-PKA* transgene expression , using a daily heat shock regimen that suppresses the dNf1 size defect [4] , does not reduce the elevated dNf1 larval brain phospho-ERK level , and neither does Ras2-Gal4 driven neuronal UAS-PKA* expression ( Figure 8D ) . Providing further mechanistic clues , our results demonstrate that dNf1 and cAMP/PKA both affect systemic growth non-cell-autonomously , but not necessarily in the same cells . Thus , we previously showed that only relatively broadly expressed neuronal Gal4 drivers restored mutant growth when driving UAS-dNf1 , whereas multiple drivers expressed in specific subsets of neurons , including several expressed in the ring gland , lacked the ability to restore dNf1 growth [5] . By contrast , using UAS-dncRNAi or a series of newly generated attenuated UAS-PKA* transgenes that avoid the toxicity associated with high level PKA expression [73] , we now show that manipulating cAMP/PKA signaling with broadly expressed neuronal Gal drivers does not affect the dNf1 size phenotype , whereas the same transgenes induced with three ring gland drivers did suppress . Intriguingly , the most potent rescue was observed when UAS-dncRNAi or attenuated UAS-PKA* transgenes were driven in AKH-producing cells at the base of the ring gland , whereas weaker rescue was also observed with two ring gland drivers that show overlapping expression in the juvenile hormone producing corpora allata . This suggests that the dNf1 growth deficiency involves a defect in processes controlled by one or both of these neuroendocrine hormones . As might be expected of a screen that used systemic growth as a read-out , our work identified a diverse set of potential modifiers . Notably , however , among a non-exhaustive set of 18 1st or 2nd chromosome genes implicated in various aspects of Drosophila body , organ , and/or cell size control ( dAlk , B4 , chico , hpo , Hr4 , Ilp6 , jeb , Mer , mir-8 , Pi3K21B , Pten , Ptth , SNF1A , sNPF , step , Tor , ush and yki; see Table S3 for details ) , only dAlk and jeb scored as dominant dNf1 pupal size modifiers , whereas the remaining 16 genes were uncovered by non-modifying deficiencies , or in the case of Ptth , by two deficiencies that altered developmental timing ( Table S2 ) . Further explaining this lack of overlap , the previously implicated PI3 kinase regulator B4 act in a recessive manner and several of the above listed genes function outside of the CNS . Our screen excluded such genes , because dNf1 controls growth non-cell-autonomously by regulating neuronal Ras [5] . As previously noted , a special case is provided by insulin pathway components chico and Pten , which affect growth antagonistically . Both genes map within 5 kb of each other on the 2nd chromosome and are uncovered by the same non-modifying deficiency . Two newly identified dNf1 growth defect suppressors , Dap160 and CCKLR-17D1 , affect synaptic architecture or functioning [36] , [56] , [57] . Because dNf1 was recently reported to function downstream of focal adhesion kinase to restrain NMJ synaptic growth and neurotransmission [16] , and because the cholecystokinin receptor related CCKLR-17D1 drosulfakinin receptor stimulates NMJ growth [36] , we analyzed whether this and three Ras signaling related dNf1 size defect suppressors also affected NMJ architecture . Our results confirm that dNf1 mutants exhibit synaptic overgrowth , and show that loss of CCKLR-17D1 suppresses this defect . Importantly , loss of jeb , dAlk , or cnk similarly suppresses both size and synaptic overgrowth defects , suggesting that both phenotypes may be related . The results presented here further support our previous conclusion that excess neuronal Jeb/dAlk/Ras/MEK/ERK signaling is the root cause of the cAMP/PKA sensitive dNf1 systemic growth defect . What happens downstream of this primary defect remains less clear , although our demonstration that increasing cAMP/PKA signaling in AKH-producing cells and other parts of the neuroendocrine ring gland suppresses the size defect provides an important new clue , not only about pathways involved in the dNf1 growth defect , but also about the likely non-cell-autonomous cause of similar growth defects of PKA-C1 or dCreb2 mutants [85] , [86] . Other questions that remain to be fully answered concern the role of the NMJ architectural defect in the dNf1 growth deficiency and the role of Jeb/dAlk signaling in the NMJ defect . We note in this respect that that C . elegans ALK ortholog , T10H9 . 2 , has been implicated in synapse formation [87] , and that recent work suggests a role for trans-synaptic Jeb/dAlk signaling in the control of neurotransmission and synaptic morphology [88] . However , while the dNf1 growth defect is due to excess dAlk signaling in neurons , NMJ synapse formation has been suggested to involve the release of presynaptic Jeb activating postsynaptic dAlk [88] . Further work will have to establish whether the suppression of the dNf1 NMJ overgrowth phenotype by jeb , dAlk and cnk involves cell autonomous roles for these genes at synapses , or non-cell-autonomous functions elsewhere in the CNS . Further work is also required to reveal the functional significance and the sites of action of other novel modifiers identified in our screen . From a clinical perspective , perhaps the most relevant questions raised by our work are whether NF1 regulated ALK/RAS/ERK signaling is evolutionarily conserved and whether excessive ALK/RAS/ERK signaling contributes to human NF1 symptoms . Much indirect evidence hints at a positive answer to both questions . First , the expression of ALK and NF1 largely overlaps in the murine nervous system [77] , [78] , same as it does in Drosophila [15] . Second , ALK functions as an oncogene and NF1 as a tumor suppressor in neuroblastoma [89]–[94] . Third , midkine , a ligand that activates mammalian ALK [95] , is produced by NF1−/− Schwann cells , present at elevated levels in NF1 patient skin and serum , and acts as a mitogen for NF1 tumor cell lines [96]–[98] . We add to this evidence by showing that shRNA-mediated NF1 knockdown renders two oncogenic ALK-driven human neuroblastoma cell lines resistant to pharmacological ALK inhibition , and by confirming that ALK mRNA is expressed in neurofibroma-derived NF1−/− human Schwann cells . These findings make a strong case that ALK should be explored as a therapeutic target in NF1 , and that loss of NF1 expression should be considered as a potential mechanism in cases of acquired resistance to ALK inhibition [99] .
The dNf1E1 and dNf1E2 alleles have been described [5] . Exelixis , DrosDel and BSC deficiencies were obtained from the Bloomington Stock Center . Transgenic RNAi lines were obtained from the Vienna Drosophila Research Center ( VDRC ) and the TRiP Collection at Harvard Medical School . Eaat1SM1 and Eaat1SM2 were provided by D . van Meyel , dALK8 and jebweli by R . Palmer , cnkXE-385 and cnkE-2083 by M . Therrien , and carΔ146 by H . Kramer , ppl06913 by M . Pankratz , hs-Ilp2 transgenic line by E . Rulifson and UAS-Rab9 DN by R . Hiesinger . Flies were maintained on agar-oatmeal-molasses medium at 25°C , unless otherwise indicated . To assess feeding , larvae at various stages of development were placed on blue food dye-stained yeast paste , removed after 20 min , washed and photographed . To analyze wandering behavior , 100 larvae ( age 40–44 hr after egg deposition ( AED ) ) were placed on an agar plate with a central blob of yeast paste , and their position after 24 hr was documented . To assess the expression of starvation-sensitive genes , larvae at 72 h AED were placed in vials with water for 16 hr , after which RNA was prepared and subjected to blot analysis . To determine developmental timing , L1 larvae were collected 24 hr AED using a 2 hr egg collection and reared at 140 animals per vial . The number of larvae that pupariated was scored at hourly intervals . To determine the larval weight , L1 larvae were collected 24 hr AED using a 2 hr egg collection . Larvae were reared at 140 larvae per vial and groups of 10 larvae were weighed at 8 hr intervals . Longevity was assessed by maintaining adult flies under standard conditions and counting the number of dead flies at regular intervals . In each of these assays , genotypes were tested in duplicate . To induce hs-Ilp2 transgene expression , culture vials were placed in a circulating water bath at 37°C for 10 min once or twice a day with an 8 hr interval . The 7500 Fast Real-Time PCR System from Applied Biosystems was used to determine Ilp mRNA levels in RNA prepared from dissected larval brains or from whole wandering stage 3rd instar larvae . Results were normalized to RpL32 . The following primers were used: IIp2-Forward , GGCCAGCTCCACAGTGAAGT , Ilp2-Reverse , TCGCTGTCGGCACCGGGCAT , Ilp3-Forward , CCAGGCCACCATGAAGTTGT . Ilp3-Reverse , TTGAAGTTCACGGGGTCCAA , Ilp5-Forward , TCCGCCCAGGCCGCAAACTC , Ilp5-Reverse , TAATCGAATAGGCCCAAGGT , Ilp6-Forward , CGATGTATTTCCCAACAGTTTCG , Ilp6-Reverse , AAATCGGTTACGTTCTGCAAGTC , Ilp7-Forward , CAAAAAGAGGACGGGCAATG , Ilp7-Reverse , GCCATCAGGTTCCGTGGTT . Expression of the distantly related Ilp8 and the midgut-expressed Ilp4 genes [21] was not analyzed . The crossing schemes in Figure 2 were used to generate dNf1E2 mutants carrying 1st and 2nd chromosome deficiencies . To avoid crowding , cultures were maintained at 100–200 pupae per culture vial . Initial scoring used calipers set at the length of dNf1 female pupae , ignoring dNf1 heterozygotes recognizable by the presence of the TM6B balancer . Next , the length of individual pupae carrying candidate modifying deficiencies was measured by determining their head-to-tail length using a microscope fitted with NIS-Elements AR 3 . 0 imaging software . Measured pupae were then placed in 96-well plates ( Falcon ) to determine their gender and , if necessary , the genotype of eclosed flies . At least 40 pupae were measured for each genotype , and only measurements of female pupae were used to calculate mean values and standard deviations . Statistical significance was assessed with a two-tailed Student's t-test . Throughout this report , single or double asterisks denote p-values<0 . 05 or <0 . 01 respectively . To identify responsible modifiers we used specific alleles or UAS-RNAi knockdown . Alleles and UAS-RNAi lines on the 1st and 2nd chromosomes were crossed into the dNf1E2 background . UAS-RNAi lines on the 3rd chromosome were recombined with dNf1E2 . UAS-RNAi lines in the dNf1E2 background were crossed to Gal4 drivers in the same background . The few deficiencies that gave rise to synthetic lethal interactions were backcrossed with dNf1E1 flies to produce Df/+; dNf1E2/dNf1E1 progeny . To test whether genetic suppression reflected the inadvertent introduction of a wild-type dNf1 allele , we used fly DNA prepared using DNAzol ( Molecular Research Inc . ) in a PCR assay with AGTCACATTAATTGATCCTG and GAGATCGTTGATAAAGAAGT primers . The second primer introduces a penultimate single nucleotide change , which together with the E2 mutation results in the introduction of an RsaI restriction site . RsaI digestion of the PCR product gives rise to 370 and 61 bp fragments for the wild-type allele , and 348 , 61 and 22 bp fragments for the dNf1E2 allele . Digests were run on 8% acrylamide gels using both wild-type ( w1118 ) and dNf1E2 controls . The Akh promoter region was amplified with Akh-FORWARD ( AGATCTAATCTCCTGAATGCCGCAGCG ) and Akh-REVERSE ( AGATCTATGCTGGTCCACTTCGATTC ) primers . The resulting PCR fragment was subcloned into the BamHI site of a GAL4 coding region containing pCaSpeR derivative . The final construct was sequenced to ensure correct orientation of the Akh promoter before being used generate transgenic flies by standard protocols . To reduce the toxicity associated with high-level PKA expression , we generated modified pUAS-T vectors containing 1 , 2 , 3 or 4 , rather than 5 Gal4-binding sites . The primers used to generate these vectors were: 1×UAS-FOR: AACTGCAGAGCGGAGTACTGTCCTCCGAGCGGAGACTCTAG; 2×UAS-FOR: AACTGCAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCG; 3×UAS-FOR: AACTGCAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCG , and UAS-REV: CTAGAGGTACCCTCGAGCGCGGCCGCAAGAT . An initial PCR was performed using the 1×UAS-FOR and UAS-REV primers with the standard pUAS-T vector as a template . The resulting amplified fragment was TA subcloned into pCR2 . 1 to make pCR2 . 1-1×UAS . The 2×UAS-FOR and UAS-REV primers were then used with pCR2 . 1-UAS ( 1× ) as a template to generate a UAS ( 2× ) clone , which was subcloned to produce pCR2 . 1-UAS ( 2× ) . Similarly , 3×UAS-FOR and UAS-REV primers in a PCR reaction with pCR2 . 1-UAS ( 2× ) as template generated pCR2 . 1-UAS ( 3× ) and pCR2 . 1-UAS ( 4× ) ) . The pCR2 . 1-UAS clones were sequenced , their inserts excised with PstI and subcloned into PstI-digested p-UAST . Correct insert orientation was verified by sequence analysis , after which the mutationally activated murine PKA* coding region [100] was subcloned into the modified vectors using XbaI and NotI . Wandering third instar larvae were dissected in Ca2+-free saline and fixed in 4% paraformaldehyde for 25 min at room temperature . Following fixation , larval pelts were washed three times in phosphate-buffered saline ( PBS ) and then blocked for one hour in PBT ( PBS+0 . 1% Triton-X 100 ) +5% normal goat serum . Larvae were incubated in primary antibody solution for three hours at room temperature . Anti-HRP 568 ( 1∶1000 , Invitrogen ) was used to visualize neurons and Alexa Fluor 488 phalloidin ( 1∶500 , Invitrogen ) was used to visualize F-actin in the musculature . Images were collected using a Yokogawa CSU-X1 spinning-disk confocal microscope with the Spectral Applied Research ( Richmond Hill , ON , Canada ) Borealis modification on a Nikon ( Melville , NY ) Ti-E inverted microscope using a 60× Plan Apo ( 1 . 4 NA ) objective . The microscope was equipped with a Prior ( Rockland , MA ) Proscan II motorized stage . Larval samples were excited with 488-nm ( for phalloidin ) and 561-nm ( for HRP ) 100-mW solid-state lasers from a Spectral Applied Research LMM-5 laser merge module and was selected and controlled with an acousto-optical tunable filter . Emission was collected with a Semrock ( Rochester , NY ) quad pass ( 405/491/561/642 nm ) dichroic mirror and 525/50 nm ( for phalloidin ) and 620/60 nm ( for HRP ) Chroma ( Bellows Falls , VT ) emission filters . Images were acquired using a Hamamatsu ORCA-ER-cooled CCD camera . Hardware was controlled with MetaMorph ( version 7 . 7 . 9 ) software ( Molecular Devices , Sunnyvale , CA . ) . Five individual animals were imaged for subsequent morphological analysis . Motor nerve terminals of muscles 6 and 7 were imaged in abdominal segments A2 and A3 and Z-stacks ( 0 . 25 µM between images ) and were captured from the top to bottom of each NMJ . Morphological analysis of the NMJ was performed using NIH Image J and was assessed by quantifying the number of synaptic boutons per square micron . The number of synaptic boutons was counted as previously described [16] , [101] and muscle area covered by the NMJ was quantified by tracing a polygon connecting each terminal branch point [102] . The retroviral RNAi vectors targeting human NF1 and expression constructs of active alleles of RAS effectors were as described previously [94] . Crizotinib ( S1068 ) and NVP-TAE648 ( S1108 ) were purchased from Selleck Chemicals . Antibody against NF1 was from Bethyl Laboratories ( A300-140A ) ; antibodies against pAKT ( S473 ) and ATK1/2 were from Cell Signalling; antibodies against p-ERK ( E-4 ) , ERK1 ( C-16 ) , ERK2 ( C-14 ) and CDK4 ( C-22 ) were from Santa Cruz Biotechnology; A mixture of ERK1 and ERK2 antibodies was used for detection of total ERK from human cell lines . Antibody against mouse PKAα-cat ( A-2 ) SC-28315 was from Santa Cruz Biotechnology , β-Tubulin E7 from Developmental Studies Hybridoma Bank . SH-SY5Y , Kelly and Phoenix cells were cultured in DMEM with 8% heat-inactivated fetal bovine serum , penicillin and streptomycin at 5% CO2 . Subclones of each cell line expressing the murine ecotropic receptor were generated and used for all experiments shown . Phoenix cells were used to produce retroviral supernatants as described at http://www . stanford . edu/group/nolan/retroviral_systems/phx . html . To measure cell proliferation , single cell suspensions were seeded into 6-well plates ( 1–2×104 cells/well ) and cultured both in the absence and presence of ALK inhibitors . At the indicated endpoints , cells were fixed , stained with crystal violet and photographed . All knockdown and overexpression experiments were done by retroviral infection as described previously [103] . The 7500 Fast Real-Time PCR System from Applied Biosystems was used to determine mRNA levels . NF1 mRNA expression levels were normalized to expression of GAPDH . The following primers sequences were used in the SYBR Green master mix ( Roche ) : GAPDH-Forward , AAGGTGAAGGTCGGAGTCAA; GAPDH-Reverse , AATGAAGGGGTCATTGATGG; NF1-Forward , TGTCAGTGCATAACCTCTTGC; NF1-Reverse , AGTGCCATCACTCTTTTCTGAAG . ALK mRNA levels in neurofibroma-derived NF1−/− Schwann cells and NF1+/− fibroblasts were analyzed using the following two primer sets: ALK-N-Forward , GGAGTGCAGCTTTGACTTCC; ALK-N-Reverse , TGGAGTCAGCTGAGGTGTTG; ALK-C-Forward , GCAACATCAGCCTGAAGACA; ALK-C-Reverse , GCCTGTTGAGAGACCAGGAG . | Neurofibromatosis type 1 ( NF1 ) is a genetic disease that affects 1 in 3 , 000 and that is caused by loss of a protein that inactivates Ras oncoproteins . NF1 is a characteristically variable disease that predisposes patients to several symptoms , the most common of which include benign and malignant tumors , reduced growth and learning problems . We and others previously found that fruit fly mutants that lack a highly conserved dNf1 gene are reduced in size and exhibit impaired learning and memory , and that both defects appear due to abnormal Ras and cyclic-AMP ( cAMP ) signaling . The former was unremarkable , but how loss of dNf1 affects cAMP signaling remains poorly understood . Here we report results of a genetic screen for dominant modifiers of the dNf1 growth defect . This screen and follow-up functional studies support a model in which synaptic defects and reduced cAMP signaling in specific parts of the neuroendocrine ring gland contribute to the dNf1 growth defect . Beyond these results , we show that human ALK is expressed in cells that give rise to NF1 tumors , and that NF1 regulated ALK/RAS/ERK signaling is evolutionary conserved . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2013 | Genetic and Functional Studies Implicate Synaptic Overgrowth and Ring Gland cAMP/PKA Signaling Defects in the Drosophila melanogaster Neurofibromatosis-1 Growth Deficiency |
The tetravalent dengue vaccine CYD-TDV ( Dengvaxia ) is the first licensed vaccine against dengue , but recent findings indicate an elevated risk of severe disease among vaccinees without prior dengue virus ( DENV ) exposure . The World Health Organization currently recommends CYD-TDV only for individuals with serological confirmation of past DENV exposure . Our objective was to evaluate the potential health impact and cost-effectiveness of vaccination following serological screening . To do so , we used an agent-based model to simulate DENV transmission with and without vaccination over a 10-year timeframe . Across a range of values for the proportion of vaccinees with prior DENV exposure , we projected the proportion of symptomatic and hospitalized cases averted as a function of the sensitivity and specificity of serological screening . Scenarios about the cost-effectiveness of screening and vaccination were chosen to be representative of Brazil and the Philippines . We found that public health impact depended primarily on sensitivity in high-transmission settings and on specificity in low-transmission settings . Cost-effectiveness could be achievable from the perspective of a public payer provided that sensitivity and the value of a disability-adjusted life-year were both high , but only in high-transmission settings . Requirements for reducing relative risk and achieving cost-effectiveness from an individual perspective were more restricted , due to the fact that those who test negative pay for screening but receive no benefit . Our results predict that cost-effectiveness could be achieved only in high-transmission areas of dengue-endemic countries with a relatively high per capita GDP , such as Panamá ( 13 , 680 USD ) , Brazil ( 8 , 649 USD ) , México ( 8 , 201 USD ) , or Thailand ( 5 , 807 USD ) . In conclusion , vaccination with CYD-TDV following serological screening could have a positive impact in some high-transmission settings , provided that screening is highly specific ( to minimize individual harm ) , at least moderately sensitive ( to maximize population benefit ) , and sufficiently inexpensive ( depending on the setting ) .
A safe and effective dengue vaccine could have a major public health impact , as dengue causes approximately 9 , 000 deaths and between 50–100 million clinically apparent cases worldwide every year [1 , 2] and has a growing geographic distribution [3] . The first licensed dengue vaccine , CYD-TDV ( Dengvaxia ) , is a tetravalent , live-attenuated vaccine that was licensed in multiple countries after demonstrating efficacy against symptomatic disease in phase-III trials [4 , 5] . Protection has been hypothesized to derive primarily from the vaccine functioning as a “silent infection” [6] . Following their first natural infection subsequent to vaccination , this mechanism would result in vaccinees with prior dengue virus ( DENV ) exposure bypassing the elevated risk of severe disease typically associated with secondary infections . Modeling analyses [6 , 7] indicated that vaccination of nine-year-old children with CYD-TDV could be cost-effective in populations in which the majority of vaccinees have prior DENV exposure . The downside of this mode of protection is an elevated risk of severe disease in vaccinees with no prior DENV exposure at the time of their first natural DENV infection [8] . Recent findings [9] confirmed this hypothesis , leading to an abrupt end to CYD-TDV use in the Philippines [10] and a revision of the World Health Organization’s ( WHO ) Strategic Advisory Group of Experts on Immunization recommendations in April 2018 on the use of the vaccine [11] . Vaccination with CYD-TDV is now recommended only for individuals with known prior DENV exposure [12–14] . Because DENV infection often results in asymptomatic infection or presents with mild , non-specific symptoms [15] , an individual’s clinical history is a poor indicator of prior exposure . Thus , serological screening must play a role in any path forward for CYD-TDV or any other future dengue vaccines with similar characteristics . Reliable inference of prior DENV exposure based on serological data can be extremely challenging , however , due to cross-reactivity among DENV serotypes and among DENV and other flaviviruses [16 , 17] . To avoid elevating the risk of severe dengue by vaccinating a DENV-naïve individual , serological screening used to inform vaccination must have high specificity ( i . e . , probability that a DENV-naïve individual tests seronegative ) . At the same time , high sensitivity ( i . e . , probability that an individual with prior DENV exposure tests seropositive ) is important for ensuring that people who could benefit from the vaccine will receive it . The balance of benefits and harms caused by vaccination with CYD-TDV following serological screening with a given sensitivity and specificity must also be weighed against the economic benefits and costs of such a strategy . Although a strategy of CYD-TDV vaccination following serological screening has been examined with mathematical modeling before [18 , 19] , those analyses were restricted to a scenario in which the screening assay had perfect sensitivity and specificity . In practice , imperfect sensitivity and specificity [20] , tradeoffs between sensitivity and specificity [21] , and cost [22] all merit consideration in analyses of serological screening in CYD-TDV vaccination programs . We applied an agent-based model of DENV transmission to identify the conditions under which a strategy of vaccination with CYD-TDV following serological screening ( hereafter , referred to together as “the intervention” ) would have positive impacts on health and be cost-effective . As with a previous study [7] involving this model and seven others , we focused our analysis on a strategy of routine intervention applied to a single age of nine years old . From both an individual and population perspective , we identified minimum requirements to achieve positive health impact and cost-effectiveness as a function of sensitivity , specificity , cost of serological screening , cost of vaccination , and prior DENV exposure among nine-year-olds ( PE9 ) . We focused on cost scenarios representative of Brazil and the Philippines , which have both licensed CYD-TDV but differ in terms of economic conditions .
Our agent-based model of DENV transmission was previously described elsewhere [23] . This model has been previously used as part of a consortium of eight modeling groups to make projections of CYD-TDV impact in the absence of serological screening [7] . Despite differences with the other models , our model showed general agreement on projections of vaccination impact . In our model , humans and mosquitoes are represented by individual agents who interact with each other through mosquito blood-feeding at the household scale . The model assumes that transmission of any of the four DENV serotypes can occur whenever an infected mosquito blood-feeds on a susceptible human or a susceptible mosquito blood-feeds on an infected human . Infected humans acquire life-long immunity to the infecting serotype and temporary immunity to other serotypes to which they have not been previously exposed . Several model features are parameterized based on extensive data collection from Iquitos , Peru , including fine-scale patterns of human mobility [24] , the demographic composition of households [25] , and the geographic arrangement of residential , commercial , and other buildings [26] . Other model features were less well known a priori: the rate at which DENV was seeded into the population , the probability of an infectious mosquito infecting a susceptible human during blood-feeding , and the emergence rate of adult female mosquitoes . For a given simulation , we parameterized these features of the model by selecting a combination of parameter values that achieved a target value of the proportion of nine-year-olds with prior DENV exposure after 40 years of simulation , or PE9 , as described in S1 Appendix . The vaccine implemented in our simulations acted as a silent DENV infection in the recipient , as has been assumed in previous CYD-TDV modeling assessments [6 , 7] . Because the vaccine is assumed to act as a silent infection , vaccination results in an elevated risk of severe disease among DENV-naïve vaccinees experiencing their first natural DENV infection , because secondary infections are associated with the highest probabilities of symptomatic disease conditional on infection and hospitalization conditional on symptomatic disease . In addition , we assumed a period of temporary cross-immunity after vaccination that waned over time . The level of protection and the waning period varied for individuals with and without previous exposure to DENV . Death was assumed to occur among a small proportion ( 0 . 0078 ) of cases of symptomatic disease . Because estimates of the rates of these outcomes are highly variable across study settings [27] , we calibrated our model such that its outputs matched the most recent estimates of vaccine protection from clinical trials [9] . We did so by simulating a virtual trial [28] similar to the trials across a range of values of ten model parameters ( Table 1 ) using a sequential importance sampling approach [29] and generalized additive models from the ‘mgcv’ [30] library in R[31] , as described in S2 Appendix . The best-fit model showed agreement with estimates of vaccine efficacy against symptomatic disease and hazard ratios for hospitalization stratified by age and baseline serostatus ( S2 Appendix ) . Consistent with recently revised WHO recommendations [12] , we simulated serological screening immediately prior to vaccination with CYD-TDV . We focused on a strategy of routine vaccination in which a proportion of children underwent serological screening , and vaccination in the event of a positive result , on their ninth birthday . One consequence of this strategy was that intervention coverage ( i . e . , the proportion of children screened ) represents an upper limit on the proportion of vaccine-eligible children . Assuming that all vaccine-eligible children were vaccinated , the vaccination coverage ( i . e . , positive serological screening result and subsequent vaccination ) was related to intervention coverage by coveragevaccination=coverageintervention×SP9 , ( 1 ) where SP9 is seropositivity among nine-year-olds and is defined as SP9=PE9×sensitivity+ ( 1−PE9 ) × ( 1−specificity ) . ( 2 ) Similar to other models of CYD-TDV , our default assumption was a three-dose schedule with 100% compliance . In the event that compliance is lower , our results would be more pertinent to a scenario with a correspondingly higher coverage , as the effects of coverage and compliance are interchangeable in this way . We performed 3 , 000 sets of simulations of intervention impact , with each simulation set involving one simulation with the intervention and one without . These simulation sets used the sobol function in the pomp library [32] in R [31] to evenly span a range of values of intervention coverage ( 10–80% ) , PE9 ( 0 . 1–0 . 9 ) , and sensitivity ( 0–1 ) and specificity ( 0–1 ) of serological screening . Each simulation lasted for 50 years , with the intervention being introduced after the first 40 years . Every year thereafter , a proportion of nine-year-olds underwent serological screening for prior DENV exposure and were vaccinated if screening returned a positive result . Both simulations in each set were initiated with the same random number seed , which allowed us to isolate the impact of the intervention to the greatest extent possible under a stochastic , agent-based model . With each set of parameter values , we calculated the proportion of cases averted over a 10-year period as proportionofcasesaverted=cumulativecasesw/ointervention−cumulativecasesw/interventioncumulativecasesw/ointervention ( 3 ) for both symptomatic and hospitalized cases . To estimate the impact of the intervention from the perspective of an individual who chose to undergo serological screening , we compared the risk of individuals from the first cohort of nine-year-olds who underwent serological screening with individuals from a comparable cohort of nine-year-olds who did not undergo serological screening . These individuals were followed for 10 years after vaccination and came from the same simulation . We calculated relative risk of symptomatic disease and hospitalization as relativerisk= ( cumulativecasesw/intervention ) / ( populationw/intervention ) ( cumulativecasesw/ointervention ) / ( populationw/ointervention ) . ( 4 ) To extract average patterns from the highly stochastic outputs from 3 , 000 simulations of our model and to interpolate across gaps in parameter space , we summarized simulation outputs with generalized additive models , as described in S3 Appendix . To assess the impact of vaccination over a longer time frame , we also evaluated effects of vaccination from both population and individual perspectives over 30 years . Results corresponding to parameter sets beyond those shown here can be explored interactively online at http://denguevaccine . crc . nd . edu . Our first goal was to quantify the health impact of vaccination with CYD-TDV following serological screening under different conditions . At the population level , we made projections of the proportion of cases averted over a 10-year period , separately for symptomatic and severe cases , under a range of values of intervention coverage , PE9 , sensitivity , and specificity . From the perspective of an individual who underwent serological screening , and vaccination in the event of a positive result , we made projections of the relative risk of experiencing a symptomatic or hospitalized case as compared to someone who forewent serological screening altogether . We examined this individual risk in aggregate and stratified by prior DENV exposure . Our second goal was to understand the conditions under which vaccination with CYD-TDV following serological screening might be cost-effective . The intervention was deemed cost-effective if costintervention<DALYsaverted×costDALY+symptomaticcasesaverted×costsymp+hospitalizationsaverted×costhospitalized+deathsaverted×costdeath , ( 5 ) where costsymp and costhospitalized reflect costs of ambulatory care and inpatient hospital care for symptomatic and hospitalized cases , respectively , and costdeath refers to the direct cost of death , such as burial expenses and disruption to family income . DALYs refer to disability-adjusted life years , which are years of healthy life lost to disease . We based calculations of DALYs averted on three components: symptomatic cases averted and the DALYs associated with a symptomatic case , hospitalized cases averted and the DALYs associated with a hospitalized case , and deaths averted and the average number of years of life lost for an individual in our model with a dengue-associated death . The cost of a DALY , costDALY , was based on a country’s gross domestic product ( GDP ) per capita , in line with WHO guidance [33] . An intervention with costintervention satisfying Eq 5 was deemed “cost-effective” when costDALY = 3 x per capita GDP and “very cost-effective” when costDALY = 1 x per capita GDP . Our assumptions about the numerical values of costs in Brazil and the Philippines are based on previous estimates used by Flasche et al . [7] and are detailed in Table 2 . We applied a 3% annual discounting rate to both costs and DALYs . We took two approaches from the perspective of the cost of the intervention , which is defined as costintervention=coverageintervention×costscreen+coveragevaccination×costvac , ( 6 ) where costscreen is the unit cost of serological screening and costvac is the cost of fully vaccinating a single person . Our first approach involved seeking the threshold cost of serological screening at which costs below that threshold would be cost-effective when combined with a costvac of 69 USD , which we based on pricing information from the Philippines [34] as explained in S4 Appendix . Our second approach involved determining whether a fixed costscreen of 10 USD ( similar to a recent estimate of 9 . 25 USD in Vietnam [22] ) would result in cost-effectiveness under three different assumptions about costvac corresponding to three , two , or one doses ( 69 , 46 , or 23 USD ) , assuming that any number of doses confers the same degree of protection . The possibility that fewer than three doses may confer protection against dengue has been suggested as a possibility but requires further investigation [35] . Under both approaches , we examined how cost-effectiveness varied as a function of intervention coverage , PE9 , and the sensitivity and specificity of serological screening . Aspects of our cost-effectiveness analysis also differed depending on the perspective of who was paying for the intervention: either a public payer ( e . g . , government or healthcare provider ) or an individual . Health benefits in terms of cases and deaths averted differ from these population and individual perspectives , with the former being of interest to a public payer . Costs from these perspectives were differentiated in two ways . First , we monetized the direct cost of death , costdeath , from the individual perspective as one year of productivity lost , as previously assumed by Flasche et al . [7] , but we assumed no additional direct costs of fatal cases from the public payer perspective . Both perspectives considered the cost of death associated with DALYs due to premature death . Second , we assumed that ambulatory care and hospitalization costs were different for the individual and the public payer . Specific assumptions about costs from these perspectives are provided in Table 2 .
The proportion of cases averted depended on the sensitivity and specificity of serological screening in different ways for different values of PE9 . In terms of symptomatic cases , the intervention resulted in a positive impact under nearly all combinations of parameters in all transmission settings . This was a consequence of the fact that calibration of our model to data from CYD-TDV trials resulted in estimates of the probability of symptomatic disease that decreased with each successive infection ( Table 1 ) . Thus , vaccinating more people , regardless of serostatus , resulted in more symptomatic cases averted ( Fig 1 , top ) . Although a lower probability of symptomatic disease in secondary infections differs from other modeling studies [6 , 7 , 41] , it is consistent with calibration of our model to the most recent trial data [9] . In terms of hospitalizations averted , the intervention resulted in a negative impact under approximately half of the scenarios we examined . Specifically , impact was more positive in settings with higher transmission and more negative in settings with lower transmission ( Fig 1 , bottom ) . With respect to screening properties , sensitivity was the dominant factor in high-transmission settings , and specificity was the dominant factor in low-transmission settings . For both symptomatic and hospitalized cases , relationships at lower values of PE9 were less smooth , due to a larger influence of stochasticity and more uncertainty in these transmission settings ( S2 Fig ) . The primary explanation for the positive relationship between screening sensitivity and cases averted in the highest PE9 setting ( 0 . 9 ) is that vaccination coverage depended almost exclusively on sensitivity and very little on specificity ( S1 Fig ) . From a population perspective , achieving high coverage in a high-PE9 setting appeared ideal , although it also appeared that high specificity had benefits in high-transmission settings by increasing the proportion of hospitalized cases averted ( 11% for sensitivity = 1 , specificity = 1 ) beyond levels achievable by high vaccination coverage alone ( 9% for sensitivity = 1 , specificity = 0 ) ( Fig 1 , bottom right ) . At low PE9 , coverage was highest when specificity was low ( S1 Fig ) , but that resulted in an increased number of DENV-naïve vaccinees who then went on to experience symptomatic disease and possibly hospitalization upon natural infection ( Fig 1 , bottom left ) . Thus , public health impact was maximized at low PE9 when specificity was high ( which minimized individual harm ) and sensitivity was also high ( which increased coverage among the few who should have been vaccinated ) . From the perspective of a nine-year-old who underwent serological screening ( and , in the event of a positive result , vaccination ) , the relative risk of symptomatic disease was generally reduced . Given that the vaccine reduces the hazard of symptomatic disease for both seropositive and seronegative individuals , relative risk of symptomatic disease lessened as the proportion of vaccination coverage increased ( Fig 2 , top ) . As with population-level impacts , the relative risk of hospitalization was reduced in medium- to high-transmission settings ( PE9≥0 . 5 ) and depended on sensitivity and specificity in other settings ( PE9<0 . 5 ) ( Fig 2 , bottom ) . Under a scenario of PE9 = 0 . 7 , relative risk of hospitalization was reduced when sensitivity was at least 0 . 4 or specificity was above 0 . 9 . This reduction was mostly driven by sensitivity when specificity was below 0 . 8 , whereas specificity modulated risk as much as sensitivity for values of specificity above 0 . 8 . The greatest benefits occurred in high-transmission settings ( PE9 = 0 . 9 ) with high sensitivity ( ≥0 . 9 ) and high specificity ( ≥0 . 8 ) , in which case relative risk was as low as 0 . 4 ( Fig 2 , bottom right ) . In low-transmission settings ( PE9<0 . 5 ) , relative risk of hospitalization was generally elevated , unless specificity was very high . Moreover , the reduction of risk in low-transmission settings was low , even with high specificity and sensitivity . Even though greater sensitivity reduced relative risk for an average person undergoing serological screening , from the point of view of a truly seronegative individual undergoing screening , relative risk of hospitalization was always elevated unless specificity was perfect ( S3 Fig , top ) . In medium- to high-transmission settings ( PE9≥0 . 5 ) , relative risk was 1 . 1 or less for specificity values above 0 . 9 , compared to relative risk higher than 1 . 3 under a scenario in which serological screening resulted in all children being vaccinated ( sensitivity = 1 , specificity = 0 ) . Under an assumption of routine vaccination , age of vaccination modulated the population-level benefits of vaccination in terms of hospitalizations averted ( S4 Fig ) . In higher transmission settings , vaccination at younger ages resulted in increased benefits , given that a large proportion of vaccinees had at least one infection at the time of vaccination ( S4 Fig , top right ) . In contrast , benefits of vaccination were higher in low-transmission settings when older children were vaccinated . Vaccination in low-transmission settings appeared to have positive impacts only when routine vaccination occurred in children 15 years of age or older and specificity was high ( S4 Fig , bottom left ) . From a public payer perspective , and assuming a cost for a full three doses of vaccine of 69 USD , our results suggest that a strategy of vaccinating seropositive nine-year-olds would be cost-effective only under limited circumstances . In simulations of medium-transmission settings ( PE9 = 0 . 5 ) and with a Brazil-like scenario about costs , vaccinating seropositive nine-year-olds was cost-effective for high values of specificity ( >0 . 8 ) and modest values of sensitivity ( >0 . 3 ) . In high-transmission settings ( PE9 ≥ 0 . 7 ) , cost-effectiveness depended on both sensitivity and specificity , with the highest thresholds for cost-effectiveness found at sensitivity and specificity above 0 . 8 ( Fig 3 , bottom right ) . In a high-transmission scenario ( PE = 0 . 9 ) , we found that the threshold cost for serological screening ( i . e . , the maximum cost at which the intervention could still be cost-effective ) was around 45 USD . Under a Philippines-like scenario about costs , vaccinating seropositive nine-year-olds was not cost-effective under any of the scenarios that we considered ( S5 Fig ) . Our results showed that cost-effectiveness was possible under a somewhat broader range of parameters when we considered lower costs of the vaccine and a fixed cost of serological screening ( 10 USD ) . We found that reducing the cost of the vaccine to 46 USD ( equivalent to two doses , assuming that they provide the same protection as three ) had little impact on which parameter combinations ( PE9 , sensitivity , specificity ) resulted in cost-effectiveness ( S6 & S7 Figs ) . In contrast , reducing the cost of the vaccine to 23 USD ( equivalent to one dose , assuming that it provides the same protection as three ) resulted in cost-effectiveness in high-transmission settings ( PE9 ≥ 0 . 7 ) under both the Brazil and Philippines scenarios about costs ( Fig 4 , S8 Fig ) . From the perspective of the parent of a nine-year-old child considering serological screening , our results suggest that the intervention would not be cost-effective in Brazil or the Philippines ( Figs 5 & S9 ) . For both countries , low coverage ( 10% ) had the effect of slightly increasing the threshold cost of serological screening relative to a scenario with high coverage ( 80% ) , but not enough to achieve cost-effectiveness under any parameters we considered for the Philippines ( S10 & S11 Figs ) . This is a result of there being more to gain by an individual opting for the intervention when coverage is lower , due to lower indirect protection from others who are vaccinated . Lowering the number of doses to two ( 46 USD ) did not improve cost-effectiveness for the Brazil-like cost scenario ( S12 Fig ) , although lowering to one dose ( 23 USD ) and assuming a cost of serological screening of 10 USD did ( Fig 6 , bottom ) . Cost-effectiveness under these scenarios in moderate transmission settings ( PE = 0 . 5 ) depended on high sensitivity ( >0 . 9 ) and moderate specificity ( >0 . 5 ) . In high-transmission settings ( PE9≥0 . 7 ) , cost-effectiveness was achieved for sensitivity values above 0 . 5 ( Fig 6 , bottom ) . None of the scenarios that we considered were cost-effective under the Philippines-like cost scenario ( S13 & S14 Figs ) . Over a 30-year period , the public health impacts of the intervention were more pronounced than over a 10-year period ( Figs S15 & 1 ) . This was true for both positive impacts in high-transmission settings and negative impacts in low-transmission settings . From an individual perspective , the magnitude of relative risk differed very little over 10-year and 30-year periods ( Figs S16 & 2 ) . From both public health and individual perspectives , positive impacts were observed across a slightly wider range of sensitivity and specificity values ( S15 & S16 Figs ) . Cost-effectiveness also increased from both of these perspectives , due to the fact that the cost of the intervention was the same over both time periods ( S17–S24 Figs ) .
Using a model consistent with seven others that informed the WHO’s initial position on CYD-TDV [7 , 42] but updated with the latest clinical trial data [9] , we assessed the potential health impact and cost-effectiveness of the recent WHO recommendation [12] for vaccination with CYD-TDV following serological screening . In some respects , our projections were similar to previous results about vaccination without serological screening; namely , positive public health impacts in areas with high previous exposure [6 , 7] . In other respects , our results provide new insights on issues unique to the context of the WHO’s pre-vaccination screening recommendation . First , our results show that high specificity is essential for reducing hospitalizations in low-transmission settings but , at the same time , leads to fewer symptomatic cases averted . The latter effect resulted from our assumption that the probability of symptomatic disease is highest in primary infections and decreases with each successive infection . Models that differ in this assumption would likely reach different conclusions about this issue . Second , our results show that sensitivity is important for achieving positive health impacts in high-transmission settings , due to the fact that higher sensitivity increases population coverage in those settings . Sensitivity appears to be less important in low-transmission settings though , from both population and individual perspectives . Third , from a public payer perspective , we conclude that cost-effectiveness is unlikely except in countries with relatively high GDP and assuming low costs of serological screening ( 10 USD ) and vaccination ( 23 USD ) . Even then , cost-effectiveness would be limited to areas with relatively high transmission intensity and to tests with relatively high sensitivity . Fourth , conditions for cost-effectiveness from an individual perspective were more limited than from a public payer perspective . In low-transmission settings or with a low-sensitivity test in high-transmission settings , this results from the fact that the many who test negative pay to get tested but receive no health benefit as a result . Like other modeling assessments of interventions under consideration for implementation [43–46] , our study focused on offering general insights . As a consequence , we were only able to explore a relatively limited range of scenarios about vaccine roll-out . In reality , CYD-TDV could be deployed in a top-down manner by governments , purchased by individuals , or some combination thereof , given that is licensed for use in individuals ranging in age from nine to 45 years . Nevertheless , certain aspects of our analysis may offer insights about a broader range of scenarios . For example , some of our results about routine vaccination in nine-year-olds may apply under alternative scenarios if our parameter for prior exposure among nine-year-olds , PE9 , is interpreted more broadly as prior exposure among vaccine recipients on the whole , at whatever age that might be . Such an extrapolation would appropriately mimic the level of prior exposure among vaccinees , but it may not accurately reflect transmission intensity in a population in which that level of prior exposure is achieved by a different age . Also , given that at younger ages our model underestimated the attack rates of hospitalization at 60 months , projections of our model at these ages would potentially underestimate hospitalizations in seronegative individuals and overestimate the cost-effectiveness of routine pre-vaccination screening strategies . However , it is unlikely that individuals in that age range would ever be vaccinated , and within the 9–16 age range for routine vaccination that we considered , results from simulations involving routine vaccination in nine-year-olds appeared reasonably robust . With respect to economic considerations , our results indicate that serological screening , and vaccination in the event of a positive result , could be cost-effective only under certain circumstances . Assuming as others have [47–49] that decisions about cost-effectiveness are made in reference to a multiplier between per capita GDP and costDALY , our results predict that cost-effectiveness could be achieved only in high-transmission areas of dengue-endemic countries with a relatively high per capita GDP , such as Panamá ( 13 , 680 USD ) , Brazil ( 8 , 649 USD ) , México ( 8 , 201 USD ) , or Thailand ( 5 , 807 USD ) [39] . In the event that CYD-TDV vaccination is recommended in a country but remains unfunded , it is likely that coverage and impact will be low , similar to varicella vaccines in Australia and Canada [50–52] . To the extent that access to CYD-TDV becomes associated with the economic means to pay for serological screening and vaccination , this could exacerbate socioeconomic disparities in dengue’s burden . It is also important to note that our analysis of cost-effectiveness does not imply affordability . Multiple studies have shown that interventions that have been deemed “very cost-effective” have nonetheless not been implemented in low- and middle-income countries due to a variety of factors , such as implications for spending on competing public health priorities [53–55] . Another approach to estimating costDALY is to refer to incremental cost-effectiveness ratios ( ICERs ) from other interventions that could be displaced by CYD-TDV , such as vaccines against rotavirus and human papillomavirus . These interventions have been shown to be very cost-effective in settings comparable to Brazil and the Philippines , with ICERs below 2 , 000 [56 , 57] . Based on our results , none of the scenarios that we considered would result in cost-effectiveness of CYD-TDV comparable to these interventions , given that that would have required cost-effectiveness to be achieved with costDALY < 2 , 000 USD . Although our analysis provides an indication of desirable characteristics of assays for serological screening , there is not yet an assay available that is simultaneously rapid , point-of-care , low-cost , and both highly sensitive and specific [58] . Neutralization assays , for example , are reasonably accurate but expensive and time-consuming , whereas assays such as IgG ELISAs are faster and relatively inexpensive , but often far less accurate [20] . Given the tradeoffs between the sensitivity and specificity of any assay , our results suggest that priority should be placed on maximizing specificity . Doing so would minimize the potential risks associated with vaccination of DENV-naïve individuals misclassified by an imperfectly-specific assay as having been previously exposed . Achieving high specificity in determining DENV serostatus is complicated by numerous sources of cross-reactivity , including prior exposure to or vaccination against Japanese encephalitis , West Nile , yellow fever , or Zika viruses [16] . Because these factors affecting cross-reactivity are population-specific , any assay used to inform vaccination with CYD-TDV should be calibrated to results from a highly specific assay ( e . g . , plaque-reduction neutralization tests ) in a given population to maximize specificity [59] . Inevitably though , maximizing specificity will come at the cost of decreased sensitivity [21] and , as we have shown , reduced population-level benefits . By considering the full range of possible sensitivities and specificities , our results offer a quantitative basis for assessing the potential impact and cost-effectiveness of any existing or future assay . In theory , a highly effective , tetravalent dengue vaccine could have a substantial impact on reducing dengue’s considerable burden , but that goal remains elusive for numerous reasons [60] . In the absence of a single intervention that is highly effective across a wide range of contexts , interest continues to grow in determining how to best combine multiple interventions in ways that are appropriate for a given local context [61] . Making that determination has become increasingly challenging due to nuanced , yet highly consequential , issues associated with use of CYD-TDV . Mathematical modeling analyses offer important capabilities for addressing this challenge due to their ability to weigh complex tradeoffs among intervention properties , as demonstrated here with respect to the sensitivity and specificity of serological screening , prior DENV exposure among vaccinees , and intervention coverage and cost . In addition , by considering both individual and population perspectives , our analysis provides information that could be informative for discourse about difficult ethical considerations surrounding the use of CYD-TDV [62] . | Among several viral diseases transmitted by Aedes aegypti mosquitoes , dengue imposes the greatest and most persistent burden on global health . Efforts to curb its spread would benefit greatly from the availability of an effective vaccine . Currently , the only licensed dengue vaccine , known as CYD-TDV or by the brand name Dengvaxia , is only recommended for use in people who are known to have been exposed to dengue virus in the past . Because symptoms of dengue can range from severe to mild to imperceptible , using clinical history alone to assess whether a person was previously exposed is unreliable . Instead , serological assays , which measure a person’s immune response to dengue virus , are necessary to confirm whether a person was previously exposed . Because serological assays can be subject to substantial error , we used a simulation model to assess how impactful CYD-TDV vaccination would be under different scenarios about the accuracy of a serological assay and the intensity of transmission in a given area . We found that the health impact and cost-effectiveness of CYD-TDV vaccination depended on the accuracy of the serological assay , its cost , and the setting in which it is deployed . | [
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] | 2019 | Model-based assessment of public health impact and cost-effectiveness of dengue vaccination following screening for prior exposure |
The exogenous RNA interference ( RNAi ) pathway is an important antiviral defense against arboviruses in mosquitoes , and virus-specific small interfering ( si ) RNAs are key components of this pathway . Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission . Using deep sequencing technology , we characterized dengue virus type 2 ( DENV2 ) -specific small RNAs produced during infection of Aedes aegypti mosquitoes and A . aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line . We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A . aegypti cells indicate that they are products of Dicer-2 ( Dcr2 ) cleavage of long dsRNA , whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity , suggesting that they originate from a different small RNA pathway . Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus ( CFAV ) corroborated these findings . An in vitro assay also showed that Aag2 A . aegypti cells are capable of siRNA production , while C6/36 A . albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA . Defective expression or function of Dcr2 , the key initiator of the RNAi pathway , might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line , which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts .
Mosquito cell cultures are used routinely in arbovirology studies to grow viruses and to elucidate aspects of viral infection and replication in mosquitoes . Many of these cell lines were established by Peleg and Singh in the 1960's [1] , [2] . A mosquito cell line designated C6/36 resulted from a clone selected by Igarashi from Singh's Aedes albopictus larval line for its ability to grow dengue and chikungunya viruses to high titers [3] , and has become one of the most commonly used of arbovirologists' tools [4] , [5] , [6] , [7] , [8] . Since Aedes aegypti is the most important vector for arboviruses such as dengue , we have used another cell line , derived from A . aegypti embryos and known as Aag2 , in several recent studies [9] , [10] . This cell line was originally established by Peleg in 1968 and was further characterized by Lan and Fallon in 1990 [11] . RNA interference ( RNAi ) has been shown to play an important role in insect antiviral immunity [12] , [13] , [14] . RNAi is a molecular pathway that is triggered by exogenous long double-stranded RNA ( dsRNA ) in the cytoplasm . Much of what we know about RNAi in insects has been elucidated in Drosophila flies and cultured cells . Dicer-2 ( Dcr2 ) is a multi-domain RNase III that recognizes and cleaves dsRNA into small interfering RNAs ( siRNAs ) to initiate the RNAi pathway . The siRNAs are usually 21 bp in length with 5′ phosphates and two nt overhangs on the 3′ hydroxyl ends [15] , [16] , [17] , [18] , [19] . siRNAs , in association with Dcr2 and the dsRNA-binding protein R2D2 , are loaded into a multi-protein RNA-induced silencing complex ( RISC ) , which contains the endonuclease Argonaute-2 ( Ago2 ) [20] , [21] , [22] . The RISC unwinds and degrades one of the siRNA strands , and retains the other strand for use as a guide to identify long single-stranded RNA ( ssRNA ) , such as viral mRNA , which is complementary to the siRNA . In the effector phase of RNAi , Ago2 cleaves the long ssRNA at the point of complementarity , leading to its further destruction [23] , [24] , [25] . Other RNA silencing pathways , including Piwi-interacting ( piRNA ) and endogenous siRNA ( endo-siRNA ) , have been discovered in Drosophila [26] , [27] , [28] , [29] , [30] , [31] , [32] . piRNAs associate with members of the Piwi clade of the Argonaute proteins , which includes Piwi , Aubergine ( Aub ) and Argonaute 3 ( Ago3 ) in Drosophila . piRNAs are approximately 24–30 nt in length and are modified by DmHEN1 ( also known as Pimet ) by 2′-O-methylation on their 3′ termini [33] , [34] . The piRNA pathway trigger appears to be single-stranded RNA since the small RNAs are almost always of a single polarity , and the biogenesis is Dcr1- and Dcr2-independent , possibly using the endonuclease activity of the Piwi proteins , at least in determining their 5′ ends [27] , [28] , [35] . The piRNA pathway is believed to have important roles in controlling the transcription of transposable elements in the genome and in development of reproductive tissues . Recently , virus-derived piRNAs were discovered in cultured Drosophila ovary somatic sheet cells [36] . Genomic analyses show that mosquitoes encode Drosophila-orthologous RNAi pathway components including Dcr2 , R2D2 , and Ago2 , as well as paralogous microRNA ( miRNA ) pathway components Drosha , Pasha , Dicer-1 ( Dcr1 ) , Loquacious ( Loqs; R3D1 ) , and Argonaute-1 ( Ago1 ) [37] . Deep sequencing analyses have characterized miRNAs in A . aegypti [38] and Culex quinquefasciatus [39] and have demonstrated altered levels of expression after blood-feeding [38] and after WNV infection [39] . Dengue virus serotypes 1-4 ( DENV1-4; genus Flavivirus; family Flaviviridae ) are the most important mosquito-borne viruses affecting humans . They are hyperendemic throughout the tropics and are transmitted primarily by A . aegypti mosquitoes in an urban cycle . The DENV genome is a single-stranded , positive-sense RNA approximately 10 . 7 kilobases ( kb ) in length with a 5′ cap structure , but no 3′ polyA tail . It encodes three structural proteins and seven non-structural proteins . Viral RNA replication occurs in the perinuclear region of the cytoplasm in membrane-enclosed replication complexes [40] , [41] . During replication , a full-length negative-sense complementary RNA is used as a template for genome synthesis , resulting in a replicative form consisting of long viral dsRNA [42] . We recently showed that injection of A . aegypti mosquitoes with dsRNA derived from A . aegypti dcr2 or r2d2 mRNA to knock-down expression of RNAi pathway components , followed two days later by oral challenge with DENV2 , resulted in increased virus titers in whole mosquitoes compared to non-injected or unrelated dsRNA ( β-gal ) -injected mosquitoes [10] , indicating a role for Dcr2 in the mosquito antiviral response . DENV2-related si-like RNAs ( viRNAs ) were also detected in DENV2-infected A . aegypti and Aag2 cells in this study [10] . West Nile virus ( WNV ) -derived viRNAs were detected in WNV-infected Culex mosquitoes via deep sequencing [43]; however , small RNAs derived from WNV RNA were not detectable in northern blots from WNV-infected C6/36 cells [8] . We know of no other reports of detection or characterization of flavivirus-derived siRNAs . RNAi has also been shown to be an important antiviral pathway in alphavirus infections of A . aegypti mosquitoes . Co-injection of dsRNA derived from A . aegypti ago2 or dcr2 mRNA with Sindbis virus ( SINV ) TR339-eGFP ( genus Alphavirus; family Togaviridae ) into A . aegypti resulted in increases in detectable viral RNA , infectious virus titers and infection rates of mosquitoes [44] . When SINV were engineered to express the B2 protein , a viral suppressor of RNAi , the viruses replicated to higher titers in mosquitoes and Aag2 mosquito cell cultures , and caused cytopathic effects in cell cultures and mortality in mosquitoes , suggesting the importance of the RNAi pathway in maintaining persistent , non-pathogenic arboviral infections of the mosquito host [9] , [45] . RNAi as an antiviral defense was also demonstrated in Anopheles gambiae mosquitoes , in which injection of dsRNA to knock-down expression of Ago2 resulted in increased replication and dissemination of another alphavirus , O'nyong-nyong virus ( ONNV ) [46] . ONNV-derived viRNAs were identified in A . gambiae mosquitoes by deep sequencing [45] . Despite the demonstration of antiviral RNAi in mosquitoes and mosquito cells , arboviruses are able to establish persistent , generally non-cytopathic infections in their natural vectors . Furthermore , the mechanistic details of small RNA production have not been confirmed in mosquitoes or mosquito cell lines . Since we have observed more robust growth of DENV2 in C6/36 than in Aag2 cells , we hypothesized that this difference was due to variations in the RNAi responses of the two cell lines . We describe here a comparative study of DENV2-specific small RNAs made during infection of Aag2 and C6/36 mosquito cell cultures and A . aegypti mosquitoes , and present evidence that C6/36 cells have an aberrant antiviral RNAi pathway .
The DENV2 strain used for infections of cells and mosquitoes was highly passaged Jamaica 1409 . DENV2 stocks used for infection of C6/36 cells and Aag2 cells were propagated in Aag2 cells . Because the Aag2 cell line is persistently infected with the mosquito-only flavivirus cell fusing agent virus ( CFAV ) , DENV2 stocks contained infectious CFAV . C6/36 ( A . albopictus ) cells were grown in L-15 medium with 10% fetal bovine serum ( FBS ) , 100 U/ml/100 µg/ml penicillin/streptomycin ( P/S ) , and L-glutamine ( L-glut ) at 28°C ( without CO2 ) . C6/36 cell infections were done in L-15 with 2% FBS , P/S , L-glut and non-essential amino acids ( NEAA ) at 28°C ( without CO2 ) . Aag2 ( A . aegypti ) cells were grown in Schneider's Drosophila medium with 10% FBS , P/S , and L-glut at 28°C ( without CO2 ) . Aag2 cell infections were done in Schneider's Drosophila medium with 2% FBS , P/S , L-glut and NEAA at 28°C ( without CO2 ) . Cells were infected with DENV2 at a multiplicity of infection ( MOI ) of 0 . 1 , and cell RNA was harvested at one and five days post DENV2- or mock- infection . LLC-MK2 monkey kidney cells were cultured in modified Eagle's medium ( MEM ) supplemented with 8% FBS , L-glut , NEAA and P/S and maintained at 37°C in the presence of CO2 . For the transfection experiments , Aag2 cells were grown as described above , while the C6/36 cell line was grown in MEM with 10% FBS , P/S , L-glut , and 0 . 015% sodium bicarbonate at 28°C in the presence of CO2 . LLC-MK2 cells were grown to confluent monolayers in 24-well plates , infected with 10-fold serial dilutions of virus for 1 hour and overlaid with an agarose-nutrient mixture . After 7 days incubation at 37°C cells were stained with 5 mg/ml MTT ( 3-[4 , 5-dimethylthiazol-2-yl]-2 , 5-diphenyltetrazolium bromide ) solution . Viral titers were determined by counting plaques [9] . A . aegypti RexD strain mosquitoes were reared at 28°C with 82% humidity . Female mosquitoes one-week post-eclosion were deprived of a sugar source overnight and were then allowed to feed on artificial bloodmeals containing defibrinated sheep blood ( 40% ) ( Colorado Serum Company , Boulder , CO ) and an infected C6/36 cell suspension ( 60% ) with 1 mM ATP for one hour . The bloodmeal was maintained at 37°C in a water-jacketed glass feeder covered with hog gut membrane , and mosquitoes fed on the blood through the membrane . The bloodmeal titer of DENV2 strain Jamaica 1409 was approximately 1×107 PFU/ml , while the mock-infected mosquitoes were given a blood and uninfected C6/36 cell mixture . Bloodfed females were selected and were maintained with water and sugar for nine days after the infection ( or mock infection ) , when RNA was harvested from 20 whole mosquitoes per group . dsRNA was prepared by in vitro transcription from a PCR product of a 498 bp region of the E . coli beta-galactosidase ( β-gal ) gene with T7 promoters on both strands . Transcription was carried out using the Megascript T7 Kit ( Applied Biosystems , Foster City , CA ) for approximately 16 hours at 37°C with approximately 9% of the UTP substrate conjugated to biotin ( Applied Biosystems ) . The reaction mixture was treated with Turbo DNase ( Applied Biosystems ) for 30 minutes , followed by a phenol/chloroform extraction and an overnight ethanol precipitation . The RNA was fractionated on a TBE-urea 6% polyacrylamide gel ( Invitrogen , Carlsbad , CA ) and small RNA was eluted overnight at room temperature . RNA was extracted with phenol/chloroform ( 5∶1 ) , followed by chloroform/isoamyl alcohol ( 24∶1 ) , and precipitated overnight at −20°C in ethanol . The RNA was quantified by spectrophotometry . Cell-free lysates were generated from Aag2 cells and C6/36 cells using a previously described protocol [47] . Briefly , cells were washed in PBS three times , then resuspended in 1X lysis buffer with protease inhibitors and 5 mM DTT . The cells were disrupted in a Dounce homogenizer , and then centrifuged at 14 , 000 rpm for 25 minutes at 4°C . The supernatant was flash frozen in a dry ice/ethanol bath and stored at −80°C . Protein concentrations were determined with the DC Protein Assay ( Bio-Rad Laboratories Inc . , Hercules , CA ) and samples were equilibrated to the same protein concentration using lysis buffer immediately before the dicing assay was set up . Dicing activity reactions contained 1/2 volume lysate , 1/3 volume 40X reaction mix and approximately 70 nanograms of 498 bp biotinylated β-gal dsRNA , with the lysate being added last . At each timepoint , 10 microliters ( µl ) of the reaction were removed , added to 2X PK buffer and flash frozen . RNA was extracted using phenol/chloroform ( 5∶1 ) , followed by chloroform/isoamyl alcohol ( 24∶1 ) , and precipitated overnight at −20°C in ethanol . RNA was electrophoresed on a TBE non-denaturing 20% polyacrylamide gel ( Invitrogen ) , electrophoretically transferred to a positively charged nylon BrightStar-Plus membrane ( Applied Biosystems ) and UV-crosslinked to the membrane . Biotinylated RNA was detected with the BrightStar BioDetect Kit ( Applied Biosystems ) and exposed to autoradiography film . In some reactions , 1 µl ( 0 . 5 unit ) of recombinant human dicer enzyme ( Genlantis Inc . , San Diego , CA ) was added to the 10 µl reaction just before addition of the lysate . The enhanced green fluorescent protein ( EGFP ) gene was amplified from the pEGFP-1 plasmid ( Clontech , Mountain View , CA ) using the forward primer EGFP-Nco I F and reverse primer EGFP-Xho I R . The amplicon was digested with Nco I and Xho I and cloned into the insect-specific expression plasmid pIEx ( Novagen , Madison , WI ) to generate the pIEx-EGFP vector . The Accell EGFP siRNA used in these experiments and the control WNV siRNA were synthetically produced by Dharmacon ( Lafayette , CO ) . WNV siRNA was complementary to a 21 nt region of the WNV genome starting at position 85 in the capsid gene . Approximately 500 bp fragments corresponding to EGFP or WNV capsid genes were amplified using primers that included a T7 promoter sequence in both the forward and reverse primers . The amplicons were PCR purified and subsequently used as templates for dsRNA transcription . Synthesis of dsRNA molecules was carried out using the T7 Megascript kit ( Applied Biosystems ) as described above with omission of biotinylated UTP . The dsRNA was re-suspended in 50 µl PBS , quantified and brought to a final concentration of 1 µg/µl . The day prior to transfection , Aag2 or C6/36 cells were seeded in 24-well tissue culture plates at a density of 5×105 cells/well . For the transfections , 250 ng/well of the pIEx-EGFP plasmid were combined with either EGFP or WNV siRNA ( to a final concentration of 50 nM ) , or 1 µg/well of EGFP or WNV dsRNA in Opti-MEM medium . Subsequently , the Attractene Transfection Reagent ( Qiagen , Valencia , CA ) was added and lipid-nucleic acid complexes were allowed to form for 15 min . at room temperature . The medium on the cells was discarded and 440 µl of Opti-MEM were added to each well followed by dropwise addition of 60 µl of the complexes . The cells remained in the presence of the transfection reagent for four hours , after which appropriate medium for each cell line was replaced . Cell viability was monitored for 48 hours post transfection , when cell images were acquired and the cells harvested . The cell pellets were re-suspended in 500 µl Trizol ( Invitrogen ) and total protein for immunoblots precipitated according the manufacturer's instructions . Images were acquired using a Nikon TE2000 inverted microscope with a Hamamatsu Orca camera and Wasabi software ( Hamamatsu Photonics , Japan ) . Representative areas as determined by cell density were photographed under 10× magnification . Fluorescent images were acquired using a 222 ms exposure without gain and the light images were acquired using a 30 ms exposure without gain . The monochrome images were subsequently pseudo-colored using the Slidebook software ( Intelligent Imaging Innovations , Denver , CO ) . Total protein recovered from transfected cell cultures was quantified using the Bradford Kit on the Bio-Rad SmarSpec Plus spectrophotometer ( Bio-Rad Laboratories Inc . , Hercules , CA ) . Fifteen micrograms of total protein were separated on 12 . 5% SDS-PAGE and transferred to a nitrocellulose membrane . The presence of EGFP was detected using a primary mouse anti-Aequorea victoria EGFP monoclonal antibody ( Clontech ) at a dilution of 1∶1000 in TBST +5% non-fat dry milk . The blot was subsequently probed with phosphatase labeled goat anti-mouse IgG at a 1∶1000 dilution ( KPL Inc . , Gaithersburg , MD ) . Detection of actin was performed with primary rabbit polyclonal antibodies at a 1∶1000 dilution in TBST +5% BSA ( Abcam , Cambridge , MA ) and phosphatase labeled goat anti-rabbit IgG secondary antibody at a 1∶1000 dilution ( KPL Inc . ) . Membranes were developed with the 1-Step NBT/BCIP reagent for 5–10 minutes at room temperature ( Pierce , Rockford , IL ) . Total RNA was extracted using TRIzol ( Invitrogen ) with manufacturer's instructions from Aag2 and C6/36 cells mock- or DENV2-infected ( MOI = 0 . 1 ) at 5 days post infection . Total RNA was extracted with TRIzol from non-infectious bloodfed and DENV2 bloodfed A . aegypti mosquitoes at nine days post bloodmeal . Small RNA was isolated using the FlashPAGE Fractionator ( Applied Biosystems ) . Small RNA libraries were made using SOLiD small RNA expression kit ( Applied Biosystems ) and were sequenced at the University of Washington on a SOLiD sequencer ( Applied Biosystems ) . Potential viRNAs were aligned to the DENV2 or CFAV genome using NextGENe software ( Softgenetics , LLC , State College , PA ) , Version 1 . 11 , running the transcriptome assembly function . CSFASTA ( color-space ) files from SOLiD sequencing of samples were used as the sample file and a FASTA file of either DENV2 Jamaica 1409 RNA from Genbank accession number M20558 . 1 or CFAV RNA Genbank accession number NC001564 . 1 was used as the reference sequence . Logo analysis was performed using WebLogo 3 located at http://weblogo . threeplusone . com in March , 2010 [48] , [49] . Reads that matched DENV2 and CFAV genomes were identified with NextGENe alignment , converted to base-space with NextGENe , and used in the WebLogo . The full length ( 35 nt ) of the matched read was used to allow comparison of all of the viRNAs at once as all reads must be the same length when analyzed with the WebLogo program . The program default settings were used , except the Y-axis scale was set to 1 bit . 5 µg of total RNA from Aag2 or C6/36 cell cultures was heated at 95°C for 5 minutes , placed on ice , then loaded onto a 1 . 25% denaturing formaldehyde agarose gel and electrophoresed in MOPS/formaldehyde buffer . The RNA was passively transferred from the gel overnight to a BrightStar-Plus positively charged nylon membrane ( Applied Biosystems ) with 10X SSC buffer . The membrane was autocrosslinked twice and pre-hybridized in 5 ml of UltraHyb Hybridization Buffer ( Applied Biosystems ) for 1 hour at 68°C . Biotinylated dcr2 antisense ssRNA probes ( nt 4919-5116 ) were added to the hybridization buffer to a final concentration of 0 . 1 nM ( approximately 80 ng of probe in 5 ml buffer ) . A . aegypti dcr2 probe was added to the membrane with the Aag2 RNA bound , and A . albopictus dcr2 probe was added to the membrane with C6/36 RNA bound and the hybridizations took place in separate tubes . Membranes and probes hybridized for 18 hours at 68°C . Membranes were then washed twice for 30 minutes in 2X SSC , 0 . 1% SDS buffer and twice for 60 minutes in 0 . 1X SSC , 0 . 1% SDS buffer . All washes were done at 68°C and A . aegypti and A . albopictus membranes remained in separate tubes . The biotinylated probes attached to the membrane were detected with the BrightStar BioDetect Kit ( Applied Biosystems ) , following manufacturer's instructions , with all washes performed for maximum recommended times . The membranes were exposed to autoradiography film for various times and the film was developed in an automatic autoradiography developer . Band intensities were compared with the BioRad Quantity One software , with adjustment for background .
DENV2-specific small RNAs from either mock- or DENV2-infected Aag2 cells , C6/36 cells , or A . aegypti mosquitoes were sequenced with the ABI SOLiD 2 sequencer and analyzed using NextGENe software . The small RNA library from DENV2-infected Aag2 cells at five days post-infection contained 1 , 612 viRNAs that aligned to the DENV2 genome from over 12×106 reads ( Table 1 ) . Although this is a much higher number than in the uninfected or DENV2-infected cells at 1 dpi , it accounted for only 0 . 01% of the total number of small RNA reads from the library , possibly due to low levels of viral replication , or to sequestration of the dsRNA trigger in cellular membrane-enclosed vesicles [40] , [41] , [50] . The DENV2-infected A . aegypti mosquito library had 6 , 029 DENV2-specific small RNAs at 9 dpi , accounting for only 0 . 05% of the total small RNA reads . Many more DENV2-specific small RNAs ( 24 , 938 from over 12×106 reads ) were found in the C6/36 cells at 5 dpi ( Table 1 ) ; this is possibly related to the ability of DENV2 to grow to 10- to 100-fold higher titers in these cells than in Aag2 cells ( Figure 1 ) . The DENV2-specific small RNAs from Aag2 cells at 5 days post DENV2-infection were 59% positive ( genome ) sense , and the small RNAs from DENV2-infected mosquitoes were 55% positive sense ( Table 1 ) . Nearly-equal ratios of positive to negative sense DENV2 small RNAs suggested that most small RNAs are derived from dsRNA replicative intermediates , rather than intrastrand secondary structures in the ssRNA genome . In C6/36 cells , DENV2-specific small RNAs were 96% positive sense , suggesting that they were derived from ssRNA , and were not generated by Dcr2 cleavage of dsRNA as in A . aegypti cell cultures and mosquitoes . The predominant size of DENV2-specific small RNAs in the Aag2 library ( 5 dpi ) and in the DENV2-infected A . aegypti mosquitoes was 21 nt , which is the expected size for Dcr2 products , confirming that the exogenous-siRNA pathway was the most likely mechanism used by these cells to target DENV2 dsRNA ( Fig . 2 ) . The most common size of DENV2 small RNAs in the C6/36 cell library was 27 nt , which is not expected from the exogenous siRNA pathway . Furthermore , the few 21 nt DENV2-derived RNAs in the C6/36 cell library were predominantly positive sense , unlike the more nearly equal sense to antisense ratio found in the Aag2 cell library . The sequences of the DENV2-specific small RNAs in the Aag2 cell library ( 5 dpi ) were distributed evenly across the entire DENV2 genome , with the exception of a higher proportion of reads from one site around 10 , 000 nt ( Fig . 3 ) , further indicating that dsRNA replicative intermediates were the target of Dcr2 cleavage . The distribution along the viral genome of the DENV2 viRNAs from infected mosquitoes was somewhat different from that seen in the DENV2-infected Aag2 cells ( Fig . 3 ) , with several ‘hot spots’ for origins of either positive-sense or negative-sense viRNA , suggesting that ssRNA secondary structures in the DENV2 genome or its complement may also have been targeted by Dcr2 , and to a greater extent in the mosquito than in cell culture . Analysis of potential secondary structures in the DENV2 RNA genome using mFold ( www . bioinfo . rpi . edu/applications/mfold ) predicts optimal-energy RNA configurations with extensive intrastrand base-pairing throughout the genome; however , most of these dsRNA structures lack perfect base-pairing over regions >21 bp so it is difficult to correlate them with siRNA “hot-spots” . The DENV2-specific small RNAs in C6/36 cells were not equally distributed , but instead were derived from a few specific regions of the genome , which might represent intrastrand secondary structure in the positive-strand virus genome . From over 14×106 reads , there were only 93 matches to the DENV2 genome in the uninfected Aag2 cell sample ( approaching 0% of total reads ) ( Table 1 ) . The Aag2 library from 1 day post-DENV2 infection also had a very low number of DENV2-specific reads ( 55 ) , suggesting that viral replication to produce the dsRNA trigger in the cell was at a low level early after infection . We developed an in vitro dicing activity assay for cultured mosquito cells based on similar methods used to prepare Drosophila cell lysates [47] . This assay was used to compare the ability of cytoplasmic preparations from Aag2 and C6/36 cells to cleave a long exogenous dsRNA into 21 bp small RNAs , indicative of Dcr2 activity . Aag2 cell preparations produced the appropriate size product ( matching the recombinant human dicer control product ) within 18 hr after 500 bp long dsRNA was added to the lysate . C6/36 cell lysates did not make a 21 nt small RNA product from this labeled dsRNA during the same time period . When human recombinant dicer was added to the lysates , a siRNA-like product was made in the C6/36 cell lysate , indicating that although the C6/36 lysate lacks endogenous Dcr2 activity , it does not inhibit exogenously provided enzyme ( Fig 4A ) . Intact C6/36 and Aag2 cells were also tested for the ability of their RNAi pathways to inhibit EGFP expression from a transfected plasmid . Each cell line was transiently transformed with a plasmid expressing EGFP along with siRNAs or long dsRNA derived from the EGFP sequence or control RNAs derived from the WNV genome sequence . Transfection of EGFP-derived-siRNAs into either cell type resulted in knock-down of EGFP expression , indicating that both have a functional RNAi pathway if pre-formed siRNA is loaded into RISC . However , transfection of cognate long dsRNA resulted in knock-down of EGFP expression only in Aag2 cells , suggesting that only this cell line was able to efficiently carry out Dcr2-mediated cleavage of dsRNA ( Fig 4B ) . Immunoblotting of fractionated cell proteins with EGFP antibodies confirmed the corresponding protein expression levels ( Fig 4C ) . These results provide further evidence that C6/36 cells are defective in Dcr2 activity and suggest that both cell lines are able to form a functional RISC . During preliminary small RNA analysis , we detected cell fusing agent virus ( CFAV ) small RNA in Aag2 cells , suggesting persistent infection by this insect-only flavivirus . The sequences of small RNA libraries prepared from Aag2 cells were aligned with a CFAV genome sequence from GenBank as reference using NextGENe software . Surprisingly , there were many more small RNAs in all Aag2 libraries that aligned to CFAV RNA than to DENV2 RNA ( Table 2 ) . CFAV was first described in the precursor cell line to Aag2 cells [51] , [52] . Neither the mock-infected nor DENV2-infected A . aegypti mosquitoes appeared to have a CFAV infection , as only a small number of reads from those libraries matched the CFAV genome ( data not shown ) . The mock infected C6/36 cells also had <60 CFAV-specific small RNAs , but >21 , 000 CFAV small RNAs were detected in the C6/36 cell culture library 5 days post DENV2-infection ( Table 2 ) . Since the DENV2 stock used to infect the C6/36 cells was grown in Aag2 cells , this was probably the source of the CFAV , which was introduced to the C6/36 cells during the DENV2 infection . Interestingly , the patterns of size , polarity , and genome distribution of the CFAV-derived small RNAs were very similar to those of the DENV2-derived small RNAs in both cell lines ( Table 2 , Fig . 5A and B ) . Aag2 cell CFAV-specific small RNAs were predominantly 21 nt in length and were 54–63% positive sense . C6/36 cell CFAV-specific small RNAs were mostly 27 nt in length and 99% were derived from the positive sense strand , similar to the characteristics observed for DENV2-specific small RNAs , indicating that the defect in Dicer activity was not limited to production of DENV2 small RNAs . The DENV2- and CFAV-specific small RNAs from both cell types were analyzed with the WebLogo 3 program ( http://weblogo . threeplusone . com ) to determine if there were preferences for specific nucleotides at certain positions . The total untrimmed 35 nt length of virus RNA-matching reads was analyzed in the program; therefore , the sequences for the six 3′-terminal nucleotides match the linker attached to the small RNAs in preparation of libraries . In both the DENV2 and CFAV viRNAs from Aag2 cells , there were no apparent preferences for specific nucleotides at any positions in the 5′ 21 nt . However , in the C6/36 cell libraries , there appeared to be a bias for adenine on the nucleotide at position 10 in both the DENV2-specific and CFAV-specific small RNAs ( Fig 6 ) . Ago3-associated Piwi-interacting RNAs ( piRNAs ) often have an adenine at the 10th position , hinting at a possible mechanism for generation of these small RNAs in C6/36 cells [35] , [53] . To examine levels of dcr2 expression in C6/36 cells as a possible explanation for defective Dcr2 activity , we compared dcr2 mRNA in Aag2 and C6/36 cells by northern blot hybridization of total RNA , using specific probes based on sequences of cDNA amplified from each cell line ( data not shown ) . dcr2 messenger of the anticipated size was detected in both Aag2 cells and C6/36 cells using their respective probes ( Fig 7 ) . Neither probe hybridized to RNA from the heterologous species ( not shown ) . Comparison of band intensities determined that the Aag2 cell band was approximately 1 . 7-fold stronger than the C6/36 cell band .
The aims of this study were to further define the mechanisms of antiviral RNA silencing in mosquito cells infected with DENV2 by characterization of the virus-specific small RNAs ( viRNAs ) produced during infection and to test the hypothesis that enhanced virus production in C6/36 cells as compared to Aag2 cells is attributable to a less effective RNAi response in the former . We present further evidence that the RNAi response initiated by Dcr2 is central to antiviral defense in A . aegypti and that defective Dcr2 activity in C6/36 cells renders them less able to control DENV2 replication . Little was previously known about the nature of the DENV2 RNA trigger of the RNAi antiviral pathway and the characteristics of resulting DENV2-specific siRNAs during the natural transmission cycle in mosquitoes . We previously reported enhancement of DENV2 replication after knock-down of dcr2 expression and presence of virus-specific small RNA in A . aegypti [10] , but our attempts to characterize these small RNAs using traditional cDNA cloning and sequencing methods yielded very few genome matches ( unpublished ) ; thus in this study we employed deep sequencing and analysis of small RNA libraries . Since DENV2 induces the production of dsRNA during its replication cycle [10] , [42] , [54] , this would be the most obvious target for Dcr2 cleavage and activation of an RNAi response . Analysis of our deep sequencing data showed that 54–60% of the DENV2-small RNAs in Aag2 cells were positive sense , close to the 1∶1 ratio that would be expected if the trigger were a double-stranded intermediate composed of long strands of positive genomic RNA annealed to a complementary negative sense strand . The slight excess in positive-sense siRNAs in Aag2 cells and A . aegypti is likely to arise from Dcr2 recognition and cleavage of intrastrand secondary structures in the DENV2 genome . The distribution of viRNAs along the DENV2 genome in Aag2 cells at 5 days post DENV2 infection is relatively uniform , also implicating a long dsRNA replicative intermediate as the main source of DENV2-specific small RNAs in Aag2 mosquito cells . In DENV2-infected mosquitoes , the positive strand: negative strand ratio was even closer to 1∶1 , with 55% of the DENV2-specific small RNAs being derived from the positive sense strand . Previous studies of another flavivirus , WNV , in Culex quinquefasciatus mosquitoes showed that approximately 74% of the virus-specific small RNAs were from the positive sense RNA strand [43] . These differences in strand polarity ratios may be due to replication strategies of the viruses themselves or to a different RNAi response in Culex mosquitoes when compared to A . aegypti . Culex mosquitoes have a duplication of the ago2 gene , which could result in differences in antiviral RNAi responses [37] . Small RNA deep sequencing of A . aegypti mosquitoes infected by the positive sense RNA alphavirus SINV showed that 54% of the virus-specific small RNAs were from the positive sense strand [45] , a very similar proportion to our findings in DENV2-infected A . aegypti . When the alphavirus ONNV was studied in A . gambiae mosquitoes , the proportion of positive sense virus-specific small RNAs was slightly higher at 64% [45] . The differences seen between these alphavirus-infected mosquitoes also may be due to differences in SINV and ONNV replication mechanisms or due to different responses in the two mosquito genera . The number of DENV2-specific small RNAs in our total RNA samples was very low . Next generation SOLiD sequencing revealed that that less than 0 . 02% of the small RNAs in the DENV2-infected Aag2 cell ( 5 dpi ) library and less than 0 . 05% of the small RNAs in DENV2-infected A . aegypti mosquitoes ( 9 dpi ) were DENV2-specific . These results appear to be typical of flavivirus-infected mosquitoes , as Culex mosquitoes infected with WNV had less than 0 . 05% WNV-specific small RNAs in the total small RNA population at 7 days post-infection and 0 . 12% WNV-specific small RNAs at 14 days post-infection [43] . This may be due to sequestration of flavivirus replication complexes in membrane-enclosed vesicles in mosquito as well as mammalian cells , preventing Dcr2 access to dsRNA replicative intermediates [40] , [41] , [50] . Alphavirus replication in mosquitoes appears to generate more virus-specific small RNAs . Approximately 10% of the 18–24 nt RNAs sequenced from SINV-infected A . aegypti mosquitoes were matches to the SINV genome [45] . Although in ONNV-infected A . gambiae mosquitoes the proportion of virus-specific small RNAs was lower , with 1 . 2% of the total small RNA reads matching the ONNV genome , still it was at least 10-fold higher than for any flavivirus reported to date [45] . These higher proportions of alphavirus small RNAs as compared to flavivirus small RNAs may be due to differences in accessibility of the replicative intermediate dsRNA to RNAi machinery during viral replication , or possibly because of more rapid viral replication to higher titers in alphavirus infected mosquitoes . Another reason for the increased numbers of alphaviral small RNAs in these studies may be that the mosquitoes were injected with SINV and ONNV , whereas infection of mosquitoes used in the DENV and WNV studies was established orally . Although we have presented clear evidence that RNAi plays an antiviral role against DENV2 [10] , the low levels of DENV2 viRNAs in infected cells and mosquitoes raise the question whether the viRNAs themselves have an important role in the RNAi response . Possibly Dcr2 cleavage of replicating viral RNA alone helps to keep the DENV2 infection from overwhelming mosquito cells and causing excessive pathology and overt mortality in the insect . The DENV2-specific viRNAs in both Aag2 cells ( 5 dpi ) and A . aegypti mosquitoes were predominantly 21 nt long with similar proportions of sense and antisense polarities , suggesting that the underlying mechanistic aspects of their RNAi responses are similar . During our small RNA analysis we also discovered many CFAV-specific small RNAs in the Aag2 cell culture samples , but only a few CFAV-matching reads in the A . aegypti mosquitoes . The Aag2 cell line is persistently infected with this insect-only flavivirus , and it appears to activate the antiviral RNAi pathway . Although the CFAV RNA-specific proportion of small RNAs was higher ( 0 . 2–0 . 7% ) , the size distribution and polarity of the CFAV-specific small RNAs in Aag2 cells were similar to the DENV2-specific small RNAs found after DENV2-infection , and these characteristics suggest that they are products of the exogenous siRNA pathway . Possible effects of CFAV persistent infection on DENV2 replication in the Aag2 cells are unknown and need further study . The sequence identity between CFAV and DENV2 ( Jamaica 1409 strain ) RNAs is only 47% , so a sequence-specific response to DENV2 infection in CFAV-persistently infected Aag2 cells seems unlikely , although a change in level of RNAi activity due to persistent CFAV may have a non-specific effect on DENV2 replication in these cells . In contrast to our findings for Aag2 cells , deep sequencing and analysis of small RNA in DENV2-infected C6/36 cells revealed abundant DENV2-specific small RNA that were longer than 21 nt and almost exclusively sense polarity , characteristics not expected of Dcr2-generated siRNAs . In addition , the C6/36 cell DENV2-specific small RNAs seemed to be generated only from specific regions of the genome . Further investigation is needed to determine if these correspond to secondary structures within the genome . Despite the greater numbers of virus-specific small RNAs in C6/36 cells , the overwhelming predominance of genome-sense small RNAs , even if they are loaded into a RISC , would result in inefficient cleavage of newly-synthesized viral genomes and a comparatively weak innate immune response . The lack of functional Dcr2 activity in C6/36 cells and production of predominantly positive-sense small RNAs may play a role in their increased ability to support the replication of arboviruses such as DENV and chikungunya virus , and may account for Igarashi's speculation that “the virus-sensitive C6/36 clone may lack efficient regulatory mechanism for virus RNA synthesis and virus production” [3] . The predominant length of DENV2-derived small RNAs in C6/36 cells was 27 nt , a size characteristic of piRNAs [28] . Production of piRNAs is Dcr1/Dcr2-independent and can be mediated by Ago3 [53] . Virus-specific piRNAs were recently described in Drosophila [36] and Zambon , et al . [55] showed that piwi-family mutants of Drosophila were more susceptible to Drosophila virus X infection . Logo analysis of DENV2- and CFAV-specific small RNAs from C6/36 cells showed a bias for adenine at the 10th position from the 5′ end , which is also characteristic of piRNAs bound by Ago3 [35] , [53] . We inadvertently co-infected the C6/36 cells with CFAV contained in the DENV2 stock , and the CFAV-specific small RNAs produced had similar properties to the DENV2-specific small RNAs , but were uncharacteristic of an exogenous siRNA pathway . The C6/36 cells also did not produce typical 21 nt viRNAs in response to infection by WNV , SINV or LACV ( Brackney , et al . , 2010 submitted ) . Earlier studies in C6/36 cells engineered to express dsRNA hairpin structures derived from DENV2 RNA showed small RNAs generated from these hairpins that migrated between 20 nt and 30 nt size markers , with a size appearing to be larger than 21 nt [56] . The cells expressing these inverted repeat transcripts were resistant to DENV2 infection , and in light of our current discovery of impaired Dcr2-like activity in C6/36 cells , we speculate that the increased resistance to DENV2 infection in this engineered cell line was probably due to a Dcr2-independent RNA silencing mechanism , such as the piRNA pathway . In the study by Chotkowski et al . [8] , northern blot hybridization using a sense-strand probe failed to detect WNV-specific siRNAs in C6/36 cells . If WNV-specific small RNAs were predominantly genome-sense , as in our study , they would be poorly detected by a positive-sense hybridization probe . Our in vitro assay indicated that C6/36 cells lack the ability to cleave long dsRNA into characteristic siRNAs . Only transfected siRNAs could be used to knock-down GFP expression from a plasmid in the cells , and long dsRNA did not . Although C6/36 cells appeared to lack efficient Dcr2 activity , addition of recombinant Dicer to the lysate resulted in production of siRNAs; it thus appeared that the lack of Dcr2 activity was not due to its inhibition in C6/36 cells . Northern blot analysis showed that dcr2 was expressed at a somewhat reduced level in C6/36 cells compared to Aag2 cells; however , the magnitude of reduction does not appear to be sufficient to account for the lack of dicing activity . A recent study by Lim et al . [57] showed that missense mutations in the Drosophila dcr2 DExH helicase domain or RNase III domain caused a loss of dsRNA processing activity . We have cloned and sequenced full-length dcr2 cDNA from Aag2 cells and a 3920 nt fragment of C6/36 cell dcr2 ( equivalent of nt ∼1200-5120 on A . aegypti dcr2 ) ( data not shown ) . The Aag2 dcr2 nucleotide sequence was >99% identical to A . aegypti dcr2; however , the C6/36 dcr2 fragment showed only 79% identity with the Aag2 full-length sequence . Translation of the nucleotide sequences revealed an apparent single nt deletion in C6/36 dcr2 at nt 1508 that resulted in a termination codon , and thus a nonsense mutation . Our detection of full-length dcr2 mRNA in C6/36 cells suggests that it does not undergo nonsense-mediated decay , as would be expected for early translation termination [58] , so it is possible that a ribosomal frame-shift allows complete translation . However , because of the lack of availability of the authentic A . albopictus sequence and the high degree of divergence of C6/36 dcr2 sequence from A . aegypti dcr2 , we are unable to pinpoint particular mutations in C6/36 dcr2 that could result in a change in phenotype . The presence of unusual DENV-specific small RNAs in C6/36 cells coupled with ineffective Dcr2 activity suggested that a compensating mechanism is used by these cells for generation of viral-specific small RNAs . The piRNA pathway may serve as a backup mechanism when the exogenous siRNA pathway is not functioning correctly . Evidence for this hypothesis was seen when the endo-siRNA pathway was disrupted by mutation of ago2 in Drosophila , resulting in the appearance of somatic cell piRNAs that possibly served as a backup in transposon surveillance [32] , [59] . In our examination of RNAi in A . gambiae mosquitoes we found that co-injection into mosquitoes of dsRNA derived from the ago3 sequence with ONNV resulted in increased ONNV titers , hinting at a possible redundant role for Ago3 in antiviral immunity in these mosquitoes [46] . Recently , viral small RNAs of various sizes other than 21 nt were found in a variety of animal cells infected with RNA viruses , suggesting roles for alternative RNA silencing pathways in antiviral defense [60] . In summary , we determined that DENV2-specific small RNAs produced during infection of A . aegypti mosquitoes and A . aegypti Aag2 mosquito cell cultures appear to be made via the exogenous siRNA pathway , but they are made in very low numbers , indicating that DENV2 may have a strategy to evade the antiviral RNAi response . In vitro studies demonstrated production of characteristic siRNA in Aag2 cells but indicated that C6/36 cells exhibit inefficient Dcr2 cleavage of long dsRNA . The C6/36 A . albopictus cell line produced more abundant DENV2-specific small RNAs , although they appeared to be generated by a different small RNA pathway , possibly through a piRNA-like mechanism , and this aberrant pattern of viral small RNA production extends to other flaviviruses , alphaviruses and bunyaviruses ( Brackney , et al . , 2010 submitted ) . The ability of C6/36 cells to support robust arbovirus replication may be due to lack of a complete , functional RNAi pathway . The evidence we have presented here indicates that C6/36 cells do not provide an accurate model for mosquito-arbovirus molecular interactions in the RNAi pathway . | Understanding how arthropod-borne viruses ( arboviruses ) establish persistent infections in mosquitoes will help us to find ways to prevent viral disease transmission by these insects . RNA silencing pathways in mosquitoes and other insects , particularly RNA interference ( RNAi ) , have been shown to be important in antiviral defense . In this study we describe small RNAs involved in RNA silencing that are derived from the genome of the arbovirus dengue virus type-2 ( DENV2 ) in infected Aedes aegypti mosquito cell lines and mosquitoes . We also show that C6/36 , a mosquito cell line from A . albopictus , appears to process DENV2 RNA for silencing differently from A . aegypti mosquitoes , revealing that other small RNA pathways in mosquito cells might have a role in antiviral immunity in this cell line and provide insight into using mosquito cell cultures to study the antiviral response to arboviruses in mosquitoes . | [
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] | 2010 | Comparison of Dengue Virus Type 2-Specific Small RNAs from RNA Interference-Competent and –Incompetent Mosquito Cells |
Leprosy control is based on early diagnosis and multidrug therapy . For treatment purposes , leprosy patients can be classified as paucibacillary ( PB ) or multibacillary ( MB ) , according to the number of skin lesions . Studies regarding a uniform treatment regimen ( U-MDT ) for all leprosy patients have been encouraged by the WHO , rendering disease classification unnecessary . An independent , randomized , controlled clinical trial conducted from 2007 to 2015 in Brazil , compared main outcomes ( frequency of reactions , bacilloscopic index trend , disability progression and relapse rates ) among MB patients treated with a uniform regimen/U-MDT ( dapsone+rifampicin+clofazimine for six months ) versus WHO regular-MDT/R-MDT ( dapsone+rifampicin+clofazimine for 12 months ) . A total of 613 newly diagnosed , untreated MB patients with high bacterial load were included . There was no statistically significant difference in Kaplan-Meyer survival function regarding reaction or disability progression among patients in the U-MDT and R-MDT groups , with more than 25% disability progression in both groups . The full mixed effects model adjusted for the bacilloscopic index average trend in time showed no statistically significant difference for the regression coefficient in both groups and for interaction variables that included treatment group . During active follow up , four patients in U-MDT group relapsed representing a relapse rate of 2 . 6 per 1000 patients per year of active follow up ( 95% CI [0·81 , 6·2] per 1000 ) . During passive follow up three patients relapsed in U-MDT and one in R-MTD . As this period corresponds to passive follow up , sensitivity analysis estimated the relapse rate for the entire follow up period between 2·9- and 4·5 per 1000 people per year . Our results on the first randomized and controlled study on U-MDT together with the results from three previous studies performed in China , India and Bangladesh , support the hypothesis that UMDT is an acceptable option to be adopted in endemic countries to treat leprosy patients in the field worldwide . ClinicalTrials . gov: NCT00669643
In 1981 , the World Health Organization ( WHO ) recommended the use of multidrug therapy ( MDT ) for leprosy . Since then , the disease prevalence dropped , but the case detection rate did not decrease and currently many countries still present high detection rates [1] . According to the WHO , in 2014 more than 200 . 000 new leprosy cases were detected worldwide . Additionally , since the implementation of MDT in early 80’s , the duration of treatment has been halved from 24 to 12 months for MB patients and from 12 to 6 months for PB patients . On the other hand , no new standard treatment scheme for leprosy patients has been proposed . Leprosy remains a poorly understood infectious disease and in several endemic countries its diagnosis , treatment and control have been carried out in large scale , yet the effectiveness of these programs is yet uncertain [2] . Leprosy is caused by Mycobacterium leprae , a highly infectious microorganism with low virulence , meaning that only a small proportion of those infected will manifest the disease . Leprosy presents a wide spectrum of clinical manifestations , reflecting the interaction of the bacilli and the immune response of the host . In 1966 , Ridley and Jopling proposed a disease classification system based on clinical , histological and bacteriological data . This classification includes two polar forms , tuberculoid ( TT ) and lepromatous ( LL ) in which TT patients present with few bacilli and strong cellular immunity response while LL ones have high bacterial load and weak cellular immunity . Additionally , three intermediary forms lie between the poles: borderline-tuberculoid ( BT ) , borderline ( BB ) , and borderline lepromatous ( BL ) [3] . Later , an early indeterminate leprosy form ( I ) was included in this classification system . In 1982 , the WHO recommended two standardized multidrug therapy ( MDT ) regimens for leprosy , one for I , TT and BT leprosy cases and the other for BB , BL and LL cases . However since this classification requires clinical , histological and bacteriological data , it was very difficult for leprosy control fieldworkers to adopt it . Therefore , the classification system for treatment purposes has been later simplified to two leprosy types: paucibacillary leprosy ( PB ) referring to patients with a low bacillary load , and multibacillary ( MB ) patients with high bacillary load , based on results from bacilloscopy of Ziehl–Neelsen stained skin smears . The WHO classification into MB or PB patients for treatment purposes proposed in 1997 is based on the number of skin lesions as a proxy for the bacteriological data and defines two different treatment regimens: MB patients ( over 5 skin lesions ) receive twelve months of daily dapsone plus clofazimine and monthly rifampicin doses while for PB patients ( up to 5 skin lesions ) , treatment consists of six months of daily dapsone plus monthly rifampicin doses . The rationale for these two regimens is that the probability of the presence of a naturally resistant bacillus , among those infecting a patient , is proportional to the bacillary load . Also , in order to avoid the selection of drug resistant bacilli , patients with high bacillary load need to be treated longer and with one additional drug [4] . On the other hand , to avoid side effects , patients with low bacillary load should not be over treated . The duration of treatment for leprosy and tuberculosis has always been a controversial issue due to the presence of persistent bacilli . In leprosy , the permanence of bacilli , despite months or years of chemotherapy is probably due to the fact that M . leprae has low multiplication rate , i . e . , low metabolism , making this pathogen less susceptible to destruction by chemotherapy . Leprosy control programs are based on early diagnosis and treatment of cases , i . e . , elimination of infectious sources and the relapse rate is considered the main treatment outcome . In this context , the operational WHO classification system based on the number of skin lesions can lead to misclassifications of MB as PB cases , consequently increasing the chances of relapses . During the chronic course of leprosy , new neurological damage leading to further physical disability can occur . In the perspective of the patient and also of the medical care staff , disability is an important clinical outcome that has never been included in leprosy chemotherapy trials [5] . The uniform treatment for leprosy ( U-MDT ) consists of daily intake of dapsone plus clofazimine and monthly rifampicin for six months , despite any type of patient’s classification . Therefore , the adoption of a uniform treatment for all cases would render disease classification unnecessary , simplifying the implementation of leprosy treatment at primary care . The need for evaluating a uniform treatment for leprosy patients was included in the WHO Technical Advisory Group report in 2002 , and in 2003 a WHO U-MDT trial without a control group was launched in India and China [6] . This original report describes for the first time , long-term results of the four main outcomes of MB patients that participated in the open label randomized Clinical Trial of Uniform Multidrug Therapy conducted in Brazil ( U-MDT/CT-BR ) , concerning: ( i ) frequency of reactions; ( ii ) trends of bacteriological index ( BI ) during treatment and follow up; ( iii ) disability progression; and ( iv ) relapse rates [7] and [8] .
This study was performed under the international ( Helsinki ) and Brazilian research regulations and was approved by the National Ethics Commission of Research ( CONEP ) of the Ministry of Health , protocol number 12949/2007 . Written informed consent was required from all the patients prior to their inclusion in the study . For patients aged six to 17 years , written parental consent was mandatory . Data confidentiality was strictly guaranteed . Patients were free to leave the study , if they desired , and opt for the R-MDT regimen outside the study . An open label randomized clinical trial was conducted , from March 2007 to January of 2015 , at two Brazilian leprosy reference centres ( Fundação Alfredo da Matta ( FUAM ) in Manaus , Amazonas State , north region and Centro de Dermatologia Dona Libânia ( CDERM ) in Fortaleza , Ceará State , northeast region ) . ClinicalTrials . gov registered its protocol under the identifier–NCT 00669643 . In this trial , all patients coming to these dermatology clinics , which are in charge of treating skin diseases in general , were examined . In this report , the study population included newly diagnosed , previously untreated PB and MB leprosy patients and returning defaulters and relapse cases , provided that the last treatment dose was taken more than five years prior to the enrollment in the study . All of the leprosy patients were between six- 65 years old . Patients were excluded if they were receiving tuberculosis/TB or steroid treatment , had overt signs of acquired immune deficiency syndrome , they did not reside permanently in the area or were unable to visit the clinic every month during the treatment and follow-up periods . Patients were classified as MB according to the criteria proposed by the WHO , i . e . , patients with more than five skin lesions . Until 2011 , the study included 613 newly diagnosed MB leprosy patients with high bacterial load and among them , 323 were randomized into the U-MDT group and 290 into the WHO regular regimen ( R-MDT ) group . In order to ensure a precise estimate of relapses among MB patients , a sample size of at least 278 MB patients in each study arm was calculated . This value is based on an alfa error of 0·05 a betta error of 0·20 , i . e . , a power of 80% , a ten years relapse risk for the U-MDT group of nine per cent , and a relapse risk of 0·03 in the R-MDT group for the same period . Before starting the randomization and the controlled clinical trial , all study protocols ( standard operational procedures/SOP ) and clinical report forms ( CRF ) were evaluated in an open and uncontrolled cohort pilot study with 78 patients , conducted from 2004–2006 at the Federal University of Minas Gerais , Brazil . Randomization was performed in order to evaluate whether there were differences in the two treatment modalities . All patients who met the inclusion criteria , independent of MB or PB status were randomized into the experimental ( U-MDT ) or the control ( R-MDT ) group . Prompt action was essential because the experimental treatment group for PB patients began treatment with three drugs while the control group was treated with two drugs . Since for MB patients the drug regimen was the same for U-MDT and R-MDT , differing only in its duration , MB patients were randomized after six months of initiating therapy when the U-MDT group discontinued treatment , while the control R-MDT group continued treatment for additional six months . A randomization table was created with codes for all patients in the study , based on a random list of numbers , using the study entrance sequence according to the CRF number . For this process , the space in the worksheet that contained the randomization code was covered with the same material used in lottery scratch cards , so that the printed numbers were not visible . This code determined the directions for treatment group of each patient as follows: when the code corresponded to an odd number , the patient was part of the experimental group 1 or 3 ( U-MDT ) , according to their classification as PB or MB , respectively . When the code corresponded to an even number , the patient was part of control group 2 or 4 ( R-MDT ) , according to the classification as PB or MB , respectively . A spreadsheet containing the codes was sent to the local coordinator of each recruiting centre , which was responsible for the allocation of the patients into the study groups . For PB patients , the randomization results were identified immediately after the inclusion of the patient into the study . The randomization code of each MB case was kept blind in the spreadsheet until the patient completed six doses of the MDT regimen , when the local coordinator disclosed the code . During this trial , the local research coordinators were responsible for managing data collection according to the eligibility criteria and for ensuring the six doses of MDT , keeping the patient randomization spread sheet under his/her responsibility and coordinating treatment for each patient . In each centre , the data manager was responsible for coordinating the preparation of the spreadsheet with the randomization codes and for maintaining a confidential copy of the spreadsheet containing the randomization results . At the first visit , the dermatologist in charge performed a complete clinical examination that included registering the number of skin lesions and affected nerves and collecting skin biopsies for histopathological examination . Health workers collected blood for liver and renal function tests , complete blood count , anti-PGL-I ML Flow test and skin smear material from six sites , including ear lobes and elbows , for bacilloscopy . In each centre , a technician with extensive experience , examined the Ziehl-Nielsen stained skin smears and generated a bacilloscopic index ( BI ) that ranged from zero to six crosses for each skin site and results were summarized as the average of all six BI ( aBI ) . During the first year of follow up , patients had a monthly appointment and thereafter , yearly . The visits included dermato-neurologic examination , blood collection to evaluate liver function and whole blood counts . Skin smears were collected at the beginning and at the end of treatment and thereafter yearly . Physicians advised all patients to come to an urgent appointment in case any sign or symptom of leprosy reaction occurred . Treatment for reaction was established by the assistant dermatologist and registered in the CRF , and followed the guidelines established by the Brazilian leprosy control program from the Ministry of Health . Recurrent leprosy was defined as the reappearance of signs and symptoms of the disease after completion of MDT , not associated with leprosy reactions , and with an increase in the bacillary index ( BI ) compared to the BI after treatment completion . Patients with suspicion of relapse were clinically reviewed by the research PI ( GOP ) , by the assistant dermatologist and by Dr . Sinesio Talhari , an expert member of the independent steering committee , when skin smears and biopsies were collected . Disability grade of each patient was the highest grade reported in either eye , foot and hand as recommended by the WHO . Neurological examination indicating disability in one of these sites that was previously unaffected was considered as disability progression ( DP ) and was used to compare neurological damage in the two study groups . The protocol , the study design , preliminary results of this trial , and the patients’ profile and satisfactions have been published [7 , 9 , 10] . We used Student t test for continuous variables and Chi-square for dichotomous ones to compare the distribution of the baseline characteristics in each study arm . We evaluated the first reaction since the beginning of treatment using a Kaplan-Meyer survival function for the experimental and the control groups and a log-rank test . The survival analysis included the first six months of treatment . To compare the number of reaction episodes between the two groups after 180 days of treatment , we fitted a Zero-inflated negative binomial regression model to the number of reaction as the dependent variable and the treatment group as the independent variable with the log of follow up days of each patient as an offset variable . In order to evaluate the BI trend over time after 180 days from the onset of treatment , we fixed a multilevel linear model with mixed effects , i . e . , a random intercept model . The aBI ( average BI ) was the independent variable and the dependent variables were time ( in days ) , initial aBI categorized as high ( aBI≥4 ) and low ( aBI<4 ) , study arm ( U-MDT and control ) , and three interaction variables combining the previous ones , two by two . For this analysis , time zero was the first day of the seventh month after the beginning of treatment , i . e . , the randomization moment for MB patients . For clarity , the categorized aBI is referred as BI level , in contrast with aBI referring to continuous measure , the average of all sites of smear collection . We evaluated the first disability progression since the beginning of treatment using a Kaplan-Meyer survival function for experimental and control groups and a log-rank test . These survival analyses included the first six months of treatment . We estimated the difference of survival proportion in fixed points of time according to Kaplan Meyer curve and its confidence interval .
Figs 2 and 3 show the Kaplan-Meyer function of the survival without reaction in both treatment arms and also stratified by BI level . The logrank test for the survival curves showed no statistically significant difference between groups . By the 180th day ( six months ) of treatment , 64 . 14% of participants in U-MDT and 62 . 23% in R-MDT group were reaction-free indicating a risk ratio for at least one reaction at the period of 1·05 , CI95% [0·8554–1·2968] . Regarding the number of leprosy reactions developed in each treatment group , the negative binomial model fitted to the data showed no statistically significant difference compared with the intercept only model ( log likelihood ratio ( LLR ) test = 2·9730 , df = 2 , p = 0·7681 ) . These results indicate lack of association between the number of reactions and the treatment group ( p value for the coefficient = 0 , 221 ) , meaning that the treatment group did not affect the number of reactions . When patients were stratified into the aBI as ≥ or < 4 , no statistically significant difference in the development of leprosy reactions was seen between the study U-MDT and control R-MDT groups . Fig 4 shows the aBI as a function of time for each MB patient , and Fig 5 shows the linear adjusted aBI as a function of time . These two figures illustrate the need for a multilevel model for analysis , as a patient aBI at a fixed time is dependent on the previous aBI measure . This analysis approach considers the BI time trend of each patient instead of the BI average of all patients in each time point representing treatment duration . The full mixed effects model adjusted for the aBI trend—independent variables: treatment group , aBI level and time , plus three interaction variables—initial aBI and group; time and group; initial aBI and time—showed no statistical significance for the regression coefficient of bacilloscopic index of treatment groups and for interaction variables that included treatment group ( ‘group X time’ and ‘group X initial aBI’ ) . The full model allowed for treatment effect on aBI value , on time trend of aBI value and on different effect according to initial aBI . The final model retained the possible effect of treatment ( group variable ) on aBI value , of initial aBI effect on aBI value and of initial aBI effect ( interaction of initial aBI and time variable ) on time trend of aBI . Table 2 shows the final model excluding these two not statistically significant interaction variables . The log likelihood ratio test comparing the two models showed no statistically significant difference in BI decrease . Fig 6 shows the daily BI decrease in MB patients in U-MDT and R-MDT after 180 days of starting treatment and the BI level , with its 95% confidence interval . No statistically significant difference was observed in the BI decrease of MB leprosy patients from the U-MDT and R-MDT groups . Figs 7 and 8 show the cumulative probability survival without disability progression as a function of time of follow up . The logrank test for the survival curves showed no statistically significant difference between the two treatment groups . At the fifth year after the beginning of the treatment ( 1825 days ) , 33 . 8% of U-MDT patients had disability progression compared with 30 . 06% of patients in the R-MDT group , 3 . 74% difference , 95% CI [- 3 . 2% , 12 . 08%] . For those with aBI < 4 , the difference was 2 . 85% and 95% CI [-6 . 11% , 11 . 81%] and for those with aBI ≥ 4 the difference was 4 . 68% and 95% CI [-2 . 11% , 11 . 48%] . No subgroup presented less than 25% disability progression . These results show no statistically significant difference in disability progression of MB leprosy patients treated with U-MDT or R-MDT regimens . Four patients in the U-MDT group relapsed representing a relapse rate of 2·6 per 1000 patients per year of follow up ( 95% CI [0·81 , 6·2] per 1000 ) during the active follow up period , which ended on April 30th , 2015 . In the R-MDT group , supposing the same relapse rate , the expected number of relapses would be five , but no relapse was observed . During passive follow up ( May 1st , 2015-June 1st 2016 ) three MB patients in U-MDT and one in R-MDT group relapsed . It was difficult to define accurately the denominator to estimate the relapse rate when passive follow up time was considered . In order to overcome this , we did a sensitivity analysis , i . e . , we estimated the rate using the follow up person-years that results in an overestimation bias . The estimated rate of relapse for U-MDT group was 4 . 46 per 1000 people per year and for R-MDT 0 . 44 per 1000 people per year . This means that in the U-MDT group the overestimated relapse risk in ten years is 4 . 4% . As the relapse risk is surely lower than 4 . 4% in ten years , we consider the U-MDT relapse rate acceptable for use . Thus far , the recruitment centres participating in the U-MDT trial continue to follow up of patients . Table 3 describes sociodemographic and clinical characteristics of the four MB patients from the U-MDT regimen who relapsed during active follow up . All of these patients had initial aBI ≥ 3·5 and were classified , according to Ridley Jopling , as lepromatous or borderline lepromatous leprosy .
Our results on the first randomized and controlled study on U-MDT , together with the results from three previous studies performed in China , India and Bangladesh , support the premise that U-MDT is an acceptable option to be adopted by leprosy endemic countries , in the field worldwide . CONSORT statements checklist | Since the introduction of multidrug therapy for leprosy in the 80’s , different classification criteria for leprosy patients have been proposed and treatment has been progressively shortened . Currently , leprosy patients are classified into paucibacillary/PB and multibacillary/MB based on the number of skins lesions . MB patients ( over 5 skin lesions ) receive three drugs ( rifampicin , dapsone , clofazimine ) for 12 months , while PB patients ( up to 5 skin lesions ) receive two drugs ( rifampicin , dapsone ) for 6 months . We conducted a randomized clinical trial to evaluate the efficacy of a uniform treatment ( U-MDT ) for both PB and MB leprosy patients , regardless any classification criteria . The current study includes results from: laboratory tests ( bacilloscopic index/BI , serology and histopathology ) , clinical evaluation during a long follow-up , and uses adequate epidemiological analysis that gives robust evidence on main parameters used to evaluate the efficacy of U-MDT . This study reports data among MB leprosy patients treated with regular/R-MDT and uniform/U-MDT regarding: ( i ) The frequency of leprosy reactions; ( ii ) BI decrease , ( iii ) Disability progression and ( iv ) Relapse . Overall , our results showed that there was no statistically significant difference in these outcomes for both treatment groups . In this sense , U-MDT can be considered as part of leprosy policy by control programs in endemic countries . | [
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] | 2017 | Uniform multidrug therapy for leprosy patients in Brazil (U-MDT/CT-BR): Results of an open label, randomized and controlled clinical trial, among multibacillary patients |
Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance . However , most parasitic nematodes , particularly those of humans , are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult . The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance . Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains , MHco4 ( WRS ) and MHco10 ( CAVR ) , into the susceptible genome reference strain MHco3 ( ISE ) using a backcrossing approach . A panel of microsatellite markers were used to monitor the procedure . We demonstrated that after four rounds of backcrossing , worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci . In addition , a single marker , Hcms8a20 , showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation ( s ) . This work presents a novel genetic approach to study anthelmintic resistance and provides a “proof-of-concept” of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms . The resulting strains provide valuable resources for candidate gene studies , whole genome approaches and for further genetic analysis to identify ivermectin resistance loci .
Parasitic nematode worms are important human and animal pathogens . Human parasites infect well over 1 billion people worldwide and livestock parasites cause major economic production loss to grazing ruminants . Control is dependent on the use of a limited number of anthelmintic drugs and intensive use of these has already led to widespread resistance in livestock parasites [1]–[5] . In recent years selection pressure has been applied to parasitic nematode populations in humans by anthelmintic treatments used in various control programs and , in the case of some filarial nematodes , eradication programs [6] . In endemic regions , parasitic nematodes often occur as mixed species infections and so the application of drug treatments to control one parasite species inevitably leads to selection pressure being applied to others . Consequently , there is increasing concern about the development of anthelmintic drug resistance in nematode parasites of humans . Unfortunately , parasitic nematodes of humans make extremely difficult experimental subjects and so there is a need to develop model systems to study potential mechanisms of anthelmintic resistance . Haemonchus contortus is a parasitic nematode of sheep which has a high propensity to develop anthelmintic . It is also one of the most amenable parasitic nematodes to experimental manipulation which , together with recent progress in sequencing its genome makes it a potentially powerful model system to study drug resistance in the strongylid nematode group [7] . In addition , genetic crossing is technically possible in this parasite and has potentially powerful applications in the study of anthelmintic resistance providing we can develop the necessary techniques and resources [8]–[11] . The genetic basis of anthelmintic resistance is still relatively poorly understood . To date , most research has focussed on the investigation of possible associations between the resistance phenotype and polymorphisms in candidate genes . This approach has been successful in identifying polymorphisms in the isotype-1 β-tubulin gene as important determinants of benzimidazole resistance [12]–[13] . However , candidate gene studies have major limitations and have yet to unequivocally identify molecular loci responsible for resistance against other anthelmintic classes [14] . Genomic resources are improving for many parasitic nematodes , including the production of high quality reference genome sequences , which will allow the application of genome-wide approaches that do not depend on prior assumptions regarding potential resistance mechanisms [14]–[19] . However , the application of such approaches is not likely to be a trivial task . Attempts to associate specific genetic differences with a drug resistant phenotype will be complicated by the high level of genetic variation that often exists within and between parasitic nematode populations [20]–[22] . Simple comparisons will potentially reveal many genetic differences between drug resistant and susceptible parasite strains that are not necessarily associated with anthelmintic resistance but with background genetic variation or with other unrelated phenotypes . Consequently , there is a need to develop experimental approaches to overcome these challenges . The artificial selection of resistance by serial passage and underdosing of susceptible laboratory strains has been undertaken by a number of groups in the past ( [23] in sheep , [24] in rodents and [25]–[28] in vitro ) . However , a major limitation of such approaches is that selection in the real world is very different to that applied in the laboratory [20] . A more powerful approach is to take strains of parasites in which resistance was originally selected in the field and genetically map the anthelmintic resistance loci . To undertake detailed genetic mapping , a number of things are necessary . Firstly , the ability to undertake genetic crossing in the organism . Secondly , to have characterised genetically distinct ( preferably isogenic ) resistant and susceptible isolates on which to undertake mapping crosses . Thirdly , a fully sequenced and assembled genome ( or at least a detailed genetic map ) for the organism . All of these are achievable for H . contortus making genetic mapping in this organism a feasible objective in the future [14] . However , a number of other genetic strategies which , although short of classical genetic mapping , can potentially improve our ability to use genome-wide approaches for the identification of anthelmitic resistance genes in the short term . One example of such an approach is the introgression of resistance genes from field derived strains into a characterized susceptible genetic background with repeated backcrossing . This would allow whole genome or candidate gene comparisons such as transcriptomics and genome-wide polymorphism analysis to be more meaningfully applied and interpreted since differences between backcrossed resistant strains and the susceptible parental isolate would be limited to those regions of the genome linked to resistance-conferring loci ( Figure 1A ) . In this paper we report the introgression of ivermectin resistance-conferring loci from two different ivermectin resistant strains , into the genetic background of the susceptible genome reference strain MHco3 ( ISE ) [29] . We have used microsatellite markers to monitor the backcrossing and to genetically validate the success of the approach . We also have preliminary evidence of potential linkage of one marker to a resistance conferring locus . This work provides an important proof of concept of this novel genetic approach for parasites and has generated powerful tools to investigate the genetic basis of ivermectin resistance .
All experimental procedures described in this manuscript were examined and approved by the Moredun Research Institute Experiments and Ethics Committee and were conducted under approved British Home Office licenses in accordance with the Animals ( Scientific Procedures ) Act of 1986 . The Home Office licence number is PPL 60/03899 and experimental IDs for these studies were E06/58 , E06/75 and E09/36 . The MHco3 ( ISE ) strain [29] is the product of multiple rounds of inbreeding , is susceptible to all main classes of anthelmintics and has been adopted as the standard genome strain for the H . contortus sequencing project at the Wellcome Trust Sanger Institute ( http://www . sanger . ac . uk/Projects/H_contortus/ ) . In spite of its inbreeding history it retains high levels of genetic polymorphism [10] , [21] . The White River [30] and Chiswick avermectin resistant [31] strains of H . contortus , were originally isolated from South Africa and Australia respectively . Subsequently they have been experimentally passaged through sheep for a number of years at the Moredun Research Institute , and these versions of the strains are designated as MHco4 ( WRS ) and MHco10 ( CAVR ) respectively throughout the manuscript . These strains were chosen for this work for several reasons: Firstly , their origins were from the field and have subsequently been well characterized in the laboratory [10] , [21] , [30] , [31] Secondly , we have previously shown that these are genetically divergent with respect to the susceptible MHco3 ( ISE ) strain . This is important as it allows us to distinguish between resistance and susceptible parental genotypes when genetic markers are analyzed in the backcross progeny . Thirdly , they were originally derived from different continents –MHco4 ( WRS ) from Africa and MHco10 ( CAVR ) from Australia- and so it is highly likely that ivermectin resistance has been independently selected in each strain . This allows a potentially interesting comparison of resistance mechanisms from two independently selected strains . The basic approach was to cross male worms of the MHco3 ( ISE ) susceptible strain with female worms from the resistant strains in the initial cross . Subsequently F1 female worms derived from each generation of backcross were crossed again with male MHco3 ( ISE ) worms ( Figure 1B ) . The F1 progeny of the first genetic crosses between MHco3 ( ISE ) and the two ivermectin resistant strains MHco4 ( WRS ) and MHco10 ( CAVR ) were designated as MHco3/4 and MHco3/10 respectively . The nomenclature for subsequent backcrosses was MHco3/4 . BCn and MHco3/10 . BCn , denoting the Moredun Research Institute ( M ) , H . contortus ( Hco ) , the unique numbers allocated to both parental strains ( 3/4 or 3/10 ) , and the backcross generation ( BC2 , 3 and 4 ) ( Figure 1 ) . Progeny of each cross were collected and cultured to L3 in the standard way . They were then used to infect donor sheep to generate L4 worms for the next backcross . These donor sheep were treated with ivermectin to ensure only worms which were phenotypically ivermectin resistant were used in the next backcross ( see following section for details ) . The passage of these larvae through three more rounds of ivermectin selection and crossing against the ivermectin susceptible isolate ( MHco3 ( ISE ) ) produced a final fourth backcross generation ( MHco3/4 . BC4 or MHco3/10 . BC4 ) ( Figure 1B ) . Crosses between two strains were performed by surgically transplanting approximately 50 late L4 male worms from one strain and 50 late L4/adult female worms from the other strain directly into the abomasum of a recipient sheep . In order to produce L4 for transplantation male worm-free donor lambs were orally dosed with between 5 , 000–10 , 000 L3 of either MHco3 ( ISE ) , or the ivermectin resistant strain to be crossed against . H . contortus donors with the ivermectin resistant strains were treated with 0 . 1 mg/kg of ivermectin ( Oramec drench for sheep; Merial ) on day 10 or 11 post infection to select for ivermectin resistant progeny prior to transplantation . Donor sheep were euthanased on day 14 post infection and worms were harvested from their abomasa ( day 14 female worms of these strains have previously been shown to be sexually immature and not produce viable progeny; [32] ) . The abomasal contents and washings containing the nematodes were first passed through a 1 mm sieve , transferred into fresh physiological saline ( 0 . 85% NaCl ) and then maintained at 37°C . Male and female H . contortus could then be picked into pre-warmed petri-dishes containing RPMI 1640 tissue culture medium in readiness for surgical transfer into the abomasa of worm-free recipient sheep . 45–100 male late L4/immature adult MHco3 ( ISE ) H . contortus and 50–100 female late L4/immature adult H . contortus were surgically transferred into the abomasa of male worm free recipient lambs , within 2 hours of recovery from the donor sheep . Recipient sheep were anaesthetised to allow a 10 cm vertical incision to be made through the skin , underlying fascia , muscle and peritoneum , over the right flank , midway between the last rib and pelvis and about 10 cm above the midline . The abomasum was located and partially exteriorised , to enable a 1 cm diameter sub-serosal purse-string suture to be placed . A stab incision was then made in the centre of the purse-string suture , through which 50 male late L4/immature adult MHco3 ( ISE ) H . contortus and 50 female late L4/immature adult MHco4 ( WRS ) , or MHco10 ( CAVR ) H . contortus ( or subsequent backcross generations ) were introduced into the abomasum in approx 5 ml RPMI , using a 5 mm diameter blunt ended , glass pipette . The purse-string suture was then closed and the surgical incision repaired allowing the completion of surgical transfers within about 2 hours from the recovery of the nematodes from the donor sheep . Sheep were routinely injected with 1 mg/kg meloxicam ( Metacam 20 mg/ml solution for injection; Boehringer Ingelheim ) for post-surgical analgesia and 7 mg/kg amoxicillin/1 . 75 mg/kg clavulanic acid ( Synulox ready-to-use injection; Pfizer ) and closely monitored on completion of the surgery . No adverse effects were noted during the course of this study . Eggs were identified in the faeces approx 3 days post surgery and collected daily and coprocultured to produce L3 . A controlled efficacy test ( CET ) using ivermectin was undertaken on the two separate fourth generation backcross strains , MHco3/4 . BC4 , and MHco3/10 . BC4 alongside the three original parental strains used in this study , namely MHco3 ( ISE ) , MHco4 ( WRS ) and MHco10 ( CAVR ) . Seventy-five parasite naïve lambs were divided equally between the five different strains . For each strain , the 15 lambs were further allocated into three treatment groups of five animals: no treatment , ivermectin ( 0 . 2 mg/kg BW ) or ivermectin ( 0 . 1 mg/kg BW ) . Lambs were initially allocated randomly to strain and treatment groupings that were subsequently balanced , where needed on the basis of sex and the weight of the animal just prior to the experiment , to ensure that groups were as similar as possible . The lambs were infected with 5 , 000 H . contortus L3 on day 0 . Faecal worm egg counts ( FEC ) were conducted [33] at the start of the controlled efficacy test to confirm their parasitic nematode-free status and on days 16 , 18 , 21 , 28 , 29 and 36 pi to monitor the counts . On day 29 post infection ( pi ) the lambs were weighed again and orally dosed where appropriate with the correct volume of ivermectin ( Oramec drench for sheep; Merial ) using a syringe [34] . All of the lambs were euthanased on day 36pi and worms harvested from their abomasa for determination of H . contortus burdens in abomasal saline washings and digests [34]–[35] . H . contortus recovered from 2% ( MHco4 and MHco10 ) and 10% ( MHco3 , MHco3/4 . BC4 and MHco3/10 . BC4 ) sub-samples of the abomasal washings and digests were counted and sexed ( only adults were seen ) , the higher sub-sample volume was examined in the backcross strains due to the smaller numbers of worms present . The percentage efficacies of each anthelmintic treatment were calculated using the equation 100 ( 1−T/C ) , where T and C are the arithmetic mean total H . contortus burdens of the treated and control groups respectively [36] . The same equation 100 ( 1−T/C ) was also used for the calculation of percentage treatment efficacy using faecal egg counts of treated and control groups that were taken 7 days post treatment , on day 36pi at necropsy . For all estimates of efficacy 95% confidence intervals ( CIs ) were calculated [36] and anthelmintic resistance was deemed to be present when the percentage efficacy of reduction of parasitic nematode burdens or FECs was less than 95% [37] . In addition , the mean treatment and upper and lower 95% confidence intervals were calculated on the FEC data using Bootstrap analysis and a resampling number of 2000 using the “BootStreat” program [38] cited in [39] . All microsatellite genotyping , on both bulk and single worm DNA lysates , was performed using the same PCR amplification methods and parameters previously described [21] . Capillary electrophoresis was performed using an ABI Prism 3100 genetic analyzer ( Applied Biosystems , Foster City , CA ) for the accurate sizing of microsatellite PCR products . The forward primer of each microsatellite primer pair was 5′-end labeled with FAM , HEX , or NED fluorescent dyes ( MWG ) and electrophoresed with GeneScan ROX 400 ( Applied Biosystems ) internal size standard . Individual chromatograms were analyzed using Genemapper Software Version 4 . 0 ( Applied Biosystems ) . Bulk worm DNA lysates were made as previously described [21] . Duplicate bulk lysates were made using approximately 500 L3 worms from each generation of the backcrossing procedure ( BC1 , BC2 , BC3 , BC4 ) , the F1 progeny of the initial genetic crosses ( MHco3/4 and MHco3/10 ) and L3 from the three parental strains MHco3 ( ISE ) , MHco4 ( WRS ) and MHco10 ( CAVR ) . In addition to the bulk worm DNA preparations , 30–40 individual ( L3 or adult ) worm DNA lysates were prepared from seven strains for more detailed genetic analysis: the three parental strains MHco3 ( ISE ) , MHco4 ( WRS ) and MHco10 ( CAVR ) ; the two backcross strains , MHco3/4 . BC4 and MHco3/10 . BC4 and the two populations of survivors ( 0 . 1 mg/kg ivermectin ) of both the backcross strains ( taken from appropriate control and the ivermectin treated animals from the controlled efficacy test experiment respectively ) . The genotyping of parasite populations/strains by amplifying a microsatellite from “bulk” DNA lysates made from a population of worms has been previously described [21] . It is a valuable approach to quickly “fingerprint” worm populations for the presence or absence of microsatellite alleles and gives an approximation as to their relative frequencies . 18 microsatellite loci were used for genotyping the bulk worm DNA lysates . These included 13 previously well characterised loci: Hcms25 , Hcms27 , Hcms33 , Hcms36 , Hcms40 [40]; Hcms8a20 , Hcms22co3 [21]; HcmsX142 , HcmsX146 , HcmsX151 , HcmsX182 , HcmsX256 , HcmsX337 [10] and five new loci Hcms3561 , Hcms18210 , Hcms26981 , Hcms40506 , Hcms18188 ( Supplementary Table S1 ) . Nine microsatellite loci , chosen for their ability to differentiate between the three parental strains , were used to genotype the individual worm lysates from the seven key H . contortus strains . These were Hcms27 , Hcms36 , Hcms40 , Hcms8a20 , Hcms22c03 and four recently identified loci , namely , Hcms3086 , Hcms22193 , Hcms44104 and Hcms53265 ( Redman et al . , in preparation ) . For the single worm genotype data , Pairwise FST values were calculated using Arlequin version 3 . 11 [41] . Data were defined as “standard” rather than microsatellite , as it did not necessarily adhere to stepwise mutation model . PCA was performed using GenAlEx version 6 [42] preserving individual worm genotypes .
The percentage efficacy of ivermectin at doses 0 . 1 mg/kg and 0 . 2 mg/kg was determined from H . contortus arithmetic mean burdens of treated and control groups ( Supplementary Figure S1 ) . Ivermectin efficacies were 100 , 91 , 78 , 22 and 18% at 0 . 1 mg/kg and 100 , 90 , 94 , 38 and 50% at 0 . 2 mg/kg against the MHco3 ( ISE ) , MHco3/10 . BC4 , MHco3/4 . BC4 , MHco10 ( CAVR ) and MHco4 ( WRS ) strains respectively ( Figure 2 and Supplementary Table S2 ) . Hence , the resistance phenotypes of the parental strains MHco4 ( WRS ) and MHco10 ( CAVR ) was confirmed as was the presence of resistant parasites in the backcross populations MHco3/10 . BC4 , MHco3/4 . BC4 . Treatment efficacies based on faecal egg count reduction for the MHco3 ( ISE ) , MHco3/10 . BC4 , MHco3/4 . BC4 , MHco10 ( CAVR ) and MHco4 ( WRS ) strains were 100 , 89 , 69 , 41 and 0% at 0 . 1 mg/kg IVM and 100 , 92 , 93 , 37 and 36% at 0 . 2 mg/kg IVM respectively when compared to untreated controls ( Supplementary Table S3 ) . The genotyping of parasite populations/strains by amplifying a microsatellite from “bulk” DNA lysates made from a population of worms has been previously described [21] . Although , this technique cannot give accurate allele frequency data for alleles present at low frequency in the parasite population , it is a rapid approach to “fingerprint” worm populations for the presence or absence of microsatellite alleles and to obtain approximate frequencies for the predominant alleles . Consequently , we used this as a means of monitoring the backcrossing procedure as it progressed . The alleles from all 18 microsatellite loci were scored as either being ‘present’ or ‘absent’ in genotypes derived from the bulk DNA lysate preparations of the parental isolate , the F1 strains and each backcross generation ( Supplementary Table S4 and S5 ) . When the parental strains MHco3 ( ISE ) and MHco4 ( WRS ) are compared , a total of 17 different isolate-specific alleles were identified across 9 different loci: 5 alleles present in MHco3 ( ISE ) but absent from MHco4 ( WRS ) and 12 alleles present in MHco4 ( WRS ) but absent from MHco3 ( ISE ) . Similarly , comparison of the parental strains MHco3 ( ISE ) and MHco10 ( CAVR ) revealed a total of 41 isolate-specific alleles across 16 loci: 19 alleles present in MHco3 ( ISE ) but absent from MHco10 ( CAVR ) and 22 alleles present in MHco10 ( CAVR ) but absent from MHco3 ( ISE ) . This is consistent with our previous report that both ivermectin resistant strains are genetically distinct from the MHco3 ( ISE ) susceptible reference strain with MHco10 ( CAVR ) being more genetically divergent than MHco4 ( WRS ) [10] , [21] . For both backcrosses , almost all MHco3 ( ISE ) specific alleles were maintained through the 4 backcross generations and were still present in the MHco3/4BC4 and MHco3/10BC4 strains . The only exceptions were the Hcms25 , 215 bp and 217 bp alleles which were present in the first backcross strain ( MHco3/10 . BC1 ) but were lost at the second backcross generation ( MHco3/10 . BC2 ) . These were relatively rare alleles in MHco3 ( ISE ) strain - allele 215 bp present at 3 . 6% and allele 217 bp present at 11 . 1% - and therefore their loss could be due to purely stochastic reasons . In contrast , almost all alleles specific to the two ivermectin-resistant strains MHco4 ( WRS ) or MHc010 ( CAVR ) , disappear during the backcross procedure and are absent in the MHco3/4BC4 and MHco3/10BC4 strains . There are only two exceptions to this . First , the HcX256 allele 243 bp , which is retained in MHco3/10BC4 . However , it was only detected at a frequency of 4 . 6% in MHco3/10BC4 compared with its original frequency of 38% in the MHco10 ( CAVR ) parental strain and so although not completely eliminated , this allele has undergone a dramatic reduction in frequency during the backcrossing procedure ( Supplementary Figure S2 ) . The second exception is the MHco10 ( CAVR ) -specific alleles , 244 bp and 248 bp and the MHco4 ( WRS ) -specific allele 244 bp of loci Hcms8a20 . These are maintained throughout all generations of both backcrosses and this is presented in more detail in the following sections . However , overall the MHco3/4 . BC4 and MHco3/10BC4 strains have a similar genetic background to the MHco3 ( ISE ) parental strain based on bulk genotyping with a panel of 18 microsatellite loci as would be predicted from the backcrossing scheme . The parental strains and final backcross populations ( MHco3/4 . BC4 and MHco3/10 . BC4 ) were analysed in more detail by genotyping 30–40 individual worms with 9 of the most discriminatory microsatellite markers ( Figure 3 ) . The data is presented separately with marker Hcms8a20 either excluded ( 8 loci data ) or included ( 9 loci data ) since this marker shows evidence of an association with the ivermectin resistance phenotype ( see next section ) . Pairwise FST estimates based on the multi-locus genotype data revealed a high level of genetic differentiation between the parental strains: MHco3 ( ISE ) and MHco4 ( WRS ) had a high level of genetic differentiation ( 8 loci FST = 0 . 2101 , 9 loci FST = 0 . 2044 , Figure 3A ) and MHco3 ( ISE ) and MHco10 ( CAVR ) an even higher level ( 8 loci FST = 0 . 4146 , 9 loci FST = 0 . 4006 , Figure 3D ) . Hence MHco10 ( CAVR ) is slightly more divergent from MHco3 ( ISE ) than is MHco4 ( WRS ) confirming previous comparative analysis of these strains [21] . This genetic differentiation between the parental strains was also demonstrated by principal component analysis of individual worm multi-locus genotypes ( Figure 3B , C , E and F ) . Both of the ivermectin resistant strains ( ( MHco4 ( WRS ) and MHco10 ( CAVR ) ) form clusters distinct from the MHco3 ( ISE ) cluster . Both the BC4 backcross strains show a very low level of genetic differentiation from the MHco3 ( ISE ) parental strain ( 8 loci FST = 0 . 0299 and 9 loci FST = 0 . 0265 for MHco3/4 . BC4 and 8 loci FST = 0 . 0045 and 9 loci FST 0 . 0040 for MHco3/10 . BC4 ) . In contrast they show a high level of genetic differentiation from the ivermectin resistant parental isolates ( 8 loci FST = 0 . 1930 and 9 loci FST = 0 . 1767 between MHco4 ( WRS ) and MHco3/4 . BC4 ( Figure 3A ) and 8 loci FST of 0 . 3821 and 9 loci FST of 0 . 3657 between MHco10 ( CAVR ) and MHco3/10 . BC4 ( Figure 3D ) ) . Indeed , this level of genetic differentiation is of a similar magnitude as that seen between the original parental strain of each cross . These results are again supported by the principal component analysis of multi-locus genotypes of single worms where both the MHco3/4 . BC4 and MHco3/10 . BC4 populations are distinct from the MHco4 ( WRS ) and MHco10 ( CAVR ) strains respectively and cluster with the MHco3 ( ISE ) strain ( Figure 3B , C , E and F ) . This demonstrates the genetic background of the 4th generation backcross strains is similar to that of the MHco3 ( ISE ) parental strain . Although the MHco3/4 . BC4 and MHco3/10 . BC4 backcross populations are ivermectin resistant based on the CET , they are significantly less resistant than the original MHco4 ( WRS ) and MHco10 ( CAVR ) parental strains . This is not unexpected given the nature of the backcrossing scheme ( see discussion below ) and means the backcross strains consist of a mixed population of worms of differing ivermectin resistant phenotypes . In order to genetically characterize those worms that were phenotypically resistant to ivermectin at a dose which is 100% effective for the MHco3 ( ISE ) isolate , we infected two sheep each with MHco3/4 . BC4 and MHco3/10 . BC4 , treated with 0 . 1 mg/kg and harvested and prepared DNA from adult worms that survived this drug treatment . These worms surviving ivermectin treatment were then individually genotyped with the 9 microsatellite markers and FST and PCA analysis was performed initially with 8 loci ( loci Hcms8a20 excluded ) and subsequently with loci Hcms8a20 included in the analysis ( 9 loci ) ( Figure 3 ) . On the basis of the eight markers , the ivermectin resistant individuals within the MHco3/4 backcross strain were genetically very closely related to the parental susceptible MHco3 ( ISE ) strain , ( 8 loci FST = 0 . 0379 ) and genetically divergent from the ivermectin resistant parental strain ( 8 loci FST of 0 . 2195 for MHco4 ( WRS ) ) ( Figure 3A ) . Similarly , the phenotypically ivermectin resistant worms within the MHco3/10 . BC4 were more genetically similar to the MHco3 ( ISE ) parental strain ( 8 loci FST = 0 . 0181 ) than to the MHco10 ( CAVR ) parental strain ( 8 loci FST = 0 . 3927 ) ( Figure 3D ) . Inclusion of the Hcms8a20 locus into the analysis produced a similar result but slightly reduced the overall genetic differentiation between the ivermectin resistant backcross worms from each of the resistant parental strains ( 9 loci FST = 0 . 2137 vs 8 loci FST = 0 . 2195 between the MHco3/4 . BC4 ivermectin survivors and MHco4 ( WRS ) strain and 9 loci FST = 0 . 3647 vs 8 loci FST = 0 . 3927 between MHco3/10 . BC4 ivermectin survivors and MHco10 ( CAVR ) ( Figure 3A and D ) ) . Conversely , inclusion of the Hcms8a20 locus increases the genetic differentiation between the ivermectin resistant backcross survivors and the susceptible MHco3 ( ISE ) parental strain to a point where it is statistically significant: 9 loci FST = 0 . 0487 vs 8 loci FST = 0 . 0379 between the MHco3/4 . BC4 ivermectin survivors and MHco3 ( ISE ) and 9 loci FST = 0 . 1129 vs 8 loci FST = 0 . 0181 between the MHco3/10 . BC4 ivermectin survivors and MHco3 ( ISE ) ( Figure 3A and D; statistically significant genetic differentiation between any pair of strains highlighted in italics ) . Consistent with the PCA and FST analysis , examination of the allele frequency data derived from the single worm genotyping revealed that for eight of the nine markers , the allele frequency histograms of the ivermectin treatment survivors of strains MHco3/4 . BC4 and MHco3/10 . BC4 were very similar to the MHco3 ( ISE ) susceptible parental strain and divergent from the resistant parental strains MHc04 ( WRS ) and MHco10 ( CAVR ) respectively ( see Supplementary Figure S3A–H ) . However , this was not the case for Hcms8a20 . In this case , for both backcross strains , an allele that was frequent in the resistant parental strain was retained in the fourth generation backcross strains ( Supplementary Table S4 , S5 and Figure 4 ) . In the case of the MHco3/4 backcross , allele 244 bp ( which was specific to the MHco4 ( WRS ) parent ) was retained at a frequency of 8% in the MHc3/4 . BC4 worms . Notably this increased to a frequency of 40% in the population of MHc3/4 . BC4 worms that survived 0 . 1 mg . kg ivermectin treatment ( Figure 4 ) . Similarly , for the MHco3/10 backcross , allele 248 bp ( which was specific to the MHco10 ( CAVR ) parent ) was retained at a frequency of 12% in MHco3/10 . BC4 worms . Again , this increased to a frequency of 78% in the MHco3/10 . BC4 worms that survived 0 . 1 mg/kg ivermectin treatment ( Figure 4 ) .
H . contortus is one of the few parasitic nematodes where genetic crosses are currently possible . Previous genetic crossing experiments have been performed to assess the level of dominance of resistance genes and test for evidence of linkage of a P-glycoprotein with the ivermectin resistance phenotype [9] , [11] , [43]–[45] . More recently , a genetic mapping approach was undertaken in which resistant F2 were selected and AFLP used to look for markers associated with resistance [8] . This was a potentially powerful approach although , in that case , the ability to analyze the F2 progeny was limited by the lack of genetic differentiation of the parental strains used . We have taken a different genetic approach in which we have successfully introgressed regions of the H . contortus genome containing loci conferring ivermectin resistance from two different ivermectin resistant strains into the genetic background of the MHco3 ( ISE ) susceptible strain . This latter strain is susceptible to the main classes of anthelmintics and is currently being used as the reference strain for the H . contortus genome sequencing project ( http://www . sanger . ac . uk/resources/downloads/helminths/haemonchus-contortus . html ) . The introgression of resistance genes into this strain was achieved by repeated backcrossing of the MHco4 ( WRS ) and MHco10 ( CAVR ) strains against the MHco3 ( ISE ) strain with the application of ivermectin selection at each backcross . A therapeutic dose of 0 . 1 mg/kg ivermectin was chosen as an appropriate discriminatory dose for selection because it is 100% effective against the parental MHco3 ( ISE ) strain ( F . Jackson , unpublished data ) . This was confirmed by our controlled efficacy test; not a single worm of the MHco3 ( ISE ) strain could be found surviving treatment at this dose rate in any of the five treated sheep ( Supplementary Figure S1 ) . In contrast , for both the backcross isolates MHco3/4 . BC4 and MHco3/10 . BC4 , a proportion of worms survived ivermectin treatment at dose both the 0 . 1 and 0 . 2 mg/kg BW dose rates demonstrating these surviving individuals were phenotypically resistant to ivermectin . Although both backcross strains contained individuals that were phenotypically resistant to ivermectin treatment at doses 0 . 1 and 0 . 2 mg/kg , the overall resistance level of the backcross strains was significantly lower than either of the parental resistant strains . This is unsurprising given the nature of the backcrossing regime and experimental design: In order to produce enough infective larvae to undertake a controlled efficacy test , the F1 progeny of the fourth backcross were used to infect a donor sheep which was not treated with drug . Since any resistance alleles would be heterozygous in the F1 of the fourth backcross , resistance alleles would segregate during sexual reproduction of the worms in the final donor sheep that was used to produce L3 for the CET . Hence , the final backcross populations used in the CET would consist of a mixture of resistant and susceptible worms . The relatively low proportion of individual backcrossed worms with an ivermectin resistant phenotype in the CET is consistent with the hypothesis that multiple additive loci contribute to ivermectin resistance since only those worms in which several resistance loci have segregated would be resistant to the doses of drug used . Of course , different alleles can differ in their magnitude of effect , their level of dominance and their expressivity and so the overall relationship between genotype and phenotype is potentially complex . The important point is that backcross strains contain a proportion of individuals that are phenotypically resistant to ivermectin ( unlike the MHco3 ( ISE ) parental susceptible strain ) . The observation that some worms in the backcross strains survive treatment at this dose demonstrates that the resistance-conferring alleles have been successfully introgressed from the parental ivermectin resistant isolates MHco4 ( WRS ) and MHco10 ( CAVR ) . Importantly , when individual worms from the MHco3/4 . BC4 and MHco3/10 . BC4 backcross strains that survive the 0 . 1 mg/kg ivermectin treatment were genotyped with our microsatellite markers , their genetic background was very similar to that of the susceptible MHco3 ( ISE ) parental strain and highly differentiated from the MHco4 ( WRS ) and MHco10 ( CAVR ) resistant parental strains ( Figure 3 ) . This demonstrates that these individuals contain resistance-conferring loci , derived from the resistant parental strains , but have a MHco3 ( ISE ) susceptible genetic background across most of the genome . Hence these strains now provide a powerful resource on which to apply functional genomic strategies to identify regions of the genome harbouring resistance loci . Comparative analysis of ivermectin resistant individuals from the MHco3/4 . BC4 and MHco3/10 . BC4 backcross strains with the parental isolates can be undertaken to identify regions of the genome derived from the MHco10 ( CAVR ) and MHco4 ( WRS ) parental strains and hence harbouring resistance conferring loci . Such analyses could include genome-wide polymorphism analysis , RNAseq analysis ( to examine expression profiles and coding-region polymorphisms ) or targeted analysis of candidate genes . It is important to note that it is likely that the introgressed regions of the MHco4 ( WRS ) and MHco10 ( CAVR ) are relatively large since just four generations of backcrossing have been performed and recombination will have had limited opportunity to break down genetic linkage . Nevertheless analysis of these strains should provide the locations of major ivermectin resistance loci in the H . contortus genome . Further backcrossing , together with improving genomic resources for this parasite , will provide the opportunity to iteratively interrogate these strains to identify the genomic location of resistance loci more accurately . Of the 18 microsatellite markers that were used to monitor the backcrossing procedure , based on the presence and absence of strain-specific alleles , there was only one in which alleles specific to the parental resistance strains were retained in the 4th generation backcross progeny for both back crosses . This was marker Hcms8a20 . Furthermore , when the FST and PCA analysis were performed using the nine most discriminatory markers , each single marker was iteratively excluded to check for distortions of the data due to any effects of single markers . The only marker whose exclusion had any discordant effect on the data was Hcms8a20 ( Data not shown ) . The exclusion of the loci Hcms8a20 from the FST and PCA analysis revealed that MHco3/4 . BC4 and MHco3/10 . BC4 ivermectin resistant worms ( survivors ) , the susceptible MHco3 ( ISE ) strain and their respective backcross strains were all genetically indistinguishable . The inclusion of the loci Hcms8a20 into the same analysis increased the level of genetic differentiation between these aforementioned strains of worms to the point of statistical significance . Examination of the individual allele frequencies for this marker confirms that the allelic profile was more similar to the resistant parental strains than the MHco3 ( ISE ) parental strain ( Figure 4 ) . Indeed , a single allele , specific to the respective resistant parental strains was retained in the two backcross populations . These are present at relatively low frequency ( 8% for allele 244 bp in MHc3/4 BC4 and 12% for allele 248 bp in MHc3/10 . B4 ) . However , these are present at much higher frequencies in the populations of backcross worms that survive 0 . 1 mg/ml ivermectin treatment ( 40% for allele 244 bp in MHc3/4 BC4 and 78% for allele 248 bp in MHc3/10 . B4 ) . It is impossible to predict the precise changes in allele frequency one would expect at a single locus during the backcrossing procedure , or following drug selection , when several loci may have differing additive contributions to the overall resistance phenotype . However , the fact that the same locus , Hcms8a20 , shows evidence of retention of alleles specific for the parental resistant isolates in both fourth generation backcross strains , together with the dramatic increase in frequency of these in the phenotypically ivermectin resistant worms ( relative to the unselected backcross populations ) provides strong evidence that this locus is linked to a resistance conferring polymorphism . The fact that different alleles appear to be selected from the two different parental resistant strains is not necessarily surprising . These two strains – MHco4 ( WRS ) and MHco10 ( CAVR ) - are genetically divergent and originally derived from disparate geographical regions . Consequently , it is entirely possible that a resistance-conferring polymorphism would be genetically linked to different haplotypes of adjacent markers . As the H . contortus genome project progresses it will be interesting to “walk out” from the Hcms8a20 marker to examine additional linked markers to define the size of the region showing evidence of linkage disequilibrium . Furthermore , we hypothesize that additional loci contribute to the ivermectin resistant phenotype of the MHco4 ( WRS ) and MHco10 ( CAVR ) parental strains . We anticipate that these may be identified as we iteratively interrogate the backcross strains with larger marker panels as they become available form the H . contortus genome sequencing project . Similarly , the backcross strains now represent a powerful genetic resource with which to determine if the various candidate genes identified from other studies contribute to the ivermectin resistance phenotype of the MHco4 ( WRS ) or the MHco10 ( CAVR ) strains . In summary , we describe the introgression of resistance-conferring loci from two independent ivermectin resistant strains into a susceptible reference strain of H . contortus . This is a novel approach that provides a powerful adjunct to both candidate gene and whole genome analysis aimed at identifying anthelmintic drug resistance loci . The continued advancement of such genetic approaches , alongside genomic resources for H . contortus , should allow this organisms to be used in an increasingly powerful manner to study the genetic basis of anthelmintic resistance in strongylid nematode parasites . | The use of drugs ( anthelmintics ) to control nematode parasites ( roundworms ) is common in both humans and animals . This has led to the widespread development of drug resistance in livestock parasites and serious concerns regarding its emergence in human parasites . Haemonchus contortus is a parasitic nematode of sheep that has a high propensity to develop resistance and is the most widely used model system in which to study anthelmintic drug resistance . Ivermectin is an extremely important drug for parasite control in both humans and animals . Here , we report a novel approach using genetic crossing to transfer a region of the H . contortus genome containing ivermectin resistance genes from resistant strains into a susceptible strain . During our backcrossing approach , we have identified a genetic marker showing evidence of genetic linkage to ivermectin resistance . The susceptible strain we have used is currently having its complete genome sequenced making the information and strains generated here extremely valuable for the identification of ivermectin resistance genes . This work represents an important proof of concept for using genetic approaches to identify genomic regions containing drug resistant genes in parasitic nematodes . | [
"Abstract",
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] | 2012 | Introgression of Ivermectin Resistance Genes into a Susceptible Haemonchus contortus Strain by Multiple Backcrossing |
Human respiratory syncytial virus ( RSV ) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide , and lacks a vaccine or effective antiviral drug . The involvement of host factors in the RSV replicative cycle remains poorly characterized . A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 ( ARP2 ) as a host factor involved in RSV infection . ARP2 knockdown did not reduce RSV entry , and did not markedly reduce gene expression during the first 24 hr of infection , but decreased viral gene expression thereafter , an effect that appeared to be due to inhibition of viral spread to neighboring cells . Consistent with reduced spread , there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection . In addition , we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent . Filopodia appeared to shuttle RSV to nearby uninfected cells , facilitating virus spread . Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia , indicating a new role for the RSV F protein , driving filopodia induction and virus spread . Thus , this study identified roles for ARP2 and filopodia in RSV-induced cell motility , RSV production , and RSV cell-to-cell spread .
RSV is the most important viral cause of severe acute pediatric lower respiratory tract illness worldwide , and also causes substantial morbidity and mortality in the elderly as well as in individuals with severe immunosuppression or cardiopulmonary disease . Despite its recognized importance , and despite decades of research , there is no licensed vaccine or specific antiviral therapy . RSV is an enveloped virus of the family Pneumoviridae [1] , and contains a single-stranded non-segmented negative-sense RNA genome ( approximately 15 . 2 kb ) with 10 genes encoding 11 proteins , including the nucleoprotein N , phosphoprotein P , matrix protein M , RNA dependent RNA polymerase L , transcription factor and second matrix protein M2-1 , polymerase factor M2-2 that is expressed from a second open reading frame ( ORF ) in the M2 mRNA , fusion glycoprotein F , attachment glycoprotein G , small hydrophobic surface protein SH , and nonstructural accessory proteins NS1 and NS2 [2] . RSV infection starts with cellular receptor binding mediated by G and F [3] . The chemokine receptor CX3CR1 has recently been identified as a receptor molecule for the RSV G protein on respiratory epithelial cells [4] . Entry of RSV is not completely defined and may involve cell surface fusion as well as macropinocytosis followed by fusion [5] , mediated by the F protein . RSV transcription and replication occur in the cytoplasm , probably in large , dense cytoplasmic inclusion bodies . Progeny virions bud from the plasma membrane [2 , 6] . In the natural human host , RSV infects respiratory epithelial cells [7] . We recently performed a genome-wide siRNA screen of more than 20 , 000 genes in human airway epithelial A549 cells infected with RSV-GFP to identify genes that affected viral expression of GFP and therefore may affect the RSV replicative cycle . This survey , which is still in progress and will be published separately , provided presumptive evidence that knockdown of the ACTR2 gene , which encodes ARP2 , resulted in a reduction of viral GFP expression , suggesting that the ARP2 protein promotes RSV infection ( For simplicity , we will refer to the ACTR2 gene and mRNA by the same name as used for the protein , ARP2 ) . ARP2 is part of the ARP2/3 complex , which plays a central role in actin polymerization [8] . Actin is a major component of the cytoskeleton , and actin rearrangement affects a multitude of intra- and intercellular processes including cell shape , structure , and motility [9] . Actin is present in globular monomeric ( G-actin ) and polymeric filamentous ( F-actin ) forms . F-actin can form polymeric structures resulting in cell membrane extensions , such as lamellipodia ( sheet-like extensions ) , filopodia and microvilli ( finger-like protrusions ) , and dot-like podosomes [10] . Cellular actins also appear to be involved in RSV gene expression , replication and morphogenesis [11–13] , but the mechanim is poorly understood , and the role of ARPs has not been studied . In the present study , we show that ARP2 knockdown reduced viral gene expression and protein production , viral yield , and cell-to-cell spread in A549 cells . These effects were most prominent at later times of infection , affecting viral spread rather than early events in RSV infection . We found that the RSV infection induces the formation of filopodia on the cell surface , which are distinct from structures representing filamentous RSV particles . Filopodia appear to shuttle RSV particles to neighboring cells , a previously unknown mechanism for RSV spread . We also found that RSV infection increases cell motility , likely contributing to cell-to-cell spread . Our results show that ARP2 contributes to RSV cell-to-cell spread in human lung epithelial cells .
To investigate the effects of ARP2 knockdown on RSV infection , A549 cells were transfected with an ARP2-specific siRNA ( siARP2 ) or a negative control siRNA ( siControl ) . At 48 hr post-transfection , the cells were infected with RSV-GFP ( that expresses GFP from an additional gene [14] ) at a multiplicity of infection ( MOI ) of 1 . 0 plaque forming unit ( PFU ) /cell . The cultures were evaluated for ARP2 and viral gene expression at 0 , 24 , 48 , and 72 hr post-infection ( hpi ) ( Figs 1–3 ) . To put this timing into context , in a growth cycle for RSV in A549 cells ( which is shown later ) , the production of progeny virus was first detected at approximately 12 hpi , increased substantially by 24–48 hpi , and was maintained or slowly increased through approximately 72 hpi . The effectiveness of siARP2-mediated knockdown on ARP2 protein expression was evaluated by Western blot analysis ( Fig 1A ) . A substantial reduction in ARP2 protein accumulation was observed 48 hr following siARP2 transfection , the time of RSV infection , and remained stable for the duration of the 72 hr infection . Quantitative ( q ) RT-PCR confirmed that knockdown of ARP2 mRNA was highly efficient and stable over the same time course ( Fig 1B ) . In the absence of siARP2 , there was no significant difference in the expression of ARP2 mRNA or protein in RSV-GFP infected versus uninfected cells , indicating that expression was unaffected by RSV infection ( Fig 1A and 1B ) . Importantly , ARP2 knockdown was achieved without compromising cell viability , as determined by alamarBlue viability assay ( Fig 1C ) . As a first step to investigate the effects of ARP2 knockdown on RSV infection , we used qRT-PCR to quantify the accumulation of the complete set of mRNAs encoded by RSV-GFP in A549 cells transfected with siARP2 or siControl ( Fig 2 ) . The input MOI in this experiment was 1 PFU/cell ( calculated by titration on Vero cells ) . Since the susceptibility of A549 cells is slightly lower than that of Vero cells , the majority ( about 2/3 ) of A549 cells was not infected by the initial inoculum and could support subsequent rounds of replication . Each of the RSV ORFs was quantified , including M2-1 and M2-2 that are expressed on a single mRNA ( Fig 2 ) . This showed that , for the first 24 hr following infection , the accumulation of each virally-encoded mRNA was similar in ARP2-knockdown and control cells , except for the RSV F and L mRNA levels which were slightly but significantly reduced in siARP2 depleted cells; unexpectedly , the NS1 and NS2 mRNA levels were slightly but significantly lower in siControl treated cells than in siARP2 cells . However , between 24 and 48 hpi , there was a much smaller increase in the accumulation of viral mRNAs in the ARP2-knockdown cells than in the control cells . By 48 hpi , NS1 , NS2 , P , SH , M2 and L mRNA levels were significantly lower in siARP2-knockdown cells; the difference in P mRNA levels as well as M2 mRNA levels measured using the M2-1 ORF specific assay were significant only at 48 hr , and the differences in GFP and F mRNA levels reached significance by 72 hpi . N , M , G mRNA levels also were lower in siARP2 transfected A549 cells at late time points after infection , but the differences to siControl transfected cells were not statistically significant . Western blot analysis showed that the accumulation of viral proteins was also reduced , shown here for P and F ( Fig 3A ) . For a more sensitive analysis of protein expression over time , the kinetics of infection and viral protein expression in siARP2-transfected cells were further investigated by flow cytometry using expression of GFP , F , and M2-1 as markers for infection . Data from a representative experiment are shown in Fig 3B . At 24 hpi , we found little difference in the percentage of RSV-infected A549 cells in ARP2-knockdown versus control cultures . In contrast , at 48 hpi , the number of GFP- , M2-1- , or F-expressing cells had substantially increased in the control cultures , while only a small increase was observed in the siARP2-treated cultures . A similar pattern for the percentage of infected cells was observed when the results were averaged from two ( M2-1 ) or three ( GFP and F ) independent experiments ( Fig 3C ) . In addition , Fig 3C shows that the average mean fluorescence intensity ( MFI ) for each of these proteins was similar between siControl- and siARP2-transfected cells at both 24 and 48 hpi . These results show that ARP2 knockdown did not reduce RSV protein expression in individual infected cells; rather , ARP2 knockdown reduced the spread of RSV infection at late time points after infection . To exclude a possible role of ARP2 in RSV entry , A549 cells were studied at earlier time points after infection ( Fig 4 ) . Flow cytometry showed that , at 16 hpi , the proportion of GFP-expressing cells in cultures infected at an MOI of 1 or 3 was similar in siARP2- and siControl-treated cells ( Fig 4A ) . To exclude possible artifacts due to transfection , a stable ARP2-knockdown A549 cell line ( ARP2/KD-A549 cells ) was generated using a lentiviral vector system expressing three small hairpin RNAs to ARP2 , and RSV-GFP infection was analyzed at 6 or 12 hpi by flow cytometry using GFP expression as a marker for infection . We found that the proportion of infected ( ARP2/KD-A549 cells ) did not differ from that in untransduced A549 cells , even at higher MOIs ( e . g . MOI = 5 ) ( Fig 4B ) . These results suggested that ARP2 does not have a role at early stages in RSV infection . Next , we evaluated a possible role for ARP2 in RSV entry using the compound CK-666 , which is an inhibitor of ARP2/3 complex-driven actin nucleation that acts by stabilizing the ARP2/3 complex in an inactive conformation [15] ( Fig 4C and 4D ) . For comparison , we also evaluated EIPA ( 5-ethylisopropyl amiloride ) , which is a potent inhibitor of macropinosome formation and has been demonstrated to inhibit RSV entry [5] , and thus is a positive control for the inhibition of entry . A preliminary dose-ranging experiment was performed in which A549 cells were incubated for 1 hr with various concentrations of EIPA , followed by 30 min further incubation with 1 mg/ml dextran fluorescein , which is a marker for uptake by macropinosomes [16] ) . Dextran uptake was quantified by flow cytometry . This identified 100 μM EIPA as the optimal concentration , reducing dextran uptake by 75% , whereas 100 μM of CK-666 ( the known effective concentration [17 , 18] ) or DMSO solvent had no substantial effect on dextran uptake ( Fig 4C ) . Live/Dead cell staining indicated a lack of cytotoxicity on A549 cells at this concentration . Therefore , A549 cells were incubated with 100 μM EIPA , 100 μM CK-666 , or DMSO ( solvent-only ) for 1 hr , followed by RSV-GFP infection ( MOI = 5 ) for 6 or 12 hr , followed by analysis of GFP expression by flow cytometry ( Fig 4D ) . The EIPA treatment reduced the number of infected cells by about 60% at 6 hpi or 50% at 12 hpi , whereas CK-666 and the DMSO control had little effect ( Fig 4D ) . These results confirm that ARP2 does not detectably affect RSV entry or gene expression in A549 cells during the first 6–16 hpi . We also investigated the production of infectious virus in response to ARP2 knockdown by siARP2 , which was done as part of the experiments described in Figs 1A , 2 and 3A . In one set of cultures , we harvested the cell culture medium alone without disturbing the cells to measure released virus over a 72 hr period . This showed that ARP2 knockdown resulted in a ~10-fold reduction in the production of free infectious virus in the supernatant ( Fig 5A ) . In a second set of cultures , the infected cells were scraped into the medium and the suspension was vortexed and clarified in order to quantify total virus production ( i . e . , cell-associated and cell-free released virus , Fig 5B ) . The titer of infectious progeny RSV was reduced in the ARP2-knockdown cultures compared to the control cells , but this difference was smaller for total RSV ( Fig 5B ) than for released RSV ( Fig 5A ) . We then evaluated the effect of ARP2 knockdown on RSV production in another human airway epithelial cell line , Calu-3 cells [19] . Similar to the results in A549 cells , we found that ARP2 knockdown did not reduce cell viability , and knockdown was stable up to 120 hr post siRNA-transfection ( S1 Fig ) . In the ARP2 depleted Calu-3 cells , production of free infectious virus was reduced at late time points after infection , and the production of total infectious progeny was reduced by about 10 fold at 72 hpi , a greater reduction than that observed in A549 cells ( S1 Fig ) . In an additional experiment , we included human parainfluenza virus type 3 ( HPIV3 ) expressing GFP ( HPIV3-GFP ) for comparison ( S2A Fig ) . Replicate cultures of A549 cells were transfected with siARP2 or siControl , infected with RSV-GFP or HPIV3-GFP , and harvested at 24 , 48 , and 72 hpi . Quantification of free infectious progeny virus in the supernatant showed that the production of released RSV-GFP was reduced by ~10-fold in siARP2-transfected cells compared to siControl-transfected cells , consistent with the results in Fig 5A . In contrast , there was little or no reduction in the release of HPIV3-GFP in siARP2 treated cells , indicating that the effect was specific to RSV . To evaluate the effects of ARP2 knockdown on RSV-induced syncytium formation , the RSV-GFP-infected cell monolayers from the experiment described immediately above ( harvested at 24 , 48 , and 72 hpi ) were fixed , permeabilized , and stained with the nuclear fluorescent stain diamidino-2-phenylindole ( DAPI ) and with rhodamine phalloidin to visualize the actin cytoskeleton . The total coverslip was scanned by fluorescence microscopy , and at least 5000 cells per treatment were analyzed in each of two independent experiments to quantify the number of nuclei that were present in GFP-positive syncytia ( syncytia were defined as containing ≥ 3 nuclei ) compared to the total number of nuclei in all RSV-infected cells ( whether in syncytia or not ) . We detected a significant reduction in the overall number of RSV-GFP infected cells in siARP2-knockdown cells ( consistent with the results in Fig 3B ) , but , when corrected for the lower rate of infected cells present in the evaluated fields , syncytium formation was not reduced in siARP2 treated cells ( S2B Fig ) . Thus , ARP2 does not seem to have a role in RSV fusion and syncytia formation . We investigated the effects of ARP2 knockdown on RSV particle morphology using transmission electron microscopy ( TEM ) ( Fig 6A ) . ARP2-transfected- or siControl-transfected A549 cells were mock-infected or infected with RSV at an MOI of 1 , and were fixed at 24 hpi . Overall , areas with accumulations of particles were substantially less frequent on ARP2-knockdown infected cells , and therefore we scanned for areas with accumulations of budding particles and made photomicrographs of these areas to compare virion morphology . In Fig 6A , panels 2 and 3 ( siControl-knockdown infected cells ) and 5 and 6 ( siARP2-knockdown infected cells ) show representative examples of cell-associated virions ( indicated by arrows ) . This comparison indicated that , while the abundance of particles was reduced in infected cells treated with siARP2 , in those areas where virions were evident there were no apparent changes in virion morphology between infected cells treated with siARP2 versus siControl . Next , we examined cell surface morphology and virion budding by immune scanning electron microscopy ( immuno-SEM ) ( Fig 6B and 6C ) . A549 cells were transfected with siARP2 or siControl , and infected with RSV at an MOI of 5 or mock-infected . At 24 hpi , the cells were fixed and subjected to immunostaining with a primary mouse monoclonal antibody ( mAb ) against the F protein and a secondary anti-mouse-IgG polyclonal antibody ( pAb ) conjugated with gold particles ( 15 nm ) , and analyzed by SEM . As shown in Fig 6B , panels 2 and 2a , the surface of the siControl-transfected RSV-infected cells contained areas with large numbers of long filamentous structures ( asterisks ) that were organized in parallel arrays that appeared to be lying flat on the infected cell surface . These structures were not evident on the surface of siControl-transfected mock-infected cells ( Fig 6B , panel 1 ) . Furthermore , these structures stained with F-specific immunogold ( Fig 6B , panel 2a , arrows ) , whereas images of siControl-transfected mock-infected cells examined in parallel exhibited a lack of staining . The dimensions of these filamentous structures were approximately 100 nm in diameter and 2–10 μm in length , which is consistent with values reported for RSV virions [20] . Thus , these appeared to be filamentous progeny virus particles . The surface of the siARP2-transfected RSV-infected cells showed a reduced number of presumptive viral filaments , suggesting that ARP2 knockdown reduced RSV budding ( Fig 6B , panels 4 and 4a ) . Some of these particles appeared to lie on the cell surface ( Fig 6B , panels 4 and 4a , asterisks ) , comparable to what was observed in siControl-transfected RSV-infected cells , whereas a number of particles appeared to be lifted off the surface in a disordered array ( indicated by arrowheads ) . In addition , the surface of siARP2-transfected infected cells contained occasional small surface protuberances that stained heavily with F-specific immunogold and might represent aberrant , incomplete budding of particles containing F protein ( Fig 6B , panel 4a , red arrow ) . Thus , while TEM suggested that ARP2 knockdown did not visibly alter the morphology of the RSV particle , immuno-SEM suggested that ARP2 knockdown resulted in virus filaments that were somewhat disorganized at the cell surface , as well as in smaller protuberances that stained well for F protein . Lower-magnification images of this same experiment revealed another difference between mock-infected and RSV-infected cells . Specifically , siControl-transfected RSV-infected cells were observed to contain prominent surface protrusions ( Figs 6C and S3 , panel 2 , cyan arrows ) that were largely absent and reduced in length in siControl-transfected mock-infected cells ( Figs 6C and S3 , panel 1 ) . These protrusions also were largely absent in siARP2-transfected RSV-infected cells ( Figs 6C and S3 , panel 4 ) . The size and nature of these protrusions identified them presumptively as lamellipodia and filopodia , which are cytoplasmic extensions involved in cell motility , sensing , and cell-to-cell interactions [21] . Lamellipodia typically are broad flat cytoplasmic protrusions containing an internal branched actin network , and filopodia are slender cytoplasmic protrusions that extend beyond the leading edge of lamellipodia and contain linear actin filaments ( F-actin ) , reviewed in [22] . The filopodia are investigated further below . We further visualized the effects of RSV infection and ARP2 knockdown on virus and cell morphology using immunofluorescence confocal microscopy . A549 cells were transfected with siARP2 or siControl for 48 hr , mock-infected or infected with RSV-wild type ( RSV-WT ) for 24 hr ( MOI = 1 ) , fixed , permeabilized , immunostained for RSV F ( green ) and beta-tubulin ( cyan ) , and stained with rhodamine phalloidin ( red ) to detect F-actin as a marker for filopodia and with the nuclear stain DAPI ( blue ) . All four colors are shown in the images in Fig 7A , and images that individually show the green , cyan , and red channels are shown in S4 Fig . In siControl-transfected RSV-infected cultures ( Fig 7A , third row of panels ) , RSV-infected cells were found to contain long surface projections that were intensely stained with rhodamine phalloidin ( red ) , indicative of F-actin content . In addition , these surface projections were deficient in tubulin ( cyan ) ( S4 Fig , third row of panels , arrows ) . The morphology of these surface filaments , and the presence of actin and relative absence of tubulin , was consistent with filopodia , whereas lamellipodia contain abundant tubulin . Filopodia were not prominent in the mock-infected siControl- or siARP2-transfected cells ( Fig 7A , top first and second rows of panels ) , consistent with the previous observations with immuno-SEM , suggesting that they were induced by RSV infection . Moreover , consistent with findings in the immuno-SEM images described above , extensive filamentous extracellular virions were observed in the RSV-infected siControl-transfected cultures ( Fig 7A , asterisks indicate free extracellular virions and arrowheads indicate examples of filopodia-associated virions ) . The filopodia were reduced in number and length in siARP2-transfected RSV-infected cells ( Fig 7A , bottom row; Fig 7B ) , consistent with the previous observation by immuno-SEM . The abundance of extracellular virions also was markedly reduced . Some variability in the extent of filopodia reduction was observed for the siARP2-transfected RSV-infected cells even within the same wells: in some cells , the cell surface appeared to be completely devoid of filopodia , while in other cells filopodia were present but were reduced in number and were much shorter . One possibility is that this reflects cell-to-cell differences in the efficiency of ARP2 siRNA transfection and knockdown . We quantified the number and length of filopodia in the siARP2- and siControl-transfected mock-infected or infected cells by automated scanning using confocal microscopy . We counted all filopodia from , on average , 100 cells per treatment per experiment , and the length of each filopodium was measured from the cell surface to the tip of the filopodium . The number and length of filopodia from two independent experiments are summarized in Fig 7B . In the siControl-transfected infected cells , the filopodial length ( measured only on the infected cells in the culture , confirmed by RSV-F-staining ) was observed to be up to 100 μm , and the longer filopodia were observed in greater numbers in the lower-density cultures , suggesting that length increased with increasing space between cells . In contrast , in siARP2-transfected RSV-infected cells , the number of longer filopodia ( measured only on the infected cells , identified by F-actin staining ) was greatly reduced ( Fig 7B ) . Compared to RSV-infected cells , fewer filopodia were present on mock-infected siControl and siARP2 cells , and filopodia were less than 20 μm in length . In mock-infected ARP2-knockdown cells , filopodia were furthest reduced in numbers and length . These results confirm that ( i ) RSV infection is associated with the formation of long filopodia , and ( ii ) that , filopodia formation involves ARP2 . To confirm these findings and to exclude off-target effects of ARP2 knockdown , we also characterized the role of Wiskott-Aldrich Syndrome protein ( WASP ) , specifically the homolog N-WASP , which has been known to bind directly ARP2/3 complex and stimulate actin polymerization [23] . N-WASP depletion by siRNA caused only a modest reduction in the viability of A549 cells , and was stable over at least 120 hr post-siRNA transfection , and it reduced RSV production in A549 cells ( S5 Fig ) . Importantly , RSV-induced filopodia in A549 cells were reduced or abolished by N-WASP depletion ( S6 Fig ) , confirming that depletion of another factor involved in filopodia formation has a similar effect to that of ARP2 depletion . We also examined RSV infection of ARP2/KD-A549 cells versus untransduced A549 cells using stimulated emission depletion ( STED ) microscopy , which provides higher resolution ( Fig 8A and 8B ) . A549 cells or ARP2/KD-A549 cells ( i . e . , stable ARP2 knockdown cells ) were infected with RSV-WT ( MOI = 1 ) , incubated for 24 hr , and fixed , permeabilized , and stained for F-actin ( red ) and RSV F protein ( green ) . In A549 cells , we observed abundant extracellular filamentous structures that stained intensely with RSV-F-specific mAb and were consistent with being RSV virions ( green; examples of free virions are indicated with arrowheads and examples of cell- and filopodia-associated virions are indicated with arrows ) , suggesting extensive virus shedding ( Fig 8A , panel 1a ) ; in contrast , in the infected ARP2/KD-A549 cells , reduced virus shedding was observed ( Fig 8A , panel 2a ) . This result was consistent with the result in the Fig 7A , bottom two rows . Two A549 cells , labeled “A” and “C” in Fig 8A , panel 1 , appear to be infected based on staining for F protein ( green ) and the presence of filopodia ( red; some examples are indicated with asterisks in panel 1a ) . Some of the filopodia from cell “A” ( Fig 8A , panels 1 and 1a ) appear to be contacting ( yellow arrow ) cell “B” that is located on the left hand side of the panel , and this area of contact in cell B has a number of virus-like particles , whereas the rest of cell B has little or no staining for RSV F ( except at the junction of filopodia-driven interaction by cell “C” , shown only in Fig 8A , panel 1 ) and has minimal filopodia and thus appears to be otherwise uninfected . Thus , the filopodia appeared to be conveying virions from infected cells to uninfected cells . RSV virions were frequently observed at the tip of the filopodia , further supporting the idea of filopodia-driven RSV cell-to-cell spread ( Fig 8B ) . In contrast , filopodia-driven RSV cell-to-cell spread was not apparent in the ARP2/KD-A549 cells ( Fig 8A , panels 2 and 2a ) . We investigated a possible role for RSV-induced filopodia in viral spread using live cell imaging . A549 cells were transfected with siControl or siARP2 , infected with RSV-GFP , and observed with confocal microscopy over time , with images taken every 6 min ( MOI = 0 . 1 , imaged from 24 to 48 hpi , S1 and S2 Movies , respectively ) . The time-lapse images showed that RSV infection resulted in increased cellular motility in the control-knockdown cells , but not in the ARP2-knockdown cells . The control-knockdown infected cells were much more active in migration and showed an increased number of filopodia ( visualized as abundant hair-like structures on the surface ) , compared to the ARP2-knockdown infected cells . As filopodia are a means of cell motility [24] , this suggests that the RSV-induced filopodia rendered the RSV-infected cells able to migrate and contact other cells , which appeared to result in the secondary cells becoming GFP-positive . This was inhibited by ARP2 knockdown . In order to visualize actin in live cell experiments , we generated a Red F-actin-A549 cell line , which stably expresses a 17-amino-acid-long actin-binding domain linked genetically to red fluorescence protein ( RFP ) . This fluorescent fusion protein binds to F-actin , reportedly without interfering with its function [25] . siControl- and siARP2-transfected cells were mock-infected ( S3 and S4 Movies , respectively ) or infected with RSV-GFP ( MOI = 0 . 1 , imaged from 24 hpi to 48 hpi , S5 and S6 Movies , respectively ) . In response to RSV-GFP infection , the control-knockdown cells formed filopodia containing red actin , and were motile and appeared to spread infection to neighboring uninfected cells . A region of interest is magnified from RSV-GFP infected Red F-actin A549 cell line ( MOI = 0 . 01 , imaged from 24 to 48 hpi ) to show viral cell-to-cell spread ( S7 Movie ) . To more closely mimic RSV infection in non-ciliated respiratory epithelial cells of the lower respiratory tract , we also performed these experiments using confluent monolayers of Red F-actin-A549 cells ( S8 , S9 , S10 , and S11 Movies ) . Time lapse imaging of siControl transfected Red F-actin-A549 cells confirmed that RSV-GFP infected cells ( S10 Movie ) were more dynamic than uninfected cells ( S8 Movie ) . Interestingly , the infected cell monolayers were disrupted over time by the cytopathic effect of RSV-GFP; any void was filled quickly by actively migrating newly RSV infected ( newly green ) cells , resulting in efficient spread of RSV infection through the monolayer ( S10 Movie ) . As observed previously in non-confluent monolayers , ARP2 knockdown reduced the motility and filopodia formation of infected cells ( S11 Movie ) . As a consequence , the cell monolayers were retained longer in siARP2 transfected cells ( S11 Movie ) than in siControl cells ( S10 Movie ) . We also used a scratch-wound assay based on A549 cells as a surrogate model for tissue damage caused by the cytopathic effect of RSV , and we measured the migration of RSV infected cells in this assay ( Fig 9 ) . Confluent siControl- or siARP2-transfected Red F-actin-A549 cell monolayers were mock-infected or infected with RSV-GFP ( MOI = 1 ) . At 24 hpi , the monolayers were scratched with a pipette and were followed by imaging every 5 min from 24 hpi to 36 hpi . To measure the migration of Red F-actin-A549 cells into the wound area , we scored the red intensity in the scratched area over time . We confirmed that RSV infection considerably increased cell motility , whereas ARP2 depletion drastically reduced RSV-induced motility ( Fig 9 ) . In vivo , RSV promotes shedding and cell death of bronchial and lung epithelial cells [26 , 27] . This suggests that the induction of ARP2-dependent mobility of RSV infected cells promotes the spread of RSV infection and cytopathogenicity in the respiratory tract . We compared the efficiency of induction of filopodia by RSV , and their effects on motility and virus spread , to that of two other common respiratory paramyxoviruses , namely HPIV3 and human metapneumovirus ( HMPV ) . A549 cells were transfected with siARP2 or siControl; infected with RSV-GFP , HPIV3-GFP , and HMPV-GFP; incubated for 24 hr; fixed , permeabilized , stained with rhodamine phalloidin ( red ) and DAPI ( blue ) ; and examined by confocal microscopy . Examination of infected cells ( green ) showed that RSV-GFP induced abundant filopodia ( Fig 10A , top row left panel; examples of filopodia are indicated with arrows ) , as expected . In contrast , HPIV3-GFP and HMPV-GFP induced few filopodia-like structures on the infected cell surface ( Fig 10A , middle and bottom rows , respectively ) . Long intracellular actin filaments were observed in each of the cultures , and were especially evident in uninfected cells , but the induction of filopodia was much more robust in response to RSV ( Fig 10B ) . As expected , RSV-induced filopodia formation was greatly reduced in siARP2-transfected , RSV-GFP-infected cells ( Fig 10A , top right ) , but ARP2 knockdown had little effect on the appearance of HPIV3-GFP and HMPV-GFP infected A549 cells ( Fig 10A , right middle and bottom panels ) . The ability of HPIV3-GFP and HMPV-GFP to induce mobility was investigated in parallel with RSV-GFP using Red F-actin-A549 cells and live cell imaging . Infection with RSV-GFP induced motility and appeared to facilitate viral spread to neighboring uninfected cells ( S12 Movie ) , whereas this was not observed with HPIV3-GFP ( S13 Movie ) , and was minimal with HMPV-GFP ( S14 Movie ) . Since the RSV F protein mediates viral fusion and thus has dramatic effects on the cell plasma membrane , we investigated whether it induces filopodia formation . We expressed RSV F from a eukaryotic expression plasmid transfected into A549 cells , in parallel with a control plasmid expressing GFP . At 12 hr post-transfection , the cells were fixed , permeabilized , and stained with DAPI , rhodamine phalloidin and mAb specific for the RSV F protein , and analyzed by confocal microscopy ( Fig 11A ) . Long intracellular actin filaments were observed in each of the cultures , but the induction of filopodia-like structures on the transfected cells ( Fig 11A , arrows ) was observed in response to RSV F but not GFP expression . We also investigated the effect of the expression of RSV F protein on filopodia formation using a chimeric bovine/human ( B/H ) PIV3 ( B/HPIV-3 ) that consists of BPIV3 in which the F and HN genes have been replaced by those of HPIV3 , and which in addition expresses RSV F from an added gene [28] . A549 cells were transfected with siARP2 or siControl followed by infection with B/HPIV3-RSV-F ( Fig 11B ) . HPIV3-GFP infected cells , included here for comparison ( Fig 11B , top row of panels ) , had minimal induction of filopodia , as already shown in Fig 10 . In contrast , the expression of the RSV F protein from B/HPIV3-RSV-F in control-knockdown cells induced filopodia-like structures on the infected cells ( Fig 11B , middle panel , arrows ) . This was largely blocked by ARP2 knockdown ( Fig 11B , bottom panel ) .
Actin is a major component of the cytoskeleton , and cell shape , motility , and a multitude of dynamic intra- and intercellular processes are dependent on actin rearrangement [9] . Actin-dependent cellular functions require precise regulation of actin polymerization . The ARP2/3 complex plays an important role in the initiation of F-actin polymerization ( also called nucleation ) during diverse cellular processes [29] . The ARP2/3 complex is one of the three major classes of factors for actin nucleation [together with formins and the tandem-monomer-binding family] [21 , 30] . Viral infection can modulate the actin cytoskeleton morphology , reviewed in [10] . Many enveloped and nonenveloped viruses interact with the actin cytoskeleton during virus entry [10]; actin involvement in RSV endocytosis or macropinocytosis has been described previously [5 , 31] . While this manuscript was in preparation , it was shown that ARP2/3 complex-dependent actin rearrangement is required for alphavirus trafficking and egress at late time points after infection [32] . Actin is also involved in RSV replication [13] , gene expression , and morphogenesis [11 , 12 , 33] . However , the mechanisms for actin involvement in RSV infection are poorly understood , and no information was available on the role of ARP proteins during RSV infection . We identified ARP2 in a comprehensive genome-wide siRNA screen targeting approximately 21 , 500 human genes , performed on RSV-infected human lung epithelial A549 cells . In the present study , we have systematically investigated the effect of ARP2 on the RSV replicative cycle . For several other viruses , actin polymerization has been shown to play a role in viral entry , and different viruses use the cellular actin system differently . For example , primate lentiviruses enter cells by membrane fusion followed by initiating ARP2/3 complex-driven actin polymerization to generate a mechanical force that allows the lentiviral core complex to pass through the cortical layer and migrate to nucleus . Even though vaccinia viruses enter cells by membrane fusion at the cell surface , cell cytoskeletal rearrangement immediately after viral attachment is required for viral entry and transport through the cytoplasm [34] . One route of RSV entry seems to be through Rab5-positive , fluid-filled macropinosomes . Specifically , RSV binding at the cell surface activates a signaling cascade for actin rearrangement , resulting in viral entry through macropinocytosis [5] . Therefore , we began the present study by investigating possible effects of APR2 on RSV entry and early steps in RSV replication . Examination of the kinetics and distribution of viral gene expression by flow cytometry showed that ARP2 knockdown had no or only minor effects on the efficiency of infection and viral gene expression during the first 24 hr following inoculation ( which approximately spans the time from the initiation of infection to the time when virus production has become robust ) but thereafter ARP2 knockdown reduced the number of infected A549 cells without affecting the magnitude of gene expression per cell . We also analyzed RSV mRNA and protein expression over time by qRT-PCR and Western blotting , and similarly showed little or no effect prior to 24 hpi , and an overall reduction in gene expression after 24 hpi . A second ARP2-specific siRNA ( Hs_ACTR2_7 , QIAGEN ) was evaluated with similar results , although the efficiency of knockdown was marginally lower . We also investigated whether the ARP2/3 complex is involved in early steps of RSV infection by using the potent ARP2/3 complex inhibitor CK-666 [17] . We found that this inhibitor did not reduce RSV entry at concentrations that had previously been shown to be effective in inhibiting ARP2/3 complex-driven actin nucleation [18] , while the macropinocytosis inhibitor EIPA did reduce RSV entry , as previously shown [5] . Thus , the ARP2/3 complex did not seem to be essential for any steps of RSV entry or early events in the viral replicative cycle , in particular for actin nucleation during macropinocytosis of RSV . However , actin nucleating factors other than the ARP2/3 complex could contribute on actin nucleation for the more localized macropinosome formation during RSV entry . Even though macropinocytosis is a transient actin dependent endocytic process , it is primarily associated with cell-wide plasma membrane ruffling , and involves formation of large vacuoles for non-selective internalization of fluid and membranes [35 , 36] . The Ras superfamily of GTPases plays an important role in regulating ruffle formation for macropinocytosis [37] , whereas other Rho GTPases such as cell division cycle 42 ( Cdc42 ) regulates ARP2/3 complex-driven actin polymerization for filopodia formation [38 , 39] . There is extensive overlap and crosstalk , and dynamic interactions are evident among Rho GTPases signaling pathways [40 , 41] . A coordinated activation of several Rho GTPases was suggested to be involved in the cytoskeleton rearrangement induced by HMPV for its cell-to-cell spread [42] . Further research is necessary to understand whether RSV modulates the signaling of different GTPases for cytoskeleton reorganization during entry . We next investigated a possible role of ARP2 in RSV production , syncytium formation , budding , and virion morphology . ARP2 knockdown resulted in a 10-fold reduction in RSV release into the supernatant of infected A549 cells when measured two and three days post-infection , while the release of HPIV3 into the supernatant was only minimally reduced in siARP2-transfected cells . A similar reduction in RSV production was observed in another respiratory cell line , Calu-3 ( S1 Fig ) when infected with RSV-WT ( MOI = 1 ) . This reduction in RSV production was partly evident by 24 hpi , suggesting that it reflected , at least in part , reduced virus yield per infected cell . The reduction was greater by 48 and 72 hpi , suggesting that it also reflected reduced secondary infection . We did not observe substantial effects of ARP2 depletion on RSV fusion , suggesting that this was one aspect of RSV spread that was not affected by ARP2 knockdown . Consistent with the reduction in RSV production , we also observed reduced extracellular RSV particles on the cell surface by microscopy . TEM and immuno-SEM showed that there were many fewer virus-like particles and filaments on the surfaces of ARP2-knockdown infected cells compared with control-knockdown infected cells . The TEM studies suggested that , while fewer RSV progeny particles were present , their general morphology appeared unchanged compared to control-knockdown infected cells . However , analysis by immuno-SEM showed that , while the general morphology of progeny virions on the surface of ARP2-knockdown infected cells was similar to that of control-knockdown infected cells , their arrangement appeared to be less orderly . A contribution of actin filaments to RSV virion maturation and egress , and to the formation of viral filaments , has been reported previously [6 , 11 , 12 , 43 , 44] . A role of actin and myosin in the transport of the viral ribonucleoprotein ( RNP ) complex has also been described previously , and the role of the ARP2/3 complex in the terminal stage of virus budding and in the process of pinching off from the cell surface has been proposed [6] . Conversely , a previous study showed that F protein trafficking to the cell surface and assembly of RSV filaments appeared to take place even when cytoskeleton rearrangement is blocked by actin inhibitors [45] . Our results suggest that efficient RSV budding indeed depends on functional ARP2/3 nucleation and actin rearrangement . Perhaps our most interesting observation was that RSV infection in A549 cells induced filopodia , which are finger-like membrane protrusions that are rich in F-actin and mostly deficient in tubulin . Lamellipodia ( which are larger , broad , flat protrusions containing branched actin and tubulin ) also appeared to be formed in response to RSV infection , although this was not further investigated in the present study . Filopodium formation is a highly orchestrated multistep process associated with actin cytoskeleton rearrangement; however , the underlying mechanism for initiation and maintenance of filopodia has not been fully characterized [46] . Compared to RSV viral filaments , filopodia are larger in diameter and largely devoid of RSV proteins . We discriminated filopodia from lamellipodia based on the former’s thin extended morphology , low microtubulin content , and intensity of F-actin staining [47] . Membrane protrusions have been shown to serve as vehicles for pathogen spread for Listeria monocytogenes and Shigella flexneri . These bacteria induce actin motility when they enter into the cytosol , which facilitates bacterial interaction with the plasma membrane . Bacteria have been observed to be transported within cell membrane protrusions that extended into invaginations in adjoining cells , contributing to cell-to-cell spread , reviewed in [48] . In the present study , STED observations of RSV particles on A549 cells suggested that clustered filamentous RSV virions are present on filopodia , similar to filopodia-associated virions which were observed on cells infected with murine leukemia virus ( MLV ) [49] and African swine fever virus ( ASFV ) [50] . We also detected filamentous RSV virions clustered at the tips of filopodia , suggesting that mature virion particles are shuttled to neighboring uninfected cells . In the STED observations , immunostaining was done on permeabilized cells . This technique does not discriminate whether mature virions were located intracellularly or extracellularly on the plasma membrane . However , immuno-SEM performed using the same Ab in non-permeabilized infected cells showed that mature RSV virions are present extracellularly on the plasma membrane of filopodia , as might have been expected . These results suggest that RSV is shuttled on the exterior of the filopodia to infect neighboring cells . However , RSV potentially might also shuttle through filopodia using intracellular vesicle transport , which is currently under investigation . We found that expression of the RSV F protein from a plasmid vector or a heterologous viral vector was sufficient to induce filopodia , although the number and length of these structures were reduced compared to RSV-infected A549 cells . Our study identifies a new mechanism by which the F protein promotes RSV spread , namely thorough inducing filopodia formation , thus promoting filopodial spread of RSV . Filopodia induction in A549 cells due to the expression of RSV F from a vector also was sensitive to ARP2 depletion , similar to the filopodia induced by RSV infection . RSV-mediated filopodia induction was not observed in either Calu-3 or Vero cells , despite the evidence that ARP2 contributes to RSV production in both cell types ( Calu-3 cells , S1 Fig ) . Further research is necessary to understand whether cell-type specificity contributes to RSV-driven filopodia induction , whether the RSV F protein uses the actin network and filopodia for intra-and/or inter-cellular transport , whether F expression activates filopodia signaling pathways , and whether it acts as a nucleation promoting factor ( NPF ) for ARP2/3 complex-driven actin polymerization . We found that in addition to filopodia formation , RSV infection increased cellular motility , which promoted virus spread to neighboring cells . Virus-induced cell migration has been reported for vaccinia virus [51] . Live cell imaging in the infected Red-F-actin A549 cells illustrated that filopodia-driven RSV spread was facilitated by cell mobility , and ARP2 knockdown reduced RSV spread not only by inhibiting the induction of filopodia , but also by restricting cell mobility . Filopodia-driven cell-to-cell spread was much more robust for RSV compared to HPIV3 and HMPV . A recent study showed that HMPV induces intercellular extensions of a branched actin network for its spread in human bronchial epithelial cells . However , a branched actin network was less obvious in A549 and Vero cells [42] . Our study shows that RSV exploits the actin cytoskeletal system of human lung epithelial cells for its spread . If our findings are relevant to the situation in vivo , they suggest that RSV may promote cell motility , in particular in areas subject to RSV cytopathic effect . Our findings also suggest that RSV induces filopodia ( through the F protein ) , and that filopodial spread allows rapid dissemination of the virus to new target cells . Filopodia may also facilitate spread in the presence of biophysical barriers such as mucous layers on mucosal surfaces [52 , 53] , and spread under these conditions may reduce exposure to virus-neutralizing antibodies [53 , 54] . In future studies , we will further investigate the possibility of virus-induced filopodia formation and filopodia-driven viral spread in primary human airway epithelial cells . Filopodial protrusions can function as sensors of the local environment in migrating cells , and the spatial-temporal dynamics of filopodia have been described [55] . The ARP2/3 complex directly contributes to filopodia formation by nucleating actin filaments in lamellipodia [56] , but the ARP2/3 complex by itself has little actin-nucleating activity . NPF binding to the ARP2/3 complex activate actin-polymerization and dictate where and when nucleation originates in the cell [8] . Indeed , we showed that a depletion of another potent NPF , N-WASP , reduced or abolished the formation of RSV-induced filopodia , and consequently reduced RSV production in A549 cells . Results of the N-WASP depletion studies confirmed that RSV modulates filopodia signaling for its direct cell-to-cell spread . However , NPF activities are highly regulated by signal-transduction pathways such as Rho-family GTPases , Cdc42 and Rac ( reviewed in [29] ) . A contribution of the small GTPase RhoA , which acts through myosin II , and affects motility independently of the ARP2/3 complex , in RSV infection has already been reported [57]; future studies are required to explore the contributions of other small GTPases Rac1 and Cdc42 to filopodia-driven RSV spread . More work is needed to evaluate if the RSV F protein , and possibly other viral proteins , interact with ARP2/3 complex-specific NPFs to initiate ARP2/3 complex-driven actin polymerization . In conclusion , ARP2 knockdown reduced RSV production , primarily by reducing viral budding and spread . There was no evidence of effects on RSV entry , or of significant effects on events in the viral replicative cycle prior to approximately 24 hpi , a time when the production of progeny virions and virus spread becomes robust . Our results show that RSV infection increases cell motility , induces filopodia formation , that filopodia promote RSV spread , and that this occurred in human lung epithelial A549 cells . ARP2 , which is part of ARP2/3 complex , was shown to be necessary for induction of filopodia in RSV-infected cells , for increased motility of RSV-infected cells , and for filopodia-mediated spread of RSV . Filopodia formation and particle formation in A549 cells were also dependent on N-WASP , which binds to ARP2/3 and stimulates actin polymerization . ARP2 depletion reduced the spread of RSV infection in confluent monolayers in vitro . If these results translate to respiratory epithelium of the lower respiratory tract , this would suggest that inhibition of actin polymerization may reduce the spread of RSV infection in the lower respiratory tract . This identifies a previously unappreciated effect of the RSV F protein , a previously unappreciated mechanism of RSV spread , a novel cellular factor for RSV spread , a new insights on the effect of RSV infection on cellular functions , and a potential therapeutic target to combat RSV infection .
Calu-3 ( ATCC HTB-55 ) and A549 ( ATCC CCL-185 ) cells were obtained from the ATCC . Calu-3 cells were maintained in EMEM ( ATCC ) , supplemented with 10% fetal bovine serum ( FBS ) ( Life Technologies ) . A549 cells were maintained in F-12 complete medium [F-12 Nutrient Mixture ( Life Technologies ) , supplemented with 10% FBS and 1% L-glutamine] . Recombinant RSV expressing enhanced GFP from an added gene , inserted between the P and M genes ( RSV-GFP ) , was described previously [14] , and is a derivative of recombinant RSV-WT ( A2 strain ) . For all experiments , RSV was grown in Vero cells and purified by centrifugation and banding in discontinuous 30% to 60% ( wt/vol ) sucrose gradients as previously described [58] . Similarly , HPIV3-GFP [59] , HMPV-GFP [60] , and B/HPIV3-RSV-F were sucrose-purified . B/HPIV3-RSV-F is a chimeric B/HPIV3 based on BPIV3 in which the fusion ( F ) and hemagglutinin-neuraminidase ( HN ) surface glycoproteins have been replaced by their counterparts from HPIV3 , and which in addition expresses a codon-optimized version of the RSV F protein from an added gene [28] . Viral infections were done at an MOI of 1 unless specified otherwise . A549 cells were seeded in 24-well plates and were incubated with virus in 100 μl of F-12 medium for 1 hr , and rinsed twice with F-12 medium . Infected cells were incubated in F-12 medium with 2% FBS and 1% L-glutamine . For viral infections of Calu-3 cells , EMEM medium was used instead of F-12 medium . For live cell imaging , infected cells were kept in media with 25 mM HEPES ( Life Technologies ) . ARP2/KD-A549 cell line was generated by using a lentiviral vector-based construct expressing three target-specific 19–25 nt ( plus hairpin ) small hairpin RNAs designed to knockdown gene expression ( sc-29737-V , Santa Cruz Biotechnologies , Inc ) according to the manufacturer's recommendations with minor modifications . Briefly , 1x105 A549 cells were infected at a range of MOIs from 0 . 1 to 0 . 01 with 6 μg/ml polybrene ( Santa Cruz Biotechnologies , Inc ) and incubated in F-12 complete medium with 10 μg/ml puromycine ( Takara Clontech ) . A clonal population was obtained from a single cell . Similarly , we generated Red F-actin-A549 cells that stably express Red F-actin , which is a fusion protein that combines an actin-binding domain with a red fluorescent protein ( RFP ) ( Ibidi ) . This was created using the lentiviral vector-based construct rLVUbi-LifeAct-TagRFP ( Ibidi ) , and clonal population was obtained . siRNA transfections were done in 24-well plates using a reverse transfection protocol ( i . e . , cells in suspension were added to wells containing the siRNA and transfection reagent ) , which was optimized using the KDalert GAPDH Assay Kit ( Life Technologies ) . Briefly , the knockdown efficiency of different concentrations of siRNA ( siGAPDH ) and RNAimax transfection reagent ( Life Technologies ) was tested on 3x104 cells . GAPDH knockdown was determined by measuring the enzymatic activity according to the manufacturer’s instructions . Transfection with 1μl of RNAiMax transfection reagent and 7 . 5 μl of siRNA ( 2 μM siRNA concentration ) reduced GAPDH activity by more than 80% without compromising cell viability . Therefore , this concentration was used for protein knockdown experiments in A549 cells unless otherwise mentioned . In Calu-3 cells , protein knockdown was done similarly by using twice the amount of siRNA on 100 , 000 cells . Reverse transfections of A549 or Calu-3 cells were performed 48 hr before virus infection and siARP2 ( s223082 , Life Technologies ) and siControl ( Silencer Select Negative Control #2 , Life Technologies ) were used for all ARP2 knockdown experiments , unless otherwise specified . For N-WASP knockdown in A549 cells , siN-WASP ( 137397 , Life Technologies ) was used . Cell viability was evaluated using resazurin ( alamarBlue , Life Technologies ) according to the manufacturer's protocol . Briefly , a 10% volume of alamarBlue was added to the cell culture media and incubated at 37°C for 3–4 hr . To evaluate the cell viability , alamarBlue fluorescence , a marker for metabolic activity , was analyzed using a Synergy 2 Multi Mode microplate reader ( BioTeK ) . Ten micrograms of total protein was separated on 4–12% Bis-tris SDS polyacrylamide gels , followed by dry blot transfer onto polyvinylidene floride ( PVDF ) membranes according to the manufacturer’s instructions ( Life Technologies ) . For viral protein detection , samples were denatured at 90°C for 10 min with 1% reducing agent before gel electrophoresis . The PVDF membranes were incubated in LI-COR blocking buffer ( 1:1 in PBS ) ( LI-COR Biosciences ) for 1 hr , followed by overnight incubation with primary Ab in blocking buffer . The membranes were washed 4x for 5 min each in wash buffer ( PBS with 0 . 1% Tween 20 , Sigma-Aldrich ) , followed by incubation with secondary IRDye Ab ( LI-COR Biosciences ) for 1 hr . After washing 4x for 5 min in wash buffer , fluorescence was analyzed using the Odessey imaging system ( LI-COR Biosciences ) . ARP2 was detected using a rabbit mAb ( ab129018 , Abcam ) and a goat anti-rabbit IRDye800 Ab ( LI-COR Biosciences ) . Alpha-tubulin was detected using a mouse mAb ( T6199 , Sigma-Aldrich ) and a goat anti-mouse IRDye680 ( LI-COR Biosciences ) . N-WASP was detected using a rabbit mAb ( 30D10 , Cell Signaling Technologies ) and a goat anti-rabbit IRDye800 Ab . RSV F and P were detected using mouse mAbs ( ab43812 and ab94965 , respectively ) for primary and respective secondary Abs ( goat anti-mouse IRDye800 and IRDye680 , respectively ) . GAPDH was detected using a primary rabbit pAb ( sc25778 , Santa Cruz Biotechnologies , Inc . ) and a goat anti-rabbit IRDye680 Ab . Cells were harvested and intracellular RNA was purified by RNAeasy ( QIAGEN ) . The RNA concentration was determined , and 100 ng of RNA was used for first-strand cDNA synthesis ( Superscript III , Life Technologies ) using oligo ( dT ) primers ( Life Technologies ) . qRT-PCR was performed using TaqMan assays ( ARP2: Hs00855199 , Life Technologies and RSV ORFs: primer sequence available upon request ) in the 7900HT Fast Real-Time PCR System ( Life Technologies ) , and fold changes were calculated using the delta-delta-CT method using 18S RNA ( Hs99999901 , Life Technologies ) or beta-actin ( Hs99999903 , Life Technologies ) as a calibrator . For quantitation of free RSV , the cell culture medium was harvested without disturbing the cells and then clarified at 300 x g for 10 min . For quantification of total RSV in the culture ( cell-associated and free virus ) , monolayers were scraped into the cell culture medium , collected and vortexed vigorously three times for ten seconds each , and clarified by centrifugation at 300 x g for 5 min . Supernatants were snap frozen on dry-ice and kept at -80°C . RSV titration was done by plaque assay on Vero cells in which viral plaques were visualized by immunostaining with three mAbs against RSV F [61] . Alternatively , in the case of RSV-GFP and HPIV3-GFP , plaques were visualized directly by GFP expression using a Typhoon Trio+ imager ( GE Healthcare ) , followed by quantification using the software ImageJ . Cells were washed in PBS and detached with 0 . 5% TrypLE Select 1x ( Life Technologies ) , incubated with FBS to neutralize trypsin activity , pelleted by centrifugation at 300 x g for 5 min , and incubated with Live/Dead near-infrared fluorescence reactive dye ( Life Technologies ) for 30 min . Cells were washed and fixed and permeabilized with BD Cytofix/Cytoperm buffer ( BD Biosciences ) . Multicolor flow cytometry was used to analyze the expression of the RSV F and M2-1 proteins and GFP simultaneously in infected cells . To detect RSV F , we used a commercial biotin-labeled mAb ( 133-1H MAB8262B-5RSV , Millipore ) , and Strepatvidin BV605 ( Biolegend ) . For RSV M2-1 , we labeled an M2-1 specific mAb ( RSV5H5 , Abcam ) with AlexaFluor647 or Site-Click R-PE antibody labeling kit ( Life Technologies ) according to the manufacturer’s instruction . Single color antibody labeled cells for each antibody were used for compensation , and fluorescence minus one controls were included to aid in setting gates . Acquisition was done until 20 , 000 live single cells were recorded and cells were gated on live singlet cells , followed by gating on GFP , F and M2-1 positive cells using a BD LSR Fortessa flow cytometer and FACSDiva software ( BD Biosciences ) , followed by analysis with FlowJo software version 9 . 7 . 2 ( FLOWJO , LLC ) . 5x105 cells per well in 6-well plates were treated for 1 hr with a final concentration of 100μM of the entry inhibitors EIPA or CK-666 ( Sigma-Aldrich ) [17 , 18] for 1 hr . Optimal , non-toxic concentrations were determined in preliminary experiments . The inhibitors were made as concentrated stock solutions in DMSO , and the final DMSO concentrations in the cultures were 1% or less , and a DMSO ( solvent-only ) control was included . Following the 1 hr incubation , cells were infected with RSV-GFP at an MOI of 5 , or with a comparable amount of UV-inactivated RSV-GFP for 6 or 12 hr , or was incubated with dextran fluorescein ( Life Technologies ) for 30 min . Plates were transferred onto wet ice to stop entry . To quantify dextran or RSV internalization by flow cytometry , cells were detached , incubated with Live/Dead near-infrared fluorescence reactive dye , and fixed with BD Cytofix/Cytoperm buffer . Dextran fluorescein or GFP single live positive cells were quantified using the FACS CANTO II ( BD Biosciences ) . FACS data analysis was done using FlowJo software version 9 . 7 . 2 . UV-inactivated RSV-GFP was prepared using a Stratalinker UV cross-linker ( Agilent ) at 0 . 5 J/cm2 . Complete inactivation was evaluated by plaque assay as described previously [62] . Infected cells grown on coverslips were fixed , permeabilized , and stained with DAPI and rhodamine phalloidin as described for confocal microscopy , and images for quantification were acquired using a Leica DMI6000 inverted wide-field microscope equipped with DAPI , GFP , and rhodamine filter cubes , a HCX Pl Fluotar 20X/0 . 4 objective , and a DFC 360FX monochrome camera . The entire coverslip was imaged in a single plane using the tiling and predictive focus features in LAS software . Using Imaris image processing software , large regions of the tiled image were selected; nuclei were automatically counted using the “Spot” feature , while syncytia and infected cells were counted manually . Per coverslip , at least 5000 cells were analyzed . Syncytia were counted when they contained ≥3 nuclei and were GFP-positive ( which was the case for the vast majority of syncytia ) . For TEM , A549 cells were seeded on Thermanox coverslips ( Electron Microscopy Sciences ) at a density of 3x104 cells per well and reverse transfected with siRNA for 48 hr , followed by infection with RSV-GFP at an MOI of 1 for 24 hr . Cells were fixed with 2 . 5% glutaraldehyde in Sorensen’s phosphate buffer ( Electron Microscopy Sciences ) . Samples were post-fixed 1 hr with 0 . 5% osmium tetroxide/0 . 8% potassium ferricyanide , 1 hr with 1% tannic acid and overnight with 1% uranyl acetate at 4C . Samples were dehydrated with a graded ethanol series , and embedded in Spurr’s resin . Thin sections were cut with a Leica UCT ultramicrotome ( Vienna ) stained with 1% uranyl acetate and Reynold’s lead citrate prior to viewing at 120 kV on a FEI Tecnai BT Spirit transmission electron microscope ( Hillsboro , OR ) . Digital images were acquired with an AMT digital camera system ( AMT ) and processed using Adobe Photoshop CS5 ( Adobe Systems Inc ) . For immuno-SEM , A549 cells were seeded on silicon chips ( Ted Pella Inc . ) at a density of 3x104 cells per well were first reverse transfected with siRNA for 48 hr , followed by infection with either RSV-GFP at an MOI of 5 for 24 hr . Cells were fixed with 4% PFA in PBS for 30 min , blocked with 3% bovine serum albumin ( BSA ) in PBS , incubated with mAb ( ab43812 ) ( 1:100 dilution in 0 . 1% BSA ) overnight at 4°C , washed with 3% BSA in PBS , followed by incubation with secondary Ab ( goat anti-mouse conjugated with 15nm colloidal gold particle ) ( EM . GMHL15 , BBInternational ) . After immuno-labeling , the specimens were fixed with 2 . 5% glutaraldehyde in Sorensen’s phosphate buffer overnight at 4C , post-fixed for 1 hr with 1% osmium tetroxide , and dehydrated in a graded ethanol series . The samples were critical-point dried under CO2 in a Bal-Tec model cpd 030 dryer ( Balzers ) , mounted on aluminum studs , and sputter coated with 35 angstroms of chromium in a model IBS/TM200S ion beam sputterer ( South Bay Technologies ) . Specimens were viewed at 10 kV in a Hitachi SU-8000 field emission SEM ( Hitachi ) using mixed backscatter and secondary imaging modes . A549 cells were seeded onto cover glasses ( Deckglaser ) at a density of 3x104 cells per well and reverse transfected with siRNA for 48 hr , followed by infection with RSV-WT at an MOI of 1 . At 24 hpi , cells were washed with PBS , fixed with 4% PFA ( Polysciences , Inc . ) in PBS for 10 min at room temp , permeabilized with 0 . 5% Triton-X100 ( Sigma-Aldrich ) for 10 min and then blocked with 3% BSA solution in PBS for 2 hr . Cells were then incubated with primary Abs ( mouse mAb , ab43812 for RSV F protein and rabbit mAb , 9F3 , Cell Signaling Technology , Inc . ) ( diluted 1:500 and 1:100 , respectively in 0 . 1% BSA solution ) overnight at 4°C , followed by incubation with secondary Abs ( anti-mouse AlexaFlour488 or anti-rabbit AlexaFlour647 ) ( diluted 1:200 in 0 . 1% BSA solution ) for 2 hr at 4°C , followed by incubation with rhodamine phalloidin ( diluted 1:500 ) ( Cytoskeleton Inc ) for 30 min at room temp in the dark , followed by nuclear staining with NucBlue Fixed cell Stain ReadyProbes ( Life Technologies ) for 20 min in the dark . Coverslips were washed with PBS before mounting on microscope slides ( Scientific Device Laboratory ) using ProLong Gold anti-fade mounting media ( Life Technologies ) . Confocal images were collected using a Leica SP8 confocal microscope ( Leica Microsystems ) enabled with 63X/1 . 4NA and 40X/1 . 25NA oil immersion objectives and hybrid ( HyD ) detectors . To visualize the details of finer structures such as fiolopodia and lamellipodia , Z stack slices of 0 . 3 to 0 . 5 μm were collected and random fields of the cover slip were acquired with automated tiling methods to get an unbiased data set of approximately 50 to 100 random fields of interest . Some confocal images were subsequently deconvolved using Huygens software ( Scientific volume imaging ) to improve resolution . Filopodial structures were discriminated from lamellipodia using microtubulin Ab staining ( absent in filopodia ) , and number of filopodia and their length was quantified using the “Measurepoint” module in Imaris image analysis software . Data was averaged from two independent experiments . RSV-WT infected A549 or ARP2/KD-A549 cells were seeded onto coverslips and fixed and permeabilized at 24 hpi as described above . Cells were stained with a mAb specific for RSV-F and secondary goat anti-mouse AlexaFlour488 or AlexaFlour647 . F-actin was stained with rhodamine phalloidin . Images were collected on a Leica TCS SP8 STED 3X system equipped with a white light excitation laser , 600 nm and 775 nm depletion lasers , HC PL APO 100x/1 . 40 oil STED White objective , and gated HyD detectors . Images were further deconvolved using Huygens de-convolution software . Ibiditreat ( Ibidi ) 8-well chambers were used to seed Red F-actin-A549 cells or parental A549 cells at a density of 3 , 000 per chamber and incubated at 37°C overnight . Cells were infected with RSV-GFP , HPIV3-GFP or HMPV-GFP at an MOI of 0 . 1 unless mentioned otherwise for 1 hr at 37°C . Monolayers were washed 2x with only F-12 medium and incubated at 37°C in F-12 medium with 2% FBS and 1% L-glutamine and 25 mM HEPES for 24 hr prior to live cell imaging . For ARP2 knockdown , reverse transfections with siRNA were done 48 hr prior to infection with RSV-GFP in F-12 medium with 2% FBS , 1% L-glutamine and 25mM HEPES . Time-lapse images were acquired on an inverted Leica SP5 confocal equipped with a Ludin environmental chamber set to 37°C with 5% CO2 , a HCX Pl APO 63X/1 . 4 oil objective , PMT detectors , Argon 488nm and DPSS 561nm lasers , and a motorized stage to enable the mark-and-find module in LAS AF software . Imaging started at 24 hpi and images were acquired approximately every 6 minutes , unless otherwise mentioned . Three randomly selected locations were imaged per sample per experiment . For imaging of the Red F-actin-A549 confluent monolayer , 30 , 000 cells were used and ARP2 knockdown was done with siARP2 for 48 hr before RSV-GFP infection ( MOI = 0 . 1 ) . To visualize nuclei in live cells , just before the start of the imaging , 50 nM Sir-DNA ( Cytoskeleton Inc . ) was added to the medium according to the manufacturer’s instructions . Imaging started at 24 hpi , and images were collected every 5 minutes for 24 hr on a Leica SP5 inverted confocal microscope equipped with a 63X/1 . 4NA objective , hybrid HyD detectors , an environmental chamber with CO2 , and argon 488nm , DPSS 561nm , and HeNe 633nm lasers . Three randomly selected locations were imaged per sample . Reverse transfection with siARP2/ siControl was done for 48 hr on 30 , 000 Red F-actin-A549 cells in Ibiditreat 8-well chambers . Cells were mock-infected or infected with RSV-GFP at an MOI of 1 for 1 hr at 37°C . Monolayers were washed 2x with F-12 medium and incubated at 37°C in F-12 medium with 2% FBS and 1% L-glutamine and 25 mM HEPES for 24 hr . Cell monolayers were scratched with a 20 μl pipette tip ( Molecular BioProducts ) , followed by imaging every 5 min for 12 hr on a Leica DMI6000 inverted wide-field microscope equipped with a 10x/0 . 4NA objective , a pco . edge sCMOS camera , adaptive focus control , and an environmental chamber with CO2 . Three randomly selected locations were imaged per sample . Cell migration was measured by quantifying the intensity of Red F-actin in the scratch , normalized to the intensity of Red F-actin in the field at each time point using the image analysis software Imaris ( Bitplane ) . | RSV is the most common viral cause of severe acute pediatric lower respiratory tract illness , including pneumonia and bronchiolitis , in young children worldwide . In a genome-wide siRNA screen in human lung epithelial A549 cells infected with RSV expressing green fluorescent protein ( RSV-GFP ) , we identified ARP2 as a cellular factor with a role in the RSV replicative cycle . ARP2 is part of the actin-related protein 2/3 ( ARP2/3 ) complex , which contributes to cell shape and motility through its role in actin polymerization . ARP2 depletion reduced the production and spread of RSV in human lung epithelial cell cultures , with the most noticeable effects at late time points after RSV infection . RSV infection induced the formation of slender actin-rich cell protrusions , called filopodia and increased cell motility . Filopodia formation and cell motility were inhibited by ARP2 knockdown . The filopodia appeared to shuttle RSV to neighboring cells , facilitating virus spread . Thus , RSV uses two previously unrecognized ARP2 dependent features to facilitate viral spread , namely cell motility and filopodia formation . | [
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] | 2016 | Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread |
L1 retrotransposons have a prominent role in reshaping mammalian genomes . To replicate , the L1 ribonucleoprotein particle ( RNP ) first uses its endonuclease ( EN ) to nick the genomic DNA . The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly ( A ) tail , a process known as target-primed reverse transcription ( TPRT ) . Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence ( degenerate 5′-TTTT/A-3′ sites ) and frequently preceded by imperfect T-tracts . However , it is currently unclear whether—and to which degree—the liberated 3′-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly ( A ) tail of the L1 RNA for efficient priming of reverse transcription . Here , we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process . First , efficient priming is detected with as few as 4 matching nucleotides at the primer 3′ end . Second , L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides . All terminal mismatches are not equally detrimental to DNA extension , a C being extended at higher levels than an A or a G . Third , efficient priming in the context of duplex DNA requires a 3′ overhang . This suggests the possible existence of additional DNA processing steps , which generate a single-stranded 3′ end to allow L1 reverse transcription . Based on these data we propose that the specificity of L1 reverse transcription initiation contributes , together with the specificity of the initial EN cleavage , to the distribution of new L1 insertions within the human genome .
Retrotransposons are highly repetitive and dispersed sequences , accounting for almost half of our DNA [1] . These elements have the ability to proliferate in genomes through an RNA-mediated copy-and-paste mechanism , called retrotransposition . LINE-1 ( L1 ) elements are the only autonomously active elements in humans and one of the most active elements in mice . They belong to the broad family of non-LTR retrotransposons ( see [2]–[6] for recent reviews ) . L1 retrotransposition starts with the transcription of a 6 kb L1 RNA driven by an internal Pol-II promoter [7] . After its export to the cytoplasm , the bicistronic L1 mRNA is translated into two proteins ( ORF1p and ORF2p ) , which associate preferentially in cis with their encoding mRNA [8]–[11] . This is a critical feature of the L1 replication mechanism since it limits the association of the L1 machinery with other cellular mRNAs , including defective L1 RNA sequences , and thus increases the specificity of the reverse transcription process . The resulting complex is a stable ribonucleoprotein ( RNP ) thought to form the core of the retrotransposition machinery [10] , [12]–[19] . Its precise composition is currently unknown but it contains at least the L1 RNA and the ORF1p and ORF2p proteins [10] , [16] , [18] , [19] . The ORF1p protein is a trimeric RNA binding protein with RNA chaperone activity [20]–[25] and the ORF2p protein shows endonuclease ( EN ) and reverse transcriptase ( RT ) activities [26] , [27] . All are essential to L1 retrotransposition [16] , [18] , [28] , [29] . The L1 RNP is imported into the nucleus where reverse transcription and integration into the host genome take place [30] . The current model for non-LTR retrotransposon integration , named target-primed reverse transcription ( TPRT ) , was originally deduced from biochemical studies on the insect R2Bm element [31] . This retrotransposon encodes a single protein with EN and RT activities and integration of new copies occurs at a specific and defined position in the rDNA [31] , [32] . The TPRT process is initiated by the formation of a nick in the genomic double-stranded DNA target . Then the R2 RT extends the newly formed 3′OH using the R2 RNA as a template [27] , [31] , [33]–[35] . Priming of reverse transcription occurs without any complementarity between the R2 RNA template and the DNA target site [36] , [37] . Non-LTR retrotransposons can be divided into several clades , which differ considerably in the machinery that they encode ( single or multiple ORFs , restriction-like or APE-endonuclease , RNaseH or not , etc… ) [38] . Despite these differences , cell culture-based retrotransposition assays and analyses of novel or recent integration sites have revealed the same overall requirement for EN and RT activities , supporting the TPRT model [28] , [39]–[43] . Intriguingly , non-LTR retrotransposon 3′ ends and preintegration sites often exhibit partial sequence identity , suggesting that annealing of the target site DNA to the RNA template might be a necessary step to prime reverse transcription , in contrast to R2 [40]–[43] . This step could significantly influence the genomic distribution of these elements , by imposing additional constraints after the initial endonuclease cleavage . As regards L1 , conclusive evidence on whether primer-template complementarities are required for efficient reverse transcription initiation is lacking . Most L1 pre-integration sites contain an EN recognition sequence ( 5′-TTTT/A-3′ ) and are often preceded by T-tracts of variable length [1] , [27] , [44]–[50] . Thus , in theory , the region covering the EN consensus and its upstream sequence has the ability to base-pair with the L1 poly ( A ) tail and to promote reverse transcription initiation . Nevertheless , target sites frequently contain nucleotides other than Ts , sometimes at the 3′ terminal end of the nicked DNA , which could severely impair interaction with the L1 RNA and extension by L1 RT . On the other hand , isolated recombinant L1 ORF2p produced in insect cells was found to equally extend any linear DNA substrate in vitro , without apparent sequence or structure requirement , or any need for primer-template complementarity [33] . Likewise , native L1 RNPs enriched from cells are able to extend oligonucleotides ending with terminal mismatches [10] , [51] , indicating that complementarity base-pairing between the 3′ end of the target DNA and the L1 RNA template is not an absolute requirement . But Kulpa and Moran also observed that primer sequence could influence RT initiation [10] . A common limitation of these previous studies was the use of PCR-based assays , which precluded a quantitative comparison of priming efficiencies and might lead to the detection of marginal products . Here , we addressed the question whether - and to which degree - the liberated 3′-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly ( A ) tail of the L1 RNA for efficient priming of reverse transcription . To achieve this goal , we validated a direct L1 extension assay ( DLEA ) to quantitatively measure the ability of native L1 RNPs to initiate reverse transcription . Then we systematically assayed more than 65 DNA substrates varying in sequence and structure , allowing us to define the preferential rules of L1 reverse transcription priming . Our results clarify the importance of base-pairing between the L1 RNA template and the target site DNA for this process and demonstrate its exceptional flexibility .
To test the DNA primer requirements for initiating L1 reverse transcription , we set up a direct L1 extension assay ( DLEA ) , which would avoid PCR and therefore would allow us to quantitate L1 priming efficiencies . The L1 retrotransposition machinery is notoriously difficult to express and to detect in most experimental systems . To obtain sufficient amounts of L1 RNPs for direct detection , we modified the protocol developed by Kulpa and Moran [10] by transiently overexpressing the canonical human L1 . 3 element [28] ( referred thereafter as hL1 ) or a codon-optimized murine L1spa element ( Orfeus [52] , referred thereafter as mL1 ) in HEK293T cells , followed by a 3-day selection of transfected cells . HEK293T cells are transfected with much higher efficiency and express higher levels of transgenes than the HeLa cells , which were used in the original protocol . Then we prepared native L1 RNPs from cell extracts by sucrose cushion ultracentrifugation as previously reported ( Figure 1A ) [10] . In parallel , we prepared RNPs from empty vector-transfected cells or with a point mutation in the RT active site ( D702A for hL1 and D709A for mL1 , referred thereafter as RT* L1 ) as negative controls . We detected the mORF1p protein in RNP preparation from mL1-transfected cells but not from hL1 or empty vector-transfected cells by immunoblotting ( Figure 1B , compare lanes 1–3 with 4–5 ) . Similarly hORF1p levels were much higher in hL1-transfected cells than in vector control cells ( Figure 1B , lanes 2–3 ) . However long exposure revealed low levels of endogenous hORF1p in all RNP preparations ( Figure 1B , lanes 1 and 4–5 ) . To evaluate the presence of L1 RT activity and L1 RNA associated with ORF1p in the RNP preparations , we used the L1 element amplification protocol ( LEAP ) in which the L1 RT first extends a primer and the resulting cDNA is subsequently amplified by PCR [10] . The PCR primers are anchored in the tail of the RT primer and in the Neomycin-resistance genetic marker inserted in the transfected L1 3′ UTR . Therefore only products produced from the transfected L1 element can be amplified . Since hL1 and mL1 share the same genetic marker , the same primers can be used for both elements . As expected from previous work [10] , [18] , we detected L1 RT activity only in the RNP prepared from wild-type hL1 or mL1 , but not in the vector or RT-defective L1 transfected cells ( Figure 1C , top panel , compare lanes 5 and 7 with 3–4 and 6 ) , even if the L1 RNA is present ( Figure 1C , middle panel ) . Sequencing of the LEAP products confirmed that hL1 or mL1 RNA was reverse transcribed . This indicated that RNPs produced in our experimental conditions contain the core of the L1 machinery and used L1 RNA as a template . Previous studies have shown that L1 RNPs enriched on sucrose cushion as prepared here co-fractionate with many other cellular RNPs , including ribosomes [10] , [16] . However , the L1 RNA is reverse transcribed at least 100 times more efficiently than other co-fractionating abundant cellular RNAs [10] , a property known as L1 cis-preference [8] , [9] . We reasoned that if L1 RNPs were active enough we should detect the extension of an oligo ( dT ) 18 primer in the presence of radiolabelled 32P-dTTP . This reaction would mimic the initiation step of L1 reverse transcription , which starts at the poly ( A ) tail of the L1 RNA . After a 4 min incubation at 37°C , we purified the reaction products and resolved them on sequencing gels . A short end-labeled oligonucleotide was added after the reaction as a recovery control ( RC ) . No or minimal extension was detected in vector or RT-defective controls consistent with the presence of only minimal amounts of endogenous hL1 activity in RNP preparations ( Figure 1E , lanes 3–6 and 9–10 , and Figure 1D ) . In contrast when wild-type hL1 or mL1 element was transfected we could easily detect the incorporation of radiolabelled dTMPs ( Figure 1D and Figure 1E , lanes 8 and 12 ) . Importantly , the amount of product formed was linearly dependent on the amount of L1 RNPs ( Figure 1D ) , showing that the levels of primer extension could be quantitatively measured under the reaction conditions employed ( linear phase , also known as initial velocity phase ) . We focused our work on reverse transcription initiation by using short extension times ( 4 min ) and by adding only 32P-dTTP to the reaction and no other dNTP . In these experimental conditions , the products were short enough to be resolved on sequencing gels and we could follow the extension at the nucleotide resolution . The linear phase ranged from 0 . 2–0 . 25 µg up to 4 µg of RNPs , which indicates a dynamic range between 10- and 20-fold ( data not shown ) . We chose to use 2 µg of RNPs , at the upper end of the linear range , for all following experiments and to set to 100% the level of extension obtained with an oligo ( dT ) 18 primer under these conditions . Based on the dynamic range of the initial RNP titration , primer extension efficiencies as low as 5% should therefore be reliably quantified . The products are heterogeneous in length , consistent with the expected products of poly ( A ) reverse transcription and range from 19 nucleotides ( nt ) to approximately 150 nt ( Figure 1E , lanes 8 and 12 ) . To further confirm that the ladder observed results directly from the reverse transcriptase activity of the transfected L1 element , we performed additional controls . RNase treatment reduced primer extension to undetectable levels ( Figure S1A , compare lanes 2 and 3 ) , showing that the detected DNA polymerase activity is RNA-dependent . If the reaction is conducted in the presence of RT inhibitors known to inhibit L1 retrotransposition and recombinant L1 RT activity [53]–[55] such as AZT or d4T , DNA polymerization is abolished ( Figure S1B , compare lanes 2 and 3–4 ) . No extension was detected in these experimental conditions with radiolabelled dATP , dGTP or dCTP in agreement with the reverse transcription of the poly ( A ) sequence ( data not shown ) . When extension time was prolonged to 1 h ( Figure S1C ) , the reaction was not in its linear phase anymore ( and the assay was no longer quantitative ) . Products were longer than the maximum poly ( A ) length in mammals ( ∼250 nt ) , which is likely to result from L1 RT slippage in the poly ( A ) track as recently reported in vivo [56] . If all four dNTPs were present in the reaction , high molecular weight products appeared , consistent with reverse transcription ongoing beyond the L1-poly ( A ) boundary ( Figure S1D ) and in agreement with the LEAP results ( Figure 1C ) . Altogether these results show that DLEA detects bona fide initiation of reverse transcription by native mammalian L1 RNPs through the direct incorporation of radiolabeled dTMP in a primer extension reaction . Importantly , DLEA is quantitative since it demonstrates a linear relationship between the signal and RNP quantities under the reaction conditions employed . In contrast to most DNA polymerases , it was previously demonstrated that the hL1 RNP is able to extend a terminal mismatched base pair using a PCR-based assay followed by sequencing of the products [10] . To determine more quantitatively the efficiency of extension of such mismatched primers , we changed the last nucleotides of the oligo ( dT ) 18 primer to a non-T nucleotide in order to prevent base-pairing of the primer 3′ end to the L1 poly ( A ) tail ( Figure 2A ) . Although decreased as compared to the oligo ( dT ) 18 primer , the hL1 RNP can extend a primer with a single or double terminal mismatch ( V1 and V2 , Figure 2B , lanes 3–4; V = not T ) or with a mismatch at the penultimate position ( VN , 15% of the oligo ( dT ) 18 extension , not shown ) , in agreement with previous reports [10] , [51] . In contrast , if the primer ends with more than two mismatched nucleotides ( V3 to V6 ) , DNA polymerization becomes undetectable under the employed reaction conditions ( Figure 2B , lanes 5–7 ) . Similarly , the hL1 RNP is not able to efficiently use an unrelated oligonucleotide ending with three Gs ( the T7 promoter primer , noted R , Figure 2A ) as a primer for its reverse transcription ( Figure 2B , lane 8 ) . Next , we measured the influence of each individual terminal base on primer extension . Although all terminal mismatches reduced the efficiency of reverse transcription initiation to some extent , a terminal G was the most detrimental , whereas a C or an A was better tolerated ( Figure 3 ) . Thus the levels of extension of a T-tract is dependent on the nature of its 3′ terminal base with the following preference: T>C>A>G . To further characterize the need for terminal matching nucleotides in the priming of hL1 reverse transcription , we added an increasing number of Ts to the R primer ( T1 to T6 ) . Initiation of reverse transcription is robustly detected only when the single-stranded primer ends with at least 4 Ts and trace activity can already be detected with 3 terminal Ts ( Figure 2B , lanes 11–13 ) . We obtained similar results with mL1 RNPs ( Figure 2C , lanes 1–7 and Figure S2 ) . In order to compare the properties of the native L1 RNPs with a retroviral RT , we tested the ability of recombinant Avian Myeloblastosis Virus ( AMV ) RT to prime reverse transcription under identical experimental conditions . In these experiments , exogenous poly ( rA ) was added as a template together with quantities of the AMV RT that lead to similar levels of extension as the L1 RNP using the ( dT ) 18 primer ( Figure 2C , compare lanes 2 and 9 ) . Under these experimental conditions , reverse transcription by AMV RT was not primed by oligonucleotides ending with terminal mismatches ( Figure 2C , compare lanes 4–5 to 11–12 ) or by oligonucleotides ending with 4 or 6 Ts ( Figure 2C , compare lanes 6–7 to 13–14 ) . These observations suggest that limited base-pairing interactions between the primer and the template might be stabilized by the L1 RNP , through direct binding of ORF1p or ORF2p to the single-stranded DNA . In addition , the extension products of the ( dT ) 18 oligonucleotide obtained with the AMV RT are much shorter than those obtained with the L1 RNP . This might suggest that the L1 RNP is more processive than the AMV RT and/or that the L1 RNP has a higher affinity for dTTP than AMV RT as shown for the R2 element [57] , [58] . However , since the templates used are not strictly similar , it is difficult to draw definitive conclusions on this aspect . It was previously reported that a nuclease activity in the RNP preparations could process primers before their extension [51] . Thus , in principle , it is possible that primers ending with terminal mismatches are first processed to eliminate the mismatch ( es ) and then extended . Against this possibility , the majority of the products observed in sequencing gels start at the expected +1 position or above ( Figure 2 and Figure S2 ) . As an additional control , we performed LEAP reactions using primers ending with the same sequence as depicted in Figure 2A . We could amplify , clone and sequence products with up to 3 terminal mismatches ( Figure S3A ) . Although a small percentage of processed primers were found ( 7 out of 160 sequences in total ) , the majority of the mismatches were directly extended ( Figure S3C ) . Thus differences of extension are not due to differential processing of the primers . We note that the levels of the nuclease activity responsible for primer processing , which co-fractionates with L1 RNPs in sucrose gradients , might dependent on the cell type used to prepare RNPs . Using the same RACE primer ending with VN , Kulpa et al . observed processing in 33/81 ( 39% ) of the analyzed clones obtained with HeLa cells , while Kopera et al . found 5/45 ( 11% ) of processed primers in CHO-derived cell lines . In comparison , we obtained 2/70 ( 3% ) clones showing a processed primer with RNPs prepared from HEK293T cells . Altogether these observations show that native L1 RNPs efficiently prime reverse transcription at DNA ending with 4–6 terminal matching nucleotides , although it can accommodate terminal mismatches with lower priming efficiencies . L1 EN-mediated nicking at a consensus target site produces a 3′-OH DNA ending with four Ts [27] , [44] . This is consistent with our observation that the L1 RT can extend primers ending with as little as four Ts . However , L1 integration sites often contain degenerate L1 EN recognition sites that differ from the consensus recognition sequence [1] , [46] , [47] . This prompted us to analyze the ability of native hL1 RNPs to extend primers which mimic bona fide insertion sites . We designed 35 primers corresponding to previously published insertion sites recovered from new hL1 retrotransposition events obtained in cultured cells [46] . The sequence and the original name of each recovered clone is indicated in Figure 4A . Levels of extension were normalized to those obtained with the primer LOU541 ( clone 10BglIIL1 . 3 ) , which corresponds to a ( dT ) 20 oligonucleotide . We observed that all sites are not equally extended ( see Figure 4A ) . The levels of extension range between 7% ( LOU535 ) and 120% ( LOU552 ) . The best primer is 17-fold more extended than the least-efficient primer . Even if we know that these target sites were used in vivo without processing [46] , we choose six of them differing from each other by the position or the nature of the mismatched nucleotides to perform LEAP ( Figure S3B ) and we sequenced the products . Again we found a small number of processed primers ( ∼5% ) , but the majority of products result from the direct extension of mismatched primers ( Figure S3 ) . We categorized primers based on their potential of extension ( Figure 4A; 0–40% , light red; 40–80% , medium red; 80–120% , dark red ) . Four primers have the ability to form stable hairpins ( Figure 4A , white bars ) , and were excluded from further analyses since hairpin formation is dependent on primer length , which was arbitrarily chosen ( the specific impact of primer structure on L1 RT initiation is presented at the end of the ‘Results’ section ) . Top ranking primers ( dark reds ) all end with at least 4 Ts , often more , and are extremely rich in Ts , in agreement with the results presented in Figure 2 . Interestingly , primers with a mismatch in the last critical four nucleotides are more efficiently extended if they are preceded by a T-rich upstream sequence . For example , primers LOU525 , LOU527 and LOU538 all end with 5′-TTTC-3′ and their respective levels of extension are LOU527<LOU538<LOU525 , which roughly follows the number of Ts close to the 3′ end . This suggests a compensation mechanism allowing the extension of primers ending with suboptimal sequences . To address the significance of this phenomenon more quantitatively , we calculated for each oligonucleotide two parameters: ( i ) the density of Ts ( number of Ts/length of the oligonucleotide ) , which simply reflects the abundance of Ts in the primer , and ( ii ) the position-weighted T-density , which is similar but the weight of each T is inversely proportional to the distance from the 3′ end ( see Material and Methods section for more details ) . Using linear regression , we found that the activity correlates significantly with both parameters ( p = 0 . 0002 and p<0 . 0001 , respectively ) but the goodness-of-fit is much better with the position-weighted T-density than with the T-density ( R2 = 0 . 7895 vs 0 . 3950 , not shown ) . To evaluate the number of terminal nucleotides that contribute to priming efficiency , we further correlated the priming efficiency with position-weighted T-density , taking into account a variable number of terminal nucleotides . The goodness-of-fit ( R2 ) increases steadily up to 10 considered nucleotides and then reaches a plateau ( Figure 4B ) . Considering nucleotides beyond position 10 ( from the 3′ primer end ) does not improve the correlation . The correlation between priming efficiency and the position-weighted T-density when only the last 10 nucleotides are considered is plotted in Figure 4C ( R2 = 0 . 8276 ) . In conclusion , we have demonstrated biochemically that complementarity between the L1 poly ( A ) tail and the last 10 nucleotides of the target DNA plays a role in extension at the target site , the last 4 nucleotides being the most critical . Suboptimal primers with a mismatch in their last 4 nucleotides are extended with a lower efficiency , which can be partially compensated by increasing the number of Ts in the upstream sequence . To illustrate these findings , we propose that the four terminal bases of the primer , which overlap with the EN nuclease recognition sequence , act as a specific snap and the upstream six bases act as a weaker velcro strap ( Figure 5A ) . When the snap is closed ( perfect terminal matches , EN consensus sequence ) , initiation is efficient , but is enhanced if the velcro strap ( upstream bases ) is also tightly fastened . Inversely , if the snap is open ( terminal mismatches ) , extension occurs preferentially if this is compensated by a tightly fastened velcro strap . The rational to distinguish snap and velcro regions is to highlight the preponderant role of the terminal nucleotides , which is also reflected in the position-weighted T-density mode of calculation . To test this model , we determined for each primer whether the snap is open or closed and whether the velcro strap is loosely or tightly fastened . A snap was considered closed only if the 3′ end of the primer was ( T ) 4 . The velcro strap was considered as tightly fastened if the position-weighted T-density score of this region was at least half of its maximum value ( see Materials and Methods section for the precise definition of these states ) . Then for each group we calculated the mean efficiency of extension by the hL1 RNP ( Figure 5B , data from Figure 4A ) . In agreement with the model , tightly fastened velcro improves the extension of target sites with a snap closed and partially rescue those with a snap open . Both snap and velcro contribute extremely significantly to the differences of extension between primers ( p<0 . 0001 , two-way ANOVA ) . A testable prediction of this model is that , in vivo , at the genomic level , L1 elements would more frequently insert at putative EN recognition sites with a closed snap and a tightly fastened velcro strap; and that a tightly fastened velcro would favor insertions as compared to similar sites with an open velcro . To test this model , we searched in the human reference genome ( hg19 ) for the position of all potential EN targets: R/TTTT , which corresponds to a closed snap; or R/VTTT , R/TVTT , R/TTVT and R/TTTV , which correspond to open snaps ( R = purine , V = not T ) . For each of them , we extracted the 10 nucleotides upstream of the nick position and categorized each on the basis of its snap/velcro status to obtain the exact frequency of each category in hg19 . Then we extracted the exact insertion sites for all the L1HS polymorphic insertions present in dbRIP [59] or in recent catalogs of somatic L1 insertions in cancer genomes [60] , [61] for which the insertion sites are annotated at nucleotide resolution . Since some insertions occurred through an EN-independent mechanism , we only kept sites with a recognizable EN target ( R/TTTT , R/VTTT , R/TVTT , R/TTVT , R/TTTV , as above ) . We categorized these sites based on their snap/velcro status . First , we determined the distribution of these categories in the human reference genome ( hg19 , Figure 5C ) or its repeat-masked counterpart ( hg19 RM , Figure 5C ) and we compared it to that of L1 insertions in each dataset ( dbRIP , Solyom and Lee , Figure 5C ) . Strikingly , the proportion of L1 insertions in sites with closed snap and/or tightly fastened velcro was significantly increased as compared to their proportion in the human genome ( Chi-square test , p<0 . 0001 for all insertion datasets ) . As an additional analysis , we calculated the frequency of each category in a given L1 insertion datasets as compared to their frequency in the human genome . We normalized this enrichment relative to the insertion sites with an open snap and a loosely fastened velcro strap . As shown in Figure 5D , L1 insertions are more frequent at sites with a closed snap or a tightly fastened velcro , and even more frequent at sites having both . Consistent with the in vitro data , given a snap status , insertions are more frequent at sites with a tightly fastened velcro than with a loosely fastened velcro . Other studies have previously reported that T-richness extends beyond four nucleotides upstream of the cleavage site [48] , [50] . Our analysis differs from these previous observations in that each position is not considered independently from the others . Altogether the distribution of polymorphic L1 insertions in vivo is consistent with the snap-velcro model at the genomic level , but it should also be stressed that , in vivo , other determinants are likely to influence L1 insertion profiles . An alternative pathway of L1 integration uses preformed double-stranded DNA lesions instead of EN-mediated cleavage . To determine whether the L1 RNP is able to directly initiate reverse transcription at blunt DNA ends , we designed model hairpins ending with four or six Ts at their 3′ terminus ( Figure 6A , primers H and H-ext ) . Notably , we used hairpins instead of two separate DNA strands to exclude the possibility that remaining free single-stranded primers could be extended ( Figure 6A ) . The expected start position of each extension product ( +1 ) , which depends on primer length ( see Figure 6A ) , is indicated by a black dot on the left side of each lane . Although we can readily detect elongation of the single-stranded ext- ( dT ) 18 primer ( Figure 6B , lane 2 ) , no mL1-specific extension was observed with these blunt substrates ( Figure 6B , compare lane 2 to 3–4 ) . The radiolabeled molecules detected below the +1 of the reverse transcription ( Figure 6B , between 40 and 56 nt and Figure 7B , below 40 nt ) result from contaminating activities , which co-fractionate with the mL1 RNP in the sucrose cushion ( see below for a detailed characterization ) . In addition , we asked whether the mL1 RNP could access and extend a stretch of 4 Ts embedded in a duplex DNA . No extension was observed when we used various hairpins with 3′ recessed ends ending with 4 Ts ( Figure 6A , 5′TT-H , 5′GC-H , 5′CTGC-H and Figure 6B , compare lanes 5–7 to 12–14 ) . Identical results were obtained with hL1 RNPs ( Figure S4A ) . Since L1 elements are believed to integrate into double-stranded genomic DNA and L1 RNPs can efficiently extend single-stranded oligonucleotides ( see above ) , we reasoned that L1 RNPs might be able to prime DNA synthesis on double-stranded primers ending with a 3′ overhang . To test this hypothesis we designed model hairpins extended by a 3′ overhang of increasing size ( Figure 7A , primers H0 to H6 ) . In contrast to reactions performed with blunt or 3′-recessed hairpin substrates , initiation of mL1 reverse transcription is easily detected as soon as the 3′ overhang reaches a length of 6 nt , as shown by the mL1-specific ladder which appears above 50 bp ( Figure 7B , compare lane 8 to 3–7 and 19 ) . Increasing the length of the overhang to 8 nt slightly increases the levels of reverse transcription , which indicates that a 6 nt 3′ overhang is necessary and sufficient for efficient extension by the mL1 RNP . In the experiments using single-stranded substrates , we demonstrated that 4 matching bases at the 3′ end of the substrate are sufficient to prime reverse transcription at detectable levels . This is also true for 3′ overhang hairpins , since a hairpin with a 6- or 8-nucleotide 3′ overhang but ending with only 4 Ts is extended , although to lower levels than a similar single-stranded primer ending with 4Ts ( Figure 7B , lanes 9–10 and Figure S2 , lane 12 ) . Identical results were obtained with hL1 RNPs ( Figure S4B ) . As mentioned above , incubation of L1 RNP fractions with hairpin primers and 32P-dTTP results in labeled products , which are shorter than the expected +1 of the reverse transcription reaction ( Figure 6B and Figure S4A , between 40 and 56 nt and Figure 7B and Figure S4B , below 40 nt ) . These products are also detected at similar levels with RT-defective L1 RNP preparations ( Figure 6B , lanes 9–14 and Figure 7B , lanes 14–22 ) and with RNPs prepared from vector-transfected cells ( data not shown ) , suggesting that they result from contaminating cellular activities , which co-fractionate with the L1 RNP in the sucrose cushion . To verify this hypothesis , we further purified the mL1 RNPs by immunoprecipitation using an antibody raised against the mORF1p protein ( Figure 8A and 8B ) , and then we performed reverse transcription reactions on the beads . As a negative control , we performed the immunoprecipitation with the preimmune serum . First , we could directly detect the mL1 RT activity in the immunoprecipitated complex ( Figure 8C , compare lanes 8 and 14 ) , reinforcing the notion that the L1 RNA , ORF1p and ORF2p form a stable complex [18] . Second , the immunopurified mL1 RNP extends the H6 hairpin primer with a 3′ overhang but not the blunt or 3′-recessed primers ( Figure 8C , compare lanes 9–12 and 15–18 ) . Third , the short products formed upon incubation with the sucrose cushion mL1 RNP preparation disappear if the mL1 RNP is further purified by immunoprecipitation ( Figure 8C , compare lanes 3–6 , dashed boxes , and 15–18 ) . Altogether these observations confirm that the bands below the +1 are indeed nonspecific products resulting from cellular contaminating activities and that the ladder-like products above ∼50 nt are bona fide L1 RNP reverse transcription products . Based on these data we conclude that native L1 RNPs preferentially extend DNA substrates ending with at least 4 Ts and a 6-nt single-stranded 3′ overhang , but does not efficiently extend blunt or 3′-recessed double-stranded DNA substrates .
Although L1 elements are responsible for a very large part of mammalian genomes and are an important source of genetic diversity and diseases [60] , [62]–[66] , detailed molecular mechanisms of their replication remain poorly studied at the biochemical level . We have developed here a direct L1 extension assay ( DLEA ) to explore the impact of primer sequence and structure on reverse transcription initiation by native L1 RNPs ( Figure 1 and Figure S1 ) . The DLEA protocol differs from previous approaches [10] , [33] , [51] , [55] , [67] because it combines native L1 RNP purification from cell extracts , by sucrose cushion ultracentrifugation or immunopurification ( Figure 8 ) , with the direct detection of extension products . Since it does not require a PCR amplification step , the DLEA allows quantitative comparisons of priming efficiencies for a large variety of substrates with different sequences and structures . A limitation of this assay is the absence of sequence information on the product . Therefore we complemented DLEA data with LEAP amplification and sequencing . By testing more than 65 different primers , including many that mimic bona fide L1 insertion sites recovered from cultured cells , we could define the rules of L1 reverse transcription initiation with an unprecedented resolution: ( i ) partial sequence complementarity between the 10 terminal nucleotides of the target site and the L1 RNA poly ( A ) tail impact reverse transcription initiation ( Figure 2 and Figure S2 , and Figure 4 ) ; ( ii ) four terminal Ts are sufficient to promote efficient extension of the target DNA ( Figure 2 and Figure S2 ) ; ( iii ) the L1 RNP can tolerate a mismatch in the crucial last 4 nucleotides if it is compensated by an increased number of matching nucleotides upstream of these bases ( Figure 2 , Figure S2 and Figure 4 ) ; ( iv ) the preferred terminal base is T>C>A>G ( Figure 3 ) . Based on these quantitative data , we propose a ‘snap-velcro’ model to illustrate the high level of flexibility of the L1 RNP toward primer use ( Figure 5A ) . This model identifies two distinct regions in the cleaved target DNA: ( i ) the terminal 3′ four nucleotides ( snap ) , which correspond to the EN recognition site , and are also essential to reverse transcription initiation; and ( ii ) the upstream six nucleotides ( velcro ) , which enhance reverse transcription efficiency and compensate potential mismatches in the snap region , when rich in Ts . Studying the properties of L1 RNPs in vitro provides detailed molecular insights into specific steps of the retrotransposition process . This is a useful complement to retrotransposition cellular assays , which offer a more global view of this mechanism . Nevertheless , a number of differences between the in vitro and in vivo situations , and between endogenously and ectopically expressed L1 , should be emphasized . First , reverse transcription initiation is uncoupled from the cleavage of the target DNA , in primer extension assays such as LEAP or DLEA . Thus , we cannot completely exclude that L1 RNPs would utilize a different priming mechanism in the context of a L1 TPRT reaction . Likewise , it is possible that the detected activity results from a minor fraction of the RNPs , which can only extend exogenous primers . This situation is reminiscent of L1 reverse transcription initiation at existing DNA lesions as hypothesized for EN-independent integration events [51] , [68]–[70] . Second , due to read-through transcription , L1 RNAs expressed from endogenous loci sometimes contain a first poly ( rA ) sequence , which is transcribed by RNA-Polymerase II from the L1 poly ( dA ) tail and can occasionally be imperfect , followed by a downstream genomic sequence , and ending with a perfect poly ( rA ) tail generated by Poly ( A ) -Polymerase [71] , [72] . Theoretically , alternative nucleotides present in such internal and imperfect poly ( A ) sequences could match perfectly to degenerate endonuclease sites , such that mismatches between primer and template would be less frequent . In contrast , L1 RNA polyadenylation in ectopically expressed constructs is generally driven by the strong SV40 polyadenylation sequence and by Poly ( A ) -Polymerase leading to perfect poly ( rA ) tails . Finally , our data suggest that target site choice is dictated not only by the specificity of the first EN cleavage , but also by the efficiency of RT priming after nicking . Interestingly , an engineered L1 endonuclease with relaxed sequence specificity in vitro has been described [73] . In vivo , L1 elements carrying this endonuclease variant still integrate in extended T-rich sequences , which shows that additional factors other than the EN specificity contribute to L1 insertion profile in vivo . Our data suggest that primer-template complementarity might be one of these factors , by promoting the initiation of reverse transcription , but it is also very likely that additional partners or inhibitors influence L1 targeting in vivo , modulating or relaxing EN or RT specificity . Indeed , L1 insertions occasionally take place at sites that do not strictly follow the rules described here ( Figure 5C , and [46] , [47] , [49] , [51] , [69] ) , suggesting that primers for which we cannot detect extension by DLEA might actually be L1 substrates . From our data we can only conclude that they are extended in vitro at least 10–20 fold less efficiently than the best target sites that were used as references in our assays . In contrast to the L1 RNP , R2 reverse transcriptase does not require sequence matching to prime DNA synthesis and does not require a 3′ overhang [74] . This might be related to the fact that specific structures in the R2 RNA allow the R2 RT to position and guide the exact start of reverse transcription at the cleavage site [36] . In this configuration , primer-template annealing is no longer a requirement to position the primer at the end of the template . Biochemical studies with non-LTR retrotransposon RT from other clades will be necessary to determine , which of these two situations is the rule and the exception . The current model of L1 retrotransposition , which has been largely inspired by studies on the R2 element , starts with a nick in the target DNA followed by the extension of this nick . Our data indicate that extension by the L1 RNP is efficient on single-stranded DNA substrates , but inefficient when the 3′ OH is embedded in duplex DNA , either at a blunt end or at a 3′ recessed end ( Figure 6B and Figure S4A ) . In contrast , it efficiently initiates reverse transcription on double-stranded DNA molecules ending with a 3′ single-stranded overhang ( Figure 7B and Figure S4B ) . Thus , our results suggest an additional step in the retrotransposition process , which generates a single-stranded 3′ end from a blunt end or from a nick to allow L1 reverse transcription . We envisage two ways in which this 3′ overhang could be established . In the first model , the L1 endonuclease directly generates a double-strand break with staggered cuts instead of acting sequentially on one strand and then on the other strand only after minus strand cDNA synthesis . Consistently , recombinant L1 endonuclease can linearize plasmid DNA in vitro [27] and ectopic L1 expression results in the activation of a DNA damage response in cultured cells [75] , [76] . In the second model , an unidentified machinery could promote unwinding of the nicked DNA or permit strand-exchange between the duplex DNA and the RNA moiety of the L1 RNP . The ORF1p protein has been proposed to play such a role through its nucleic acid chaperone activity [20] , [24] . Indeed , nucleic acid chaperone activities promote reverse transcription in retroviruses and LTR-retrotransposons through several mechanisms , including primer annealing to the template RNA [77]–[80] . All the experiments described here use native L1 RNP preparations , which contain ORF1p ( Figure 1 and Figure 8 ) . However , in our experimental conditions , we were unable to detect extension of blunt or 3′ recessed double-stranded substrates . Thus , if such a DNA remodeling machinery is involved , it has to be of cellular origin . Nevertheless , it should be noted that , in primer extension assays , as performed in LEAP or DLEA experiments , the initiation of reverse transcription is uncoupled from the cleavage of the target DNA , in contrast to the TPRT process . Thus , we cannot completely exclude that the L1 RNP would utilize a different priming mechanism in the context of a L1 TPRT reaction . The requirement of a 3′ overhang could also be relevant to alternative L1 integration pathways . Indeed , L1s can initiate reverse transcription at preformed DNA lesions or at telomeric ends and thus insert into the genome independently of their EN activity [51] , [68]–[70] . EN-independent retrotransposition was only observed in cell lines deficient in the nonhomologous end-joining ( NHEJ ) pathway [68] . Interestingly , binding of NHEJ components to DNA ends interferes with end resection [81] . As a result of this competition , end resection ( the first step of homologous recombination ) is increased in NHEJ-deficient cell lines . Thus , we speculate that EN-independent retrotransposition might require the 5′ to 3′ end resection step , which initiates HR , to generate a 3′ overhang suitable for L1 reverse transcription initiation . The link between end resection factors ( such as the MRN complex , CtIP , Exo1 , BLM , Dna2 , etc . ) and the ability of L1 to engage in EN-independent insertions will be an important direction for future studies . Similarly , the L1 RNP is also able to prime cDNA synthesis at dysfunctional telomeres in NHEJ-deficient hamster cells [51] , [69] . Telomeres end with a 3′ overhang [82] , [83] , the formation of which is highly regulated and involves a specialized set of factors [84] . Telomeres can also be extended by a specialized cellular RNP with reverse transcriptase activity , called telomerase [85] , [86] . Like L1 , telomerase requires a 3′ single-stranded overhang to extend double-stranded DNA [87] . Thus our observations reinforce the notion that these two endogenous reverse transcriptases , which are evolutionary related [88]–[90] , share common mechanistic properties [51] . In conclusion , our data demonstrate that partial sequence complementarity between the target site and the L1 RNA facilitates L1 reverse transcription priming and highlight the flexibility of the L1 RT . Interestingly , EN cleavage and RT priming appear to target the same TTTT sequence , suggesting that these two L1 biochemical activities have co-evolved . We speculate that their exceptional flexibility has participated in the evolutionary success of the L1 family and in its wide spread distribution within mammalian genomes .
Plasmids JM101/L1 . 3 and JM105/L1 . 3 respectively contain WT and RT-mutated ( D702A ) versions of the human L1 . 3 element in a pCEP4 backbone ( a kind gift of N . Gilbert ) [9] . Plasmid pWA121 contains a codon-optimized version of the mouse L1spa element in a pCEP4-Puro backbone ( a kind gift of J . D . Boeke ) [91] . A fragment containing mORF2p was amplified by PCR from pWA121 using oligonucleotides LOU266 and LOU267 . The purified attB PCR product was cloned into pDONR207 using BP Clonase II under the manufacturer's conditions ( Gateway system , Life Technologies ) to obtain plasmid pVan239 . A point mutation in the RT domain ( D709A ) was introduced in this construct using the QuikChange II XL Site-Directed Mutagenesis Kit ( Agilent Technologies ) and the DNA primer pair LOU419-LOU420 to generate pVan330 ( mORFeus RT* ) . The RT* mutation introduces a new SacII restriction site in ORF2 , allowing quick screening of the mutation . The latter was confirmed by sequencing . A SdaI-NruI DNA fragment containing part of ORF2p from this entry clone was inserted back into the original pWA121 plasmid digested by the same enzymes . A full list of the oligonucleotides used in this study is provided as Table S1 . Peptides corresponding to the C-termini of mouse ( N-CNQYKNGNNALEKTRR-C ) or human ( N-CERNNRYQPLQNHAKM-C ) ORF1p were synthesized and coupled to the KLH protein as a carrier . The first cysteine ( underlined ) is not present in the ORF1p sequence but was added for the coupling reaction with the carrier protein . KLH-coupled peptides were used to immunize rabbits ( Eurogentec ) . For immunoblotting the mORF1p antiserum ( SE-0560 ) , the hORF1p antiserum ( SE-6798 ) , and the S6 protein antibody ( Cell signaling , #2217 ) were used at a dilution of 1∶2000 . One hundred micrograms of each lyophilized oligonucleotide was dissolved in 10 µl of 98% deionized formamide , 1 mM EDTA , 0 . 01% ( w/v ) xylene cyanol and 0 . 01% ( w/v ) bromophenol blue and resolved in 10% polyacrylamide-urea denaturing gels . Full length oligonucleotides were visualized by UV shadowing , excised from the gel and eluted overnight at 37°C in 0 . 3 M sodium acetate , 0 . 1% SDS and 10 mM MgCl2 . Eluted oligonucleotides were precipitated with ice-cold ethanol ( 3v ) . After centrifugation for 30 min at 4°C at 16'000 g , the pellets were washed with 70% ethanol , air-dried and dissolved in 10 mM Tris-HCl pH 8 . 0 , 1 mM EDTA . L1 RNPs were produced in HEK293T cells grown in Dulbecco's Modified Eagle Medium ( DMEM , Life Technologies ) containing 2 mM L-Glutamine , 4500 mg/L D-Glucose , 1 mM Sodium Pyruvate , 10% ( v/v ) fetal bovine serum ( Life Technologies ) and 100 units/mL penicillin/streptomycin ( Life Technologies ) . Cells were plated at 3×106 cells per 10 cm Petri dish . Twenty-four hours after plating , the cells were transfected with 24 µg of plasmid DNA ( see plasmids above ) per dish using the calcium phosphate method . Growth medium was changed 5 hours later . One day post-transfection , cells were split into two plates in growth medium supplemented with 1 . 5 µg/mL puromycin ( mORFeus , Life Technologies ) or 100 µg/mL hygromycin ( L1 . 3 , Life Technologies ) . Cells were collected 4 days post-transfection by trypsinization , pooled and washed in PBS . Cell pellets were lysed in 500 µL of CHAPS lysis buffer ( 10 mM Tris-HCl [pH 7 . 5] , 1 mM MgCl2 , 1 mM EGTA , 0 . 5% ( w/v ) CHAPS , 10% ( v/v ) Glycerol , supplemented before use with Complete EDTA-free protease inhibitors cocktail ( Roche ) and 1 mM DTT ) . After incubation at 4°C for 15 min , cell debris was removed by spinning down extracts at 4°C for 10 min at 16'000 g . Supernatants were transferred to clean tubes and 500 µL of lysis buffer were added to each of them . L1 RNPs were prepared as previously described [10] . In brief , a sucrose cushion was prepared with 8 . 5% and 17% ( w/v ) sucrose in 20 mM Tris-HCl [pH 7 . 5] , 80 mM NaCl , 5 mM MgCl2 , 1 mM DTT and Complete EDTA-free protease inhibitors cocktail ( Roche ) . For each sucrose cushion , 1 mL of cell lysates , prepared as described above , was used . Samples were centrifuged for 2 h at 178'000 g at 4°C and the pelleted material was resuspended in 100 µL H2O . Total protein concentration was determined by Bradford assay ( Biorad ) . The samples were diluted in 50% ( v/v ) glycerol , quick frozen in liquid nitrogen and stored at −80°C until use . Protein A-Sepharose beads ( Sigma ) were blocked overnight at 4°C in PBS containing 0 . 5 mg/mL of bovine serum albumin ( BSA ) and washed twice in 1 mL of IP buffer ( 10 mM Tris-HCl [pH 7 . 5] , 150 mM NaCl ) . Eight microliters of preimmune or anti-mORF1p serum were bound to 70 µl of blocked beads for 3 h at 4°C . For each immunoprecipitation , 200 µL of L1 RNPs ( 2 µg/µL ) were diluted 1∶1 ( v/v ) in IP buffer . The RNPs were precleared with blocked beads for 1 h at 4°C and incubated for 3 h at 4°C with antibody-bound beads on a rotating wheel . After 4 washes in IP buffer , the bead slurry was split equally into 7 tubes ( 6 for RT reactions and 1 for immunoblotting ) . Beads were pelleted for 5 min at 4°C at 750 g , supernatants were removed and the RT reaction mixture was directly added to the beads ( see below ) . Reverse transcriptase assays were carried out for 4 min at 37°C in 25 µL reactions containing 2 µg of RNPs , 400 nM of primer , 50 mM Tris-HCl [pH 7 . 5] , 50 mM KCl , 5 mM MgCl2 , 10 mM DTT , 0 . 05% ( v/v ) Tween-20 and 10 µCi of α-32P-dTTP ( 3000 Ci/mmol , PerkinElmer ) . In reactions using the Avian Myeloblastosis Virus RT ( AMV RT , Promega ) , the RNPs were replaced by 0 . 04 U of AMV RT and 250 ng of poly ( rA ) template ( Roche ) . Reactions were stopped by the addition of 8 . 3 mM EDTA and 0 . 83% SDS final . Trace amounts of a 32P-labelled 14- or 30-mer DNA oligonucleotide were added as recovery control ( noted RC ( 14 ) or RC ( 30 ) in the figures ) . Products were purified by phenol-chloroform extraction and ethanol precipitation with 10 µg of glycogen as a carrier and 0 . 1 mM sodium acetate [pH 5 . 2] . DNA pellets were resuspended in 98% deionized formamide containing 10 mM EDTA , 0 . 02% ( w/v ) xylene cyanol and 0 . 02% ( w/v ) bromophenol blue , heated to 95°C for 5 min , and analyzed on 13% polyacrylamide-urea sequencing gels . After drying , gels were exposed to a PhosphorImager screen . For primers used in Figure 4 , we first resolved the products on sequencing gels to verify that the profiles of the products were similar to those obtained with other linear oligonucleotides and that nonspecific products were not generated . In a second time , to facilitate quantification of a large number of reactions performed in parallel , we spotted 5 µL of each reaction onto DE-81 paper immediately after the 4 min incubation , in triplicate . DE-81 paper is an ion exchange paper , which retains the incorporated nucleotides , but not the free dNTPs . Papers were next washed 5 times with 200 mL of 2x saline-sodium citrate ( SSC ) solution and exposed to a PhosphorImager screen . We tested the complete set of primers three times . For gel or spot quantification , the reaction without primer obtained with a given RNP preparation was used as background and was subtracted from the reaction with primers . Only the signal above the primer size was quantified for the hairpin oligonucleotides . To determine whether 32P incorporation was RNase sensitive ( Figure S1A ) , we incubated reaction mixes in the presence of 30 µg of RNase A and 150 U of RNase I ( New England BioLabs ) , or of 40 U of RNasin ( Promega ) as a negative control , for 1 h at 37°C before adding 32P-dTTP and primer . RT inhibitors ( AZT and d4T , also known as Stavudin ) as triphosphate derivatives were obtained from Biocentric . They were added to reactions at a final concentration of 10 µM ( Figure S1B ) . LEAP was performed as previously described [10] with only minor modifications . Briefly , L1 reverse transcription was carried out for 1 h at 37°C in 50 µL reactions containing 0 . 75 µg L1 RNP ( 50% ( v/v ) glycerol ) , 50 mM Tris-HCl [pH 7 . 5] , 50 mM KCl , 10 mM DTT , 5 mM MgCl2 , 0 . 05% ( v/v ) Tween-20 , 20 U RNasin ( Promega ) , 200 µM dNTP , and 0 . 4 µM LEAP primer . Eventually , unextended primers were eliminated through an S-400HR size-exclusion spin column ( GE Healthcare ) . Reverse transcription products ( 1 µL of the LEAP reaction ) were PCR-amplified in 50 µL reactions containing 1 U of Platinum Taq DNA Polymerase ( Life technologies ) , 0 . 2 µM of primers LOU851 and LOU312 , 200 µM dNTP , 3 mM MgCl2 in the Platinum Taq buffer . A first step at 94°C for 2 min was followed by 35 cycles of [30 s at 94°C , 30 s at 60°C and 30 s at 72°C] . The final extension was at 72°C for 5 min . PCR products were analyzed by 2% agarose gel electrophoresis in 1x TBE . Gels were stained by SYBR Safe ( Life technologies ) or ethidium bromide . LEAP products were gel-purified with a gel extraction kit ( Macherey Nagel ) and cloned into the pGEM-T-easy vector ( Promega ) , according to manufacturer's protocol . Clones from isolated colonies were sequenced by GATC . Regions with low quality ( Phred<Q20 ) were trimmed or filtered out using Geneious 5 . Total RNA was extracted from 30 µg of L1 RNP using TRIzol extraction ( Molecular Research Center Inc ) following the manufacturer's instruction . RNA was resuspended in 20 µL of milliQ water and quantified by Nanodrop . One microgram of RNA was digested by 1 U of RNase-free RQ1 DNase ( Promega ) in 10 µL reaction in the manufacturer's buffer at 37°C for 30 min . DNase was heat-inactivated for 10 min at 65°C . Then , cDNA synthesis was performed at 50°C for 1 h in 20 µL reactions containing 6 µL of the DNase reaction , 200 U of SuperScript III Reverse Transcriptase ( Life technologies ) , 500 µM dNTP , 50 pmol of RACE primer , 40 U RNAseOUT ( Life technologies ) , 50 mM Tris-HCl [pH 8 . 0] , 75 mM KCl , 3 mM MgCl2 and 5 mM DTT . Primer pairs used for PCR were LOU851/LOU312 ( mOrfeus or L1 . 3 ) or LOU852/LOU312 ( GAPDH ) . PCR products were resolved by 2% agarose gel electrophoresis in 1x TBE . The T-density is calculated by dividing the number of Ts in the oligonucleotide by the length of the oligonucleotide . The position-weighted T-density gives more weight to Ts which are close the 3′ extremity of the primer . The weight is inversely proportional to the distance from the 3′ end . For example: Primer LOU519 has a position-weighted T-count equal to: Primer LOU541 has a position-weighted T-count equal to: The position-weighted T-density of a given primer is calculated by dividing the position-weighted T-count of this primer to the maximum position-weighted T-count . Thus the position-weighted T-density of LOU519 is equal to 2 . 23/3 . 60 = 0 . 62 and the position-weighted T-density of LOU541 is equal to 3 . 60/3 . 60 = 1 The snap is considered open if the 4 terminal nucleotides contain a non-T nucleotides and closed if the last four nucleotides are 4 Ts . We calculated a position-weighted T-count for the upstream 6 nucleotides ( velcro region ) and we divided it by the maximum value ( 1/5 ) + ( 1/6 ) +…+ ( 1/10 ) = 0 . 84563492 to obtain the velcro position-weighted T-density . We consider a velcro as fastened if its position-weighted T-density is ≥0 . 5 ( half of the maximum ) and opened otherwise . All putative integration sites with a perfect or degenerate EN recognition sequence ( from 3′ to 5′ , R/TTTT , R/VTTT , R/TVTT , R/TTVT , R/TTTV ) were recovered from both strands of the reference human genome ( hg19 ) or from its repeatmasked version ( hg19 RM ) . For each putative EN site , snap and velcro status were defined as described above . The C++ program used to achieve this task is available in Protocol S1 . Polymorphic L1 insertions were extracted from dbRIP [59] or from cancer genome whole-genome sequences [60] , [61] . Only insertion sites with an identifiable EN recognition site as defined above were kept for the analysis . This filtering step was necessary to eliminate internal initiation events most likely related to EN-independent insertions or other forms of structural variation and insertion sites which position was not precise at nucleotide resolution . Raw data are provided in Table S2 . For each dataset , we calculated the frequency of each category and we normalized first to hg19 count and second to the “open snap/tightly fastened velcro” category to evaluate the effect of a closed snap and/or velcro . We compared observed ( polymorphic L1 insertions ) and expected ( hg19 ) frequencies by Chi-squared test . We used the Graphpad Prism 6 . 00 software for Mac for all statistical analyses . | Jumping genes are DNA sequences present in the genome of most living organisms . They contribute to genome dynamics and occasionally result in hereditary genetic diseases or cancer . L1 elements are the only autonomously active jumping genes in the human genome . They replicate through an RNA–mediated copy-and-paste mechanism by cleaving the host genome and then using this new DNA end as a primer to reverse transcribe its own RNA , generating a new L1 DNA copy . The molecular determinants that influence L1 target site choice are not fully understood . Here we present a quantitative assay to measure the influence of DNA target site sequence and structure on the reverse transcription step . By testing more than 65 potential DNA primers , we observe that not all sites are equally extended by the L1 machinery , and we define the rules guiding this process . In particular , we highlight the importance of partial sequence complementarity between the target site and the L1 RNA extremity , but also the high level of flexibility of this process , since detrimental terminal mismatches can be compensated by an increasing number of interacting nucleotides . We propose that this mechanism contributes to the distribution of new L1 insertions within the human genome . | [
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] | 2013 | The Specificity and Flexibility of L1 Reverse Transcription Priming at Imperfect T-Tracts |
Altered daily patterns of hormone action are suspected to contribute to metabolic disease . It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism . Here , we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq , NMR spectroscopy and liquid chromatography-based methods . We observed dysregulation of metabolic pathways including glutaminolysis , the citrate and urea cycles and glyoxylate detoxification . Constant , non-rhythmic glucocorticoid treatment rescued many of these changes , with some notable exceptions among the amino acid related pathways . Surprisingly , the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns . A combination of E-box and glucocorticoid response elements is enriched in the rescued genes . This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure , revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription . Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency , even under glucocorticoid replacement therapy . Moreover , we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism .
The circadian clock is an endogenous oscillator that regulates daily changes of behavior , physiology and metabolism [1] . The molecular basis of the circadian clock is a transcriptional-translational feedback loop , a central part of which are E-box enhancer elements [2] . To generate physiological rhythms , “peripheral” clocks in almost all tissues interact with signals produced by a “central” pacemaker , the hypothalamic suprachiasmatic nucleus . A key target of circadian clock control is metabolism , with circadian rhythms present in many metabolites and enzyme activities [3] . In addition , hormones with metabolic functions are regulated by the circadian clock . This includes glucocorticoids ( GCs ) , steroid hormones mainly produced by the adrenal gland [4] . GC production shows higher basal levels in the morning in humans and at night in rodents . GCs were also shown to interact with clock factors in the transcriptional regulation of metabolic gene expression [5] . However , the global role of the interaction between the circadian clock and GCs in the regulation of physiology and metabolism and its underlying mechanisms are only incompletely understood . Patients suffering from adrenal insufficiency ( AI ) have inadequate GC amounts either because of defects in the adrenal gland itself ( primary AI ) or due to deficient input from the pituitary gland ( secondary AI ) [6] . Patients with secondary AI have an increased risk to develop metabolic syndrome , and abnormal glucose tolerance is observed upon long-term therapy with current GC replacement regimes . This may be linked to an inadequate replication of the natural circadian GC rhythm [7] . Only limited information is available on the metabolic changes present in patients with AI [8 , 9] , particularly with respect to their temporal dynamics . Animal models , preferentially with a diurnal lifestyle , could contribute to improve therapy by providing a mechanistic understanding of metabolic dysregulation in AI . The zebrafish is a well-established model system for human disease including metabolic diseases [10] and has proven useful for chronobiology and endocrinology studies [11 , 12] . Zebrafish embryos and larvae are well-suited for in vivo bioimaging and drug screenings [13] . Similar to humans , zebrafish are diurnal and use cortisol as the main GC hormone , whereas laboratory rodents are nocturnal and use corticosterone . We previously described a mutation that leads to GC deficiency in homozygous larvae . rx3 mutants of both a weak and a strong allele show a severe eye defect [14] . The strong allele additionally presents a severe reduction of ACTH producing corticotrope pituitary cells , leading to reduced cortisol amounts which also lack a diurnal rhythm ( Fig 1A ) [15] . Thus , this mutant condition resembles secondary AI . Intriguingly , a clock output rhythm , the circadian fluctuation of cell proliferation , is attenuated in the mutant larvae . These rhythms can be rescued by constant treatment with the synthetic GC , dexamethasone ( DEX ) [15] , and are thus not dependent on the diurnal glucocorticoid release pattern . It is currently not understood how a constant GC signal integrates with circadian clock function to generate such GC-dependent clock output rhythms . Here , we examined diurnal changes in the transcriptome and metabolism of rx3 strong mutants with or without continuous DEX exposure . A surprisingly large part of diurnal gene expression is rescued by this constant DEX treatment , which also relieves specific metabolite changes in the mutants . Analysis of gene regulation revealed a combined simple enhancer element that is sufficient to mediate diurnal GC-dependent transcription . Besides providing mechanistic insight on GC-circadian clock crosstalk , our study reveals widespread changes in metabolism in an animal model of GC deficiency . These findings will help to better understand morbidities in patients with AI and identify metabolic pathways that could be used for monitoring of therapy efficiency .
To examine if and how diurnal patterns of transcription are perturbed in the GC deficiency model , we measured diurnal transcriptome changes in rx3 strong mutant zebrafish larvae and their wild-type siblings ( Fig 1A ) at four time points over 24 h ( Fig 1B; S1 Table , for quality control and validation experiment results see S1A–S1C Fig ) . To control for eye absence in the strong allele , we included the equally eyeless rx3 weak mutant larvae , which have normal diurnal cortisol levels . Statistical analysis based on harmonic linear regression [16] grouped genes into models according to their rhythmic or non-rhythmic expression behaviour under the three conditions ( Fig 1C–1E , S2A Fig , S1 Table; see Materials and Methods for details ) . Genes classified into model 1 did not exhibit rhythmicity under any condition , while genes grouped within the other 14 models showed rhythmic expression in at least one condition . Genes which did not fulfill our statistical cutoff criteria to fit within these models were named “ambiguous” . Several models were of particular interest for our aim to identify genes with GC dependent diurnal patterns of transcription . Model 11 was rhythmic in all conditions with the same rhythmic parameters ( Fig 1C and 1E ) ; we will refer to this group of genes as “unaffected” . By contrast , models 5 , 6 , 14 and 15 showed either a lack of rhythmic expression ( model 5 , 6 ) or a change in amplitude or phase ( model 14 , 15 ) in the strong allele ( Fig 1D and 1E ) . For these four models , global gene expression amplitudes are not significantly different between wild-type and rx3 weak allele mutants , while they are significantly reduced ( model 14 and 15 ) or absent ( model 5 and 6 ) compared with the wild-type in strong allele mutants ( S2D–S2G Fig ) . Furthermore , global comparison of phases shows that they are more perturbed when comparing wild-type and strong allele mutants than when comparing wild-type and weak allele mutants ( S2H–S2K Fig ) . We will refer to the genes of models 5 , 6 , 14 and 15 as “affected” . These four models define a set of 5970 genes that are candidates for mediating GC-dependent circadian functions . Representing 43 . 6% of all genes showing rhythmic expression and 23 . 4% of all detected genes , they constitute a surprisingly large category of the diurnal transcriptome ( Fig 1F ) . Gene Ontology ( GO ) analysis showed enrichment in GO terms for metabolic processes in this set ( S1E Fig ) . Indeed , 39 . 9% of the temporal profiles of metabolic genes are assigned to model 5-6-14-15 genes ( Fig 1F ) . By contrast , circadian clock and cell cycle genes are less affected in their temporal expression in rx3 strong mutants . 75% of all circadian clock genes are not altered in their rhythmicity and belong to model 11 ( Fig 1F , S3A Fig ) . There is also enrichment for model 11 within the group of cell cycle genes ( 23 . 8% , Fig 1F , S3B Fig ) . Fitting this observation , model 11 genes are enriched for GO terms related to the cell cycle ( S1D Fig ) . These findings show that many cell cycle related genes have a rhythmic expression pattern which does not change in the rx3 strong mutants . Still , 25 . 4% of cell cycle genes belong to model 5-6-14-15 ( Fig 1F ) . Interestingly , there is no statistically significant alteration in oscillation amplitude between weak and strong allele mutants across both all cell cycle genes and all circadian clock genes ( Fig 1G ) . However , the amplitudes of oscillation are significantly reduced across the metabolic genes in rx3 strong mutants ( Fig 1G ) , further indicating a higher degree of attenuated rhythm within this group . The “affected” set of genes also encompasses a larger number of enriched metabolic pathways than the “unaffected” set ( compare S1F Fig with S1G Fig ) , again underlining the strong effect present in this group on the diurnal expression of metabolic genes . Next , we asked whether and to what extent affected gene expression rhythms can be restored in the mutants by constant GC treatment . To determine how the diurnal transcriptome changes under chronic DEX treatment ( Fig 2A ) , we carried out RNA-Seq analysis of treated rx3 strong mutants and wild-type siblings . To evaluate whether the treatment leads to a rescue of rhythmic expression in the mutants , we analyzed the two treated conditions for rhythmic parameters of gene expression as done for the untreated samples . This allowed us to evaluate if differences in expression between the genotypes were abolished by the treatment , even if other general DEX effects on transcription affected both conditions similarly . Our statistical analysis yielded five models ( S2B Fig ) , of which two ( D and E ) exhibit rhythmicity in both wild-types and mutants . Therefore , these models could indicate a rescue of dysregulated patterns among the “affected” gene group ( Fig 2B and 2C; S4 Fig ) . Model D regroups those genes in which there is no difference in rhythmic expression between mutants and wild-type under DEX treatment . Model E indicates those genes in which a difference in phase or amplitude between mutant and wild-type is still present under DEX treatment . We classified as rescued all genes which were not rhythmic in the mutants before ( model 5 and 6 ) and in which rhythms have been restored ( model D or E ) , or genes of models 14 and 15 in which a phase or relative amplitude difference in the untreated condition has been abolished ( model D ) or reduced ( model E ) . By contrast , we did not count as rescued all those genes of model E in which phase or amplitude differences were not reduced by the treatment ( named model E* ) . Applying these rescue criteria , 46% of the model 5-6-14-15 genes were rescued ( Fig 2D , “all affected genes” ) . Strikingly , 58% of the metabolic genes of model 5-6-14-15 are rescued ( Fig 2D ) . Interestingly , even though 68% of affected cell cycle genes do not show convergence of mutant and wild-type expression patterns in the DEX treated condition ( Fig 2D , S3B Fig ) , this treatment restores cell cycle rhythms in the rx3 strong mutants [15] . In summary , chronic DEX treatment is able to restore rhythmic expression patterns matching the wild-type in nearly half of all rhythmic genes affected in the mutants and in about 60% of the metabolic genes . This is a striking finding , showing that a large proportion of affected genes do not require rhythmic GC input for the GC-dependent regulation of their rhythmic transcription . Our RNA-Seq analysis revealed a high proportion of metabolism-related genes among the set with GC-dependent diurnal transcription . Therefore , to determine metabolite changes , we examined extracts from rx3 strong and rx3 weak mutant zebrafish larvae and their wild-type siblings at five time points over 24 h ( Fig 2A ) by NMR spectroscopy . We recorded 1D spectra for quantitation and additionally 2D J-resolved spectra for unambiguous identification of compounds . Principal component analysis ( PCA ) of the NMR spectra ( Fig 3A ) shows that rx3 weak and wild-type samples cluster together , illustrating that the metabolomes of rx3 weak mutants and wild-type larvae are more similar to each other than to the strong allele samples . Under DEX treatment , the wild-type and rx3 strong mutants cluster much closer together than the control samples ( Fig 3B ) . Betaine , creatine , lactate and glutamine appear as major contributors to the main principal components ( S5A and S5B Fig ) . Indeed , glutamine showed a strong accumulation in rx3 strong larvae at all examined time points , which was rescued by DEX treatment ( S5C Fig ) . Glutamine plays an important role in amino acid and central carbon metabolism , pathways which were also found to be enriched among the rescued gene set ( Fig 2E ) . Therefore , we measured a set of amino acids and TCA cycle related metabolites by UPLC-FLR and IC-CD analysis , and evaluated rhythmicity of these data using the harmonic linear regression based model selection approach ( S2C Fig , S2 Table ) . Rhythmicity behaviour and changes in mean levels of the metabolites indicated three groups of particular interest . In the first group , which includes branched chain amino acids ( BCAAs ) and aromatic amino acids ( AAAs ) , there is an accumulation of compounds in rx3 strong mutants which is not rescued by DEX treatment ( Fig 3C , S5D–S5G Fig and S2 Table ) . Generally , compound levels in the wild-type show a stronger overall decrease over time than in the mutants . Also , rhythmicity behaviour in this group is not dramatically affected by GCs ( models II [Tyr] and VI [Val , Leu , Ile , Phe] ) . These findings only partly correlate with gene expression pattern changes in the first degradation steps of the corresponding pathways . In the AAA pathway , half of the affected genes are rescued ( S6A Fig ) , and in the BCAA pathway , all three key enzymes are rescued ( S6B Fig ) . Here , metabolite accumulation seems to reflect other processes that are independent of GC regulation or not rescued by constant DEX application . Such processes may include posttranscriptional and -translational regulation of these enzymes , or BCAA accumulation due to increased degradation of BCAA containing proteins . The second group of metabolites equally accumulates in the mutants , but here their levels are rescued by DEX treatment . Glyoxylate , lysine and the ornithine-urea cycle ( OUC ) compounds ornithine , citrulline and arginine belong to this group ( Fig 3D , S5H–S5K Fig and S2 Table ) . Rhythmicity behavior varies in this group ( models I [glyoxylate] , III [Cit] , VII [Arg] or ambiguous [Lys , Orn] ) . Patterns of gene expression in the corresponding pathways seem to be more closely correlated with metabolite changes than in the first group: glyoxylate metabolizing enzymes are downregulated , while those producing glyoxylate are upregulated in rx3 strong mutants , and changes in both pathways are rescued by DEX treatment ( S6C Fig ) . A similar behavior is seen in the OUC pathway ( S6D Fig ) . The last group contains only one compound , glutamine . Here , accumulation in rx3 strong is rescued by DEX treatment , as in the second group . Additionally , glutamine shows a diurnal rhythm in the wild-type which is slightly flattened and shifted in the rx3 strong mutant ( log2 amplitude 0 . 40 and peak at ZT22 . 2 compared with 0 . 47 and ZT17 . 5 in wild-type ) . Under DEX treatment , both mutant and wild-type exhibit strong rhythmic glutamine concentrations and the phase difference is reduced ( model IX , Fig 3E , S5C Fig , S2 Table ) . Remarkably , glutamine is the only compound of the set which shows such a rescue of both overall levels and circadian rhythmicity by DEX treatment . Glutamine forms part of several pathways enriched in the rescued gene set . For example , it is a required source of nitrogen for purine and pyrimidine synthesis . Interestingly , an entire chain of enzymes downstream of glutamine entry into the purine biosynthesis pathway shows dysregulation in rx3 strong mutants , which is rescued by DEX ( S6E Fig ) . These enzymes act upstream of IMP ( inosine 5'-monophosphate ) dehydrogenase 2 ( impdh2 ) , which has recently been suggested to be involved in the regulation of circadian rhythms of cell proliferation [18] . impdh2 expression is also dysregulated in the mutants and rescued by DEX . As genes in many other branches of the purine synthesis pathway are equally rescued ( S1 and S3 Tables ) , GCs seem to regulate a large part of the diurnal transcription within this pathway . Glutamine is also important for refilling ( anaplerosis ) of the TCA cycle with α-ketoglutarate when it is deprived of intermediates ( Fig 4A ) . We chose this glutamine-TCA cycle connection for a proof-of-principle analysis . Examination of the cumulated levels of six TCA intermediates shows that citrate levels are higher in the mutants , while succinate levels are reduced ( Fig 4B ) . This finding is consistent with reduced anaplerosis at the level of α-ketoglutarate , leading to an upstream block and downstream depletion of cycle intermediates . In DEX treated conditions the levels are normalized , indicating restored flow . The TCA cycle connects glutaminolysis , glycolysis and gluconeogenesis , and indeed , many TCA cycle and glycolysis enzymes as well as key enzymes of gluconeogenesis show dysregulated expression rescued by DEX treatment ( Fig 4A , 4C and 4D ) . Among them , phosphoenolpyruvate carboxykinase 1 ( soluble , pck1 ) removes OAA from the TCA cycle ( cataplerosis ) and channels it into gluconeogenesis . It has been suggested that this cataplerotic PCK1 function balances anaplerotic refilling of the TCA cycle by glutamine metabolism [19] . Expression of dysregulated genes in this anaplerotic pathway , such as glutaminase 2 ( gls2 ) , is also restored by chronic DEX treatment ( Fig 4A , 4C and 4E ) . Therefore , DEX treatment likely restores the cataplerosis/anaplerosis balance of the TCA cycle , thereby normalizing TCA compound and glutamine levels . PCK1 has long been described as a GC target gene and was reported to show circadian expression in mammals [5] . Interestingly , there are two Glucocorticoid Response Elements ( GREs ) and an E-box in the zebrafish pck1 promoter region , which are conserved across evolution ( Fig 5A ) . Our RNA-seq study identified gls2 as another GC inducible circadian gene , and it equally contains both GREs and E-boxes . To test whether this is a typical feature of GC regulated circadian genes , the putative promoter sequences ( -1000 +500bp ) of the model 5-6-14-15 genes were examined for concomitant presence of both E-box and GRE elements . We observed an enriched co-occurrence of E-boxes and GREs in the rescued genes ( hypergeometric test , p = 0 . 04 ) , while no significant enrichment was seen in the non-rescued ones ( p = 0 . 97; Fig 5B ) . Importantly , this enrichment is also observed in the mouse orthologues of the zebrafish genes , indicating evolutionary conservation of this regulatory module . These findings indicate that E-box/GRE modules , which allow for direct transcriptional regulation by both the circadian clock and GCs , are a characteristic feature of GC-dependent diurnal genes . Can a simple combination of GREs and E-box enhancer elements drive GC dependent circadian patterns of gene expression ? Exploiting the direct light sensitivity of zebrafish cells [20] , we transfected cells with luciferase reporter constructs driven by different combinations of E-boxes and GREs and exposed them to light-dark cycles at different concentrations of DEX in otherwise GC-depleted culture medium ( Fig 5C–5H ) . Of all combinations , only the E-box/GRE combination ( Fig 5G ) drove GC dependent circadian transcription , while the other constructs were not sensitive to DEX ( 1x E-box , 4x E-box ) or did not show rhythmic expression ( 1x E-box , 1x GRE , 4x GRE ) . Thus , a synergistic interaction between a single E-box and a GRE is sufficient to drive GC-dependent circadian gene expression . To begin to explore mechanistic aspects of this interaction between E-Boxes and GREs , we examined the bioluminescence patterns driven by the combined E-box/GRE element upon entrainment by different light intensities of the light part of the LD cycle and upon changing distances between the two elements . Interestingly , at the highest light intensity examined , oscillation behaviour was less pronounced ( S7A and S7B Fig ) . Two-way ANOVA analysis indicated that there was no interaction between GC dose ( 10–60 nM ) and light intensity effects ( 150–1500 lux , S7B Fig ) . Examination of constructs with different E-box/GRE spacings revealed a slightly less robust oscillation behaviour when the two elements were separated by 10 bps , as in the original construct , than at the other distances ( 5 , 15 and 20 bp; S7C and S7D Fig ) . These results indicate that the E-box/GRE module is sensitive to light conditions and that interaction between the two elements might be hampered when they are placed relatively nearby on the same side of the DNA helix . To examine whether the interaction between E-boxes and GREs also occurs in vivo , we generated larvae carrying stable genomic insertions of the 1xE-box/GRE reporter . Both in wild-type siblings and in rx3 strong mutants , the construct drove rhythmic expression ( Fig 5I ) , indicating that the low levels of cortisol present in the mutants [15] are still sufficient for activation of the construct . Importantly however , the amplitude of expression was clearly reduced in the mutants ( Fig 5J ) . Strikingly , the amplitude difference between mutants and wild-type siblings disappeared when the larvae were treated with DEX during the experiment ( Fig 5I and 5J ) . Thus , E-box/GRE driven bioluminescence rhythms mimic the rhythmicity behavior of many metabolic genes from the RNA-Seq analysis ( Fig 1F ) , suggesting that the E-box-GRE combinations enriched in the GC-dependent diurnal gene set mediate highly efficient regulatory inputs for this expression pattern .
Our data reveal a strong impact of GCs on diurnal gene transcription and key metabolite levels . About 44% of all genes with diurnal expression patterns are dysregulated in GC deficient larvae , and almost half of these can be rescued by constant chronic DEX treatment . This result shows that a surprisingly large part of the GC-dependent diurnal transcriptome does not depend on the diurnal pattern of GC levels themselves . Thus , GCs may have a permissive rather than instructive role in the diurnal expression of these genes . This mechanism of regulation may prevent these genes from being inappropriately regulated by irregular GC rises , such as those during acute stress responses . Recent studies have described direct interactions of circadian clock factors with GRs , leading to circadian modulation of GC signaling [21 , 22 , 23] . For example , circadian Pck1 expression was attributed to direct inhibitory regulation of the GR by CRY binding [22] . Interestingly , the Pck1 promoter also contains an E-box element which is conserved between fish and mammals . Our in vivo reporter gene analysis shows that a simple E-box-GRE combination is sufficient to mediate GC dependent circadian luciferase expression . However , we did not observe circadian regulation of reporter expression driven by four concatemerized GREs . Endogenous Cry levels in zebrafish fibroblasts may not be high enough to mediate transcriptional repression of the 4xGRE reporter in vivo , in contrast to the elevated CRY levels upon overexpression used in mouse fibroblasts studies reported previously [22] . Remarkably , GR binding to the GRE in the mouse Pck1 gene is highest at the time when CRYs reach peak levels and reduced in CRY double knock-out mice [22] . Stable CRY-GR interactions may occur at the promoter itself , where the Clock-Bmal1 bound to the E-box may increase the local concentration of CRY proteins and thereby facilitate their interactions with GRs binding a neighbouring GRE . The increased co-occurrence of E-boxes and GREs in the genes rescued by constant DEX treatment argues in favor of such a mechanism . Of note , zebrafish Cry1a has been implicated in mediating a negative regulatory influence of light on the circadian clock by its interaction with the Clock-Bmal dimer [24] , a function that may underlie our observation of reduced oscillations upon exposure to light-dark cycles with higher light intensity . Furthermore , our data indicate that interactions between the GR and the clock machinery may be hampered when the two elements are located nearby on the same side of the DNA helix , as elements separated by 10 bp show slightly less robust oscillations than those separated by 5 bp or 15 bp . Further studies targeted at identifying the full set of factors involved in regulation of transcription by the variants of the element will likely reveal more mechanistic details of the interactions between GCs and the clock in transcriptional regulation . While the constant DEX treatment clearly influenced gene expression and metabolite patterns , it did not lead to any visible perturbation of the larval phenotype , nor did it affect circadian rhythms of S-phase in the wild-type [15] . Effects of prolonged DEX treatment may appear only at later developmental stages or in adulthood , and only be noticeable upon challenges to the organism . This is seen in humans or other mammals exposed to high DEX levels during development , which later in life show increased disease susceptibility and altered responses to stress [25 , 26] . The DEX-treatment related changes to metabolites or gene expression patterns revealed within our data set may provide interesting starting points for understanding the mechanistic principles of such long-term effects . Constant DEX treatment rescues circadian rhythms of S-phase in rx3 strong mutants [15] . Our transcriptome analysis reveals that a number of cell cycle genes , related to all phases of the cell cycle , show a similar pattern , as do numerous genes acting in metabolic pathways implicated in the regulation of cell proliferation . Surprisingly , fewer cell cycle genes than metabolic genes were affected by the loss of GCs . Also , more of the affected metabolic genes were rescued by constant DEX treatment . This indicates that GC dependent metabolic control may play a more important role in circadian cell cycle rhythms than GC regulation of cell cycle genes . Strikingly , of the metabolites examined , only glutamine showed a restoration of both overall levels and rhythmicity by constant DEX treatment . Glutamine plays a major role in cell proliferation related pathways such as purine synthesis or anaplerosis of the TCA cycle . Thus , it emerges as a potential key metabolite in the circadian orchestration of cell proliferation . The presence of a circadian rhythm of glutamine levels in human blood [27] and in rat liver [28] suggests that such functions are conserved across evolution . Intriguingly , we observed a strong accumulation of glyoxylate in rx3 strong mutants , which was prevented by DEX treatment . This glyoxylate accumulation may reflect the bypassing of part of the disturbed TCA cycle by the so-called glyoxylate cycle . However , apart from C . elegans , the presence of this cycle in metazoans is controversial , and orthologues of the glyoxylate cycle enzymes isocitrate dehydrogenase and malate synthase are reported to be absent or pseudogenes in vertebrates [29] . Interestingly , the zebrafish genome contains a potentially functional malate synthase-like sequence ( ENSDARG00000074684 ) , which showed DEX rescue of a dysregulated expression pattern in rx3 strong mutants ( S1 Table ) . Also peroxisomal and mitochondrial glyoxylate detoxification is dysregulated in the mutants , involving decreases in expression of glyoxylate metabolizing enzymes and increases in glyoxylate producing enzymes . Importantly , three of the dysregulated genes encode enzymes linked with human genetic disorders of glyoxylate detoxification ( alanin-glyoxylate amino transferase [agxtb] , glyoxylate reductase/hydroxypyruvate reductase [grhprb] and 4-hydroxy-2-oxoglutarate aldolase 1 [hoga1] , which are involved in human primary hyperoxalurias type I-III , respectively [17] ) . With one exception ( grhprb ) , constant DEX rescues expression patterns of all these genes . Thus , GCs may be a promising candidate for treatment of patients in which insufficient amounts of functional protein are produced [17 , 30] . Another intriguing finding is our observation that the aberrant accumulation of both BCAA and aromatic AA levels in the mutants could not be restored by DEX treatment . Dysregulation of these metabolites may reflect a perturbation of other regulatory inputs in addition to GCs in the secondary AI model . Alternatively , our temporally constant GC replacement by DEX may not be sufficient for proper functional restoration of all pathways influencing BCAA and AAA levels . It will be interesting to explore if other treatment schemes are more efficient in restoring BCAA and AAA levels , and whether these amino acids also show disturbed regulation in human patients . In summary , our study establishes zebrafish larvae as an easily accessible model for studies targeting metabolic aspects of GC related disease and GC therapy . In addition , our work reveals a massive impact of GCs on the diurnal patterns of gene transcription . Surprisingly , a large part of this diurnal regulation does not require changing levels of GCs themselves , and we provide a model based on simple enhancer element interactions that can explain this behavior .
Animal experiments were conducted in accordance with German animal protection regulations and approved by local regulatory authorities ( Regierungspräsidium Karlsruhe , approval number Aktenzeichen 35–9185 . 81/G-83/14 and 35–9185 . 81/G-242/15 ) . Fish ( AB wild-type and the mutant lines rx3t25327/t25327 [rx3 strong] and rx3t25181/t25181 [rx3 weak] [14] ) were raised and bred as described [31] . Total RNA extraction , cDNA synthesis and qPCR were carried out as described [32] . Primer sequences used were: β-actin: fw: 5’-gcctgacggacaggtcat-3 , rv:’ 5’-accgcaagattccataccc-3’; apoa1: fw: 5’-cttgacaacctggacggaac-3 , rv: 5’-gcatattcctggagcttggt-3; arntl1a: fw: 5’-tagagcgctgtttgctgatg-3’ , rv:5’-gacccgtggacttcagtgac-3; cyp3a: fw: 5’-ccaaagacaacacgaagcag-3’ , rv: 5’-acaagatctcgtggtcactcag-3’; rbp4: fw: 5’-ccgaagatccagctaagttca-3 , rv:’ 5’-caatgatccagtggtcgtca-3’; si:dkey-18a10 . 3: fw: 5’-ctttgtgcgccaactcaac-3’ , rv: 5’-tttaggcaagccggagtcta-3’; pck1: fw: 5’-tgacgtcctggaagaacca-3’ , rv: 5’-gcgtacagaagcgggagtt-3’; gls2a: fw:5’-gacatgacagcagctcttgact-3’ , rv: 5’-tgcctgactcacatgtcacc-3’ . Synthesis of probes against lhx4 , apoa1 , and zgc:158293 and whole-mount in situ hybridization was carried out following Armant et al . [33] . 1 dpf rx3 mutant and wildtype sibling embryos from pooled clutches were separated and transferred into cell culture flasks containing E3 medium ( 5 mM NaCl , 0 . 17 mM KCl , 0 . 33 mM CaCl2 , 0 . 33 mM MgSO4 , 0 . 1% methylene blue ) . For GC treatment , the medium was supplemented with either 25 μM Dexamethasone ( DEX ) solved in 0 . 1% DMSO or 0 . 1% DMSO alone as a control . Embryos/larvae were kept at 28°C in an incubator under a 12 h light:12 h dark ( LD ) exposure . Larvae were sampled in liquid nitrogen at the indicated Zeitgeber times ( ZT , ZT3 = 3 hours after lights on ) starting at 5 dpf and stored at -80°C until further processing . To ensure efficient and reproducible sample preparation for NMR metabolomics of zebrafish larvae , we have established an extraction protocol that generates highly reproducible data . 50 larvae per sample were collected in homogenization tubes ( PeqLab , #91-PCS-CK14 ) , snap-frozen in liquid nitrogen and lyophilized overnight to avoid recovery of enzymatic activity . Lyophilized samples were stored at -80°C for a maximum of 2 days . For extraction , 1 ml of acetonitrile/water ( 1:1 ) and ceramic beads ( #91-PCS-CK14 , PeqLab ) were added to the larvae and extracted with a liquid nitrogen cooled cell shaker ( Precellys 24 , PeqLab ) according to the manufacturer’s instructions , with the following settings: 6 , 000 rpm , 4x20 s , 120 s . Homogenates were vortexed for 1 min and incubated on ice for 10 min . Next , the samples were transferred into fresh vials , and were briefly centrifuged at 4°C to remove debris . 750 μl of supernatant were transferred into fresh vials , and 620 μl of ultrapure water ( HPLC grade ) were added . Samples were vortexed before lyophilization overnight . For measurements , 650 μL of D2O/buffer ( 1 . 5 M KH2PO4 , 2 mM NaN3 , 0 . 1% ( v/v ) TSP ( = 3 , 3- ( trimethylsilyl ) -2 , 2' , 3 , 3'-tetradeuteropropionic acid ) in D2O ) ( 9:1 ) were added to the extracts , and 600 μl of this mixture were transferred into a 5 mm standard NMR tube . Spectra were recorded on a Bruker Avance III 600 spectrometer equipped with a 1H , 13C , 15N-TCI triple resonance cryoprobe . 1D spectra were recorded with 64k data points , 90 . 5 receiver gain and 32 scans for comparing the phenotypes and 64 scans for comparing the DEX treatment at 300 K using a 1D NOESY experiment with presaturation for water suppression . A mixing time of 10 ms and a prescan delay of 10 s were used . Pulse length was determined automatically by the Bruker AU program pulsecal and presaturation was set corresponding to a 25 Hz pulse . Irradiation frequency for water suppression was optimized prior to acquisition . Spectra were processed identically with an exponential apodization function with line broadening of 0 . 3 Hz . Automatic phasing , baseline correction and referencing were done by the Bruker AU program apk0 . noe . Additionally , J-resolved spectra were recorded with identical pulse lengths , presaturation , irradiation frequency and receiver gain as the corresponding 1D NOESY . Spectra were recorded with 8192 data points in the direct dimension and 40 increments in the indirect dimension . One scan was acquired per increment . Spectra were processed with 16k × 256 data points and a sine window function in both dimensions . We employed ultra-performance liquid chromatography with fluorescence detection ( UPLC-FLR ) for targeted quantification of amino acids and ketoacids ( S8 Fig ) and ion chromatography with conductivity detection ( IC-CD ) for quantification of other organic acids . 30 larvae per sample ( in triplicates ) were collected for absolute quantification of amino acids and α-ketoacids ( glyoxylate ) and for organic acid content each . For extraction of free amino acids and α-ketoacids 300μl 0 . 1M HCl was used . Derivatization and separation of amino acids was performed as described by Yang et al . [34] . For derivatization of α-ketoacids 150 μl of the acidic extract was mixed with an equal volume of 25 mM OPD ( o-phenylendiamine ) solution and incubated at 50°C for 30 min . The derivatized α-ketoacids were separated using an Acquity HSS T3 column ( 100 mm x 2 . 1 mm , 1 . 7 μm , Waters ) connected to an Acquity H-class UPLC system . Prior separation , the column was heated to 40°C and equilibrated with solvent A ( 0 . 1% formic acid in 10% acetonitrile ) at a flow rate of 0 . 55 ml/min . Separation of α-ketoacid derivates was achieved by increasing the concentration of solvent B ( acetonitrile ) in solvent A as follows: 2 min 2% B , 5 min 18% B , 5 . 2 min 22% B , 9 min 40% B , 9 . 1min 80% B and hold for 2min , and return to 2% B in 2 min . The separated derivates were detected by fluorescence ( Acquity FLR detector , Waters , excitation: 350 nm , emission: 410 nm ) and quantified using ultrapure standards ( Sigma ) . For quantification , a linear seven-point calibration curve ranging from 0 . 3–15 pmol on column was used ( R2 > 0 . 99 ) . Data acquisition and processing was performed with the Empower3 software suite ( Waters ) . Organic acids were extracted with 700 μl ultra-pure water for 20 min at 95°C . These compounds were separated using an IonPac AS11-HC ( 2mm , ThermoScientific ) column connected to an ICS-3000 system ( Dionex ) and quantified by conductivity detection after cation suppression ( ASRS-300 2mm , suppressor current 95–120 mA ) . Prior separation , the column was heated to 30°C and equilibrated with 5 column volumes of solvent A ( ultra-pure water ) at a flow rate of 0 . 38 ml/min . Separation of anions and organic acids was achieved by increasing the concentration of solvent B ( methanol ) and solvent C ( 100mM NaOH ) in buffer A as follows: 8 min 4% C , 11 min 10% C , 18 . 2 min 20% B / 18 . 1% C , 27 . 5 min 20% B / 21% C , 32 min 24% C , 43 min 30% C , 47 min 40% C , 48 min 90% C for 8 min , and return to 4% C in 9 min . A linear three-point calibration curve was used for quantification of organic acids ( 0 . 5–5nmol on column; R2 > 0 . 99 ) Data acquisition and processing was performed with the Chromeleon 6 . 7 software ( Dionex ) . Rhythmic properties of metabolite levels were assessed with the model selection based method described in Atger et al . [16] ( see also below , Rhythmicity assessment in different genotypic backgrounds ) . Library preparation and sequencing were performed by the IGBMC Microarray and Sequencing Platform and by the Next Generation Sequencing and Genomics facility of the BioInterfaces research programme at KIT . RNA integrity numbers measured with a 2100 Bioanalyzer ( Agilent ) were 10 for all samples . cDNA libraries were generated using the directional mRNA-seq sample preparation kit ( #15018460 , Rev . A , October 2010 , Illumina ) . Single-end 54 nt reads were obtained with a Genome Analyzer IIx . Zebrafish cells were maintained as described [44] in Leibowitz’s ( L-15 ) medium ( Life Technologies , #11415–049 ) supplemented with antibiotics and 15% ( PAC2 cells ) or 17% ( AB . 9 GRE:Luc cells ) Fetal bovine serum ( %[v/v] , FBS , Biochrom AG , #S0115 ) . Bioluminescence studies for cells and larvae were carried out as reported previously [32 , 44 , 45] . In case of multiple comparisons , p-values were adjusted using the Benjamini-Hochberg method [40] . | Glucocorticoids , steroid hormones of the adrenal gland , are important regulators of metabolism and the stress response . They are also widely used as anti-inflammatory drugs . Production and release of glucocorticoids show a diurnal pattern regulated by the circadian clock . Importantly , altered daily patterns of hormone action are thought to contribute to metabolic diseases . Here , we examined diurnal patterns of gene expression and metabolism in a zebrafish model of glucocorticoid deficiency . We observed that a surprisingly large number of genes show glucocorticoid-dependent diurnal patterns of transcription . This behaviour is particularly pronounced in metabolic genes , and metabolites of various central metabolic pathways are dysregulated . Interestingly , non-rhythmic glucocorticoid treatment restored many of these metabolite changes . It also restored expression of almost half of the dysregulated genes . Using in vivo bioluminescence assays , we provide evidence that this rescue is mediated via combined glucocorticoid–circadian clock activity on a simple regulatory DNA module enriched in the genomic vicinity of these genes . Our work provides mechanistic insight into how the circadian clock and glucocorticoids cooperate in the regulation of daily patterns of gene expression and metabolism . Furthermore , it highlights metabolic pathways that may contribute to disease mechanisms in patients with a disturbed glucocorticoid hormone system . | [
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] | 2016 | Extensive Regulation of Diurnal Transcription and Metabolism by Glucocorticoids |
During development , progenitor expansion , lineage allocation , and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function . Pancreatic dysfunction results in some of the most debilitating and fatal diseases , including pancreatic cancer and diabetes . Several transcription factors regulating pancreas lineage specification have been identified , and Notch signalling has been implicated in lineage allocation , but it remains unclear how these processes are coordinated . Using a combination of genetic approaches , organotypic cultures of embryonic pancreata , and genomics , we found that sphingosine-1-phosphate ( S1p ) , signalling through the G protein coupled receptor ( GPCR ) S1pr2 , plays a key role in pancreas development linking lineage allocation and specification . S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification . S1pr2-mediated stabilisation of the yes-associated protein ( YAP ) is essential for endocrine specification , thus linking a regulator of progenitor growth with specification . YAP stabilisation and endocrine cell specification rely on Gαi subunits , revealing an unexpected specificity of selected GPCR intracellular signalling components . Finally , we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels , thus regulating lineage allocation . Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage . These findings identify S1p signalling as a novel key pathway coordinating cell survival , lineage allocation , and specification and linking these processes by regulating YAP levels and Notch signalling . Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches .
The pancreas is the origin of some of the most debilitating and fatal diseases , including pancreatic cancer and diabetes . Understanding the signalling pathways and gene regulatory networks underlying pancreas development will shed light in the origins of these diseases and suggest novel therapeutic approaches . Mouse reverse genetics and studies in humans have uncovered multiple transcription factors that regulate formation of the pancreatic anlagen and its subsequent expansion , branching morphogenesis , and cell specification into the endocrine , acinar , and ductal lineages [1 , 2] . Several extracellular signals have also been implicated [2 , 3] , but the molecular mechanisms coordinating lineage allocation with lineage specification remain elusive . The early pancreatic multipotent progenitor cells ( MPCs ) emerge at the posterior foregut region of the definitive endoderm and are defined by the expression of the transcription factors Pdx1 , Ptf1a , and Sox9 [4–8] . Maintenance of high Notch signalling is necessary for the expansion of MPCs to form a tree-like branched epithelium and prevent early differentiation [9–12] . The Hippo signalling effectors , the transcription factor TEAD1 and its coactivator yes-associated protein ( YAP ) , activate key pancreatic signalling mediators and transcription factors to regulate expansion of pancreatic progenitors [13] , but the signal ( s ) regulating YAP stability are not known . Decreased Notch activity at the tips of the epithelium and the antagonistic functions of Ptf1a and Nkx6 transcription factors delineate the acinar progenitor and endocrine/duct bipotent trunk territories [14–17] . In the trunk , differential Notch signalling enables progenitors to differentiate into ductal and endocrine cells . High Notch levels divert cells to the duct fate through repression of the expression of the Ngn3 transcription factor . Cells escaping high Notch levels induce Ngn3 and become endocrine progenitors [18] . A Notch-mediated posttranslational mechanism for Ngn3 stabilisation has been proposed [19] . Endocrine progenitors provide Notch-dependent and Notch-independent feedback to maintain proliferative growth of the bipotent cell population [20–22] . Despite the central role of Notch signalling , it is not understood whether levels of Notch activity are intrinsically regulated as a function of time or whether unidentified extracellular signals play a role in this process . We used genetic approaches and organotypic cultures of mouse embryonic pancreata to reveal the implication of sphingosine-1-phosphate ( S1p ) signalling in progenitor survival , differentiation and lineage allocation . Our data show that S1p signals through the S1p receptor 2 ( S1pr2 ) and YAP to up-regulate connective tissue growth factor ( CTGF ) that participates in mediating survival of endocrine and acinar progenitors . Signalling specifically through Gαi and YAP stabilisation is necessary for endocrine specification . Additionally , S1p signalling attenuates Notch signalling to regulate lineage allocation . Both YAP stabilisation and Notch attenuation are necessary for endocrine and acinar specification . Taken together , the data uncover a novel mode of coordinating lineage allocation and cell specification through S1p signalling and link these processes with tissue growth control .
S1p is a lipid mediator secreted in the extracellular milieu , where it interacts with the S1p G protein-coupled receptors to regulate different cellular responses such as migration , survival , and differentiation [23 , 24] . Because S1pr2-mediated S1p signalling guides the migration of endocrine progenitors to form islets [25] , we decided to examine its implication in earlier stages of pancreas development . We first tracked its expression during mouse pancreas development by following β-galactosidase activity in heterozygous S1pr2tm1lacz [26] ( S3 Table ) embryos and by quantitative PCR ( qPCR ) analysis in wild-type ( wt ) embryos . Consistent with an early role for this receptor in mesenchymal growth [27] , S1pr2 tm1lacz expression was predominantly mesenchymal from 9 . 5 days post coitum ( dpc ) to 12 . 5 dpc ( S1 Fig ) . During secondary transition , its expression was significantly up-regulated , and in the developing epithelium , it was colocalised with both trunk and tip progenitors ( Fig 1A–1C , S1 Fig ) . By 16 . 5 dpc , Lacz expression was eliminated in nascent endocrine cells and significantly reduced in the rest of the developing organ , whereas it was completely abolished by postnatal day 1 ( P1 ) ( S1 Fig ) . qPCR analysis confirmed this pattern , clearly indicating that S1pr2 expression in the developing pancreas peaked during secondary transition ( Fig 1C ) . To further investigate the epithelial expression of S1pr2 , we isolated by fluorescence-activated cell sorting ( FACS ) the epithelial and mesenchymal components of the developing pancreas at 13 . 5 , 14 . 5 , and 15 . 5 dpc ( S1 Fig ) . Consistent with the S1pr2tm1lacz expression pattern , we found that S1pr2 expression also peaked at 14 . 5 dpc in the epithelium ( S1 Fig ) . S1p is a systemically circulating sphingolipid generated by platelets and endothelial cells but it may also be generated in situ . Sphingosine kinases ( Sphk ) 1 and 2 catalyse the formation of secreted S1p using endogenous cellular sphingosine [28] . Analysis of Sphk1 and Sphk2 expression by qPCR showed that their expression peaked during secondary transition and suggested a predominantly epithelial expression ( Fig 1F , S1 Fig ) . Immunostainings at 12 . 5 , 14 . 5 . and 16 . 5 dpc confirmed the predominantly epithelial expression of Sphk during that time ( Fig 1D and 1E and S1 Fig ) . Taken together , the temporal expression profile of S1pr2 and Sphks suggested that this signalling pathway may act as an autocrine signal to mediate pancreas specification . Expression of both S1pr2 and Sphks in the developing epithelium peaked at 14 . 5 dpc , suggesting that this time point was optimal to address their possible role in pancreas specification . To address the role of S1pr2 in pancreas development , we first compared the RNA Seq gene expression profiles of S1pr2tm1Rlp ( S3 Table ) null [29] embryonic pancreata ( S2 Fig ) with those of 13 . 5 , 14 . 5 , and 15 . 5 dpc wt embryonic pancreata . Principal component analysis ( PCA ) suggested that S1pr2 null pancreata were developmentally delayed ( Fig 2A ) . Endocrine and acinar lineage commitment genes were strongly down-regulated . Ductal specification was much less affected , and the expression of important duct markers such as Hnf1b , Car2 , and Slc9a1 was not affected ( Fig 2B–2D , S1 Table ) . Surprisingly , expression of transcription factors and other genes implicated in epithelial progenitor specification and maintenance was not significantly affected ( S2 Fig , S1 Table ) [30] . Consistent with that , organ size , epithelial proliferation assessed by pH3 immunofluorescence , and cell survival assessed by TUNEL assays were not affected ( S2 Fig ) . These findings suggested that expansion of the progenitor population in the S1pr2tm1Rlp null pancreata was not affected , but lineage commitment was severely delayed . S1pr2 null embryos develop to full term but show perinatal mortality and display a range of phenotypes , particularly in the hematopoietic and vascular systems [26 , 29] . Surprisingly , immunofluorescence analysis of S1pr2 tm1Rlp null pancreata at P1 showed no major defects in the development of the endocrine , acinar , and ductal lineages . Additionally , fasting glucose levels and glucose tolerance tests in the surviving adults failed to uncover glucose homeostasis defects , suggesting no gross alterations , at least concerning endocrine cell function ( S2 Fig ) . To investigate whether systemic signals were necessary for functional compensation , we used serum free air–liquid interface ( ALI ) cultures of embryonic pancreata . ALI cultures mimic normal embryonic development and thus also give a means to temporally manipulate signalling conditions and elucidate their effects on lineage specification [25 , 31] . Wt 14 . 5 dpc pancreata in ALI cultures continue their developmental program over the course of 6 d , giving rise to acinar , endocrine , and duct cells ( S3 Fig ) . However , S1pr2tm1Rlp 14 . 5 dpc null pancreata failed to develop normally , because both the endocrine and acinar lineages were severely compromised and there was a strong increase in the number of duct-like cells ( S3 Fig ) . This was consistent with the strong down-regulation of endocrine and acinar differentiation markers at 14 . 5 dpc ( Fig 2B and 2C ) and in contrast to the eventually normal development of S1pr2tm1Rlp null pancreata in utero . We next looked for up-regulated related receptors that may mediate functional compensation of the S1pr2 null mutation and rescue the early pancreatic phenotype of the S1pr2 nulls . S1pr2 belongs to the family of the closely related lysophospholipid ( LPL ) receptors ( LPLrs ) that have similar expression patterns and overlapping functions [32 , 33] . RNA Seq data showed that only S1pr3 and lysophosphatidic receptor 1 ( Lpar1 ) , which are functionally related to S1pr2 [32] , were expressed to any appreciable extent and to levels comparable to that of S1pr2 in the 14 . 5 wt pancreata ( S1 Table ) . Interestingly , S1pr3 and Lpar1 expression increased in the 14 . 5 dpc S1pr2tm1Rlp null total pancreata ( S2 Fig ) . To independently confirm that this up-regulation was a consequence of abrogating S1pr2 signalling , we acutely blocked S1pr2 function by injecting intraperitoneally ( ip ) pregnant dams at 13 . 5 , 14 . 5 , and 15 . 5 dpc with either 2 or 5 mg/Kg body weight of the specific S1pr2 antagonist JTE013 [34] . In both treatments , expression of both S1pr3 and Lpar1 was up-regulated in 16 . 5 dpc pancreata ( S2 Fig ) . Expression of S1pr3 and Lpar1 is primarily mesenchymal in 13 . 5 , 14 . 5 , and 15 . 5 dpc wt pancreata ( S2 Fig ) , but , strikingly , their expression was selectively up-regulated , by 5- and 7-fold , respectively , in the epithelium of 14 . 5 dpc S1pr2 null pancreata ( Fig 2E and S2 Fig ) . To establish that these receptors mediate the recovery of the endocrine lineage , we blocked their function in ALI cultures by applying for 3 d the specific S1pr3 and Lpar1 inhibitors VPC23019 and Ki16425 at 50 μM and 20 μM , respectively , in ALI cultures of 13 . 5 dpc wt and S1pr2 null pancreata . Either inhibitor on its own was not sufficient to block endocrine specification , but when used in combination , they essentially shut off endocrine specification , specifically in S1pr2 null pancreata but not in wt pancreata ( Fig 2F–2J ) , demonstrating that S1pr3 and Lpar1 mediate the functional compensation in the S1pr2 nulls . These findings established an important role for S1p signalling in the specification of pancreas progenitors and uncovered extensive functional compensation among specific LPL receptors in pancreas development . To fully address the role of S1pr2 signalling in pancreas development , we decided to isolate systemic signals mediating the functional compensation in S1pr2 null embryos using ALI cultures . The presence of 15 μM JTE013 in ALI cultures of 14 . 5 dpc S1pr2 null pancreata did not alter their growth , survival , and differentiation patterns , further confirming its specificity ( S3 Fig ) . In contrast , blocking S1pr2 function in wt pancreata resulted in obvious morphological defects , most notably the absence of light-reflective , dense cell clusters ( S3 Fig ) . The progression of the developmental program in ALI cultures of wt 14 . 5 dpc pancreata is apparent 2 d following culture initiation . The segregation of the endocrine and ductal lineages from the Nkx6 . 1+ bipotent progenitors has progressed and is shown by the reduction in the numbers of Nkx6 . 1+ and Ngn3+ cells , the increase of strongly labelled Pdx1+ cells , and the appearance of numerous C-pep+ and Gcg+ as well as CK19+ cells . Acinar cells become numerous , as seen by the appearance of Ptf1a+/Pdx1- and Amy+ cells ( compare Fig 3A–3D with 3E–3H , see also S4 Fig and Fig 3M–3P ) [16] . However , in the absence of S1pr2 signalling , development of both acinar and endocrine lineages was blocked , as shown by the loss of Amy+ and C-Pep+ , whereas the development of CK19+ duct-like cells did not appear affected ( S4 Fig ) . These findings correlated with the persistence of a large number of Nkx6 . 1+ cells , the loss of Ptf1a+/Pdx1- cells , the reduction of strongly labelled Pdx1+ cells as well as the dramatic reduction in the number of Ngn3+ cells ( compare Fig 3E–3H with 3I–3L , see also S4 Fig and Fig 3M–3P ) . Additionally , there was a marked decrease of epithelial proliferation and the appearance of apoptotic cells ( S4 Fig ) . To reveal the full extent of the changes , we analysed the transcriptome of ALI pancreatic explants cultured for 2 d . Similarly to the transcriptome analysis of S1pr2 null pancreata at 14 . 5 dpc , the expression of progenitor markers , upon S1pr2 blocking with 15 μM of JTE013 , was only mildly affected , with the striking exception of the transcription factor Nkx6 . 2 and the Notch membrane-bound ligand Dll1 , two genes that are important for the segregation of the bipotent trunk population from the acinar progenitor pool and Notch-mediated lineage segregation ( S4 Fig , S1 Table ) [16 , 18] . On the other hand , and in agreement with the transcriptome analysis of S1pr2 null pancreata at 14 . 5 dpc , there was coordinated repression of transcription factors and terminal differentiation markers of the endocrine and acinar lineages . In contrast , the ductal lineage was much less affected because , similarly to S1pr2 null phenotype at 14 . 5 , expression of several important genes , such as Hnf1b , Car2 , and Slc9a1 , was not affected ( Fig 3Q–3S , S1 Table ) . Consistent with an important role of S1pr2 signalling for lineage commitment during secondary transition , the total number of regulated genes in its absence increased by 2 . 5-fold at 14 . 5 dpc + 2 d ( S4 Fig ) . After 6 d in culture in the absence of S1pr2 signalling , beta cells and acinar cells were virtually eliminated , and the explants were taken over by a greatly expanded number of CK19+ duct-like cells ( compare Fig 4A–4C , Fig 4E–4G and 4N ) . Additionally , cell survival was severely compromised at that stage , as shown by TUNEL assays ( Fig 4D and 4H ) . To establish that S1p is the ligand mediating these effects , we supplemented the S1pr2-blocked ALI cultures with 20 μM S1p and achieved a rescue of both the acinar and endocrine lineages ( S3 Fig , S7 Fig ) . These findings showed that S1p signalling , through S1pr2 , was necessary for both cell survival and lineage commitment . To address whether these effects were distinct from one another , we sought means to rescue the cell survival independently of cell specification . CTGF was a plausible candidate to participate in mediating survival , as it has been implicated as such a signal downstream of S1p signalling and it is expressed in the developing pancreas in both the epithelium and the mesenchyme ( S5 Fig ) [35] . Importantly , its expression was significantly repressed ( 3 . 5-fold ) upon blocking S1pr2 signalling for 2 d in ALI 14 . 5 dpc explants ( S5 Fig ) . Addition of 50 ng/ml CTGF at the onset of the ALI culture in the absence of S1pr2 signalling eliminated cell death 6 d later ( Fig 4D , 4H and 4L ) . However , this was not sufficient to reverse the early differentiation block , and results were very similar in this respect . After 2 d in culture , there were again very few Ngn3+ cells , no Ptf1a+/Pdx1- cells , the number of strongly labelled Pdx1+ cells did not increase compared to the 14 . 5 dpc stage , and proliferation rates in the epithelium remained low ( compare Fig 3G , 3K , 3H and 3L with S5 Fig and S5 Fig with S4 Fig ) . Consistent with that , after 6 d in culture , the endocrine cells , beta cells in particular , and acinar cells were again missing , whereas the numbers of duct-like cells remained greatly expanded ( compare Fig 4A and 4B to 4E , 4F , 4I and 4J , see also Fig 4N ) . These findings , the loss of Ptf1a+ cells , and the persistence of a large number of Pdx1+ and Nkx6 . 1+ cells ( Fig 4C , 4G and 4K , S5 Fig and Fig 4M ) suggest that cells are stabilised in a trunk bipotential identity when S1pr2 signalling is blocked but survival is rescued by CTGF . To further corroborate that cell survival was mediated independently of cell fate commitment , we compared gene expression changes of 14 . 5 + 2 d ALI embryonic pancreata cultured in conditions of S1pr2 block in the presence or absence of 50 ng/ml CTGF . Gene expression changes of progenitor , endocrine , acinar , and duct markers [30] were strikingly similar in the two conditions as compared to untreated controls ( S5 Fig , S1 Table ) . Furthermore , PCA showed that S1pr2 blocked explants with or without CTGF clustered much closer together ( PC2 , 16% variance ) than with control pancreata ( PC1 , 68% variance ) ( S5 Fig ) . To establish that S1p signalling regulates expression of CTGF , we used 20 μM of S1p in combination with 15 μM of JTE013 in 14 . 5 dpc ALI cultures and showed that CTGF expression was restored to normal levels by S1p ( S5O Fig ) . These data showed that S1p signalling through S1pr2 mediates cell fate commitment to the endocrine and acinar lineages . Independently of that , it promotes progenitor cell survival at least partly through up-regulation of CTGF . As shown above , genetic inactivation of S1pr2 results in strong developmental delay , particularly for the endocrine and acinar lineage specification , but this is compensated for by up-regulation of the expression of the related receptors S1pr3 and Lpar1 specifically in the epithelium . To bypass this compensation and identify the downstream components of S1pr2 signalling , we first targeted the function of Gαi subunits that are common mediators among these receptors [32] and can be specifically inactivated by pertussis toxin A ( PTX ) -mediated adenosine diphosphate ( ADP ) -ribosylation [36 , 37] . We previously showed that conditional expression of a single allele of the ROSA26LSLPTX transgene [38] ( S3 Table ) in endocrine progenitors using the Ngn3-Cre driver mouse line TgNgn3Cre [39] ( S3 Table ) or a low ( 5 μg/ml ) PTX dose in embryonic pancreatic explants resulted in disruption of endocrine cell migration to form islets [25] . Expression of a single allele of the ROSA26LSLPTX transgene in epithelial progenitors using the Pdx1-Cre driver TgPdx1Cre [40] ( S3 Table ) had a similar effect ( S6 Fig ) , but neither treatment had any effect on pancreas lineage specification [25] . Strikingly , activation of two alleles of the ROSA26LSLPTX transgene using the same driver resulted in specific and strong loss of C-pep+ and Gcg+ endocrine but not Amy+ acinar or CK19+ duct cells at P1 ( Fig 5A and 5B , S6 Fig ) . ALI cultures of 14 . 5 dpc embryonic pancreata expressing two PTX alleles in Pdx1+ progenitors also generated very few endocrine cells after 6 d in culture without loss of Amy+ acinar or CK19+ duct cells ( Fig 5C–5E , S6 Fig ) . Loss of endocrine cells was preceded by a dramatic reduction in the number of Ngn3+ cells ( Fig 5F and 5G , S6 Fig ) . To assess whether loss of the Ngn3+ progenitors in the TgPdx1Cre/ROSA26LSLPTX/LSLPTX embryos was due to disruption of signalling events directly responsible for Ngn3 induction or defective competence of Pdx1+ progenitors acquired in earlier stages of pancreas development , we cultured 14 . 5 dpc wt embryonic pancreata in ALI culture in the presence of 10 μg/ml dose of PTX . The effects of this treatment closely matched the genetic experiment , as the number of C-Pep+ and Gcg+ cells was also dramatically reduced , but the number of Amy+ acinar or CK19+ duct cells was not affected ( Fig 5C and 5H , S6 Fig ) . Again , this was preceded by a dramatic reduction in the number of Ngn3+ cells ( Fig 5I , S6 Fig ) . Thus , Gαi-mediated signalling controls both endocrine specification and migration in a dose-dependent manner . Irrespective of the dose , neither acinar nor ductal specification was affected in either the genetic or ALI paradigms . The data described so far showed that S1p signalling through S1pr2 plays an important role in pancreas progenitor survival at least partly through CTGF transcriptional activation and in triggering progenitor specification . Additionally , our findings show that endocrine specification in particular is specifically mediated by Gαi subunits . We next sought to determine the transcriptional mediator of this signalling cascade . The transcriptional responses following S1p activation are not fully understood , but recent work has shown that S1p receptors can stabilise the transcriptional coactivator YAP , a central player in tissue growth control [41–45] . YAP is a component of the Hippo signalling cascade that phosphorylates it , leading to its retention in the cytoplasm and its degradation by the proteasome [46] . Additionally , CTGF is a direct target of YAP transcriptional activation , mediating cell survival in different cellular contexts [47–49] . Importantly , the TEAD obligatory YAP transcriptional partners have been shown to bind to multiple enhancer elements of genes implicated in pancreas development [13] . Expression of YAP can be detected from the early stages of pancreas development , but , similarly to S1pr2 and Sphk expression , it peaks during secondary transition , and transcript levels are significantly higher in the epithelium ( S7 Fig ) . Immunostaining with E-cadherin clearly showed that the protein is stabilised and detectable only in the epithelium ( Fig 6A ) . Therefore , we asked whether S1pr2 signalling regulates YAP levels and , therefore , YAP-mediated transcriptional regulation . RNA Seq and qPCR results clearly showed that YAP expression was not affected in either S1pr2 nulls , S1pr2 blocked ALI cultures , or Gαi blocked ALI cultures ( S7 Fig ) . In contrast , YAP protein levels were significantly reduced in both S1pr2 null pancreata at 14 . 5 dpc and 14 . 5 pancreatic explants subjected to S1pr2 signalling block for 2 d in culture ( Fig 6A , 6B , 6D , 6E and 6G ) . YAP target genes such as CTGF , Cdk6 , and Ccnd1 identified in other contexts [47 , 50 , 51] as well as Sox9 and CTGF in pancreas development [13] were transcriptionally down-regulated in S1pr2 blocked explants ( S2 Table ) . To show direct dependence of YAP stability upon S1p signalling , we titrated the amount of exogenous S1p and found that 20 μM S1p in explants treated with 15 μM JTE013 was sufficient to rescue YAP protein stability ( Fig 6D–6F and 6G ) . The determined binding affinities are slightly higher for S1p 34 , 52 and therefore in these concentrations S1p is in small molecular excess . Additionally , S1p , being the natural ligand , should diffuse faster in the explant tissue . This finding further confirmed that YAP protein stability depends on S1p signalling through S1pr2 . S1pr2 signalling can be conveyed through Gαq , Gα12/13 , and Gαi subunits [52] , and we sought to determine whether selective blocking of each of these Ga subunits would affect YAP protein stability . Blocking of the Gαq subunits with 50 uM of the specific Gαq antagonist-2A ( GP2A ) [53] or blocking the function of Gα12/13 subunits with 5 ug/ml of the C3 exoenzyme [54] in explants had no effect on YAP stability ( S7 Fig ) . In contrast , inactivation of Gαi subunits by 10 μg/ml PTX resulted in a dramatic loss of YAP protein stabilisation , showing that signalling specifically through Gαi subunits is an important component of YAP stabilisation ( Fig 6C and 6G ) . To address the potential role of YAP in pancreas specification , we first sought to rescue the protein stability in ALI embryonic pancreas explants where S1pr2 signalling was blocked . Addition of 2 d of 1 μM of the reversible proteasome inhibitor MG132 , used previously to restore YAP stability [55] , resulted in the YAP stabilisation ( Fig 6G ) . Strikingly , this restored specifically endocrine but not acinar specification ( Fig 6H and 6I , S7 Fig ) , suggesting that S1pr2 signalling acts through Gαi and YAP stabilisation to promote the endocrine lineage . To directly address this , we used the TgPdx1CreERT2 Cre driver and the YAPfl/fl allele ( S3 Table ) to temporally inactivate YAP and bypass its early role in the expansion of the pancreatic epithelium [13] . Following tamoxifen administration permissible for normal pregnancy and embryo development [56] , the TgPdx1CreERT2 [57] allele partially labelled all three lineages in the presence of a conditional ROSA26LSLtdTomato reporter allele ( S3 Table ) at similar efficiencies ( S7 Fig ) . Thus we used TgPdx1CreERT2 and tamoxifen administration to inactivate YAP ( S7 Fig ) in a temporally delayed manner . Whereas there was no effect on the number of Ngn3+ cells at 14 . 5 dpc ( S7 Fig ) , the number of endocrine Cpeptide+ and Gcg+ cells was reduced by nearly 40% at P1 compared to controls ( Fig 6J–6L ) . The number of Amy+ acinar and CK19+ duct cells was not affected , demonstrating the specific role of YAP in endocrine specification ( S7 Fig ) . There was no cell death in these pancreata , suggesting that the remaining YAP+ epithelial cells generated sufficient secreted CTGF and/or other secreted factors to maintain cell survival . To further confirm that S1p signalling acts through YAP in endocrine specification , we treated 14 . 5 dpc YAP-deleted pancreata with 20 μM S1p for 6 d in culture . This treatment failed to restore the loss of endocrine cells , confirming that YAP mediates the S1p effects on endocrine specification ( S7 Fig ) . Taken together , these results reveal a distinct mechanism whereby S1p signalling acts through Gαi subunits to stabilise YAP , which in turn is necessary for endocrine specification . Importantly , YAP destabilisation does not affect the number of Ngn3+ cells , suggesting that a distinct S1p-dependent pathway regulates Ngn3 expression . Disruption of S1pr2 signalling dramatically reduced the number of Ngn3+ cells ( Fig 3G , 3K and 3O ) . YAP destabilisation could not account for this effect , as shown above by the conditional inactivation of YAP . Additionally , after S1pr2 signalling disruption , Ngn3 gene expression was only marginally affected , and there was a large increase in the number of duct-like cells ( Figs 3G , 3K , 3O , 3Q , 4B , 4F and 4N ) . Both of these effects could be a result of high Notch activity that has been shown to destabilise Ngn3 and induce the formation of supernumerary duct cells [18 , 19 , 58 , 59] . To investigate this , we activated Notch signalling above the endogenous levels using 10 μM of the soluble Notch ligand peptide Delta-Serrate/lag-2 ( DSL ) in ALI cultures of 14 . 5 dpc pancreata . This peptide is derived from the Jag-1 ligand domain , and it also corresponds to the conserved , receptor binding part of the other Notch ligands , thus eliciting canonical Notch signalling [60 , 61] . This activation resulted in a very similar phenotype to that of blocking S1pr2 signalling , disrupting endocrine and acinar specification . Ngn3+ cells were essentially eliminated ( S8 Fig and Fig 7B ) , and the number of endocrine and acinar cells was drastically reduced in favour of a greatly expanded population of duct-like cells ( compare Fig 4A , 4B , 4E and 4F with S8 Fig , see also Fig 7C ) . Therefore , we next determined whether S1pr2 signalling was necessary to down-regulate Notch signalling . Notch levels normally drop , concurrently with differentiation , on 14 . 5 dpc explants maintained for just 1 d in culture , as shown by Notch intracellular domain ( NICD ) and Hes-1 immunofluorescence ( Fig 8B , 8C , 8E and 8F ) . Notch down-regulation failed to take place in explants with S1pr2 signalling block ( Fig 8H and 8I ) but was restored by the addition of 20 μM of S1p ( S8 Fig ) , further establishing the down-regulation of Notch signalling by S1p . Because transcript levels of Notch signalling components did not point to an overall transcriptional regulation of Notch by S1pr2 signalling ( S1 Table ) , we investigated whether such regulation may occur posttranscriptionally . Sel-1 suppressor of lin-12-like ( Sel1l ) is a member of the endoplasmic reticulum ( ER ) -associated protein degradation ( ERAD ) pathway , first identified in Caenorhabditis elegans as a negative posttranscriptional regulator of Notch signalling [62 , 63] . Sel1l is expressed in the epithelium of the developing pancreas [64] ( Fig 8A ) , and its disruption resulted in loss of endocrine and acinar differentiation and prolongation of the progenitor cell state [65] , a phenotype strikingly similar to that of the S1pr2 signalling block . Consistent with a role in differentiation , Sel1l is up-regulated in differentiating pancreas explants ( Fig 8D ) . S1pr2 signalling block resulted in the loss of the Sel1l protein , concomitant with maintenance of Notch signalling ( Fig 8G ) . Further confirming this finding , activation of the Notch signalling above the endogenous levels with the addition of 10 μM of DSL resulted in a strong decrease of Se1l1 levels ( S8 Fig ) . Moreover , addition of 20 μM of S1p restored Se1l1 levels , confirming its dependence upon S1p signalling ( S8 Fig ) . Se1l1 protein levels were also restored in the absence of S1pr2 signalling by 1 μM of the specific proteasome inhibitor MG132 ( Fig 8J ) , and this was sufficient to restore attenuation of Notch signalling ( Fig 8K and 8L ) . Therefore , S1pr2 signalling stabilises the Sel1l protein that in turn mediates the Notch signalling down-regulation that is necessary for acinar and endocrine differentiation . These results suggested that S1pr2 signalling mediates Notch down-regulation to induce endocrine and acinar differentiation but did not clarify whether this was sufficient and whether Notch signalling affected YAP levels . To answer these questions , we first blocked Notch signalling without affecting endogenous S1pr2 signalling using 10 μM of the gamma-secretase inhibitor DAPT in ALI cultures of 14 . 5 dpc pancreata [19 , 66] . This induced a large increase in the number of Ngn3+ cells and resulted in preferential endocrine differentiation ( Fig 7B and 7D ) ( compare Fig 4A , 4B , 4E and 4F with Fig 7E and 7F and compare S4 Fig with S8 Fig , see also Fig 7C ) . We then addressed whether Notch down-regulation in the absence of S1pr2 signalling would be sufficient to initiate and maintain differentiation . We found that despite the large number of Ngn3+ cells at 14 . 5 dpc + 2 d explants ( Fig 7B and 7G ) and the transient initiation of differentiation ( compare S4 Fig with S8 Fig ) , neither endocrine nor acinar cell differentiation were maintained ( Fig 7F and 7I ) , suggesting that Notch down-regulation is not sufficient but acts in concert with other effectors of S1pr2 signalling . We have shown above that with regard to endocrine specification YAP plays an important role . Importantly , we have further found that YAP levels depend upon S1pr2 but not Notch signalling . YAP levels did not change when Notch signalling alone was inhibited or when Notch signalling was inhibited concurrently with S1pr2 inhibition ( Fig 7A ) . This suggested that YAP levels are regulated by S1pr2 signalling through the Gαi function and not Notch attenuation ( Fig 6G ) . Taken together , these experiments showed that S1pr2 signalling attenuates Notch activity through the protein stabilisation of its negative regulator Sel1l . Both this attenuation and the YAP stabilisation are necessary for the specification of the endocrine and acinar lineages .
During development , cell fate decisions need to be coupled to the implementation of differentiation programs so that different cell types in each organ are generated in the appropriate numbers . Notch signalling is widely employed as a regulator of binary cell fate decisions , and it also plays a key role in sequential lineage segregation during pancreas development [9–12 , 14 , 18 , 19] . How Notch activity is coordinated with the implementation of specification programs is not well understood . Furthermore , signalling molecules that trigger lineage differentiation remain largely elusive . Our findings revealed the implication of the S1p signalling pathway in key pancreas developmental decisions . We found that the S1p signalling pathway has a dual role in pancreas development by regulating lineage allocation and differentiation and thus coordinating these processes . Consistent with the notion that S1p is an important signalling molecule for pancreas development , expression of the S1p generating enzymes at both the protein and the mRNA levels is substantially higher in the epithelium and gradually increases during the secondary transition . We found that S1pr2 is the main S1p receptor expressed in the developing epithelium . Development of the acinar and endocrine lineages was severely delayed in S1pr2 null embryos , but specific up-regulation of the functionally related receptors S1pr3 and Lpar1 in the epithelium compensated and eventually restored pancreas development . Acute S1pr2 blocking in wt explants prevented this compensation , resulting in complete loss of the endocrine and acinar lineages and a massive appearance of duct-like cells . Down-regulation of Sox9 expression may contribute to the loss of endocrine and acinar lineages [67] . The remaining cells are clearly of ductal identity , although ductal gene expression is perturbed consistent with the interdependence of pancreatic lineage development [22] . This experimental set up provided us with a means to unravel the downstream events . Specific blocking of the S1pr2 downstream effector Gαi , either in explants or genetically , dramatically and selectively reduced endocrine lineage specification , suggesting that different Ga subunits mediate distinct aspects of pancreas development that could be exploited in stem cell differentiation protocols . Loss of S1pr2 signalling or Gαi function resulted in destabilisation of the YAP transcriptional coactivator . Inactivation of the Hippo pathway by pancreas selective disruption of the Mst1/2 kinases led to increased levels of YAP in acinar cells and their subsequent ductal metaplastic conversion [68 , 69] . However , the role of YAP during pancreas development has not been genetically explored . Genetic temporal inactivation of YAP specifically reduced endocrine specification without affecting the appearance of Ngn3+ cells . YAP stabilisation by proteasome inhibition in the absence of S1pr2 signalling restored endocrine specification . Furthermore , additional S1p signalling could not restore endocrine specification levels in the YAP-deleted pancreata , establishing that an S1p to S1pr2 , Gαi , and YAP stabilisation cascade is necessary for the endocrine differentiation program to be triggered ( Fig 9 ) . YAP mRNA levels are significantly higher in the epithelium , and these findings explain the selective importance of the S1p signalling/YAP axis for the development of the epithelium . S1p signalling block was accompanied by cell death and down-regulation of CTGF , a secreted survival factor and known YAP target gene [47–49] . Exogenous addition of CTGF in S1pr2 blocked pancreata rescued the cell death but not the specification defects underlining distinct functions of S1p signalling in progenitor survival and specification ( Fig 9 ) . Several effects of S1pr2 signalling loss would be consistent with maintenance of high Notch activity , raising the possibility that S1p signalling regulates Notch . We found that S1p signalling is necessary for the maintenance of Sel1l protein levels , a member of ERAD pathway , and negative Notch regulator [62 , 63] . Sel1l deficiency impaired endocrine and acinar differentiation and maintained the progenitor cell state [65] . Consistent with that , S1p signalling attenuates Notch activity , a necessary step to trigger differentiation of the acinar and endocrine lineages [9–12 , 14 , 18 , 19] . Rescue of the Sel1l protein levels by proteasome inhibition restored Notch down-regulation in the absence of S1pr2 signalling . However , Notch down-regulation on its own was not sufficient to sustain endocrine and acinar differentiation in the absence of S1pr2 signalling , demonstrating that another effector of the latter is also necessary for endocrine specification , and we have shown that this effector is Gαi , which mediates YAP stabilisation ( Fig 9 ) . Interestingly , Ngn3 protein levels are dependent upon Notch signalling but not on YAP , suggesting that S1p-dependent YAP stabilisation may be acting as a primer of the endocrine program that is then realised in the cells that stabilise Ngn3 protein levels . Developmental pathways are often employed in adult tissues during regeneration , and their application has also been pivotal in the generation of glucose responsive beta-like cells from human pluripotent stem ( PS ) cells [70–72] . During pancreas development , TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors to regulate the expansion of pancreatic MPCs that will generate all the pancreatic lineages . After the onset of differentiation , YAP is rapidly degraded in acinar and endocrine cells but is retained in ductal cells [13 , 68 , 69] . Our findings suggested an additional role for YAP in activating the endocrine differentiation program and identified S1p as an important signal that maintains its stability through the secondary transition . This work identified a new signalling pathway in pancreas development that leads to YAP stabilisation and Notch attenuation in the developing pancreas , thus linking lineage allocation and differentiation with a molecular player central for tissue growth control . These findings have important implications for the efficient generation of endocrine cells from pluripotent stem cells and for pancreas regeneration .
Animal maintenance and experimentation were conducted in accordance with the FELASA recommendations and the ethical and practical guidelines for the care and use of laboratory animals set by the competent veterinary authorities in the authors’ institutions . Animal maintenance and experimentation were conducted in compliance with the FELASA recommendations and the ethical and practical guidelines for the care and use of laboratory animals set by the competent veterinary committees in the author’s institutions . Mouse mutant and transgenic lines used were S1P2tm2Ytak [73] , YAPfl/fl [74] , TgPdx1CreERT2 [57] , S1pr2tm1Rlp [29] , and Gt ( ROSA ) 26Sortm1 ( ptxA ) Cgh [38] purchased from the Mutant Mouse Regional Resource Center ( MMRRC; Chapel Hill , NC ) and Gt ( ROSA ) 26Sortm9 ( CAG-tdTomato ) hZE [75] and Tg ( Pdx1-cre ) 6Tuv [76] purchased from The Jackson Laboratory ( Bar Harbor , Maine ) ( S3 Table ) . All mouse strains used were interbred onto the same genetic background ( C57BL/6J ) . Genotyping was performed by conventional PCR on genomic DNA isolated from mouse-tails using standard procedures . Briefly , mouse tails were dissolved in Tail buffer ( 100 mM Tris-HCl pH 8 . 0 , 200 mM NaCl , 5mM EDTA , and 0 . 2% SDS ) with 50 μg/ml Proteinase K ( Sigma ) overnight at 55°C . Following a protein extraction step with Phenol/Chloroform ( Sigma ) , genomic DNA was precipitated from the aqueous phase with 100% ethanol and finally resuspended in TE buffer ( 10 mM Tris-HCl pH 8 . 0 and 1 mM EDTA ) . PCR conditions and primers used are provided in S4 Table . Tamoxifen ( Sigma ) was diluted in corn oil and injected ip in pregnant dams for 6 consecutive days starting at 10 . 5 dpc with 1 mg , followed by 0 . 5 mg in the following days . To ensure homogeneity in the analysis , all newborns were collected either following delivery or by caesarean section at 19 . 5 dpc ( P1 ) . JTE013 was diluted in PBS and injected ip at 2 or 5 mg/Kg body weight in pregnant dams at 13 . 5 , 14 . 5 , and 15 . 5 dpc . Embryos were collected at 16 . 5 dpc for analysis . Dorsal pancreatic buds were dissected under a light stereoscope ( Leica MZ75 ) at 13 . 5 or 14 . 5 dpc and cultured for 1 , 2 , 3 , or 6 d on 0 . 4-μm pore diameter filters ( Millicell-CM; Millipore ) in DMEM ( Gibco ) supplemented with N2 ( Gibco ) and streptomycine-penicillin-glutamine ( Gibco ) . Treatments were with JTE013 ( Tocris ) at 15 μM , CTGF ( Peprotech ) at 50 ng/ml , PTX ( Calbiochem ) at 10 μg/ml , DSL ( CDDYYYGFGCNKFCRPR ) ( AnaSpec , AS-61298 ) at 10 μM , DAPT ( Sigma ) at 10 μM , C3 exoenzyme ( Biomol ) at 5 μg/ml , GP antagonist-2A ( Enzo ) at 50 μM , VPC23019 ( Avanti Lipids ) at 50 μM , Ki16425 ( Sigma ) at 20 μM , MG132 ( Sigma ) at 1 μM and S1P ( Tocris ) at 20 μM . Controls were treated with the same amount of solvent where appropriate . Chemicals were replenished with each medium change daily for 2-day and 3-day cultures or every second day for 6-day cultures . Dissected pancreata were fixed in 4% paraformaldehyde ( PFA; Sigma ) , washed in PBS , and dehydrated in 30% sucrose ( Sigma ) overnight at 4°C . Tissues were embedded in optimal cutting temperature ( OCT ) compound ( BDH ) , cut into sections of 12-μm thickness and mounted onto Superfrost slides ( VWR ) for storage at −80°C . For immunostainings , cryosections were post-fixed in 4% PFA for 5 min and blocked for 1 h at room temperature in 10xBlocking solution ( 10% goat serum , 0 . 1% BSA , and 0 . 3% Triton X-100 in PBS ) . Primary antibodies were diluted in 1xBlocking solution and incubated o/n at 4°C , whereas secondary antibodies , also diluted in 1xBlocking solution , were incubated for 2 h at room temperature . All washes were done in PBST ( PBS with 0 . 3% Triton X-100 ) . After the final washes , slides were mounted with DAPI ( Vectashield; Vector or Prolong Gold antifade reagent with DAPI; Molecular Probes ) and taken for fluorescent microscopy . Dissected pancreata were fixed in 10% formalin ( Sigma ) for 1 h at 4°C , washed in PBS , and taken into 70% ethanol . Tissues were then gradually dehydrated in a series of ethanol concentrations ( 70% , 95% , and 100% for 10 min each ) , before transferring in xylene for 10 min and then into liquid paraffin for an additional 10 min . Sections of 6-μm thickness were cut on a Leica RM2265 microtome and mounted onto polylysine-treated slides ( VWR ) . For immunofluorescence , slides were incubated with Xylene for 20 min for deparafinisation and then immersed in a reverse series of ethanol concentrations ( 100% , 95% , 70% , and 50% ) for rehydration followed by washes in PBS . Sections were then processed for immunofluorescence as described for cryosections , with the addition of an antigen-retrieval and a signal-amplification step . For antigen retrieval , slides were immersed in 10 mM Sodium citrate pH 6 . 0 , boiled in a microwave oven at 600 W for 4 min , and then left to simmer at 250 W for 15 min . Slides were allowed to cool at room temperature for 40 min and then washed in distilled water and finally in PBS before proceeding with blocking . For amplification , a biotinylated secondary antibody ( Vector ) was used , followed by incubation with an avidin/biotin-HRP enzyme complex ( ABC kit; Vector ) for 1 h at room temperature . Slides were then washed in PBST and incubated with Tyramide-Cy3 diluted at 1:50 in TSA buffer ( TSA kit; Perkin Elmer ) for 3 min at room temperature to achieve HRP signal amplification . After a final wash in PBST , slides were mounted with DAPI ( Vectashield ) and taken for fluorescent microscopy . Morphometric analysis on embryonic pancreata and pancreatic explants was performed using immunofluorescent images taken at saturation and the ImageJ ( Fiji ) software . More specifically , the “Analyse Particles” tool of ImageJ was used to quantitate the number of pixels that correspond to signal area on an immunofluorescent image . For the 14 . 5 dpc pancreata and all pancreatic explants , analysis was performed using at least three 12-μm-thick cryosections per sample 24 μm apart , spanning the entire tissue . For the 19 . 5 dpc and P1 pancreata , analysis was performed using at least six 12-μm-thick cryosections per sample , 120 μm apart , spanning the entire tissue . For endocrine , acinar , and ductal cell quantitation , the signal area of C-peptide/Glucagon- , amylase- , and Cytokeratin19-positive immunofluorescence , respectively , was measured and divided by the corresponding total signal area for DAPI , thus normalising for mass . The same procedure was followed for Pdx1- , Ptf1a- , Nkx6 . 1 , and YAP-signal area quantitation . For quantitation of epithelial mitotic activity , PH3-positive nuclei were counted and divided over the DAPI area of the epithelium as marked by E-cadherin co-staining . Similarly , for endocrine progenitor quantitation , Ngn3-positive nuclei were counted and divided over total DAPI area . To quantify the recombination efficiency , double Tdt + each marker ( Cpeptide+Glucagon , Amylase , CK-19 ) positive cells were counted and this number divided for the total number of cells expressing each marker . For each quantitation , at least three pancreata of each genotype and/or condition were analysed . Total RNA was prepared using the RNeasy kit with on-column genomic DNA digestion following the manufacturer’s instructions ( Qiagen ) . First strand cDNA was prepared using Superscript II RT ( Invitrogen ) . Real-time PCR primers were designed using the Primer 3 software ( SimGene ) , specificity was ensured by in silico PCR , reactions were performed with SYBR-Greener ( Invitrogen ) using an ABI PRISM 7000 machine or a Roche LC480 machine , and primary results were analysed using the machine’s software . Reactions were carried out from at least four independent samples with the exception of embryonic pancreata from JTE013 injected dams where three samples were used . Absolute expression values were calculated using the ΔCt method using β-actin for normalisation except for the embryonic pancreata from JTE013 injected dams where Eif3 was used . Primers used were further evaluated by inspection of the dissociation curve . Primer sequences were as follows: S1pr2 F: CACTACAATTACACCAAGGAGAC , R: CAGCACAAGATGATGATGAAGG; Sphk1 F: AGAAGGGCAAGCATATGGAA , R: ACCATCAGCTCTCCATCCAC; Sphk2 F: ACTGCTCGCTTCTTCTCTGC , R: GCCACTGACAGGAAGGAAAA; Lpar1 F: TCTTCTGGGCCATTTTCAAC , R: TCCTGGGTCCAGAACTATGC; S1pr3 F: GGGAGGGCAGTATGTTCGTA , R: GGCATCATATGGCCTCATCT; Yap1 F: GCGGTTGAAACAACAGGAAT , R: TGCTCCAGTGTAGGCAACTG; Pdx1 F: TCCACCACCACCTTCCAG , R: CAGGCTCGGTTCCATTCG; Ctgf F: AGCGTCCAGACACCAACCT , R: GGTAGGAGGATGCACAGCAG; β-actin F: TGGCTCCTAGCACCATGA , R: CCACCGATCCACACAGAG Eif3 F: TCTCCGGCCGTACCGGCTAA , R: GAGCTGGCGTGGATGGGGTG Embryonic pancreata ( 13 . 5 , 14 . 5 , or 15 . 5 dpc ) from up to three pups of the same genotype were pooled to generate one sample . For ALI cultures , up to three pancreata having undergone the same treatment ( normal medium , JTE013 or JTE013 , and CTGF supplemented ) were pooled to generate one sample . Three independent samples from each stage/genotype or ALI condition were used as biological replicates . Total RNA was prepared using the RNeasy kit with on-column genomic DNA digestion ( Qiagen ) , and only RNA with an integrity number of ≥8 was used . mRNA was isolated from 1 ug total RNA by poly-dT enrichment using the NEBNext Poly ( A ) mRNA Magnetic Isolation Module according to the manufacturer’s instructions . Final elution was done in 15 ul 2x first strand cDNA synthesis buffer ( NEBnext , NEB ) . After chemical fragmentation by incubating for 15 min at 94°C , the sample was directly subjected to the workflow for strand-specific RNA-Seq library preparation ( Ultra Directional RNA Library Prep , NEB ) . For ligation , custom adaptors were used ( Adaptor-Oligo 1: 5'-ACA-CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-3' , Adaptor-Oligo 2: 5'-P-GAT-CGG-AAG-AGC-ACA-CGT-CTG-AAC-TCC-AGT-CAC-3' ) . After ligation , adapters were depleted by an XP bead purification ( Beckman Coulter ) adding bead in a ratio of 1:1 . Indexing was done during the following PCR enrichment ( 15 cycles ) using custom amplification primers carrying the index sequence indicated with ‘NNNNNN’ . ( Primer1: Oligo_Seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT , primer2: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT , primer3: CAAGCAGAAGACGGCATACGAGAT NNNNNN GTGACTGGAGTT ) . After two more XP beads purifications ( 1:1 ) , libraries were quantified using Qubit dsDNA HS Assay Kit ( Invitrogen ) . Libraries were equimolarly pooled and sequenced on an Illumina HiSeq 2500 , resulting in ca . 16–20 million single end reads per library . After sequencing , FastQC ( http://www . bioinformatics . babraham . ac . uk/ ) was used to perform a basic quality control on the resulting reads . As an additional control , library diversity was assessed by redundancy investigation in the aligned reads . Alignment of the short reads to the mm10 reference was done with GSNAP ( v 2014-12-17 ) [77] , and Ensembl gene annotation version 69 was used to detect splice sites . The uniquely aligned reads were counted with featureCounts ( v1 . 4 . 6 ) [74] and the same Ensembl annotation . Normalisation of the raw read counts based on the library size and testing for differential expression between the different conditions was performed with the DESeq2 R package ( v1 . 6 . 2 ) [78] . To calculate the number of regulated genes between two conditions , we accepted a maximum of 10% false discoveries ( padj ≤ 0 . 1 ) and considered only genes with normalised counts >100 in either condition and fold change <0 . 6 or >1 . 6 . Unknown transcripts , pseudogenes , and RNAs other than mRNAs were not considered . For the PCA , the raw read counts of the conditions to be analysed and their replicates were transformed with the variance stabilising transformation of the DESeq2 package . The top 500 genes that showed the highest variance were selected and used for the PCA calculation . TUNEL assays on cryosections were performed using a fluorescent cell death detection kit ( Roche ) according to the manufacturer’s instructions . Briefly , cryosections were washed in PBS and incubated in Permeabilisation solution ( 0 . 1% Triton X-100 in 0 . 1% Na-citrate ) for 2 min at 4°C . For labelling of DNA strand breaks , slides were incubated with 50 μl of TUNEL reaction mixture ( fluorescein-labelled nucleotides mixed with terminal deoxynucleotidyl transferase ) for 1 h at 37°C . Slides were finally rinsed in PBS , mounted with DAPI ( Vectashield ) , and analysed by fluorescent microscopy . Slides that were incubated with DNase I prior to the labelling reaction were used as a positive control . Protein extraction , gel electrophoresis , and immunoblotting procedures were according to standard protocols . For protein extraction , samples were lysed in RIPA buffer ( 50 mM Tris-HCl pH 7 . 4 , 1% NP-40 , 0 . 5% Na-deoxycholate , 0 . 1% SDS , 150 mM NaCl , and 2 mM EDTA ) and supplemented with protease and phosphatase inhibitor cocktails ( Sigma ) . Proteins were loaded at 30 μg/lane on a polyacrylamide gel for electrophoresis , and protein transfer was subsequently performed on a standard nitrocellulose membrane ( Amersham ) . Blocking was performed in 5% milk in TBST ( 50 mM Tris-HCl pH 7 . 6 , 150 mM NaCl , and 0 . 05% Tween-20 ) for 1 h at room temperature , and primary and secondary antibodies were diluted in 1% milk in TBST and incubated overnight at 4°C or for 2 h at room temperature , respectively . Signal was developed using the ECL kit ( Perkin Elmer ) according to the manufacturer’s instructions . Primary antibodies used were rabbit anti-Yap1 ( 1:1000; Proteintech ) , rabbit anti-S1P2 ( 1:500; Thermo Fisher Scientific ) , rat anti-E-Cadherin ( 1:2 , 000; Zymed ) , and mouse anti β-actin ( 1:5 , 000; Santa Cruz ) . Secondary antibodies were anti-rabbit , anti-rat , and anti-mouse horseradish peroxidase ( HRP ) -conjugated goat antibodies ( 1:5 , 000; Dako ) . The epithelial and mesenchymal components of embryonic and cultured pancreata were separated by FACS based on the epithelial expression of Cd49f as described [79] . Briefly , pancreata were dissected and incubated in a 0 . 05% Trypsin/EDTA solution ( Gibco ) for 5 min at 37°C . The reaction was terminated with 10% FBS ( Sigma ) , tissues were dispersed with mild pipetting , and single cells were collected by centrifugation ( 400 g; 4 min ) . Cells were resuspended in PBS with 0 . 5% FBS , and after blocking with rat IgG ( Abcam ) for 15 min , cells were incubated with a Cd49f-FITC ( BD Pharmigen ) antibody , added at 1:50 dillution for 1 h on ice with gentle rocking . Cells were collected by centrifugation , resuspended in PBS with 0 . 5% FBS , and sorted by FITC fluorescence intensity on a BD FACS Aria III , following standard procedures [79] . Two fractions were collected consisting of fluorescent ( epithelial ) and non-fluorescent ( mesenchymal ) cells , which were subsequently processed for RNA isolation and real-time PCR analysis . The efficiency of the separation was independently confirmed by determining Pdx1 expression in the two fractions . Cryosections of embryonic pancreata were washed in PBS and incubated with 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside ( X-Gal; Roche ) in staining solution [5mM K3Fe ( CN ) 6 , 5mM K4Fe ( CN ) 6 , 2 mM MgCI2 , and 5 mM EGTA] for 8 h at 37°C . For double stainings , X-gal–treated sections were rinsed in PBS , incubated in blocking solution ( 10% normal goat serum , 0 . 3% Triton X-100 ) for 1 h at room temperature , and subsequently processed for immunofluorescence as described . For imaging , X-gal stainings were photographed in bright field , and the image was inverted and assigned to the green colour of the RGB composite . This was then merged with the immunofluorescent image ( in red ) and the DAPI image ( in blue ) of the corresponding field photographed under UV . Statistical significance in morphometric analyses and real-time PCRs was determined by the Student’s t test for two-tailed distributions of unpaired groups . Error bars represent the standard error of the mean ( SEM ) . p < 0 . 05 was considered significant . Primary antibodies used were rabbit anti-Ngn3 ( 1:100; Acris ) , rat anti-E-cadherin ( 1:400; Zymed ) , rabbit anti-C-peptide ( 1:200; Linco ) , rabbit anti-Amylase ( 1:300; Sigma ) , rabbit anti-Ptf1a ( 1:3 , 000; Gift from B . Breant ) , rat anti-Cytokeratin19 ( 1:250; DSHB ) , mouse anti-Glucagon ( 1:500; Sigma ) , rabbit anti-Pdx1 ( 1:5 , 000; Gift from C . Wright ) , mouse anti-Pdx1 ( 1:250; DSHB ) , rabbit anti-Sphk ( 1:250; Abcam ) , mouse anti-Nkx6-1 ( 1:1 , 000; DSHB ) , mouse anti-Insulin ( 1:1 , 000; Sigma ) , rabbit anti-PH3 ( 1:500; Cell Signaling ) , rabbit anti-Yap1 ( 1:200; Cell Signaling ) , rabbit anti-NICD ( 1:100; Cell Signaling ) , rat anti-Hes1 ( 1:500; MBL-Biozol ) , and rabbit anti-Sel1l ( 1:250; Abcam ) . Secondary antibodies were anti-mouse , anti-rabbit , and anti-rat Alexa-488- , Alexa-568- , and Alexa-633-conjugated goat antibodies ( 1:500; Molecular Probes ) , as well as anti-rabbit and anti-rat biotinylated goat antibodies ( 1:100; Vector ) . For Nkx6 . 1 and Pdx1 mouse antibody staining together with Sphk , a Goat anti-Mouse IgG affiniPure Fab Fragment from Jackson ImmunoResearch ( 115-007-003 ) was used at 1:50 dilution following the instructions of the manufacturer . | The pancreas develops from a field of progenitor cells localised in a restricted region of the embryonic endoderm . These progenitor cells proliferate and eventually differentiate to generate the three distinct lineages comprising the endocrine ( which include insulin-producing β cells ) , acinar , and ductal cells . The molecular pathways implicated in the early generation of pancreas progenitors and their proliferation are well understood . It is also known that the Notch signalling pathway is implicated in sequential binary cell fate decisions that generate the three lineages; however , other signals that may regulate this process remain unknown . Here , we show that a phospholipid , sphingosine-1-phosphate ( S1p ) , generated by the progenitor cells themselves , acts as a signal necessary to define the acinar and endocrine lineage . We observe that in the absence of S1p only duct cells are generated and the survival of pancreas progenitors is compromised . The function of this signalling pathway in the generation of the endocrine cells is two-fold . Firstly , it stabilises yes-associated protein ( YAP ) , a transcriptional gene coactivator known to regulate pancreatic progenitor proliferation , and we show that this stability is necessary for the activation of the endocrine specification program . Secondly , it attenuates Notch signalling , allowing the generation of endocrine and acinar cells . Notch attenuation is necessary for the stabilisation of the transcription factor Ngn3 , which is required for the generation of endocrine cells . We conclude that S1p acts as an autocrine signal regulating YAP stabilisation and Notch attenuation to mediate pancreas specification . Understanding lineage allocation and specification in the pancreas will shed light on the origins of pancreatic diseases and may suggest novel therapeutic approaches . | [
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] | 2017 | Pancreas lineage allocation and specification are regulated by sphingosine-1-phosphate signalling |
Individual metazoan transcription factors ( TFs ) regulate distinct sets of genes depending on cell type and developmental or physiological context . The precise mechanisms by which regulatory information from ligands , genomic sequence elements , co-factors , and post-translational modifications are integrated by TFs remain challenging questions . Here , we examine how a single regulatory input , sumoylation , differentially modulates the activity of a conserved C . elegans nuclear hormone receptor , NHR-25 , in different cell types . Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C . elegans SUMO ( SMO-1 ) as an NHR-25 interacting protein , and showed that NHR-25 is sumoylated on at least four lysines . Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1 , demonstrating that sumoylation has been strongly conserved within the NR5A family . We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription , and that NHR-25 activity was enhanced in vivo upon loss of sumoylation . Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells ( VPCs ) during development . Importantly , however , overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate . Moreover , SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs . That is , accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development , but as cells began dividing , a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs , intermediate levels in 2° fated VPCs , and low levels in 3° fated VPCs . We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner .
Tissue-specific and cell type-specific transcriptional networks underlie virtually every aspect of metazoan development and homeostasis . Single TFs , operating within gene-specific regulatory complexes , govern distinct gene regulatory networks in different cells and tissues; thus , combinatorial regulation underpins tissue- and cell type-specific transcription . Determining the precise mechanisms whereby such specificity arises and how networks nevertheless remain flexible in responding to environmental and physiological fluctuations is an interesting challenge . TFs integrate signaling information from co-factors , chromatin , post-translational modifications , and , in the case of nuclear hormone receptors , small molecule ligands , to establish transcription networks of remarkable complexity . Here , we approach this problem by studying a covalent modification of a nuclear hormone receptor ( NHR ) in C . elegans , a simple metazoan with powerful genetic tools , a compact genome , and an invariant cell lineage leading to well-defined tissues . NHRs are DNA-binding TFs characterized by a zinc-finger DNA binding domain ( DBD ) and a structurally conserved ligand binding domain ( LBD ) [1] . The genome of C . elegans encodes 284 NHRs while humans only have 48 NHRs [1] . Of the 284 NHRs , 269 evolved from an HNF4α-like gene [2] , and 15 have clear orthologs in other species . NHR-25 is the single C . elegans ortholog of vertebrate SF-1/NR5A1and LRH-1/NR5A2 , and arthropod Ftz-F1 and fulfills many criteria for the study of tissue-specific transcriptional networks [1] . NHR-25 is broadly expressed in embryos and in epithelial cells throughout development [3] , [4] . It is involved in a range of biological functions such as molting [3]–[5] , heterochrony [6] , and organogenesis [7] . Furthermore , both NHR-25 and its vertebrate orthologs regulate similar processes . SF-1 and NHR-25 promote gonadal development and fertility [8] , [9] , while NHR-25 and LRH-1 both play roles in embryonic development and fat metabolism [4] , [10]–[12] . The pleiotropic phenotypes seen following RNAi or mutation of nhr-25 highlight the broad roles of the receptor , and its genetic interaction with numerous signaling pathways ( β-catenin , Hox , heterochronic network ) [6]–[8] make it an excellent model to study combinatorial gene regulation by NHRs . SUMO ( small ubiquitin-like modifier ) proteins serve as post-translational modifiers and are related to but distinct from ubiquitin [13]; we show here that NHR-25 is sumoylated . Sumoylation uses similar enzymology as ubiquitination to conjugate the SUMO protein onto substrate lysines [13] . Briefly , SUMO is produced as an inactive precursor . A SUMO protease activates SUMO by cleaving residues off the C-terminus to expose a di-glycine [13] . A heterodimeric E1 protein consisting of UBA2 and AOS1 forms a thioester bond with the exposed diglycine and then transfers SUMO to an E2 enzyme ( UBC9 ) , also through a thioester bond [14] . The E2 enzyme then either directly conjugates SUMO onto a target lysine , or an E3 ligase can enhance the rate of sumoylation; that is , unlike in ubiquitination , E3 ligases are not always required . Like many post-translational modifications , sumoylation is reversible and highly dynamic . The same SUMO protease that initially activated SUMO cleaves the isopeptide linkage that covalently attaches SUMO to the target protein [14] . Indeed , global failure to remove SUMO from substrates compromises viability in mice and S . pombe [15] , [16] . The extent of sumoylation of a given target can be regulated by varying the expression , localization , stability or activity of components of the sumoylation machinery in response to external and internal cellular cues [14] . SUMO-regulated processes include nuclear-cytosolic transport , DNA repair , transcriptional regulation , chromosome segregation and many others [14] . For example , sumoylation of the glucocorticoid receptor prevents synergy between two GR dimers bound at a single response element [17] . In this sense , SUMO is analogous to the small hydrophobic hormones and metabolites that serve as noncovalent ligands for nuclear receptors , except it associates both covalently and non-covalently with its targets . Sumoylation modulates the activities of multiple classes of cellular proteins , such as transcriptional regulators , DNA replication factors and chromatin modifiers . Elucidating how a single nematode NHR integrates cellular signals to regulate specific genes in distinct tissues will advance our understanding of metazoan transcription networks . To this end , we examined how sumoylation regulates the C . elegans nuclear hormone receptor NHR-25 , and the physiological relevance of this nuclear hormone receptor-SUMO interaction . Using a combination of genetics , cell biology , and in vitro biochemistry we sought to understand how signaling through sumoylation impacts NHR-25's role in animal development , and how sumoylation affects the NHR-25 transcriptional network .
We identified an interaction between NHR-25 and the single C . elegans SUMO homolog ( SMO-1 ) in a genome-wide Y2H screen using the normalized AD-Orfeome library , which contains 11 , 984 of the predicted 20 , 800 C . elegans open reading frames [18] . SMO-1 was the strongest interactor in the screen on the basis of two selection criteria , staining for β-galactosidase activity and growth on media containing 3-aminotriazole ( Figure 1A ) . To assess the selectivity of the SMO-1–NHR-25 interaction , we tested pairwise combinations of SMO-1 with full-length NHR-25 , an NHR-25 isoform β that lacks the DNA-binding domain , and each of seven additional NHRs: NHR-2 , NHR-10 , NHR-31 , NHR-91 , NHR-105 , FAX-1 , and ODR-1 ( Figure S1A ) . The NHR-25-SMO-1 interaction proved to be selective , as SMO-1 failed to bind the other NHRs tested . NHR-25 also interacted with the GCNF homolog , NHR-91 ( Figure S1A ) . SMO-1 was an enticing NHR-25 interacting partner to pursue . SUMO in C . elegans and other eukaryotes regulates TFs and chromatin , thus is well positioned to impact NHR-25 gene regulatory networks . Furthermore , spatial and temporal expression patterns of smo-1 and nhr-25 during development largely overlap [3] , [4] , [19] . SUMO interacts with the mammalian homologs of NHR-25 , suggesting that the interaction is likely evolutionarily conserved [20] , [21] . Among its many phenotypes , smo-1 loss-of-function ( lf ) mutants display a fully penetrant protruding vulva ( Pvl ) phenotype , reflecting disconnection of the vulva from the uterus [19] ( Figure 1B , C ) . smo-1 RNAi or mutation also cause low penetrance of ectopic induction of vulval cells , which can generate non-functional vulval-like structures known as multivulva ( Muv ) [22] ( Figure 1B , C ) . Similar to smo-1 mutants , nhr-25 reduction-of-function leads to a Pvl phenotype , but does not cause Muv [7] . This nhr-25 Pvl phenotype results from defects in cell cycle progression , aberrant division axes of 1° and 2° cell lineages , and altered vulval cell migration ( Table 1 , Figure 2 , Bojanala et al . , manuscript in preparation ) . Because at an earlier stage NHR-25 is also necessary for establishing the anchor cell ( AC ) [8] , which secretes the EGF signal that initiates vulval precursor cell ( VPC ) patterning , our RNAi treatments were timed to allow AC formation and examination of the effect of nhr-25 depletion on later developmental events . When smo-1 and nhr-25 were simultaneously inactivated , animals exhibited a fully penetrant vulvaless ( Vul ) phenotype and an exacerbated Muv phenotype ( Figure 1B , C ) . The ectopically induced vulval cells expressed an egl-17::YFP reporter , indicating that 3°-fated cells aberrantly adopted 1° and 2° fates in these animals ( Figure S2B ) . This egl-17::YFP reporter allowed us to monitor 1°/2° fate induction despite the cell division arrest phenotypes of nhr-25 ( RNAi ) and smo-1 ( lf ) ;nhr-25 ( RNAi ) animals . Lineage analyses showed that following simultaneous inactivation of both smo-1 and nhr-25 , daughters of all VPCs normally responsible for vulva formation , ( P5 . p , P6 . p and P7 . p ) failed to undergo the third round of vulval cell division ( Table 1 ) resulting in premature cell division arrest and the Vul phenotype . Although P5 . p , P6 . p and P7 . p VPCs were induced , the execution of 2° fate was abnormal: in both smo-1 ( ok359 ) and smo-1 ( ok359 ) ;nhr-25 ( RNAi ) backgrounds , the expression of the 1° marker , egl-17::YFP exhibited ectopically high expression in P5 . p and/or P7 . p ( Figure S2A ) at the 4-cell stage . Moreover , in smo-1;nhr-25 ( RNAi ) animals , the P ( 3 , 4 , 8 ) . p cell , which normally divides only once and fuses into the hypodermal syncytium , kept dividing ( Table 1 ) . This continued division enhanced the Muv induction phenotype seen in smo-1 mutants . Thus , reduction of SMO-1 activity enhanced cell division defects in 1° and 2° nhr-25 mutant VPCs , while reduction of NHR-25 activity enhanced the smo-1 mutant Muv phenotype in 3° fated cells . NHR-25 and SMO-1 interact physically in Y2H assays and genetically in vivo , consistent with their overlapping expression patterns [4] , [19] . Furthermore , the mammalian NHR-25 homologs are sumoylated , suggesting that SMO-1-NHR-25 interactions are conserved and physiologically important . Y2H interactions with SUMO can reflect non-covalent binding , or covalent sumoylation where the SUMO protein is coupled onto the substrate through an isopeptide bond . These two possibilities can be distinguished genetically . Mutations in the β-sheet of SUMO interfere with non-covalent binding , whereas deletion of the terminal di-glycine in SUMO selectively compromises covalent sumoylation [23] . As can be seen in Figure 3A , deletion of the terminal di-glycine residues of SMO-1 ( ΔGG ) completely abrogated the interaction with NHR-25 . The SMO-1 V31K mutation predicted to disrupt the conserved β-sheet of SMO-1 hampered the Y2H interaction between NHR-25 and SMO-1 , although not as severely as the SMO-1 ΔGG mutation ( Figure 3A ) . These findings are similar to those with DNA thymine glycosylase and the Daxx transcriptional corepressor , both of which bind SUMO non-covalently and are also sumoylated [24] , [25] . The V31K β-sheet mutant was competent to bind the C . elegans SUMO E2 enzyme , UBC-9 , confirming its correct folding ( Figure S3A ) . Together , these results suggested that NHR-25 is both sumoylated and binds SMO-1 non-covalently; conceivably , the two modes of interaction confer distinct regulatory outcomes . As our Y2H data suggested that NHR-25 was sumoylated , we identified candidate sumoylation sites within NHR-25 using the SUMOsp2 . 0 prediction program [26] . The sumoylation consensus motif is ψ-K-X-D/E , where ψ is any hydrophobic amino acid , K is the lysine conjugated to SUMO , X is any amino acid , and D or E is an acidic residue [14] . Three high scoring sites reside in the hinge region of the protein: two are proximal to the DBD ( K165 and K170 ) and one ( K236 ) is near the LBD ( Figure 3C ) . We mutated these sites , conservatively converting the putative SUMO acceptor lysine residues to arginine to block sumoylation . Single mutation of any of the three candidate lysines had no apparent effect on the NHR-25 interaction with SMO-1 in Y2H assays , whereas the three double mutants had modest effects , and the NHR-25 3KR triple mutant ( K165R K170R K236R ) abrogated binding ( Figure 3D ) . A fourth candidate sumoylation site ( K84 ) located in the DBD was completely dispensable for the Y2H interaction ( data not shown ) . To verify that the 3KR mutations blocked the interaction with SMO-1 specifically , rather than causing NHR-25 misfolding or degradation , we confirmed that NHR-25 3KR retained the capacity to bind NHR-91 ( Figures S1 , Figure 3B ) . These data suggested that either non-covalent binding is dispensable for the SMO-1-NHR-25 interaction and that this was a rare case in which the SUMO β-sheet mutation impaired sumoylation , or that the three lysines in NHR-25 were important for both the covalent and non-covalent interaction with SMO-1 . To ensure that our Y2H results indeed reflected NHR-25 sumoylation , we turned to in vitro sumoylation assays . As both human and C . elegans sumoylation enzymes were used in these experiments , we distinguish them with prefixes “h” and “Ce” . As a positive control , we expressed and purified recombinant hE1 , hUBC9 , hSUMO1 , and hSENP1 from E . coli . We also purified a recombinant partial hinge-LBD fragment of mouse SF-1 from E . coli; this fragment contains a single sumoylation site in the hinge region . SF-1 is a vertebrate ortholog of NHR-25 and the fragment that we used is a robust sumoylation substrate ( Figure S4A ) [27] . We then purified an N-terminally hexahistidine-Maltose Binding Protein ( 6×His-MBP ) tagged fragment of NHR-25 ( amino acids 161–541 ) containing most of the hinge region and ligand-binding domain , including all three candidate SUMO acceptor lysines . Coomassie staining and immunoblotting revealed three slower-migrating species , which were collapsed by the addition of the SUMO protease , hSENP1 ( Figure 4A , S5A ) . We detected sumoylation of the same 6×HisMBP-NHR-25 fragment when it was expressed in rabbit reticulocyte lysates , followed by incubation with hE1 , hE2 and hSUMO1 ( Figure 4B ) . We further tested NHR-25 substrates containing two ( 2KR; K170R K236R ) or three arginine substitutions ( NHR-25 3KR ) . When only one predicted acceptor lysine was available ( 2KR ) , we detected a single dominant sumoylated species , whereas for NHR-25 3KR , sumoylation was abrogated ( Figure S5B ) . We performed sumoylation reactions on in vitro transcribed and translated wild type NHR-25 , NHR-25 3KR , and NHR-25 3EA . In NHR-25 3EA ( E167A E172A E238A ) the acidic glutamic acid residues within the three consensus sumoylation sites were mutated to alanine . NHR-25 3EA leaves the acceptor lysines available , but is predicted to inhibit sumoylation by impairing interaction with UBC9 . While wild type NHR-25 was clearly sumoylated , the 3EA mutation severely impaired sumoylation ( Figure 4B ) . When sumoylation reaction times were extended 5–20 fold , additional species of sumoylated NHR-25 were generated ( Figure S6A ) . These species could reflect sumoylation of NHR-25 on other sites or formation of hSUMO1 chains . To distinguish between these possibilities , we used methyl-hSUMO1 , which can be conjugated onto a substrate lysine , but chain formation is blocked by methylation . Long incubations with methyl-hSUMO1 resulted in only three sumoylated NHR-25 bands , as determined by NHR-25 immunoblotting , indicating that there are indeed only the three major acceptor lysines ( Figure S6A ) . hSUMO2 , which readily forms polySUMO chains , was included as a control in this experiment . Even with extended incubation times , we observed only three dominant sumoylated forms of NHR-25 , suggesting that additional bands in reactions using hSUMO1 or CeSMO-1 reflect inefficient chaining . We conclude that NHR-25 is sumoylated in vitro on three lysines and that C . elegans SMO-1 does not readily form polySUMO chains , unlike yeast SMT3 and mammalian SUMO2 . All studies of C . elegans sumoylation to date have used hE1 , hUBC9 , and hSUMO proteins [19] , [28] , [29] . We purified recombinant CeE1 , CeUBC-9 and CeSMO-1 from E . coli and tested their activity in in vitro sumoylation assays . Our CeE1 preparation was inactive , but was effectively substituted by hE1 . Under those conditions , our CeUBC-9 and CeSMO-1 catalyzed sumoylation of the SF-1 hinge-LBD fragment ( Figure S4B ) . Similar to hUBC9 and hSUMO1 , recombinant CeUBC-9 and CeSMO-1 yielded three sumoylated species using the 6×His-MBP-NHR-25 substrate ( Figure 4C , S4C ) . To determine the kinetics of the three SUMO modifications of NHR-25 , we performed a time course of standard sumoylation reactions with hUBC9/CeUBC-9 and hSUMO1/CeSMO-1 proteins . In both cases , we detected a single band by 15 minutes , followed by two and then three sumoylated species as the reaction progressed ( Figure S6B–E ) . These data imply that the three sumoylation sites are modified sequentially , in a particular order . All of our reactions were performed without addition of an E3 ligase . The high efficiency of SF-1 sumoylation in the absence of E3 ligase is in part due to a direct interaction with UBC9 [30] . Surprisingly , we failed to detect an interaction between NHR-25 and CeUBC-9 either by Y2H assays or through immunoprecipitation of purified proteins ( Figure S3B; data not shown ) . However , when we performed a yeast three-hybrid assay , where untagged CeSMO-1 was added to the system , we observed a weak interaction between NHR-25 and CeUBC-9 , suggesting either that CeSMO-1 bridges NHR-25 and CeUBC-9 or that NHR-25 recognizes a CeSMO-1-bound CeUBC-9 species ( Figure S3B ) . To begin to investigate how sumoylation affects NHR-25-dependent transcriptional activity , we employed a HEK293T cell-based assay . We used a luciferase reporter driven by four tandem Ftz-F1 ( Drosophila homolog of NHR-25 ) consensus sites , previously shown to be responsive to NHR-25 [8] . When Myc-tagged wild type NHR-25 was transfected , reporter expression was enhanced ( Figure 5A ) , and the sumoylation-defective mutant NHR-25 ( 3KR ) activated the reporter more strongly ( Figure 5A ) . Anti-Myc immunostaining indicated no detectable increase in protein level or nuclear localization ( Figure 5B ) . To better characterize NHR-25-dependent transcriptional activity and generate reporters that could subsequently be used for in vivo assays , we generated a construct based on the canonical , high affinity SF-1 regulatory elements derived from the Mullerian inhibiting substance ( MIS ) and CYP11A1 ( CYP ) genes . We assessed NHR-25 binding to these elements using yeast one-hybrid ( Y1H ) and electrophoretic mobility shift assays ( EMSAs ) . The Y1H assays indicated that NHR-25 bound the MIS and CYP11A1 elements ( Figure S7A , B ) . Mutations in the MIS binding site that block SF-1 binding ( MIS MUT ) [27] prevented NHR-25 binding ( Figure S7B ) . Moreover , the NHR-25 L32F ( ku217 ) mutant , which has impaired DNA binding in vitro [7] , displayed reduced activity in the Y1H experiment ( Figure S7B ) . Consistent with the Y1H data , we found that a 6×His-MBP tagged fragment of NHR-25 ( amino acids 1–173 ) purified from E . coli clearly bound MIS and CYP11A1 sites singly ( Figure S7C ) or in combination ( 2×NR5RE WT , for nuclear receptor NR5 family Response Element; Figure S7D ) but only weakly to the mutant sites ( Figure S7C–D , 7A ) . Sumoylation of SF-1 regulates binding to specific DNA sequences [27] . Therefore , we asked whether sumoylation could similarly affect DNA binding capacity of the 6×His-MBP tagged fragment of NHR-25 . We found that this fragment , which encompasses the DBD and part of the hinge region of NHR-25 ( amino acids 1–173 ) , was an even more potent sumoylation substrate than the hinge-LBD fragment , as almost all of the DBD substrate could be sumoylated ( Figure 6A ) . Unlike SF-1 [27] , NHR-25 DNA binding did not inhibit sumoylation ( data not shown ) . Use of methyl-hSUMO1 in our in vitro sumoylation assays indicated that there were three sumoylation sites within the 6×His-MBP tagged fragment of NHR-25 DBD substrate ( Figure 6B ) . These corresponded to the hinge region K165 and K170 acceptor lysines , which are analogous to the SF-1 fragment used by Campbell et al . ( 2008 ) , and a third SUMO acceptor lysine ( K84 ) within the DBD region between the second zinc finger and the conserved Ftz-F1 box ( Figure 6C ) . This acceptor lysine is conserved in D . melanogaster Ftz-F1 as well as the mammalian LRH-1 ( Figure 6C ) [31] . EMSAs indicated that sumoylation diminished binding of the NHR-25 DBD fragment to the MIS and CYP derived binding sites ( Figure S7D ) . Modifying the EMSAs such that the sumoylation reaction preceded incubation with the 2×NR5RE oligos severely impaired binding ( Figure S7E ) . These in vitro findings are consistent with the notion that , as in mammals , sumoylation could diminish NHR-25 DNA binding . We next wanted to assess the effects of sumoylation on NHR-25-dependent transcription in vivo . To enhance the sensitivity of our assays , we constructed a reporter carrying four tandem repeats derived from each of MIS and CYP genes ( Figure 7A , eight SF-1/NHR-25 binding sites designated as 8×NR5RE ) . The binding sites were spaced ten base-pairs apart to facilitate potential cooperative binding [32] . We generated transgenic C . elegans carrying the 8×NR5RE positioned upstream of a pes-10 minimal promoter and driving a 3×Venus fluorophore bearing an N-terminal nuclear localization signal . In wild type animals , reporter expression was not detected ( Figure 7B ) , whereas after smo-1 RNAi , strong expression was detected in developing vulval cells , the hypodermis , seam cells , the anchor cell ( Figure 7B ) and embryos ( not shown ) , tissues in which NHR-25 is known to be expressed ( Figures 7F ) and functional [4] , [7] , [33] . Reporter expression was especially prominent during the L3 and L4 stages . Mutation of the binding consensus , 8×NR5RE ( MUT ) abolished reporter expression in a smo-1 ( RNAi ) background ( Figure 7E ) , as expected for NHR-25-dependent reporter expression . Moreover , genetic inactivation of nhr-25 either by RNAi ( smo-1 , nhr-25 double RNAi ) or by use of nhr-25 ( ku217 ) , a reduction-of-function allele of nhr-25 , abrogated reporter expression even in smo-1 knockdown animals ( Figure 7C , D ) . We conclude that sumoylation of NHR-25 strongly reduces its transcriptional activity in vivo . To examine functionally the consequences of NHR-25 sumoylation , we returned to the roles of nhr-25 and smo-1 in vulval organogenesis . Noting that smo-1 mutants but not nhr-25 reduction-of-function mutants display a Muv phenotype , we investigated whether this might reflect enhanced NHR-25 activity due to its reduced sumoylation . We therefore generated transgenic animals expressing tissue-specific NHR-25 and/or SMO-1 driven by three different promoters; egl-17 for the VPCs , grl-21 for the hypodermal hyp7 syncytium , and wrt-2 for the seam cells . These transgenes included ( i ) wild type NHR-25; ( ii ) NHR-25 3KR; or ( iii ) SMO-1 alone . Although egl-17 is typically used as a 1° and 2° cell fate marker during vulva development , it is expressed in all VPCs in earlier stages [34] ( Figure S2C ) . We used the egl-17 promoter rather than commonly used VPC driver , lin-31 , because the heterodimeric partner of LIN-31 is sumoylated and directly involved in vulva development [28] . Muv induction was scored by observing cell divisions of the six VPCs with the potential to respond to the LIN-3/EGF signal , which promotes differentiation . Normally , only P5 . p , P6 . p , and P7 . p are induced while P3 . p , P4 . p and P8 . p each produce no more than two cells as they are destined to fuse with the surrounding hyp7 syncytium ( Figure 2 ) . In wild type animals , overexpression of NHR-25 in the VPCs ( egl-17 promoter ) but not in hyp7 or seam cells ( grl-21 and wrt-2 promoters , respectively ) drove Muv induction at the P8 . p position , mimicking smo-1 RNAi ( Figure 8 , Table S1 ) . Thus , high level NHR-25 acted cell-autonomously to produce a Muv phenotype . Overexpression of the NHR-25 3KR mutant in the VPCs resulted in an even more penetrant Muv phenotype and greater induction of P3 . p , P4 . p , and P8 . p ( Figure 8A ) . In contrast , overexpression of SMO-1 alone did not produce the Muv phenotype . These overexpression experiments implied that excess unsumoylated NHR-25 altered 3° VPC fate , permitting extra divisions that produce the Muv phenotype . If sumoylation of NHR-25 normally constrains its activity , animals with decreased sumoylation activity would be expected to enhance the Muv phenotype . To test this hypothesis , we assessed the effect of smo-1 RNAi in animals expressing a low-copy , integrated transgene expressing C-terminally GFP-tagged NHR-25 [35] . This transgene likely recapitulates the expression pattern of endogenous nhr-25 , since the construct includes the complete 20 kb intergenic region upstream of nhr-25 , and the entire nhr-25 gene and 3′-UTR; the animals display normal vulvas . However , exposure to smo-1 RNAi caused the Muv phenotype in about 30% of animals carrying the nhr-25::gfp transgene , which exceeded the 12% Muv frequency in smo-1 RNAi controls ( Figure 8 ) . This extra vulva induction was seen in the P4 . p . lineage in addition to P8 . p . Together , our findings strongly suggest that in wild type animals , NHR-25 sumoylation prevents ectopic vulva induction in 3° fated cells . One interpretation of our genetic and biochemical data is that the in vivo ratio of sumoylated to non-sumoylated NHR-25 specifies or maintains the 3° VPC fate . We were therefore interested in how NHR-25 sumoylation was regulated . SMO-1 is expressed at constant levels throughout vulval development [19] , so we examined whether NHR-25 levels were regulated in VPCs during development . The low-copy , integrated NHR-25::GFP translational fusion allowed us to examine the developmental pattern of NHR-25 expression . NHR-25::GFP was evenly distributed prior to the first division in all VPCs , whereas after the first division the pattern became graded: highest in 1° P6 . p daughters , lower in 2° P5 . p and P7 . p daughters , and lowest in 3° P ( 3 , 4 , 8 ) . px ( Figure 9A , B ) . After the third round of cell divisions NHR-25::GFP expression continued in all 22 P ( 5–7 ) . pxxx cells and remained high during early vulva morphogenesis ( Figure 9D ) until it temporarily disappeared by the “Christmas tree stage” ( data not shown ) . smo-1 RNAi caused ectopic NHR-25::GFP expression in P ( 4 , 8 ) . pxx cells ( Figure 9E ) , which displayed the strongest Muv induction in NHR-25::GFP;smo-1 ( RNAi ) , and Pegl-17::NHR-25 ( 3KR ) backgrounds ( Figure 8 ) . In wild type animals , NHR-25::GFP was normally expressed in the anchor cell at the time of the first VPC divisions , and subsequently decreased ( Figure 9D ) . Interestingly , we noted that in nine of ten smo-1 ( RNAi ) animals NHR-25::GFP was re-expressed in the AC at the “bell stage” ( Figure 9F ) . Subsequently , no AC invasion occurred and the AC remained unfused . Therefore , in addition to restricting NHR-25 activity in 3° cells ( previous section ) , sumoylation also limits NHR-25 accumulation in cells that are destined to assume the 3° fate . The resultant NHR-25 gradient combined with constant levels of SMO-1 may account for the observed pattern of NHR-25 sumoylation .
Supporting the notion that sumoylation can constrain NHR-25 activity , we found that a reporter fusion responsive to NHR-25 was strongly upregulated upon depletion of smo-1 by RNAi ( Figure 7B ) . Our in vitro findings suggested that sumoylation of NHR-25 diminished DNA binding ( Figure S7 ) , while our in vivo studies suggested that reduction of smo-1 caused ectopic accumulation of NHR-25 ( either synthesis or impaired degradation ) in VPCs P4 . p and P8 . p ( Figure 9 ) . These data suggest two modes , not mutually exclusive , through which sumoylation can regulate NHR-25 . Moreover , overexpression of either NHR-25 or its sumoylation-defective form ( NHR-25 3KR ) led to multivulva induction in cells that normally adopt the 3° fate ( Figure 8 ) . Together , our data support a model in which proper differentiation of VPCs depends on the appropriate balance of sumoylated and unsumoylated NHR-25 ( Figure 10 ) . Importantly , NHR-25 affects VPC specification cell-autonomously , as overexpression of NHR-25 in other epidermal cells , such as the seam cells or hyp7 , did not cause a Muv phenotype ( Table S1 ) . Furthermore , NHR-25 appears to form a gradient across the VPC array , accumulating to high levels in 1° fated cells , intermediate levels in 2° fated cells and low levels in 3° fated cells ( Figure 9 ) . Our findings indicate that sumoylation promotes a specific pattern of NHR-25 activity in differentially fated VPCs and the relative level of NHR-25 sumoylation is critical for promotion and/or maintenance of the 3° cell fate ( Figure 10 ) . The role ( s ) of NHR-25 and SMO-1 in vulval induction are likely pleiotropic . Multiple vulval development factors are sumoylated [22] , [28] , [29] , [36] , including LIN-11 , which is responsible in part for promoting vulval-uterine fusion [19] . Based on expression pattern and phenotypes , NHR-25 likely acts in other cell-types ( hyp7 , 1°/2° VPCs , or AC ) and at different developmental time points to regulate vulval induction . The Muv phenotype of smo-1-deficient animals was enhanced by nhr-25 RNAi ( Figure 1 ) . Synthetic multivulva ( synMuv ) genes inhibit lin-3 activity in the syncytial hyp7 cell to prevent aberrant vulva induction in the neighboring 3° cells [37] . Yet , overexpression of NHR-25 in the hyp7 syncytium did not cause Muv induction ( Table S1 ) , thus it is unlikely that NHR-25 acts through this pathway . Our overexpression data indicates that NHR-25 acts cell-autonomously in the VPCs ( Figure 8 ) , and likely interacts with canonical signaling pathways that promote VPC fate . The NHR-25 expression gradient is reminiscent of the LIN-3/EGF gradient which promotes vulval induction through Ras activation and subsequent Notch signaling [38] . nhr-25 appears to act downstream of LET-60/Ras signaling , as gain-of-function LET-60/Ras causes elevated NHR-25 expression ( data not shown ) . However , regulation of lin-3 by NHR-25 in the anchor cell has also been suggested [39] . Ectopic expression of NHR-25 in the AC following smo-1 RNAi is unlikely to cause Muv induction since , developmentally , this expression occurs much later than VPC fate determination . In wild type animals , NHR-25 levels are therefore downregulated in the AC , which may be required for proper completion of AC invasion and/or fusion . Additionally , the cell division arrest seen in nhr-25 RNAi leading to the Pvl phenotype was enhanced by inactivation of smo-1 ( Figure 1 ) . For instance , the Pvl phenotype can arise from nhr-25 reduction of function , which causes defective 1° and 2° cell divisions ( Figure 1 , Table 1 ) , or from smo-1 ( lf ) , which impairs uterine-vulval connections [19] . Thus , an exquisite interplay between various sumoylated targets as well as the balance between sumoylated and unsumoylated NHR-25 collaborate to ensure proper vulval formation . How could unsumo∶sumo NHR-25 balance regulate 3° cell fate ? Sumoylation might alter NHR-25 levels or activity in a manner that shifts the unsumo∶sumo NHR-25 ratio , which in turn acts as a switch to determine NHR-25 output . The activities of a mammalian nuclear hormone receptor have been shown to shift dramatically with signal-driven changes in levels of receptor activity [40] . Another possibility is that the sumoylated and unsumoylated versions of NHR-25 regulate distinct targets , and the unsumo∶sumo ratio in different cells thereby determines the network of NHR-25-regulated genes . Indeed , sumoylation appears to affect the genomic occupancy of the NHR-25 ortholog SF-1 [27] . We note that NHR-25 sumoylation could be context-dependent . Sumoylation could increase NHR-25 activity at particular response elements . Accordingly , sumoylation positive regulates the activity of the nuclear hormone receptors RORα and ER [41] , [42] . The finding that overexpression of NHR-25 strongly provoked a Muv phenotype suggests that sumoylation state of NHR-25 in VPCs is exquisitely regulated . Such regulation might be accomplished by subtle changes in availability of SUMO in different VPCs , not detected by our assays , or by the relative activities of the sumoylation machinery and the SUMO proteases . A similar competition for constant levels of SUMO regulates Epstein-Barr virus infections , where the viral BZLF protein competes with the host PML protein for limiting amounts of SUMO1 [43] . It is intriguing to consider SMO-1 as an NHR-25 ligand parallel to hormones or metabolites bound noncovalently nuclear hormone receptors in other metazoans , and by the C . elegans DAF-12 receptor . Indeed , such expansion of the concept of signaling ligands could “de-orphan” many or all of the 283 C . elegans nuclear hormone receptors for which no traditional ligands have been identified . Detection of noncovalent ligands is very challenging; numerous mammalian NHRs remain “orphans” despite intensive efforts to find candidate ligands and evidence that the ancestral NHR was liganded [44] . In principle , SUMO can be conjugated to its target sequence motif anywhere on the surface of any protein , whereas classic NHR ligands bind only stereotyped pockets within cognate NHR LBDs . Viewed in this way , SUMO may directly regulate many NHRs ( and other factors as well ) , whereas classical NHR ligands act more selectively on only one or a few NHRs . The multifactorial regulation of NHRs would provide ample opportunity for gene- , cell- or temporal-specificity to be established in cooperation with the SUMO ligand . There are three ways in which SUMO can potentially interact with target proteins: i ) non-covalent binding , where a protein binds either free SUMO or SUMO conjugated onto another protein; ii ) sumoylation , where SUMO associates covalently with a target protein through an isopeptide linkage; and iii ) poly-sumoylation , where chains of SUMO are built up from an initially monosumoylated substrate . In C . elegans , SMO-1 can bind proteins non-covalently [45] or can be covalently linked to substrates ( Figure 4 ) . Polysumoylation occurs through SUMO modification of acceptor lysines within SUMO proteins [46] . In our assays , we saw no robust polyCeSMO-1 chains compared to the hSUMO2 control , even after prolonged reaction times ( Figure S6 ) . Consistent with this result , sumoylation motifs were predicted within hSUMO1 , 2 and 3 , and yeast SMT3 but not in CeSMO-1 . PolySUMO chains in yeast and vertebrates can be recognized by SUMO targeted ubiquitin ligases ( STUbLs ) that polyubquitinate the polySUMO chain and direct it for degradation by the 26S proteasome [46] . Judging from BLAST analysis , there are no evident homologs of the known STUbLs hsRNF4 or yeast SLX5–8 in C . elegans . As both S . cerevisiae SUMO ( SMT3 ) and vertebrate SUMO2 and SUMO3 form polySUMO chains , it appears that C . elegans has lost the ability to form polySUMO chains . The mammalian homologs of NHR-25 ( SF-1 and LRH-1 ) are sumoylated on two sites within the hinge region of the protein , between the DBD and LBD [21] , [27] , [47] . These SUMO acceptor sites occur at corresponding positions in NHR-25 , with the site near the DBD being duplicated ( Figure 6C ) . Additionally , our DBD sumoylation experiments suggest the presence of a fourth sumoylation site in NHR-25 , conserved with D . melanogaster Ftz-F1 and mammalian LRH-1 ( Figure 6C ) [7] , [31] . Thus , NHR-25 appears to have sumoylation sites that are conserved in both SF-1 and LRH-1 as well as at least one site that is only conserved in LRH-1 . Similarly , NHR-25 seems to combine regulation of processes that in mammals are either regulated by SF-1 only or LRH-1 only . Additionally , human SUMO1 can be conjugated onto NHR-25 and C . elegans SMO-1 can be conjugated onto SF-1 ( Figure 4 , S4 ) . Therefore , despite the 600–1200 million years of divergence since the common ancestor of humans and nematodes , regulation of NR5A family by sumoylation appears to be incredibly ancient . There are also , however , notable differences . For instance , while LRH-1 and SF-1 strongly interact with UBC9 , providing a mechanism for robust , E3 ligase-independent sumoylation [20] , this did not appear to be the case for NHR-25 . As indicated above , we also did not find evidence for polysumoylation of NHR-25 . Having established SUMO as an NHR-25 signal that regulates cell fate , it will be exciting to further explore how sumoylation affects the NHR-25 gene regulatory network . It will be essential in future work to identify direct NHR-25 target genes by ChIP-seq , to determine how sumoylation impacts NHR-25 response element occupancy , and to mutate sumoylation sites and response elements with genome editing technologies , such as CRISPR/Cas9 [48] . The compact C . elegans genome facilitates unambiguous assignment of putative response elements to regulated genes , a daunting challenge in vertebrate systems . Further , the extensive gene expression and phenotypic data accessible to the C . elegans community will allow identification of candidate NHR-25 target genes directly responsible for regulating animal development and physiology . Understanding how NHR-25 sumoylation regulates specific genes , and how this information is integrated into developmental circuits will advance our understanding of combinatorial regulation in metazoan gene regulatory networks .
cDNAs and promoters/binding sites were Gateway cloned ( Invitrogen ) into pDONR221 and pDONR-P4P1r , respectively . Mutations were introduced into the nhr-25 cDNA using site-directed mutagenesis with oligonucleotides carrying the mutation of interest and Phusion polymerase ( NEB ) . cDNAs and promoters were then moved by Gateway cloning into destination vectors . NHR-25 ( amino acids 161–541 ) and NHR-25 ( amino acids 1–173 ) were moved into the bacterial expression vector pETG-41A , which contains an N-terminal 6×His-MBP tag . CeUBC-9 and CeSMO-1 cDNAs were moved into the bacterial expression vector pETG-10A , which contains an N-terminal 6×His tag . The CeUBC-9 construct also carried an N-terminal tobacco etch virus ( TEV ) cleavage site for removal of the 6×His tag , similar to the hUBC9 bacterial expression construct . For Y1H experiments , 2×SF-1 binding sites were Gateway cloned into pMW2 and pMW3 [49] . For Y2H experiments , cDNAs were moved into pAD-dest and pDB-dest [18] , which contain the Gal4 activation domain and DNA binding domain , respectively . For Y3H , smo-1 was moved into pAG416-GPD-ccdB-HA [50] , which results in constitutive expression . For luciferase experiments , cDNAs were moved into pDEST-CMV-Myc . For our C . elegans expression experiments , cDNA constructs were Gateway cloned into pKA921 along with either the egl-17 , wrt-2 , or grl-21 promoter . The egl-17 promoter was PCR cloned from N2 genomic DNA . The wrt-2 and grl-21 promoters ( pKA279 and pKA416 , respectively ) were previously cloned [12] . pKA921 contains a polycistronic mCherry cassette to allow monitoring of construct expression . For our 3×Venus reporters , three-fragment Gateway cloning into pCFJ150 [51] was performed . The 8×NR5RE-pes-10Δ promoter fragments were cloned into pDONR-P4P1r . C . elegans codon optimized 3×Venus was cloned from Prnr::CYB-1DesBox::3×Venus [52] and an NLS was added on the 5′ end of the gene and NLS-3×Venus was Gateway cloned into pDONR221 . The unc-54 3′-UTR in pDONR-P2rP3 was a gift from the Lehner lab . Primer sequences are provided in Table S2 . Plasmids generated for this study are listed in Table S3 . Yeast transformations and Y2H assays were carried out as described by Deplancke et al . [53] . For the Y2H screen , S . cerevisiae strain MaV103 carrying a pDB-nhr-25 construct was transformed with 100 ng of the AD-Orfeome cDNA library , in which 58% of the known C . elegans open reading frames are fused to the Gal4 activation domain [18] . Six transformations were performed per screen and 149 , 800 interactions were screened , representing 12 . 5-fold coverage of the library . Positive interactions were selected for by growth on SC dropout plates lacking leucine , tryptophan , and histidine; these plates were supplemented with 20 mM of the histidine analog 3-aminotriazole . Interactions were confirmed by β-galactosidase staining . We identified 42 candidate interactors , but only smo-1 was recovered multiple times ( seven independent isolations ) . Moreover , upon cloning and retesting the candidate interactor cDNAs , only smo-1 was confirmed as an interactor . The screen identified no other components of the SUMO machinery or known SUMO binding proteins . Generation of Y1H bait strains and Y1H analyses were performed as described [53] . pDB constructs carrying NHR-2 , NHR-10 , NHR-31 , NHR-91 , NHR-105 , FAX-1 , and ODR-1 cDNAs were a gift from Marian Walhout . Recombinant hE1 , hUBC9 , hSUMO1 , hSUMO2 , hSENP1 , and murine SF-1 LBD were purified as described [27] , [54]–[56] . 6×His-CeSMO-1 and 6×His-TEV-CeUBC-9 were expressed in BL21 ( λDE3 ) E . coli and purified using a similar scheme as used to purify their human counterparts [55] , [56] . 6×His-MBP-NHR-25 ( amino acids 161–541 ) was freshly transformed into BL21 ( λDE3 ) E . coli . A 1 L culture was grown to an OD600 of ∼0 . 8 , induced with 0 . 2 mM isopropylthio-β-galactoside ( IPTG ) , and shaken at 16°C for four hours . Bacteria were lysed using a microfluidizer in 20 mM Tris-HCl pH 8 . 0 , 350 mM NaCl , 20 mM imidazole containing EDTA-free Protease Inhibitor Cocktail III ( EMD Millipore ) . 6×His-MBP-NHR-25 was then purified using nickel affinity chromatography ( 5 ml His Trap FF column , GE Healthcare ) . Peak fractions were pooled , dialyzed into 20 mM HEPES ( pH 7 . 5 ) , 1 mM EDTA , and 2 mM CHAPS {3-[ ( 3-cholamidopropyl ) -dimethylammonio]-1-propanesulfonate} , and purified by anion-exchange chromatography using a MonoQ column ( GE Healthcare ) and eluted with a 1 M ammonium acetate gradient . Peak fractions were pooled , concentrated and 6×His-MBP-NHR-25 was purified by size-exclusion chromatography using an S200 column ( GE Healthcare ) . Peak fractions containing 6×His-MBP-NHR-25 were pooled , concentrated , dialyzed into 20 mM Tris pH 7 . 5 , 50 mM NaCl , 10% glycerol , flash frozen in liquid nitrogen , and stored at −80°C . Later purifications used only nickel affinity chromatography . Using this preparation in sumoylation assays produced results similar to those obtained using the preparations purified over the three aforementioned columns . 6×His-MBP-NHR-25 ( amino acids 1–173 ) was expressed and purified using a single nickel affinity chromatography step , as described above for the 6×His-MBP-NHR-25 ( amino acids 161–541 ) fragment . Reactions were performed as described by Campbell et al . [27] . Briefly , 50 µl sumoylation reactions were set up with 0 . 1 µM E1 , 10 µM UBC9 , and 30 µM SUMO in a buffer containing 50 mM Tris-HCl ( pH 8 . 0 ) , 100 mM NaCl , 10 mM MgCl2 , 10 mM ATP , and 2 mM DTT . Substrates were added at 1 µM and when required , 2 . 5 µg of hSENP1 SUMO protease was added . When in vitro transcribed proteins were used as substrates , 50 µl reactions were generated using a TnT T7 Quick Coupled Transcription/Translation System ( Promega ) . 16 µl of this reaction was then used as a substrate in a 25 µl sumoylation reaction using the same molarities as described above . When SUMO protease was required , 1 . 25 µg of hSENP1 was added . Reactions were incubated at 37°C for the desired time , and stopped by boiling in protein sample buffer ( 10% Glycerol , 60 mM Tris/HCl pH 6 . 8 , 2% SDS , 0 . 01% bromophenol blue , 1 . 25% beta-mercaptoethanol ) . Proteins were resolved by SDS-PAGE on either 4–12% Bis-Tris gradient gels ( Invitrogen ) or 3–8% Tris acetate gels ( Invitrogen ) followed by either Coomassie staining or immunoblotting . For immunoblotting , anti-NHR-25 , anti-guinea pig-HRP ( Santa Cruz ) , and anti-guinea pig-IR800 ( Li-Cor ) antibodies were used . Blots were developed using a LAS500 imager ( GE Healthcare ) or an Odyssey laser scanner ( Li-Cor ) . Reactions were performed as described by Campbell et al . [27] with the following alterations . We added 400 µg/ml of bovine serum albumin to the EMSA buffer ( 50 mM Tris ( pH 8 . 0 ) , 150 mM NaCl , 10 mM MgCl2 , 10 mM DTT , 10 mM ATP , and a 1 µM concentration of double-stranded oligonucleotide ) . Sequences of oligonucleotides are provided in Table S2 . Oligonucleotides were annealed and then centrifuged in an Amicon Ultra 0 . 5 ml centrifugal filter ( MWCO 50 ) . Sumoylation reactions were set up on ice and added directly to the annealed oligonucleotides ( 20 µl final volume ) . Standard reactions used 500 nM of unmodified NHR-25 substrate , titration experiments added NHR-25 in 100 nM increments from 200–700 nM . At this point SENP1 ( 0 . 5 µl ) was added when appropriate . We incubated these reactions at room temperature for 30 minutes to allow both sumoylation and DNA binding to occur . Half of the EMSA reaction ( 10 µl ) was removed and added to 2 µl of 4× protein sample buffer and denatured by boiling for five minutes . Sumoylation products in the input were analyzed by immunoblotting using anti-MBP ( NEB ) and anti-mouse-IR800 ( LiCor ) antibodies . Blots were imaged using an Odyssey laser scanner . The remaining EMSA reaction was resolved on a 4–20% TBE polyacrylamide gel ( Invitrogen ) at 200 volts and stained with 1× SYBR Gold ( Molecular Probes ) in 0 . 5× TBE . Gels were then imaged using a Typhoon laser scanner ( GE Healthcare ) . C . elegans was cultured at 20°C according to standard protocols and the wild type strain is the N2 Bristol strain [57] . The following mutant and transgenic strains were used in this study: PS3972 unc-119 ( ed4 ) syIs90 [egl-17::YFP+unc-119 ( + ) ] , OP33 unc119 ( ed3 ) ; wgIs33 [nhr-25::TY1::EGFP::3×FLAG ( 92C12 ) +unc-119 ( + ) ] , VC186 smo-1 ( ok359 ) /szT1[lon-2 ( e678 ) ]; +/szT1 , MH1955 nhr-25 ( ku217 ) . The following transgenic strains were generated for this study: HL102 jmEx102[Pegl-17::Myc::NHR-25_mCherry+rol-6 ( su1006 ) ] , HL107 , HL108 , HL110 are independent lines carrying jmEx107[Pegl-17::Myc::NHR-25 ( 3KR ) _mCherry+rol-6 ( su1006 ) ] , HL117 jmEx118 [Pegl-17::Myc::SMO-1_mCherry+rol-6 ( su1006 ) ] , HL111 and HL112 are independent lines carrying jmEx111[Pgrl-21::Myc::NHR-25_mCherry+rol-6 ( su1006 ) ] , HL121 jmEx121[Pgrl-21::Myc::SMO-1_mCherry+rol-6 ( su1006 ) ] , HL113 and HL114 are independent lines carrying jmEx113[Pwrt-2::Myc::NHR-25_mCherry+rol-6 ( su1006 ) ] , HL115 and HL116 are independent lines carrying jmEx115[Pwrt-2::Myc::SMO-1_mCherry+rol-6 ( su1006 ) ] , HL153 jmEx153[8×NR5RE ( WT ) :pes-10Δ:NLS-3×Venus:unc-54 3′-UTR+Pmyo-2::tdTomato] , HL155 jmEx155[8×NR5RE ( MUT ) :pes-10Δ:NLS-3×Venus::unc-54 3′-UTR+Pmyo-2::tdTomato] , HL170 nhr-25 ( ku217 ) ; jmEx153 . The following Gateway-based constructs were generated in pKA921: pJW522[Pegl-17 ( 1914 bp ) ::Myc::NHR-25_polycistronic_mCherry] , pJW774 [Pegl-17 ( 1914 bp ) ::Myc:: NHR-25 ( 3KR ) _polycistronic_mCherry] , pJW773 [Pegl-17 ( 1914 bp ) ::Myc::SMO-1_polycistronic_mCherry] , pJW526 [Pgrl-21 ( 746 bp ) ::Myc::NHR-25_polycistronic_mCherry] , pJW775 [Pgrl-21 ( 746 bp ) ::Myc::SMO-1_polycistronic_mCherry] , pJW524[Pwrt-2 ( 1380 bp ) ::Myc::NHR-25_polycistronic_mCherry] , pJW776[Pwrt-2 ( 1380 bp ) ::Myc::SMO-1_polycistronic_mCherry] . The following Gateway-based constructs were generated in pCFJ150 [51]: pJW1109 [8×NR5RE ( WT ) :pes-10Δ:NLS-3×Venus:unc-54 3′-UTR] and pJW1110 [8×NR5RE ( MUT ) :pes-10Δ:NLS-3×Venus::unc-54 3′-UTR] . Plasmids were prepared using a PureYield Plasmid Midiprep System ( Promega ) followed by ethanol precipitation , or a Qiagen Plasmid Midi kit ( Qiagen ) . Transgenic strains were generated by injecting 50 ng/µl of each plasmid into the C . elegans gonad [58] with the co-injection marker pRF4 [59] . For 8×NR5RE reporter strain generation , N2 animals were injected with 30 ng/µl of the reporter plasmid and 5 ng/µl of co-injection marker Pmyo-2::tdTomato [60] . Feeding RNAi was performed as described , with the indicated alterations to the protocol [61] . dsRNA was initially induced for four hours in liquid culture using 0 . 4 mM IPTG , before bacteria were concentrated and seeded on plates also containing 0 . 4 mM IPTG . Bacteria carrying pPD129 . 36 without an insert were used for control RNAi . For nhr-25 RNAi , synchronized L2 larvae ( 19–20 hours after hatching ) were fed on bacteria expressing nhr-25 dsRNA to bypass the anchor cell ( AC ) defect . smo-1 RNAi was performed on late L4 or young adults . For in vivo reporter assays , sodium hypochlorite-treated eggs were placed on RNAi plates seeded with dsRNA induced bacteria . To score vulva induction , nematodes were anesthetized in 10 mM levamisole , mounted onto 5% agar pads ( Noble agar , Difco ) and the number of daughter cells for each VPC were counted under differential interference contrast ( DIC ) optics . For lineaging analyses , the division pattern was followed under DIC from the two to eight cell stages [62] . Animals were mounted onto 5% agar pad with bacteria in S-basal medium without anesthesia . Olympus Fluoview FV1000 and Zeiss Axioplan microscopes were used for observation and imaging . A peptide-based anti-NHR-25 antibody was raised in guinea pig ( Peptide Specialty Laboratories , GmbH , Germany ) . Animals were immunized against four short peptides in the hinge and LBD regions: PEHQVSSSTTDQNNQINYFDQTKC ( 24 a . a . 141–163 ) ; SLHDYPTYTSNTTNC ( 15 a . a . 250–263 ) ; TSSTTTGRMTEASSC ( 15 a . a . 283–296 ) RYLWNLHSNXPTNWEC ( 16 a . a . 507–521 ) . Human embryonic kidney ( HEK ) cell line 293T was maintained in Dulbecco's modified Eagle's medium ( DMEM , Gibco ) , supplemented with 10% fetal bovine serum . Transfections were performed with polyethyleneimine ( 25 kDa , Sigma ) . The transcriptional activity of NHR-25 was tested with a luciferase vector carrying a CMV basic promoter driven by two copies of the Ftz-F1 binding consensus sequences TGAAGGTCA and TCAAGGTCA ( total of four binding sites , 2×TGA-TCA::Luc ) [8] , [63] . Cells were seeded onto 24-well plates and the next day were transfected for three hours with a polyethylenimine mixture containing 50 ng of pTK-Renilla plasmid ( Promega ) as an internal control , 300 ng of the luciferase reporter plasmid , and 150 ng of the appropriate expression vector . The total amount of DNA was kept constant ( 1 µg ) by adding empty expression vector where necessary . Forty hours post-transfection , the cells were harvested and processed using the Dual Luciferase Reporter Assay System ( Promega ) . Eight independent biological replicates from three independent experiments were assayed , and data were presented as average values with standard deviations after normalization against the Renilla luciferase activities . For immunocytochemistry , transfected cells were fixed with 4% formaldehyde ( Sigma ) for 10 min . After washing with PBS , cells were permeabilized with PBS containing 0 . 2% TritonX-100 in ( PBST ) , washed with TBST buffer ( 25 mM Tris-HCl , pH 7 . 5 , 136 mM NaCl , 2 . 7 mM KCl and 0 . 1% TritonX-100 ) , incubated in blocking solution ( 2 . 5% skim milk and 2 . 5% BSA in TBST ) . Anti-Myc 9E10 antibody ( Sigma; 1∶2000 dilution ) was added and incubated for overnight at 4°C . Following washing , goat-anti-mouse-TRITC conjugated 2° antibody ( Jackson ImmunoResearch; 1∶2000 dilution ) was added and incubated at room temperature for two hours . Cells were counterstained with DAPI ( 1 µg/ml ) to visualize the nucleus . | Animals precisely control when and where genes are expressed; failure to do so can cause severe developmental defects and pathology . Transcription factors must display extraordinary functional flexibility , controlling very different sets of genes in different cell and tissue types . To do so , they integrate information from signaling pathways , chromatin , and cofactors to ensure that the correct ensemble of genes is orchestrated in any given context . The number of regulatory inputs , and the complex physiology and large numbers of cell and tissue types in most experimentally tractable metazoans have rendered combinatorial regulation of transcription nearly impenetrable . We used the powerful genetics and simple biology of the model nematode , C . elegans , to examine how a single post-translational modification ( sumoylation ) affected the activity of a conserved TF ( NHR-25 ) in different cell types during animal development . Our work suggests that sumoylation constrains NHR-25 activity in order to maintain proper cell fate during development of the reproductive organ . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2013 | Sumoylated NHR-25/NR5A Regulates Cell Fate during C. elegans Vulval Development |
HIV-associated subacute meningitis is mostly caused by tuberculosis or cryptococcosis , but often no etiology can be established . In the absence of CT or MRI of the brain , toxoplasmosis is generally not considered as part of the differential diagnosis . We performed cerebrospinal fluid real time PCR and serological testing for Toxoplasma gondii in archived samples from a well-characterized cohort of 64 HIV-infected patients presenting with subacute meningitis in a referral hospital in Indonesia . Neuroradiology was only available for 6 patients . At time of presentation , patients mostly had newly diagnosed and advanced HIV infection ( median CD4 count 22 cells/mL ) , with only 17 . 2% taking ART , and 9 . 4% PJP-prophylaxis . CSF PCR for T . Gondii was positive in 21 patients ( 32 . 8% ) . Circulating toxoplasma IgG was present in 77 . 2% of patients tested , including all in whom the PCR of CSF was positive for T . Gondii . Clinically , in the absence of neuroradiology , toxoplasmosis was difficult to distinguish from tuberculosis or cryptococcal meningitis , although CSF abnormalities were less pronounced . Mortality among patients with a positive CSF T . Gondii PCR was 81% , 2 . 16-fold higher ( 95% CI 1 . 04–4 . 47 ) compared to those with a negative PCR . Toxoplasmosis should be considered in HIV-infected patients with clinically suspected subacute meningitis in settings where neuroradiology is not available .
In settings of Africa and Asia , the most common cause of subacute meningitis in patients with advanced HIV infection is either tuberculous or cryptococcal infection [1] , [2] . However , in many patients , the etiology of subacute meningitis cannot be established [1] , [3] . In line with a large retrospective cohort of adult meningitis patients in South Africa , where 52 . 8% had no definite diagnosis despite extensive microbiological testing [1] , we could not identify the causative pathogen in 48 . 9% of HIV-infected meningitis patients in an Indonesian setting [4] . Toxoplasmosis is a common and serious central nervous system ( CNS ) infection in patients with advanced HIV infection [5]–[8] , although its incidence has decreased with introduction of antiretroviral treatment ( ART ) [6] , [9] . Cerebral toxoplasmosis mostly presents as cerebral mass lesions with headache , confusion , fever , lethargy , seizures , cranial nerve palsies , psychomotor changes , hemiparesis and/or ataxia [10] . Some of these symptoms may also mimic meningitis , but cerebral toxoplasmosis is generally not considered as a differential diagnosis of subacute meningitis in HIV-infected patients . This is especially the case in low-resource settings where no CT or MRI can be performed . We have therefore examined if toxoplasmosis can be diagnosed in HIV-infected patients presenting with subacute meningitis of unknown origin in Indonesia , using cerebrospinal fluid ( CSF ) PCR for T . gondii .
Anonymized CSF and blood samples were used from an already-existing hospital collection , from a cohort of patients collected as part of a project ‘Optimization of diagnosis of meningitis’ , approved by the Ethical Committee of Hasan Sadikin Hospital/Medical Faculty of Universitas Padjadjaran , Bandung , Indonesia ( No . 85/FKUP-RSHS/KEPK/Kep/EC/2006 ) . As this study was done using already existing sample collection , no separate consent was asked for this study . HIV testing is done routinely with oral informed consent for all patients with suspected meningitis in Hasan Sadikin hospital , after 24% were found HIV-positive in a previous cohort study of 185 patients in the same hospital [4] . Consent is obtained from closest relatives ( husband/wife or parents ) for those patients who are unstable or unconscious at time of presentation . With approval from the ethical committee HIV testing was done anonymously afterwards for those who had died before consent could be obtained . We included adult patients presenting with suspected meningitis at Hasan Sadikin Hospital , the top referral hospital for West Java Province , Indonesia , between December 2006 and October 2010 . Clinical data including outcome was recorded in individual case report form . Definite TB meningitis was diagnosed if CSF culture or real time PCR were positive for M . tuberculosis , cryptococcal meningitis if either CSF India Ink examination , culture or cryptococcal antigen testing were positive , and toxoplasmosis if CSF T . gondii PCR was positive . HIV testing is done routinely for patients presenting at this hospital , but cerebral CT-scanning is rarely done in this setting and is not covered by the government health insurance for the poor . CSF cell count and differentiation , protein and glucose were measured . CSF microscopy was done for cryptococci , acid-fast bacilli and bacterial pathogens . CSF was cultured for Mycobacterium tuberculosis ( solid Ogawa and liquid MB-BacT , Biomerieux ) , bacterial pathogens ( blood agar , chocolate agar , and brain-heart infusion ) and fungi ( Sabouraud ) . Cryptococcal antigen ( CALAS , Meridian Diagnostics ) testing was done on CSF samples following the manufacturer's instructions . Five to 7 ml CSF samples were used for molecular testing . After centrifugation of CSF samples at 3000×g for 10 minutes , DNA was extracted from 200 µl of CSF sediment by using QIAmp DNA mini kit ( Qiagen , USA ) . CSF M . tuberculosis real time PCR was done using IS6110 , a repeated insertion sequence specific for M . tuberculosis , as a target [11] . Measurement of CD4-cell count for HIV-patients only became available during the time of the study and was measured only for those who survived for more than 4 days . Real time PCR for T . gondii , using the multicopy B1 gene of the T . gondii as the target as described elsewhere [12] , was performed to archived CSF samples at Radboud University Nijmegen Medical Centre . CSF specimens from 22 HIV-negative meningitis patients ( 16 with definite TB meningitis , 2 with bacterial meningitis , and 4 with no definite diagnosis ) , and nine patients with non-infectious CNS diseases , all recruited at Hasan Sadikin Hospital , were used as controls for T . gondii PCR . These samples were collected during the study period over a similar time scale compared to the case CSF samples . Toxoplasma immunoglobuline G ( toxoplasma IgG ) were measured by electro chemiluminescent assay ( ECLIA , Elecsys , Roche ) in archived serum samples of patients included in the study . Characteristics of patients with definite tuberculosis , cryptococcosis and toxoplasmosis were compared using Chi-square test for proportions and Mann-Whitney U test for continuous variables . Progression to death using 2-month mortality data was examined by Kaplan–Meier estimates .
During the period , 401 patients presented with clinical meningitis , 76 were diagnosed with HIV infection , and 64 had archived CSF samples and were included in this study . Patients included in the study presented after a median 7 days , with meningismus ( 86 . 0% ) , headache ( 80 . 8% ) , lowered consciousness ( 33 . 3% ) , fever ( 28 . 8% ) , hemi- or tetraparesis ( 28 . 6% ) , cranial nerve palsies ( 12 . 5% ) , and seizures ( 10 . 9% ) . HIV was newly diagnosed in 53 patients ( 82 . 8% ) . All 11 patients previously diagnosed with HIV were taking ART , and 6 were using co-trimoxazole as Pneumocystis jiroveci ( PJP ) prophylaxis at time of presentation . The median CD4 cell count was 22 cells/mL , and less than 200 cells/mL in 22 out of 23 patients tested ( 96% ) . CSF T . gondii PCR was positive in 21 of 64 HIV-infected patients ( 32 . 8% ) , with a median Ct-value of 36 . 0 ( IQR: 34 . 2–39 . 3 ) . None of the 22 HIV-negative control and 9 non-infectious CNS disease patients had a positive T . gondii PCR . Archived serum sample was not available in 14 patients . Toxoplasma IgG was positive in 78% of patients tested , including all patients with positive CSF T . gondii PCR . Toxoplasma IgG titers were higher among patients with a positive CSF T . gondii PCR ( p = . 017 ) . A definite diagnosis of TB meningitis was established in 21/64 patients ( 32 . 8% ) . Out of 21 patients with positive T . gondii PCR , five had combined tuberculosis and toxoplasmosis . Cryptococcosis was diagnosed in 15/64 patients ( 23 . 4% ) , including two who were also diagnosed with tuberculosis . In 14 patients ( 21 . 9% ) no causative pathogen was isolated . Neck stiffness , headache and fever , the classical signs of meningitis , were equally common in patients diagnosed with toxoplasmosis , cryptococcosis and tuberculosis , as were most other signs and symptoms , except hemiparesis ( Table 1 ) . None of the patients with toxoplasmosis had received ART or co-trimoxazole prophylaxis prior to admission with meningitis . CT scans were available for 6 patients , including 4 with a positive T . gondii PCR . Three showed signs of hydrocephalus , one a hypodense lesion that showed no enhancement using contrast , and two were normal . No mass lesions typical for cerebral toxoplasmosis were seen . CSF cell count and protein were normal or mildly elevated in patients with toxoplasmosis , and hypoglycorrhachia was less common compared with tuberculosis or cryptococcosis ( Table 1 ) . CD4 counts , missing in two-thirds of patients due to early death or the unavailability of CD4 cell testing during the initial phase of the cohort study , were low in all but one patient . Table 2 lists the CSF findings of individual patients , Figure 1 is a graphic representation of the CSF cell count , protein and glucose ratio , showing the overlap in CSF findings between patients with toxoplasmosis , cryptococcal and tuberculosis CNS infection . Patients with confirmed cryptococcosis received amphotericine B , followed by fluconazole; all others received empiric tuberculosis treatment combined with adjunctive corticosteroids [13] . No toxoplasmosis treatment was given , as T . gondii PCR was performed retrospectively and was not available at time of presentation . Eight patients were lost to follow up and were not included in Kaplan Meier analysis . Mortality among those with positive CSF T . gondii PCR was 2 . 16-fold ( 95% CI 1 . 04–4 . 47 ) higher compared to those who had a negative PCR result; median survival was 7 days for toxoplasmosis , 7 days for tuberculosis meningitis , 110 days for cryptococcosis , and 32 days for patients with an unknown cause of meningitis ( Figure 2 ) .
In our cohort of HIV-infected patients presenting with clinical signs and symptoms of CNS infection , CSF T . gondii PCR was positive in 32 . 8% of patients , sometimes in conjunction with tuberculosis . In the absence of CT or MRI of the brain , toxoplasmosis could not be distinguished from tuberculosis or cryptococcosis . Mortality in this cohort of newly diagnosed and advanced HIV infection was extremely high and associated with a positive T . gondii PCR . Cerebral toxoplasmosis typically causes space occupying lesion ( s ) , leading to subacute or acutely developing confusion , with or without focal neurological deficits [14] . In the absence of CT or MRI of the brain , common findings like headache , fever , hemiparesis and decreased level of consciousness [10] may mimic those of meningitis [4] , [15]–[17] . In previous series of cerebral toxoplasmosis [18] , [19] , meningeal signs have been reported in 3 to 16% of the patients , although in many reports neck stiffness is not mentioned [14] . Although rare , cases of spinal cord toxoplasmosis have also been reported [20] . No typical mass lesions were found in 6 patients with an available CT scan . This is not surprising , as this study depended on the availability of CSF samples , that would not have been obtained if typical mass lesions had been found . We used T . gondii PCR for diagnosis of cerebral toxoplasmosis . In previous studies CSF T . gondii PCR had a sensitivity of 50–60% to confirm cerebral toxoplasmosis in HIV-infected patients [21] , [22] . The sensitivity is possibly higher among patients with meningoencephalitis compared to those with space-occupying lesions only , but this has not been examined . Specificity of T . gondii PCR is high , between 97 and 100% [22]–[24] . The positivity rate of 32 . 8% in our study might therefore be an underestimate , especially in the category of patients in whom no other pathogen was isolated despite extensive microbiological testing . In our cohort , toxoplasmosis could not be distinguished clinically from tuberculosis and cryptococcosis . From our previous series [4] , CSF samples were available for the current study for 36/47 HIV-infected patients . Ten out of 17 patients who were diagnosed with ‘probable TB meningitis’ and ‘unknown’ in the previous study were found to have a positive T . gondii PCR ( and no bacteriological confirmation of tuberculosis ) in the current study . Diagnosis of cerebral toxoplasmosis is usually based on clinical findings and CT or MRI of the brain . However , if cerebral imaging is lacking , toxoplasmosis may not be considered . Positive toxoplasma serology , which has a high sensitivity but very poor specificity , is helpful to exclude but not to confirm cerebral toxoplasmosis , although some reports suggest that high toxoplasma IgG titers are only found in patients with symptomatic toxoplasmosis [25] . Indeed , in our study , patients with a positive T . gondii PCR had a higher IgG titers compared to those who had a negative PCR . An autopsy study from India provides further support for the notion that cerebral toxoplasmosis is not always considered; among 233 HIV patients , toxoplasmosis accounted for 6 . 8% of deaths , but in none of these cases toxoplasmosis had been suspected clinically [26] . The incidence of cerebral toxoplasmosis varies between countries [14] and is related to the seroprevalence of toxoplasmosis in the general population [19] , [25] . In the United States , toxoplasma seroprevalence varies from 3% to 30% , whereas in France 73%–90% of the population is infected [10] . Reported seroprevalence rates were varied from 13–31% in the general population , and 45–68% in HIV patients in studies from several developing countries [8] , [27] , [28] . In our study , 78% of patients had detectable toxoplasma IgG , but this does not reflect the seroprevalence in the general population or among unselected HIV-infected patients , as only meningitis patients were examined . Mortality in this cohort of patients was very high , higher compared to reported rates in other series [8] , [9] , [29] . One explanation is that patients mostly presented with advanced and untreated HIV infection . In addition , no toxoplasmosis treatment was provided , as PCR was done retrospectively on archived samples . In our previous study , HIV infection was associated with a 2 . 5-fold increased mortality among patients presenting with meningitis [4] . Data from the current study suggests that this may is at least in part attributable to a high prevalence of ( unrecognized and untreated ) toxoplasmosis . Future studies should examine the benefit of timely diagnosis and/or empiric treatment of toxoplasmosis for patients in settings like ours . Empiric treatment for subacute meningitis in HIV-infected patients should probably also include tuberculosis , which is difficult to exclude as culture is slow and microscopy and commercial PCR assays have insufficient sensitivity [30] . Our study suffers from several limitations . Most importantly , no CT or MRI of the brain could be performed . In addition , clinical data , CD4 cell counts and other laboratory parameters were missing in a number of patients . Despite these limitations the data strongly suggest that toxoplasmosis should be included in the differential diagnosis of HIV-infected with clinically suspected subacute meningitis , and that molecular testing or empiric treatment for toxoplasmosis should be considered in these patients , especially if no CT or MRI can be performed . Obviously , timely diagnosis and treatment of HIV will help prevent this severe opportunistic infection . | If HIV-infected patients present with seizures , focal neurological symptoms or confusion , a CT-scan or MRI of the brain is normally made . If mass lesions are found ( and the CD4 cell count is sufficiently low ) , cerebral toxoplasmosis is suspected , and often treated empirically . However , some of the symptoms of cerebral toxoplasmosis may mimic those of subacute meningitis . Therefore , in settings where no cerebral imaging can be performed , HIV-associated cerebral toxoplasmosis may be under-diagnosed . We retrospectively looked for toxoplasmosis in a cohort of HIV-infected patients presenting with subacute meningitis in an Indonesian hospital , where neuroradiology was not available for most patients . Patients mostly came with newly diagnosed and advanced HIV infection and few were on HIV-treatment or PJP-prophylaxis . Molecular testing of cerebrospinal fluid ( CSF ) was positive for Toxoplasma gondii in 32% of patients , serology was positive in 78% . Clinically , in the absence of neuroradiology , toxoplasmosis was difficult to distinguish from tuberculosis or cryptococcal meningitis . A positive CSF T . gondii PCR was associated with a two-fold increased mortality . We conclude that toxoplasmosis should be considered in HIV-infected patients with clinically suspected subacute meningitis in settings where neuroradiology is not available . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [
"medicine",
"infectious",
"diseases",
"global",
"health",
"neurology",
"neurological",
"disorders"
] | 2013 | Cerebral Toxoplasmosis Mimicking Subacute Meningitis in HIV-Infected Patients; a Cohort Study from Indonesia |
miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets . Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates , but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes . To provide genetic insight from Drosophila , we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation . On the other hand , we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene , including short lifespan , increased dendrite variation , impaired larval locomotion , and aberrant synaptic release at the NMJ . These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions . Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals , purified on the basis of mir-124 promoter activity , revealed broad upregulation of direct miR-124 targets . However , in contrast to the proposed mutual exclusion model for miR-124 function , its functional targets were relatively highly expressed in miR-124–expressing cells and were not enriched in genes annotated with epidermal expression . A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway , whose activation in neurons increases synaptic release at the NMJ , similar to mir-124 mutants . Derepression of the direct miR-124 target network also had many secondary effects , including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature . Altogether , these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology .
microRNAs ( miRNAs ) are ∼22 nucleotide ( nt ) regulatory RNAs that function primarily as post-transcriptional repressors . In animals , miRNAs have propensity to target mRNAs via 6–7 nt motifs complementary to their 5′ ends , termed “seed” regions [1]–[4] . This limited pairing requirement has allowed most miRNAs to capture large target networks . Analysis of multigenome alignments indicates that typical human miRNAs have hundreds of conserved targets , and that a majority of protein-coding genes are under miRNA control [5] , [6] . The extraordinary breadth of animal miRNA:target networks has been extensively validated by transcriptome and proteome studies [7] . miR-124 is strictly conserved in both primary sequence and spatial expression pattern , being restricted to the nervous system of diverse metazoans , including flies [8] , nematodes [9] , Aplysia [10] , and all vertebrates studied [11]–[13] . Such conservation implies substantial functions of miR-124 in controlling neural gene expression . miR-124 has been a popular model for genomewide investigations of miRNA targeting principles . For example , studies of miR-124 yielded the first demonstration of the downregulation of hundreds of direct targets detected by transcriptome analysis , and that this activity was driven by the miRNA seed region [14] . In addition , miR-124 provided one of the first illustrations of spatially anticorrelated expression of a miRNA and its targets [15] , and was exploited for analysis of Ago-bound target transcripts [16]–[19] and direct identification of Ago-bound target sites [20] . Functional studies have connected vertebrate miR-124 to various aspects of neural specification or differentiation . Studies in chick ascribed miR-124 as a proneural factor that inhibits the anti-neural phosphatase SCP1 [21] . However , no substantial effect of miR-124 on chick neurogenesis was found in a parallel study [22] , although miR-124 was observed to repress neural progenitor genes such as laminin gamma1 and integrin beta1 . In the embryonic mammalian brain , miR-124 was reported to direct neural differentiation by targeting polypyrimidine tract binding protein 1 ( PTBP1 ) , a global repressor of alternative splicing in non-neural cells [23] . In the adult mammalian brain , miR-124 promoted neural differentiation of the immediate progenitors , the transit-amplifying cells ( TAs ) . Here , miR-124 directly targets the transcription factor Sox9 , which maintains TAs and is downregulated during neural differentiation [24] . Other mammalian studies bolster the concept that miR-124 promotes neurogenesis [25] or neural differentiation [26] . One mechanism involves direct repression by miR-124 of Baf53a , a neural progenitor-specific chromatin regulator that must be exchanged for a neural-specific homolog to consolidate neural fate [27] . However , complicating the picture is the recent report that Xenopus miR-124 represses neurogenesis by directly targeting the proneural bHLH factor NeuroD1 [28] . All vertebrate miR-124 loss-of-function studies have relied on antisense strategies and have yet to be validated by bona fide mutant alleles . However , as the three vertebrate mir-124 loci are co-expressed in the nervous system , analysis of the null situation will require a triple knockout . So far , a mir-124 knockout has only been described in C . elegans , which harbors a single copy of this gene [29] . Like most other miRNA mutants in this species , the loss of miR-124 did not cause obvious developmental , physiological or behavioral phenotypes . Nevertheless , comparison of gene expression in mir-124-expressing cells from wildtype and mir-124 mutant animals revealed strong enrichment in miR-124 target sites amongst upregulated transcripts , revealing the impact of miR-124 on neuronal gene expression [9] . The broad , but phenotypically-tolerated , misregulation of miR-124 targets in this species is potentially consistent with the “fine-tuning” model for miRNA regulation . Here , we analyze a knockout of the sole mir-124 gene in D . melanogaster . Although this mutant is viable and exhibits grossly normal patterning , we documented numerous phenotypes , including short lifespan , increased variation in the number of dendritic branches of sensory neurons , decreased locomotion and aberrant synaptic release at CNS motoneuron synapses . All of these phenotypes were rescued by a single copy of a 19 kilobase ( kb ) genomic transgene encompassing the mir-124 locus . We generated a transcriptional reporter of mir-124 that recapitulated the CNS expression of endogenous pri-mir-124 , and used this to purify mir-124-expressing cells from stage-matched wild-type and mir-124-mutant embryos . Transcriptome analysis revealed strong enrichment of direct miR-124 targets amongst genes upregulated in mir-124-mutant cells . The miR-124 target network included coordinate repression of multiple components in the retrograde BMP signaling pathway , whose activity controls synaptic release . Loss of miR-124 further correlated with increased activity of other neural miRNAs and the neural translational regulator Pumilio , and had the net effect of impairing transition from the neuroblast to neuronal gene expression signature . Altogether , we demonstrate that endogenous miR-124 has substantial impact on CNS gene expression , which underlie its requirement for organismal behavior and physiology .
Northern analysis first detected mature miR-124 at 4–6 hrs of development ( Figure 1A ) , corresponding approximately to embryo stages 9–10 . Its level peaked during 12–24 hrs , declined during the first and second larval stages , and was then upregulated in the third instar through adulthood . The apparent temporal fluctuation in miR-124 levels appeared to be a consequence of its tissue-specificity . For example , most miR-124 in the adult was present in the head ( Figure 1A ) , consistent with comparison of head and body small RNA data [30] . We therefore used in situ hybridization to primary miRNA transcripts to analyze expression of Drosophila mir-124 at the cellular level [8] . Close examination showed that its primary transcription , as reflected by nuclear dots of elongating pri-mir-124 transcripts ( Figure 1B , inset ) , was first detected in the ventral nerve cord around stage 8 during germband elongation ( Figure S1 ) and became more prominent in subsequent stages . Its expression in the ventral nerve cord and brain was maximal in the fully germband retracted embryo from stage 13 onwards ( Figure 1B–1D ) . To facilitate analysis of mir-124 expression , we generated a transcriptional reporter . We fused 4 . 2 kb of sequence upstream of the mir-124 hairpin , including ∼1 kb more genomic sequence than the previously studied mir-124:Gal4 transgene [31] , to a nuclear DsRed gene in the insulated H-Red-Stinger vector . Multiple transgenic lines exhibited identical expression in the embryonic nervous system that recapitulated endogenous pri-mir-124 expression . Similar to endogenous pri-mir-124 , the mir-124:DsRed transgene was faintly active at stage 8 ( Figure S1 ) , and exhibited nearly completely colocalization with the pan-neuroblast marker Deadpan in the stage 9 CNS ( Figure 1E , 1E′ ) ; at this stage mature neurons have not yet been specified . Neuroblasts ( NBs ) divide to regenerate the NB as well as a ganglion mother cell ( GMC ) . GMCs can be marked by Prospero , and these cells were similarly labeled by mir-124:DsRed ( Figure 1E , 1E″ ) . We continued to observe DsRed expression in NBs and GMCs as development proceeded ( Figure 1F , 1G ) . GMCs divide to generate sibling cells and neurons , and neuronal commitment is marked by expression of Elav . mir-124:DsRed was active in the full complement of neurons in the CNS , but Elav alone was highly expressed in the peripheral nervous system ( Figure 1H , 1I ) . We used ends-out homologous recombination to replace the endogenous mir-124 hairpin with a white+ marker flanked by loxP sites ( Figure 2A ) . We established several knockouts from independent insertions of the original targeting vector , so that we could query trans-heterozygous deletion combinations . We also deleted the white+ marker to obtain clean deletions of the locus . As these behaved similarly to the white+ alleles ( not shown ) , most subsequent analyses utilized the latter alleles since the marker facilitated the construction of recombinant lines . We used Northern analysis to verify that multiple independent mir-124 knockout alleles did not express mature miR-124 ( Figure 2B ) , demonstrating that these are truly null backgrounds . The mir-124 mutant alleles were viable and fertile , and exhibited normal external morphology . However , they were not easily kept as homozygous stocks , potentially reflecting detrimental effects of mir-124 deletion . Because homologous recombination in Drosophila can induce unlinked aberrations , which might theoretically be shared by independent targeting events , we were cautious in the comparison of trans-heterozygous mutants to wildtype . We therefore generated a P[acman] insertion of 19 kb of mir-124 genomic DNA ( 39N16 , Figure 2A ) , a region lacking annotated protein-coding genes; note that it contains mir-287 , but this locus has not been confirmed in largescale sequencing [30] , [32] . We recombined the 39N16 rescue with mir-124 deletion alleles , and used Northern analysis to validate that this transgene restored a normal level of miR-124 to mutant adults ( Figure 2B ) . We subsequently focused on phenotypes evident in trans-heterozygous animals compared to heterozygotes , that were rescued by the mir-124 genomic transgene . We observed that 60–70% of mir-124 deletion embryos of various genotypes failed to hatch , and that embryonic lethality was substantially ( although not fully ) rescued by the mir-124 genomic transgene ( Figure S2 ) . Following embryogenesis , we did not observe substantial differences in viability between the mir-124 mutant and wildtype , at larval/pupal/adult stages ( Figure S2 ) . However , mir-124 mutant adult males exhibited substantially shortened lifespan , and this defect was completely rescued by introduction of the mir-124 genomic transgene ( Figure 2C ) . These observations suggest that miR-124 is detectably required for organismal fitness . Because of the specific expression of mir-124 in the CNS , we were interested to see if we could uncover any defects in neural development . We analyzed a number of CNS markers , but did not detect obvious changes across a panel of neuroblast and GMC markers , including Deadpan and Prospero ( Figure 3A , 3B ) and Hunchback and Miranda ( Figure S3 ) . Careful quantification of the numbers of Deadpan+ neuroblasts did not reveal differences within either thoracic or abdominal segments ( Figure 3C ) . The overall pattern of Elav was also normal ( Figure 3A , 3B ) . Since many cells express Elav , we also checked Even-skipped , which is expressed in small populations of neurons and sibling cells , but these also appeared relatively normal ( Figure 3D , 3E and Figure S3 ) . We further analyzed the glial marker Repo , which was reported as a direct miR-124 target with anti-correlated expression [15] , [33] , but its pattern was not substantially altered ( Figure 3F , 3G ) . Finally , mir-124 mutants exhibited grossly normal axonal architecture in the late embryo , as marked by 22C10 ( Figure S4 ) . To assess a possible phenotype in later development , we also examined the larval CNS . We detected abundant activity of mir-124:DsRed in the larval CNS , including both the brain and ventral nerve cord ( Figure S5A ) . Within the brain , activity or mir-124:DsRed was highest in the central complex ( Figure S5B ) . However , Deadpan/Elav staining showed relatively normal patterns of neuroblasts and neurons in the mir-124 mutant brain ( Figure 3H , 3I ) . Finally , we assessed the proliferation of larval neuroblast clones using the MARCM technique . Using this strategy , the neural progeny of single neuroblasts can be labeled in situ ( Figure S5C ) . We observed that mir-124 mutant neuroblast clones appropriately maintained a single neuroblast and could undergo multiple divisions to generate many neurons ( Figure 3J ) . This level of analysis does not address potential quantitative defects in neuroblast clones , nor does it rule out that a subpopulation of cells may have developed abnormally . However , pan-CNS Drosophila miR-124 does not appear to be required for bulk aspects of neurogenesis or differentiation , as has been concluded for its vertebrate counterparts . Since we did not observe substantial defects in neural development , we checked for functional defects in the central nervous system . An informative assay involved tracking the locomotion of third instar larvae . We examined the movements of cohorts of larvae in 1 minute movies , and quantified total distance traveled and crawling speed . Different trans-heterozygous mir-124 mutant combinations exhibited a clear defect in both parameters , and these were fully rescued by the mir-124 genomic transgene ( Figure 4A–4C and Figure S6 ) ; the differences were highly statistically significant ( Figure 4D ) . Therefore , miR-124 is required for normal locomotion . To gain functional insight into the basis of this defect , we first tested for a possible role of miR-124 in synaptic structure . We analyzed the arborization of neuromuscular junctions ( NMJs ) of CNS motoneurons in third instar larvae ( Figure 4E , 4F ) , but this did not reveal significant changes in the number of NMJ boutons or the area arborized ( Figure 4G ) . We therefore went on to analyze the activity of these synapses . Using the two-electrode voltage clamp technique , we measured both spontaneous miniature excitatory junctional currents ( mEJCs ) and evoked junctional currents ( EJCs ) from control and mir-124 mutant larvae ( Figure 4H–4J ) . mEJCs were indistinguishable in the two groups , but mir-124 mutants showed an increase in the average EJC amplitudes , indicating a significant elevation in quantal content at the NMJ ( Figure 4K ) . The increase in EJCs was fully rescued when we included a mir-124 genomic transgene in the homozygous mutant larvae , indicating that the increase in EJCs and quantal content was attributable to the mir-124 deletion . Therefore , miR-124 serves to limit synaptic activity . Expression of Drosophila mir-124 was confidently detected only in CNS , but did not exclude potential expression in the PNS . Of note , C . elegans mir-124 is predominantly expressed in sensory neurons [9] . We therefore checked for PNS phenotypes , judging that defects that were rescuable should reflect endogenous requirements for miR-124 . This analysis revealed a defect in the differentiation of dendrites in a subset of sensory neurons ( Figure 5A–5C ) . On the one hand , the average number of dendritic branches in mir-124 mutants did not show a statistical difference from that in wildtype larvae . However , the variation in dendrite numbers was substantially increased in mir-124 mutants ( Figure 5D ) ; this was especially noticeable for ddaD . This defect was rescued by the mir-124 genomic transgene , indicating that miR-124 suppresses variability in dendritic branching numbers . We also tested the effect of misexpressing miR-124 in class I neurons , building on our observation that ectopic miR-124 reduces dendrite numbers in wild-type [31] . Misexpression of miR-124 in mir-124 mutants , using a newly constructed UAS-DsRed-mir-124 transgene , recapitulated this defect ( Figure 5C ) . Despite the gain-of-function phenotype of reduced dendrite number , the variation in dendrite numbers across the population was rescued ( Figure 5D ) . These results suggest that miR-124 helps maintain the consistency of dendritic branching patterns of specific neurons . Such a function has not yet been reported from the study of other dendrite mutants [34] . Having established a variety of clear phenotypes in mir-124 mutants , we wished to query changes in gene expression in the mutant cells . Because this miRNA is only expressed in the nervous system , we did not expect to be able to make specific measurements using whole embryos . Instead , we took advantage of the mir-124:DsRed reporter to isolate mir-124-expressing cells from dissociated embryos using fluorescence activated cell sorting ( FACS ) . We introduced mir-124:DsRed into the mir-124 mutant background , so that we could isolate the relevant mutant cells ( Figure 6A ) . Consistent with the lack of substantial neural specification defects in the mutant , the expression of the mir-124 reporter was similar in the presence or absence of the miRNA . This suggested that transcriptional profiling by this strategy was not likely to be substantially affected by the absence of cell types whose specification might require miR-124 , or that might fail to be isolated because of positive autoregulatory feedback of miR-124 onto its own transcription . We note that analogous mir-124 promoter fusions in nematode and zebrafish were correctly expressed in the absence of endogenous mir-124 and Dicer , respectively [9] , [35] . Recognizing that substantial manipulation is incurred during embryo dissociation and cell sorting , we were interested to obtain confidence that potential changes in gene expression in our measurements could be specifically attributed to miR-124 activity . Although some degree of non-autonomous regulatory effect is plausible , for example due to miRNA targeting of signaling factors , a general expectation is that the direct regulatory effects of a miRNA should be cell autonomous . Therefore , we sought to gauge the specificity of gene expression changes by comparing cells that normally express the miRNA with those that do not . To do so , we separated miR-124:DsRed+ and DsRed− cells from stage 13–16 embryos ( ∼10–16 hrs of development ) that were wildtype or deleted for mir-124 . We chose this as a temporal window that was late enough to permit the full pattern of miR-124 expression to be established , but putatively early enough to minimize highly indirect changes in gene expression ( i . e . , that might arise during the remainder of embryogenesis from 16–22 hrs ) . Post-sort analysis showed that ∼80% of the selected cells were DsRed+ , and qPCR analysis of these sorted wild-type cells confirmed that the DsRed+ cells specifically expressed pri-mir-124 ( Figure 6B ) . We then examined a panel of transcripts with high-ranking TargetScan scores ( http://www . targetscan . org/ ) and conserved miR-124 target sites in their 3′ UTRs ( Figure 6C ) . We could indeed validate many such targets as being upregulated in miR-124:dsRed+ cells isolated from mir-124 mutants by qPCR ( Figure 6C ) . In contrast , we observed very few changes in these same transcripts in mir-124 mutant cells that did not express miR-124:DsRed , indicating that their deregulation was likely a direct consequence of miR-124 activity . With these data in hand , we moved to transcriptome-wide analysis . We purified three biologically independent samples of miR-124:DsRed+ cells from dissociated wildtype and mir-124 mutant stage 13–16 embryos and profiled them using Affymetrix microarrays . We generated sufficient RNA from purified cells so that only a single amplification step was required . The triplicate wild-type and mir-124-mutant transcriptomes were highly segregated by unsupervised hierarchical clustering ( Figure 6D ) , indicating that major changes in expression profiles were due to genotype and not to technical variation . Although several genomewide studies in vertebrates demonstrated upregulation of direct targets upon miRNA depletion or knockout [36]–[39] , in some cases a genomewide signature was not recovered with mutants of single-copy , tissue-specific , miRNAs ( e . g . mir-182 ) [40] . Therefore , broad upregulation of targets in a miRNA mutant is not a given . We plotted the cumulative distribution function ( CDF ) of various sets of genes , comparing their levels in the mir-124 mutant relative to wildtype . Indeed , transcripts bearing miR-124 sites predicted by mirSVR [41] exhibited a highly statistically significant shift to higher levels in mir-124 mutants ( p-value<1 . 11e-12 ) ( Figure 6E ) ; i . e . shifted to the right in the CDF plot . Therefore , endogenous miR-124 strongly influences the transcriptome of the Drosophila nervous system . Moreover , derepression of direct targets accounted for a substantial proportion of the most deregulated genes in mir-124 mutants , since 24/59 genes upregulated >2-fold with p-value<0 . 05 bore miR-124 seed sites ( Table S1 ) . We further divided targets into a poorly-conserved cohort ( site alignment is confined , at most , to the five melanogaster group species ) and a well-conserved cohort ( target site is aligned in both melanogaster group and non-melanogaster group Drosophilids ) . As has been observed in vertebrate systems , well-conserved targets of fly miR-124 were overall repressed more potently than poorly-conserved targets ( Figure 6F ) . Nevertheless , recently-evolved miR-124 target sites exerted palpable regulatory impact in the intact animal , since transcripts with such sites were detectably shifted in their expression relative to background . We also subdivided targets by category ( 7mer , 6mer and non-canonical sites with seed-mismatches ) and observed that these conferred progressively less regulation ( Figure 6G ) . To validate the capacity for direct targeting of these transcripts by miR-124 , we assayed the response of luciferase-3′ UTR sensors to ectopic miR-124 in S2 cells . Analysis of 8 such sensors , bearing single conserved 7mer or 6mer sites , showed that all were significantly repressed upon transfection of ub-Gal4 and UAS-mir-124 expression constructs ( Figure 6H ) . The distribution of repression values confirmed that 7mers generally yielded greater repression than 6mers . In summary , this first transcriptome-wide analysis of target expression in purified cell populations in a Drosophila miRNA mutant supports general notions of target site activity from vertebrate studies . A general principle of miRNA targeting emerged from comparing the spatial expression of tissue-specific Drosophila miRNAs with their predicted targets . A bias for spatial anti-correlation of such miRNAs and their targets was observed , termed “mutual exclusion” [15] . For example , neural genes were depleted of miR-124 target sites while epidermal genes were enriched for miR-124 target sites . Since all of these cell types derive from a common progenitor , the neuroectoderm , this led to the model that expression of miR-124 helps to repress epidermal potential in neurons [15] . In principle , such a pattern might reflect an active role of miR-124 to suppress the epidermal program in neurons , or might reflect a fail-safe program that is secondary to transcriptional mechanisms . We were in a position to test this using our gene profiling data from wildtype and mutant miR-124-expressing cells . We first tested whether we could reproduce the mutual exclusivity principle amongst miR-124 target genes , as defined by an independent set of miRNA target predictions generated using mirSVR [41] and multiZ alignments of twelve Drosophila genomes [42] . Together , this analysis incorporates more information on miRNA targeting and more genomes than were available earlier [15] . Indeed , cross-referencing these target predictions against in situ annotations catalogued from Drosophila embryogenesis [43] confirmed that epidermal genes were enriched amongst miR-124 targets at stages 11–12 and 13–16 ( Figure 7A ) , as reported earlier [15] . However , when performing a similar analysis using our data from functional derepression in mir-124 mutant cells , we failed to observe broad derepression of epidermal target genes , either amongst well-conserved or poorly-conserved target sets ( Table S2 ) . There were certainly individual miR-124 targets that are expressed and/or function in epidermal development , but this was not an overall trend amongst derepressed miR-124 targets ( Figure 6E–6G ) . We also did not observe overall enrichment for epidermal genes amongst all upregulated genes ( thus including both direct and indirect effects , Table S2 ) , and only a few transcripts with miR-124 targets were absent in wild-type miR-124:DsRed+ cells and now present in mir-124 mutant cells ( 17/204 putative targets , but only 2 of these bore conserved sites; Table S1 ) . Overall , these observations suggested that mutual exclusion of miR-124 and target accumulation is not a feature actively driven by miRNA activity . We investigated this further by examining the absolute levels of predicted miR-124 targets in miR-124-expressing cells . miR-124 targets exhibited a strong trend to be amongst the more highly expressed genes compared to non-targeted transcripts; this was true not only in the mir-124 mutant but also in wildtype ( Figure 7B and Figure S7 ) . Moreover , well-conserved miR-124 targets were generally more highly expressed than poorly-conserved targets , even in wild-type miR-124-expressing cells ( Figure 7B ) . We conclude that evolutionary selection of miR-124 target sites in miR-124-expressing cells is biased for transcripts that accumulate to above-average levels , even though the presence of miR-124 target sites clearly decreases the endogenous levels of these target transcripts ( Figure 6E–6G ) . To complement these quantitative data with cellular data , we examined the expression of the miR-124 target Repo [33] , which we confirmed to be directly responsive to miR-124 ( Figure 6H ) . The spatial expression of miR-124 and Repo was previously reported to be mutually exclusive [15] , and we confirmed exquisite exclusion of their domains in the ventral ectoderm , where miR-124 is active in neurons and Repo in glia ( Figure 7C–7C′″ ) . Only in rare cells could we observe co-expression of these markers , and these might potentially be due to reporter perdurance . Looking more ventrally into the progenitor layer , we observed strong co-expression of miR-124:DsRed with neuroblasts marked by Deadpan ( Figure 7D ) , as noted earlier ( Figure 1E–1G ) . However , this layer also contained strongly Repo-positive cells ( Figure 7D′ ) that colabeled with miR-124:DsRed but were exclusive of Deadpan; we infer these to be glioblasts . As these cells are progenitors , perdurance does not appear to explain co-expression of miRNA reporter and target . We infer that a phase of coexpression of miR-124 and repo precedes the adoption of their mutually-exclusive state . Overall , these data indicate a substantial trend for co-expression of miR-124 and its targets genomewide , as similarly deduced from studies of miR-124 in zebrafish [35] and C . elegans [9] . Furthermore , while we could confirm that mutual exclusion with epidermal genes is clearly a feature of the target network selected by Drosophila miR-124 , it does not seem to be a major determinant in directing neuronal-specific programs of gene expression , since epidermal genes were not overall substantially upregulated in the absence of the miR-124 . Given that we failed to observe substantial contribution of mutual exclusion to the functional miR-124 target network , we sought connections between de-repressed miR-124 targets and mutant phenotypes . Amongst neural genes upregulated ∼2-fold in mir-124 mutant cells and contain miR-124 binding sites in their 3′ UTRs were multiple members of the retrograde BMP signaling pathway , including the receptors saxophone ( sax ) ( Figure 6C ) and wishful thinking ( wit ) , and the transcription factor Mad ( Tables S1 and S2 ) . Further inspection showed that another BMP receptor thickveins ( tkv ) and the co-Smad Medea also contain highly conserved miR-124 binding sites , although tkv mRNA was not upregulated in the microarray and Medea was not detected by this platform ( even though it has a critical function in neurons ) . These five genes are core positive components of the retrograde BMP signaling pathway ( Figure 8A ) , by which the target muscle activates BMP signaling in the neuron to control NMJ development and synaptic physiology [44] . Although many of these sites in BMP pathway targets were only 6mers ( matching positions 2–7 of miR-124 ) , all of them except the Mad site were well-conserved across Drosophilid evolution ( Figure 8B and Figure S8 ) , implying their functional constraint . Moreover , both sax and tkv contained closely paired sites that are predicted to function cooperatively [45] . We conducted sensor assays to examine the response of these targets to miR-124 , and observed that all five targets were indeed repressed by ectopic miR-124 , with especially strong repression of the sax and tkv sensors that contained conserved paired sites ( Figure 8C ) . Since coordinate regulation of multiple aspects of an entire pathway by an individual miRNA is only rarely observed [1] , [46] , [47] , this property is a distinctive aspect of the miR-124 target network . Notably , we recently showed that misexpression of activated Sax and Tkv receptors in motoneurons increases synaptic activity without affecting NMJ structure [48] , [49] , similar to mir-124 mutants . We conducted further experiments by expressing activated Tkv alone in motoneurons using BG380-Gal4 . Activated Tkv did not affect spontaneous synaptic activity , as measured by miniature EJCs , but did increase both evoked EJCs and quantal content by 50% ( Figure 8D–8F ) . These defects phenocopied the electrophysiological defects of mir-124 mutant synapses ( Figure 4E–4K ) . Although deregulation of other targets likely contributes to the observed mir-124 mutant phenotypes , the similarity in electrophysiological defects upon deletion of miR-124 and overactivity of retrograde BMP signaling suggests that deregulation of this pathway may contribute to aberrant physiology of mir-124 mutant synapses . The bioinformatic analyses presented thus far focused specifically on motifs of interest , e . g . miR-124 seeds . A complementary strategy is to assess what sequence motifs best explain global shifts in gene expression between control and experimental conditions . The miREDUCE algorithm performs an unbiased search for motifs that correlate with patterns of upregulated or downregulated expression changes [50] . Amongst 7-nt motifs associated with transcripts that increased in the mir-124 mutant nervous system , the highest-scoring motif ( p-value = 0 ) corresponded to the miR-124 seed region ( positions 2–8 ) , while the next highest-scoring motifs amongst globally upregulated transcripts corresponded to variations of 2–7 miR-124 seeds ( Figure 8G ) . These encompassed larger gene cohorts than the canonical seed cohort ( 227 and 174 , compared to 155 canonical seed targets ) , but were associated with more modest overall target over-accumulation , consistent with the directed CDF analysis ( Figure 6G ) . The fourth-highest scoring motif ( GCGCGCC ) amongst up-regulated transcripts did not match a continuous region of miR-124 , but exhibited notable similarity . It is not clear if such matching is biologically relevant , or a statistical anomaly related to its GC-rich character . In any case , these data provide clear evidence that the derepression of direct miR-124 targets is the major determinant causing gene upregulation in mir-124 mutant cells . The miREDUCE analysis also revealed several motifs associated with transcripts that were downregulated in the absence of miR-124 . Two of these were seeds for K box miRNAs and for the Hox miRNA miR-10-5p ( Figure 8G ) . Interestingly , we have earlier shown that a cluster of three K box miRNAs ( mir-2c , mir-13a and mir-13b-1 ) is specifically expressed throughout the embryonic CNS [8] , and other Hox miRNAs ( e . g . mir-iab-4 and mir-iab-8 ) are restricted to specific anterior-posterior domains in the CNS of germband-retracted embryos [51] . Therefore , the loss of the abundant CNS miRNA miR-124 may result in the overactivity of other CNS miRNAs . Amongst motifs that did not match known miRNA seeds , we were struck by the enrichment of UGUAAAU amongst down-regulated transcripts , at a p-value = 0 ( Figure 8G ) . This motif corresponds exactly to the Pumilio binding site [52] . Drosophila Pumilio was originally characterized as a critical translational repressor during embryonic patterning , but was later recognized to be re-expressed and regulate gene expression in neurons [53]–[55] . The FlyAtlas database confirmed high expression of pumilio in the larval central nervous system and adult head ( http://www . flyatlas . org/ ) . Pum transcript was only mildly upregulated in mir-124 mutant cells , and available antibodies were not suitable for immunostaining ( not shown ) . Nevertheless , the strong enrichment of Pumilio binding sites amongst transcripts downregulated in mir-124 mutants suggests its overactivity . Interestingly , Pumilio is also known to regulate neuronal excitability [55] , in addition to BMP signaling . Therefore , direct and indirect consequences may both contribute to electrophysiological defects caused by the absence of miR-124 . Having documented both primary and secondary effects of loss of miR-124 on neural gene expression , we asked whether such gene deregulation exerted a coherent overall effect on cell identity . Despite bioinformatic evidence for the mutual exclusion model ( Figure 7A ) [15] , we do not find evidence for encroachment of epidermal characteristics within mir-124 mutant neurons . Nevertheless , gene deregulation in mir-124 mutant cells could be interpreted as a failure to consolidate the neural gene expression signature . Since mir-124 is activated in neuroblasts and maintained in differentiated neurons ( Figure 1 ) , we hypothesized that the absence of miR-124 might be manifest in the transition from the neuroblast to neural state . To study this , we took advantage of larval neuroblast and neuronal gene expression signatures defined by comparison of normal and various brain tumor mutants , which generate a high proportion of neuroblasts [56] . This yielded clusters of 1109 and 1415 unique genes that were mostly restricted to neuroblasts and neurons , respectively , of which 1002 and 1269 were expressed in miR-124+ cells . These gene lists overlapped rather poorly with direct miR-124 targets , and that the number of direct targets in the neuroblast and neuronal clusters was comparable ( 51 and 74 , respectively ) . Therefore , miR-124 does not seem to have an overarching theme in , for example , directly targeting neuroblast genes . Nevertheless , we observed strikingly opposite behavior of neuroblast and neuronal genes as a whole , in the absence of mir-124 ( Figure 8H ) . Neuronal gene expression was globally decreased in miR-124:DsRed cells isolated from mir-124 mutants compared to wild-type ( p<2 . 2E-16 ) . Reciprocally , we observed that neuroblast gene expression was globally increased in these mutant cells ( p<2 . 2E-16 ) . We infer from these gene expression patterns that the derepression of the miR-124 target network , impedes the normal transition of gene expression from neuroblasts to differentiated neurons in mir-124 mutants . Altogether , our analyses reveal a complex set of primary and secondary effects on neuronal gene expression in mir-124 mutants , which are collectively associated with behavioral dysfunction in larval and adult stages .
Our studies of Drosophila mir-124 demonstrate that its loss is compatible with grossly normal neural development and differentiation , despite broad changes in gene expression and global upregulation of direct miR-124 targets . Nevertheless , we detected many clear defects in these mutants , including short lifespan of adult males , defective larval locomotion , and aberrant synaptic transmission . The latter phenotype is perhaps reminiscent of reports that inhibition of Aplysia miR-124 similarly results in an increase in evoked EPSP amplitude [10] . We confirmed these phenotypes to be due to miR-124 loss , as shown by their rescue by a mir-124 genomic transgene . Importantly , these phenotypes were obvious even under optimal culture conditions , demonstrating palpable requirements for this miRNA in the intact animal . It remains to be seen if synaptic overactivity in the mir-124 mutant can be directly linked to the behavioral defects we observed at the organismal level ( Figure 4 ) . The electrophysiological defects in mir-124 mutants phenocopy activation of BMP signaling at the synapse , and miR-124 directly targets multiple components of this pathway ( Figure 8 ) . Still , it remains possible that the many other gene expression changes in mir-124 mutant neurons ( Figure 6 , Figure 7 , Figure 8 ) contribute to its loss of function phenotype . Our detailed in vivo transcriptome-wide analysis of endogenous miR-124 targets sets the stage for future studies of how individual targets might affect different settings of miR-124 function . Only a handful of other miRNA mutants are lethal or exhibit overt morphological defects [29] , [57] , suggesting that many miRNAs serve as robustness factors . For example , a Drosophila mir-7 mutant exhibits minor cell specification defects , but these are enhanced by heat shock [58] . In addition , the introduction of many C . elegans “benign” miRNA mutants into genetically sensitized backgrounds uncovers a high frequency of phenotypes [59] . Interestingly , miR-124 is not required for normal dendrite formation per se , but its absence caused a broader distribution of dendrite numbers on ddaD and ddaE neurons , i . e . a “robustness” defect . We speculate that environmental or genetic stress may reveal additional requirements for miR-124 in development and differentiation of the nervous system . In light of the broad roles ascribed to endogenous miR-124 in neurogenesis , neural differentiation , and neural physiology [60] , all from antisense strategies , the extensive negative data from our Drosophila mir-124 knockout are equally compelling . While we may not have examined the relevant neural subpopulation , our studies indicate that miR-124 is not required for gross aspects of neurogenesis and differentiation in the embryonic and larval nervous system . Similarly , C . elegans deleted for mir-124 , which is expressed mostly in ciliated sensory neurons , do not reveal obvious defects in neural development [9] . Given that these invertebrate orthologs of miR-124 are identical in sequence to their vertebrate counterparts , and are highly and specifically expressed in their respective nervous systems , there is not strong reason a priori to suspect that miR-124 should not have comparable requirements amongst different animals . The analysis of vertebrate mir-124 knockouts is therefore highly anticipated . The Drosophila system has been critical for elucidating fundamental features of miRNA target recognition in animals [3] , [15] , [46] , [61]–[63] , and for studying specific miRNA-target interactions that mediate phenotype [64] . However , it has been little-used to analyze the effects of miRNA-mediated gene regulation in the animal at the transcriptome-wide level . Perhaps the clearest example is the broad upregulation of maternal transcripts in early embryos lacking the mir-309 cluster [65] . However , most miRNAs are tissue or cell-specific , and while it is much simpler to profile transcripts from whole flies , the inclusion of irrelevant cells can mask the action of the miRNA . For example , only 4/200 transcripts upregulated in mir-8 mutant pupae appeared to be direct conserved targets [66] . By purifying cognate miRNA-expressing cells from wild-type and miRNA-mutant backgrounds , we were able to assess transcriptome-wide effects of genetic removal of miR-124 with precision . Our data provide a new perspective on the utilization of “anti-targeting” in Drosophila . Previously , miR-124 was selected as a particularly compelling case in which its Drosophila targets were depleted for in situ terms related to nervous system development , and enriched for terms related to epidermal development [15] . Since these tissues derive from a common developmental progenitor , the neuroectoderm , this led to a model in which miR-124 may solidify the neural fate by widespread suppression of epidermal genes that should be absent from neurons . We could confirm this bioinformatic correlation using an independently-derived set of miRNA targets ( Figure 7A ) . Nevertheless , two observations suggest that the feature of mutual exclusion in the Drosophila miR-124 network is of subtle consequence . First , derepressed target genes were not enriched for epidermally-expressed genes . This is consistent with the view that on the transcriptome-wide level , the exclusion of epidermal genes from miR-124-expressing cells is primarily enforced by transcriptional mechanisms . Second , miR-124 targets were preferentially amongst the higher-expressed transcripts in miR-124+ cells , even in wild-type . Moreover , as well-conserved targets were expressed at overall higher absolute levels than poorly-conserved targets in miR-124+ cells , we conclude that a dominant feature of the miR-124 target network has selected for substantial co-expression of the miRNA and its targets , perhaps to fine-tune their levels . This viewpoint is consistent with analyses of miR-124 targets in human [50] , zebrafish [35] and C . elegans [9] , indicating a unifying theme for this particular miRNA across animals . Early manifestations of the miRNA world emerged from pervasive control of the C . elegans heterochronic pathway [67] and the D . melanogaster Notch pathway [1] , [46] by miRNAs , and a few similar situations have been documented , i . e . direct targeting throughout the branched amino acid catabolism pathway by miR-277 [47] or repression of multiple components of fatty acid metabolism by miR-33 [68] . Nevertheless , it is rare for such dedicated target networks to be seen amongst the miRNA oeuvre . Amongst the broad network of miR-124 targets , we are struck by the coordinate targeting of multiple components of the retrograde BMP signaling pathway [44] , including all three receptors ( Sax/Tkv/Wit ) , the downstream transcription factor ( Mad ) and its cofactor ( Medea ) . We recently showed that misexpression of activated Sax and Tkv receptors in motoneurons increases evoked excitatory junctional potentials without affecting spontaneous activity , very similar to that of mir-124 mutants [48] . We extended this finding by analysis of activated Tkv alone ( Figure 8D–8F ) . Therefore , deregulation of BMP signaling may contribute to the electrophysiological defects observed in mir-124 mutants . Still , a “one size fits all” description of miR-124 activity is not appropriate , since we certainly do observe a number of functional miR-124 targets whose predominant activities are in epidermal or other non-neural derivatives . Thus , the large miR-124 network accommodates a range of target properties [69] , [70] . Derepression of a sufficient number of such non-neural transcripts may contribute collectively to the incomplete capacity of mir-124 mutant cells to transition from a neuroblast to neuronal gene expression signature ( Figure 8H ) . One may speculate that dysfunction of miRNAs , which have large networks of targets , may trigger global changes in other modes of gene regulation . For example , overexpression of individual miRNAs or siRNAs can de-repress endogenous regulation via non-cognate miRNAs , possibly reflecting a titration mechanism [71] . In addition to a global effect on neuroblast-to-neural transition , we observed that genes downregulated upon in vivo loss of miR-124 were enriched for seeds of K box miRNAs and miR-10-5p ( Figure 8G ) . This is potentially consistent with a model in which absence of this abundant miRNA frees up AGO1 complexes to accept other neural miRNAs , yielding their overactivity . Another plausible mechanism might be that miR-124 represses a transcriptional repressor of these other miRNAs . We also observed that Pumilio binding sites were strongly associated with downregulated transcripts in mir-124 mutants . Pumilio is well-characterized as a neural RNA binding protein and translational regulator , and affects synaptic function and dendrite morphogenesis [53]–[55] , which we also observed to be miR-124-regulated settings . Predictions of conserved miRNA binding sites ( e . g . TargetScan or mirSVR ) did not identify miR-124 target sites in the annotated pumilio 3′ UTR or CDS; however modENCODE data [72] revealed that pumilio transcription extends >2 kb downstream of its annotated 3′ end . The regulatory potential of such long pumilio 3′ UTR isoforms remains to be studied . Other possibilities are that miR-124 regulates a transcriptional regulator of pumilio , or that Pumilio activity is altered in mir-124 mutants . Future studies should address the cross-talk of post-transcriptional regulation in neurons mediated by miR-124 , neuronal miRNAs and Pumilio .
Deletion alleles of mir-124 were generated using ends-out recombination [73] . ∼4 kb left and right homology arms were amplified using PCR ( Table S3 for primer sequences ) and cloned into pW25 . 2 donor targeting vector , and injected into w[1118] ( BestGene , Chino Hills CA ) . Donor insertions on chromosome X or III were used for mir-124 targeting , and were crossed to flies carrying heat shock-inducible FLP recombinase and I-SceI endonuclease , to mobilize the miRNA targeting element from the donor chromosome and linearize the excised fragment . Adult flies collected from larvae subjected to 1 hr heat shock at 37°C were crossed with balancer flies that contain second and third chromosome markers that allow mapping of mini-white . For flies in which mini-white mapped to chromosome II , PCR was performed to verify the integration of the targeting construct at the mir-124 locus using primers that bind outside the left homology arm and within unique vector sequence downstream of the left homology arm but upstream of the mini-white gene . Only flies with correct targeting produce a ∼4 . 5 kb PCR fragment . Excision of the mini-white gene using hs-Cre recombinase was verified by PCR generating a diagnostic ∼500 bp fragment . Primer sets for mir-124 validation are listed in the Table S3 . The mir-124 rescue transgene was generated by injection of P[acman] clone CH322-39N16 into attP16 strain [74] ( Genetic Services Inc . , MA ) . miR-124:dsRed was generated by cloning 4 kb upstream of the hairpin into Red-H-Stinger [75] . ∼400 bp genomic fragment containing the pre-mir-124 sequence was cloned into the UAS-dsRed [47] to generate the UAS-dsRed-mir-124 transgene . To analyze larval neuroblast clones , we heat-shocked hsflp , tubGal4 , UAS-GFP; FRT40A , tubGal80/FRT40A mir-124[6] for 37°C for 90 minutes at 24 hr ALH ( after larval hatching ) and dissected at 96 hr ALH . DNA templates were generated by PCR amplification of ∼1 kb genomic sequences containing the miRNA hairpin; T7 promoter was attached to the antisense strand primers . See Table S3 for primer sequences . Antisense digoxigenin-labeled RNA probes were generated by in vitro transcription with the DNA template and T7 polymerase according to the standard protocol ( Roche ) . Embryos were fixed and prepared as described previously [8] . For immunostaining , embryos were dechorionated in bleach and fixed in 4% formaldehyde for 20 min followed by devitellination . Fixed embryos were stored in −20°C at least one overnight before staining . Embryos were rehydrated in 50% methanol , washed in PBSTw ( 0 . 1% Tween-20 in PBS ) and then blocked in 0 . 5% PBSBT ( 0 . 5% BSA and 0 . 1% Triton X-100 in PBS ) . Both primary and secondary antibodies were incubated overnight in 4°C . The following primary antibodies were used: rabbit-anti-dsRed ( 1∶500 , Clontech ) , rat-anti-Elav ( 1∶250 , DSHB ) , rat-anti-deadpan ( 1∶50 , Doe lab ) , rabbit-anti-Hunchback ( 1∶200 , Doe lab ) , guinea pig anti-Miranda ( 1∶500 , Doe lab ) , mouse-anti-Prospero ( 1∶20 , DSHB ) , mouse-anti-Eve ( 1∶5 , DSHB ) , mouse-anti-Repo ( 1∶20 , DSHB ) , mouse-anti-22C10 ( 1∶100 , DSHB ) . Alexa Fluor-488 , 568 , 647 secondary antibodies were from Molecular Probes and used at 1∶500 . For staining of larval neuromuscular junction , 3rd instar wandering larvae were dissected as described [76] . Alexa Fluor-568 phalloidin ( 1∶400 , Invitrogen ) and FITC-HRP ( 1∶250 , Jackson ImmunoResearch ) were used to visualize the F-actin and NMJ . Images were captured with a Leica TCS confocal microscope . Synaptic boutons and NMJ expansion were quantified with the Leica software . Lethal phase analysis . Flies were cultured in 25°C and allowed to lay eggs for 12 hours . For each genotype , 100 embryos were collected and transferred to an apple juice plate and each plate was scored for the number of hatched larvae and pupae . The number of eclosed adults was scored everyday from day 8 to 13 for each genotype . Experiments were repeated five times . Life span assay . Male flies were collected within 24 hr of eclosion and maintained in 29°C in low density ( 5 males/vial , 20 vials per genotype ) . Flies were transferred to fresh vials every 2∼3 days and scored for survivors across the timecourse . Larval locomotion assay . Larval locomotion was assayed as described [77] but without odor source . Briefly , single mid-3rd instar larva was placed on a 96 well plate lid covered with 3% agarose and animal locomotion was recorded by a CCD camera for 1 min since its first movement . Data was collected and analyzed with the Ethovision software ( Noldus ) . 15∼30 animals were tested for each genotype . Gal4221 driver was used to label ddaD and ddaE neurons with mCD8-GFP and drive the expression of transgenes . The dendritic morphology of GFP-labeled dorsal sensory neurons was recorded by confocal ( Nikon , D-Eclipse C1 ) . One ddaD neuron and one ddaE neuron were recorded from A3 segment of each larva and their dendrites were counted as described [78] . Briefly , dendritic ends of ddaD or ddaE neurons were identified visually and highlighted with dots , which were counted using Adobe Photoshop . The data were analyzed by the Wilcoxon test and F test . Wandering third instar larvae were dissected in cold HL3 solution without Ca2+ following standard protocol [79] , using the mir-124 genotypes described above and BG380-Gal4>UAS-TkvA [80] . The spontaneous ( mEJC ) and evoked ( EJC ) membrane currents were recorded from muscle 6 in abdominal segment A3 with standard two-electrode voltage-clamp technique [80] . All the recordings were performed at room temperature in HL3 solution containing 0 . 5 mM Ca2+ . The current recordings were collected with AxoClamp2B amplifier ( Molecular Devices Inc . ) and stored on a desk top computer using Clampex 9 . 2 software ( Molecular Devices Inc . ) . The nerve stimulation was delivered through a suction electrode , which held the cut nerve bundle . In all voltage clamp recordings , muscles were held at −80 mV . The holding current was less than5 nA for 90% of the recordings and we rejected any recording that required more than 10 nA current to maintain the holding potential . The amplitudes of mEJC and EJC were measured using Mini Analysis 6 . 0 . 3 software ( Synaptosoft ) and verified by eye . QC was calculated by dividing the mean EJC amplitude by mean mEJC amplitude . The recording traces were generated with Origin 7 . 5 software ( Origin Lab ) . Data are presented as Mean ± SEM ( n = number of NMJs unless otherwise indicated ) . Histograms were generated using Excel software ( Microsoft Corporation ) . Statistical significance was determined using PASW 7 . 0 software ( SPSS Inc . ) . Each data set was first subjected to a variance test . In the absence of a significant difference , One-way ANOVA followed by Tukey post-hoc test was applied . If there were differences in variance , Games-Howell post-hoc test was applied . Wild type ( mir-124:dsRed ) and mutant ( mir-124[del12/12]; mir-124:dsRed ) flies were raised in collection cages at 25°C . 10∼16 hours embryos were collected and dechorionated in house bleach solution for 2∼3 min . Then embryos were washed in 80% ethanol for 5 min with occasional vortex and rinsed in modified Schneider media supplemented with 2% FBS , 0 . 1% Pen/Strep and 0 . 005 mg/ml Gentamicin for 3 times . Embryos were transferred to supplemented Schneider media ( 20% FBS ) and homogenized in a 7 ml tissue grinder ( Wheaton #357542 ) until no large clumps were visible . Homogenate was transferred to an eppendorf tube and spun at 5000 rpm for 5 min . Pellets were resuspended in 0 . 01% trypsin in unsupplemented Schneider media ( without FBS ) and incubated for 5 min . Dissociated cells were purified by passage through a cell strainer cap ( BD Falcon #352235 ) twice and finally resuspended in 20% FBS supplemented Schneider media . Fluorescence activated cell sorting was carried out immediately after preparation using a MoFlo flow cytometer ( Cytomation ) in the MSKCC Flow Cytometry Core Facility . Total RNA from the sorted cells was extracted using Trizol LS ( Invitrogen ) . To enhance precipitation , RNA was precipitated with glycogen ( Ambion ) . RNA samples including 3 biological replicates for each genotype were labeled and hybridized to the GeneChip Drosophila Genome 2 . 0 Array ( Affymetrix ) by the MSKCC Genomics Core Laboratory . Primers for qPCR validation of Dsred and pri-mir-124 are listed in Table S3 . 3′ UTRs of predicted miR-124 targets were cloned into the psiCHECK-2 vector ( Promega ) using cold fusion cloning ( System Biosciences ) . Sensor plasmid and ub-Gal4 were cotransfected with UAS-DsRed-miR-124 or empty pUAST vector into S2-R+ cells using Effectene ( Qiagen ) . Luciferase activities were measured by Dual-Glo Luciferase assay ( Promega ) . To verify several gene expression changes in microarray , qRT-PCR were performed using SYBR Green reagent ( Applied Biosystems ) and the CFX96 Real-Time PCR detection system ( Bio-Rad ) . Primers used for cloning 3′ UTR sensors and performing qPCR are listed in Table S3 . Microarray data were normalized using the GCRMA bioconductor package and log enrichment values were computed using the limma package with p-values adjusted for multiple hypothesis using FDR . For genes with multiple probes , the probe with lowest adjusted p-value was selected . Targets were predicted and scored using miRanda-mirSVR method [41] . Predicted target sites were restricted to include perfect seed complementarity ( positions 2–7 ) and non-canonical sites with favorable mirSVR scores ( <−0 . 1 ) . Empirical cumulative distributions were computed using the R ecdf function on mutually exclusive gene sets and P-values were computed by the Kolmogorov-Smirnov non-parametric test . Detection of sequence motifs that are correlated with log-fold expression changes was performed using miReduce [50] with motif length parameter of 7 and p-value cutoff< = 0 . 05 . The predicted miR-124 target sites were partitioned into well-conserved and poorly-conserved based on the Multiz 15 fly species alignment in the UCSC genome browser [42] . Target sequences where at least 5 sequences ( including D . pseudoobscura ) from D . sechellia , D . simulans , D . yakuba , D . erecta , D . ananassae and D . pseudoobscura were identical to D . melanogaster were considered well-conserved , all other sequences were labeled poorly-conserved . Enrichment of Gene Ontology annotations and in-situ gene expression profiles [43] were computed with Fisher's exact test , using the Bonferroni correction for multiple hypothesis testing . Up- and down regulated genes were required to have fold change >30% and p-value<0 . 05 . | microRNAs are abundant ∼22 nucleotide RNAs inferred to mediate pervasive post-transcriptional control of most genes . Still , relatively little is understood about their endogenous requirements and impact , especially in animal systems . We analyzed a knockout of Drosophila mir-124 , which is conserved in sequence and neuronal expression across the animal kingdom , and predicted to have hundreds of mRNA targets . While dispensable for gross neural specification and differentiation , deletion of mir-124 caused short lifespan , increased variation in dendrite numbers , impaired larval locomotion , and aberrant synaptic release at the NMJ . These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions . Loss of miR-124 broadly upregulated its direct targets but did not support the proposed mutual exclusion model , as its functional target genes were relatively highly expressed in neurons . One notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway , whose activation in neurons phenocopies loss of miR-124 . Derepression of the direct miR-124 target network had many secondary effects , including over-activity of other post-transcriptional repressors and impaired transition from neuroblast to neuronal transcriptome signatures . Altogether , we demonstrate complex requirements for this conserved miRNA on gene expression and neurophysiology . | [
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] | 2012 | Neurophysiological Defects and Neuronal Gene Deregulation in Drosophila mir-124 Mutants |
A high level of HER2 expression in breast cancer correlates with a higher tumor growth rate , high metastatic potential , and a poor long-term patient survival rate . Pertuzumab , a human monoclonal antibody , can reduce the effect of HER2 overexpression by preventing HER2 dimerization . In this study , a combination protocol of molecular dynamics modeling and MM/GBSA binding free energy calculations was applied to design peptides that interact with HER2 based on the HER2/pertuzumab crystal structure . Based on a β hairpin in pertuzumab from Glu46 to Lys65—which plays a key role in interacting with HER2—mutations were carried out in silico to improve the binding free energy of the hairpin that interacts with the Phe256-Lys314 of the HER2 protein . Combined the use of one-bead-one-compound library screening , among all the mutations , a peptide ( 58F63Y ) with the lowest binding free energy was confirmed experimentally to have the highest affinity , and it may be used as a new probe in diagnosing and treating HER2-positive breast cancer .
Human epidermal growth factor receptor 2 ( HER2 ) is a prominent target in breast cancer diagnosis and treatment , as approximately 20–30% of patients with breast cancer overexpressing the HER2 receptor [1 , 2] , a 185-kD transmembrane glycoprotein with 1 , 255 amino acids [3] . The HER2 gene is a proto-oncogene that maps to chromosome 17q21 . HER2 contains four domains ( I , II , III , and IV ) that comprise a ligand-binding extracellular portion , a single transmembrane helix , a tyrosine kinase domain closely related to the Janus kinases , and a C-terminal tail with a number of tyrosine phosphorylation sites that serve as a scaffold for adaptor molecules and enzymes in facilitating downstream signaling [4] . The heterodimerization of HER2 with any of the other three HER family receptors results in autophosphorylation of the terminal carboxyl segment and initiates a variety of signaling pathways that regulate cell growth , proliferation , and metastasis [5–7] . Currently , a number of therapeutic approaches have been developed to antagonize the effects of HER2 overexpression; these approaches include the humanized monoclonal antibodies trastuzumab and pertuzumab [8] . Trastuzumab demonstrates clinical benefits in the treatment of HER2-positive breast cancer , in both early and metastatic stages . One year of trastuzumab therapy is recommended for all patients with HER2-positive breast cancer who are also receiving chemotherapy [9] . However , as trastuzumab becomes a routine therapy , resistance can develop following an initial robust response; a lack of response to initiation has also been observed among patients [10 , 11] . The other antibody drug , pertuzumab , has received US Food and Drug Administration approval for the treatment of HER2-positive metastatic breast cancer . Trastuzumab and pertuzumab bind to different epitopes in the extracellular domain of HER2 , and their mechanisms of action differ . Pertuzumab binds the pocket of domain II , inhibits HER2 dimerization with other receptors , and leads to slowed tumor growth . Trastuzumab , on the other hand , binds to subdomain IV [12] , and works by inhibiting the PI3K/Akt , Mirk , and hKIS pathways and promoting proteolytic cleavage of the extracellular domain [13] . However , both drugs have been shown to stimulate the antibody-dependent cellular cytotoxicity mechanism [14] . It has been commonly recognized that , compared to antibody drugs , small peptides are cost-effective , have good tissue and membrane permeability , high target specificity , and low toxicity . Moreover , specific modifications to targeting peptides can be employed to provide diverse biosensing functions; this strategy has been leveraged to develop a method by which to detect metastatic tumor cells in primary tumors [15] . The science of molecular dynamics ( MD ) has been widely applied to chemical physics , materials science , and the modeling of biomolecules—such as interactions between ligands and receptors [16 , 17]—by simulating the physical movement of atoms and molecules based on a family of molecular mechanics force fields . The MM/GBSA method is often used to estimate the free energy of solute–solvent interactions . In this method , a generalized Born ( GB ) model is used to approximate the Poisson–Boltzmann equation , based on modeling a molecule as a set of spheres . The accessible surface area ( SA ) approximates the experimental value of the averaged behavior of many highly dynamic solvent molecules between the transfer free energy and the surface area of a solute molecule [18] . The one-bead-one-compound ( OBOC ) [19–21] library method can be used to systematically synthesize and screen the peptide library of a target protein . It is a simple means of rapidly identifying small molecules that bind with high affinity to receptor molecules . The strategy has been modified by many researchers to overcome the several limitations inherent in the original approach [22] . Our research group has previously advanced it to a lab-on-chip system that embraces the whole peptide screening process—from single bead trapping to the final sequencing of peptides—by using MALDI–TOF–MS [23 , 24] . In this study , we use a combination protocol comprising MD , MM/GBSA binding free energy calculation [25–27] to derive peptides that interact with HER2 protein based on the HER2/pertuzumab crystal structure ( PDB entry: 1S78 ) . In silico mutations were performed to screen for peptides with the lowest binding free energy , and OBOC peptide library screening was then carried out . Both the binding free energy calculation and the OBOC library screening found the peptide 58F63Y to have the highest affinity to HER2 . 58F63Y , together with five other peptides , were selected for further analysis and experimental validation . All results show that the peptide 58F63Y binds most favorably to HER2 , with a dissociation constant ( KD ) of 536 nmol/L . The results of ex vivo and in vivo experiments using mouse xenografted tumors confirm that this peptide has strong affinity and high specificity to HER2 . Binding free energy decomposition analysis [28–30] and distances calculation using Pymol found that there are more paired residues with low binding free energy and distances of less than 5 Å , which may explain the high affinity . Compared to other peptides that target HER2 [31] , peptide 58F63Y is unique in that it is acquired based on simulation using a different primary model with binding sites on domain II of the HER2 protein . Given its low toxicity , this peptide may be used as an alternative probe in the diagnosis and treatment of HER2-positive breast cancer and contributes to the HER2-targeting peptide library .
Pertuzumab is a monoclonal antibody marketed by Genentech for the treatment of HER2-positive breast cancer . Pertuzumab binds to HER2 at the center of domain II , sterically blocking the pocket essential to receptor dimerization and signaling . The HER2/pertuzumab crystal structure was obtained from the Protein Data Bank . In this structure , the soluble extracellular domain of HER2 [32] was crystallized in complex with the Fab fragment of the disulfide anti-HER2 monoclonal antibody pertuzumab [33] . Based on the calculation of distances for all residues between HER2 and pertuzumab and the selection of those within 5 Å , we found a peptide fragment of 20 residues in length ( sequence: EWVADVNPNSGGSIYNQRFK ) with a beta folding layer structure , named 4665 , that plays an important role in the interactions ( Fig 1A ) . The HER2/fragment 4665 complex was chosen for further simulation analysis . MM/GBSA free energy was calculated based on 500 snapshots from 7 to 10 ns of MD simulation trajectories ( Fig 1B ) for each complex , as described in the Materials and methods section . The results show that the predicted binding free energy between 4665 and HER2 is –48 . 53 kcal/mol with the van der Waals ( ΔEvdw ) contribution being a main component . To improve the affinity of 4665 against HER2 , we undertook single and double mutations and performed MD simulations to estimate the binding affinity . The properties of the interacting amino acids in both HER2 and peptides , as well as the space among the interactions , were considered . As shown in Fig 1C , Glu46 , Trp47 and Lys65 have low energy contributions to the HER2/4665 complex . In addition , Asn52-Asn54 has almost no contributions to the binding . Therefore , mutations were made mostly in two beta strands in 4665: one is from Val48 to Val51 with high hydrophobicity , and the other is from Ser55 to Phe64 . The basic rules are that mutations should favor electrostatic and van der Waals interactions , and do not cause steric overlap . Briefly , the van der Waals ( ΔEvdw ) contribution is a main component in HER2/4665 interactions as suggested from the free energy calculation . As a consequence , residues in the strand of Ser55-Phe64 were preferably mutated to nonpolar amino acids ( Ser55 , Gly56 , Gly57 and Gln62 ) to increase the van der Waals ( ΔEvdw ) contribution . Moreover , residues with large side chains are mutated into amino acids with similar side chain groups , such as Val48 , Ala49 , Gly56 and Gly57 . We also took into account of the inter spaces between the peptide and HER2 . For example , Ser58 is located on a beta strand that is close to the HER2 fragment Phe256-Lys314 but with a large spatial distance , so Ser58 was mutated to the residues with larger side chain groups or nonpolar amino acids . Arg63 is also located on a beta strand that is closer to the HER2 fragment Phe256-Lys314 . Considering its distance ( 3 . 9 Å ) to Phe257 , Arg63 is mutated into residues with side chain groups no larger than benzol methyl or nonpolar residues . Finally , we first carried out 59 single mutations based on the 4665 sequence by performing MD simulations , and binding free energies were calculated for each mutant . Seventeen single mutations with binding free energies < –48 . 53 kcal/mol were selected to create combinations of double mutations , thus resulting in another 56 mutants . All mutations and their binding free energies are shown in Tables 1 and 2 . Among these mutations , 34 sequences have lower binding free energies than the original 4665 peptide; peptide 58F63Y ( sequence: EWVADVNPNSGGFIYNQYFK ) is the lowest , and so it is expected to bind most tightly to HER2 . The results of our previous work show that when receptor–ligand interactions are similar—save for only a few residue differences—computational binding free energy calculations can closely reflect the relative affinity of peptide binding [31 , 34] . In the current study , to determine whether the above in silico screening correctly identified a peptide with affinity among the highest ones , the OBOC peptide library approach ( Fig 2A ) was later performed . We designed the peptide library based on the calculated binding free energies of peptides/HER2 with single and double mutations , using MD simulations ( Tables 1 and 2 ) . Mutations with binding free energies lower than the wild type 4665 ( ΔGtot < –50 kcal/mol ) were selected , and the intersection of single and double mutations from 55 to 64 was used in library construction ( Fig 2B ) , resulting in a library of 5184 sequences . Biological screening of the OBOC peptide library is routinely carried out as described in the Materials and methods section . Three positive beads were identified , and following MALDI–TOF–MS/MS analysis , one of them was found to be the same as the peptide 58F63Y , which has the lowest binding free energy with MD simulation ( Fig 2C ) . After combining the results , peptide 58F63Y was selected for further experimental validation . Another double mutant 55V63Y and its single mutations 58F , 63Y , and 55V—as well as the original wild type 4665—were also selected to facilitate better comparison . The sequences of these six peptides were aligned using Clustal Omega; the results are shown in S1 Fig . Surface plasmon resonance imaging ( SPRi ) —which has been previously used to estimate interactions between molecules for the purposes of disease diagnosis , drug discovery , and peptide screening [23 , 24 , 35–37]—was used in this study to estimate the dissociation constants of peptides binding to HER2 , as described in the Materials and methods section . The dissociation constant was calculated from kinetic constants obtained by fitting association and dissociation curves to real-time binding and washing data . While none of the four peptides shows any affinity to the HSA protein ( S2 Fig ) , Fig 3 indicates that the KD values of the peptides 4665 , 58F , 63Y , 55V , 58F63Y , and 55V63Y with HER2 protein are 9 . 86 μmol/L , 1 . 32 μmol/L , 1 . 54 μmol/L , 64 . 6 μmol/L , 0 . 536 μmol/L , and 8 . 16 μmol/L , respectively . We can see that , to some extent , the KD values agree with the binding free energies from the simulation , which range from –48 kcal/mol to –66 kcal/mol . This finding is consistent with that of our previous work , in which it was found that computational binding free energy from MM/GBSA can be used to estimate the relative affinity of peptide binding . Based on the SPRi results , three peptides ( 58F , 63Y , 58F63Y ) with the highest affinity , as well as the starting peptide 4665 , were chosen for later confocal fluorescence imaging analyses . Four tumor cell lines ( SKBR3 , MCF7 , MDA-MB-468 , and 293A ) , each with a different HER2 expression level , were used in confocal fluorescence imaging analysis to confirm the binding specificity of peptides to the HER2 protein . Among these cell lines , the expression of HER2 was found to be high in SKBR3 , medium in MCF7 , and low in MDA-MB-468 and in 293A . As shown in Fig 4 and S3 Fig , when treated with Cy5 . 5-peptides , fluorescent intensities are strong in SKBR3 cells , but weaker in MCF7 cells and absent in MDA-MB-468 and 293A cells . Especially , SKBR3 cells treated with Cy5 . 5-58F63Y show the strongest fluorescence , thus indicating that 58F63Y binds with the highest affinity to HER2 ( Fig 4B ) ; this finding is consistent with the aforementioned MD calculation and SPRi analytical results . All these results confirm that peptides can specifically bind at the cellular level to the extracellular domain of the HER2 protein . Peptide 58F63Y ( which had the highest affinity ) and the wild type 4665 were chosen for subsequent in vivo studies . Toxicity of peptides 58F63Y and 4665 to HUVEC and SKBR3 cells was also measured . 4665 and 58F63Y shows no toxicity to both cell lines ( S4 Fig ) . To investigate the affinity and specificity of peptides to HER2-positive tumors in vivo , nude mice bearing subcutaneous SKBR3 tumor xenografts were intravenously injected with Cy5 . 5-labeled peptides and Cy5 . 5 as the control; they were then subjected to whole-body optical imaging , using a small animal in vivo imaging system ( CRI Maestro 2 ) . Fig 5A shows clear differences between the tumor images of mice with Cy5 . 5–58F63Y or Cy5 . 5–4665 and those of the control mice . The intensities are plotted in bar charts ( Fig 5C ) that indicate that binding affinity increases 5 . 09-fold for 58F63Y and 3 . 52-fold for 4665 , relative to the control . Moreover , fluorescence images of the dissected organs of the experimental mice , taken 30 min postinjection with Cy5 . 5-labeled peptides or control Cy5 . 5 , were acquired for further examination . Both images and quantification in the bar charts ( Fig 5B and 5D ) show that tumors treated with 58F63Y and 4665 have significantly high fluorescence signals , compared to those in the controls . Among all the organs , the kidney was found to have the highest background signal for both peptides and control , probably due to the toxic effect of Cy5 . 5 [38 , 39] . Taken together , all the results demonstrate that 58F63Y has high specificity and affinity for HER2-positive tumors . To better understand the reason of the high affinity of 58F63Y to HER2 , a more detailed in silico analysis was performed . Another four mutants—as well as the original wild type 4665—were also selected for a more comprehensive comparison . To verify peptide structure stability after binding to HER2 , the root mean square deviation ( RMSD ) values of the backbone atoms of the initial structure and of successive simulated structures were calculated for all six HER2/peptides complexes . S5 Fig shows that for all six complexes , the RMSD values become stable after about 5000 ps in the MD trajectories , thus indicating the convergence of each peptide and the complex structures towards an equilibrium state . A glance at the results of the free energy decomposition analysis of the peptides found that more residues have low energies in the mutants than in 4665 . Specifically , for 4665 , except Tyr60 , no other residue contributes energy < –2 . 5 kcal/mol ( Fig 6A ) . However , in the mutants ( Fig 6B–6F ) —besides Asn54 and Ser55 in all mutants—the following were found to contribute energy < –2 . 5 kcal/mol: Tyr60 and Gln62 in 58F; Ser58 , Ile59 , and Tyr63 in 63Y; Gln62 in 55V; Ser58 , Phe59 , Gln62 , and Arg63 in 58F63Y; and Asn52 and Ile59 in 55V63Y . That is to say , each mutant has more than three residues that are favorable to HER2 binding . From S6 Fig , we can see that all six peptides overlay in the same pocket of the HER2 protein , thus indicating that the binding sites of these peptides remain the same as those in the wild type . MM/GBSA binding free energy calculation and decomposition analysis of the HER2 protein reveals that fragment 236–314 has major interactions with these peptides , and that for each mutant , more than three residues have binding free energies < –3 kcal/mol ( S7 Fig ) . Together , the beneficial changes of each mutant contribute to a lower binding free energy than that in wild type 4665 . Models from PyMOL were used to better visualize the key interacting residues in peptides/HER2 complexes ( S8 Fig ) . In Fig 7 , 18 residues pairs have distances of less than 5 Å in 58F63Y/HER2 ( 236-314 ) , compared with 10 pairs in 4665/HER2 ( 236-314 ) . As a summary , more residues pairs with lower binding free energy and close distances in the 58F63Y/HER2 complex may contribute to the high affinity of 58F63Y . In summary , based on the crystal structure of HER2/pertuzumab , we acquired a peptide that was 20 residues in length ( i . e . , 58F63Y ) that targets the HER2 protein; these findings were derived through the use of mutations and the computational calculation of the affinity with a combination protocol of molecular dynamics modeling , MM/GBSA binding free energy calculations , as well as the screening of an OBOC peptide library based on the mutations from the in silico modeling . This work proves that MM/GBSA binding free energy can be used to reflect the relative affinity of peptide binding closely . The peptide 58F63Y has a KD value of 536 nmol/L and binds to HER2 at the same site as the parent fragment of pertuzumab . Through confocal fluorescence imaging and in vivo and ex vivo studies , the peptide was found to have high affinity and specificity for the extracellular domain of HER2 . We expect this peptide to serve as an alternative probe that can be used in combination with others to improve the early detection , diagnosis , and targeted therapy of HER2-positive breast cancer .
The primary sequences of the pertuzumab fragment from 46 to 65 ( named 4665 ) and its mutants 58F , 63Y , 55V , 58F63Y , and 55V63Y were aligned by using the Clustal Omega program , which is available on EMBnet website ( http://www . ebi . ac . uk/Tools/msa/clustalo/ ) . The model for the HER2/4665 complex derives from the crystal structure of the HER2 extracellular region and pertuzumab in the RCSB PDB . The model for HER2/4665 was constructed based on the crystal structure of HER2/pertuzumab , by keeping related amino acids in pertuzumab . Other models were constructed based on HER2/4665 . The AMBER03 force field was used to investigate the potentials of the complexes in the following molecular mechanics minimizations and MD simulations [40] . Missing atoms were added by using the tleap program . The whole system was solvated with TIP3P [41] water molecules in a truncated octahedron box with a minimum solute box-edge distance of 12 Å [42] . Then , the largest negative coulombic potential around the protein was randomly neutralized with counter-ions Na+ placed on the grids . The numbers of water molecules and Na+ ions in each system are listed in the supporting information ( S1 Table ) . To remove poor-quality contacts between the complex and the solvent molecules , three-step energy minimization was performed by using the sander module of AMBER12 prior to undertaking the MD simulations . First , the whole protein was fixed and the water molecules and counter-ions were minimized; second , the backbone atoms of the protein were fixed and the side chains were minimized using the same settings as above; third , the whole system was minimized without any constraints . The first two stages consisted of a 5 , 000-cycle steepest descent and a 2 , 500-cycle conjugate gradient minimization; the final step consisted of 10 , 000 cycles of steepest descent and 5 , 000 cycles of conjugate gradient minimization . The SHAKE [43] method was applied to constrain covalent bonds related to hydrogen atoms , with a tolerance of 10−5 Å . Particle Mesh Ewald [44] was employed to adequately deal with long-range electrostatic interactions , and in the MD simulations , the cutoff distances for nonbond energy interactions were set to 12 Å [45] . Then , the entire system was gradually heated from 0 to 310 K in seven steps [46 , 47] over 60 ps [46 , 48] in the NVT ( canonical ensemble ) . Finally , 10 ns MD simulations were implemented with a 2 fs [49] time step under the constant temperature of 310 K . During the sampling process , the trajectories were saved every 0 . 2 ps , and the conformations generated from the simulations were used in further analysis . MM/GBSA [50] serves as an effective computational tool in analyzing biomolecular interactions . When used with knowledge-based energy terms , MM/GBSA can help determine the binding free energies of all systems , based on the calculation of the average free energies of solvation ( ΔGbind ) between targeted protein and ligands over trajectories of MD simulation . The MM/GBSA method can be summarized as the following equation . In which , ΔGbind represents the binding free energy in solution consisting of the molecular mechanics free energy ( ΔEMM ) , the conformational entropic effect to binding ( −TΔS ) in the gas phase , and the solvation free energy containing polar contribution ( ΔGGB ) and nonpolar contribution ( ΔGSA ) . The ΔEMM term includes ΔEele ( electrostatic ) and ΔEvdw ( van der Waals ) energies and was calculated by the sander module of AMBER12 . The polar contribution was calculated by using the GB [51] mode , with solvent and the solute dielectric constants set to 80 and 4 , respectively . Additionally , the nonpolar energy was estimated , with a solvent-probe radius of 1 . 4 Å: ΔGSA = 0 . 0072 × ΔSASA [52] , by the LCPO method [50] based on the SASA model [53] . For each ligand–protein , 500 snapshots were taken from 7 to 10 ns on the MD trajectories . Due to the low prediction accuracy and the high computational cost [54 , 55] upon the nmode module in AMBER12 as well as their similar values in analogical system [31 , 34] , the entropic contribution was ignored in the calculation of the predicted total binding free energy ( ΔGtot* means that ΔGtot does not contain -TΔS energy ) . The specific inhibitor-residue interaction spectra were generated by using MM/GBSA decomposition analysis [28 , 56] undertaken through the mm_pbsa program of AMBER12 . Four kinds of energy were found—namely , ΔEvdw , ΔEele , ΔGGB , and ΔGSA—and each contributed to the binding interaction of each ligand–residue pair . The ΔEvdw and ΔEele energy terms were calculated by the sander module of AMBER12 . The polar contribution ( ΔGGB ) to solvation energy was calculated by using the GB module and the parameters for the GB calculation were developed by Onufriev et al . [57] . The nonpolar solvation contribution ( ΔGSA ) part was computed based on the SASA determined through the ICOSA method [52] . All energy components were calculated by using 500 snapshots extracted from the last 3 ns of the MD trajectories . After undertaking the decomposition process , the free energy contribution could be allocated to each residue from the association between the receptor and the ligand . Graphic visualizations and presentations of protein structures were generated by using PyMOL [58–60] . The OBOC peptide library was designed based on binding free energies derived from the MD simulations of single and double mutants that are lower than the HER2/4665 complex . These mutations are in the 4665 fragment of pertuzumab from 55 to 64: EWVADVNPNX55X56X57X58X59X60N61X62X63X64K . According to computational calculations , the V , S , and M mutants have lower energies for X55 . This is also the case for G , M , and Y for X56; G , A , and V for X57; S , F , and H for X58; I and R for X59; Y and W for X60; Q and F for X62; R , W , Q , and V for X63; and F and R for X64 . The result is a 3*3*3*3*2*2*2*4*2 = 5184 library capacity . The OBOC library synthesis and screening was performed as per the previously used method [61–64] . Briefly , HBTU ( 4 mmol ) and Fmoc-amino acid ( 4 mmol ) reagent was dissolved in 0 . 4 mol/L N-Methyl morpholine in N , N-dimethylformamide and coupled with the solid phase supporting materials for 40 min during the coupling step . A 20% piperidine was used to remove the Fmoc group for 10 min in the deprotection step . During the OBOC library synthesis , the amino acid coupling process was carried out in the “split” step , while the deprotection process was carried out in the “pool” step . After elongation , a trifluoroacetic acid cleavage reagent was introduced to cleave the side chain protection group of each residue . Afterwards , the solid phase supporting materials were incubated with 5% milk , then with HER2/biotin complex , and then with monodispersed magnetic streptavidin microspheres . Each step was performed in an incubator at 37°C for 2 h , and followed by three washes with PBS . The HER2 protein was biotinylated using a biotinylation kit ( Solulink Inc . , USA ) . Positive beads with dark colors were picked out for the in situ chemical cleavage before MALDI–TOF–MS/MS analysis , and a 30 mg/mL cyanogen bromide solution was used overnight . Peptides were synthesized using Fmoc strategy solid phase peptide synthesis [65–67] . Unsophisticated peptides were purified using a Hitachi HPLC system ( L-7100 , Japan ) on a TSK gel ODS-100V reversed-phase column . Peptides were eluted with a linear gradient of 5–80% acetonitrile containing 0 . 1% trifluoroacetic acid at a flow rate of 2 mL/min within 25 min . Peptides were then subjected to MALDI–TOF–MS ( Bruker Daltonics ) analysis . Purified peptides were dried in vacuum desiccators and then stored at –20°C until further use . 2- ( 1H-benzotriazole-1-yl ) -1 , 1 , 3 , 3-tetramethyluronium hexafluorophosphate was purchased from GL Biochem ( China ) . Trifluoroacetic acid and fluorescein 5-isothiocyanate were acquired from Sigma-Aldrich Co . LLC ( USA ) . N-Methyl morpholine and N , N-dimethylformamide were acquired from a Beijing chemical plant ( China ) . For SPRi analysis , a cysteine residue linked to the amino terminal of all peptides was used for interacting with a bare gold chip bearing a 47 . 5-nm thickness . First , 1 μL peptides at 1 mg/mL was added to the gold surface of the chip and incubated overnight at 4°C . The chip was then washed with PBS and deionized water three times , and 5% nonfat milk was applied to block overnight at 4°C . After the chip was washed again with PBS and water , it was dried with nitrogen for later use . Human serum albumin ( HSA ) protein ( Sigma-Aldrich Co . LLC ) was used as the control . HER2 ( Sino Biological Inc . , China ) and HSA proteins ( Sigma-Aldrich Co . LLC ) were dissolved in PBST and diluted to 10 , 5 , 2 . 5 , 1 . 25 , or 0 . 625 μg/mL . The SPRi analytical procedure was carried out on the prepared SPRi chip by running PBST buffer for baseline stabilization , followed by the protein sample , a PBST running buffer for washing , and finally 0 . 5% H3PO4 in deionized water for regeneration . This cycle was repeated for each concentration of HER2 and HSA protein at 20 , 10 , 5 , 2 . 5 , 1 . 25 , and 0 . 625 μg/mL . Real-time binding signals were recorded and analyzed through the use of a PlexArray HT system ( Plexera LLC , Bothell , WA , USA ) . The dissociation constant was calculated by fitting the association–dissociation curves . Four cell lines ( SKBR3 , MCF7 , MDA-MB-468 , and 293A ) were seeded at a density of 3000 cells/mL into culture dishes and allowed to culture overnight with 5% CO2 at 37°C . Cell nuclei was stained with 1 mM Hoechst 33342 in 200 μL cell culture medium and incubated at 37°C for 15 min . Then , cells were incubated in culture medium with 50 μM Cy5 . 5-labeled peptide at 4°C for 20 min . Finally , cells were washed three times with cold PBS for observation . An Olympus FV1000-IX81 confocal-laser scanning microscope was used for confocal fluorescence imaging . An FV5-LAMAR 633 nm laser was used as the excitation source , and the emission wave length was collected at 690 nm . Hoechst 33342 was excited by a FV5-LD405-2 405 nm laser and collected within the range of 422–472 nm . All microscope parameters were identical for all observations of the binding ability of the various peptides . All animal experiments were conducted in compliance with the Beijing University Animal Study Committee’s requirements vis-à-vis the care and use of laboratory animals . The Beijing University Animal Study Committee approved the experimental protocols . Five to six-week-old Balb/c female nude mice were subcutaneously administered approximately 1 × 107 SKBR3 cells into the right hind leg , to establish xenografted tumors . Thereafter , tumor size was periodically measured with a caliper , and mice with tumors of 6−8 mm in diameter were selected for the following small animal experiments . Cy5 . 5–NHS ( Lumiprobe ) was used to label peptides . Either Cy5 . 5-peptides or the control Cy5 . 5 ( 1 μM , 200 μL ) was intravenously injected into tumor-bearing nude mice via the tail vein . The mice were anesthetized and fluorescence signals measured using the small animal in vivo imaging system 30 min postinjection . Three mice were used for each peptide and for control . Near-infrared fluorescence imaging of tumor-bearing nude mice were taken with an exposure time of 50 ms , using the Cy5 . 5 filter sets ( excitation: 673 nm; emission: 707 nm ) , and the intensities were quantified using the same software . Then , fluorescence images of the main organs and of tumors dissected from nude mice were individually taken as above . | Many therapeutic approaches , including the human monoclonal antibodies trastuzumab and pertuzumab , target the human epidermal growth factor receptor 2 ( HER2 ) of any breast cancer that features HER2 overexpression . Compared to these antibodies , peptides have many advantages , including lower cost , easier synthesis , high affinity , and lower toxicity . Here , we first designed peptides that interact with HER2 protein based on the HER2/pertuzumab crystal structure ( PDB entry: 1S78 ) , using a combination protocol of molecular dynamics modeling , molecular mechanics/generalized Born solvent-accessible surface area ( MM/GBSA ) binding free energy calculations . Then , combined with the peptide library screening , six peptides were selected for further analysis and experimental validations . The results of ex vivo and in vivo experiments confirmed that one peptide ( 58F63Y ) in particular has a strong affinity and high specificity to HER2-overexpressing tumors . This may due to more paired residues and lower binding free energy in peptide 58F63Y and HER2 complex based on free energy decomposition analysis and distances calculation . While both in silico and in vitro screenings point to the same high-affinity peptide , the findings suggest that in silico screening based on calculated binding free energies is rather reliable . Additionally , based on the calculation of binding free energies among mutants , we can reduce the library capacity of one-bead-one-compound screening . In summary , we present a rather simple and rapid means of deriving a peptide with a clear binding site to its target protein . | [
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] | 2017 | Peptide probes derived from pertuzumab by molecular dynamics modeling for HER2 positive tumor imaging |
Insulin resistance and obesity are associated with reduced gonadotropin-releasing hormone ( GnRH ) release and infertility . Mice that lack insulin receptors ( IRs ) throughout development in both neuronal and non-neuronal brain cells are known to exhibit subfertility due to hypogonadotropic hypogonadism . However , attempts to recapitulate this phenotype by targeting specific neurons have failed . To determine whether astrocytic insulin sensing plays a role in the regulation of fertility , we generated mice lacking IRs in astrocytes ( astrocyte-specific insulin receptor deletion [IRKOGFAP] mice ) . IRKOGFAP males and females showed a delay in balanopreputial separation or vaginal opening and first estrous , respectively . In adulthood , IRKOGFAP female mice also exhibited longer , irregular estrus cycles , decreased pregnancy rates , and reduced litter sizes . IRKOGFAP mice show normal sexual behavior but hypothalamic-pituitary-gonadotropin ( HPG ) axis dysregulation , likely explaining their low fecundity . Histological examination of testes and ovaries showed impaired spermatogenesis and ovarian follicle maturation . Finally , reduced prostaglandin E synthase 2 ( PGES2 ) levels were found in astrocytes isolated from these mice , suggesting a mechanism for low GnRH/luteinizing hormone ( LH ) secretion . These findings demonstrate that insulin sensing by astrocytes is indispensable for the function of the reproductive axis . Additional work is needed to elucidate the role of astrocytes in the maturation of hypothalamic reproductive circuits .
Reproduction is essential for species survival . Because energy is required to locate a mate , maintain a pregnancy , and rear young , fertility is modulated by the status of energy stores [1–3] . Excessive energy expenditure or insufficient caloric intake in humans and rodents delays the pubertal transition and reduces fertility [4 , 5] . Moreover , diseases that cause metabolic disturbances , such as thyroid disease , chronic inflammatory states , and malnutrition , are associated with a disruption of the normal timing of puberty [6] . The pancreatic hormone insulin serves as one metabolic signal linking hypothalamic function with metabolic state [7–9] . Postnatal deletion of insulin receptors ( IRs ) in glial fibrillary acidic protein ( GFAP ) -expressing cells decreased the activation of pro-opiomelanocortin ( POMC ) neurons by glucose [10] . Additionally , mice with IR ablated from astrocytes in the mediobasal hypothalamus became insulin and glucose intolerant [10] . These findings suggest that IRs on hypothalamic astrocytes play a role in regulating glucose metabolism . Insulin is a key regulator of the gonadotropin-releasing hormone ( GnRH ) network that controls fertility [8 , 11–14] . Insulin increases GnRH-dependent luteinizing hormone ( LH ) secretion in adult male mice [2 , 15] . Similarly , hyperinsulinemic clamps in women significantly increase LH pulsatility [2 , 16 , 17] . Insulin signaling in the brain may also provide a prerequisite signal for the initiation of puberty [18 , 19] . Insulin increases in children around the time of adrenarche in association with increasing circulating insulin-like growth factor 1 ( IGF1 ) levels [2] . Administering metformin to girls with precocious pubarche to reduce their insulin levels results in a delay in the onset of puberty [20 , 21] . However , the specific mechanisms underlying insulin modulation of pubertal timing are largely unknown . A seminal paper by Brüning and colleagues [8] showed that 50% of mice lacking the IR in cells expressing nestin ( NIRKO mice ) displayed hypogonadotropic hypogonadism in adulthood . Targeted deletion of IRs in specific neuronal populations , however , has failed to induce the subfertile phenotype and GnRH network dysregulation of NIRKO mice [2 , 3 , 6 , 22 , 23] . For instance , Divall and colleagues found that mice with IR deletion in GnRH neurons experienced normal pubertal timing and fertility [6] . Mice with IR deletion in kisspeptin neurons displayed a 4–5 day delay in pubertal onset but normal fertility and gonadal hormonal levels in adulthood [2] . In another example , mice with IR deletion in gamma-amino butyric acid ( GABA ) -ergic or glutamatergic cells showed normal pubertal progression , estrous cyclicity , and fertility [23] . More widespread deletion of IR in Ca2+/calmodulin-dependent protein kinase-expressing neurons , located in the dentate gyrus , cortex , olfactory bulb , amygdala , striatum , thalamus , and hypothalamus [24] , also produced mice with normal reproductive maturation and fertility [3] . These numerous negative results suggest that insulin action in neurons does not play an essential role in hypothalamic-pituitary-gonadal ( HPG ) axis function . Alternatively , it has been suggested [3] that the hypothalamic hypogonadism observed in NIRKO mice results from the chronic absence of insulin signaling in glia rather than neurons . Indeed , the nestin-cre line drives deletion of IR in both neuronal and non-neuronal cells [8 , 25–27] . Glial cells , which include astrocytes and tanycytes , are known to play an important role in the puberty onset , estrus cyclicity , and fecundity [28 , 29] . Therefore , we hypothesized that astrocytic insulin sensitivity is required for normal GnRH release during the pubertal period and in adulthood . We tested this hypothesis by using the cre-lox system to examine the effect of chronic astrocyte IR deletion on fertility .
To generate mice with IR deletion in astrocytes , we crossed IRloxp and GFAP-cre mouse lines . To assess whether Cre expression was restricted to astrocytes in the resulting mice , we crossed experimental mice with tdTomato-loxP reporter mice , which express red fluorescent protein ( RFP ) in a cre-dependent manner . RFP was found in IRKOGFAP brains but in not those of control mice that carried only the IRloxp allele ( Fig 1A ) . Our data confirm the specificity and selectivity of IR gene and transcript deletion to the brain and not other tissues , including the gonads ( S1 Fig ) . Double immuno-staining labeling of GFAP and tdTomato showed sufficient cre activity to drive tdTomato expression in 94% of GFAP positive cells . When neurons were labeled with the neuronal nuclear antigen NeuN , there was no colocalization with cre-driven tdTomato expression ( Fig 1B ) . We performed immuno-staining colocalization studies in various regions of the brain , including the arcuate nucleus ( ARC ) , anteroventral periventricular nucleus ( AVPV ) , and the cortex to further confirm the wide-spread deletion of IR in astrocytes ( S2 Fig ) . Fluorescence-activated cell sorting ( FACS ) was performed on isolated brain cells using tdTomato as a marker of cre expression . The data show that 46 . 0% of isolated brain cells were positive for astrocyte cell surface antigen-1 ( ACSA-1 ) and tdTomato , whereas 11 . 2% of cells were positive for ACSA-1 yet negative for tdTomato in the IRKOGFAP mice . In addition , very few cells ( 0 . 7% ) were positive for tdTomato and negative for ACSA-1 in brain cells isolated from IRKOGFAP mice ( Fig 1C ) . Astrocytes isolated by this method ( tdTomato+ allopycocyanin+ [APC] ) showed a substantial reduction in IR mRNA levels in IRKOGFAP mice when compared to IRloxp ( tdTomato− APC+ ) ( Fig 1C ) . Meanwhile , the expression levels of IR mRNA in the isolated nonastrocyte cells ( tdTomato− APC− ) from IRKOGFAP mice were comparable to the IRloxp group , confirming the specificity of the deletion ( Fig 1C ) . Previous studies have suggested that tanycytes near the third ventricle express GFAP [30] . Therefore , to further verify the purity of astrocytic FACS isolation , we measured gene expression of different markers of neuronal , tanycytic , microglia , and endothelial markers and confirmed the specific isolation of astrocytes via FACS ( S3 Fig ) . In addition , western blotting of brain tissues confirmed decreased levels of IR protein in IRKOGFAP mice when compared to the IRloxp group ( Fig 1D ) ( S4 Fig ) . Because it is still unclear if astrocytes are derived from erythromyeloid progenitors , the same lineage that produces macrophages in the periphery [31 , 32] , we tested whether macrophages , which originate as monocytes produced in bone marrow , exhibited loss of IRs . Expression of IRs and GFAP was not different in macrophages from IRloxp and IRKOGFAP mice ( S5 Fig ) . Balanopreputial separation serves as an indicator of the initiation of puberty in males . IRKOGFAP male mice showed a significant delay in the postnatal day ( PND ) of balanopreputial separation ( PND 33 . 36 ± 0 . 67 ) when compared to IRloxp control mice ( PND 28 . 44 ± 0 . 36 ) ( Fig 2A ) . In contrast , we found that the GFAP-cre mouse line alone has no phenotype in comparison to IRloxp mice ( S6 Fig ) . To assess the progression of puberty in female mice , vaginal opening and timing of the onset of estrus cycling were measured . IRKOGFAP mice exhibited a delay in vaginal opening of approximately 4 days ( PND 34 . 08 ± 0 . 69 ) when compared to IRloxp mice ( PND 29 . 44 ± 1 . 05 ) ( Fig 2B ) . IRKOGFAP mice showed a significant delay in the age of first estrus by approximately 5 days ( PND 42 . 55 ± 0 . 45 ) when compared to IRloxp mice ( PND 36 . 00 ± 1 . 01 ) ( Fig 2C ) . In addition , no differences were seen in body weight or body growth at 3 weeks of age between IRKOGFAP and IRloxp mice ( S7 Fig ) . IRKOGFAP females exhibited irregular cyclicity and longer estrous cycles . The estrus cycle length was approximately 2 days longer in IRKOGFAP females ( PND 6 . 25 ± 0 . 21 ) when compared to IRloxp mice ( PND 4 . 80 ± 0 . 13 ) ( Fig 2D ) . IRKOGFAP mice spent significantly less time in estrus and a longer time in diestrus when compared to IRloxp females ( Fig 2E ) ( S8 Fig ) . To assess fertility in IRKOGFAP mice , pregnancy rate , litter size , and mating success were measured . IRKOGFAP males produced fewer pregnancies when paired with fertile wild-type ( WT ) females ( 54% induced pregnancies ) , while IRloxp males were 90% successful in producing pregnancies ( Fig 2F ) . IRKOGFAP females , when paired with fertile WT males , exhibited a significantly reduced pregnancy rate of 45% , compared to 89% for IRloxp females ( Fig 2L ) . The interval from mating to birth did not differ between groups ( Fig 2H and 2N ) . However , IRKOGFAP male and female mice exhibited a significant decrease in litter size when compared to IRloxp mice ( litter size for IRloxp 7 . 44 ± 0 . 97 versus IRKOGFAP 2 . 55 ± 1 . 02 ) ( Fig 2G and 2M ) . We next assessed the function of the HPG axis in adult male and randomly cycling female mice by measuring LH , follicle-stimulating hormone ( FSH ) , and sex steroid levels between 8 and 10 AM . IRKOGFAP males showed a significant decrease in LH and testosterone levels ( Fig 2I and 2K ) but no change in FSH when compared to IRloxp mice ( Fig 2J ) . LH , FSH , and estradiol levels were significantly decreased in IRKOGFAP females when compared to IRloxp mice ( Fig 2O–2Q ) . LH pulse amplitude and frequency have been reported to be reduced on estrus , although basal levels of LH are similar on all days of the cycle [33] . Since IRKOGFAP female mice spent less time in estrus yet had lower LH levels , mouse cycle stage is unlikely to explain these findings . Gonadal morphology was examined in both sexes . There was a reduction in the sperm count per seminiferous tubule cross-section in all stages ( Fig 3A and 3B ) . Spermatogonia , spermatocytes , spermatid , and spermatozoa counts were significantly reduced in the seminiferous tubules of IRKOGFAP males ( 128 . 3 ± 16 . 53 , 128 . 0 ± 7 . 16 , 209 . 0 ± 15 . 76 , and 138 . 3 ± 12 . 61 ) when compared to IRloxp mice ( 212 . 0 ± 13 . 72 , 229 . 0 ± 14 . 01 , 361 . 0 ± 48 . 30 , and 278 . 0 ± 31 . 10 ) ( Fig 3C–3F ) . IRKOGFAP female mice exhibited altered ovarian morphology when compared to IRloxp mice ( Fig 3G and 3H ) . Similarly , the number of primary follicles , preovulatory follicles , and corpora lutea per ovary cross-section were significantly lower ( 3 . 00 ± 0 . 57 , 1 . 67 ± 0 . 33 , and 3 . 33 ± 0 . 33 ) when compared to IRloxp mice ( 7 . 50 ± 0 . 64 , 4 . 00 ± 0 . 57 , and 7 . 00 ± 1 . 00 ) ( Fig 3I–3M ) . Primordial and secondary follicle numbers were not different between groups . Because astrocytic insulin signaling has been linked to depressive-like behavior [69] , we examined sexual behavior in these mice to determine whether reduced fertility in IRKOGFAP mice could be partially attributed to reduced sexual motivation or performance . IRKOGFAP and IRloxp females were paired with WT gonadectomized males , and multiple parameters were measured , including lordosis , mounting attempts , lordosis quotient , and latency to first lordosis . IRKOGFAP and IRloxp female mice showed no differences in any of these parameters ( S9 Fig ) . Likewise , IRKOGFAP and IRloxp male mice showed no differences in mounting attempts , latency to first mount , and latency to first intromission when paired with control females ( S9 Fig ) . Astrocytes release specific growth factors that stimulate the secretion of GnRH . In particular , prostaglandin E2 ( PGE2 ) release stimulates the secretion of GnRH; Clasadonte and coworkers investigated the firing activity of GnRH neurons in mice with deficient PGE2 synthesis in astrocytes and found the excitability of these neurons significantly decreased [34] . We therefore measured protein levels of prostaglandin E synthase 2 ( PGES2 ) , which catalyzes the conversion of prostaglandin H2 to prostaglandin E2 , in isolated astrocytes from IRKOGFAP and control mice . IRKOGFAP astrocytes exhibited a significant reduction in PGES2 levels when compared to IRloxp astrocytes ( Fig 4A–4C ) .
Astrocytes assist neurons through nutritional and structural support and by promoting neurotransmitter release and recycling . They also appear to contribute to information processing by the brain [35 , 36] . Astrocytes possess a dense network of fine processes whose membranes contain potassium channels [37 , 38] , aquaporins [39] , glutamate transporters [40] , and lactate transporters [41] . These processes enwrap neuronal synapses and ensure effective synaptic transmission . Astrocytes also display increased intracellular calcium ( but not electrical excitability ) in response to chemical and neuronal cues [42] , which is believed to lead to the release of gliotransmitters , such as adenosine , polyphosphate , D-serine , glutamate , GABA , and lactate , that can alter neuronal activity [43–48] . As one critical element of the blood–brain barrier , astrocytes are readily able to sense circulating metabolic and endocrine signals [49 , 50] . Notably , insulin acts on IRs in primary human astrocytes , promoting glycogen synthesis [51] . Astrocytes are also able to release vasoactive molecules to regulate cerebral blood flow and to ensure a sufficient supply of oxygen and glucose to active neurons [52] . Astrocytes are therefore believed to play a critical role as central nervous system ( CNS ) metabolic sensors [53] . The current study demonstrates that insulin is a critical metabolic signal acting through astrocytes to permit reproductive competency via the GnRH network; astrocyte insulin signaling prevented hypogonadism and allowed normal fertility in adulthood . Similar to NIRKO mice [54] , IRKOGFAP mice exhibited impaired spermatogenesis , folliculogenesis , and ovulation , resulting in an almost 50% decrease in pregnancy rate and a nearly 69% reduction in litter size . IRKOGFAP mice also showed a significant decrease in LH and testosterone levels in males and LH , FSH , and estradiol levels in females . These findings indicate that disruption of astrocytic insulin signaling leads to hypogonadotropic hypogonadism [55 , 56] . Given that IRKOGFAP mice exhibit a delay in vaginal opening and first estrous in females and balanopreputial separation in males , disruption of astrocytic insulin action also serves as a critical role in the maturation of the HPG axis . Astrocytes have the potential to control GnRH release in several ways . GFAP-immunoreactive astrocyte processes have been shown to ensheath GnRH cell bodies in the rostral preoptic area of the rat [57] and GnRH cell bodies in the medial basal hypothalamus of monkeys [58 , 59] . In addition , GnRH processes in the median eminence are apposed largely by astrocytes , with the support of tanycytes [60] . The structural relationships at both sites are dynamic and regulated by gonadal steroids in rodents and rhesus monkeys [57 , 58 , 61 , 62] . GnRH neurons adhere to astrocytes using heterophilic ( contactin/RPTPβ ) and homophilic synaptic cell adhesion molecule ( SynCAM ) interactions; these molecules have signaling capabilities , suggesting they can activate intracellular signaling cascades in astrocyte and GnRH neurons [63] . Indeed , transgenic mice that express a dominant negative SynCAM1 under the control of a human GFAP promoter had a delayed onset of puberty , disrupted estrous cyclicity , and reduced fecundity associated with low GnRH release [29] . Astrocytes also synthesize and release factors that regulate GnRH secretion [28] . Astrocytes are believed to produce growth factors such as basic fibroblast growth factor IGF1 and transforming growth factor ( TGF ) -β1 that act directly on GnRH neurons to stimulate production of GnRH . In addition , in vitro evidence suggests that their production of growth factors of the epidermal growth factor family ( TGFα and neuroregulins ) causes glial release of mediators like PGE2 that stimulate GnRH release [64] . Mice expressing a dominant-negative Erbb2 receptor tyrosine kinase 4 receptor , which responds to EGFs , under the control of the GFAP promoter exhibit delayed sexual maturation and a diminished reproductive capacity in early adulthood due to impaired release of GnRH [65] . Interestingly , human hypothalamic hamartomas associated with sexual precocity in humans contain numerous astrocytes expressing TGFα and erbB1 receptors [66] . Astrocytes also release substances , like calcium , glutamate , and ATP , capable of stimulating GnRH release [67 , 68] . Cai and coworkers ( 2018 ) recently found that insulin signaling can target astrocyte-specific soluble NSF attachment protein receptors to regulate exocytosis of ATP [69] . Thus , IR deletion in IRKOGFAP mice may lead to impaired tyrosine phosphorylation of mammalian uncoordinated-18 , leading to decreased astrocytic ATP exocytosis [69] . Finally , neurons require glial-provided precursors such as glutamine to synthesize glutamate and GABA . This mechanism allows astrocytes to influence neuronal glutamate production and availability at the synaptic cleft by expressing glutamine synthase [70 , 71] . This regulation is responsive to estradiol levels and pubertal progression [72 , 73] . Overall , these studies demonstrate that astrocytes can influence GnRH release through multiple pathways . Studies have shown that hypothalamic astrocytes release PGE2 in response to cell–cell signaling . PGE2 release stimulates the secretion of GnRH to regulate the pituitary release of LH and FSH [34] . Our work shows decreased levels of astrocytic PGES2 protein levels in knockout mice when compared to controls , suggesting reduced production and release of PGE2 . Interestingly , PGE2 release is mediated by exocytosis . Shimada and colleagues have shown that solute carrier organic anion transporter family member 2A1 , a PGE2 transporter , is responsible for loading intracellular PGE2 into lysosomes in macrophages; PGE2 is then released via exocytosis induced by Ca2+ influx [74] . Future studies should therefore investigate whether impaired insulin-dependent exocytosis could also affect PGE2 release from astrocytes . Another important consideration for future study is the role of astrocyte insulin action during development versus its actions in the adult animal . Indeed , insulin and IGFs may directly influence brain development and neuronal survival [75–77] . While the contribution of astrocyte insulin signaling to the establishment of neuroendocrine function is unknown , it may play a role during the organization of reproductive circuitry . In summary , our findings suggest that impaired insulin sensing in astrocytes delays the initiation of puberty and dramatically reduces adult reproductive success . These effects are due to dysfunction of the HPG axis , leading to hypogonadotropic hypogonadism , and are associated with decreased PGES2 levels in astrocytes . This model is the first to recapitulate the effects of brain IR deletion on fertility . Our findings emphasize the importance of astrocytic signaling in the regulation of reproduction and lay the foundation for future studies addressing this communication at different stages of development . Additional studies are warranted to investigate the mechanism of how insulin action on astrocytes modulates the GnRH network .
All procedures were approved by the Institutional Animal Care and Use Committee ( IACUC ) of the University of Toledo College of Medicine and Life Sciences in Toledo , Ohio . All experiments were performed in accordance with the relevant guidelines and regulations described in the IACUC-approved protocol number 106448 . To create an astrocyte-specific deletion of IR ( IRKOGFAP mice ) , GFAP-Cre mice ( C57Bl/J6 ) ( Frederick National Laboratory for Cancer Research , Frederick , Maryland , United States ) were crossed with IRloxp mice ( C57Bl/J6 ) in which exon 4 of the IR gene was flanked by loxP sites [22] . GFAP is the main intermediate filament protein in mature astrocytes and an important component of the cytoskeleton in astrocytes during development [78 , 79] . After the first generation of the breeding , GFAP-Cre , IRloxp mice were crossed with homozygous IRloxp mice to generate the experimental mice . IRloxp mice littermates lacking Cre expression were used as controls; comparisons between IRloxp mice and GFAP-Cre mice were also performed where specified . Where noted , the mice also carried the tdTomato gene inserted into the Gt ( ROSA ) 26Sor locus to serve as a reporter under the control of Cre recombinase expression . Mice were housed in the University of Toledo College of Medicine animal facility at 22°C–24°C on a 12-hour light/dark cycle and were fed standard rodent chow . Mice were weaned on postnatal day ( PND ) 21 . Genotyping was performed by Transnetyx , Inc . ( Cordova , Tennessee , US ) using a real-time RTPCR–based approach . Mice were sacrificed via ketamine/xylazine injections , and the brain and other tissues were removed . Total RNA was extracted using an RNeasy Lipid Tissue Mini Kit ( Qiagen , Valencia , California , US ) . Single-strand cDNA was synthesized by a high-capacity cDNA Reverse Transcription Kit ( Applied Biosystems ) . Bone marrow–derived macrophages were obtained , as previously described [80] . Specifically , femurs and tibias were collected and flushed with medium containing sterile RPMI , 1% penicillin/streptomycin , and L929‐conditioned medium to isolate bone marrow cells . These cells were then allowed to differentiate for 7 days ( 37°C , 5% CO2 atmosphere ) with a change of media on day 4 . Then , RTPCR was performed [81] . Briefly , total RNA was prepared from BMDMs using Perfect Pure RNA Tissue kit ( 5Prime kit ) according to manufacturer's instructions . cDNA was synthesized with random primers and reverse transcriptase ( Applied Biosystems ) using 1 μg of total RNA . cDNA was evaluated with quantitative RTPCR using True Amp SYBR green qPCR Supermix ( Applied Biosystems ) . The relative amount of mRNA was calculated by comparison to the corresponding controls and normalized relative to Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) . RQ is expressed as means ± SE relative to IRloxp . Sequences of primers used are as follows: IR: Forward—CCCCAACGTCTCCTCTACCA , Reverse—TGTTCACCACTTTCTCAAATG; GFAP: Forward—ACATCGAGATCGCCACCTAC , Reverse—ATGGTGATGCGGTTTTCTTC; CD68: Forward—TCCAAGCCCAAATTCAAATC , Reverse—ATATGCCCCAAGCCTTTCTT; MAP-1: Forward—AGTGAGAAGAAAGTTGCCATCATC , Reverse—TTAATAAGCCGAAGCTGCTTAGG; CD11b: Forward—TGCCAAGACGATCTCAGCAT , Reverse—GCCTCCCACCACCAAAGT; Hes-1: Forward—CAACACGACACCGGACAAAC , Reverse—GTGGGCTAGGGACTTTACGG; Hes-5: Forward—GGTACAGTTCCTGACCCTGC , Reverse—AGAGGGTGGGCCCTGATTAT; vWF: Forward—CTACCTAGAACGCGAGGCTG , Reverse -CATCGATTCTGGCCGCAAAG; GAPDH: Forward—CCAGGTTGTCTCCTGCGACT , Reverse—ATACCAGGAAATGAGCTTGACAAAGT . Mice were sacrificed via ketamine/xylazine injections , and brains were collected . The hypothalami were then excised and minced with a razor blade on an ice-cold glass plate and placed in a microfuge tube with 1 ml of hibernate A ( HA-LF; Brian Bits , Springfield , Illinois , US ) . Hibernate A was then replaced with 1 ml Accutase ( SCR005 , Millipore , Temecula , California , US ) , and tubes were rotated for 30 minutes at 4°C . Samples were centrifuged at 425 x g for 2 minutes and each pellet was resuspended in 250 μl of ice-cold Hibernate A [82] . For cell dissociation , samples were triturated 10 times with a large Pasteur pipet and then placed on ice . Large pieces were allowed to settle , and 600 μl of supernatant was transferred to a 15-ml Falcon tube on ice . 600 μl of Hibernate A was added to the original tube , and the same procedure was repeated with medium and small Pasteur pipets . The collected supernatants were transferred to a 15-ml Falcon tube . Lastly , 750 μl of Hibernate A was added to the original tube , and 800 μl of supernatant was added to the 15-ml Falcon tube . Large debris was removed from the cell suspension by serial filtration through 100-μm and 40-μm cell strainers into 50-ml Falcon tubes , respectively ( Falcon 352360; Falcon 352340; BD Biosciences , San Jose , California ) [82] . The cell suspension was then centrifuged at 300 x g for 10 minutes and supernatant was aspirated completely . 100 μl of buffer ( PBS +5% FBS ) per 106 nucleated cells was added to the pellet . Then , 10 μl of ACSA-1 antibody ( MACS Cat . #130-095-814 ) was added , mixed well , and incubated for 10 minutes in the dark . Cells were washed by adding 1 ml of buffer and centrifuged at 300 x g for 10 minutes . The supernatant was then aspirated completely . Lastly , the cell pellet was resuspended in 500 μl of buffer . Cells were sorted in FACSAria ( BD Biosciencs , San Jose , California ) using tdTomato and ACSA-1-APC appropriate wavelengths ( 581 nm and 660 nm , respectively ) [83] . Astrocytes were isolated from IRloxp ( tdTomato− APC+ ) , and IRKOGFAP ( tdTomato+ APC+ ) . In addition , nonastrocyte cells were isolated from IRKOGFAP ( tdTomato− APC−/ tdTomato+ APC−/ tdTomato− APC+ ) . RNA from these cells were purified to determine IR gene expression [84] . Mice were sacrificed via ketamine/xylazine injections , and brains were collected , then excised and minced with a razor blade on an ice-cold glass plate and placed in a microfuge tube with 1 ml of hibernate A ( HA-LF; Brian Bits , Springfield , Illinois ) . A similar procedure was followed to isolate brain cells , as previously described in the FACS method section . Then , astrocytes expressing NA+-dependent glutamate transporter ( GLT-1 ) were positively selected using rabbit anti GLT-1 antibody ( Cat . #OSE0004W , ThermoFisher Sci ) and goat antirabbit IgG magnetic beads ( Cat . #S1432S , Biolabs ) . Full details of the procedure were described previously [85] . For protein expression , isolated astrocytes were lysed in RIPA buffer ( Cat . #SC-24948 , Santa Cruz Biotech ) . Lysate was centrifuged , followed by BCA assay to determine protein concentration . The primary antibodies used were as follows: IRβ ( Cat . #3025S , Cell signaling ) ; PGES2 ( Cat . #bs-2639R , Bioss ) [86 , 87]; and GADPH ( Cat . # SC-32233 , Santa Cruz Biotechnology ) . Secondary antibodies used were as follows: goat antirabbit-800 ( LI-COR , P/N 925–32211 ) and donkey antimouse-680 ( LI-COR , P/N 925–68075 ) . Images were captured using the LI-COR odyssey infrared imaging system , and only the contrast and brightness were adjusted for this purpose . Adult males and females ( in diestrus ) were perfused at the age of 7–8 months . Brains of the mice were collected and postfixed with 10% formalin at 4°C overnight , followed by immersion in 10% , 20% , and 30% sucrose for 24 hours each . A sliding microtome was used to cut sections ( 35–40 μm ) of the brain into five series [2 , 88] . For immunofluorescence , these sections were permeablized in 1 x PBS / 0 . 4% Triton x 100 for 1 hour at room temperature . Then , they were blocked in 1% BSA/5% normal donkey serum in 1 x PBS/Triton 0 . 4% at room temperature for 1 hour . After that , tissues were incubated with primary antibodies in blocking buffer at 4°C overnight , followed by five washes in PBST , with each wash lasting 10 minutes . Then , the tissues were incubated with secondary antibodies in blocking buffer for 2 hours at room temperature , followed by five washes in PBST . Sections were mounted on slides , air-dried overnight , and coverslipped with fluorescence mounting medium containing DAPI ( Vectasheild , Vector laboratories , Inc . Burlingame , California ) . Brain sections were visualized for the expression of tdTomato , GFAP , and NeuN fluorescence in IRKOGFAP mice using Total Internal Reflection Microscopy ( B&B microscopy limited Olympus IX-81 ) and Confocal Microscopy ( Leica ) and captured via Metaphore for Olympus Premier software . The primary antibodies used are as follows: anti-dsred 1° antibody ( [1:50] Clone Tech , Cat . #632496 ) , rabbit anti-GFAP polyclonal antibody-FITC conjugated ( Bioss , Cat# bs-01994-FITC ) , and rabbit anti-NeuN ( [1:100] abcam , Cat . #ab177487 ) . The secondary antibodies used are as follows: Alexa Fluor 594 ( 1:1 , 000 , Life Tech , Lot #1256153 ) and Alexa Flour 488 ( 1:1 , 000 , Thermofisher Scientific , Cat . #A-21206 ) . Only the contrast and brightness were adjusted during imaging . Males and females were checked for onset of puberty daily starting after weaning at 3 weeks of age . Balanopreputial separation in males was checked by attempting to manually retract the prepuce with gentle pressure . For females , vaginal opening was checked daily [89] . Thereafter , vaginal lavages were collected from experimental mice for at least 3–4 weeks . Cytology of collected cells was examined to assess estrus stages . Predominance of leukocyte cells was taken to indicate a diestrous stage , predominance of nucleated cells a proestrous stage , and predominance of cornified epithelial cells an estrous stage [90 , 91] . First estrous was defined as the first day of predominant cornified epithelial cells after the completion of one initial estrous cycle . For fertility studies , adult control IRloxp and IRKOGFAP females 3–4 months old were placed with WT males . Length of time until birth of the first litter and litter size were then determined [2] . The mice were paired for 8 days , and copulatory plugs were observed for evidence of successful mating . After that , mice were separated , and the delivery date was recorded . Similar procedures were used for IRloxp and IRKOGFAP male mice paired with WT females . IRloxp and IRKOGFAP male mice were paired with WT females on the day the female was in proestrus . IRloxp and IRKOGFAP females were paired with experienced vasectomized males . Mating behavior was captured using infrared cameras ( Swann ) placed beside individual cages . Mice were placed in the procedure room at 1 PM to acclimate to the new environment and then the lights were turned off at 6 PM to begin the dark phase . After 2 hours in the dark ( 8 PM ) , a female in proestrus was introduced into each cage with a single male . Filming began at 8 PM and continued until 2 AM . The following morning , the female mice were checked for copulatory plugs , as previously described [92] . The video files were collected and analyzed for specific hallmarks of female sexual behavior , such as lordosis events and latency to first lordosis , as well as indicators of male sexual interest , such as latency to first mount and number of mounting attempts . A single-blinded rater completed the analysis to ensure consistency and reliability . Submandibular blood was collected from IRloxp and IRKOGFAP diestrus female and male mice between 8–10 AM in randomly cycling mice to avoid the rise in LH that occurs on proestrus afternoon . LH and FSH levels were measured using multiplex testing performed by the University of Virginia Center for Research in Reproduction ( Charlottesville , Virginia ) . Multiplex LH and FSH levels were measured with intra-assay CV < 20% and reportable range of 0 . 24–30 ng/ml for LH and 2 . 4–300 ng/ml for FSH . Female serum estradiol was measured using ELISA ( Calbiotech . Spring Valley , California ) with sensitivity of 3 pg/ml and intra-assay CV < 10 . 5% . Male serum testosterone levels were measured by ELISA ( Calbiotech . Spring Valley , California ) with sensitivity of 0 . 1 ng/ml and intra-assay CV of 3 . 17% [93] . At 6–7 months of age , adult males and diestrous females were perfused with 10% formalin and organ tissues including the testis or ovary were collected and postfixed immediately in 10% formalin overnight . Next , the tissues were kept in 70% ethanol overnight . Then , tissues were embedded in paraffin , cut into sections , and stained by hematoxylin and eosin [2] . Histological section were visualized via Olympus BX61US microscope ( X-cite 120 LED boost EXCELITAS technology ) and captured via OlyVia 2 . 9 software . Ovary sections ( 4 per mouse ) were analyzed by evaluating follicle maturation , including counting the number of primordial , primary , secondary , and preovulatory follicles and corpora lutea . Testes sections were analyzed by evaluating sperm stages , including counting the number of spermatogonium , spermatocytes , spermatid , and spermatozoa . Sperm and follicle counts are reported per seminiferous tubule/ovary cross-section . Only the contrast and brightness were adjusted during imaging . Data are presented as the mean ± SEM . Two-tailed , unpaired t testing was used for comparisons of two groups . One-way ANOVA was used to compare three groups , followed by Bonferroni multiple comparison test . Chi-squared test was used to analyze statistical differences in fertility studies . Data were analyzed using Prism 6 software ( GraphPad ) . P < 0 . 05 was considered statistically significant . The numerical data used in all figures are included in S1 Data . | Astrocytes are a major cell type in the central nervous system , yet their impact on the neuroendocrine circuits that control fertility is under appreciated . Here , we show in mice that ablation of insulin signaling in astrocytes leads to delayed puberty , hypothalamic-pituitary-gonadotropin ( HPG ) axis dysfunction , and reduced fertility . These findings are the first demonstration that astrocytes and a metabolic signal collaborate to permit the maturation of the reproductive axis and adult fertility . | [
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] | 2019 | Ablating astrocyte insulin receptors leads to delayed puberty and hypogonadism in mice |
The DNA repair enzyme polynucleotide kinase/phosphatase ( PNKP ) protects genome integrity by restoring ligatable 5’-phosphate and 3’-hydroxyl termini at single-strand breaks ( SSBs ) . In humans , PNKP mutations underlie the neurological disease known as MCSZ , but these individuals are not predisposed for cancer , implying effective alternative repair pathways in dividing cells . Homology-directed repair ( HDR ) of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells , but the critical HDR protein Rad51 is not required in PNKP-null ( pnk1Δ ) cells of Schizosaccharomyces pombe . Here , we report that pnk1Δ cells have enhanced requirements for Rad3 ( ATR/Mec1 ) and Chk1 checkpoint kinases , and the multi-BRCT domain protein Brc1 that binds phospho-histone H2A ( γH2A ) at damaged replication forks . The viability of pnk1Δ cells depends on Mre11 and Ctp1 ( CtIP/Sae2 ) double-strand break ( DSB ) resection proteins , Rad52 DNA strand annealing protein , Mus81-Eme1 Holliday junction resolvase , and Rqh1 ( BLM/WRN/Sgs1 ) DNA helicase . Coupled with increased sister chromatid recombination and Rad52 repair foci in pnk1Δ cells , these findings indicate that lingering SSBs in pnk1Δ cells trigger Rad51-independent homology-directed repair of collapsed replication forks . From these data , we propose models for HDR-mediated tolerance of persistent SSBs with 3’ phosphate in pnk1Δ cells .
Maintenance of genome integrity depends on the accurate repair of DNA lesions that sever one or both strands of the double-helix . Single-strand breaks ( SSBs ) are by far the most abundant DNA scission , occurring at frequencies of thousands/cell/day in proliferating human cells [1] . SSBs are formed by many mechanisms , including oxidative attack of the sugar-phosphate backbone by endogenous reactive oxygen species ( ROS ) , by base and nucleotide excision repair , through the activity of anti-cancer drugs such as camptothecin or bleomycins , or by exposure to other DNA damaging agents . These SSBs often have 5’-hydroxyl or 3’-phosphate termini that prevent ligation . Polynucleotide kinase phosphatase ( PNKP ) is a bifunctional enzyme that restores 5’-phosphate and 3’-hydroxyl to these DNA ends [2 , 3] . PNKP’s importance is indicated by its conservation throughout eukaryotic evolution , although some species such as Saccharomyces cerevisiae have only retained the phosphatase domain [4] . The consequences of eliminating PNKP activity varies dramatically in eukaryotes . At one extreme , deleting the PNKP gene in mice causes early embryonic lethality [5] . PNKP probably plays an equally important role in humans , as a rare autosomal recessive disease characterized by microcephaly , early-onset intractable seizures and developmental delay ( denoted MCSZ ) was traced to partial loss-of-function mutations in the PNKP gene [6–8] . MCSZ is not associated with cancer; indeed , neurodegeneration in the absence of cancer predisposition appears to be a typical consequence of SSB repair defects in humans [9] . In contrast to mammals , S . cerevisiae cells lacking the DNA 3’ phosphatase encoded by TPP1 display no obvious phenotypes or sensitivity to DNA damaging agents [10] . However , requirements for Tpp1 are revealed when other DNA repair pathways are inactivated . Most notably , in cells lacking the apurinic/apyrimidinic ( AP ) endonucleases Apn1 and Apn2 , deletion of TPP1 increases cellular sensitivity to several DNA damaging agents , including the DNA alkylating agent methyl methanesulfonate ( MMS ) and the topoisomerase I inhibitor camptothecin ( CPT ) [10 , 11] . These AP endonucleases process DNA ends with various 3’-terminal blocking lesions , including 3’ phosphoglycolate ( 3’-PG ) , 3′‐unsaturated aldehydic , α , β‐4‐hydroxy‐2‐pentenal ( 3′‐dRP ) , and 3’-phosphates . PNKP is not essential in the fission yeast Schizosaccharomyces pombe , but pnk1Δ cells are sensitive to a variety of DNA damaging agents , most notably CPT [12–14] . These phenotypes were attributed to loss of Pnk1 phosphatase activity , as they are rescued by expression of TPP1 or kinase-null mutations of pnk1 , but not pnk1 alleles that eliminate phosphatase activity [14] . In contrast to S . cerevisiae , in which tpp1Δ apn1Δ apn2Δ cells display no obvious growth defect [10] , in S . pombe pnk1Δ apn2Δ cells are inviable [14] . If SSBs with 5’-hydroxyl or 3’-phosphate are left unrepaired in PNKP-deficient cells , progression through S-phase should lead to replication fork collapse by replication runoff , resulting in one-ended double-strand breaks ( DSBs ) [1] . These DNA lesions are subject to homology-directed repair ( HDR ) , which initiates when an endonuclease consisting of Mre11-Rad50-Nbs1 ( MRN ) protein complex and Ctp1 ( CtIP/Sae2 ) binds the DSB and progressively clips the 5’ strand , generating a 3’ single-strand DNA ( ssDNA ) overhang [15–17] . This ssDNA is coated with Replication Protein A ( RPA ) , which is then replaced by Rad51 recombinase by a mechanism requiring Rad52 strand-annealing protein [18] . Rad51 catalyzes the homology search and invasion of the intact sister chromatid , culminating in restoration of the replication fork . This fork repair mechanism produces a DNA joint molecule ( JM ) , either a D-loop or Holliday junction ( HJ ) , that must be resolved to allow chromosome segregation during mitosis . Replication-coupled single-strand break repair ( RC-SSBR ) as outlined above has been widely proposed as an alternative mechanism for repairing SSBs in PNKP-deficient cells [1 , 19 , 20] . However , data supporting this model are weak . Notably , Rad51 is not required in pnk1Δ mutants of fission yeast [14] . Nor does elimination of TPP1 cause any reported phenotype in rad52Δ cells of budding yeast , although significant growth defects appear when AP endonucleases are also eliminated in this genetic background [10] . Most critically , it is unknown whether HJ resolvases are required in PNKP-deficient cells , which is a decisive prediction of the RC-SSBR model . Brc1 is a fission yeast protein with 6 BRCT ( BRCA1 C-terminal ) domains that is structurally related to budding yeast Rtt107 and human PTIP [21 , 22] . The C-terminal pair of BRCT domains in Brc1 bind phospho-histone H2A ( γH2A ) , equivalent to mammalian γH2AX , which is formed by Tel1 ( ATM ) and Rad3 ( ATR/Mec1 ) checkpoint kinases at DSBs and damaged or stalled replication forks [22–24] . Brc1 is not required for DSB repair but it plays an important role in recovery from replication fork collapse [13 , 23 , 25–29] . We recently discovered a synergistic negative genetic interaction involving brc1Δ and pnk1Δ [13] , suggesting that pnk1Δ cells suffer increased rates of fork collapse . This result was curious , because as mentioned above , the critical HDR protein Rad51 is not required for the viability of pnk1Δ cells [14] . Here , we investigate the genetic requirements for surviving PNKP deficiency in fission yeast , uncovering crucial roles for key HDR proteins such as Mre11 , Rad52 and Mus81 in a variant mechanism of RC-SSBR that does not require Rad51 .
Epistatic mini-array profiling ( E-MAP ) screens identified synergistic negative genetic interactions involving brc1Δ and pnk1Δ , indicating that Brc1 helps to maintain cell viability when Pnk1 activity is lost [13 , 30 , 31] . We confirmed this synthetic sick interaction in spot dilution assays in which the colony size of pnk1Δ brc1Δ mutants were reduced compared to either single mutant ( Fig 1 , untreated panels ) . The growth defect of pnk1Δ brc1Δ cells was also verified in liquid growth assays that measured doubling times ( Fig 1 ) . The growth defect of pnk1Δ brc1Δ cells was enhanced in the presence of MMS or CPT , which produce DNA lesions that can be processed to yield SSBs with 3’ phosphate ( Fig 1 ) . Failure to repair these SSBs before entry into S-phase would be expected to increase the frequency of replication fork collapse . Brc1 is thought to act as a scaffold protein to promote replication fork stability and repair [22 , 29] . These activities partially depend on the ability of Brc1 to bind γH2A through its C-terminal pair of BRCT domains . The crystal structure of these domains bound to γH2A peptide allowed us to design T672A and K710M mutations that specifically disrupt the γH2A-binding pocket in Brc1 and abolish Brc1 foci formation [22] . These mutations did not cause an obvious growth defect in the pnk1Δ background but they strongly enhanced sensitivity to MMS or CPT ( Fig 1 ) . From these data , we conclude that Brc1 binding to γH2A is critical when pnk1Δ cells are treated with genotoxins that cause formation of SSBs with 3’ phosphate . The requirement for Brc1 binding to γH2A in pnk1Δ cells suggested that unrepaired SSBs in these cells triggers a DNA damage response involving the master checkpoint kinase ATR , known as Rad3 in fission yeast [32] . Indeed , pnk1Δ rad3Δ colony size was reduced and doubling time increased compared to either single mutant ( Fig 2A ) . This negative genetic interaction became more obvious when pnk1Δ rad3Δ cells were grown in the presence of CPT , MMS or the replication inhibitor hydroxyurea ( HU ) ( Fig 2A ) . Elimination of Brc1 further impaired growth in pnk1Δ rad3Δ cells ( Fig 2A ) , which is consistent with previous studies indicating that Brc1 has both Rad3-dependent and independent activities [13] . Rad3 phosphorylates the checkpoint kinase Chk1 in response to replication fork collapse [33–35] . Immunoblot assays that detect phospho-Chk1 confirmed that Chk1 is activated even in the absence of genotoxin treatment in pnk1Δ cells , providing molecular evidence of increased DNA lesions in these cells ( Fig 2B ) . Chk1 phosphorylation in response to HU treatment was also increased in pnk1Δ cells compared to wild type ( Fig 2B ) . No negative genetic interaction between pnk1Δ and chk1Δ was evident in the absence of genotoxins , indicating that the spontaneous DNA lesions causing Chk1 activation in pnk1Δ cells are efficiently repaired in the time frame of a normal G2 phase ( Fig 2C , untreated panel ) . These data suggest that the Chk1-activiating lesions are occurring early in the cell cycle , such as when replication runoff at lingering SSBs forms Chk1-activating DSBs . However , genotoxin treatment revealed a synergistic negative genetic interaction between pnk1Δ and chk1Δ that was most evident in cells treated with CPT . Chk1 was also critical in pnk1Δ cells treated with HU , which was consistent with the enhanced Chk1 phosphorylation in HU-treated pnk1Δ cells ( Fig 2B ) . Elimination of Chk1 also enhanced the CPT and MMS sensitivity of pnk1Δ brc1Δ cells ( Fig 2C ) . Rad3 phosphorylates Cds1 checkpoint kinase , homologous to mammalian Chk2 , in response to replication fork arrest caused by HU [36] . We did not observe negative genetic interactions between pnk1Δ and cds1Δ , either in the absence or presence of genotoxins ( HU or CPT ) ( Fig 2D ) . These data indicate that pnk1Δ cells do not suffer from high frequencies of replication fork arrest . However , the triple mutant pnk1Δ cds1Δ chk1Δ displayed modestly increased HU sensitivity relative to cds1Δ chk1Δ , and modestly increased CPT sensitivity relative to pnk1Δ chk1Δ ( Fig 2D ) . From these results , we conclude that pnk1Δ cells accumulate DNA lesions that activate a Rad3-dependent checkpoint response leading to activation of Chk1 . This response becomes especially critical when Brc1 is absent or when cells are treated with genotoxins that create SSBs . These data indicated that pnk1Δ cells accumulate DNA lesions that activate DNA damage responses . To further test this proposition , we monitored foci formation of Rad52 , which is normally essential for all forms of homology-directed repair in fission yeast . Mutants that suffer increased rates of replication fork collapse , or are unable to efficiently repair collapsed forks , typically display increased numbers of Rad52 nuclear foci [37–40] . For these studies , we monitored Rad52 tagged with yellow fluorescent protein ( Rad52-YFP ) expressed from the endogenous locus . As observed previously [22] , the frequency of cells with Rad52-YFP foci was significantly increased in brc1Δ cells ( 12 . 5% ) compared to wild type ( 5 . 6% ) ( Fig 3A ) . The incidence of cells with Rad52-YFP foci was higher in the pnk1Δ strain ( 19% ) , and there was a further significant increase in the brc1Δ pnk1Δ strain ( 35 . 1% ) ( Fig 3A ) . Cell cycle phase analysis indicated that in all strains most of the cells with Rad52-YFP foci were in S-phase or early G2 phase , which suggests fork collapse as a primary source of these lesions . It was noteworthy that there was a large increase in mid- to late-G2 phase cells with Rad52 foci in the brc1Δ pnk1Δ strain ( 8 . 1% ) compared to either single mutant ( 3 . 2% or 2 . 5% ) , respectively ( Fig 3A ) . These data suggest Brc1 is required to efficiently repair lesions that accumulate in pnk1Δ cells , which could explain why Rad3 and Chk1 are crucial in pnk1Δ brc1Δ cells ( Fig 2 ) . In a separate experiment , we assessed foci formation by RPA , which is the major single-stranded DNA binding activity in eukaryotes . For these studies , we used strains that expressed the largest subunit of RPA , known as Ssb1 or Rad11/Rpa1 , with a green fluorescent protein ( GFP ) tag ( Fig 3B ) . The frequency of RPA-GFP foci was moderately increased in brc1Δ ( 11 . 6% versus 5 . 5% in wild type ) , further increased in pnk1Δ cells ( 15 . 7% ) , and even further increased in brc1Δ pnk1Δ ( 26 . 4% ) . As seen for Rad52 , in all strains the RPA foci were predominantly observed in cells that were in S or early G2 phase , although combining the brc1Δ and pnk1Δ mutations did result in a substantial increase in mid- or late-G2 phase cells with RPA foci ( 4 . 8% ) , versus 1 . 6% in brc1Δ cells or 1 . 5% in pnk1Δ cells ( Fig 3B ) . These results are consistent with the synergistic growth defect and genotoxin sensitivity observed in brc1Δ pnk1Δ cells , and suggest that efficient repair of unligatable SSBs that accumulate in the absence of Pnk1 depends on Brc1 . Our studies suggested that lingering SSBs in pnk1Δ cells are converted to DSBs by replication runoff . To further investigate this possibility , we assessed the requirements for the two major DNA end-binding protein complexes in fission yeast . The Ku70/Ku80 heterodimer has a high affinity for DSBs . It promotes nonhomologous end-joining ( NHEJ ) , which is critical for DSB repair in G1 phase when cells lack sister chromatids required for HDR [41] . The pku80Δ mutation did not impair the growth of pnk1Δ cells ( Fig 4 ) . These findings show that NHEJ does not play a significant role in an alternative pathway for repairing SSBs in the absence of PNKP . The Mre11-Rad50-Nbs1 ( MRN ) endonuclease complex also binds DSBs , whereupon it endonucleolytically liberates Ku and initiates 5’-3’ resection to generate ssDNA tails required for HDR [42] . These activities depend on Ctp1 ( CtIP/Sae2 ) , which is only expressed in S and G2 phases in S . pombe [43 , 44] . E-MAP studies indicated that both Mre11 and Ctp1 are likely to be important in the absence of Pnk1 [31 , 45] . Indeed , we found that pnk1Δ mre11Δ and pnk1Δ ctp1Δ double mutants grew very poorly compared to the respective single mutants ( Fig 4 ) . The requirement for MRN and Ctp1 to initiate resection of DSBs can be substantially alleviated by genetically eliminating Ku , which allows Exo1 exonuclease to access DSBs and initiate resection [43 , 44 , 46] . To investigate whether Exo1 effectivity substitutes for MRN-Ctp1 in the absence of Ku , we introduced the pku80Δ mutation into pnk1Δ mre11Δ and pnk1Δ ctp1Δ backgrounds . This analysis revealed that eliminating Ku partially restored growth and genotoxin resistance in these genetic backgrounds ( Fig 4 ) . In the case of pnk1Δ ctp1Δ cells , we confirmed that suppression by pku80Δ depended on the presence of Exo1 ( Fig 4 ) . These findings indicate that the DSB resection activity of MRN-Ctp1 is critical in pnk1Δ cells . The genetic requirements for Mre11 and Ctp1 strongly suggested that HDR resets replication forks that collapse at lingering SSBs in pnk1Δ cells . However , the critical HDR recombinase Rad51 was reported to be nonessential in these cells [14] . We investigated these seemingly contradictory findings and confirmed that pnk1Δ rad51Δ cells are viable ( Fig 5A ) . Assays that measured doubling times in liquid media indicated a modest growth defect in pnk1Δ rad51Δ cells relative to the single mutants ( Fig 5A ) . Growth in the presence of HU or MMS revealed a more obvious negative genetic interaction between pnk1Δ and rad51Δ ( Fig 5A ) . These findings suggest that many of the spontaneous SSBs with 3’ phosphate that accumulate in pnk1Δ cells are repaired by an MRN-Ctp1-dependent HDR mechanism that does not require Rad51 . Previously , Whitby and co-workers reported that ~50% of CPT-induced collapsed replication forks are repaired by a Rad51-independent mechanism of HDR that requires Rad52 [47] . Similarly , we found that elimination of the Swi1-Swi3 replication fork protection complex leads to collapse of replication forks that are repaired by a mechanism requiring Rad52 but not Rad51 [48] . We set out to test whether Rad52 is critical in pnk1Δ cells . Genetic crosses involving rad52Δ are complicated by the frequent appearance of suppressors caused by loss of the F-box helicase Fbh1 [49] . Therefore , we generated pnk1Δ rad52Δ or rad52Δ cells that were complemented by a pRad52 plasmid containing rad52+ and the ura4+ selectable marker ( Fig 5B ) . Both strains grew relatively well in LAH medium that selects for the ura4+ marker , but the pnk1Δ rad52Δ cells grew much more poorly in 5-FOA media that counter-selects against the ura4+ marker ( Fig 5B ) . These data show that Rad52 is critical for cell viability in the pnk1Δ background . These results indicate that many of accumulated spontaneous SSBs in pnk1Δ cells are repaired by a Rad52-dependent mechanism that does not require Rad51 . In mitotic fission yeast , homology-directed repair of two-ended DSBs , for example as generated by ionizing radiation ( IR ) , proceeds by synthesis-dependent strand annealing ( SDSA ) . In SDSA , joint molecules do not mature into Holliday junctions , which explains why Mus81-Eme1 resolvase is not required for IR resistance in fission yeast [50–52] . In contrast , HDR-mediated restoration of a broken replication fork produces a joint molecule ( JM ) , either a D-loop or Holliday junction ( HJ ) , that must be resolved to allow chromosome segregation in mitosis , hence the acute requirement for Mus81 in conditions that increase replication fork collapse [47 , 51 , 53] . We mated pnk1Δ and mus81Δ strains and found that the large majority of double mutant spores failed to yield viable colonies . The few viable double mutants were extremely sick ( Fig 6A ) . We obtained the same results when we attempted to create pnk1Δ eme1Δ strains ( Fig 6B ) . If Rad51 is not required for JM formation during repair of collapsed replication forks in pnk1Δ cells , loss of Rad51 should not rescue the synthetic lethal interaction of pnk1Δ and mus81Δ . Indeed , genetic crosses showed that rad51Δ did not suppress pnk1Δ mus81Δ synthetic lethality ( Fig 6C ) . We also investigated Rad54 , which interacts with Rad51 and is required for Rad51-dependent HDR [54] , but not the Rad52-dependent HDR of CPT-induced DNA damage that occurs independently of Rad51 [47] . As predicted by our model , elimination of Rad54 failed to rescue the pnk1Δ mus81Δ synthetic lethality ( Fig 6D ) . Rqh1 is a RecQ family 3’-5’ DNA helicase that is orthologous to human WRN ( Werner syndrome ) and BLM ( Bloom syndrome ) DNA helicases , and S . cerevisiae Sgs1 DNA helicase [55 , 56] . Rqh1 is involved in multiple genome protection pathways and is particularly notable for its essential function in the absence of Mus81 [51] . Strikingly , we found that Rqh1 is essential in the pnk1Δ background ( Fig 6E ) . In S . cerevisiae , the viability of tpp1Δ apn1Δ apn2Δ cells depend on Rad10-Rad1 3’ flap endonuclease , which is orthologous to human ERCC1-XPF [11] . These results suggest that Rad10-Rad1 provides an alternative mechanism for eliminating 3’-phosphates from DNA termini through endonucleolytic cleavage of 3’ DNA flaps . In fission yeast , pnk1Δ apn2Δ cells are inviable [14] , but it remained possible that the 3’ flap endonuclease Swi10-Rad16 [57 , 58] , orthologous to budding yeast Rad10-Rad1 , played an important role in repairing lingering SSBs in pnk1Δ cells . We found that pnk1Δ swi10Δ cells were viable and displayed no obvious growth defect relative to the respective single mutants ( Fig 7 ) . The pnk1Δ swi10Δ strain displayed slightly more sensitivity to HU and CPT but not MMS , but these genetic interactions did not appear to be synergistic . Thus , unlike key HDR proteins , Swi10-Rad16 3’ flap endonuclease is not part of a critical back-up mechanism for repairing SSBs with 3’ phosphate . Finally , to explore whether Pnk1 deficiency creates perturbations to replication fork progression that increase recombination , we performed a mitotic intrachromosomal recombination assay . This assay determines the spontaneous frequency of Adenine positive ( Ade+ ) colonies arising by recombination between two ade6 heteroalleles flanking the his3+ gene [59] . Two classes of recombinants can be distinguished: deletion-types ( Ade+ His- ) and conversion-types ( Ade+ His+ ) ( Fig 8A ) . Total spontaneous recombination frequencies ( deletion + conversion types , reported as events per 104 cells ) were increased ~3 . 4-fold in pnk1Δ cells ( 4 . 78 ± 1 . 16 ) compared with wild-type ( 1 . 41 ± 0 . 57 ) . Although earlier studies indicated that spontaneous recombination frequencies in brc1Δ cells were strongly reduced [29] , in our assays the spontaneous recombination frequencies of brc1Δ cells were not significantly different from wild type ( Fig 8B ) . The spontaneous recombination frequency in brc1Δ pnk1Δ cells ( 3 . 91 ± 2 . 3 ) was moderately decreased compared to pnk1Δ cells ( Fig 8B ) . Interestingly , conversion-type recombinants predominated in pnk1Δ cells even though Rad51 is required for this mechanism of repair [47] , suggesting that Rad51 often participates in repair of collapsed forks at this locus even though it is not essential in pnk1Δ cells . The statistically significant decrease of total recombinants in brc1Δ pnk1Δ cells compared to pnk1Δ was caused by a loss of conversion-type recombinants . Collectively , these data indicate that a Pnk1 deficiency increases HDR-mediated genome instability .
In this study , we have investigated how fission yeast cells tolerate the loss of polynucleotide kinase/phosphatase . In principle , a PNKP deficiency should result in lingering SSBs if there is no other efficient alternative mechanism for repairing SSBs with 3’ phosphate . Note that for this discussion we are presuming that genetic interactions involving pnk1Δ are caused by loss of 3’ phosphatase activity , as this defect is responsible for the DNA damage sensitivities of pnk1Δ cells , although it is formally possible that loss of 5’ kinase activity also contributes to these genetic interactions [14] . SSBs can be converted into broken replication forks during S-phase . Broken forks in PNKP-deficient cells are proposed to be restored by homology-directed repair , also known as RC-SSBR [1] , thus key HDR proteins should be critical in the absence of PNKP . Surprisingly , there is scant evidence for this assumption , and that which exists in fission yeast contradicts this model . Notably , the critical HDR protein Rad51 is not required in pnk1Δ mutants of fission yeast [14] . We investigated this conundrum . Our experiments confirm that pnk1Δ rad51Δ cells are viable; indeed , eliminating Pnk1 only moderately impairs growth in the rad51Δ background . However , we have found that other HDR proteins become crucial for cell viability in the absence of Pnk1 . Our studies established that Rad52 is critical in pnk1Δ cells . Similarly , pnk1Δ mre11Δ and pnk1Δ ctp1Δ strains grow very poorly . As the principal role of MRN complex and Ctp1 is to initiate resection of DSBs , these data strongly suggest that defective SSB repair in pnk1Δ cells is rescued by a mechanism that involves homology-directed repair of DSBs . Another key finding was the requirement for Mus81-Eme1 resolvase in pnk1Δ cells . As discussed above , Mus81-Eme1 is not required for survival of IR-induced DSBs , but it is crucial for recovery from replication fork breakage [51 , 53 , 60] . Thus , our data strongly support the idea that lingering SSBs in pnk1Δ cells trigger replication fork collapse . This conclusion is further supported by the large increase in RPA and Rad52 foci in pnk1Δ cells , and cell cycle phase analysis indicating that most of the cells with these foci were in S-phase or early G2 phase . The nature of the accumulating DNA lesions in pnk1Δ cells are also indicated by the negative genetic interaction with brc1Δ . As previously reported , brc1Δ cells are largely resistant to IR but quite sensitive to CPT , indicating that Brc1 functions in S-phase to assist the repair of collapsed replication forks [22] . Thus , a defect in efficiently repairing collapsed replication forks most likely accounts for the synthetic sickness observed in pnk1Δ brc1Δ cells . Brc1 function partially depends on its ability to bind γH2A [22] , hence it is noteworthy that mutations that specifically disrupt this binding show a synergistic negative genetic interaction with pnk1Δ when cells are exposed to CPT . Recent studies with budding yeast revealed that the putative Brc1 ortholog Rtt107 plays a role in promoting the activation of Mus81-Mms4 resolvase ( orthologous to fission yeast Mus81-Eme1 ) by several cell cycle-regulated protein kinases [61 , 62] . In fission yeast , Mus81-Eme1 is regulated by the master cell cycle regulator Cdc2 ( CDK1 ) protein kinase and Rad3 ( ATR ) checkpoint kinase [63] . If Brc1 promotes Cdc2- or Rad3-mediated activation of Mus81-Eme1 , this mechanism could partly explain the requirement for Brc1 in pnk1Δ cells . The absence of an obvious negative genetic interaction involving pnk1Δ and chk1Δ mutations in cells grown without genotoxins , despite the evident activation of Chk1 , also provides clues about the DNA lesions that accumulate in the absence of PNKP . Chk1 delays the onset of mitosis by inhibiting Cdc25 , which is the activator the cyclin-dependent kinase Cdc2 [64] . Fission yeast has a naturally long G2 phase , thus activating a cell cycle checkpoint that delays mitosis should be less important if all DSBs are formed early in the cell cycle during S-phase . These facts explain why chk1Δ cells are relatively tolerant of moderate doses of genotoxins such as CPT , in which toxicity is mainly caused by breakage of replication forks , unless homology-directed repair is slowed by partial loss-of-function mutations in HDR proteins [65] . These observations are consistent with a model in which replication forks break when they encounter lingering SSBs in pnk1Δ cells . Neither pnk1Δ or chk1Δ mutants are strongly sensitive to 1 or 2 μM CPT , yet the pnk1Δ chk1Δ double mutant is acutely sensitive ( Fig 2C ) . A similar genetic relationship is observed for pnk1Δ and rad3Δ ( Fig 2A ) . These heightened requirements for checkpoint responses in pnk1Δ cells suggest that alternative repair pathways for repairing SSBs with 3’ phosphate are slow or inefficient . This interpretation is consistent with the increased level of Chk1 phosphorylation observed in the absence of genotoxin exposure in pnk1Δ cells ( Fig 2B ) . What is the explanation for the Rad51-independent repair of broken replication forks in pnk1Δ cells ? As previously proposed , replication fork collapse caused by the replisome encountering a single-strand break or gap can generate a broken DNA end and a sister chromatid with a single-strand gap [47] . This gap will tend to persist when it has a 3’ phosphate , as described below . The ssDNA gap may provide access to a DNA helicase that generates unwound donor duplex that participates in Rad52-mediated strand annealing [47] . This process would not require Rad51 . An alternative explanation concerns the location of lingering SSBs in pnk1Δ cells . In fission yeast , the ~150 copies of the ribosomal DNA locus are arranged in tandem repeats at each of chromosome III . We have previously reported that Slx1-Slx4 structure-specific endonuclease helps to maintain rDNA copy number by promoting HDR events during replication of the rDNA [66] . Strikingly , these HDR events require Rad52 but not Rad51 . Moreover , Mus81 and Rqh1 have crucial roles in maintaining rDNA in fission yeast [35 , 63] . If a large fraction of the lingering SSBs in pnk1Δ cells occur in the rDNA , this property could explain why Rad52 , Mus81 and Rqh1 are required in pnk1Δ cells , but Rad51 is dispensable . We note that both gene conversion and deletion types increase between the ade6- heteroalleles in pnk1Δ cells ( Fig 8 ) , suggesting that Rad51 often participates in this repair , even if it is not essential in pnk1Δ cells . The 3’ phosphate responsible for the persistence of a SSB in pnk1Δ cells can itself be a barrier to the completion of homology-directed repair when the SSB is converted to a broken replication fork [67] . Here , we consider models for tolerance of persistent SSBs with 3’ phosphate ( Fig 9 ) . When a replication fork collapses upon encountering a SSB with 3’ phosphate in the lagging strand template , the product is a one-ended DSB containing a 3’ phosphate ( Fig 9 , step 1a ) . Resection generates a single-strand overhang that invades the sister chromatid , but the 3’ phosphate blocks priming of DNA synthesis and restoration of an active replication fork ( step 1b ) . This barrier to DNA synthesis might favor dissolution of the JM , but its resolution by Mus81-Eme1 would stabilize the sister chromatid junction , allowing completion of replication by the converging fork ( step 1c ) . The final product is a replicated chromosome containing a small ssDNA gap with the 3 ‘phosphate ( step 1d ) . When a replication fork collapses upon encountering a SSB with 3’ phosphate in the leading strand template , the product is a one-ended DSB containing a 3’ hydroxyl opposite a sister chromatid with a ssDNA gap with 3’ phosphate ( Fig 9 , step 2a ) . As previously noted [67] , the SSB in the sister chromatid will block homology-directed repair , but replication by the converging fork will lead to replication fork collapse , leaving a DSB with a 3’ phosphate ( step 2b ) . At this point repair can proceed by SDSA ( step 2c ) , eventually leading to one intact chromosome and the other containing a single-strand gap with a 3’ phosphate ( step 2d ) . Plans are underway to test these models . In summary , these studies establish that polynucleotide/kinase phosphatase plays a crucial role in preventing the accumulation of SSBs that trigger replication fork collapse and genome instability in fission yeast , with the special property that many of these broken replication forks are repaired by an HDR mechanism that requires Mre11 , Rad52 and Mus81 , but not Rad51 . With the recent evidence that Rad52 plays a crucial role in repair of broken replication forks in mammalian cells [68 , 69] , it will be of special interest to evaluate the importance of Rad52 in PNKP-deficient mammalian cells .
The strains used in this study are listed in S1 Table . Standard fission yeast methods were used [70] . Deletion mutations strains were constructed as described [71] . The pnk1::KanMX6 strains were created from the wild-type strains using the PCR-based method and the primers , pnk1 . G ( 5′-GTATGTTATTGAAACCACCCATTTTCATTGCTATGCAATTATAATATAGCTAACTCAATTACCAAGTCCCATTTAGTATTCGGATCCCCGGGTTAATTAA-3′ ) and pnk1 . H ( 5′-ATAATTTTTATAAACGTTTGGTTTTAGTGGGATCAATAACTATATATTTTTGAAATTAATGCAATTTAATAATTTCTTAG GAATTCGAGCTCGTTTAAAC-3′ ) . The nucleotide sequences in boldface overlap to the KanMX cassette of plasmid pFA6a-kanMX4 . Successful deletion of these genes was verified by PCR . Tetrad analysis was performed to construct double mutants and verified by PCR . DNA damage sensitivity assays were performed by spotting 10-fold serial dilutions of exponentially growing cells onto yeast extract with glucose and supplements ( YES ) plates , and treated with indicated amounts of hydroxyurea ( HU ) , camptothecin ( CPT ) , and methyl methanesulfonate ( MMS ) . For UV treatment , cells were serially diluted onto YES plates and irradiated using a Stratagene Stratalinker UV source . Cell survival was determined after 3–4 days at 30°C . Doubling times were performed with cell grown in YES liquid media at 32°C . Values are an average 3 cultures . For Chk1 shift , whole cells extracts were prepared from exponentially growing cells in standard NP-40 lysis buffer . Protein amounting to ~100 mg was resolved by SDS-PAGE using 10% gels with acrylamide:bis-acrylamide ratio of 99:1 . Proteins were transferred to nitrocellulose membranes , blocked with 5% milk in TBST ( 137 mM Sodium Chloride , 20 mM Tris , pH 7 . 6 , 0 . 05% Tween-20 ) and probed with anti-HA ( 12CA5 ) antibody ( Roche ) . Cells were photographed using a Nikon Eclipse E800 microscope equipped with a Photometrics Quantix charge-coupled device ( CCD ) camera and IPlab Spectrum software . All fusion proteins were expressed at their own genomic locus . Rad52-yellow fluorescence protein ( YFP ) expressing strains were grown in EMM until mid-log phase for focus quantification assays . Quantification was performed by scoring 500 or more nuclei from three independent experiments . Mitotic recombination was assayed by the recovery of Ade+ recombinants from the strains containing the intrachromosomal recombination substrate . Spontaneous recombinant frequencies were measured as described by fluctuation tests [59 , 72] . Frequencies of fifteen colonies were averaged to determine the mean recombination frequency . Error bars indicate standard deviation from the mean . Two sample t-test were used to determine the statistical significance of differences in recombination frequencies . | DNA is constantly damaged by normal cellular metabolism , for example production of reactive oxygen species , or from exposure to external DNA damaging sources , such as radiation from the sun or chemicals in the environment . These genotoxic agents create thousands of single-strand breaks/cell/day in the human body . An essential DNA repair protein known as polynucleotide kinase/phosphatase ( PNKP ) makes sure the single-strand breaks have 5’ phosphate and 3’ hydroxyl ends suitable for healing by DNA ligase . Mutations that reduce PNKP activity cause a devastating neurological disease but surprisingly not cancer , suggesting that other DNA repair mechanisms step into the breach in dividing PNKP-deficient cells . One popular candidate was homology-directed repair ( HDR ) of replication forks that collapse at single-strand breaks , but the crucial HDR protein Rad51 was found to be non-essential in PNKP-deficient cells of fission yeast . In this study , Sanchez and Russell revive the HDR model by showing that SSBs in PNKP-deficient cells are repaired by a variant HDR mechanism that bypasses the requirement for Rad51 . Notably , Mus81 endonuclease that resolves sister chromatid recombination structures formed during HDR of collapsed replication forks was found to be essential in PNKP-deficient cells . | [
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] | 2017 | Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast |
The role of calcium ( Ca2+ ) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage ( HKM ) apoptosis has been reported . Here , we report the pro-apoptotic involvement of calmodulin ( CaM ) and calmodulin kinase II gamma ( CaMKIIg ) in the process . We observed significant increase in CaM levels in A . hydrophila-infected HKM and the inhibitory role of BAPTA/AM , EGTA , nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent . Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg . Using the CaMKIIg-targeted siRNA , specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A . hydrophila-infected HKM . Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression . CaMK Kinase ( CaMKK ) , the other CaM dependent kinase exhibited no role in A . hydrophila-induced HKM apoptosis . We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production . Using siRNA to PKACA , the catalytic subunit of PKA , anti-PKACA antibody and H-89 , the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A . hydrophila . Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 ( ERK 1/2 ) . We conclude that the alteration in intracellular Ca2+ levels initiated by A . hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM .
Aeromonas hydrophila , a Gram-negative , rod-shaped , facultatively intracellular bacterium is commonly found as part of the normal microbial flora in the aquatic environment [1] . The pathogenicity of A . hydrophila is complex and multi-factorial . It induces a plethora of symptoms in fish characterized by severe open dermal ulcers , anaemia , visceral granulomata , septicaemia , failure of osmoregulatory balance and death , which together comprise the ulcerative disease syndrome or UDS [2] . A . hydrophila is also known for its wide range of host tropism that includes amphibians , reptiles as well as mammals [2] . In humans , this bacterium is frequently associated with individuals suffering from gastroenteritis , wound infections , septicemia and immunodeficiency disorders [1] . Pathogen-induced alterations in intracellular Ca2+ with varied effects have been well documented . In several instances an alteration in the intracellular Ca2+-levels was found to be a pre-requisite for pathogen-induced apoptosis of different cell types including macrophages [3] . Studies have also documented that initiation of Ca2+-influx delays the onset of apoptosis facilitating pathogen survival and growth inside the host macrophages [4] . In this context , it is worth mentioning that all such observations were based on studies in the mammalian systems and that there is scarcely any information on the role of Ca2+ or its dependent kinases in host-pathogen interactions in fish . Calmodulin is one of the most abundant and well characterised Ca2+ sensor proteins [5] . It regulates numerous Ca2+-mediated cellular functions such as cell growth , differentiation , proliferation and apoptosis in divergent models including macrophages [6] . The increased cytosolic Ca2+ binds to CaM and the resulting Ca2+-CaM interaction leads to activation of several protein kinases including CaM-dependent kinases ( CaMKs ) [7] . The CaMK cascade includes CaMKII and CaMKK . CaMKII is one of the best characterised of CaMK family of kinases . It has been suggested that CaMKII , a serine/threonine kinase , is activated by Ca2+-CaM binding , followed by rapid autophosphorylation on Thr286 , which abolishes its auto-inhibition [8] . As a consequence , a transient elevation in Ca2+ leads to a prolonged activation of CaMKII . Among the CaMKII isoforms , the gamma-isoform of CaMKII ( CaMKIIg ) has been reported in macrophage functioning [6] , [9] and implicated both as pro- [6] and anti-apoptotic [10] in different studies . The cyclic nucleotide , cAMP , is generated from ATP by adenylyl cyclases ( ACs ) , whereas the phosphodiesterases ( PDEs ) catalyse its hydrolytic degradation . In eukaryotic cells cAMP requires the protein kinase A complex ( PKA ) as an intermediate to carry out its effects . Studies indicate that cAMP/PKA-pathway regulates a broad range of cellular responses that includes its central role as both a pro- and anti-apoptotic regulator [11] . There are varied pathways by which cAMP/PKA mediates its action and activation of MAPKs is one of them [12] . The MAPK family is composed of the extracellular signal-regulated kinase 1 and 2 ( ERK 1/2 ) , p38 and stress activated protein kinase/c-Jun N-terminal kinase ( SAPK/JNK ) pathways [13] . This family of kinases is important in a wide spectrum of cellular functions like proliferation , cytokine biosynthesis , cytoskeletal organization [14] . Amongst the MAPK family , activation of ERK 1/2 in pathogen-induced macrophage apoptosis is well characterized [15] . Mononuclear phagocytes such as macrophages are essential to the innate immune response against invading microorganisms . It has been suggested that one mechanism by which A . hydrophila induces pathogenicity in fish is through initiation of host macrophage apoptosis . In fish , especially teleosts , head kidney ( HK ) represents the anterior part of kidney . It is analogous to the mammalian bone marrow and the primary site of definitive hematopoiesis . The HK appears in the pre-hatching embryos on either side of the pharynx as renal units , each with a single glomerulus and short renal tubule . However , during the course of development the renal elements get degraded and it becomes primarily involved in hematopoiesis , antibody production and regulating immune-endocrine axis [16] , [17] . The HKM serves as the first line of defence against invading pathogens . We have shown that HKM are crucial in the pathogenesis of A . hydrophila at the cellular level [18] . Recently , we demonstrated that live A . hydrophila infection leads to apoptotic death of HKM , involving calpain activation [19] . Here , we have identified and characterized other signalling effectors and executors that are involved in A . hydrophila-HKM interactions . Clarias gariepinus was selected as our model because of its availability round the year , ability to adapt to laboratory conditions and for having easily identifiable immune organs .
We earlier reported A . hydrophila-induced intracellular Ca2+ influx and subsequent calpain-2 activation in the infected HKM [19] . This study was designed to identify the role of other key molecules of Ca2+-pathway on A . hydrophila-induced HKM apoptosis and CaM was a rational candidate . At the outset , we checked for CaM expression at mRNA level following infection with A . hydrophila . In the absence of genome sequence of C . gariepinus degenerate primers for CaM were designed using the homologous stretch across vertebrates as the template . The PCR product was cloned and sequenced and the sequence showed 100% identity with CaM-mRNA sequence of channel catfish , Ictalurus sp . Primers for real time analysis were designed from this sequence and the real-time data revealed maximum induction of CaM at 2 h post-infection ( p . i . ) at the mRNA level ( P<0 . 05 ) ( Figure S1A ) . The next step was to check A . hydrophila-induced CaM protein expression using CaM assay kit . We observed maximum CaM protein expression at 2 h p . i . ( P<0 . 05 ) ( Figure S1B ) and hence selected this time point for subsequent studies on CaM . At further time points CaM mRNA and protein levels decreased significantly reaching basal levels at 24 h p . i . ( data not shown ) . CaM activity was assayed in presence of the CaM antagonist-CMZ . This CaM antagonist binds reversibly to CaM thus inhibiting CaM-mediated enzyme activation [20] . Pre-treatment with CMZ inhibited CaM activation ( P<0 . 05 ) in infected HKM ( Figure 1A ) which clearly suggested the role of CaM on A . hydrophila-induced HKM-pathology . Further , pre-treatment with Ca2+-chelators BAPTA/AM , EGTA and the Ca2+-channel blockers Nf and Vp led to significant reduction in CaM activity at the protein levels indicating CaM expression to be Ca2+-dependent in the infected HKM ( Figure 1A ) . Our next step was to correlate CaM with HKM apoptosis . The HKMs were pre-treated with CMZ and apoptosis assessed 24 h p . i . by Hoechst , AV-PI staining and caspase-3 activation using specific assay kit . Pre-treatment with CMZ inhibited ( P<0 . 05 ) apoptotic death and attenuated caspase-3 activity ( P<0 . 05 ) ( Figure 1B and 1C ) in infected HKM implicating the role of CaM on initiating A . hydrophila-induced HKM apoptosis . These results were also confirmed using CaM-siRNA . Transfection with CaM-siRNA down-regulated CaM expression at mRNA level ( Figure 1D ) , protein level ( Figure 1A ) at 2 h p . i . and also attenuated A . hydrophila-induced HKM apoptosis at 24 h p . i . ( Figure 1B and 1C ) . Our results , for the first time , indicated the pro-apoptotic role of CaM in A . hydrophila-induced HKM apoptosis . Among several downstream events that are initiated due to altered CaM activity the activation of CaMKs are believed to be important . We studied the role of CaMKK and CaMKII , two important CaMKs in our model . The HKM were pre-treated with specific pharmacological inhibitors of CaMKK and CaMKII and apoptosis was evaluated 24 h p . i . by Hoechst and AV-PI staining . First , we used the CaMKK inhibitor STO-609 which specifically inhibits the CaMKK activity by suppressing Ca2+-dependent signalling [21] . STO-609 had no preventive effect on A . hydrophila-induced HKM apoptosis ( Figure 2A and 2B ) . Subsequently , we used the CaMKII specific inhibitor KN-93 and its structural analogue KN-92 to investigate the role of CaMKII on the process . KN-93 competitively blocks CaM binding to the kinase whereas KN-92 is a congener of KN-93 without CaM kinase inhibitory activity and is used as an experimental control [22] . We observed that pre-treatment with KN-93 attenuated caspase-3 activity and conferred significant protection to the infected HKM from A . hydrophila-induced apoptosis ( Figure 2A and 2B ) . The inactive analogue KN-92 failed to inhibit HKM apoptosis suggesting a mediatory role of CaMKII on initiating apoptosis of infected HKM . To validate these observations qRT-PCR and EIA were done to evaluate the status of the specific transcript and protein . Degenerate primers for CaMKIIg were designed using the homologous stretch across vertebrates as the template . The PCR product was cloned , sequenced and the sequence showed 100% identity with CaMKIIg of zebrafish , Danio rerio . Primers for real time analysis were designed from this sequence for quantifying CaMKIIg levels . We observed that A . hydrophila infection led to significant increase in CaMKIIg mRNA expression with maximum levels being at 2 h p . i . ( Figure S2A ) , which decreased thereafter reaching basal levels at 24 h p . i . ( data not shown ) . We checked CaMKIIg concentrations using specific EIA kit 24 h p . i . as CaMKII is detected for prolonged period even after the Ca2+ signal has decayed [8] . A significant increase in CaMKIIg levels ( P<0 . 05 ) was noted in the infected HKM which was inhibited ( P<0 . 05 ) in presence of CMZ and KN-93 , antagonists to CaM and CaMKIIg respectively ( Figure 2D ) but not by KN-92 in A . hydrophila-infected HKM . Using siRNAs specific to CaM and CaMKIIg that significantly down-regulated CaMKIIg at mRNA and protein levels ( Figure 2C and 2D ) , significant attenuation of HKM apoptosis was also observed ( Figure 2A and 2B ) . Taken together our data implicates Ca2+-induced CaM-CaMKIIg expression to be a critical event in A . hydrophila-induced HKM apoptosis . We studied cAMP/PKA signalling in A . hydrophila-induced HKM apoptosis as several macrophage activities appear to be controlled by this pathway [23] . Lysates from infected HKM were collected at different time intervals and assayed for intracellular cAMP production . It was observed that A . hydrophila-infection led to significant increase in intracellular cAMP levels at all time points studied with maximum production recorded at 24 h p . i . ( Figure 3A ) . The cell-permeable cAMP analogue ( 8-Br-cAMP ) when used as positive control elevated intracellular cAMP level and induced HKM apoptosis ( data not shown ) . Our results thus implicate the role of cAMP in the pathophysiology associated with A . hydrophila infection . We next studied the role of CaMKIIg on initiating the production of cAMP in infected HKM since Ca2+-dependent signalling molecules often converge on cAMP [24] . As the cAMP levels were maximally induced at 24 h p . i . , this time point was chosen for subsequent studies . It is evident from Figure 3B that A . hydrophila-induced cAMP levels were significantly reduced following pre-treatment with CaMKII specific inhibitor-KN-93 . This suggests CaM-activated CaMKIIg plays an important role in cAMP generation in the infected HKM . Protein kinase A is an important mediator of the cAMP dependent signalling pathway . The cyclic nucleotide cAMP causes PKA activation by binding to the regulatory subunit , triggering release , activation and nuclear translocation of PKACA [25] , [26] . Therefore , our next step was establishing the involvement of PKACA in the pathogenicity of A . hydrophila . Degenerate primers specific for PKACA were designed the PCR product cloned , sequenced and nBLAST analysis suggested 100% identity with PKACA of Ictalurus sp . We used real time primers based on the sequence obtained and observed that A . hydrophila-infection induced a several fold increase in PKACA transcripts with maximum expression recorded by 2–6 h p . i . ( P<0 . 05 ) ( Figure S2B ) and thereafter it started declining ( data not shown ) though significant level of PKACA mRNA expression was noted till 24 h p . i . This decline in PKACA transcripts at later time points probably serves as controlling mechanism to prevent the overshoot of cAMP dependent downstream events in the infected cells . CaMKIIg and PKACA targeted siRNAs also led to significant inhibition of PKACA at the transcript level ( Figure 4A ) . We then studied PKACA expression and nuclear translocation by immunofluorescence 24 h p . i . in infected HKM . The results with anti-PKACA antibody clearly suggested increased PKACA expression in nuclei of A . hydrophila-infected HKM ( Figure 3C ) . We extended the study by including PKA specific inhibitor H-89 , CaMKII specific inhibitor-KN-93 or transfected the HKM with CaMKIIg-siRNA and PKACA-siRNA . The inhibitor H-89 blocks PKA actions through competitive inhibition of the adenosine triphosphate ( ATP ) site on the PKA catalytic subunit [27] . We observed decreased expression and restricted nuclear movement of PKACA in the infected HKM at 24 h p . i . in presence of H-89 , KN-93 , CaMKIIg-siRNA and PKACA-siRNA ( Figure 3C ) . Our results for the first time established the role of CaMKIIg on PKACA activation in A . hydrophila-infected HKM . The presence of H-89 and PKACA-siRNA also led to significant alleviation ( P<0 . 05 ) of apoptotic death and diminished caspase-3 activity in the infected HKM ( Figure 4B and 4C ) suggesting the critical role of PKA pathway on the pathogenicity induced by the bacteria . We next studied the link between the activation of PKA and intra-cellular survival of A . hydrophila . Pre-incubation of HKM with H-89 or PKACA-siRNA reduced ( P<0 . 05 ) recovery of intracellular bacteria ( Figure 4D ) . Besides , we found that H-89 addition till 60 mins p . i . reduced the bacterial load and beyond that exhibited little inhibitory effect ( Figure S3 ) . No direct effect of H-89 on A . hydrophila was observed as adding the inhibitor at the particular concentration did not affect the viability , growth and initial uptake by HKM ( data not shown ) . The ERK 1/2 pathway has been implicated both as pro-and anti-apoptotic in host macrophage responses . We aimed to investigate the involvement of ERK 1/2 activation in A . hydrophila-induced HKM apoptosis . The HKM were infected with A . hydrophila in presence or absence of specific inhibitors , lysed at indicated time intervals and the changes in the levels of total and phosphorylated ERK 1/2 measured using specific EIA kits . It is evident from Figure 5A that there was significant increase in pERK 1/2 levels in the A . hydrophila-infected HKM though no noticeable change was observed in total-ERK 1/2 levels . The role of cAMP/PKA on ERK 1/2 activation has been reported [28] . To investigate this possibility the HKM were pre-treated with PKA specific inhibitor H-89 and changes in total and pERK 1/2 levels studied . We observed that H-89 significantly inhibited pERK 1/2 levels in the infected HKM implicating the role of cAMP/PKA pathway in initiating the kinase activity . U0126 is a non-ATP competitive inhibitor of mitogen-activated protein kinase kinase ( MEK ) which exerts its effect by inhibiting the ability of MEK to phosphorylate downstream ERK 1/2 [29] . Hence it served as an effective negative control in the study . To further substantiate the role of cAMP/PKA pathway on ERK 1/2 activation we used PKACA-siRNA and our results ( Figure 5A ) confirmed ERK 1/2 activation to be downstream to cAMP/PKA in A . hydrophila-infected HKM . We next investigated the contribution of ERK 1/2 activation on HKM apoptosis . For that HKM were pre-treated with or without U 0126 prior to infection with A . hydrophila and checked for apoptotic death using Hoechst 33342 , AV-PI staining and caspase-3 activity 24 h p . i . It is clear from Figure 5B and 5C that inhibition of ERK 1/2 significantly inhibited ( P<0 . 05 ) A . hydrophila-induced HKM apoptosis and lowered caspase-3 activity . Taken together our observations suggest ERK 1/2 activation is downstream event of cAMP/PKA pathway and is pro-apoptotic in A . hydrophila-infected HKM .
Calmodulin plays a significant role in microbial pathogenicity . Its role has been implicated in the pathogenicity of Mycobacterium sp . [28] , Clostridium perfingens [30] and for survival of Pneumocystis-infected alveolar macrophages [31] . The presence of well conserved CaM is well documented in fish [32] , [33] . As we observed increased intracellular Ca2+-levels in infected HKM [19] and CaM being a well-known Ca2+-sensor , we hypothesised a role of CaM on the pathogenicity of A . hydrophila . Indeed , results obtained from qRT-PCR , specific EIA assays , siRNA and pharmacological inhibitors conclusively demonstrated the importance of Ca2+-induced CaM expression in the initiation of A . hydrophila-induced HKM apoptosis . The kinetics of CaM expression , both at transcript and protein level demonstrated maximum expression at 2 h p . i . This suggested CaM expression at protein level closely follows pattern at transcript level , a phenomenon observed for many signalling molecules [34] . To our knowledge , this is the first report that clearly documents the pro-apoptotic involvement of CaM in A . hydrophila infections . We had earlier reported the role of calpain-2 in A . hydrophila-induced HKM apoptosis . We questioned whether the two Ca2+-dependent molecules crosstalk to initiate HKM apoptosis . Contrary to our expectations we did not observe any crosstalk between calpain-2 and CaM activity ( data not shown ) , which suggests two possibilities: ( a ) CaM is resistant to the lytic action of calpains [35] and ( b ) CaM and calpain activity are independent to each other in our model . Although there is little information regarding the mechanisms of CaM involvement in apoptosis , there are reports suggesting pro-apoptotic roles of CaM-dependent enzymes including CaMKs [8] . We reasoned that downstream activation of CaMKs is important for the initiation of apoptosis and inhibiting the kinase activities would confer protection to the infected HKM . To investigate this , we investigated the roles of CaMKK and CaMKII in initiating A . hydrophila-induced HKM apoptosis . We selected these two kinases as they are conserved , well characterised , have wide tissue distribution including macrophages and work through distinct pathways regulating diverse biological functions [8] . Our inhibitor studies indicated CaMKII involvement and clearly ruled out the involvement of CaMKK in the process . This observation also ruled out the involvement of CaMKI and CaMKIV in the process as both kinases require upstream phosphorylation of CaMKK for getting activated [7] . CaMKII has been reported to be both pro-apoptotic [6] and anti-apoptotic [10] . To investigate the role of CaMKII in A . hydrophila-induced HKM apoptosis we selected the gamma-isoform , CaMKIIg as it is largely reported in macrophage functioning [6] , [9] . Use of siRNAs confirmed the involvement of CaMKIIg and its modulation by CaM in HKM due to A . hydrophila infection . It is worth mentioning that there is little information on the involvement of CaMKIIg in pathogen-induced apoptosis except for its role in the pathogenicity induced by Mycobacterium sp . [28] . These results clearly indicated CaMKIIg to be important in Ca2+-mediated apoptotic signalling downstream of CaM in A . hydrophila-infected HKM . CaMKs are considered to be the likely substrates for calpain proteolysis [35] . Calpain-mediated CaMKIV activation is well documented in the apoptosis of SH-SY5Y cells [36] and cerebellar granule cells [37] . In contrast , there is no report on the role of calpains on CaMKIIg expression especially in fish macrophages . In this connection , it is worth mentioning that pre-treatment with calpain-2 inhibitor led to significant decline in CaMKIIg expression in infected HKM . Our finding for the first time suggested the existence of an alternate or calpain-mediated pathway for CaMKIIg expression in fish macrophages following A . hydrophila-infection . Further studies are needed to understand the crosstalk between CaMKIIg and activated calpains . The role of cAMP varies between the prokaryotic and eukaryotic system . In eukaryotes cAMP signalling is important in diverse biological processes such as metabolism , memory formation and apoptosis [38] . To begin with , we initially looked for the changes of cAMP and observed significant increase in cAMP levels following A . hydrophila infection . Earlier studies with Brucella suis [39] , Bordetella pertussis [40] , Bacillus anthracis [41] , Mycobacterium sp [28] , Pseudomonas aeruginosa [42] and Porphyromonas gingivalis [43] also reported increased cAMP production via different mechanisms implicating cAMP in the pathogenicity of these microbes . It is interesting to note that the levels of A . hydrophila-induced cAMP observed by us is comparable to those recorded in many of these microbes which make us believe that such an A . hydrophila-induced effect could also be important for the pathogenicity of this bacteria . Besides , we also observed a time-dependent increase in cAMP production following A . hydrophila infection which corroborates with the earlier reports of Chopra et al . , ( 2000 ) [44] in which a gradual increase in cAMP production was seen in the murine macrophage cell line RAW 264 . 7 , treated with the A . hydrophila-toxin Act . There are also reports suggesting cAMP to be both pro- and anti-apoptotic [11] . A time-dependent increase of cAMP levels in infected HKM suggested that downstream cAMP-dependent pathways ultimately determine the outcome of infection in infected cell . This also led us to the identification that elevated cAMP levels are indeed due to CaM-dependent CaMKIIg activity in the A . hydrophila-infected HKM . Determining how the CaM-CaMKIIg axis is connected with cAMP production would be an interesting direction of the future . Previous studies have suggested that some isoforms of AC can be activated by Ca2+-CaM-CaMK by phosphorylation of the serine residues on AC [45] . It remains to be seen if this is also true in our system . On establishing the role of cAMP we looked for its downstream targets in the infected HKM . Protein kinase A being an important intermediary in cAMP-dependent signalling , was an attractive candidate . Our observations using qRT-PCR , siRNA , cAMP/PKA-specific inhibitor , H-89 and PKACA antibody suggested that activation of cAMP/PKA is a downstream consequence of CaMKIIg activation . Pre-treatment with H-89 did not influence CaMKIIg activation in the infected HKM ( data not shown ) . The cAMP/PKA pathway attributed to the virulence of A . hydrophila as inhibition of PKA activity led to increased HKM viability with concomitant decline in the number of intracellular bacteria . If PKA activation is essential for survival of A . hydrophila inside the HKM at what stage of infection is it important ? We suggest PKA activation is critical at initial stages of infection as inhibiting PKA activity at late stages of infection had little effect on the replication of intra-cellular A . hydrophila . Our observations are similar to those reported by Gross et al . , ( 2003 ) [39] for Brucella suis and we believe that A . hydrophila alike B . suis initiates an early cAMP/PKA-mediated virulence trait that helps in eluding macrophage-microbicidal responses critical at the onset of infection . Although , it is not possible from this study to conclude on how A . hydrophila affects cAMP/PKA pathway; we speculate that the bacteria could be releasing toxins which directly phosphorylate ACs or inhibit the activation of PDEs leading to increased accumulation of the cyclic nucleotide inside the HKM . The participation of MAPKs has been widely observed during pathogenic invasion [46] . The genes encoding the MAPK family proteins are well conserved and have been reported in fish to be regulating various downstream targets such as transcription factors and heat shock proteins [47] . The involvement of MAPK cascade is beginning to be understood in the pathogenicity of A . hydrophila in fish [48] . Amongst the MAPK family members , ERK 1/2 has been reported to act both as pro- and anti-apoptotic factor under different conditions of pathogenic-stress [46] . We sought to understand its role in A . hydrophila-infection and our results conclusively demonstrate pro-apoptotic role of ERK 1/2 in A . hydrophila-infected HKM . ERK 1/2 activation is also reported in A . hydrophila pathogenicity though in mammalian system [1] . Interestingly , we also noted pro-apoptotic role of JNK and p38 , the other members of the MAPK family in initiating A . hydrophila-induced HKM apoptosis ( unpublished observations ) . This suggests that A . hydrophila uses a common signalling mechanism to activate the MAPK cascade to induce apoptosis in different hosts . Thus , our observations contradict those reported in Mycobacterium [27] , Pseudomonas [49] or Yersinia [50] where the differential activation of MAPK family was reported to be important for pathogenicity . Caspases are members of a family of cysteine proteases that are involved in apoptosis induced by various stimuli in different cell types , including macrophages . We recently reported A . hydrophila-induced HKM apoptosis to be caspase-3 mediated [19] . Presently , we aimed to determine what other signals initiated out of Ca2+ wave have a role in the activation of caspase-3 following A . hydrophila infections . From the available literature it is evident that a relationship between MAPKs and caspase-3 exist with reports suggesting MAPK activating caspase-3 [51] and vice versa [52] . Interestingly , our results clearly showed caspase-3 to be downstream of MAPK ( ERK 1/2 ) in A . hydrophila-infected HKM . The means by which MAPKs regulate caspase-3 activity remain unclear and further investigation in this direction will aid in understanding the pathogenicity of this bacteria . To conclude , our findings emphasise the unrecognized role of CaM in initiating A . hydrophila-induced HKM apoptosis . We propose that the Ca2+-influx initiated by A . hydrophila activates pro-apoptotic CaM and downstream CaMKIIg in infected HKM . Calpains also appear to have some role on CaMKIIg expression . CaMKIIg in turn initiates a network of signals resulting in the downstream activation of cAMP/PKA and MAPK pathways respectively and the cascade of events culminate in caspase-3 activation and apoptosis of infected HKM ( Figure 6 ) .
All animal experiments described in the present study were approved by the Animal Ethics Committee of University of Delhi ( DU/ZOOL/IAEC-R/2013/33 ) and carried out in accordance with animal experimentation protocols approved by The Prevention of Cruelty to Animals Act , Govt . of India . Catfish ( Clarias gariepinus , Siluriformes , 100±20 g ) were maintained in 50-L glass tanks ( 2–3 fish per tank ) under natural conditions . The water quality , dissolved oxygen content and pH were monitored regularly in each tank . The fish were fed boiled chicken liver ad libitum and acclimatized to laboratory conditions for 15 days before use for experimental purpose . During this period and when the experiments were conducted , fish health was routinely monitored by appearance and pathological examinations as mentioned earlier [53] . The A . hydrophila ( Strain 500297 ) was a gift from Dr . T . Ramamurthy , National Institute of Cholera and Enteric Diseases ( NICED ) , India . The bacteria were grown to late log phase ( 12 h ) in brain heart infusion broth ( BHI , HiMedia ) containing 100 μg mL-1 ampicillin at pH 7 . 4 overnight at 30°C with aeration and maintained in nutrient agar slants at 4°C . The pathogenicity of the strain was confirmed in vitro by hemolysin and cytotoxicity assays and by the ability to induce characteristic UDS lesions when injected into the fish [18] . Head kidneys were aseptically removed and placed in RPMI-1640 ( Gibco-Invitrogen ) with phenol-red indicator and supplemented with 25 mM HEPES ( Gibco-Invitrogen ) containing 1% penicillin-streptomycin ( complete-RPMI ) . Single cell suspensions of each pair of head kidney were prepared using 100 μm wire mesh , diluted and the total cell population pelleted by centrifugation at 400×g for 10 mins at 4°C . The supernatant was discarded and the re-suspended pellet layered on discontinuous percoll density gradient ( 34/51 ) and centrifuged at 400×g for 20 mins at 4°C . The phagocyte rich fraction appearing above the 34/51 interface was collected , washed and enriched for macrophages as described earlier [19] . The purity of the HKM was checked by staining with Wright Giemsa Stain and viability determined using 0 . 4% trypan blue dye exclusion method . The HKM were seeded in 24 well plates . The A . hydrophila were grown to mid log phase , harvested and infected into HKM at a multiplicity of infection ( MOI ) of 50 for 60 mins . To kill extracellular bacteria , the infected HKM cultures were incubated for a further 1 h in RPMI containing 30 μg mL−1 chloramphenicol . Exposure to 30 μg mL−1 chloramphenicol for 1 h was sufficient to kill 100% of microorganisms but had no effect on macrophage viability . Uninfected HKM served as control and maintained in complete-RPMI for the same time . The siRNA transfection was done using HiPerFect Transfection Reagent ( Qiagen ) as per manufacturer's instructions . The siRNA-Hiperfect complex ( 5 μl each ) was mixed gently in Opti-MEM ( Invitrogen ) and incubated for 20 mins to allow complex formation . This complex was added to the isolated HKM maintained in Opti-MEM , mixed properly and incubated at 30°C and 5% CO2 . Cell viability was continuously monitored and after 5 h of incubation , media was changed to complete-RPMI . At 24 h post-transfection , HKM were washed , placed in fresh complete-RPMI and infected with A . hydrophila as mentioned earlier and proceeded for apoptotic studies , qRT-PCR and protein assays to confirm knockdown . Targeted [ ( CaM , SENSE-5′-CCAUUACGACCAAAGAGUU-3′ and ANTISENSE-5′-AACUCUUUGGUCGUAAUGG-3′ ) ; ( CaMKIIg , SENSE-5′-GGACAUUUGGGCUUGUGGA-3′ and ANTISENSE-5′-UCCACAAGCCCAAAUGUCC-3′ ) ; ( PKACA , SENSE-5′-CAGUAAAGGCUACAAUAAA-3′ and ANTISENSE-5′-UUUAUUGUAGCCUUUACUG-3′ ) ] plus siCONTROL non-targeting siRNA pool ( HS Number 29349990700 ) 5 nM each were used . For inhibitor studies , HKM were pre-treated with or without 10 μM caspase-3 inhibitor ( Acetyl-Asp-Glu-Val-Asp-aldehyde , Ac-DEVD-CHO , Promega ) , 50 μM calpain-2 inhibitor ( N-acetyl-leucyl-leucyl-methioninal , calpain-2i ) , 4 nM CaM inhibitor ( calmidazolium chloride , CMZ ) , 20 μM CaMKII inhibitor ( KN-93 , Calbiochem ) , 20 μM inactive analogue of KN-93 ( KN-92 ) , 20 μM cAMP dependent PKA inhibitor ( H-89 ) for 1 h or 5 mM intracellular calcium chelator {[1 , 2-bis- ( o-aminophenoxy ) ethane-N , N , N′ , N′-tetraacetic acid tetra ( acetoxymethyl ) ester] , BAPTA/AM ) } , 10 mM extracellular calcium chelator [Ethyleneglycol-bis ( β-aminoethyl-N , N , N′ , N′-tetraacetic acid ) , EGTA] , 10 μM calcium channel blocker nifedipine ( Nf ) , 5 μM calcium channel blocker verapamil ( Vp ) , 100 μM CaMKK inhibitor ( STO-609 ) , 20 μM ERK 1/2 inhibitor ( U 0126 , Calbiochem ) for 2 h followed by A . hydrophila infection as mentioned above and checked for apoptotic studies and protein assays . The doses of different inhibitors were selected on the basis of inhibitor specificity and cytotoxicity . The HKM treated with the indicated concentrations of the inhibitors remained as viable as control HKM at all time points as determined by the trypan blue ( 0 . 4% ) dye exclusion method and were maintained during the entire course of the experiment . For apoptosis studies the HKM ( 1×106 ) were pre-treated separately with or without indicated concentrations of targeted or scrambled siRNAs , indicated concentrations of inhibitors for different time periods then infected with A . hydrophila at an MOI of 50 as described above . The HKM were subjected to Hoechst 33342 and Annexin-V-fluorescein isothiocyanate-Propidium Iodide ( AV-FITC-PI , BD Pharmingen ) staining 24 h p . i . as mentioned earlier [19] . For the Hoechst 33342 study , HKM were collected , washed and fixed with 3 . 7% paraformaldehyde solution at room temperature . After fixation , the HKM were stained with Hoechst 33342 ( 2 μg mL−1 in 1×PBS ) and visualized under fluorescence microscope ( ×40 , Nikon Eclipse 400 ) within 30 mins of adding the stain . A total of 100 cells were studied in each field and three such fields were included to determine the percentage of apoptotic HKM . For the AV-FITC-PI study the HKM were collected , washed , fixed with 3 . 7% paraformaldehyde solution and stained with AV-FITC-PI following manufacturer's instructions . The HKM were observed under the fluorescence microscope ( ×40 , Nikon Eclipse 400 ) within 30 mins of adding the dye . A total of 100 cells were studied in each field and three such fields were included to determine the percentage of apoptotic HKM . The HKM ( 2×107 ) transfected separately with or without indicated concentrations of targeted or scrambled siRNA were infected with A . hydrophila and at indicated time point p . i . , culture was terminated , total RNA was isolated using TRIZOL according to manufacturer's instructions . Total RNA dissolved in diethylpyrocarbonate ( DEPC ) water was treated with deoxyribonuclease I ( RNase-free , MBI Fermentas ) at 37°C for 30 mins and the DNase was inactivated by incubation with 50 mM EDTA at 65°C for 10 mins . One microgram of total RNA from each sample was reverse transcribed using first strand cDNA synthesis kit as per manufacturer's instructions ( MBI Fermentas ) . The resulting cDNA was then subjected to PCR amplifications using degenerate primers for each gene ( Table 1 ) . The amplified product was then gel extracted using QIA quick Gel Extraction Kit ( Qiagen ) and cloned into pGEM-T EASY vector ( Promega ) and sequenced ( Macrogen ) . The sequences obtained ( Table 2 ) were aligned to nBLAST and have been submitted to NCBI database . Real-Time PCR for quantitation was done for CaM , CaMKIIg and PKACA using SYBR green PCR Master Mix ( Applied Biosystems ) according to the manufacturer's instructions ( ABI ViiA , Applied Biosystems ) using gene specific primers ( CaM forward: 5′-AAGATGGAGATGGCACCATTA-3′ and reverse: 5′-TGGTCAGGAACTCTGGGAAG-3′; CaMKIIg forward: 5′- TTGTTGACATCTGGGCATGT-3′ and reverse: 5′- CATAAGCTCCGCTTTGATCT-3′; PKACA forward: 5′-AGGTTACGGATTTCGGCTTT-3′ and reverse: 5′-GATCTGAATGGGCTGGTCTG-3′; β-actin forward: 5′- CGAGCAGGAGATGGGAACC-3′ and reverse: 5′-CAACGGAAACGCTCATTGC-3′ ) . The PCR mixture ( total volume 10 μl ) contained 5 μl of Power SYBR Green , 1 μl of the first-strand cDNA ( diluted to 1/100 of the original concentration ) and 0 . 20 μM of forward and reverse primers . Amplification and detection of all genes was performed with ABI ViiA using the following thermal cycling conditions: one cycle of 95°C for 10 mins , 40 cycles of 95°C for 15 s , 60°C for 1 mins . Reactions were performed with cDNAs from six independent experiments and the expression of each transcript was quantified by the comparative ΔΔCT method and normalized to those of β-actin chosen as endogenous control . The cell lysates of HKM ( 1×106 ) were pre-treated separately with or without indicated concentrations of CaM-siRNA , scrambled siRNA and inhibitors including BAPTA/AM , EGTA , Vp , Nf for different time periods then infected with A . hydrophila at an MOI of 50 as described earlier . The HKM were collected at indicated time point p . i . and checked for CaM concentration by Enzyme Immunoassay Kit ( EIA , USCN Life Sciences ) as per manufacturer's instruction with brief modifications . Briefly , the lysates along with the standards provided were diluted with diluents , placed at the bottom of each well in triplicate and incubated overnight at 4°C . The liquid of each well was removed carefully and 100 μL of Detection Reagent A was added to each well and incubated for 1 h at 30°C . The solutions were aspirated carefully and each well washed with 200 μL of wash buffer . The Detection Reagent B ( 100 μL ) was added to the wells for 30 mins at 30°C and washed with 200 μL of wash buffer . Ninety microliters of substrate solution was added to each well and the reaction terminated with the addition of 50 μL of stop solution after 25 mins of incubation and the readings taken at 450 nm . The amount of CaM was interpolated from the standard curves obtained by plotting the O . D . of the standards . All chemicals used in the assay were provided with the kit . The HKM ( 1×106 ) were pre-treated separately with or without indicated concentrations of CaM-siRNA , CaMKIIg-siRNA , scrambled siRNA and inhibitors including CMZ , KN-93 and KN-92 for different time periods then infected with A . hydrophila at an MOI of 50 as described earlier . The HKM were collected at 24 h p . i . and quantitative estimation of CaMKIIg was done in cell lysates using EIA Kit for CaMKIIg ( USCN ) as per manufacturer's instruction . Briefly , HKM were collected by centrifugation , re-suspended in chilled lysis buffer provided with the kit and incubated on ice for 30 mins . Following incubation the cell lysates were collected by centrifugation at 16 , 000×g for 20 mins at 4°C . Hundred microlitres each of standards and cell lysates were added into the wells and incubated for 5 h at 30°C . Following incubation the liquid from each well was removed and 100 μl of Detection Reagent A added to each well and further incubated for 1 h at 30°C . The Detection Reagent A was removed and the wells washed several times with 200 μl of 1× wash solution followed by addition of 100 μl of Detection Reagent B to each well and incubated for 30 mins at 30°C . The wells were washed , 90 μl of substrate added to each well and incubated for 30 mins at room temperature . The reaction was stopped by adding 50 μl stop solution , the readings taken at 450 nm in ELISA plate reader ( BMG labtech ) and the amount of CaMKIIg interpolated from the standard curves obtained by plotting the O . D . of the standards . All chemicals used in the assay were provided with the kit . The cell lysates of HKM ( 1×106 ) were pre-treated separately with or without indicated concentrations of inhibitors including KN-93 and KN-92 for different time periods then infected with A . hydrophila at an MOI of 50 as described and checked for intracellular cAMP level by ELISA kit ( Enzo Life Sciences ) as per manufacturer's instruction . Briefly , HKM were collected by centrifugation and resuspended in 100 μl of 0 . 1 N HCl for 10 mins at room temperature followed by centrifugation at 800×g to pellet cellular debris . Hundred microlitres each of standards and supernatant was added to the bottom of appropriate wells , 50 μL each of conjugate and antibody then added and incubated on a plate shaker at room temperature for 4 h . The plate was washed , 200 μL of substrate added to each well and incubated for 1 h at room temperature . The reaction was finally stopped by adding 50 μl of stop solution and reading taken at 405 nm ( BMG Labtech ) . All chemicals used in the assay were provided with the kit . The HKM ( 4×106 ) were pre-treated separately with or without indicated concentrations of CaMKIIg-siRNA , PKACA-siRNA , scrambled siRNA and inhibitors including KN-93 and H-89 for different time periods then infected with A . hydrophila at an MOI of 50 as described earlier . The HKM were collected at 24 h p . i . , fixed in methanol/acetic acid ( 1∶1 , v/v ) for 30 mins on ice and subsequently incubated with blocking and permeabilizing solution ( PBS , 2 mg/ml BSA , 0 . 2 mg/ml saponin ) . The cells were washed and incubated with primary antibody for PKACA ( 1: 200 , Sc-903 , anti-PKACA raised in rabbit , Santacruz ) overnight at 4°C . The HKM were washed , incubated with secondary antibody ( 1: 250 , FITC-conjugated , anti-rabbit , Cell Signalling Technology ) for 1 h at room temperature and mounted on microslide with cover slips using fluoroshield . Nuclear staining was done with DAPI ( 1 μg/ml ) . The expression and nuclear translocation of PKACA was studied under fluorescence microscope ( ×100 oil immersion , Zeiss ) . Total ERK was measured using ERK 1/2 EIA ( Enzo Life Sciences ) and pERK was measured with pThr202/Tyr204 ERK 1/2 EIA kit ( Enzo Life Sciences ) [54] using chemicals supplied with the kit . Briefly , HKM ( 1×106 ) were pre-treated separately with or without indicated concentrations of PKACA-siRNA , scrambled siRNA and inhibitors including H-89 and U 0126 for different time periods then infected with A . hydrophila at an MOI of 50 as described earlier . Total ERK 1/2 and pERK 1/2 activities were measured 24 h p . i . The HKM were collected by centrifugation and re-suspended in chilled lysis buffer and incubated on ice for 30 mins . The cell lysate was collected by centrifugation at 16 , 000×g for 20 mins at 4°C . The assay was performed in a total volume of 100 μl in microtiter plate coated with mouse monoclonal antibody specific to ERK 1/2 provided with the kit . Hundred microliters of the samples were pipetted to the wells and incubated for 2 h at room temperature with shaking . The contents of the wells were washed and 100 μl of polyclonal ERK 1/2 and pERK 1/2 antibody were added separately into respective well and incubated overnight at 4°C . The plates were washed , incubated for 45 mins with HRP-conjugated secondary antibody at room temperature following which 100 μl of TMB substrate containing hydrogen peroxide added to each well and incubated for 30 mins . Finally , 100 μl of stop solution was added and colour development studied at 450 nm . Triplicate sets were prepared containing serially diluted standards , blank ( no cell extract ) , negative control ( extract from untreated cells ) and A . hydrophila-infected HKM . The amounts of ERK 1/2 and pERK 1/2 in the cell lysates were interpolated from the standard curves obtained by plotting the O . D . of the standards . All chemicals used in the assay were provided with the kit . The caspase-3 ( DEVDase ) assay was performed using caspase-3 assay kit ( Promega ) [19] . Briefly , HKM ( 1×106 ) were pre-treated separately with or without indicated concentrations of CaM-siRNA , CaMKIIg-siRNA , PKACA-siRNA , scrambled siRNA and inhibitors including CMZ , KN-93 , KN-92 , STO-609 , H-89 and U 0126 for different time periods then infected with A . hydrophila at an MOI of 50 as described earlier . The HKM were collected 24 h p . i . and re-suspended in 50 μL of chilled cell-lysis buffer followed by incubation on ice for 10 mins . The cell lysate were then collected by centrifugation at 10 , 000×g for 5 mins at 4°C . The caspase-3 ( DEVDase ) assay was performed in a total volume of 100 μL in 96 well plates . Triplicate wells were prepared containing blank ( no cell extract ) , negative control ( extract from untreated cells ) and A . hydrophila-infected cells . In 10 μL cell extract 32 μL caspase buffer , 2 μL DMSO , 10 μL DTT ( 100 mM ) and 2 μL of the DEVD-pNA substrate were added . The plates were incubated at 37°C for 5 h and absorbance read at 405 nm ( BMG Labtech ) . All chemicals used in the assay were provided with the kit . The PKA specific inhibitor , H-89 was added to HKM ( 1×106 ) at different time point viz . , pre-infection , simultaneous as well as post-infection to A . hydrophila infection . In another set of experiment , HKM were transfected with PKACA-siRNA or scrambled siRNA then infected with A . hydrophila as described earlier . To quantify the number of intracellular A . hydrophila , the HKM were lysed 24 h p . i . with Triton X-100 at a final concentration of 0 . 1% ( v/v ) in sterile distilled water . Serial dilutions of lysate from each well were prepared and 0 . 1 mL of each dilution was plated on nutrient agar and CFU determined after 24 h of incubation at 30°C . Data are presented as mean ± SE of number of experiments performed , as indicated in the corresponding figure . Pair-wise comparison was done between group employing paired t-test with P<0 . 05 as the minimum significant level . | Aeromonas hydrophila is a natural fish pathogen and is known to induce apoptosis of HKM . Head kidney is an important immune-organ in fish and HKM are critical for immunity against the invading pathogen . The mechanisms of cell death induced by A . hydrophila are incompletely characterized . We have studied the role of Ca2+-dependent signalling pathways in the induction of A . hydrophila-induced HKM apoptosis . We observed that A . hydrophila infection led to increased CaM expression in infected HKM which was Ca2+-dependent . The inhibitor and siRNA studies suggested CaM to be pro-apoptotic and triggered CaMKIIg expression in the infected HKM . Calpain-2 appeared to influence CaMKIIg expression . However , further studies are needed to understand the process . We report that the CaM-CaMKIIg pathway is important for initiating cAMP production within the infected HKM . The pro-apoptotic activation of cAMP dependent PKA was quite evident . The activation of ERK 1/2 was observed in the HKM and results clearly suggested the pro-active role of cAMP/PKA in the process . Thus we conclude that CaM-CaMKIIg initiates the cAMP/PKA pathway that induces ERK 1/2 phosphorylation to promote caspase-3 mediated apoptosis of the A . hydrophila-infected HKM . | [
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] | 2014 | Role of Calmodulin-Calmodulin Kinase II, cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages |
The PTEN-induced kinase 1 ( PINK1 ) is a mitochondrial kinase , and pink1 mutations cause early onset Parkinson's disease ( PD ) in humans . Loss of pink1 in Drosophila leads to defects in mitochondrial function , and genetic data suggest that another PD-related gene product , Parkin , acts with pink1 to regulate the clearance of dysfunctional mitochondria ( mitophagy ) . Consequently , pink1 mutants show an accumulation of morphologically abnormal mitochondria , but it is unclear if other factors are involved in pink1 function in vivo and contribute to the mitochondrial morphological defects seen in specific cell types in pink1 mutants . To explore the molecular mechanisms of pink1 function , we performed a genetic modifier screen in Drosophila and identified aconitase ( acon ) as a dominant suppressor of pink1 . Acon localizes to mitochondria and harbors a labile iron-sulfur [4Fe-4S] cluster that can scavenge superoxide to release hydrogen peroxide and iron that combine to produce hydroxyl radicals . Using Acon enzymatic mutants , and expression of mitoferritin that scavenges free iron , we show that [4Fe-4S] cluster inactivation , as a result of increased superoxide in pink1 mutants , results in oxidative stress and mitochondrial swelling . We show that [4Fe-4S] inactivation acts downstream of pink1 in a pathway that affects mitochondrial morphology , but acts independently of parkin . Thus our data indicate that superoxide-dependent [4Fe-4S] inactivation defines a potential pathogenic cascade that acts independent of mitophagy and links iron toxicity to mitochondrial failure in a PD–relevant model .
Parkinson's disease ( PD ) is the most frequent neurodegenerative movement disorder , but the pathways that explain disease pathology remain poorly understood [1] , [2] . While the most recognized pathological feature of PD is the preferential loss of dopaminergic ( DA ) neurons , one of the earliest observations in post mortem PD brains was the accumulation of iron in the substantia nigra ( SN ) [3] , [4] . Iron-mediated toxicity may thus contribute to DA neuron dysfunction but the mechanism has not been established . Mitochondrial dysfunction is thought to be an important aspect of PD progression . Mitochondrial toxins have been linked to sporadic forms of the disease and mitochondrial defects have been described in many cell types , also in SN mitochondria of PD patients [5] , [6] . Likewise some of the genetic factors linked to the disease also point to a role for mitochondria . PD-associated mutations in pink1 and parkin , both affect mitochondrial function in genetic model organisms and in mammalian cells [7] , [8] , but how mitochondrial dysfunction and iron toxicity are linked remains elusive . Pink1 and Parkin have been implicated in the clearance of dysfunctional mitochondria , a process dubbed mitophagy . In support , loss of parkin or pink1 in different cell types in flies , results in the accumulation of swollen and clumped mitochondria [9] , [10] , believed to be the result of defective mitophagy [11] . Furthermore , expression of factors that promote mitochondrial fission and , as a consequence , also indirectly promote mitophagy ( gain of drp1 or loss of opa1 or mfn ) partially rescue defects seen in pink1 and parkin mutants [12]–[14] . Further studies indicate that mitochondrial depolarization triggers the recruitment Parkin to mitochondria in a Pink1-dependent manner , facilitating mitophagy [15] . In line with this idea , over expression of Parkin in pink1 mutants , alleviates pink1-associated defects [9]–[16] . Hence , Pink1 acts with Parkin to regulate mitophagy . In parallel , pink1 may also harbor supplementary roles . Expression of Parkin or Drp1 , or loss of opa1 or marf only partially rescue pink1-associated defects , suggesting additional pathways are contributing to the phenotype . Furthermore , loss of pink1 function causes defects in the electron transport chain in fly and mouse cells [17] , [18] that are not [19] or only partially [20] rescued by expression of Drp1 . Finally , bypassing Complex I dysfunction , by expressing a yeast Complex I equivalent protein Ndi1 partially rescues the defects in pink1 mutants , but not those seen in parkin mutants [19] . Hence , Pink1 may play multiple roles in mitochondria , but the relative contribution of these different pathways to the pink1-dependent phenotypes , including the accumulation of swollen , clumped mitochondria remains to be determined . In an unbiased genetic screen for heterozygous suppressors of Drosophila pink1 [21] we identified mitochondrial aconitase ( acon ) that encodes an enzyme catalyzing the first step of the Krebs Cycle [22] . Acon harbors an iron-sulfur [4Fe-4S] cluster [23] and we show that oxidative inactivation of this cluster in pink1 mutants is a major cause of iron toxicity that contributes to mitochondrial swelling and clumping in pink1 mutants . Our data are most consistent with acon acting downstream of pink1 and affecting mitochondrial morphology independently of parkin-mediated mitophagy . Thus oxidative inactivation of Aconitase is a source of iron toxicity that leads to mitochondrial defects in pink1 mutants and we propose a model where different pathways controlled by Pink1 , including mitophagy and the maintenance of ETC activity can contribute to mitochondrial failure in specific cell types .
Pink1 mutants show a severe defect to fly caused by mitochondrial dysfunction [19] , [21] . To identify genetic modifiers of pink1 , we have tested a collection of 193 chemically induced ( EMS ) recessive lethal mutants that have been pre-selected for defects in mitochondrial function and neuronal communication [24]–[26] , for their ability to modify the pink1 null mutant flight defect . At the 1% significance level we isolated 5 suppressors ( p<0 . 01 ) [21] and to reveal mechanisms by which the modifiers affect Pink1 , we mapped one of these recessive lethal suppressors to aconitase ( acon ) and named it acon1 . This mutant fails to complement a deletion that uncovers acon as well as a lethal transposon insertion in acon that we named acon2 ( Figure S1A ) . Sequence analysis of acon1 reveals a nonsense mutation in exon 2 ( Figure S1A ) . In addition , semi-quantitative RT-PCR and Western blot analysis indicates severely reduced mRNA and protein levels in animals that are homozygous for either acon allele ( Figure S1C and S1D ) , indicating that both are loss of function alleles . Moreover we can rescue the lethality and phenotypes associated with acon1/acon2 using a 20 kb genomic fragment encompassing the acon locus , yielding normal adult flies that do not show obvious behavioral abnormalities ( Figure S1A , S1B ) . Likewise ubiquitous expression of acon cDNA is also able to rescue acon1/acon2-associated lethality ( Figure S1B ) . Thus , one of the suppressors of pink1 harbors a lethal lesion in acon and the lethality in the mutants is solely due to disruption of acon . Heterozygosity of acon significantly suppresses the flight defect associated with pink1B9 mutants ( Figure 1A , 1B ) . The extent of rescue we obtained by removing acon , is similar to previously reported conditions that suppress pink1 flight defects , including adding a copy of drp1 ( drp1+ ) that facilitates mitochondrial fission , removing a copy of opa1 ( opa1S3 ) , reducing mitochondrial fusion ( Figure S2A , S2B ) , expression of Parkin , expression of yeast NDI1 that bypasses Complex I of the electron transport chain ( ETC ) , or feeding pink1 mutants ubiquinone or menaquinone that boost ETC function [9] , [10] , [13] , [19] , [21] . To test if the rescue that we observe is solely due to partial loss of acon ( and not due to second site interactors on the chromosome ) , we determined flight but also ATP levels of pink1 mutants with one copy of a mutant acon allele . While heterozygous acon1 and acon2 mutants alone do not show defects ( Figure 1C ) , we find that one copy of either acon1 or acon2 significantly rescue the reduced ATP levels in pink1 mutants ( Figure 1D ) . This effect in pink1 mutants is specific to loss of acon as introduction of a genomic copy encompassing wild type acon in pink1B9;acon2/+ flies completely reverses both the flight and ATP level phenotypes to pink1B9 mutant levels ( Figure 1B and 1D ) . Thus , pink1 mutant phenotypes are specifically rescued by partial loss of acon expression . To further quantify the effect of acon on pink1 mutant phenotypes we also analyzed mitochondrial morphology in adult indirect flight muscles using transmission electron microscopy . As previously described [9] , [10] , the flight muscles of pink1 mutants exhibit swollen mitochondria with disorganized and fragmented cristae when compared to flight muscles from control flies or when compared to heterozygous acon mutants that do not show mitochondrial morphological defects ( Figure 1E and Figure S1F ) . Partial loss of acon in pink1 mutant results in a substantial rescue of the mitochondrial morphological defects in flight muscles , displaying substantially more intact cristae and less swollen mitochondria compared to pink1 mutants ( Figure 1E ) . Hence , also at the ultrastructural level , partial loss of acon significantly alleviates mitochondrial morphological defects in pink1 mutant muscles . Pink1 mutants also show swollen and clumped mitochondria in dopaminergic neurons in the adult brain [9] , [10] . To test if loss of acon can also rescue this defect , we expressed mitoGFP in pink1 mutant flies and in pink1 mutant animals heterozygous for acon . In line with the electron microscopy data of muscles , mitochondria in muscles labeled by mitoGFP ( expressed using the ubiquitous da-GAL4 ) are spherical and aggregated in pink1 mutants and this defect is significantly rescued by partial loss of acon ( Figure 1F and 1G ) . Next , we expressed mitoGFP in dopaminergic neurons using ple-Gal4 ( also called TH-Gal4 ) . While mitochondria are organized in a tubular network in wild type dopaminergic neurons , pink1 mutant mitochondria appear mostly as fragmented spherical aggregates in all dopaminergic neuron clusters analyzed ( Figure S1H ) [9] , [10] . We quantified size and number of mitochondrial aggregates in the PPM3 cluster ( Figure S1H and Methods ) . While heterozygous acon1 and acon2 mutants do not show defects compared to controls ( Figure 1H , 1I ) , we find that both one copy of either acon1 or acon2 significantly rescue the increased size and number of mitochondrial aggregates in pink1 mutants ( Figure 1H , 1I and Figure S2A ) . This rescue in pink1 mutants is specific to the partial loss of acon as introduction of a genomic copy encompassing wild type acon in pink1B9; acon1/+ flies reverses these phenotypes back to pink1B9 mutant levels ( Figure 1F–1I ) . Furthermore , we confirm that protein levels are reduced by about 50% in pink1B9; acon1or2/+ compared to pink1 mutants and are restored in flies expressing a genomic copy of wild type acon ( Figure S1E ) . Thus , together our data indicate that morphological defects of mitochondria in pink1 mutants are significantly rescued by partial loss of acon expression and the mitochondrial morphological defects in pink1 mutants are dependent on acon expression . acon is predicted to encode mitochondrial Aconitase ( Acon ) , an iron sulfur cluster containing protein , that catalyzes the formation of isocitrate in the first step of the Krebs cycle [22] . To assess whether Acon localizes to mitochondria we fractionated fly tissue in cytoplasmic and mitochondrially enriched fraction and performed Western blotting using anti-Acon antibodies . Acon is enriched in the mitochondrial fraction ( Figure S1G ) . Acon harbors a single unligated iron atom in its [4Fe-4S]2+ cluster , and the enzyme is in this respect unique in mitochondria . Such an unligated iron atom is particularly sensitive to superoxide ( O2− ) -dependent oxidation [27]–[29] that results in cluster instability . Oxidation is followed by the release of Fe2+ and H2O2 that may contribute to oxidative damage and mitochondrial morphological defects through the formation of the potent hydroxyl radical ( OH . ) by the Fenton reaction [30] . Thus , the specific configuration of the Acon [4Fe-4S]2+ cluster in combination with its proximity to mitochondrially generated superoxide place Acon as a major mediator of oxidative stress in mitochondria . We therefore wondered if O2− leaking from defective pink1 mutant mitochondria could be a source of Acon inactivation resulting in morphological defects . To test if also in fruit flies the loss of pink1 function results in increased O2− production , we incubated mitochondrial preparations from pink1 mutant flies and controls with Complex I substrates ( pyruvate/malate ) and used the fluorogenic probe dihydroethidium ( DHE ) to measure O2− production [31] , [32] . Similar to wild type mitochondria in the presence of AntimycinA , known to induce O2− production ( Figure 2A ) , pink1B9 mitochondria show a significant increase in DHE fluorescence compared to controls ( Figure 2A ) . These data indicate that pink1 loss leads to increased O2− production . If the increased O2− in pink1 mutants can act via the Acon [4Fe-4S] cluster to cause mitochondrial swelling , we expect ( 1 ) that partial loss of acon does not rescue the increased O2− production in pink1 mutants; ( 2 ) that Acon enzymatic activity normalized to total Acon protein is reduced in pink1 mutants; ( 3 ) that H2O2 and Fe2+ levels are increased in pink1 mutants as a result of Acon inactivation , and ( 4 ) that this defect is rescued by partial loss of acon . First we assessed O2− in pink1 mutants heterozygous for acon1 or acon2 that we showed rescues mitochondrial morphological defects in pink1B9 . However , in line with our model , heterozygosity for acon does not reduce pink1B9-induced O2− production ( Figure 2A ) , indicating that increased O2− production per se does not induce mitochondrial morphological defects . Next we measured Acon activity in pink1 mutant mitochondria and we find that Acon activity normalized to total Acon protein levels is significantly reduced compared to the controls . These data are in line with increased Acon inactivation in pink1 mutants ( Figure 2B ) , likely as a result of the increased O2− . Further testing our model , we also measured H2O2 and Fe2+ content . To measure H2O2 and its radical derivatives we incubated fly lysates with the fluorescent probe dichlorofluorescein diacetate ( DHCF-DA ) [33] . We find a 50% increase in fluorescence in pink1 mutant lysates compared to the control ( Figure 2C ) . Thus , pink1 mutants accumulate H2O2 and/or derivatives thereof . We also measured mitochondrial Fe2+ content by incubating mitochondrial enriched fractions with Rhodamine B-[ ( 1 , 10-phenanthrolin-5-yl ) aminocarbonyl]benzyl ester ( RPA ) [34] . In the presence of Fe2+ , RPA fluorescence quenches and in pink1B9 mitochondria , we observe a significant increase in RPA quenching compared to controls ( Figure 2D ) . These data indicate increased mitochondrial Fe2+ levels in pink1B9 mutants . This effect is specific , as incubating mitochondria of controls and mutants in Rhodamine B 4-[ ( Phenanthren-9-yl ) Aminocarbonyl]benzyl ester ( RPAC ) that consists of the same fluorophore as RPA but without iron-chelating properties , does not show quenching in pink1B9 or in controls ( Figure 2D ) . Thus , our data indicate that pink1B9 mutants harbor increased levels of Fe2+ and of H2O2 and/or its radical derivatives . Next we tested if increased mitochondrial Fe2+ and H2O2 accumulation in pink1 mutants is a consequence of Acon[4Fe-4S] inactivation by O2− . We therefore measured Fe2+ and H2O2 and its derivatives levels in mitochondria of pink1 mutants heterozygous for acon1 or acon2 . While the increased O2− production in pink1B9 mutants was not reduced by heterozygous acon , as shown above ( Figure 2A ) , we find that compared to pink1B9 , mitochondrial Fe2+ and H2O2 levels are significantly lower in pink1B9 heterozygous for acon1 or acon2 ( Figure 2C and 2D ) . Thus , these data are consistent with the possibility that mitochondrial Fe2+ and H2O2 and/or its radical derivatives-accumulation in pink1 mutants is caused by oxidative inactivation of Acon . Our biochemical data support a model in which oxidative inactivation of Acon and ensuing Fe2+ and H2O2 accumulation contributes to the mitochondrial morphology defects in pink1 mutants . We reasoned that if partial loss of acon protects against mitochondrial stress in pink1 mutants , increased levels of Acon expression may predispose cells to develop mitochondrial morphological defects , provided sufficient O2− is around . We therefore created transgenic animals that overexpress wild type Acon ( Figure 3A ) resulting in increased Acon activity ( Figure 3B ) . We then determined mitochondrial morphology using mito-GFP and the ple-GAL-4 driver upon expression of Acon in DA neurons . While mitochondria in DA neurons of control flies organize in a long tubular network , mitochondria in DA neurons that overexpress Acon form fragmented spherical aggregates ( Figure 3C , 3D and Figure S2A ) . Hence , in contrast to partial loss of acon that rescues mitochondrial defects in pink1 mutants , overexpression of Acon causes mitochondrial morphological defects and swelling of mitochondria in DA neurons . Based on the finding that increased expression of Acon causes mitochondrial morphological defects we tested if pink1 mutant flies upregulate Acon expression . We measured acon mRNA and protein levels in pink1 mutants , but in contrast to our expectation , we find a significant downregulation of both acon mRNA and Acon protein levels in pink1 flies ( Figure S2C , S2D ) suggesting that an adaptive mechanism already acts in pink1 mutants to down regulate Acon expression . Thus , the pink1B9-induced stress response results in lower Acon levels and , as shown above , further reducing Acon expression ( using heterozygous acon mutants ) is protective against mitochondrial defects in pink1 mutants . Taken together , the data are consistent with Acon being a dosage sensitive modifier of morphological defects in mitochondria . To test if the mitochondrial morphological defects in DA neurons following Acon overexpression are induced by increased Acon catalytic activity or by the presence of an [4Fe-4S] cluster we generated transgenic flies that either overexpress a catalytic inactive Acon ( AconS677A ) that still harbors its [4Fe-4S] cluster , or flies that overexpress an Acon without its [4Fe-4S] cluster ( AconC459S ) and is thus also catalytically inactive [22] , [35] . Western blotting indeed indicates overexpression of the mutant Acon proteins ( Figure 3A ) , and as expected , Acon enzymatic activity measured in fly head lysates is only increased when wild type Acon is expressed , and not when AconS677A or AconC459S are expressed ( Figure 3B ) . While overexpression AconS677A in DA neurons results in obvious mitochondrial morphological defects similar to the overexpression of wild type Acon , overexpression of AconC459S is inert ( Figure 3C , 3D and Figure S2A ) . Hence , the Acon [4Fe-4S] cluster predisposes DA neurons to mitochondrial morphological defects . Our data are in line with a model where oxidative inactivation of the Acon [4Fe-4S] cluster by O2− contributes to mitochondrial morphological defects . To find further evidence for this idea we expressed Drosophila mitochondrial Ferritin ( Fer3HCH ) [36] in DA neurons of pink1B9 , using the ple-GAL4 driver and assessed mitochondrial morphology using mito-GFP . We find that expression of Fer3HCH significantly rescues defects in mitochondrial morphology in pink1B9 mutants ( Figure 4E , Figure 2F , and Figure S2A ) , suggesting that iron toxicity causes mitochondrial defects in pink1 mutants . Consistent with this model , expression of Fer3HCH in flies that over express Acon also results in a significant rescue of the mitochondrial morphological defects in the DA neurons ( Figure S2E , S2F ) . Hence , the mitochondrial swelling as a result of Acon overexpression is at least in part mediated by iron . Together these data indicate that Acon is a critical source of Fe2+-mediated mitochondrial toxicity . Mitochondrial dynamics and mitophagy are critical processes in maintaining a healthy population of mitochondria . Pink1 has been implicated to regulate mitochondrial homeostasis via several mechanisms . Deregulation of these pathways may be a source of O2− , responsible for Acon inactivation . While Pink1 has been found to maintain the activity of Complex I in the ETC [17]–[20] , the protein has also been linked to mitophagy in a pathway involving Drp1 and Parkin [11]–[15] , [20] , [37]–[40] . Dysfunctional mitochondrial parts may be segregated by the fission factor Drp1 [41] , [42] . Pink1 stabilized on depolarized mitochondria then mediates Parkin recruitment causing the ubiquitination of mitochondrial proteins and activation of the autophagic machinery [41] , [42] . To test if enlarged and swollen mitochondria upon Acon over expression are a consequence of defective remodeling or mitophagy we co-overexpressed Parkin , a protein that ubiquitinates mitochondrial targets , or Drp1 , a mitochondrial fission factor , two conditions thought to facilitate mitophagy . While over expression of Parkin or Drp1 -as expected- result in fragmentation of mitochondria , these conditions do not rescue the defect in mitochondrial swelling and clumping induced by expression of Acon or AconS677A ( Figure 3E , 3F and Figure S2A ) . Hence , our data suggest that the defects in mitochondrial morphology induced by Acon expression are at least in part caused independently from defects in remodeling and mitophagy . Given that pink1 mutants display reduced Complex I activity [17]–[20] and this feature may also be a source of increased O2− we tested if mitochondrial swelling and clumping seen in animals where we downregulated an evolutionary conserved Complex I component , NDUFA8 , can be rescued by partial loss of acon . First , we confirm increased O2− production and find a concomitant inactivation of Acon activity upon RNAi-mediated downregulation of NDUFA8 ( Figure 4A and 4B ) . Second , we believe that this O2− is produced at least partly independently from defects in mitochondrial remodeling because expression of Drp1 in DA neurons with reduced NDUFA8 function does not fully rescue the mitochondrial swelling and clumping phenotypes in PPM3 DA neurons ( Figure 4C , 4D and Figure S2A ) . Next , we tested the ability of heterozygous acon to modulate the mitochondrial morphological defect induced by NDUFA8 RNAi and find that heterozygous acon is more effective than expression of Drp1 in rescuing the mitochondrial deficits in DA neurons ( Figure 4C , 4D and Figure S2A ) . Likewise , and in line with our model , expression of mitoferritin ( Fer3HCH ) also alleviates mitochondrial defects in animals that express RNAi to NDUFA8 in DA neurons ( Figure 4E , 4F and Figure S2A ) . Hence , our data suggest that Acon is inactivated by ETC-derived O2− causing oxidative stress . Our work suggests that mitochondrial morphological defects in pink1 mutant DA cells can be of different origin: both O2−-dependent Acon inactivation or loss of Parkin-dependent mitophagy yield swollen and clumped mitochondria . Alleviating the defects induced by either pathway using heterozygous acon or expressing Drp1 or Parkin both rescue the mitochondrial morphological defects in pink1 mutants ( this work; [12]–[14] , [16] ) . To further support this notion , we first assessed if mitochondrial defects in parkin mutants can be rescued by partially removing acon function . parkin mutants display enlarged and swollen mitochondria in muscles and DA neurons , many of the flies also fail to fly and animals harbor lower ATP levels . In contrast to removing acon function in pink1 mutants , heterozygosity for acon fails to rescue the inability of parkin mutants to fly , their reduced ATP levels and their defects in mitochondrial morphology ( Figure 5A–5D ) . Hence , our data suggest that acon acts independently from defects in Parkin-dependent mitophagy . Finally if our model is correct , we reasoned that the combination of Drp1 expression and acon heterozygosity in pink1 mutants should yield additive ‘super rescue’ . We therefore tested the ability of these flies to fly and find that they fly significantly better than pink1 mutants or than pink1 mutants partially rescued by either Drp1 expression or by heterozygous acon ( Figure 5E ) . Hence , these data are in line with Pink1 controlling different mitochondrial pathways that can be targeted largely independently . We speculate that increased O2− derived from a defective Complex I in pink1 mutants is an important contributor to Acon inactivation , but other sources of O2− may contribute to mitochondrial failing as well .
Iron accumulation in the substantia nigra , systemic mitochondrial dysfunction and oxidative stress have all been implicated in PD pathology; however , a link between these factors remains elusive . Here we show that oxidative inactivation of Acon generates iron-mediated oxidative stress that contributes to mitochondrial swelling in Drosophila pink1 mutants ( Figure 6 ) . Inactivation of Acon[4Fe-4S] clusters could contribute to mediating O2− toxicity by simultaneous release of Fe2+ and H2O2 [43] that combine in the Fenton reaction to generate highly toxic hydroxyl radicals [30] , [44] ( Figure 6 ) . Hydroxyl radicals can induce mitochondrial permeability transition and swelling [45]–[47] , in line with electron microscopic analyses of pink1 mutants where mitochondria appear swollen and show disorganized cristae [9] , [10] ( Figure 1 ) . Four major findings support that this iron-mediated toxic mechanism is an additional important aspect of mitochondrial dysfunction in pink1 mutants . First , we find increased O2− production , increased Acon inactivation and more Fe2+ and H2O2 accumulation in pink1 mutants ( Figure 2 ) . Second , partial loss of Acon reduces Fe2+ and H2O2 accumulation and alleviates pink1-associated phenotypes including mitochondrial morphological defects in muscle and DA neurons ( Figure 1 ) . Third , overexpression of wild type Acon in dopaminergic neurons produces a mitochondrial morphological defect and this effect is completely dependent on the presence of the [4Fe-4S] cluster in Acon ( Figure 3 ) . These data also indicate mitochondrial integrity is sensitive to Acon [4Fe-4S] cluster dosage . Finally , chelating iron by expressing mitochondrial Ferritin is sufficient to rescue pink1 mitochondrial morphological defects ( Figure 4 ) . Thus , our data suggest that inactivation of Acon and iron accumulation might be a pathogenic mechanism triggered by loss of pink1 and increased superoxide , linking iron accumulation and mitochondrial failure . Acon inactivation is dependent on O2− that , amongst other sources ( see below ) , may be produced in defective mitochondria . While various mitochondrial insults can result in increased O2− production , our work is most consistent with Parkin-dependent mitophagy being not the major source of Acon inactivation in pink1 mutants . The mitochondrial morphological defects induced by Acon overexpression were not strongly rescued by expressing Drp1 , a condition that indirectly promotes mitophagy and parkin mutants were not majorly rescued by partial loss of acon ( Figure 3 and Figure 5 ) . In contrast , mitochondrial morphological defects in DA neurons of flies with reduced Complex I activity are significantly rescued when acon is heterozygous ( Figure 4 ) . Hence , Acon seems to act in a Pink1-dependent pathway that can operate largely independently of mitophagy ( Figure 6 ) . Defects at the level of Complex I are often associated with increased leaking of the toxic O2− [48] , [49] , and likewise , systemic inhibition of Complex I mimics features of PD in animal models [50]–[53] . Previous work in flies or mice has indicated reduced ETC function [17]–[20] in pink1 mutants , and we show that this condition results in mitochondrial morphological defects in an Acon-dependent manner . Similar to pink1 mutants , RNAi-mediated knock down of an evolutionary conserved Complex I component , NDUFA8 , also results in an increased production of superoxide as well as in Acon inactivation . We show that these biochemical changes correlate with mitochondrial morphology defects in dopaminergic neurons that can be rescued by partial loss of acon or by over-expression of mitoferritin that scavenges the released Fe2+ [36] ( Figure 4 ) . It is interesting to note that increased O2− production per se is not sufficient to generate mitochondrial morphological defects , and that the presence of sufficient amounts of Acon is required . Indeed , our data indicate that pink1 mutants heterozygous for acon show increased levels of O2− but normal mitochondrial morphology . Our data also indicate that upstream events in pink1 mutants that result in increased O2− production contribute to mitochondrial morphological defects because of oxidative inactivation of Acon . In line with this , overexpression of the mitochondrial superoxide dismutase 2 ( SOD2 ) that scavenges O2− , successfully rescues mitochondrial swelling phenotype of pink1 in DA neurons [54] . Given that both genetic forms of PD as well as sporadic cases of PD show ETC defects [5] , [6] , [17]–[19] , our work may be relevant for idiopathic cases that suffer from mitochondrial dysfunction as well . Acon inactivation and iron-mediated toxicity might thus have a more general role in the pathogenesis of PD . While pink1 loss affects numerous cell types , our data also start to provide insight as to why DA neurons in the substantia nigra are more vulnerable in PD . While overexpression of Acon or downregulation of Complex I produces mitochondrial morphological defects in DA neurons , in Drosophila flight muscles mitochondria appear morphologically largely normal ( data not shown ) . These data suggest a tissue-specific response in that Acon inactivation has a stronger impact in DA neurons than in muscle cells . Each cell type is exposed to various sources of O2− , but DA neurons in particular are exposed to dopamine-induced oxidative stress that is a source of O2− [55]–[57] . Furthermore , the substantia nigra in humans is naturally rich in iron [58] and this feature may lower the threshold for hydroxyl radical production in the Fenton reaction that is facilitated by Acon inactivation . Pink1 mutations or environmental factors in some sporadic cases of PD already result in increased levels of O2− , but we hypothesize that in DA neurons , additional dopamine-induced oxidative stress may facilitate Acon inactivation and hydroxyl radical production providing insight into one of the pathways underlying mitochondrial failure in pink1 mutants .
Flies were raised on standard cornmeal and molasses medium at 25°C . w1118; UAS-mitoGFP , w1118; daGal , w1118; pleGal4 , w; UAS-4EBP and w1118; Mi{ET1}AconMB09176/SM6a ( acon2 ) and were obtained from Bloomington stock center ( Indiana , USA ) . w1118 pink1B9 and w1118 pink1RV , parkin1 and parkinRV [59] were provided by Jongkyeong Chung ( Advanced Institute of Science and Technology , Korea ) [10] . parkinΔ21 mutant flies were a gift from Graeme Mardon ( Baylor College of Medicine ) [60] and drp1+ genomic rescue constructs were provided by Hugo Bellen ( Baylor College of Medicine ) [61] w1118; UAS-Fer3HCH was provided by Dr Fanis Missirlis ( Qeen Mary University of London , UK ) . w1118; UAS-CG3683RNAi ( w1118; P{GD16787}v46799/CyO ) was from the Vienna Drosophila RNAi Center ( VDRC ) [62] . The genomic clone CH322-18I04 was obtained from BACPAC Resources ( Children's Hospital Oakland ) . UAS-Aconwt was generated by PCR amplification of BDGP cDNA clone LD24561 using primers: AconcDNA . F ( 5′ ATGGCTGCGAGATTGATGAACG ) and AconcDNA . F ( 5′ TTACTGGGCCAGCTCCTTCATGC ) . The S677A and C459S mutations were introduced in the primers and the mutated cDNAs were generated by overlap extension PCR using the following primers: S677A . F ( 5′GAcgCACCCTCGCCGTAGTTCTCATC ) S677A . R ( 5′AACTACGGCGAGGGTGcgTC ) , C459 . F ( 5′GGTCCCtCcATTGGACAGTGGGATCG ) and C459 . R ( 5′CGATCCCACTGTCCAATgGaGGGACC ) . All constructs were cloned into the EcoRI and NotI restriction sites of pUAST-attB [63] . Following sequencing , transgenic flies were created at GenetiVision Inc . ( Houston , USA ) using PhiC31 mediated transgenesis in the VK1 docking site ( 2R , 59D3 ) [64] . For quantitative RT-PCR , total RNA was isolated using TRI Reagent ( Sigma-Aldrich ) according to the manufacturer's protocol . Subsequently , the RNA samples were cleaned up using the RNeasy Mini Kit with the on-column DNAse treatment ( Qiagen ) . 1 µg of total RNA was used as a template for synthesis of oligodT-primed double stranded cDNA using the SuperScriptIII First-Strand Synthesis System ( Invitrogen ) . 20 ng cDNA of each sample was used for acon SYBR Green PCR Master mix ( Applied Biosystems ) and the following primers were used: aconRT-F ( 5′ TCGTGCCATTATCGTCAAGTC ) and aconRT-F ( 5′ AGGTTGAGCAGGGAGATTTTG ) . All experiments were performed in triplicate and run on a Roche LC480 system . The data were normalized utilizing RP-49 , a ribosomal gene , using following primers: RP-49-F ( 5′ ATCGGTTACGGATCGAACAA ) and RP-49-R ( 5′ GACAATCTCCTTGCGCTTCT ) . For Western blots , flies were homogenized in cold T-PER buffer ( ThermoScientific ) with complete protease inhibitor mixture ( Roche ) . Protein concentration was determined by BCA protein quantification kit ( Pierce ) . Samples were diluted in 2-mercaptoethanol 10% SDS loading buffer and boiled for 5 min and 15 µg of proteins were separeted on pre-cast 4–12% NuPage Bis-Tris gels ( Invitrogen ) . Following transfer to nitrocellulose , blots were probed with primary antibodies: 1∶5000 Anti-ACO2 ( AbGent ) , 1∶1000 anti-Tubulin ( B5–12 , Sigma ) and 1∶1000 HRP coupled secondary antibodies ( Jackson immunolabs ) . Blots were developed with Western-Lightning-ECL ( PerkinElmer ) and imaged . Quantification was performed using gel analyzer tool in ImageJ software from the US National Institute of Health ( http://rsb . info . nih . gov/ij/ ) . Batches of 5 days old male flies were transferred to an empty vial ( 5 cm D , 10 cm H ) . Flies were allowed to climb above a marked line at 9 cm height; the vial was gently tapped and visually scored for flying flies . Flies at the bottom were removed and the remaining flies were retested . Flies that fly twice were assigned a score of 1 , the others a score of 0 . ATP content was determined as described [10] . 5 days-old flies with abdomen dissected out were homogenized in 50 µl of 6 M guanidine-HCl 100 mM Tris and 4 mM , EDTA , pH 7 . 8 . These homogenates were snap-frozen in liquid nitrogen and then boiled for 3 min . Samples were then centrifuged and the supernatant was diluted ( 1/50 ) in extraction buffer , mixed with luminescent solution ( ATP Determination Kit , Invitrogen ) and luminescence was measured on an EnVision Multilabel Reader ( Perkin Elmer ) . ATP ( nmol ) was determined using a standard curve and normalized to protein content ( mg ) measured by BCA assay ( Pierce ) . Thoraxes were fixed in paraformaldehyde/glutaraldehyde , postfixed in osmium tetroxide , dehydrated and embedded in Epon . Sections 80 nm thick were stained with uranyl acetate and lead citrate and subjected to TEM analysis . H2O2 was measured as described [33] . 4–5 adult flies were homogenized in 50 µl cold lyses buffer T-PER ( Thermo scientific ) and the homogenate were cleared by centrifugation at 1000×g for 5 min at 4°C . 140 µL of PBS containing 50 µM of DCFH-DA ( molecular probe ) were added to 10 µL of lysate in a 96-well plate format and incubated at 25°C for 10 minutes in the dark . DCFH-DA fluorescence ( 485exc/530em ) was measured using Wallac Victor2 1420 ( Perkin Elmer ) . Fluorescence intensity was normalized to the protein amount ( BCA , Pierce ) and expressed as relative to the control . Fifty flies were gently crushed in 1 ml chilled mitochondrial isolation medium ( Mitosciences ) by using a porcelain mortar and pestle , then spun twice at 1 , 000×g for 5 min at 4°C to remove debris . The supernatant was then spun at 12 , 000×g , for 15 min at 4°C . The pellet , containing the mitochondria , was washed with 1 ml of isolation medium and resuspended in 40 µl of isolation medium supplemented with complete protease inhibitor mixture without EDTA ( Roche ) . Mitochondrial Superoxide production was measured as described [32] . 10 µg of mitochondria were incubated in experimental buffer ( EB: 125 mM KCl , 10 mM Tris-MOPS , 1 mM KPi , 10 µM EGTA-Tris , pH 7 . 4 , 25°C ) supplemented with 1 . 25 mM Pyruvate/1 . 25 mM malate and 5 µM DHE ( Molecular probe ) in a 96-well plate format for 10 min . The fluorescence was measured ( 485exc/590em ) using Wallac Victor2 1420 ( Perkin Elmer ) . Fluorescence intensity was normalized to the initial value and expressed as relative to the control . 10 µM antimycin A was used to induce superoxide production in control mitochondria . For mitochondrial ferrous iron level measurements , 10 µg of mitochondria were resuspended in isolation buffer ( Mitosciences ) and incubated with 20 µM of RPA or RPAC ( Squarix Biotechnology ) in a 96-well plate format at room temperature for 10 min . RPA/RAPC fluorescence ( 560 exc/600 em ) was measured using Wallac Victor2 1420 ( Perkin Elmer ) . Quenching was calculated as percent of initial fluorescence . Aconitase enzyme activity microplate kit ( Mitosciences ) was used according to the manufacturer's protocol to measure Aconitase activity . 20 µg of mitochondria were incubated with assay buffer and the activity was measured by following conversion of isocitrate to cis-aconitate as in increased in 240 nm UV absorbance . Measurements were recorded over 30 min . at 1 min intervals and aconitase activity were calculated from the linear increase in absorbance and normalized to the amount of aconitase , determined by western blot , in the same mitochondrial preparation . Values were reported as relative activity to the control . Brain dissection and whole-mount immunohistochemistry for tyrosine hydroxylase ( TH ) was performed as described [65] . Primary 1∶100 antibody against TH ( Chemicon ) and secondary alexa 555 ( Invitrogen ) were used . Brains were imaged on a Zeiss LSM 510 META confocal microscope using a 63xoil NA 1 . 4 lens . Mitochondrial tagged GFP ( mito-GFP ) was visualized using 488 nm laser and 500–530 band pass emission filter . Because mitochondrial morphology is sensitive to environmental conditions , variations did occur from batch to batch . We only compared flies of different genotypes if normal mitochondrial morphology was observed in the control samples ( Figure S1H–S1H″ ) in the same batch . For quantification of mitochondrial aggregates size and numbers , DA neurons of PPM3 cluster ( Figure S1H–S1H″ ) were scored . Quantification of aggregate size was done using “analyzing particles” plugin in ImageJ ( http://rsb . info . nih . gov/ij/ ) : rounded particles were automatically detected and the average surface area of aggregates in each neuron was determined as total area occupied by aggregates/number of aggregates . Adult flies were fixed in PBS with 5% formaldehyde and 0 . 4% Triton for 3 hours . Thoraxes were dissected in PBS and mounted in vectashield ( Vector Laboratories ) and were imaged on a Zeiss LSM 510 META confocal microscope using a 63xoil NA 1 . 4 lens . Mitochondrial tagged GFP ( mito-GFP ) was visualized using 488 nm laser and 500–530 band pass emission filter . For muscle section with same area were scored and quantification of mitochondrial aggregates was performed as described above . | In this work we provide mechanistic insight linking together two of the earliest observations in Parkinson's disease: the excessive build-up of iron in diseased substantia nigra neurons and mitochondrial dysfunction particularly increased reactive oxygen species production at the level of Complex I . We identify aconitase mutants as strong genetic suppressors of Parkinson-related pink1 mutant phenotypes , both at the organismal and at the cellular/mitochondrial level . We show that the mitochondrial dysfunction in pink1 mutants that includes Complex I dysfunction results in superoxide-dependent inactivation of the Aconitase iron-sulfur cluster , leading to the release of iron and peroxide that combine to produce hydroxyl radicals and mitochondrial failure . Consequently , scavenging free iron using expression of mitoferritin or decreasing the levels of aconitase both rescue pink1 mutants; while increased wild-type Aconitase , but not a mutant that does not harbor an iron-sulfur cluster , results in severe mitochondrial defects . Given that reduced electron transport chain activity , increased oxidative stress , and natural iron build-up in the substantia nigra are common factors in sporadic and familial forms of Parkinson's disease , we believe that oxidative inactivation of Aconitase may represent an important pathogenic cascade underlying neuronal dysfunction in Parkinson's disease . | [
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] | 2013 | Aconitase Causes Iron Toxicity in Drosophila pink1 Mutants |
The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens , such as Vibrio cholerae , the cause of cholera . All organisms in this family have two chromosomes , and replication of the smaller one depends on rctB , a gene that is restricted to the Vibrionaceae . Given the increasing prevalence of multi-drug resistance in pathogenic vibrios , there is a need for new targets and drugs to combat these pathogens . Here , we carried out a high throughput cell-based screen to find small molecule inhibitors of RctB . We identified a compound that blocked growth of an E . coli strain bearing an rctB-dependent plasmid but did not influence growth of E . coli lacking this plasmid . This compound , designated vibrepin , had potent cidal activity against V . cholerae and inhibited the growth of all vibrio species tested . Vibrepin blocked RctB oriCII unwinding , apparently by promoting formation of large non-functional RctB complexes . Although vibrepin also appears to have targets other than RctB , our findings suggest that RctB is an attractive target for generation of novel antibiotics that only block growth of vibrios . Vibrio-specific agents , unlike antibiotics currently used in clinical practice , will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms .
The Vibrionaceae are a diverse family of bacteria that includes more than 80 species [1] , [2] . Vibrios are gram-negative rods that usually inhabit aquatic habitats , often in association with eukaryotes . This large family includes important human pathogens such as V . cholerae , V . parahaemolyticus and V . vulnificus , which can cause gastrointestinal disorders and other illnesses [2] , [3] . Vibrio species are also pathogenic for several economically important marine organisms; for example farmed shrimp are harmed by V . harveyi and V . nigripulchritudo [2] , [4] , [5] . One notable attribute of the Vibrionaceae is their bipartite genomes . The genomes of most other γ-proteobacteria ( such as E . coli ) consist of single circular chromosomes , whereas the genomes of vibrios are comprised of two circular chromosomes [6] . Studies of V . cholerae , the agent of cholera , have revealed that different proteins initiate replication of its two chromosomes . Initiator proteins bind to and melt origins of replication and also recruit components of the replisome to the origin [7] . DnaA , a conserved AAA+ ATPase , is thought to be the initiator of chromosome DNA replication in most eubacteria [8] , [9] , and several observations support the idea that DnaA serves as the initiator of replication of the large V . cholerae chromosome ( chrI ) as well . The origin of replication of chrI , oriCI , is similar in sequence to oriC , the origin of replication of the E . coli chromosome [10] , and both contain several binding sites for DnaA . Replication of oriCI-dependent minichromosomes is DnaA-dependent [10] , and overexpression of DnaA leads to overinitiation of oriCI [11] . Finally , DnaA can unwind oriCI but can't unwind oriCII , the origin of replication of the small V . cholerae chromosome ( chrII ) [12] . Several observations suggest that RctB , a 658 amino acid protein that lacks any known motifs or similarity to characterized initiators , is the initiator of chrII replication . First , RctB binds to several sites within oriCII [10] . Second , overproduction of RctB in V . cholerae promotes overinitiation of chrII and not chrI [11] . Third , RctB is necessary and sufficient to enable replication of oriCII-based minichromosomes in E . coli , and the copy number of such minichromosomes increases as the level of RctB is raised [12] , [13] . Finally , RctB can unwind oriCII and not oriCI [12] . rctB homologues are encoded by all vibrios that have been tested but are not found outside the Vibrionaceae [10] . As a conserved and essential gene product restricted to the Vibrionaceae , RctB might be an attractive target for new vibrio-specific antibiotics . Given the increasing prevalence of multi drug resistance in pathogenic vibrios [14]–[18] , there is a growing need for new drugs to combat these organisms . Here , we carried out a high throughput cell-based screen to find small molecule inhibitors of RctB . We identified a compound that blocks growth of an E . coli strain bearing an rctB-dependent plasmid but does not influence growth of E . coli lacking this plasmid . This compound , designated vibrepin , also has potent cidal activity against V . cholerae and inhibits growth of all vibrio species tested . Genetic and biochemical evidence strongly suggest that this compound , designated vibrepin , targets RctB . Our findings suggest that RctB is a useful drug target for the creation of Vibrionaceae-specific antimicrobial agents .
We developed a high throughput cell-based screen to identify small molecule inhibitors of RctB . This assay relied on pYB289 , a small plasmid that contains only oriCII , rctB and a gene ( aph ) that confers resistance to kanamycin ( Figure 1A ) . This plasmid can replicate in E . coli , since expression of rctB in E . coli is sufficient to enable replication of oriCII-dependent plasmids in this heterologous host [12] , [13] . E . coli harboring pYB289 exhibit kanamycin resistance , and we used kanamycin resistance as a marker of this plasmid's replication in our screen . We screened a library of ∼138 , 000 small molecules for compounds that inhibited growth of E . coli Mach1 harboring pYB289 in the presence of kanamycin but did not inhibit growth of Mach1 without the plasmid . Several candidate RctB inhibitors were identified , and one - 3 ( 3 , 4dichlorophenyl ) cyclopropane 1 , 1 , 2 , 2 tetracarbonitrile ( Figure 1B ) , designated here as vibrepin ( for vibrio replication inhibitor ) - was selected for further study , since its predicted pharmacologic properties were superior to the others . Vibrepin had no effect on the growth ( indicated by increased OD600 nm in cultures ) of E . coli strain DH5α ( Figure 1C ) . In addition , it did not influence growth in the presence of kanamycin of DH5α harboring pWSK129 or pYB190 , plasmids with distinct non-RctB dependent origins of replication ( pSC101 and pUC , respectively ) that contain aph cassettes , and so does not appear to interfere with establishment of kanamycin resistance ( Figure 1G and data not shown ) . However , 16 µg/ml vibrepin completely inhibited growth in the presence of kanamycin of DH5α containing the RctB-dependent plasmid pYB289 for 6 hr ( Figure 1D ) . Some growth of this strain was detectable after 6 hr , probably because the drug was no longer present , since these cells did not appear to be resistant to vibrepin if tested in fresh media . Although vibrepin-resistant DH5α/pYB289 did not arise in these assays , growth of DH5α containing one of several derivatives of pYB289 ( pYB340 or pYB344 ) that encode variants of RctB with single amino acid substitutions ( RctB L365I and P516Q , respectively , which were identified in an unrelated study , see materials and methods ) was not impaired by vibrepin ( Figure 1E and F ) . These strains grew as well in the presence of vibrepin as did DH5α lacking a plasmid . Since the only differences between strains DH5α/pYB289 , DH5α/pYB340 and DH5α/pYB344 are single amino acid differences in RctB , these observations strongly support the idea that vibrepin targets RctB . We also assessed the compound's effect on growth of DH5α/pYB289 in the absence of kanamycin . We anticipated that vibrepin would not inhibit bacterial growth under these conditions , since RctB activity should not be required in the absence of kanamycin . Unexpectedly , we found that vibrepin impaired the growth of DH5α/pYB289 even when plasmid replication was not required . Addition of 16 µg/ml vibrepin to cultures of this strain lacking kanamycin prevented an increase in OD600 nm for ∼2 hr and caused a decrease in the number of viable cells ( Figure 2B ) but did not influence growth of DH5α lacking this plasmid ( Figure 2A ) . Vibrepin also stimulated the loss of pYB289 from DH5α in the absence of kanamycin selection ( Figure 2D ) . Vibrepin even had a mild inhibitory effect on the growth of DH5α harboring pYB376 , a pSC101-based vector containing rctB ( Figure 2C ) . Collectively , these results suggest that production of wild type RctB in the presence of vibrepin may have toxic effects , at least in E . coli , and that such effects may contribute to the growth inhibition observed in the assay used above . However , since vibrepin was a less potent inhibitor of DH5α/pYB289 growth in the absence of kanamycin than in its presence , it is likely that vibrepin inhibits growth of this strain by more than one mechanism . To assess vibrepin's influence on RctB's activity as a replication initiator , we tested the compound's effects on unwinding of oriCII by RctB , using a P1 nuclease-based assay . In this assay , the single-strand specific P1 endonuclease cleaves a plasmid containing oriCII if it becomes unwound; linearized plasmid is then detected by agarose gel electrophoresis [12] . As seen in Figure 3A , unwinding of oriCII by RctB was markedly inhibited by vibrepin . DMSO , the solvent used to dissolve vibrepin , did not influence RctB unwinding activity ( data not shown ) . These observations are consistent with the hypothesis that vibrepin interferes with RctB function as an initiator of replication by blocking its ability to unwind oriCII . We also explored whether vibrepin influences the oligomeric state of RctB . In our ongoing studies of the mechanism of action of RctB , we found that purified RctB does not pellet after centrifugation at 20 , 000×g for 30 min in the absence of DNA . However , in the presence of 10 µg/ml of vibrepin , ∼50% of RctB was found in the pellet fraction after centrifugation; when higher amounts of vibrepin were added , most of the RctB added to the assay pelleted ( Figure 3B ) . In contrast , RctB [P516Q] , which appeared to be resistant to in vivo inhibition by vibrepin ( Figure 1F ) , was less susceptible than wild type RctB to vibrepin-induced aggregation in the pelleting assay ( Figure 3C ) . Furthermore , vibrepin did not promote the aggregation of either CpxR [19] ( Figure 3D ) or ParA2 ( data not shown ) , DNA-binding proteins unrelated to RctB . Thus , vibrepin does not indiscriminately aggregate proteins . The vibrepin solvent DMSO also did not promote the pelleting of RctB . Together , these findings suggest that vibrepin leads to the formation of high molecular weight complexes of RctB that are no longer soluble . Consistent with these observations , we found that vibrepin increased the apparent radius of RctB complexes approximately 4-fold in dynamic light scattering ( DLS ) assays ( from 153 to 588 , Figure 3E ) . The range of radii of the RctB complexes after addition of vibrepin was narrow , suggesting that vibrepin promotes the formation of RctB complexes of a particular stoichiometry rather than random aggregation of this protein . Although the apparent radius of RctB [P516Q] was greater than that of wild type RctB , vibrepin only had a minor effect ( from 257 to 370 , Figure 3F ) . Collectively these observations suggest that vibrepin may interfere with RctB oriCII unwinding by promoting the formation of non-functional RctB complexes . RctB is required for replication of V . cholerae chrII and is hypothesized to govern chrII replication initiation in all other vibrio species as well , since it is highly conserved . We therefore assessed whether vibrepin could inhibit the growth of vibrio species . Vibrepin prevented growth of V . cholerae at doses as low as 1 . 0 µg/ml ( Figure 4A ) . Used at this concentration , vibrepin induced stasis; however , at higher doses vibrepin had cidal activity . At concentrations of 4 µg/ml , vibrepin reduced the numbers of N16961 colony forming units ( CFU ) by more the 5 orders of magnitude within 30 minutes ( Figure 4A ) . Vibrepin also prevented growth of all additional vibrio species tested ( Table 1 ) . Drug concentrations ranging from 0 . 4 to 0 . 8 µg/ml were sufficient to inhibit the growth of the three major human vibrio pathogens V . cholerae , V . parahaemolyticus and V . vulnificus , as well as the shrimp pathogen V . nigripulchritudo ( Table 1 ) . In contrast , vibrepin concentrations <13 µg/ml did not inhibit growth of any of the E . coli strains we tested , including several pathogenic strains , and most E . coli strains were resistant to at least 16 µg/ml of vibrepin ( Table 1 ) . Together these observations are consistent with the idea that vibrepin targets RctB , a vibrio-specific essential protein . However , we also observed that vibrepin inhibited growth of Bacillus subtilis and Staphylococcus aureus ( Table 1 ) , two Gram-positive species that lack RctB homologues . Thus , it appears that some organisms contain vibrepin targets other than RctB . The vibrepin target ( s ) in these Gram-positive bacteria has yet to be defined . Several approaches were taken to confirm the target of vibrepin in V . cholerae . First , we repeatedly screened for V . cholerae mutants that had spontaneously acquired resistance to vibrepin; however , resistant colonies were never obtained . We were not able to introduce point mutations in the chromosomal copy of rctB . However , we transformed V . cholerae with plasmids encoding alleles of RctB that were resistant to vibrepin in E . coli ( pYB303 and pYB345 , encoding RctB L365I and P516Q , respectively ) , and assessed whether they conferred resistance . Exogenous production of RctB from these plasmids ( which did not alter V . cholerae's growth rate ) did not render cells resistant ( data not shown ) , suggesting that the presence of wt RctB results in dominant sensitivity , as might be expected given viprepin's toxicity in DH5α/pYB289 even in the absence of plasmid selection . Alternatively , V . cholerae may contain vibrepin targets in addition to RctB , which confer sensitivity even in the presence of resistant rctB alleles . Such targets may be related to the non-RctB targets that must exist in Gram-positive organisms . We used an RctB-GFP fusion protein to explore whether vibrepin altered the subcellular distribution of RctB in V . cholerae . We constructed a strain where rctB-gfp is expressed from the native rctB promoter by introducing a gene encoding GFP in-frame at the 3′ end of rctB . Since this rctB-gfp fusion is the only copy of rctB in the cell and this strain had wild type growth ( data not shown ) , the RctB-GFP fusion protein must be functional . In this strain , the distribution of RctB-GFP was generally diffuse , though small foci , usually near mid cell , were occasionally observed ( Figure 4B ) . One hour after addition of 1 µg/ml of vibrepin , the diffuse pattern of RctB-GFP fluorescence was no longer observed; instead , large puncta of RctB-GFP were seen ( Figure 4C ) . In control experiments , we found that vibrepin treatment did not alter the pattern of untagged GFP fluorescence ( Figure 4D and E ) , consistent with our observation that vibrepin does not lead to indiscriminate aggregation of proteins in vitro . These data suggest that the RctB complexes induced by vibrepin in vitro may be a reflection of its mode of action in vivo , and that vibrepin's toxicity for V . cholerae may result , at least in part , from induction of RctB aggregation . Different vibrepin targets could recognize distinct moieties in this compound . In an initial structure activity study of vibrepin , we identified a compound [3- ( 3-dimethylamino-phenyl ) -cyclopropane-1 , 1 , 2 , 2-tetracarbonitrile] ( referred to as C2 ) , that is similar in structure to vibrepin and that contains the same highly substituted cyclopropane moiety linked to a phenyl group ( compare Figures 5A and 1B ) . However , C2 did not inhibit growth of DH5α/pYB289 ( Figure 5C ) or V . cholerae ( Figure 5D ) . Since C2 lacks the chlorine substitutions on the phenyl group that are present in vibrepin , these observations may suggest that the chlorines in vibrepin are important for its targeting/inhibition of RctB . However , C2 is not void of antibiotic activity . This compound inhibited growth of B . subtilis ( Figure 5E ) and S . aureus ( Figure 5F ) , albeit with lower potency than vibrepin . Together , these observations raise the possibility that the additional ( non-RctB ) targets of vibrepin may interact with moieties of this compound that are chemically distinguishable from the parts of the molecule that inhibit RctB .
The development of new agents to combat emerging multidrug resistant pathogens is a critical challenge for infectious disease research [20] , [21] . In recent years , multidrug resistance in V . cholerae , V . vulnificus , and V . parahaemolyticus , important human pathogens [14]–[18] , and in vibrio species that damage shellfish and other marine organisms raised in aquaculture facilities [22] has been reported . Since RctB is required for replication of the vibrio second chromosome and conserved among the Vibrionaceae , we reasoned that RctB could be a target for development of antibiotics that specifically target vibrios . Our cell-based screen for small molecules that inhibited growth of an E . coli strain containing an oriCII- and RctB-dependent plasmid yielded vibrepin . Evidence that this compound targets RctB includes the observations that vibrepin did not inhibit growth of E . coli lacking the RctB-dependent plasmid or E . coli strains bearing nearly identical oriCII-dependent plasmids that contained single amino acid substitutions in RctB . Furthermore , vibrepin blocked unwinding of oriCII by RctB , apparently by promoting the formation of non-functional RctB complexes . Finally , vibrepin inhibited the growth of all vibrio species tested and had potent cidal activity against V . cholerae . Although vibrepin also appears to have targets other than RctB , our findings suggest that RctB is an attractive target for generation of antibiotics that only block growth of vibrios . One mechanism by which vibrepin appears to inhibit RctB function is by promoting formation of RctB complexes . Vibrepin induction of RctB complexes is relatively specific , as the compound hardly affected the oligomeric state of RctB[P516Q] and did not induce multimerization of two DNA-binding proteins unrelated to RctB , CpxR and ParA2 . There may be several consequences of RctB aggregation . First , since vibrepin inhibited RctB-mediated unwinding of oriCII , RctB complexes may be unable to initiate chrII replication . Second , since vibrepin impaired growth of DH5α/pYB289 in the absence of kanamycin , formation of RctB complexes may be toxic to cells even when RctB-dependent replication is not required . To date , the mechanism underlying such toxicity is unknown . Vibrepin also has targets other than RctB , since this compound inhibited the growth of bacterial species that do not encode RctB orthologues . The multiple effects of vibrepin on RctB as well as the possible existence of a non-RctB vibrepin target ( s ) in V . cholerae likely explains our inability to isolate vibrepin-resistant V . cholerae mutants . Bacteria-specific mediators of DNA replication might be expected to be attractive targets for antimicrobial agents . Recently , O'Donnell and colleagues identified a small molecule that inhibits the interaction of the E . coli β-clamp with DNA polymerases using an in vitro biochemical screen [23] . This compound did not inhibit the interaction of the yeast clamp with polymerase and hence should not target eukaryotes; however , it is not known whether this compound inhibits bacterial growth . Inhibitors of DnaA , the initiator of chromosome DNA replication in almost all eubacteria , might also have potential as broad spectrum antibiotics and Skarstad and colleagues reported the development of a high throughput cell-based assay to identify such inhibitors [24] . However , no antibiotics are currently in use that directly target bacterial chromosome replication . We found that RctB activity can be inhibited and thus showed that this initiator of replication of the second vibrio chromosome is a reasonable target for development of new Vibrionaceae-specific antimicrobial agents . Even though vibrepin has targets besides RctB , we anticipate that it will be possible to modify vibrepin or find new compounds that only target RctB . All antibiotics used in the clinic today are relatively broad spectrum and target highly conserved cellular processes . Therefore , these compounds inevitably select for resistant organisms in the normal human flora as well as in environmental microorganisms after antibiotics are shed into the environment . Genes that confer resistance can then be horizontally transmitted from bystander bacteria to pathogens . Vibrionaceae-specific antibiotics will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms . Thus , in principle , genes mediating resistance to these compounds will not arise in and be transferred from non-vibrios to vibrios , perhaps postponing the development of resistance . Vibrio–specific agents may be useful as new agents for aquaculture and in the prevention and treatment of human vibrioses .
High throughput screening for small molecule inhibitors of growth of YBA685 in the presence of kanamycin was carried out at the NSRB screening facility at Harvard Medical School . YBA685 is E . coli strain Mach1 containing pYB289 , an rctB-dependent vector ( Figure 1A ) . Mach1 ( Invitrogen ) , which grows faster than most laboratory E . coli strains , was used for the screening phase of our study to minimize the time required to detect growth inhibition . For screening , an overnight culture of YBA685 was inoculated at a 1∶500 dilution into LB broth containing kanamycin ( 50 µg/ml ) ; the culture was grown at 37°C until reaching an OD600 nm of ∼0 . 1; then , 30 µl aliquots of the culture were transferred into 384-well plates . The compound library ( 100 nl of 5 mg/ml in DMSO , final concentration 16 . 7 µg/ml ) was pin-transferred to the plates in duplicate . After the plates were incubated at 37°C for 3 hours , the OD600 nm of the wells was measured . We used Z-scores [25] to evaluate the growth inhibition of the compounds tested . The mean and standard deviation of the OD600 nm values from the experimental wells in each plate were obtained; then Z-scores for each well were calculated as the difference between the OD600 nm values in each well and the average OD600 nm divided by the standard deviation . Compounds with Z-scores below −3 in duplicate plates were considered positive hits . There were 149 positive hits among 137 , 694 compounds screened . These compounds were counter-screened to exclude molecules that inhibited the growth of Mach1 in the absence of pYB289 . Ultimately , we identified four compounds that inhibited growth of YBA685 in the presence of kanamycin but did not inhibit Mach1 growth . Vibrepin ( CID 2803695 ) and C2 were purchased in milligram quantities from Maybridge ( Tintagel , UK ) and Sigma-Aldrich ( St . Louis , MO ) , respectively . All compounds were dissolved at 50 mg/ml concentration in DMSO and stored at −20°C . The strains and plasmids used in this study are listed in Table 2 and 3 , respectively . As part of an on-going project to study oriCII-based replication , we have isolated a series of mutant oriCII-based plasmids that contain rctA , a negative regulator of RctB , and mutations in rctB ( see Materials and Methods of ref . [12] ) . These mutant rctB alleles can support replication of oriCII , at least in E . coli . We tested the sensitivity of DH5α harboring several of these mutant oriCII plasmids to vibrepin and found that plasmids A-52 and A-57 containing rctB[L365I] and [P516Q] respectively confer resistance to the compound . rctA was deleted from A-52 and A-57 , yielding pYB340 and pYB344 respectively , using the QuickChange XL Site Directed Mutagenesis Kit ( Stratagene ) . The later 2 plasmids are otherwise identical to pYB289 , which contains wild type rctB . The copy numbers of pYB289 , pYB340 and pYB344 in E . coli DH5α were approximately the same and the growth rates of DH5α bearing any of these 3 plasmids were indistinguishable . To create rctB [L365I] or rctB [P516Q] expression vectors , the relevant rctB variant was PCR amplified and then inserted into pGZ119EH as previously described [12] . rctB [P516Q] was inserted into pET28b ( Novagen ) ( yielding pYB346 ) , for high level expression of C-terminal His tagged RctB [P516Q] . All the relevant DNA sequences of all vectors used in this study were determined . The sequences of the PCR primers used in this study are available upon request . A V . cholerae strain containing a rctB-gfp translational fusion in the rctB locus and under the control of the native rctB promoter ( YBB874 ) and a strain harboring gfp gene inserted in the lacZ locus under the control of the Plac promoter ( YBB182 ) were constructed by allele exchange techniques using pCVD442-based plasmids ( pYB364 and pJZ111 , respectively ) as described [26] . A SynergyHT microplate reader ( BioTek , Winooski , VT ) was used to determine the growth kinetics shown in Figures 1 and 5 . In these experiments , overnight cultures were diluted 1∶200 and then incubated for 9 hours . For the growth curves shown in Figures 2 and 4A , overnight cultures were diluted into fresh media in test tubes and OD600 nm and CFU were determined at the indicated time points . All cultures were grown in LB media at 37°C except Tryptic Soy Broth ( BD ) was used for S . aureus cultures and LB containing 0 . 5 M NaCl was used for V . nigripulchritudo cultures which were grown at 30°C . The copy numbers of oriCII-based plasmids pYB340 and pYB344 relative to pYB289 were measured by quantitative PCR as described previously [12] . For Figure 2D , Southern hybridization of total genomic DNA was used to quantify pYB289 plasmid DNA ( relative to E . coli chromosomal DNA ) in vibrepin treated and untreated cells . A ∼600 bp DNA fragment of rctB was used as a probe for pYB289 and a similar sized narW fragment was used as a probe for the E . coli chromosome . Probe preparation and detection were carried out with the ECL Direct nucleic acid labeling and detection system ( GE Healthcare ) according to the manufacture's instructions . Bands were quantified using a Fujifilm FLA-5100 imager . The P1 nuclease-based assays for RctB unwinding of oriCII-containing plasmid substrates were performed as described previously [12] . Briefly , different concentrations of C-terminal His-tagged versions of RctB was mixed with 150 fmol of pOriII in 50 µl of a solution composed of 10 mM Hepes-KOH ( pH 7 . 6 ) , 8 mM magnesium acetate , 30% glycerol and 320 µg/ml BSA . After 10 min at 37°C , 1 . 2 units of P1 nuclease was added to each reaction for 30 seconds; the reactions were stopped with 40 µl of stop buffer ( 25 mM EDTA , 1% SDS ) . For quantification of the linearized fraction of the pOriII substrate DNA , an aliquot of the reaction was electrophoresed on a 0 . 8% agarose gel and then stained with ethidium bromide; the proportion of the plasmid DNA linearized by P1 nuclease was determined using densitometry . Purified C-terminal His-tagged RctB or RctB[P516Q] ( 10 µM ) was incubated in 20 µl buffer F ( 30 mM Tris pH 8 , 5 mM MgSO4 , 100 mM KCl , 2 mM DTT ) in the absence or presence of compounds for 10 min at 30°C . Reactions were then centrifuged for 30 min at 4°C at 20 , 000×g in a refrigerated table top centrifuge . The supernatant and pellet fractions were resuspended in 1X loading dye , electrophoresed on SDS PAGE gels and then the amount of protein in each fraction was determined by coomassie staining . DLS assays with a Viscotek 802 ( He–Ne laser , 633 nm ) were used to evaluate the effects of vibrepin on the apparent molecular mass of RctB and RctB[P516Q] . To remove any protein aggregates prior to the DLS assays , the proteins were centrifuged for 30 min at 20 , 000×g and then the supernatants were collected and filtered through a 0 . 1 µm ultrafree MC Milipore filter . In each assay , 300 ng of protein was added to a 12 µl reaction in sedimentation buffer F in a quartz cuvette in presence or absence of compounds . The amplitude plotted on the y-axis in Figure 3E and F is reflective of the intensity measurements generated in these experiments; intensity is proportional to the size and concentration of the scattering particles . Amplitude values were calculated using Omnisize 3 . 0 software . Each curve is representative of at least 5 measurements . | Multi-drug resistant bacteria continue to emerge and there is a pressing need for the development of new antibiotics . Here , we carried out a cell-based high throughput screen to identify inhibitors of RctB , the initiator of replication of the second chromosome found in all the species of the Vibrionaceae . This family of bacteria includes several human pathogens , including Vibrio cholerae , the cause of cholera , as well as several species that damage economically important marine organisms . We identified a compound—designated vibrepin—that has potent cidal activity against V . cholerae and inhibited growth of all vibrio species tested . Vibrepin blocked RctB unwinding of the origin of replication of the second V . cholerae chromosome , apparently by promoting the formation of large non-functional RctB complexes . Vibrepin represents a new class of antibiotic that specifically targets a particular family of microorganisms ( the Vibrionaceae ) . Such targeted agents will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms . Thus , in principle , genes mediating resistance to these compounds will not arise in and be transferred from non-vibrios to vibrios , perhaps postponing the development of resistance . | [
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] | 2009 | Targeting the Replication Initiator of the Second Vibrio Chromosome: Towards Generation of Vibrionaceae-Specific Antimicrobial Agents |
The obligate intracellular pathogen Chlamydia trachomatis is the most common cause of bacterial sexually transmitted diseases in the United States . In women C . trachomatis can establish persistent genital infections that lead to pelvic inflammatory disease and sterility . In contrast to natural infections in humans , experimentally induced infections with C . trachomatis in mice are rapidly cleared . The cytokine interferon-γ ( IFNγ ) plays a critical role in the clearance of C . trachomatis infections in mice . Because IFNγ induces an antimicrobial defense system in mice but not in humans that is composed of a large family of Immunity Related GTPases ( IRGs ) , we questioned whether mice deficient in IRG immunity would develop persistent infections with C . trachomatis as observed in human patients . We found that IRG-deficient Irgm1/m3 ( -/- ) mice transiently develop high bacterial burden post intrauterine infection , but subsequently clear the infection more efficiently than wildtype mice . We show that the delayed but highly effective clearance of intrauterine C . trachomatis infections in Irgm1/m3 ( -/- ) mice is dependent on an exacerbated CD4+ T cell response . These findings indicate that the absence of the predominant murine innate effector mechanism restricting C . trachomatis growth inside epithelial cells results in a compensatory adaptive immune response , which is at least in part driven by CD4+ T cells and prevents the establishment of a persistent infection in mice .
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes frequent infections in humans and significant morbidity throughout the world [1] . Ocular infection with C . trachomatis is the leading cause of preventable blindness worldwide and genital infection with C . trachomatis is the most common bacterial sexually transmitted infection ( STI ) in the United States [2] , [3] . The major complications of C . trachomatis genital tract infections arise primarily in women . Acute genitourinary infections with C . trachomatis remain asymptomatic in a high proportion of infected individuals , and therefore often go untreated . In a substantial number of infected untreated women C . trachomatis can establish persistent infections , which over time result in pelvic inflammatory disease and tubal scarring and can ultimately cause infertility [4] , [5] . C . trachomatis is a highly specialized , human-adapted pathogen with a narrow host range . Like many other pathogens with a very restricted host range , C . trachomatis has evolved to cause persistent infections in its preferred host enabling C . trachomatis to establish reservoirs for new infections and assure its survival as a pathogen within the human population [6] . Generally speaking , if a highly specialized pathogen enters a non-typical or “accidental” host , the non-typical host will either succumb to the infection and die or , more commonly , will rapidly clear the infection [7] . However , it is extremely rare for chronic infection to develop in a non-typical , immune-competent host . This basic principle holds true for experimental infections of laboratory mice with C . trachomatis . In contrast to human infections , C . trachomatis is rapidly cleared from mice when the organisms are instilled in the vagina or directly into the uterus [8] , [9] . If it were possible to model elements of human C . trachomatis pathogenesis in mice – with a mouse model of chronic human infection - it would accelerate the study of this disease and therapies to combat it . A first step toward this goal is to understand the underlying mechanisms that promote persistent C . trachomatis infections in the human host and prevent the establishment of chronic C . trachomatis infections in the murine host . A milestone in dissecting the basis for host tropisms of C . trachomatis was the discovery that IFNγ-induced cell-autonomous resistance in epithelial and other non-hematopoietic cell types like fibroblasts fundamentally differs between mice and humans . In human epithelial cells , IFNγ exerts its antimicrobial effect on C . trachomatis predominantly through the induction of indole-2 , 3-dioxygenase ( IDO ) . The enzyme IDO degrades intracellular tryptophan stores , thus starving C . trachomatis , a natural auxotroph for tryptophan , of this essential nutrient [10] , [11] . In contrast to human cells , most IFNγ-activated murine epithelial cells express insufficient amounts of IDO to restrict bacterial growth , with the notable exception of alveolar epithelial cells [10] , [12] , [13] , [14] . Accordingly , IDO is not required for the clearance of vaginal C . trachomatis infections in mice [10] , [15] . Instead , mice restrict Chlamydia species through a cell-autonomous resistance system that is executed by members of a large family of IFNγ-inducible GTPases called Immunity Related GTPases or IRGs [15] , [16] , [17] , [18] , [19] , [20] . Remarkably , the divergent IFNγ responses of these two host species , mice and humans , are reflected in the counter-immune mechanisms that exist in two closely related Chlamydia species with distinct host tropism . Whereas genital strains of the human pathogen C . trachomatis can utilize exogenous indole to produce tryptophan to overcome IDO-mediated growth restriction , the rodent-adapted species Chlamydia muridarum has evolved a mechanism to evade IRG-driven immune responses [19] , [21] . Given that the human pathogen C . trachomatis is highly susceptible to an IRG-driven immune response that is absent in its typical host , humans , but present in epithelial cell and fibroblasts of its non-typical host , mice , we investigated in this study whether the removal of the IRG resistance system would render mice permissive for persistent C . trachomatis genital infections . Here , we report that mice deficient for the expression of two pivotal IRG regulatory proteins , Irgm1 ( also called Lrg-47 ) and Irgm3 ( also called Igtp ) , initially develop high bacterial burden after genital infection compared to wildtype mice . However , in spite of the initial delay in immune clearance , Irgm1/m3 ( -/- ) mice are ultimately able to resolve a C . trachomatis infection as rapidly as wildtype mice . An exacerbated CD4+ T cell response is essential for the efficient clearance of genital C . trachomatis infections in Irgm1/m3 ( -/- ) mice and the prevention of persistence . Our data show that the absence of early innate immune defenses and the resulting unrestricted expansion of C . trachomatis trigger an amplified T cell response that results in sterilizing immunity .
We previously reported that Irgm1 and its paralog Irgm3 are required for resistance to C . trachomatis in an in vivo systemic infection model [19] . It is well established that IRG genes like Irgm1 and Irgm3 mediate a cell-autonomous antimicrobial response that directly targets vacuolar pathogens like C . trachomatis [22] , [23] , [24] . More recently it has been shown that at least one IRG family member , Irgm1 , is also required for the proper expansion of CD4+ T cells [25] . To determine if Irgm1 and Irgm3 deficient mice were more susceptible to C . trachomatis infections due to an intrinsic T cell deficiency , we first tested whether expression of Irgm1 and/or Irgm3 in T cells was required for the activation and expansion of CD4+ T cells during an intrauterine infection with C . trachomatis . Towards this goal we crossed the C . trachomatis-specific , MHC class II restricted T cell receptor transgene NR1 onto the Irgm1-/- , Irgm3-/- and Irgm1/m3 ( -/- ) genetic backgrounds . From these mice we derived naïve T cells and transferred these T cells into recipient wildtype mice . Although transfer of an antigen experienced or pre-activated population of NR1 cells can accelerate bacterial clearance , the naïve activation state and the low number of C . trachomatis-specific T cells transferred in these experiments did not result in accelerated immune clearance , as shown previously [26] . Therefore , the transfer of a relatively small number of IRG-deficient NR1 cells into a wildtype host allowed us to monitor pathogen specific immunity without altering the normal course of the immune response [26] , [27] . One day after NR1 cell transfer , we directly instilled C . trachomatis into the uterus of these mice by a transcervical infection method . Six days post-infection we monitored the activation and expansion of the transferred NR1 cells in the uterus and the draining lymph nodes of the genital tract . The transferred NR1 cells expressed an allele of the congenic surface marker CD90 that was distinct from the allele expressed by T cells derived from the recipient mice allowing us to specifically detect transferred T cells . We observed that the IRG genotype of the NR1 cells had no apparent impact on the expression of surface activation marker CD62L and CD25 ( Fig . 1A ) or on the expression of the markers CD127 , CD69 and CD44 ( data not shown ) . Similarly , transferred NR1 cells of distinct IRG genotypes were indistinguishable in regards to the proportion of cells expressing the cytokines IFNγ , TNFα and IL-2 ( data not shown ) . However , Irgm1-/- NR1 cells accumulated to significantly lower numbers in the uterus than wildtype NR1 cells did ( Fig . 1B ) , and showed a trend towards lower cell numbers in the draining lymph nodes of the genital tract ( Fig . 1B ) . These data are consistent with the previous observation that Irgm1-deficient CD4+ T cells fail to expand following an infection in mice [25] . Remarkably , we also found that the expansion defect of Irgm1-/- CD4+ cells was reversed by the concomitant removal of Irgm3 ( Fig . 1B ) , suggesting that Irgm1/m3 ( -/- ) T cells could be fully immune-competent . To directly determine whether Irgm1/m3 ( -/- ) NR1 cells could convey protection to an intrauterine infection with C . trachomatis , we transferred Th1 skewed wildtype and Irgm1/m3 ( -/- ) NR1 cells into Ifng-/- mice and subsequently instilled C . trachomatis directly into the uterus of these mice . In these experiments the early clearance of the infection is solely dependent on IFNγ secreted by the transferred NR1 cells , since the recipient mice are deficient for IFNγ production [26] . We found that NR1 cells doubly deficient in Irgm1 and Irgm3 conveyed protection against intrauterine C . trachomatis infection with an efficiency similar to wildtype NR1 cells ( Fig . 2 ) . In sum , these data indicated that the removal of Irgm3 salvaged the T cell intrinsic defect of Irgm1-/- cells and that Irgm1/3 ( -/- ) CD4+ T cells were fully functional . The inability of Irgm1-/- NR1 cells to expand to similar numbers as wildtype NR1 cells at the site of the infection could be explained by the propensity of Irgm1-deficient mature CD4+ T cells to prematurely die when activated for proliferation [25] . Considering that the concomitant removal of Irgm3 ‘rescues’ the T cell expansion defect of Irgm1-/- NR1 cells , we hypothesized that the simultaneous deletion of Irgm3 might serve to reverse the premature cell death phenotype of Irgm1-/- CD4+ T cells . To test this idea , we labeled wildtype , Irgm1-/- , Irgm3-/- and Irgm1/m3 ( -/- ) CD4+ T cells with CFSE and then activated them for proliferation using plate-bound anti-CD3 and anti-CD28 antibodies for 72 hours . As reported previously [25] , we observed increased total cell death of Irgm1-/- T cells compared to wildtype cells using propidium iodide incorporation as a measure for cell death ( Fig . 3 ) . The increase in cell death in Irgm1-/- T cells compared to wildtype cells was most pronounced in the population of CFSEhigh cells that had only undergone a few rounds of cell division . However , Irgm1/m3 ( -/- ) T cells showed a significant decrease in cell death compared Irgm1-/- T cells and the total percentage of dead Irgm3-/- and Irgm1/m3 ( -/- ) T cells was similar to wildtype T cells ( Fig . 3 ) . Taken together , these data showed that the deletion of Irgm3 reverses the T cell expansion and cell survival defect found in Irgm1-deficient T cells . IFNγ-activated cells lacking Irgm1 expression are impaired in their ability to contain the intracellular growth of various intracellular pathogens including Salmonella typhimurium , Toxoplasma gondii and C . trachomatis [22] , [23] , [24] . Recently , it has been shown that Irgm1-deficient cells can regain the ability to fully restrict growth of S . typhimurium when an additional genetic lesion in Irgm3 is introduced , suggesting that Irgm1 and Irgm3 are not essential to restrict S . typhimurium inside an infected cell [28] . In contrast , IFNγ-activated cells doubly deficient in Irgm1 and Irgm3 remain at least as susceptible to a T . gondii infection as cells harboring single Irgm gene deletions [28] . To determine whether Irgm1 and Irgm3 are essential for IFNγ-induced cell-autonomous resistance to C . trachomatis , we generated Irgm1/m3 ( -/- ) mouse embryonic fibroblasts ( MEFs ) . MEFs lacking both Irgm1 and Irgm3 displayed a near complete loss of IFNγ-induced resistance to C . trachomatis growth . Whereas only very few C . trachomatis inclusion were detectable in IFNγ-activated wildtype MEFs , the number of inclusions found in IFNγ-treated and untreated Irgm1/m3 ( -/- ) MEFs was similar ( Fig . 4A ) . To more accurately quantify C . trachomatis replication in these MEFs , we harvested DNA from infected cells at 4 and 30 hours post-infection ( hpi ) and measured the amount of Chlamydia DNA using qPCR . Neither IFNγ treatment nor IRG deficiency had any appreciable effect on C . trachomatis burden at 4 hpi , suggesting normal bacterial attachment and entry under all conditions . By 30 hpi IFNγ activation , C . trachomatis yields were reduced by nearly 2 logs in wildtype MEFs . In contrast , we only observed a 2-fold reduction in IFNγ-treated Irgm1/m3 ( -/- ) MEFs ( Fig . 4B ) . Because it was formally possible that C . trachomatis was able to replicate but unable to differentiate into infectious elementary bodies in IFNγ-activated Irgm1/m3 ( -/- ) MEFs , we also assayed the release of infectious EBs by monitoring the production of inclusion forming units ( IFUs ) in these cells . Consistent with our findings by qPCR ( Fig . 4B ) , we found that the ability of IFNγ-activated MEFs to restrict the production of infectious C . trachomatis progeny was severely compromised in Irgm1/m3 ( -/- ) MEFs ( Fig . 4C ) . Collectively , our results showed that Irgm1/m3 ( -/- ) MEFs have lost their ability to efficiently restrict growth of C . trachomatis upon IFNγ activation . Because these experiments were conducted in tryptophan-rich culture media , we could not dismiss a possible role for the tryptophan-degrading enzyme IDO in restricting C . trachomatis growth in IFNγ-activated MEFs . To test for such a function of IDO , we first measured induction of mouse IDO1 expression upon IFNγ activation . In contrast to the approximately 1000-fold induction of Irgm3 and Irgb10 mRNA expression , IDO1 mRNA was only moderately induced ( 5-fold ) , and more importantly , inhibition of C . trachomatis growth upon IFNγ treatment remained unchanged in the presence or absence of tryptophan in the culture media ( Fig . S1A and B ) . Because ectopic expression of IDO resulted in significant restriction of C . trachomatis growth in MEFs ( Fig . S1C ) , we conclude that endogenous IDO expression levels are too low in IFNγ-stimulated MEFs to restrict C . trachomatis growth . Since MEFs isolated from Irgm1/3-/- mice failed to constrain C . trachomatis growth in the presence of IFNγ , we expected that these mice would also be unable to control C . trachomatis genital infections . To test this idea , we infected wild type , Ifngr1-/- , and Irgm1/m3 ( -/- ) mice transcervically with C . trachomatis and measured the bacterial burden in the genital tract over 45 days . As expected , mice lacking the gene encoding the IFNγ receptor ( Ifngr1-/- ) showed greater bacterial burden than wildtype mice over the entire time course ( Fig . 5 ) . Initially Irgm1/m3 ( -/- ) mice also developed high bacterial burden similar to the burden observed in Ifngr1-/- mice , yet by day 15 post-infection the number of organisms present in Irgm1/m3 ( -/- ) mice was reduced to levels similar to the ones found in wildtype mice ( Fig . 5 ) . By day 45 post-infection , C . trachomatis was still detectable in Ifngr1-/- mice but undetectable in wildtype and Irgm1/m3 ( -/- ) mice . These data show that although Irgm1/m3 ( -/- ) mice were initially defective in clearing C . trachomatis infections , they ultimately were able to control the infection . Therefore , we conclude that Irgm1/m3 ( -/- ) mice do not exhibit an infection of extended duration as had been observed with the Ifngr1-/- mice . We then began to investigate how Irgm1/m3 ( -/- ) mice were able to clear C . trachomatis infections despite the initially high bacterial burden observed at day 5 and earlier . One possible explanation was that the increased antigen burden in the Irgm1/m3 ( -/- ) mice stimulates the expansion and activation of C . trachomatis-specific effector T cells . To test the idea that host Irgm1/m3-deficiency could impact the development of wildtype T cells , we transferred naïve , CFSE-labeled , wildtype C . trachomatis-specific NR1 cells into wildtype , Ifngr1-/- or Irgm1/m3 ( -/- ) host mice . Although we had already shown that Irgm1/m3 ( -/- ) T cells appear to be indistinguishable from wildtype T cells ( Fig . 1 and 2 ) , we opted to use wildtype NR1 cells in these experiments in order to exclude any cell-autonomous effects on T cells that could be caused by the presence of the Ifngr1-/- or the Irgm1/m3 ( -/- ) alleles . One day after the transfer of wildtype NR1 cells into mice of the indicated genotypes , mice were infected with C . trachomatis in the uterus . We found that C . trachomatis infection stimulated robust proliferation of transferred NR1 cells regardless of the genotype of the recipient mice ( Fig . 6A , top panel ) . Though the genotype of the recipient mouse had no discernable impact on NR1 cell proliferation based on CSFE dye dilution , we did observe a significant increase in the total number of NR1 cells in the iliac lymph node ( Fig . 6A and 6B ) and the uterus ( Fig . 6C ) of Irgm1/m3 ( -/- ) mice on day 6 post-infection compared to Ifngr1-/- and wildtype mice . The greater expansion of NR1 cells in Irgm1/m3 ( -/- ) mice corresponded to an increase in the population of NR1 cells expressing the IL-2 high affinity receptor CD25 on NR1 cells resident in C . trachomatis-infected Irgm1/m3 ( -/- ) mice compared to infected Ifngr1-/- and wildtype mice ( Fig . 6A ) . Additionally , a significantly larger proportion of NR1 cells expressed IFNγ , the hallmark cytokine of Th1 activation , and the proinflammatory cytokine TNFα in combination with IFNγ , when transferred into Irgm1/m3 ( -/- ) recipient mice compared to Ifngr1-/- and wildtype recipient mice ( Fig . 6D ) . As expected for conventional Th1 cells , we did not detect significant IL-17 expression in NR1 cells transferred into either wildtype or Irgm1/m3 ( -/- ) mice on day 6 post-infection ( data not shown ) . These results suggested that enhanced expansion of C . trachomatis-specific T cells and a boost in their activation status may compensate for the loss of innate immune restriction due to the Irgm1 and Irgm3 mutations . To prove that a compensatory amplification of the T cell response was responsible for clearance of C . trachomatis from the genital tract , we subjected groups of Irgm1/m3 ( -/- ) and wildtype mice to multiple treatments with either anti-CD4 depleting or control antibodies and then determined bacterial burden in the uterus at 15 days post-infection . As expected , depleting CD4+ T cells resulted in greater bacterial burden in both wildtype as well as Irgm1/m3 ( -/- ) mice relative to control mice of the same genotype , confirming that the adaptive immune response contributes to immune clearance at later times in the course of the infection ( Fig . 7 ) . Depletion of CD4+ T cells , however , had a greater effect on bacterial burden in Irgm1/m3 ( -/- ) mice than it did in wildtype mice , increasing bacterial yield in the uterus by two logs instead of one log . These data confirmed that Irgm1/m3 ( -/- ) mice compared to wildtype mice developed an exacerbated T cell response that was instrumental in the rapid clearance of genital infection with C . trachomatis . We then began to investigate the mechanism by which an exacerbated CD4+ T cell response clears C . trachomatis infections in mice lacking cell-autonomous IRG resistance in epithelial cells . Because of the recent demonstration that Chlamydia-specific T cells can control Chlamydia replication inside epithelial cells by a Nos2-dependent mechanism [29] , we tested whether Nos2 was responsible for the elimination of C . trachomatis in Irgm1/m3 ( -/- ) mice . To test this hypothesis , we generated Irgm1/m3 ( -/- ) Nos2 ( -/- ) triple knockout mice . Although C . trachomatis-infected Irgm1/m3 ( -/- ) Nos2-/- mice trended towards higher bacterial burden compared to Irgm1/m3 ( -/- ) or wildtype mice , this effect was not statistically significant in our experiments ( Fig . S2 ) . While exploring alternative CD4-dependent immune mechanisms targeting C . trachomatis in Irgm1/m3 ( -/- ) mice , we identified an increase in the uterine population of cells expressing the neutrophil surface marker GR1 relative to wildtype mice at day15 post-infection . An increase in the number of GR1+ cells was also observed in the iliac lymph nodes but not in the spleen of Irgm1/m3 ( -/- ) mice ( Fig . 8A ) . Similar to the depletion of CD4+ T cells , the depletion of the GR1+ cell population or the simultaneous depletion of CD4+ and GR1+ cells in Irgm1/m3 ( -/- ) mice resulted in a dramatic increase in bacterial burden ( Fig . 8B ) , suggesting an important role for neutrophils in the elimination of C . trachomatis in Irgm1/m3 ( -/- ) mice . Lastly , we explored the question as to why Irgm1/m3 ( -/- ) mice cleared C . trachomatis infections more efficiently than Ifngr1-/- mice did ( Fig . 5 ) . Both Irgm1/m3 ( -/- ) and Ifngr1-/- mice lack IRG-mediated cell-autonomous resistance , yet differ in the number and activation status of their Chlamydia-specific T cell population ( Fig . 6 ) . To determine whether T cells in conjunction with neutrophils are responsible for the relatively more efficient elimination of C . trachomatis in Irgm1/m3 ( -/- ) mice compared to Ifngr1-/- mice , we treated Irgm1/m3 ( -/- ) , Ifngr1-/- and wildtype mice with anti-CD4 and anti-GR1 depleting antibodies , and determined bacterial burden at 15 days post infection . As expected , bacterial burden was dramatically elevated in all CD4/GR1-depeleted animals regardless of their genetic background . However , the most dramatic effect on bacterial burden was observed in Irgm1/m3 ( -/- ) mice ( Fig . S3 ) elevating the bacterial yield to the same levels observed in similarly treated Ifngr1-/- mice . These results suggest that IFNγ directs T cell- and neutrophil-dependent clearance of C . trachomatis infections .
Genital infections with C . trachomatis are among the most common STI worldwide and constitute the most frequent bacterial STI in the United States . Asymptomatic and consequently unrecognized and untreated C . trachomatis infections can ascend from the cervix to the fallopian tube and establish persistence resulting in irreversible tissue damage and infertility [30] , [31] . In contrast to humans , mice clear genitourinary infection with C . trachomatis rapidly and members of the IRG gene family play an important role in conveying resistance to C . trachomatis infections in the mouse . Whereas the importance for IRG proteins in resistance to C . trachomatis infections in mice is undisputed , the function of the constitutively expressed human ortholog IRGM in the pathogenesis of human C . trachomatis infections is less clear . Although IRGM induces antimicrobial xenophagy in human cells [32] , [33] , [34] , [35] , most human cells restrict growth of C . trachomatis by an IDO-dependent and apparently IRGM-independent mechanism ( [10] , [11] , [15] and Fig . S1B ) . Although IRGM may still prove to be important in providing resistance to C . trachomatis infections in some cell types or tissues , IRGM is less likely to play a prominent role in the pathogenesis of human C . trachomatis infections . For these reasons , we sought to determine whether mice deficient in IRG-dependent resistance to C . trachomatis - and thus resembling humans in that regard - would develop persistent C . trachomatis infections . We have previously shown that two members of the IRG gene family , Irgm1 and Irgm3 , mediate resistance to C . trachomatis in an in vivo model of systemic infection [19] . Because the gene Irgm1 not only conveys cell-autonomous resistance to vacuolar pathogens inside an infected host cell but is also required to prevent the premature cell death of activated , proliferating CD4+ T cells , we first tested the hypothesis that the inability of IRG-deficient mice to efficiently restrict C . trachomatis infections could partly be due to a diminished CD4+ T cell response . We found that Irgm1-/- CD4+ T cells specific for a C . trachomatis epitope failed to efficiently expand in the uterus of genitally infected animals and prematurely died when activated for proliferation ex vivo . These results are consistent with the previously described expansion defect of Irgm1-/- CD4+ T cells [25] . However , we also found that the simultaneous removal of Irgm3 in Irgm1/m3 ( -/- ) CD4+ T cells ‘rescued’ the phenotype of Irgm1-/- CD4+ T cells . Two alternative models may account for these observations: the first model is based on the published finding that IFNγ-induced IRG proteins form large protein aggregates in the absence of Irgm1 expression [36] , [37] . These protein aggregates are not found in IFNγ-activated wildtype cells and are likely to have cytotoxic properties . The concomitant removal of Irgm3 has been shown to reduce the levels of IRG protein aggregates inside a cell , possibly below a threshold level of toxicity [28] . Because T cells have a relatively small cytoplasmic volume compared to most differentiated cells , they may be particularly susceptible to the cytotoxic effects of IRG protein aggregates . According to the “aggregate model” recently proposed by Hunn and Howard [38] , the expansion defect of Irgm1-/- CD4+ T cells could be due to the cytotoxicity of IRG protein aggregates that form when the finely balanced network of IRG protein interactions is artificially disrupted by the Irgm1 gene deletion . In a second , alternative , model IFNγ-induced IRG mediated cell death in T cells is a regulated biological process that plays an important role in fine-tuning T cell homeostasis . Our observations can be reconciled with a model in which Irgm3 protein acts as an inducer of cell death whereas Irgm1promotes cell survival , functioning as an Irgm3 antagonist . According to this model , regulated changes in the expression of Irgm1 relative to Irgm3 or post-translational modifications of either Irgm protein could shift the balance towards either T cell survival or T cell death in wildtype T cells . Removal of the pro-survival factor Irgm1 in Irgm1-/- T cells would result in uncontrolled cell death due to the unrestricted action of Irgm3 . Further removal of the pro-death factor Irgm3 in Irgm1/3 T cells would restore cell survival . In support of the latter model , we found that anti-CD3 stimulated Irgm3-/- CD4+ T cells showed improved cell viability compared to wildtype T cells at least during the first few rounds of anti-CD3 stimulated cell division ( Fig . 2 ) , suggesting that Irgm3 may indeed function as a pro-cell death molecule . Additionally , we observed that Irgm3-/- mice ( but not Irgm1-/- or wildtype mice ) expressing the NR1 transgene relatively frequently developed lymphoma with advanced age ( JC and MNS , unpublished data ) , indicating a potential role for Irgm3 as a tumor suppressor gene . Beyond the uncertainty of the biological functional importance of IRG proteins in murine T cells , the additional unanswered question remains as to whether IRGM , the single human IRG ortholog constitutively and ubiquitously expressed in human cells , plays any role in regulating T cell homeostasis . Because Irgm1/3 double deficiency does not intrinsically affect T cell expansion or function , we were able to investigate whether the absence of IRG-mediated resistance in epithelial cells in an otherwise immune-competent host would impact the course and duration of a genital C . trachomatis infection . We found that Irgm1/m3 ( -/- ) mice initially failed to effectively clear C . trachomatis during the early course of an infection . This observation can be explained with a nearly complete defect in IFNγ-induced cell-autonomous resistance in cells doubly deficient in Irgm1 and Irgm3 ( Fig . 3 ) . At later time points , however , Irgm1/m3 ( -/- ) mice cleared genital C . trachomatis infections rapidly due to an exacerbated C . trachomatis-specific CD4+ T cell response . The observation that Irgm1/m3 ( -/- ) mice developed a more pronounced T cell response towards C . trachomatis than Ifngr1-/- mice did , can be explained by the important role IFNγ plays in stimulating the adaptive immune response , for example , through improved antigen presentation allowing for direct cytolysis by degranulating CD4 T cells [29] and/or enhanced neutrophil recruitment and survival [39] , [40] , [41] , [42] . It has also been reported that IFNγ acts as a positive regulator of CD25 expression on CD4+ T cells in a mouse model of myocarditis , though it is unclear whether IFNγ directly or indirectly controls CD25 expression in CD4+ T cells [43] . The boost in CD25 surface expression on wildtype NR1 cells resident in C . trachomatis-infected Irgm1/m3 ( -/- ) mice compared to C . trachomatis-infected Ifngr1-/- mice may therefore be the net result of high antigen burden and simultaneous IFNγ activation of antigen presenting cells . In addition to an increase in CD25 expression on NR1 cells , we also observed greater accumulation of NR1 cells in C . trachomatis infected Irgm1/m3 ( -/- ) mice compared to C . trachomatis infected wildtype or Ifngr1-/- mice . It seems likely that these two phenotypes are linked: the surface protein CD25 is identical to the alpha chain of the high-affinity , trimeric IL-2 receptor and expression of CD25 has been shown to be upregulated on activated conventional T cells [44] , [45] . Although signaling through the IL-2 receptor is not essential for T cell effector function in vivo , IL-2 is known to promote T cell survival and expansion [46] , [47] , [48] , [49] . The increase in CD25 expression in NR1 cells and the resulting boost in IL-2 mediated cell survival may therefore be the underlying cause for the greater expansion of NR1 cells that we observed in C . trachomatis infected Irgm1/m3 ( -/- ) mice . Collectively , these data indicate that the virtual absence of cell-autonomous host resistance during the early course of a C . trachomatis infection triggers a compensatory adaptive immune response in an otherwise fully immune-competent host and ultimately prevents the establishment of a sustained genital infection with C . trachomatis in mice . These results prompt the question of how C . trachomatis can prevent clearance by the adaptive immune response in its natural host , humans , which lack a robust IRG-dependent immune response that targets C . trachomatis directly . We propose that the answer to this question lies in the adaption of C . trachomatis to a different cell-autonomous IFNγ response – one found in humans where the pathogen has evolved but absent from urogenital epithelial cells in mice [50] . A wealth of in vitro experimental data shows that IFNγ activation of human epithelial cells results in the expression of IDO , which leads to the depletion of intracellular tryptophan stores [10] , [51] , [52] , [53] , [54] . In response to tryptophan starvation , C . trachomatis transforms from an active , replicating , state into a quiescent form [55] . Replication of these organisms stops , yet they endure in a quiescent form until immune response wanes and tryptophan becomes available again [4] , [5] , [55] , [56] , [57] . The IDO-induced , non-replicating quiescent organisms are less likely to trigger a strong adaptive immune response due to reduced antigenic burden . Additionally , the quiescent bacterium may be more resistant to immune effector mechanisms than its replicating form . We therefore propose that in order to avoid clearance by the adaptive immune response , C . trachomatis has evolved to co-opt the IDO-driven cell-autonomous immune response as an inducer of bacterial quiescence . Accordingly , a humanized mouse model for chronic C . trachomatis infections would require both the removal of the murine IRG response and the recreation of IDO-mediated cell-autonomous immunity in urogenital epithelial cells .
All experiments were approved by the Institutional Animal Care and Use Committee of Harvard Medical School . Harvard maintains an animal care and use program certified by The Association for the Assessment and Accreditation of Laboratory Animal Care ( AAALAC ) and all procedures are conducted in accordance with guidelines established by the American Veterinary Medical Association . All mice were maintained and bred under specific pathogen-free conditions . Control C57BL/6J ( wildtype ) B6 . Ifngr1-/- and B6 . Ifng-/- mice were obtained from The Jackson Laboratory . The targeted gene deletions of Irgm1 and Irgm3 , mice doubly deficient for Irgm1 and Irgm3 and NR1 mice expressing a TCR transgene specific for the C . trachomatis antigen Cta1 have been described previously [27] , [28] , [58] , [59] . The NR1 T cell receptor transgene was crossed onto the Irgm1-/- , Irgm3-/- and Irgm1/m3 ( -/- ) genetic backgrounds . C . trachomatis serovar L2 434/Bu were propagated in McCoy cells and purified as described [17] . Cells were routinely cultured in high glucose DMEM ( Gibco ) supplemented with 10% fetal calf serum . For those experiments in which we monitored the antimicrobial effects of IDO-mediated tryptophan depletion , cells were transferred into DMEM F-12 culture medium lacking tryptophan ( US Biological ) supplemented with 3% fetal calf serum . Limiting the amount of calf serum was necessary to reduce the amount of serum-derived tryptophan to levels at which IFNγ-activated HeLa cells maximally restricted growth of C . trachomatis . Where indicated media was supplemented with tryptophan at a final concentration of 0 . 05 mg/ml . To quantify the bacterial load in Chlamydia-infected cells and in the uteri of infected animals , a previously described quantitative PCR assay was applied [17] . Briefly , total nucleic acid from infected cells or spleen homogenates was prepared using the QIAamp DNA mini kit from Qiagen . Chlamydia 16S DNA and mouse GAPDH DNA content of individual samples was then quantified by qPCR on an ABI 7000 sequence detection system using primer pairs and dual-labeled probes . Standard curves were generated from known amounts of Chlamydia and mouse DNA , and these curves were used to calculate the mass of Chlamydia DNA per unit mass of mouse DNA in the samples . Alternatively , infected cells in culture were lysed by sonication and combined with their culture supernatants . Serial dilutions of the lysate were applied to McCoy cell monolayers . Inclusions were counted by immunofluorescence microscopy 30 hours post-infection . Mouse embryonic fibroblasts were generated from the indicated mouse strains as previously described [17] . Cells were treated with 100 U/ml recombinant mouse IFNγ ( Chemicon International ) over night before infection or left untreated . Cells were infected with C . trachomatis at a multiplicity of infection ( MOI ) of 2 in SPG buffer ( 220 mM sucrose , 12 . 5 mM phosphate , and 4 mM L-glutamic acid ( pH 7 . 5 ) by centrifugation at 1928 x g for 1 h at 37°C and then returned to standard medium . Chlamydia inclusions were detected in infected cells using mouse anti-Hsc70 antibody ( Abcam ) , followed by anti-mouse secondary fluorescently labeled antibody . Cell nuclei were visualized with 4′ , 6-diamidino-3-phenylindole ( DAPI ) staining and epifluorescent images were acquired with a Nikon Eclipse TE2000-U microscope using a Nikon Plan Apo 20x /0 . 75 N . A . Phase 2 objective . Images were saved as TIFF files and imported into Adobe Illustrator for labeling . Before transfer , C . trachomatis-specific CD4+ T cells were isolated from peripheral lymphoid tissues of mice transgenic for the NR1 TCR ( Vα2+ , Vβ8 . 3+ ) and labeled with 5 µM carboxyfluorescein-diacetate-succinimidyl-ester ( CFSE ) in serum-free medium as described [26] . One day before an intrauterine infection with C . trachomatis recipient CD90 . 2+ mice were injected i . v . with 106 lymphocytes derived from NR1 transgenic animals of the indicated genetic backgrounds and congenically marked with the surface marker CD90 . 1+ . To infect the genital tract , mice were treated with 2 . 5 mg of medroxyprogestrone acetate s . c . and one week later 2*106 inclusion-forming units of C . trachomatis L2 were instilled into the uterus using a commercially available non-surgical embryo transfer device ( Paratechs ) . In order to deplete CD4+ T cells , mice were injected intraperitoneally with 0 . 5 mg anti-CD4+ mAb ( GK 1 . 5 ) two days prior to infection and with 0 . 25 mg of the same antibody on days 0 , 2 , 5 , 7 , 9 and 12 post-infection . To deplete GR1+ cells , mice were injected with the anti-GR1+ depleting antibody RB6-8C5 and control mice were injected with the isogenic rat IgG2b antibody LTF-2 ( BioXCell ) following the same injection regiment . At the indicated times post-infection , lymph nodes and uteri were collected . Uteri were digested with 1 mg/ml type XI collagenase and 50 , 000 units/ml DNase for 45 minutes at 37°C . Single-cell suspensions were prepared for staining via mechanical disaggregation . Tissues were mechanically disaggregated and immediately stained for activation markers or stimulated for 5 h with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of brefeldin A to determine intracellular cytokine staining . Cells were preincubated with anti-FcRγ ( Bio X-Cell ) before staining with anti-CD4 Pacific Blue ( Biolegend ) , anti-CD90 . 1 peridinin chlorophyll-a protein ( BD Bioscience ) , and Live/Dead Aqua ( Invitrogen ) . For activation marker analysis , we examined anti-CD62L allophycocyanin-Alexa 750 ( Ebioscience ) , and anti-CD25 allophycocyanin ( BD Bioscience ) . For intracellular staining , the following antibodies were used: anti-IFNγ-PE or -Alexa 700; anti-IL2-PE or -allophycocyanin; and anti-TNFα-PE or -PE-CyChrome 7 ( BD Biosciences ) . Cells were permeabilized with the Cytofix/Cytoperm Plus kit according to the manufacturer's instructions ( BD Bioscience ) . In all samples , an unbiased total of 106 lymphocytes were collected based on forward and side scatter gating . Post-acquisition , lymphocytes were gated based on forward and side scatter , dead cells were excluded , and NR1+ cells were delineated by gating on CD4+ , CD90 . 1+ , Vα2+ , Vβ8 . 3+ T cells events . Data were collected on a modified FACSCalibur ( Cytek Development ) or an LSRII ( BD Bioscience ) and analyzed using Flow Jo ( Tree Star ) . Spleens were mechanically disaggregated , red blood cells were lysed and cell suspension was enriched for CD4+ T cells using the Dynal Mouse CD4+ Negative Isolation Kit ( Invitrogen ) according to the manufacturer's instructions . Isolated cells were CFSE labeled and 105 cells were plated out in wells of a 96-well flat bottom plates coated with anti-CD3 ( 500 ng/ well ) and anti-CD28 ( 50 ng/ well ) antibodies . Three days later cell viability was assessed by flow cytometry with propidium iodide staining . CD4+ T cells were purified from NR1 mice using a mouse CD4+ isolation kit ( Dynal; Invitrogen ) per the manufacturer's directions . The T cells were cultured in RPMI 1640 ( Invitrogen ) supplemented with 10% FCS , L-glutamine , HEPES , 50 µM 2-ME , 50 U/ml penicillin , and 50 µg/ml streptomycin . To stimulate the T cells , irradiated feeder splenocytes were pulsed with 5 µM Cta1133–152 peptide and co-cultured with the CD4+-enriched NR1 cells at a stimulator to T cell ratio of 4∶1 . To polarize T cells towards Th1 , T cells were incubated with 10 ng/ml IL-12 ( Peprotech ) and 10 µg/ml anti-IL-4 ( Biolegend ) for 5–7 days . 107 Th1-skewed C . trachomatis-specific CD4+ T cells were transferred into mice , and 24 h later mice were infected in the uterus as described above . Uteri were harvested 6 days after infection . To assess the protective capacity of the skewed cells , uteri from infected mice were homogenized , and DNA was prepared as described above and used for qPCR . All groups were evaluated for statistical significance through the use of unpaired two-tailed t tests . Where it appeared necessary to highlight significant differences between data points , the level of significance is depicted as: * , p<0 . 05; ** , p<0 . 01; and *** , p<0 . 005 . | Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease and can lead to pelvic inflammatory disease , ectopic pregnancy , infertility , and other complications in women . These serious complications have been difficult to study experimentally in laboratory animals because C . trachomatis only causes these complications in humans . Previous work in our laboratory has identified two mouse genes responsible for resistance to C . trachomatis . Here we consider whether mice lacking the two resistance genes might succumb to the same complications of infection observed in humans . Although initial infection of the mice lacking the two resistance genes is greater in magnitude compared to normal mice , eventually the mice resist serious infection . We found that one component of the mouse immune system , the T cells , expands more robustly to compensate for the lack of the resistance genes . | [
"Abstract",
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] | [
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] | 2011 | Compensatory T Cell Responses in IRG-Deficient Mice Prevent Sustained Chlamydia trachomatis Infections |
Two key biological features distinguish Trypanosoma evansi from the T . brucei group: independence from the tsetse fly as obligatory vector , and independence from the need for functional mitochondrial DNA ( kinetoplast or kDNA ) . In an effort to better understand the molecular causes and consequences of these differences , we sequenced the genome of an akinetoplastic T . evansi strain from China and compared it to the T . b . brucei reference strain . The annotated T . evansi genome shows extensive similarity to the reference , with 94 . 9% of the predicted T . b . brucei coding sequences ( CDS ) having an ortholog in T . evansi , and 94 . 6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater . Interestingly , several procyclin-associated genes ( PAGs ) were disrupted or not found in this T . evansi strain , suggesting a selective loss of function in the absence of the insect life-cycle stage . Surprisingly , orthologous sequences were found in T . evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T . brucei , although a small number of these may have lost functionality . Consistent with previous results , the F1FO-ATP synthase γ subunit was found to have an A281 deletion , which is involved in generation of a mitochondrial membrane potential in the absence of kDNA . Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T . evansi reads . Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T . evansi strains with worldwide distribution that also includes isolates classified as T . equiperdum . At least three other types of T . evansi or T . equiperdum have emerged independently . Overall , the elucidation of the T . evansi genome sequence reveals extensive similarity of T . brucei and supports the contention that T . evansi should be classified as a subspecies of T . brucei .
Trypanosomatid parasites Trypanosoma evansi and T . equiperdum are responsible for animal diseases with extensive pathological and economic impact and closely related to the T . brucei group [1] , [2] . The latter includes three subspecies: the human parasite T . b . rhodesiense , the zoonotic parasite T . b . gambiense , and the animal parasite T . b . brucei . Together T . brucei , T . evansi , and T . equiperdum comprise the subgenus Trypanozoon . The exact nature of the phylogenetic relationship between these three species has been the subject of ongoing debate , with some evidence suggesting that T . evansi and T . equiperdum are monophyletic and other evidence suggesting that they are polyphyletic and have emerged multiple times from T . b . brucei [3]–[6] . Trypanosomatids are a family within the protist group Kinetoplastida , the eponymous feature of which is a large and complex network of circular DNAs ( kinetoplast or kDNA ) inside their single mitochondrion . Two key biological features distinguish T . evansi and T . equiperdum from the T . brucei group . Firstly , their transmission is independent from the tsetse fly as obligatory vector . T . evansi is predominantly transmitted by biting flies and causes surra in a wide variety of mammalian species ( the name of the disease varies with geographical area ) , while T . equiperdum causes a sexually transmitted disease called dourine in horses [1] , [3] , [7] . The altered mode of transmission has enabled both parasites to escape from the sub-Saharan tsetse belt and become the pathogenic trypanosomes with the widest geographical distribution . Secondly , all strains of T . evansi and T . equiperdum investigated so far are dyskinetoplastic , i . e . , lacking all ( akinetoplastic ) or critical parts of their kDNA [8] . The loss of kDNA is thought to lock T . evansi and T . equiperdum in the bloodstream life cycle stage , presumably because the absence of kDNA-encoded components of the oxidative phosphorylation system prevents ATP generation in the tsetse midgut [9] . Nonetheless , whether dyskinetoplasty preceded the switch to tsetse-independent transmission or vice versa is unresolved [8] , [10] , [11] . The kDNA that comprises the mitochondrial genome in T . brucei consists of numerous concatenated circles of two kinds: maxicircles that encode genes primarily involved in oxidative phosphorylation and minicircles that encode guide RNAs ( gRNAs ) [12] . The majority of maxicircle mRNAs undergo RNA editing to insert or delete uridine residues as specified by template gRNAs in a process catalyzed by multiprotein complexes called editosomes [13]–[15] . One kDNA-encoded transcript that requires editing is the F1FO-ATPase subunit 6 , which is essential in both bloodstream and insect stage T . brucei [16]–[19] . However , it has recently been shown that mutations found in the nuclear-encoded ATPase subunit γ of some T . evansi and T . equiperdum strains can compensate for the loss of kDNA , explaining their viability [20] . In an effort to better understand the causes and consequences of tsetse-independent transmission and kDNA-independent viability , we sequenced the genome of T . evansi strain STIB805 . This strain was isolated in 1985 from an infected water buffalo in the Jiangsu province of China , shown to completely lack kDNA ( i . e . to be akinetoplastic ) , and suggested to belong to a possibly clonal group of T . evansi with worldwide distribution [4] , [21] , which is why it was chosen for this study . The comparative genome analysis between this strain and the T . b . brucei TREU 927/4 strain reference genome [22] revealed extensive similarities . While the sizes of the chromosomes differ between T . evansi and T . brucei , the gene content within their respective genomes are largely similar , as 92 . 7% of T . evansi CDS have an identifiable ortholog in T . brucei . Analysis of T . evansi variant surface glycoprotein ( VSG ) sequences shows broad conservation of N-terminal sub-types , with extensive phylogenetic similarity and no evidence of any species-specific expansion of clades . An analysis of T . evansi CDS corresponding to the identified T . brucei mitochondrial proteome revealed that virtually all are retained , despite the lack of requirement in an akinetoplastic trypanosome for respiratory complexes I-IV or any proteins involved in maintenance or expression of the mitochondrial genome . Phylogenetic analyses with several genetic markers conclusively show that extant strains of T . evansi or T . equiperdum are not monophyletic and evolved on at least four independent occasions . Together , the results presented here show few critical differences between T . evansi and T brucei , indicating that dyskinetoplasty and concomitant tsetse-independent transmission are significant phenotypic changes underpinned by relatively subtle genomic alterations .
The rearing of animals was regulated by Czech legislation ( Act No 246/1992 Coll . ) . All housing , feeding and experimental procedures were conducted under protocol 90/2013 approved by Biology Centre , Czech Academy of Sciences and Central Commission for Animal Welfare of the Czech Republic . Trypanosomes ( T . evansi strain STIB805 ) were purified from mice by DEAE ( DE52 ) cellulose [23] . Total DNA was extracted as described elsewhere [24] . Briefly , the cells were lysed using SDS , and incubated with proteinase K and RNase . DNA was harvested after phenol extraction and ethanol precipitation . Four runs of single-end 454 sequencing plus 2 runs of paired-end 454 sequencing were obtained using GS FLX ( + ) System following the manufacturer's instruction ( Roche ) and generated 1 , 904 , 327 reads ( 225 , 826 paired end , 1 , 678 , 501 single end ) [25] . Approximately 10 µg of genomic DNA was sheared by nebulization into desired fragments sizes ( ∼400 bp for single-end 454 , ∼3 kb for paired-end ) and adaptor oligos ligated to create the library for sequencing . Additional sequence data was obtained by shearing genomic DNA to ∼200–300 bp fragments sizes for sequencing on an Illumina GAIIx producing 19 , 701 , 740 tags with an ordered read length of 76mers . Illumina reads for T . b . brucei strains TREU 927/4 and Lister 427 ( provided by the Wellcome Trust Sanger Institute , Hinxton , UK ) were downloaded from the European Nucleotide Archive ( accession nos . ERX009953 and ERX008998 ) . Genome assemblies and identification of sequence polymorphisms ( SNPs and indels ) were carried out with CLC Genomics Workbench ( CLC bio ) . Reads for T . evansi STIB805 , T . b . brucei TREU 927/4 and T . b . brucei Lister 427 were mapped against the T . brucei TREU 927/4 version 4 reference ( Tb927 ) using the following mapping parameters: global alignment , similarity fraction = 0 . 9 , length fraction = 0 . 5 , insertion cost = 3 , deletion cost = 3 , mismatch cost = 2 . De novo assemblies for each strain were created using either all reads or those binned during the reference-based assemblies , using the following parameters: automatic word size = yes , bubble size = 50 , similarity fraction = 0 . 9 , length fraction = 0 . 5 , deletion cost = 3 , insertion cost = 3 , mismatch cost = 2 . De novo contigs were aligned to Tb927 using standalone NCBI BLAST version 2 . 2 . 25 and Artemis Comparison Tool release 12 . 0 [26] . For RPKM ( reads per kilo base per million ) analysis , aligned STIB805 and TREU 927/4 reads were assigned to sequential 1-kb bins along the length of the Tb927 reference . For each chromosome , the log2 ratio of binned reads from the two read-sets was calculated for each bin , and normalized to a median log2 ratio of 0 by offsetting all values by the median log2 ratio . SNPs and indels were called with CLC bio using the following parameters: minimum coverage = 10 , maximum coverage = 100 , minimum variant frequency = 30% , minimum central quality = 20 , minimum average quality = 15 . Coding sequence prediction of T . evansi genome was done using a combination of de novo gene prediction approach and reference based gene transfers . The Rapid Annotation Transfer Tool ( RATT ) was used to transfer the gene boundaries and functional annotations from Tb927 onto T . evansi chromosomes ( target hereafter ) [27] . To prevent the transfer of paralogs to the same target region , thus resulting in multiple overlapping/duplicate gene calls , the annotation transfer was performed pairwise between one chromosome from Tb927 and the corresponding chromosome from the target . This resulted in the transfer of more than 96 . 4% ( 9427/9776 ) of genes from Tb927_v4 onto TevSTIB805ra . Though we observed extensive conservation of synteny between Tb927 and target genomes , this approach would likely have missed genes in targets that were shuffled by chromosomal fission and fusion that occurred during evolution . We performed another RATT transfer considering all Tb927 chromosomes and one target chromosome at a time . We then parsed out genes that were predicted uniquely by this approach ( i . e . from regions where the initial RATT transfer failed to predict any genes ) . Combining these two approaches allowed us to predict genes that were shuffled across the chromosomes while avoiding predictions that overlap each other . We then used an in-house consensual de novo gene prediction suite called 'AutoMagi' to predict protein coding genes from T . evansi [28] . AutoMagi internally uses three gene prediction algorithms ( genescan , testcode , codonusage ) and predicts a consensus gene model out of individual gene predictions . The codon usage table required by AutoMagi was generated by ‘cusp’ using the RATT output of T . evansi STIB805 assembly [29] . We then used an in-house built prolog system to combine the de novo gene predictions and reference-based gene predictions . The genes that are unique to RATT predictions were automatically included in the final set . Genes that are unique to AutoMagi were compared with the NCBI non-redundant ( NR ) database ( accessed 23 February 2011 ) using 'BLASTp' ( default criteria: EXPECT = −10 , WORD SIZE = −3 , MATRIX_NAME = BLOSUM62 , GAP COST = Existence:11 Extension:1 ) algorithm . All of the 'BLASTp' results were reviewed manually , and genes meeting the following two criteria were retained: ( a ) an E-value of 5e-6 or lower to the matched NR sequence; ( b ) coding strand identical to nearest neighbors on either side ( i . e . on same poly-cistronic unit ) . This manual curation removed 165 putative CDS from making into the finalized TevSTIB8805ra . The gene calls that overlapped between RATT and AutoMagi were subdivided into the following 4 groups . 1 ) Identical: overlapping exactly , 2 ) AM_subsetof_RATT: AutoMagi prediction is entirely contained within RATT prediction , 3 ) RATT_subsetof_AM: RATT prediction is entirely contained within AutoMagi prediction 4 ) StaggeredOverlap: both predictions overlap in a staggered fashion . In the first two cases ( Identical & AM_subsetof_RATT ) , AutoMagi gene calls were ignored and RATT models were retained . In the third case ( RATT_subsetof_AM ) coordinates from AutoMagi were combined with annotation information from the RATT model . Genes in the fourth category ( Staggered ) were subjected to a thorough manual review process . The review process involved ( i ) 'BLASTp' search against NCBI's NR database to identify coordinates for queries and subject; ( ii ) a ClustalW multiple sequence alignment of both candidate genes with their potential homolog ( s ) from T . brucei . Manual review of BLAST and ClustalW was performed in each case to decide either to split/merge/choose one of the AutoMagi/RATT predictions . These newly derived coordinates were then combined with the annotation information from the RATT model . GeneIDs of the final set of protein coding genes were unified and ordered from left to right end of the chromosome . Entire genome and all the predicted genes are publicly available at TriTrypDB ( http://tritrypdb . org/tritrypdb/ ) . The fastq files containing the Illumina read data for T . evansi STIB805 are available at: http://www . ebi . ac . uk/ena/data/view/ERA000101 . Bloodstream parasites at 2×108 cells/ml were purified using DEAE cellulose ( DE52 ) chromatography , and subsequently used to prepare chromosome blocks as previous described [30] . DNA from T . b . evansi and T . b . brucei cells was embedded in low-melt agarose blocks ( final concentration of 5×107 cells/ml ) according to [31] , and was resolved using a 1% Megabase agarose ( Bio-Rad ) gel with 0 . 5X TBE buffer in the CHEF-DRIII system ( Bio-Rad ) . S . cerevisiae DNA was used as a size marker . Pulse field gel electrophoresis ( PFGE ) was run at 14°C under the following conditions: switch time A increased from 28 . 6 s to 228 s for 24 hrs , followed by switch time B , with increase from 28 . 6 s to 1 , 000 s for another 24 hrs . The angle was set to 120° and voltage gradient to 3 V/cm . The PFGE gel was stained with ethidium bromide after the run . VSG sequences were extracted from the genome assembly using hidden markov models ( HMM ) and HMMER 3 . 0 [32] . HMMs were constructed for a- and b-type VSG respectively using multiple sequence alignments of T . brucei TREU 927/4 and T . b . gambiense DAL972 sequences [33] . All open reading frames>100 bp were marked up and the predicted amino acid sequences were searched for matches to either HMM using HMMER 3 . 0 . Significant matches were checked manually to ensure that each VSG was complete and gene boundaries were correct . Intact VSG were extracted and aligned approximately using Clustal X [34] . The aligned sequences were combined with existing alignments of T . brucei TREU 927/4 a- and b-type VSG [35] and modified by eye . C-terminal domains were trimmed ( due to recombination these present an inconsistent phylogenetic signal ) , resulting in a- and b-VSG alignments of 470 and 492 characters , respectively . Neighbour-joining trees were estimated for each amino acid sequence alignment using PHYLIP v3 . 6 , with a JTT rate matrix and 100 non-parametric bootstrap replicates . A frequency distribution of species-specific clade sizes , ( i . e . three T . evansi VSG clustered together with a T . brucei TREU 927/4 sequence as the sister lineage has a clade size of 3 ) was calculated to express the degree of intercalation of sequences from the two strains . Putative VSG orthologs were extracted from the phylogenies . In situations where single VSG from T . evansi STIB805 and T . brucei TREU 927/4 were sister taxa , supported by a bootstrap value>95 , these genes were interpreted as orthologs . The rates of synonymous and non-synonymous nucleotide substitutions per site were estimated for these orthologous pairs . The ratio of these rates ( ω ) was estimated using Ka/Ks Calculator v1 . 2 [36] using GY and MS methods . For comparison , this was repeated for 151 pairs of non-VSG orthologs chosen at random . The dihydrolipoamide dehydrogenase ( LipDH ) CDS ( Tb927 . 11 . 16730 ) was PCR-amplified from total parasite DNA using primers 5′-ATA AAG CTT ATG TTC CGT CGC TGC-3′ ( forward ) and 5′-ATA AGA TCT TTA GAA GTT GAT TGT TTT GG-3′ ( reverse ) and Phusion polymerase ( New England Biolabs , NEB ) . In cases where direct sequencing of the amplicon revealed heterozygosity , sequence information for individual alleles was obtained after cloning . After removal of the primer sequences , LipDH CDSs were aligned using ClustalX [37] . Phylogenies were reconstructed using the program MrBayes [38] via the Topali platform [39] , implementing the GTR substitution model and a discrete gamma rate distribution model with four rate categories ( to account for rate heterogeneity among sites ) as the most appropriate nucleotide substitution model . Four independent MCMC chains of 1×106 generations were sampled every 100th generation . A 50% majority rule consensus tree was derived after the first 25% of trees were discarded as burn-in . The ATP synthase γ subunit sequence ( Tb927 . 10 . 180 ) was PCR-amplified from total parasite DNA with primers 5′-GCG GAA TTC GAA GCA GAT GAC ACC TAA-3′ ( forward ) and 5′-GCG GAA GAC CTT GCT GCG GAG CCA CTC T-3′ or 5′-GGC GAC ATT CAA CTT CAT-3′ ( reverse ) and Phusion polymerase ( NEB ) . The sequence was determined by direct amplicon sequencing or , in cases of heterozygosity , after cloning . A partial 812-bp sequence of cytochrome oxidase 1 ( COX1 ) was obtained by PCR and used for phylogenetic analysis as previously described [40] . Briefly , we assessed phylogenetic relationships among T . equiperdum and T . brucei isolates using a haplotype network constructed using the statistical parsimony approach implemented in TCS v . 1 . 21 [41] . Subnetworks were created with 95% confidence limit and then unconnected subnetworks>10 mutations apart were connected by relaxing the confidence limit . To verify that the haplotypes containing T . equiperdum isolates were on phylogenetically distinct branches , we estimated a phylogenetic tree using the Bayesian approach implemented in MrBayes v . 3 . 2 [42] . PartitionFinder v . 1 . 0 . 1 [43] determined the Hasegawa , Kishino and Yano nucleotide substitution [44] with invariant sites ( HKY+I ) without partitioning by codon positions as the most appropriate model for the MrBayes analysis . Microsatellite genotyping was carried out exactly as described previously [40] . Briefly , isolates were typed for eight microsatellite markers [45] . Principal component analysis ( PCA ) was performed in R using the package adegenet , as described [40] .
PFGE was performed to visualize the pattern of chromosomes in the akinetoplastic T . evansi STIB805 strain . Multiple bands corresponding to megabase chromosomes ranging in size from ∼1 to ∼5 Mb could be visualized ( Fig . 1 ) . The most noticeable differences in the chromosomal pattern in comparison to T . brucei are the five intermediate chromosome bands that range from ∼300 kb to ∼800 kb . Additionally , two size groups of minichromosomes ( ∼100 kb and ∼200 kb ) were observed in T . evansi STIB805 . Such a degree of variability is within the range observed among strains of T . b . brucei [46] . Genomic DNA isolated from T . evansi STIB805 was subjected to both 454 and Illumina sequencing , generating a combined total of 21 , 445 , 221 reads . Using the T . brucei TREU 927/4 genome sequence [22] ( version 4; from here onwards abbreviated Tb927 for convenience ) as a scaffold , a reference-based assembly of these T . evansi reads was generated . This assembly , called TevSTIB805ra , incorporates 17 , 856 , 165 reads and has an average coverage of 57 . 2 reads per nucleotide across the entire assembly and of 48 . 4 reads per nucleotide for the ‘core regions’ [i . e . regions excluding telomeres , subtelomeres , and internal regions that consist of repetitive coding sequences such as VSGs , expression site-associated genes ( ESAGs ) or retroposon hot spot genes ( RHS ) ] . An assembly approach based on the genome of a closely related reference strain allows the reliable identification of homologous gene pairs , of any differences that might exist between these sequences , and of genes that are missing or highly diverged in the genome of interest . This approach has limited power in identifying structural differences between genomes and , by definition , cannot identify sequences that are present in the genome of interest but not in the reference genome . For that reason we have supplemented the reference-based approach with the analysis of contigs that were assemble de novo . As detailed below , this allowed the confirmation of Tb927 genes that are absent in in T . evansi STIB805 and the identification of candidate genes that might be present in the latter but absent in the former . However , genome rearrangements that are not associated with differences in gene content will have been missed and the T . evansi STIB805 genome as published on TriTrypDB may indicate gene synteny where it does not exist . Annotation of the TevSTIB805ra T . evansi genome was performed using a combination of RATT and AutoMagi , followed by manual curation . RATT ( Rapid Annotation Transfer Tool; [27] ) identified likely orthologs in the T . brucei reference genome and transferred their functional annotations to the T . evansi genome , while AutoMagi predicted genes de novo using the consensus of three gene prediction algorithms ( genescan , testcode , codonusage ) [28] . A total of 10 , 110 CDS were identified , with 9368 CDS annotated as T . brucei orthologs by RATT and subsequent manual inspection ( Table 1 , columns C , E and F; S1 Table ) , and 742 CDS uniquely predicted by AutoMagi ( Table 1 , column D; S2 Table ) . Thus , 92 . 7% of the identified T . evansi CDS have an identified ortholog in T . brucei , while 7 . 3% were uniquely detected by de novo gene prediction . Analysis of the 742 AutoMagi CDS by BLAST searching of the GenBank non-redundant database revealed at least one hit ( E-value ≤5e-6 ) for each of these CDS to a gene from a Trypanosoma species ( T . b . gambiense 568 CDS; T . b . brucei 168 CDS; T . equiperdum , T . vivax , or T . congolense 6 CDS ) . The most common annotations among these BLAST hits were for ‘hypothetical unlikely’ ( 415 CDS ) or other hypothetical ( 150 CDS ) sequences ( S2 Table ) . Orthologous sequences had not been annotated in the Tb927 reference , presumably due to more stringent criteria for gene calling . Thus , most if not all of the de novo predicted genes in TevSTIB805ra are also present in T . brucei . Of the 9368 T . evansi CDS identified as T . brucei orthologs , 8421 CDS were classified as non-repetitive genes; the remaining 947 CDS were VSG , ESAG , RHS , or duplicate sequences . A comparison of the 8421 non-repetitive CDS between T . brucei and T . evansi revealed 7970 ( 94 . 6% ) had a nucleotide identity of 95% or greater , 320 ( 3 . 8% ) had a nucleotide identity between 70–95% , and 131 ( 1 . 6% ) had a nucleotide identity less than 70% ( Fig . 2; S1 Fig . ) . After RATT annotation transfer , a total of 503 ( 5 . 1% ) Tb927 GeneIDs did not have identified orthologs in the TevSTIB805ra annotated genome ( Table 1 , column G; S2 Table ) . The majority of these ( 406 ) represent repetitive genes ( e . g . VSG , ESAG , and RHS ) or ‘hypothetical unlikely’ genes , which were not analyzed further . Five CDS correspond to predicted pseudogenes . T . evansi STIB805 orthologs for 49 Tb927 CDS were shown to be wholly or partially missing in TevSTIB805 reads that mapped to these gene loci ( see below ) . T . evansi homologs for the remaining 43 Tb927 GeneIDs were ‘missed’ by RATT and AutoMagi , but were subsequently identified by manual examination of the T . evansi sequence . Thus , the majority of T . brucei CDS have extremely similar T . evansi orthologs , and very few T . brucei CDS were not found in T . evansi . This result is consistent with a very close phylogenetic relationship between these parasites . The initial RATT approach only identified T . evansi CDS with an annotated Tb927 homolog in the syntenic position . To identify potential CDS in TevSTIB805ra that are syntenic to unannotated sequences in Tb927 , and are homologous to annotated CDS in other ( non-syntenic ) locations , RATT was performed a second time by comparing each TevSTIB805ra chromosome to the entire Tb927 genome . This approach complemented de novo gene calling by AutoMAGI and identified 112 CDS in TevSTIB805ra ( Table 1 , columns E and F; S2 Table ) . The majority of these CDS were annotated as hypothetical proteins ( 64 CDS ) or repetitive sequences such as VSG , ESAG , and RHS ( 39 CDS ) . For almost all loci , manual inspection revealed that either ( i ) a syntenic CDS existed in Tb927 that was unannotated or ( ii ) a syntenic , annotated CDS did exist in Tb927 but the presence of a highly similar sequence elsewhere caused a RATT artefact . An exception was TevSTIB805 . 6 . 770 , where the syntenic sequence in Tb927 was found to be disrupted by frame-shifts , but in T . b . gambiense DAL972 to be annotated as Tbg972 . 6 . 420 ( hypothetical protein , unlikely ) Two approaches were used to find genes in the T . evansi genome that may not be present in the T . brucei genome . Firstly , reads from T . evansi that did not match Tb927 were de novo assembled to make TevSTIB805dn and putative CDS called with AutoMagi ( Table 2 and S2 Table ) . The 22 CDS with a homolog in Tb927 that was not a repetitive or hypothetical gene included multiple copies of pseudogenes for putative UDP-Gal/UDP-GlcNAc-dependent glycosyltransferase and lytic factor resistance-like protein , an adenosine transporter , calmodulin , a DNA topoisomerase , a leucine-rich repeat protein , ribulose-phosphate 3-epimerase , major surface protease B , and a galactokinase pseudogene . 36 of the 202 CDS that did not get a hit against Tb927 had a BLASTx hit in the NCBI non-redundant database: 23 CDS matched hypothetical Trypanosoma spp . and 9 appeared to be contaminants from other - mostly bacterial - organisms . The remaining 4 BLAST hits were for T . vivax RNA-dependent DNA polymerase and T . evansi VSGs ( 2 hits ) and diminazene-resistance-associated protein . Because Tb927 was created using a traditional sequencing approach and only covers the 11 large chromosomes [22] , sequences found in the de novo assembly of T . evansi deep sequencing reads might not be unique to this strain or species , but rather reflect a difference in sequencing methodology or stem from intermediate-sized or mini-chromosomes . To address this , Illumina reads from sequencing T . brucei TREU 927/4 that also did not match Tb927 were de novo assembled into Tb927dn . Comparison of Tb927dn to TevSTIB805dn showed that 45 . 6% of the binned T . evansi reads matched to Tb927dn . The remaining T . evansi reads were then assembled into TevSTIB805dn_sub and putative CDS called with AutoMagi ( Table 2 and S2 Table ) . Of the 84 BLASTp matches to non-repetitive genes , 71 are annotated as hypothetical proteins , 2 are lytic factor resistance-like proteins , 1 is a putative transporter , and 10 are annotated as putative ( or pseudogene ) UDP-Gal or UDP-GlcNAc-dependent glycosyltransferase . Glycosyltransferases in T . brucei are frequently found in subtelomeric regions that are difficult to assemble due to repetitive sequences , and are known to be highly variable among trypanosome strains [33] , [47] . Of the 117 TevSTIB805dn_sub CDS for which no match was identified in Tb927 , 18 had a BLASTx hit in the NCBI non-redundant database: 9 CDS matched hypothetical genes from various Trypanosoma species and 1 matched a diminazene resistance-associated protein that was previously suggested to convey diminazene aceturate ( Berenil ) resistance to certain strains of T . evansi [48] . Thus , gene prediction found very few CDS in de novo T . evansi assemblies that are not present in T . brucei , and analysis of these CDS revealed differences reminiscent of those observed among strains of the same species . When T . evansi STIB805 de novo contigs were filtered for minimum coverage of at least 5x , the number of genes without obvious homologs in Tb927 was reduced considerably ( Table 2 ) . To test the potential absence of these genes in T . b . brucei more rigorously , short read data sets for strains TREU 927/4 and Lister 427 were searched for matches to the 22 candidates from TevSTIB805dn and the 6 candidates from TevSTIB805dn_sub . Only seven CDS candidates remained where the sequence was either entirely absent from both T . b . brucei datasets , or did not contain an undisrupted ORF ( S2 Table , highlighted ) . Whether these candidates are indeed functional CDS , and whether they are generally absent from T . brucei ssp . and present in T . evansi , and therefore are of potentially diagnostic value , requires further investigation . A non-redundant list of the ORFs that did not have a BLASTx hit in NCBI ( 200 ORFs in total ) is provided as S1 Data File . A total of 49 CDS found in Tb927 were not identified in TevSTIB805ra . These loci were analyzed individually by ( i ) specifically searching for matching T . evansi STIB805 reads ( S2 Table ) ; ( ii ) comparing reads per kilobase per million ( RPKM ) coverage plots of these regions for T . brucei and T . evansi; ( iii ) aligning contigs of a full T . evansi STIB805 de novo assembly to the respective regions in Tb927 . These analyses confirmed the absence or disruption of several of these CDS in T . evansi STIB805 ( including the iron/ascorbate oxidoreductase loci , the Tb927 . 9 . 7950/Tb927 . 9 . 7960 repeat , the Tb927 . 4 . 3200- . 3270 region , both Tb927 . 8 . 490 and Tb927 . 8 . 500 , and the Tb927 . 8 . 7300- . 7330 region ) , as illustrated in S2–S9 Fig . , with the procyclin loci described in detail below . These cases have in common that the loci in question show considerable variation among Trypanozoon strains and CDS appear to be absent in T . b . brucei Lister 427 and/or T . b . gambiense DAL972 as well [33] . The differences observed in T . evansi STIB805 compared to Tb927 therefore are unlikely to be relevant for kDNA loss or tsetse-independent transmission . In T . evansi STIB805 , the procyclin loci either lack or have disrupted versions of several CDS found in T . brucei . GPEET and EP procyclins and associated genes are encoded in loci on chromosomes 6 and 10 , respectively , in various T . brucei strains [49] . Procyclin proteins are GPI-anchored coat glycoproteins that are expressed exclusively in the procyclic insect form of T . brucei , and they have been hypothesized to be involved in protection against tsetse fly midgut hydrolases [50] . Experiments in T . brucei have shown that knocking out all of the procyclin genes ( Null mutants of GPEET and EP3 on chromosome 6; EP1 and EP2 on chromosome 10 ) causes no growth defect in vitro and permits completion of the entire life cycle , but causes a selective disadvantage during co-infection with wild type cells in the tsetse fly midgut [51] . These procyclin loci also contain procyclin-associated genes and a gene related to expression site associated gene 2 ( PAG3 and GRESAG2 on chromosome 6; PAG1 , 2 , 2* , 4 , and 5 on chromosome 10 ) [49] . The functions of the PAG proteins and GRESAG2 are unknown; although transcripts of PAG1–3 have been shown to increase during differentiation to procyclic forms , published experiments using cell lines with all PAG genes knocked out reported no obvious abnormal phenotypes in vitro or in vivo [49] . In multiple T . brucei strains , the chromosome 10 procyclin loci are heterozygous , with one chromosome containing EP1/EP2/PAG1/PAG5/PAG2*/PAG4 and the other chromosome containing EP1/EP2/PAG2/PAG4 ( PAG2 being a fusion of the 5′ part of PAG1 and the 3′ part of PAG2* ) [49] , [52]–[56] . In T . evansi STIB805 , chromosome 10 appears to be homozygous , with only the EP1/EP2/PAG2/PAG4 locus present , and the associated absence of the segment containing the 3′ part of PAG1 , PAG5 and the 5′ part of PAG2* ( Fig . 3 ) . The full STIB805 de novo assembly contained a single 14 . 9 kb contig corresponding to the EP1/EP2/PAG2/PAG4 locus ( S2 Fig . ) . Also on chromosome 10 , the EP2 in T . evansi contains a stretch of 12 divergent amino acids in the domain N-terminal to the EP repeat; this region is highly conserved in T . brucei [57] , [58] . Although the function of this domain is unknown , these 12 amino acids are found in all sequenced T . brucei genomes with very few variations . The chromosome 6 procyclin locus contains a triplication of three genes ( EP3/PAG3/GRESAG2 ) in T . brucei TREU 927/4 , with GPEET present only in front of the last unit ( Fig . 3 ) . Copy numbers of these genes may vary among T . brucei strains , as Southern analysis showed that these are single copy genes in the AnTat1 . 1 strain [49] . In TevSTIB805ra , coverage of this locus is much reduced ( Fig . 3 , S3 Fig . ) , indicating either divergence , reduced copy number , or both . The locus did not assemble into a single contig in the de novo approach and because of the repeat nature of this locus in Tb927 , assigning sequence differences to particular gene copies was not possible . Nonetheless , several non-synonymous mutations in EP3 are evident , and the PAG3 and GRESAG2 genes have frameshifts and deletions . In contrast , analysis of a subset of other T . evansi orthologs to CDS shown to be upregulated in PF relative to BF T . brucei ( Tb927 . 1 . 2310 , Tb927 . 1 . 2350 , Tb927 . 1 . 2560 , Tb927 . 1 . 580 , Tb927 . 10 . 10260 , Tb927 . 10 . 10950 , Tb927 . 10 . 4570 , Tb927 . 11 . 16130 , Tb927 . 11 . 8200 , Tb927 . 4 . 1800 , Tb927 . 4 . 1860 , Tb927 . 4 . 4730 , Tb927 . 5 . 1710 , Tb927 . 5 . 2260 , Tb927 . 6 . 510 , Tb927 . 8 . 5260 , Tb927 . 4 . 4730 , Tb927 . 8 . 8300 , Tb927 . 9 . 15110 , and Tb927 . 9 . 8420 ) [59] detected no notable changes . Any Tb927 gene encoding a product that is exclusive involved in ( i ) maintenance or expression of kDNA , ( ii ) life cycle progression , or ( iii ) insect-stage specific energy metabolism , e . g . oxidative phosphorylation , is redundant in T . evansi . Although many of these genes encode mitochondrial proteins , numerous other mitochondrial activities are expected to remain essential even in an akinetoplastic bloodstream trypanosome ( see Discussion ) . Extensive proteomic analyses have identified 978 mitochondrial proteins in T . brucei [60]–[63] . A search of TevSTIB805ra and TevSTIB805dn identified orthologous gene sequences for all of these proteins ( S6 Table ) . A detailed examination of a subset of genes representing categories i-iii above ( with the exception of the F1 subunits of respiratory complex V , which remain essential in T . evansi [19] ) suggests that the vast majority , if not all of these genes remain functional ( see S6 Table for details ) . To obtain further evidence for or against mutational decay in these genes , we calculated Ka , Ks , and Ka/Ks for the following orthologous gene pairs in Tb927 and T . evansi: 208 CDS associated with kDNA expression or function; 942 unambiguous orthologous gene pairs for mitochondrial proteins; and 6331 unambiguous orthologous gene pairs corresponding to non-VSG and non-mitoproteome annotations ( Table 3 ) . These orthologous gene pairs were selected using greater stringency ( reciprocal best matches in BLASTp searches with a minimum e-value threshold of 1×10−4 ) than the analysis shown in Fig . 2 , to ensure that paralogous gene pairs were excluded ( these would possibly increase Ka and Ks estimates and skew the distribution; S6 Table ) . We found no evidence of relaxed selection for T . evansi CDS associated with kDNA expression or function or the mitoproteome as a whole . Both Ka and Ks were lower for CDS associated with mitochondrial expression , and Ka/Ks was also lower relative to the genomic background , indicating that purifying selection is generally stronger among mitoproteome CDS , perhaps suggesting a higher proportion of essential genes compared to the control set . Furthermore , we made the same comparison between Tb927 and T . b . gambiense DAL972 ( both of which retain functional kDNA ) and the values for Ka , Ks and Ka/Ks show no significant differences ( by t-test ) with the values for T . evansi . The predicted protein structures of VSGs in T . evansi STIB805 conform to the canonical structures in T . b . brucei and T . b . gambiense , and include all five recognized N-terminal sub-types ( N1-5 ) . In T . evansi STIB805 we identified 525 a-type VSGs ( i . e . N-1-3 and 5; S2 Data File ) and 505 b-type VSGs ( i . e . N4; S3 Data File ) ; of these 453 ( 86% ) and 451 ( 89% ) , respectively , are full-length . Given that the assembly of subtelomeric and mini-chromosomal regions is fragmentary , these numbers may underestimate the real number of VSGs , although the total is comparable with both T . b . brucei TREU 927/4 [22] and T . b . gambiense DAL972 sequences [33] . The VSG repertoire is largely conserved between the T . b . brucei TREU 927/4 reference and T . evansi STIB805 , as the VSGs are interspersed among each other in neighbour-joining molecular cladograms ( S10 Fig . ) . Another indication of the similarity of the VSG repertoire is that very few clades of strain-specific VSG are larger than 2–3 ( Fig . 4a and b ) . Large clades of VSG from a single genome would suggest divergence of VSG repertoire through gene duplication . Hence , 376 and 384 ( = 85 . 1% ) T . evansi STIB805 and T . b . brucei TREU 927/4 a-VSGs , respectively , are most closely related to an ortholog in the other strain , or are paraphyletic to a clade of such sequences ( i . e . strain-specific clade size = 1 ) . Only 40 T . evansi STIB805 VSGs and 51 T . b . brucei TREU 927/4 VSGs form a clade with a paralog from the same genome ( i . e . strain-specific clade size = 2 ) , suggesting a single gene duplication since the strains separated . The same patterns occur with b-VSGs . Orthology between VSGs does not mean that they are unaffected by recombination , only that enough sequence homology persists for two orthologs to cluster together . Indeed , among 151 putative a-VSG orthologs 33 ( 21 . 8% ) have dissimilar C-terminal types , while among 112 b-VSG orthologs 32 ( 28 . 6% ) showed similar evidence for recombination having occurred since these two strains split from their common ancestor . Thus , analysis of the VSG repertoire reveals no evidence for the evolution of specific VSG gene clusters or subfamilies in T . evansi , similar to what had been observed for T . b . gambiense [33] . This comparison of orthologous VSGs from T . evansi STIB805 and T . b . brucei TREU 927/4 provides an overview of the molecular evolutionary forces incident on the VSG archive . Fig . 4c and d show that substitution rates among orthologous VSGs are typically higher than the background defined by comparison to the 6331 non-VSG orthologous gene pairs previously used in the mitoproteome analysis above . The mean average rate for VSG is significantly greater than other genes ( p<0 . 05 ) and comparison of Ka/Ks ( ω ) explains this . The distribution of ω is left-skewed for non-VSG genes , which reflects the strong purifying selection on non-synonymous substitutions that typifies most genes that perform essential functions . The distribution for VSG is normal , indicating that purifying selection is much weaker on average , although still in effect . This difference in the distribution of ω is significant ( p<0 . 005 ) and shows that VSGs evolve in a more neutral fashion than other genes , resulting in higher substitution rates . However , there is no more evidence for positive selection among VSGs than for 'normal' genes , which reflects our current understanding that VSG sequence evolution in T . brucei is driven predominantly by the diversifying effects of recombination [35] , rather than directional selection driven by host immune responses . Consistent with earlier reports [64] that have suggested RoTat 1 . 2 ( NCBI accession AF317914 ) as a predominant and diagnostic T . evansi VSG , we identified a closely related ORF in STIB805 . The first 1250 bp of a 1431-bp ORF on a 3 . 9-kb de novo contig are nearly identical to the published RoTat1 . 2 sequence , while the 200 bp at the 3′ end are nearly identical to Tb927 . 8 . 240 , annotated as ‘VSG , degenerate’ in TriTrypDB ( S11 Fig . ) . Thus , the gene in STIB805 had most likely obtained a different C-terminal region through recombination , an important mechanism in VSG evolution [35] . We did not identify any close homologs to the 5′ region of RoTat1 . 2 in the publicly available T . b . brucei TREU 927/4 , T . b . brucei Lister 427 or T . b . gambiense DAL972 datasets . A comparison of the TevSTIB805ra to the Tb927 reference genome revealed 354 , 809 single nucleotide polymorphisms , 68 , 938 of which were non-synonymous in identified CDS . 32 , 892 of these non-synonymous SNPs were found in 904 CDS classified as repetitive ( e . g . RHS , VSG , ESAG ) , while 36 , 046 were found in 6630 CDS from non-repetitive genes ( S3 Table ) . This number is slightly lower than the ∼45 , 000 non-synonymous SNPs reported for T . b . gambiense orthologs of non-repetitive T . b . brucei TREU 927/4 genes [33] . Additionally , there were 45 , 269 short indels ( insertions/deletions of up to eight nucleotides ) , of which 5544 were in CDS regions . Of these , 4963 ( 89 . 5% ) were not an indel of mod 3 , and would therefore be expected to cause a frameshift . 3247 ( 58 . 6% ) of the indels were found in 656 CDS classified as repetitive , while 2297 ( 41 . 4% ) were found in 1091 CDS from non-repetitive genes ( S4 Table ) . The latter consisted of 1154 insertions , 1000 deletions , and 143 that were complex ( a combination of insertion , deletion and/or SNP ) . The Tb927 reference contains information for one allele only for any heterozygous locus . We therefore also identified SNPs and indels for TREU 927/4 using publicly available Illumina reads and the same criteria that were applied for STIB805 . We identified 26 , 522 non-synonymous SNPs and 1630 indels . Of these , 814 and 58 , respectively , were homozygous , thus reflecting discrepancies between the original Sanger sequencing data and the Illumina reads . The cause of these discrepancies , which could arise from a number of potential sources , including DNA isolation from different cultures of the same strain , is unknown . Only 1267 non-synonymous allele variations ( SNP or indel ) in CDSs ( repetitive or non-repetitive ) were shared by STIB805 and TREU 927/4 , 8710 variations affected the same position , but were different , and 89 , 043 affected a different position . All non-synonymous SNPs and indels identified in STIB805 and TREU 927/4 are compared in S5 Table , listed by chromosome and gene . In summary , while numerous small sequence variations exist between T . evansi STIB805 and the T . b . brucei reference genome , these are comparable in number to differences between the T . b . brucei and T . b . gambiense subspecies . The phylogenetic relationship of various T . evansi and T . equiperdum isolates to each other and to isolates of T . brucei subspecies is controversial . We compared T . evansi STIB805 CDSs with their T . b . brucei TREU 927/4 orthologs as retrieved from Tb927 in order to identify a genetic marker that would likely be informative for the elucidation of the phylogenetic origins of T . evansi and T . equiperdum , and for determining their evolutionary relationships with established T . brucei sub-species . Selection criteria included ( i ) sufficient SNPs over a length of 1–3 kb ( to minimize number of internal primers necessary for amplification and sequencing ) , ( ii ) lack of paralogs , and ( iii ) minimal alignment gaps . One promising candidate , the LipDH gene ( Tb927 . 11 . 16730 ) , a shared component of four mitochondrial multi-enzyme complexes [65] , was selected for sequence analysis . Both LipDH CDS alleles were amplified and sequenced from a total of 40 isolates from various geographical locations ( 13 T . b . brucei , 3 T . b . gambiense group 1 , 4 T . b . rhodesiense , 5 T . equiperdum , 15 T . evansi; S7 Table ) . Since mutations in subunit γ of the mitochondrial ATP synthase complex were recently identified as important factors in the viability of T . evansi and T . equiperdum , the sequence for both γ alleles was also determined for all isolates . 32 unique LipDH haplotypes were identified . The majority of strains ( 36/41 ) had two different alleles and four strains appeared to be homozygous at this locus , all of which were T . b . brucei ( although it cannot be ruled out that the primers used were selective for one allele in these cases ) . Phylogenetic analysis ( Fig . 5 ) showed all but three of the haplotype sequences fell into one of five major clusters with strong support ( posterior probabilities ≥0 . 9 ) , which we refer to as clades V , W , X , Y and Z . Importantly , 14 different haplotypes are derived from the T . evansi/T . equiperdum isolates , which are found in three different clades as well as outside of the clades . This feature is incompatible with monophyly of T . evansi or T . equiperdum . Some subspecies did have relatively restricted phylogenetic diversity , for example T . b . gambiense type 1 had only five closely related haplotypes and certain clades contained sequences from a restricted number of the subspecies; e . g . clade X only contained T . b . rhodesiense and T . b . brucei; clade Y included only T . b . gambiense type 1 and T . b . brucei . This may reflect relatively more recent origins of certain populations , bottleneck events and/or sampling bias , and is in line with most previous work ( [40] and references therein ) . Eight unique T . evansi/T . equiperdum genotypes ( haplotype pairs ) were found which , by disregarding minor sequence differences , were reduced to four major genotypes: W/Z , V/V , V/Z , or atypical ( Fig . 5; S7 Table ) . These LipDH genotypes correlated very well with the four ATP synthase γ genotypes found in these strains ( Fig . 5B; see also S7 Table ) . The largest group ( Group 1 ) , consisting of 13 T . evansi isolates ( including STIB805 ) and 2 T . equiperdum isolates , is characterized by LipDH genotype W/Z and γ genotype A281Δ/WT . The single exception , Tev48 , had two Z haplotypes ( Hap24+Hap29 ) distinguished by two SNPs , which may be a result of loss of heterozygosity ( LOH ) followed by two mutations , or it may represent a cloning artefact . Non-T . evansi/T . equiperdum strains that had sequences within these clades originated from both East and West Africa in the case of clade Z , and the T . b . gambiense samples from Ivory Coast in the case of clade W . The remaining T . evansi/T . equiperdum strains were split into three further groups according to the LipDH/ATP synthase γ genotypes as follows . Group 2 is characterized by LipDH V/Z and γ genotype A273P/A273P . The two T . equiperdum strains carrying this genotype ( Teq21 and Teq22 ) were heterozygous for two alleles derived from well-separated clades: one ‘V’ allele ( Hap17 or Hap18 , 1 SNP difference ) and one ‘Z’ allele ( Hap14 or Hap15 , 1 SNP difference ) . The divergence between the V and Z alleles ( 11 or 12 discriminating SNPs ) , suggests that these strains have evolved out of a T . b . brucei background that had relatively old and distinct LipDH alleles , possibly via a recombination event . Interestingly , the most closely related non-T . evansi/T . equiperdum alleles are found in current East African populations as well as in three T . b . brucei from Ivory Coast . Group 3 is characterized by LipDH V/V and γ genotype WT/WT . The one T . equiperdum strain with a WT γ genotype ( Teq23 = STIB841; probably synonymous with the OVI strain [5] ) was heterozygous with two similar ‘V’ clade alleles that are closely related to the V alleles in Teq21 , Teq22 , Tbr02 ( Kenya ) and Tbb08 ( Zambia ) ( S7 Table ) . Group 4 is characterized by atypical LipDH and γ genotype M282L/WT . This genotype was found in a single T . evansi strain ( Tev42 = KETRI 2479 ) with Hap27+Hap28 and could not be reliably grouped with any other strains . An interesting aspect of the T . evansi/T . equiperdum genotypes is that most strains possess two divergent alleles that are more similar to those found in other , non-Tev/Teq strains than they are to each other . These non-Tev/Teq strains are themselves heterozygous , with second alleles that are clearly distinct from either of the Tev/Teq alleles . A clear example is the most common Teq/Tev LipDH genotype , W/Z , that is carried by 13 out of 15 T . evansi analyzed and 2 out of 5 T . equiperdum analyzed . This finding is incompatible with allelic divergence due to long-term clonal evolution , rather it shows that recombination between ancestral parasites occurred either at or immediately prior to the origin of this genotype which has subsequently undergone clonal expansion in the absence of the possibility of sexual exchange in the tsetse vector . Three of the four T . b . rhodesiense strains ( Tbr01 , 03 , 04 ) also exhibit alleles split across different clades as does Tbb06 . Interestingly , all Tbb except Tbb06 have alleles from the same clade , possibly indicative of inbreeding . The COX1 gene , encoded in the mitochondrial maxicircle DNA , was recently used as a highly informative marker to investigate the phylogeography of T . brucei subspecies [40] . Although most , if not all , T . evansi isolates lack a maxicircle , the presence of at least one T . equiperdum isolate with at least a partial maxicircle in three of the above groups prompted us to determine the COX1 haplotypes for these strains and to investigate their relationship with the Trypanozoon isolates from the earlier study ( S7 Table ) . A maximum parsimony network analysis strongly supported the notion of three independent evolutionary origins for these three groups ( Fig . 6 ) . T . equiperdum STIB818 ( Teq24; COX1 haplotype 23 ) links Group 1 with COX1 clade A , which is composed of isolates of all three T . brucei subspecies found across all of sub-Saharan Africa [40] . Although Groups 2 and 3 , which share a related ‘V’ LipDH genotype , are both linked to COX1 clade C ( composed of T . b . brucei and T . b . rhodesiense isolates from eastern and southern Africa ) , they are well separated within this clade , indicating independent evolutionary origins . T . equiperdum STIB841 ( Teq23 ) shares both its COX1 haplotype 14 and its LipDH haplotype 19 with Tbr02 from Zambia and Tbb08 from Kenya , both members of the Kiboko group , suggesting relatively recent common ancestry . Note that Tev42 ( KETRI2479 ) , the lone representative of the ‘atypical’ LipDH genotype , is not represented in this network since this isolate lacks a maxicircle and therefore the COX1 gene . Incorporation of microsatellite data for a subset of T . evansi/T . equiperdum isolates into an established PCA network [40] also gave results that are inconsistent with monophyly of either species ( Fig . 7 ) . Most T . evansi isolates , together with Teq24 ( STIB818 ) , formed a cluster ( grey circle ) related to , but somewhat distinct from , non-Kiboko T . b . brucei ( light blue circle ) and T . b . rhodesiense ( red circle ) . The single exception among T . evansi was again Tev42 ( KETRI2479 ) , which localized near the centre of the non-Kiboko cluster . Teq21 ( BoTat1 . 1 ) was also more related to non-Kiboko T . b . brucei , but relatively distant from Tev42 . The PCA analysis , consistent with the COX1 and LipDH data , suggested a relatively close evolutionary relationship of T . equiperdum STIB841/OVI ( Teq23 ) with the Kiboko group of T . b . brucei ( dark blue circle ) . The results of the phylogenetic analyses are summarized in Table 4 , along with the ATPase subunit γ genotypes and , where known , dominant minicircle class and RoTat 1 . 2 VSG genotype . Combined , these markers suggest that the T . evansi/T . equiperdum isolates investigated in our study can be arranged into four distinct groups of independent evolutionary origin ( see Discussion )
Taken together , the genome analysis and accompanying phylogenetic studies presented in this work revealed important insights into the biology and evolution of T . evansi STIB805 and dyskinetoplastic trypanosomes in general . Their re-classification as subspecies of T . brucei , i . e . T . b . evansi and T . b . equiperdum , seems clearly justified considering the vast similarities observed at the genome level and the lack of monophyly confirmed by phylogenetic analyses . Important questions that remain include the molecular basis of tsetse-independent transmission and the evolutionary timescale of the appearance of the T . evansi/T . equiperdum groups . The genome data presented here provide an important tool for future studies aimed at resolving these questions . | The single-cell parasite Trypanosoma evansi is the disease-causing trypanosome with the widest geographical distribution . The disease , called surra , has significant economic impact primarily due to infections of cattle , horses , and camels . Morphologically the parasite is indistinguishable from bloodstream stage T . brucei , a parasite causing sleeping sickness in humans and the disease nagana in animals . T . brucei , however , is strictly bound to sub-Saharan Africa where its obligate vector , the tsetse fly , resides . The lack of a complete mitochondrial genome in T . evansi further distinguishes this parasite from T . brucei . Important questions regarding the biology of T . evansi include how it escaped from Africa , whether this has happened more than once , and how exactly it is related to T . brucei . To help answer these questions we have sequenced the T . evansi nuclear genome . Our phylogenetic analysis demonstrates that T . evansi , and the closely related horse parasite T . equiperdum , evolved more than once from T . brucei . We also demonstrate extensive similarity to T . brucei , including the maintenance of numerous genes that T . evansi no longer requires . Therefore , despite the significant functional and pathological differences between T . evansi and T . brucei , our analysis supports the notion that T . evansi is not an independent species . | [
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] | 2015 | Genome and Phylogenetic Analyses of Trypanosoma evansi Reveal Extensive Similarity to T. brucei and Multiple Independent Origins for Dyskinetoplasty |
Cytotoxic CD8+ T cells ( CTLs ) play a critical role in controlling viral infections . HIV-infected individuals develop CTL responses against epitopes derived from viral proteins , but also against cryptic epitopes encoded by viral alternative reading frames ( ARF ) . We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope , Q9VF , encoded by an HIVgag ARF and presented by HLA-B*07 . Using PBMCs of HIV-infected patients , we first cloned and sequenced proviral DNA encoding for Q9VF . We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid ( Q9VF/5D ) and a majority encoding an asparagine ( Q9VF/5N ) . We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients . Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients , suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals . We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N . Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes . The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides . We then dissected the cellular mechanisms involved in the presentation of Q9VF variants . As expected , cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs . In contrast , Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs . Using in vitro proteasomal digestions and MS/MS analysis , we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope , leading to a dramatic reduction of Q9VF epitope production . Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes .
Multiple lines of evidence suggest that CD8+ cytotoxic T lymphocytes ( CTLs ) play a critical role in controlling HIV-1 replication . During acute infection , expansion of HIV-specific CD8+ T cells ( HS-CTL ) , before appearance of neutralizing antibodies , is associated with decreased viremia [1] and most likely determines the viral set point during chronic infection [2] , [3] . Resistance to disease progression correlates with the detection of Gag-specific CTLs and with the presence of particular HLA alleles , such as HLA-B*57 and –B*27 [4] , [5] . HIV rapidly mutates to evade virus-specific CD8+ T lymphocyte responses , underlying the selection pressure exerted by CTLs [2] , [6] , [7]–[11] . In large part due to its error prone reverse transcriptase activity , HIV possesses a unique capacity to mutate and evade CTL responses . During acute and chronic HIV infection , CTL escape mutations have been well documented [9] , [12] , [13] . In most cases , these mutations are intra-epitopic and affect HLA binding and/or alter TCR interactions leading to loss of CTL activation or more subtle effects [14] . However , interference with antigen processing may also lead to a reduced generation of precursor peptides and consequently peptide/MHC-I complex formation and T cell activation . This could occur at any stage of the processing pathway . Mutations in epitope-flanking regions might affect proteasomal processing or N-terminal trimming leading to escape from CTL recognition [15]–[20] . CTLs recognize peptides originating from proteasomal processing of viral proteins or truncated misfolded viral polypeptides , also called DRiPS ( for defective ribosomal products ) [21]–[23] . These viral polypeptides are classically derived from the fifteen HIV-1 viral proteins encoded by the nine primary open reading frames [24] . However CTLs also target peptides translated from alternative reading frames or ARFs ( also called cryptic epitopes ) . ARF-derived peptides ( ARFPs ) result from a differential usage of the three-letter codon alphabet during protein synthesis . How this change of reading frame occurs remains elusive but various mechanisms have been proposed . Ribosomes can initiate translation at an internal initiation codon ( Met or Cys ) , change reading frame by shifting , or translate alternatively spliced mRNA . Nonetheless , ARF polypeptides are processed in cells and thus constitute an important source of cryptic epitopes for MHC-I presentation [25] . CTL responses directed against these cryptic epitopes have been detected in autoimmune disease [26] , in tumors [27] , [28] but also in several infectious diseases , including influenza virus [29] , murine AIDS [30] , SIV [31] and importantly HIV infections [32]–[35] . We previously described six ARFPs presented by HLA-B*0702 overlapping the alternative reading frames of HIV-1 gag , pol or env genes [32] . CTL responses specific for these ARF-derived peptides were detected in the blood of HIV+ patients . In addition , HIV-infected cells were recognized by CTLs specific for the gag-overlapping ARF epitope ( so called Q9VF/5D epitope ) . Importantly , we showed that the introduction of a stop codon within gag-ARF abrogated Q9VF/5D epitope generation and Q9VF/5D–specific CTL activation [32] . Recent studies further highlighted the in vivo relevance of ARFP-specific CTL responses [33] , [34] , [36] . In two independent cohorts studies , Bansal et al . and Berger et al . investigated the association between specific HLA alleles and HIV sequence polymorphisms within ARFs . This “HLA class I footprint approach” allowed the prediction of numerous ARFPs within the HIV-1 genome , both from sense and antisense transcripts . On a restricted number of ARFPs , they also demonstrated that these cryptic epitopes induced CTL responses during natural infection that might contribute to viral control in vivo [33] , [34] . In the present work , we bring to light a novel mechanism of CTL escape altering the processing and presentation of the Q9VF epitope encoded by the gag-overlapping ARF . In PBMCs of HLA-B*07+ and HLA-B*07- HIV-infected individuals , we first compared the prevalence of QPRSNTHVF ( Q9VF/5N ) and QPRSDTHVF ( Q9VF/5D ) variants of the gag-ARFP . To this end , we PCR amplified and sequenced twenty HIV proviral genomes per individuals . We noticed that the proportion of proviruses encoding Q9VF/5D was significantly lower in HLA-B*07+ than in HLA-B*07- patients , suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals . In HLA-B*07+ and HLA-B*07- patients , we analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N and we dissected the immunogenicity of Q9VF variants . We observed that cells infected with HIV-1 strains encoding Q9VF/5N were neither recognized by Q9VF/5N- nor Q9VF/5D-specific CTLs . We demonstrate that this single amino acid ( AA ) variation is responsible for the lack of CD8+ T cell recognition . We show that HIV can escape CTL surveillance by introducing mutations leading to epitope destruction by proteasomes .
Q9VF was originally predicted from the sequence of the consensus HIVHxB2 ( HIVLAI ) isolate [32] . HIVLAI bears an asparagine ( N ) to aspartic acid ( D ) substitution at position 5 ( Q9VF/5D ) representing less than 5% of HIV-1 clade B strains retrieved from Genbank . We decided to extend these observations by sequencing HIV proviral sequences isolated from 10 HLA-B*07+ and 10 HLA-B*07- patients . HLA-typing , virological and clinical characteristics of these patients are presented in Table 1 . Both groups were age-matched and did not present any significant differences in terms of CD4 counts , viral loads or treatments ( not shown ) . From the PBMCs of each patient , we cloned and sequenced at least 20 HIV-proviral sequences encompassing the gag-ARF DNA region ( Figure 1A and Supplementary Figure S1 ) . The isolated HIV sequences encoded either Q9VF/5N ( present in 16 out of 20 patients , representing 62% of all isolates ) , Q9VF/5N variants ( exhibiting within the epitope an additional AA difference from the consensus sequence , 9 out of 20 patients , 14% of all isolates ) or Q9VF/5D ( 7 out of 20 patients , 15% of all isolates ) and Q9VF/5D variants ( 2 out of 20 patients , 1% of all isolates ) ( Table 2 ) . Between Q9VF/5N and Q9VF/5N-variants , Q9VF/5N was the major variant representing 80% of proviral sequences in this group . Q9VF/5D was the major sequence representing 94% of proviral sequences among Q9VF/5D and Q9VF/5D-variants . Note that these mutations did not impact the translation of classical gag ORF ( Supplementary Figure S1 and not shown ) . In contrast , HIV proviruses harboring a STOP codon prior to Q9VF ( 8% of all isolates ) that most likely abolishes Q9VF translation were also identified ( Figure 1A ) . HIV proviral sequences encoding Q9VF/5N and Q9VF/5N-variants were predominant in both HLA-B*07+ and HLA-B*07- patients . Q9VF/5D or Q9VF/5D-variant HIV proviral sequences could be retrieved in two out of the ten HLA-B*07+ patients and in six out of the ten HLA-B*07- donors . Taking into consideration the diversity of HIV sequences per donor with regard to their HLA-B7 status , we observe a significant lower proportion of Q9VF/5D+ HIV strains in HLA-B*07+ than in HLA-B*07- donors ( p<0 . 04 , mean value 3% vs 29% of proviral sequences in HLA-B*07+ and HLA-B*07- donors , respectively , Figure 2B ) . Altogether , these results suggested that Q9VF/5D-encoding HIV strains might be under negative selective pressure in HLA-B*07+ donors . We thus analyzed CTL responses directed against Q9VF/5D and Q9VF/5N epitopes in PBMCs of patients including the 10 HLA-B*07+ patients used for the analysis of HIV proviral sequences . PBMCs from 31 HLA-B*07+ patients were loaded with various peptides and submitted to IFNγ-ELISpot ( Figure 1C and not shown ) . Incubations with peptides corresponding to well-characterized HLA-B*0702-restricted immunodominant epitopes from HIV-1 Gag classical ORF ( SPRTLNAWV , TPQDLNTML , YPLASLRSLF ) induced a significant IFNγ-release , demonstrating that in the course of natural infection the donors mounted CTL responses to HIV-1 antigens . Five out of the 31 HLA-B*07+ donors showed a low but significant activation with Q9VF/5D and Q9VF/5N peptides ( Figure 1C ) . Note that donors reacted to both peptides or reacted to none and that the frequencies of CTL responding to Q9VF/5D and Q9VF/5N peptides were in the same order of magnitude ( from 150 to 300 CTL per million of PBMCs ) , suggesting that the reactivity to one or the other peptide might be due to cross reactivity . We previously demonstrated that CTL lines raised against Q9VF/5N were indeed cross-reactive on Q9VF/5D and vice versa ( [32] and Supplementary Figure S2 ) . Viruses encoding Q9VF/5D were not isolated from PBMCs of the five Q9VF responders ( Figure 1 ) , with the exception of patients P1 that harbored proviruses encoding a Q9VF/5D variant ( QPRGDTHVF , representing 16% of sequences in this donor ) . These data prompt us to study the immunogenicity of the Q9VF/5N and Q9VF/5D epitope variants . We asked whether the Q9VF/5N epitope was processed and presented to HS-CTLs by HIV-infected cells . HLA-B*0702+ cells were infected with HIVLAI and HIVNL-AD8 strains encoding Q9VF/5D or Q9VF/5N respectively . Five days post-infection ( pi ) , 50 and 47% of the cells were productively infected by HIVLAI and HIVNL-AD8 respectively ( as monitored by intracellular Gag-p24 FACS-staining ( not shown ) ) . Infected cells were then co-cultured with HIV-specific CTL lines and T cell activation measured using IFNγ-ELISpot assays ( Figure 2 ) . HLA-transgenic mice offer a rapid and convenient model to identify human T cell epitopes [24] and to generate CTL lines specific for peptides of unknown immunogenicity in humans , such as Q9VF/5N . For this reason , Q9VF/5D- and Q9VF/5N-specific CTL lines were generated by peptide immunization of HLA-B*0702+ transgenic mice and in vitro restimulations [32] , [37] . As expected , Q9VF/5D- and Q9VF/5N- specific CTLs secreted high levels of IFNγ in response to Q9VF/5D and Q9VF/5N peptide loaded cells respectively ( Figure 2A ) . Note that Q9VF/5D- and Q9VF/5N-specific CTL lines displayed similar capacity to recognize peptide-loaded cells ( Supplementary Figure S2 ) , suggesting that the Q9VF/5N variant affects neither MHC nor TCR binding of the peptide . As we previously reported [32] , HIVLAI-infected cells induced a robust activation of Q9VF/5D-specific CTLs . Due to their capacity to cross-react on Q9VF/5D peptide ( Supplementary Figure S2 and [32] ) , Q9VF/5N-specific CTLs were also stimulated by HIVLAI-infected cells , thus demonstrating that these CTL lines are fully competent in recognizing HIV-infected cells . In contrast , Q9VF/5D- and Q9VF/5N-specific CTLs were not activated upon co-culture with HIVNL-AD8-infected cells ( Figure 2A ) . This is not due to the incapacity of HIVNL-AD8-infected cells to activate HS-CTLs since CTL clones specific for an HLA-B*0702-restricted HIV-1 Nef epitope ( F10LR ) , raised as a control in these experiments , were activated upon co-culture with HIVLAI- and HIVNL-AD8-infected cells . To extend these observations to other HIV-1 isolates , HLA-B*0702+ cells were also infected with HIVMN that encodes for Q9VF/5N and used as target cells to activate Q9VF/5D- and Q9VF/5N-specific CTLs ( Supplementary Figure S3 ) . HIVNL-AD8- and HIVMN-infected cells did not induce Q9VF/5D- nor Q9VF/5N-specific CTL activation . Overall , these results suggested that HIV-infected cells did not present the Q9VF/5N peptide . Epitope flanking regions have a direct impact on antigen processing and presentation [38] . Thereafter , to exclude the possibility that HIV sequence variations outside the Q9VF/5N peptide might be responsible for the lack of presentation , we introduced in HIVLAI a D to N mutation within the Q9VF epitope ( so called HIVLAI-5D>5N ) . This mutation did not affect the primary open reading frame of Gag ( Supplementary Figure S1 ) and did not alter viral replication in T cell lines or primary CD4+ T cells ( Figure 2B ) . However , cells infected with HIVLAI-5D>5N could not activate Q9VF/5D- nor Q9VF/5N-specific CTLs ( Figure 2C ) . Thereafter , this single amino acid substitution was sufficient to abrogate CTL recognition , thus indicating that this asparagine alters Q9VF MHC-I presentation . We then sought to dissect the mechanism responsible for the lack of Q9VF/5N MHC-I presentation . The capacity of antigenic peptides to bind to a given HLA allele is determined by the so-called anchor residues [39] . Mutating an anchor residue abrogates peptide HLA-binding and subsequent T cell activation , a strategy often used by viruses to escape viral-specific T cell responses . The anchor residues of HLA-B*0702 reside at position 2 and 9 of the peptide-ligands . Thereafter , the D to N substitution at position 5 was not predicted to influence Q9VF peptide binding to HLA-B*0702 [40] . However , besides anchor residues , auxiliary residues might affect peptide binding , we thus compared the capacity of Q9VF/5D and Q9VF/5N peptides to bind HLA-B*0702 . To this end , T2-HLA-B*0702 cells were loaded O/N with Q9VF/5D or Q9VF/5N peptides and binding to HLA-B*0702 molecules at the cell surface monitored by FACS ( Figure 3A , left panel ) . Q9VF/5D and Q9VF/5N peptides exhibited similar capacities to bind HLA-B*0702 with a relative affinity ( RA , based on the reference peptide ) of 2 . 6 and 1 . 5 respectively ( Figure 3A , left panel ) . To further characterize the impact of the 5D to 5N substitution on peptide-MHC interactions , we compared the capacity of the peptides to stabilize HLA-B*0702 molecules at the cell surface of T2-HLA-B*0702 ( Figure 3A , right panel ) . To this end , T2-HLA-B*0702 were cultured O/N at 26°C to allow surface expression of peptide-receptive MHC molecules , loaded with a high concentration of peptides , shifted to 37°C and the stability of HLA-B*0702-peptide complexes monitored by FACS at various time points . An exponential regression of HLA-B*0702 mean fluorescence intensity ( MFI ) vs . time reveals that the stability ( t1/2 ) of HLA-B*0702 pulsed with an irrelevant peptide ( S9L ) is 22 min while binding of Q9VF/5D and Q9VF/5N peptides prolongs the t1/2 to 211 and 641 min respectively ( Figure 3A , right panel ) . Thereafter , Q9VF/5D and Q9VF/5N peptides are very good HLA-B*0702-binders and 5D to 5N substitution tends to prolong surface expression of HLA-B*0702 . Precursor peptides are transported by the TAP pumps ( transporter associated with antigen processing ) from the cytosol into the endoplasmic reticulum ( ER ) , and then loaded on nascent MHC-I molecules [41] . N-terminally extended peptide precursors are also transported and further trimmed in the ER by the endoplasmic reticulum aminopeptidase ERAAP and bound to MHC-I molecules [42] , [43] . We asked whether the absence of Q9VF/5N peptide presentation by HLA-B*0702 within infected cells might be the result of inefficient ER-translocation of the Q9VF/5N epitope and/or Q9VF/5N-peptide precursors by TAP . Hence , we used a TAP-binding assay [44] to evaluate the affinities of Q9VF/5D and Q9VF/5N and their precursors with TAP . Q9VF/5D and Q9VF/5N exhibited a poor affinity for TAP ( Figure 3B ) , most likely due to the presence of a proline at position 2 that negatively impacts on TAP-mediated peptide transport [44] . In contrast , their N-terminally extended peptide precursors EGF-Q9VF/5D and EGF-Q9VF/5N showed at least a two-log increased efficiency to compete for TAP with an equal 1/IC50 of 0 . 15 . Whatever the precursor , Q9VF/5D and Q9VF/5N containing peptides did not show differences in their capacity to bind human TAP molecules . Overall , these data demonstrated that the D to N substitution within Q9VF does not impact on TAP transport and HLA binding . In contrast , the 5N substitution might prolong epitope presentation on the cell surface . The proteasomes , that are the major catalytic enzymes involved in antigen processing , generate the carboxyl termini of most MHC-bound peptides [38] , [45] . We thus asked whether the generation of Q9VF/5D was dependent on proteasomal processing . To this end , HLA-B*0702+ cells were infected with HIVLAI . Five days pi , infected cells were incubated with a potent and selective proteasome inhibitor , epoxomicin [46] , treated with a citrate-phosphate buffer to remove residual MHC-peptide complexes , washed and cultured with Q9VF/5D-specific CTLs as previously described . Epoxomicin treatment abolished the capacity of HIVLAI-infected cells to activate Q9VF/5D-specific CTLs , as measured in IFNγ-ELISpot ( Figure 3C , left panel ) . Note that epoxomycin inhibition affected neither MHC-density ( as monitored by FACS , not shown ) nor the capacity of treated cells to present exogenous peptide ( at 0 . 1 µg/ml ) ( Figure 3C , right panel ) . Thereafter , these results demonstrated that the generation of Q9VF epitope depends on proteasomal processing . Proteasomes might also destroy CTL epitopes by generating aberrant cleavages within the epitope [47] or in epitope-flanking regions [19] , [48] . We thus asked whether aberrant proteasomal cleavages might be responsible for the lack of Q9VF/5N presentation . The proteasome is a large multicatalytic protease composed of standard and inducible subunits that replace the standard subunits upon exposure to IFNγ and form the so-called “immunoproteasomes” ( IP ) . IP is found in most cell types after IFNγ-exposure , but is constitutive in APCs and is induced in HIV-infected T cells [49] . Standard ( SP ) and IP proteasomes display discrete differences in their capacity to cleave a given peptide substrate [50] . We submitted the full-length polypeptides from the gag-overlapping ARF to IP processing . 27mer peptides encompassing Q9VF/5D or Q9VF/5N peptides were synthesized and incubated with IP purified from T2 . 27 cells [51] . After 1 h incubation , the digestions were analyzed by mass spectrometry ( RP-HPLC SI ) and peptide fragments identified by MS/MS ( Figure 4A ) . IP digestion of Q9VF/5D encompassing peptide showed the presence of major proteasomal cleavage sites after amino acids F10 , F19 , I22 and R24 representing around 80% of total cleavages . The cleavage at position F19 generated the C-terminal cut of the N-extended precursors of Q9VF ( M1-F19 ) . After 1 h incubation , when comparing the IP digestion profiles of Q9VF/5D and Q9VF/5N encompassing peptides , we noticed the presence of a new cleavage site within the Q9VF/5N epitope . This cut at position N15 was the most prevalent among Q9VF/5N representing up to 28% of total IP cleavages . These results demonstrated that the D to N substitution introduces a major cleavage site within the Q9VF/5N epitope . Nonetheless the C-terminal cut necessary for the generation of Nt-extended Q9VF/5N precursors was also detected following 1 h of proteasomal digestion . Thereafter , we sought to evaluate the amount of cleavage products generated during Q9VF/5D and Q9VF/5N digestions . To this end , we performed kinetics of IP digestion where aliquots were regularly collected and submitted to mass spectrometry analysis as before ( Figure 4B ) . To compare the amounts of cleavage products , we used the MS fragment intensity as a surrogate marker for quantity since these two parameters correlate significantly [15] . The variations among the different fragments generated are presented as the relative intensity of peptides that exhibit a Q9VF C-terminal cut ( epitope or precursors ) or peptides issued from cleavages within the Q9VF epitope ( referred to as the antitopes ) ( Figure 4B ) . Kinetics of digestion of peptides encompassing either Q9VF/5D or Q9VF/5N were identical: 24% , 59% and 96% of both substrates was degraded after 30 min , 1 h and 2 h respectively . At latter time points , both 27mers were undetectable . In the course of Q9VF/5D substrate digestion , the precursor ( M1-F19 ) was readily produced starting from 30 min with a peak at 4 h digestion ( representing 20% of digested products ) . The epitope was detected starting from 1 h digestion and accumulated reaching 13% of all peptide fragments at time 18 h . At latter time points , Q9VF/5D epitopes and precursors represented up to 14% of all peptide fragments detected . An antitope corresponding to a cleavage at position S14 was also generated but represented less than 2% of detected fragments at each time point . In contrast , during Q9VF/5N substrate digestion , the antitopes corresponding to the cleavage at position N15 were already produced after 30 min of digestion and reached around 77% of all peptides from 4 to 18 h , further demonstrating that N15 is a major cleavage site within Q9VF/5N . Interestingly , during Q9VF/5N digestion , the epitope was barely detected even at latter time points ( less than 2% of digested products ) . The precursor M1-F19 accumulated from 30 min to 2 h ( 8% of digested products ) but was undetectable after 4 h , suggesting that the cleavage at position N15 destroyed this peptide . Overall , the amounts of Q9VF/5N epitope and precursors produced were markedly reduced as compared to Q9VF/5D digestion . Taken together , these results demonstrate that the Q9VF/5D epitope is efficiently produced by proteasomes and accumulates with time . In contrast , the D to N substitution introduces a major cleavage site within the epitope leading to the destruction of the Q9VF/5N epitope and thus the absence of MHC-I binding and presentation .
The three-letter codon alphabet allows protein synthesis in six possible overlapping reading frames . A vast number of ARFs have the potential to encode proteins or epitopic peptides ( ARFPs ) . Using an “HLA class I footprint” approach , Bansal et al and Berger et al recently predicted the existence of numerous ARFPs within HIV-1 genome [33] , [34] . We have previously shown that ARFP-specific CTLs are induced during natural infection [32] . These CTL responses might contribute to viral control driving HIV evolution at the population level . ARFPs can mutate during the first year of infection , suggesting a possible selection of escapes variants [33] , [34] . Such a scenario has been highlighted in the macaque model of SIV infection [31] . Mamu-B*17+ macaques generate strong CTL responses against SIV ARF-encoded epitopes leading to ARF mutation affecting epitope binding to Mamu-B*17 molecules and subsequent SIV replication rebound [31] . In the present study , we characterized a novel mechanism of ARFP-specific CTL escape resulting from HIV epitope destruction by the proteasomes . We suggest that ARFP-specific CTLs exert a selection pressure leading to negative selection of targeted HIV strains . Overall , our work shows that CTL escape mutations are not limited to epitopes encoded by classical ORF , highlighting the role of ARFP-specific CTLs in the control of HIV infection . We previously identified a panel of epitopes encoded by ARFs within HIV-1 gag , pol and env genes [32] . The gag-overlapping ARF encoding for the Q9VF epitope presented by HLA-B*0702 drew our attention due to its polymorphism . In a cross-sectional cohort study , we report that proviruses encoding the Q9VF/5D epitope ( and 5D variants ) are rare and significantly under-represented in PBMCs of HLA-B*07+ patients , thus suggesting Q9VF/5D-specific CTLs might exert a negative selection pressure on HIV strains encoding Q9VF/5D variants . In HIV-1 gag ARF , the virus might escape CTL immune pressure by introducing a 5D to 5N substitution or Stop codons but prior the epitope . We thus analyzed CTL responses directed against Q9VF/5D and Q9VF/5N epitopes in PBMCs of patients . Q9VF/5D and Q9VF/5N peptides induced CTL responses in 16% of HLA-B*07+ individuals tested . Donors reacted to both peptides or reacted to none . The frequencies of CTLs responding to Q9VF/5D and Q9VF/5N peptides were about the same magnitude , suggesting that the reactivity to one or the other peptide might be due to cross reactivity . The frequency and magnitude of Q9VF/5D responses in HLA-B*07+ patients were rather low as compared to immunodominant HLA-B*07-restricted responses ( Figure 1 and [24] ) . This might be due to the fact that the patients included in the study were under retroviral therapy that might affect the expression of ARF during residual HIV-1 translation ( Table 1 ) . Alternatively in our assays , we are most likely monitoring memory responses to Q9VF/5D that are usually of low magnitude . This possibility is supported by the observation from Bansal et al that ARFP encoding sequences mutate during the first year of infection [33] . Overall , the low representation of Q9VF/5D encoding HIV proviral sequences in PBMCs of HLA-B*07+ individuals and the low frequency and magnitude of CTL responses to Q9VF/5D strongly supported our initial hypothesis that 5N substitution is an escape mutation . We dissected the immunogenicity of the Q9VF/5N epitope . We showed that cells infected with HIV-1 strains encoding Q9VF/5N ( HIVNL-AD8 and HIVMN ) were not recognized by Q9VF/5N-specific CTLs . In contrast , Q9VF/5N- and Q9VF/5D-specific CTLs were activated by HIV-1 strains encoding Q9VF/5D ( HIVLAI ) . We demonstrated that the single AA substitution from 5D to 5N in HIVLAI sequence is sufficient and required to abrogate CTL recognition of HIV-infected cells . Thereafter , the acquisition of this 5N mutation by HIV might help the virus to interfere with Q9VF epitope expression or processing and presentation . Viruses can interfere with antigen expression to escape CTL lysis [23] . Various mechanisms have been proposed for the biosynthesis of ARF-derived polypeptides . Ribosomes can scan through conventional initiation codons [29] , initiate translation at an internal initiation non-AUG-codons ( Leu or Cys ) [34] , [52] , change reading frame by shifting [53] , or translate alternatively spliced mRNA ( for review see [25] ) . We previously described the presence of a conserved slippery motif ( UUUAAAU ) upstream of gag-ARF start codon that may facilitate ribosomal slippage and thus Q9VF synthesis [32] . Interestingly , a structured region ( hairpin ) in HIV-1 RNA has been identified downstream of this slippery motif [53] . This highly structured RNA region might cause ribosomal pausing during gag translation thus facilitating ribosomal slippery and Q9VF expression . The D to N substitution within the Q9VF epitope is translated from a codon that is located in the flexible loop of the RNA hairpin structure [53] . Although it remains to be formally proven , this D to N substitution most likely does not impact the RNA structure and hence Q9VF expression . Viruses also manipulate antigen processing and presentation to escape CTL responses . Interference with antigen presentation could arise at any stage in the pathway , including processing by proteasomes , binding of epitope-precursors to TAP , destruction of these precursors by peptidases in the ER or cytosol and peptide binding to the MHC-I molecule . HIV-specific CTL responses have been shown repeatedly to select for intra-epitope mutations that affect HLA-binding or TcR recognition . In addition , HIV escape mutations outside the epitope ( extra-epitope mutations ) can interfere with antigen processing by proteasomes [17]–[19] , [47] , [54] , [55] or by the ER aminopeptidase ERAAP [16] . To our knowledge , intra-epitope mutations affecting antigen processing have not been described thus far . Several studies proposed that intra-epitope variation might affect processing but did not provide a mechanism [34] , [20] . The only evidence that intra-epitope mutations might affect proteasomal processing of viral antigens comes from mouse models [47] , [56] . We provide several lines of evidence strongly suggesting that the D to N substitution within the Q9VF epitope impacts neither TcR recognition nor MHC binding: i ) Q9VF/5N- and Q9VF/5D-specific CTLs can be generate upon peptide immunization of HLA-B*07-transgenic mice and cross-react to the alternate peptide ( [32] and Supplementary Figure S2 ) ; and ii ) Q9VF/5N and Q9VF/5D peptides bind HLA-B*0702 ( Figure 3A ) . In addition , we show that Q9VF/5N and Q9VF/5D peptide and their precursors ( elongated on the N-termini ) efficiently bind TAP , thus demonstrating that the D to N substitution does not affect peptide translocation into the ER . As previously observed with peptides bearing a proline at position 2 [44] , the optimal Q9VF/5N- and Q9VF/5D epitopes had a reduced capacity to bind TAP as compared to their Nt-extended precursors ( Figure 3B ) , suggesting that in the ER peptide-trimming is required for proper HLA-B*0702 binding . The ER aminopeptidase ERAAP provides peptides for many MHC-I molecules but has been also implicated in the destruction of CTL epitopes [16] . However , ERAAP cannot process X-P motifs in peptide sequences [42] . Thereafter , though it cannot be formally excluded , a role of ERAAP in the destruction of Q9VF/5N is very unlikely . Overall , these data support the concept that the intra-epitope D to N substitution interferes with proteasomal processing . Using in vitro proteasomal digestions , we demonstrate that the D to N substitution introduces a major cleavage site within the Q9VF epitope ( at position N15 ) . Note that at 1 h-digestion time point we identify mainly primary cleavage products since less than 50% of the peptide substrates ( the 27mer ) have been digested ( Figure 4A ) . To further highlight the potential impact of this N15 cleavage site in the generation of the Q9VF epitope , we performed kinetics of peptide digestion using IP . We observed that amounts of Q9VF/5N epitope and precursors produced were markedly reduced as compared to Q9VF/5D . These results strongly suggest that proteasome cleavages at position N15 destroy the Q9VF/5N epitope and precursors resulting in the lack of MHC-I presentation and CTL activation . In conclusion , a single amino acid variation within HIV epitope can result in epitope destruction and absence of HIV-specific CTL activation . Mutation in HIV-1 genome can be silent or can differentially impact the fitness of the virus . Due to the redundancy of the codon alphabet , the 5D to 5N substitution in Q9VF does not impact the primary gag-ORF and thus viral replication ( Figure 2B ) . Nevertheless , considering the multitude of existing ARFs , some mutations within ARF encoding sequences most likely affect viral fitness and these ARF sequences might be unavoidably conserved throughout HIV-1 isolates . Thereafter , the great diversity of ARF epitopes produced during HIV infection offers a vast panel of therapeutic targets to stimulate CTL responses . It is interesting to note that ARF-specific CD8+ T cells can performed multiple functions [33] , [34] and control viral replication in vitro , characteristics that correlate with slow disease progression [57] . In addition , CTLs targeting ARF-derived epitopes can be induced upon vaccination [58] and tumor infiltrating CTLs specific for ARFPs have been also identified in various cancers , including melanoma and breast cancers [25] . Such responses against crytptic epitopes represent a great potential for future immunotherapeutic strategies .
HIV-1-infected peripheral blood mononuclear cells ( PBMCs ) were obtained from HCV ( Hepatitis C virus ) negative French ALT-ANRS-CO15 cohort patients [59] . The 31 HLA-B*07+ and 10 HLA-B*07- individuals were identified using the anti-HLA-B*07 antibody ME . 1 . HLA status was further confirmed by genotyping using PCR [60] or using the Luminex xMAP technology [61] . HLA-typing , virological and clinical characteristics of the ten HLA-B*07+ and ten HLA-B*07- patients included in the study are presented in Table 1 . Patient samples were collected according to French Ethical rules . Written informed consent and approval by institutional review Board at the Pitié-Salpêtrière Hospital were obtained . Animals were bred at the Pasteur Institute . The Office of Laboratory Animal Care at Pasteur Institute reviewed and approved protocols for compliance with the French and European regulations on Animal Welfare and with Public Health Service recommendations ( Directive 2010/63/EU ) . PBMCs were isolated by ficoll-centrifugation , pulsed with Q9VF peptides ( 1 µM , 1 h at 37°C ) , and submitted to IFNγ-ELISpot assays as previously described [46] . The HLA-B*0702-restricted peptides used were: HCV-derived epitope G9AT ( GPRLGVRAT ) , CMV-derived epitope T10AM ( pp65 417TPRVTGGGAM426 ) used as negative and positive control respectively and a pool of known Gag HIV-1-derived epitopes ( p24 16SPRTLNAWV24 , p24 48TPQDLNTML56 , p2p7p1p6 121YPLASLRSLF130 ) as control for HIV reactivity [24] . Responses were considered positive when IFNγ production was superior to 50 spots/106 PBMCs and at least threefold higher than background ( measured with the HCV peptide ) . Mouse CTL lines were derived from splenocytes of peptide immunized HLA-B*07mα3 transgenic mice . In brief , these mice express HLA-B*0702 heavy chain with a murine α3 domain and their H-2Kb and H-2Db class Ia genes have been inactivated [37] . Cytolytic activity of splenocyte cultures was first assessed in a51Cr release assay [32] . Peptide specific CTL lines were stimulated in vitro ( 5 µg/mL of peptide ) and cultured in RPMI 1640 medium supplemented with 10% FCS , 0 . 5 µM 2-β-mercaptoethanol ( Sigma ) , 100 IU/mL penicillin and 100 µg/mL streptomycin ( Gibco-BRL ) . Ten days later , 2×103 , 400 and 80 CTLs in triplicates were stimulated by 105 HIV-1-infected T1-B7 cells and IFNγ release was detected by ELISpot assay . Cross-reactivity of Q9VF/5D- and Q9VF/5N-specific CTLs was tested in IFNγ-ELISpot and Cr51-release assays [32] using T1-B7 peptide-loaded cells . Mouse CTL lines specific for the HLA-B*0702-restricted HIV-1 Nef-derived epitope F10LR ( Nef 68FPVTPQVPLR77; [22] ) were used as controls . When stated , HIV-infected T1-B7 cells were treated with epoxomicin ( 6 h , 1 µg/ml , Calbiochem ) . To remove residual MHC-peptide complexes , epoxomycin-exposed cells were treated with a citrate-phosphate buffer ( pH 3 . 3 ) containing 1% BSA and washed twice , prior co-culture with CTLs for an additional 8 h . HIVLAI 5D>5N was generated by a single amino acid mutation in HIVLAI provirus . The GAT codon ( D ) of Gag-ARF ( AA in position 15 ) was replaced by an AAT codon ( N ) without affecting the primary Gag AA coding sequence , using the following primer ( 5′-GGC TTT CAG CCC AGA AGT AAT ACC CAT GTT TTC AGC ) and Quickchange XL Site-directed Mutagenesis Kit ( Stratagene ) . HIVLAI , HIVLAI-5D>5N , HIVNL-AD8 and HIVMN were produced by transfection of 293T cells using routine procedures [62] . T1 cells ( 174xCEM , CCR5+ LTR-GFP+ ) stably transfected with the HLA-B v T1-B7 cells , [53] ) were infected and used as antigen-presenting cells . 5×106 T1-B7 cells were infected with 500 ng of p24 for 3 h in culture medium containing 10 mM Hepes and 4 µg/ml DEAE-dextran . 2 to 5 days p . i . , infected T1-B7 cells were used as antigen-presenting cells in IFNγ-ELISpot assay . For the infection kinetics , T1-B7 cells were infected with the indicated viruses according to the same procedure using 1 , 10 or 100 ng/ml of p24 . Primary CD4+ T cells were isolated from the blood of healthy donors using ficoll centrifugation and magnetic beads ( Miltenyi ) and activated using PHA ( 1 µg/ml , PAA ) and rhIL-2 ( 50 IU/ml , Chiron ) [62] . Seven days post activation , CD4+ PHA blasts were infected with various doses of HIV ( from 1 to 100 ng/ml of p24 ) . HIV infection was monitored by FACS ( Becton Dickinson ) using intracellular HIV p24 staining ( KC57 Ab , Beckman Coulter ) or p24-Elisa ( PerkinElmer ) . Total DNA was extracted from PBMCs of HLA-B*07+ and HLA-B*07- HIV+ patients using QIAampblood DNA minikit ( Qiagen ) . To analyze the diversity of HIV-1 proviruses in the PBMCs of patients , a 267-bp fragment encompassing the Gag-ARF coding sequence was amplified by nested PCRs as followed: 5 min of initial denaturation at 94°C , 1 min at 94°C , 1 min at 57°C , and 1 min at 72°C for 30 cycles , followed by 7 min at 72°C . The outer primer pair used was ( 5′- ATC AAG CTT GCA CAG CAA GCA GCA GCT GAC ) and ( 5′- CAG GAA CTA CTA GTA CCC TTC AGG AAT TCG G ) , and the inner primer pair was ( 5′- TAC CCT ATA GTG CAG AAC ATC CAG GG ) and ( 5′- GAT AGA GTG CAT CCA GTG CAT GCA ) . Samples were treated separately and negative controls were systematically included . Purified PCR products were cloned using a TOPO-TA cloning kit ( Invitrogen ) . Twenty clones per patient were isolated and gag-ARF inserts from each clonal DNA plasmid were amplified by PCR using M13 primers and sequenced ( Applied Biosystem ) . The capacity of the peptides to bind HLA-B*0702 was determined using a classical HLA stabilization assays with the TAP-deficient cell line T2 HLA-B*0702+ [37] . Briefly , cells were incubated overnight with 100 , 10 , 1 and 0 . 1 µM of peptide in serum-free medium at room temperature . Cells were then stained with the anti-HLA-B*07 ME . 1 antibody and HLA-B*07 surface expression analyzed by FACS ( Becton Dickinson ) . The concentration needed to reach 50% of the maximal fluorescence ( as defined with the R10TV peptide ( CMV pp65 265RPHERNGFTV274 ) was calculated ( IC50 ) . The relative affinity ( RA ) is the IC50 ratio of the tested and R10TV reference peptide ( the lower the relative affinity , the stronger the binding ) . The HLA-A*02-restricted peptide S9L ( HIV-1 p17 77SLYNTVATL85 ) was used as negative control . To monitor the capacity of the peptides to stabilize HLA-B*0702 , T2-HLA-B*0702 were cultured O/N at 26°C and pulsed the last 2 h with peptide ( 100 µM ) in presence of β2-microglubilin ( Sigma , 1 µg/ml ) and brefeldin-A ( BFA , Sigma , 10 µg/ml ) . Cells were then shifted to 37°C for 1 h , washed to remove unbound peptides and incubated at 37°C in presence of BFA ( 0 . 5 µg/ml ) . Samples were removed to 0°C at the indicated time points . Cells were then stained at 4°C using the ME . 1 antibody and analyzed by FACS . Data ( HLA-B*0702 expression ) are expressed as MFI vs . time . The capacity of each peptide to stabilize HLA-B*07 ( t1/2 ) is deduced from an exponential regression ( one phase decay ) using Prism software . A constrain corresponding to the MFI value obtained for the irrelevant peptide ( S9L ) at the latest time point was applied to the plateaus . T10AM ( pp65 417TPRVTGGGAM426 ) and T9ML ( p24 48TPQDLNTML56 ) peptides were used as positive controls . The capacity of the peptides to bind TAP was measured in a competitive binding assay as described previously [44] . Briefly , microsomes were purified from Sf9 insect cells expressing human TAP1–TAP2 complexes , pulsed with the iodinated reporter peptide R9L ( RRYNASTEL ) at 300 nM , and loaded with a dilution of competitor test peptides ( 0 . 1 to 1 , 000 fold molar excess relative to radioactive reporter peptide ) . TAP affinities were determined as the concentrations required to inhibit 50% of reporter peptide binding ( IC50 ) . Results are expressed as 1/IC50 ratios and are mean values from three independent experiments . The highest the 1/IC50 ratio , the highest the affinity . Immunoproteasomes were isolated from T2 . 27mp cells ( that stably express all three immunosubunits ) as previously described [51] . Purified proteasomes were analyzed by SDS-PAGE . The yield was calculated at 90–95% . The 27mer peptides encompassing Q9VF/5D or Q9VF/5N were synthesized using standard Fmoc method on an Applied Biosystems 433A automated synthesizer . The peptides were purified by HPLC and analyzed by mass spectrometry . Three nmol of peptides were digested in vitro using 1 µg of proteasomes ( for 0 . 5 , 1 , 2 , 4 , 8 and 18 h ) in 100 µl of buffer containing 20 mM Hepes/KOH , pH 7 . 8 , 2 mM magnesium acetate and 2 mM dithiothreitol . Reactions were stopped by the addition of trifluoroacetic acid to a final concentration of 0 . 3% . The digestions were analyzed , by mass spectrometry ( RP-HPLC ESI ) and the products were identified by MS/MS . A standard two-tailed nonparametric Mann-Whitney U-test ( with P<0 . 05 considered significant ) was used to perform statistical comparison of HIV-1 proviral sequences frequencies using statistical analysis Prism software ( GraphPad ) . | In addition to the classical open reading frames encoding for the well characterized HIV proteins , HIV exhibits a vast number of alternative reading frames that have the potential to encode proteins or polypeptides . We have previously shown that such reading frames within gag , pol and env genes express T cell epitopes . In the present work , we further characterized the role of T-cell responses targeting the gag-overlapping reading frame in the selection of HIV variants in vivo . We demonstrate that under CD8+ T cell immune pressure , HIV escapes by introducing mutation that affects T-cell recognition of HIV-infected cells . We characterized the mechanism of CTL-escape and demonstrate that HIV manipulates antigen processing and presentation . Our results highlight the importance of CTL targeting these alternative reading frame-encoded antigens in the control of HIV replication . | [
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] | 2011 | CTL Escape Mediated by Proteasomal Destruction of an HIV-1 Cryptic
Epitope |
In endothermic species , heat released as a product of metabolism ensures stable internal temperature throughout the organism , despite varying environmental conditions . Mitochondria are major actors in this thermogenic process . Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport , but a substantial proportion is released as heat . Using a temperature-sensitive fluorescent probe targeted to mitochondria , we measured mitochondrial temperature in situ under different physiological conditions . At a constant external temperature of 38 °C , mitochondria were more than 10 °C warmer when the respiratory chain ( RC ) was fully functional , both in human embryonic kidney ( HEK ) 293 cells and primary skin fibroblasts . This differential was abolished in cells depleted of mitochondrial DNA or treated with respiratory inhibitors but preserved or enhanced by expressing thermogenic enzymes , such as the alternative oxidase or the uncoupling protein 1 . The activity of various RC enzymes was maximal at or slightly above 50 °C . In view of their potential consequences , these observations need to be further validated and explored by independent methods . Our study prompts a critical re-examination of the literature on mitochondria .
As the main bioenergetically active organelles of nonphotosynthetic eukaryotes , mitochondria convert part of the free energy released by the oxidation of nutrient molecules into ATP and other useful forms of energy needed by cells . However , this energy conversion process is far from being 100% efficient , and a significant fraction of the released energy is dissipated as heat . This raises the hitherto unexplored question of the effect of this heat production on the temperature of mitochondria and other cellular components . To address this issue , we made use of the recently developed , temperature-sensitive fluorescent probe ( S1 Fig ) , MitoThermo Yellow ( MTY ) [1] . Because the fluorescence of many molecular probes is known to be sensitive to diverse factors , we investigated whether the changes in MTY fluorescence that we observed in human embryonic kidney ( HEK ) 293 cells could be influenced by altered membrane potential or by associated parameters , such as pH , ionic gradients , or altered mitochondrial morphology . As a major conclusion of this study , based on the fluorescence changes of MTY , we found that the rise in mitochondrial temperature due to full activation of respiration is as high as about 10 °C ( n = 10 , range 7–12 °C , compared to 38 °C , the temperature of the cell suspension medium ) . We also showed that respiratory chain ( RC ) activities measured in intact mitochondria can be increased up to threefold when assayed at the inferred mitochondrial temperature of intact cells .
We first confirmed MTY targeting to mitochondria in both HEK293 cells and primary skin fibroblasts , based on colocalization with the well-characterized dye MitoTracker Green ( MTG ) ( Fig 1A ) . It was previously shown that the initial mitochondrial capture of MTY was dependent on the maintenance of a minimal membrane potential [1] . The exact sub-mitochondrial location of the probe is yet to be established , although it has been postulated to reside at the matrix side of the inner membrane [1] . MTY fluorescence from mitochondria was retained over 45 min , regardless of the presence of RC inhibitors , whilst full depolarization with an uncoupler as carbonyl cyanide m-chlorophenylhydrazone ( m-Cl-CCP ) led to an irreversible MTY leakage from mitochondria after only 2 min ( S3 Fig ) . Fluorescence remained stable over 2 h in HEK293 cells , although the degree of mitochondrial MTY retention varied between cell lines , with probe aggregation observed in the cytosol in some specific lines ( S3 Fig ) . In HEK293 cells , which were selected for further study , we observed no toxicity of MTY ( 100 nM in culture medium ) over 2 days ( S5 Fig ) . We initially calibrated the response of MTY to temperature in solution . Its fluorescence at 562 nm ( essentially unchanged by the pH of the solution buffer in the range 7 . 2–9 . 5 ) ( S2 Fig ) , decreased in a reversible and nearly linear fashion as temperature was increased: a temperature rise from 34 to 60 °C decreased fluorescence by about 50% , whilst 82% of the response to a 3 °C shift at 38 °C was preserved at 50 °C ( Fig 1Ba and 1Bb ) . Using a thermostated , magnetically stirred , closable 750-μl quartz-cuvette fitted with an oxygen-sensitive optode device [2] , we simultaneously studied oxygen consumption ( or tension ) and changes in MTY fluorescence ( Fig 1C ) . Adherent cells were loaded for a minimum of 10 min with 100 nM MTY , harvested , and washed , then kept as a concentrated pellet at 38 °C for 10 min , reaching anaerobiosis in <1 min . When cells were added to oxygen-rich buffer , they immediately started to consume oxygen ( red trace; Fig 1C ) , accompanied by a progressive decrease of MTY fluorescence ( blue trace; phase I ) ( Fig 1C ) . In the absence of any inhibitor , the fluorescence gradually reached a stable minimum ( phase II ) . At that point , either due to a high temperature differential between the mitochondria and the surrounding cytosol ( about 10 °C ) and/or changes in membrane permeability leading to decreased thermal insulation , the temperature of the probe-concentrating compartment appeared to reach equilibrium . We computed the energy released as heat by the RC in this experiment as 1 . 05–1 . 35 mcal/min , based on the measured rate of oxygen consumption ( 11 . 3 ± 1 . 8 nmol/min/mg protein ) and assuming that heat accounts for the difference between the 52 . 6 kcal/mol released by the full oxidation of NADH and the 21 kcal/mol conserved as ATP under a condition of maximal ATP synthesis of 3 molecules per molecule of NADH oxidized . This should be sufficient to ensure the observed thermal equilibrium ( about 50 °C after 20 min ) ( See S1 Text ) . Once all the oxygen in the cuvette was exhausted ( red trace ) , the directional shift of MTY fluorescence reversed ( phase III ) , returning gradually almost to the starting value ( phase IV ) . To calibrate the fluorescence signal in vivo , the temperature of the extracellular medium was increased stepwise ( green trace ) . MTY fluorescence returned to the prior value when the medium was cooled again to 38 °C ( Fig 1C , phase V ) . This in vivo calibration was consistent with the response of MTY fluorescence in solution up to 44 °C , although further direct calibration steps in vivo are not possible without compromising cell viability . However , we confirmed that the response of MTY fluorescence to increased temperature deviates ( but only slightly ) from linearity in vivo in the same manner as in aqueous solution , namely that at maximal mitochondrial warming , extrapolated as being close to 50 °C , the response to a 2 °C temperature shift is approximately 80% of that at 38 °C ( S6 Fig ) . We therefore estimate the rise in mitochondrial temperature due to full activation of respiration as about 10 °C ( n = 10 , range 7–12 °C ) . At the lowest ( phase II ) and highest fluorescence values ( 38 °C , imposed by the water bath ) ( phase IV ) , the signal was proportional to the number of added cells in a given experiment ( Fig 1Da ) . Cell number did not affect the maximal rate of fluorescence decrease ( computed from phase I ) . However , once anaerobic conditions had been reached , the initial rate of fluorescence increase ( phase III , initial ) was inversely related to the number of cells ( Fig 1Db ) . To confirm that the observed fluorescence changes were due to mitochondrial respiration and not some other cellular process , we depleted HEK293 cells of theirmitochondrial DNA with ethidium bromide ( EtBr ) to a point at which cytochrome c oxidase activity was less than 3% of that in control cells ( Fig 1Ea , S1C Fig ) . In EtBr-treated cells , no MTY fluorescence changes were observed upon exposure to oxygenated buffer , and cyanide treatment had no effect ( Fig 1Eb ) . As an additional control , we used the structurally related dye A15 ( S1 Fig ) , which is much significantly less thermoresponsive than MTY [1] . Tested under similar conditions in HEK293 cells as MTY , no decrease of A15 fluorescence ( in fact , an initial increase ) was observed to accompany the activation of mitochondrial respiration by oxygenation of the medium ( S8 Fig ) , in contrast to MTY . Because MTY is derived from a membrane potential–sensitive dye , the fluorescence of which is essentially unaffected by temperature ( Fig 1Bb ) , we investigated whether the observed changes in MTY fluorescence could be influenced by altered membrane potential or by associated parameters . We took advantage of the fact that cyanide or oligomycin exert opposite effects on membrane potential ( Fig 2C , S1 Fig ) as well as on pH [3] and on the condensed versus orthodox state of the mitochondrial matrix [4 , 5] , and we compared the response of MTY fluorescence to these inhibitors ( Fig 2A and 2B ) . To avoid the possibly confounding effect of anaerobiosis , the quartz cuvette was kept uncapped in this experiment , with the oxygen tension rather than the rates of oxygen uptake being recorded ( red traces ) . Once MTY fluorescence was stabilized ( assumed to represent maximal mitochondrial heating ) and the suspension buffer re-oxygenated , cyanide was added , causing a progressive increase in MTY fluorescence to the starting value ( Fig 2A ) . Note that , when cyanide was initially present , fluorescence changes and oxygen uptake were both abolished ( Fig 2A , dotted lines ) . Adding oligomycin in lieu of cyanide also decreased oxygen consumption and , as observed with cyanide , brought about a similar increase in MTY fluorescence ( Fig 2B ) . If added first , oligomycin progressively decreased oxygen uptake , simultaneously abolishing the decrease in MTY fluorescence ( Fig 2B , dotted lines ) . Taken together , these experiments imply that electron flow through the RC rather than membrane potential or any related factor influences MTY fluorescence , which can thus be interpreted as a surrogate for mitochondrial temperature . Examining the respective kinetics of membrane potential changes ( tens of seconds ) and MTY fluorescence changes ( tens of minutes ) supports this conclusion . A similar effect was observed with two other respiratory inhibitors ( S1 Fig ) , affecting either RC complex I ( CI; rotenone , Fig 2D ) or III ( CIII; antimycin , Fig 2E ) . Despite their different effects on the redox state of the various RC electron carriers , these inhibitors blocked oxygen uptake and again triggered an increase in MTY fluorescence . Importantly , these two inhibitors ( and oligomycin ) have been shown to trigger increased production of superoxide by the RC [6] , but their effects on MTY fluorescence are similarly determined by oxygen consumption as for cyanide inhibition , which decreases superoxide production at complex III . This excluded any interference from superoxide in the observed MTY fluorescence changes . Taking advantage of cyanide removal from cytochrome c oxidase to form cyanohydrin in the presence of α-ketoacids under aerobiosis [7] , we confirmed that the blockade of the RC did not result in MTY leakage from the mitochondria because pyruvate addition resulted in the resumption of oxygen uptake and a renewed decrease in MTY fluorescence , both of which were inhibited by a further addition of antimycin ( Fig 2F ) . Leakage of the probe from mitochondria of other cell lines was reflected in a decreased ability of cyanide to restore MTY fluorescence to its initial value ( S4 Fig ) . Note that changes in MTY fluorescence cannot be attributed to altered mitochondrial morphology , since 45 min of MTY treatment had no detectable effect on the mitochondrial network ( S3 Fig , panels i , j ) , nor did any of the inhibitors used in the above experiments ( S3 Fig , panels a–f ) . Similarly , MTY fluorescence changes appear to be independent of oxygen tension as ( 1 ) a similar loss of fluorescence was observed , regardless of whether oxygen tension was maintained at a constant level ( capped or uncapped cell; e . g . , Figs 1C versus 2A or 2B ) ; ( 2 ) MTY fluorescence was not modified by mechanical re-oxygenation of the medium ( Fig 2A ) ; and ( 3 ) using cells expressing the cyanide-insensitive nonproton motive alternative oxidase ( AOX ) from C . intestinalis , MTY fluorescence remained stable after the addition of cyanide , even though oxygen continued to be consumed ( Fig 3B after cyanide addition ) . Importantly , the fluorescence of the endoplasmic reticulum ( ER ) -targeted probe ER thermo yellow [8] in both HEK293 cells and skin fibroblasts was essentially unaffected by the activity of the mitochondria when modulated by cyanide , pyruvate , or antimycin ( S7 Fig ) , although it was sensitive to externally imposed ( water-bath ) temperature changes . We next studied MTY probe behavior in HEK293 cells , in which CI was inhibited by the addition of varying amounts of rotenone ( Fig 3A ) . The rate of change of MTY fluorescence was proportional to the residual respiratory electron flux whilst the maximal temperature , as implied by MTY fluorescence at equilibrium ( phase II ) , was essentially unchanged ( Fig 3A , inset ) . We next tested the effect of expressing AOX ( Fig 3B , S1 Fig ) , the activity of which is unmasked in the presence of cyanide [8] . Before cyanide addition , the decrease in MTY fluorescence in AOX-expressing cells was similar to control cells , consistent with previous inferences that the enzyme does not significantly participate in uninhibited cell respiration [9] . However , upon cyanide addition , AOX-endowed cells maintained low MTY fluorescence ( Fig 3B , blue trace ) , whilst the rate of oxygen consumption decreased by more than 50% ( red trace ) . The implied increase in the ratio of heat generated to respiration is consistent with the predicted thermogenic properties of AOX . Subsequent addition of 0 . 1 mM propylgallate , which inhibits AOX , almost completely abolished the residual respiration and brought MTY fluorescence back to its starting value ( corresponding to 38 °C ) . So as to circumvent the fact that we were not able to use chemical uncouplers with this probe [1] , we used HEK293 cells engineered to express the uncoupling protein 1 ( UCP1 ) ( Fig 3C , S1 Fig ) , which diminishes membrane potential and shifts the balance of respiratory energy conversion from ATP synthesis towards heat production . As expected , UCP1 conferred an increased rate of respiration , which was only partially inhibited by oligomycin ( red trace ) and accompanied by an even greater drop in MTY fluorescence . Based on the internal calibration at the end of the experiment , this is equivalent to a temperature of about 12 °C above the cellular environment . HEK293 cells expressing UCP1 also exhibited a faster rate of MTY fluorescence decrease compared with control HEK293 cells , about twofold during the first 5 min . Importantly , the expression of UCP1 did not slow down the rate of MTY fluorescence decrease as would be predicted if this decrease were dependent on membrane potential or pH gradient ( Fig 3D ) . The surprisingly high inferred mitochondrial temperatures prompted us to test the dependence on assay medium temperature of RC enzyme activities measured under Vmax conditions in crude extracts , in which mitochondrial membrane integrity is maintained ( Fig 4A and 4B ) . Antimycin-sensitive CIII , malonate-sensitive succinate cytochrome c reductase ( CII+CIII ) , and cyanide-sensitive cytochrome c oxidase ( CIV ) activities all showed temperature optima at or slightly above 50 °C , whilst these activities tended gradually to decrease as the temperatures were raised further ( Fig 4A ) . This was not so for those enzymes whose activities can be measured in vitro only after osmotic disruption of both outer and inner mitochondrial membranes ( Fig 4B ) . Oligomycin-sensitive ATPase ( CV ) activity was optimal around 46 °C , whereas rotenone-sensitive NADH quinone-reductase ( CI ) activity declined sharply at temperatures above 38 °C . After disruptive treatment , the activities of CIV and CII of mitochondria , as revealed by native electrophoresis and in-gel activity ( IGA ) ( Fig 4C ) , were also impaired at high temperature . Taken together , these findings strongly suggest a vital role for the integrity of the inner mitochondrial membrane structure in the stabilization of the RC complexes at high temperature . We next analyzed the temperature profile of RC activity of primary skin fibroblasts . For CII+CIII , CIII , and CIV ( Fig 4D ) as well as CV ( Fig 4E ) , similar temperature optima were observed as in HEK293 cells , whilst MTY fluorescence ( Fig 4F ) also indicated mitochondria being maintained at least 6–10 °C above environmental temperature .
Our data raise many questions: have we excluded all possible artifacts and confounding factors influencing MTY fluorescence ? Is the inference that mitochondria operate at temperatures of 48 °C or more credible , in light of theoretical considerations ? Are the findings consistent with those reported elsewhere ? What are their implications for the structure , function , and pathology of mitochondria ? For several years , an intense debate has addressed the possibility of maintaining temperature gradients in isolated cells , considering the small volumes involved [11 , 12 , 13 , 14 , 15] . Largely based on theoretical considerations , it has been suggested that factors other than temperature could account for the large fluorescence changes observed using thermosensitive probes [14] . For mitochondria , these potential factors include membrane potential changes ( and related changes in pH , ionic gradients , and matrix morphology ) , altered mitochondrial superoxide production , varying oxygen concentration , changes in probe conformation ( especially for fluorescent proteins ) , or probe leakage from mitochondria . Using indirect methods , we were able to exclude most of these factors from significantly influencing intracellular MTY fluorescence under our experimental conditions . Previous studies of MTY in aqueous solution already excluded changes in pH , viscosity , metal ions , and oxygen species from affecting its fluorescence [1] , although it remains formally possible that this nonresponsiveness is modified under cellular conditions . More direct measurements , as successfully implemented for the ER-targeted probes ER thermo yellow [8] and its derivative ERthermAC [17] in regard to Ca2+ levels and pH , are precluded by the fact that MTY requires a minimal membrane potential to be taken up and retained inside mitochondria , preventing meaningful studies from being carried out using fixed cells . Despite this caveat , we observed a consistent and robust correlation between MTY fluorescence and temperature , unaffected by or unrelated to implied changes in any other parameter . However , these observations will need to be validated and explored further by independent methods . Uncertainties regarding micro- and nanoscale physical parameters [15] render purely theoretical considerations questionable when considering the complex and dynamic structure of mitochondria . Most models have treated mitochondria as isolated , undifferentiated balls , floating in an aqueous medium , the cytosol . In considering temperature diffusion , the situation is often depicted as a thin membrane separating two compartments , whilst the MTY-containing space should be more accurately depicted as a narrow zone sandwiched between heat-producing membranes . Structural oversimplification may underestimate significant temperature differences between mitochondria and the cytosol . Furthermore , mitochondria in vivo typically form an interconnected filamentous network , variously packed according to cell type . Assuming MTY to be localized to the inner face of the inner membrane or the adjacent pockets of matrix , the heated compartments would be juxtaposed to each other rather than to the colder cytosol . Moreover , compaction of the cytosol in domains rich in mitochondria , as observed in HEK293 cells , would also limit heat conduction out into the rest of the cell . The molecular heterogeneity of the various submitochondrial compartments must also be considered . The inner membrane is rich in proteins ( protein-to-lipid ratio is 80:20 , compared to 50:50 for the outer membrane ) , including those that are sources of heat , and has a distinct lipid composition , including cardiolipins . The intermembrane space , the phospholipid-rich outer membrane , the cytosol , plus the plasma membrane potentially represent additional layers of insulation from the cell suspension medium , with many relevant parameters unknown , including heat conductance and geometry of the various compartments . Measuring how the lipid composition of the mitochondrial membranes influences the thermal stability of the RC complexes and the temperature-responsiveness of other mitochondrial properties , such as swelling and the permeability transition , will shed light on these issues . It will also be interesting to compare the content of cardiolipins and other lipids in mitochondrial membranes from homeotherms and from poikilotherms living at much colder environmental temperatures . A 6–9 °C temperature shift between mitochondria and the surrounding cytosol , induced by the addition of an uncoupler ( 10 μM carbonyl cyanide-4- ( trifluoromethoxy ) phenylhydrazone ) was recently reported in HeLa cells [16] using a genetically encoded , green fluorescent protein-derived ratiometric fluorescent probe . Although carried out under quite different conditions ( confocal microscopy of a single cell ) and without determining mitochondrial activity under these conditions , the data are consistent with our own findings . In addition , the investigation of the temperature of the ER in activated brown adipocytes using ERthermAC as a probe [17] concluded that it was able to reach temperatures over 15 °C hotter than the surrounding medium . Noting that the ER in these cells is in close juxtaposition to mitochondria , which are the source of heat , this would be consistent with brown fat mitochondria being maintained at temperatures even exceeding those we inferred in the present study . The physical , chemical , and electrical properties of the inner mitochondrial membrane and of mitochondria in general should be carefully considered in light of our findings . Most previous literature reflects experiments conducted far from our inferred physiological temperature , implying a need to expand this voluminous body of work to take account of it . Traditional views of the lipid component of the respiratory membrane as a lake in which the RC complexes are freely diffusing , or as a sealant occupying the space between tightly packed proteins [17] , may need to be revised to consider it as a glue that maintains the integrity of the respiratory complexes . Organisms survive across a wide range of temperatures , ranging from below 0 °C to above 100 °C , and their enzymes and membranes have adapted to function accordingly [20 , 21] , in addition to other adaptations that insulate them from the external environment . For example , the NADP-dependent isocitrate dehydrogenase of thermophilic bacteria functions efficiently above 70 °C [18] . Thus , there is no theoretical reason why enzymes of mammalian mitochondria could not tolerate the temperatures implied by our findings . Currently , rather little information is available on the thermal stability , temperature optima , and thermosensitive properties of mitochondrial enzymes . One well-documented case indicates that the thermal stability of purified NADP-dependent isocitrate dehydrogenase from nonthermophiles [19] is dependent on its substrate , isocitrate , and on its cofactor , magnesium , leading to full protection of activity at 60 °C for 2 h , conditions that otherwise readily inactivate it . It will obviously be instructive to evaluate the relevant parameters of mammalian mitochondrial enzymes under conditions as close as possible to those encountered physiologically and to try to interpret such data in light of the actual range of temperatures of different submitochondrial and subcellular compartments , once temperature-responsive probes for these locations become available . The effects of respiratory dysfunction may need to be reconsidered , taking account of temperature changes that could impact membrane fluidity , electrical conductance , and transport . The organization of RC supercomplexes [20 , 21] should be re-examined using methods less disruptive than CNE The subcellular distribution of mitochondria ( e . g . , perinuclear , or synaptic ) has previously been considered to reflect ATP demand , but mitochondria should also be regarded as a source of heat , potentially relevant in specific cellular or physiological contexts , not just in specifically thermogenic tissues like brown fat . Furthermore , temperature differences should be considered as an additional possible dimension to the intracellular functional heterogeneity of mitochondria . Fully resolving how heat is conducted inside the cell will require the development of fluorescent or fluorescently tagged temperature-responsive probes tightly targeted to specific subcellular and submitochondrial locations , to report on their temperature and how it changes under specific physiological conditions .
Human cells derived from embryonic kidney , HEK293 , hepatoma tissue culture , HTC-116 , and large-cell lung cancer , NCI-H460 ( American Type Culture Collection , Manassas , VA ) , as well as primary skin fibroblasts derived from healthy individuals and HEK293 cells expressing C . intestinalis AOX [22] or UCP1 [23 , 24] were cultured in DMEM medium containing 4 . 5 g/L glucose and 2 mM glutamine ( glutamax; Gibco Thermo Fisher Scientific , MA ) , 10% fetal calf serum , 200 μM uridine , 2 mM pyruvate , 100 U/mL each penicillin and streptomycin . The trypan blue exclusion test was used to determine the number of viable and dead HEK293 cells [25] . For western blot analysis , mitochondrial proteins ( 50 μg ) were separated by SDS–PAGE on a 12% polyacrylamide gel , transferred to a nitrocellulose membrane , and probed overnight at 4 °C with antibodies against the protein of interest , AOX 1:10 , 000 [26] , UCP1 1:10 , 000 [27] . Membranes were then washed in TBST and incubated with mouse or rabbit peroxidase-conjugated secondary antibodies for 2 h at room temperature . The antibody complexes were visualized with the Western Lightning Ultra Chemiluminescent substrate kit ( Perkin Elmer ) . For the analysis of RC complexes , mitochondrial proteins ( 100 μg ) were extracted with 6% digitonin and separated by hrCN-PAGE on a 3 . 5%–12% polyacrylamide gel . Gels were stained by IGA assay detecting CI , CII , and CIV activity , as described [28] . Cells were seeded on glass coverslips and grown inside wells of a 12 well plate for 48 h in standard growth media at 37 °C , 5% CO2 . The culture medium was replaced with prewarmed medium containing fluorescent dyes , namely 100 nM MTG ( Invitrogen M7514 ) and 100 nM MTY [1] or 100 nM ER thermo yellow [29] . After 10 min , the staining medium was replaced with fresh prewarmed medium or PBS and cells were observed immediately by Leica TCS SP8 confocal laser microscopy . The measurement of RC activities was carried out using a Cary 50 spectrophotometer ( Varian Australia , Victoria , Australia ) , as described in [30] . Protein was estimated using the Bradford assay . Detached subconfluent HEK293 , NCI-H460 , or HTC-116 cells ( 25 cm2 flask ) or trypsinized subconfluent skin fibroblasts ( 75 cm2 flask ) were treated for a minimum of 10 min with 100 nM MTY ( or 100 nM compound A15 [1] ) in 10 mL DMEM and recovered by centrifugation at 1 , 500 gmax for 5 min . The pellet was washed once in 1 mL PBS , then maintained as a concentrated pellet . After anaerobiosis ( checked by inserting an optic fiber equipped with an oxygen-sensitive fluorescent terminal sensor [Optode device; FireSting O2 , Bionef , Paris , France] ) was established ( 10 min incubation of the pellet at 38 °C ) , cells ( 1 mg prot ) were added to 750 μl PBS thermostatically maintained at 38 °C . The fluorescence ( excitation 542 nm , emission 562 nm for MTY; excitation 559 nm , emission 581 nm for ER thermo yellow; excitation 500 nm , emission 520 nm for A15 ) , the temperature of the medium in the cuvette , and the respiration of the intact cell suspension were simultaneously measured in a magnetically stirred , 38 °C-thermostated 1-mL-quartz cell using a Xenius XC spectrofluorometer ( SAFAS , Monaco ) . Oxygen uptake was measured with an optode device fitted to a handmade cap , ensuring either closure of the quartz cell yet allowing micro-injections ( hole with 0 . 6-mm diameter ) , or leaving the quartz cell open to allow for constant oxygen replenishment . Alternatively , untreated HEK293 cells ( 250 μg protein ) were added to 750 μL of buffer consisting of 0 . 25 M sucrose , 15 mM KCl , 30 mM KH2PO4 , 5 mM MgCl2 , 1 mM EGTA , pH 7 . 4 , followed by the addition of rhodamine to 100 nM and digitonin to 0 . 01% w/v . The permeabilized cells were successively given a mitochondrial substrate ( 10 mM succinate ) and ADP ( 0 . 1 mM ) to ensure state 3 ( phosphorylating ) conditions , under which either 5 μM oligomycin or 0 . 8 mM cyanide was added . Data are presented as mean ±SD . Statistical significance was calculated by standard unpaired one-way ANOVA with Bonferroni posttest correction; a p value <0 . 05 was considered statistically significant ( GraphPad Prism ) . | To ensure a stable internal temperature , endothermic species make use of the heat released during the final steps of food burning by the mitochondria present in all cells of the organism . Indeed , only a fraction of the energy released by the oxidation of respiratory substrates is used to generate ATP , while a substantial proportion is released as heat . Using a temperature-sensitive fluorescent probe targeted to mitochondria , we measured the temperature of active mitochondria in cultured intact human cells . Mitochondria were found to be more than 10 °C warmer when the respiratory chain was functional . This differential was abolished in cells depleted of mitochondrial DNA or by respiratory inhibitors but preserved or enhanced by the expression of thermogenic enzymes such as Ciona alternative oxidase or by uncoupling protein 1 . The activity of various respiratory chain enzymes was found to be maximal near 50 °C . Note that in view of their potential consequences , the observations reported here need to be validated and explored further by independent methods . | [
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] | 2018 | Mitochondria are physiologically maintained at close to 50 °C |
Since Kaposi's sarcoma associated herpesvirus ( KSHV ) establishes a persistent infection in human B cells , B cells are a critical compartment for viral pathogenesis . RTA , the replication and transcription activator of KSHV , can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors . In addition , the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity . To analyze the contribution of CBF1/CSL to reactivation in human B cells , we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV . 219 and selected for viral maintenance by selective medium . Both lines maintained the virus irrespective of their CBF1/CSL status . Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines , which also failed to produce detectable levels of infectious virus . Induction of immediate early , early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL . To identify additional viral RTA target genes , which are directly controlled by CBF1/CSL , we analyzed promoters of a selected subset of viral genes . We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL . Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging . ORF65 encodes the small capsid protein essential for capsid shell assembly . Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production . Thus , CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade .
Kaposi's sarcoma associated herpesvirus ( KSHV ) establishes a persistent infection in the human host . Infected human B cells in the circulation of the infected host are likely to constitute a major latent reservoir , from where KSHV can reactivate and spread . In addition , the strong association of KSHV with primary effusion lymphoma ( PEL ) and the plasma cell variant of multicentric Castleman's disease strongly suggests a causative role of the virus in the pathogenesis of these B cell diseases [1]–[4] . Thus , human B cells are likely to comprise a very important compartment for the persistent KSHV infection . The study of latent and lytic life cycle in B cells has been the focus of many studies in the past . The replication and transcription activator ( RTA ) is a KSHV immediate early protein that activates a broad spectrum of lytic viral genes and thereby induces lytic reactivation . RTA can either directly bind to RTA-responsive elements or use cellular DNA binding factors like Ap-1 , C/EBP-α , Oct-1 and CBF1/CSL as adaptors to DNA as reviewed [5] . The DNA binding factor CBF1/CSL ( C-promoter binding factor , Suppressor of hairless , and Lag1 also designated CSL or RBP-Jκ ) is a highly conserved ubiquitously expressed protein and the major effector of Notch receptor signaling . In the following we will use the term CBF1 for this protein . It can serve as adaptor for transactivators but also can recruit corepressor complexes and thereby silence gene expression [6] , [7] . Two general experimental strategies focussing on the cellular interaction partner or the viral genome have been used to study the functional implications of the RTA-CBF1 interaction . Initially , infection and virus production of CBF1 proficient and deficient murine fibroblasts was studied in isogenic systems . These studies suggested lytic reactivation is blocked while establishment of latency is not impaired in cells lacking CBF1 [8] , [9] . Alternatively , computational prediction of potential CBF1 binding sites in the viral genome was followed by biochemical and mutational analysis of the respective viral promoters in promoter reporter assays or in the context of the viral genome by recombinant virus technologies . In addition , activation of viral targets by activated Notch defined further sets of CBF1 dependent promoters . In summary , these studies convincingly showed , that CBF1 contributes to the activation of ORF6 , ORF8 , ORF19 , ORF47 , ORF50 , ORF57 , ORF59 , ORF61 , ORF70 , K2 , K5 , K6 , K8 , K14 and PAN as well as the latency transcript cluster ( summarized and referenced in Table 1 ) . Importantly , these genes control multiple stages in the viral latent and lytic life cycle . The relevance of the contribution of CBF1 to target gene control varied among the individual genes that were studied . Thus , the question remained to which extent the activity of CBF1 contributes to the process of viral reactivation and virus production in human B cells . In vitro a broad spectrum of cell lines derived from diverse tissues can be infected with KSHV but human B cells and B cell lines appeared to be refractory to infection [4] , [10]–[13] . Most recently , the productive infection of primary B cells could be accomplished but did not lead to long term proliferation of the infected cells [14]–[17] . After infection of the lymphoblastoid B cell line BJAB with cell-associated recombinant virus and cultivation of these infected BJAB cells in selective medium stable latently infected cultures were generated . However , viral reactivation was attenuated and virion production was almost blocked in these human B cells , rendering them unsuitable to address the role of CBF1 in lytic reactivation [18] . The goal of this study was to analyze the contribution of CBF1 to viral lytic reactivation in KSHV infected human B cells . To this end , we have infected isogenic CBF1 deficient and proficient human B cell lines with recombinant KSHV and compared them for viral reactivation and virus production . Our results indicate that viral reactivation is initiated in both CBF1 proficient and deficient B cells but viral gene expression is severely attenuated and virus production is below the detection level in the absence of CBF1 . In addition to the viral genes already known to be controlled by CBF1 , we could identify two further direct CBF1 dependent RTA target genes , ORF29a and ORF65 .
In order to analyze the contribution of CBF1 to viral reactivation in KSHV infected B cells we used a CBF1 negative human somatic B cell line . This cell line had been generated previously by gene targeting of the cellular CBF1 gene in the EBV negative Burkitt's lymphoma B cell line DG75 [19] . For infection of CBF1 proficient parental ( DG75 wt ) and CBF1 deficient ( DG75 CBF1 ko ) DG75 cells we used cell culture supernatants of the recombinant virus rKSHV . 219 produced in Vero cells . The recombinant virus rKSHV . 219 is a derivative of the KSHV strain obtained from JSC-1 cells [20] . rKSHV . 219 encodes for red fluorescent protein ( RFP ) under the control of a CBF1 independent fragment of the KSHV early lytic PAN promoter , constitutively expresses green fluorescent protein ( GFP ) , and carries a puromycin resistance cassette as a selectable marker . DG75 wt and DG75 CBF1 ko cells were infected with rKSHV . 219 at a multiplicity of infection ( MOI ) of factor 5 . GFP expression was monitored by flow cytometry during a time course of 12 weeks . A GFP positive cell population could be detected 1 week post infection . The intensity of the GFP signals recorded by flow cytometry was initially weak but increased over time in both , CBF1 proficient and deficient B cell lines ( Fig . 1A ) . The results suggest that stable homogenous KSHV infected B cell cultures are generated within 4–6 weeks post infection irrespective of the CBF1 status . High GFP expression remained stable as long as the cultures were maintained in media containing puromycin ( Fig . 1A ) . If the established rKSHV . 219 infected DG75 wt and DG75 CBF1 ko cell lines ( K-DG75 wt and K-DG75 CBF1 ko ) were cultivated in the absence of puromycin a GFP negative population developed . Only 30% of CBF1 proficient and 22% of CBF1 deficient B cells were still GFP positive after they had been cultivated in puromycin free media for 10 weeks ( Fig . 1B ) . Hence , viral maintenance required selection in both B cell lines . Viral genome copy numbers of established K-DG75 wt and K-DG75 CBF1 ko cell lines were determined and compared to data obtained from two independent KSHV infected PEL cell lines , BCBL-1 and BC-1 . As expected 50 to 60 intracellular genomes were detected in BCBL-1 and BC-1 cells [21] , [22] . K-DG75 wt and CBF1 ko cells carried approximately 10 viral copies per cell ( Fig . 1C ) . Both K-DG75 cell lines , CBF1 proficient and deficient , expressed the KSHV latent marker genes ORF73/LANA and K10 . 5/vIRF3 at similar levels ( Fig . 1D ) . ORF73/LANA is consistently expressed in latently infected cells of different tissue origin and required to maintain episomal viral genomes during cell division [23]–[25] . Latent expression of K10 . 5/vIRF3 is only seen in KSHV infected B cells [26] . The relative transcript levels of both viral genes were lower than in BCBL-1 and BC-1 cells but readily detectable . These distinct expression levels might partially reflect the lower viral genome copy numbers in the K-DG75 cell lines compared to BCBL-1 and BC-1 . Next we examined if the virus can reactivate in K-DG75 cell lines . Upon treatment with sodium butyrate ( NaB ) the surrogate early lytic marker gene RFP was induced and expressed with similar frequencies in two independent K-DG75 wt and K-DG75 CBF1 ko cell lines ( Fig . 2A ) . The combination of 12-O-Tetradecanoylphorbol-13-acetate ( TPA ) and NaB further enhanced RFP expression with a negligible difference between wt and CBF1 ko cells ( Fig . 2B ) . Initiation of lytic reactivation thus does not appear to be a CBF1 dependent process in these B cells . We next analyzed if CBF1 proficient and deficient K-DG75 cell lines exhibit similar rates of viral replication upon lytic reactivation by TPA/NaB treatment . The rate of dead cells was determined for treated and untreated cell populations by trypan blue exclusion assays . The TPA/NaB treatment caused similar rates of dead cells in both populations ( Fig . S1A ) . Viable subpopulations were defined by forward and sideward scatter signals ( Fig . S1B ) and RFP+/GFP+ cells were separated from RFP−/GFP+ cells by cell sorting . Equal numbers of sorted cells were compared for the number of intracellular viral genomes . Viral copy numbers increased in CBF1 proficient and deficient cells , but the increase in CBF1 proficient cells was two fold stronger ( Fig . 2C ) . Since lytic viral replication was impaired in CBF1 deficient B cells we investigated whether virus production was also attenuated . K-DG75 B cells were treated with TPA and NaB for 4 days , supernatants were harvested , concentrated 100-fold and used to infect HEK293 cells . These HEK293 cells were cultured either in the presence or absence of puromycin for 48 h and subsequently analyzed for GFP expression by fluorescence microscopy ( Fig . 2D ) . In addition , cellular viability was monitored by flow cytometry using forward and sideward scatter to visualize viable and dead cell populations ( Fig . 2E ) . GFP positive HEK293 cells were readily detected after infection with K-DG75 wt cell supernatants . K-DG75 CBF1 ko cells produced no or extremely low numbers of virions that conferred GFP expression . As a second marker for successful infection we tested whether the viral supernatants conferred puromycin resistance ( Fig . 2D and E ) . Indeed , HEK293 cells infected with viral supernatants harvested from K-DG75 wt cells could be expanded in the presence of puromycin while HEK293 cells infected with supernatants from K-DG75 CBF1 ko cells lost their typical shape and their adherent phenotype and subsequently died ( Fig . 2E ) . These results suggest that supernatants derived from CBF1 deficient K-DG75 cells contain no or only low amounts of infectious virus . To further confirm this assumption , extracellular viral genome copy numbers in K-DG75 wt and CBF1 ko derived supernatants were quantified and compared to virion DNA produced by BC-1 . Virus production was induced ∼165-fold upon chemical reactivation of K-DG75 wt cells , approximately 3-fold higher than in BC-1 cells . In contrast , virion production by CBF1 deficient cells was barely detectable ( Fig . 2F ) . In summary , our data show that K-DG75 cells can produce infectious virus . Genome replication was attenuated in CBF1 deficient K-DG75 cells and later processes required for morphogenesis of infectious virus were blocked . In order to examine how the differences in viral reactivation were reflected by alterations in viral gene expression patterns we analyzed lytic viral gene expression in CBF1 proficient and deficient K-DG75 cells before and 2 , 4 , 8 , 16 or 32 h post induction by NaB . The viral gene expression profile was analyzed using a previously developed real-time RT-PCR array for KSHV that represents 86 viral genes [27] , [28] . The results of this genome wide analysis of viral reactivation in CBF1 proficient and deficient K-DG75 cells are shown as heat map ( Fig . 3 ) . The genes were arranged into groups of latent , early and late lytic genes as reviewed [29] . Induction of lytic viral genes could be observed in CBF1 proficient and deficient K-DG75 cells but was severely attenuated and delayed in CBF1 deficient cells . The difference between CBF1 proficient and deficient K-DG75 cells was most pronounced 32 h post induction . A selection of viral genes was retested by real-time RT-PCR choosing primers distinct from those primers used for the array analysis ( Fig . 4A ) . These results confirmed that all lytic genes were induced in CBF1 proficient and deficient K-DG75 cells but the degree of induction was different . Again the difference was best seen at late time points , when viral replication had already been initiated . The latent ORF73/LANA expression was not changed dramatically by chemical induction . Latent K10 . 5/vIRF3 was only induced in CBF1 proficient K-DG75 cells . As already seen by the array analysis , CBF1 dependent gene induction was not confined to a specific gene class . Thus , CBF1 appears to be required at multiple stages during the reactivation process . Chemical induction of KSHV infected cells typically leads to reactivation of only a subset of the cellular population . We thus sorted RFP−/GFP+ latent and RFP+/GFP+ lytic populations and tested them for induction of the immediate early gene ORF50/RTA and the early lytic genes ORF57 and ORF59 . As expected the difference in gene expression between CBF1 proficient and deficient K-DG75 cells was more pronounced if selected subsets were analyzed ( Fig . 4B ) . In order to confirm our results we reintroduced a Flag-tagged version of the CBF1 protein into K-DG75 CBF1 ko cell lines . The expression construct , pRTS-2 , we used carries a bidirectional doxycycline responsive promoter [30] . Induction of the gene of interest can be monitored by flow cytometry of a surrogate marker , a truncated version of the NGF-receptor , on the cell surface . Stable cell lines carrying the Flag-CBF1 expression construct showed doxycycline dependent NGF-receptor and Flag-CBF1 expression while cells carrying a control vector only expressed the NGF-receptor ( Fig . 5A and B ) . Next the induction of a series of lytic viral genes ( ORF50/RTA , ORF57 , ORF59 , ORF65 , ORF29a and ORF4 ) was tested in K-DG75 CBF1 ko cells in which Flag-CBF1 was induced by doxycycline . Two independent inducible cell lines were tested and compared to two control cell lines . Induction of lytic viral genes was restored by Flag-CBF1 and even exceeded the levels reached in K-DG75 wt ( Fig . 5C ) . This observation indicates that the endogenous cellular CBF1 protein level is rate limiting for viral reactivation in DG75 cells . ORF50/RTA is the major transactivator of ORF57 and ORF59 . In addition , both , ORF57 and ORF59 , are known to enhance RTA functions . ORF57 is a multifunctional protein which enhances splicing and translation efficiencies and supports the export of intronless viral mRNA and stabilizes transcripts [31] . ORF59 is one of the DNA polymerase processivity factors which are necessary for origin dependent viral replication [32] . Since ORF50/RTA induction was diminished in CBF1 deficient cells this defect in RTA expression could already cause a severe phenotype in CBF1 deficient DG75 cells . We hence wanted to ask if ORF50/RTA expression might rescue the deficiency of the CBF1 deficient K-DG75 cells . We thus expressed ORF50/RTA in K-DG75 cells and induced the lytic cycle by NaB . ORF50/RTA could strongly enhance ORF57 and ORF59 expression in NaB treated CBF1 proficient cells but induction in the absence of CBF1 was weak ( Fig . 6 ) . These results suggest that the lack of induction of ORF50 is not the only rate limiting factor for viral reactivation in CBF1 deficient B cells . In summary , the analysis of viral gene expression profiles had shown that the induction of lytic genes of all classes was not blocked but attenuated in CBF1 deficient B cells . Viral genes that had been previously defined as genes which need CBF1 to recruit either RTA or Notch are listed in Table 1 . In order to identify additional CBF1 dependent viral target genes the promoters of 4 selected genes which carried at least one CBF1 binding site predicted by using the MatInspector software of Genomatix were analyzed . Since the transcriptional start site of these genes had not been defined experimentally in the past a fragment of 1000 bp upstream of the translational start site was tested as a putative promoter . Transactivation of these putative promoter fragments by ORF50/RTA was tested in transient reporter gene assays using KSHV negative DG75 cells . For comparison the promoter of ORF59 , a well known CBF1 dependent target gene , was included in the analysis . All promoters responded to ORF50/RTA even in the absence of CBF1 in a dose dependent manner indicating that there is no absolute requirement for CBF1 to recruit RTA to these target genes and activate transcription . Promoter activation of ORF59 , ORF29a and ORF65 was significantly diminished in CBF1 deficient DG75 cells while transactivation of the ORF9 and ORF62 promoters was not impaired ( Fig . 7A ) . By reintroducing CBF1 , transactivation of the ORF59 , ORF29a and ORF65 promoters was strongly enhanced confirming that CBF1 is a rate limiting factor for efficient activation of these promoters ( Fig . 7B ) . Since we wanted to prove that CBF1 is recruited to the promoters of ORF29a and ORF65 we performed chromatin immunoprecipitations followed by real-time PCR for viral promoter fragments ( Fig . 7C ) . While CBF1 could be readily detected on the promoter of its cellular target gene CD23 in KSHV positive and negative DG75 wt cells , as expected CBF1 was seen on viral promoters in KSHV infected DG75 wt cells only . In summary , these results suggest that the promoters of ORF29a and ORF65 are directly bound by CBF1 .
The CBF1 protein is a DNA binding factor which is highly conserved in evolution . In mammals CBF1 is ubiquitously expressed in all tissues . Thus , CBF1 provides a central hub in every mammalian cell that is used by the cell to integrate information transmitted by external and internal stimuli . The two human γ-herpesviruses , EBV and KSHV , use CBF1 as a DNA adaptor for viral nuclear transcription factors to control the activity of viral and cellular target genes . EBV requires the CBF1 protein to enter and maintain the latent life cycle . In contrast , lytic infection and reactivation are CBF1 dependent processes in KSHV infected cells [8] , [9] . RTA , the initiator of viral lytic replication , can bind to DNA directly but also uses CBF1 as an adaptor to regulatory elements of target genes . RTA expression is controlled by CBF1 binding sites within the RTA promoter . Thereby RTA initiates a positive feed back loop driving lytic reactivation [33] . On the other hand , LANA expression is also controlled by RTA via CBF1 dependent activation while LANA represses RTA expression via binding to CBF1 at the RTA promoter . However , subsequent studies using infective recombinant KSHV which lacked CBF1 binding sites in the RTA promoter demonstrated that these viruses had an enhanced capacity to infect and establish latency in primary human B cells in short term experiments [33] . In summary , CBF1 serves as the mediator of a negative and positive feed back loop that balances RTA and LANA expression and hence lytic and latent life cycle of the infected cell [34] , [35] . Consequently , targeting CBF1 signaling by antiviral drugs could be highly attractive if critical stages of the virus life cycle relevant for pathogenesis could be efficiently blocked . The functional analysis of CBF1 dependent processes has been hampered by the ubiquitous expression of CBF1 in human cells . Thus , the first report which described the interaction of RTA with CBF1 used CBF1 deficient fibroblasts derived from knock-out mice to study the contribution of CBF1 to establishment of latency , reactivation and lytic infection [9] , [36] . While establishment of latency was not impaired in mouse fibroblasts reactivation was blocked at the stage of delayed early gene expression . In summary , our data confirm and extend the results of previous studies . Unlike murine fibroblasts , KSHV infected CBF1 deficient B cells can enter the lytic cycle with low efficiency but no specific block before the onset of viral replication or late gene expression is installed . Using the identical recombinant rKSHV . 219 virus it has been described most recently that viral reactivation is blocked at multiple stages before viral DNA synthesis in CBF proficient murine fibroblasts [37] . These multiple blocks attenuated the induction of the lytic cascade in CBF1 deficient murine fibroblasts in the previous study and most likely explain why reactivation in human B cells can still be initiated . The goal of our study was to identify CBF1 dependent processes that are rate limiting for viral reactivation and production of infectious viruses specifically in B cells . Both , CBF1 proficient and CBF1 deficient KSHV infected DG75 B cell lines could be established , carried similar numbers of intracellular viral genomes , and expressed similar amounts of the latent marker genes ORF73/LANA and K10 . 5/vIRF3 . During the first weeks post infection GFP expression was low but detected readily by flow cytometry and increased during the following weeks ( Fig . 1 ) . Since it was not relevant for the process of viral reactivation we have not analyzed the switch from GFP low to GFP high cultures in detail . At this point of our studies we do not want to exclude that GFP was transferred passively by viral particles and measured by flow cytometry early after infection . Future studies should reveal whether GFP expression levels changed during in vitro cell culture due to changes in viral genome copy numbers or were caused by epigenetic modifications of the viral chromatin . Since all KSHV infected DG75 cells were grown in selective media the infection process as well as establishment of the latent state in these cells may not reflect all features of the infection process under physiological conditions . Thus , at present the DG75 infection system cannot yet be used to study the potential role of CBF1 during early phases of the establishment of latency . While CBF1 proficient and deficient B cell lines could induce the RFP reporter gene controlled by the early lytic PAN promoter of rKSHV . 219 , only CBF1 proficient DG75 B cells could produce infectious virus . Virus produced from K-DG75 wt cells induced a bright GFP signal but also conferred puromycin resistance to infected HEK293 cells ( Fig . 2 ) . Reactivation of the lytic cycle was initiated in both cell lines . However , activation of lytic genes was delayed in CBF1 deficient cells and did not reach the same expression levels as the CBF1 proficient cells 32 h post induction . Even 4 days after induction neither extracellular viral genomes nor release of infectious virus was detectable . Transcription of CBF1 dependent genes ( ORF50/RTA , ORF57 , ORF59 , ORF65 , ORF29a or ORF4 ) could be rescued or even enhanced by conditional expression of CBF1 . Thus , we could formally prove that the phenotype of CBF1 deficient K-DG75 cells is truly caused by the lack of CBF1 . Ectopic expression of ORF50/RTA in K-DG75 CBF1 ko cells induced ORF57 and ORF59 expression weakly but induction rates never reached the levels obtained in K-DG75 wt cells . Thus , ORF50/RTA induction in K-DG75 CBF1 ko cells is essential but not the single rate limiting factor for reactivation . Our results suggest that the attenuated lytic gene expression levels are caused by additive effects of genes that can only be weakly activated by RTA in the absence of CBF1 . While virus replication is still detected in K-DG75 CBF1 ko cells at reduced levels , virion production is entirely abolished . Interestingly , we could not identify a single gene that is not activated in K-DG75 CBF1 ko cells during reactivation and thus completely dependent on CBF1 for activation ( Fig . 3 ) . We conclude that the lack of CBF1 has pleiotropic effects during the reactivation process caused by direct and indirect effects triggered by ORF50/RTA or perhaps other viral CBF1 binding proteins like ORF73/LANA or vIRF4 [38] , [39] . While LANA is an antagonist of RTA function , vIRF4 cooperates and enhances RTA activities [40] . Whether this cooperation of vIRF4 and RTA is CBF1 dependent still needs to be clarified . As summarized in Table 1 there is a growing list of lytic viral genes requiring CBF1 for activation . In search for additional viral ORF50/RTA responsive and CBF1 dependent promoters , the putative promoters of ORF29a , ORF65 , ORF9 and ORF62 were analyzed in reporter gene assays . The promoter of ORF59 was studied in parallel since ORF59 is a well characterized target gene of RTA ( Fig . 7A ) . While activation of the endogenous ORF59 gene requires CBF1 , ORF59 promoter activation is attenuated if CBF1 binding sites are deleted but RTA DNA binding sites are retained [9] , [41] . Our experiments confirm that the requirement for CBF1 is much more pronounced if endogenous ORF59 gene expression is studied . These results might suggest that the chromatin state of the ORF59 gene has an important impact on promoter responses to RTA . If so , this could be relevant for the biology of the virus since the viral DNA in the infectious virus is chromatin free and unmodified . While activation of the endogenous transcripts of ORF9 and ORF62 was CBF1 dependent , activation of the promoter reporter constructs of these genes by RTA was CBF1 independent . For ORF9 and ORF62 we cannot exclude that we have used incomplete promoter fragments which recruit RTA directly but do not carry the relevant CBF1 responsive elements . Perhaps these CBF1 binding sites are located in remote enhancers as it has been demonstrated recently for the CBF1 interaction partner Epstein-Barr virus nuclear antigen 2 in the context of the cellular genome [42] . Alternatively , the CBF1 dependent effects measured on endogenous gene expression were chromatin dependent , post-transcriptional , and caused by the attenuated lytic cascade in CBF1 deficient cells . In contrast , RTA activation of the promoters of ORF29a and ORF65 was strongly impaired in the absence of CBF1 but could be rescued by ectopic expression of CBF1 . In addition , CBF1 binding to these promoters could be demonstrated . Thus , ORF29a and ORF65 are RTA target genes which require direct binding of CBF1 to promoter sites . Both are late viral genes which are critical for viral morphogenesis and virus production . For this study , we have developed a new cell culture model to study KSHV reactivation in human B cells . The DG75 human B cell line will be a versatile tool to study KSHV mutants in a cellular background that permits to inactivate genes by gene targeting . In the future individual KSHV loss of function mutants can be tested and these experiments can be combined with specific DG75 variants deficient for selected cellular proteins . In summary , the results obtained with this novel B cell system strongly suggest that CBF1 signaling in human B cells has pleiotropic effects that coordinate and enhance the course of KSHV lytic viral gene induction at multiple stages . As exemplified by the two novel CBF1 dependent late genes ORF29a or ORF65 it appears that CBF1 is a global player , required at multiple stages to coordinate the lytic cascade . If antivirals targeting CBF1 signaling could be established we would expect that KSHV reactivation is severely impaired .
The cell lines BC-1 [1] , BCBL-1 [22] , HEK293 [43] , DG75 wt [44] , and DG75 CBF1 ko ( SM224 . 9 ) [19] have been described . Routinely all cell lines were grown in RPMI 1640 supplemented with 10% fetal calf serum ( FCS ) , penicillin ( 100 U/ml ) , streptomycin ( 100 µg/ml ) , and glutamine ( 4 mM ) and maintained at 37°C in a 5% CO2 atmosphere . BC-1 and BCBL-1 were cultured in media containing 20% FCS . K-DG75 wt a ( BS532 . 2a ) and b ( BS854 . 2b ) and K-DG75 CBF1 ko a ( BS532 . 1a ) and b ( BS648 . 1e ) were cultured in media containing 20% FCS and puromycin ( 4 µg/ml ) . K-DG75 CBF1 ko tet-CBF1 a ( BS1177 . 3 ) and b ( BS1177 . 7 ) or tet-ctrl a ( BS1247 . 8 ) and b ( BS1247 . 9 ) were cultured in media containing 20% FCS , puromycin ( 4 µg/ml ) and hygromycin B ( 400 µg/ml ) . Vero cells containing rKSHV . 219 were kindly provided by J . Vieira ( University of Washington ) [20] and grown in DMEM supplemented with 10% FCS and 5 µg/ml puromycin . The Gateway compatible destination vectors pHACR3 [45] as well as the CBF1 expression vector ( AJ247 ) have been published previously [19] . The vector pcDNA3 . 1-lacZ ( Invitrogen ) is commercially available . The pENTRY construct encoding ORF50/RTA has been published [46] . For expression in mammalian cells ORF50/RTA was transferred into destination vector pHACR3 by LR reaction ( Invitrogen ) . A Triple-Flag-CBF1 ORF was cloned into pRTS-2 containing a hygromycin B resistance cassette and a bidirectional doxycycline ( Dox ) -regulated promoter which drives the simultaneous expression of a truncated NGF-receptor and Flag-CBF1 after addition of Dox [30] . In order to generate KSHV promoter constructs ( ORF59-p , ORF9-p , ORF29a-p , ORF62-p and ORF65-p ) fragments of 1000 bp upstream of the translational initiation codon of the KSHV genes were amplified by PCR using genomic DNA isolated from BC-1 cells as template and specific primers containing restriction sites . The PCR products were ligated into a luciferase reporter plasmid carrying a minimal silent promoter ( Ga50-7 ) . Primers used for cloning are listed in supplementary Table S1 . The prediction of potential CBF1 binding sites in these KSHV promoter fragments was done by using the MatInspector software provided by Genomatix ( matrices V$RBPJK . 01 ( cgTGGGaa ) and V$RBPJK . 02 ( gTGGGaaa ) , core similarity 1 . 0 and matrix similarity >0 . 8 ) . The position of the promoter fragments and the potential CBF1 binding sites in the viral genome are listed in supplementary Table S2 . For lytic cycle reactivation cells were treated with sodium butyrate ( NaB ) and/or 12-O-Tetradecanoylphorbol-13-acetate ( TPA ) as indicated . 1 . 5×105 cells per ml were induced . Virus was harvested as described before [47] . Briefly , 4 days post induction cells were pelleted and the supernatant was filtered through a 0 . 45 µm pore-size filter . The virus was concentrated and washed twice in RPMI 1640 media without supplements by ultracentrifugation at 25 , 000 rpm in a Beckman SW28 rotor for 3 hours . Finally , the virus pellet was resuspended in 1/100 volume of the initial volume in cell culture medium . To determine the number of infectious virus HEK293 cells were infected with serial dilutions of the viral supernatants and the number of GFP positive cells was counted [20] . 2×105 DG75 wt or CBF1 ko cells per ml were seeded in a volume of 100 µl in a 96-well plate . The following day cells were infected with virus supernatant of Vero-rKSHV . 219 cells with a multiplicity of infection ( MOI ) of factor 5 . The culture plate was centrifuged at 300 g for 40 min at 32°C . One day post infection medium was replaced . 7 days post infection selection for rKSHV . 219 positive cells was started by adding puromycin ( 1 µg/ml ) . Every 7 days half of the culture medium was replaced with medium supplemented with an increasing concentration of puromycin up to 4 µg/ml . Stable GFP positive cell lines were designated K-DG75 wt a ( BS532 . 2a ) and b ( BS854 . 2b ) or K-DG75 CBF1 ko a ( BS532 . 1a ) and b ( BS648 . 1e ) . K-DG75 CBF1 ko cells were transfected with the Triple-Flag-CBF1 pRTS-2 vector or a Triple-Flag control pRTS-2 vector by electroporation . Cells were selected in the presence of hygromycin B ( 400 µg/ml ) and puromycin ( 4 µg/ml ) . Stable cell lines were designated K-DG75 CBF1 ko tet-CBF1 a ( BS1177 . 3 ) and b ( BS1177 . 7 ) or tet-ctrl a ( BS1247 . 8 ) and b ( BS1247 . 9 ) . Digital images were acquired using the Openlab acquisition software ( Improvision ) and a microscope ( Axiovert 200 m , Carl Zeiss MicroImaging , Inc . ) connected to a 5 charge-coupled device camera ( ORCA-479 , Hamamatsu ) . Infection of DG75 wt or CBF1 ko cells by rKSHV . 219 or of HEK293 cells was monitored by GFP expression . NGF-receptor expression of K-DG75 CBF1 ko tet-CBF1 or tet-ctrl cells after Dox treatment was analyzed using a primary α-NGF-receptor antibody ( HB8737-1 , ATCC ) or an isotype control and a Cy5-coupled secondary antibody ( Dianova ) . Fluorescence of cells was detected and analyzed using a FACSCalibur system and CellQuest Pro software ( BD Bioscience ) . To determine the percentage of lytically induced RFP+/GFP+ K-DG75 cells or to separate RFP−/GFP+ and RFP+/GFP+ K-DG75 cells , cells were sorted and analyzed using FACSAria III cell sorter ( BD Bioscience ) and FlowJo software ( version 7 . 6 . 4 ) . For determination of intracellular KSHV DNA copy numbers 1×106 cells were harvested , washed in PBS , resuspended in solution A ( 10 mM Tris-HCl , pH 8 . 3 , 100 mM KCl , 2 . 5 mM MgCl2 ) and lyzed in solution B ( 10 mM Tris-HCl , pH 8 . 3 , 2 . 5 mM MgCl2 , 1% Tween 20 , 1% NP-40 ) supplemented with RNase A ( 0 . 2 µg/µl ) and Proteinase K ( 1 . 5 µg/µl ) and incubated for 30 min at 37°C and subsequently for 60 min at 50°C . DNA was purified by phenol-chloroform extraction . Extracellular virion-associated KSHV genomes in culture supernatants of lytically induced cells were isolated as described [48] . Intracellular and extracellular viral DNA was analyzed by real-time PCR as described below . For quantification a standard curve with defined numbers of PCR fragments of the KSHV genome corresponding to the ORF50 promoter region and β-actin was generated and analyzed in parallel . The intracellular KSHV copy number per cell was determined after normalization to β-actin . Primers used for real-time PCR are listed in supplementary Table S3 . K-DG75 wt or CBF1 ko cells were lytically induced with 3 mM NaB for 0 , 2 , 4 , 8 , 16 or 32 hours . RNA of was extracted , mRNA was enriched and cDNA was synthesized by reverse transcription as described [27] . The KSHV real-time PCR Array was performed in collaboration with D . Dittmer ( Lineberger Comprehensive Cancer Center , Chapel Hill ) as described previously [27] . dCt values of each primer pair of 86 KSHV genes were normalized to β-actin . Heat map representation of the viral gene expression profile was generated using the software Genesis [49] . RNA of 5×106 cells was extracted , treated with DNase and cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit ( Applied Biosystems ) according to the manufacturer's protocol . Relative quantification of the transcripts by real-time PCR was performed with the LightCycler 480 II system and the data were processed by LightCycler 480 software , version 1 . 5 . 0 . 39 ( Roche ) . cDNA was amplified using the LightCycler 480 SYBR Green I Master mix according to the manufacturer's protocol ( Roche ) . Cycling conditions were 1 cycle of 95°C for 10 min and 45 cycles of denaturation ( 95°C for 2 s ) , annealing ( 63°C for 10 s ) , and extension ( 72°C for 20 s ) . All PCR products were examined by melting curve analysis and the expected PCR fragment size was verified by agarose gel electrophoresis . To account for differences in reaction efficiencies , a standard curve was generated for each primer pair by using the serial dilutions of PCR products as templates for amplification and plotting the crossing points versus the known dilutions . All data were normalized to the relative abundance of the β-actin transcript . Primers used for real-time RT-PCR are listed in supplementary Table S3 . 1×107 DG75 wt or CBF1 ko cells were transfected with the indicated plasmid DNA by electroporation ( 220 V , 950 µF ) using a gene Pulser II ( Bio-Rad ) . Luciferase reporter gene assays were performed as described previously [39] . Briefly , cells were transfected with 3 µg luciferase reporter construct , 1 µg pcDNA3 . 1-lacZ and the indicated expression plasmid . The DNA amounts were adjusted by adding the corresponding empty vector . Cells were harvested 48 h after transfection and luciferase and β-galactosidase activity was measured . Transfections were done in triplicates and results were normalized to ß-galactosidase activity derived from the reporter construct pcDNA3 . 1-LacZ included in each sample . Western blot analysis was performed as described [19] . The α-CBF1 rat monoclonal antibody RBP-7A11 ( produced in collaboration with E . Kremmer , Helmholtz Center Munich ) has been published [19] . The α-Flag ( Sigma-Aldrich ) and α-GAPDH ( Millipore ) antibodies were purchased . . ChIP analysis was performed as described [50] with minor modifications using a mixture of hybridoma supernatant of the α-CBF1 rat monoclonal antibodies RBJ-1F1 and RBJ-6E7 ( see supplementary text and Table S4 for details ) . | Kaposi's sarcoma associated herpesvirus ( KSHV ) establishes a life-long persistent infection in B cells , which constitute the viral reservoir for reactivation and production of progeny virus . Viral reactivation is associated with multiple AIDS related malignancies including Kaposi's sarcoma , an endothelial tumor , and two B cell lymphoproliferative malignancies , the primary effusion lymphoma and the multicentric Castleman's disease . CBF1/CSL is a cellular DNA binding protein that can recruit transactivators or repressors to regulatory sites in the viral and cellular genome . The replication and transcription activator ( RTA ) plays an essential role in the switch between latency and lytic reactivation . RTA can either bind to DNA directly or is recruited to DNA via anchor proteins like CBF1/CSL and activates transcription . In this study we used a novel cell culture model to analyze the contribution of the CBF1/CSL protein to the process of viral reactivation in human B cells . Two isogenic CBF1/CSL proficient or deficient B cell lines were latently infected with recombinant KSHV . Lytic viral gene expression , viral replication and virus production were compared . Our results suggest that viral lytic gene expression is severely attenuated but not abolished at multiple stages before and after the onset of lytic replication while virus production is below detection levels in CBF1/CSL deficient B cells . | [
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] | [
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] | 2013 | Abortive Lytic Reactivation of KSHV in CBF1/CSL Deficient Human B Cell Lines |
Plaque vulnerability , defined as the likelihood that a plaque would rupture , is difficult to quantify due to lack of in vivo plaque rupture data . Morphological and stress-based plaque vulnerability indices were introduced as alternatives to obtain quantitative vulnerability assessment . Correlations between these indices and key plaque features were investigated . In vivo intravascular ultrasound ( IVUS ) data were acquired from 14 patients and IVUS-based 3D fluid-structure interaction ( FSI ) coronary plaque models with cyclic bending were constructed to obtain plaque wall stress/strain and flow shear stress for analysis . For the 617 slices from the 14 patients , lipid percentage , min cap thickness , critical plaque wall stress ( CPWS ) , strain ( CPWSn ) and flow shear stress ( CFSS ) were recorded , and cap index , lipid index and morphological index were assigned to each slice using methods consistent with American Heart Association ( AHA ) plaque classification schemes . A stress index was introduced based on CPWS . Linear Mixed-Effects ( LME ) models were used to analyze the correlations between the mechanical and morphological indices and key morphological factors associated with plaque rupture . Our results indicated that for all 617 slices , CPWS correlated with min cap thickness , cap index , morphological index with r = -0 . 6414 , 0 . 7852 , and 0 . 7411 respectively ( p<0 . 0001 ) . The correlation between CPWS and lipid percentage , lipid index were weaker ( r = 0 . 2445 , r = 0 . 2338 , p<0 . 0001 ) . Stress index correlated with cap index , lipid index , morphological index positively with r = 0 . 8185 , 0 . 3067 , and 0 . 7715 , respectively , all with p<0 . 0001 . For all 617 slices , the stress index has 66 . 77% agreement with morphological index . Morphological and stress indices may serve as quantitative plaque vulnerability assessment supported by their strong correlations with morphological features associated with plaque rupture . Differences between the two indices may lead to better plaque assessment schemes when both indices were jointly used with further validations from clinical studies .
Cardiovascular diseases ( CVD ) , especially acute coronary syndromes , are closely associated with atherosclerotic plaque progression and rupture . Accurate assessment of plaque vulnerability is of ultimate importance to CVD research , diagnosis , prevention and proper treatment . Plaque vulnerability is defined as the likelihood that a plaque would rupture . While this vulnerability concept is well accepted , its quantification is extremely difficult due to lack of actual plaque rupture and clinical event data . In this paper , morphological plaque vulnerability index ( morphological index ) and stress-based plaque vulnerability index ( stress index ) will be introduced as plaque assessment using morphological and mechanical data . These indices provide computable quantitative plaque vulnerability measures for patient screening purpose using morphological and mechanical data while the absolute vulnerability is not available . Correlations between these indices and key plaque features including cap thickness and necrotic lipid-rich core ( referred to as lipid thereafter ) will also be investigated . Atherosclerotic plaque progression and rupture are believed to be associated with morphological factors , plaque components , material properties , and mechanical stress/strain conditions , etc . [1–7] . More and more studies have shown that the mechanical forces play an important role in plaque progression and rupture process and should be considered with plaque morphology and compositions together for better plaque vulnerability assessment [1 , 8–12] . Considerable advances in medical imaging technology have made it possible to construct image-based computational models integrating plaque morphology , components , and mechanical stress/strain conditions and obtain plaque vulnerability assessment based on more complete information [12–13] . Based on in vivo magnetic resonance imaging ( MRI ) carotid plaque data with actual plaque rupture indicated by ulceration ( n = 12 , 5 with ulceration ) , Tang et al . reported that mean plaque wall stress ( PWS ) from all ulcer nodes in ruptured plaques was 86% higher than that from all non-ulcer nodes ( 123 . 0 vs . 66 . 3 kPa , p<0 . 0001 ) . Mean flow shear stress ( FSS ) from all ulcer nodes in ruptured plaques was 170% higher than that from all non-ulcer nodes ( 38 . 9 vs . 14 . 4 dyn/cm2 , p<0 . 0001 ) [12] . Mean critical PWS ( CPWS ) from the 5 ruptured plaques was 126% higher than that from the 7 non-ruptured ones ( 247 . 3 vs . 108 kPa , p = 0 . 0016 using log transformation ) [12] . Using finite element analysis to calculate the plaque structural stress ( PSS ) for 4429 intravascular ultrasound ( IVUS ) frames from 53 patients , Teng et al . found PSS was higher in non-calcified thin-cap fibroatheroma and in patients with the factors contributing to the acute syndrome such as plaque burden > = 70% , mean lumen area < = 4mm2 [14] . Based on a comparative study of 40 patients with 20 symptomatic and 20 asymptomatic , Li et al . reported that lumen curvature and fibrous cap thickness were the major factors affecting plaque stress distribution . Furthermore lumen curvature in plaques of symptomatic patients was significantly larger compared to that of asymptomatic patients [15] . Based on the simulation results of endothelial shear stress ( ESS ) in the human coronary of 7 patients , Hetterich et al . claimed plaque prevalence was highest in areas of low ESS ( 49 . 6% ) and followed by high ESS quartile ( 34 . 8% ) while in parts exposed to intermediate-low and intermediate-high ESS quartiles , few plaques were found ( 20 . 0% and 24 . 0% ) ( p<0 . 001 ) [16] . In a comparison study between 54 asymptomatic and 45 acutely symptomatic patients , Zhu et al . reported that plaque wall stresses at critical vulnerable sites were significantly higher in acutely symptomatic group comparing to the asymptomatic group ( median , inter quartile range: 198 . 0 kPa ( 119 . 8–359 . 0 kPa ) vs 138 . 4 kPa ( 83 . 8–242 . 6 kPa ) , P = 0 . 04 ) [17] . In another multi-patient study based on in vivo MRI data of carotid plaques , Huang et al . demonstrated that mean PWS at the hemorrhage nodes were higher than that from the non-hemorrhage nodes ( 75 . 6 vs . 68 . 1 kPa ) , and mean FSS at hemorrhage nodes were also higher than that from the non-hemorrhage nodes ( 15 . 0 vs . 11 . 5dyn/cm2 ) [18] . A very noticeable finding for vulnerable plaque research was concerned with microcalcifications [19–23] . Vengrenyuk et al . [19–20] , Bluestein et al . [21] , Maldonado et al . [22] , Cardoso and Weinbaum [2] , and Kelly-Arnold et al . [23] demonstrated that plaque cap with micro-calcification inclusions are associated with elevated stress levels and may be related to plaque rupture . For plaque progression studies , in a multi-patient IVUS-based follow-up study ( n = 20 ) , Samady et al . divided slices into low , intermediate , and high wall shear stress ( WSS ) groups and found that low-WSS segments demonstrated greater reduction in vessel ( P<0 . 001 ) and lumen area ( P<0 . 001 ) , and high-WSS segments demonstrated an increase in vessel ( P<0 . 001 ) and lumen ( P<0 . 001 ) area [24] . In a follow-up study of 506 patients with acute coronary syndrome ( ACS ) to assess plaque natural history , Stone et al . reported that increase in plaque area was predicted by baseline large plaque burden; decrease in lumen area was independently predicted by baseline large plaque burden and low wall shear stress [25] . In this paper , in vivo intravascular ultrasound ( IVUS ) data were acquired to construct 3D models with fluid-structure interactions ( FSI ) and cyclic bending and obtain plaque morphological and mechanical stress/strain data for analysis . Six hundred and seventeen ( 617 ) slices were obtained from 14 patients . Based on morphological features available from IVUS Volcano Histology ( VH ) , three morphological characteristic-related indices ( lipid index , cap index , and morphological index ) were introduced consistent with American Heart Association ( AHA ) plaque classifications [26] . Flow shear stress ( FSS ) and plaque wall stress/strain ( PWS/PWSn ) were collected at the critical sites in each slice to investigate their correlations with the morphological related factors . The Linear mixed-effect ( LME ) Models were used to analyze the correlation between each of the mechanical condition and each of the indices .
Table 3 summarized the correlation results between the three mechanical factors ( CPWS , CPWSn , and CFSS ) and 5 morphological factors: min cap thickness , cap index , lipid percentage , lipid index and morphological index . CPWS correlated significantly with each of the five morphological factors . There was a significant correlation between CPWS and the morphological index with correlation coefficient r = 0 . 7411 , p-value<0 . 0001 . Cap index ( r = 0 . 7852 ) showed a slightly stronger correlation with CPWS than min cap thickness ( r = -0 . 6414 ) . The correlations showed opposite sign because thinner cap thickness had higher cap index . Lipid percentage and lipid index yielded weaker correlation than CPWS with correlation coefficient r = 0 . 2445 and r = 0 . 2338 , respectively . Fig 4 shows the distribution of the CPWS with respect to cap index , min cap thickness , lipid index and morphological index . Statistically significant correlations were also observed between the CPWSn and 5 morphological factors ( min cap thickness , cap index , lipid percentage , lipid index and morphological index ) based on results from the 617 slices ( see Table 3 ) . Correlation results between CPWSn and min cap thickness , cap index and morphological index are similar to those of CPWS , but much weaker . CPWSn had negative correlation with lipid percentage and lipid index which is different from the results of CPWS . CFSS had significant correlations with lipid percentage and lipid index with r = 0 . 1144 and r = 0 . 0825 , respectively . However , CFSS did not have significant correlations with min cap thickness , cap index , and morphological index . It is natural that min cap thickness and CPWS correlate strongly . However , lipid content does not have similar strong correlation because some lipid core could have very thick cap . CFSS and min cap thickness did not have significant correlation because they do not correspond very well . Individual patient correlations between CPWS and min cap thickness , cap index , lipid percentage , lipid index , morphological index were obtained for each of the 14 plaques and results are summarized in Table 4 . For all 14 plaques , CPWS had significant positive correlations with cap index and morphological index , and negative correlation with min cap thickness . On the other hand , correlations between CPWS and lipid percentage , lipid index showed mixed results for the 14 patients . The number of patients showing positive/no significant/negative correlation between CPWS and lipid percentage were 4/10/0 respectively , while only 5 out of 14 patients showed significant positive correlation between CPWS and lipid index and the remaining 9 showed no correlation . Table 5 shows that correlation results of the stress index is similar to those of CPWS . There are significant correlations between stress index and all 5 morphological indices and factors . The correlation coefficient between stress index and cap index is the highest with r = 0 . 8185 . There is a strong correlation between stress index and morphological index with r = 0 . 7715 . The correlation coefficients between stress index and min cap thickness , lipid percentage and lipid index were r = -0 . 7127 , 0 . 3139 , 0 . 3067 respectively , all with p< 0 . 0001 . For the 617 slices , each slice was assigned a stress index and 3 morphological related indices: cap index , lipid index and morphological index . Table 6 gives the agreement rates between stress index and cap index , lipid index , and morphological index . The matching rates ( two indices have the same value for a slice ) between stress index and cap index , lipid index and morphological index were 57 . 37% , 25 . 93% and 66 . 77% respectively . The result demonstrated the stress index has a strong agreement with morphological feature closely related to the plaque vulnerability , and could be used to aid the assessment of plaque vulnerability and plaque rupture .
Quantification of plaque vulnerability by its strict definition based on in vivo data is not currently realistic due to its statistical nature and lack of available in vivo data for plaque rupture and rupture-related clinical events . The morphological and stress-based plaque vulnerability indices introduced in this paper provided a possible approach for quantitative plaque vulnerability assessment using features known to be closely associated with plaque rupture . These indices may get closer to serve as appropriate approximations to plaque vulnerability as more plaque rupture and clinical event data are investigated and continued research adds more supporting evidence for the use of those alternative indices . Currently , histology is serving as the gold standard for plaque vulnerability assessment . It has been well established that cap thickness and lipid size are closely related to plaque vulnerability . Our stress index was introduced trying to have best match with the morphological index . The stress index had a 66 . 77% agreement rate with morphological index . It should be emphasized that the disagreement between stress index and morphological index may lead to better plaque assessment strategies when both indices were used together to supplement each other . Large lipid core gives a higher morphological index . However , if its cap is thick , the slice may have a lower stress index . Large curvature causes higher CPWS which leads to a high stress index , which the morphological index may be low due to cap thickness and lipid size features . Further investigations are needed to investigate the differences of the two indices and seek for potential improvement on plaque assessment techniques and patient screening tools . It is clear that CPWS and min cap thickness have strong correlations . However , CFSS did not have significant correlations with min cap thickness and cap index . CFSS showed weak positive correlation with lipid percentage and lipid index . This may be related the fact the higher CFSS values are associated to lumen narrowing , thicker vessel wall and larger lipid core . In general , CFSS is only one value for a slice and FSS from entire vessel should be used for further investigations [8] . Several factors such as cap thickness and lumen surface inflammation are known to be related to plaque vulnerability and quantitative measurements of those features will bring improvement to our index accuracies . Among several modalities , Optical Coherence Tomography ( OCT ) may help advance our ability to diagnose vulnerable from non-vulnerable plaques to enhance future risk prediction and effective treatment for patients . OCT uses light rather than ultrasound; has a resolution of 15–20 microns and therefore can measure fibrous cap thickness; can classify plaque as fatty , fibrotic , calcific , or thrombotic; and can detect thin-cap fibroatheromas and plaque erosions in patients presenting with an acute coronary syndrome . The major limitations of OCT are penetration so that overall plaque burden cannot be measured and the inability to image through blood or penetrate red thrombus . We are currently working on techniques combining IVUS with OCT to obtain good overall plaque 3D geometry with better cap thickness quantifications . Some other limitations of this study include: a ) Patient-specific and tissue-specific material properties were not available for our study . We are currently investigate other approaches to measure patient-specific material properties in vivo; b ) Currently , IVUS resolution is at 100–150 microns , mostly depending on the transducer frequency , which is not enough to clearly identify the thin caps with thickness under 100 micron . Optical coherence tomography can be used jointly with IVUS procedures and can provide spatial resolution of 10–20 microns . These combined approaches are also under investigation; c ) While patient-specific angiography was used to extract vessel curvature variations in cardiac cycle for our cyclic bending , only angiography from one view angle was used . 3D curvature re-constructed from multiple angiography images with different ( preferably orthogonal ) view angles should be used in our future models [24]; d ) Because IVUS data does not contain the adventitial layer of the vessel , our model also did not include that , as this is done in the current literature . It is arguable if simply adding a layer without actual data support would give us a better model . The adventitial layer should have considerable effect on stress/strain calculations . So our results should be understood with that simplification; e ) Microcalcifications were not included in the current morphological index due to limitations of imaging; f ) Interaction between the heart and vessel were not considered in this model . A model coupling heart motion and coronary bending would be designed when required data become available . | Cardiovascular diseases are closely related to atherosclerotic plaque progression and rupture . Early detection of vulnerable plaques and prediction of potential plaque rupture and related clinical events are of vital importance . Plaque vulnerability , defined as the likelihood that a plaque would rupture , is difficult to measure due to lack of in vivo plaque rupture data . Morphological and stress-based plaque vulnerability indices were introduced in this paper as alternatives to obtain quantitative vulnerability assessment with potential improvement of patient screening tools . In vivo intravascular ultrasound data were acquired from patients and computational coronary plaque models were constructed to obtain data for analysis and index assignments . For the 617 slices from the 14 patients , morphological and stress indices were assigned to each slice using methods consistent with American Heart Association plaque classification schemes . Correlation analyses were performed for all the morphological and mechanical factors considered . The stress index has 66 . 77% agreement with morphological index . Morphological and stress indices may serve as quantitative plaque vulnerability assessment supported by their strong correlations with morphological features associated with plaque rupture . Differences between the two indices may lead to better plaque assessment schemes when the complementary indices were jointly used with further validations from clinical studies . | [
"Abstract",
"Introduction",
"Results",
"Discussion"
] | [] | 2015 | Morphological and Stress Vulnerability Indices for Human Coronary Plaques and Their Correlations with Cap Thickness and Lipid Percent: An IVUS-Based Fluid-Structure Interaction Multi-patient Study |
A subset of high-risk Human Papillomaviruses ( HPVs ) are the causative agents of a large number of human cancers , of which cervical is the most common . Two viral oncoproteins , E6 and E7 , contribute directly towards the development and maintenance of malignancy . A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif ( PBM ) at its C-terminus , which confers interaction with cellular proteins harbouring PDZ domains . Here we show that this motif allows E6 interaction with Sorting Nexin 27 ( SNX27 ) , an essential component of endosomal recycling pathways . This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets , SNX27 is not targeted for degradation by E6 . Rather , in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner . Analysis of a SNX27 cargo , the glucose transporter GLUT1 , reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery . Furthermore , knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27 , both in terms of GLUT1 expression levels and its vesicular localization , with a concomitant marked reduction in glucose uptake , whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions . These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules , one consequence of which is modulation of nutrient availability in HPV transformed tumour cells .
Human Papillomaviruses ( HPVs ) are the causative agents of a large number of human malignancies , chief among which is cervical cancer , with over 500 , 000 reported cases worldwide annually [1 , 2] . There are currently more than 150 known types of HPVs , but not all of them are etiological agents of carcinomas . The cancer-causing HPVs are classified as “high-risk” types and these include HPV-16 and HPV-18 , among others [3] . A hallmark of HPV induced-malignancy is the continued expression of the viral oncoproteins E6 and E7 throughout the course of tumour development [4 , 5] . Inhibiting the expression of either oncoprotein in cells derived from cervical tumours results in cell growth arrest and induction of apoptosis , demonstrating a continued requirement for E6 and E7 in the maintenance of the transformed phenotype [6] . Both viral oncoproteins act cooperatively , where E7 reprograms the infected cell to enter S phase by targeting , in part , the pRb family members , thus allowing the E2F family of transcription factors to transactivate various cell cycle genes [7–9] . The E6 oncoprotein complements the action of E7 by curbing the cell’s pro-apoptotic response to unscheduled DNA replication and targets pro-apoptotic proteins such as p53 [10] and Bak [11] for proteasome-mediated degradation via the action of the E6AP ubiquitin ligase [12] . However the ability of both E6 and E7 to contribute to cancer development depends upon a large number of other important interactions . In the case of the high-risk E6 oncoproteins a typical example is interaction with cellular PDZ ( PSD-95/DLG/ZO-1 ) domain containing proteins . A unique characteristic of the cancer-causing E6 oncoproteins is the presence of a PDZ binding motif ( PBM ) on their carboxy termini [13] . An intact E6 PBM is important for the ability of E6 to cooperate with E7 in the generation of tumours in transgenic mouse models , and also has transforming potential in some tissue culture models [14–16] . In the context of the whole viral genome , loss of E6 PBM function results in a defective replicative life cycle , with reduced levels of viral DNA amplification and , ultimately , loss of the viral episomes [17 , 18] . A large number of cellular PDZ domain-containing targets of E6 have been reported , with some of the best-characterised being a group of proteins involved in the regulation of cell polarity [19] . These include the Discs Large and Scribble proteins , which are key regulators of cell polarity and potential tumour suppressor proteins [20 , 21] . In addition , MAGI-1 would also appear to be a relevant target of E6 , affecting the integrity of tight junctions in HPV-positive cells [22] . Recent high throughput screens have identified many further potential PDZ domain-containing targets of E6 , with Sorting Nexin 27 ( SNX27 ) being one such intriguing candidate [23 , 24] . A critical element in epithelial organisation is the regulation of polarity , and whilst key elements of the pathway , such as Scrib and Dlg , are well defined , it is clear that endocytic transport pathways also play important roles in epithelial organisation and polarity control [25–27] . Within these pathways , the retromer plays an important part in the transport of proteins from endosomes to the trans-Golgi network [28] . The retromer is an oligomeric protein complex formed by a Vacuolar protein sorting ( Vps ) subcomplex , consisting of Vps26 , Vps29 and Vps35 , and a heterodimer of sorting nexins ( SNXs ) . SNXs are proteins characterized by the presence of a phosphoinositide binding PX domain , which targets them to phosphatidylinositol-3-monophosphate-rich membranes of the endosomes [29] . Many members of the SNX family also contain a number of protein-protein interaction domains , through which they interact with their respective cargoes and ensure appropriate trafficking [29–32] . Of particular interest from the HPV E6 point of view is SNX27 . It contains a C-terminal Ras Association/FERM like domain , which has been shown to associate with the Ras GTPase [33] , and SNX27 is the only SNX that contains a PDZ domain [30 , 32 , 34] . Recent proteomic studies have indicated that HPV-16 and HPV-18 E6 are potential interacting partners of SNX27 [23 , 24] . The SNX27 PDZ domain mediates the interaction with various PBM-containing cargoes and is thought to regulate the trafficking of these proteins through the endosomal pathway [35–38] . SNX27-mediated recycling of PBM-containing cargoes from endosomes to the plasma membrane is dependent on its interaction with the retromer , and recent studies have shown that the SNX27-retromer link is crucial for the retrieval and recycling of various transmembrane proteins . These include proteins required for maintenance of cellular homeostasis and growth , among which is GLUT-1 , which is essential for glucose uptake [39 , 40] . We therefore initiated a series of studies to investigate whether E6 can associate with SNX27 and , if so , to ask what are the potential implications of this interaction in the recycling function of the SNX27-retromer complex in the context of GLUT-1 as a cargo .
Proteomic screens for cellular interacting partners of HPV E6 identified the PDZ domain containing protein SNX27 as a potential novel target of the high risk HPV E6 proteins [23 , 24] . To verify if this is indeed a bona-fide interaction , a series of in vitro binding assays were performed . The E6 proteins derived from the high-risk HPV-16 , 18 , 31 , 33 , 51 and 58 were expressed as GST fusion proteins and purified . These were then incubated with in vitro translated and radiolabelled SNX27 for 2 hours at 4°C . MAGI-1 , which has been shown previously to be a strongly bound PDZ domain-containing target of HPV-16 and HPV-18 E6 was used as a positive control [41] . After thorough washing of the beads , the level of binding was determined by SDS-PAGE and autoradiography . The results in Fig 1A show that SNX27 is indeed a strong interaction partner of all the high-risk E6 types tested . The E6 proteins from low-risk HPV types lack PBMs , therefore we wished to determine whether the ability of E6 to interact with SNX27 was restricted to high-risk types . To do this , pull-down assays were performed using purified GST-HPV18 E6 and GST-HPV11 E6 fusion proteins with in vitro translated and radiolabeled SNX27 . The results in Fig 1B demonstrate very strong association between HPV-18 E6 and SNX27 , with much weaker , albeit still detectable , association with HPV-11 E6 . To determine whether the interaction between the high-risk E6 proteins and SNX27 required the E6 PBM , the assay was repeated using wild type HPV-18 E6 and the HPV-18 E6 T156E mutation , which has been shown previously to abolish PDZ recognition [42] . The results in Fig 1C demonstrate that the T156E mutation dramatically reduces the ability of E6 to interact with SNX27 , although some residual interaction is still detectable . Taken together these results demonstrate that HPV-18 E6 interacts with SNX27 primarily through its PBM , although other residues within E6 are also most likely to be involved , and this is reflected in a weak degree of association between HPV-11 E6 and SNX27 . Since SNX27 is a PDZ domain-containing protein , we wanted to determine if the interaction between SNX27 and E6 is through PBM-PDZ recognition . To do this , various C-terminal mutants of HPV-18 E6 [42] were translated and radiolabelled in vitro and used in pull down assays with purified GST-SNX27 . The results in Fig 2A show that the mutation V158A , which destroys an essential part of the HPV-18 E6 PBM [42] , greatly reduces the interaction with SNX27 . Furthermore , the E155A mutation also severely compromises E6 recognition of SNX27 , whilst the other mutants had either minimal effects or increased the level of association . This differential contribution of specific residues within the E6 PBM is in agreement with how E6 recognises other PDZ domain-containing substrates [43] . Taken together , these results demonstrate that HPV-18 E6 recognises SNX27 via sequences within the E6 PBM although , in agreement with the T156E mutation , other residues are also likely to be involved . To confirm that the PDZ domain of SNX27 is necessary for E6 binding , PDZ deletion mutants of SNX27 were used in a series of pull down assays with GST-HPV18 E6 fusion proteins . As can be seen from Fig 2B , wild type SNX27 binds to HPV-18 E6 very strongly , but this interaction is greatly reduced with the HPV-18 E6ΔPBM mutant . The deletion of the retromer binding region of SNX27 , spanning amino acids 67–77 [44] in the outer extended loop of the SNX27 PDZ domain , does not affect the interaction with HPV-18 E6 . Interestingly , deletion of amino acids 97–107 and 113–121 in the core of the SNX27 PDZ domain markedly reduces the interaction with wild type HPV-18 E6 , but does not abolish it . When the SNX27 mutants are used in binding assays with the E6ΔPBM mutant , interaction is found at a level similar to that seen with the wild type HPV-18 E6 . These results demonstrate that the major site of recognition between HPV-18 E6 and SNX27 involves a classic PBM-PDZ interaction , although a second weaker interaction site also exists . Many E6 interacting partners are targeted for ubiquitin-dependent proteasomal degradation . However , in an extensive series of in vitro and in vivo assays , we found no evidence that SNX27 was a degradation substrate for either HPV-16 or HPV-18 E6 . Previous studies have reported E6 to be largely expressed in the nucleus , whilst SNX27 exists primarily within the early endocytic compartments in the cytoplasmic and membrane compartments of the cell ( 35 ) . We were therefore first interested in determining whether HPV-18 E6 and SNX27 were present within similar cellular compartments . To do this we performed cell fractionations of HPV-18 positive HeLa cells , in the presence and absence of the proteasome inhibitor MG132 to rescue any pools of the two proteins that might be subject to proteasome-mediated degradation . In this analysis we also made use of a DOX-inducible shRNA to ablate SNX27 expression , to ascertain whether SNX27 might have any effect on the pattern of E6 expression . The results obtained are shown in Fig 3 . As expected , the bulk of SNX27 is found within the membrane fractions of the cell ( 35 ) . Most interestingly , the bulk of HPV-18 E6 in HeLa cells is also localised within the membrane compartment , suggesting a potentially similar location to SNX27 , although loss of SNX27 has no major effect on the subcellular distribution of E6 . In addition , the membrane-bound pool of E6 appears to be unaffected by proteasome inhibition , whilst there is a clear evidence of proteasome mediated regulation of E6 levels in the cytoplasmic , nuclear and cytoskeletal fractions . Having found that E6 and SNX27 exist within the membrane fraction of HeLa cells , we were then interested in determining whether E6 could in any way modulate the subcellular localisation of SNX27 . To do this , we analysed the distribution of SNX27 in HPV-18 positive HeLa cells , in the presence and absence of HPV-18 E6 . The cells were transfected with control siRNA against Luciferase and siRNA against HPV-18 E6/E7 and E6AP as an alternative means of reducing E6 levels of expression [45] . After 72h , the cells were fixed and immunofluorescence analyses performed to detect SNX27 , and also p53 as a control for the silencing of E6/E7 expression . We also analysed the distribution of the retromer component Vps35 . The results in Fig 4 show a diffused speckled pattern of SNX27 distribution in control transfected cells , in agreement with previous studies ( 35 ) , but a marked perinuclear accumulation , with increased levels of co-localisation with Vps35 following silencing of E6/E7 or E6AP , where a strong nuclear p53 staining can also be observed . Taken together , these results suggest that E6 contributes to the maintenance of a dispersed pattern of Vps35 and SNX27 expression in HeLa cells , and that its loss results in a significant increase in Vps35/SNX27 co-localization at sites close to the nuclear membrane . To determine whether this change in the distribution of SNX27 is related to the ability of E6 to interact with SNX27 , we analysed the distribution of SNX27 in the immortalised keratinocyte cell line NIKS , expressing either wild type HPV-16 E6 or a HPV-16 E6ΔPBM mutant . The cells were fixed and stained for SNX27 and Vps35 as described above . The results obtained in Fig 5 show significant co-localisation of Vps35 and SNX27 in the control NIKS not expressing E6 , whilst this becomes much more dispersed in the wild type HPV-16 E6 expressing cells with limited areas of co-localisation . In contrast , the pattern of SNX27 and Vps35 distribution in the HPV-16 E6ΔPBM cells , shows increased levels of co-localisation of SNX27 and Vps35 , reflecting more the pattern seen in the control NIKS . These results indicate that changes in the subcellular distribution of SNX27 and Vps35 in the presence of HPV E6 are in part dependent upon on intact E6 PBM . The above results indicate that E6 expression can alter the subcellular distribution of SNX27 , and can affect the degree of its co-localisation with components of the retromer . We next wanted to determine whether E6 could modulate the degree of interaction between SNX27 and Vps35 , and , furthermore , whether E6 could also be found in the SNX27/Vps35 complex in vivo . To do this , co-immunoprecipitation assays were performed where endogenous E6 from HeLa cells was immunoprecipitated using an anti-HPV-18 E6 monoclonal antibody . The immune complexes were then adsorbed on Protein A Sepharose beads and analyzed by SDS-PAGE followed by Western blotting for E6 , SNX27 and Vps35 . As can be seen from Fig 6A ( left hand panel ) , both SNX27 and the Vps35 component of the retromer co-immunoprecipitate with E6 , indicating that endogenous E6 is found in close proximity with the retromer . Likewise , HPV-18 E6 also co-immunoprecipitates with SNX27 when anti-SNX27 antibodies are used , and the specificity of this interaction is further demonstrated by loss of signal when the cells are transfected with siRNA against E6/E7 ( Fig 6B ) . In order to determine whether E6 can affect association between SNX27 and Vps35 , we performed an SNX27 immunoprecipitation from HeLa cells , which had been previously transfected with control siRNA luciferase or siRNA E6 to ablate E6 expression . The results in Fig 6A ( right hand panel ) show no major difference in the levels of SNX27/Vps35 interaction in the presence or absence of HPV-18 E6 . Previous studies have shown that the glucose transporter GLUT-1 requires both SNX27 and Vps35 for its efficient recycling back to the cell surface , with loss of SNX27 resulting in a marked decrease in the levels of GLUT-1 expression owing to its enhanced lysozomal degradation [40] . Since we observed that loss of E6 leads to alterations in the distribution of SNX27 and Vps35 , we wanted to determine whether this might be reflected in changes in expression of a well-defined SNX27 cargo . To investigate this , we performed a series of assays to directly monitor the levels of expression of GLUT-1 in HeLa cells following ablation of either HPV-18 E6 or SNX27 expression . Cells were transfected with siRNA against Luciferase or siRNA against E6 , or were treated with DOX to induce the SNX27 shRNA for 72h . After this time the cells were harvested and the levels of GLUT-1 expression analysed by western blotting . As a control for E6 ablation we monitored p53 , and SNX27 was also analysed to ensure efficacy of the inducible shRNA . The results in Fig 7 demonstrate that GLUT-1 migrates as multiple bands , which is in agreement with previous studies . Most importantly however , there is a clear reduction in the levels of expression of the higher molecular weight , functional forms of the protein [46 , 47] , following loss of E6 or SNX27 . These results demonstrate that removal of E6 in HeLa cells has a very similar effect upon GLUT-1 expression levels as removal of SNX27 . The above results demonstrate that GLUT-1 levels are reduced in HeLa cells lacking either E6 or SNX27 . We were therefore interested in determining whether E6 had any influence on the composition of the endocytic compartments and their associated cargoes . To do this HeLa cells were transfected with siRNA against E6 or Luciferase for 72h and cell extracts subsequently fractionated on 5–25% OptiPrep gradients as described previously [48] . The fractions were then collected and analyzed by Western blotting . As can be seen in Fig 8 , there are marked changes both in the distribution of cargoes and in the endocytic compartments following E6 ablation . In control transfected cells , the early endosome marker Rab4 appears to follow very closely the distribution of both SNX27 and Vps35 . However , upon loss of E6 there is a clear shift in the distribution of both Rab4 and SNX27 within the gradient , whilst Vps35 appears to be relatively unchanged . Likewise , there is a corresponding marked shift in the distribution of the SNX27 cargo GLUT-1 , and there is also an overall reduction in the levels of expression of the higher molecular weight forms of GLUT-1 , similar to that seen in Fig 7 . Interestingly there is no significant alteration in the mobility of the late endosomal marker LAMP2 . These results demonstrate that in HeLa cells loss of E6 has quite a profound effect on the distribution of SNX27 within the cargo transporting apparatus , in support of the notion that E6 can modulate SNX27 function . The above data suggest a significant shift in the distribution of several endocytic compartments and of at least one associated cargo , GLUT-1 , following the ablation of E6 expression in HeLa cells . We were therefore interested in determining whether this alteration in the pattern of GLUT-1 expression could be visualised by immunofluorescence analysis . Cells were transfected with siRNA against Luciferase or E6 and , after 72h , analysed by immunofluorescence for GLUT-1 and Vps35 localisation , with p53 serving as a positive control for E6 ablation . As can be seen from Fig 9B , most of the GLUT-1 appears to be localized on the cell periphery in the control cells , while the Vps35 remains distributed throughout the cytoplasm as seen before . In contrast , upon E6 ablation , the majority of GLUT-1 is located in intracellular vesicle-like structures with a pool showing significant co-localization with Vps35 . Similar results are also obtained in SiHa cells following ablation of E6AP expression , which we have shown previously to result in a loss of E6 [45] . In addition , analysis of GLUT-1 distribution within NIKS cells ( Fig 9A ) also shows a clear E6 PBM-dependent effect upon the pattern of GLUT-1 expression , although the specific distribution varies between the different cell types analysed . Taken together , these results indicate that the loss of E6 leads to an increased cytoplasmic expression of GLUT-1 , a subset of which accumulates in close proximity to a component of the retromer complex . The results in Fig 8 indicated a change in the localisation of the early endosomal marker Rab4 . We therefore proceeded to investigate whether this was also reflected in a change in its subcellular distribution following ablation of E6 expression . The results in Fig 9C indeed show a significant alteration in the pattern of Rab4 distribution in HeLa cells following ablation of either E6 or E6AP expression . Once again there appears to be increased accumulation in close proximity to the Vps35 component of the retromer . The above data demonstrate that E6 can modulate the pattern of SNX27 association with the endosomal transport machinery in a PBM-dependent manner . A consequence of this appears to be reflected in changes to GLUT-1 association with endosomal recycling complexes , with loss of E6 resulting in lower levels of GLUT-1 expression and altered intracellular distribution . Therefore we next wanted to determine whether this alteration in GLUT-1 transport was also reflected in an alteration of its function as a glucose transporter . To do this , HeLa cells were treated with siRNA against E6 , or Luciferase as control , for 72h and glucose uptake was then measured using the analog 2-deoxyglucose ( 2-DG ) . As can be seen from Fig 10A , there is a significant decrease in the amount of glucose that is taken up by HeLa cells when E6 is depleted , compared with the Luciferase control . These data show that the depletion of GLUT-1upon E6 ablation is reflected in a reduction in glucose uptake by these cells , and reflects reports of similar results seeing loss of GLUT-1 expression following SNX27 knockdown [40] . This demonstrates a direct functional consequence for HPV-18 E6 modulation of the SNX27-retromer cargo transport pathway . The above results indicate that SNX27 plays an important role in glucose uptake , an activity that is modulated by E6 . They also suggest that under conditions of low glucose availability , loss of SNX27 might result in reduced rates of cell proliferation . To investigate this possibility , control HeLa cells and shRNA SNX27 HeLa cells were plated in low and high glucose , both in the absence and presence of DOX to induce the SNX27 shRNA . The cells were then counted over a period of days and the results obtained are shown in Fig 10B . As can be seen , loss of SNX27 in conditions of low glucose has a marked inhibitory effect upon continued cell proliferation , whilst high glucose availability largely mitigates these effects . These results confirm that SNX27 contributes directly towards nutrient uptake in HPV-18 positive HeLa cells .
In this study we have identified a novel activity of the high risk HPV E6 oncoproteins , linking them to the modulation of endosomal transport pathways . This appears to be mediated , at least in part , through a direct interaction between the high risk HPV E6 oncoproteins and the cellular SNX27 , a protein that controls cargo fate determination in endocytic recycling . One consequence of this interaction is modulation of the endocytic transport of the glucose transporter GLUT-1 , which subsequently affects the amount of glucose uptake in HPV-positive tumour cells . These results suggest that E6 can directly affect the nutrient balance in HPV infected cells , through modulation of endocytic recycling pathways to maintain sufficient nutrients for cell survival during the HPV life cycle and in progression to malignancy . Previous proteomic analyses had suggested that HPV-18 E6 could potentially interact with different components of the endocytic sorting machinery , including SNX27 and the retromer components Vps35 and Vps26 , indicating that E6 might be in close proximity to the retromer complex [23 , 24] . To investigate this in more detail we first performed a series of in vitro interaction assays and found that multiple high risk HPV E6 oncoproteins all share a similar ability to interact with SNX27 . Mutational analyses demonstrated that the principal mode of recognition was through classic PBM-PDZ recognition , although there were some subtle differences from other reported E6 interactions with PDZ domain-containing substrates . In particular , the number of residues in the region of the E6 PBM that were critically required for SNX27 PDZ recognition appeared to be fewer than those required for MAGI or Dlg recognition . In addition , ablation of the E6 PBM or the core region of the SNX27 PDZ domain still allowed a low level of interaction , suggesting the existence of additional means by which E6 can interact with SNX27 . In support of this , a weak but consistent interaction was also observed between HPV-11 E6 and SNX27 , suggesting that modulation of endocytic transport might also be a feature of low risk HPV types , and this is currently a subject for further investigation . In agreement with the proteomic analyses ( 23 ) , we also found that HPV-18 E6 could co-immunoprecipitate both SNX27 and the Vps35 component of the retromer from cells , confirming a close association of E6 with the retromer complex . Indeed , cell fractionation studies demonstrated that the bulk of endogenously expressed E6 in HeLa cells is actually present within the membrane fraction of the cell , which is very similar to the localisation seen for SNX27 . Throughout , we found no compelling evidence to suggest that E6 can influence the turnover of SNX27 through proteasome-dependent pathways . However , it was interesting to note that the membrane-bound forms of E6 were not subject to proteasome-mediated degradation , as there was no change in the levels of E6 expression in this fraction of the cell following proteasome inhibition . This contrasts with the situation with the cytoplasmic , nuclear and cytoskeletal pools , where all these forms of E6 were rescued following proteasome inhibition . It should also be mentioned that when E6 is overexpressed using exogenous transfection of expression plasmids , we invariably detect the bulk of E6 within the cytoskeletal compartment of the cell , emphasising the need to study the endogenously expressed E6 for these types of analyses . Having defined SNX27 as a novel interacting partner of HPV-18 E6 , we then proceeded to investigate the potential consequences of this interaction . Clearly , removal of E6 from HPV-18 positive HeLa cells has quite a marked effect on the subcellular distribution of SNX27 , with significant accumulation in regions proximal to the nucleus and an apparent increase in the co-localisation with Vps35 . To confirm that these alterations in the pattern of SNX27 expression were PBM-dependent , we also analysed the distribution of SNX27 in control NIKS , and in NIKS expressing HPV-16 E6 or HPV-16E6 ΔPBM . Again , there was significant co-localisation between SNX27 and Vps35 in the control NIKS and HPV-16 E6ΔPBM lines , but this was greatly decreased in the wild type E6-expressing cells . Taken together these studies suggest that the E6 association with SNX27 can modulate its subcellular distribution . To further investigate this we then analysed the distribution of SNX27 in different endosomal/lysosomal fractions using OptiPrep gradients to fractionate the subcellular membrane compartments in HeLa cells . Again , loss of E6 resulted in a marked shift in the distribution of SNX27 within the gradients . SNX27 remained in close proximity to the early endosomal marker Rab4 , but shifted with respect to some of the Vps35 component of the retromer . Albeit less clearly , the GLUT-1 SNX27 cargo also appeared to shift in the gradient , suggesting that loss of E6 could also affect the distribution of an SNX27 cargo . These results were independently verified by immunofluorescence analyses , which also demonstrated an alteration in the co-localisation of Rab4 with Vps35 , together with an increase in cytoplasmic GLUT-1 in cells where E6 expression had been ablated . Having defined a role for the E6 PBM in modulating components of the endosomal transport machinery , we then proceeded to assess the effects on the function of GLUT-1 . In both total cell extracts and in the gradient analyses , loss of E6 resulted in a marked decrease in the levels of expression of the functional higher molecular weight forms of GLUT-1 . Similar effects were also observed following loss of SNX27 expression . Functionally , this loss of E6 resulted in a reduction in the levels of glucose uptake , suggesting that a direct consequence of E6 modulation of SNX27 function is an increase in the rates of glucose uptake , thereby favouring a more efficient use of available nutrients for continued cell proliferation and cell survival . In order to confirm that SNX27 does indeed play a significant biological role in HeLa cells we also measured cell growth following ablation of SNX27 expression under different growth conditions , and clearly found a reduction in cell proliferation when cells were grown in a low glucose medium . Taken together these results demonstrate that the association between E6 and SNX27 can modulate the endocytic transport of the GLUT-1 glucose transporter , one consequence of which is maintenance of cell proliferation under low nutrient conditions . Several major questions arise from these studies . The first relates to the precise mechanism by which E6 can modulate SNX27 function . The interaction is largely PBM-PDZ mediated , although not exclusively . It seems unlikely that E6 and GLUT-1 could occupy the SNX27 PDZ pocket simultaneously; however recent studies have shown that the GLUT-1 affinity for the SNX27 PDZ domain is significantly higher than that of E6 [49] . This suggests that E6 association might be transitory , and be replaced in the PDZ pocket by high affinity cargoes . It remains to be determined whether the weaker association of E6 with SNX27 might also then contribute to a modulation of the sorting process , or whether the E6 PBM helps recruit SNX27 to certain endocytic compartments , favouring faster recycling . It is also possible that other , more weakly bound , cargoes of SNX27 might actually be out-competed for binding SNX27 by E6 , thereby inhibiting their recycling . Recent studies have shown an important role for the retromer in the function of SNX27 , and certainly E6 appears to exist in a complex with SNX27 bound to Vps35 , without apparently affecting the biochemical levels of either SNX27 or Vps35 [40 , 44] . Nonetheless depletion of E6 does seem to affect the degree of co-localisation of SNX27 with Vps35 , both in immunofluorescence experiments and in gradient fractionations . In one of these assays , this modulation is shown to be PBM dependent . This suggests that E6 can modulate the endocytic compartment to which SNX27 is recruited , suggesting that the time spent in close proximity with Vps35 is reduced in the presence of E6 . Dissecting the molecular basis for this will be an important avenue for future investigation . Taken together , these studies indicate the existence of a novel activity for the HPV E6 oncoproteins , linking them directly to the modulation of endosomal transport pathways , and suggesting a completely novel way of modulating the cellular homeostasis , both during viral infection and in the development of malignancy .
HeLa ( ATCC ) and HeLa S4 shSNX27 ( 38 ) and SiHa ( ATCC ) cells were maintained in Dulbecco’s Modified Eagles’s Medium ( DMEM ) supplemented with 10% Fetal Calf Serum ( Life Technology ) , penicillin-streptomycin ( 100 U/ml ) and glutamine ( 300 μg/ml ) . Cells were cultured at 37°C with 10% CO2 . The NIKS ( Normal Immortalised Keratinocytes [50] ) control , NIKS 16 E6 and NIKS 16 E6ΔPBM [51] cells were maintained in F medium ( 0 . 66 mM Ca2+ ) composed of 3 parts Ham's F12 medium to 1 part Dulbecco's modified Eagle's medium and supplemented with the following components: 5% fetal bovine serum ( FBS ) , adenine ( 24 μg/ml ) , cholera toxin ( 8 . 4 ng/ml ) , epidermal growth factor ( 10 ng/ml ) , hydrocortisone ( 2 . 4 μg/ml ) , and insulin ( 5 μg/ml ) . HeLa cells were transfected with siRNA using Lipofectamine RNAiMAX transfection reagent ( Invitrogen ) . HeLa S4 shSNX27 cells were treated with 0 . 2 mg/ml Doxycycline to induce the shRNA . The following siRNAs were used: HPV-18 E6 and HPV-18 E6/E7 were custom synthesised by Dharmacon whilst the E6AP and SNX27 siRNAs were a Dharmacon Smart Pools . GST fusion proteins were generated from pGEX2T plasmids expressing HPV-11 E6 , 16 E6 , 18 E6 , 31 E6 , 33 E6 , 51 E6 and 58 E6 proteins , SNX27 [37] , HPV-18 E6ΔPBM and HPV-18 E6 T156E as described previously [52] . The SNX27 deletion mutants ( Δ67–77 , Δ97–110 and Δ113–121 ) were prepared using primers against the specified regions using the pCI Neo Myc tagged SNX27 as template . The HPV-18 E6 C-terminal mutants were used for the in vitro binding assays as described previously [41] . All expression constructs were transformed into E . coli strain DH5α . Purified GST fusion proteins were incubated with in vitro translated and radiolabelled proteins as indicated for 2 hours at room temperature . Proteins were translated in vitro using a Promega TNT Rabbit Reticulocyte Lystate kit and radiolabelled with [S35] Cysteine or [S35] Methionine ( Perkin Elmer ) . Equal amounts of translated proteins were mixed with the GST fusion proteins immobilized on glutathione agarose beads and incubated on a rotating wheel . The beads were then washed thrice with PBS containing 1% Triton X-100 and analyzed by SDS-PAGE followed by autoradiography . For endogenous protein co-immunoprecipitation assays , HeLa cells were seeded in 10 cm dishes and either left untreated or treated with siRNA against Luciferase or E6 as indicated for 72 hours . Cell lysates were prepared either in Lysis Buffer ( 20mM Tris pH 7 . 5 , 150mM NaCl , 1mM EDTA , 1mM EGTA , 1% Triton X-100 ) containing protease inhibitors ( Calbiochem Protease Cocktail 1 ) ; or they were extracted using the ProteoExtract Cell Fractionation Kit ( Calbiochem ) . Then extracts were incubated with either anti-HA antibody ( Roche ) , anti 18E6 antibody ( ArborVita ) or anti-SNX27 antibody ( Abcam ) as indicated overnight on a rotating wheel at 4°C . The immune complexes were captured using Protein A Sepharose beads and analyzed by SDS-PAGE followed by Western Blotting using anti-SNX27 antibody ( Abcam ) , anti-Vps35 antibody ( Abcam ) and anti-18E6 antibody ( ArborVita ) . For cell fractionation studies , HeLa S4 shSNX27 cells were seeded on 6cm dishes at a density of approximately 1 . 2 x 105 and treated with Doxycycline or DMSO as indicated for 72 hours . The cells were then treated with MG132 or DMSO as indicated for 3 hours . Cells were collected by trypsinization and fractionated into cytoplasmic , membrane , nuclear and cytoskeletal fractions using the ProteoExtract Cell Fractionation Kit ( Calbiochem ) according to the manufacturer’s instructions and fractions were analyzed by SDS-PAGE followed by Western Blotting using anti-SNX27 antibody ( Abcam ) , anti-18E6 antibody ( ArborVita AVC#399 ) , anti-α tubulin antibody ( Sigma Aldrich ) , anti-p84 antibody ( Abcam ) , anti-Transferrin Receptor antibody ( Santa Cruz ) or anti-Vimentin antibody ( Santa Cruz ) as indicated . Cells were seeded on glass coverslips at a density of approximately 1 . 2 x 105 cells and transfected with siRNA against Luciferase , E6 or E6/E7 as indicated for 72 hours . The cells were fixed using 4% Paraformaldehyde and permeabilized using PBS containing 0 . 1% Triton X-100 . Immunostaining was performed by incubating the coverslips in PBS containing antibodies against SNX27 ( Abcam ) , Vps35 ( Abcam ) , GLUT-1 ( Abcam ) , p53 ( Santa Cruz ) or Rab4 ( Abcam ) as indicated overnight in a humidified chamber at 4°C . The coverslips were then washed thrice with PBS and incubated with the respective fluorophore conjugated secondary antibodies as indicated for 1 hour in a humidified chamber at 37°C . The coverslips were then washed thrice with PBS and twice with distilled water and mounted onto glass slides . The images were captured using the LSM510 META Confocal Microscope ( Carl Zeiss ) and co-localisations were quantified using the Velocity Software and Pearson’s Correlation Coefficient ( PCC ) calculated for each set of images , where values closer to 1 indicate closer degrees of colocalisation , whilst a value of zero would indicate no co-localisation . HeLa cells were seeded in 10cm dishes and transfected with siRNA against Luciferase or E6 for 72 hours . Optiprep gradients ( 5%-25% ) were prepared as described previously [44] and equilibrated at room temperature for 3 hours . Cell extracts were prepared in the homogenization buffer containing protease inhibitors as described previously [44] , and the lysates were syringed to ensure breakdown of the cells . The lysates were centrifuged at 3000 x g for 5 minutes at 4°C and the post nuclear extracts ( supernatants ) were loaded onto the equilibrated gradients . The gradients were centrifuged at 32 , 000 rpm for 18 hours at 4°C and fractions were collected using a peristaltic pump . The collected fractions were mixed with chilled acetone and incubated at -20°C overnight to precipitate the proteins . The precipitates were centrifuged at 14 , 000 rpm for 10 minutes at 4°C and washed once with chilled absolute ethanol . The precipitates were air dried and dissolved in 2x Laemmeli’s buffer . The fractions were loaded onto 12% SDS polyacrylamide gels and the endocytic profiles were analyzed by Western Blotting using anti-GLUT-1 antibody ( Abcam ) , anti SNX27 antibody ( Abcam ) , anti-p53 antibody ( Santa Cruz ) , anti- Vps35 antibody ( Abcam ) , anti- Rab4 antibody ( Abcam ) and anti- LAMP2 antibody ( Abcam ) as indicated . HeLa cells were seeded at a density of approximately 1 . 2 x 105 cells in 6cm dishes and transfected with siRNA against Luciferase or E6 as indicated using the Lipofectamine RNAiMAX transfection reagent ( Invitrogen ) for 72 hours . Glucose uptake was measured using the glucose analog 2-Deoxyglucose ( 2-DG ) which can be taken up by the cells but is not metabolized . The uptake of 2-DG was measured colorimetrically using the Glucose Uptake Assay Kit ( Abcam ) as per the manufacturer’s instructions . The glucose uptake is measured as picomoles/μl and the measurements from three independent assays were used to generate the graph and standard deviations . Control HeLa cells and HeLa cells ( S4 ) containing DOX inducible SNX27 shRNA were plated in high ( 4 . 5g/l ) or low glucose ( 1g/l ) in the presence or absence of DOX to induce SNX27 knockdown . Cell numbers were then counted over a period of 4 days . | A unique feature of the high risk Human Papillomavirus ( HPV ) E6 oncoproteins is the presence of a PDZ binding motif ( PBM ) on its extreme C-terminus . This motif confers on E6 an ability to interact with a number of cellular proteins which possess PDZ domains , and this activity of E6 is important during the viral life cycle and contributes towards HPV-induced malignancy . In this study we describe a novel activity of high risk HPV E6 oncoproteins involving the direct regulation of endocytic transport pathways . This activity is dependent upon the E6 PBM and involves interaction with the endocytic cargo sorting machinery via sorting nexin 27 ( SNX27 ) . One of the consequences of this interaction is a redistribution of SNX27 with respect to components of the retromer complex and this in turn affects the composition of the endocytic transport machinery . This impacts directly upon rates of cargo recycling and in the case of HPV transformed cells , contributes towards maintaining high levels of glucose uptake . This study therefore describes a new function for the E6 oncoproteins and sheds light on how HPVs can modulate endocytic transport pathways . | [
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] | 2016 | Interaction of the Human Papillomavirus E6 Oncoprotein with Sorting Nexin 27 Modulates Endocytic Cargo Transport Pathways |
A proportion of homologous recombination ( HR ) events in mammalian cells resolve by “long tract” gene conversion , reflecting copying of several kilobases from the donor sister chromatid prior to termination . Cells lacking the major hereditary breast/ovarian cancer predisposition genes , BRCA1 or BRCA2 , or certain other HR-defective cells , reveal a bias in favor of long tract gene conversion , suggesting that this aberrant HR outcome might be connected with genomic instability . If termination of gene conversion occurs in regions lacking homology with the second end of the break , the normal mechanism of HR termination by annealing ( i . e . , homologous pairing ) is not available and termination must occur by as yet poorly defined non-canonical mechanisms . Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells . We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4 . We observe obligatory use of microhomology ( MH ) -mediated end joining and/or nucleotide addition during rejoining with the second end of the break . Notably , non-canonical HR termination is associated with complex breakpoints . We identify roles for homology-mediated template switching and , potentially , MH-mediated template switching/microhomology-mediated break-induced replication , in the formation of complex breakpoints at sites of non-canonical HR termination . This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer .
Double strand breaks ( DSBs ) are dangerous lesions , the misrepair of which can contribute to genomic instability and cancer predisposition , premature aging and immunological deficiency in mammals [1–3] . A major trigger to chromosome breakage occurs during attempted replication across a damaged DNA template [4–8] . Such replication-associated DSBs may be repaired by sister chromatid recombination ( SCR ) —a potentially error-free pathway of homologous recombination ( HR ) in which the broken chromosome uses the neighboring sister chromatid as a template for repair [9–12] . Germ line mutation of HR genes contributes to hereditary breast/ovarian cancer susceptibility , Fanconi anemia and other cancer-prone or developmental disorders [1 , 13–15] . Other recognized DSB repair pathways include classical non-homologous end joining ( C-NHEJ ) , alternative end-joining ( A-EJ , i . e . , end-joining in the absence of one or more C-NHEJ genes ) and single strand annealing ( SSA ) [2] . A-EJ is characterized by the dominant use of microhomology ( MH ) -mediated end joining ( MMEJ ) —rejoining events in which the two DNA ends share short stretches of homology at the breakpoint [16 , 17] . Cancer genomes commonly reveal complex patterns of chromosomal rearrangement . This complexity may take the form of multiple breakpoints at the site of a chromosome rearrangement with insertion of short stretches of DNA sequence derived from ectopic loci [18–20] . The breakpoints of cancer rearrangements frequently reveal MH , but homeologous breakpoints ( i . e . , breakpoints with extensive but imperfect homology ) and breakpoints with untemplated nucleotide addition ( N-addition ) are also observed [18] . Such complex rearrangements could entail rejoining of simultaneously arising chromosome breaks , break-induced copying from ectopic templates , or both [21] . A major pathway of HR repair in somatic cells is “Synthesis-dependent strand annealing” ( SDSA ) [22] . SDSA entails DNA end resection , loading of the Rad51 recombinase onto single stranded ( ss ) DNA and Rad51-mediated homologous invasion of the donor DNA molecule , such as the neighboring sister chromatid , by one of the two DNA ends . Extension of the invading/nascent strand by repair synthesis is followed by its release ( “displacement” ) and termination of SDSA normally occurs by annealing ( i . e . , homologous pairing ) of the displaced nascent strand with complementary ssDNA sequences on the resected second end of the DSB . The majority of HR events triggered by a DSB resolve by “short tract” gene conversion ( STGC ) , which typically entails repair synthesis of <100 base pairs from the donor [23–25] . A proportion of HR events resolve as “long tract” gene conversions ( LTGC ) , in which several kilobases ( up to ~10 kb ) of the neighboring , undamaged sister chromatid are copied into the break site of the damaged chromosome [26 , 27] . LTGC and crossing over can produce similar rearrangements in the context of an HR reporter . Where studied , these outcomes have proven to be mediated by LTGC and not by crossing over [26 , 28–30] . Genetic inactivation of the major hereditary breast/ovarian cancer predisposition HR genes BRCA1 or BRCA2 , or of other HR genes such as the Rad51 paralogs Rad51C , XRCC2 or XRCC3 biases HR in favor of LTGC [28–34] . Thus , understanding the mechanisms underlying LTGC in mammalian cells may yield insight into mechanisms of genomic instability in HR-defective hereditary breast/ovarian cancer-predisposition syndromes . Very long gene conversions in Saccharomyces cerevisiae are mediated by break-induced replication ( BIR ) , which can copy >100 kilobases from the donor molecule [35–37] . The BIR copying mechanism in S . cerevisiae is conservative , rather than the semi-conservative mechanism of a conventional replication fork [38 , 39] . BIR in S . cerevisiae is dependent on the Pif1 helicase and entails a migrating bubble mechanism [39 , 40] . Gene conversions in S . cerevisiae that ultimately resolve as BIR may reveal homologous template switches during the early stages of the process , suggesting that the initial steps of BIR can be mediated by less robust copying mechanisms [41] . Further , spontaneous somatic gene conversions in S . cerevisiae reveal a bimodal distribution of tract lengths , with median peaks at 6 kb and >50 kb [42] . Taken together , these studies suggest that classical BIR and LTGC , although topologically similar processes , retain some mechanistic differences . If the site of HR termination lacks homology with the second ( non-invading ) end of the DSB , the classical SDSA mechanism of termination by annealing with the resected second end of the DSB is not available . Under these circumstances , HR termination may be mediated by end joining mechanisms [26 , 27 , 43 , 44] . Breakpoints of non-canonical HR termination often reveal MH , suggesting a role for A-EJ in this process [43 , 44] . However , the genetic regulation of non-canonical HR termination in mammalian cells is currently undefined . In Drosophila melanogaster , non-homologous termination of HR repair of a transposase-induced break is independent of the C-NHEJ gene LIG4 and is mediated by the error-prone DNA polymerase PolΘ , encoded by the POLQ gene [45 , 46] . Here , we use a previously described mammalian reporter of LTGC between sister chromatids [27] to analyze mechanisms of non-canonical LTGC termination in XRCC4 conditional and isogenic XRCC4 null mouse embryonic stem ( ES ) cells [47 , 48] . Our work reveals that non-canonical termination of HR in mammalian cells is independent of XRCC4 and can lead to the formation of complex breakpoints , mediated by template switching . This suggests that non-canonical termination of HR may contribute to the formation of complex breakpoints in the cancer genome .
We previously described a HR reporter that enables positive selection of both short tract ( STGC ) and long tract gene conversions ( LTGC ) between sister chromatids in response to a site-specific DSB induced by the rare-cutting homing endonuclease I-SceI ( Fig 1 ) [27] . Briefly , we positioned two artificial exons of the gene encoding blasticidin S deaminase ( here termed “BsdR” ) in a non-productive orientation between the two GFP copies of an HR reporter . Parental cells , or products of STGC , remain blasticidin sensitive ( BsdR–; Fig 1A ) . In contrast , LTGC duplicates the BsdR cassette , thereby allowing expression of wild type ( wt ) BsdR by splicing ( Fig 1A ) . LTGC is experimentally defined here as a gene conversion of >1 . 03kb—sufficient to duplicate exon B of the blasticidin cassette . The most abundant I-SceI-induced HR product is STGC , in which the broken copy of GFP is converted to wild type GFP , leaving the reporter structure otherwise unchanged ( Fig 1A ) . In wild type cells , approximately 5% of all I-SceI-induced GFP+ products resolve by LTGC [28 , 29 , 47 , 49] . LTGC frequently results in triplication of the GFP copies within the repaired sister chromatid ( Fig 1A ) . However , a small proportion of I-SceI-induced LTGCs are terminated in regions lacking homology with the second end of the DSB [26 , 27 , 29] . These LTGCs must be terminated by non-canonical mechanisms ( Fig 1A ) . To study the contribution of C-NHEJ to non-canonical HR termination , we introduced the above-noted “long tract” HR/SCR reporter into mouse embryonic stem ( ES ) cells carrying biallelic conditional ( “floxed” ) alleles of XRCC4 ( XRCC4fl/fl ES cells ) [48 , 50] . We identified individual clones in which a single , intact copy of the reporter had been integrated into the ROSA26 locus , as described previously and in Materials and Methods [49] . We transduced two distinct XRCC4fl/fl HR/SCR reporter ES cell clones with adenovirus encoding the Cre recombinase and screened Cre-treated cells for derivative clones that either had or had not undergone biallelic Cre-mediated deletion of XRCC4 . Southern and western blotting identified XRCC4Δ/Δ and XRCC4fl/fl derivatives of these cells ( examples in Fig 1B ) . We transfected XRCC4fl/fl and , in parallel , XRCC4Δ/Δ HR/SCR reporter ES cells with I-SceI ( with appropriate controls as described in Materials and Methods ) , and scored HR products as the frequency of I-SceI-induced GFP+ and BsdR+ events ( LTGCs ) . The ratio LTGC:Total HR ( BsdR+ GFP+: Total GFP+ ) is a measure of the probability that a given HR event will resolve as LTGC . This value was ~3% in each cell type , suggesting that XRCC4 does not directly influence the probability of engaging LTGC during I-SceI-induced HR . We amplified I-SceI-induced BsdR+ colonies from two XRCC4fl/fl HR/SCR reporter clones ( n = 163 ) and two isogenic XRCC4Δ/Δ HR/SCR reporter clones ( n = 211 ) , prepared genomic DNA ( gDNA ) , and analyzed the underlying structure of the LTGC product by Southern blotting , as described in Materials and Methods—results summarized in Table 1 . We noted examples of non-canonical LTGC termination in both XRCC4fl/fl ( 6/163; 3 . 7% ) and XRCC4Δ/Δ ( 5/211; 2 . 4% ) HR/SCR reporter cells ( difference not significant by Fisher’s exact test ) . This establishes that non-canonical HR termination can occur in the absence of the C-NHEJ gene XRCC4 . A proportion of LTGCs produced aberrant Southern blot patterns , either in the form of off-size bands or additional GFP-hybridizing bands , which defied easy interpretation . 14/211 ( 6 . 6% ) of all LTGCs examined in XRCC4Δ/Δ HR/SCR reporter cells were aberrant; the equivalent proportion in XRCC4fl/fl HR/SCR reporter cells was 2/163 ( 1 . 2% ) ; ( P = 0 . 0102 by Fisher’s exact test ) . The higher proportions of aberrant LTGCs noted in XRCC4Δ/Δ HR/SCR reporter cells is consistent with the known role of XRCC4 in suppressing chromosomal rearrangements [51 , 52] . Analysis of one of these aberrant LTGCs in XRCC4Δ/Δ HR/SCR reporter cells is presented below . Table 1 summarizes Southern blot analysis of I-SceI-induced blasticidin-resistant clones in XRCC4fl/fl ( n = 163 ) and XRCC4Δ/Δ ( n = 211 ) SCR reporter cells . Fisher’s exact test XRCC4fl/fl vs . XRCC4Δ/Δ for GFP triplication vs . non-canonically terminated LTGC: not significant . Fisher’s exact test XRCC4fl/fl vs . XRCC4Δ/Δ for GFP triplication vs . aberrant LTGCs ( excludes non-canonically terminated LTGC products ) : P = 0 . 0102 . The unrearranged parental reporter and the major “GFP triplication” LTGC product produce predictable patterns of hybridization following gDNA digestion with a panel of restriction endonucleases ( Fig 2 ) . We made the assumption that non-canonical termination of LTGC normally entails rejoining with the second end of the DSB and used the specific pattern of Southern blot hybridizations to deduce the likely site of non-canonical LTGC termination in XRCC4fl/fl or XRCC4Δ/Δ LTGC clones . Two such examples are shown in Fig 3 . We were able to clone the breakpoints of six XRCC4fl/fl and three XRCC4Δ/Δ non-canonical LTGC termination products ( see Materials and Methods ) . The cloned breakpoints did indeed reflect rejoining to the second end of the DSB , which had undergone varying degrees of resection ( Fig 4 ) . Each breakpoint revealed use of MMEJ or untemplated nucleotide addition ( N-addition ) at the breakpoint . It has been suggested that N-addition breakpoints of the type observed here might also be products of MMEJ-type rejoining [45] . There were no blunt-ended non-homologous breakpoints in this limited sample and no breakpoints were suggestive of dual homologous invasions by both ends of the original I-SceI-induced DSB . Thus , non-canonical termination of HR can occur in the absence of the C-NHEJ gene XRCC4 and entails use of MMEJ/N-addition rejoining mechanisms , implicating A-EJ as a contributing mechanism . We used a similar restriction mapping approach to analyze one aberrant LTGC product identified in XRCC4Δ/Δ HR/SCR reporter cells . As discussed above , aberrant LTGC products characteristically reveal off-size or additional GFP-hybridizing bands by Southern blotting . One such aberrant clone is shown in Fig 5 . Southern analysis appeared to show two groups of GFP-hybridizing bands with distinct intensities . Importantly , these groups were not separated by recloning of the cells , indicating that all the GFP fragments visualized by Southern blotting reside within one nucleus . We interpret the Southern blot pattern as a case of non-canonical LTGC termination ( blue arrow-heads Fig 5 ) in which LTGC termination occurred between the SacI and HindIII sites within the reporter . However , all restriction fragments involving enzymes beyond the SacI site ( i . e . , HindIII , EcoRI and SpeI ) reveal off-size GFP-hybridizing bands ( Fig 5B ) . These fragments do not match restriction fragment patterns of ROSA26 sequence up to 50 kb beyond the second end of the DSB . This suggests that LTGC termination in this case entailed incorporation of ectopic chromosomal sequences . We interpret the fainter GFP-hybridizing bands in this Southern blot ( orange arrow-heads ) as possible products of the second end of the break ( Fig 5C ) . If so , the rearrangement underlying this aberrant LTGC product could entail a gross chromosomal rearrangement ( GCR ) initiated by non-canonical LTGC termination . Alternatively , the ectopic sequences ( grey bars ) depicted in Fig 5A and 5B might be part of one single insertion of several kilobases between the site of LTGC termination and the second end of the break . In this regard , the solitary ~9 kb SpeI fragment in Fig 5A , which appears to have a higher intensity than all other bands , could potentially span this insertion , while retaining GFP sequences from both sides of the termination breakpoint . However , our attempts to amplify such a putative insertion product between the two ends of the break have not yet been successful . The notion that non-canonical LTGC termination might lead to GCR is consistent with the expected greater availability of free DNA ends in XRCC4Δ/Δ cells , where efficient C-NHEJ mechanisms are compromised . This clone is an example of non-canonical LTGC termination that presents with an aberrant LTGC pattern by Southern blotting . However , until this and other aberrant LTGC products are mapped and sequenced , it would not be valid to conclude that all aberrant LTGC outcomes arise from non-canonical LTGC termination . In one XRCC4fl/fl clone in which LTGC had been terminated by non-canonical mechanisms , sequencing revealed two distinct breakpoints: one homologous and one N-addition breakpoint . The homologous breakpoint reflected incorporation of sequences from the episomal I-SceI expression vector within the repaired sister chromatid ( Fig 6A ) . The vector sequence had been incorporated at a site of perfect and extensive homology between the chromosomally integrated HR/SCR reporter and the episomal plasmid , based upon shared rabbit β-globin intron sequences [27 , 53] . Following LTGC using the sister chromatid as template , a template switching mechanism allowed the displaced nascent strand to invade homologous sequences on the episomal plasmid . After further nascent strand synthesis of ≥342 bp ( the exact point of homologous invasion of the episomal plasmid is not definable ) , the newly extended nascent strand was displaced from the plasmid template and was joined to the second end of the I-SceI-induced chromosomal break , with insertion of one nucleotide at this second ( non-homologous ) breakpoint ( Fig 6A ) . Thus , non-canonical termination of LTGC can entail homologous template switching—a phenomenon known to be associated with LTGC and BIR in S . cerevisiae [41 , 54] . A second complex breakpoint of non-canonical LTGC termination was present in one XRCC4Δ/Δ clone . Sequencing of the breakpoint revealed an inversion/duplication rearrangement of the second end of the DSB ( Fig 6B; Southern blot analysis of this clone is shown in Fig 3B ) , involving at least two breakpoints in close proximity to one another . The first breakpoint entailed a 21bp insertion at the site of non-canonical LTGC termination , showing 16bp identity with several heterologous loci in the mouse genome ( if templated , this 21bp insertion could represent two independent breakpoints ) . The second was a 4bp MH breakpoint generated during ligation to the second end of the DSB , with an accompanying complex deletion/inversion/duplication rearrangement of the second end of the DSB . Although the mechanisms underlying this complex rearrangement are a matter of speculation , the rearrangement suggests that the nascent strand , having been displaced from the donor sister chromatid during LTGC termination , underwent further rounds of MH-mediated template switches and short nascent strand extension—a process termed “microhomology-mediated BIR” ( MMBIR ) [55] . Fig 7 depicts how this MMBIR rearrangement could have arisen through a fork stalling and template switching ( FoSTeS ) mechanism [56] . Notably , the 146 bp inversion fragment ( Fig 6B ) is of a size consistent with FoSTeS-type copying from a lagging strand donor .
We used the positive selective power of a HR/SCR reporter to capture rare LTGCs in which HR had been terminated by non-canonical mechanisms in XRCC4fl/fl and XRCC4Δ/Δ mouse ES cells . Rejoining with the second end of the chromosomal break entails use of XRCC4-independent MMEJ ( i . e . A-EJ ) , in agreement with previous studies in D . melanogaster [45 , 46] . A notable finding of the current study is that non-canonical HR termination in mammalian cells may entail homologous template switching or MH-mediated template switching ( i . e . , MMBIR ) prior to rejoining with the second DNA end , leading to the formation of complex breakpoints at the site of HR termination . Long gene conversions during gap repair in D . melanogaster have been proposed to entail cycles of invasion and displacement of the nascent strand , with an implied potential for template switching [57] . Both homologous template switches and MMBIR have been described in S . cerevisiae during LTGC/BIR , suggesting that these error-prone mechanisms of HR termination are evolutionarily conserved [41 , 54 , 58] . Our findings provide direct evidence of homologous template switching during mammalian HR , highlighting the extreme reactivity of the displaced nascent strand and its potential significance as an instigator of genomic instability . Given the likely importance of template switching mechanisms in the formation of complex breakpoints in cancer cells , our findings suggest that aberrant HR termination may underlie some of the complex breakpoints observed in cancer genomes [18–21] . A striking feature of the breakpoints associated with non-canonical LTGC termination is the frequent use of MMEJ/insertional rejoining mechanisms . The channeling of repair into an MMEJ mechanism is likely best explained by the DNA structures that are presented for rejoining . Both the displaced nascent strand and the resected second end of the break possess extended 3’ ssDNA tails . These are poor substrates for Ku binding and , hence , for C-NHEJ-mediated rejoining , leading to a preference for A-EJ [59] . Completion of non-canonical LTGC by MMEJ-mediated rejoining to the second end of the DSB may suppress more deleterious outcomes , such as template switching , BIR and chromosome translocation , at sites of non-canonical HR termination . Direct testing of this hypothesis must await the development of more readily quantifiable systems for studying non-canonical HR termination in mammalian cells . However , this idea is strongly corroborated by work on the A-EJ mediator PolΘ , which suppresses genomic instability in mammalian cells and prevents large deletions at sites of replication arrest or at transposase-induced gaps in model organisms [46 , 60–64] . Conversely , unrestrained LTGC in BRCA mutant and other HR-defective cells might channel HR towards these deleterious outcomes as a mechanism of genomic instability in tumorigenesis [28–30] . In the cell lines studied here , non-canonical LTGC termination accounts for ~3% of all LTGCs in XRCC4fl/fl cells , corresponding to ~0 . 1% of all measured GFP+ I-SceI-induced HR events . These low frequencies may nonetheless be highly significant for genomic instability and cancer predisposition , since cancer initiation and progression result from stochastic events on a “per cell” basis . The significance of non-canonical termination of LTGC may be greater than is suggested by the above calculations , since the repetitive structure of the HR reporter used here presents two opportunities for HR termination by annealing: during STGC and in the termination of LTGC by “GFP triplication” ( Fig 1 ) . In contrast , when gene conversion occurs within non-repetitive sequences , STGC alone provides an opportunity for HR to be terminated by annealing . In this more natural setting , presumably all LTGCs must resolve either by non-canonical termination mechanisms or by BIR . In this regard , it is relevant that mammalian cells lacking the major hereditary breast/ovarian cancer predisposition genes BRCA1 or BRCA2 or other cancer predisposition HR genes reveal a bias towards LTGC [28 , 31–34] . This bias is even more marked at stalled replication forks , where >80% of HR events may resolve as LTGCs in BRCA/HR-defective cells [30] . In this setting , the arrival of a converging replication fork and the activity of stalled fork endonucleases may be additional determinants of genomic instability [65] . The work described here identifies mechanisms by which dysregulated LTGC may contribute to genomic instability in BRCA/HR-defective cells and in general tumorigenesis .
Plasmids—The sister chromatid recombination reporter was previously characterized . Expression plasmids for I-SceI and GFP were described previously [27 , 49] . New constructs described here were generated by standard cloning procedures . Cell Lines and Cell Culture—XRCC4fl/fl mouse embryonic stem ( ES ) cells were obtained from Catherine Yan and Frederick Alt and have been described previously [48] . ES cells were maintained in ES medium on either irradiated MEF feeder cells or gelatinized plates . To generate SCR reporter stable lines , 20μg of KpnI-linearized SCR reporter plasmid was electroporated into 2x107 XRCC4fl/fl ES cells and cells were seeded into 60mm dishes with neomycin resistant feeder mouse embryonic fibroblasts and 400μg/mL G418 ( Sigma-Aldrich ) was added to the medium 1 day after electroporation . Beginning 1 week after continuous selection , G418-resistant colonies were isolated and screened by Southern blotting for single-copy SCR reporter integration . To generate isogenic XRCC4fl/fl , XRCC4fl/Δ and XRCC4Δ/Δ SCR cell lines , adeno-Cre infection was performed as described previously [49] , followed by screening of derivative cell lines by Southern blotting . Recombination Assays—1 . 6x105 trypsinized ES cells were transfected with 0 . 5μg plasmid DNA using Lipofectamine 2000 ( Invitrogen ) in a 24-well plate . Transfection efficiency was measured by parallel transfection of wtGFP expression vector ( at 1:10 dilution in empty vector ) . GFP+ frequencies were measured 72 hr post-treatment by flow cytometry using an FC500 ( Beckman Coulter ) as described previously [27] . To assay LTGC events , cells were counted and replated at 1-3x105 cells per gelatinized 100mm dish in triplicate into media containing 5μg/mL blasticidin ( Invitrogen ) . Approximately 2 weeks later , blasticidin resistant colonies were stained and counted or expanded for molecular analysis . Plating efficiency was determined by plating 3-5x102 cells per gelatinized 100mm dish in triplicate into media lacking selection . HR measurements were corrected for background levels of HR events , transfection efficiency and plating efficiency , as described previously [49] . Southern Blotting—Genomic DNA was extracted from 5-20x106 cells using the ArchivePure Cell/Tissue Kit ( 5 PRIME ) . GFP and XRCC4 Southern blots were carried out as previously described [27 , 47 , 50 , 66] . Western Blotting—Cell lysates were prepared using RIPA buffer ( 50 mM Tris-HCl [pH 8 . 0] , 1 . 0% NP-40 , 150 mM NaCl , 0 . 5% sodium deoxycholate , 0 . 1% SDS ) containing protease inhibitors ( Roche ) . Protein concentration was estimated using Bradford’s Reagent ( Sigma-Aldrich ) . Cellular proteins were resolved by SDS-PAGE on NuPAGE Novex Bis-Tris Gels ( Invitrogen ) , transferred to nitrocellulose membrane ( Bio-Rad semi-dry transfer system , 40 mA overnight ) . The membrane was blocked with 5% nonfat milk in 0 . 05% PBST ( 0 . 05% Tween 20 , in PBS ) and incubated with rabbit polyclonal anti-XRCC4 1:200 ( Sigma-Aldrich ) or mouse monoclonal anti-β-tubulin 1:200 ( Abcam ) at room temperature for 3 hrs . Membranes were washed in 0 . 05% PBST , incubated with peroxidase-conjugated Protein A ( GE Healthcare ) or goat anti-mouse antibody ( Jackson ImmunoResearch ) and developed using high-sensitivity ECL ( PerkinElmer ) . PCR and Sequencing—Breakpoints were amplified using AccuPrime Taq DNA Polymerase High Fidelity ( Invitrogen ) according to manufacturers instructions . The PCR products were excised from the gel and purified using the QIAquick Gel Extraction Kit ( QIAGEN ) and subsequently cloned into the pGEM-T Easy vector ( Promega ) . Sequencing was performed at the Dana-Farber/Harvard Cancer Center DNA Resource Core . | Complex breakpoints are a recognized feature of cancer genome rearrangements , but the mechanisms that lead to their formation are undefined . Although homologous recombination ( HR ) is considered a potentially error-free pathway , cells lacking critical HR genes , such as the major hereditary breast/ovarian cancer predisposition genes , BRCA1 or BRCA2 , frequently engage error-prone homologous recombination mechanisms in which HR termination does not occur in a timely fashion . We show here that aberrant termination of HR in mammalian cells involves the use of error-prone alternative end joining mechanisms and can lead to the formation of complex breakpoints by means of template switching mechanisms . This suggests that defective termination of homologous recombination underlies some of the complex breakpoints observed in cancer cells . | [
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] | 2016 | Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells |
Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase , and catalase activity thought to be important in protection against host immune responses to infection . In this study , we generated a monoclonal antibody ( 48Eg ) against haemoglobin of the nematode Anisakis pegreffii , and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection . Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts , which were identified as haemoglobins by peptide mass fingerprinting and MS/MS . Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N . brasiliensis . Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting . Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice , while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera . Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode , Psuedoterranova decipiens , which lacks the four –HKEE repeats of Ascaris haemoglobin important in octamer assembly . The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis , Ascaris and N . brasiliensis . Anisakis haemoglobin was strongly expressed in the L3 stage , unlike Ascaris haemoglobin , which is reportedly mainly expressed in adult worms . Passive immunization of mice with 4E8g prior to infection with N . brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens . The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection .
Soil-transmitted intestinal nematodes infect more than 1 billion people worldwide , with detrimental effects ranging from anaemia and impaired mental and physical development to potentially fatal intestinal blockages that can result in anywhere from 12 000–135 000 deaths per year [1] , [2] . Worm infestations can also increases susceptibility to unrelated infections such as malaria [3] , HIV [4] cholera [5] and tuberculosis [6] by skewing immune responses . Evidence exists that nematode infections may also impair vaccine efficacy [7] , [8] . The most common soil transmitted nematode species are roundworms ( Ascaris lumbricoides ) , whipworms ( Trichuris trichurius ) and hookworms ( Necator americanus and Ancylostoma duodenate ) [2] , [9] . Nematodes have adapted to survive in particular host species over millions of years , and have evolved numerous mechanisms to adapt to their typically hypoxic ecological niche in the host and survive host defences . One group of molecules , the nematode haemoglobins , are intriguing both from an evolutionary standpoint [10] , [11] , [12] , [13] and because of their unusually high affinity for oxygen [12] , [14] . Ascaris haemoglobin has been best explored , and is an octameric molecule that binds oxygen nearly 25 000 times more tightly than human haemoglobin [14] . Another high-oxygen affinity haemoglobin was identified in Pseudoterranova decipiens [15] , a marine nematode that parasitizes seals and is closely related to Anisakis species , which can accidentally infect humans . The function of Ascaris haemoglobin ( and other nematode haemoglobins ) is unknown , as it is considered to bind oxygen too tightly to be involved in its delivery , and may rather sequester oxygen to maintain an anaerobic environment [12] , [14] . Furthermore , nematode haemoglobin can bind and break down nitric oxide ( NO ) and hydrogen peroxide , suggesting that it may provide protection against host oxidative defences [12] , [14] , [16] . Its association with sterols also suggests a potential role in egg production , which requires oxygen [12] . In the present study , a monoclonal antibody ( 4E8g ) was found to recognize highly immunogenic excretory-secretary haemoglobins of both Anisakis and Ascaris , as well as haemoglobin of the more distantly related murine hookworm Nippostrongylus brasiliensis , commonly used in mouse models of nematode infection . Passive immunization of mice with anti-Hb reduced worm burdens after N . brasiliensis infection , demonstrating that haemoglobin of some nematodes may be a potential target for new treatments or vaccines against nematode infections .
This study was performed in strict accordance with the South African code of practice for laboratory animal procedures , and all mouse experiments were performed according to protocols approved by the Animal Research Ethics Committee of the Health Sciences Faculty , University of Cape Town . Research on the human serum samples was approved by the University of Cape Town Research Ethics committee , and written informed consent was obtained from all participants . Live Anisakis pegreffii ( A . pegreffii ) larvae ( L3 ) were removed from the gut of parasitized fish ( Thyrsites atun ) obtained at a fish market , and washed in alternating volumes of PBS and acetic acid ( 4% ) . Adult Ascaris lumbricoides worms were collected after surgical removal at Red Cross Children's Hospital , Cape Town . Syphacia obvelata adult worms were collected from mice as previously described [17] . Nematodes were killed by freezing then homogenized in PBS , and the extracts were sonicated , centrifuged and filter sterilized using a 0 . 20 µm filter ( Sartorius , Goettingen , Germany ) . Nippostrongylus brasiliensis extract was made from N . brasiliensis L3 by snap freezing larvae in liquid nitrogen , homogenizing the extract and centrifuging to remove debris . Anisakis ES products were produced by incubating Anisakis larvae at 37°C for 5 days in saline solution . The ES products were then concentrated using Millipore protein concentration tubes with a 5 kDa cut-off ( Amicon Ultra ) . The BCA Protein Estimation Kit ( Pierce , Rockford ) was used to measure protein concentrations . Heligosmoides polygyrus ( adult worm ) and Contracaecum sp ( stage L3 ) extracts were kind gifts from Dr . Rick Maizels ( University of Edinburgh , U . K . ) and Dr . Shokoofeh Shamsi ( Charles Sturt University , Australia ) respectively . A BALB/c mouse was immunized subcutaneously with 20 µg of Anisakis extract in Freund's complete adjuvant . At weeks 3 and 6 the mouse was boosted with 20 µg Anisakis extract in Freund's incomplete adjuvant . At week 9 the mouse was boosted with 20 µg Anisakis extract in PBS , and three days later the mouse was killed and the spleen removed . Isolated lymphocytes were fused with AgX63 . 653 murine myeloma cells using 50%PEG 6000/15% DMSO in DMEM-20 . Fused cells were plated in DMEM-20-HAT at 5×105 cells/ml in 96 well plates and incubated at 37°C . After 7–10 days , supernatant from wells containing clones was tested for Anisakis-specific IgG antibody by ELISA . Positive clones were sub-cloned and weaned off HAT by culturing in DMEM-20-HT , followed by culturing in DMEM-20 . The positive subclone 4E8g ( IgG1 isotype ) was chosen for further studies based on its stability and cross-reactivity to Ascaris lumbricoides . The Seize X Protein G Immunoprecipitation kit ( Pierce , Rockford ) was used according to the manufacturer's protocol to isolate the antigens binding to 4E8g in Anisakis and Ascaris extracts . Eluted proteins were analyzed by SDS-PAGE and immunoblotting using 4E8g . Electrophoresed proteins were stained with GelCode Blue ( Pierce , USA ) and excised from the gel . Gel slices were destained with 200 mM NH4HCO3∶acetonitrile ( Romill , Sigma ) 50∶50 until clear . Samples were dehydrated and dessicated with a SpeedVac SC110 ( Savant ) before reduction with 5 mM Tris- ( 2-carboxyethylphosphine ) ( TCEP , Fluka ) in 100 mM NH4HCO3 for 30 minutes at 56°C . Excess TCEP were removed and the gel pieces again dehydrated . Cysteines were carbamidomethylated with 100 mM iodoacetamide ( Sigma ) in 100 mM NH4HCO3 for 30 minutes at room temperature in the dark . After carbamidomethylation the gel pieces were dehydrated and washed with 50 mM NH4HCO3 followed by another dehydration step . Proteins were digested by rehydrating the gel pieces in proteomics grade trypsin solution ( 20 ng/uL ) ( Roche ) and incubating at 37°C overnight . Peptides were extracted from the gel pieces with 50 µL 0 . 1% trifluoroacetic acid ( TFA ) ( Sigma ) . The samples were desiccated and 50 µL water was added , then concentrated to less than 20 µL to remove residual NH4HCO3 The eluted peptides were spotted using the dried droplet technique with two times 0 . 5 uL overlay . The matrix was 10 mg/mL α-cyano-4 h-ydroxycinnamic acid ( Fluka ) with 20 mM NH4H2PO4 ( Fluka ) in 80% acetonitrile , 0 . 2% TFA for a final concentration of 5 mg/mL matrix in 40% acetonitrile , 0 . 1% TFA , 10 mM NH4H2PO4 ( Fluka ) . Mass spectrometry was performed with a 4800 MALDI ToF/ToF ( Applied Biosystems ) . All MS spectra were recorded in positive reflector mode . Spectra were generated with 400 laser shots/spectrum at laser intensity of 3800 ( arbitrary units ) with a grid voltage of 16 kV . All peptide containing spots were internally calibrated using trypsin autolytic fragments . Database interrogation was performed with the Mascot algorithm using the MSDB database on a GPS workstation ( www . matrixscience . com ) . Search parameters were as follows: Species – all entries , Enzyme – trypsin , Maximim number of missed cleavages - −1 , Fixed modifications – carbamidomethyl ( C ) , Variable modifications – oxidation ( M ) , Precursor tolerance – 50 ppm . Protein scores >65 were considered significant . Sequences obtained from MS/MS analysis were analyzed using the BLAST search engine . For immunoblot analysis , nematode extracts or purified haemoglobins were separated on 10% acrylamide gels and transferred to a HybondC+ nitocellulose membrane ( Amersham , Biosciences , UK ) at a constant voltage of 300 mA for 1 h . Haemoglobin was detected using 4E8g diluted 1∶2000 in 1% milk/TBS and incubated overnight at 4°C . An alkaline phosphatase-labelled goat anti-mouse IgG1 secondary antibody ( Southern Biotech , USA ) diluted 1∶1000 in 1% milk/TBS was used with 5-bromo-4-chloro-3-indolyl phosphate-4-nitroblue tetrazolium ( Sigma-Aldrich , Germany ) . For the detection of haemoglobin-specific mouse IgG and IgE , serum collected from Anisakis-infected mice in a previous study [18] was used with AP-labelled goat anti-mouse IgG1 ( Southern Biotech , USA ) and goat anti-mouse IgE ( Southern Biotech , USA ) as secondary antibodies . For the detection of haemoglobin-specific human IgG , sera were used from individuals with elevated specific IgE to Anisakis and Ascaris [19] . Anti-Ascaris specific IgE ranged from 0 . 9 kU/L–17 . 9 kU/L in these subjects while anti-Anisakis specific IgE ranged from 0 . 6 kU/L–16 . 9 kU/L . Negative control sera had 0 kU/L specific IgE . Antibodies were detected with AP-labelled anti-human IgG and IgE ( Sigma ) . Binding of 4E8g to mouse blood was measured by measured by ELISA . Whole mouse blood was collected in EDTA to prevent coagulation and used in the ELISA . Sample preparation was based on commercially available mouse haemoglobin ELISA protocols ( Abcam ) . Plates were coated with Anisakis extract ( positive control ) or mouse blood using concentrations from 0 . 4 ng/ml to 4 mg/ml , and blocked with 4% milk powder/PBS . Monoclonal antibody 4E8g was added overnight at 4°C at a concentration of 30 µg/ml , and detection was carried out using alkaline phosphatase labelled anti-mouse IgG1 ( Southern Biotechnology ) with P-nitrophenylphosphate substrate ( Sigma-Aldrich ) . Absorbance was measured at 405 nm with 492 nm as a reference wavelength . Total RNA was extracted from Anisakis larvae using the Nucleospin RNA II kit ( Macherey-Nagel , Germany ) and cDNA was generated using the Transcriptor First Strand cDNA Synthesis Kit ( Roche , Germany ) . Degenerate PCR was performed using primers designed with Primer3 ( http://frodo . wi . mit . edu ) [20] , using highly conserved regions of aligned Ascaris lumbricoides and Pseudoterranova decipiens haemoglobin sequences . The following forward primers: 5′-CACGTTITITGCGCCAC ( A/C ) TACGA-3′ and reverse primer: 5′-TGTCCTTGTTT ( A/C ) ACGAAGAA-3′ were used . PCR products were analysed by agarose gel electrophoresis ( 1 . 6% ) and fragments of the correct size were excised and purified using the Nucleospin extract II kit ( Macherey-Nagel , Germany ) . Cloning was carried out using the pGEM-T easy cloning system ( Promega , USA ) with the pGEM-T easy vector and JMIO9 High Efficiency Competent Cells ( E . coli ) for transformation . Plasmid DNA was extracted from the cells using the SV Miniprep DNA Purification Kit ( Promega , USA ) . The presence of the insert of correct size in the plasmid was confirmed by PCR using the pGEM-T easy primers ( Sp6 and T7 ) . Plasmids were sequenced by automated sequencing based on the dye-terminator sequencing method at the Molecular and Cellular Biology Department at UCT . Obtained sequences were used to design gene specific primers for 5′ ( 5′-GAAGACCACGATGGACGAGCCAACC-3′ ) and 3′ ( 5′-CACGTCCTCAGCCGTGTAGTTCTCG-3′ ) rapid amplification of cDNA ends ( RACE ) . Amplification of the 5′ and 3′ regions of the haemoglobin sequence was conducted using the SMART RACE CDNA Amplification Kit ( Clontech ) with the Primescript Reverse Transcriptase ( Takara , Japan ) . The resulting PCR products were cloned and sequenced as described above . An additional round of RACE-PCR was then performed on the extended sequence using SMARTER-RACE PCR kit with the following primers: 3′ RACE: CCGACGTTCGATTTCTTCGTTGAC , 5′ RACE: GCGAGCAGGATGTTTTGACCTTGT , CTCATGCAATGGTCACGAAC and CGAAGAACTCGTCCTTCTGC . Final gene sequences were analyzed on the ClustalW program ( www . ebi . ac . uk/clustalw/ ) to assess their homology to the known Ascaris and Pseudoterranova haemoglobin sequences . A phylogenetic tree was constructed using MEGA5 with neighbour joining and the minimum evolution method [21] . Amino acid sequences of the haemoglobins from these nematodes were aligned using MUSCLE ( http://www . ebi . ac . uk/Tools/muscle/index . html ) and analyzed on Jalview [22] . For histology , specimens of Anisakis ( L3 ) , A . lumbricoides ( adult worm ) , N . brasiliensis ( adult worms ) and small intestines from mice with N . brasiliensis infection ( day 7 post infection ) were preserved in 4% phosphate-buffered formalin overnight , cut in paraffin sections 5 to7 µm , washed in alcohol and pre-blocked with 3% H202 . Antigen retrieval was performed for 2 min in 0 . 1 M Citrate Buffer pH6 in a pressure cooker , sections were blocked with normal serum and Avidin/Biotin using a Vector Blocking Kit ( Dako ) , and then stained with haemoglobin-specific 4/E8g antibody or a mouse IgG1 isotype control ( anti-TNP mouse IgG1 , a kind gift from Dr Reece Marillier and Dr Jacques van Snick , Ludwig Institute for Cancer Research , Brussels , Belgium ) . Detection was then carried out using mouse Envision ( Dako ) with DAB substrate ( Dako ) and slides were counterstained with Mayers Haematoxylin . Wildtype BALB/c mice were housed in independently ventilated cages under specific pathogen free conditions in the University of Cape Town Animal Facility . Mice were injected with 200 µg of 4E8g in 200 µl PBS intraperitoneally one day prior to infection with 500 N . brasiliensis L3 . Control mice were injected with anti-TNP mouse IgG1 isotype control antibody ( a kind gift from Dr Reece Marillier and Dr Jacques van Schnick , Ludwig Institute for Cancer Research , Brussels , Belgium ) . Mice were killed at 12 , 24 , 48 and 72 hours and 5 , 7 and 10 days post-infection , and intestines and lungs were collected for worm counts . Whole lungs were collected at 12 , 24 , 48 , 72 and 120 hours post-infection , cut into 2–5 mm pieces and placed in a sterile gauze . The gauze was placed in a 15 ml Falcon tube filled with 0 . 9% NaCl and left overnight at 37°C allowing the worms to migrate out of the lungs . Intestines were collected at days 3 , 5 , 7 and 10 in 0 . 9% NaCl and incubated for 3 hours at 37°C for worm counts . The following day the gauze with lung was removed and worms were counted under a dissecting microscope . Mesenteric lymph nodes were collected and single cell suspensions were restimulated with anti-CD3 for 72 hours . Cell supernatants were then collected for cytokine ELISAs , as previously described [18] . Mouse mast cell protease ( MMCP-1 ) was quantified in serum from infected mice as previously described [18] . Data is given as mean ± SEM . Statistical analysis was performed in GraphPad Prism ( Prism software; http://www . prism-software . com ) using the unpaired Student's t test ( * , p≤0 . 05; ** , p≤0 . 01; *** , p≤0 . 001 ) .
Using immunoblotting , the monoclonal antibody 4E8g was found to bind to a 37 kDa protein present in both somatic and excretory-secretory extracts of A . pegreffii L3 , and a 40 kDa protein ( as well as an 80 kDa protein presumed to be a dimer [23] ) present in A . lumbricoides worm whole protein extract ( Figure 1A ) . 4E8g also recognized a 37 kDa protein in N . brasiliensis L3 and a higher MW protein in Contracaecum spp . L3 , but did not recognize proteins in H . polygyrus or S . obvelata worm extracts , indicating that the antibody is cross-reactive among several nematodes but not all ( Figure 1B ) . An immunoprecipitation kit was then used to identify the antigens bound by 4E8g . Affinity-based purification of the antigen was very effective , with a distinct 37 kDa antigen visible following elution of 4E8g binding proteins in both somatic Anisakis extract and Anisakis ES ( Figure 1C and D ) . Immunoprecipitation to purify the 4E8g binding protein from both A . lumbricoides extract resulted in elution of a 40 kDa band respectively ( data not shown ) . Trypsin digestion and MS/MS analysis was used to identify the eluted proteins . The Ascaris 40 kD protein matched with high confidence with a score of 420 ( protein score >65 is significant; p<0 . 05 ) to pig roundworm ( Ascaris suum ) haemoglobin . Ascaris suum and Ascaris lumbricoides are extremely closely related both morphologically and biochemically [24] . There was no exact match for the eluted Anisakis protein , which was not in the database , but PMF showed a match with the haemoglobin protein of Pseudoterranova decipiens with low significance ( score of 61 ) . In addition , MS/MS followed by a MSBLAST search showed a high peptide match ( HMFEHYPHMR ) to Pseudoterranova decipiens haemoglobin . Pseudoterranova and Anisakis are closely related marine nematodes belonging to the same family , indicating that 4E8g recognizes Anisakis haemoglobin . Anisakis and Ascaris haemoglobins were purified using the 4E8g antibody ( anti-Hb ) with the Seize X Protein G immunoprecipitation kit , electrophoresed and transferred onto nitrocellulose membranes . Immunoblotting with serum from mice that had been infected with live Anisakis larvae showed that mice had generated both specific IgG ( Figure 2A ) and specific IgE ( Figure 2B ) to Anisakis haemoglobin . IgG from most of the mice also recognized Ascaris haemoglobin by cross-reactivity . Interestingly , none of the mouse IgE recognized Ascaris haemoglobin ( data not shown ) . In addition , immunoblotting was carried out using sera from subjects who had tested positive for specific IgE against Ascaris and Anisakis . Specific IgG antibodies against Ascaris haemoglobin seemed to be present in the majority of Ascaris-positive subjects ( Figure 2C ) , while specific IgE against haemoglobin could also not be detected in these human sera ( data not shown ) . Anisakis haemoglobin was not recognized by any of the sera . Degenerate PCR resulted in a partial nucleotide sequence of 428 bp , which was used to design gene specific primers for 5′ and 3′ rapid amplification of cDNA ends . Despite repeated RACE reactions , the sequence for the initial 22 amino acids in the 5′ region was not obtained . However , complete sequencing at the 3′ end demonstrated that Anisakis haemoglobin is similar to Pseudoterranova haemoglobin and lacks the four –HKEE repeats of Ascaris haemoglobin that promote the assembly of tetramers [12] ( Figure 3A ) . Like both Ascaris and Pseudoterranova haemoglobins , the Anisakis haemoglobin consists of two similar domains , each with a haem binding site , and has the B10 tyrosine , E7 glutamine and F8 histidine that are important for high oxygen avidity [12] . 4E8g also recognized the more distantly related N . brasiliensis haemoglobin ( Figure 1B ) . Unlike ascarid haemoglobins , which contain two similar domains , N . brasiliensis haemoglobin contains one domain which occurs in two isoforms , a body globin isoform and a cuticular globin isoform [23] . This is thought to be due to gene duplication event similar to the one that resulted in the two domain gene of ascarid haemoglobins [23] . This one-domain protein tends to dimerize to a 37 kDa protein , as seen in Figure 1B , and shows high similarity to the second domain of Anisakis haemoglobin , explaining why the monoclonal antibody is cross-reactive ( Figure 3A ) . A phylogenetic tree assembled using published haemoglobin sequences from various organisms shows that Anisakis haemoglobin branches off with Pseudoterranova haemoglobin , but is also closely related to Ascaris haemoglobin ( Figure 3B ) . The monoclonal antibody 4E8g allowed for immunohistochemical staining to identify the location of haemoglobin expression in Anisakis , Ascaris and N . brasiliensis ( Figures 4–6 ) . Staining of Anisakis sections confirmed immunoblotting results , indicating that Anisakis haemoglobin is an excretory-secretary protein ( Figure 4 ) . Haemoglobin is highly concentrated in the intestinal lumen of the Anisakis larvae ( Figure 4A , B ) , but is also found in the surrounding muscle cells and just below the cuticle . The staining demonstrates that haemoglobin is an ES product that is expressed in the gut and passes via the excretory glands into the environment ( Figure 4C , D ) . Sections stained with isotype control antibody did not show any staining in these areas ( Figure 4 E–F ) . Ascaris haemoglobin also appears to be expressed from the gut through excretory cells ( Figure 5 ) and is also found within muscle cells . Similarly , in N . brasiliensis worms , haemoglobin was associated with excretory glands near the intestine ( Figure 6A , B ) , suggesting that this nematode species may also secrete haemoglobin . Staining of haemoglobin in cross-sections of N . brasiliensis worms that were present in infected mouse small intestine also demonstrated the presence of haemoglobin in peripheral areas such as the fluid-filled median zone of the cuticle ( Figure 6B , C ) . Nematode and vertebrate haemoglobins are very dissimilar [23] ( Figure 7A ) , with nematode haemoglobins showing only 10–15% homology to vertebrate haemoglobins [13] 4E8g was shown not to bind mouse haemoglobin by ELISA and western blotting ( Figure 7B ) . To determine whether nematode haemoglobin could be targeted for immunization strategies , mice were injected with 4E8g one day prior to N . brasiliensis infection . Parasite burdens were counted in the lungs and intestine at different timepoints post infection . N . brasiliensis L3 migrate to the lungs after injection and moult into L4 larvae between 19–32 hours [25] The L4 remain in the lung till approximately 50 hours post infection before penetrating the alveoli , being coughed up and swallowed into the intestine . In the intestine they moult into adult worms at about 90–108 hours post infection . The number of parasites in the lungs was similar in anti-Hb and isotype control treated groups at 24 h and 48 h post infection ( Figure 8A ) . Parasite numbers were low in the lungs in both groups at 72 h due to migration out of the lungs , although in anti-Hb treated mice the parasite numbers were significantly lower . In the intestine , equivalent worm burdens were found in both groups of mice at 72 h post infection . However at both day 5 and day 7 post infection , worm burdens were significantly reduced in anti-Hb treated mice . Protection was associated with increased levels of MMCP-1 , a marker of mast cell degranulation , in the serum ( Figure 8B ) and increased production of Th2 cytokines by restimulated mesenteric lymph node cells ( Figure 8 C–F ) . Together these data suggest that anti-Hb primarily confers protection against the adult worm , either directly or by priming the immune system to increase expulsion or destruction of the parasites .
Nematode haemoglobins have unusually high oxygen affinity and a range of studies indicate that they may represent an important component of parasite adaptation to co-existence with the host . Haemoglobin sequestering of oxygen has been suggested to aid the parasite in maintaining a locally anaerobic environment while their ability to break down nitric oxide ( NO ) and hydrogen perdoxide produced by innate immune cells would also aid parasite survival [12] , [14] . Ascaris haemoglobin may also play a role in egg production , as oxygen is required for sterol synthesis [12] . As such , parasite haemoglobins represent an attractive target for vaccines . In the present study , a monoclonal antibody was generated against a nematode haemoglobin , and conferred protection against N . brasiliensis in a mouse model . While cheap and effective drugs exist for the treatment of nematode infections , rapid re-infection is a frequent problem , and drug resistance is increasing among nematodes of livestock [2] . Understanding host protective responses against nematodes is critical for the design of effective vaccines for humans or livestock [26] . Expulsion of nematode parasites is typically considered to be dependent on T cell mediated immunity and production of Th2 cytokines , particularly IL-13 [27] . However , increasing evidence shows that antibodies also play a protective role [27] , [28] , [29] , [30] . Many studies using a wide range of helminths have shown that transfer of immune sera or purified parasite specific IgG can be protective , depending on the species and the quality of antibody used [28] , [30] . Highly specific , affinity-matured antibodies that arise after multiple infections are required for optimal protection . When B cell deficient mice were able to effectively expel N . brasiliensis in both primary and secondary infections , it was concluded that antibodies were unlikely to play a significant role in protection against this species , although the authors did note that immune serum could contribute to protection [31] . In the present study , passive immunization of N . brasiliensis infected mice with anti-Hb antibody significantly reduced worm burdens , indicating that while antibodies may be redundant in clearing infection , they can certainly play a protective role , especially when highly specific . After initial experiments demonstrated protection , kinetic experiments were performed in order to determine at which stage of infection the anti-Hb had an effect , since N . brasiliensis matures as it migrates through the host and the different stages may require different mechanisms of immunity [32] . Similar numbers of larvae were found in the lungs of treated and untreated groups in the early stages post infection , whereas worm counts in the intestine were significantly lower in the anti-Hb treated mice . This suggests that anti-Hb responses provide protection against the adult intestinal-dwelling stage of the helminth life cycle . Although previous authors did not find expression of globin cDNA in N . brasiliensis pre-infection L3 larvae using northern blotting [23] , we did detect the globin in extract derived from L3 using 4E8g , which may be a more sensitive method of detection . However since globin cDNA was readily detected by the same authors in parasite stages isolated from the mammalian host , this suggests that expression of N . brasiliensis globins is higher in adults , and this may explain why passive immunization with 4E8g was effective only against the adult stage . It was suggested that the increased in size after moulting , the tendency of the larger adult nematodes to lie further into the lumen , where the partial pressure of oxygen is lower , and the advent of egg production all lead to an increased requirement for oxygen that may increase the need for globins [23] . Furthermore , the two N . brasiliensis isoforms were found to be differentially expressed , with the body isoform expressed in all stages within the host but the cuticular isoform only expressed after the L4-adult moult [23] . It is therefore possible that the protective effect of 4E8g specifically involves the cuticular isoform of N . brasiliensis globin . It is possible that the anti-haemoglobin antibody acts by recruiting immune cells such as basophils , eosinophils , mast cells and macrophages to attack the parasite . Fcγ cross-linking results in the release of cytokines , chemokines , proteases and free radicals that can enhance immunity and damage the parasite [28] . An increase in Th2 cytokine production and mast cell proteases was observed , indicating that the protective effect could be due to immune system activation . Previously it was shown that passive sensitization with IgG1 antibodies increased allergic inflammation , which has similar characteristics to helminth induced inflammation , possibly by priming mast cells to degranulate after subsequent exposure to antigen [33] , [34] . Activated mast cells , basophils and eosinophils release a variety of cytokines and chemokines that can stimulate immune responses , including IL-4 and IL-13 , which promote Th2 differentiation and mediate Th2 effector responses , respectively ( Metcalfe 2008 ) . Since mast cell proteases , an indicator of mast cell degranulation , were increased in passively immunized mice , this could explain why Th2 responses increased and worm burdens decreased after treatment . However , as hookworms feed on the blood of the host , it is also possible that they ingested anti-Hb antibody , which could then directly bind to haemoglobin in the intestinal lumen , perhaps interfering with its function . Like ascarid haemoglobins , N . brasiliensis globins have oxygen affinities 100-fold higher than haemoglobins of their hosts [35] , which is thought to help them survive in their anaerobic environment [23] . As nematode haemoglobin from Ascaris has been shown to bind NO , it would be interesting to determine whether N . brasiliensis haemoglobin has the same property , and if blocking the haemoglobin would thus affect parasite survival by inhibiting its ability to detoxify NO . It was also shown for the liver fluke , Fasciola hepatica , that vaccination with haemoglobin induced protection against infection in cattle [36] . Efficacy was further enhanced by combining the liver fluke haemoglobin with another antigen , cathepsin L2 . This combination vaccine was as effective as some flukicides , and not only reduced pathology and fluke growth , but also had a direct effect on egg production , which requires oxygen in this species . Variable protection was also obtained when vaccinating cattle against Ostertagia ostertagi with globin enriched from the parasite , with two out of four trials showing some protection [37] . Depending on the expression and function of haemoglobin within a particular parasite species , haemoglobin may therefore be a useful vaccine candidate , particularly in combination with other proteins . Recently , Ascaris haemoglobin was purified by chromatography from pseudocoleomic fluid of Ascaris suum and used to vaccinate pigs , after which they were challenged with Ascaris suum eggs [38] . Vaccinated pigs showed no difference in faecal egg outputs or worm burdens , but had an increase in white spots on the liver , previously associated with growing resistance to parasite infection [38] , [39] . This study also found that haemoglobin was not detectable at the protein level and barely detectable at the transcriptional level in freshly hatched L3 larvae , which may explain why the haemoglobin vaccination was not protective in Ascaris infection . In contrast to Ascaris , Anisakis L3 strongly express haemoglobin , which is therefore an interesting difference between the two species . Sequencing of Anisakis haemoglobin showed that it is most similar to haemoglobin of the closely related marine nematode , P . decipien , although haemoglobins of both these nematodes are similar to that of Ascaris . Both Ascaris and Pseudoterranova haemoglobins were found to be extremely oxygen avid and consist of two globin domains followed by a short COOH- terminal tail . The tail of Ascaris has four direct repeats of His-Lys-Glu-Glu ( HKEE ) , whereas the Pseudoterranova tail has only one HKEE repeat and is 7 amino acids shorter than that of Ascaris [40] . In both nematodes the tails are involved in octamer assembly and stability , but the differences in sequence are thought to imply a different mode of oligermisation [12] , [41] . Immunoblotting with serum from infected mice demonstrated that IgG antibodies , but not IgE antibodies , were cross-reactive between Anisakis and Ascaris . IgE antibodies against Ascaris haemoglobin were also not detected in human sera , although IgG antibodies were present . This may suggest that Anisakis but not Ascaris haemoglobin is an IgE-binding protein , which should be investigated in patients with gastroallergic anisakiasis . As neither IgE nor IgG antibodies were detected in the human sera of subjects who had Anisakis specific IgE and were occupationally exposed to Anisakis proteins , it would also be interesting to see whether live infection with Anisakis is required for the development of antibodies to Anisakis haemoglobin . This may reveal whether antibodies against Anisakis haemoglobin are a useful diagnostic marker of infection . Measurement of anti-Hb antibodies in pigs by indirect ELISA was recently shown to be a reliable means of diagnosing ascariasis in pigs , with higher sensitivity than faecal examination ( 99 . 5% versus 59 . 5% at week 7 and 100% verus 68 . 4% at week 14 ) [42] . Detection of IgG antibodies against haemoglobin in human sera could therefore also be investigated as a more sensitive and convenient method of establishing infection with A . lumbricoides . In conclusion , we have generated a specific monoclonal antibody , 4E8g , against Anisakis haemoglobin , that can also be used to detect Ascaris and N . brasiliensis haemoglobin , and may be useful in the detection of haemoglobins from other nematode species that have not yet been tested . 4E8g may be useful in further studies to examine the role and function of nematode haemoglobins and in diagnostic assays for nematode infection . Addtionally 4E8g further demonstrates the potential for anti-haemoglobin antibodies in protecting against certain helminth infections . | Nematode haemoglobins are fascinating molecules with unusually high affinity for oxygen . This is one example of many unique adaptations that nematodes have acquired to survive in their hosts , as nematode haemoglobin is thought to sequester oxygen to maintain an anaerobic environment , and can break down nitric oxide ( NO ) and hydrogen peroxide produced by host defences . This study describes the characterization of nematode haemoglobins using a novel monoclonal antibody ( anti-Hb ) generated against Anisakis haemoglobin , which was found to be highly expressed in stage 3 larvae and associated with the excretory-secretary ducts . Anisakis haemoglobin is an IgE-binding molecule in infected mice , while Ascaris haemoglobin was recognized by IgG but not IgE in human sera . Finally , passive immunization of mice with anti-Hb provided protection against Nippostrongylus brasiliens ( rodent hookworm ) , with mice showing reduced worm burden and enhanced Th2 responses , showing that haemoglobin may be a good vaccine target in some nematodes . The monoclonal antibody generated in this study will be useful in further studies to examine the biology of nematode haemoglobins . | [
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] | [
"medicine",
"infectious",
"diseases",
"soil-transmitted",
"helminths",
"hookworm",
"infection",
"neglected",
"tropical",
"diseases",
"immunology",
"biology",
"hookworm",
"ascariasis",
"parasitic",
"diseases",
"helminth",
"infection"
] | 2013 | A Cross-Reactive Monoclonal Antibody to Nematode Haemoglobin Enhances Protective Immune Responses to Nippostrongylus brasiliensis |
In nature , stressful environments often occur in combination or close succession , and thus the ability to prepare for impending stress likely provides a significant fitness advantage . Organisms exposed to a mild dose of stress can become tolerant to what would otherwise be a lethal dose of subsequent stress; however , the mechanism of this acquired stress tolerance is poorly understood . To explore this , we exposed the yeast gene-deletion libraries , which interrogate all essential and non-essential genes , to successive stress treatments and identified genes necessary for acquiring subsequent stress resistance . Cells were exposed to one of three different mild stress pretreatments ( salt , DTT , or heat shock ) and then challenged with a severe dose of hydrogen peroxide ( H2O2 ) . Surprisingly , there was little overlap in the genes required for acquisition of H2O2 tolerance after different mild-stress pretreatments , revealing distinct mechanisms of surviving H2O2 in each case . Integrative network analysis of these results with respect to protein–protein interactions , synthetic–genetic interactions , and functional annotations identified many processes not previously linked to H2O2 tolerance . We tested and present several models that explain the lack of overlap in genes required for H2O2 tolerance after each of the three pretreatments . Together , this work shows that acquired tolerance to the same severe stress occurs by different mechanisms depending on prior cellular experiences , underscoring the context-dependent nature of stress tolerance .
All organisms must respond to stressful stimuli that result from external environmental changes or internal defects caused by mutation and disease . Decades of research have characterized the mechanisms for surviving individual stresses , by mapping downstream protection systems as well as upstream signaling pathways that mediate these responses [1]–[7] . However , much less is known about the effects of combinatorial stress treatments and how cells defend against compound stresses . For example , stressful environmental changes in nature likely occur together , either simultaneously or in close succession , especially for microbes living in natural conditions . How the mechanisms of stress defense differ when cells experience successive stresses rather than a single insult is poorly understood . Successive stress treatments can cause cells to acquire resistance to a severe ( ‘secondary’ ) stress after experiencing an initial mild ( ‘primary’ ) dose of stress . Acquired stress resistance can occur if the mild and severe treatments represent the same stressor but also across different mild and severe stresses ( known as ‘cross-stress’ protection ) . Acquired stress resistance has been observed in diverse organisms , including yeast , bacteria , archaea , plants , flies , and mammals including mice and humans [8]–[20] . A better understanding of how cells are able to increase their resistance to further insults has potential medical application for decreasing cell death and improving human recovery from stressful events such as chemotherapy treatments and ischemia following heart attack or stroke [21]–[23] . In yeast , it had been suggested that acquired stress resistance in general , and cross-stress protection specifically , may be due to activation of the Environmental Stress Response ( ESR ) [24]–[30] . The ESR is a gene expression response commonly activated by a wide variety of stressful conditions [24] , [25] . It includes induced expression of ∼300 genes involved in stress defense , and reduced expression of ∼600 genes broadly involved in protein synthesis and growth . However , we previously showed that ESR activation alone is insufficient to explain cross-stress protection [31] . Moreover , the ‘general-stress’ transcription factors MSN2 and MSN4 are conditionally required for acquired stress resistance , depending on the precise combination of mild and severe stress treatments [31] . These results revealed that the mechanism of acquired stress resistance is more complex than previously suspected and suggested that the response occurs through different mechanisms depending on the mild stress pretreatment . Many studies have identified genes required to survive a single dose of oxidative stress , and several studies characterized increased tolerance after preconditioning ( reviewed in [5] , [6] , [32] ) . The majority of these studies used single-gene approaches , though several used the yeast deletion collection to interrogate the entire genome [33]–[36] . Kelley et al . ( 2009 ) identified genes required to survive an acute dose of H2O2 and genes necessary to acquire H2O2 resistance following a mild H2O2 pretreatment . They found that the genes required for acquisition of H2O2 tolerance only partially overlapped the genes required to survive the acute dose alone , indicating that the mechanism of acquired H2O2 tolerance is distinct from the mechanism of basal H2O2 resistance [34] . The mechanism of cross-stress protection , in which the mild pretreatment is a different stressor than the subsequent severe stress , is largely unexplored . Here , we leveraged the power of yeast genetics and high-throughput analysis to identify genes and processes important for acquired resistance to severe H2O2 stress after each of three mild pretreatments ( mild NaCl , heat shock , or DTT treatment ) . We used the pooled yeast deletion collection [37] , [38] , including ∼4 , 800 homozygous diploid nonessential genes ( homozygous profiling ) , ∼1 , 300 heterozygous diploid essential genes ( haploinsufficiency profiling ) , and 1 , 140 strains harboring DAmP alleles of the essential genes ( in which the transcript is destabilized due to insertion of a drug marker into the 3' UTR [39] ) to query the vast majority of the yeast genome in a single experiment . We found that , although each pretreatment provided similar levels of subsequent H2O2 resistance , different genes and processes were required depending on the mild stress used . Functional analysis of the genes required during each pretreatment provided new insights into the relationships between regulators and processes . Acquired stress resistance thus serves as a unique phenotype through which to uncover new insights into stress biology .
We exposed the pooled yeast deletion libraries [37]–[39] to severe doses of H2O2 after pretreatment with one of three mild stresses ( Figure 1 ) . These mild stresses were chosen because they each produce increased H2O2 tolerance in wild type but present different initial challenges to the cell . The pooled library was exposed to either 60 min of 0 . 7 M NaCl , 60 min at 40C after a 30°C–40°C heat shock , or 2 h exposure to 2 . 5 mM DTT - each treatment produces roughly equivalent levels of subsequent H2O2 tolerance in wild-type cells . After the pretreatment , cells from the culture were washed and then exposed for 2 hours to either 1 . 0 mM or 1 . 2 mM H2O2 . Exposure to these H2O2 doses kills >85% of untreated wild-type cells but results in >80% viability in cells previously exposed to mild stress ( data not shown ) . To identify mutant strains with defects in acquired H2O2 tolerance , an aliquot of the pooled library was removed from stress at each sample point ( Figure 1 ) and outgrown for precisely 10 generations to dilute dead cells from the population . Relative strain abundances were then measured by quantifying the unique ‘barcode’ sequences ( identified by microarray and/or deep sequencing analysis ) . A defect in acquired H2O2 tolerance was identified based on the log2 change in strain abundance before and after treatments ( see Figure 1 and Materials and Methods for details ) . We also identified 202 strains that were sensitive to a low dose of 0 . 4 mM H2O2 in the absence of any pretreatment ( e . g . Sample 2 versus Sample 1 , false discovery rate ( FDR ) <0 . 05 , Table S1 ) ; these included many genes and regulators known to be important for the H2O2 response [33]–[35] . Because we were interested only in genes important for the acquisition of stress tolerance , we removed from consideration strains with equal fitness defects at both the low and ‘secondary’ doses of H2O2 and strains sensitive to the mild stress treatment alone ( identified by comparing Sample 3 versus Sample 1 ) . Strains that met all of these criteria in replicate experiments were defined as having a specific defect in acquiring resistance to H2O2 . A substantial fraction of the yeast genome was required for acquisition of normal H2O2 resistance after at least one of the three pretreatments . In all , 841 strains ( ∼13% of measured genes ) displayed a defect in acquiring H2O2 tolerance , with 225 strains identified following mild NaCl treatment , 308 after heat shock , and 497 after DTT treatment ( Table S2 ) . Validation experiments were performed for 48 strains , the majority of which were predicted to have a defect after one or more pretreatments and three that were predicted to have no defect after any pretreatment . We measured mutant phenotypes in response to all three mild stresses , allowing us to quantify false positive and false negative rates , by competing each identified strain or the isogenic wild type against a GFP-marked strain ( see Materials and Methods for details ) . This defined an upper limit of ∼25% false positives and ∼25% false negatives; however , these values are almost certainly inflated , because our validation assay does not precisely mirror the selection experiments and was performed using the haploid deletion library . Nonetheless , the results validate that the majority of our strain identifications are accurate . There was surprisingly little overlap between the genes necessary for acquired H2O2 resistance following each mild stress ( Figure 2A ) – only 28 strains had defects following all three primary-stress conditions ( Table 1 ) . This observation cannot be explained by the nominally high false-negative rate: of the 48 strains validated , 34 were predicted to have conditional defects - only two of these 34 ( 6% ) proved to have a universal defect in the validation experiments . There was also low overlap between the genes necessary following these primary stresses compared to genes required after mild H2O2 pretreatment [34] . There is little functionality in common to the 28 shared genes , with a few exceptions . There were several genes involved DNA damage repair and vacuolar processes , along with negative regulators of Ras ( IRA1 ) and TOR ( NPR2 and NPR3 ) signaling , which themselves suppress the stress response ( reviewed in [4] , [40] ) . However , even among the 28 strains with universal defects , the magnitude of their fitness defects varied dramatically depending on the initial mild stress used ( Figure 2B ) . Thus , even the genes necessary in all three cases were not equally important following each mild-stress pretreatment . We also found limited overlap in functional processes enriched in each group of required genes ( Table 2 ) . Genes necessary for acquired H2O2 tolerance after NaCl pretreatment were involved in proteolysis as well as HOG signaling , a pathway well known to respond to NaCl . By contrast , genes important following heat shock were enriched for DNA damage repair , protein transport , and late endosome to vacuole transport . Functions enriched in the group of the DTT-required genes included ubiquitin-dependent and -independent protein catabolism , ribosomal proteins , and regulation of translation . Other processes were shared for two of the three mild stressors ( Table 2 ) . Considering this and the above results , we conclude that genes and processes necessary to acquire H2O2 resistance are largely distinct and determined by each pretreatment . Previous studies showed little correlation between a gene's expression change during stress and its requirement to survive prolonged treatment with that stressor [37] , [41]–[46] . However , we and others showed that gene expression changes are not required to survive the initial stress treatment , but rather are critical for acquired resistance to the secondary stress [31] , [47]–[49] . We therefore wondered if gene expression changes were more correlated with genes' involvement in acquired , rather than basal , stress tolerance . However , we too found low correlation between a gene's fitness effect and its expression change during the mild-stress treatment . Roughly 24% of genes necessary for acquired H2O2 tolerance after mild NaCl or heat shock were induced in expression during pretreatment ( a slight enrichment above that expected by chance , p = 0 . 048 ) . In fact , genes necessary for acquired H2O2 tolerance after DTT treatment were actually enriched for DTT-repressed genes ( p = 0 . 0003 ) . Conversely , the majority of genes whose expression increased during each mild stress treatment played no role in subsequent H2O2 tolerance . Thus , gene induction is a poor predictor of gene requirement for both basal [37] and acquired stress tolerance ( see Discussion ) . Initiation of the yeast ESR was originally proposed to give rise to cross-stress protection [24]–[30]; however , we showed that initiation of the ESR cannot explain acquired stress resistance [31] . Consistent with this notion , we observed little enrichment of ESR genes in any of the gene lists identified above ( with the exception of repressed-ESR genes among those required after DTT pretreatment ) . While individual ESR genes can contribute substantially to the acquisition of stress tolerance ( see below ) , the ESR as a whole seems not to be the sole determinant of the resistance acquired . The results above indicate that acquired H2O2 tolerance occurs through distinct modes , rather than a common mechanism , for each mild-stress pretreatment . We were interested in exploring the possible reasons for the low overlap in required genes . Below we present example cases of three models that explain the low overlap in required genes . One possibility is that different upstream signaling pathways mediate the cellular response , even if the downstream effectors of acquired H2O2 tolerance may be the same across pretreatments . Indeed , an example of condition-specific signaling is seen if NaCl is the pretreatment . Several transcriptional regulators and signaling molecules were important for acquired H2O2 resistance after NaCl stress , including the stress-activated transcription factor MSN2 [25]–[27] , [50] and the majority of HOG signaling components ( including HOG1 , PBS2 , SSK2 , SSK1 , STE50 , and CDC42 ) ( Figure S1 ) . Notably , none of the corresponding deletion strains was sensitive to a 1 h exposure to 0 . 7 M NaCl ( data not shown ) , but all had major defects in acquired H2O2 tolerance . The Hog1 pathway regulates expression of stress-responsive genes specifically during osmotic shock and related stresses but not other conditions ( J . Clarke and APG , unpublished data ) . Consistently , none of the HOG mutant strains validated with an acquired-stress defect after other mild stresses ( although the hog1Δ strain had a general recovery defect , perhaps due to its separate role in cell-cycle progression [51]–[55] ( Table 1 ) ) . Thus , Hog1 components are required for acquisition of H2O2 tolerance if NaCl is the mild treatment but not after pretreatments that do not activate the pathway . This explanation also holds for other pretreatments . The transcription factor Hsf1p , a critical regulator of the heat-shock response that plays an overlapping role with Msn2p [56] , [57] , was required for full acquisition of H2O2 tolerance following heat , but not NaCl or DTT , pretreatments . Interestingly , several regulators not previously known to respond to DTT exposure were required after this pretreatment . These included RTG transcriptional regulators ( RTG1 , RTG2 , RTG3 ) and members of the Snf1p signaling system ( GAL83 , STD1 , and SNF3 ) that respond to mitochondria-to-nucleus retrograde signaling and nutrient availability , respectively [58] , [59] . This suggests that additional , novel regulators of the primary responses are likely being uncovered . Although different upstream regulators were involved in each mild-stress response , we wondered if the same downstream effectors might be universally required for subsequent H2O2 tolerance . We focused on the cytosolic catalase Ctt1p , which reduces H2O2 to water and oxygen , as an obvious mechanism for detoxifying H2O2 . CTT1 was the most important gene for acquiring H2O2 resistance after mild NaCl treatment ( Figure S1 ) . Importantly , cells lacking CTT1 had no observable sensitivity to H2O2 in the absence of pretreatment , consistent with the low basal expression of this gene ( [60] and S . Haroon and APG , data not shown ) . Somewhat surprisingly , CTT1 was not universally required for acquisition of H2O2 tolerance: although the gene was critical if NaCl was the mild stressor , CTT1 was completely dispensable after heat shock or DTT pretreatments ( Figure S2 ) . Instead , both heat shock and mild DTT treatments required the glutathione system for acquisition of H2O2 tolerance . Glutathione peroxidases provide an independent mode of H2O2 reduction that is coupled to glutathione oxidation [5] . Deletion of either of the glutathione peroxidases GPX1 or GPX2 did not result in an acquired stress defect ( likely due to their known functional redundancy [61] ) . However , deletion of genes involved in glutathione metabolism , including GSH1 that encodes the first step of glutathione synthesis and the glutathione reductase Glr1p that recycles the oxidized peptide , produced a defect after heat shock or DTT pretreatments but not NaCl . Thus , cells appear to rely on different modes of H2O2 detoxification after NaCl versus heat or DTT pretreatments . We wondered why cells would utilize different detoxification mechanisms for different pretreatments . At least part of the answer lies in the gene-expression response . Although CTT1 transcript was induced by all three mild stresses ( albeit to different levels ) , Ctt1 protein accumulated to significant levels only after NaCl treatment ( Figure S3 ) . Neither Gsh1p nor Glr1p increased in abundance after any treatment ( data not shown ) . However , glutathione peroxidases did increase under different conditions: Gpx2p was induced nearly 2 . 5-fold in response to DTT but only marginally ( 1 . 3-fold ) after heat or NaCl exposure ( Figure S3C ) . We were unable to measure Gpx1p levels by Western , although GPX1 transcript increased after heat and NaCl treatments ( data not shown and Figure S3A ) . These results show that the differential requirement for CTT1 and genes involved in glutathione metabolism correlates with the conditional induction of Ctt1p or Gpx2p . Consistent with this result , we found that a double mutant lacking CTT1 and GSH1 had no additional defect in acquired H2O2 tolerance compared to the single mutants ( data not shown ) . A third model for condition-specific mechanisms of acquired H2O2 tolerance is that the mode of resistance depends on the unique cellular conditions after each pretreatment . This model implies that the cell may experience H2O2 differently depending on its internal status immediately before treatment . As an example , we focused on the stress-specific poly-ubiquitin Ubi4p , which was necessary for acquired H2O2 tolerance after heat and DTT treatments but dispensable following NaCl . Ubi4p plays an important role in protein degradation and turnover in response to heat shock ( reviewed in [2] and [62] ) . Consistent with previous observations [63] , cells lacking UBI4 were not sensitive to mild heat shock , based on viability ( data not shown ) or growth rate ( Figure 3A ) . Interestingly , the ubi4Δ strain was able to acquire H2O2 resistance after heat shock , since it had wild-type viability after secondary-stress treatment ( Figure S4 ) . However , the mutant had a significant growth defect upon recovery from H2O2 stress that persisted until ∼8 h after removal from H2O2 ( Figure 3B and 3C ) . The temporary recovery defect recapitulated the ubi4Δ fitness defect observed in the selection experiments . To assess why Ubi4p was required after heat shock but not NaCl treatment , we measured free ubiquitin levels before , during , and after stress treatments ( Figure 3D ) . Mono-ubiquitin was diminished but measurable in the ubi4Δ strain exposed to mild NaCl or heat shock alone . In contrast , free ubiquitin was virtually undetectable in cells treated with heat shock followed by H2O2 ( Figure 3D ) . Mono-ubiquitin levels were again observable in the ubi4Δ strain 8 h after removal from H2O2 , when the growth rate recovered . In contrast to the case of heat pretreatment , mono-ubiquitin was not depleted in the ubi4Δ strain treated with successive NaCl and H2O2 . Thus , the combined effects of heat followed by H2O2 treatment require ubiquitin synthesis from the UBI4 gene to supplant the consumed ubiquitin . Another possible example of context-dependent stress defense was seen when H2O2 stress followed mild DTT treatment , which invoked a large number of unique genes . To examine the connections between these genes , we constructed a network based on their genetic or physical interactions ( Figure 4 ) . The resulting network was heavily connected and pointed to a few key processes . Ribosomal proteins and proteins involved ubiquitin metabolism showed a large number of physical interactions , both within and between processes , while proteins involved in chromatin biology and actin cytoskeleton/cell wall showed the most genetic connections . This highly interconnected network demonstrates that the long list of genes important after DTT treatment can be collapsed into a smaller subset of processes . To delineate whether the roles of these processes were related to DTT's reducing potential or to specific effects on ER function through the unfolded protein response ( UPR ) , we repeated the selection using tunicamycin as a primary stress , to induce the UPR by blocking N-glycosylation in the ER [64] . We found that some , but not all , of the genes important after DTT treatment were also required after tunicamycin pretreatment ( Table S2 ) . Most notably , ribosomal proteins were important after both pretreatments ( p = 6×10−7 ) . Other genes required after DTT pretreatment were in fact necessary for tunicamycin survival; these were enriched for vacuolar/lysosomal transport ( p = 8×10−6 ) and protein deubiquitination ( p = 6×10−6 ) , and included several genes linked to RNA processing . Why genes related to ribosome synthesis and protein and RNA metabolism are necessary for acquired H2O2 resistance after DTT , and to some extent tunicamycin , treatment remains unclear . However , hints from the literature suggest a connection between ER function and RNA catabolism [65]–[70] . These processes may be particularly susceptible to H2O2 attack if ER function is already disrupted ( see Discussion ) .
Our results show that the genes and processes necessary to acquire resistance to the same severe stress ( H2O2 in this case ) are distinctly different depending on the mild stress to which cells are previously exposed . Although there were some shared processes required for pairs of pretreatments , there were surprisingly few genes required for acquisition of H2O2 tolerance after all three mild-stress treatments . Even among these shared genes , their contributions varied dramatically depending on the pretreatment . Thus , the vast majority of genes function in a condition-specific manner to produce the same end result - increased H2O2 tolerance . We have presented three different models explaining the low degree of mechanistic overlap , including 1 ) condition-specific signaling , 2 ) use of different downstream effectors that enact the same roles , and 3 ) application of entirely different defense strategies based on each pretreatment . Furthermore , we note that the genes and processes involved in acquired stress resistance could function in two fundamentally different ways . Induced production and/or function of some gene products may be sufficient to boost H2O2 resistance . For example , an exogenous pulse of CTT1 expression in the absence of stress is sufficient to increase H2O2 tolerance ( S . Haroon and APG , unpublished ) . Alternatively , some genes and processes may be necessary , but not sufficient on their own , for acquired H2O2 resistance . Their action may instead be important to combat compounded stress , which may render some cellular processes more susceptible to oxidative attack . This model may explain the requirement for fundamental cellular processes , including RNA metabolism , ribosome biogenesis , and actin cytoskeleton , when DTT is the pretreatment . These processes are unlikely to produce H2O2 tolerance , but may instead become sensitive to H2O2 attack after DTT . Indeed , a prior study showed that ribosomal proteins are particularly prone to DTT-induced aggregation when the thioredoxin defense system is abolished [71] . Furthermore , the recent links between genes involved in RNA catabolism , P-body formation , and normal ER function [65]–[70] , [72]–[74] may explain why mild stresses that trigger the UPR uniquely require these genes for subsequent stress survival . Previous studies showed that <1% of genes required for long-term NaCl treatment showed increased expression in response to that condition [37] . Here we found that up to 24% of genes necessary for subsequent H2O2 tolerance are induced during the NaCl pretreatment . This enrichment was not true for all pretreatments , particularly DTT exposure during which most important genes showed reduced expression . Nonetheless , it suggests that gene expression is more closely correlated with , but still a relatively poor predictor of , a gene's requirement in acquired stress tolerance . The low correlation could reflect pervasive post-transcriptional regulation during mild-stress treatment . Alternatively , many genes necessary but not sufficient for acquired H2O2 tolerance may not be actively regulated in response to stress , but rather are already present at a required basal activity . Many other genes are induced during pretreatment but unnecessary for survival of either the mild stress or severe H2O2 treatment ( [37] , [75] and this study ) . It is likely that subsets of these genes ( including many in the ESR ) are important for acquiring resistance to other secondary stresses [31] . Beyond the mechanisms that underlie acquired stress resistance , a remaining question is its purpose . Cross-stress protection may simply be a byproduct of the overlapping effects of two stresses . For example , very high doses of NaCl can produce oxidative damage [76]; it is possible that oxidative defense mechanisms are induced during mild NaCl treatment to prepare for severe NaCl treatment rather than H2O2 . Alternatively , cells may have evolved to prepare for impending stress if successive stressful environments are frequently encountered in nature , or if surviving infrequent compound stresses provides a sufficient selective advantage [31] , [77] , [78] . In E . coli , stresses that occur sequentially as bacteria travel through the gastrointestinal tract can provide cross-stress protection , and this acquired resistance is lost if cells evolve in the absence of sequential exposure [77] , [78] . The role of acquired stress resistance in nature will become clearer as more is learned about the natural ecology of yeast . In the meantime , acquired stress resistance serves as an important phenotype to provide new insights into stress resistance and the complex relationship between phenotype and environment .
Strains used are shown in Table S3 . We used normalized pools of the diploid homozygous non-essential yeast knockout ( YKO ) collection ( BY4743 MATa/α his3Δ1/his3Δ1 leu2 Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0 background ) , diploid heterozygous essential YKO collection ( BY4743 ) , and DAmP yeast library ( derived from BY4741/Y6683 strains ( MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 ura3Δ0/ura3Δ0 met15Δ0/met15Δ0 CYH2+/cyh2 ) [39] . GFP-marked strain AGY0231 ( MATa ura3Δ0 lys2Δ0 dORF-SWH1::Ptdh3-yEGFP-Tcyc1 ) used for competition experiments was graciously provided by Barry Williams . Unless otherwise noted , cells were grown in batch culture in YPD ( 1% yeast extract , 2% peptone , 2% glucose ) at 30°C . Pools of the three deletion collections were grown separately for ∼7 . 5 generations in YPD to an optical density ( OD600 ) of 0 . 3 . The cultures were then mixed such that each strain was roughly equally represented in the resulting pools of barcodes , and an aliquot was removed as the unstressed , time 0 control sample ( Sample 0 ) . One fraction of the culture was outgrown in YPD for 10 generations ( with washing and dilution in fresh YPD medium after 2 , 5 , and 10 generations to maintain log-phase growth ) . The resulting outgrown culture was collected as Sample 1 and compared to Sample 0 to identify slow-growing strains ( Figure 1 ) . A second fraction of the original culture was exposed to 0 . 4 mM H2O2 for two hours , centrifuged , washed , returned to fresh YPD , and outgrown 10 generations before collection as Sample 2 . The remainder of the culture was exposed to one of three primary stresses , including 1 h exposure to 0 . 7 M NaCl , 2 h exposure to 2 . 5 mM DTT , or 1 h growth at 40°C after a 30°C culture was collected and resuspended in fresh 40°C medium . Following primary-stress exposure cells were centrifuged , washed , and a fraction of the culture was outgrown for 10 generations and collected as each respective Sample 3 . The remaining culture was exposed to secondary stress ( 1 . 0 mM or 1 . 2 mM H2O2 ) for 2 hours , then centrifuged , washed and returned to fresh YPD medium for 10 generations outgrowth before collection as Sample 4 ( 1 . 0 mM H2O2 ) or Sample 4A ( 1 . 2 mM H2O2 ) . Comparing Sample 4 to Sample 3 identified strains with a defect in acquiring resistance to severe H2O2 after mild-stress pretreatment . Most experiments were performed in at least duplicate from start to finish , with the exception of essential-gene mutants done once for NaCl and heat shock pretreatments ( see Table S4 ) . All samples were characterized by microarray analysis , and two of each experiment were also interrogated by deep sequencing ( see below ) . Selections were also performed as above using 20 µM Tunicamycin ( Sigma ) for four hours as a primary stress , in biological duplicate . Fitness scores for all experiments are listed in Table S5 . The barcode microarrays were performed as in Pierce et al . 2007 . Briefly , ‘up’ and ‘down’ barcodes were separately amplified from genomic DNA using common primers , and resulting PCR products were hybridized to 16K TAG4 barcode microarrays ( Affymetrix part no . 511331 ) as previously described [79] . Each ‘up’ and ‘down’ barcode tag is represented five times on the array , for a total of 10 measurements per deletion strain . For each array , signal intensities of ‘up’ and for ‘down’ tags were averaged separately , excluding clear outliers . Quantile normalization was performed across all arrays and done separately for averaged ‘up’ and ‘down’ tag signal intensities . Following normalization , a correction factor was applied to correct for feature saturation [79] , and the relative abundance of each barcoded deletion strain was then determined . Negative log2 ratios of strain abundance signify decreased strain fitness . Strains with positive log2 values , which may represent a fitness advantage , were generally not confirmed in validation assays and are not discussed further ( data not shown ) . The sequencing protocol was adapted from [80] . Barcodes were amplified from genomic DNA using primers that included the common YKO barcode amplification sequences , the Illumina anchor sequences , and multiplex indexes for sample multiplexing ( sequences available in Table S6 ) , using Herculase II Fusion DNA polymerase ( Agilent ) . PCR products of ∼150 bp were purified using the e-Gel gel purification system and SybrGreen ( Invitrogen ) . Two biological replicates of Samples 1 , 2 , 3 , and 4 were sequenced for each selection . Finished libraries were sent to the University of Wisconsin Sequencing Facility for Illumina sequencing . Briefly , quality and quantity of the finished libraries were assessed using an Agilent DNA 1000 series chip assay and QuantIT PicoGreen dsDNA Kit ( Invitrogen ) , respectively . Each library was standardized to 10 µM , then 12 uniquely indexed upstream barcode libraries and 12 uniquely indexed downstream barcode libraries were pooled in each lane ( representing ‘up’ and ‘down’ tags from 12 different Samples above ) . Cluster generation was performed using a standard Cluster Kit ( v4 ) and the Illumina Cluster Station , or a standard cBot Kit ( v4 ) and the Illumina cBot . Single-end 50 bp or 75 bp reads were collected using standard SBS kits ( v4 ) and SCS 2 . 5 software , on an Illumina Genome Analyzer IIx . Images were analyzed using the standard Illumina Pipeline , version 1 . 5 , and the sequence reads were mapped back to YKO barcode sequences using custom scripts , allowing one mismatch per 6-bp multiplex sequence and two mismatches per 20 bp ‘up’ or ‘down’ tag ( discarding mismatches that did not map uniquely ) . Survival experiments were performed as in [31] , except viability was scored using an EasyCyte flow cytometer ( Millipore ) and LIVE/DEAD Fungalite Yeast Viability Kit ( Invitrogen ) . Briefly , cells were exposed to mild stress or mock treatment in flasks , collected by centrifugation , and resuspended in YPD . Cells were then exposed to 12 doses of H2O2 ( 0–5 mM ) in 96-well plates for 2 hours , and incubated with dye for 30–60 min before fluorescence was scored at each H2O2 dose . Survival scores shown in the figures were based on the fraction of pretreated cells that survived each dose , minus the fraction of mock treated cells that survived that dose – a single score was then computed as the sum of those values across all doses of secondary stress [31] . CTT1 results were also validated in an independent ctt1Δ::URA3 strain that was then complimented with CTT1 on a plasmid ( data not shown ) . GFP competition experiments were performed by competing a GFP-marked strain against either wild-type BY4741 or single-gene deletion strains from the haploid yeast deletion library ( Open Biosystems ) . Cells were grown separately overnight to early log phase ( OD600 0 . 3 ) and mixed at a 1∶5 ratio of GFP-marked: unmarked cells . Mixed cultures were exposed to no stress , primary stress alone , 0 . 4 mM H2O2 alone , or primary stress followed by 1 . 0 mM H2O2 for 2 hours; cells were then washed with YPD and grown 10 generations in YPD in the absence of stress . Relative strain abundance was inferred based on the proportion of GFP-expressing cells assayed using the EasyCyte flow cytometer ( Millipore ) before and after outgrowth [81] . The proportion of GFP-expressing cells when mixed with a given deletion strain was compared to proportion of GFP-expressing cells mixed with wild-type BY4741; an increase in the number of GFP-marked: mutant cells , relative to the wild-type control , indicated a competition defect in the deletion strain of interest . BY4741 and ubi4Δ cells were grown at least 7 generations to early log phase and a sample of each culture was collected for an unstressed control . The culture was exposed to 30–40°C heat shock or 0 . 7 M NaCl for one hour , then washed and exposed to 1 . 0 mM H2O2 for 2 hours , and washed and outgrown in YPD . Cell samples were collected before and after pretreatment and at 1 h and 8 h during YPD outgrowth . Whole-cell lysate was assayed by Western analysis with the following primary antibodies: polyclonal rabbit anti-ubiquitin ( kindly provided by R . Vierstra ) , monoclonal mouse anti-FLAG ( F3165 , Sigma ) , polyclonal rabbit anti-TAP ( CAB1001 , Open Biosystems ) , or monoclonal mouse anti-actin ( MAB1501; Millipore , Billerica , MA ) . Secondary antibodies included LiCor ( Lincoln , NE ) IRDye 680LT goat anti-rabbit ( 926–68021 ) or goat anti-mouse ( 926–32210 ) fluorescent antibodies . Blots were visualized and analyzed using an Odyssey Infrared Imaging System v3 . 0 . 21 . Free ubiquitin , FLAG-Ctt1p , or Gpx2-TAPp were normalized to actin in each lane . Quantitative PCR was done as previously described [82] using iQSYBR Green Supermix ( Bio-Rad , Hercules , CA ) on a MyiQ2 Bio-Rad Cycler . Primers spanned a 3' 100–200 bp region of each ORF . Cycle numbers were normalized ERV25 mRNA as an internal control unaffected by stress . For each strain , fitness after a particular treatment was taken as the log2 change in strain abundance between each Sample and its corresponding control ( see Figure 1 ) . Strains were identified as defective in acquired stress resistance if they met the following criteria in the microarray and/or sequencing experiments: 1 ) Strains displayed a fitness defect in response to 1 . 0 mM H2O2 following primary treatments ( e . g . Sample 4 compared to Sample 3 ) that was at least 1 standard deviation from the mean of all strains . 2 ) The fitness defect following primary-stress treatment alone ( Sample 3 versus Sample 1 ) was <1 standard deviation from the mean of all strains . 3 ) The fitness defect in response to 0 . 4 mM H2O2 ( Sample 2 versus Sample 1 ) was less than the defect in 1 . 0 mM ( or 1 . 2 mM ) H2O2 . 4 ) These criteria were true in at least two replicates . These stringent lists were expanded by manually adding strains whose fitness phenotypes were highly correlated with identified mutants . For libraries with only one replicate ( for example , the heterozygous deletion collection used in the NaCl selection ) , identified strains were required to meet the stringent criteria for both 1 . 0 mM and 1 . 2 mM H2O2 doses or in corresponding mutants from multiple libraries ( e . g . a significant defect in both the heterozygous-gene deletion strain and DAmP strain ) . Clustering was done in Cluster 3 . 0 ( http://bonsai . hgc . jp/~mdehoon/software/cluster/software . htm ) using hierarchical clustering and uncentered Pearson correlation as the metric [83] . Enrichment of gene functional categories was performed using the hypergeometric distribution in Excel or the program Funspec [84] with Bonferroni-corrected p-values <0 . 01 taken as significant . Network graphs were constructed using Cytoscape 2 . 8 [85] . Genetic and physical interactions were downloaded from BioGRID release 3 . 0 . 66 [86] . Enrichment of genetic or physical interactions , compared to random chance , was determined for 1000 randomly sampled networks with the same number of genes and assessing the number of trials with equal or greater number of total pairwise connections to the observed networks . Genes with defects in acquired stress resistance were defined as induced or repressed during pretreatments if the average ( n> = 3 ) expression change was greater than 1 . 5X higher or lower than unstressed cells 45 min after 0 . 7 M NaCl or 15 min after a 30–37°C heat shock [31] , or 90 min after 2 . 5 mM DTT ( S . Topper and APG , unpublished ) . | Cells experience stressful conditions in the real world that can threaten physiology . Therefore , organisms have evolved intricate defense systems to protect themselves against environmental stress . Many organisms can increase their stress tolerance at the first sign of a problem through a phenomenon called acquired stress resistance: when pre-exposed to a mild dose of one stress , cells can become super-tolerant to subsequent stresses that would kill unprepared cells . This response is observed in many organisms , from bacteria to plants to humans , and has application in human health and disease treatment; however , its mechanism remains poorly understood . We used yeast as a model to identify genes important for acquired resistance to severe oxidative stress after pretreatment with three different mild stresses ( osmotic , heat , or reductive shock ) . Surprisingly , there was little overlap in the genes required to survive the same severe stress after each pretreatment . This reveals that the mechanism of acquiring tolerance to the same severe stress occurs through different routes depending on the mild stressor . We leveraged available datasets of physical and genetic interaction networks to address the mechanism and regulation of stress tolerance . We find that acquired stress resistance is a unique phenotype that can uncover new insights into stress biology . | [
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] | 2011 | Multiple Means to the Same End: The Genetic Basis of Acquired Stress Resistance in Yeast |
The factors that determine the pattern and rate of spread of influenza virus at a continental-scale are uncertain . Although recent work suggests that influenza epidemics in the United States exhibit a strong geographical correlation , the spatiotemporal dynamics of influenza in Australia , a country and continent of approximately similar size and climate complexity but with a far smaller population , are not known . Using a unique combination of large-scale laboratory-confirmed influenza surveillance comprising >450 , 000 entries and genomic sequence data we determined the local-level spatial diffusion of this important human pathogen nationwide in Australia . We used laboratory-confirmed influenza data to characterize the spread of influenza virus across Australia during 2007–2016 . The onset of established epidemics varied across seasons , with highly synchronized epidemics coinciding with the emergence of antigenically distinct viruses , particularly during the 2009 A/H1N1 pandemic . The onset of epidemics was largely synchronized between the most populous cities , even those separated by distances of >3000 km and those that experience vastly diverse climates . In addition , by analyzing global phylogeographic patterns we show that the synchronized dissemination of influenza across Australian cities involved multiple introductions from the global influenza population , coupled with strong domestic connectivity , rather than through the distinct radial patterns of geographic dispersal that are driven by work-flow transmission as observed in the United States . In addition , by comparing the spatial structure of influenza A and B , we found that these viruses tended to occupy different geographic regions , and peak in different seasons , perhaps indicative of moderate cross-protective immunity or viral interference effects . The highly synchronized outbreaks of influenza virus at a continental-scale revealed here highlight the importance of coordinated public health responses in the event of the emergence of a novel , human-to-human transmissible , virus .
Seasonal and pandemic influenza remains one of the most important infectious diseases of humans and is associated with high levels of both morbidity and mortality [1] . Influenza epidemics occur annually due to the continual accumulation of small changes in surface antigens of influenza virus that escape host immunity and hence allow reinfection [2] . At present , four different forms of influenza virus co-circulate in human populations: the H3N2 and H1N1 subtypes of influenza A virus and the Victoria and Yamagata lineages of influenza B virus . Influenza-related mortality is highest in years when A/H3N2 viruses dominate , the rapid evolution of which also leads to the frequent emergence of antigenically distinct variants [2–4] . Influenza in temperate regions is characterized by an annual winter epidemic , whereas tropical regions experience less distinct annual patterns with sporadic outbreaks throughout the year [5] . Defining the seasonal and climatic drivers of influenza virus in both temperate and tropical regions has proven problematic and been the focus of much recent research [6 , 7] . However , a detailed characterization of the spread of influenza virus through time and space at the scale of individual countries and/or continents has been hindered by the lack of spatially refined incidence data . Indeed , our understanding of influenza transmission has relied heavily on observations of influenza-like illness ( ILI ) . Unfortunately , the accuracy of clinical diagnosis of influenza based on ILI alone is limited due to the considerable overlap of symptoms caused by other respiratory pathogens [8] , with influenza virus reported to cause as few as 29% of all ILI infections [9] . Nevertheless , the detailed analysis of ILI data from the United States has shown that the nationwide spatial transmission of influenza virus is principally driven by workflow commuting [10] . In addition , both international and domestic air travel has been suggested as an important driver of influenza introduction and subsequent spread [11] . Much of this work suggests a high geographical correlation of influenza epidemics both at a local level in the United States [2 , 10] , and at the international level across the northern hemisphere [12] . The island continent of Australia offers a unique and opposing exploration of influenza dissemination . Australia’s human population is geographically concentrated in urban centers within two widely separated coastal regions that span over 4 , 000 kilometers . Hence , Australia is simultaneously one of the world’s most highly urbanized populations and least densely populated countries . The continent spans tropical to temperate latitudes and experiences highly variable climatic conditions . Human populations in temperate regions of Australia generally experience seasonal outbreaks of influenza infection between May and October [13] . Nevertheless , inter-seasonal influenza has been shown to persist locally with sustained transmission , particularly in tropical and sub-tropical regions such as Darwin and Townsville [6] . Hence , as well as antigenic variation , it is necessary to consider Australia’s climate complexity to inform its national influenza vaccination strategy [14] . The national and international spread of influenza virus is clearly complex , with the onset , duration and disease severity being largely dependent on the circulating virus strains , population immunity , human mobility and climatic factors . However , teasing apart the individual contributions of these factors has proven difficult , particularly given the diagnostic uncertainty associated with ILI data . Quantifying the spatiotemporal spread of influenza virus is particularly important for identifying the factors that contribute to its epidemic spread and for the precise targeting of control interventions . Here , we utilize large-scale laboratory-confirmed influenza incidence data collected by the National Notifiable Disease Surveillance System ( NNDSS; http://www . health . gov . au/internet/main/publishing . nsf/content/cda-surveil-nndss-nndssintro . htm ) , which is under the auspices of the Communicable Disease Network Australia ( CDNA ) , as well as viral genome sequence data collected on a global scale , to determine the correlation of influenza spread through time and space within Australia during the period 2007–2016 . In particular , we sought to reveal the extent of epidemiological synchronicity within such a large and geographically diverse country , and what this means for understanding the determinants of influenza spread .
The data provided by the Australian NNDSS are characterized by both spatial and temporal richness . The data set includes date of diagnosis , postcode of residence at the time of the test and , in the vast majority ( 98% ) of cases , the influenza virus type detected . Although the overall aim for this surveillance is for complete case ascertainment , there are clearly some caveats in that only those cases for which health care was sought and a laboratory test conducted were represented . Nevertheless , this is one of the most well defined laboratory-confirmed influenza data sets at a nationwide level currently available globally . The data set included the number of influenza cases per day between 2006 and 2016 , by age group and virus type/subtype ( S1 Fig ) . The majority ( 75% ) of viruses were influenza A virus; however , in 70% of these cases the subtype was unspecified , with the remaining influenza A cases defined as H3N2 ( 8% ) and H1N1 ( 22% ) . The number of un-subtyped influenza A virus cases varied among years . For this reason , we necessarily considered all influenza A cases in the main analysis as a single group rather than considering individual influenza A virus subtypes . Other viruses isolated included: A and B ( 0 . 01% ) ; B ( 25% ) ; C ( 0 . 0007% ) ; and unknown ( 0 . 002% ) cases . Data included the five-year age group of patients , which ranged from 0 to 85+ years old . In all years , the age group reporting the highest number of cases was the 0–4 year old group , with the exception of 2009 and 2015 in which the highest number of cases were reported from the 10–14 year old and 5–9 year old groups , respectively . Since data collection increased markedly from 2007 onwards , cases in 2006 and prior were excluded from further analysis . We examined the number of cases of influenza A and B , the most common influenza viruses in the data set , over the ten-year sampling time ( Fig 1 ) . Influenza C was not examined because of the small number of cases reported reflecting the fact that few laboratories in Australia currently test for influenza C . Maps show that the proportion of influenza A compared to influenza B varied each year between 2007–2016 . As expected , very few influenza B cases were reported during the 2009 H1N1 pandemic , while in other years more locations reported higher proportions of influenza B cases . Influenza B dominated in 2015 with 60% of the total influenza cases , while only 39% were influenza A and the remaining cases were mixed infections of A and B . In all other years , influenza A was the dominant virus . During 2012 , influenza A cases were concentrated in the southeast of Australia , while influenza B cases were largely concentrated in northern and western regions . While the majority of seasons displayed a co-circulation of influenza A and B , these two virus types often occupied and dominated different postcodes ( Fig 1A ) . It is important to note , however , that many remote postcodes often reported very few cases each year , such that these patterns may be a reflection of low sample sizes in these locations . Despite this caveat , this spatial pattern might indicate that while one virus dominated in a particular geographic region , the other virus may have failed to establish , perhaps indicative of moderate cross-protective immunity or interference caused by the first establishing virus . We next analyzed the time series of each influenza virus . These displayed a strong seasonal signature and an average annual peak at week ~34 ( i . e . mid-August ) for both influenza A and B , but was delayed in 2010 until week 39 for both viruses , likely due to the large-scale A/H1N1 outbreak during 2009 . We also investigated the possibility of cross-protective immunity among influenza A and B by analyzing the time series in more detail ( Fig 1 , lower right panel ) . This reveals that , on occasion , there is a single dominant type annual peak during a seasonal cycle , again compatible with moderate cross-protective immunity or possibly residual immunity from previous seasons infection or vaccination . We next estimated the timing of epidemic onset of influenza in Australia . To this end we first compared epidemic onset timing , which corresponds to the breakpoint in the piecewise regression , of influenza A across ten years from 2007–2016 ( Fig 2 ) . This comprised fitting linear regressions to the number of cases as a function of time , where the break-point marked the change in the slope of the line . Overall , the timing of the nationwide epidemic onset ranged from week 20 . 6 in 2009 to week 30 . 9 in 2010 . To determine whether the timing of influenza outbreaks varied across Australia , we also estimated the epidemic onset timing for each sampling location . Estimation of epidemic onset timing required an established virus outbreak above baseline and is hence reliant on the strength of the influenza signal in each location . The total number of locations with established epidemics was therefore only a fraction of those locations with reported influenza cases , and ranged from 53 locations in 2008 to 679 locations in 2016 ( Table 1 ) . The time for influenza to reach all locations ranged from 28 weeks in 2009 to 43 weeks in 2013 . Maps of epidemic onset timing show variable spatial patterns across all ten years ( Fig 3A ) . In six out of ten years , the first established epidemic occurred in the southern cities ( i . e . Melbourne , Adelaide , Canberra and Perth ) . In 2010 , however , the first established outbreak was observed on Thursday Island in far north Queensland in the second week of January . Australia , which covers climatically diverse latitudes , often experiences sporadic yet sustained inter-seasonal outbreaks that have been shown to persist in tropical and sub-tropical regions [6] . Indeed , the data analyzed here show that epidemics in northern , tropical regions often precede and follow epidemics in more temperate regions ( Fig 3B ) . During 2013 and 2016 there was a significant negative correlation between epidemic onset time and latitude; hence , northern regions experienced outbreaks that were , on average , earlier than southern regions ( p<0 . 01 ) . This was largely driven by isolated inter-seasonal outbreaks in Darwin and Townsville ( Fig 3B ) . Conversely , the mean epidemic onset time in Hobart was consistently later than most other cities , although this may be due to the small sample size of metro postcodes in this area and was only significantly later in 2013 and 2016 ( p<0 . 05 ) . Overall , our analysis revealed that latitude was a poor predictor of epidemic onset time , and there was no consistent association even with those postcodes located along Australia’s east coast ( i . e . >145° longitude ) , where most of the human population resides . Only in 2009 and 2012 did the onset of epidemics in southern latitudes precede those in northern latitudes ( p<0 . 05 ) ( Fig 4A ) . There were no obvious radial patterns of virus dispersal from the location of the first outbreak each year . Rather , it was striking that epidemic onset was largely synchronized in the major cities , particularly during 2009 ( Fig 3B ) . This result suggests dissemination of influenza through long-distance domestic air travel , likely coupled with multiple entries of influenza into Australia within a short period of time . We also found that onset timing of influenza A and B epidemics occurred around the same time each year , with the exception of the 2009 A/H1N1 pandemic season . In 2009 , the mean epidemic onset timing of influenza B was week ~7 due to a few sporadic , inter-seasonal cases that preceded the emergence and takeover of the pandemic A/H1N1 ( S2A Fig ) . Although the important caveats regarding the sampling inconsistencies of influenza A subtypes ( noted in the Materials and methods ) prevented detailed analysis , both A/H1N1 and A/H3N2 also appeared to be largely synchronized in their epidemics , with the exception of 2009 and 2010; likely a consequence of the emergence of A/H1N1 pandemic strain in 2009 ( S2B Fig ) . To help reveal the determinants of influenza spread , we next investigated the role of population density and volume of air traffic between Australia’s domestic airports on the timing of epidemic onset . This revealed that there was no association between population density per postcode and the onset of epidemics ( Fig 4B ) . Similarly , we found no linear relationship between the distance to the nearest domestic airport and the epidemic onset in a given postcode . Although the timing of epidemics was more clustered ( i . e . synchronized ) at postcodes close to airports with a higher number of domestic passengers ( Fig 4C ) , busy domestic airports are located in major cities , which we have already shown to be well-synchronised with respect to epidemic onset ( Fig 3B ) . To determine the role of distance on the spatial spread of influenza in Australia , we investigated the difference in onset timing between pairs of locations and their pairwise distance ( Fig 5 ) . Spatial synchrony of epidemic onset timing was highly variable . In 2009 , all locations experienced epidemic onset around the same time at , on average , week 23 with an interquartile range of week 21–26 , suggesting very strong synchrony . In addition , epidemics were overall well-synchronized in 2012 , 2014 and 2016 ( all A/H3N2 dominant years ) compared to other years . Importantly , epidemic onset timing was not correlated with geographic distance . In fact , the furthest points tended to display synchrony in onset timing suggesting that influenza reached both coastal extremes of Australia at approximately the same time . For example , across all years sampled , there was no significant difference in epidemic onset timing between Sydney and Perth , which are separated by a distance of 3 , 300 kilometers . In addition , points that fall on the grey vertical bars seem to be close to zero , meaning that there is little difference in onset time between pairwise locations . Although there appears to be a synchrony break at about 1750–2000 km , this reflects the fact that there are few populous postcodes that have this range of pairwise distances . Overall , these results suggest a highly synchronized epidemic onset timing within the most populous Australian cities that may be enhanced during years with antigenic novelty . To determine the relationship of those influenza viruses present in Australia and those circulating globally , we inferred phylogenetic trees for three subtypes over key years: ( i ) influenza A H1N1 during 2009; ( ii ) influenza A H3N2 during 2014; and ( iii ) influenza B during 2015 ( Fig 6 ) . Because of their relatively large data sets , we utilized global hemagglutinin ( HA ) gene sequence data comprising 19 , 482 sequences and analyzed the phylogeographic patterns present in the data , particularly the positions of the Australian sequences . For each of these influenza seasons there was little clustering of Australian sequences . Indeed , Australian sequences were dispersed across all the phylogenies , indicating multiple introductions into Australia from the global population as is common in other localities [15] . For example , the 224 Australian sequences available from the outbreak of H1N1 influenza A virus in 2009 fell into 41 phylogenetically distinct clusters or single lineages . The largest clades comprising Australian-only sequences were ( i ) H1N1 , 2009 = 52 ( of 224 ( 23% ) Australian samples ) ; ( ii ) H3N2 , 2014 = 23 ( of 196 ( 12% ) Australian samples ) ; and ( iii ) influenza B , 2015 = 30 ( of 164 ( 18% ) Australian samples ) . In addition , these Australian clades were sampled from many different locations within Australia , highlighting the importance of virus spread within Australia ( S3 Fig ) as noted previously [6] . For example , in 2014 , A/H3N2 appears to enter the state of Victoria and from this location spreads to both the east ( i . e . Sydney , Brisbane and Newcastle ) and the west ( i . e . Perth ) coasts ( S3C and S3D Fig; note that only sequences with known sampling locations within Australia are shown ) .
We examined the spatial and temporal spread of influenza viruses in Australia over ten years between 2007–2016 using a unique data set of spatially refined laboratory-confirmed influenza cases that comprises >450 , 000 entries . While many studies of the spatial spread of influenza are based on ILI data , in which accuracy is often highly variable and not well measured , our data set offers a rare opportunity to study laboratory-confirmed influenza ( based mostly on real-time PCR testing ) . We focused on the onset time of established epidemics in each postal region ( by postcode ) across Australia in which data were reported . Importantly , establishment of an epidemic requires sustained transmission compared to a baseline number of cases , because the number of cases at baseline may not necessarily be associated with a transmission event leading to an epidemic . Detection of established epidemics was therefore reliant on virus outbreaks above baseline and thus the strength of the epidemic signal in each location . The most prominent result of our study was that despite its huge climatic variation sustained influenza epidemics in Australia were often highly synchronized , especially during years that were observed to be associated with antigenically distinct strains . Although we were unable to assess pairwise synchrony by A subtype or B lineage ( due to lack of data ) , we observed that the onset of nationwide epidemics was more synchronized during years in which there was emergence of novel strains or distinct antigenic changes , including 2009 ( pandemic A/H1N1 ) , 2012 ( a new A/H3N2 variant ) and 2014 ( another new A/H3N2 variant ) . However , due to the low level of exact influenza A subtype data we could not assess whether the pairwise synchrony was entirely driven by these subtypes . For example , although 2009 and 2012 were dominated by A/H1N1 and A/H3N2 , respectively , more precise subtype data suggested that both A/H1N1 and A/H3N2 viruses were present , with A/H1N1 circulating early in the season and A/H3N2 circulating later in the season [16] . Despite the obvious importance of domestic travel in driving the spread of influenza , it is likely that onset synchronicity has been enhanced by the multiple introductions of global influenza viruses into Australia , a process that was clearly apparent in our large-scale phylogeographic analysis . We also believe that this pattern is robust to ascertainment bias . In particular , although the rate of spread during 2009 was much higher compared to other years , the mean week of epidemic onset across all locations was later , at 23 weeks , compared to 21 weeks in 2011 and 2013 ( Table 1 ) . In addition , that all years studied exhibited greater nationwide synchrony than previously observed in the US [10] suggests that the pattern is indeed genuine and reflects the intrinsic dynamics of influenza in Australia . This synchronicity occurs despite the diverse array of climate types present across the Australian continent , suggesting that such seasonal epidemic dynamics override considerable climatic variation . The onset of epidemics during the 2009 influenza season exhibited the greatest synchrony . Famously , 2009 was dominated by the emergence of a novel influenza A/H1N1 virus that resulted in a global pandemic and displaced the previous lineage of A/H1N1 that had circulated in the human population since 1977 . We found that the time from the 5th to the 95th percentile of locations to be infected was less than 10 weeks in 2009 , compared to an average time of ~25 weeks for the other years studied here . In Australia , up to 65% of ILI clinical isolates tested positive for influenza A and by early July 2009 , A/H1N1 accounted for 90% of influenza A isolates [17] . Indeed , following its initial detection in the United States in April 2009 , pandemic A/H1N1 rapidly spread and genetically diversified throughout the global human population [18] . Influenza seasons in 2012 , 2014 and 2016 were also characterized by highly synchronous epidemics and were dominated by A/H3N2 viruses , and often associated with distinct antigenic changes . Specifically , 2012 saw the emergence of A/Victoria/361/2011-A/Texas/50/2012-like viruses ( 3C . 1 clade ) , and in 2014 the emergence and co-circulation of three genetically distinct viral clades; the A/Hong Kong/4801/2014-like viruses ( also referred to as 3C . 2a clade ) , A/Switzerland/9715293/2013-like viruses ( 3C . 3a ) and A/Newcastle/22/2014-like viruses ( 3C . 3b ) . Following both influenza seasons , there was a change in the H3 vaccine recommendation for the following year ( 2013 SH vaccine: A/Victoria/361/2011-like virus and for 2015 SH vaccine: A/Switzerland/9715293/2013-like virus ) [19] . In addition in 2016 , a highly prevalent new clade ( 3C . 2a1 ) also circulated . Although no major antigenic changes were detected that were distinct from the Hong Kong/4801/2014-like viruses that had circulated in 2015 , 2015 was dominated by influenza B such that relatively few A/H3N2 cases were detected compared to previous years . Consequently , the population was less exposed to A/H3N2 during 2015 , likely leading to lower levels of population immunity in years immediately following . We found a distinct lack of radial spread from the first point of virus entry; instead , populous cities tended to have well-synchronized epidemics . While data on work flow patterns in Australia were unavailable for this study , this national synchrony suggests that transmission patterns driven by work flow may have played a relatively minor role in influenza epidemic spread . Rather , these synchronized epidemics across Australian cities suggest rapid dissemination either through domestic flight traffic and/or by multiple global introductions . Indeed , our phylogeographic analysis of global influenza gene sequence data from key years shows that there have been multiple introductions of influenza into Australia annually , which were then able to establish transmission chains across the country , similar to that seen in other localities [20] . These continual introductions from the global population are emphasized by the relatively small number of clades exclusively comprised of Australian isolates . Overall , this pattern again highlights the fluidity with which influenza viruses spread both nationally and globally [21] , and which acts to give annual epidemics a distinct degree of synchrony . The apparent lack of radial virus dispersal observed in Australia also suggests that short-range commuter transmission has not played a major role in epidemic spread , or that it cannot be resolved in the scale of the data analyzed here . This sits in marked contrast to previous studies of more homogeneously-populated countries such as the United States , in which the analysis of ILI data revealed that virus dispersal was predominantly localized with distinct radial patterns from an infected location , with the estimated risk of transmission decreasing sharply with geographic distance [10] . Although it is possible that some of this difference reflects underlying differences in the data collected ( i . e . laboratory-confirmed influenza versus ILI ) , the degree of synchronicity that we have observed during novel antigenic influenza seasons supports continued highly coordinated public health responses across Australia’s populous cities in the event of the emergence of a novel , directly transmissible , virus . Accordingly , in light of the speed with which novel viruses spread , the most populous cities experience synchronized epidemics , likely to be driven by strong domestic connectedness and international travel . These observations may be used to inform prospective pandemic planning efforts both in Australia and likely in other highly urbanized localities .
Laboratory-confirmed influenza notifications data were requested from CDNA . These data can be requested from CDNA ( http://www . health . gov . au/cdna ) , pending appropriate human research ethics committee approval and from the data custodians in each jurisdiction . Ethical approval for this project was granted by The University of Sydney , project number 2015/625 . Laboratory-confirmed influenza is a notifiable disease in Australia , with notifications made by health care professionals and laboratories to jurisdictional health departments . The data set included 454 , 800 entries from 2 , 510 distinct postal areas , collected between 1st January 2006 and 31st December 2016 . These entries included the patient’s age ( within a five-year age group ) and , in 98% of cases , the influenza virus type ( i . e . A , B or C ) detected . The ‘diagnosis date’ represented either the onset date or , where the date of onset was not known , the specimen collection date or the notification date . Data collected in 2006 were excluded from further analysis since data collection in all locations became more consistent from 2007 . This left a final data set size of 451 , 480 entries spanning ten years collected between 2007 and 2016 . We first analyzed the number of laboratory-confirmed influenza cases of influenza viruses A and B to determine the dominant viruses present in Australia between 2007–2016 . All cases specified as influenza A and B were included in this analysis . Only 33% of influenza A notifications included the subtype ( i . e . A/H1N1 or A/H3N2 ) , precluding further analysis by subtype . Since ‘diagnosis date’ might represent the time from the onset of symptoms or the date at which the specimen was collected , we aggregated the data by the number of cases per week and thus considered the weekly time series of these laboratory-confirmed cases . We explored both the proportion and number of cases of each virus across Australia . For seasonal smoothing , data were de-trended and a stable seasonal filter was applied and subtracted from the time series data ( using Matlab v . 2016b ) . By removing the seasonality of the time series , only the long-term trends and the noise components of the data were exposed . We estimated the ‘epidemic onset timing’ , defined as the timing of the break-point in influenza incidence [10] , across the ten-year period for which sufficient incidence data were available ( i . e . 2007–2016 ) . For each year , we therefore fitted piecewise linear models to determine the break-point in influenza incidence using the Segmented package in R [22] . Importantly , the break-point represents the time at which an epidemic can be considered established–in other words , the time of epidemic onset–rather than the time at which the virus first entered Australia each year . The establishment of an epidemic requires the sustained transmission of influenza , whereas an introduction simply represents the first case of influenza each year regardless of whether that first introduction triggered an outbreak . To this end , the onset timing of all influenza A ( regardless of subtype ) epidemics for each year was determined since these represented the majority of cases in the data set . With these data in hand we aimed to better understand the spatial and temporal spread of influenza in Australia . To this end we estimated the epidemic onset timing in each postcode area for all cases of influenza A between 2007–2016 . We investigated the synchrony of epidemic onset timing between pairs of locations and their pairwise geographical distance within a 1 kilometer range . Finally , we compared the epidemic onset timing between major cities in Australia ( Sydney , Melbourne , Brisbane , Adelaide , Townsville , Darwin , Hobart and Perth ) , and between laboratory-confirmed influenza A and B viruses . Next , we performed additional quantitative analyses of the strength of association between particular socio-economic parameters and epidemic onset timing . First , we investigated the role of population density within postcodes and the timing of epidemic onset . Accordingly , population size per postcode ( taken from the 2016 Australian census data available at http://www . abs . gov . au/census ) and postcode geographic data ( available from the Australian Bureau of Statistics; http://www . abs . gov . au/ were used to calculate population density ( i . e . number of people per square kilometer ) . Second , to explore the association between the extent of air travel and epidemic onset , we calculated the number of domestic passengers on inbound and outbound flights from the busiest 96 airports in Australia ( data obtained from https://bitre . gov . au/statistics/aviation/ ) , as well as the distance ( in kilometers ) from each postcode to its nearest airport . To further investigate the introduction and subsequent spatial spread of influenza virus in Australia , particularly the number of introductions in any one influenza season and the presence of Australia-specific clades , we estimated the phylogenetic relationships of influenza virus A and B on a global scale . For this we utilized global gene sequence data available from the GISAID EpiFlu database ( platform . gisaid . org ) [23 , 24] ( note that these data are unlinked to the influenza incidence data analyzed here ) . All available HA genetic sequences , with a minimum length of 500 nucleotides , were downloaded for years in which global influenza data were abundant: ( i ) 2009 H1N1 ( n = 10 , 016 ) ; ( ii ) 2014 H3N2 ( n = 5 , 584 ) ; and ( iii ) 2015 influenza B virus ( n = 3 , 882 ) . Each sequence data set was aligned using the multiple-sequence alignment method available in the MAFFT program , using the FFT-NS-1 strategy [25] . Phylogenetic inference utilized the maximum likelihood ( ML ) method available in RAxML ( v8 . 2 . 10 ) [26] , applying the general time reversible ( GTR ) nucleotide substitution model with a gamma ( Γ ) distribution of among-site rate variation . Support for individual nodes was assessed using a bootstrap procedure with 100 replicates and phylogenetic trees were annotated in FigTree ( v1 . 4 . 3 ) . | We use a unique combination of large-scale laboratory-confirmed influenza surveillance and genomic sequence data to determine the pattern and determinants of influenza spread through time and space at a continental scale , utilizing 450 , 000 data entries gathered across Australia over a 10-year period ( 2007–2016 ) . Our results reveal a remarkable epidemiological synchronicity across this large and geographically diverse continent , and in the face of enormous climatic variation , from the tropical north to the temperate south . Such synchronicity was especially marked during years associated with the emergence of antigenically distinct strains . This pattern of continental synchronicity highlights the importance of a highly coordinated responses in the event of the emergence of a novel , human-to-human transmissible , virus , and hence has broad-scale public health implications . | [
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] | 2018 | Continental synchronicity of human influenza virus epidemics despite climactic variation |
Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size , which facilitates full-length sequencing and genetic manipulation . Previous work has shown that animal and plant RNA viruses are very sensitive to mutation . Here , we characterize mutational fitness effects in single-stranded ( ss ) DNA and ssRNA bacterial viruses . First , we performed a mutation-accumulation experiment in which we subjected three ssDNA ( ΦX174 , G4 , F1 ) and three ssRNA phages ( Qβ , MS2 , and SP ) to plaque-to-plaque transfers and chemical mutagenesis . Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups . Second , we used site-directed mutagenesis to obtain 45 clones of ΦX174 and 42 clones of Qβ carrying random single-nucleotide substitutions and assayed them for fitness . In ΦX174 , 20% of such mutations were lethal , whereas viable ones reduced fitness by 13% on average . In Qβ , these figures were 29% and 10% , respectively . It seems therefore that high mutational sensitivity is a general property of viruses with small genomes , including those infecting animals , plants , and bacteria . Mutational fitness effects are important for understanding processes of fitness decline , but also of neutral evolution and adaptation . As such , these findings can contribute to explain the evolution of ssDNA and ssRNA viruses .
Mutational fitness effects are important for understanding the genetic variability of populations , the relative roles of natural selection and drift , the origin of sex and recombination , or the ability to produce evolutionary innovations , among other processes [1]–[4] . Further , they are of practical relevance to several fields , including complex human disease [5] and conservation genetics [6] . A simple parameter describing mutational fitness effects is the mean selection strength , defined as the average change in fitness caused by random mutations . However , it is also important to determine their variance and the shape of their statistical distribution . A classic approach to achieve these goals is the mutation-accumulation experiment , in which lines derived from a founder clone are propagated at the smallest possible population size to minimize selection , thereby allowing mutations to accumulate [7] , [8] . A more direct and powerful approach consists of genetically engineering random mutants , although this has been done far less often due to the greater difficulty of the task [9] . Some progress has been made in characterizing mutational fitness effects . For instance , in Escherichia coli more than 90% of gene knock-outs are viable [10] and random insertions caused by transposition reduce fitness by 3% on average [11] . In nematodes , the vast majority of nucleotide substitutions have very small fitness effects [12] , whereas in humans few amino acid substitutions have effects greater than 10% and about 30% evolve neutrally [9] . However , the distribution of mutational fitness effects is complex and large differences between species may exist [9] . For example , in some RNA viruses random nucleotide substitutions reduce fitness by nearly 50% on average and up to 40% are lethal [13] , [14] . This extreme mutational sensitivity contrasts with the greater robustness of cellular organisms . Mutational robustness is thought to emerge from the presence of alternative metabolic pathways , genetic redundancy , or modularity , but these buffering mechanisms are usually not found in RNA viruses due to their compact genomes [15] . Viruses are a unique experimental system for characterizing mutational fitness effects because it is relatively easy to engineer sets of random single-nucleotide substitutions . This has been done previously for single-stranded ( ss ) RNA viruses , but not for ssDNA viruses . These two types of viruses have similar genome sizes and therefore might be expected to be equally sensitive to mutation . On the other hand , given the obvious ecological [16] , evolutionary [17] , and genetic [18] differences between ssDNA and ssRNA viruses , differences in robustness would not be surprising . For instance , ssRNA viruses show higher mutation rates [19]–[21] , possibly selecting for greater robustness [22]–[24] , although this might have also promoted genome compression [25]–[27] . Second , replicase genes represent a large portion of the genome of RNA viruses but are not encoded by ssDNA viruses , which use host DNA polymerases . Since replicase genes are typically highly constrained , this might also lead to differences in robustness between ssRNA and ssDNA viruses . Finally , some ssDNA viruses encode two scaffolding proteins , apparently introducing some redundancy in the process of capsid assembly [28] . Here , we compare mutational fitness effects in ssDNA and ssRNA viruses using six phages which can infect the same E . coli strain under identical environmental conditions [29]: the microviruses ΦX174 and G4 , the inovirus F1 ( ssDNA viruses ) , the alloleviviruses Qβ and SP , and the levivirus MS2 ( ssRNA viruses ) ( Figure 1 ) . First , we carried out a mutation-accumulation experiment in which we subjected phages to plaque-to-plaque passages and chemical mutagenesis , quantified changes in fitness , and sequenced the ancestral and evolved genomes . Second , we used site-directed mutagenesis to determine the mean strength of selection more accurately and to characterize the distribution of fitness effects in ΦX174 and Qβ . We demonstrate that ssDNA and ssRNA phage exhibit very similar mutational fitness effects .
Preliminarily , we adapted each phage to our laboratory conditions by performing serial passages at high population sizes , until all lineages reached stable fitness values . For each phage , three independent mutation-accumulation lineages were initiated by picking single plaques at random from the adapted populations . Phage supernatants from plaques were subjected to chemical mutagenesis using nitrous acid and plated to isolate new plaques , a protocol that was continued until plaque sizes became drastically reduced . Under these conditions , selection is minimized and therefore , except for highly deleterious or lethal mutations , random genetic drift and mutational pressure are the only factors driving the fixation of mutations [9] . Growth rates were then assayed and the fitness values relative to the non-mutated genotype were obtained as growth rate ratios . The change in fitness was homogeneous among species ( Table 1 , nested ANOVA: P = 0 . 769 ) and there were no significant differences between the DNA and RNA groups ( P = 0 . 605 ) . After sequencing the six ancestral and the 18 evolved genomes ( GenBank accession numbers GQ153912-GQ153935 ) we found that DNA and RNA phages had accumulated 157±3 and 146±1 nucleotide substitutions in total , respectively ( Table 1 , Figure 1 , nested ANOVA: P = 0 . 802 ) . We calculated the per-mutation effect by dividing the fitness loss of each lineage by the number of mutations accumulated and again , this did not reveal significant differences between DNA and RNA phages ( P = 0 . 870 ) . We therefore conclude that selection strength is similar in the two groups . F1 appeared to show the highest level of robustness , but differences between species were non-significant ( P = 0 . 088 ) and a Tukey's post-hoc test indicated that the six phages formed a single coherent group . However , the number of passages required to reach similar fitness losses or mutation numbers was higher on average for DNA phages ( Table 1 , nested ANOVA , P = 0 . 010 ) . This might be due to their lower spontaneous mutation rates or to lower susceptibility to the mutagen . In both groups , the proportion of transitions was high , and this excess was significantly more marked in DNA phages ( 94 . 9% versus 84 . 9% , Fisher's exact test: P = 0 . 004 ) . Since nitrous acid induces mainly transitions , this suggests that , in DNA phages , most substitutions were caused by the mutagen , whereas in RNA phages there was a greater contribution of spontaneous mutation . This could explain why more passages were required in the former group , although differences in susceptibility to the mutagen or in the proportion of transitions among spontaneous mutations cannot be discarded . Importantly , the higher transition/transversion ratio of DNA phages did not result in a significantly higher fraction of synonymous substitutions ( nested ANOVA , P = 0 . 484 ) and therefore is unlikely to have biased the above selection strength estimates . We constructed 45 clones of ΦX174 and 42 clones of Qβ carrying single-nucleotide substitutions , choosing the target site and the substitution at random . First , we amplified the viral genomes by PCR using mutagenic primers , transfected E . coli with the PCR products , picked single plaques , and verified the presence of the mutation by sequencing . Some transfected cultures failed to form plaques , suggesting that the engineered mutation was lethal for the virus . We first sequenced the region of the mutagenesis PCR product flanking the target site to verify that there were no additional changes . Then , control assays were carried out in which the entire mutagenesis protocol was repeated using PCR primers that did not carry the mutation . By comparing the numbers of plaques formed in mutagenesis and control assays , we showed that 9 and 12 mutations were lethal in ΦX174 and Qβ , respectively ( Figure S1 ) , i . e . a lethal fraction of pl = 0 . 20 and pl = 0 . 29 , respectively . These two proportions did not differ significantly ( Fisher's exact test , P = 0 . 454 ) . All lethal mutations found in ΦX174 produced amino-acid changes , whereas in the case of Qβ , one was synonymous ( U2379A ) and one intergenic ( G1329A ) . We measured the growth rate of viruses carrying viable substitutions to obtain the distribution of mutational fitness effects . Growth rates were also determined for the above control samples and corrected means and variances were obtained by subtracting the mean and variance of the control group from the grand mean and variance of the mutants . In ΦX174 , this yielded a mean selection strength of = −0 . 301 including all ( lethal or viable ) mutations , with variance V ( s ) = 0 . 162 . For viable mutations only , = −0 . 126 and V ( sv ) = 0 . 047 . In Qβ , = −0 . 359 , V ( s ) = 0 . 181 , = −0 . 103 and V ( sv ) = 0 . 018 ( Table 2 ) . There were no significant differences in mean selection strength between the two phages ( nested ANOVA for viable mutations: P = 0 . 633; Mann-Whitney test for all mutations: P = 0 . 336 ) . The above correction using controls implies that our inferences of the mean and variance should be free of experimental error or bias resulting from the presence of additional mutations or changes in the assay conditions , although it is still possible that the fitness values of some individual mutants were altered by the presence of additional mutations . The distribution of fitness effects of viable mutations was highly skewed , with deleterious substitutions of small effect being more abundant than those of large effect ( Figure 2 ) , a property shared by most model systems studied [9] . Focusing on viable mutations with negative s-values , we performed non-linear regression to characterize the shape of the distribution . In ΦX174 , an exponential model with an expected mean selection strength against deleterious mutations of = −0 . 186±0 . 005 described the data satisfactorily ( R2 = 0 . 989 ) and was as accurate as a Gamma ( R2 = 0 . 989 ) or a Beta ( R2 = 0 . 985 ) model ( partial F-test , P>0 . 25 in both cases ) . Other statistical models were not considered here . In Qβ , the data were better described by the Gamma distribution ( R2 = 0 . 975 ) with an expected = −0 . 136±0 . 008 . The Beta model provided a similarly good fit ( R2 = 0 . 973 ) , whereas the exponential model was slightly worse ( R2 = 0 . 965 , P = 0 . 025 ) . Finally , we must emphasize that mutants with positive values were not used for these inferences , which implies that they were assumed to be neutral or beneficial . Neglecting beneficial mutations , the fraction of deleterious to total mutations should thus be pd = ( −pl ) /sd and the fraction of neutral to total mutations simply pn = 1−pl−pd ( Table 2 ) . However , classifying mutations as neutral or deleterious is somewhat unnatural since the difference between a deleterious mutation of infinitesimally small effect and a neutral mutation is merely formal .
Previous site-directed mutagenesis studies have shown that most random nucleotide substitutions are strongly deleterious in animal and plant ssRNA viruses . In Vesicular stomatitis virus ( VSV ) , pl = 0 . 40 , = −0 . 48 and = −0 . 13 [14] , whereas in the unrelated Tobacco etch virus ( TEV ) , pl = 0 . 41 , = −0 . 49 , and = −0 . 13 [13] . After performing similar experiments with phage Qβ , we confirm that ssRNA viruses are extremely sensitive to mutation in general . Roughly speaking , the probability that a random single nucleotide substitution is lethal for an ssRNA virus is one third or higher , while viable mutations reduce fitness by 10–13% on average . The observed fraction of lethals was lower in Qβ than in VSV or TEV , which might reflect real biological differences or may be a consequence of methodological issues . Concerning the shape of the distribution , viable mutations of small effect are more abundant that those of large effect . The specific statistical model that better describes the data varies , but the Gamma and the Beta distributions are generally accurate . However , larger mutant collections would be needed to increase the statistical power of inferences about the distribution of mutational fitness effects . We analyzed ΦX174 using the same methodology and under the same environment as Qβ to make the comparison between ssDNA and ssRNA viruses . The two phages did not significantly differ in the fraction of lethal mutations or the average strength of selection , suggesting that ssDNA and ssRNA viruses are similarly sensitive to mutation . The strong parallel shown by viruses as different as ΦX174 and Qβ probably stems from the fact that both have small genomes with few and short non-coding regions , several multifunctional proteins , and little genetic redundancy [15] , [30] . Differences between ΦX174 and Qβ were only minor . For instance , deleterious ( non-lethal ) effects seemed to follow a simple exponential law in ΦX174 whereas Qβ deviated from this model , probably because there were more mildly deleterious mutations and fewer strongly deleterious ones ( Figure 2 , Table 2 ) , although the fraction of lethals might be higher in the latter . The similar robustness shown by ssDNA and ssRNA viruses was also supported by the mutation-accumulation experiment , in which we examined three phage species of each group and found no significant differences in mean selection strength . Differences between species within the two groups were weak or absent , although F1 appeared to be the most robust phage . Related to this , it is worth noting that F1 has fewer overlapping genes . This might be related to the fact that inoviruses have filamentous capsids , which are structurally less constrained than icosahedral ones and can hence accommodate larger genomes [31] , [32] . However , further work is needed to elucidate whether F1 really differs from the other phages in terms of robustness . It is possible to compare directly the mean selection strengths estimated by mutation-accumulation and site-directed mutagenesis , since the same viruses and the same environment were used . Because lethal mutations cannot be sampled during plaque-to-plaque passages we expected the former to be lower . However , even after excluding lethals , the values obtained by site-directed mutagenesis experiments were approximately twofold higher . The most likely explanation for this discrepancy is that , since the population size within plaques necessarily exceeds one , some degree of selection is inevitable in mutation-accumulation experiments . Also , given that several mutations accumulated in each lineage , the observed fitness values were dependent on genetic interactions . In RNA viruses , mutations tend to have weaker effects as they accumulate ( antagonistic epistasis ) and the same might hold for ssDNA viruses and small genomes in general [33] , probably leading to an underestimate of the mean selection strength in mutation-accumulation experiments . The fitness effects of random mutations are relevant to evolution in many ways . For instance , they determine the fraction of nucleotide sites that evolves neutrally and thus the rate at which populations diverge through random genetic drift . Further , neutral and deleterious mutations can also determine the rate of adaptive evolution indirectly . The observation that mutations tend to be highly deleterious indicates that there are no efficient buffering mechanisms , which might result in large phenotypic variation and strong selection for beneficial mutations too [34]–[36] . Consistent with this view , the fixation of big-benefit mutations has been reported in several phages [37]–[39] and low mutational robustness has been associated with faster adaptation in VSV [40] . Also , everything else being equal , greater effects of deleterious mutations might result in faster adaptation because strongly deleterious mutations are removed more efficiently from populations , favoring the spread of beneficial ones [41] . The connection between mutational robustness and evolvability is controversial , however . By reducing phenotypic variation , robustness should facilitate the accumulation of genetic variation and may foster evolutionary innovation upon changes in the environment or the genetic background over the long-term [4] , [36] , [42]–[44] , a prediction that has also been supported experimentally [42] , [45] . The fitness effects of random mutations are not only relevant to neutral evolution and adaptation , but obviously , also to processes of fitness decline such as Muller's ratchet [46] or lethal mutagenesis [47] . Viruses experience frequent transmission bottlenecks during which random mutations may fix in the population . The consequences of this process for viral fitness are highly dependent on how deleterious are these mutations on average [46] . A substantial proportion of the variation observed in natural populations of RNA viruses comprises transient deleterious mutations , but most fail to reach fixation [48] , possibly due to their strongly negative impact on fitness . Mutational fitness effects are also important for understanding viral extinction through mutagenesis [47] and the clinical use of mutagens as ribavirin to treat viral infections makes this subject of practical importance . A conceptual dichotomy between DNA and RNA viruses has been established based on the observation that the latter evolve faster [49] . However , it has been shown more recently that some ssDNA viruses , including parvoviruses [50] , anelloviruses [51] and geminiviruses [52] can match the evolutionary rates of RNA viruses and rapidly adapt to new hosts ( although this has not been tested experimentally yet ) . Despite mutating faster than double-stranded DNA viruses or cellular organisms , ssDNA viruses are less error-prone than ssRNA viruses [19]–[21] . Therefore , the rate at which spontaneous mutations occur may not fully explain why ssRNA and ssDNA viruses evolve at similar rates . Additional factors as , for instance , the fitness effects of mutations , should be considered .
Bacteriophages ΦX174 , G4 , F1 , Qβ , SP , and MS2 and the E . coli C strain IJ1862 [29] were obtained from Prof . James J . Bull ( University of Texas ) . The Qβ infectious clone pBRT7Qβ was provided by Dr . René C . Olsthoorn ( Leiden University ) . General biology of the six phages can be found elsewhere [31] . Each phage was serially passaged at high population sizes in IJ1862 cells . For each transfer , ∼105 particle forming units ( pfu ) were inoculated into 0 . 5 mL LB medium containing IJ1862 cells at their exponential growth phase . The appropriate cell density varied depending on the virus lytic activity and growth rate ( OD600 = 0 . 7 for ΦX174 and G4 , 0 . 15 for F1 , Qβ , and MS2 , and 0 . 05 for SP ) . Infected cultures were incubated in agitation ( 650 rpm ) at 37°C in a Thermomixer 24-tube shaker ( Eppendorf ) and harvested during the late exponential growth phase of the virus ( ∼109 pfu/mL ) . Cells were removed by centrifugation and supernatants were aliquoted , stored at −70°C , and titrated using LB medium solidified with soft agar ( 0 . 7% ) . Initial and final titers were used to calculate growth rates and to adjust sampling times for the next passage accordingly . We performed 60–80 passages for each phage . In all cases , no significant changes in growth rate were observed during the last 30 passages , indicating that phages had reached a quasi-maximal fitness under these experimental conditions . Three independent mutation-accumulation lines were seeded for each phage by picking random plaques from the high-fitness populations . Each plaque was resuspended in 50 µL LB and stored at −70°C . Lines were propagated plaque-to-plaque as previously described [53] and after each passage , phages were mutagenized with nitrous acid . To do so , four volumes of 0 . 3 M acetate buffer pH 4 . 3 were mixed with one volume of 5 M sodium nitrite and 50 µL of this solution were immediately added to 4 µL of phage-containing supernatant . The exposure time was adjusted to obtain a maximal titer loss . The mutagenesis reactions were quenched by adding 200 µL of 1 M acetate buffer pH 4 . 3 containing 0 . 1 mg/mL BSA and 100 µL of this final mix were plated without dilution . After incubation at 37°C , a single lysis plaque was picked , resuspended in 50 µL , aliquoted , stored at −70°C , titrated , and used for the next round of chemical mutagenesis . The High Pure Plasmid Isolation kit ( Roche ) was used to purify ΦX174 DNA and the pBRT7β plasmid from partially lysed E . coli IJ1862 cells and from an overnight culture of E . coli K12 previously transformed with this cDNA , respectively . For PCR-based mutagenesis , full-length PCR amplicons were obtained from 500 pg of template using Phusion high-fidelity DNA polymerase ( New England Biolabs , error rate provided by the manufacturer: 4 . 4×10−7 per base per replication round ) and a pair of adjacent , divergent , 5′ phosphorylated primers , one of which carried the desired nucleotide substitution . PCR products were circularized using the Quick T4 ligase ( New England Biolabs ) and E . coli IJ1862 competent cells were transfected by the heat-shock method ( 42 C , 30 s ) in the presence of CaCl2 100 mM . The transfected cells were immediately plated onto LB plates using soft agar and individual plaques were picked after 5-9 hours of incubation at 37°C , resuspended in LB , and stored at −70°C . To verify that the desired mutation had been introduced and that no additional changes were present in the region flanking the target site , ( RT ) -PCR was performed directly from the resuspended plaque . Moloney murine leukemia virus reverse transcriptase ( Fermentas ) was used for cDNA synthesis and Phusion DNA polymerase for PCR . The products were column-purified and sequenced using sequence-specific primers . In cases where transfection yielded no plaques or phages recovered from plaques had not incorporated the mutation , the entire protocol was repeated and , after three consecutive failures , the mutation was classified as a candidate lethal . In all of these cases , the number of plaques obtained from transfection assays was abnormally low , suggesting that the mutation was lethal for the virus and that the few observed plaques came from the template wild-type DNA . To confirm lethality , we first sequenced the region of the PCR-based mutagenesis product flanking the target site to check that the mutation was present and that no additional changes had appeared in this region . Then , we designed new primers that did not carry the nucleotide substitution but were otherwise identical to those used for the mutagenesis reaction , and performed the PCR , circularization and transfection steps exactly as above . Lethality was assessed based on the comparison between the numbers of plaques recovered from mutagenesis versus control reactions ( Figure S1 ) . Similar control assays were carried out to estimate the contribution of non-desired mutations and other sources of error to the inferred distribution of mutational fitness effects . For each phage , we transfected three of the above control PCR-based mutagenesis products and picked 11–12 random plaques from each , yielding 36 and 33 plaques in total for ΦX174 and Qβ , respectively . The relative fitness of each clone was determined as above and we obtained the mean and variance for each phage . These values were subtracted from the means and variances obtained for the real mutants . This allowed us to account for experimental error and , in particular , to control for any potential mutations present in the DNA templates , arising during PCR amplification , or during plaque growth . To measure growth rates , ∼104 pfu were inoculated into 0 . 5 mL of LB medium containing IJ1862 cells at their exponential growth phase . Infected cultures were incubated in agitation ( 650 rpm ) at 37°C in a Thermomixer 24-tube shaker and harvested when the wild-type reached an estimated titer of 108 pfu/mL . Cells were removed by centrifugation and the supernatants were aliquoted , stored at −70°C and titrated . The growth rate ( r ) was calculated as the increase in log-titer per hour . Relative fitness was obtained as W = ri/r0 , where i and 0 refer to the mutant and wild-type , respectively ( notice that this fitness measure is in log-scale ) , and the fitness effect ( selection coefficient ) was calculated as s = W−1 . In all cases , mutant and wild-type samples were assayed in the same experimental block , and experimental blocks were done in triplicate . Sequences were obtained using the Sanger method ( Applied Biosystems ) . In general , we sequenced column-purified plaque- ( RT ) -PCRs directly . In cases where this yielded low-quality readings , we cloned the PCR fragments using the Zero Blunt TOPO PCR Cloning kit ( Invitrogen ) and sequenced the inserts with vector-based and internal primers , discarding mutations that were not present in at least 3/5 clones . For the mutation-accumulation experiment , we used a univariate linear model with two factors: Genetic material ( G , main fixed factor ) and Phage species ( S , random factor nested within G ) , whereas the line was the experimental replicate . The variables analyzed were the number of passages , the number of mutations fixed , and the expected fitness effect per mutation . For each variable v , the model can be written as νijk = μ+Gi+Sij+lijk , where μ is the grand mean and l is the line ( error term ) . To test for differences in average fitness between the ΦX174 and Qβ mutant collections , we used a univariate linear model with three factors: Phage species ( S , main fixed factor ) , Assay type ( A , site-directed mutagenesis or control , fixed factor nested within S ) , and Plaque ( P , each of the mutant or control plaques , random factor nested within A ) . Hence , each experimental determination can be expressed as sijkl = μ+Si+Aij+Pijk+εijkl , where μ is the grand mean , is positive for mutagenesis assays and negative for control assays , and ε is the error term . Non-linear regressions were performed to estimate and V ( sd ) using the Levenberg-Marquardt algorithm implemented in SPSS v12 . | The fitness effects of mutations are the raw material for natural selection . It has been shown that point mutations typically have strongly deleterious effects in plant and animal RNA viruses , whereas cellular organisms are comparatively more robust . Here , we characterize the fitness effects of random mutations in DNA viruses and compare them with those found in RNA viruses , using six phage species of similar genome sizes . To achieve this goal , we introduced mutations by chemical and site-directed mutagenesis , identified the genetic changes by sequencing , and quantified their fitness effects using growth-rate assays . In all cases , mutations had a strong average impact on fitness . We conclude that mutational sensitivity is a general property of viruses with small genomes and discuss the evolutionary implications of these findings . | [
"Abstract",
"Introduction",
"Results",
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"and",
"Methods"
] | [
"genetics",
"and",
"genomics/microbial",
"evolution",
"and",
"genomics",
"evolutionary",
"biology/microbial",
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"genomics",
"microbiology/microbial",
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"genomics",
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"genomics/population",
"genetics"
] | 2009 | The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages |
Burkholderia dolosa is a member of the Burkholderia cepacia complex ( BCC ) , which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis ( CF ) and can be associated with outbreaks carrying high morbidity and mortality . While investigating the genomic diversity of B . dolosa strains collected from an outbreak among CF patients , we previously identified fixL as a gene showing signs of strong positive selection . This gene has homology to fixL of the rhizobial FixL/FixJ two-component system . The goals of this study were to determine the functions of FixLJ and their role in virulence in B . dolosa . We generated a fixLJ deletion mutant and complemented controls in B . dolosa strain AU0158 . Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species . In a murine pneumonia model , the B . dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B . dolosa strain AU0158 at 7 days post infection . Interestingly , the fixLJ deletion mutant made more biofilm , albeit with altered structure , but was less motile than strain AU0158 . Using RNA-seq with in vitro grown bacteria , we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158 . Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant , so we also evaluated virulence of a fliC deletion mutant , which lacks a flagellum . We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158 , suggesting factors other than flagella caused the phenotype of decreased persistence . We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells . In conclusion , B . dolosa fixLJ is a global regulator that controls biofilm formation , motility , intracellular invasion/persistence , and virulence .
Burkholderia dolosa is a member of the Burkholderia cepacia complex ( BCC ) , which is a group of related Gram-negative bacilli that can be dangerous respiratory pathogens for patients with cystic fibrosis ( CF ) [1 , 2] . BCC can also cause outbreaks of bacteremia or respiratory infection in hospitalized non-CF patients , including the recently discovered cluster of cases of BCC respiratory infection linked to contaminated stool softener ( docusate ) [3] . BCC are also common pathogens for individuals with chronic granulomatous disease [4] . Among CF patients in the United States colonized with BCC , the species most commonly seen are B . cenocepacia ( 45% ) , B . multivorans ( 35% ) , B . vietnamiensis ( 6% ) , B . cepacia ( 6% ) , and B . dolosa ( 3% ) , although there is significant variability based on geographic region and institution [5] . A number of studies have shown an association between infection with BCC and clinical pulmonary deterioration of CF patients [6–10] . The so-called “cepacia syndrome” refers to the clinical presentation of fevers , leukocytosis , elevated erythrocyte sedimentation rate ( ESR ) , and progressive severe pneumonia , sometimes with bacteremia , in CF patients occurring relatively soon after acquiring BCC , with a mortality rate initially reported at 62% [11] . BCC infection in CF patients can be transmitted person-to-person , and multiple outbreaks have been described , including one of a highly antibiotic resistant strain of the BCC species B . dolosa among almost 40 CF patients at Boston Children’s Hospital [12] . This outbreak has been associated with accelerated decline of lung function and decreased survival [13] . BCC in general are commonly multidrug resistant ( MDR ) or extensively drug resistant ( XDR ) pathogens , highlighting the necessity for novel approaches for treatment of these bacteria [14] . In previous work , we sequenced the genomes of 112 B . dolosa isolates collected from 14 individuals over 16 years from the Boston Children’s Hospital outbreak [15] . We found that a subset of genes having 3 or more point mutations contained 18 times as many non-synonymous mutations than expected by neutral drift and were thus under strong positive selection ( dN/dS = 18 , 95% CI: 4 . 9–152 . 7 ) [15] . Positive selection here refers to the type of mutations that were seen ( non-synonymous versus synonymous ) , not mutations that necessarily make a protein more active . One of these genes ( BDAG_01161 , AK34_969 ) , which had a remarkable 17 non-synonymous mutations , encodes a protein with homology to FixL of the FixLJ two-component system of Sinorhizobium and Caulobacter . FixL in those species is a sensory histidine kinase that detects oxygen tension and phosphorylates the transcription factor FixJ under low oxygen conditions , which subsequently induces transcription of fixK [16] . The predicted domains of B . dolosa FixL and FixJ ( BDAG_01160 , AK34_968 ) are depicted in Fig 1 . Both proteins have domains consistent with their putative function as a two-component system [17] . FixL is predicted to contain PAS domains and a heme-binding pocket where oxygen binding to FixL likely occurs [16] . Notably , B . dolosa fixK ( BDAG_04180 , AK34_4936 ) has a B . cenocepacia homolog ( BCAM0049 ) that was found to be up-regulated during growth in a low-oxygen environment in two separate studies [18 , 19] . A high number of non-synonymous mutations in the B . multivorans fixL homolog ( BMD20_10585 ) was recently reported in another whole-genome sequencing study of isolates recovered from chronically infected CF-patients [20] , suggesting that the FixLJ system has a similar role in all 3 BCC species commonly seen in CF patients . The FixLJ system in Rhizobium and Caulobacter induce expression of genes required for fixing of atmospheric nitrogen in the absence of oxygen [21] . While B . vietnamiensis and Burkholderia species outside of the BCC can fix nitrogen , most members of the BCC , including B . cenocepacia , B . multivorans , and B . dolosa lack the genes necessary for nitrogen fixation [22–24] . In this study , we sought to determine the functions of B . dolosa FixLJ and their role in pathogenesis . We hypothesized that the FixLJ system senses oxygen depletion and regulates genes involved in virulence based on its function in other species . We show that B . dolosa lacking fixLJ are unable to induce transcription of a fixK-lacZ reporter grown in low oxygen , have altered expression of ~11% of the genome , and are cleared faster in a murine lung infection model . Interestingly , the fixLJ deletion mutant is less motile and less invasive but makes more biofilm than the parental strain . Non-motile B . dolosa lacking flagella show no defect in the murine infection model at comparable dosages , indicating that a reduction in flagella expression is not the mechanism for decreased persistence seen in the fixLJ deletion mutant . Overall , the fixLJ system regulates a large number of genes and is critical for B . dolosa pathogenicity .
To determine the stimulus that induces the fixLJ pathway , we constructed B . dolosa fixK promoter-driven LacZ reporter plasmid using the pSCrhaB2 backbone [26] . B . dolosa fixK ( AK34_4936 ) is the homolog of S . meliloti fixK , which is a target of the fix pathway [16] . We conjugated this plasmid into the BCC strains and measured activation of the pathway when grown in low oxygen ( <5% ) relative to growth in ambient oxygen . There was a ~5 fold induction in the fix pathway ( measured by fixK-driven activity of LacZ ) when grown in low oxygen ( Fig 2 ) . This induction was specific for the B . dolosa fixK promoter sequence as it was lost when using a reporter plasmid containing the S . meliloti fixK promoter sequence to drive LacZ expression ( which served as a negative control ) . The fixLJ deletion mutant was unable to induce fix activity in response to low oxygen . When the fixLJ deletion mutant was complemented chromosomally with fixLJ under the control of its own promoter , induction of fix activity in response to low oxygen was restored , demonstrating that induction of the pathway was fixLJ-specific . We found that the fixLJ deletion mutant has a mild ( ~7 . 5 fold ) growth defect when grown in ambient and low ( <5% ) oxygen relative to strain AU0158 ( S1 Fig ) . The fixLJ DNA sequences in B . dolosa ( strain AU0158 ) , B . cenocepacia ( strains J2315 & K56-2 ) , and B . multivorans ( strain ATCC17616 ) are nearly identical ( 94–95% identity ) , suggesting FixLJ has the same function in all species . We conjugated the B . dolosa fixK reporter plasmid into B . cenocepacia ( strain J2315 ) and B . multivorans ( strain ATCC17616 ) and found the same induction in response to low oxygen , demonstrating this pathway functions similarly in these two more commonly encountered BCC species . To determine the role of the B . dolosa fixLJ system in virulence , we infected C57BL/6 mice with ~4x108 CFU/mouse of B . dolosa strain AU0158 or its fixLJ deletion mutant and measured the bacterial load within the lungs and the spleen at 1 and 7 days post infection . As early as 1 day after infection , there was a significant three-fold reduction in the amount of bacteria recovered from the lungs of mice infected with the fixLJ deletion mutant compared to mice infected with the wild-type strain AU0158 ( Fig 3A ) . At 7 days , there was a 18-fold reduction of viable counts of the fixLJ deletion mutant compared to strain AU0158 ( Fig 3B ) . One day after infection there was no significant difference in the amount of bacteria recovered from the spleens of mice infected with AU0158 or the fixLJ deletion mutant ( Fig 3C ) , suggesting there is no defect in the ability of the fixLJ deletion mutant to disseminate into the spleen . Seven days after infection there was a significant , 16-fold reduction in the number of bacteria recovered from the spleen of mice infected with the fixLJ deletion mutant relative to strain AU0158 ( Fig 3D ) . Mice infected with the fixLJ deletion mutant chromosomally complemented with fixLJ under the control of its own promoter had significantly higher bacterial loads within the lungs ( Fig 3E ) and spleen ( Fig 3F ) at 7 days post infection compared to mice infected in fixLJ deletion mutant chromosomally complemented with an empty vector , demonstrating that this reduction of in vivo fitness is fixLJ-specific . To ensure that the bacteria are causing an infection and not merely colonizing the lungs , we examined histopathology of the lungs at day 7 after infection . Mice infected with either strain AU0158 or the fixLJ deletion mutant showed signs of inflammation consistent with bacterial pneumonia ( S2 Fig ) . Slides were score by a rodent pathologist who saw no difference in severity of lung inflammation between infection groups . To better understand the mechanism of reduced persistence seen in the fix deletion mutant , we compared the phenotype of the fixLJ deletion mutant to the parental strain in the ability to produce biofilm on PVC plates after 48 hours of growth . Interestingly , we found that the B . dolosa fixLJ deletion mutant forms significantly more biofilm than the wild-type strain AU0158 ( Fig 4A ) . This phenotype was quite drastic as we are able to see approximately 3-fold more biofilm in wells that were inoculated with 100-fold fewer fixLJ deletion mutant CFU compared to B . dolosa AU0158 ( 2x105 vs . 2x107 CFU/well ) . Since the fixLJ deletion has the mild growth defect ( S1 Fig ) , the magnitude of the increase in biofilm formation becomes much larger when accounting for bacteria growth ( S3 Fig ) . The fixLJ deletion mutant complemented with fixLJ under the control of its own promoter formed significantly less biofilm than the fixLJ deletion mutant carrying an empty vector ( Fig 4B ) , demonstrating this phenotype is fix-specific . We also chromosomally complemented the fixLJ deletion mutant under the control of a rhamnose-inducible promoter . When grown in the presence of rhamnose , expression of genes controlled by this promoter are induced , while growth in glucose inhibits expression of genes controlled by this promoter [26] . The rhamnose-inducible complementation of fixLJ was able to produce significantly more biofilm when grown in glucose compared to growth in rhamnose ( Fig 4C ) . The fixLJ deletion mutant complemented with the empty vector containing the rhamnose-inducible promoter produced significantly more biofilm in both rhamnose and glucose containing media . Here , we determined biofilm measurements relative to the amount of bacterial growth ( O . D . 600 ) since the addition of glucose/rhamnose had differing effects on bacterial growth . We analyzed the biofilm structure grown on glass slides stained with a live/dead stain by confocal fluorescent microscopy . After 48 hours of growth , strain AU0158 had drastically fewer bacteria adherent to the slide , and those that were adherent were in dome-like structures ( Fig 4D ) . The dome-like structures of strain AU0158 had heights of 10–15 μm ( Fig 4D , inset ) , while the fixLJ deletion mutant adhered to the glass evenly across the entire surface in a nearly single-cell layer 2–3 μm high ( Fig 4E ) . To better understand the mechanism of biofilm formation and the components of biofilms in strain AU0158 and the fixLJ deletion mutant , we treated established biofilm with DNase I , proteinase K , or dispersin B ( which cleaves the biofilm polysaccharide PNAG ) [27] . We found that strain AU0158 biofilms were susceptible to treatment with any of the three enzymes while biofilms formed by the fixLJ deletion mutant were only susceptible to treatment with proteinase K ( S3C Fig ) . Since swimming motility has been correlated with virulence in B . cenocepacia [28] , we next assessed the impact of fixLJ deletion on swimming motility . We plated overnight cultures of various B . dolosa constructs onto low density ( 0 . 3% ) agar plates and measured the swimming diameter after 48 hours . The fixLJ deletion mutant had significantly lower swimming motility compared to AU0158 ( Fig 5A ) . Full motility was restored in the fixLJ deletion mutant complemented with fixLJ under the control of its own promoter . Using the above-described rhamnose-inducible system to complement the fixLJ deletion , we also found that motility was restored when bacteria were grown in the presence of rhamnose but not glucose ( Fig 5B ) . In order to determine the mechanism ( s ) that are responsible for the decreased persistence in vivo , increased biofilm , and decreased motility seen in the fixLJ deletion mutant , we compared the transcriptomes of the fixLJ deletion mutant to the parental strain AU0158 using RNA-seq . Differentially expressed genes were identified to have at least a 2-fold change ( log2 >1 ) and a q value <0 . 05 ( Fig 6 ) . Of the 5717 genes in the annotated genome of strain AU0158 , 300 genes were significantly up- regulated ( denoted as green ) and 285 genes were significantly down-regulated ( denoted as red ) , in the fixLJ deletion mutant relative to AU0158 . A sampling of selected genes meeting these criteria is shown in Table 1 , and the full list of these genes is in S1 Table . Genes appearing in Table 1 were chosen for their magnitude of change and/or potential role in virulence based on roles in related bacteria . We confirmed differential expression of a subset of these genes using qRT-PCR ( S4 Fig ) . In those experiments , we also confirmed that the differential expression could be complemented in the fixLJ deletion mutant carrying fixLJ compared to the empty vector ( S4 Fig ) . We found that fixK was significantly down-regulated in fixLJ deletion mutant compared to the parental AU0158 by qRT-PCR ( S4 Fig ) . We found that fliC ( BDAG_00084 , AK34_2913 ) expression was significantly down-regulated in the fixLJ deletion mutant ( S4B Fig ) , which correlated with the reduction in motility seen in Fig 5 . We also found that the master regulator of flagella assembly , flhD ( AK34_2903 ) , was expressed significantly lower in the fixLJ deletion mutant compared to the wild-type strain AU0158 . Components of a putative type IVa pilus ( AK34_2364- AK34_2368 ) were down-regulated in the fixLJ deletion mutant ( Table 1 ) . In contrast , multiple subunits of a putative type IVb pilus ( AK34_1641- AK34_1653 ) were actually up-regulated in the fixLJ deletion mutant relative to the wild-type strain AU0158 , and this was confirmed for the flp gene in the qRT-PCR experiments ( S4C Fig ) . A putative type III secretion system ( AK34_3647- AK34_3658 ) was also up-regulated in fixLJ deletion mutant ( Table 1 ) . Notably , several transcription regulators were significantly up- or down-regulated indicating that the fix regulon is complex . Since other groups have found that flagella and motility are associated with virulence in B . cenocepacia [28 , 29] and we found decreased motility ( Fig 5 ) and decreased expression of flagella-associated genes ( Table 1 and S4 Fig ) in the fixLJ deletion mutant , we tested the hypothesis that the decreased expression of the flagella in the fixLJ deletion mutant is a major mechanism for the reduction of in vivo persistence seen after lung infection with this mutant ( Fig 3 ) . To do this , we generated a fliC deletion mutant in B . dolosa strain AU0158 background . As expected this mutant was completely non-motile when plated on low-density LB agar plates ( Fig 7A ) . We also saw no difference in the ability of the fliC deletion mutant to form biofilm on PVC plates after 48 hours compared to strain AU0158 ( Fig 7B ) . We next infected C57BL/6 mice with ~5x108 CFU/mouse of strain AU0158 or its fliC deletion mutant via the intranasal route and examined bacterial loads in the lungs and spleen 7 days after infection . There was no statistically significant difference in levels of viable bacteria in the lungs ( Fig 7C ) or the spleen ( Fig 7D ) . Since we observed multiple pili systems and flagella associated genes to be differentially expressed in the fixLJ deletion mutant in our RNA-seq experiments ( Table 1 , S1 Fig , S1 Table ) we next determined the ability of the fixLJ deletion mutant to invade and persist within A549 lung epithelial cells and THP-1-derived macrophages . Multiple pili systems and flagellum have been described to be involved in invasion in BCC and other ands Gram-negative bacteria [29–32] . We infected A549 cells or THP-1-derived macrophages with B . dolosa ( M . O . I . of ~10:1 ) for two hours , after which extracellular bacteria were killed by the addition of kanamycin . There was ~2-fold reduction in the amount of fixLJ deletion mutant bacteria within A549 lung epithelia cells compared to the parental strain , AU0158 ( Fig 8A ) . We also found that the fixLJ deletion mutant has ~4 fold reduction compared to parental AU0158 in the ability to survive within THP-1 cells that had been previously treated with PMA to induce differentiation into macrophage-like cells ( Fig 8B ) . Similar to the fixLJ deletion mutant , we found a ~2-fold reduction in invasiveness of the fliC deletion mutant compared to strain AU0158 in A549 lung epithelial cells ( Fig 8A ) . In contrast , when THP-1 derived macrophages were infected with fliC deletion mutant , we saw an increase in the number of intracellular bacteria relative to strain AU0158 ( Fig 8B ) . To understand the mechanism of decreased bacterial loads in THP-1 derived macrophages infected with the fixLJ deletion mutant , we varied the amount of time the bacteria had to invade before kanamycin treatment or the time of kanamycin treatment . When infected THP-1 derived macrophages were treated with kanamycin for 24 hours to kill extracellular bacteria , there was a 30-fold reduction in the number bacteria inside fixLJ deletion mutant-infected cells compared to AU0158-infected cells ( Fig 8C ) . To determine if the fixLJ deletion mutant is less able to become internalized than strain AU0158 , we varied the length of infection time before the addition of kanamycin . We found that both the fixLJ deletion mutant and the parental AU0158 could equally become internalized at the early time points ( 15 & 30 min , Fig 8D ) and there was no significant difference until 2 hours ( Fig 8D ) . When we did the complementary experiment to measure the ability of the bacteria to replicate intracellularly by varying the amount of time of kanamycin exposure after a 2 hour infection period , we found there was a significant ~2-fold increase in the number of intracellular bacteria at 3 and 4 hours post infection in THP-1 derived macrophages infected with strain AU0158 relative to 1 hour post infect that was not seen in macrophages infected with the fixLJ deletion mutant ( Fig 8E ) . Across all time points , there were significantly greater numbers of bacteria in strain AU0158-infected cells compared to cells infected with the fixLJ deletion mutant ( Fig 8E ) . To address the possibility of the B . dolosa constructs causing differing amounts of cytotoxicity , we measured the cytotoxic effects of the live bacteria on A549 and THP-1 derived macrophages . We saw minimal amounts of LDH release above unstimulated cells and found no statistically significant difference in cells infected with strain AU0158 versus the fixLJ deletion mutant ( ΔfixLJ ) , ΔfixLJ + fixLJ , ΔfixLJ + empty vector ( EV ) , or strain AU0158 vs . the fliC deletion mutant in LDH release after 6 hours of exposure ( S5 Fig ) .
A recent study using whole-genome sequencing of multiple strains from an outbreak of B . dolosa in CF patients at Boston Children’s Hospital discovered that the fixLJ genes show evidence of strong positive selection . These findings were recently corroborated to another BCC species , B . multivorans [20] . In the current study , we report the functional significance of the fixLJ genes in B . dolosa , finding not only that the pathway is induced by low oxygen , but also that it regulates a large number of genes and is critical for pathogenicity in vivo and intracellular invasion in vitro . Our findings that the B . dolosa fix pathway is activated by low oxygen fit well with prior reports that the B . cenocepacia homolog of fixK ( BCAM0049 ) was up-regulated during growth in a low-oxygen environment [18 , 19] . FixL homologs in other pathogenic Gram negative bacteria include one in Brucella , which was found to be necessary for intracellular survival [33] . Pseudomonas aeruginosa has a FixL homolog called BfiS ( biofilm initiation sensor ) along with a FixJ homolog , BfiR , which , as their names suggest , have been shown to be critical for the irreversible attachment phase of biofilm development [34 , 35] . The environmental signal that activates BfiS has not yet been elucidated . One recent study using Tn-seq found that BfiS and BfiR were critical for virulence in a murine model of GI tract colonization as fitness was 24- or 60-fold reduced compared to in vitro grown bacteria , respectively , and BfiS and BfiR transposon mutants were unable to disseminate after these colonized mice were made neutropenic [36] . We found that the fixLJ deletion mutant was less able to persist within the murine model ( Fig 2 ) . At 1 day after infection , we saw lower numbers of bacteria within the lungs but similar levels of in the spleens of mice infected with the fixLJ deletion mutant , demonstrating that there was no defect in the ability of the fixLJ deletion mutant to disseminate . Dissemination from the lungs to the spleen could occur via an intracellular or an extracellular mechanism . In Fig 8 , we show that the fixLJ deletion mutant was less able to survive within THP-1 derived macrophages , which suggests that fixLJ deletion mutant will be less able to disseminate via an intracellular mechanism . These findings suggest that B . dolosa dissemination occurs via an extracellular mechanism in this model . Lung histology showed evidence of significant inflammation , consistent with the bacteria causing an infection opposed to colonization ( S2 Fig ) . There was no difference in lung pathology induced by strain AU0158 versus the fixLJ deletion mutant , but this is likely related to the relatively high bacterial load seen in both infection groups . Interestingly the B . dolosa fixLJ deletion mutant made significantly more biofilm than the parental strain , which is the opposite of what was seen when the P . aeruginosa bfiSR pathway was inactivated [34] . Confocal microscopy images were consistent with the crystal violet staining , where the fixLJ deletion mutant produced more biofilm likely due to the increased number of bacteria attached to the glass ( Fig 4 ) . These results are interesting because other groups have shown that non-motile P . aeruginosa and BCC are less likely to form biofilms [37 , 38] , although non-motile BCC seem to be able to produce similar amounts of biofilm compared motile wild-type strains if given enough time [38] . The clinical relevance of BCC biofilm production is unclear . Indeed , one study found no correlation between the ability of BCC isolates to form biofilm and clinical outcomes [39] . In another study that examined P . aeruginosa and/or BCC infected CF lung tissue removed after transplant using species-specific antibodies to stain the lung tissue , BCC bacteria were rarely found in biofilm-like structures while P . aeruginosa were often found in such structures [40] . These reports along with our findings suggest that biofilm production may not be beneficial for BCC infection . Using RNA-seq , we found that ~11% of the B . dolosa genome was differentially expressed within the fixLJ deletion mutant , demonstrating the size of the FixLJ regulon . Multiple components of the flagella assembly pathway were found to be down-regulated in the fixLJ deletion mutant . To evaluate the reduction in flagella expression as the mechanism of reduced persistence in vivo seen in the fixLJ deletion mutant , we also tested a fliC deletion mutant . We found that there was no reduction in the ability of the fliC deletion mutant to persist in the murine lung or spleen , demonstrating that flagella are not required for B . dolosa virulence in our model . This was a surprising result as other groups have found that BCC lacking flagella ( or otherwise non-motile ) are less invasive , less able to form biofilms , and less lethal in murine models [28 , 29 , 41] . This finding suggests that the virulence factors and mechanism of infection of B . dolosa are in some respects different from other members of the BCC . The ability of BCC to invade and/or survive within epithelial cells and macrophages is thought to represent an important aspect of BCC pathogenesis [42–44] . We found that the B . dolosa fixLJ deletion mutant was less invasive towards A549 epithelial cells and THP-1 derived macrophages ( Fig 8 ) . It is possible that the fixLJ deletion mutant is either less able to invade the cell or less able to survive within the cell , or both . We found that the fixLJ deletion mutant could equally become internalized as the parental AU0158 at early time points while the fixLJ deletion mutant was unable to replicate intracellularly . The decreased numbers of the fixLJ deletion mutant seen at the late time points in Fig 8D is likely due to decreased ability of the fixLJ deletion mutant to survive and/or replicate intracellularly . These findings suggest that lower intracellular survival seen in the fixLJ deletion mutant is responsible for the majority of the decrease in invasiveness seen in Fig 8A and 8B . We hypothesize that the fixLJ deletion mutant is unable regulate appropriate gene expression to survive within the intracellular environment since it is unable to detect the lower oxygen concentration found there . Compared to strain AU0158 , the fliC deletion mutant was less able to invade/survive within A549 cells , but it was better able to survive within THP-1 derived macrophages . Both cell types were centrifuged after addition of bacteria , eliminating the possibility of the fliC deletion mutant being unable to swim to the human cell . Mohr and co-workers also reported that B . cepacia flagella deletion mutants were less able to invade A549 cells [29] . Our finding that the B . dolosa fliC deletion mutant had an increased number of intracellular bacteria in THP-1 derived macrophages likely relates to the absence of TLR-5 mediated activation of macrophages by flagella in the fliC deletion mutant . Our data overall suggest that the ability of B . dolosa to survive within THP-1 derived macrophages is a better surrogate than survival within A549 epithelial cells for in vivo fitness since results with the THP-1 derived macrophages matched well with the murine model ( fixLJ deletion mutant attenuated but fliC deletion mutant not attenuated ) . Our RNA-seq data suggest other pathways that might explain the phenotypes seen in the fixLJ mutant . We found genes encoding a putative type IVa pilus that is downregulated in the fixLJ deletion mutant . A similar type IVa pilus in Burkholderia pseudomallei has been described to bind to human epithelial cells and be required for virulence in nematode and murine models [30] . Other studies have found the homolog of the pilin ( pilE ) that was down-regulated in the fixLJ deletion mutant to be critical for attachment of pathogenic Neisseria species to human cells [31 , 32] . We also identified genes encoding a putative type IVb pilus that is upregulated in the fixLJ deletion mutant . Type IVb pili have been described to be involved in tight adhesion to substrates and biofilm formation [45] , so this pathway might explain the high biofilm production of the fixLJ deletion mutant . Consistent with this hypothesis , we found that the fixLJ deletion mutant biofilm was susceptible to proteinase K treatment , suggesting dependency on proteins such as the flp pilus for biofilm formation . Further supporting this hypothesis , we found that the fixLJ deletion mutant was able to adhere to the entirety of the glass surface , which is perhaps due to the increased expression of the flp pilus in the fixLJ deletion mutant compared to strain AU0158 , which primarily formed biofilms with dome-like structures ( Fig 4 ) . Interestingly , homologs of the up-regulated component of the type IVb pilus ( flp subunit ) were found to be down-regulated in vivo in a chronic B . cenocepacia infection model in rats [46] , suggesting that this pilus is detrimental to in vivo fitness during long-term infections . We also saw that the RNA-binding protein Hfq was upregulated in the fixLJ deletion mutant , suggesting a role for sRNA in modulation of the fix regulon . We also found a large number of regulatory proteins , suggesting the fixLJ regulon is complex and composed of elements that are both directly and indirectly regulated by fixLJ . We also used the RNA-seq data to examine pathways that have been identified in other BCC to mediate the phenotypes we observed . We found 2 sigma factors , rpoN ( AK34_313 ) and rpoE ( AK34_2044 ) , which were 1 . 9 and 2 . 3 , respectively upregulated in the fixLJ deletion mutant relative to AU0158 . Both of these sigma factors were found to be critical for B . cenocepacia survival within macrophages , and rpoN was also critical for biofilm formation [41 , 47] . In Table 1 we identify a putative T3SS that was upregulated in the fixLJ deletion mutant relative to AU0158 . Tomich et al . found that B . cenocepacia T3SS mutants are less fit a murine model [48] . In both cases we would have expected the sigma factors and T3SS to be downregulated in the fixLJ deletion mutant if they were responsible for the phenotypes observed . The fact that these previously published findings are not consistent with our findings demonstrates that these phenotypes are complex , with multiple factors involved , and warrant further investigation . BCC produce an exopolysaccharide , cepacian , that is required for mature biofilm formation [39] . We saw no differential expression in any of the genes involved in the production of cepacian [49] suggesting that other mechanisms such as the flp pilus are responsible for the increased biofilm production . Whole-genome sequencing of isolates from an outbreak of B . dolosa led to the identification of the fixLJ pathway being under strong positive selection during chronic infection [15 , 50] . In this study , we suggest that the B . dolosa FixLJ system is an oxygen-sensing mechanism that regulates biofilm formation , motility , intracellular invasion , and virulence . It appears that fixLJ is required for in vivo persistence by limiting biofilm formation and allowing for survival within macrophages , which is known to be a low-oxygen environment . Future studies will better characterize the FixLJ pathway and identify individual components that are required for in vivo persistence .
All animal protocols and procedures were approved by the Boston Children’s Hospital Institutional Animal Care and Use Committee ( assurance number A3303-01 ) . The specific protocol number is 15-02-2889 . All animal protocols are complement with NIH Office of Laboratory Animal Welfare , Guide for the Care and Use of Laboratory Animals , The US Animal Welfare Act , and PHS Policy on Humane Care and Use of Laboratory Animals . All strains used and generated in this study are listed in Table 2 . B . dolosa strain AU0158 was obtained from John LiPuma ( University of Michigan ) and is an early isolate from the index patient from the B . dolosa outbreak ( about 3 years into the outbreak ) . BCC and E . coli were grown on LB plates or in LB medium and supplemented with following additives: ampicillin ( 100 μg/mL ) , kanamycin ( 50 μg/mL for E . coli , 1 mg/mL for BCC ) , trimethoprim ( 100 μg/mL for E . coli , 1 mg/mL for BCC ) , gentamicin ( 15 or 50 μg/mL ) , chloramphenicol ( 20 μg/mL ) , or diaminopimelic acid ( 200 μg/mL ) . Plasmids that were used in this study are listed in Table 3 . Human lung epithelial cells A459 and human monocyte line THP-1 were obtained from ATCC . Both cell lines were grown at 37°C with 5% CO2 . A459 cells were grown in RPMI 1640 with L-glutamine and 10% heat-inactivated fetal calf serum ( FCS , Gibco ) . Penicillin and streptomycin were added for routine culture but were removed the day before and during experiments . THP-1 cells were cultured in RPMI-1640 medium containing 2 mM L-glutamine , 10 mM HEPES , 1 mM sodium pyruvate , 4500 mg/L glucose , and 1500 mg/L sodium bicarbonate , supplemented with 10% heat-inactivated FCS and 0 . 05 mM 2-mercaptoethanol . Human THP-1 monocytes were differentiated into macrophages by seeding 1 mL into 24 well plates at 8x105 cells/mL with 200 nM phorbol 12-myristate 13-acetate ( PMA ) and incubated for 72 hours then washed two times with media lacking PMA . Low oxygen environments were generated by the CampyGen Gas Generating System ( Thermo-Fisher ) , and the low-oxygen concentration ( <5% ) is based on the manufacture specifications . An in-frame , clean deletion of fixLJ in B . dolosa AU0158 was generated using the suicide plasmid pEXKm5 [51] . Briefly , 1 . 4 kbp and 1 . 1 kbp flanking upstream and downstream , respectively , were PCR amplified to introduce SmaI sites on the 5’ and 3’ ends of the upstream and downstream fragments , respectively , and 16 bp overlap between the 3’ end of the upstream fragment and the 5’ end of the downstream fragment . The two fragments were joined using the NEBuilder HiFi DNA Assembly Master Mix ( NEB ) per manufacturer’s protocol and sub-cloned into pGEM-T ( Promega ) . The fragment was digested with SmaI and cloned into pEXKm5 at the SmaI site and was named pFIXKO . pFIXKO was Sanger sequenced and transformed into RHO3 E . coli and then conjugated into B . dolosa AU0158 along with the conjugation helper plasmid pRK2013 [54 , 55] . Conjugants were selected for on LB with kanamycin at 1 mg/mL . Insertion of the plasmid was confirmed by PCR . To resolve merodiploidy , conjugants were counterselected against by plating on LB with 15% w/v sucrose and incubated for 2 days at 30°C . Resolution and confirmation of fixLJ deletion was confirmed by PCR and Sanger sequencing . To complement the fixLJ deletion mutant , we generated stable chromosomally integrated constructs using the mini-Tn7 system , which integrates into an attTn7 site [52 , 55] . B . dolosa strain AU0158 fixLJ along with 670 bp upstream was amplified using PCR with primers that generated NsiL and KpnI sites at the 5’ and 3’ ends respectively . This fragment was cloned into pUC18T-mini-Tn7T-Tp at the NsiL and KpnI sites , generating pfixLJ . A rhamnose-inducible FixLJ expression construct was generated by replacing the arabinose operator of pTJ-1 with rhamnose operator of pSCrhaB2 using NEBuilder HiFi DNA Assembly Master Mix ( NEB ) per manufacturer’s protocol and primers generated using NEBuilder Assembly tool , generating pTJrha . B . dolosa AU0158 fixLJ was amplified by PCR with primers that generated NcoI and HindIII sites at the 5’ and 3’ ends respectively . This fragment was cloned into pTJrha at the NcoI and HindIII sites generating the plasmid pFixLJrha . All insertions were verified by PCR and Sanger sequencing . The fixLJ complementation vectors and the corresponding empty vector controls were conjugated into the fixLJ deletion mutant with pRK2013 and pTNS3 using published procedures [54 , 55] . Conjugants were selected for by plating on LB agar containing trimethoprim ( 1 mg/mL ) and gentamicin ( 50 μg/mL ) . Insertions into the attTn7 site downstream of BDAG_4221 was confirmed by PCR . To generate the fixK-lacZ reporter plasmid the first 23 bp of fixK and the immediate 243 bp upstream of the start codon were PCR amplified to generate a HindIII and KpnI sites at the 5’ and 3’ ends respectively . The fragment was subcloned into the HindIII and KpnI sites of S . meliloti PfixK-lacZ reporter plasmid [21] generating an in-frame fusion of PfixK with lacZ . The S . meliloti and B . dolosa PfixK-lacZ fusion was removed by digestion with HindIII and BamHI , and cloned into pSCrhaB2 at the HindIII and BamHI sites which resulted in pSmfixK-reporter and pfixK-reporter , respectively . The insertion was verified by PCR and occurred in the opposite orientation of the rhamnose operator , which is in pSCrhaB2 . For use in some experiments the trimethoprim resistance gene of pfixK-reporter in the pSCrhaB2 backbone was replaced with the kanamycin resistance gene of pEXKm5 using NEBuilder HiFi DNA Assembly Master Mix ( NEB ) per manufacturer’s protocol and primers generated using NEBuilder Assembly tool generating pfixk-reporterKm . The pfixK-reporter and pSmfixk-reporter were conjugated into B . dolosa AU0158 , B . cenocepacia J2315 , and B . multivorans ATCC 17616 in triparental matings with pRK2013 [54 , 55] . Conjugants were selected for by plating on LB agar containing trimethoprim ( 1 mg/mL ) and gentamicin ( 50 μg/mL ) . Conjugants were maintained in LB containing trimethoprim ( 1 mg/mL ) . An in-frame , clean deletion mutation in the B . dolosa fliC gene was generated using similar methods . Briefly , 700 bp upstream and 700 bp downstream of BDAG_00084 was amplified by PCR . These templates were used in a second PCR as templates to generate fused amplicons . These amplicons were restricted and ligated into in the XmaI and EcoRI sites of pEXKm5 , generating pEXKm5-fliCdel . For use in B . dolosa we cloned in the tet gene from miniCTX plasmid ligated into the SpeI sites of pEXKm5-fliCdel . Sm10λ were transformed with pEXKm5Tet-fliCdel and then transferred into B . dolosa by conjugation , and complemented isolates were counterselected on LB agar supplemented with kanamycin ( 50 μg/ml ) + tetracycline ( 75 μg/ml ) + ampicillin ( 100 μg/ml ) . Insertion of the plasmid into the chromosome was verified by PCR . Counter-selection , to resolve merodiploidy was performed by plating the transconjugants on LB medium + 10% sucrose . Resolution and confirmation of fliC deletion was confirmed by PCR and Sanger sequencing . Burkholderia carrying a fixK-lacZ report plasmid were grown overnight in LB with kanamycin or trimethoprim ( 1 mg/mL ) . Cultures were subcultured in LB in ambient air or LB that had been degassed in CampyGen Gas Generating System ( Thermo-Fisher ) . Cultures were grown in ambient oxygen with shaking ( 200 rpm ) at 37°C or within CampyGen Gas Generating System at 37°C for 4–6 hours . The level of fix-driven LacZ activity was measured by determining Miller Units following published procedures [21] . Female C57BL/6 mice 6–8 weeks of age were obtained from Taconic Biosciences . Mice were maintained at the animal facilities at Boston Children’s Hospital . Mice were anesthetized with ketamine ( 100 mg/kg ) and xylazine ( 13 . 3 mg/kg ) given intraperitoneally . While the mice were held in dorsal recumbency , 10 μL of inoculum was instilled in each nostril ( 20 μL total ) . The inoculum consisted of log-grown B . dolosa washed in PBS and diluted to a concentration of ~2x1010 CFU/mL ( 4x108 CFU/mouse ) . Mice were euthanized 1 or 7 days after infection by CO2 overdose , when lungs and spleen were aseptically removed . Lungs and spleens were weighed and placed into 1 mL protease peptone , homogenized , and then serially diluted and plated on Oxidation/Fermentation-Polymyxin-Bacitracin-Lactose ( OFPBL ) plates . At 7 days after infection , mice infected in parallel were euthanized by CO2 overdose; 1 mL 1% paraformaldehyde ( Boston BioProducts ) was instilled into the lungs for 5 min . Lungs were removed and fixed in 1% paraformaldehyde for 2 hours . Lungs were stored in 70% ethanol until paraffin embedded and 5 micron sections were made and hematoxylin and eosin stained . A rodent pathologist scored the slides for degree of lung inflammation . The ability to form biofilm on PVC plates was determined using published methods [56] . Briefly overnight cultures were diluted in TSB with 1% glucose and pipetted into wells of a 96-well PVC plate . Plates were incubated 48 hours at 37°C , when unattached bacteria were washed with DI water . Biofilms were stained with 0 . 5% crystal violet and excess stain was washed away , and stain was solubilized with 33% acetic acid . The solution was transferred to a flat bottom plate , and then staining ( biofilm amount ) was quantified by measuring O . D . 540 . To measure biofilm dispersal , 48 hour biofilms were washed with DI water and treated with 120 u/mL DNase I , 3 . 18 mAu/mL proteinase K , or 50 μg/mL dispersin B for 24 hours at 37°C . Biofilm was then measured as described above . Overnight cultures of strain AU0158 or its fixLJ deletion mutant were diluted 1:100 in TSB with 1% glucose and 200 μL was pipetted into 4 wells of an 8-well glass chamber slide ( Lab-Tek ) and incubated for 48 hours at 37°C . Non-adherent bacteria were removed by two washes with PBS . Bacteria were stained with Live/Dead BacLight Bacterial Viability Kit ( Molecular Probes ) using STYO 9 and propidium iodide per the manufacturer’s protocol . Stained slides were washed with PBS twice and fixed with 4% paraformaldehyde for 15 minutes at room temperature and then visualized Zeiss Axiovert Spinning Disk Confocal Microscope . Mosaic images consisting of 16 fields of view and 1 μm Z-stack images were acquired analyzed using Slidebook ( 3i , Intelligent Imaging Innovations Inc . ) . The ability B . dolosa to swim was measured in low density LB agar using published methods [57] . Briefly , 10 μL of overnight B . dolosa culture was plated in the center of low density ( 0 . 3% agar ) LB plate . Plates were incubated agar side down for 48 hours at 37°C when swimming diameter was measured . RNA was isolated from log phase B . dolosa ( two biological replicates for AU0158 and three biological replicates for AU0158 fixLJ deletion mutant ) using the Ribopure Bacterial RNA Purification Kit ( Ambion ) per manufacturer’s protocol , contaminant DNA was removed using provided DNase . RNA was cleaned using Trizol Reagent ( Fisher Scientific ) per manufacturer’s protocol . rRNA was depleted from 10 μg of total RNA using the MICROBExpress Bacterial mRNA Enrichment Kit ( Ambion ) and then the RNA was fragmented using NEBNext Magnesium RNA Fragmentation Module ( NEB ) , both per manufacturer’s protocols . The NEBNext Multiplex Small RNA Library Prep Set for Illumina was used to prepare the library with each replicate labeled with a unique barcode enabling multiplexing . Samples were sequenced using single end 50 bp reads using the Illumina HiSeq . Data analysis occurred within in the Galaxy platform ( usegalaxy . org ) [58] . Reads ( 9–12 million reads per replicate ) were trimmed using the Trimmomatic tool [59] and mapped to the B . dolosa AU0158 genome ( GenBank assembly accession GCA_000959505 . 1 ) [60] using BowTie2 using very sensitive local preset settings [61] . Differentially expressed genes were identified using CuffDiff using Benjamini–Hochberg procedure to determine the q value ( p value corrected for multiple comparisons ) [62] . The ability of B . dolosa to invade and persist within human epithelial cells and macrophages was determined using published protocols [42 , 63 , 64] . A549 cells were grown to confluence in 24-wells plates . Human THP-1 monocytes were differentiated into macrophages by 72 hour PMA treatment . A549 or THP-1 derived macrophages were infected with log-phase grown B . dolosa washed in RPMI three times at ~2x106 CFU/well ( MOI of ~10:1 ) . Plates were spun at 500 g for 5 minutes to synchronize infection and then incubated 15 minutes -2 hours at 37°C with 5% CO2 . To determine the total number of bacteria , wells were treated with 100 μL of 10% Triton-X100 lysis buffer ( final concentration 1% Triton-X100 ) , serially diluted , and plated to enumerate the number of bacteria . To determine the number of intracellular bacteria , separate infected wells were washed two times with PBS and then incubated with RPMI + 10% heat-inactivated FCS with kanamycin ( 1 mg/mL ) for 1–24 hours . Monolayers were washed three times with PBS and lysed with 1% Triton-X100 , serially diluted , and plated to enumerate the number of bacteria . cDNA was synthesized from 2 μg RNA using the ProtoScript II First Strand cDNA Synthesis Kit ( NEB ) per manufacture’s protocol . cDNA was cleaned using QIAquick PCR Purification Kit ( Qiagen ) . Genes were amplified using oligos listed in S2 Table and FastStart Essential DNA Green Master Mix ( Roche ) per manufacturer’s protocol . Expression was determined relative to AU0158 normalized by gyrB ( AK34_3072 ) or rpoD ( AK34_4533 ) expression using the ΔΔCt method [65] . Both gyrB and rpoD had similar expression by RNA-seq between AU0158 and the fixLJ deletion mutant , and these genes have been used to normalize expression in B . cenocepacia in other studies [49 , 66] A549 cells were grown to confluence in 24 wells plates . Human THP-1 monocytes were differentiated into macrophages by 72 hour PMA treatment . A549 or THP-1 derived macrophages were infected with B . dolosa washed in RPMI three times at ~1x107 CFU/well ( M . O . I . of ~100:1 ) . Plates were spun 500 g for 5 minutes to synchronize infection and incubated 6 hours at 37°C with 5% CO2 . In other experiments , THP-1 derived macrophages were infected with B . dolosa washed in RPMI three times at ~1x106 CFU/well ( M . O . I . of ~10:1 ) . Plates were spun 500 g for 5 minutes to synchronize infection and incubated 2 hours at 37°C with 5% CO2 . Plates were washed two times with PBS and then incubated with RPMI + 10% heat-inactivated FCS with kanamycin ( 1 mg/mL ) for 24 hours . All plates were spun 500 x g for 5 minutes and supernatants were removed and froze at -80°C . LDH release was measured within supernatants using CytoTox 96 Non-Radioactive Cytotoxicity Assay ( Promega ) per manufacture’s protocol . For a positive control untreated wells were treated with 10X lysis buffer ( provided with kit ) during the last 30 minutes of incubation . Percent cytoxicity was determined relative to maximum LDH release from cells treated with lysis buffer . | In people with cystic fibrosis ( CF ) , infection with bacteria in the Burkholderia cepacia complex ( BCC ) is often associated with clinical deterioration . In a whole-genome sequencing study of the BCC species B . dolosa , we previously identified the fixL gene of the FixL/FixJ two-component system called FixLJ to be under strong positive selective pressure during chronic infection . In this study we show that low oxygen levels activate FixLJ , and that a mutant of B . dolosa in which the fixLJ genes are deleted is less able to persist in the lungs and spread to the spleen in a lung infection model in mice . The fixLJ deletion mutant has defective motility and intracellular survival within epithelial cells and macrophage cell lines . However , a flagella mutant is fully infectious , suggesting that low motility is not responsible for the fixLJ deletion mutant’s inability to persist within the host . Analysis of global RNA expression shows that the fixLJ system regulates many genes , indicating that multiple pathways likely contribute to the low virulence of the fixLJ deletion mutant . In conclusion , B . dolosa FixLJ compose an oxygen sensor that regulates the ability of the bacteria to survive inside host cells . | [
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] | 2017 | An Oxygen-Sensing Two-Component System in the Burkholderia cepacia Complex Regulates Biofilm, Intracellular Invasion, and Pathogenicity |
Leishmania ( Viannia ) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions . Factors leading to this diversity of clinical presentations are not clear , but parasite factors have lately been recognized as important in determining disease progression . Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection , we evaluated the activity of these enzymes in promastigotes from 23 L . braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease . Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides . Furthermore , we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate . In addition , we found that L . ( V . ) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions . One isolate with high ( PPS6m ) and another with low ( SSF ) ecto-nucleotidase activity were chosen for further studies . Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate . In addition , PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages . Finally , we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages . Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis .
Leishmania is the etiological agent of leishmaniasis , a parasitic disease with diverse clinical manifestations in human beings and other mammals . The parasite presents two main stages in their life cycle: the flagellated mobile promastigote , which multiply in the midgut of the sandfly vector and non-motile amastigotes , obligate intracellular forms that live inside host macrophages . This cell differentiation involves numerous changes and is crucial for Leishmania pathogenicity [1] , [2] . Leishmania braziliensis is the species responsible for the majority of cases of human cutaneous leishmaniasis ( CL ) in Brazil; usually it causes single self-limited cutaneous ulcers at the site of parasite delivery; however , parasites may also metastasize and produce mucosal lesions , usually in mouth , nose , pharynges , and larynges . The mucosal involvement may occur simultaneously to the cutaneous disease ( mucocutaneous form ) or months to years after the spontaneous or treatment-induced healing of the cutaneous lesion ( mucosal form ) . In both situations mucosal involvement is serious but the latter more often leads to destructive mucosal involvement with disfiguring scars [3] . In humans , resistance to infection by L . ( V . ) braziliensis is associated with the early establishment of a type 1 immune response along with the control of exacerbated inflammatory responses [4]–[6] . In murine models of infection , interferon gamma ( IFN-γ ) has been shown to act in synergy with another macrophage derived cytokine , tumor necrosis factor alpha ( TNF-α ) , in activating macrophages to synthesize nitric oxide ( NO ) , a potent microbicidal agent that leads to killing of intracellular parasites [7] , [8] . Factors or mechanisms leading to the diversity of clinical presentations are not well known . Although the involvement of host immune response [4] , [5] , [9]–[12] , and , a limited number of parasite factors also have lately been recognized as important [13] , [14] . Several virulence factors have been associated with the establishment of Leishmania infection , including lipophosphoglycan ( LPG ) , gp63 and other proteases . These factors are involved in the establishment of intracellular parasitism as well as in the inhibition of host immune response [15] , [16] . Components of extracellular ATP metabolism pathway are emerging candidates to determine the virulence of these parasites , since ATP and adenosine ( Ado ) , a product of AMP hydrolysis , are able to influence the immunological response of the host and , in consequence , the parasite establishment [17]–[21] . Due to their incapacity to de novo synthesize purine nucleotides , Leishmania parasites need to obtain extracellular nucleosides to feed the salvation pathway for purine nucleotides synthesis . This is achieved through the action of extracellular enzymes [22] , amongst them , the ecto-nucleoside triphosphate dyphosphohydrolase ( E-NTPDase or apyrase ) which will hydrolyze ATP to ADP and then to AMP and the 5′-nucleoside monophosphate phosphohydrolase ( 5′-nucleotidase or 5′-NT ) , which produces Ado by removing the phosphate group from AMP . Ado is , then internalized via specific transporters [23] . An increase in the levels of extracellular ATP is interpreted by the immune system as a danger signal and triggers an inflammatory response [24] . On the other hand , extracellular Ado , acting on P1 receptors , modulates the inflammatory response by increasing the intracellular cAMP concentration [25] . Previous studies from our group corroborate the hypothesis that enzymes involved in the extracellular metabolism of nucleotides can also act as virulence factors for parasites . In a comparative study with metacyclic promastigotes of Leishmania ( Leishmania ) amazonensis , Leishmania ( Leishmania ) major and L . ( V . ) braziliensis , a potential correlation between enzymatic activity and virulence in vivo was demonstrated [18] . More recently , we demonstrated that long-term culture of L . ( L . ) amazonensis promastigotes results in a decreased ability to hydrolyze nucleotides that is associated with loss of virulence [17] . Since ecto-nucleotidases have a crucial role in metabolism of extracellular nucleotides , which can be correlated to parasitism and the development of infection , we focused our study on the activity of these enzymes in L . ( V . ) braziliensis isolates from patients as a possible parasite-related factor that could influence the clinical presentation of disease . Moreover , we also examined whether ecto-nucleotidases can be correlated with the control of immune response in the infection of C57BL/6J mice and J774-macrophages , as well as the infection and activation of dendritic cells ( DC ) . Our results show that parasites with high ecto-nucleotidase activity are able to modulate the host immune response by inhibiting macrophage microbicidal mechanisms and DC activation . In addition , we observed that promastigotes from the L . ( V . ) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from cutaneous lesions , indicating that differences in the enzymatic activity may influence disease outcome in patients with L . ( V . ) braziliensis infection .
Female C57BL/6J mice ( 4–8 weeks old ) were obtained from the University's animal facility . Animals were given water and food ad libitum . The L . ( Viannia ) braziliensis parasites were obtained from the Oswaldo Cruz Institute Leishmania collection ( Coleção de Leishmania do Instituto Oswaldo Cruz , CLIOC ) , Leishmaniasis Immunobiologic Bank ( Leishbank ) of the Brazilian Mid-West region in the Tropical Pathology and Public Health Institute , Federal University of Goias ( UFG ) and Centro de Referências em Leishmanioses do Centro de Pesquisas René Rachou-Fiocruz ( CRL-CPqRR ) ( Table 1 ) . Clinical forms used in the study were those defined by the cell bank from where the isolates were obtained . Parasites were cultured in Grace's insect medium ( Sigma-Aldrich , St . Louis , MO , USA ) supplemented with 10% heat-inactivated fetal calf serum ( FCS – LGC , Cotia , SP , Brazil ) , 2 mM l-glutamine ( Gibco BRL , Grand Island , NY , USA ) and 100 U/mL penicillin G potassium ( USB Corporation , Cleveland , OH , USA ) , pH 6 . 5 , at 25°C . Parasites were kept in culture for no more than twenty passages . Metacyclic promastigotes were purified by gradient centrifugation of parasites at the stationary phase of culture ( day 5 ) over Ficoll 400 ( Sigma-Aldrich ) , as previously described [18] . The amastigote forms were obtained from J774 infected macrophages as previously described [26] except for the modification of the incubation temperature of the suspension containing the macrophages and the parasites ( 33°C ) . The protocols to which animals were submitted were approved by the Universidade Federal de Ouro Preto Ethical Committee on Animal Experimentation ( OFíCIO CEP N° . 005/2009 ) and followed the guidelines from the Canadian Council on Animal Care . All isolates were obtained from pre-established collections from anonymized samples . IRB approval is not required for the use of the parasite isolates and hence not sought . ATPase , ADPase and 5′-nucleotidase activities were measured by incubation of intact parasites for 1 hr at 30°C in a mixture containing 116 mM NaCl , 5 . 4 mM KCl , 5 . 5 mM D-glucose , 5 mM MgCl2 , and 50 mM Hepes–Tris buffer , in the presence of ATP , ADP or AMP ( Sigma-Aldrich ) 5 mM [27] . The reaction was started by the addition of living stationary phase promastigotes or amastigotes isolated from infected J774 macrophages and terminated by the addition of ice cold HCl 0 . 2 M [28] . Nonspecific hydrolysis was determined by adding the parasites after the reaction was stopped . Parasite suspensions were pelleted and aliquots of supernatant were used for the measurement of released inorganic phosphate ( Pi ) as previously described [29] . Enzymatic activities were expressed as nmol of Pi released by 1 . 0×108 parasites in 1 hr . C57BL/6J mice were inoculated in the left hind footpad with 1 . 0×107 stationary phase promastigotes and lesion development was followed weekly with a dial micrometer ( Serie 7; NO . 7301; Mitutoyo ) . The results were expressed as the difference between measures of infected and contra lateral non-infected footpad [30] . The number of parasites in the footpad was estimated by a limiting dilution assay [30] . Mice were sacrificed and the whole lesion was removed and ground in Grace's insect medium , pH 6 . 5 , in a glass tissue grinder . Tissue debris was removed by centrifugation at 50× g at 4°C/1 min , and supernatant was transferred to another tube and centrifuged at 1540× g at 4°C/15 min . The pellet was resuspended in 0 . 5 mL Grace's insect medium supplemented with 10% heat-inactivated FCS , 2 mM l-glutamine and 100 U/mL penicillin G potassium , pH 6 . 5 . Parasite suspension was , then , serially diluted in duplicates in a final volume of 200 µL in 96-well plates . Pipette tips were replaced for each dilution . Plates were incubated for 15 days at 25°C and examined under an inverted microscope for the presence of parasites . Results were expressed as −log of the parasite titer corresponding to the last dilution in which parasites were detected . Footpad lesions from C57BL/6J mice were harvested , embedded in paraffin and 4 µm-thick sections stained with hematoxylin and eosin ( HE ) and examined under a light microscope . Deparaffined slides were hydrated and incubated with 4% hydrogen peroxide ( 30vv ) in 0 . 01 M Phosphate Buffered Saline ( PBS; pH 7 . 2 ) to block endogenous peroxidase activity , followed by incubation with normal goat serum ( 1∶100 dilution ) to block non-specific immunoglobulin absorption . Heterologous hyperimmune serum from rabbit inoculated with Leishmania infantum extract [31] was diluted 1∶800 with BSA 0 . 1% and employed as the primary antibody . Slides were incubated in a humid chamber at 4°C for 18–22 h , washed with PBS , incubated with biotinylated goat anti-mouse and anti-rabbit Ig ( Dako , Carpinteria , CA , 192 USA; LSAB2 kit ) , washed in PBS , and incubated with streptavidin-peroxidase complex ( Dako; LSAB2 kit ) for 20 min at room temperature . Slides were treated with 0 . 024% diaminobenzidine ( Sigma-Aldrich ) and 0 . 16% hydrogen peroxidase ( 30vv ) , dehydrated , cleared , counterstained with Harris's hematoxylin and mounted with cover slips [31] . The images were captured in a Leica DM5000B microscope with a coupled camera DFC300FX using the program Leica Application Suite ( version 2 . 4 . 0 R1 , Leica Microsystems Ltd . , Heerbrugg , Switzerland ) . J774 cells were plated at 1×106 cells/mL onto round coverslips in Dulbecco's minimal essential medium ( Sigma-Aldrich ) containing 10% FCS , 2 mM l-glutamine , 100 U/mL penicillin G potassium , 25 mM N-2-hydroxiethylpiperazine-N′-2-ethanosulfonic acid ( HEPES; Sigma-Aldrich ) and 50 µM β-mercaptoetanol ( Pharmacia Biotech AB , Uppsala , Sweden ) in 24-well plates . Cells were incubated for 90 min at 37°C , 5% CO2 . Non-adherent cells were removed by washing with warm phosphate-buffered saline ( PBS ) . Promastigotes or amastigotes isolated from infected J774 macrophages were added to the culture at a 5∶1 parasite to cell ratio . After 3 hr co-culture , cells were washed with PBS to remove non-internalized parasites and coverslips were collected to evaluate infectivity . Fresh medium was added to the cultures and the macrophages were stimulated with 10 U/mL IFN-γ and 100 pg/mL lipopolysaccharide ( LPS ) . After 72 hr , coverslips were collected for evaluation of infectivity and supernatants were collected for measurement of nitrite and interleukin-10 ( IL-10 ) production . Coverslips were fixed in methanol for 10 min ( Vetec Fine Chemistry ) , dried and stained using the kit Panótico Rápido ( Laborclin , Pinhais , PR , Brazil ) , according to manufacturer's instructions . The analysis was performed using an Olympus BX50 optical microscope ( Olympus , Center Valley , PA , USA ) . The number of infected and uninfected cells and the number of parasites present in infected cells were determined . A minimum of 200 macrophages per coverslip was examined . Quantification of NO produced by the cells was performed by the indirect Griess method to detect nitrite [32] , and the production of IL-10 levels was evaluated by indirect enzyme-linked immunosorbent assay ( ELISA ) ( PeproTech Inc . , Rock Hill , NJ , USA ) , according to manufacturer's specifications , in 72 hr supernatants . Bone-marrow derived dendritic cell ( BMDC ) were obtained from C57BL/6J bone marrow as previously described [33] . Briefly , bone marrow cells were isolated from the femur and tibia of C57BL/6J mice . The suspension was centrifuged and cells cultured in RPMI-1640 ( Sigma-Aldrich ) supplemented with 10% FCS , 2 mM l-glutamine , 100 U/mL penicillin G potassium , and 50 µM β-mercaptoetanol , pH 7 . 2 . Cells were plated in Petri dishes at a concentration of 3×105 cells/mL , and incubated at 37°C/5% CO2 . Granulocyte-Macrophage Colony-Stimulating Factor ( GM-CSF ) ( R&D Systems , Minneapolis , MN , USA ) was added to each plate on the days 0 , 3 and 6 , at the concentration of 3 ng/mL ( 1050 U/mL ) . Non adherent DC were collected on the 9th day of culture . 5 ( 6 ) -carboxyfluorescein diacetate N-succinimidyl ester ( CFSE ) -labeled metacyclic promastigotes and BMDCs were co-incubated ( 1∶3 cell to parasite ratio ) at 33°C/5% CO2/3 hr . To ensure full activation of these cells , LPS ( Sigma-Aldrich ) at a concentration of 2 µg/mL was then added to the culture and the cells were incubated at 37°C/5% CO2 for up to 17 hr . Infected BMDCs were submitted to analysis by flow cytometry . For flow cytometry analysis , cells at a concentration of 1×107 cells/mL in PBS/1% bovine serum albumin ( BSA ) were submitted to FcγR blocking in the presence of anti-mouse CD16/CD32 ( eBioscience , San Diego , CA , EUA ) . Twenty-five µL of the cell suspension were incubated with a combination of desired antibodies at 4°C/30 min , protected from light . The antibodies used were: anti-mouse CD11c ( HL3 clone – BD Pharmingen , San Diego , CA , EUA ) , anti-mouse MHCII ( M5 114 . 15 . 2 clone ) , anti-mouse CD86 ( GL1 clone – eBioscience ) , and their respective isotype controls . The suspension was centrifuged and the cells were washed in PBS , pH 7 . 2 and resuspended in a solution of 1% paraformaldehyde , 47 . 7 mM sodium cacodylate , and 113 mM NaCl , pH 7 . 2 . The samples were analyzed in BD FACSCalibur™ flow cytometer . Cell acquisition was performed using BD CellQuest™ Pro software . Data analysis was performed using FlowJo software ( Tree Star , Ashland , OR , USA ) . Student's t-test , ANOVA analysis with Bonferroni post-test and Spearman's test were performed using Prism 5 . 0 software ( GraphPad Software , La Jolla , CA , USA ) . p<0 . 05 was considered statistically significant .
Infection by L . ( V . ) braziliensis can cause distinct clinical manifestations , amongst which the development of often mutilating mucosal lesions that may cause permanent impairments to the digestive and respiratory tracts . The reasons for the development of the different clinical manifestations are poorly understood and may involve the host immune response as well as parasite associated virulence factors . Although mucosal leishmaniasis is the most devastating consequence of L . ( V . ) braziliensis infection in humans , consistent correlation between the source of the clinical isolate and the size of lesions developed in mice infected with different isolates of L . ( V . ) braziliensis obtained from cutaneous or mucosal/mucocutaneous lesions has yet not been evaluated . We decided to compare the course of infection in C57BL/6J mice of isolates obtained from mucosal/mucocutaneous lesions to those from cutaneous lesions . Thus , C57BL/6J mice were inoculated with each of the 23 isolates of L . ( V . ) braziliensis ( Table 1 ) and lesion development monitored weekly . As shown in Figure 1 ( A and B ) no consistent differences in the lesion size after 11 weeks of infection were observed between the two groups of isolates . With the exception of a few isolates from each group , which developed small but permanent lesions , mice inoculated with isolates from either group were , in general , able to control infection and resolve the lesion . Comparison of the lesion size after eleven weeks of infection showed no statistical difference between the two groups ( Figure 1C ) . In addition , parasitism at the site of infection was similar in both groups ( Figure 1D ) . Thus , our data demonstrated that the ability to develop severe mucosal lesions in patients does not correlate with the development of larger lesions in the murine model . Several hypotheses have been proposed for the function of ecto-ATPases in trypanosomatids , which include acquisition of Ado from the media , necessary for normal growth , modulation of parasite infection and virulence , and involvement in cellular adhesion [17] , [27] , [34]–[43] . It has been suggested that the ecto-nucleotidase activity of promastigotes correlates with infectivity of Leishmania parasites . This correlation has been observed both among parasites from different species and as well as among isolates from the same species or clones from a single isolate [17] , [18] , [35] . In these studies , however , some of the strains used were isolated from infected sand flies making it impossible to correlate the ecto-nucleotidase activity with clinical features of the infected host . Thus , promastigotes from the 23 L . ( V . ) braziliensis isolates were compared in their ability to hydrolyze adenine nucleotides . Figure 2 shows that promastigotes from these isolates present a large variation in the hydrolysis of ATP , ADP and AMP . Statistical analysis of the ecto-nucleotidase activity showed that some isolates were capable of hydrolyzing significantly more nucleotides than others . In Figure 2 the results are presented in the order of increasing capacity of hydrolysis of nucleotides . It is noteworthy that the promastigotes from isolates that have high activity for ATP hydrolysis also show high hydrolytic activity for ADP and AMP ( Figure 2 ) . Thus , promastigote forms of these parasites are able not only to reduce the concentration of ATP , but also to increase the extracellular Ado concentration . The reasons for the variability in nucleotide hydrolysis are currently unknown , however , the presence of polymorphisms in the ecto-NTPDase ( our unpublished data ) or ecto-5′-nucleotidase genes or differences in the expression of the enzymes on the parasite surface cannot be excluded and will be evaluated in the future . In fact , differences in the activity of ecto-NTPDase and 5′-nucleotidase have been observed in clinical isolates of Trichomonas vaginalis [44] . In addition , mutations in the genes of ecto-NTPDases are known to interfere with the enzyme activity and substrate specificity [45]–[48] . Our results do not show a correlation between the levels of ecto-nucleotidase activity and the ability of parasites to control lesion development ( data not shown ) . However , when observing the course of infection of the different isolates we noted that some of them presented a peak of lesion development at an earlier time point than others ( Figures 1A and B ) . Analysis of lesion development and ecto-nucleotidase activity of the promastigote forms from the 23 isolates demonstrated a positive correlation between the time necessary for the establishment of the peak of lesion development in C57BL/6J and enzymatic activity of the isolate ( Figure 3 ) . This correlation was significant for all three nucleotides analyzed . This result suggests that parasites with greater capacity to hydrolyze ATP , ADP and AMP can control the host immune response at the beginning of the infection , which would favor the multiplication of the parasite . Interestingly , when we segregate the isolates based on the clinical manifestation , we were able to show that isolates from mucosal/mucocutaneous lesions also showed a delayed time for peak of lesion development when compared to isolates from cutaneous lesions ( Figure 4 ) . The correlation between clinical manifestation and delayed lesion development raised the question of whether promastigotes from mucosal/mucutaneous lesions showed different ecto-nucleotidase activity than those from cutaneous lesions . In agreement with the findings above , our data ( Figure 5 ) demonstrates that promastigotes from ML/MCL presented higher ecto-nucleotidase activity than those from CL , suggesting that the delayed lesion development in infections by ML/MCL parasites is due to the increased ecto-nucleotidase activity of the promastigotes . The delay in lesion development as a result of the increased ecto-ATPase activity of the parasite was recently demonstrated in a study using L . ( L . ) amazonensis isolates obtained from patients with different clinical forms [21] . Our results expand on this observation indicating that not only the reduction of extracellular ATP , but also , the increase in extracellular Ado may play an important role in stalling the immune response . In fact , previous data from our group demonstrated that the addition of Ado at the moment of L . ( V . ) braziliensis inoculation in C57BL/6J induced an increased lesion development and parasitism , resulting in a delay in the control of the infection [18] . The combined action of the promastigote's ecto-nucleotidases may contribute to the delay in the immune response both by decreasing the extracellular ATP concentration as well as increasing the levels of extracellular Ado . It has been demonstrated that the hydrolysis of extracellular ATP and ADP by ecto-NTPDases present in the host cells reduces the concentration of these nucleotides decreasing the activation of P2 receptors which are important stimulators of the immune system . In addition , the subsequent increase in the concentration of Ado may increase the stimulation of P1 receptors , especially the A2A and A2B receptors , which generates an immunosuppressive response [25] , [49] , [50] . Thus , reduction of extracellular ATP with subsequent increase in Ado levels by the promastigote's enzymes may facilitate the persistence of Leishmania in the host , allowing their multiplication and dissemination to other sites of body , favoring the establishment of infection . In support for this hypothesis , a recent study demonstrated that a decrease in type 1 immune response in patients with disseminated leishmaniasis may account for parasite dissemination due to decreased control of parasite growth [51] . In addition to these ecto-nucleotidases , Leishmania parasites also express a bifunctional enzyme called 3′-nucleotidase/nuclease that may play a significant role in the generation of Ado that may contribute to regulation of the host immune response [52] , [53] . In order to further investigate the mechanisms underlying the delayed lesion development by parasites with higher ecto-nucleotidase activity , two isolates ( PPS6m and SSF ) were chosen for subsequent studies of infectivity and immune response in the murine model . As previous studies raised the idea that ecto-nucleotidase activity could be related to virulence in Leishmania [17] , [18] , [35] , [41] , [54] as well as Toxoplasma gondii [55] , Trypanosoma cruzi [42] , [56] and Entamoeba histolytica [57] , we inoculated C57BL/6J mice with promastigotes PPS6m and SSF isolates and followed lesion development during 12 weeks . As shown in Figure 6 , mice inoculated with PPS6m developed a peak of lesion between the seventh and ninth weeks of infection while mice inoculated with SSF presented their maximum lesion sizes between the third and fifth weeks of infection . In addition , the lesions caused by PPS6m presented a larger parasite load than lesions caused by the SSF isolate at 12 weeks of infection . It has been demonstrated that , in the murine model of cutaneous leishmaniasis , lesion development , rather than being a direct measure of parasite proliferation , is more related to cell migration and development of an immune response [58]–[60] . Given the differences in lesion development between PPS6m- and SSF-infected mice , we evaluated the levels of parasite proliferation and cellular infiltration at 4 and 8 weeks of infection by histological and immunohistochemical evaluation . Our results show that , at four weeks of infection , mice inoculated with the PPS6m isolate showed fewer lymphocytes at the site of infection than mice infected with SSF isolate ( Figures 7A and E ) . In addition , at four weeks of infection , parasitism was higher in mice inoculated with PPS6m than in animals infected by SSF ( Figures 7C and G ) . Consistent with a delayed establishment of an immune response , at 8 weeks of infection , mice inoculated with PPS6m presented an intense lymphocytic infiltrate ( Figure 7B ) which was still associated with elevated parasitism ( Figure 7D ) . At this time point , lesions from SSF inoculated mice , although still presenting a lymphocytic infiltrate , demonstrated evidence of tissue remodeling with very low parasitism ( Figures 7F and H ) . These results corroborate our hypothesis that the level of ecto-nucleotidase activity present in the promastigote modulates the immune response of the host , causing a delay in the migration of lymphocytes to the lesion site . In view of the critical role DC play in orchestrating the innate and adaptive components of the immune system , we decided to investigate whether the differences in the establishment of the immune response could be attributed to differences in the ability of each isolate to interfere with DC activation and expression of co-stimulatory molecules . To this aim , we infected BMDC with CFSE-labeled metacyclic promastigotes from both isolates and evaluated infectivity and the expression of activation markers after LPS treatment ( Figure 8 ) . Our results show that PPS6 infected twice as many BMDC than SSF . In addition , while both isolates decreased DC activation , inhibition of MHCII and CD86 expression by PPS6m was significantly higher than by SSF . It has been shown that infection of DC by Leishmania parasites is associated with the inhibition of cell activation [61]–[63] . Our results corroborate these findings by showing that L . ( V . ) braziliensis-infected DC are refractory to further activation by LPS , since the expression of MHCII and CD86 was decreased when compared with uninfected cells . IL-10 has been suggested as a possible factor associated with DC inhibition [64] , [65] . However , no differences in IL-10 production were detected in macrophages infected by the two isolates ( data not shown ) . Extracellular nucleotides and nucleosides have been shown to affect DC activation and migration . For example , extracellular ATP induces DC maturation and priming of Th1 cells while Ado has been shown to inhibit DC activation and pro-inflammatory cytokine production [66]–[68] . Activation of A2B Ado receptors has been shown to impair DC maturation and , consequently , decrease their ability to activate other immune cells [25] . Our results show that the isolate with high ecto-nucleotidase activity ( PPS6m ) presents greater capacity to inhibit the activation of BMDC , showing a possible correlation between the decreased expression of activation markers on infected DC and extracellular Ado production by ecto-nucleotidases present in L . ( V . ) braziliensis promastigotes . Macrophages play a double role during Leishmania infection . At the same time they are responsible for parasite clearance , they also harbor the parasite allowing its multiplication in the susceptible host . To further characterize the interactions between the parasites and the host , we evaluated the in vitro infectivity of isolates with different ecto-nucleotidase activity in J774-macrophages ( Figure 9 ) . No differences were observed between the isolates PPS6m and SSF with regard to their ability to proliferate in J774 cells in the absence of activation by IFN-γ plus LPS . Macrophage activation prevented multiplication of parasite from both isolates ( Figure 9B ) , however , the percentage of macrophages infected by the SSF isolate after 72 hr after activation by IFN-γ plus LPS was significantly reduced ( Figure 9A ) indicating that some cells were able to completely eliminate their parasites . The reasons for this ability of some cells to completely eliminate infection while other cells remain infected are unclear . Importantly , the reduction in the percentage of infected macrophages after activation was not observed in PPS6m infected cells . No differences in IL-10 production by cells infected with both isolates were observed ( data not shown ) . However , while activation of macrophages led to a decreased production of nitric oxide in PPS6m infected cells , this was not observed in SSF infected macrophages . Taken together , these results suggest that PPS6m inhibits macrophage activation , even in the presence of IFN-γ plus LPS , which leads to increased parasite survival although with limited capacity to multiply within these cells . On the other hand , macrophage activation in SSF-infected cells seems to be more efficient thus leading to decreased percentage of infected cells . PPS6m and SSF isolates , in addition to present high and low ecto-nucleotidase activity , were isolated , respectively , from mucosal and cutaneous lesions . In order to verify whether the observed effects on parasite survival and NO production were related to the clinical manifestation of the isolate , we performed the same experiments with two other isolates . Isolate HPV6 ( obtained from cutaneous lesion ) presents high ecto-nucleotidase activity while isolate JBC8m ( from mucosal lesion ) presents the lower ectonucleotidase activity amongst the mucosal/mucocutaneous isolates . Analysis of Figures 9A to 9C shows that HPV6 presents similar behaviour to PPS6m despite being isolated from cutaneous lesions . On the other hand , JBC8m was susceptible to macrophage activation and did not inhibit NO production similarly to what has been demonstrated to SSF . These results confirm our hypothesis that the level of ecto-nucleotidase activity of the promastigote rather than the clinical manifestation of the isolate is responsible for the resistance to macrophage activation . The mechanisms leading to inhibition of NO production during Leishmania infection are not completely clear , however , molecules present at the parasite surface such as LPG and glycoinositolphospholipids ( GIPL ) have been shown to interfere with the expression of inducible oxide nitric synthase ( iNOS ) or NO production after IFN-γ+LPS stimulation [69] . We now propose that , the ecto-nucleotidase activity of the promastigote , either directly or indirectly , via the production of Ado , is also involved in macrophage modulation by inhibiting NO production thus contributing to the establishment of infection . Taken together , the results from macrophage and DC infection by L . ( V . ) braziliensis isolates further corroborate our hypothesis [17] , [18] that lesion development and parasite multiplication within the host are associated with the level of ecto-nucleotidase activity of the promastigote . Furthermore , the observation that the PPS6m isolate is able to modulate the immune response of the host by inhibiting macrophage and DC activation confirms previous findings of our laboratory with other Leishmania species [17] , [62] and extends these results to L . ( V . ) braziliensis isolates . The inhibition of BMDC activation could explain the delay in mounting an immune response at the beginning of the infection in C57BL/6J mice thus causing the delay in lesion development . In addition , resistance to the initial activation of the macrophage would also allow for extended parasite survival . Leishmania organisms have a relatively simple life cycle , characterized by two principal stages: the flagellated mobile promastigotes living in the gut of the sandfly vector and the immobile amastigotes within phagolysosomal vesicles of the vertebrate host macrophages . Our results indicate the ecto-nucleotidase activity of the promastigote is associated with decreased immune response by the host at the establishment of infection . A limited number of studies evaluated the ectonucleotidase activity in amastigotes . It has been shown that in vitro derived amastigotes of L . ( L . ) amazonensis present higher levels of ATP hydrolysis than promastigotes [41] suggesting the possibility of an even more pronounced effect of Ado production on the modulation of the immune response during the chronic phase of the infection . However , no direct assessment of the role of the amastigote's enzymes on the course of infection has been performed . To determine the role of ecto-nucleotidases in the propagation of the disease , we decided to evaluate the ecto-enzymes activity of amastigotes isolated from infected J774 macrophages . Contrary to what has been shown for axenic amastigotes [41] , our results show that amastigotes obtained from macrophages show very low ecto-nucleotidase activity . The reason from this discrepancy is not known , but could be related to the parasite species ( L . ( L . ) amazonensis versus L . ( V . ) braziliensis ) . Our results also show that PPS6m and SSF amastigotes do not differ in their ability to hydrolyze adenine nucleotides . Furthermore , PPS6m amastigotes presented decreased ecto-nucleotidase activity when compared to the promastigote form of this isolate . ( Figures 10A–10C ) . Evaluation of amastigote infectivity in J774 macrophages showed that activation of these cells by IFN-γ and LPS was able to control parasite proliferation from both isolates as previously shown for promastigotes ( Figure 10E ) . In addition , amastigotes , which presented less ecto-nucleotidase activity than promastigotes , were not able to down modulate NO production by activated macrophages ( Figure 10F ) . Curiously , however , this inability to reduce NO production did not correlate with a decreased percentage of infection as shown for promastigotes ( Figure 10D ) . These results suggest that a different mechanism of parasite control might be triggered depending on whether the infection is initiated by promastigotes or amastigotes . It has been demonstrated that the various life-cycle stages have different sensitivities to reactive oxygen species ( ROS ) and provoke different oxidative responses of the macrophage . Although promastigotes and amastigotes enter the macrophages by phagocytosis , the concomitant oxidative burst is substantially different . For both stages , an increase in macrophage superoxide production is seen after infection , but the response is much higher to promastigotes compared to amastigotes [2] , [70] . In addition , whereas infections of macrophages by promastigote forms of Leishmania mexicana pifanoi induce the production of superoxide , infections by amastigotes barely induce superoxide production [71] . Nitric oxide combines with superoxide to form peroxynitrite which has been suggested to be more toxic for the amastigote than NO [2] . Also , in vitro studies confirmed that peroxynitrite is cytotoxic to amastigotes forms of parasites whereas nitric oxide is cytostatic [72] . On the other hand in vitro infections with promastigotes of Leishmania major show that the killing of parasite is mediated only by NO [73] . These observations support our results since NO production is inhibited in macrophages infected with promastigotes with high ecto-nucleotidase activity resulting in increased parasite survival . In infections by amastigotes , which have low ecto-nucleotidase activity , NO production is not inhibited , confirming the role of these enzymes in modulating macrophage activation . However , parasites may survive in the presence of NO , due to the incapacity of this parasite stage to induce ROS production . Most of the prior studies on the factors that determine the development of mucosal lesions in leishmaniasis focused on the host immune response and the ability of host cells or cytokines to influence the outcome of Leishmania infection . In general , patients with mucosal leishmaniasis present an exacerbated inflammatory response associated with high TNF-α and IFN-γ and decreased IL-10 production [4] . In addition , decreased response to IL-10 has also been suggested as a possible cause for the intense inflammation at the site of infection [5] . Furthermore , the involvement of Th17 cells associated with the presence of neutrophils [12] , high levels of pro-inflammatory monocyte chemoattractant protein ( MCP-1 ) to recruit monocytes [10] , exacerbated CD8+ activity [11] and activity of matrix metalloproteinases such as MMP-9 have been described as so important in the pathogenesis of mucosal leishmaniasis . The intrinsic capacity of MMP-9 activation of each individual might influence the intensity of macrophage efflux and dissemination of L . ( V . ) braziliensis infection to different anatomic areas [9] . Taken together , these studies indicate that patients with mucosal lesions develop a highly inflammatory response to the parasite which would seem to contradict our hypothesis . However , due to the intrinsic nature of these studies , they analyzed the patient's response after the establishment of the mucosal lesions and , therefore , cannot account for the role of the parasite in the initial phases of the infection . The fact that different strains of the same parasite species are able to cause distinct outcomes in isogenic mice ( this study and [21] , [74] ) indicates that parasite specific factors may also contribute to the result of the infection . According to Vendrame et al . ( 2010 ) , the high arginase activity of isolates from mucosal cases suggests that this characteristic favored the development of mucosal lesions and contributed to the survival and proliferation of Leishmania in a hostile environment [14] . In addition , isolates obtained from patients with mucosal leishmaniasis are more resistant to NO when compared to isolates obtained from patients with cutaneous leishmaniasis [13] . More recently , it was shown that the infection of Leishmania by RNA virus-1 ( LRV1 ) subverted the immune response to infection favoring promote parasite persistence and metastasis [75] . We propose high ecto-nucleotidase activity of the promastigote stage of the parasite as another parasite-related factor that could influence the clinical presentation of disease . As suggested above , high ecto-nucleotidase activity would decrease extracellular ATP and increase Ado during the initial contact of the parasite with the host at the site of infection , delaying the establishment of the response which would allow for the dissemination of the parasite . Later in the course of infection , the immune response would eventually be established and parasite control achieved . Depending on host related factors , such as decreased IL-10 receptor expression [5] , mucosal destruction due to uncontrolled response would occur . Previous results from our group suggest that early events that occur prior to amastigote transformation have an important role in the course of infection . Thus , inoculation of Ado together with L . ( V . ) braziliensis promastigotes caused an increase in lesion size and parasitism and also a delay in lesion resolution [18] . Furthermore , we also showed that the addition of Ado to L . ( L . ) amazonensis promastigotes culture medium decreases the ecto-nucleotidase activity of the parasite which correlates with decreased lesion size and parasitism [17] . Another intriguing question related to our results is the existence of isolates from cutaneous lesions with high ecto-nucleotidase activity ( Figure 5 ) . It is important to note that development of mucosal or mucocutaneous forms of leishmaniasis is generally preceded by single cutaneous lesions . It is tempting to speculate that some of the isolates from CL patients that present high ecto-nucleotidase activity would be able to induce mucosal lesions given the proper host environment . A close monitoring of these patients for decades would be needed to solve this issue . In summary , our findings suggest that the ecto-nucleotidase activity of L . ( V . ) braziliensis isolates influences lesion development in C57BL/6J mice . The degradation of ATP and subsequent production of Ado is able to create an anti-inflammatory environment that culminates in the inhibition of the activation of DC and macrophage microbicidal mechanisms creating an environment that favors the multiplication of the parasite inside the host cell and its dissemination to other sites of the body . Although the correlation between the activity of ecto-NTPDases and parasite virulence has already been proposed , our results expand on this concept by demonstrating that Ado production may also be important and that this combination may interfere with the clinical outcome of disease . This allows us to suggest that the ecto-nucleotidases can be characterized as a virulence factor of the parasite , indicating not only a marker for the development of the mucosal/mucocutaneous clinical forms , but also a possible target for future therapeutic interventions against Leishmania parasites . | Cutaneous leishmaniasis is a widespread tropical disease caused by different species of Leishmania protozoa that are transmitted by infected sandflies . Clinical presentations are extremely diverse and dependent on a variety of parasite and host factors that are poorly understood . Leishmania ( V . ) braziliensis infection may result in a devastating disease manifestation characterized by the development of destructive lesions in the oral , nasal , and pharyngeal mucosal . Ecto-nucleotidases are enzymes that are involved in the hydrolysis of extracellular nucleotides . These enzymes have been shown to correlate with virulence of Leishmania parasites . In this work , we evaluated the ecto-nucleotidase activity of promastigotes from the twenty three different L . braziliensis isolates . We demonstrated that isolates obtained from mucosal lesions present higher levels of ecto-nucleotidase activity than those from cutaneous lesions . In addition , we show that in the murine model of cutaneous leishmaniasis , promastigote forms of parasite with higher activity induce a delayed/decreased immune response that may correlate with spreading of the parasites throughout the body . Thus , we propose that the level of ecto-nucleotidase activity of promastigotes may be a marker for the development of severe clinical forms of cutaneous leishmaniasis and also a possible target for future therapeutic intervention . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"and",
"Discussion"
] | [
"biochemistry",
"enzymes",
"parasitology",
"immunology",
"biology",
"microbiology",
"host-pathogen",
"interaction",
"immune",
"response"
] | 2012 | Ecto-Nucleotidase Activities of Promastigotes from Leishmania (Viannia) braziliensis Relates to Parasite Infectivity and Disease Clinical Outcome |
An important layer of plant innate immunity to host-adapted pathogens is conferred by intracellular nucleotide-binding/oligomerization domain-leucine rich repeat ( NB-LRR ) receptors recognizing specific microbial effectors . Signaling from activated receptors of the TIR ( Toll/Interleukin-1 Receptor ) -NB-LRR class converges on the nucleo-cytoplasmic immune regulator EDS1 ( Enhanced Disease Susceptibility1 ) . In this report we show that a receptor-stimulated increase in accumulation of nuclear EDS1 precedes or coincides with the EDS1-dependent induction and repression of defense-related genes . EDS1 is capable of nuclear transport receptor-mediated shuttling between the cytoplasm and nucleus . By enhancing EDS1 export from inside nuclei ( through attachment of an additional nuclear export sequence ( NES ) ) or conditionally releasing EDS1 to the nucleus ( by fusion to a glucocorticoid receptor ( GR ) ) in transgenic Arabidopsis we establish that the EDS1 nuclear pool is essential for resistance to biotrophic and hemi-biotrophic pathogens and for transcriptional reprogramming . Evidence points to post-transcriptional processes regulating receptor-triggered accumulation of EDS1 in nuclei . Changes in nuclear EDS1 levels become equilibrated with the cytoplasmic EDS1 pool and cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites . We propose that coordinated nuclear and cytoplasmic activities of EDS1 enable the plant to mount an appropriately balanced immune response to pathogen attack .
In animals and plants , innate immune responses of individual cells constitute a major barrier to pathogen infection . Recognition of microbe- or damage-associated molecules is mediated by germ line encoded receptors whose activation is transduced by intracellular signaling systems to an anti-microbial response . Plant innate immunity is expressed as several layers [1] . Membrane pattern recognition receptors ( PRRs ) with external ligand recognition domains and intracellular kinase domains detect conserved pathogen molecules ( Microbe Associated Molecular Patterns or MAMPs ) in a similar manner to non-self recognition in animals [2] . PRR activation triggers a resistance response known as MAMP-triggered immunity ( MTI ) that is normally sufficient to resist colonization by non-adapted microbes . Successful pathogens have evolved effectors that dampen MTI and raise thresholds for activation of defense thereby allowing host invasion [1] , [2] . During infection , many pathogen effectors are delivered inside host cells and plants possess intracellular recognition systems mediated by nucleotide-binding and oligomerisation domain ( NB or NOD ) -leucine rich repeat ( LRR ) immune receptors [1] . Structurally related NOD-LRR proteins , known also as CATERPILLER , NACHT-LRR or NOD-like receptors ( NLRs ) , serve as pathogen and damage sensors in innate immune responses and cell death control in mammals [3] . Plant NB-LRR genes are often located within polymorphic gene clusters [4] . Consistent with a high genetic diversity , particular plant NB-LRRs recognize specific pathogen effectors or their actions on host molecular targets in a process known as effector-triggered immunity ( ETI ) [1] . The activation of NB-LRRs potentiates host defenses , accelerates defense-associated transcriptional reprogramming and often leads to programmed cell death at attempted infection sites as part of a hypersensitive response ( HR ) [5] , [6] , [7] . NB-LRR proteins and host cell death are necessarily under strict control . The biochemical mechanisms underlying receptor activation reveal that NB-LRR proteins behave as molecular switches which are structurally constrained in their inactive forms and activated by release from repression [3] , [8] . However , little is known about events between NB-LRR activation and defense induction or the mechanisms which limit resistance signaling to prevent auto-immune reactions . Recent studies of several plant NB-LRR receptors reveal that they partially localize to and function inside nuclei to trigger innate immune responses [9] , [10] , [11] . A powdery mildew effector-triggered interaction was observed between barley MLA10 receptor and members of the WRKY family of plant transcription factors [11] . Also , the tobacco N receptor recognizing tobacco mosaic virus ( TMV ) interacts with certain plant Squamosa Promoter-like ( SPL ) transcription factors [12] , suggesting a close association between some NB-LRRs and the transcription machinery . N resistance requires a host chloroplastic sulfurtransferase that becomes partially relocalized to the cytosol and nucleus by the TMV p50 effector [13] . Thus , dynamic signaling between the cytoplasm and nucleus is likely to be important for innate immune responses [14] . In line with this , Arabidopsis plants carrying mutations in genes encoding components of the nucleocytoplasmic trafficking machinery such as the nucleoporins MOS3 ( Modifier of snc1 , 3 ) /SAR3/Nup96 and MOS7/Nup88 , and importins MOS6/AtImpα3 and AtImpα4 , display defects in resistance to pathogens [15] , [16] , [17] , [18] . The plant immune regulator EDS1 ( Enhanced Disease Susceptibility1 ) is an essential component of basal resistance to virulent ( host-adapted ) biotrophic and hemi-biotrophic pathogens [19] , [20] , [21] , forming complexes in the cytoplasm and nucleus with its defense co-regulators PAD4 ( Phytoalexin Deficient4 ) and SAG101 ( Senescence Associated Gene101 ) [20] , [22] . EDS1 is also required for resistance conditioned by NB-LRRs that possess an N-terminal Toll/Interleukin-1 Receptor ( TIR ) domain ( known as TIR-NB-LRR receptors ) [20] , [22] , [23] , [24] . While Arabidopsis TIR-NB-LRR receptor RPS4 functions inside nuclei , EDS1 is not necessary for its steady state accumulation or nuclear accessibility [10] . Instead , EDS1 was found to signal after TIR-NB-LRR immune receptor activation and upstream of the transcriptional reprogramming of defense genes , production of resistance hormone salicylic acid ( SA ) and host cell death [10] , [20] . These data are consistent with EDS1 transducing signals generated by activated immune receptors to trigger defense and cell death programs . Convergence of numerous TIR-NB-LRR receptor activities on EDS1 raises the question of how diverse stimuli are coordinated inside cells to produce a measured immune response . To address this , we have examined where in the cell EDS1 signals in Arabidopsis TIR-NB-LRR-conditioned resistance and cell death . We report that there is an increase in the EDS1 nuclear pool during RPS4-triggered resistance to avirulent Pseudomonas syringae bacteria which precedes or coincides with EDS1-dependent transcriptional induction and repression of host genes . We also provide evidence for an essential role of nuclear EDS1 in basal and TIR-NB-LRR-conditioned immunity and in reprogramming defense gene expression . While nuclear EDS1 directs transcriptional changes , our data highlight the need also for cytoplasmic EDS1 to induce a complete immune response .
The Arabidopsis snc1 ( suppressor of npr1-1 , constitutive1 ) mutant displays EDS1-dependent constitutive resistance and dwarfism due to auto-activation of a TIR-NB-LRR protein [25] , [26] . We assessed whether this deregulated immune response is associated with a change in relative amounts of cytoplasmic and nuclear EDS1 that might reflect EDS1 activity in one compartment . As expected , combining Col eds1-2 [5] with snc1 ( in accession Col-0 ) to produce a snc1/eds1-2 double mutant led to full suppression of snc1 dwarfism ( Figure S1A ) and resistance ( data not shown ) . Western blot analysis of EDS1 protein revealed that total EDS1 amounts were higher in snc1 compared to wild type ( wt ) ( Figure 1A ) . However , a proportionate increase in EDS1 accumulation was observed in both nuclei-depleted and nuclear-enriched snc1 fractions . A stable eds1-2 transgenic line expressing EDS1 driven by its native promoter and fused at the C-terminus to yellow fluorescent protein ( YFP ) was selected ( EDS1-YFP ) . It complemented the eds1 defect in basal resistance ( Figure S1E ) and was detectable by fluorescence in the cytoplasm and nucleus using a confocal laser-scanning microscope ( Figure 1B ) . Fractionation of EDS1-YFP leaf tissues showed integrity of the EDS1-YFP fusion protein and a similar nucleo-cytoplasmic distribution on Western blots of the same samples probed with anti-EDS1 or anti-GFP antibodies ( Figure S1B ) . Since the native EDS1 ( Figure 1A ) and EDS1-YFP proteins displayed a similar distribution , we reasoned that EDS1-YFP fluorescence imaging could be used as a reliable indicator of EDS1 localization in leaf cells . The EDS1-YFP line was crossed into snc1/eds1-2 and the subcellular localization of EDS1-YFP protein examined in vivo by confocal imaging . Consistent with the distribution of native EDS1 protein ( Figure 1A ) , EDS1-YFP fluorescence was higher in both the cytoplasm and nuclei of snc1/eds1-2 leaf cells compared to eds1-2 ( Figure 1B ) . These results show that constitutive TIR-NB-LRR resistance increases accumulation of EDS1 protein in both cell compartments . We tested whether the constitutive resistance of snc1 plants could be explained by increased levels of EDS1 . As in snc1 , the EDS1-YFP line ( Figure 1B ) and a selected Col eds1-2 line expressing a functional EDS1-HA fusion under control of the constitutive CaMV 35S promoter , accumulated higher amounts of EDS1 in both nuclei-depleted and nuclei-enriched fractions compared to wt ( Figure S1C ) . However , neither of the two lines displayed dwarfism ( Figure S1D ) or enhanced basal resistance to virulent Pseudomonas syringae pv tomato strain DC3000 ( Pst DC3000 ) bacteria ( Figure S1E ) . Therefore , raising EDS1 steady state levels does not per se produce an auto-immune phenotype . We concluded that the constitutive resistance of snc1 depends on additional signals generated by the activated TIR-NB-LRR protein . We examined whether there is a change in EDS1 subcellular distribution at an early time point after triggering TIR-NB-LRR resistance that may be short-lived or masked by constitutive activation of the immune pathway . Leaves of wt plants were spray-inoculated with 10 mM MgCl2 ( mock treatment ) , virulent Pst DC3000 or avirulent Pst DC3000 expressing the effector AvrRps4 ( Pst DC3000 AvrRps4 ) recognized by TIR-NB-LRR receptor RPS4 [27] . As expected , Pst DC3000 AvrRps4 infection produced leaf-spot disease symptoms on eds1-2 but not on wt leaves after 2–3 days . Total protein , nuclei-enriched and nuclei-depleted fractions were prepared at 0 , 1 , 3 and 8 h after inoculation . Western blot analysis revealed an increase in EDS1 nuclear amounts 3 h and 8 h after inoculation with Pst DC3000 AvrRps4 that was not reflected in changes of EDS1 levels in total or nuclei-depleted fractions ( Figure 2 ) . No nuclear enrichment of EDS1 was observed at these time points in response to virulent Pst DC3000 or mock treatment ( Figure 2 ) . We concluded that an early and potentially important host response to avirulent bacteria involves a change in EDS1 leading to its increase in the nuclear compartment . Transcriptional profiles of wt and eds1 responses to Pst DC3000 AvrRps4 bacteria infiltrated into leaves [5] were analyzed and candidate genes selected whose expression ( at 6 h after infection ) was induced or repressed in an EDS1-dependent manner ( Tables S1 and S2 ) . Prominent among the EDS1-dependent upregulated genes are components of SA biosynthesis and signaling ( ICS1 , PBS3 and CBP60g ) [28] , [29] , [30] and FMO1 which positively regulates an SA-independent branch of EDS1 defense [5] , [31] . PR1 , a commonly used SA response marker [32] , was also identified in this group . Among the genes showing EDS1-dependent repression was DND1 , a negative component of plant innate immunity encoding a cyclic nucleotide-gated channel [33] and ERECTA , a receptor-like kinase required for resistance to the bacteria Ralstonia solanacearum and necrotrophic fungi [34] , [35] . A transcription factor ( MYB48 ) and predicted nucleic acid binding protein gene ( At1g66140 ) , both with unknown functions , were also selected for analysis . In order to validate the expression trends and measure transcriptional changes in relation to EDS1 nuclear accumulation , we quantified transcript levels of the chosen EDS1-dependent up- and down-regulated genes in the same leaf tissue extracts that were used for analysis of EDS1 protein ( Figure 2 ) . The genes displayed significant EDS1-dependent induction ( Figure 3A ) or repression ( Figure 3B ) at 8 hpi with Pst DC3000 AvrRps4 . Induction of the EDS1-dependent genes in response to virulent Pst DC3000 was not observed at 8 hpi but was seen at 24 hpi , consistent with Pst DC3000 triggering a slower transcriptional response [6] . We concluded from these data that Pst DC3000 AvrRps4-triggered EDS1 nuclear accumulation precedes or coincides with EDS1-dependent transcriptional reprogramming of defense-related genes . An EDS1-dependent increase in PAD4 transcripts and EDS1 induction also occurred at 8 hpi with Pst DC3000 AvrRps4 ( Figure 3A ) . Thus , EDS1 nuclear enrichment observed 3 h after pathogen challenge ( Figure 2 ) is unlikely to be due to increased EDS1 gene expression but rather to a post-transcriptional mechanism . We reasoned that EDS1 may need to attain a certain concentration in nuclei to fulfill its defense signaling function downstream of TIR-NB-LRR receptor activation . This is supported by the higher nuclear EDS1 amounts observed in snc1 immune-activated tissues ( Figure 1 ) and lower levels of nuclear EDS1 in an Arabidopsis mos7 mutant which has a defective Nucleoporin 88 and displays compromised immune responses [16] . In animals , Nup88 modulates CRM1-mediated nuclear export of proteins containing a leucine-rich-type nuclear export sequence ( NES ) [36] . Thus , EDS1 might possess a functional leucine-rich-type NES and be exported from the nucleus via the Arabidopsis CRM1 homolog XPO1 [37] as a mechanism to control nuclear accumulation . The EDS1 amino acid sequence contains two predicted bipartite nuclear localization signals ( NLS ) at positions 366 and 440 [19] and a putative leucine-rich nuclear export sequence ( NES ) around amino acid 530 ( Figure S2A ) that might enable nucleo-cytoplasmic movement . However , mutation of core residues in the EDS1 NLS or NES ( Figure S2B ) did not lead to obvious relocalization of YFP-tagged EDS1 protein in transient plant expression assays ( data not shown ) . The functionality of an NES can also be determined by assessing protein localization after treatment with the nuclear export inhibitor Ratjadone A ( RatA ) which inhibits plant and animal XPO1/CRM1 exportins [37] , [38] . We examined mesophyll protoplasts generated from the EDS1-YFP line for EDS1-YFP nuclear and cytoplasmic accumulation in the presence or absence of RatA . There was a marked shift in EDS1-YFP fluorescence to nuclei in RatA-treated compared to mock-treated protoplasts ( Figure 4 ) , consistent with EDS1 normally being shuttled out of the nucleus via NES-driven nuclear export . As a control , protoplasts generated from a transgenic line expressing YFP under the CaMV 35S promoter did not respond to RatA treatment ( Figure 4 ) because this protein is able to diffuse between the cytoplasm and nucleus [9] . To establish whether EDS1 operates in the nucleus or cytoplasm or both compartments in transducing resistance signals , we attempted first to reduce EDS1 nuclear accumulation by increasing the rate of protein nuclear export through fusion of an additional functional NES sequence [11] , [39] . An NES ( LALKLAGLDI ) or mutated ‘nes’ ( LALKAAGADA ) [39] was attached to the C-terminus of EDS1-YFP and this fusion protein expressed stably in Col eds1-2 under the control of the EDS1 native promoter . Multiple independent transgenic lines were selected that expressed the EDS1-YFP-NES/nes fusions at levels similar to EDS1 in wt , as monitored on a Western blot ( Figure 5A ) . EDS1-YFP-NES fluorescence was detected in the cytoplasm and in a low proportion of nuclei whereas EDS1-YFP-nes accumulated in both compartments of leaf epidermal cells ( Figure 5B and S3A ) . Protoplasts derived from an EDS1-YFP-NES line exhibited fluorescence in the cytoplasm and at the nuclear rim ( Figure 4 ) consistent with the NES fusion increasing the rate of EDS1 export from nuclei . There was a strong increase in EDS1-YFP-NES nuclear accumulation in this line after RatA treatment ( Figure 4 ) , indicating that the NES-tagged protein has the capacity to enter nuclei and that RatA treatment inhibits NES-driven nuclear export . We monitored the distribution of EDS1-YFP-NES ( in lines #2–10 and #2–11 ) and EDS1-YFP-nes ( line #1–2 ) compared to EDS1-YFP in nuclei-depleted and nuclei-enriched fractions on a Western blot probed with anti-EDS1 antibody . This did not reveal obvious differences in nuclear accumulation between the EDS1-YFP-NES and EDS1-YFP-nes extracts ( Figure S3B ) , contrasting with the distinct in vivo EDS1-YFP-NES/nes fluorescence patterns ( Figure 5B ) . To investigate reasons for this discrepancy , we imaged fluorescence in sections through individual nuclei in EDS1-YFP-NES line #2–11 and EDS1-YFP-nes line #1–2 on a confocal microscope and compared with the EDS1-YFP line . EDS1-YFP-NES protein fluorescence was detected mostly in the cytoplasm , at the nuclear rim ( as seen before in the protoplasts; Figure 4 ) and inside nuclei in ∼5% of epidermal cells ( Figure S3C ) . EDS1-YFP-nes and EDS1-YFP fluorescence was observed inside the nuclear compartment in most imaged cells ( Figure S3C ) . A similar distribution was found in eds1-2 epidermal cells transiently expressing EDS1-YFP-NES/nes constructs after particle bombardment ( Figure S3D ) . Together , the data suggest that addition of a functional NES to EDS1-YFP reduces its accumulation inside nuclei but the NES does not allow complete release of EDS1-YFP from the nuclear envelope and associated structures . This may account for its fractionation with nuclei during biochemical separation . We examined whether resistance mediated by the TIR-NB-LRR genes RPS4 [27] or RPP4 [40] is affected by increased removal of EDS1 from inside nuclei . The EDS1-YFP-NES lines displayed reduced RPS4 resistance to Pst DC3000 AvrRps4 , measured by bacterial growth ( Figure 5C ) , and RPP4 resistance to H . arabidopsidis isolate Emwa1 , monitored by trypan blue staining of leaves for pathogen structures and plant cell death ( Figure 5D ) . By contrast , the EDS1-YFP-nes lines were fully resistant ( Figure 5C and D ) . Basal resistance to virulent Pst DC3000 ( Figure 5E ) and H . arabidopsidis Noco2 ( data not shown ) was also compromised in the EDS1-YFP-NES transgenics but was unaffected in EDS1-YFP-nes lines . These data suggest that EDS1 needs to accumulate to a sufficient level inside nuclei to signal a full innate immune response against virulent and avirulent pathogens . With both pathogens , a substantial degree of resistance remained in the EDS1-YFP-NES lines compared to the complete susceptibility of eds1 mutants . We reasoned that the residual pathogen resistance in the EDS1-YFP-NES lines ( Figure 5 ) might be conferred by a low amount of EDS1-YFP-NES protein initially entering nuclei before it is exported or partially trapped at the nuclear envelope ( Figure 4 and S3 ) , arguing for an entirely nuclear function of EDS1 . Alternatively , the intermediate resistance could reflect a contribution of cytoplasmic EDS1 to the immune response . We therefore used a different strategy to control EDS1 localization inside cells by fusing the C-terminus of EDS1-YFP or YFP alone ( as control ) to the steroid binding domain of the mammalian glucocorticoid receptor ( GR ) in a cassette driven by the CaMV 35S promoter [41] . Proteins fused to GR are normally retained in the cytoplasm through association with an Hsp90 chaperone complex [42] and treatment of plant cells with the steroid hormone Dexamethasone ( Dex ) drives nuclear localization of the GR fusion protein [41] , [43] . From multiple independent transgenic lines expressing EDS1-YFP-GR or YFP-GR in an eds1-2 background ( in Arabidopsis accession Ler ) two lines ( #1 and #4 ) were selected that had detectable EDS1-YFP-GR protein on a Western blot of leaf extracts probed with anti-GFP antibody ( Figure 6A ) . Extracts were also probed with anti-EDS1 antibody which gave weaker signals than with anti-GFP and showed that EDS1-YFP-GR protein accumulated at a rather low level ( Figure S4A ) despite being driven by the 35S promoter ( Figure S4B ) . A single line expressing YFP-GR ( Figure S4C ) was taken as control . Before Dex treatment , weak YFP fluorescence was observed in the cytoplasm of the EDS1-YFP-GR lines , as shown for line #4 ( Figure 6B ) . YFP fluorescence was also detected in nuclei at 5 h ( not shown ) and 13 h ( Figure 6B ) after spraying leaves with 30 µM Dex . At 13 h ( after Dex ) this was associated with an increase in EDS1-YFP-GR protein in total , nuclei-depleted and nuclei-enriched fractions ( Figure 6A ) but not with a change in EDS1-YFP-GR transcript levels ( Figure S4B ) . The EDS1-YFP-GR transgenic lines developed normally before or after treatment with the steroid hormone . We therefore used them to investigate the roles of cytoplasmic and nuclear EDS1 in the plant immune response . EDS1-YFP-GR plants were first analyzed for their response to avirulent Pst DC3000 AvrRps4 . Leaves were pre-treated with 30 µM Dex for 5 h before bacterial inoculation and bacterial titers were counted at day 4 . In the absence of Dex , the EDS1-YFP-GR lines displayed resistance to Pst DC3000 AvrRps4 that was intermediate between wt and eds1-2 ( Figure 6C ) . Treatment with Dex reduced bacterial numbers by ∼1000-fold to levels below those seen in wt ( Figure 6C ) . Wt ( Ler ) and control YFP-GR ( Ler eds1-2 ) backgrounds exhibited , respectively , similar levels of resistance and susceptibility with or without Dex treatment ( Figure 6C ) . Also , Dex application to wt plants did not alter levels of native EDS1 protein ( Figure S4D ) , indicating that the resistance of the EDS1-YFP-GR lines is conditioned by Dex-induced release of EDS1 . Resistance mediated by a different EDS1-dependent TIR-NB-LRR gene ( RPP5 ) [44] to H . arabidopsidis isolate Noco2 was also conditional on Dex treatment , with pathogen growth being efficiently contained by a plant hypersensitive response at infection sites ( HR; Figure 6D ) . As with Pst DC3000 AvrRps4 bacteria , the EDS1-YFP-GR lines displayed partial resistance to H . arabidopsidis in the absence of Dex . Notably , this was associated with spreading cell death at infection foci ( Figure 6D ) . Without Dex treatment , basal resistance in these lines to virulent Pst DC3000 was suppressed to similar levels as in eds1-2 and was equivalent to resistance in wt after Dex application ( Figure 6E ) . The EDS-YFP-GR signal in nuclei-enriched fractions was strongest when Dex treatment was followed by inoculation with Pst DC3000 AvrRps4 ( Figure 6A and S4A ) . Since EDS1-YFP-GR is expressed under the control of a constitutive promoter , it is unlikely that the increase is due to transcriptional up-regulation . Indeed , no significant change in expression of the EDS1-YFP-GR transgenes was observed in leaves 8 h after challenge with Pst DC3000 AvrRps4 , with or without Dex pretreatment ( Figure S4B ) . These data suggest that recognition of AvrRps4 or another pathogen stimulus enhances nuclear accumulation of Dex-released EDS1-YFP-GR . We monitored expression of genes displaying EDS1-dependent transcriptional changes 8 h after Pst DC3000 AvrRps4 inoculation ( Figure 3 ) in the EDS1-YFP-GR transgenic lines with or without 5 h Dex pre-treatment . The 13 h time point after Dex application is when EDS1-YFP-GR protein signals were monitored on Western blots and by fluorescence imaging ( Figure 6A and B ) . The results show Dex-dependent reprogramming of EDS1-induced and EDS1-repressed genes in response to DC3000 AvrRps4 ( Figure 7 ) . Whereas cytosolic retention of EDS1-YFP-GR substantially reduced pathogen-induced expression changes , Dex-induced nuclear accumulation of EDS1-YFP-GR allowed similar or larger transcriptional changes than in DC3000 AvrRps4-challenged wt plants ( Figure 7 ) . We concluded that nuclear EDS1 is needed to drive pathogen-induced reprogramming of transcription .
We used a combination of biochemical fractionation of leaf tissue extracts and in vivo imaging of fluorescent-tagged proteins in leaf cells to assess EDS1 accumulation in the cytoplasmic and nuclear compartments . Enhanced EDS1 expression in snc1 mutants ( with constitutive resistance due to an auto-activate TIR-NB-LRR protein ( Figure 1 ) ) [26] or reduced EDS1 in mos7 mutants ( with compromised resistance because of a defect in Nup88-mediated nuclear retention [16] ) resulted in similar EDS1 nuclear and cytoplasmic ratios to those found in healthy wt plants . We also observed higher EDS1 accumulation in both cell compartments at late stages of infection with virulent H . arabidopsidis ( data not shown ) , suggesting that nuclear and cytoplasmic EDS1 pools need to be equilibrated during prolonged activation of defense and development . These EDS1 accumulation patterns , together with our earlier finding that EDS1 forms different complexes with its signaling partners PAD4 and SAG101 in the cytoplasm and nuclei of healthy leaf cells [22] , suggest distinct but cooperative roles of EDS1 cytoplasmic and nuclear pools . Analysis of mesophyll protoplasts derived from the EDS1-YFP and EDS1-YFP-NES transgenic lines ( Figure 4 ) showed that EDS1 is capable of nuclear transport receptor ( CRM1/XPO1 ) -mediated shuttling between the cytoplasm and nucleus through the nuclear pores . It is therefore likely that cytoplasmic and nuclear EDS1 pools communicate through the nuclear pores to coordinate resistance and cell death programs . There may be a requirement for optimal cycling of EDS1 between the nuclear and cytoplasmic compartments , consistent with dynamic signaling across the nuclear envelope being critical for animal and plant immune responses [14] . An increase in nuclear EDS1 accumulation either by pathogen infection triggering TIR-NB-LRR activation ( Figure 2 ) or by Dex-induced release of a EDS1-YFP-GR fusion to nuclei accompanying a pathogen stimulus ( Figure 6 ) correlated with the induction or repression of particular host genes ( Figures 3 and 7 ) , supporting a role for nuclear EDS1 in driving transcriptional reprogramming during plant defense . Among the EDS1-induced genes are components of SA biosynthesis and signaling ( e . g ICS1 , PAD4 , PBS3 and CBP60g ) [28] , [29] , [30] , [47] ( Table S2 ) . Hence , a key step of EDS1 nuclear action is to stimulate the SA pathway . Although SA has a minor role in local TIR-NB-LRR triggered resistance and cell death [20] , [40] , [48] it is a central component of plant systemic resistance to biotrophic pathogens [49] . Accordingly , EDS1 is required for systemic signaling beyond pathogen infection sites [50] , [51] . EDS1-directed repression of genes such as DND1 [33] and ERECTA [34] , [35] which have a negative impact on resistance to biotrophic pathogens but contribute to resistance to some necrotrophic pathogens , suggests a ‘master’ role of EDS1 complexes in coordinating gene expression outputs . Control of ERECTA expression may be of particular significance since this gene affects plant growth and development as well as responses to environmental stimuli through a network of cis- and trans-regulation [52] . The precise mode of action of EDS1 inside nuclei remains unclear . The primary EDS1 amino acid sequence does not have obvious DNA-binding domains [19] and analysis of nuclear EDS1 has not so far revealed specific association with chromatin ( S . Blanvillain-Baufumé , R . P . Huibers and J . E . Parker , unpublished ) . However , interactions have been found between EDS1 and a number of transcription factors in yeast 2-hybrid assays ( S . Blanvillain-Baufumé , R . P . Huibers and J . E . Parker , unpublished ) , suggesting a mechanism by which EDS1 could modulate transcription . Nuclear EDS1 complexes may work by binding transcription factors and/or repressors in the nucleoplasm to guide activities and associations with the DNA . Restraining proteins away from their site of action has emerged as an important mechanism for controlling transcriptional activators such as NF-KB in mammalian cells [53] and a number of transcriptional regulators in plants ( such as bZIP10 , WRKY33 , NPR1 ) , and emphasizes the extent of intracellular protein dynamics during stress signaling [14] . The role of the EDS1 signaling partners PAD4 and SAG101 in this process is also not clear . They form transient complexes with EDS1 that distribute differently between the cytoplasm and nucleus and , together , are essential for immune response and cell death activation [20] , [22] . One model is that PAD4 and SAG101 help EDS1 to circulate between the cytoplasm and nucleus to coordinate the binding and release of transcription factors . It will be important to test whether EDS1 mislocalization affects association with PAD4 or SAG101 and if these components participate in EDS1 interactions with transcription factors in the cell . Evidence points to EDS1 operating downstream of activated RPS4 [10] . We therefore postulate that a change in EDS1 nucleo-cytoplasmic status leading to transcriptional reprogramming is triggered by RPS4 recognition of AvrRps4 . However , we cannot exclude the possibility that an alteration in EDS1 occurs as part of the plant response to bacterial MAMPs . An early MAMP-induced change might be effectively dampened by virulent DC3000 bacteria and reinstated ( or amplified ) by RPS4 responding to AvrRps4 [1] . Whatever the stimulus , a rise in EDS1 alone seems insufficient to trigger resistance because a mild increase in EDS1 levels ( in the pEDS1:EDS1-YFP transgenic line ) or strong over expression of EDS1 ( in the p35S:EDS1-HA line ) resulting in over-accumulation of EDS1 in the nuclear and cytoplasmic compartments did not produce an auto-immune response ( Figure S1C–E ) . These data suggest that R protein activation is a necessary step for EDS1 inside nuclei to reprogram transcription . In this regard , nuclear EDS1 levels appear to be controlled post-transcriptionally . This holds for the early nuclear accumulation of EDS1 after RPS4 activation because it occurs before changes in EDS1 transcript levels are observed ( Figures 2 and 3 ) . It is therefore distinct from pathogen-induced increases in EDS1 and PAD4 mRNAs and total protein at later time points which are attributed to a positive feedback loop involving SA to amplify host resistance [5] , [20] , [47] . Post-transcriptional regulation likely also accounts for the rise in EDS1-YFP-GR steady state levels after its Dex-induced release to the nucleus because bacterial and Dex treatments did not alter EDS1-YFP-GR mRNA abundance ( Figure S4B ) . The low level of EDS1-YFP-GR protein accumulating in the cytoplasm before Dex treatment ( Figure 6 and S4 ) may be a consequence of impeding nuclear access or nucleo-cytoplasmic cycling of EDS1 . In line with the tendency for nuclear and cytoplasmic EDS1 to equilibrate , we postulate that an initial rise in nuclear EDS1-YFP-GR causes a rapid adjustment of the cytoplasmic EDS1 pool , thereby permitting a complete immune response to be activated ( Figure 6 ) . Modification of EDS1 leading to increased protein stabilization likely alters its functions inside nuclei to permit reprogramming of transcription . A post-transcriptional process was also proposed to account for lowered total cellular accumulation of EDS1 and the SA response regulator NPR1 in the Arabidopsis mos7 ( nup88 ) nucleoporin mutant since their corresponding transcript levels were not reduced [16] . A growing body of evidence shows that targeted degradation by the proteasome coordinates the exchange of transcription components on and off the chromatin , allowing dynamic shifts from repression to activation of genes [54] , [55] . A recent analysis indeed shows that proteasome-mediated turnover of NPR1 in the nucleus is necessary for its function as a transcriptional co-activator in systemic resistance [56] . Partial resistance to bacteria and oomycete pathogens exhibited by the EDS1-YFP-NES ( Figure 5 ) and EDS1-YFP-GR lines ( in the absence of the Dex release stimulus; Figure 6 ) points to a role of cytoplasmic EDS1 in promoting an efficient immune response . It is possible that transient EDS1 nuclear pools in the EDS1-YFP-NES lines ( Figure 4 ) or leakiness in EDS1-YFP-GR cytoplasmic retention ( though undetectable in the microscope – Figures 6B ) could account for the residual resistance . Also , fusion of an additional NES to EDS1 may have unexpected consequences since , although it reduces levels of EDS1-YFP inside nuclei , it appears to impede efficient release of EDS1 from the nuclear rim ( Figure 4 and S3 ) . Nevertheless , the fact that EDS1 is actively shuttled from the nucleus to the cytoplasm where it forms different complexes [22] and a cytoplasmic EDS1 pool is maintained throughout infection and development , argue for EDS1 functions in the cytoplasm . A role for cytoplasmic EDS1 is most evident in TIR-NB-LRR resistance to Pst DC3000 AvrRps4 ( Figures 5C and 6C ) and H . arabidopsidis ( Figures 5D and 6D ) but less clear in EDS1-dependent basal resistance ( Figures 5E and 6E ) . The cytoplasmic EDS1 pool may counter-balance activities of nuclear EDS1 by , for example , sequestering transcription factors outside the nuclei . Alternatively , but not exclusively , EDS1 cytosolic complexes might have a unique signaling function needed for a complete immune response . Particularly noticeable was the expansion of cell death lesions at sites of infection by the obligate biotrophic pathogen H . arabidopsidis in EDS1-YFP-GR lines in the absence of Dex ( Figure 6D ) . These lesions extended beyond obvious pathogen structures and were therefore different from the characteristic trailing necrotic phenotype observed in Arabidopsis mutants with relaxed TIR-NB-LRR resistance [20] . No cell death was observed in the EDS1-YFP-GR lines before pathogen inoculation indicating that the cell death requires a pathogen stimulus . One scenario is that failure to restrict host cell death in these lines is due to diminished EDS1 nuclear function in regulating genes that suppress or contain cell death . Supporting this , a recent study showed that SA antagonism of EDS1-driven cell death initiation is needed for a complete immune response to biotrophic pathogens [57] . It is possible that cytosolic EDS1 actively promotes cell death in response to oxidative stress signals emanating from the chloroplasts [57] , [58] , [59] and this is counter-balanced by transcriptional reprogramming in the nucleus to moderate potentially destructive cellular events . Future work aims to characterize the cytoplasmic and nuclear activities of EDS1 complexes which seem to be carefully poised in the cell for optimal responsiveness to biotic stress .
Arabidopsis wild type accessions , eds1-2 [5] and rps4-2 [10] mutants have been described . Plants were grown in soil in controlled environment chambers under a 10 h light regime ( 150–200 µE/m2s ) at 22°C and 65% relative humidity . Pst DC3000 and Pst DC3000 AvrRps4 strains were grown for 24 h at 28°C on NYGA solid medium supplemented with the corresponding antibiotics . For bacterial growth assays and expression analyses , 4-week-old plants were spray-inoculated with bacterial suspensions at 4×108 cfu/ml in 10 mM MgCl2 with 0 , 04% ( v/v ) Silwet L-77 ( Lehle Seeds ) or mock-treated with 10 mM MgCl2 containing 0 , 04% ( v/v ) Silwet L-77 . In planta bacterial titers were determined at the indicated time points after inoculation by shaking leaf discs in 10 mM MgCl2 with 0 , 01% ( v/v ) Silwet L-77 at 28°C for 1 h [60] . At least five plants per genotype were used for each sampling . Bacterial numbers were compared between lines using a two-tailed Student's t-test . H . arabidopsidis isolates Emwa1 and Noco2 were maintained and inoculated onto 2-week-old plants at 4×104 spores/ml as described [22] . Plant cell death and H . arabidopsidis infection structures were visualized under a light microscope after staining leaves with lactophenol trypan blue [23] . Binary vectors suitable for Gateway cloning ( Invitrogen ) and protein localization studies were generated . Monomeric yellow fluorescent protein ( YFP ) was PCR-amplified from vector pcDNA3-mYFP ( obtained from Dr . Irine Prastio , Howard Hughes Medical Institute , UC San Diego , CA ) and ligated into the binary vector pXCS-HisHA containing a CaMV 35S promoter [61] , generating pXCS-YFP . A Gateway recombination cassette ( reading frame B as EcoRV-fragment , Invitrogen ) was ligated and the CaMV 35S promoter removed , resulting in the Gateway destination vector pXCG-YFP . Genomic Ler EDS1 sequence including 1 . 4 kb of endogenous promoter and 2 . 1 kb of coding sequence without stop codon was cloned in pENTR/D-TOPO ( Invitrogen ) and an LR reaction was performed to generate the vector pXCG-pEDS1-EDS1-YFP . Constructs were used to transform Col eds1-2 plants [5] using the floral-dip method [62] . Several independent EDS1-YFP transgenic lines were generated that fully complemented the eds1-2 mutation and a representative line used for further analysis . Functional NES from PKI ( LALKLAGLDI ) and the non-functional nes ( LALKAAGADA ) [39] were attached to the C-terminus of mYFP through PCR amplification of vector pcDNA3-mYFP . The same strategy as described above was followed to generate construct pXCG-pEDS1-EDS1-YFP-NES/nes and stable Arabidopsis Col eds1-2 stable transgenic lines . To generate transgenic plants expressing EDS1-HA under the CaMV 35S promoter , an LR reaction was made between the pENTR/D-TOPO ( Invitrogen ) vector containing genomic Ler EDS1 coding sequence [22] and the pXCS-3xHA vector [61] . Constructs were transferred to A . tumefaciens strain GV3101 ( pMP90RK ) and transformed into Arabidopsis Col eds1-2 plants . To generate transgenic plants expressing StrepII-3xHA-YFP driven by the CaMV 35S promoter , the plasmid pENS-StrepII-3xHA-GW was made using the vector pXCS-3xHA [61] . YFP was PCR-amplified from vector pcDNA3-mYFP and cloned in pENTR/D-TOPO ( Invitrogen ) and an LR reaction performed to obtain the pENS-StrepII-3xHA-YFP plasmid . Constructs were transformed into Arabidopsis Col plants , as described above . GR fusions were generated using the vector pBI-ΔGR [41] and cEDS1-YFP amplified from the pEXG-cEDS1-YFP vector . Constructs were transferred to A . tumefaciens strain GV3101 ( pMP90 ) and used to transform Ler eds1-2 plants [19] . Total protein extracts were prepared by grinding leaf material in liquid nitrogen . Samples were resuspended in equal volumes of 2× Laemmli loading buffer , boiled for 5 min and centrifuged to remove cell debris . Proteins were separated by SDS-PAGE and electroblotted to nitrocellulose membranes for protein gel blot analysis . Equal loading was monitored by staining membranes with Ponceau S ( Sigma-Aldrich ) . Nuclear fractionation of Arabidopsis tissue was performed as previously described [22] using 4-week-old plants . Nuclei-enriched fractions were 30× more concentrated than nuclei-depleted fractions based on the final volume of each fraction . Antibodies used for immunoblot analysis were as described: anti-EDS1 [22] , anti-PEPC ( Rockland; [63] ) , anti-Histone H3 ( Abcam; [22] ) , anti-PICKLE [64] and anti-CSN4 ( BIOMOL International ) . Arabidopsis leaf mesophyll protoplasts were prepared from 4-week-old plants grown in a normal light/dark regime , according to Asai et al [65] with some modifications . Leaf strips were digested in enzyme solution ( 0 . 4 M Mannitol; 20 mM KCl; 20 mM MES pH 5 . 7 ) with 1 . 5% cellulase ( Onozuka R-10 , Merck , Darmstadt , Germany ) and 0 . 4% Macerozyme ( R-10 , Serva , Heidelberg , Germany ) . The solution was vacuum infiltrated for 3 min , incubated for 30 min with vacuum pressure and then for 2 h with gentle shaking . The protoplast solution was filtered through a 62 µm nylon mesh and washed with W5 solution ( 154 mM NaCl , 25 mM CaCl2·2H2O , 5 mM KCl , 2 mM MES pH 5 . 7 ) . Isolated protoplasts were resuspended in Mannitol solution ( 0 . 4 M Mannitol , 15 mM MgCl2·6H2O , 4 mM MES , pH 5 . 8 ) . The nuclear export inhibitor Ratjadone A ( Alexis Biochemicals ) was dissolved in methanol ( 10 ng/µl ) and added to protoplasts to a final concentration of 15 ng/ml . Control samples were mock treated-with an equal concentration of methanol . Arabidopsis leaves or protoplast solutions were examined with a confocal laser-scanning microscope Leica TCS 4D . Total RNA was extracted from plant leaves using TRI-reagent ( SIGMA ) and RNA was reverse transcribed into cDNA using SuperScriptII ( Invitrogen ) following the manufacturer's instructions . Quantitative RT-PCR experiments were performed in an iQ5 Real-Time PCR Detection System ( Bio-Rad ) using Brilliant SYBR Green QPCR Core Reagent ( Stratagene ) as dye . Experiments were performed using three independent biological samples . Relative transcript levels were calculated using the iQ5 Optical System Software ( Version 2 . 0 ) . Ubiquitin UBQ10 ( At4g05320 ) transcript levels were used as internal reference . Primers used in these experiments are available on request . | Plants have evolved a multilayered innate immune system to recognize and respond to potentially destructive microbes in the environment . Resistance to invasive biotrophic and hemi-biotrophic pathogens often involves transcriptional mobilization of defenses and programmed death of host cells at infection sites . However , these processes disturb normal metabolism and growth and therefore have to be tightly controlled . In this study , we examine resistance signaling events inside Arabidopsis cells after pathogen activation of intracellular immune receptors . We show that the nucleo-cytoplasmic protein EDS1 acts as an important regulator of transcriptional reprogramming in the immune response by allowing the induction and repression of particular defense-related genes . We provide evidence that EDS1 accomplishes its role as a defense signaling ‘hub’ through coordinated activities in the cytoplasm and nucleus . Maintaining a balance between these two EDS1 pools is probably important for resistance and cell death to a range of infectious microbes and to not ‘overshoot’ defense activation which would be detrimental for the plant . | [
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] | 2010 | Balanced Nuclear and Cytoplasmic Activities of EDS1 Are Required for a Complete Plant Innate Immune Response |
Madurella mycetomatis is the main causative organism of eumycetoma , a persistent , progressive granulomatous infection . After subcutaneous inoculation M . mycetomatis organizes itself in grains inside a granuloma with excessive collagen accumulation surrounding it . This could be contributing to treatment failure towards currently used antifungal agents . Due to their pivotal role in tissue remodelling , matrix metalloproteinases-2 ( MMP-2 ) and 9 ( MMP-9 ) or tissue inhibitor of metalloproteinases ( TIMP ) might be involved in this process . Local MMP-2 and MMP-9 expression was assessed by immunohistochemistry while absolute serum levels of these enzymes were determined in mycetoma patients and healthy controls by performing ELISAs . The presence of active MMP was determined by gelatin zymography . We found that both MMP-2 and MMP-9 are expressed in the mycetoma lesion , but the absolute MMP-2 , -9 , and TIMP-1 serum levels did not significantly differ between patients and controls . However , active MMP-9 was found in sera of 36% of M . mycetomatis infected subjects , whereas this active form was absent in sera of controls ( P<0 . 0001 ) . MMP-2 , MMP-9 , and TIMP-1 polymorphisms in mycetoma patients and healthy controls were determined through PCR-RFLP or sequencing . A higher T allele frequency in TIMP-1 ( +372 ) SNP was observed in male M . mycetomatis mycetoma patients compared to controls . The presence of active MMP-9 in mycetoma patients suggest that MMP-9 is activated or synthesized by inflammatory cells upon M . mycetomatis infection . Inhibiting MMP-9 activity with doxycycline could prevent collagen accumulation in mycetoma , which in its turn might make the fungus more accessible to antifungal agents .
Madurella mycetomatis is the most prevalent causative organism of eumycetoma , a persistent , progressive granulomatous infection involving subcutaneous tissues and bones [1] . Mycetoma lesions are characterized by subcutaneous masses , sinuses and fungal grains , which commonly progress under inappropriate treatment resulting in deformation and disabilities of infected body parts [1] . To treat eumycetoma , a combination of surgery and treatment with antifungal agents is required [2] . Treatment with the currently used antifungal agents , ketoconazole and itraconazole , only facilitates surgical removal of mycetoma lesions as they induce encapsulation of the fungal grain with fibrous tissue [3] , [4] . Encapsulation of the fungal grain by excessive collagen accumulation could be contributing to the in vivo observed treatment failure towards antifungal agents [1] , [2] , [5] . Collagen accumulation occurs due to a disrupted equilibrium of extracellular matrix ( ECM ) synthesis and degradation in which Matrix Metalloproteinases ( MMPs ) and Tissue Inhibitors of Matrix Metalloproteinases ( TIMPs ) play a pivotal role [6] . MMPs are classified into distinct groups according to their substrate specificity: collagenases ( MMP-1 , -8 , -13 ) , gelatinases ( MMP-2 , -9 ) , stromelysins ( MMP-3 , -10 , -11 ) , matrilysin ( MMP-7 , -26 ) , macrophage metalloestase ( MMP-12 ) , and membrane-type MMP ( MMP-14 to MMP-25 ) [6] . MMP-2 and MMP-9 have the ability to degrade a variety of ECM constituents ( e . g . gelatin , elastin , and various types of collagen ) [6] , [7] . Since both MMP-2 and MMP-9 are zymogens , proteolytic activation is prerequisite to become completely active [7] . Although it seems paradoxical , inhibition of MMP by a synthetic inhibitor decreased collagen accumulation in peritoneal sclerosis rats and bleomycin-induced pulmonary fibrotic rats [8] , [9] . In addition , accumulation of collagen is correlated with MMP-2 or MMP-9 in several pathological conditions such as atherosclerosis [10] , cardiac fibrosis in diabetic patients [11] , and granulomatous fibrosis of rats with Angiostrongylus cantonensis infection [12] , suggesting that collagen deposition can be promoted by gelatinases . Although the exact mechanism ( s ) explaining these observations have to be clarified , it is hypothesized that MMPs induce de novo ECM accumulation through its digestion of ECM constituents . Another explanation might be that MMPs provoke collagen accumulation via another pathway than ECM digestion . Neither TIMP-1 nor MMP-2 and MMP-9 have been described to be involved in mycetoma pathogenesis . In this study , it is determined if MMP-2 and MMP-9 were expressed locally in the lesion by immunohistochemistry . Furthermore , MMP activity and absolute levels of MMP-2 , MMP-9 , and TIMP-1 in sera were assessed in both mycetoma patients and healthy endemic controls . In addition , polymorphisms in promoter regions of MMP-2 , MMP-9 , and TIMP-1 were compared in both groups . The results obtained show that MMP-9 is associated with mycetoma .
Genomic DNA of 125 M . mycetomatis infected patients from Sudan ( 72 . 8% male; 27 . 2% female ) and 103 healthy endemic controls without M . mycetomatis infection ( 73 . 8% male; 26 . 2% female ) were used for genotyping . Sera from another 44 male Sudanese M . mycetomatis mycetoma patients and 44 male healthy endemic controls were used to determine levels of MMP-2 , MMP-9 , and TIMP-1 and to determine gelatinolytic activity . Tissue sections from the foot were obtained in 1998 from Sudanese patients with M . mycetomatis infection . The patients' demographic characteristics were recorded and that included gender , duration of disease , lesion size and site of infection . To localize collagen fibres , tissue sections of 8 M . mycetomatis infected subjects were stained with Sirius red staining and subsequently photographed . Immunohistochemical staining was used to assess whether MMP-2 and MMP-9 are expressed around the fungal grain . Deparaffinised tissue sections of the same 8 M . mycetomatis infected subjects used for the Sirius red staining were treated with 0 . 3% hydrogen peroxidase for 30 minutes to quench endogenous peroxidase activity . To inhibit aspecific binding of primary antibodies , specimens were incubated for 1 hour with 2% normal goat serum in PBST ( 0 . 05% Tween20 ( Sigma , Zwijndrecht , The Netherlands ) in PBS ) . Tissue sections were incubated overnight at 4°C with primary antibodies against MMP-2 ( 40 µg/ml; IM33 Calbiochem ) and MMP-9 ( 40 µg/ml; IM09L Calbiochem ) . After 1 hour incubation with goat anti-mouse IgG HRP-conjugated antibody ( 1∶200; Dako , Heverlee , Belgium ) at RT , immunoreactivity was visualized using 3-amino-9-ethyl-carbazole ( AEC; Sigma , Zwijndrecht , The Netherlands ) . Mayer's hematoxylin ( Sigma , Zwijndrecht , The Netherlands ) was used for counterstaining . As a negative control , tissues were stained without the primary antibodies being used . Absolute serum levels of MMP-2 and MMP-9 in serum of M . mycetomatis infected patients ( n = 36 ) and healthy controls ( n = 36 ) were determined utilizing Human MMP-2 and Human MMP-9 enzyme-linked immunosorbent assay ( ELISA ) kits ( cat#: RAB0365 , Sigma-Aldrich , Zwijndrecht , The Netherlands; and cat#: KHC3061 , Invitrogen , Breda , The Netherlands ) . Human TIMP-1 ELISA kit ( Cat#: OK-0163 , Assay Biotechnology , Breda , The Netherlands ) was used to assess the serum level of TIMP-1 in both study populations ( n = 44 for both populations ) . Experiments were conducted according to the manufactures instructions . Gelatinolytic activity in sera of M . mycetomatis infected patients and healthy endemic controls were determined by gelatin zymography . One µl serum was electrophoresed under non-reducing conditions on a 10% SDS-polyacrylamine gel co-polymerized with 1 mg/ml gelatin ( Fluka Analytical , Zwijndrecht , The Netherlands ) . As a positive control 0 . 4 ng activated proenzyme MMP-2 and 0 . 1 ng activated proenzyme MMP-9 ( Enzo Life Sciences , Antwerpen , Belgium ) were used . After incubating the gel four times in 2 . 5% Triton X-100 ( v/v ) ( Sigma , Zwijndrecht , The Netherlands ) for 15 minutes , the gel was incubated in developing buffer ( 50 mM Tris ( pH 7 . 5; Sigma , Zwijndrecht , The Netherlands ) , 200 mM NaCl ( Merck , Amsterdam , The Netherlands ) , 5 mM CaCl2 ( Merck , Amsterdam , The Netherlands ) and 0 . 02% BRIJ35 ( Calbiochem , San Diego , USA ) ) for 65 hours at 37°C . The gel was stained with 50% methanol ( Fisher Scientific , Landsmeer , The Netherlands ) , 20% acetic acid ( J . T . Baker , Deventer , The Netherlands ) , and 0 . 125% Coomassie Brilliant Blue R-250 ( Sigma , Zwijndrecht , The Netherlands ) and destained with destaining solution ( 30% methanol and 1% formic acid ( J . T . Baker , Deventer , The Netherlands ) ) until transparent lysis bands were visible . Functional SNPs in promoter regions of MMP-2 ( −1306 C/T ) , MMP-9 ( −1562 C/T ) , and TIMP-1 ( +372 C/T ) , associated with altered transcriptional activity [13]–[15] , were genotyped utilizing genomic DNA of 125 M . mycetomatis infected patients and 103 healthy controls . To determine MMP-2 ( −1306 C/T ) genotype , DNA was isolated as described before [16] , [17] and amplified using primers 5′-CTTCCTAGGCTGGTCCTTACTGA-3′ and 5′-CTGAGACCTGAAGAGCTAAAGAGCT-3′ . The PCR reaction consisted of 40 cycles of 30 s denaturation at 94°C , 30 s annealing at 58°C and 30 s elongation at 72°C . The genotype of the resulting amplicon was determined by restriction fragment length polymorphism ( PCR-RFLP ) with SphI . To determine the MMP-9 ( −1562 C/T ) genotype , DNA was amplified using primers 5′-GCCTGGCACATAGTAGGCCC-3′ and 5′CTTCCTAGCCAGCCGGCATC-3′ . The PCR reaction was similar to the one described for the MMP-2 ( −1306 C/T ) polymorphism , only the annealing temperature was changed to 65°C . The genotype of the resulting amplicon was determined by restriction fragment length polymorphism ( PCR-RFLP ) with BfaI . The TIMP-1 ( +372 C/T ) genotype was identified by sequencing after amplification with primers 5′-GCACATCACTACCTGCAGTC-3′ and 5′-GAAACAAGCCCACGATTTAG-3′ . Deviation from Hardy-Weinberg equilibrium for each polymorphism was calculated by the Pearson's χ2 test . Differences in categorical variables and continuous variables between M . mycetomatis infected patients and reference group were tested with Fisher's exact or Mann-Whitney test respectively . Statistical comparisons were carried out using GraphPad Prism 5 . 0 or GraphPad InStat 3 . 0 ( GraphPad Software , San Diego California USA ) . P<0 . 05 was considered to be statistically significant . Written informed consent was obtained from all participants and ethical clearance was obtained from Soba University Hospital Ethical Committee , Khartoum , Sudan .
Collagen accumulation around the fungal grain was assessed by staining specimens of M . mycetomatis infected subjects by Sirius red . A representative photomicrograph of a Sirius red stained tissue section shows that the fungal grain is encapsulated with collagen deposition ( Figure 1b ) . After this first collagen deposition ring , often a denser collagen capsule is seen at some distance of the grain . In that capsule typical collagen fibres are noted . In order to determine if the gelatinases MMP-2 and MMP-9 play a role in the encapsulation of the mycetoma grain , the presence of these two MMPs was demonstrated by immunohistochemical staining of tissue sections of patients infected with M . mycetomatis ( Figures 1c and 1d ) . As is seen in figure 1c and 1d , both MMP-2 and MMP-9 were detectable as red cytoplasmatic staining in cells , mainly in zone 2 surrounding the grain . In the neutrophil zone ( zone 1 ) , little expression of either metalloproteases was noted , although in some patients also this zone showed expression of MMP-2 and MMP-9 . Strikingly expression was mainly found in areas where little collagen deposition was seen . If there was heavy collagen deposition hardly any MMP-2 and MMP-9 expression was noted ( figure 1 ) . In slides where primary antibodies were omitted , coloration was absent ( not shown ) . In order to determine if the MMP-2 and MMP-9 expression was also found in serum , ELISAs were performed to determine the concentrations of MMP-2 and MMP-9 in sera of mycetoma patients and healthy controls . It appeared that MMP-2 was hardly detected in sera of either patients or controls ( Figure 2A , MMP-2 median 0 ng/ml for both groups ) . There were no differences between the patients and the healthy controls ( Mann-Whitney , p = 0 . 42 ) . Also , similar concentrations of MMP-9 were found in sera of both M . mycetomatis infected patients and controls ( Figure 2B , median concentration 451 . 6 ng/ml versus 461 . 2 ng/ml , respectively; Mann-Whitney , p = 0 . 57 ) . The drawback by measuring MMP-2 and MMP-9 concentrations by ELISA is that it is not possible to distinguish between inactive and active MMP-2 and MMP-9 . In order to distinguish between active and inactive gelatinase in sera of M . mycetomatis infected patients and healthy endemic controls , gelatin zymography was used . Characteristic gelatinolytic patterns due to the presence of pro-active and active forms of MMP-2 and MMP-9 in sera of M . mycetomatis infected patients and healthy controls are depicted in Figure 3 . Active MMP-9 of 84 kDa was found in sera of 36% of M . mycetomatis infected subjects , whereas this active form was not present in sera of the control population ( Fisher Exact , p<0 . 0001 ) . No correlation was found between the presence of active MMP-9 and lesion size or disease duration ( data not shown ) . Active MMP-2 ( 62 kDa ) was absent in both groups . The pro-active forms of MMP-2 and MMP-9 , 72 and 92 kDa respectively , were present in sera of all M . mycetomatis infected patients and healthy controls . Since mycetoma patients had more often active MMP-9 in their sera , while the total amount of MMP-9 ( both active and inactive ) did not differ , it was investigated if TIMP-1 levels differed between patients and healthy endemic controls by ELISA . This was done since TIMP-1 is known to block protease activity of both MMP-2 and MMP-9 . It appeared that TIMP-1 serum levels of both groups did not significantly differ ( Figure 2C , median 195 . 8 ng/ml for M . mycetomatis infected patients vs . 170 . 0 ng/ml for controls; Mann-Whitney , p = 0 . 99 ) . Ratios of MMP-9 to TIMP-1 were comparable and did not reach statistical significance ( Figure 2D; Mann-Whitney , p = 0 . 59 ) . In order to determine if the difference in active MMP-9 levels was the result of genotypic differences between patients and healthy controls , we determined whether allele frequencies in functional polymorphisms in MMP-2 , MMP-9 and TIMP-1 differed between M . mycetomatis infected patients and healthy controls by SNP analyses . All studied genotypes did not show deviation from Hardy-Weinberg equilibrium ( p>0 . 05 ) . Allele frequencies of MMP-2 , MMP-9 , and TIMP-1 polymorphisms were compared between M . mycetomatis infected patients and healthy controls ( Table 1 ) . Since the TIMP-1 gene is X-chromosome located , genotype analyses were stratified according to gender . The allele distributions for MMP-2 ( −1306 C/T ) and MMP-9 ( −1562 C/T ) polymorphisms did not significantly differ between M . mycetomatis infected subjects and controls ( p = 0 . 39 and p = 1 . 00 respectively ) . The T allele frequency in TIMP-1 ( +372 C/T ) polymorphism was significantly higher in male M . mycetomatis infected patients compared to the male reference group ( 46% versus 26% ) ( p = 0 . 0004 ) . In female M . mycetomatis infected patients the allelic distribution in TIMP-1 ( +372 C/T ) polymorphism did not significantly differ with female control subjects ( p = 0 . 53 ) .
Eradication of M . mycetomatis mycetoma remains challenging as in vivo treatment failure towards currently used antifungal agents is frequently observed . It has been reported that collagen accumulation contributes to limited penetration of chemotherapeutic agents into the granuloma [18] , suggesting that a dense collagen network might influence drug accessibility . Therefore , diminished response upon antifungal treatment might be partly caused by excessive collagen accumulation in the mycetoma lesion . Unravelling the mechanism behind observed changes in tissue architecture around the fungal grain could direct to novel therapeutic options . In this study we investigated suitable candidates , MMP-2 , MMP-9 , and TIMP-1 , as they are participants in ECM remodeling . Both MMP-2 and MMP-9 were found to be expressed in the mycetoma lesion , and both were highly expressed locally surrounding the fungal grain . Constitutive expression of MMP-2 takes place in various cell types and is barely induced under pro-inflammatory conditions [19] . Furthermore , one of the characteristics of mycetoma is that during grain formation high amounts of neutrophils are recruited to the site of M . mycetomatis infection [17] . MMP-9 is constitutively expressed and stored in high quantities in granules of neutrophils , and several chemotactic chemokines and cytokines are able to induce degranulation of MMP-9 containing granules [20] , [21] . Furthermore , inflammatory stimuli are able to upregulate MMP-9 expression in a wide range of inflammatory cell types , such as lymphocytes , monocytes , and neutrophils [19] , [22] . Absolute MMP-2 , MMP-9 , and TIMP-1 serum levels were comparable between M . mycetomatis infected patients and healthy controls and did not reach statistical significance . However , since the ELISA measured both the pro-active and the active forms of MMP-2 and MMP-9 , these observations have only a limited value . Therefore , MMP-2 and MMP-9 activity was tested by gelatin zymography . Despite comparable absolute serum levels in both groups , MMP-9 activity was significantly higher in the M . mycetomatis mycetoma population . A higher MMP-9 activation could be the result of a higher MMP-9 expression or a lower TIMP-1 expression . TIMP-1 inhibits MMP-9 activity by forming a 1∶1 stoichiometric non-covalent complex [6] . Disruption of MMP-9:TIMP-1 complexes result in release and activation of MMP-9 . Several other participants in MMP-9 activation have been described , including protease-based activators ( e . g . trypsin [23] and neutrophil-derived elastase [24] ) and other MMPs [21] . Although we only found activated MMP-9 found in mycetoma patients , there was still a large proportion of the patients in which we did not find the activated form . Similar findings were reported for patients with severe sepsis [25] . Only in 10 out of 20 patients with severe sepsis on the intensive care unit , activated MMP-9 was found [25] . Again no correlation with disease severity was noted [25] . Why we measured in one patient active MMP-9 and the other not is not clear . Several reasons could be attributing . First of all , we only took one time-point and these time-points differ for each patient . MMP-9 expression could be dependent on the disease stadium , although we did not find a correlation with the disease duration or the size of the lesion , other factors might be responsible such as if the patient had at the time of sampling discharging sinuses or not . Furthermore we did not record if the patient had other infections . Furthermore , it is also plausible that co-infections could play are role since they are frequently reported in mycetoma [26] and a correlation between mycetoma and schistomiasis was also recently reported [27] . Differences in MMP-9 expression between the M . mycetomatis infected patients individually and between patients and healthy endemic controls as a group could also be caused by genetic differences . We therefore genotyped functional polymorphisms in the promoter regions of MMP-2 ( −1306 C/T ) , MMP-9 ( −1562 C/T ) , and TIMP-1 ( +372 ) . While no significant difference in allele distributions in the MMP-9 ( −1572 C/T ) polymorphism was found , other SNPs in the promoter region of MMP-9 or MMP-9 itself were not investigated and could contribute to increased MMP-9 activation in M . mycetomatis infected patients . In contrast , a genetic difference between both groups was found for TIMP-1 ( +372 SNP ) . The T allele frequency in TIMP-1 SNP in male M . mycetomatis infected patients was significantly higher compared to healthy controls . In man , T allele associated transcriptional activity of TIMP-1 is lower than C allele associated transcriptional activity [15] , suggesting that TIMP-1 production and thereby MMP inhibition in these subjects is reduced . This finding might explain previously reported male predominance in mycetoma [1] . Due to lower T allele associated transcriptional activity of TIMP-1 , we expected reduced TIMP-1 serum levels in M . mycetomatis infected subjects , but this was not the case . However , since mycetoma is a localized infection , a localized reduction of TIMP-1 could result in higher MMP-9 levels in the lesion , which , in its turn , could be found in serum . In this study we showed that collagen is indeed encapsulating the grain and MMP-9 is the collagenase activated during M . mycetomatis infection . The question remains what the exact function of grain encapsulation is . Is this encapsulation beneficial to the host , by keeping the M . mycetomatis infection localized and preventing the spread of infection ? Or prevents the collagen capsule surrounding the fungal grain the penetration of drugs into the grain ? If the latter would be the case , one could consider adding the antimicrobial agent doxycycline to the currently used therapeutic strategy . Doxycycline is a potent MMP inhibitor which is able to reduce MMP-2 and MMP-9 mRNA expression and MMP-2 production in vitro and thereby attenuates collagen accumulation in pulmonary fibrosis [28] . By attenuating the collagen deposition around the grain , ketoconazole and itraconazole might be able to better penetrate to the fungus . In summary , the results obtained in the present study show increased MMP-9 activity during M . mycetomatis infection , suggesting that MMP-9 is associated with M . mycetomatis mycetoma . | Eumycetoma , mainly caused by the fungus Madurella mycetomatis , is a chronic infection which , without treatment , results in deformation of the infected body part . Inside the body , the fungus organises itself in grains which are surrounded by collagen . This collagen could act as a natural barrier for antifungal agents . Since collagen modulation is regulated by matrix metalloproteinase-2 ( MMP-2 ) , MMP-9 and tissue inhibitors of metalloproteinases ( TIMPs ) , these enzymes could play a role in the formation of the collagen capsule surrounding the fungal grain . Indeed , we demonstrated that MMPs were found surrounding the mycetoma grain and that measurable levels of both MMPs were found in serum of both mycetoma patients and healthy controls . Only in mycetoma patients the active form MMP-9 was found . The presence of active MMP-9 in the serum of mycetoma-patients was not the result of lower levels TIMP-1 but more likely from differences in allele frequencies in the TIMP-1 gene . In conclusion , our results showed an increased MMP-9 activity in mycetoma patients . We hypothesize that inhibition of MMP-9 activity by doxycycline will result in breakdown of the collagen capsule surrounding the grain , which in turn will make the entrance of antifungal drugs into the grain easier . | [
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] | 2014 | Active Matrix Metalloprotease-9 Is Associated with the Collagen Capsule Surrounding the Madurella mycetomatis Grain in Mycetoma |
The endoplasmic reticulum ( ER ) responds to changes in intracellular homeostasis through activation of the unfolded protein response ( UPR ) . Yet , it is not known how UPR-signaling coordinates adaptation versus cell death . Previous studies suggested that signaling through PERK/ATF4 is required for cell death . We show that high levels of ER stress ( i . e . , ischemia-like conditions ) induce transcription of the ubiquitin ligases Siah1/2 through the UPR transducers PERK/ATF4 and IRE1/sXBP1 . In turn , Siah1/2 attenuates proline hydroxylation of ATF4 , resulting in its stabilization , thereby augmenting ER stress output . Conversely , ATF4 activation is reduced upon Siah1/2 KD in cultured cells , which attenuates ER stress-induced cell death . Notably , Siah1a+/−::Siah2−/− mice subjected to neuronal ischemia exhibited smaller infarct volume and were protected from ischemia-induced death , compared with the wild type ( WT ) mice . In all , Siah1/2 constitutes an obligatory fine-tuning mechanism that predisposes cells to death under severe ER stress conditions .
The endoplasmic reticulum ( ER ) plays a central role in the folding , assembly , and modification of secretory and cell membrane proteins [1] , [2] . Deregulated protein folding affects diverse cellular processes , including transcription , translation , cell cycle , and cell death [3] , [4] . The ER responds to exogenous and endogenous stressors that can disrupt protein folding by increasing its protein folding capacity through specialized signaling pathways that are collectively known as the unfolded protein response ( UPR ) [3]–[6] . The UPR increases transcription of many genes encoding functions in protein folding and secretion , and thus constitutes a coordinated regulatory mechanism that restores protein-folding fidelity in the ER and reestablishes normal cellular homeostasis [1]–[4] , [7] . The UPR is coordinated by three main ER-proximal sensors that respond to increased levels of unfolded proteins: ATF6α ( activating transcription factor 6 ) , IRE1α ( inositol-requiring protein 1 ) , and PERK ( PKR-like ER kinase ) [3] , [4] , [7] . ATF6α is proteolytically cleaved upon trafficking to the Golgi to generate the soluble active product , which initiates a transcriptional program to relieve ER stress [8] , [9] . IRE1 undergoes autophosphorylation , which activates its intrinsic RNase activity and leads to splicing of XBP1 mRNA to produce the active transcription factor sXBP1 [10] , [11] . Activated PERK phosphorylates the eukaryotic initiation factor 2 on the alpha subunit ( eIF2α ) , resulting in an overall attenuation of mRNA translation [12] , [13] . Although global protein production is reduced following UPR , the translation of a select group of mRNAs , including the transcription factor ATF4 , is increased following PERK activation , via alternative AUG initiation codon selection that occurs when eIF2α is inactivated by phosphorylation [13]–[15] . ATF4 counters the UPR by inducing the expression of genes that improve ER protein folding capacity , facilitate amino acid biosynthesis and transport , and reduce oxidative stress , as well as the pro-apoptotic factor C/EBP homologous protein CHOP [14] , [16]–[19] . ER stress response , as part of the UPR , can facilitate the restoration of cellular homeostasis , via the concerted activation of ERAD and the respective transcriptional program ( i . e . , chaperones ) induced by the ATF4 , sXPB1 and ATF6α [4] , [7] . However , severe ER stress often results in the activation of the cell death program , which is mediated by the UPR transducers CHOP and PUMA [20] , [21] . Notably , the mechanism underlying the ability of the UPR to divert cellular survival to death pathways is not well understood . Here we identify the ubiquitin ligases Siah1/2 as important regulatory components in the UPR , which serves as rheostats that can dial up the degree of ER stress response to induce cellular changes that promote cell death . Our data establish the role of Siah1/2 ubiquitin ligases in fine-tuning of the cellular UPR . Siah1/2 are RING finger ubiquitin ligases that are evolutionarily conserved from Drosophila melanogaster to vertebrates [22] . The two human isoforms , Siah1 and Siah2 , and the three murine homologues ( Siah1a , Siah1b , and Siah2 ) are implicated in ubiquitin-dependent degradation of proteins that play key roles in hypoxia and MAPK signaling pathways [23]–[25] . Siah1/2 controls the Ras/Raf signaling pathways and contributes to tumorigenesis by controlling the regulatory protein Sprouty2 [26] , [27] . Regulation of the scaffolding protein AKAP121 by Siah2 controls mitochondrial fission under low oxygen conditions , which contributes to ischemia in a mouse myocardial infarction model [28] . Siah1/2 plays an important role in the control of the overall hypoxia response through its effects on the stability of HIF1α , as by affecting the level of HIPK2 ( homeodomain-interacting protein kinase 2 ) and FIH ( factor-inhibiting hypoxia-inducible factor ) , which controls HIF1α own transcription and activity , respectively [29] , [30] . HIF1α degradation requires prolyl hydroxylation by the PHD enzymes that enable its recognition and subsequent degradation by pVHL [31] , [32] . Whereas PHD2 plays key roles in HIF1α hydroxylation under normoxia , PHD3 and PHD1 , which are regulated by Siah2 , contribute to the control of HIF1α availability under physiological hypoxic conditions ( 3–6% O2 ) [25] , [33] , [34] . Correspondingly , Siah1/2 mutant mice exhibit phenotypes resembling those seen in HIF1α heterozygous animals [33] . Siah1/2 deletion in mice attenuates the growth or progression of prostate or melanoma tumors , in a HIF1α-dependent manner [27] , [35] and attenuates breast , lung , and pancreatic cancer development [36]–[38] . Genetic inactivation of Siah1/2 also protects from acute myocardial infarction in mice , and affects life span in worms [28] . Mechanisms underlying the regulation of Siah1/2 transcription are confined to Siah1 ( but not Siah2 ) , which is transcriptionally induced by p53 following DNA damage [39]–[42] . Here , we identify a mechanism that establishes Siah ubiquitin ligases as integral components of the ER stress response , which fine-tunes the magnitude of UPR , directing cell death programs in response to severe ER stress conditions .
To identify physiological conditions that affect Siah1/2 transcription , we examined cellular responses to key stimuli that are known to influence Siah1/2 substrates or Siah1/2-dependent processes . We considered the UPR as one such condition , given that the Siah1/2 substrates TRAF2 , Sprouty2 and PHD3 are implicated in the UPR as part of the ER stress response [43]–[45] . To determine whether conditions that induce UPR also affect Siah2 regulation , we examined Siah2 transcription and protein expression following exposure of cells to the glycosylation inhibitor tunicamycin ( TM ) , which is commonly used to induce UPR . Siah2 protein is barely detectable under normal growth conditions , due to its efficient self-degradation [33] , [46] . TM treatment increased Siah2 protein expression in mouse embryo fibroblasts ( MEFs ) from WT but not Siah1a−/−::Siah2−/− knockout ( KO ) mice ( Figure 1A ) . Similarly , Siah2 protein expression was increased following treatment of 293T cells with TM ( not shown ) . Since Siah1 is often found to augment Siah2 activity , we also monitored the possible induction of Siah1 by UPR . Siah1 protein expression was found to increase following treatment of MEF cells with TM ( Figure 1B ) . Noteworthy are the levels of ATF4 , which appear to correspond to Siah1/2 expression , implying a possible regulatory link between Siah1/2 and ATF4 . We observed a dose-dependent increase in levels of Siah2 mRNA in both MEFs ( >4-fold; Figure 1C ) and 293T cells ( not shown ) following TM treatment . Addition of TM also caused a notable increase in Siah2 mRNA levels in several other cell lines including HeLa , and the melanoma cell line LU1205 ( >3-fold; Figure 1D ) . Collectively , these results indicate that conditions that induce the UPR stimulate Siah2 expression at the RNA and protein levels . Siah1 transcription was also induced following treatment by TM , but to a lower degree ( ∼2 . 5-fold ) compared with that of Siah2 ( ∼12-fold , Figure 2A ) . Of note , murine Siah1 consists of two isoforms ( Siah1a and Siah1b ) , which share 93% homology at the mRNA level . Thus , the ability to distinguish between the two Siah1 murine isoforms , at the protein or RNA levels is limited . To determine whether Siah1a or Siah1b ( or both ) are induced following TM treatment , MEFs from Siah1a−/−::Siah2−/− KO mice ( where Siah1a gene is deleted ) were treated with TM . Addition of TM did not cause any induction of Siah1 in Siah1a−/−::Siah2−/− KO cells , implying that Siah1b transcription is not induced after TM treatment ( Figure 1B ) . We therefore concluded that the changes we monitor in the WT MEFs are likely to be of the Siah1a isoform ( data shown below , Figure 2A ) . To explore the newly identified link between the UPR and Siah2 expression we asked whether Siah2 transcription was regulated by transducers of the UPR . ATF4 is one of several key UPR transcriptional activators [14] , [47] , [48] , suggesting it may play a role in Siah2 transcription . Indeed , we found that TM-stimulated Siah2 transcription was reduced from 8-fold in WT MEFs to ∼2 . 2-fold in MEFs from Atf4−/− mice ( Figure 1E ) . These findings provide genetic evidence for a role of ATF4 in the regulation of Siah2 transcription in response to UPR . Hypoxia , which was previously shown to stimulate Siah2 transcription [33] , elicited a less pronounced effect than TM on Siah2 transcription ( 2 . 2-fold vs . 8-fold; Figure 1E ) . Corresponding changes in ATF4 transcriptional targets , CHOP and VEGFA were observed under these conditions ( Figures 1F , G ) . To confirm that ATF4 is critical for Siah2 transcription in response to UPR , we re-expressed ATF4 in Atf4−/− cells . Indeed , forced expression of ATF4 ( Figure 1H ) enhanced induction of Siah2 transcription following treatment with TM ( Figure 1I ) , suggesting that ATF4 is required for induction of Siah2 transcription following UPR stimuli , mediated here by TM . To determine the degree of ATF4-dependent activation of Siah1 and Siah2 following UPR stimuli , MEFs from ATF4 WT and KO genotypes were subjected to TM treatment . TM induced Siah2 mRNA to a greater degree than Siah1 , and ATF4−/− MEFs exhibited a more pronounced reduction of Siah2 transcription ( Figure 2A ) . These data indicate that the Siah1 and Siah2 genes are transcriptionally induced by the UPR through ATF4 , with Siah2 being the primary responder to the UPR . We next determined whether other major UPR transducers , IRE1α/sXBP1 and ATF6α , another key UPR transcription factor [49] , play a role in the activation of Siah transcription . Whereas TM- and more effectively thapsigargin ( TG ) -induced Siah2 transcription to a greater degree ( 8- and ∼13-fold , respectively ) than Siah1 transcription ( ∼2 . 7–5 and ∼4-fold , respectively ) , in Ire1α−/− MEFs Siah2 activation was attenuated to a greater degree , compared with Siah1 ( Figure 2B ) . Notably , re-expression of sXBP1 in Ire1α−/− cells effectively rescued expression of Siah2 ( Figure 2C ) . Similar analysis in Atf6α−/− MEFs revealed a role of ATF6α in Siah2 transcription ( Figure 2D , right panel ) , albeit lower than was observed for ATF4 and IRE1α . In contrast , ATF6α did not have an effect on Siah1 transcription ( Figure 2D , left panel ) . Collectively these data suggest that the UPR induced Siah2 transcription to a greater degree than the transcription of Siah1 , and that among the UPR sensors , ATF4 is the most potent mediator of this activation , followed by sXBP1 , and to lesser degree ATF6α . Since hypoxia was also found to affect Siah2 transcription , albeit to lower degree , we assessed whether the activation of Siah1/2 transcription upon UPR may be mediated by HIF1α . To this end we assessed the degree of TM/TG activation of Siah1/2 in WT versus HIF1α−/− MEFs . Notably , the lack of HIF1α did not elicit a marked effect on the level of Siah1/2 transcriptional activation in response to UPR stimuli ( Figure 2E ) , suggesting that the induction of Siah1/2 transcription under these conditions is HIF1α-independent . We next asked whether the effect of ATF4 on Siah2 transcription was reflected in Siah2 ubiquitin ligase activity . Overexpression of ATF4 increased the protein levels Siah2 , with concomitant decrease in the protein levels PHD3 and OGDH , representing Siah2 substrates ( Figure 3A ) . In agreement , re-expression of ATF4 in Atf4−/− cells that were maintained under hypoxic conditions increased expression levels of HIF1α ( PHD3 substrate regulated by Siah2 ) while reducing the expression of a Siah2 substrate AKAP121 ( Figure 3B ) , consistent with increased Siah2 availability ( Figure 3B ) . This finding supports a role for the UPR-induced transcription factor ATF4 in the regulation of Siah2 ubiquitin ligase activity . The finding that PERK/ATF4 and IRE1α/sXBP1 increase the level of Siah1/2 mRNA prompted us to search for ATF4 and sXBP1 response elements within the Siah2 promoter region . To this end , we cloned a 2 kb fragment ( position −1652; +620 ) including a region upstream of the start site and the 5′UTR , and two intronic regions into a luciferase reporter construct ( Figure 3C ) . The luciferase activity driven by the 2 kb promoter region containing the 5′-UTR revealed a marked ( 10-fold ) ATF4-dependent increase in ( Figure 3D ) . Within this region we mapped two putative ATF4 response elements ( TTxCATCA; Figure 3C ) , which were then mutated individually . Mutation of only one of the two elements , which are conserved in human and mouse ( +308 relative to the transcription start position; ATF4 M ) , resulted in a marked decrease in ATF4-dependent luciferase activity ( Figure 3D ) . Chromatin immuneprecipitation ( ChIP ) experiments confirmed that ATF4 bound directly to the Siah2 promoter element within 308–317 following TG treatment ( Figure 3E ) . Together , these data establish that expression of Siah2 mRNA is regulated by ATF4 , and that such regulation plays an important role in upregulating Siah2 expression in response to ER stress . The Siah2 promoter also contains two putative HIF1α response elements ( HRE ) that can be occupied by HIF1α , which overlaps the sXBP1 binding site ( Figure 3C ) . To map putative sXBP1 sites , we monitored luciferase activity driven by Siah2 promoter , which was mutated on the primary putative sXBP/HRE sites . Luciferase activities identified the sXBP1 response element at position +287 , but not the one at position +272 , as the site responsible for the activation of Siah2 transcription ( Figure 3F ) . Mutation of the 287 site attenuated sXBP1-dependent activation of Siah2 promoter-driven luciferase activity ( Figure 3F ) . Finally , ChIP experiments confirmed that sXBP1 bound to Siah2 promoter following treatment of cells with TG ( Figures 3C , G ) . These data establish the regulation of Siah2 mRNA levels by two of the three UPR signaling sensors—PERK/ATF4 and IRE1α/sXBP1 . Notably , ATF4 and sXBP1 occupy distinct sites within the Siah2 promoter . Similar analysis , performed for the Siah1a promoter , identified ATF4 binding within the first intron of Siah1a ( +4710 , Figure 3H ) , consistent with previous ChIP-Seq reports [19] . These findings demonstrate the regulation of Siah1a/2 transcription by the UPR transducers ATF4 and sXBP1 . Gene expression profiling was performed to identify Siah1/2-dependent changes in cells subjected to ER stress , hypoxia ( 1% O2 ) , and combined glucose/oxygen deprivation . To this end , we compared gene expression profiles of WT and Siah1a−/−::Siah2−/− MEFs that were subjected to TM , TG , hypoxia ( oxygen deprivation ) , glucose deprivation , or glucose+oxygen deprivation . When comparing all six experimental conditions using Ingenuity Pathway Analysis ( IPA; canonical pathway analysis ) , the most significant changes in gene expression between WT and Siah1a−/−::Siah2−/− expressing cells were in the HIF1α/hypoxia signaling pathway ( Figure 4A ) , consistent with our earlier studies showing that Siah2 affects this pathway through its regulation of PHD1/3 stability [33] . Significantly , this analysis also supports the role of Siah1/2 in the regulation of genes implicated in the ER stress response . As shown in the general heat map , major clusters identified to be Siah1/2-dependent included diabetes and metabolism , in addition to hypoxia signaling . Notably , clusters associated with pathogen infection , which were previously associated with the ER stress response [50]–[52] , were also identified ( Figure 4A; Table 1 ) . The Venn diagram ( Figures 4B , C ) represents the overlaps of significantly down- ( green ) , and up- ( red ) regulated genes ( listed in Tables 2–3 ) involved in the functional groups as indicated in the heatmap . A principal component analysis ( PCA ) for the microarray data set confirmed the clustering of the individual arrays based on their treatment groups ( Figure 4D ) . Approximately 35 genes were up- or down-regulated between the hypoxia ( OD ) , hypoxia and glucose deprivation ( OGD ) and control groups , among the WT and Siah1a−/−::Siah2−/− cells . Those genes were primarily clustered within the hypoxia and diabetes signaling pathways . Somewhat similarly , the comparison among the WT and Siah1a−/−::Siah2−/− groups subjected to TG or TM versus control identified 27 up-regulated and 13 down-regulated genes , which primarily clustered within the cellular movement and development functional groups . qPCR analysis of representative genes from each of the major three clusters , confirmed the changes predicted by the expression array ( Figures 4E–H ) . We next compared the expression of genes that were significantly altered in a Siah2- dependent manner with previously reported datasets for ATF4- , PERK- , and HIF1α-dependent gene expression . Gene Set Enrichment Analyses ( GSEA ) confirmed the effect of Siah1/2 on expression of genes associated with ER stress , metabolic signaling and ER-Golgi transport ( Table 4 ) . Representative genes from each of these clusters were confirmed by qPCR ( Figures 4I–K ) . Taken together , this analysis confirmed changes associated with ER stress that had not previously been associated with Siah1/2 signaling , substantiating Siah1/2 as important coordinator of ER stress through the ATF4 and sXbp1 pathways . Independent studies demonstrated that the prolyl hydroxylases PHD1 and 3 negatively regulate the protein level and transcriptional activity of ATF4 [45] , [53] , [54] . We thus tested whether the regulation of Siah1/2 transcription by ATF4 constitutes a feed-forward loop in which Siah2 degradation of PHD1/3 directly increases the availability and transcriptional activity of ATF4 . Given the greater effect of the UPR on Siah2 transcription , ATF4 protein and transcript levels were measured in cells transfected with Siah2 . We found that ATF4 protein levels were increased in cells expressing ectopic Siah2 ( Figure 5A ) , with minor changes in ATF4 transcript levels ( Figure 5B ) . Notably , the UPR-induced increase in ATF4 protein levels was attenuated in Siah1a−/−::Siah2−/− MEFs and in primary keratinocytes from Siah1a+/−::Siah2−/− mice ( Figure 5C ) , suggesting that Siah1/2 increases ATF4 protein expression in response to UPR . Ectopic expression of Siah2 ( Figure 5D ) also increased the transcription of the ATF4 target genes CHOP and VEGFA in an ATF4-dependent manner , as the Siah2 effect was largely abolished in Atf4−/− cells ( Figures 5E , F ) . Whereas hypoxia did not induce a notable increase in the levels of CHOP mRNA , it induced VEGF mRNA , to a similar degree as observed upon Siah2 expression alone ( Figures 5E , F ) , suggesting that the Siah2-ATF4 axis also contributes to the increase in VEGFA mRNA levels under hypoxia . Moreover , the elevated ATF3 mRNA levels following TM treatment were attenuated in Siah1a−/−::Siah2−/− MEFs compared with WT MEFs ( Figure 5G ) . These data point to the contribution of Siah2 to ATF4-dependent transcription in the UPR , thereby constituting a feed forward mechanism for the UPR . Because PHD3 reduces ATF4 transcriptional activity [45] , [53] , [54] , we next assessed whether the effect Siah2 elicited on ATF4 activity is PHD3-dependent . Therefore , we monitored changes in the levels of ATF4 target genes in Siah1a−/−::Siah2−/− MEFs transfected with PHD1 and PHD3 shRNA either alone or in combination . Expression of PHD1/3 shRNA in Siah1/2 DKO cells reduced PHD1/3 mRNA level by >50% ( Figures 6A , B ) while increasing the expression of CHOP mRNA following TM ( Figure 6C ) or hypoxia treatment ( Figure 6D ) . Consistent with earlier reports [33] , PHD3 protein levels increased in Siah1a−/−::Siah2−/− MEFs ( Figure 6E ) . These findings confirm the role of Siah2 , via its regulation of PHD3 stability , in the feed-forward regulation of ATF4 . We next determined whether PHD1/3 affects ATF4 by prolyl hydroxylation , similar to its control of HIF1α stability . ATF4 , which enriched by immunoprecipitation from HEK293T cells that were transfected with both Flag-ATF4 and Myc-PHD3 , was subjected to liquid chromatography tandem mass spectrometry ( LC-MS/MS ) . This analysis identified two proline hydroxylation sites at P235 and P60 ( Figure 6F ) , of which the P235 site is conserved among species ( data not shown ) . ATF4 with proline mutated to alanine at each individual or both sites was assessed for altered expression level . Notably , mutation of either P60 or P235 increased the steady state levels of ATF4 , with the double mutant revealing a more pronounced increase ( Figure 6G ) . Further , inactivation of PHD ( DMOG ) or proteasomes ( MG132 ) further increased the expression levels of ATF4 , suggesting that additional factors may contribute to its overall expression ( both transcriptionally and post translationally ) . To determine the significance of Siah activation by- and its augmentation of- the UPR we monitored the changes in Siah activation and its possible contribution to different forms of ER stress . Oxidative and ER stresses both effectively induced Siah2 mRNA levels , with TG and histidinol eliciting the greatest and the least effects , respectively ( Figure 7A ) . To determine the conditions required for Siah activation by the UPR , we monitored the degree of its transcriptional activation following different levels of ER stress . Relatively mild levels of ER stress , which are elicited upon the inducible expression of coagulation factor VII [55] ( Figure 7B ) , increased the transcript levels of CHOP ( Figure 7C ) and ATF3 ( not shown ) , but not of Siah1 ( Figure 7D ) or Siah2 transcripts ( Figure 7E ) . Similarly , the treatment with increasing doses of TM revealed that only concentrations in excess of 0 . 25 µg/ml resulted in notable activation of Siah1/2 transcription ( Figure 7F ) . These observations suggest that low levels of ER stress do not engage Siah1/2 in the ER stress response . Conversely , exposure to severe ER stress conditions resulted in marked increase of Siah1/2 transcription . Severe ER stress is often seen under conditions of glucose and oxygen deprivation , as occurs in cachexia , ischemia , or tumorigenesis [56]–[62] . Therefore , we examined the effects of oxygen and glucose deprivation on Siah2 and ATF4 expression . While there was limited change in Siah2 or ATF4 mRNA levels in cells deprived of glucose alone , deprivation of both glucose and oxygen resulted in a marked increase ( 6-fold ) in Siah2 mRNA levels ( Figure 7G ) . To further substantiate the link between Siah and the UPR we assessed possible changes in Siah2 activation in cells with homozygous Ser51Ala knock-in mutation at the phosphorylation site in eIF2α , a key component in the PERK–ATF4 pathway . Phosphorylation mutant of eIF2α abolishes ATF4 synthesis , while causing severe ER stress due the inability to attenuate protein synthesis and the lack of compensatory mechanisms activated by ATF4 . Thus , we assessed whether activation of Siah2 following UPR would mirror that of ATF4 and whether this activation is dependent on the phosphorylation of eIF2α . Strikingly , Siah2 expression at both the Siah2 protein ( Figure 7H ) and RNA levels ( Figure 7I ) phenocopied that of ATF4 , as it was no longer observed following ER stress in the phosphomutant eIF2α cells . These findings further substantiate the integral role of eIF2α phosphorylation-dependent ATF4 synthesis with a concomitant effect on Siah2 transcription and contribution to the UPR . The induction of Siah2 by glucose/oxygen deprivation prompted us to determine the effects of these conditions on ATF4 target gene expression . Notably , the mRNA levels of ATF3 , CHOP , and VEGFA were markedly increased under conditions of glucose and oxygen deprivation ( 4- to 6-fold ) . Significantly , this effect was Siah1/2-dependent because the increase was essentially abrogated in Siah1/2 DKO cells ( Figures 8A–C ) , consistent with attenuated expression of ATF4 protein ( Figure 9A ) . The contribution of Siah2 ( Figure 8D ) was more pronounced as compared with that of Siah1 ( Figure 8E ) , consistent with our earlier experiments . There were marginal differences in ATF4 mRNA expression under the same experimental conditions between Siah1a+/+:Siah2+/+ and Siah1a−/−:Siah2−/− cells ( Figure 8F ) . To determine the effects of Siah1/2 on PHD1 and PHD3 protein levels during UPR , WT and Siah1a−/−::Siah2−/− ( DKO ) MEFs were subjected to glucose and oxygen deprivation . Notably , glucose and oxygen deprivation caused increase in PHD3 protein levels in Siah1/2 DKO MEFs , but not in WT MEFs . Further , PHD1 protein level decreased in WT , but not in Siah1/2 DKO MEFs subjected to OGD ( Figure 8G ) . Cycloheximide chase analysis allowed us to follow changes in the half-life of the PHD3 protein in Siah1/2 WT and DKO MEFs that were subjected to OGD . The short half-life of PHD3 was detectable in Siah1/2 DKO MEFs ( at the 15 minute time point ) , but not in the WT MEFs ( Figure 8H ) . Correspondingly , ATF4 half-life was also shorter in the Siah1/2 DKO MEFs , following ODG treatment ( Figure 8H ) . These finding substantiate the role of Siah1/2 in determining PHD3 as well as ATF4 stability , following OGD . Given the Siah2-dependent expression of ATF4 in response to the UPR , we monitored changes in both ATF4 protein and ATF4 target gene CHOP levels , as well as in the degree of apoptosis following different levels of ER stress . The Siah2-dependent increase in ATF4 , as well as in CHOP and HIF1α protein levels was enhanced in response to glucose/oxygen deprivation ( Figure 9A ) . This increase was notably blunted in the Siah1/2 DKO MEFs . HIF1α expression levels were reduced during OGD , compared with hypoxia alone , ( Figure 9A ) , consistent with the inhibition of HIF1α translation during severe ER stress conditions [63] , [64] . Time course analysis following TM treatment revealed that ATF4 and its target gene CHOP were upregulated to a lesser degree in the Siah2 KO MEFs ( Figure 9B ) at the 6 h time point . Whereas the degree of ATF4 activation was reduced 24 h after TM treatment , the level of CHOP expression was comparable , and the expression of the apoptotic marker cleaved Caspase 3 were increased . Notably , although CHOP and ATF4 expression were still observed in the Siah1/2 DKO MEFs , Caspase 3 was not activated in these cells ( Figure 9B ) . Additionally , the expression of ATF4 , and its target gene CHOP , was Siah-dependent at an early time point following exposure to ER stress stimuli of increasing severity , represented here by TM ( 6 h; Figure 9B ) , the more severe OGD ( 8 h; Figure 9A ) or the most severe ischemia-like condition ( 0 . 5 , 2 h; ODG+ lack of nutrients; Figure 9D ) . Moreover , at these early time points there was no evidence for Caspase 3 cleavage or the induction of apoptosis . Only at later time points , including 24 h after TM , 48 h after OGD ( Figures 9B , C ) , or 16 h after ischemic conditions ( Figure 9d ) , was cleaved Caspase 3 observed in the WT but not the Siah1/2 DKO cells , indicating that Siah1/2 are required for UPR-induced cell death ( Figures 9B–D , respectively ) . Of interest , at these later time points , the level of ATF4 was increased in the Siah1/2 DKO cells , ( Figures 9B–D ) , suggesting that ATF4 facilitates survival rather than death programs in the absence of Siah1/2 . Consistent with the finding that cell death following UPR requires Siah1/2 , flow cytometric analyses confirmed that Siah1/2 DKO cells were significantly more protected from early and advanced cell death , as indicated by PI or Annexin+PI staining ( Figures 9E , F ) . Analysis performed at a later time point ( 48 h ) revealed that the Siah1/2 DKO cells were protected from OGD induced apoptosis , reflected by the levels of PI and Annexin V uptake ( Figure 9E ) . Similarly , the exposure of cells to ischemic conditions resulted in a lower degree of cell death in the Siah1/2 DKO cells , at the early time points ( 16/24 h ) ( Figure 9F ) . These findings establish the importance of Siah2 in conferring cell commitment to apoptosis in response to severe ER stress conditions . To further determine the role of Siah1/2 in cell death induced by severe ER stress such as ischemia , we used the middle cerebral artery occlusion model ( MCAO ) , a surgical model of cerebral ischemia known to induce severe ER stress and cell death [65] , [66] . Notably , 44% of the WT but none of the Siah1a+/−::Siah2−/− mice died within 24 h following permanent MCAO , indicating that WT mice are more sensitive to brain ischemia compared with the Siah1a+/−::Siah2−/− mice ( Figure 10A ) . Consistently , the infarct volumes of WT mice were higher , compared to those observed in the Siah1a+/−::Siah2−/− mice [mean of 138 . 84 vs . 34 . 13 mm3] ( Figure 10B ) . TUNEL staining of the most affected slices ( slices #3 and #4 from the anterior side ) revealed the presence of TUNEL-positive dead cells within the ischemic area of WT animal-derived brains ( Figure 10C ) . Consistent with independent findings indicating that increased infarct size results in more pronounced cell death in WT brain under ischemia , ATF4 and its downstream gene , CHOP , were highly expressed in areas adjacent to the ischemic cores of brains from WT mice but not within equivalent areas of the brains of the Siah1a+/−::Siah2−/− that were subjected to the same procedure ( Figure 10D ) . Our analysis was confined to the adjacent areas due to extensive cell death within the ischemic core regions; those regions have been previously reported to express reduced level of ER stress-related proteins ( 58 ) . Taken together , these results establish the role of Siah in the fine-tuning of ER stress during brain ischemia , as reflected by the degree of protection from cell death seen in the Siah1a+/−::Siah2−/− mice .
The UPR constitutes a tightly controlled signaling network that dictates the ability of a cell to cope with stress and ultimately determines its fate . This well-orchestrated network is primarily regulated by three ER-anchored sensors , IRE1α , PERK , and ATF6α , which engage in crosstalk and feedback mechanisms to drive distinct cellular response programs . In spite of our current understanding of UPR and related pathways , mechanisms underlying cell decision to undergo adaptation versus death programs , in response to UPR of differing magnitudes , has been poorly understood . Here , we demonstrate that the Siah1/2 ubiquitin ligases are an integral part of the UPR , as they are ( i ) activated by UPR transducers ATF4 and sXBP1 , ( ii ) contribute to the degree of ATF4 transcriptional activation thereby increasing the ATF4 output , and ( iii ) primarily mediate the severe UPR stimuli by conferring cell death programs . Our experiments suggest that Siah1/2 ubiquitin ligases constitute an obligatory fine-tuning mechanism that favor cell death under severe UPR conditions . The role of UPR in the regulation of Siah1/2 transcription constitutes an important addition to the previously established link between DNA damage ability to activate Siah1 ( but not Siah2 ) through p53 binding to the Siah1 promoter [39]–[42] . As p53 is also induced by UPR and the greater ER stress program [67] , our findings offer important insight into the role of diverse ER stress-inducible programs , namely , ATF4 , sXPB1 and p53 in the activation of Siah1/2 genes . In this newly identified regulatory axis , Siah2 is activated by two of the three UPR signaling pathways , PERK–ATF4 and IRE1α–sXBP1 , in response to a mild to severe but not low level of ER stress . Thus , a threshold mechanism exists for ATF4 or sXBP1 induction of Siah2 mRNA , potentially upon cooperation with additional transcription factors , or following select post-translational modifications occurring following more severe UPR conditions . Once activated , Siah1/2 ligases attenuate the level of PHD3-dependent prolyl-hydroxylation of ATF4 , which includes proline residues 60 and 235 . Indeed , ATF4 , which is mutated at these residues , is more stable enabling greater activation of downstream target genes . Correspondingly , the overexpression of Siah increases , whereas the inhibition of Siah reduces , the levels of ATF4 in a PHD3-dependent manner . Thus , the activation of Siah1/2 augments the expression and activity of ATF4 , thereby increasing the overall level of the UPR . Our findings also clarify a long-standing debate underlying the mechanism of PHD1/3 regulation of ATF4 availability and activity . Whereas one report points to the importance of PHD3 catalytic activity in the control of ATF4 [45] , another suggests that the catalytic activity may not be required for ATF4 regulation [53] , pointing to the possible recruitment of additional factors that contribute to ATF4 stability and activity . Here , we identified two previously unknown sites on ATF4 that are subject to prolyl hydroxylation by PHD1/3 and demonstrate the role of this hydroxylation in the Siah2-dependent stabilization of ATF4 . Important to note that we identified the prolyl-hydroxylation sites on ATF4 in vivo , as oppose to earlier attempts , that were carried out in vitro . The latter suggest that additional post-translational modifications may be required for PHD3-hydroxylation of ATF4 . Previous reports identified the SCF complex component βTrCP as an ATF4 ubiquitin ligase . Since βTrCP requires ATF4 phosphorylation for its association and ubiquitination , it is of interest to determine the relationship between hydroxylation and phosphorylation of ATF4 , particularly as the P235 site we identified is positioned within the phosphorylation sites mapped for recognition by βTrCP . The significance of the Siah ubiquitin ligases to the overall UPR is reflected in their impact on cellular commitment to undergo death . Severe forms of ER stress , including exposure to higher levels of TM , OGD , or ischemia ( where nutrients are also absent ) , results in cell death programs , which were markedly attenuated in the Siah1/2 KO cells . The evidence for the requirement of Siah1/2 for cell death programs was substantiated by biochemical ( absence of cleaved caspase 3 ) and cellular ( FACS analysis ) read-outs , and more so , by using the genetic Siah1+/−::Siah2−/− animals . Ischemic brain injury was reported to occur in response to ER stress conditions , in an ATF4-dependent manner [65] , [66] . Notably , ischemic brain injury was attenuated in the Siah1+/−::Siah2−/− animals , providing further evidence for the requirement of Siah2 in fine tuning of ER stress/ATF4-dependent cell death programs . How does Siah contribute to cell death programs in response to ER stress ? In the presence of Siah2 , a higher level and activity of ATF4/CHOP induces cell death programs ( PUMA , BAX ) . Under such conditions Siah2 also contributes to an increased rate of mitochondrial fission , as demonstrated in our earlier studies [28] . In the absence of Siah2 , the expression of ATF4/CHOP is insufficient to induce the death signaling pathways , and instead promotes survival pathways ( i . e . , autophagy ) . Further , in the absence of Siah2 the rate of mitochondrial fission is reduced , contributing to protective mechanisms [28] . The latter is consistent with the finding that ATF4 is required for mitochondrial dynamics [68] . Correspondingly , Siah2 control of ATF4 expression may explain changes that underlie the switch from cell survival to death programs , under severe ER stress conditions . These findings highlight a mechanism underlying the fine-tuning of the UPR , addressing a long sought quest for mechanisms that distinguish the role of ER stress in recovery of normal homeostasis versus the activation of cell death . Our studies also offer an initial link between UPR and hypoxia pathways . Through its common substrate , PHD3 , Siah1/2 regulates key components in hypoxia ( HIF1α ) and UPR ( ATF4 ) . Noteworthy is that the cross talk among the different ER stress sensors may further augment the mechanism described in our studies . Namely , upregulation of Siah2 mRNA by sXbp1 points to a mechanism by which the IRE1 sensor may augment the activity of the PERK sensor . Conditions under which IRE1/sXBP1 are activated will induce Siah1/2 , which in turn will augment ATF4 availability . Of interest is to note that the sXBP1 binding site on the Siah2 promoter overlaps with the hypoxia response element ( HRE ) , raising the possibility that stress ( ER or oxidative ) that is sufficient to trigger either ATF4 or sXbp1 transcription may constitute the initial signal for Siah2 transcription . Subsequently , Siah1/2 stabilization of HIF1α could replace sXBP1 on Siah1/2 promoter , to sustain Siah1/2 mRNA levels . Alternatively , sXBP1 and HIF1α may cooperate on promoters where their response elements overlap , as seen for Siah1/2 promoters . Thus , Siah1/2 ligases contribute to key cellular pathways , hypoxia and the UPR , while being regulated by their primary components as part of a feed forward loop mechanism . We showed that under conditions of glucose/oxygen deprivation , or ischemia , Siah1/2 ligases are required for ER stress/ATF4 activation of cell death programs . In agreement , the increased expression of Siah1 coincided with decreased PHD3 expression following cerebral ischemia [69] . Moreover , we expect that the role of Siah ligases in fine-tuning the UPR would be relevant to an array of pathological conditions associated with neuronal ischemia , fatty liver , and heart diseases [70]–[74] . Overall , our studies identify Siah2 as an important regulatory component of the UPR , which serves to fine-tune the degree of UPR , and in doing so , to impact the outcome of the UPR ability to initiate cell death programs .
All animal work has been conducted according to relevant national and international guidelines in accordance with recommendations of the Weatherall report and approved . Our study protocols using Siah1a+/+::Siah2+/+ and Siah1a+/−::Siah2−/− mice ( 129/BL6 background ) were generated as previously described [75] and approved by the Institutional Animal Care and Use Committee ( IACUC ) of the Sanford-Burnham Medical Research Institute . HEK293T , CHO HeLa , Lu1205 , and HEK 293T cells were maintained in Dulbecco's modified Eagle's medium ( DMEM , Invitrogen ) with 10% fetal bovine serum ( FBS; Sigma ) and 1% penicillin–streptomycin ( Invitrogen ) at 37°C . The melanoma cell line Lu1205 was a kind gift from Meenhard Herlyn . Siah1a/2 WT , Siah1a/2 KO , IRE1α WT , IRE1α KO , ATF4 WT , and ATF4 KO MEFs [10] , [75] , [76] were similarly maintained with the addition of 55 µM β-mercaptoethanol ( Invitrogen ) and 1× nonessential amino acids ( Invitrogen ) . Cells were seeded and cultured to ∼60–80% confluency . On the day of the experiment , cells were treated with vehicle ( DMSO ) , or 1–2 µg/ml tunicamycin ( Sigma ) in DMSO , or 1 µM of thapsigargin ( Sigma ) in DMSO , or subjected to hypoxia ( 1% O2 ) . Glucose deprivation was performed by incubating the cells in complete DMEM or in DMEM lacking glucose ( Invitrogen ) and containing 10% dialyzed FBS ( Invitrogen ) . Primary mouse keratinocytes were isolated from newborn mouse epidermis as described [77] and seeded at a density of 5×106 cells per 60-mm dish ( or equivalent concentrations ) in a defined serum-free keratinocyte specific medium ( Invitrogen ) . Doxycycline was purchased from Sigma . Antibodies to Siah2 , GRP94 , ATF4 , XBP1 , AKAP121 , myc , β-actin , CHOP were from Santa Cruz Biotechnology . Anti-HIF-1α was a generous gift from Dr . Robert Abraham . Anti-Flag and anti-Siah1 were from Sigma , while anti-PHD1 and anti-PHD3 were from Abcam . All antibodies were used according to the suppliers' recommendations . MG132 was from Calbiochem . DMOG was from Sigma . Immunohistochemical analysis of ATF4 expression was performed using anti-ATF4 antibody from Abcam . Samples were prepared from ATF4 immunoprecipitates ( antibodies described above ) using standard procedures . Briefly , protein samples were reduced , alkylated , trypsin-digested , desalted and dried in a speed vac . Samples were re-suspended in 0 . 1% formic acid/5 . 0% acetonitrile and analyzed using a MS2 HPLC , HTC-PAL autosampler , Captive Spray Source ( Bruker-Michrom ) and a LTQ Orbitrap Velos Pro mass spectrometer equipped with electron transfer dissociation ( Thermo Fisher Scientific; [78] ) . Database searches were as described previously [78] against a mouse protein database ( mouse . ipi . 3 . 73 ) with the exception that proline hydroxylation ( +15 . 99491 Daltons ) was included as a differential modification . The accuracy of the proline hydroxylation site identifications was manually confirmed ( Figure 6F ) . The data were analyzed by Student's -test . p<0 . 05 was considered statistically significant . To verify that the number of mice used in the study was sufficient to provide the statistical power we calculated the sample size using G*Power 3 . 1 software for power analysis , further aided by using the t-test for assuring sufficient difference between two independent groups . The human Siah2 promoter fragments were amplified from HeLa cell genomic DNA by PCR and cloned into the pGL3-luciferase reporter vector ( Promega ) . All constructs were verified by DNA sequencing . PHD3 tagged with myc , OGDH and Siah2 tagged with Flag were cloned as previously described [27] , [33] , [46] , [79]–[81] . The luciferase reporter vector containing the ATF4 response element was constructed by cloning the promoter region of mouse Trib3 gene ( −2000 to +30 bp of transcription start site ) into pGL3-luciferase . Mutagenesis of ATF4 and XBP1 binding site on the Siah2 promoter and mutagenesis of ATF4 on the hydroxylation sites were performed using QuickChange II XL mutagenesis kit ( Invitrogen ) . The sites of mutation for ATF4 and XBP1 are respectively shown in small letters: 5′-GTCCGCGCGCGCCCCCGGGGCcTGtcTCAGCGGCTGTTCCAGAAG-3′ ( ATF4 ) , 5′-CGTGTCCAGGCGTattaTCCGCGCGCGCCCCCGG ( XBP1 ) . The primers used for mutagenesis on mouse ATF4 of the two proline in position 60 and 235 were the following: for the mutation of proline in position 60: 5′-GGCTCCTCGGAATGGgCGGCTATGGATGATGGCTT-3′ ( in lower case is the mutated nucleotide ) ; for the mutation of proline in position 235: 5′-TCTCCCCAGCATAGCgCCTCCACCTCCAGGGCCCCA-3′ ( in lower case is the mutated nucleotide ) . Adenoviruses expressing WT ATF4 , and spliced XBP1 were previously reported [82] , [83] . Adenoviruses-expressing green fluorescent protein or β-Gal were used as a control . MEFs were infected with adenoviruses at a multiplicity of infection of 20 . Twenty hours after infection , the adenovirus was removed , cells were harvested and total RNA and protein were extracted for analysis . The shRNA vectors for silencing of Siah1a or Siah2 were previously described [28] . The sequences used for scramble , Siah2 and Siah1a shRNA were respectively: caacaagatgaagagcaccaa ( scramble ) , ccttggaatcaatgtcacgat ( mSiah2 ) , atttagctcaagtcgatatgc ( mSiah1a ) . For silencing of the murine PHD1 and PHD3 genes , shRNAs were from Sigma ( TRCN0000273085 , TRCN0000009744 TRCN0000009746 ) . For transduction , viral particles were harvested after transfection of HEK293T cells with the plasmid of interest and matched packaging plasmids using Jet Prime ( Polyplus Transfection ) . Target cells were infected with viral particles by inoculation in the presence of polybrene ( 4 µg/ml , Sigma ) . Stable clones were established by growing cells in media containing puromycin ( 1 µg/ml , InvivoGen ) . For the preparation of PHD3 shRNA #2 , nucleotides corresponding to 357–375 nt of mouse PHD3 coding sequence were synthesized and cloned into pSuper vector ( pSup-PHD3 ) as previously described [33] . Empty pSuper vector was used as a control . RNAi expression vectors were introduced into MEFs by transfection ( jetPrime , ) . After 48 h , cells were treated with or without hypoxia and harvested . To extract whole cell lysate , cells were harvested using RIPA buffer ( 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 1% Triton X-100 , 0 . 1% SDS , 0 . 1% Na-deoxycholate , 1 mM EDTA , 1 mM sodium orthovanadate , 1 mM PMSF , 10 µg/ml aprotinin , and 10 µg/ml leupeptin ) . Cell lysates were subjected to SDS-PAGE and proteins transferred onto a nitrocellulose membrane ( Osmonics Inc . ) . The membrane was probed with primary antibodies ( described above ) followed by a secondary antibody conjugated with fluorescent dye and detected using the Odyssey detecting system ( LI-COR Bioscience ) . Cells were exposed to hypoxia ( 1% O2 ) in a hypoxia workstation ( In Vivo 400; Ruskinn Corp . ) and then processed immediately on ice . Cells in 24-well plates were transfected with 100 ng of Luciferase vector containing the Siah2 promoter region or the Luciferase vector containing the ATF4 response element and 80 ng of β-Gal vector using jetPrime in triplicate . After 24 h , cells were infected either with ATF4 , spliced XBP1 , or GFP virus for 24 h , prior to collection of cell lysate using 60 µl of reporter lysis buffer ( Promega ) . Cell lysates ( 10 µl ) were loaded onto a 96-well plate and luciferase activity was measured using a Veritas Microplate Luminometer ( Turner Biosystems ) according to manufacturers' instructions . β-Gal assay was used to normalize transfection efficiency . For the β-Gal assay , 10 µl of cell lysate was incubated in reaction buffer ( 100 mM NaH2PO4 pH 7 . 5 , 0 . 1% O-Nitrophenyl-β-D galactopyranoside ( ONPG ) , 1 . 2 mM MgCl2 and 50 mM β-mercaptoethanol ) at 37°C for 20 min , and the reaction was stopped by addition of 1 M Na2CO3 . The OD at 420 nm measured with a spectrophotometer was used to reflect β-Gal activity . The luciferase activity of each sample was divided by the β-Gal activity to calculate the relative luciferase activity . ChIP assays were performed using a Magna CHIPTM kit ( Upstate ) . Triplicate biological samples were processed and analyzed . Briefly , cells ( 1 . 5×107 ) were maintained in normoxia or hypoxia ( 1% O2 ) for 6 h , or treated with 1 µM of TG for 5 h , and crosslinked using 1% formaldehyde for 10 min at RT . The crosslinking was stopped by 5 M glycine . Cells were lysed and sonicated to obtain 200–500 bp chromatin fragments . Chromatin was immunoprecipitated with 5 µg of antibodies ( Santa Cruz ) and 20 µl of protein A/G magnetic beads in a total volume of 0 . 5 ml overnight at 4°C . After four washes , crosslinking of protein/DNA complex was reversed , DNA was purified using spin column , and subjected to qPCR analysis . The following primers corresponding to mouse Siah2 5′-untranslated region and spanning the HRE , ATF4 and XBP1 binding sites were as follows: 5′-GCGATCGACTCATTCAAGGGCTC-3′ and 5′-GCGTTCTGGTGCGCAGAG-3′ . WT and Siah1a/Siah2 KO MEF cells were treated with TM or TG for 6 h , or subjected for 12 h to OD , GD or OGD , in duplicate . Total RNA ( 500 ng ) was used for synthesis of biotin-labeled cRNA using an RNA amplification kit ( Ambion ) . The biotinylated cRNA was labeled by incubation with streptavidin-Cy3 to generate probe for hybridization with the Mouse-6 Expression BeadChip ( Illumina ) that contain 48 K probes corresponding to mouse gene symbols . We analyzed the BeadChips using the manufacturers BeadArray Reader and collected primary data using the supplied Scanner software . Illumina BeadArray scanned data were pre-processed and normalized by Illumina GenomeStudio software ( Illumina Inc , San Diego , CA ) . Probesets that are absent in all the study samples were removed from further analyses . To identify differentially expressed genes , the linear modeling approach and empirical Bayes statistics as implemented in the limma package [84] were employed . The Benjamini–Hochberg method was used to correct for the multiple comparison errors [85] . Principal component analysis ( PCA ) was performed with Partek Genomics Suite ( Partek Inc . St . Louis , MO ) , and hierarchical clustering and other statistical analyses were performed using R/Bioconductor [86] . Genes with at least a 2-fold change at the 95% confidence level were considered as significant . The selected statistically significant genes in each of these experimental groups were than analyzed for functional enrichments using the Ingenuity Pathway Analysis ( IPA ) platform ( Ingenuity Systems Inc . , Redwood City , CA ) . The Gene Set Enrichment Analysis ( GSEA ) approach was also applied for the functional enrichment analyses [87] . A Fisher's exact test was employed to calculate the significant value , which determined the probability that an association between the genes within the dataset and the functional pathway could be explained by chance alone . Functional groups ( or pathways ) with a p-value<0 . 05 were considered to be statistically significant . The data obtained through our gene expression analysis has been deposited in the GEO public dataset ( GSE39244 ) . Total RNA was extracted using a total RNA miniprep kit ( Sigma ) and digested with DNase I . cDNA was synthesized using oligo-dT and random hexamer primers for SYBR Green qPCR analysis . 18S rRNA and H3 . 3A were used as internal controls . Triplicate biological samples were used for the qPCR analysis . The PCR primers were designed using Primer3 and their specificity was checked using BLAST ( NCBI ) . The PCR products were limited to 100–200 bp . The primers used for qPCR analysis were as follows: mouse VEGFA forward 5′-ATCTTCAAGCCGTCCTGTGT-3′ and reverse 5′-GCATTCACATCTGCTGTGCT-3 . ′; mSiah2 forward 5′-GCTGAGAACTTTGCCTACAG-3′ , and reverse 5′-GCTATGCCCAAATAACTTCC-3′; 18S rRNA forward 5′-GAGCGAAAGCATTTGCCAAG-3′ , and reverse 5′-GGCATCGTTTATGGTCGGAA-3; mCHOP forward 5′-AAGCCTGGTATGAGGATCTGC-3′ , and reverse 5′-GGGGATGAGATATAGGTGCCC-3′; mATF3 forward 5′-AGAGCTGAGATTCGCCATCC-3′ , and reverse 5′-TGTTGACGGTAACTGACTCCA-3′;m spliced XBP1 forward 5′-CTGAGTCCGAATCAGGTGCAG-3′ ( original CAG sequence was mutated to AAT to reduce the background signal from unspliced XBP-1 ) and reverse 5′-GTCCATGGGAAGATGTTCTGG-3′; mSiah1a forward 5′-GCTGAAAATTTTGCATATCG-3′ and reverse 5′-CCAGGAAAGTTTTAGGTTGG-3′; mSiah1b forward 5′-GAGATGAGCCGTCAGGCTGCTA-3′ and reverse 5′-GAATAGGTGGCAACACATAG; mATF4 FORWARD 5′-CCTGAACAGCGAAGTGTTGG-3′ and reverse 5′-TGGAGAACCCATGAGGTTTCAA-3′; mPHD1 forward 5′-AGTCCTTGGAGTCTAGCCGAG-3′ and reverse 5′-TGGCAGTGGTCGTAGTAGCA-3′; mPHD3 forward 5′-TTATGTTCGCCATGTGGACAA-3′ and reverse 5′-gcgtcccaattcttattcaggta-3′; hFactor VII forward 5′-AACCCCAAGGCCGAATTGT-3′ and reverse 5′-CGCGATCAGGTTCCTCCAG -3′ . mMMP10 forward 5′- GAGCCACTAGCCATCCTGG , and reverse 5′- CTGAGCAAGATCCATGCTTGG-3′; mWNT5a forward 5′- CTCCTACAGTGTGGTTGTCAGG-3′ , and reverse 5′- GCGCATCCATAAAGAGTCTTGA-3′; mWNT6 forward 5′- CTCCTACAGTGTGGTTGTCAGG-3′ , and reverse 5′- GCGCATCCATAAAGAGTCTTGA-3′; mSOX11 forward 5′- CGAGCCTGTACGACGAAGTG-3′ , and reverse 5′- AAGCTCAGGTCGAACATGAGG-3′; mPDGFC forward 5′- GCCAAAGAACGGGGACTCG-3′ , and reverse 5′- AGTGACAACTCTCTCATGCCG-3′; mNRP1 forward 5′- ACCTCACATCTCCCGGTTACC-3′ , and reverse 5′- AAGGTGCAATCTTCCCACAGA-3′; mALDH1 forward 5′- TTCCCACCGTCAACCCTTC-3′ , and reverse 5′- CCAATCGGTACAACAGCCG-3′; mIGFBP6 forward 5′- TGCTAATGCTGTTGTTCGCTG-3′ , and reverse 5′- CACGGTTGTCCCTCTCTCCT-3′; mGRB10 forward 5′- GGACAAATCGGAAGAGTGATCG , and reverse 5′- CATCCGTGTGCTCCCGTTAC-3′; mGPX7 forward 5′- TCCGAGCAGGACTTCTACGAC-3′ , and reverse 5′- TCTCCCTGTTGGTGTCTGGTT-3′; mCYP7b1 forward 5′- GGAGCCACGACCCTAGATG-3′ , and reverse 5′- GCCATGCCAAGATAAGGAAGC-3′; mCBR2 forward 5′- GGGCAGGGAAAGGGATTGG-3′ , and reverse 5′- CCACACACACGGGCTCTATTC-3′; mIGF2 forward 5′- GTGCTGCATCGCTGCTTAC-3′ , and reverse 5′- ACGTCCCTCTCGGACTTGG-3′; mPTGIS forward 5′- GCCAGCTTCCTTACCAGGATG-3′ , and reverse 5′- GAGAACAGTGACGTATCTGCC-3′; mRab6 forward 5′- GGAGACTTCGGGAATCCGC-3′ , and reverse 5′- ACTGTCATACATGAATCGGGTGA-3′; mGOLT1b forward 5′- ATGATCTCCCTCACGGATACG-3′ , and reverse 5′- TCGAGACCAATTACAAAAGCCAA-3′; mHSPA5 forward 5′- ACTTGGGGACCACCTATTCCT , and reverse 5′- GTTGCCCTGATCGTTGGCTA-3′ . Siah1a+/+::Siah2+/+ and Siah1a+/−::Siah2−/− mice ( male , 8–9 weeks old with 22–25 mg body weight ) were subjected to induction of focal cerebral ischemia through intraluminal MCAO . Mice were anesthetized with 2 . 0–2 . 5% isoflurane administered by mask and MCAO was performed as described [88] . Briefly , unilateral MCAO was induced by insertion of silicone rubber-coated monofilament ( Doccol Corp . ) . The silicone suture ( size 6-0 , diameter 0 . 09–0 . 11 mm ) introduced from the common carotid artery was advanced into the internal carotid artery ( 10–11 mm from the common carotid artery bifurcation site ) . The filament was left in place for 24 h for pMACO . The post-ischemic mice were sacrificed , the brain was collected and placed at −20°C for 12 min . Then , coronal slices with 1 mm thickness were obtained by cutting using brain matrix . Sections were stained with 2% 2 , 3 , 5-triphenyltertrazolium chloride ( TTC ) solution for 25 min at 37°C . The sections were fixed with 10% neutral buffered formalin solution ( Sigma-Aldrich ) at 4°C . Coronal brain slices were scanned with a high-resolution scanner ( HP ScanJet G4010 ) . The healthy non-ischemic area and infarct area were measured using Image J1 . 44 ( NIH ) . To avoid an edema effect , infarct area was calculated by subtracting healthy area of ipsilateral hemisphere from total area of contralateral hemisphere as described [82] . Infarct volume was calculated by adding all brain sections with 1 mm thickness . To verify that the number of mice used in the study is sufficient to provide the statistical power we calculated the sample size using G*Power 3 . 1 software for power analysis , which was further aided using the t-test to assure sufficient difference between two independent groups . To have a 95% probability of detecting a difference in means of 4 . 3 standard deviations , a sample size of three mice in each treatment group was calculated to be necessary . Accordingly the sample size used in these experiments is sufficient to support statistically significant conclusions . Of note , we used a larger group size for the WT animals since 44% of the WT mice died within 24 h following permanent MCAO . The increased group size for the WT enabled to secure the number of mice needed for statistical power . Brain slices from Siah1a+/+::Siah2+/+ and Siah1a+/−::Siah2−/− mice were obtained after 24 h of pMCAO . Among the slices , #3 and #4 slices were further sectioned through paraffin-embedded method and stained using ApopTag-red ( Millipore ) according to manufacturer's direction . Immunohistochemistry analyses of adjacent slices were performed for expression of ATF4 and CHOP . Tissue collected from Siah1a+/+::Siah2+/+ and Siah1a+/−::Siah2−/− mice were fixed in Z-fix ( buffered zinc formalin fixatives , Anatech ) overnight . After fixation , tissues were washed twice with PBS and processed for paraffin embedding . Brain embedded in paraffin blocks were sliced at 5 µm and stained with hematoxylin and eosin . For ATF4 and CHOP staining , all cryosections were fixed with Z-fix ( buffered zinc formalin fixatives , Anatech ) and followed by a blacking of non-specific binding sites ( Dako/Agilent Inc . ) for 30 min at room temperature . Antigen retrieval was performed in a pressure cooker ( Decloaking chamber , Biocare Medical ) in citrate buffer ( pH 6 . 0 ) and used for CHOP and ATF4 immunostaining . Antibodies/dilutions for the following markers were used in antibody diluent ( Dako ) overnight at 4°C . For CHOP analysis secondary antibody labeled with Alexa Fluor 488 , was placed on tissue sections for 1 h at room temperature ( 1∶400 , Molecular Probes ) . Nuclei were counterstained using SlowFade Gold Anti-fade reagent with 4′ , 6-diamidino-2-phenylindole ( DAPI; Vector Laboratories ) . For immunohistochemistry analysis of ATF4 the staining was visualized using an alkaline phosphatase technique ( Vector red alkaline substrate kit I , Vector Laboratories ) . Siah1a/2 WT and double knockout MEFs were grown under control ( normoxic , high glucose ) , OD ( 1% O2 ) and OGD ( 1% O2 , glucose-free for 48 h ) , or ischemic condition ( no nutrient media for 16 h ) . The cells were then harvested and immediately stained with Annexin V-FITC and propidium iodide using the Biovision Annexin V-FITC Apoptosis Kit ( Biovision , USA; K101-100 ) . The cells were subjected to FACS analysis using a FACSCanto ( BD Biosciences , USA ) and FlowJo software ( TreeStar , USA ) ( n = 10 , 000 cells per replicate ) . The data shown represent experiments performed in triplicate . | Maintaining a balanced level of stress ( protein folding , reactive oxygen radicals ) is important for keeping cellular homeostasis ( the ability of a cell to maintain internal equilibrium by adjusting its physiological processes ) . The accumulation of stress ( external or internal ) will trigger a well-orchestrated machinery that attempts to restore homeostasis , namely , the unfolded protein response ( UPR ) . The UPR either restores balance to the cells or induces a cell death program , which clears the damaged cell . How this machinery activates cell survival versus cell death is not entirely clear . Here we identify a new layer in the regulation of the UPR , which determines the magnitude of this response . We demonstrate the importance of this newly identified regulatory component for cell death commitments , in response to the more severe conditions ( ischemia , lack of oxygen and nutrients ) . Our findings highlight an undisclosed mechanism that is important for the cell death decision following severe stress conditions , while pointing to the ability to fine tune cellular response to stress . | [
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] | 2014 | Fine Tuning of the UPR by the Ubiquitin Ligases Siah1/2 |
A number of paramyxoviruses are responsible for acute respiratory infections in children , elderly and immuno-compromised individuals , resulting in airway inflammation and exacerbation of chronic diseases like asthma . To understand the molecular pathogenesis of these infections , we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 ( hPIV3-C ) . We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2 , whereas C proteins from measles or Nipah viruses failed to do so . Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling , but the interaction with GRB2 was unexpected . This adaptor protein bridges Epidermal Growth Factor ( EGF ) receptor to MAPK/ERK pathway , a signaling cascade recently found to be involved in airway inflammatory response . We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF , as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2 , 40S ribosomal subunit protein S6 and translation initiation factor 4E ( eIF4E ) . Furthermore , inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells . Altogether , our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors .
Viruses need to interact with host macromolecules to hijack the cellular machinery and replicate . These interactions are essential for viruses to target endocytic pathways and penetrate host cells , to recruit cellular transcription and/or translation machinery , and to achieve intracellular migration and viral particles assembly . But viruses also encode virulence factors that induce a substantial alteration of host cell functions and genetic programs to increase virus replication and spreading . For example , specific viral factors stimulate survival pathways to prevent apoptosis of infected cells or inhibit cell signaling involved in immune response . Among these pathways , IFN-α/β signaling represents a prime target for viruses because of its critical role in the induction of both innate and adaptive antiviral immune responses [1] . IFN-α/β transduce signals through direct binding to a cell surface receptor composed of two transmembrane subunits , IFNAR1 and IFNAR2c [2] . This interaction activates IFNAR1/IFNAR2c associated kinases Tyk2 and Jak1 that subsequently phosphorylate STAT2 and STAT1 transcription factors . Activated STAT1 and STAT2 , altogether with IRF9 , form the Interferon-Stimulated Gene Factor 3 that binds IFN-stimulated response element ( ISRE ) promoter sequences to induce a large antiviral gene cluster . As a consequence , most viruses that are pathogenic in vertebrates have evolved virulence factors both to block IFN-α/β expression and signal transduction downstream of IFN-α/β receptor . Human parainfluenza virus type 1 ( hPIV1 ) and human parainfluenza virus type 3 ( hPIV3 ) are important human pathogens that belong to Respirovirus genus ( Paramyxoviridae family; [3] ) . These viruses are responsible for upper respiratory tract infections and colds , but often spread to the lower respiratory tract causing bronchitis , bronchiolitis and pneumonia in young children and immuno-compromised patients . hPIV3 infection is also suspected to exacerbate chronic airway inflammatory diseases like asthma [4] . Sendai virus and bovine parainfluenza virus type 3 ( bPIV3 ) are animal counterparts of hPIV1 and hPIV3 that infect mouse and cattle , respectively . Respirovirus genome is a single-strand , negative-sense RNA molecule that encodes six structural proteins ( Mononegavirales order ) . While hemagglutinin-neuraminidase ( HN ) and fusion ( F ) are membrane glycoproteins associated with the envelop of hPIV3 particles , the nucleoprotein ( N ) , the phosphoprotein ( P ) and the viral polymerase ( L ) form the ribonucleocapsid complex . The matrix protein ( M ) is at the interface between glycoprotein tails and ribonucleocapsids . The P gene of Respirovirus encodes for P but also for a panel of accessory proteins by site-specific editing of P mRNA and usage of overlapping open reading frames ( ORFs ) . In all Respirovirus except hPIV1 , the co-transcriptional insertion of one G residue at an editing motif midway of P mRNA leads to the expression of a chimeric protein called V . The V proteins of bPIV3 and Sendai virus bind MDA5 and suppress double-stranded RNA-stimulated IFN-β production , thereby contributing to the virus evasion of host immune response [5] . Surprisingly in hPIV3 , multiple stop codons localized downstream of the editing site prevent the normal expression of a full-length V protein . As a result , P mRNA molecules edited by the addition of one G residue encode for the 242 amino acid ( AA ) -long N-terminal residues of P followed by only six additional AA ( see Materials and Methods and [6] ) . But P mRNA molecules edited by the addition of five G residues encode for D , a protein exhibiting a large and specific C-terminal domain of unknown function ( Figure 1A ) . Besides co-transcriptional edition , an overlapping ORF embedded in the first half of the P mRNA allows the expression of a single C protein ( hPIV3 and bPIV3 ) or a nested set of four proteins called C′ , C , Y1 and Y2 ( Sendai virus and hPIV1 ) . The C proteins of Sendai virus and hPIV1 have a high degree of sequence homology and have been studied in details . They are involved in the regulation of viral RNA synthesis [7] , [8] , the inhibition of innate immune response [9] and potentially contribute to the budding of viral particles [10]–[12] . In particular , the C protein of Sendai virus both inhibits IFN-β production [13] , [14] and blocks interferon signaling downstream of IFN-α/β and IFN-γ receptors [15]–[17] . The C proteins of hPIV3 and bPIV3 only share ∼35% of sequence homology with the C proteins of Sendai virus and hPIV1 , but they have also been shown to target interferon expression and signaling [5] , [18] . Although expression of the C protein of hPIV3 ( hPIV3-C ) is essential to virulence in vitro and in vivo [19] and explains hPIV3 ability to block IFN-α/β signaling [20] , host proteins that bind hPIV3-C remain unknown . In an attempt to answer this question , we performed a yeast two-hybrid screen and we report here the identification of STAT1 and GRB2 as direct interactors of hPIV3-C . Although binding to STAT1 accounts for hPIV3-C ability to block IFN-α/β signaling , the interaction with GRB2 was unexpected . This adaptor protein bridges growth factor receptor tyrosine kinases ( RTKs ) , like Epidermal Growth Factor ( EGF ) receptor , to the mitogen-activated protein kinase/extracellular signal-regulated kinase ( MAPK/ERK ) pathway . Upon engagement by their ligands , RTKs autophosphorylate on tyrosine residues to recruit adaptor proteins containing phosphotyrosine binding ( PTB ) or Src homology 2 ( SH2 ) domains like GRB2 [21] . Once associated to RTKs by its SH2 domain , GRB2 recruits the guanine nucleotide-releasing factor son-of-sevenless ( SOS ) to activate Ras . Downstream events include MAPK/ERK kinase ( MEK1/2 ) activation , which in turn phosphorylates ERK1/2 . Finally , phosphorylated ERK1/2 directly or indirectly activates numerous cellular targets including transcription factors ( e . g . Elk1 , SAP1 , SAP2 , c-Fos , CREB , SRF ) but also cellular factors that control mRNA translation like eukaryotic initiation factor 4E ( eIF4E ) or small ribosomal subunit S6 protein [22] , [23] . Growth factor binding to RTKs regulates a multiplicity of cellular processes including proliferation , differentiation and survival . In the respiratory tract , this signaling cascade has been shown to trigger inflammation and mucus secretion by epithelial cells [24]–[27] , suggesting a critical role in innate immunity [28] . However , excessive activation of this pathway could benefit to virus replication by inhibiting IFN-α/β signaling [29] and promoting infected cell survival [25] . Altogether , these data provided a rational to investigate the functional impact of hPIV3-C expression on IFN-α/β vs EGF receptor and MAPK/ERK signaling pathways .
To identify cellular targets of hPIV3-C , this viral protein was used as bait in the yeast two-hybrid system to screen a human cDNA library . The screen was performed at saturation with a 10-fold coverage of the library ( 50 . 10+6 diploids ) , and positive yeast colonies growing on selective medium were analyzed by PCR and sequencing to identify binding partners of hPIV3-C . STAT1 and GRB2 were the main interactors of hPIV3-C identified in the screen with 5 and 150 yeast colonies corresponding to these cellular proteins , respectively . In both cases , cDNA clones retrieved from the screen corresponded to full-length STAT1 and GRB2 in frame with the Gal4-AD transactivation domain . To validate these interactions in human cells , GST-tagged hPIV3-C was expressed in HEK-293T cells and purified with glutathion-sepharose beads . As shown in Figure 1B and 1C , endogenous STAT1 and GRB2 co-purified with hPIV3-C . Highly divergent C proteins from measles virus ( MV-C ) and Nipah virus ( Nipah-C ) failed to do so , thereby demonstrating the specificity of identified interactions . Binding to STAT1 provides molecular basis to the inhibition of IFN-α/β signaling by hPIV3-C [18] , and parallels the interaction previously identified between Sendai virus C protein and mouse STAT1 [15] . Altogether , this suggests that STAT1 is a specific cellular interactor of Respirovirus C proteins . In contrast , binding to GRB2 is unexpected and suggests a new function for hPIV3-C that we decided to investigate . The adaptor protein GRB2 plays a critical role in coupling signal from growth factor receptors to MAPK/ERK signaling pathway . To address the question of hPIV3-C interference with this pathway , we used a trans-reporter gene assay that measures Elk1 activation by ERK1/2 . In this system , Elk1 transcription factor is fused to the DNA binding domain of Gal4 ( Gal4-DB ) and binds the promoter sequence of a luciferase reporter gene . Upon stimulation with a growth factor like EGF , Elk1 is activated as assessed by a significant increase in luciferase expression . Surprisingly , we observed a 6-fold enhancement in this cellular response to EGF when 3×FLAG-tagged hPIV3-C was expressed in HEK-293T cells ( Figure 2A ) . Same results were obtained when using hPIV3-C without a tag ( 14-fold enhancement ) or tagged with the red fluorescent protein Cherry ( 7-fold enhancement ) . In contrast to hPIV3-C , neither MV-C nor Nipah-C enhanced Elk1 activity upon EGF stimulation ( Figure 2A ) whereas expression levels of hPIV3-C , MV-C and Nipah-C were similar in this system ( Figure 2F , left panel ) . Elk1 activity was also enhanced by hPIV3-C expression in Vero and Hela cells as well as BEAS-2B and A549 , two epithelial cell lines that originate from the respiratory tract , which is the tissue targeted by hPIV3 in vivo ( Table 1 ) . The effect of hPIV3-C in these different cell lines was highly significant ( see p-values in Table 1 ) although relatively modest when compared to HEK-293T cells . This is probably because our reporter system requires the co-transfection of four plasmids and Vero , Hela , BEAS-2B and A549 cells are more difficult to transfect than HEK-293T . In parallel experiments , cellular response to IFN-α/β was monitored using a cis-reporter gene , of which expression is controlled by five ISREs . As previously reported [18] , we found that hPIV3-C efficiently blocked IFN-α/β signaling ( Figure 2B ) as opposed to what we observed for the EGF pathway . Again , MV-C or Nipah-C was unable to do so . Altogether , these results show that hPIV3-C enhances the cellular response to EGF in addition to its ability to block IFN-α/β signaling . We also determined if similar effects on the EGF pathway were observed in infected cells . HEK-293T cells were infected with hPIV3 ( MOI = 3 ) and then transfected with Elk1 activity reporter plasmids . Infection of HEK-293T cells was confirmed by anti-hPIV3 hemagglutinin-neuraminidase ( hPIV3-HN ) immunostaining and flow cytometry analysis ( Figure 2E ) . Like hPIV3-C alone , hPIV3 infection enhanced Elk1 activity upon EGF stimulation ( Figure 2C ) . Interestingly , hPIV3 infection induced a significant level of Elk1 activity in the absence of EGF stimulation . This suggests that in addition to hPIV3-C interaction with GRB2 , other mechanisms modulate MAPK/ERK pathway during hPIV3 infection . Finally , to demonstrate that enhancement of Elk1 activation by hPIV3-C is completely dependent on ERK1/2 activation , HEK-293T cells were pre-treated with MEK1/2 inhibitor U0126 before stimulation with EGF . This molecule targets MEK1/2 and totally abrogates downstream phosphorylation and activation of ERK1/2 [30] . As shown in Figure 2D , Elk1 activation was blocked by U0126 , whereas hPIV3-C expression was maintained ( Figure 2F , right panel ) . This demonstrates that hPIV3-C is acting through ERK1/2 stimulation . Altogether , these results support a model where hPIV3-C interaction with GRB2 enhances cellular response to growth factors as assessed by an increased activation of MAPK/ERK pathway . To further document hPIV3-C impact on MAPK/ERK signaling pathway , we compared the kinetic of ERK1/2 phosphorylation in HEK-293T cells expressing hPIV3-C or not . Cells were transfected with 3×FLAG-tagged hPIV3-C or a control plasmid and 24 h post transfection , they were starved before stimulation with EGF . ERK1/2 phosphorylation was determined at 10 , 30 and 120 min after stimulation . As illustrated by one representative experiment in Figure 3A , EGF stimulation induced ERK1/2 phosphorylation in control cells but signal was markedly and reproducibly increased by hPIV3-C expression at maximum phosphorylation time point ( 1 . 4 to 2 . 8 fold increase; p = 0 . 005; n = 4 ) . We then determined the phosphorylation level of two downstream targets of this pathway that are involved in the control of mRNA translation , the translation initiation factor eIF4E and the ribosomal protein S6 ( Figure 3B ) . Before EGF stimulation , low levels of phosphorylated eIF4E and S6 were detectable in mock-treated cells ( Figure 3B and 3D ) . hPIV3-C expression had virtually no effects on this background . Thus , eIF4E and S6 phosphorylation levels were determined at different time-points after EGF stimulation . Because ERK1/2 activation precedes eIF4E and S6 phosphorylation , maximal phosphorylation occurs at later time points and was determined at 30 min , 2 h , 6 h and 24 h after stimulation . As observed for ERK1/2 , phosphorylation levels of eIF4E and S6 were enhanced by hPIV3-C expression when stimulating the cells with EGF . To validate these observations in infected cells , HEK-293T cells were infected with hPIV3 ( MOI = 3 ) and 24 h later , cells were starved for 12 h before stimulation with EGF . Like hPIV3-C expression alone , hPIV3 infection enhanced ERK1/2 phosphorylation at the peak of induction , i . e . 10 min after adding EGF to the cells ( Figure 3C ) . Interestingly , hPIV3 infection of A549 cells also enhanced ERK1/2 phosphorylation but the induction profile was different . Indeed , ERK1/2 phosphorylation was not significantly increased at the peak of induction , but the signal was boosted by hPIV3 infection at late time points ( Figure S1 ) . The same profile was observed when eIF4E and S6 phosphorylation levels were analyzed in infected HEK-293T cells . hPIV3 infection sustained the phosphorylation of these two translation factors at late time points , but showed no increase at the peak of stimulation , i . e . 30 min after adding EGF to the cells ( Figure 3D ) . This could relate to the fact that hPIV3 infection also induces low levels of eIF4E and S6 phosphorylation in the absence of EGF stimulation ( Figure 3D ) . This is reminiscent to what was observed for Elk1 ( Figure 2C ) , and suggests that hPIV3 infection induces a basal activation of MAPK/ERK pathway leading to the constitutive phosphorylation of downstream targets . Altogether , these data demonstrate that hPIV3 infection or hPIV3-C expression alone both enhance MAPK/ERK pathway activation in EGF-stimulated cells . Several RNA viruses require an activated MAPK/ERK pathway to produce viral components and replicate properly ( for review see [31] ) . To test if the same was true for hPIV3 , cells were treated for 2 h with MAPK/ERK pathway inhibitor U0126 and infected with hPIV3 ( MOI = 1 ) . Two days after infection , cell surface expression of hPIV3-HN was detected by immunostaining and flow cytometry . U0126 completely blocked the expression of hPIV3-HN in hPIV3-infected cells ( Figure 4 ) , whereas the same inhibitor had no effect when cells were infected with MV ( Figure S2 ) . Altogether , this demonstrates that MAPK/ERK signaling is essential for the expression of hPIV3 proteins and suggests that hPIV3 manipulates this pathway to increase replication efficiency . To better understand how hPIV3-C targets both the IFN-α/β and EGF signaling pathways , we characterized the functional domains of hPIV3-C that bind STAT1 and GRB2 . To do so , we generated by PCR a full matrix of hPIV3-C overlapping fragments and tested their ability to interact with either STAT1 or GRB2 in the yeast two-hybrid system ( Figure 5 and 6 ) . Both forward and reverse primers were designed every 75 nucleotides along hPIV3-C sequence and fused to appropriate tails to allow gap-repair recombination with linearized Gal4-DB yeast two-hybrid vector ( Figure 5A ) . All possible combinations of forward and reverse primers were used to amplify hPIV3-C fragments . Finally , corresponding PCR products were transformed in a yeast strain expressing Gal4-AD fused to either STAT1 or GRB2 , and growth on selective medium was used to detect potential interactions . A 124 ( AA ) -long peptide encompassing position 76 to 199 located in the C-terminal half of hPIV3-C was sufficient to bind STAT1 ( Figure 5B ) or GRB2 ( Figure 6A ) . In an iterative process , we then generated a second , a third and a fourth set of hPIV3-C fragments corresponding to one-by-one AA deletions ( Figure 5C–E and Figure 6B–D ) , allowing to further reduce the STAT1 and GRB2 binding motifs to minimal peptides . A 106 AA peptide encompassing residues 90 to 195 of hPIV3-C was sufficient to observe the interaction with STAT1 ( Figure 5E ) . The binding region to GRB2 was virtually the same , encompassing AA 97 to 195 ( Figure 6D ) . The C-terminal region of hPIV3-C required to bind STAT1 and GRB2 in the yeast two-hybrid system is highly conserved among Respiroviruses ( Figure 7A ) and suspected to fold into a structured coiled-coil domain [18] . Furthermore , virtually the same C-terminal region of Sendai virus C protein ( AA 85-204 ) was previously reported to mediate the interaction with mouse STAT1 [32] . To further validate our observations performed in the yeast two-hybrid system , we retested by co-affinity purification the ability of hPIV3-C fragment encompassing AA 90-195 ( hPIV3-C90-195 ) to interact with STAT1 and GRB2 in HEK-293T cells . GST-tagged hPIV3-C90-195 was expressed together with 3×-FLAG-tagged STAT1 or GRB2 , and purified with glutathion-sepharose beads . Full-length hPIV3-C and the N-terminal region encompassing AA 1-89 ( hPIV3-C1-89 ) were used as positive and negative controls , respectively . As shown in Figure 7B and 7C , hPIV3-C90-195 interacted with STAT1 and GRB2 , whereas hPIV3-C1-89 did not . Although hPIV3-C90-195 interacted with GRB2 as efficiently as full-length hPIV3-C ( Figure 7C ) , interaction with STAT1 was weaker suggesting that more residues contribute to the stabilization of this interaction ( Figure 7B ) . Altogether , these results confirm that AA 90-195 of hPIV-3 include both STAT1 and GRB2 binding sites . Although STAT1 and GRB2 essentially bind to the same region of hPIV3-C as demonstrated above , it remained unclear whether these interactions are mutually exclusive . To answer this question , a competition experiment was designed where GST-tagged hPIV3-C was co-expressed with STAT1 in the presence or absence of GRB2 ( Figure 7D ) . In this setting , GRB2 expression prevents STAT1 co-purification together with GST-tagged hPIV3-C . This validates our finding that STAT1 and GRB2 interact with the same region of hPIV3-C , and demonstrates that STAT1 and GRB2 compete for hPIV3-C binding . Interestingly , GRB2 interaction with hPIV3-C was not affected by STAT1 expression ( Figure 7D and data not shown ) , suggesting that GRB2 has a higher affinity for hPIV3-C than STAT1 . We finally tested if hPIV3-C90-195 was able , like full-length hPIV3-C , to block IFN-α/β signaling and enhance cellular response to EGF stimulation . First , cells were transfected with 3×FLAG-tagged hPIV3-C , hPIV3-C90-195 or hPIV3-C1-89 together with the IFN-α/β reporter plasmid , and stimulated 24 h later with recombinant IFN-β . Reporter gene expression was determined 24 h post transfection and found to be inhibited exclusively by full-length hPIV3-C ( Figure 8A ) . Although this may reflect the weakness of hPIV3-C interaction with STAT1 ( Figure 7B ) , this also indicates that both the N-terminal and C-terminal regions of hPIV3-C are required to block IFN-α/β signaling , even if only the C-terminal region is required for the binding to STAT1 . The same constructs were tested using the Elk1 activity reporter plasmids ( Figure 8B ) . Again , only full-length hPIV3-C was able to enhance Elk1 activation upon EGF stimulation whereas full-length hPIV3-C and hPIV3-C90-195 were expressed at similar levels in transfected cells ( Figure 8B , upper right panel ) . Because GRB2 binding to hPIV3-C and hPIV3-C90-195 were essentially equivalent in co-affinity purification experiments , we hypothesized that the N-terminal region of hPIV3-C was required for its proper subcellular localization . Thus , hPIV3-C , hPIV3-C90-195 and hPIV3-C1-89 were expressed in fusion downstream of the red fluorescent protein Cherry . As shown in Figure 8C , Cherry alone or fused to hPIV3-C90-195 localized both in the nucleus and the cytoplasm of transfected cells . In contrast , full-length hPIV3-C essentially accumulated at the cellular membrane whereas hPIV3-C1-89 was in the nucleus . Although we have no explanation for this unexpected localization of hPIV3-C1-89 , these observations show that only full-length hPIV3-C is able to target the cellular membrane where both IFN-α/β and EGF signaling are triggered .
Paramyxoviridae have evolved various mechanisms to block IFN-α/β response , in particular signaling downstream IFNAR1/IFNAR2c receptor [20] , [33] . Although members of Pneumovirinae subfamily have specific genes to encode inhibitors of IFN-α/β signaling pathway , those expressed by other Paramyxoviridae ( i . e . Paramyxovirinae subfamily ) are encoded by overlapping reading frames embedded within the gene P . Rubulaviruses express V proteins that target STAT1 and/or STAT2 for ubiquitination and degradation , while Morbilliviruses and Henipaviruses V proteins essentially impair STAT1/2 phosphorylation , activation and nuclear translocation . In addition , Morbilliviruses and Henipaviruses also encode for C proteins of which role in the inhibition of IFN-α/β response has been a matter of debates [34]–[37] . Recent reports showed that Morbillivirus C proteins only have a minor role in the inhibition of IFN-α/β signaling [37] , but are essential to block IFN-α/β induction [38] . Whether Henipavirus C proteins can directly block IFN-α/β or promote viral replication through alternative mechanisms is unclear [35] . In contrast , it has been clearly established that Respirovirus C proteins are potent inhibitors of IFN-α/β signaling [5] , [18] , [39]–[41] . In this report , we show that hPIV3-C , but not MV-C or Nipah-C , directly interacts with STAT1 and efficiently inhibits IFN-α/β signaling . In addition , we identified a minimal STAT1 binding domain that encompasses AA 90-195 of hPIV3-C , a region suspected to fold into a coiled coil . Interestingly , this conserved domain is localized within the STAT1 binding region shared by all four isoforms of Sendai virus C protein [32] . Together , these results confirm the capacity of hPIV3-C to block IFN-α/β signaling pathway [18] , provide molecular basis to this inhibition and clarify the fact that Respirovirus C proteins are functionally distinct from Morbillivirus and Henipavirus C proteins . In addition to STAT1 , we show that hPIV3-C interacts directly with GRB2 and enhances MAPK/ERK signaling downstream of EGF receptor ( EGFR ) . Our data give the first example of a Paramyxoviridae protein that contributes to the stimulation of EGFR and MAPK/ERK pathway and provides molecular basis to this activity . This pathway has been known for decades as a prime target of DNA tumor viruses and oncogenic retroviruses , and its activation represents an essential step toward carcinogenesis [3] , [42] . But recent data demonstrate that non-oncogenic RNA viruses also activate this signaling cascade to support viral replication and spreading [31] . Whether it is activated upon EGFR engagement or other means , MAPK/ERK pathway regulates a multiplicity of cellular processes including proliferation , differentiation , development , cell survival and inflammation . As a consequence , how the activation of MAPK/ERK pathway promotes viral replication is a complex question . Interestingly , two non-oncogenic RNA viruses associated with acute respiratory tract infections have been recently reported to modulate the EGFR pathway . Both human respiratory syncytial virus ( hRSV ) , a member of Paramyxoviridae like hPIV3 , and a rhinovirus that belongs to Picornaviridae family activate EGFR and MAPK/ERK pathway [25] , [27] . Infection of epithelial cells by these viruses stimulates the processing and activation of EGFR ligands by membrane matrix metalloproteinase and subsequent engagement of EGFR through autocrine/paracrine mechanisms . Experiments performed on rhinovirus show that viral replication and TLR3 engagement by viral RNA are both required to activate the EGFR and MAPK/ERK pathway [27] . In this report , we show that hPIV3 infection also activates MAPK/ERK pathway in the absence of external stimuli , a phenomenon that possibly relies on the engagement of pathogen recognition receptors . Although hPIV3-C alone is unable to activate this pathway , our data suggest that expression of this virulence factor enhances MAPK/ERK activation above normal level in infected cells , thereby contributing to viral replication and pathogenesis . Induction of MAPK/ERK pathway by RNA viruses has numerous consequences on cell biology . First , it results in increased expression of inflammatory factors , in particular cytokines and chemokines that recruit cellular effectors of immunity [27] , [43]–[47] . MAPK/ERK pathway was also reported to block the antiviral response induced by IFN-α/β , making its activation beneficial to virus replication [29] . Another consequence of MAPK/ERK pathway activation is the induction of mucin production by infected epithelial cells [24] , [27] . Although mucin expression is a critical innate defense system , excessive production of mucus results in the obstruction of airways and delays the elimination of pathogens . Finally , it has been demonstrated in vitro that upon hRSV infection , activation of EGFR and MAPK/ERK pathway sustains viral replication by retarding the death of infected cells [25] . Altogether , these data suggest that although a moderate activation of MAPK/ERK pathway contributes to the innate response against viruses , an excessive activation leads to deleterious inflammation , inhibition of IFN-α/β response , airway obstruction and infected cell survival [28] . Therefore , it is tempting to speculate that hPIV3-C interaction with GRB2 and EGFR pathway participates in such deregulation of airway epithelium homeostasis to promote hPIV3 replication and spreading . A consequence of such perturbations could be an aggravation of chronic inflammatory airway diseases like asthma or chronic obstructive pulmonary disease as already suggested by epidemiological links with Paramyxoviridae infections and in vivo models [48] , [49] . Besides its effects on immune response , activation of MAPK/ERK pathway has direct consequences on viral replication as assessed by in vitro experiments . It is now well documented that MAPK/ERK pathway inhibition with U0126 or PD098059 deeply impairs the replication of numerous RNA viruses including hRSV ( [50]; and for review see [31] ) . Similarly , we show in this report that MAPK/ERK pathway inhibition prevents hPIV3 protein expression in infected cells as assessed by hPIV3-HN detection . In influenza virus infected cells , membrane accumulation of influenza virus hemagglutinin ( HA ) induces lipid-rafts clustering that leads to MAPK/ERK pathway activation and nuclear export of viral ribonucleoprotein complexes to achieve viral particles assembly [51]–[53] . Because hPIV3 replication cycle is only cytoplasmic , mechanisms involved are necessarily distinct . A possible link between MAPK/ERK pathway and hPIV3 protein expression lies in the fact that among downstream targets of this pathway are essential factors of cellular translational machinery . We show that hPIV3-C expression enhances the phosphorylation of S6 and eIF4E . The small ribosomal subunit protein S6 is the major phosphoprotein of eukaryotic ribosomes with five phosphorylation sites ( Ser235 , Ser236 , Ser240 , Ser244 , and Ser247 ) . Two families of serine/threonine kinases phosphorylate S6 in vitro: S6K1/2 and p90 ribosomal S6 kinase ( RSK ) . Recently it has been shown that MAPK/ERK signaling pathway activates RSK family members that contribute to S6 phosphorylation on Ser235/236 thereby stimulating cap-dependent translation [23] . In addition , eIF4E that interacts with the cap structure and brings translation initiation factors together with the small ribosomal subunit via the scaffold protein eIF4G , undergoes regulated phosphorylation on Ser209 upon MAPK/ERK pathway activation . This phosphorylation event is dependent on eIF4G-associated MAPK signal-integrating kinases , Mnk1 and Mnk2 [22] . eIF4E is believed to be the least abundant of all initiation factors and therefore considered as a perfect target to regulate protein synthesis . Even though there is no direct link between eIF4E phosphorylation and the enhanced translation observed , the fraction of phosphorylated eIF4E dramatically increases following treatment of the cells with growth factors , hormones and mitogens . Therefore , eIF4E phosphorylation has been associated with increased translation rates . hPIV3 mRNAs are capped and polyadenylated like their host counterparts . Thus , S6 and eIF4E phosphorylation together with a high level of viral gene transcription may contribute to a rapid switch toward viral protein synthesis within infected cells . Specific biochemical investigations are still required to decipher how hPIV3-C can both inhibit IFN-α/β signaling and enhance EGFR and MAPK/ERK pathway . When searching the literature for viral proteins that target GRB2 , we found specific reports on NS5A from hepatitis C virus and ORF3 from hepatitis E virus [54] , [55] . Although NS5A inhibits MAPK/ERK activation induced by exogenous EGF , ORF3 was described as an activator of MAPK/ERK pathway like hPIV3-C . Both NS5A and ORF3 exhibit a proline-rich motif ( PXXP ) to bind the Src homology 3 ( SH3 ) domains of GRB2 , but there is no such motif in hPIV3-C suggesting that other mechanisms mediate STAT1 and GRB2 binding . Interestingly , these two cellular proteins exhibit SH2 domains . Such domains typically bind a phosphorylated tyrosine residue in the context of a longer peptide motif within a target protein . Although there is no evidence that hPIV3-C becomes phosphorylated , we have tested hPIV3-C interaction with mutant STAT1 and GRB2 exhibiting SH2 domains disabled for the interaction with phosphotyrosine residues . These mutants were not affected for the interaction with hPIV3-C ( data not shown ) . This suggests that hPIV3-C either binds distinct regions of STAT1 and GRB2 , or interacts with a region of the SH2 domain that does not involve the phosphotyrosine binding site . Finally , our results also show that full-length hPIV3-C is required to modulate IFN-α/β and MAPK/ERK pathways since AA 90-195 that bind STAT1 and GRB2 are unable to do so when expressed alone . Full-length hPIV3-C was also required to observe a localization at the cell membrane , suggesting a link with its activity . Interestingly , the N-terminal 23 residues of Sendai virus C protein act as a membrane targeting signal [56] . But the N-terminal residues of hPIV3-C ( AA 1-89 ) were unable to do so , and sequence analysis did not show any conservation with the C protein of Sendai virus . Thus , hPIV3-C tertiary structure is apparently required to target this protein at the cell membrane . This specific localization could both sequester STAT1 to prevent the stimulation of IFN-target genes and contribute to the aggregation of GRB2-SOS complexes to enhance MAPK/ERK signaling [57] . Altogether , this suggests that hPIV3-C interaction with STAT1 and GRB2 represents a potential target for the development of antiviral molecules against hPIV3 and possibly other members of Respirovirus genus .
P-encoding sequence from hPIV3 wild-type strain ( DF042505 ) was amplified by RT-PCR ( Titan One tube; Roche Applied Science ) from total RNA purified from infected cells ( RNeasy kit; Qiagen ) . Amplification was performed using the following hPIV3-P specific primers flanked with Gateway cloning sites: 5′-ggggacaactttgtacaaaaaagttggcatgGAAAGCGATGCTAAAAACTATCAAA and 5′-ggggacaactttgtacaagaaagttggttaTTGGCAATTATTGACATCTTCATTGAAC . PCR products were cloned using TOPO TA Cloning kit ( Invitrogen ) into TOPO vector . A total of 21 clones were analyzed to establish the sequence of hPIV3-P ( GenBank ID: EU719627 ) . Interestingly , 8 clones were not edited , 11 clones were edited by the addition of one G residue , and 2 clones were edited by the addition of 5 G residues . One of the plasmids containing the unedited sequence of hPIV3-P was selected and subsequently used as a template to clone hPIV3-C . DNA sequences encoding full-length hPIV3-C or fragments corresponding to AA 1-89 or 90-195 were amplified by PCR from p ( hPIV3-P ) -TOPO and cloned by in vitro recombination into pDONR207 ( Gateway system; Invitrogen ) as previously described [58] . Similarly , MV-C was amplified from p ( + ) MV323 that contains the full genome of measles virus wild-type strain Ichinose ( kindly provided by Dr . K . Takeuchi , [59] ) . Nipah-C was amplified from NiV-P plasmid ( kindly provided by Dr . TF . Wild; [60] ) . GRB2 coding sequence was amplified from the human spleen cDNA library used to perform the yeast two-hybrid screen ( Invitrogen ) . The pDONR207 plasmid containing STAT1 was previously described [58] . Viral or cellular coding sequences were subsequently transferred by in vitro recombination from pDONR207 into different Gateway-compatible destination vectors ( see below ) following manufacturer's recommendation ( LR cloning reaction , Invitrogen ) . To perform yeast two-hybrid experiments , coding sequences were recombined into pPC86 ( Invitrogen ) to be expressed in fusion downstream of the activation domain of Gal4 ( Gal4-AD ) or into pDEST32 to be expressed in fusion downstream of the DNA binding domain of Gal4 ( Gal4-DB ) . In mammalian cells , GST-tag and 3×FLAG-tag fusions were achieved using pDEST27 ( Invitrogen ) or pCI-neo-3×FLAG vector , respectively [61] . We also used pCI-neo ( Promega ) and pmCherry-C1 ( Clontech ) to express proteins without a tag or in fusion downstream of Cherry , respectively . These two plasmids were made Gateway-compatible using the Gateway vector conversion system ( Invitrogen ) . HEK-293T , Hela and Vero cells were maintained in Dulbecco's modified Eagle's medium ( DMEM; Gibco-Invitrogen ) containing 10% fetal bovine serum , penicillin , and streptomycin at 37°C and 5% CO2 . A549 and BEAS-2B cells were maintained in F-12K medium ( Gibco-Invitrogen ) containing 10% fetal bovine serum , penicillin , and streptomycin at 37°C and 5% CO2 . hPIV3 ( strain C243 ) was amplified and titrated on Vero cells following recommendations of ATCC ( American Type Culture Collection ) . Recombinant MV-EGFP virus used in Figure S2 has been previously described [62] . Infections were performed for 2 h at 37°C in Optimem ( Gibco-Invitrogen ) . Later on , cells were washed and incubated in fresh culture medium for 24 or 48 h . To detect viral replication , cells were recovered and incubated in PBS-paraformaldehyde 3 . 2% for 20 min . After extensive washing with PBS , cells were permeabilized with PBS-Triton 0 . 05% for 15 min , and then incubated with a monoclonal antibody specific to hPIV3-HN ( M02122321 , Abcam ) . Cells were washed and incubated in the presence of an anti-mouse Cy3-conjugated antibody ( Jackson Immunoresearch ) . After extensive washing , cellular immuno-staining was analyzed using a FACSCalibur flow cytometer ( BD ) . When specified , cells were pre-treated with MEK1/2 specific inhibitor U0126 ( 20 µM final; Promega ) for 2 h before , during and after infection to study the impact on hPIV3 infection . To perform co-affinity purification experiments , cloned ORFs were transferred from pDONR207 to pDEST27 expression vector ( Invitrogen ) to achieve GST fusion , and to pCI-neo-3×FLAG vector [61] for 3×FLAG-fusion . Cell transfections were performed using Lipofectamine 2000 ( Invitrogen ) . Unless specified otherwise , 5×105 HEK-293T cells were dispensed in each well of a 6-well plate , and transfected 24 h later with 600 ng of each plasmid DNA per well . Two days post transfection , HEK-293T cells were washed in PBS , then resuspended in lysis buffer ( 0 . 5% Nonidet P-40 , 20 mM Tris–HCl at pH 8 , 120 mM NaCl and 1 mM EDTA ) supplemented with Complete Protease Inhibitor Cocktail ( Roche ) . Cell lysates were incubated on ice for 20 min , and then clarified by centrifugation at 14 , 000×g for 10 min . For pull-down analysis , 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads ( Amersham Biosciences ) to purify GST-tagged proteins . Beads were then washed 3 times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer ( Invitrogen ) . Purified complexes and protein extracts were resolved by SDS-polyacrylamide gel electrophoresis ( SDS-PAGE ) on 4–12% NuPAGE Bis–Tris gels with MOPS running buffer ( Invitrogen ) , and transferred to a nitrocellulose membrane . Proteins were detected using standard immunoblotting techniques . 3×FLAG- and GST-tagged proteins were detected with a mouse monoclonal HRP-conjugated anti-3×FLAG antibody ( M2; Sigma-Aldrich ) and a rabbit polyclonal anti-GST antibody ( Sigma-Aldrich ) , respectively . Specific antibodies were used to detect endogenous STAT1 ( clone-1; BD Biosciences ) , GRB2 ( clone-81; BD Biosciences ) , phospho-ERK1/2 ( clone-12D4; Upstate ) , ERK1/2 ( CT; Upstate ) , phospho-eIF4E ( Ser209; Cell Signaling ) , eIF4E ( Cell Signaling ) , phospho-S6235-236 ( Ser235/236; Cell Signaling ) , S6 ( clone-54D2; Cell Signaling ) and hPIV3-HN ( M02122321; Abcam ) . Secondary anti-mouse and anti-rabbit HRP-conjugated antibodies were from GE-Healthcare . Densitometric analysis of the gels was performed using a specific module of Photoshop CS3 Extended ( Adobe Systems Inc . ) . Our yeast two-hybrid protocols have been described in details elsewhere [58] . Briefly , pDEST32 plasmid encoding Gal4-DB fused to hPIV3-C was transformed in AH109 yeast strain ( Clontech ) , and used to screen by mating a human spleen cDNA library cloned in the Gal4-AD pPC86 vector ( Invitrogen ) and previously established in Y187 yeast strain ( Clontech ) . Yeast cells were plated on a selective medium lacking histidine and supplemented with 10 mM 3-amino-triazole ( 3-AT; Sigma-Aldrich ) to select for interaction-dependent transactivation of HIS3 reporter gene . AD-cDNAs from [His+] colonies were amplified by PCR and sequenced to identify the host proteins interacting with hPIV3-C . The gap-repair procedure was used to map the minimal portion of hPIV3-C interacting with STAT1 and GRB2 . As previously described [63] , both forward and reverse PCR primers were designed along the sequence of hPIV3-C and fused to specific tails allowing yeast-based recombination in Gal4-DB two-hybrid vector . Matrix combinations of forward and reverse primers were used to amplify fragments of hPIV3-C by PCR . AH109 yeast cells expressing AD-fused STAT1 or GRB2 were co-transformed with 5 µL of each PCR product in the presence 50 ng of linearized pDEST32 vector to achieve recombinatorial cloning by gap-repair . Fragments of hPIV3-C fused to Gal4-DB were then tested for interaction with AD-STAT1 or AD-GRB2 by plating yeast cells on selective medium lacking histidine and supplemented with 10 mM of 3-AT . HEK-293T , Hela or Vero cells were plated in 24-well plates ( 2×105 cells per well ) . One day later , cells were transfected with either pISRE-Luc ( 0 . 3 µg/well; Stratagene ) or pFA2-Elk1 ( 0 . 3 µg/well; Stratagene ) and pGal4-UAS-Luc plasmids ( 0 . 3 µg/well; provided by Dr . Y . Jacob ) together with pRL-CMV reference plasmid ( 0 . 03 µg/well; Promega ) . Cells were simultaneously co-transfected with 0 . 3 µg/well of pCI-neo-3×FLAG , pCI-neo or pmCherryC1 expression vectors encoding viral proteins as specified . 24 h after transfection , cells were stimulated with IFN-β ( Biosource ) at 1000 IU/ml or starved for 6 h then stimulated with EGF ( Upstate ) at 100 ng/ml . 24 h post transfection , cells were lysed , and both firefly and Renilla luciferase activities in the lysate were determined using the Dual-luciferase Reporter Assay System ( Promega ) . Reporter activity was calculated as the ratio of firefly luciferase activity to reference Renilla luciferase activity , and normalized so that positive control activity equals 100 . When indicated , cells were treated with U0126 ( Promega ) at 20 µM final concentration upon EGF stimulation . 24-well plates containing coverslips were seeded with HEK-293T cells ( 2×105 cells per well ) . One day later , cells were transfected with pmCherryC1 expression vector alone or encoding hPIV3-CFL , hPIV3-C1-89 or hPIV3-C90-195 . 36 h after transfection , cells were incubated with PBS-PFA 4% for 20 min at RT , then treated with PBS-Triton 0 . 05% for 15 min at RT to permeabilize the cells . Finally , cells were incubated for 10 min at RT in a PBS-PFA 4% solution containing DAPI ( 4′-6-Diamidino-2-phenylindole ) at 10 µg/ml . Preparations were mounted using Fluoromount-G ( Southernbiotech ) , and imaging performed using a SP5 confocal miscroscope ( Leica ) . | Respiroviruses are important pathogens responsible for acute respiratory tract infections associated with severe airway inflammation in children , elderly and immuno-compromised individuals . Their RNA genome encodes for structural proteins that compose viral particles , but also for virulence factors that alter cell biology to enhance virus replication and spreading . Among them , the C protein plays a critical role by blocking cellular response to type I interferons , the main antiviral cytokines secreted during virus infections . To provide molecular basis to this activity , we found that the C protein of human parainfluenza virus type 3 ( hPIV3-C ) , the most frequent human Respirovirus , interacts with STAT1 , a key component of type I interferon receptor complex . But hPIV3-C was also found to interact with GRB2 , an adaptor molecule involved in cellular response to Epidermal Growth Factor ( EGF ) , and to enhance cell response to this cytokine . This pathway increases protein translation , promotes cell survival and contributes to airway inflammation and mucus secretion . Thus , our findings show that hPIV3-C not only inhibits the antiviral response but also stimulates cellular response to EGF , which benefits virus replication and induces an excessive inflammation of airways during infection . | [
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] | 2009 | Differential Regulation of Type I Interferon and Epidermal Growth Factor Pathways by a Human Respirovirus Virulence Factor |
The human genome represents a fossil record of ancient retroviruses that once replicated in the ancestors of contemporary humans . Indeed , approximately 8% of human DNA is composed of sequences that are recognizably retroviral . Despite occasional reports associating human endogenous retrovirus ( HERV ) expression with human disease , almost all HERV genomes contain obviously inactivating mutations , and none are thought to be capable of replication . Nonetheless , one family of HERVs , namely HERV-K ( HML-2 ) , may have replicated in human ancestors less than 1 million years ago . By deriving a consensus sequence , we reconstructed a proviral clone ( HERV-KCON ) that likely resembles the progenitor of HERV-K ( HML-2 ) variants that entered the human genome within the last few million years . We show that HERV-KCON Gag and protease proteins mediate efficient assembly and processing into retrovirus-like particles . Moreover , reporter genes inserted into the HERV-KCON genome and packaged into HERV-K particles are capable of infectious transfer and stable integration in a manner that requires reverse transcription . Additionally , we show that HERV-KCON Env is capable of pseudotyping HIV-1 particles and mediating entry into human and nonhuman cell lines . Furthermore , we show that HERV-KCON is resistant to inhibition by the human retrovirus restriction factors tripartite motif 5α and apolipoprotein B mRNA-editing enzyme , catalytic polypeptide-like ( APOBEC ) 3G but is inhibited by APOBEC 3F . Overall , the resurrection of this extinct infectious agent in a functional form from molecular fossils should enable studies of the molecular virology and pathogenic potential of this ancient human retrovirus .
A characteristic that is unique to retroviruses is their propensity to integrate their genomes into host-cell DNA as an essential part of their replication cycle . Thus , if the target cell population of a given retrovirus includes germ cells or their progenitors , retroviral genomes can be inherited in a Mendelian manner as so-called “endogenous” forms ( see [1] for review ) . Indeed , endogenous retroviruses have accumulated over time in the genomes of many organisms and are extraordinarily common in mammals , comprising approximately 8% of human DNA [2] . Nonetheless , while some avian , murine , and primate species harbor replication-competent retroviruses within their genomes , intact retroviruses are relatively infrequent and almost all endogenous retroviruses are obviously defective due to the presence of stop codons and frameshifts in one or more genes . Among the numerous families of defective human endogenous retroviruses ( HERVs ) found in modern human DNA , the human mouse mammary tumor virus–like 2 ( HML-2 ) subfamily of HERV-K proviruses is of special interest . Even though replication-competent forms of HERV-K ( HML-2 ) have not been found , some proviruses were deposited in the human genome after speciation and represent some of the youngest HERVs known [3–6] . Also , occasional reports link their expression with human disease [7] . The age of an endogenous provirus can be roughly estimated by comparing sequence of the two long terminal repeats ( LTRs ) . At integration , the two proviral LTRs should be identical , but during host DNA replication , each LTR independently accumulates mutations as a function of age , and it is estimated that one difference between two LTRs should occur every approximately 200 , 000 to 450 , 000 y . Several HERV-K ( HML-2 ) proviruses have been identified in human DNA that have less than five differences between the two LTRs , suggesting deposition perhaps less than 1 million y ago [3–6] . HERV-K ( HML-2 ) –related proviruses are found only in Old World primates genomes , and many are unique to humans , with nonhuman primate genomes containing empty preintegration sites at orthologous loci . Compellingly , polymorphism exists in humans with respect to the presence or absence of proviruses at some HERV-K integration sites , indicating insertion relatively recently in human evolution [3–6] . Furthermore , many of the younger HERV-K ( HML-2 ) proviruses contain a subset of open reading frames ( ORFs ) with a few or no mutations [3 , 6 , 8] . However , all known HERV-K proviruses are replication defective . There are several ways in which a defective provirus can proliferate in a host's genome , including via exogenous infection events following complementation in trans , where functional proteins are supplied by other endogenous or exogenous viruses . Alternatively , for some retroelements , envelope-independent retrotransposition can occur in cis , where an element copies itself and inserts into a new genomic locus within the same cell , forgoing the normal extracellular phase of the retroviral life cycle . Defective proviruses can also be proliferated as a result of long interspersed element retrotransposition [9] . However , most HERV-K ( HML-2 ) replication appears to have been a consequence of autonomous infection by extracellular virions [10 , 11] . This conclusion is based on the comparatively low number of stop codons and ratio of nonsynonymous to synonymous changes ( dN/dS ) in HERV-K ORFs , indicating a purifying selection on all proteins . Notably , this finding holds for HERV-K Env [10] , which should be required for replication that includes an extracellular step but not for any other mode of provirus proliferation . Ancient retroviruses are of interest , in part because they likely imposed selective pressure on host defenses in human ancestors . Indeed , the tripartite motif ( TRIM ) 5α and apolipoprotein B mRNA-editing enzyme , catalytic polypeptide-like ( APOBEC ) 3 proteins that provide part of the host defense against modern retroviruses have been under positive selection for much of primate evolution [12–16] . As a retrovirus that appears to have replicated in the ancestors of modern Old World monkeys , apes , and humans , HERV-K may be partly responsible for this pressure . Moreover , it is conceivable that HERV-K exists today in an undetected replication-competent form in rare humans [4] . However , no studies of the virology or pathogenic potential of this ancient human virus have been possible because a contemporary , replication-competent HERV-K strain has not been identified and may not exist at all . Despite some functional degradation due to mutation during deposition or during human DNA replication , HERV-K ( HML-2 ) proviruses that have been deposited in human DNA in the past few million years should be reasonably well preserved and have relatively few inactivating mutations . Indeed , various studies have shown that individual proteins from certain HERV-K proviruses can function in vitro [17–25] . We reasoned that it might be possible to resurrect HERV-K ( HML-2 ) in replication-competent form using proviruses that are thought to have most recently entered the human genome as a template . Therefore , we constructed a HERV-K strain whose genome sequence is a consensus of a subset of HERV-K ( HML-2 ) proviruses . Importantly , we demonstrate that all viral proteins necessary for viral replication encoded by this provirus are functional and that proteins and genomes based on the reconstructed HERV-K ( HML-2 ) viral genome can be used to generate infectious exogenous retrovirus particles .
Initially , our attempts to construct an infectious HERV-K provirus were based on the sole HERV-K provirus ( HERV-K113 ) that has apparently intact ORFs for all viral proteins [6] . This provirus , believed to be among the youngest human-specific HERV-K proviruses , is present in the genomes of a minority of humans . Unfortunately , construction of Gag , Gag-protease ( PR ) , and Gag-PR-Pol expression plasmids based on HERV-K113 resulted in proteins that were poorly expressed and were inefficiently processed and released as virus-like particles ( VLPs ) ( unpublished data ) . Thus , because presence of intact ORFs did not necessarily imply intact function , we took an alternative approach . Specifically , we adopted a strategy that was based on the assumption that any inherent replication defects that are encoded within HERV-K ( HML-2 ) proviruses present in contemporary human DNA are either unique to each provirus or shared only by a minority of recently integrated proviruses . If this assumption was correct , then each individual defect should be absent from a sequence representing the consensus of a collection of proviruses , even if each provirus that contributes to the consensus is defective . We selected a group of ten full-length HERV-K ( HML-2 ) proviruses to derive a consensus HERV-K sequence . Specifically , the ten proviruses with the best scores following BLAST searching of human DNA with full-length HERV-K113 sequence were chosen . As well as HERV-K113 itself , this search yielded HERV-K101 , HERV-K102 , HERV-K104 , HERV-K107 , HERV-K108 , HERV-K109 , HERV-K115 , HERV-K11p22 , and HERV-K12q13 . All of these proviruses are known to be unique to humans , indicating integration into the germ-line within the last 6 million y , when the human lineage is believed to have diverged from the chimpanzee lineage [3–6] . Moreover , several show insertional polymorphism in humans , with intact preintegration sites present in a fraction of the human population , suggesting even more recent replication . While all except HERV-K113 encoded an obvious defect in at least one ORF , all of the selected proviruses also had an intact ORF for at least one of the putative HERV-K proteins ( Figure 1A ) . The nucleotide encoded by the majority of each of the ten proviruses was deduced for each of 9 , 472 nucleotide positions to derive HERV-KCON . Thereafter , using a set of synthetic , approximately 60 base oligonucleotides spanning the entire HERV-KCON sequence and a PCR- based strategy to progressively link them together , we first constructed a plasmid containing the HERV-KCON proviral genome . The complete proviral consensus sequence and the viral proteins that it encodes are shown in Figure S1 . As expected , the HERV-KCON sequence was positioned close to the root of a phylogenetic tree constructed using HERV-KCON itself and each of the ten proviruses used to derive it ( Figure 1B ) . Thus , we reasoned that HERV-KCON represented a reasonable approximation to the ancestor of HERV-K sequences that integrated into the human genome within the past few million years and might , therefore , be capable of replication . Interestingly , pairwise comparisons indicated that the majority of nucleotide differences between HERV-KCON and each of the ten contributing proviruses were either G-to-A or C-to-T changes , or vice versa ( Figure 1C ) . This finding hints at a possible role of cytidine deaminases in driving HERV-K evolution in humans , and perhaps contributing to the inactivation of contemporary proviruses . To determine whether the major HERV-KCON structural proteins and enzymes were capable of assembling into retrovirus-like particles , we constructed plasmids expressing the consensus Gag , Gag-PR and Gag-PR-Pol ORFs . The HERV-K genome has an unusual nucleotide composition in that it is relatively A-rich . This feature , which is also characteristic of lentiviruses such as HIV-1 , is partly responsible for the nuclear retention of HIV-1 mRNAs and contributes to the requirement for Rev in mediating export of incompletely spliced HIV-1 transcripts . Indeed , HERV-K encodes a functional ortholog of the Rev protein , termed K-Rev or Rec , that mediates nuclear export of HERV-K RNAs [18–20 , 26] . Therefore , because of the likely requirement for a Rev-like post-transcriptional activator for efficient HERV-K mRNA export , cDNAs encoding the HERV-KCON structural proteins were inserted into a previously described expression vector , termed pCRV1 [27] , that provides an HIV-1 Rev response element to the expressed mRNA in cis and the HIV-1 Rev protein in trans . Transfection of pCRV1-based plasmids expressing HERV-KCON Gag , Gag-PR , or Gag-PR-Pol resulted in the expression of a protein of approximately 70 to 80 kDa , detected by Western blotting using a commercially available antibody raised against HERV-K Gag ( Figure 2A ) . This approximated to the size expected ( 74 kDa ) of the intact HERV-K Gag precursor . A concurrent analysis of proteins pelleted from culture supernatant through 20% sucrose revealed that Gag expression alone could efficiently generate extracellular particles ( Figure 2A ) . In addition to the 74-kDa Gag precursor , a protein of approximately 40 kDa that reacted with the HERV-K Gag antibody was detected in lysates of cells transfected with Gag-PR and Gag-PR-Pol expression plasmids . While the precise identity of the 40-kDa protein is unknown , it likely represents a proteolytically processed form of Gag and , therefore , this finding suggested that the HERV-KCON protease was active . Consistent with this notion , Western blot analysis of extracellular particles generated following Gag-PR and Gag-PR-Pol expression did not contain detectable Gag precursor but did contain the 40-kDa apparently processed Gag species ( Figure 2A ) . Analysis of total protein in extracellular particles by SDS-PAGE and silver staining revealed that HERV-KCON Gag expression alone generated particles composed of a single dominant protein of the size predicted for the HERV-K Gag precursor , as expected ( Figure 2B ) . Particles generated by Gag-PR contained a dominant protein of 30 kDa , which based on previous studies likely represents HERV-KCON capsid ( CA ) [24 , 28 , 29] . A smaller protein or proteins of 20 kDa were also observed in Gag-PR particles , which presumably represents other mature Gag processing product or products such as matrix or nucleocapsid ( Figure 2B ) . Additionally , a protein of 40 kDa that likely corresponded to the 40-kDa band detected by Western blotting was also observed on silver-stained gels . However , the 40-kDa protein was a minor species in Gag-PR particles , and it is therefore possible that this protein represents a partly processed intermediate . HERV-K Gag-PR-Pol expression also yielded particles containing the same apparently processed Gag proteins as those generated by Gag-PR but at slightly lower levels ( Figure 2B ) . The appearance of the 30-kDa putative CA protein on silver-stained gels ( Figure 2C ) was abolished when three predicted active site residues ( Asp-Thr-Gly ) in the HERV-KCON protease ORF were mutated to Ala-Ala-Ala . Additionally , a higher-molecular-weight protein , possibly representing the Gag-PR precursor , was observed in particles harvested from cells expressing the mutant Gag-PR protein ( Figure 2C ) . Although their low abundance relative to contaminating extraneous cellular proteins and the lack of available antibodies precluded unambiguous identification of Pol proteins in SDS-PAGE analyses of HERV-KCON VLPs , supernatants of 293T cell cultures transfected with the HERV-KCON Gag-PR-Pol expression plasmid contained quite high levels of reverse transcriptase activity , as detected by an ELISA-based assay designed for the detection of HIV-1 reverse transcriptase ( Figure 2D ) . As controls , no reverse transcriptase activity was detected in cultures transfected with HERV-KCON Gag or Gag-PR expression plasmids . Coexpression of HERV-KCON Gag and Gag–green fluorescent protein ( GFP ) fusion proteins in 293T cells revealed that HERV-KCON Gag localized predominantly to the plasma membrane , where numerous fluorescent puncta were observed ( Figure 2E ) . Moreover , electron microscopic examination of 293T cells expressing HERV-KCON Gag-PR revealed the presence of cell-associated retrovirus-like particles and structures that appeared to represent assembly intermediates ( Figure 2F ) . Most particles appeared as 100 to 150 nm , apparently spherical immature virions , with a minority assembled as aberrant particles that appeared as two or more connected , partly assembled , virions . While we did not observe unambiguously mature virions associated with the surface of Gag-PR–expressing cells , it is possible that full maturation , which was clearly indicated by the biochemical analysis of extracellular VLPs ( Figure 2B and 2C ) , occurred only after the completion of particle release from cells . Completely or incompletely assembled particles appeared exclusively at the plasma membrane with a morphology resembling partly assembled alpharetroviruses or gammaretroviruses . Even though betaretroviruses represent HERV-Ks closest exogenous retrovirus relatives , no cytoplasmic , nonenveloped particles , typically observed in betaretroviruses , were found . To determine whether particles containing the HERV-KCON genome , Gag , PR , and Pol proteins were capable of infectious transfer of the HERV-KCON genome to target cells , we inserted a reporter gene cassette ( cytomegalovirus [CMV]-GFP ) into the env gene of the HERV-KCON proviral plasmid . Additionally , because the HERV-K LTR promoter is extremely weak in 293T cells ( unpublished data ) , we replaced U3 sequences 5′ to the TATA box with corresponding sequences from the promoter/enhancer of CMV . This construct was named CHKCG ( Figure 3A ) . As expected , transfection of this Env-defective CHKCG construct in 293T cells resulted in GFP expression in transfected 293T cells , but inoculation of target cells with 0 . 2-μm filtered supernatant harvested from these cells did not result in infectious transfer of the reporter gene . However , when an envelope protein from vesicular stomatitis virus ( VSV-G ) was expressed in trans , clear GFP expression was observed in rare foci of target cells inoculated with filtered supernatant from CHKCG-transfected cells . Moreover , by boosting HERV-K protein expression , the yield of infectious virions was improved ( Figure 3B–3E ) . Indeed , when K-Rev/Rec was expressed in trans with CHKCG and VSV-G , infectious particle yield was in excess of 102 IU/ml ( Figure 3B and 3E ) . Similarly , when the HERV-KCON Gag-PR-Pol expression plasmid was provided in trans , the yield of infectious particles also increased to greater than 102 IU/ml ( Figure 3D ) . The combined expression of CHKCG , VSV-G , HERV-K Gag-Pol , and K-Rev/Rec yielded the highest infectious titers ( up to 103 IU/ml , Figure 3C and 3E ) , and this combination of plasmids was used to generate infectious HERV-KCON ( VSV-G ) pseudotyped particles in subsequent studies . While this infectious titer is low compared to that generated by many exogenous retroviruses ( e . g . , murine leukemia virus [MLV] and HIV-1 ) , the yield of infectious HERV-K particles was of the same order as that obtained with similarly constructed human T-cell lymphotropic virus-I–based vector systems ( [30] and unpublished data ) . To verify that transfer of reporter gene expression by HERV-KCON particles was via bona fide retrovirus-based transduction , we inoculated 293T cells with HERV-KCON ( VSV-G ) particles containing the CHKCG genome in the presence of azidothymidine ( AZT ) , a reverse transcriptase inhibitor . AZT is a thymidine analog chain terminator and is known to inhibit reverse transcriptases from a wide variety of retroviruses [31] . As can be seen in Figure 3F , application of AZT to target cells inhibited HERV-K–mediated reporter gene transduction by approximately 30-fold . Thus , reporter gene transfer by HERV-KCON was clearly dependent on reverse transcription . In some cases , low levels of reporter gene expression mediated by retroviral gene transfer can be mediated by reverse-transcribed but nonintegrated retroviral DNA , which can exist as linear or circular forms in target cells [32–34] . However , these retroviral DNA forms are diluted during cell division and eventually lost . Stable retrovirus-mediated gene transfer that is transferred to both daughter cells requires that retroviral DNA be integrated into the target cell genome . While the formation of clear multicellular foci of GFP-positive cells suggested the reporter gene was maintained in daughter cells , integration events are most effectively assayed by daughter cell colony formation under antibiotic selection using retroviral genomes that carry resistance markers . Therefore , we constructed a variant of the CHKCG genome ( Figure 3A ) in which the CMV-GFP cassette was replaced by one carrying a CMV-driven puromycin resistance gene , termed CHKCP . We generated HERV-KCON ( VSV-G ) particles carrying CHKCP in the same way as previously ( Figure 3 ) and found that puromycin-resistant colonies were formed following exposure of 293T target cells to these virions and antibiotic selection ( Figure 4A ) . Indeed , the infectious titers of puromycin resistance transducing particles were similar to that of GFP-transducing particles . To further demonstrate that HERV-KCON genomes were capable of integration , hamster ( CHO745 ) cells were infected with HERV-KCON ( VSV-G ) particles carrying the CHKCP genome and four single cell clones were derived from the resulting puromycin-resistant cell population . Cellular genomic DNA was extracted following expansion of the clones for 2 wk in culture and analyzed for the presence of integrated HERV-K DNA using a PCR-based strategy ( Figure 4B ) . Hamster CHO745 cells were used for these experiments , because they were found to be as sensitive as human cells to HERV-KCON ( VSV-G ) infection ( see below ) , but unlike human cells , they lack endogenous HERV-K proviruses that would complicate detection and analysis of de novo HERV-K integration events . As can be seen in Figure 4C , PCR analysis using HERV-K gag specific PCR primers revealed that each of the CHKCP-transduced clones , but not parental CHO745 cells , carried HERV-K DNA . Next , sequences flanking the integrated provirus were identified using a PCR-based strategy ( GenomeWalker kit; Clontech , http://www . clontech . com ) and in each case revealed the presence of a six-nucleotide duplicated sequence immediately flanking the provirus ( Figure 4D ) . For three CHKCP-transduced CHO745 cell clones , PCR primers were designed that targeted hamster DNA sequences flanking the integrated HERV-KCON provirus ( Figure 4B and 4E ) , and these were used to authenticate the presence of the intact preintegration site in uninfected hamster cells ( e . g . , Figure 4E ) . Moreover , PCRs using combinations of the hamster DNA-specific and HERV-K–specific PCR primers were used to authenticate the presence HERV-K provirus/hamster cellular DNA junctions in three of the CHKCG-transduced clones ( e . g . , Figure 4E ) . Overall , these experiments demonstrate that HERV-K genomes can be replicated via exogenous infection in a reverse transcriptase–dependent manner , resulting in stable and authentic integration into the target cell genome . Next , we determined whether VSV-G pseudotyped HERV-KCON particles could transduce reporter genes into cells other than 293T and CHO745 . As can be seen in Figure 5A , several target cells of human , squirrel monkey , feline , and rodent origin could be infected by HERV-KCON ( VSV-G ) . However , it was noticeable that murine NIH3T3 cells and squirrel monkey Pindak cells were somewhat less sensitive to HERV-KCON ( VSV-G ) , compared to the human and feline cells . The human cells were each quite similar in their sensitivity to HERV-KCON ( VSV-G ) even though 293T cells display little or no TRIM5α-dependent resistance to retroviruses such as EIAV or N-tropic MLV , while TE671 and HT1080 exhibit strong TRIM5α-dependent resistance to N-tropic MLV and EIAV . This finding suggested that HERV-KCON may not be sensitive to human TRIM5α . Additionally , to test whether the HERV-KCON envelope sequence was functional , it was inserted into the HIV-1–based expression vector pCRV1 and expressed along with HIV-1 Gag-Pol proteins and the packageable GFP-expressing HIV-1 vector CSGW . This transfection mixture should generate HIV-1 particles , putatively pseudotyped with the HERV-KCON envelope protein . Notably , HIV ( HERV-KCON ) particles were capable of infecting 293T cells , with titers of around 3 × 102 IU/ml ( Figure 5B ) , while particles generated in the absence of HERV-KCON Env were noninfectious . Inoculation of cells from a small panel of mammalian species revealed that several , including those of human , squirrel monkey , murine , and feline origin , could be infected with HIV-1 ( HERV-KCON ) pseudovirions ( Figure 5B ) . While attempts were made to generate infectious particles that contained both HERV-KCON cores and Env proteins , we were not able to detect infection events using this combination . Nevertheless , these experiments indicate that the HERV-KCON genome contains all functional components required to complete an exogenous retroviral replication cycle . To test the sensitivity of HERV-K to retrovirus restriction factors that it might encounter in human cells and might be responsible for attenuation or extinction of replication therein , we first challenged unmodified , or human TRIM5α-expressing , hamster ( CHO ) -derived cell lines with HERV-KCON ( VSV-G ) . Despite the fact that the human TRIM5α-expressing cell line was greater than 100-fold resistant to N-tropic MLV relative to the control cell line or B-tropic MLV ( Figure 6A ) , HERV-KCON infected unmanipulated and human TRIM5α-expressing cells with nearly identical efficiency ( Figure 6B ) . Additionally , CHO cells expressing rhesus macaque TRIM5α or the unique owl monkey variant of TRIM5 ( TRIM-Cyp ) were also similarly sensitive to HERV-KCON ( VSV-G ) infection as unmanipulated control cells ( Figure 6A ) . This was despite the fact that CHO cells expressing rhesus monkey TRIM5α and owl monkey TRIMCyp were about 30-fold and 100-fold , respectively , resistant to HIV-1 infection compared to HIV-1 carrying an SIVMAC CA ( Figure 6A ) . Next , we tested whether APOBEC3G and APOBEC3F were capable of inhibiting HERV-KCON replication . These cytidine deaminases are the major inhibitors of Vif-deficient HIV-1 infectivity , although APOBEC3G is a significantly more potent inhibitor of HIV-1 replication than is APOBEC3F . Surprisingly , APOBEC3G expression during particle production only marginally inhibited HERV-KCON ( VSV-G ) infection , while APOBEC3F more potently reduced infectivity , reducing titers by about 50-fold ( Figure 6C ) . This was despite approximately equivalent levels of HERV-KCON Gag expression and generation of viral particles in the presence or absence of APOBEC3G or APOBEC3F ( Figure 6D ) . Overall , of the restriction factors tested that are likely to be encountered in human cells , HERV-KCON appeared to be resistant to human TRIM5α and APOBEC3G proteins but sensitive to APOBEC3F .
Here , we constructed a HERV-K provirus whose sequence resembles that of an ancestral human-specific HERV-K ( HML-2 ) . We demonstrate that all viral proteins encoded by this provirus are capable of functioning in the context of a retroviral replication cycle . While some recent studies have reconstituted “live” viruses from synthetic DNA [35 , 36] , this and a similar study of HERV-K which appeared online while this manuscript was in review [37] are the first examples in which the replication cycle of a virus has been reconstituted using a group of sequences that represent ancient fossils and are demonstrably defective . The methods used here are conceptually similar to those applied to the reconstitution of the transposable element Sleeping Beauty , in which a functional Tc1/mariner-type transposon present only in defective forms in fish DNA was reconstituted [38] . Successful reconstitution in that study was achieved using a majority consensus sequence to synthesize an active trasposase protein and selecting cis-acting sequences from a representative element that closely resembled those of the majority consensus sequence [38] . HERV-K likely replicated in the ancestors of humans for approximately 30 million y but is not known to exist as a replication-competent virus today . Indeed , it is possible , even likely , that HERV-K has not replicated as a retrovirus for hundreds of thousands of years . It was not obvious what the optimal approach to reconstitute functional HERV-K sequences would be , since variation in HERV-K sequence could arise through natural variation via error-prone reverse transcription , mutational degradation after deposition in the primate germ-line , or cytidine deamination before , during , or after during initial germ-line deposition ( see below ) . Moreover , it was possible that the population of proviruses accessible to us in modern DNA represented a highly biased sample of HERV-K genomes where defects might have been positively selected during primate evolution . Thus , rather than attempt reconstruct the evolutionary history of HERV-K in primates , we adopted a conservative approach to reconstitute functional sequences , selecting ten proviruses that were most similar to a relatively young and comparatively intact HERV-K provirus ( HERV-K113 ) , reasoning that these were the least likely to have undergone substantial sequence degradation . Moreover , all of the selected proviruses were unique to human DNA , and some were polymorphic in humans , suggesting comparatively recent replication . While it was possible that all of the selected proviruses would have a common lethal defect , this appeared not to be the case . Indeed , by compiling a simple majority consensus sequence , we successfully removed individual lethal defects represented in the group of proviruses that contribute to the consensus sequence , allowing replication of the consensus genome in a bona fide reverse transcription–dependent manner that resulted in the stable integration of HERV-KCON genomes into target cells . Analysis of the replication cycle of HERV-KCON in human cells allowed preliminary characterization of aspects of HERV-K biology that have heretofore been refractory to investigation . Assembly of HERV-K virions at the plasma membrane is notable , given that the exogenous retroviruses that are most closely related to HERV-K include mouse mammary tumor virus and Mason-Pfizer monkey virus , both of which are betaretroviruses that assemble complete capsids within the cytoplasm of infected cells . Nonetheless , previous analyses have suggested that the small number of human cell lines that express HERV-K exhibit plasma membrane localized assembly intermediates [28 , 29] , as was observed here for HERV-KCON . Moreover , previous work has shown that a single amino acid mutation in MPMV Gag protein can change its assembly characteristics from cytoplasmic to plasma membrane associated assembly [39] . Thus , it should not be surprising that HERV-K assembly appears morphologically different to that of its betaretrovirus relatives . Two major components of intrinsic defense against retrovirus and retroelement replication in primate cells are the TRIM5 and APOBEC3 gene products [40–42] . Analysis of these genes in modern primates indicates that these genes have likely been under positive selection pressure for significant portions of primate evolution [12–16] . As an endogenous retrovirus that has also apparently replicated exogenously and has been active for much of Old World primate evolutionary history , HERV-K is an excellent candidate for an agent that has imposed sustained evolutionary pressure on antiretroviral defenses present in modern primates . Nonetheless , HERV-K infection was not inhibited by the TRIM5 proteins that were tested . In the case of human TRIM5α , this was not unexpected , because HERV-KCON was derived from human-specific proviruses that must , by definition , have replicated in humans at some point in their evolution and may , therefore , have evolved resistance to human TRIM5α . However , HERV-KCON was also resistant to rhesus monkey TRIM5α and also TRIM-Cyp , a form of TRIM5 that is unique to owl monkeys [43 , 44] , a New World monkey species that does not carry HERV-K . At present , therefore , there is no evidence that TRIM5 proteins and HERV-K have exerted reciprocal evolutionary pressure during primate evolution . However , analysis of CA sequences reconstructed from more ancient groups of HERV-K proviruses and inserted into HERV-KCON , as well as inclusion of more TRIM5α variants , may be illuminating . The studies described herein suggest that such approaches to study interactions between ancient retroviruses and their hosts should be feasible . It was notable that the process of generating a consensus HERV-K genome , in effect , primarily involved the replacement of A and T nucleotides in modern defective proviruses with G and C nucleotides , respectively . The position of the consensus sequence near the root of the phylogenetic tree suggests that a G or C “ancestral” state at many nucleotide positions in human-specific HERV-K genomes has been replaced by A and T nucleotides in modern defective proviruses . While these results do not necessarily lead to the conclusion that cytidine deaminases are responsible for the reduction or extinction of HERV-K replication in humans , they hint that this may have been the case . At a minimum they suggest that cytidine deamination events have impacted HERV-K evolution in humans . While G-to-A changes were the most frequently represented in comparisons of HERV-KCON with contemporary proviruses , plus-strand C-to-T changes also appeared to be overrepresented . While most APOBEC-induced mutation is thought to result from deamination of minus strand cytidines during reverse transcription [45–48] , at least some APOBEC proteins are also are capable of inducing C-to-T changes on the plus strand of proviruses , by catalyzing deamination of viral RNA or perhaps dsDNA [49] . HERV-K replication was sensitive to APOBEC3F but only marginally affected by APOBEC3G . This was somewhat surprising and clearly distinguishes HERV-K from Vif-deficient HIV-1 which is more sensitive to APOBEC3G than to APOBEC3F [50–53] . Indeed , an overall view is emerging that a variety of APOBEC3 proteins can be selectively active against various retroelements and retroviruses [54] but HERV-K has yet to be analyzed . Of note is the observation that the patterns of apparent cytidine deamination events in modern HERV-K proviruses do not precisely match those predicted if APOBEC3F was primarily responsible for G-to-A and C-to-T mutation therein . Therefore , further analysis of HERV-K sequences in humans and Old World primates and the impact of their various APOBEC proteins on HERV-KCON replication may illuminate an evolutionary history of ancient host–retrovirus conflicts .
The complete HERV-K113 proviral sequence was used to search human genome sequence using National Center for Biotechnology Information nucleotide-to-nucleotide BLAST . Multiple entries of the same HERV-K proviruses were identified by inspection of flanking genomic sequence and excluded , and the most recently sequenced entries were used for the alignment . The top ten hits were aligned using AlignX program of Vector NTI Advance 10 . 0 . 1 ( Invitrogen , http://www . invitrogen . com ) to derive a consensus sequence that was termed HERV-KCON . HERV-KCON sequence was compared to other HERV-K ( HML-2 ) proviruses using HYPERMUT ( http://www . hiv . lanl . gov/content/hiv-db/HYPERMUT/hypermut . html ) [55] . The complete HERV-KCON proviral sequence was synthesized using overlapping oligonucleotides of approximately 60 bases spanning the entire genome . Oligonucleotides were assigned to 13 groups corresponding to 13 HERV-KCON fragments of approximately 700 nucleotides and assembled using sequential PCRs . In the first round , all oligonucleotides were included in the reaction and 15 cycles of synthesis were executed using Pfu DNA polymerase ( 94 °C for 10 s , 45 °C for 20 s , 72 °C for 30 s ) . Thereafter , an aliquot of the reaction product was subjected to amplification using the 5′ and 3′ oligonucleotides in each group ( 94 °C for 20 s , 45 °C for 20 s , 72 °C for 3 min; 15 cycles ) . Fragments from regions of the HERV-K genome lacking convenient restriction sites were assembled into longer fragments of up to 1 . 5 kb via overlap extension PCR . A derivative of the low-copy-number plasmid vector , pXF3 , was derived by inserting a synthetic oligonucleotide encoding the restriction sites ClaI , XhoI , EcoRV , SpeI , AgeI , SacI , DraIII , XbaI , and NheI that corresponded to convenient restriction sites in the HERV-KCON genome . These were used to sequentially insert the various synthetic DNA fragments , thereby generating the final pXF3/HERV-KCON proviral plasmid . CHKCG was created from the pXF3/HERV-KCON proviral plasmid by first replacing HERV-K U3 sequences 5′ to the TATA box with CMV promoter/enhancer sequences using overlapping PCR and ClaI and EcoRV restriction sites to generate pXF3/CMVP HERV-KCON . In parallel , an EGFP cDNA ( Clontech ) was inserted into pCR3 . 1 ( Invitrogen ) , and then the CMVP-EGFP cassette was PCR amplified and inserted into KpnI site of pXF3/CMVP HERV-KCON to create CHKCG . Similarly , a Puro cDNA was digested from pMSCVPuro ( Clontech ) with HinDIII and XbaI and inserted into pCR3 . 1 . Thereafter , a CMVP-Puro cassette was PCR amplified and cloned into pXF3/CMVP-HERV-KCON to create the CHKCP genome . pCRVI/Gag , pCRVI/Gag-PR , and pCRVI/Gag-PR-Pol were generated by insertion of the respective ORFs from pXF3/HERV-KCON into the NotI restriction site of pCRVI . Similarly , a PCR-amplified HERV-KCON Env-encoding fragment was inserted using EcoRI and NotI restriction sites , generating pCRVI/Env . A mutant form of pCRVI/Gag-PR was generated by substituting the conserved putative active site residues ( DTG ) to AAA . The two K-Rev/Rec exons were PCR amplified from BAC RP11-33P21 ( Invitrogen ) containing the HERV-K108 sequence , which happens to encode a K-Rev/Rec protein that is identical to the consensus sequence , joined using overlapping PCR , and inserted into EcoRI and XhoI of pCR3 . 1 to generate pCR3 . 1/K-Rev . The 293T , MDCK , Pindak , TE671 , HeLa , CRFK , and HT1080 cells were maintained in DMEM supplemented with 10% fetal calf serum and gentamicin . CHO745 cells and TRIM5-expressing derivatives were cultured in Ham's F-12 medium with the same additives . The 293T cells were transfected in 10-cm plates at 6 × 106 cells per plate or in six-well plates at 1 × 106 cells per well using polyethylenimine as previously described [56] . Medium was changed 24 h after transfection , and virus-containing supernatants were collected after an additional 24 h . The 293T cells were transfected with 10 μg of pCRVI/Gag , pCRVI/GagP-PR , pCRVI/Gag-PR-Pol , or pCRVI . Two days after transfection , the supernatant was collected , filtered ( 0 . 2 μm ) , and ultracentrifuged through a 20% sucrose layer at 100 , 000g for 90 min at 4 °C . The pelleted VLPs and corresponding transfected cells were resuspended in SDS-PAGE loading buffer and separated on 10% SDS-PAGE gels ( Bio-Rad , http://www . biorad . com ) . Proteins were transferred onto nitrocellulose membrane and probed with an anti–HERV-K Gag antibody ( Austral Biologicals , http://www . australbiologicals . com ) . Alternatively , VLPs were separated on 4% to 20% gradient or 10% SDS-PAGE gel ( Bio-Rad ) and silver stained using a kit , as per the manufacturer's instructions ( Sigma-Aldrich , http://www . sigmaaldrich . com ) . Reverse transcriptase activity in 293T culture supernatants was measured using a commercially available reverse transcriptase assay ( Cavidi , http://www . cavidi . se ) in which BrdUTP is incorporated into a plate-bound oligo ( dT ) /poly ( rA ) substrate . Thereafter , solid phase polymerized BrdU is detected using an anti–BrdU–alkaline phosphatase conjugate and a colorimetric substrate . Activity is standardized using a recombinant HIV-1 RT standard . To generate VSV-G pseudotyped HERV-KCON particles , 293T cells in six-well plates were transfected with 1 . 3 μg of CHKCG or CHKCP , 1 μg of pCRVI/Gag-PR-Pol , 0 . 5 μg of pCR3 . 1/Rec , and 0 . 2 μg of VSV-G . Empty control vectors were transfected when necessary . In some experiments , 1 μg of plasmids expressing myc-tagged human APOBEC3F and APOBEC3G [53] was also transfected . Alternatively , 293T cells in 10-cm dishes were transfected with 6 . 5 μg of CHKCG , 4 μg of pCRVI/Gag-PR-Pol , 3 μg of pCR3 . 1/Rec , and 1 . 5 μg of VSV-G . At 24 h after transfection , the transfection mixture was replaced with of fresh media containing 5 μM sodium butyrate . To generate HIV-1 ( HERV-KCON ) pseudotypes , 293T cells in 10-cm plates were transfected with 6 μg of HIV-1-GagPol , 6 μg of CSGW , and either 3 μg of pCRVI/HERV-KCON Env or empty pCRVI as a control . Filtered ( 0 . 2 μm ) supernatant from HERV-KCON ( VSV-G ) – or HIV-1 ( HERV-KCON ) –producing cells was placed onto target cells seeded in 24-well plates along with fresh media in the presence of 5 μg of polybrene/ml for 24 h . Two days after infection , GFP+ target cells were quantified either by counting foci microscopically or by FACS analysis . In some experiments , 50 μM AZT was added to the medium at the time of infection . CHO745 cells were infected with CHKCP-carrying HERV-KCON ( VSV-G ) virus stock and transduced cells selected in 2 . 5 μg/ml puromycin for approximately 10 d . From the puromycin-resistant population comprising several hundred colonies , four single cell clones were derived by limiting dilution and expanded in culture ( approximately 2 wk ) . Cellular DNA extracted from each clone ( Qiagen extraction kit , http://www . qiagen . com ) was subjected to PCR analysis using HERV-KCON gag-specific primers Gag-S ( nucleotides 1236 to 1262 , Figure S1 ) and Gag-AS ( nucleotides 1991 to 1946 , Figure S1 ) . Additionally , host DNA sequences flanking the integrated CHKCP proviral DNA were cloned using the GenomeWalker kit ( Clontech ) according to the manufacturer's instructions and PCR primers directed to the HERV-KCON LTRs . Specifically , LTR-AS ( GCA AGA GAG ATC AGA TTG TTA CTG TGT CTG ) and LTR-S ( TAC GAG AAA CAC CCA CAG GTG TGT AGG ) oligonucleotides were used to clone sequences flanking the 5′ and 3′ LTRs , respectively . Additional PCR primers , targeting flanking hamster DNA sequences identified via the GenomeWalker approach , were used to authenticate the presence of preintegration sites in uninfected CHO745 cells and integrated provirus in three CHKCP transduced cell clones . In the example ( clone No . 1 ) shown in Figure 4E , the primers Ham-S ( GCT ACC CTG AAG ATT TGA GCC AGT GTG C ) and Ham-AS ( TCT TGC AAG TTG TCC TGT GGC ATG G ) were used . For all PCRs , 30 cycles of amplification were completed using 200 ng of cellular DNA , with no DNA , uninfected CHO cell DNA , or human DNA templates analyzed as negative and positive controls , as appropriate . | Retrovirus genomes integrate into the genomes of host cells . If the target cells of a particular retrovirus include germ-line cells , e . g . , sperm or egg cells , then retroviral genomes can be inherited like cellular genes . So-called “endogenous” retroviruses have accumulated throughout evolution in the genomes of many organisms , including humans . While all known endogenous retroviruses of modern humans are unable to replicate as retroviruses , the human genome represents a fossil record of ancient retroviruses that once infected our ancestors . In this study , a collection of “dead” endogenous retroviral genomes in modern human DNA was used to deduce the approximate sequence of an ancestral retrovirus , human endogenous retrovirus ( HERV ) -K , that is now thought to be extinct . A pseudo-ancestral HERV-K DNA sequence was synthesized and used to produce viral proteins and RNA that could reconstitute the HERV-K replication cycle . Thus , the replication and biology of a once-extinct retrovirus can now be studied in the laboratory . Interestingly , reconstituted HERV-K replication experiments , and comparison of the reconstituted HERV-K DNA sequence with the dead HERV-Ks in modern human DNA , suggests that HERV-K may have been extinguished in humans in part by host defenses that induce mutation of retroviral DNA and that the reconstitution of the pseudo-ancestral HERV-K reversed these changes . | [
"Abstract",
"Introduction",
"Results",
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] | [
"viruses",
"virology"
] | 2007 | Reconstitution of an Infectious Human Endogenous Retrovirus |
There is solid evidence that rare variants contribute to complex disease etiology . Next-generation sequencing technologies make it possible to uncover rare variants within candidate genes , exomes , and genomes . Working in a novel framework , the kernel-based adaptive cluster ( KBAC ) was developed to perform powerful gene/locus based rare variant association testing . The KBAC combines variant classification and association testing in a coherent framework . Covariates can also be incorporated in the analysis to control for potential confounders including age , sex , and population substructure . To evaluate the power of KBAC: 1 ) variant data was simulated using rigorous population genetic models for both Europeans and Africans , with parameters estimated from sequence data , and 2 ) phenotypes were generated using models motivated by complex diseases including breast cancer and Hirschsprung's disease . It is demonstrated that the KBAC has superior power compared to other rare variant analysis methods , such as the combined multivariate and collapsing and weight sum statistic . In the presence of variant misclassification and gene interaction , association testing using KBAC is particularly advantageous . The KBAC method was also applied to test for associations , using sequence data from the Dallas Heart Study , between energy metabolism traits and rare variants in ANGPTL 3 , 4 , 5 and 6 genes . A number of novel associations were identified , including the associations of high density lipoprotein and very low density lipoprotein with ANGPTL4 . The KBAC method is implemented in a user-friendly R package .
Currently there is great interest in investigating the etiology of complex disease due to rare variants [1]–[6] . Until recently , indirect mapping of common variants has been the emphasis of complex trait association studies . It has been demonstrated that common variants tend to have modest phenotypic effects while rare variants are likely to have stronger phenotypic effects [7] , although not strong enough to cause familial aggregation [8] . For mapping complex diseases due to common variants , instead of genotyping functional variants , tagSNPs are genotyped which act as a proxy for the underlying causal variants . For rare variant association studies , indirect mapping is not an optimal approach due to low correlations ( ) between tagSNPs and rare variants . Instead , direct mapping should be used , where functional variants are analyzed . In order to implement direct mapping , variants must first be identified . Large scale sequencing efforts have begun including the 1000 Genome Project , which will provide a better understanding of the allelic architecture of the genome and a detailed catalog of human variants . Next-generation sequencing technologies e . g . Roche 454 , ABI SOLiD , and Illumina HiSeq , have made it feasible to carry-out rare variant association studies of candidate regions , exomes and genomes . Gene interactions are believed to be involved in a broad spectrum of complex disease etiologies [9] . Although a number of methods have been developed to detect gene interactions between common variants [10]–[13] , their detection has been limited [10] . There is evidence that rare variant interaction also plays a role in disease etiology . In direct association mapping of rare variants , one or more genetic loci are commonly jointly analyzed in order to aggregate information , for example genes with similar functions or residing in the same pathway [3] , [4] . Therefore it is necessary to account for potential interactions between rare variants in different loci [14] and interactions between common and rare variants [15] , [16] . Ideally , when carrying out direct mapping , only causal variants should be tested for associations . When DNA samples are sequenced , both causal and non-causal variants are uncovered . Bioinformatics tools [17] , [18] or filters [1] can be used to predict functionality of variants , although tools such as PolyPhen [18] or SIFT [17]can have low sensitivity and specificity [6] , [19] . Empirical studies have shown that predictive errors can be as high as 47% and 37% for PolyPhen and SIFT respectively [6]; therefore , their usefulness in selecting variants to be included in association analysis is limited . Even when functionality can be correctly inferred , whether the identified variants affect the phenotype of interest is still unknown . Two types of misclassifications of variant causality can frequently arise: 1 . ) non-causal variants are included in the analysis: a . ) sequencing incorrectly identifies monomorphic sites as variant sites ( false positive SNP discovery ) , b . ) variants are falsely predicted to be functional or c . ) variants are functional but non-causal; 2 . ) causal variants are excluded from the analysis: a . ) due to locus heterogeneity , not all loci containing causal variants are included in the analysis , b . ) region not sequenced , e . g . intronic variants , c . ) variants not detected by sequencing assay ( false negative SNP discovery ) or d . ) causal variants are falsely predicted to be non-functional . Driven by the advancement of sequencing technologies and availability of data , statistical and computational methods are needed for analyzing sequence data . It has been demonstrated that methods used to analyze common variants are low powered when applied to the analysis of rare variants [20] , [21] . Methods to analyze rare variants have been proposed [20] , [21]; although they have clear advantages over implementing common variant analysis approaches , more powerful and robust methods need to be developed to analyze rare variant data , especially in the presence of variant misclassification or gene interactions . The Kernel Based Adaptive Cluster ( KBAC ) was developed to overcome the problems of detecting rare variant associations in the presence of misclassification and gene interaction . Under the KBAC framework , data-based adaptive variant classification and testing of association are unified . The sample risk of a multi-site genotype is modeled using a mixture distribution with two components , where one component represents the distribution of sample risk of genotype if it is non-causal and the other component represents distribution of sample risks of causal genotypes . Ideally , if distributions for causal components were known , classification could first be performed and only the causal genotypes would be used in association studies . However , when searching for genotype-phenotype associations , it is usually unknown which variants are causal . Instead of performing an unrealistic two-step procedure , variant classification and association testing are unified in the KBAC framework . Continuous adaptive weighting which is implemented in the KBAC is preferable , particularly for low frequency alleles , than classifying variants and carrying out a stratified analysis , because increasing classification and shrinking size of strata can increase both type I and II error . For the KBAC , adaptive weighting procedure is implemented using the cumulative distribution functions for the multi-site genotype counts . Distributions of multi-site genotype counts are compared between cases and controls . Those multi-site genotypes that are enriched in cases will be up-weighted . Under the null hypothesis , the assigned weights asymptotically follow a uniform distribution . While under the alternative hypothesis , disease causal multi-site genotypes tend to be more frequent in cases than in controls . Therefore they are more likely to be adaptively up-weighted . The weighted multi-site genotype frequencies are aggregated and contrasted between cases and controls . In order to evaluate whether there is an association , significance of the KBAC can be assessed using either permutation or Monte Carlo approximation ( See Methods and Figure S1 ) . The performance of the KBAC was compared to the weighted sum statistic ( WSS ) [21] the combined multivariate and collapsing ( CMC ) method [20] , and the comparison of rare variants found exclusively in cases to those found only in controls ( RVE ) [3] using simulated data sets . Forward time simulation [22] assuming infinite-site Wright-Fisher model was used to generate population genetic data . Demographic change and purifying selection were both incorporated in the simulation , using parameters estimated from re-sequencing datasets from studies of African Americans ( AA ) and European Americans ( EA ) [23] . In addition to forward time simulation , population genetic data was also generated according to estimated site frequency spectrums ( SFS ) in AA and EA from the Dallas Heart Study ( DHS ) re-sequencing data of the ANGPTL3 , 4 , 5 , and 6 genes . For the simulated population data phenotypes were generated separately and motivated by epidemiological disease studies . Two types of main effects phenotypic model are considered: 1 . ) constant genetic effects for each causal variant and 2 . ) genetic effects inversely correlated with minor allele frequencies ( MAF ) of causal genetic variants . In order to evaluate the impact of variant misclassification , a variety of scenarios were examined where 1 . ) different proportions of non-causal variants were included in the analysis and 2 . ) different proportions of causal variants were excluded from the analysis . Two disease models of gene interactions were also evaluated . The example of with-in gene interaction was motivated by Hirschsprung's disease [15] , [16] , where an interaction between a common polymorphism in the promoter region and multiple rare non-synonymous ( NS ) mutations in exonic regions of the RET gene is hypothesized [15] , [16] . The example of between gene interaction is based on the observation that rare variants within the CHEK2 gene increase risk of breast cancer in the absence of BRCA1 and BRCA2 mutations , but because of a shared pathway , the same CHEK2 variants in the presence of high risk BRCA variants do not further increase risk [14] , [24] , [25] . Under each of the above scenarios , phenotype-genotype association testing is performed for rare NS variants . It is demonstrated that the KBAC has a clear advantage in power and robustness over other existing methods and this benefit is especially strong , when rare variant data is analyzed where there is either variant misclassification or gene interactions . In order to further illustrate applications of the KBAC and other statistical methods , i . e . , WSS , CMC , and RVE to carry-out association studies , energy metabolism traits and rare variants in ANGPTL 3 , 4 , 5 and 6 genes obtained from sequence data were analyzed . In addition to identifying the originally reported association between triglyceride levels and ANGPTL 4 , KBAC identified associations for a . ) body mass index and ANGPTL 5 , b . ) diastolic blood pressure with ANGPTL 6 , c . ) high density lipoprotein with ANGPTL 4 , d . ) triglyceride levels with ANGPTL 3 e . ) very low density lipoprotein with ANGPTL 3 and ANGPTL 4 .
Rare NS variants carrier information is summarized ( Table 1 ) for replicates used in power comparisons in the presence of misclassifications . Under the phenotypic model with variable genetic effects , when all variants ( both non-causal and causal variants ) were analyzed , 5 . 5% of cases and 3 . 4% of controls are carriers , with carrier frequency in cases 61% higher than in controls . When only causal variants are included , the fractions of carriers in cases and in controls are 3 . 8% and 1 . 7% respectively . The case rare variant frequency is approximately 2 . 3 times of the controls frequency , which implicates that average ORs of uncovered rare variants lie between 2 to 3 . For the phenotypic model with fixed genetic effects , the results are similar . The carrier frequency observed in cases is around 2 . 5 times the frequency in controls . Compared to the model with fixed effects , lower frequency rare causal variants have larger ORs for variable effects model . The probability that these low frequency rare variants are uncovered in a case-control sample is higher . Therefore , in all scenarios examined , more rare variants sites are uncovered for the model with variable effects . When all the variants are included , 11% more rare NS variants sites are uncovered for the model with variable effects . The number of rare variants sites that are exclusive to cases or controls is also higher under the variable effect model . For example , when 100% of the variant sites are included in the analysis , 47 . 4% and 41 . 1% of the sites are found exclusively in either cases or controls for the variable and the fixed effects model , respectively . For both models , within a single gene , very few cases and controls carry more than one rare variant . For the within gene interaction model ( Table 2 ) , similar patterns of NS variants sites and carrier frequencies are observed . When 100% of the rare variants are causal , 5 . 5% of the cases and 3 . 2% of the controls are carriers on average for a case-control sample . Due to interaction , frequency differences between cases and controls are mitigated . In the between gene interaction model ( Table 2 ) , higher case carrier frequency and more rare variants sites are observed for the high risk gene than for the low risk gene . The proportions of rare variants carriers for the two genes combined can be high , e . g . when 100% of the variants are causal , up to 12% of the cases can be rare variant carriers . Rare variants distributions can be found in the ( Text S1 ) for main effects models ( Table S1 ) and within and between gene interactions models ( Table S2 ) using simulated SFS for EA , and for main effects models using estimated SFS for AA ( Table S3 ) and EA ( Table S4 ) with re-sequencing data from ANGPTL3 , 4 , 5 , and 6 genes . When permutation was used to evaluate significance for the KBAC , type I error was well controlled , because p-values were obtained empirically . Additionally , in order to ensure that the type I error for RVE is well controlled permutation is also used to obtain empirical p-values . For the WSS [21] , CMC [20] method , it was previously demonstrated that for the analysis of rare variants , their type I errors are well controlled [20] . For moderate sample sizes e . g . 400 cases/400 controls , the distributions of p-values for the Monte Carlo approximation are very close to those obtained using permutations and theoretical expectations ( Figure 1 ) and additionally type I error is well controlled . In order to further illustrate the application of KBAC and other rare variant analysis methods ( i . e . WSS , CMC and RVE ) , rare variants in the ANGTPL 3 , 4 , 5 and 6 genes were analyzed to determine whether they are associated with energy metabolism traits ( Table 3 ) . As in the original DHS study [26] , the association of rare variants in the ANGPTL3 , 4 , 5 and 6 genes with triglyceride ( TG ) , low density lipoprotein ( LDL ) , very low density lipoprotein ( VLDL ) , high density lipoprotein ( HDL ) , cholesterol , glucose , body mass index ( BMI ) , systolic ( SysBP ) and diastolic blood pressure ( DiasBP ) were investigated . In the original DHS study , NS variants were analyzed using RVE , and significant associations were found between ANGPTL3 , ANGPTL 4 and TG as well as between ANGPTL 6 and cholesterol [5] , [6] . In this article NS variants , most of which are very rare [5] , [6] , were analyzed . Individuals with confounding factors ( lipid lowering drugs , diabetes mellitus and heavy alcohol use ) were removed for all analyses . Multiple associations were identified with KBAC but not with other approaches , i . e . the novel associations between ANGPTL 6 and DiaBP , as well as between ANGPTL 3 and TG levels . Additionally multiple novel associations were observed for analyses carried out with KBAC , WSS and CMC: 1 . ) ANGPTL4 and VLDL ; 2 . ) ANGPTL5 and BMI ; 3 . ) ANGPTL4 and HDL and 4 . ) the previously reported association between ANGPTL4 and TG levels . It should be noted that HDL and TG levels are negatively correlated ( −0 . 42 ) and individuals with HDL levels in the lower quartile had an excess of rare variants in ANGPTL4 compared to those individuals with HDL levels in the upper quartile , while those individuals with TG levels in the upper quartile had an excess of rare variants in ANGPTL4 compared to those with TG levels in the lower quartile . The association detected by KBAC between ANGPTL4 and VLDL and between ANGPTL5 and BMI remains significant after correcting for multiple testing . RVE , on the other hand , detected associations between ANGPTL 5 , 6 and glucose while the other three methods did not . We further investigated this association by applying a more stringent MAF cutoff 0 . 1% for the NS variants analyzed in ANGPTL 5 and 6 . Using this new criterion both associations were detected by all methods ( for ANGPTL 5 , and for ANGPTL 6 , ) .
The KBAC method developed for association mapping of rare variants combines genotype classification and hypothesis testing in a coherent framework . The risk of each multi-site genotype is modeled as a mixture distribution with two components , among which only the component representing a non-causal genotype is known and is used in the adaptive weighting . Each multi-site genotype is continuously weighted using the non-causal component . The power of the KBAC as well as the other methods investigated can be affected by inclusion of non-causal mutations or exclusion of causal variants in the sample , to a varying degree . When non-causal variants are included in the analysis , the difference in rare variant carrier frequencies observed between cases and controls is mitigated . On the other hand , when causal variants are excluded from the association analysis , the marginal effect size of existing variants can vary considerably depending on whether missing causal variants exist on the same multi-site genotype . As a result , treating each variant ( or multi-site genotype ) interchangeably will incur loss of power , the severity of which will depend on the proportion of misclassified variants in the data . The performance of the KBAC is superior to the other approaches that were examined . Bioinformatics tools [17] , [18] and filters [1] can be used to determine which rare variants are potentially functional and should be included in the association analysis [1] . Their predictive accuracy , which can be low , is dependent on the amount of information available for the gene understudy . If bioinformatics tools are used to predict variant functionality and determine which variants should be included in the analysis it is best to loosen stringency , because the exclusion of causal variants is more detrimental to power than inclusion of non-causal variants . Whether or not bioinformatics tools are used as a screening tool , misclassification will occur therefore the robustness of KBAC to misclassification is particularly beneficial . Additionally in order to avoid potentially erroneous exclusion of causal variants due to locus heterogeneity , joint analysis of multiple putative genetic loci that carry similar functions or reside in the same pathway can be valuable . It is of great interest to evaluate gene×gene interactions in the study of complex diseases . The KBAC analyzes multi-site genotypes ( or multi-locus genotype ) , which can be beneficial in detecting gene interactions [11] . This property is especially important when multiple genetic loci are jointly analyzed in order to aggregate rare variants . Interactions are more likely to occur between genes involved in the same pathways . In addition , it has been hypothesized that functions of rare variants can be modulated by common variants [8] . Since the KBAC uses adaptive weighting instead of a fixed model , unknown patterns of gene interaction can be automatically integrated into the analysis . Through models motivated by Hirschsprung's disease and breast cancer , it is shown that in the presence of interactions the KBAC outperforms other approaches . An additional advantage of the KBAC is that kernel weights computed for adaptive weighting provide a measure with which the relative risk of each multi-site genotype can be assessed , for further replication studies . The RVE method which compares the occurrence of variants which are exclusively observed in cases to those which are only observed in controls has the lowest power among all tests evaluated . The RVE method possesses undesired statistical properties by excluding those variants which are observed in both cases and controls . For all variants that are not fully penetrant , when sample size is large , they tend to appear in both case and control samples and would thus be excluded from the analysis using RVE . As a result , the RVE method is not asymptotically consistent; with increasing sample size power may be even lower than for smaller sample sizes [27] . Forward time simulations of locus genetic data incorporated both population demographic change and purifying selection . Both factors are known to impact SFS for observed rare variants ( especially NS variants ) . Only NS variants were analyzed for comparing different methods , as it has been suggested that using NS variants will concentrate variations on functionally significant class of alleles , and increase signal to noise ratio [27] . There have been a number of studies on complex diseases which identified associations with NS variants [3] , [5] , [6] . When synonymous mutations are also considered in the analyses , higher proportions of non-causal variants may be introduced , so the adaptive property and the robustness of KBAC will be more advantageous . Whether or not phenotypic effects of causal rare variants are inversely correlated with their MAF is unknown . Deleterious functional variants tend to have low frequencies [28] , but the functional effect of a deleterious mutation may not be associated with the disease . On the other hand , for mutations involved in complex traits , they may not be at selective disadvantage due to the fact that most complex traits are late on-set and may not cause reductions in reproductive fitness . For both types of models , the advantage of KBAC is apparent . WSS and RVE perform better under the variable effects models , when only causal variants are present . This is because high risk causal variants are assigned higher weights . However , as low frequency non-causal variants also receive larger weights that negatively affect power , there are no measurable improvements of WSS compared to the model with fixed genetic effects . On the other hand , due to the adaptive nature of KBAC , the method performs consistently the best under both classes of models . The KBAC test statistic does not have a closed form distribution; therefore it is necessary to evaluate significance either through permutation or using Monte Carlo approximation . For small sample sizes i . e . ∼≤400 cases and 400 controls , permutation is recommended , because it can be more reliable than Monte Carlo approximation . For larger sample sizes , Monte Carlo approximation not only controls type I error , but also the estimates of power do not differ from those obtained using permutations ( data not shown ) . Permutation can be computationally intensive for large samples and/or genome-wide data where a large number of genetic regions are analyzed; therefore Monte Carlo approximation can be particularly advantageous to evaluate significance due to its computational efficiency . A well known problem of genetic association studies is spurious findings due to population substructure and/or population admixture . For rare variant association analysis this problem can occur when study subjects are sampled from different populations and the distribution of non-causal variant sites and/or aggregate frequencies of non-causal variants differ between the sampled populations . To control for population stratifications , KBAC can be coupled with principal components analysis ( PCA ) [29] approach and eigenvector ( s ) can be included as covariates in the analysis ( see Methods: Controlling for Confounders ) . PCA approach has been shown to be a powerful tool to accurately infer geographical locations [30] , [31] . In addition , KBAC can also be used with clustering/matching based methods , such as structured association [32] , [33] to control for population stratification . The application of KBAC as well as WSS , CMC and RVE were further illustrated by the analyses of genes in ANGPTL family . In the analyses , all individuals with potentially confounding factors i . e . diabetics , alcoholics , and individuals treated with lipid lowering drug were excluded . In the original studies individuals were excluded based upon both their quantitative trait values and the confounding factors . For example , only individuals treated with lipids lowering drugs in the lower quartile of TGs were removed , but those in the upper quartile were included in the analysis . We believe excluding individuals based upon their quantitative trait values should not be done instead all individuals meeting the exclusion criteria should be removed from the analysis . KBAC performs consistently well , and identifies the most phenotype-genotype associations among all the approaches compared . The effects of mutant ANGPTL genes on lipoprotein lipase ( LPL ) have been studied through in vitro functional studies and in vivo mice studies . LPL has been known to affect glucose metabolism [34] , cholesterol level [34]–[37] , and blood pressure [38] . This biological evidence strengthens the support of the identified associations . Additionally , the association between variants in ANGPTL4 gene and triglyceride levels were successfully replicated using an independent dataset [5] , [6] . Although the examples given are for the analysis of single regions and interaction between two regions , the KBAC can also be used to analyze entire exomes ( or genomes ) . In order to control for family-wise error rate ( FWER ) , it is sufficient to use a Bonferroni correction , since there will be little or no linkage disequilibrium between rare variants in different genes . It is thus not necessary to control the FWER using permutations . If exome sequencing is carried out and analysis is implemented gene by gene , given that human genome contains ∼20 , 000 genes , a significance level can be applied . The correction necessary for gene based association mapping of rare variants is less than the threshold currently used for genome-wide association studies [39] which is usually . The KBAC is a powerful tool to detect main association effects and gene interactions in large sequence data sets of candidate genes , exomes and in the future entire genomes . The KBAC is implemented in a user friendly R package and is available from the authors .
Total sample size is denoted as , among which there are affected ( A ) and unaffected ( U ) . It is assumed that there are sites within the candidate region where rare variants are observed . The rare variant multi-site genotype for each “individual” is contained in a vector , with the entry being the number of rare variants observed at site , i . e . has value 2 if the site is homozygous for the rare allele , 1 if the site is heterozygous , 0 if the site is homozygous wild-type for the common major alleles . It is further assumed that distinct multi-site genotype vectors , i . e are observed , where are multi-site genotypes with at least one rare variant and represents the wild-type genotype without any rare variants ( i . e . a vector of all 0's ) . The sample risk for multi-site genotype is defined aswhich is a consistent estimator of the ratio The ratio increases with disease penetrance of and provides a sample based measure of the relative risk . The sample risk for multi-site genotype is modeled using a mixture distribution with two components , . The component represents the distribution of the sample risk when multi-site genotype is non-causal and is known , while represents the unknown distribution of sample risk when is causal . If the null hypothesis holds , all genotypes are non-causal , therefore , . Under the alternative hypothesis , each genotype can be either causal or non-causal and the probabilities in the probabilistic mixtures are unknown . If the mixture distribution under the alternative were known , then each genotype could be classified and only the causal genotypes would be used in the analysis . However , in disease gene mapping , the causality of variants is unknown . Instead of trying to ‘estimate’ and which are unknown , each multi-site genotype is adaptively weighted using only the known component , . Each is called a kernel . The term kernel is borrowed from density estimation , where the density being estimated is spanned by a linear combination of kernel functions . The weight each rare genotype carries is given by the area under the curve which can be calculated as a generalized integralwhere is the estimated sample risk for multi-site genotype . Thereby , under the null hypothesis , the weights are uniformly distributed and under the alternative , greater weights can be placed on the multi-site genotypes that are enriched in cases . The genotypes with high sample risks will be given higher weights which can potentially separate causal from non-causal genotypes . Instead of classifying genotypes in a rigid manner with unknown likelihoods , this method weighs each genotype in a continuous fashion using only the known component from the mixture density . The adaptive weighting procedure in the KBAC attains a good balance between classification accuracy and the number of parameters which are estimated . Three types of kernels can be used to assign weights to each rare genotype; they are asymptotically equivalent . For small to moderate sample sizes , binomial and hyper-geometric likelihoods tend to work best , while for large sample sizes the asymptotic normal kernel is computationally efficient . All examples shown in this article were carried out using the hyper-geometric kernel . Each “individual” with multi-site genotype in the sample will be assigned weight . The weight is given by the kernel functions depending on the estimated sample risk i . e . . The weights assigned to rare genotypes are aggregated and contrasted between cases and controls . The KBAC statistic is defined as , which compares the difference of weighted multi-site genotype frequencies between cases and controls . When a one sided alternative hypothesis is tested , e . g . the enrichment of causal variants in cases , a corresponding one sided version of KBAC can be used , i . e . . In this article , all power comparisons were based upon two sided tests for each method . Standard permutation procedure is used to obtain empirical p-values for small sample sizes and for large sample sizes significance can be obtained through the Monte Carlo approximation . A graphical illustration of the KBAC statistic can be found in ( Figure S9 ) . In order to control for sample heterogeneities such as population stratification/admixture , it is desirable to be able to incorporate covariates in the association analysis . The kernel weights computed for the KBAC statistic can be used with logistic regression . For an individual with multi-site genotype , we define a variable for the kernel weight , i . e . . The logistic regression model for association testing has the formwhere are the covariates such as age , sex or eigenvectors for genotypes . A score statistic to test can be computed in closed form . Due to the complexities involved in computing kernel weights , the score statistic does not follow a normal distribution . Standard permutation procedure can be applied to evaluate the significance . When no additional covariates are controlled , the score function satisfies [40] . Simple algebraic manipulations will lead to the equivalence of the score function and the KBAC statistic ( up to a constant scalar ) . In addition , when common variants in the gene are also hypothesized to play a role in the etiology of the phenotype of interest , their genotypes can be included as covariates and tested in a similar manner as for the CMC [20] . The power of WSS , CMC and RVE were compared to KBAC in the article . A sketch of each method is provided here . More detailed descriptions can be found in the cited original reference . WSS was developed by Madsen and Browning [21] . It was designed to test for the differences of the number of mutations between cases and controls . Each mutation was weighted according to its frequency in controls , and lower frequency variants will be assigned higher weights . The statistical significance for the WSS statistic is obtained empirically through permutations . CMC was developed by Li and Leal [20] . When applied to testing rare variant associations , multiple rare variants in the gene region are collapsed and carrier frequencies are compared between cases and control using Pearson's Chi-square test . The RVE [3] , [4] was first introduced in the analysis of sequence data from Dallas Heart Study . It compares frequency of carriers of rare variants that are found exclusively in cases or controls using Fisher's exact test . The DHS dataset is a multi-ethnic population based probability sample [1830 AA , 601 Hispanics ( H ) , 1045 EA , and 75 from other ethnicities] from Dallas County residents whose lipids and glucose metabolism have been characterized and recorded [26] , [43] . In order to investigate how sequence variations in ANGTPL3 , 4 , 5 and 6 influence energy metabolism in humans , coding regions of the four gene were sequenced using DNA samples obtained from 3551 participants in DHS [5] . A total of 348 nucleotide sites of sequence variations were uncovered in all four genes . Most of them are rare and 86% of them have MAFs below 1% [5] . Individuals with diabetes mellitus , heavy alcohol use , or who were taking lipids lowering drugs were removed from the all the analyses because these factors could be potential confounders . Additionally individuals who do not belong to the AA , H or EA ethnic groups were removed from the analysis . Following the original study [5] , and to control for potential confounders [44] we stratified the sample by race , sex , and quantitative trait level . For each quantitative trait , to test if the rare variants are enriched in the expected extremes , individuals from bottom and top quartiles are used to mimic a case-control type of design . The KBAC , WSS , CMC and RVE were applied to carry-out the association analysis . | It has been demonstrated that both rare and common variants are involved in complex disease etiology . Until recently it was only possible to perform large scale analysis of common variants . With the development of next-generation sequencing technologies , detection and mapping of rare variants have been made possible . However , methods used to analyze common variants are not powerful for the analysis of rare variants . To address the problems of rare variant analysis working in a novel framework , the kernel-based adaptive cluster ( KBAC ) method was developed to perform gene/locus based analysis . The KBAC combines variant classification and association testing in a coherent framework . Through simulations motivated by population genetic and disease data , it is demonstrated that the KBAC has superior power to other rare variant analysis methods , especially in the presence of variant misclassification and gene interaction . Using data from the Dallas Heart Study , the KBAC method was applied to test for associations between energy metabolism traits and rare variants in ANGPTL 3 , 4 , 5 and 6 genes . A number of novel associations were identified . The KBAC method is implemented in a user-friendly R package . | [
"Abstract",
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] | [
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] | 2010 | A Novel Adaptive Method for the Analysis of Next-Generation Sequencing Data to Detect Complex Trait Associations with Rare Variants Due to Gene Main Effects and Interactions |
The sessile plants have evolved diverse intrinsic mechanisms to control their proper development under variable environments . In contrast to plastic vegetative development , reproductive traits like floral identity often show phenotypic robustness against environmental variations . However , it remains obscure about the molecular basis of this phenotypic robustness . In this study , we found that eg1 ( extra glume1 ) mutants of rice ( Oryza savita L . ) showed floral phenotypic variations in different growth locations resulting in a breakdown of floral identity robustness . Physiological and biochemical analyses showed that EG1 encodes a predominantly mitochondria-localized functional lipase and functions in a high temperature-dependent manner . Furthermore , we found that numerous environmentally responsive genes including many floral identity genes are transcriptionally repressed in eg1 mutants and OsMADS1 , OsMADS6 and OsG1 genetically act downstream of EG1 to maintain floral robustness . Collectively , our results demonstrate that EG1 promotes floral robustness against temperature fluctuation by safeguarding the expression of floral identify genes through a high temperature-dependent mitochondrial lipid pathway and uncovers a novel mechanistic insight into floral developmental control .
The sessile plants have evolved various exquisite adaptive strategies to cope with environmental changes [1 , 2] . Among them , phenotypic plasticity is the ability of a single genotype capable of producing different phenotypes in response to varying environments [3–6] . For an integral high fitness , morphologies of vegetative organs of a single plant , such as roots , leaves and stems , require a high phenotypic plasticity [7–10] , whereas that of reproductive organs , such as flowers , fruits and seeds , are always associated with low plasticity also known as phenotypic robustness/stability [11–15] . Thus , plants must coordinate the developments of these organs . Compared with progresses in understanding the molecular mechanisms of high phenotypic plasticity [10 , 16–19] , very little is known about the molecular basis of phenotypic robustness [20 , 21] . Recent studies have shown that there are a group of specific genes regulating the degree of phenotypic plasticity and determining the reaction norm of a trait among various environments , which are termed plasticity genes [22–24] . However , most of the identified plasticity genes are high plasticity-associated [16 , 25–27] , only few promote phenotypic robustness [28–30] . Members of HSP90 ( HEAT SHOCK PROTEIN 90 ) family , as central hubs of numerous biological pathways , are required for maintenance of phenotypic robustness in both animals and plants [28 , 31–34] . MSH1 ( MutS HOMOLOG1 ) , a homolog of bacterial mismatch repair protein MutS , has been reported to repress the developmental plasticity of plant architecture , leaf morphology and flowering time in several dicot and monocot plants [29 , 35] . A nuclear protein RPL1 ( RICE PLASTICITY1 ) in rice also appears to promote the relatively stable plant architecture and panicle structure between different environments [30] . Despite these discoveries , we still know very little about the molecular mechanisms of phenotypic robustness , especially that of plant reproductive traits . In addition to the known epigenetics-dependent transcriptional regulation and hormone signaling [20 , 29–31 , 35] , lipid homeostasis is also known to influence phenotypic robustness [36 , 37] . Coordinated regulations of cellular lipid homeostasis are crucial to organisms’ adaptive robustness under severe temperatures [37–40] . Furthermore , lipid-related synthetases and lipases can also be regulated at transcriptional and posttranslational levels to influence lipid homeostasis [41 , 42] . Among them , mitochondria-associated lipid metabolism is key to the lipid homeostasis [43] . For instance , Arabidopsis seedlings with decreased cardiolipin in mitochondrial membrane are easier to turn yellow and necrotic under extended darkness or heat due to a failure of mitochondrial morphogenesis , showing a lowered stability [41 , 44 , 45] . However , it remains unclear whether mitochondria also mediate the phenotypic robustness in plant reproductive organs . Flower morphology , as a gold standard in plant taxonomy , has the most remarkable robustness within and between individuals of the same population [11 , 46] , making it an ideal trait for studies on the molecular basis of phenotypic robustness against environmental fluctuation . Nevertheless , so far no gene has been identified to regulate the phenotypic robustness of floral identity , although several environment-dependent floral mutants have been reported [47–52] . We previously found that a rice floral mutant eg1 ( extra glume1 ) exhibited a floral variation in different growth conditions [53] , implying that EG1 is likely involved in floral robustness . Recent studies have shown that EG1 encodes a putative lipase regulating rice floral identity and meristem determinacy [53] . It also functions in JA ( jasmonic acid ) biosynthesis to promote the expression of floral identity gene OsMADS1 through an EG2/OsJAZ1 , OsCOIb and OsMYC2- mediated JA signaling pathway [54] , similar to its homologous genes AtDAD1 ( DEFECTIVE IN ANTHER DEHISCENCE1 ) and AtDGL ( DONGLE ) in Arabidopsis [55 , 56] . In this study , we find that EG1 is a predominantly mitochondria-localized functional lipase and promotes floral robustness against temperature fluctuation in a high temperature-dependent manner . Collectively , our results reveal a novel molecular mechanism underlying floral phenotypic robustness .
Previously , we found that eg1 displayed a floral identity variation possibly influenced by growth conditions [53] . To examine if this variability was mainly due to the environmental alterations , we analyzed the spikelet phenotypes of eg1-1 ( in indica ZF802 background ) and eg1-2 ( in japonica ZH11 background ) in two groups of separate environments ( Fig 1 ) and found that the floral phenotypic variability of eg1 is likely caused by both genotype and environment . To define the phenotypic variability , we divided the spikelet phenotypes of eg1 into six groups , which were called variable phenotypes , including Wl ( WT-like ) , eg ( extra glume ) , pl ( palea to lemma ) , sp ( smaller pa ) , le ( long empty glumes ) and rs ( reiterated spikelets ) ( Fig 1A , S1 Fig and S1 Table ) . The results showed that sp and rs of eg1-1 as well as Wl , le , eg and sp of eg1-2 exhibited significant plasticity between two environments , especially le of eg1-2 , which displayed the highest plasticity ( Fig 1B and 1C ) , suggesting that environment also contributes to the variations of floral phenotypes of eg1 . To further examine the relationships of genotype ( G ) , environment ( E ) , genotype-environment interaction ( GxE ) and the variable phenotypes , we calculated their effects on phenotypes by two-factor ANOVA and found that Wl and pl were affected mainly by G , eg by both E and G but rarely by GxE , sp , le and rs by all three factors , and among them , le could serve as a marker for the phenotypic plasticity of eg1-2 due to its large proportion in a panicle and opposite phenotypes between two environments ( Fig 1A ) . Taken together , these results showed that eg1 shows higher floral plasticity , suggesting that EG1 promotes the floral robustness in rice . To further examine the influence of genotype on the floral plasticity of eg1 , we swapped the genetic backgrounds of two eg1 alleles . eg1-1 in a largely japonica background showed high phenotypic plasticity especially for le and rs phenotypes , similar to eg1-2 ( ZH11 ) , whereas eg1-2 in an indica-dominant background showed low floral plasticity similar to eg1-1 ( ZF802 ) ( Fig 2A ) , indicating that genetic backgrounds also influence the phenotypic plasticity of eg1 spikelets . To verify this finding , we further used CRISPR/Cas9 technology to construct eg1 alleles in Nipponbare ( japonica ) and Dular ( indica ) backgrounds . Two types of spikelet phenotypes were found in eg1-4 allele with Dular background and both showed low plasticity ( Fig 2B ) , while eg1-5 and -6 alleles in Nipponbare background showed relatively higher plasticity than alleles in two indica backgrounds ZF802 and Dular , similar to that in japonica ZH11 ( Fig 2C and S2 Fig ) , suggesting that the floral plasticity of eg1 alleles in japonica backgrounds tend to be higher than that in indica backgrounds . In another aspect , eg1 alleles in indica backgrounds had severer floral disturbance than that in japonica concerning Wl and rs phenotypes ( Fig 2 and S1 Fig ) , suggesting that EG1 has functions in both floral robustness and identity , which are differentiated in two subspecies . To explore the possible causes of these differentiation , we compared the cis-elements and expressional level of EG1 in several japonica and indica varieties and discovered the correlative differences in both cis-elements and expressional levels between japonica and indica ( two types ) varieties ( S3 Fig and S2 Table ) , which implied that transcriptional differences may be a crucial cause of functional differentiation of EG1 in subspecies . All these results indicated that both eg1 allelic variations and their genetic backgrounds regulate the floral plasticity of eg1 . In order to find out the environmental factors mediating the plastic development of eg1 spikelets , we first compared the growing conditions for phenotypic analysis and found a marked difference in daily high temperatures of two environments ( S4 Fig ) , suggesting that the temperature variation between two environments could be a major environmental factor influencing the eg1 plasticity . To verify this prediction , floral plasticity of wild-types and eg1 alleles were examined in two artificial growth chambers with 35°C light 12 hr / 20°C dark 12 hr and 25°C light 12 hr / 20°C dark 12 hr respectively , while other growth conditions were kept identical . The low plasticity of eg1-1 and nearly 70% le phenotypes of eg1-2 showed that the floral plasticity of eg1 in the chambers was similar to and even higher than that under natural growth conditions ( Fig 3 ) . These results showed that temperature is a major environmental factor mediating the floral plasticity of eg1 . Previously , EG1 was shown to be localized in chloroplasts in transient expression assays [54] . However , EG1-like lipases appear to have variable subcellular locations [56–59] . To examine the subcellular localization of EG1 in vivo , two different EG1 and GFP fusion proteins driven by 35S promoter were first expressed in rice protoplasts and were found to be co-localized with both mitochondrial specific dye Mito Tracker Red and mitochondrial maker protein MTS-mOrange [60] but hardly with chloroplast auto-fluorescence , and an EG1-GFP fusion protein driven by native promoter was also detected in mitochondria ( Fig 4A and 4B ) , suggesting that EG1 protein is mainly , if not all , localized in mitochondria . To compare this finding with the previous one , the reported vector pCAMBIA1301-Pro35S:EG1-GFP [54] was also examined in our transient system and a similar localization was detected ( S5 Fig ) . To further verify the EG1 localization , subcellular fractionations of one-week seedlings of eg1-2 complementation lines with FLAG-EG1 ( S6 Fig ) were successively carried out and the EG1 fusion protein was predominately co-fractionated with mitochondria ( Fig 4D ) , confirming the mitochondrial localization of EG1 in vivo . Taken together , these results showed that EG1 encodes a protein predominately localized in mitochondria . To explore the biochemical function of EG1 , we tested its lipase activity [53] in vitro and found that both the full-length EG1 and truncated EG1 without predicted targeting peptides showed significant lipase activity ( Fig 4E ) , indicating that EG1 encodes a functional lipase . Taken together , our results showed that EG1 functions as a predominately mitochondria-localized lipase . The dependence of the eg1 floral plasticity on environmental temperature raised a possibility that either EG1 or its product or both are likewise regulated by temperature . To examine these possibilities , some heat/cold responsive cis-elements were discovered in the 2 kb genomic sequence upstream of the start codon of EG1 ( S3 Table ) , implying that its expression could be induced by extreme temperatures . To examine this possibility , one-week wild-type seedlings were treated under different temperatures and the EG1 transcript was found to accumulate gradually , to an extremely high extent under heat shock ( 42°C ) as well as usual high temperature 35°C for rice ( Fig 5A ) , but to some extent suppressed under cold stress ( 4°C ) ( S7A Fig ) , indicating the high temperature-induced expression of EG1 . A similar result was obtained by using young inflorescences in which EG1 has a high expression ( S7B Fig ) . To examine whether EG1 protein was also influenced by high temperatures , accumulation of FLAG-EG1 fusion protein in eg1-2 complementation lines , with a temperature-insensitive promoter ( S7C and S7E Fig ) , was detected under different temperatures and found it was significantly induced at extreme high temperature 42°C than 25°C and 35°C ( Fig 5B and S7D Fig ) , indicating a stabilization of EG1 protein under heat stress . Furthermore , we detected that lipase activity of EG1 fusion proteins increase as temperature rising ( Fig 5C ) , consistent with the assumption of EG1’s function required under high temperatures . Additionally , we also examined the effect of high temperatures on EG1 subcellular localization , and found no obvious translocation in protoplast system ( S8A Fig ) , while failed to detect EG1 protein in the subcellular fractions of complementation lines except under heat stress for its minute amount ( S8B Fig ) , suggesting that temperature does not significantly influence the subcellular localization of EG1 . The increased the transcriptional level , protein stability and lipase activity of EG1 under high temperatures , implying its more significant role under high temperatures . To verify this hypothesis , we observed the floral phenotypes of eg1 mutants under extremely high temperatures and found much severer spikelets in eg1 mutants especially in eg1-2 , with multilayer lemma-like organs and undetermined inflorescence meristem , which have never been found in other temperature conditions ( Fig 5D ) , showing a more significant function of EG1 at higher temperatures in floral robustness . eg1 was also found to grow faster than wild-type during primary growing days [54] , and we detected this phenotype was much severer under extremely high temperature than others compared with wild-type , which was consistent with the floral phenotype ( S9 Fig ) . Therefore , we concluded that EG1 functions in a high temperature-dependent manner to regulate the floral robustness . Since the floral plasticity of eg1 was influenced by both genotype and environment , to examine the molecular mechanism of genotype-environment interaction in floral plasticity , transcriptomes of inflorescences of two eg1 alleles ( eg1-1 and eg1-2 ) and their wild-types ( ZF802 and ZH11 ) in Beijing and Lingshui were analyzed . First , to evaluate the reliability of the transcriptomic data , we divided all transcripts into 33 modules by co-expression network analysis and analyzed their correlations with six variable phenotypes , and it turned out that the relationships among the variable phenotypes derived from these correlations were quite similar to their morphological correlations ( S10 Fig ) , showing a good reliability of the transcriptomic data . Second , through overall comparisons of all transcriptomes , we discovered that the expression patterns of floral transcriptomes of eg1 alleles between two environments were significantly different from their wild-types ( S11A Fig ) , indicating a role of EG1 in regulating expressions of environmentally responsive genes . The numbers of environmentally responsive genes in eg1 mutants were much larger than that of wild-types , in contrast to the similar numbers between two wild-types or two eg1 alleles ( Fig 6A and S11B Fig ) , implying that EG1 negatively regulates the responses of its downstream genes to environment . To verify this finding , we analyzed the effects of G , E , and GxE on transcriptomes of eg1 and wild-type by two-way ANOVA and found that the number of genes significantly affected by E and GxE in eg1 were significantly larger than that in wild-types ( Fig 6B , S11C Fig and S4 Table ) , displaying a switch of many genes from G-affected to E/GxE-affected ones ( Fig 6C ) , indicating that EG1 represses its downstream genes not only to respond to , but also to interact with environment . Furthermore , the effects of the three factors on several important pathways varied significantly between eg1 and wild-type , including the pathways related to temperature response , lipid metabolism and floral development ( Fig 6D and S5 Table ) , indicating that EG1 mediates a crosstalk of these pathways with environment . Since EG1 was reported to regulate JA biosynthesis [54] , in order to analyze its effect on the floral robustness control , we examined the expressional patterns of JA biosynthesis and signaling associated genes in our transcriptome data , and found that the transcriptional responsive patterns to environment or transcriptional level of several JA signaling genes ( JAZ7 and JAZ8 ) and JA biosynthesis genes ( four methyltransferase genes ) are varied in eg1 mutants ( S12 Fig ) , implying a possible role of JA in the EG1-associated floral robustness regulation but different from previously reported [54] . Taken together , these results revealed that EG1 mediates the transcriptional responses of downstream genes and pathways to environmental fluctuation . The significant transcriptional effects on the floral development pathways based on the G , E and GxE analysis in eg1 mutant suggested that floral identity genes are likely involved in EG1-dependent floral robustness regulation . To examine this , expressional variation of thirteen floral identity genes between two environments were further analyzed , and seven ( OsMADS1 , OsMADS6 , OsG1 , OsMADS4 , OsMADS7 , OsMADS8 , OsMADS58 ) of them showed both varied environment-dependent expression patterns and repressed expression levels in eg1 ( S13 Fig ) , indicating their positive regulatory roles in the floral robustness . To further examine this possibility , genetic analysis between EG1 and three of them ( OsMADS1 , OsMADS6 and OsG1 ) were performed . OsMADS1 and OsMADS6 are two major genes regulating glume identity and floral determinacy in rice [61–69] , and their expressions were significantly varied in eg1 ( Fig 7E and S13 Fig ) , indicating that EG1 is required for their expressions . To examine their genetic relationships with EG1 , a double mutant of OsMADS1 mutant allele nsr [61] and eg1-1 was generated and it exhibited longer and leafy lemmas/paleas similar to nsr , with all inner three whorls replaced by half-developed inflorescences or inflorescence primordia , which is severer than both single mutants ( Fig 7B ) , indicating that OsMADS1 functions downstream of EG1 in lemma/palea identity and they may together regulate the determinacy of inner three whorls . In addition , the double mutant of eg1-1 and OsMADS6 mutant allele osmads6-5 [66] showed abnormal paleas , with all transformed into one or two small lemma-like glumes and mostly with inflorescence primordia inside the spikelets ( Fig 7C ) , indicating that OsMADS6 functions downstream of EG1 in specifying palea but may also regulate floral determinacy together with EG1 . osmads6-5 exhibited weaker floral disturbance in the F2 population when crossed with eg1-1 ( ZF802 ) , with lemma-palea mosaic paleas and usually normal inner whorls ( Fig 7C ) , showing its floral phenotype is also greatly influenced by genetic backgrounds . To further examine the relationships among EG1 , OsMADS1 and OsMADS6 especially in floral determinacy , nsr osmads6-5 and eg1-1 nsr osmads6-5 were successively generated . The floral meristems of these two mutants similarly generated continuous glume primordia or became inactive before inner three whorls developed ( Fig 7D ) , which were more dedifferentiated than the inflorescence primordia of eg1-1 nsr and eg1-1 osmads6-5 , supporting the findings that both OsMADS1 and OsMADS6 act downstream of EG1 and they together control the floral differentiation of inner three whorls . Compared with eg1-1 , the rs plasticity of eg1-1 nsr osmads6-5 totally disappeared when these two MADS genes were both deficient ( Fig 7B and 7D ) , supporting the important roles of OsMADS1 and OsMADS6 in the rs plasticity regulation . Additionally , the spikelet phenotype of eg1-1 nsr [54] and nsr osmads6-5 [68 , 69] were consistent to the previously reported , and eg1 was linked with the lemma-like ( lel ) structure , and not affected by the deficiency of OsMADS1 and OsMADS6 ( Fig 7b–7d ) , implying that it is probably a special organ different from lemma and palea . Taken all these results together , we concluded that MADS1 and MADS6 together function downstream of EG1 to control the determinacy of inner three whorls of rice floret as well as to mediate the rs plasticity of eg1-1 . Furthermore , the le phenotype ( long empty glume ) of eg1-2 has the highest plasticity among all variable phenotypes ( see Figs 1–3 ) , and OsG1 ( Long Sterile Lemma ) and OsMADS19/34 are two crucial genes suppressing the elongation of empty glumes in rice [70–72] . The expression levels and patterns of OsG1 but not OsMADS19/34 appeared to be aberrant in eg1-2 ( Fig 7E and S13 Fig ) , implying that OsG1 may contribute to the le phenotype of eg1-2 . To confirm this , g1-ele allele of OsG1 [72] was used to obtain a double mutant by crossing with eg1-2 , and it turned out that almost all empty glumes of eg1-2 g1-ele were elongated to lemma-like organs similar to g1-ele , exhibiting much lower plasticity compared with eg1-2 ( Fig 7F and 7G ) , indicating that OsG1 functions downstream of EG1 in regulating empty glume development and contributes to the plastic le phenotype of eg1-2 . Taken together , these results show that OsMADS1 , OsMADS6 and OsG1 all act downstream of EG1 to mediate the floral robustness regulation .
To our knowledge , no plasticity genes have been confirmed to function in floral robustness in flowering plants [47–52] . Given the sessile nature of plant species , uncovering this class of genes and dissecting their molecular mechanisms are crucial for understanding the biology of flower development and evolution . In this study , we have shown that EG1 encodes a mitochondria-localized lipase functioning as a plasticity gene to regulate the rice floral robustness by a coordinated transcriptional regulation of temperature , lipid metabolism and flower development pathways . First , eg1 alleles showed floral variations under both natural and artificial conditions and five eg1 alleles produced increased floral plasticity . Second , RNA expression , protein stability and lipase activity of EG1 can respond to environment enhancing its function significantly in severe conditions such as heat stress . Third , EG1 appears to possess a “buffering” function of repressing environmental stimuli to interfere the target genes , and when environmental pressure becomes severer such as heat stress , the strengthened EG1 function induced by heat is enough to buffer the stronger and more deleterious effect of heat on the responsive transcriptional pathways . Last , EG1 influences the expression of numerous floral identity genes , which are the direct contributors of plastic development of eg1 spikelets . Thus , EG1 is the first plasticity gene regulating plant floral robustness against environmental fluctuation . Our finding indicates that flowers retain a system containing EG1 to sense and respond to environmental stimuli to maintain its stable development , and suggests a mechanism of transition from high plasticity to robustness in flower by recruiting plasticity-repressing genes , which ensures the coordinated development of organs with different plasticity in one organism . Lipid metabolisms are known to be involved in adaptive plasticity of organisms [36 , 37 , 41 , 42] , and mitochondria , as the energy factory of eukaryotes and one of subcellular compartmentations of lipid metabolism , have been reported to be crucial to the adaptive stability of plant vegetative traits [41 , 43 , 44] . As EG1 is a predominately mitochondria-localized functional lipase , our results suggest that the mitochondria-mediated lipid metabolism plays an important role in the regulation of floral robustness against temperature fluctuation . However , it remains unclear how this could be carried out . It is likely that mitochondria-related lipid homeostasis could serve as a “buffer” to relieve the effect of environmental stimuli and mediate the temperature-dependent floral plasticity regulation ( Fig 8 ) . Recently , EG1 has been shown to influence JA synthesis , and JA signaling pathway regulates the transcription of floral identity gene OsMADS1 [54] , showing that JA potentially mediates the crosstalk of EG1 and flower development related transcriptional factors . In fact , the most homologous gene of EG1 in Arabidopsis DGL has been verified to function in JA biosynthesis [55 , 56 , 73] , though its chloroplast location was questioned [55] , suggesting that a non-chloroplast localized lipase is likely to function in JA production ( Fig 8 ) . On the other side , we may fail to detect EG1 protein due to its minute amounts in chloroplasts . Furthermore , according to the much severer phenotype of eg1 and eg2-1D ( a mutant allele of EG2/OsJAZ1 ) double mutant compared with two single [54] , we noticed that JA signaling may be not the only pathway activated by EG1 to mediate the signal transduction from outside to inside of nucleus in the EG1-associated floral regulation , other lipid-related pathways and regulatory mechanisms are also possible ( Fig 8 ) . In our study , eg1 alleles showed a higher floral plasticity in japonica than that in indica varieties , while a severer floral disturbance in indica , revealing a functional divergence of EG1 in rice subspecies . No differences in coding sequences but cis-elements of EG1 in two subspecies were detected ( S14 Fig ) , implying that this subspecific variation might be caused by an unknown differential responsive capability of promoters to environmental or endogenous stimuli . Besides , since genetic backgrounds are known to influence developmental outcomes via phenotypic modifiers [74 , 75] , there also may be some indica-/japonica-specific modifiers to modulate EG1 function due to their different genetic backgrounds , which can be either epistatic genes or specific lipid substrates of EG1 required for floral developmental robustness . Genetic dissection of these subspecific modifiers of plasticity will provide further insights into the molecular mechanism of floral development . So far , all reported EG1 homologs in dicots have no apparent plasticity function in flowers [56–59] , and EG1 homologs in monocots can be mainly divided into two clades , one similar to that in dicots and the other unique to monocot species based on the phylogenetic tree and predicted subcellular localizations ( S13 Fig ) , we thus speculate that EG1 may have acquired a monocot-specific neofunctionalization in promoting floral robustness . Detailed genetic and biochemical analyses of these genes would provide additional clues to when and how the plasticity function and the subspecific divergence of EG1 arose in monocot species . In conclusion , our results revealed a novel function of EG1 in floral developmental robustness against environmental fluctuation by mediating the mitochondrial lipid metabolism . Our finding also provide a genetic means to maintain the stable flower development under environmental stress ensuring grain yield stability in rice and potentially in other monocot species . Further studies should unlock the molecular crosstalk between mitochondria and nucleus in regulating floral developmental robustness .
Five rice eg1 recessive alleles were used in this study . eg1-1 and eg1-2 were from our previous work [53] , in backgrounds of O . sativa L . spp . indica Zhefu 802 ( ZF802 ) and O . sativa L . spp . japonica Zhonghua 11 ( ZH11 ) respectively . eg1-4 was generated from indica Dular , and eg1-5 and -6 from japonica Nipponbare by CRISPR/Cas9 technology [76 , 77] . Besides , ZF802>ZH11 and eg1-1 ( ZF802>ZH11 ) were isolated from an F2 population of ZF802 wild-type or eg1-1 backcrossed with ZH11 three times , and ZH11>ZF802 and eg1-2 ( ZH11>ZF802 ) were isolated from an F2 population of ZH11 wild-type or eg1-2 backcrossed with ZF802 once . Other rice mutants , nsr [61] and g1-ele [72] were kindly provided by Dr . Zhukuan Cheng , and osmads6-5 also from our previous work [66] . Plants were grown in the natural conditions of Beijing ( China ) from Mar . to Oct . and Lingshui ( Hainan province , China ) from Dec . to Apr . ( year 2014~2015 ) . Weather data of 2014 . 7 . 28~2014 . 8 . 17 of Beijing and 2014 . 3 . 15~2014 . 4 . 4 of Lingshui , and 2014 . 2 . 1~2014 . 2 . 20 and 2014 . 4 . 1~2014 . 4 . 20 of Lingshui were shown respectively , which were ranged from ten days before spikelet meristem formation ( ~2 mm ) to ten days after that . Artificial conditions in chambers were 35°C light 12 hr / 20°C dark 12 hr , 25°C light 12 hr / 20°C dark 12 hr and 40°C light 12 hr / 30°C dark 12 hr , respectively , with identical light intensity 50 μmol m-2 s-1 and relative air humidity 60% . Spikelets of eg1 were divided according to phenotypes of outer glumes ( lemma , palea , empty glumes and extra glumes ) following two rules: ( 1 ) the most significant mutant phenotype of eg1 is in the outer glumes; ( 2 ) outer glumes have linkages with inner organs: normal palea always linked with normal inner organs , lemma-like palea with increased stamens and pistils , smaller palea with decreased stamens , multilayer glumes linked with rs . Main panicles/inflorescences of plants at booting stage were used for phenotypic analysis . Percentages of all variable phenotypes in one panicle were calculated for comparisons , and in some cases more than one phenotype appeared in a single spikelet . Data were statistically analyzed by using one/two-way ANOVA ( Excel ) . Effects of genotype , environment and genotype-environment interaction on all variable phenotypes were calculated using phenotypic statistic data of eg1 and wild-type grown in Beijing summer and Lingshui winter by two-way ANOVA . In CRISPR experiment , more than six independently homozygous lines were generated in each background , and their phenotypes were quite similar , thus only one/two lines were statistically analyzed and shown in our results . Full-length EG1 CDS was inserted into N- or C-terminal of GFP sequence of vector pBI221-35SPro:GFP , and full-length EG1 CDS with 1 . 5 kb native promoter into pCAMBIA1301-GFP . MTS-mOrange and Pro35S:COX11-GFP plasmids were kindly provided by Dr . Yaoguang Liu [60] , and the reported vector pCAMBIA1301-35SPro:EG1-GFP was provided by Dr . Dabing Zhang [54] . All plasmids of high quality were prepared for protoplast transfection . Rice protoplast preparation from 2-week-seedlings grown in light and polyethylene glycol ( PEG ) -mediated transfections were performed as described by Bart et al . [78] . Images were captured by a confocal microscope ( FluoView 1000 , Olympus ) . Floral disorder of eg1-2 was complemented by genetic transformation using vector pTCK303-ProUBIQUTIN:FLAG-EG1 . One-week seedlings of EG1 complementation lines were used for subcellular fractionation . Fractionations of mitochondria and chloroplasts were performed as described by Rodiger et al . [79] . After precipitating the organelle fractions , western blots were performed with α-FLAG ( Sigma ) , mitochondria specific antibodies α-AOX1/2 and α-COXII ( Agrisera ) , and chloroplasts specific antibodies α-RbcL and α-PsbA ( Agrisera ) . Fractionation assays were performed with two independent complementation lines . Full-length EG1 CDS and a truncated EG1 CDS without predicted targeting sequence ( 135 bp ) with engineered N-terminal SUMO tag were separately cloned into pET-30a ( Novagen ) to generate His6-SUMO-tagged fusion proteins . Mitochondrial targeting sequence was predicted with MitoProt II online [80] . DGL CDS without targeting sequence [81] was introduced into vector PMAL-C2X ( NEB ) . All the fusion proteins were expressed in E . coli BL21 ( DE3 ) . The His6-SUMO-tagged fusion proteins were purified using Ni-NTA ( Novagen ) and eluted with buffer containing 25 mM Tris-HCl ( pH 7 . 4 ) , 150 mM NaCl and 250 mM imidazole . The MBP-tagged fusion proteins were purified using amylose resin ( NEB ) and eluted with buffer containing 50 mM Tris-HCl ( pH 7 . 4 ) and 10 mM amylose . Imidazole in protein solutions was removed with desalting columns ( Thermo Scientific ) before lipase activity analysis . p-nPB ( p-nitrophenyl butyrate , Sigma ) was used as the substrate of lipase analysis in vitro . Colorimetric assays for lipase activity of fusion proteins were performed as described by Seo et al . [82] with some modifications . A solution containing 1 . 11 mg/mL p-nPB ( dissolved with isopropanol ) and B solution containing 50 mM Tris-HCl ( pH 7 . 4 ) and 0 . 1% Arabic gum were first prepared . Reactive solution was composed of 1 volume A solution and 9 volumes B solution with 2% Triton X-100 . About 20 μl purified proteins ( ~5 μg ) and 180 μl reactive solution were used for each reaction . After incubated under different temperatures for 30 min , p-nitrophenol formation from p-nitrophenyl butyrate was determined spectrophotometrically at 405 nm by an ELISA microplate reader . DGL and porcine pancreatic lipase ( Sigma ) were used for positive controls , and p-nitrophenol ( Sigma ) for the standard curve . Cis-elements in the genomic sequence upstream of the start codon of EG1 was analyzed by PLACE online [83] . Wild-type ZF802 , ZH11 and EG1 complementation lines were grown at 25°C for one week after germination , and wild-type ZH11 seedlings were grown in a consistent condition till 2 mm inflorescence meristem developed before being treated under different temperatures for different time . The transcripts of EG1 and FLAG-EG1 were analyzed by RT-QPCR or RT-PCR . The root and shoot phenotypes of seedlings were statistically analyzed after growing under consistent temperatures for six days after germination . Other condition parameters of physiological experiments were daylight 12 hr , light intensity 50 μmol m-2 s-1 and relative air humidity 60% . We use 42°C as an extremely high temperature for short treat but 40°C for long treat by considering the tolerance of plants . FLAG-EG1 protein in EG1 complementation lines was enriched by immunoprecipitation for its small amount and detected by western blot with α-FLAG ( Sigma ) . Two mm inflorescence meristems of eg1-1 , eg1-2 and their wild-types ZF802 and ZH11 planted in Beijing summer and Lingshui winter were used for RNA sequencing , and two biological replicates were performed . Total RNAs were isolated with TRIzol kit ( Invitrogen ) . Illumina sequencing libraries were prepared according to the manufacturer’s instructions ( Illumina Part # 15026495Rev . D ) , and sequenced with Illumina system Hiseq2500 . Analysis of RNA-seq data was conducted following the standard protocol as described by Trapnell et al . [84] . The raw reads of RNA-seq were mapped to MSU_IRGP_V7 ( japonica ) and Oryza_indicaASM465 v1 . 23 ( indica ) by Tophat [85] . Cuffdiff [85 , 86] was used to identify the differentially expressed genes between different genotypes or different environments . Co-expression network analysis was performed using R packages WGCNA . Enrichment pathways of genes significantly and specifically affected by G , E or GxE was analyzed and the -log10 ( P-values ) was tested by Fisher's exact test with Bonferroni correction as described by Lu et al . [87] . Amino acids sequences of EG1 homologs in different plants were aligned with CLUSTAL W and maximum likelihood tree was constructed with MEGA6 . 0 . Subcellular localizations of proteins were predicted with five sorts of software online . Among them , TargetP [88] ( http://www . cbs . dtu . dk/services/TargetP/ ) has the best consistency compared with MitoProt II-v1 . 101 [80] ( https://ihg . gsf . de/ihg/mitoprot . html ) , iPSORT [89] ( http://ipsort . hgc . jp/ ) , ProtComp 9 . 0 ( http://linux1 . softberry . com/berry . phtmtopic=protcompan&group=help&subgroup=proloc ) and WoLF PSORT ( http://www . genscript . com/wolf-psort . html ) online . Predicted localizations with TargetP were shown in our results , a/b in which means the protein is more likely in “a” location than in “b” although both are possible . | Various mechanisms have evolved to ensure proper organ formation under variable environments in order to complete one organism’s life cycle . In angiosperms , vegetative and reproductive organs show a differential plastic development between varied environments , with a low plasticity or high robustness for flower formation , but little is known about its intrinsic mechanism . Here we report that gene EG1 ( EXTRA GLUME1 ) can enhance the floral robustness against temperature fluctuation in rice . EG1 encodes a predominantly mitochondria-localized functional lipase and its loss of function disrupts floral development in a high temperature-dependent manner . In consistent , both EG1 and its lipase activity are positively induced by high temperature . Transcriptomic and genetic analyses revealed that EG1 functions upstream of several floral identity genes , eg , OsMADS1 , OsMADS6 and OsG1 . Taken together , our results uncover a novel mitochondria-mediated lipid metabolic pathway to promote floral developmental robustness . Our findings may help to genetically improve floral traits of rice to maintain a stable yield when planted in different locations and/or under heat stress conditions . | [
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] | 2016 | A High Temperature-Dependent Mitochondrial Lipase EXTRA GLUME1 Promotes Floral Phenotypic Robustness against Temperature Fluctuation in Rice (Oryza sativa L.) |
In food webs , many interacting species coexist despite the restrictions imposed by the competitive exclusion principle and apparent competition . For the generalized Lotka-Volterra equations , sustainable coexistence necessitates nonzero determinant of the interaction matrix . Here we show that this requirement is equivalent to demanding that each species be part of a non-overlapping pairing , which substantially constrains the food web structure . We demonstrate that a stable food web can always be obtained if a non-overlapping pairing exists . If it does not , the matrix rank can be used to quantify the lack of niches , corresponding to unpaired species . For the species richness at each trophic level , we derive the food web assembly rules , which specify sustainable combinations . In neighboring levels , these rules allow the higher level to avert competitive exclusion at the lower , thereby incorporating apparent competition . In agreement with data , the assembly rules predict high species numbers at intermediate levels and thinning at the top and bottom . Using comprehensive food web data , we demonstrate how omnivores or parasites with hosts at multiple trophic levels can loosen the constraints and help obtain coexistence in food webs . Hence , omnivory may be the glue that keeps communities intact even under extinction or ecological release of species .
Fueled by ongoing rapid decline of biodiversity [1] , ecology is in the midst of a lively debate on the effect of species loss or introduction on food web stability [2 , 3] . In food webs , complexity arises from combining a large number of species ( the nodes ) and a large number of relations between these species ( the links ) . Addressing the latter , recent attention was devoted to the structure of links using e . g . the random , cascade and niche models [4 , 5] , stirring a prolific debate on the role of the link distribution regarding food web stability [2 , 3 , 6 , 7] . We take a complementary approach: Using standard consumer-resource equations , we demonstrate fundamental constraints on node diversity in a food web , termed food web assembly rules . For consumer-resource relationships , the competitive exclusion principle states that when two consumers compete for the exact same resource within an environment , one consumer will eventually outcompete and displace the other [8 , 9] . It is known that the number of coexisting species cannot exceed the number of resources these species compete for [10] . Expressed more generally , the number of coexisting species cannot be greater than the number of distinct regulating factors in the community [11] . For trophic communities of several levels , it was subsequently stated that the number of species on any trophic level could not exceed the sum of the numbers on adjacent levels [12] . Experimental studies do demonstrate strong correlations between consumer and resource diversity [13–16] . These observations highlight that also the consumer plays a critical role in shaping the network of species , even when direct interaction between resource species is absent , an observation captured in Holt’s paradigm of apparent competition [17] . Despite the existing theoretical constraints and empirical findings , a selective theory for stable coexistence of many species in food webs is currently lacking . This lack may partially be due to the complexity of the many-species interactions , yielding an uncontrollable number of parameters and hampering direct calculations or simulations of sufficient generality . Notwithstanding these complications , progress can be made when necessary conditions are demanded . For an ecology , consisting only of a resource and a consumer level , we have recently shown that coexistence requires that the species richness of both levels is balanced and that a cascade of parameter values must be maintained [18] . Examples of such systems may be the phage-bacteria ecology in the Atlantic Ocean [19 , 20] or laboratory ecologies . However , in food webs , a subset of trophic levels can generally not be considered in isolation . A species’ niche is determined by its entire set of interactions , which generally may be composed of both beneficial and harmful interactions , i . e . the species may act both as a consumer or resource . Further , many food webs contain omnivorous interactions , i . e . those where one species preys on several other species that are located at more than one trophic level . To derive necessary conditions for coexistence in food webs , a more general starting point is required . Based on the generalized Lotka-Volterra equation [21] , we here show that in sustainable food webs each species must be part of a non-overlapping pairing . We define a non-overlapping pairing as a topological pattern for a directed network , where each species must contribute to a closed loop and none of the loops may overlap ( Details: Methods ) . Mathematically , this is a consequence of demanding that the determinant of the interaction matrix be nonzero . This allows us to formulate the principle of competitive exclusion for food webs with multiple trophic levels . We term the resulting constraints the food web assembly rules . For trophically coherent food webs [22] the assembly rules predict that species richness must be largest at intermediate trophic levels , but relatively small at the top and bottom . We then show that any food web that obeys the assembly rules can be dynamically stable and feasible , given that parameters are chosen appropriately . Using the assembly rules , we make predictions for circumstances under which secondary extinctions must occur . By investigating seven highly-resolved empirical food web data sets we finally assess the potentially stabilizing effect of parasitism and omnivory .
For consumer-resource interactions in food webs , the generalized Lotka-Volterra equations [21] are S ˙ i ( 1 ) / S i ( 1 ) = k i ( 1 ) 1 - ∑ j = 1 n 1 p j i S j ( 1 ) - α i ( 1 ) - ∑ k = 1 n 2 η k i ( 2 , 1 ) S k ( 2 ) ( 1 ) for primary producers and S ˙ k ( l ) / S k ( l ) = ∑ m = 1 n l - 1 β k m l , l - 1 · η k m l , l - 1 · S m ( l - 1 ) - ∑ p = 1 n l + 1 η p k l + 1 , l · S p ( l + 1 ) - α k ( l ) ( 2 ) for species at trophic levels l > 1 . We distinguish a species by the set of links that connect it to predators and prey or nutrients and the strength of these links ( Details: Sec . S4 in S1 Text ) . In Eqs 1 and 2 , Si ( l ) with i = 1 , … , nl are the densities of species residing at trophic level l , nl is the species richness at level l , k i ( 1 ) denote the maximal growth rates of S i ( 1 ) , pji describe differential consumption of the basic resources by the S i ( 1 ) , α i ( l ) denote the decay rate of species S i ( l ) , η k i ( l , l - 1 ) are the interaction coefficients between a species S k ( l ) with species S i ( l - 1 ) , and β k m l , l - 1 are the efficiencies of reproduction of species S k ( l ) when consuming species S m ( l - 1 ) . In the steady state , the time derivatives S ˙ i ( 1 ) and S ˙ k ( l ) on the LHS of Eq 1 , respectively Eq 2 , vanish and we have the equations ∑ j = 1 n 1 p j i S j ( 1 ) + ∑ k = 1 n 2 η k i ( 2 , 1 ) k i ( 1 ) S k ( 2 ) = k i ( 1 ) - α i ( 1 ) k i ( 1 ) ≡ k i ˜ ( 1 ) , respectively ( 3 ) ∑ m = 1 n l - 1 β k m ( l , l - 1 ) η k m ( l , l - 1 ) S m ( l - 1 ) - ∑ p = 1 n l + 1 η p k ( l + 1 , l ) S p ( l + 1 ) ≡ α k ( l ) . ( 4 ) Collecting all constant coefficients ( RHS of Eqs 3 and 4 ) in the vector k and all interaction coefficients on the LHS in the interaction matrix R , we have the linear matrix equation R · S = k , where S is the vector of all species densities . For completely shared nutrients , the competition factors pji = 1 . ( Details: Secs S1 and S2 in S1 Text ) . Parasites have complex life-cycles that can demand several hosts [23 , 24] . Notwithstanding these complications , we here formally treat them as consumers , respectively resources of independently acting other species . Also , we simplify concomitant links in terms of simple linear responses ( details: Sec . S9 . 3 . 3 in S1 Text ) . d e t ( R ) ≠ 0 can be fulfilled if every species is paired with another species or nutrient ( this constitutes a perfect matching [25] ) . For food webs with exclusively sharp trophic levels ( as in Figs 1 , 2 and 3 , but not Fig 4 ) , the network is bipartite and therefore it is required that such a perfect matching exists . When species with variation in food chain length are present , one may generally obtain nonzero d e t ( R ) by covering the entire network with closed loops of directed pairings ( i . e . cycles ) . This is a sequence of nonzero matrix elements R i j , R j k , R k l , … , R m i ( Sec . S6 in S1 Text ) , i . e . a chain of directed pairings where the direction is maintained and the last element connects to the first . A directed pairing represents one nonzero matrix element , whereas a pairing also includes the symmetric element . We use high-resolution data on seven food webs including free-living and parasite species: The North American Pacific Coast webs Carpinteria Salt Marsh ( CA ) , Estero de Punta Banda ( PB ) , Bahia Falsa ( BF , Fig 5 ) [26 , 27]; the coastal webs Flensburg Fjord ( FF ) [28] , Sylt Tidal Basin ( ST ) [29] , and Otago Harbor ( OH ) , New Zealand [30] , as well as the Ythan Estuary ( YT ) , Scotland [23] . These food webs describe consumer-resource interactions between basal , predatory and parasite species . A compilation of all seven food webs has recently been provided [31 , 32] . Specifically , the data distinguish three types of links: ( i ) links between free-living species only ( “Free” ) , ( ii ) additional links between parasites and other species ( “Par” ) and ( iii ) links from free-living consumers to the parasites of their resources ( “ParCon” ) , i . e . so-called concomitant links . Details on data analysis: Section S9 in S1 Text . For the empirical data , the lack of niches , i . e . nullity d ≡ S - rank ( R ) , was computed by using random values for all nonzero entries of the respective matrix R . Basal species were each given an individual nutrient source . In “ParCon sym” a subset ( 20 percent ) of concomitant links were randomly selected to be symmetric ( Details: Sec . S9 . 3 . 3 in S1 Text ) . In the data analysis and simulations ( Figs 5 and 6 ) , the trophic level of a species is defined by its prey-averaged food chain length ( Sec . S9 . 2 . 1 in S1 Text ) . We perform two types of simulations: ( i ) In-silico assembly of a tree-like food web ( Fig 3c ) , where parameters are chosen according to constraints discussed in Sec . S2 in S1 Text . The numerical values of the parameters are: For the interaction and growth coefficients η = β = k = 1 for all links present ( solid black arrows in Fig 3b ) , as well as the decay coefficients {α1 , … , α8} = { . 1 , . 1 , . 16 , . 1 , . 12 , . 15 , . 1 , . 1} , where the labels are as indicated in Fig 3b . Each new species is introduced at low density and time-integration is continued until steady state is reached ( using Mathematica NDSolve method ) . ( ii ) An idealized food web was constructed by using the average species counts at levels ni obtained from all empirical data sets and initially assuming sharp trophic levels for all species . Sharp trophic levels were obtained by rounding each species’ chain length to the nearest integer value ( Fig 6a ) . With the constraint of these trophic levels , a number of links was assigned to match the empirical average for free-living food webs ( Fig 6b ) . When adding further species , the empirical average of parasite species count was used ( 47 species ) . To obtain Fig 6 , initially , each parasite received one link . In the cases ( c ) and ( d ) , this link connected the parasite to any existing free-living species . In the cases ( e ) and ( f ) , this link connected the parasite to any existing species at trophic levels 3 or 4 . For any subsequent link , a parasite was chosen at random . A link was then formed in three ways: Case c: randomly to connect with another existing species; Case d: randomly , but only to species at the same trophic level as for the initial link of that parasite; Case e: randomly to other existing species at levels 3 , level 4; Case f: randomly to other existing species at levels 3 , level 4 or another parasite ( More detail: Sec . S10 in S1 Text ) .
We describe the interaction of species on L trophic levels by the generalized Lotka-Volterra equations [21 , 33] . Basal species are constrained by the system carrying capacity while the consumers are assumed not self-limiting , and trophic interactions occur through the linear type-I functional response ( Methods ) . Such equations have been widely used in community assembly models , where food web networks are assembled by numerically analyzing the equations to find parameter sets with stable and/or permanent coexistence solutions [34–36] . Here we take an alternative path by first finding a necessary condition for coexistence in terms of species richness , which results in the food web assembly rules that constrains the network topology . We subsequently show that when these assembly rules are fulfilled , there always exist parameters for which feasible and stable network structures can be obtained . In the steady state , we have the matrix equation R · S = k , where S is the vector of all species densities , R is the interaction matrix between the species , and k is the vector of growth and decay coefficients . Note that R has a block structure with nonzero entries only for interactions between neighboring trophic levels but not within the same level , and that the positions of these matrix elements are symmetric due to mutual interaction between predator and prey ( Fig 1a ) . Stable/permanent coexistence requires that a feasible solution S * ≡ R - 1 k > 0 exists [7 , 35] . Structural stability , i . e . robustness against parameter perturbations , of a feasible coexistence solution requires that d e t ( R ) be nonzero ( Sec . S2 in S1 Text ) , a condition required for the existence of the matrix inverse in a linear equation [37] . In the following , we analyze what this basic condition means for the species richnesses at the different trophic levels of a food web . We then discuss also the stability of a given solution . We first specialize to the case of a single shared basic nutrient , e . g . sunlight , used by all primary producers S i ( 1 ) , with n1 the species richness on the first trophic level and i ranging from 1 to n1 . This corresponds to setting the competition terms pji = 1 for all 1 ≤ i , j ≤ n1 in Eq 1 , thereby yielding a block of n1 × n1 unit entries in the lower right block . ( Example: Sec . S4 in S1 Text ) . Nonzero determinant is achieved if it is possible to identify a path of matrix elements that only contains elements from the non-zero sub-matrices bordering the diagonal ( Fig 1a ) . This is equivalent to demanding that every species be part of a consumer-resource pair connecting neighboring trophic levels and none of these pairs overlap ( known as perfect matching of a bipartite graph in graph theory [25] ) . The pairing guarantees that no species share exactly the same niche , i . e . a particular set of interactions with resources and consumers ( Sec . S4 in S1 Text ) , and manifests the competitive exclusion principle ( Fig 1bi and 1bii ) . For primary producers , pairings may involve the nutrient source ( Fig 1a , inset ) . In that case , at least n1 − 1 species at level two are required for pairing of the remaining basal species . We term this structure resource-limited . In this case , n2 ≥ n1 − 1 is required . In the example ( Fig 1a ) this condition can indeed be fulfilled , because a sufficient number of species exists at level two . If pairing with nutrients is not used , n2 ≥ n1 is required—a consumer-limited configuration due to the biomass restriction imposed by consumer predation ( Fig B in S1 Text ) . In turn , those species left unpaired at level two must be paired by species at level three . Defining No and Ne as the sums of node richness at odd and even levels , respectively , it is then easy to check that the general constraints become Δ ≡ N o - N e ∈ { 0 , 1 } , ( 5 ) which encompasses the competitive exclusion principle [8 , 9 , 18] . Defining L as the top trophic level , for each of the two options in Eq 5 a set of L − 1 nested inequalities arises relating the species counts ni: ( i ) ( i i ) ( i i i ) ( i v ) n 1 ≥ Δ n 2 ≥ n 1 - Δ n 3 ≥ n 2 - n 1 + Δ n 4 ≥ n 3 - n 2 + n 1 - Δ ⋮ ( 6 ) This sequence continues until nL is reached . The case Δ = 0 is consumer-limited , whereas Δ = 1 signals resource limitation . Note that these rules allow simple assessment on which food webs can have coexistence-solutions . To illustrate this , we give two simple examples of food webs that cannot coexist by Eqs 5 and 6 ( Fig 1biii and 1biv ) . Our rules thereby are more selective than those in previous work [12] . There , a requirement was stated for trophic communities of several levels , where the number of species on any level could not exceed the sum of the numbers on adjacent levels—a condition that would e . g . not rule out the two simple webs ( Fig 1biii and 1biv ) , hence not detect the lack of niches . The conditions ( Eqs 5 and 6 ) rule out stable coexistence when violated , yet they leave unanswered if all populations can be positive or stable when they are met . For any choice of species counts fulfilling Eqs 5 and 6 , a feasible solution can be obtained by starting from the complete non-overlapping pairing of species , i . e . a subset of links . A tree-like backbone of links will be obtained , which preserves the complete non-overlapping pairing . Additional links give each species access to nutrients . Note that a tree is a maximally trophically coherent network of species [22] . For this structure , we find that the parameters can always be assigned to yield positive steady-state populations for all species . This can be demonstrated by selecting all interaction strength equal to unity . Subsequently the decay rates αi can be selected to be small and to fulfill constraints at each branching point of the tree , thereby obeying trade-offs between the species in the respective branches . As the number of these constraints is less than the number of available decay coefficients , these inequalities can always be fulfilled ( details: [38] , in prep . ) . An example for possible assembly is shown in the subsequent section . Recognizing the existence of solutions may then serve as a starting point for investigations into the volume of parameter space of feasible solution for Lotka-Volterra systems [39] . The existence of a Lyapunov function is a sufficient criterion for global stability [40] . Using this , in extension of the analysis of simple chains of species [41] , we demonstrate that tree like food webs that are feasible are always stable , i . e . the Lyapunov function decreases monotonically with time after the initial perturbation ( Sec . S2 . 2 in S1 Text ) . We thereby establish that for each combination of species richnesses consistent with the conditions ( Eqs 5 and 6 ) , a feasible and stable food web exists . In practice , there may be several basic nutrients , such as different chemical compounds or spatial or temporal subdivision [42] . If nS > 1 separate nutrients are available , the assembly rules yield nS+1 sets of conditions analogous to those in Eq 6 where any Δ ∈ {0 , nS} is allowed ( Sec . S7 in S1 Text ) . The presented theory assumes simple predator-prey couplings . Non-linear interactions ( i . e . type-II response ) and cannibalism can be included by adding diagonal matrix elements in Fig 1a , corresponding to species that pair with themselves ( Secs S7 , S8 in S1 Text ) . Another future extension of the theory could be that of incorporating frequency-dependent predation [43] . We return to a single nutrient ( nS = 1 ) and now discuss species richness at the different trophic levels . In the consumer-limited case , Eq 6 ( i ) restricts n1 to an upper bound given by n2 . Eq 6 ( ii ) conversely restricts n2 to numbers equal to the sum of the total species richness in both neighboring trophic levels , hence the limitation to the abundance of n2 is much weaker compared to that of n1 . Accordingly , the basal level cannot constitute the global maximum of node richness within a level . Moving further , n3 could again exceed n2 but possible limitations stemming from the count of top predator species become noticeable . When starting at the top predator level L , by symmetry , similar constraints as in Eq 6 hold: nL − nL−1 ≤ 0 . Together with Eq 6 ( i ) we have for the consumer-limited state , n1 + nL ≤ n2 + nL−1 , i . e . species at intermediate trophic levels generally dominate food web biodiversity ( Fig 2 ) . The condition is similar for the resource-limited state , where the limit is shifted by one species . For increasing L , these equations predict further increase in the contribution of intermediate species to total biodiversity . Given these general constraints , we can now discuss food web assembly . Consider a simple food web and its interaction matrix ( Fig 3a ) . Graphically , an allowed structure is again manifested by permitting non-overlapping pairing of species . Food web growth is characterized by alternating transitions between resource- and consumer-limited states . Initial community growth requires the presence of a single primary producer ( Fig 3b ) , hence n1 = 1 . The only possible addition is then a consumer , preying on the existing producer . This entails immediate addition of a trophic level ( n1 = n2 = 1 ) and transition to a consumer-limited state . The assembly rules subsequently permit additions at different trophic levels , but alternation between consumer and resource limitation must be preserved . For each food web consistent with the conditions in Eqs 5 and 6 , parameters can be assigned in a way that food web evolution is possible , i . e . that feasibility and stability are even achieved after each addition of species . To exemplify this , we assign parameters for all eight species of the example , tree-like , food web ( Fig 3b ) . Using in-silico simulations , we mimic the evolution of this web , starting from only a single species ( Fig 3c ) . Any addition of new species leads to a transient disruption of population densities , but a new feasible steady state is eventually established ( Fig 3c ) . To quantify stability , we further compute a Lyapunov function , and verify that it monotonically decreases after each addition within each stage of the food web ( Sec . S2 in S1 Text ) . We have checked that addition of further weak links is compatible with stability and feasibility , hence in principle allowing arbitrary link structure . Our assembly rules are easily generalized to food webs containing omnivores or parasites with hosts at several trophic levels ( Sec . S6 in S1 Text ) , with the limitation that parasites are here mathematically treated as omnivores , and thus not as species that require multiple hosts to complete their life cycle . Generalizations from our theory would result , when considering nonlinear responses , such as the ones discussed elsewhere [44] . Consider again Fig 3a , but imagine that another species is added on trophic level 4 , causing a violation of the assembly rules . This violation can be rectified by a single generalist omnivore ( Fig 4 ) , corresponding to an additional row and column of nonzero coefficients in the interaction matrix . The omnivore can be interpreted as a consumer preying on all trophic levels . Hence , one can choose a level i and let its species count ni be increased by one unit to again satisfy the assembly rules . In general , omnivory or parasitism can make the graph non-bipartite . If so , the non-overlapping pairing to achieve d e t ( R ) ≠ 0 is extended to covering the entire network with closed loops of directed pairings ( Sec . S6 in S1 Text ) . The potential for stabilization due to omnivory has also been addressed by Pillai et al . within a metacommunity model [45] . There , coexistence of species on multiple patches in space can be obtained when an omnivore preys on a given basal species on one patch , while it preys on one of the consumers of that basal species on another patch . Our findings provide criteria for sustainable food webs within each patch , i . e . without the notion of spatial separation . In our model , increased diversity could then be obtained by considering partially isolated spatial locations as representing additional basal resources . What do the assembly rules teach us about real food webs ? For seven detailed empirical food webs ( Details: Materials and Methods ) containing both free-living and parasite species , we determine the difference between the respective total number of species S and the maximum number of linearly independent rows , d ≡ S - rank ( R ) ( Details: Methods ) . Linear dependence can be seen as the sharing of a specific niche by several species , therefore d measures the lack of niches in the given food web . When d = 0 , the assembly rules are satisfied ( d e t ( R ) ≠ 0 ) . As done previously [31] , we distinguish webs formed by: links between free-living species only; links between all free-living and parasite species; with additional concomitant links ( Methods and Sec . S9 . 3 . 3 in S1 Text ) . The free-living webs have substantial structure , an example is given in Fig 5a and others in Sec . S9 in S1 Text . Many species are located at sharp trophic levels ( Fig G in S1 Text ) , a feature that manifests itself by the blocks of white spaces , i . e . absent interactions ( Fig 5a ) . The organization into sharp trophic levels entails stricter demands on the combinations of species richnesses ( Eqs 5 and 6 ) , and many sub-webs consisting only of free-living species do not seem to fulfill these demands . This is quantified by an associated lack of niches in most free-living webs , i . e . d > 0 . Additional analysis reveals that all free-living webs are in a consumer-limited state , i . e . species richness in even trophic levels dominates ( Sec . S9 in S1 Text ) . We contrast these findings with simple models , namely the cascade [4] and niche models [5] . Using number of species and links from the empirical datasets , we generate network samples ( Fig 5b and 5c ) . The resulting interaction matrices are characterized by very little structure in terms of trophic levels ( the white blocks are all but missing ) . When repeating the simulations for all seven webs ( Sec . S9 in S1 Text ) and obtaining the corresponding rank deficiencies , we find that the networks simulated using the cascade and niche models consistently give d ≈ 0 and are less structured than empirical data ( Sec . S9 in S1 Text ) . We further quantify the organization of species by the chain length distributions for the empirical and modeled networks , where much broader distributions are found for the models . Quantifying omnivory by the standard deviation of chain lengths [46] , modeled networks consistently yield substantially higher fractions of omnivory . We now consider the webs involving parasites ( Fig 5d and 5e ) . At the edge of the panels we indicate by a color-coding , where , in terms of trophic level , parasites enter and how the free-living species are re-organized . Notably , parasites predominantly enter at high trophic levels , the lower section ( approximately levels one and two , red to green colors ) remains nearly unaltered by the inclusion of parasites . Specifically , interactions of a given parasite generally involve several free-living species at multiple trophic levels ( Sec . S9 in S1 Text ) , which in our linear formalism tend to loosen the structure at the higher levels ( n3 and n4 , compare Fig 5a and 5d ) and reaching agreement with the assembly rules ( Sec . S6 in S1 Text ) . In other words , some of the parasites can be seen as effectively acting as odd-level species , thereby relaxing the initially consumer-dominated free-living webs to a more balanced state . Concomitant links ( Fig 5e ) cause further entanglement of trophic levels , open additional options for possible pairings and systematically increase sustainability of food webs . Concomitant links require additional consideration , as they generally are directed links where a parasite is consumed by its host’s predator , i . e . they denote a detrimental effect on the parasite population . A positive impact on the predator’s population may however not always result . We call such links asymmetric concomitant links . If the predator’s population does benefit from the consumption , such as observed in some studies [46 , 47] , we use the term symmetric concomitant link . Nonetheless , such directed links can lead to additional non-overlapping pairings , when a closed loop of directed links is formed , e . g . a triangle ( loop of length three ) . In the empirical webs it is noticeable that inclusion of asymmetric concomitant links only rarely yields rank improvement . Investigating this further , we find that many food webs with parasite interactions already contain sufficient numbers of loops to allow pairings between parasites and free-living species . The limitation arises because a surplus of parasites exists . Each loop will involve at least two free-living species but only one parasite , making it impossible to find non-overlapping pairings for all parasites . For those webs , only the inclusion of symmetric concomitant links leads to an additional improvement of rank , since then each parasite can be paired with a single free-living species . In two webs , where loops are rare , even asymmetric concomitant links help improve the rank . Details and simulations are available in SI sections S9 . 3 . 3 and S10 . 4 in S1 Text . For all empirical food webs , we summarize the effect of the different link additions on rank deficiency d ( Fig 5f ) . We find a general decrease of d as more parasite links are added . Notably , full rank is sometimes not achieved , even when all available concomitant links are included . Indeed , empirical food web datasets may often be incomplete , as some links can be difficult to detect . Our findings may serve as a means of identifying possibly missing data , most notably in the Ythan Estuary food web , where overall link density is low and parasite-parasite links are completely absent ( further details on individual food webs: Sec . S9 . 3 . 2 and S10 . 3 in S1 Text ) . As mentioned above , existing food web models generally produce network structures lacking rank deficiency ( d ≈ 0 ) , even for the free-living webs . When instead starting from model networks with similar link density as the empirical webs but sharp trophic levels , we obtain d substantially larger than zero ( Fig 6 ) . Parasites , with their complex life-cycles [23] , often consume species at varying trophic levels during different stages of their lives . Adding species that each interact with species at multiple trophic levels ( Fig 6c ) , i . e . mimicking the addition of parasites , our modeled webs give systematic decrease of d as links are added ( black line in Fig 6g ) . When adding species that each interact with a single ( Fig 6d ) , or exclusively higher trophic levels ( Fig 6e ) , saturation at d > 0 occurs ( blue and red lines in Fig 6b ) . When simulating also parasite-parasite interaction , d is also found to decrease ( Fig 6f , Simulation details: Methods and Sec . S10 in S1 Text ) . Overall , it takes species with interaction to multiple trophic levels to understand the observed rank improvement by parasites . We have further explored addition of omnivorous links to the free-living web . The rank deficiency d is reduced rapidly if the addition happens randomly for all trophic levels , but the reduction is limited if omnivorous links occur mostly at the trophic level 3 as in the real data ( Sec . S10 . 2 in S1 Text ) . We have also performed extensive simulations on the effect of concomitant predation , which further emphasizes the importance of parasite-parasite interaction in achieving coexistence for some webs ( Sec . S10 . 4 in S1 Text ) .
The food web assembly rules generalize the competitive exclusion principle to food webs of any number of species and trophic levels . They quantify which combinations of species richness at the different trophic levels can yield coexistence solutions . We show that for any of these combinations , there are stable and feasible network structures . Demanding full rank of the food web interaction matrix expresses the simple notion that each species must occupy a unique niche and leads to biologically plausible combinations of species richnesses at the different trophic levels . The requirement , i . e . non-overlapping pairing or equivalently nonzero determinant , is simple and directly allows us to evaluate the self-consistency of empirical data . The rules help explain that actual food web networks are far from random and more structured than those obtained from the traditional niche and cascade models . While some food web datasets do fulfill our conditions , networks known to lack interactions , e . g . the Ythan Estuary web [23] , stand out as particularly far from reaching agreement with the rules . One immediate consequence for food webs with species predominantly organized according to trophic levels , e . g . many free-living webs , is that species richness at the basal and top-predator levels should be limited by the species richness of the respective neighboring levels ( compare Eq 6 ) . This can explain the observations , both for terrestrial [48] and marine food webs [49] , which report greatest species richness at intermediate trophic levels while top predators and basal species contribute little . Similar conclusions were further drawn from semi-analytical work [50] , where a maximum in biodiversity at an intermediate trophic level was predicted . Another consequence for such webs is that additions of species are generally not possible at any trophic level , if sustainable ecologies are to be achieved . Even when the addition satisfies the assembly rules , its presence might cause substantial redistribution of biomass , i . e . shifts between consumer and resource limitation . In practice , it may be precisely these dramatic transitions that explain the profound and abrupt impacts on species abundance and energy flow patterns which are sometimes observed in the field . E . g . the introduction of opossum shrimp into a lake caused a cascade of trophic disruptions by reduction of salmon numbers and subsequent depletion of eagle and grizzly bear [51] . On the other hand , our rules also describe the circumstances , under which removal of a species must trigger additional extinctions . The assembly rules thus allow predictions of secondary extinction , resulting either from addition or removal of species . If the modified food web obeys the assembly rules , the food web might be stable . Indeed , in some observed cases , ecological release of new species into a habitat has had relatively gentle effects [52] . However , a violation of the assembly rules ( Eqs 5 and 6 ) by addition of a new species can have one of two effects: Either the new species will not be competitive and collapse , or a number of species will collapse ( possibly including the species itself ) to restore the food web to a permitted state . For removal of a species that leads to violation of the assembly rules , secondary extinctions [53] must be triggered to re-gain a sustainable state . We find a consistent pattern , when considering species removal in empirical food webs: E . g . in consumer-limited webs , such as the free-living empirical webs , secondary extinctions are more likely triggered by removal of resource than consumer species ( Sec . S9 and Fig L in S1 Text ) . Community omnivory [2 , 54–56] and parasitism [24 , 31 , 57] have been suggested as contributing to food web stability . Our approach provides theoretical support for this claim . We indeed find that full rank of the food web interaction matrix is difficult to achieve for species that are organized at strict trophic positions . Species that consume resources at different trophic positions , e . g . omnivores or some parasites , are shown to loosen the constraints and make it easier to comply with the assembly rules , i . e . finding a non-overlapping pairing of species . | Human impact currently induces rapid reductions in global biodiversity . Assessing the consequences of such modifications requires that ecological science better understand the conditions under which the species in a community can coexist and when not . Fundamentally , two species can not coexist indefinitely when they exclusively compete for the same prey—one must inevitably become extinct . This paradigm is known as the competitive exclusion principle . We consider communities of any number of species and multiple trophic levels , i . e . the average number of steps between a predator and basic nutrient , e . g . sunlight or sugars . We show that the extension of the competitive exclusion principle to such large systems means that each species must be part of a “non-overlapping pairing” . Such pairings are exclusive connections between two species , e . g . a predator and a prey . We demonstrate that a stable food web can always be obtained if a non-overlapping pairing exists . The food web assembly rules are explicit conditions that specify sustainable combinations of species at the different trophic levels . As also seen in field data , our rules imply high species numbers at intermediate levels and few at the top and bottom . We further show that omnivorous species—those with hosts at multiple trophic levels—may take a special role in stabilizing food webs , as they combine several trophic levels . | [
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] | 2016 | Food Web Assembly Rules for Generalized Lotka-Volterra Equations |
Amphotericin B has emerged as the therapy of choice for use against the leishmaniases . Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control . Understanding the risks and mechanisms of resistance is therefore of great importance . Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease . Metabolomic analysis demonstrated that ergosterol , the sterol known to bind the drug , is prevalent in wild-type cells , but diminished in the resistant line , where alternative sterols become prevalent . This indicates that the resistance phenotype is related to loss of drug binding . Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells , but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway , sterol 14α-demethylase ( CYP51 ) . The mutation , N176I , is found outside of the enzyme’s active site , consistent with the fact that the resistant line continues to produce the enzyme’s product . Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis , showing that the mutation is indeed responsible for resistance . The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress . This work reveals the power of combining polyomics approaches , to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself .
The leishmaniases are a complex of diseases caused by parasitic protozoa of the genus Leishmania which are transmitted between people via the bite of an infected sandfly [1] . The specific disease caused by the parasites depends upon which Leishmania species is responsible and ranges from a self-limiting cutaneous form , through a mucocutaneous disease and a frequently fatal visceral form [2] . Control is largely dependent upon the use of chemotherapy . In recent years the polyene amphotericin B ( AmB ) has emerged as the treatment of choice where available , particularly in the liposomal formulation which abrogates some of the toxic effects associated with the parent compound itself [3] . The specificity of AmB relates to its mode of action being mediated through a binding to the membrane sterol ergosterol , which is the primary sterol of fungal and Leishmania membranes , while binding with less avidity to cholesterol [3] , the principal sterol of mammalian host membranes . It was suggested that AmB molecules polymerise at membranes where they bind , forming pores that cause membrane leakage to various ions and this has been proposed as a key cause of death [4] , although binding to ergosterol alone is sufficient to cause death in fungi [5 , 6] . AmB in a liposomal formulation , AmBisome , has emerged as a treatment of choice because of the enhanced efficacy of the drug against macrophage-resident Leishmania parasites and the accompanying reduction in host toxicity [7 , 8] . Several trials using the drug as a combination with other leishmanicides [9] indicate that AmB containing combinations offer promise for future therapies [10] . Other trials [11] have indicated that a single injection of AmBisome is efficacious , particularly in India where other drugs , including pentavalent antimony [12] and miltefosine [13] are suffering from treatment failure and increasing resistance . The fact that the incidence of resistance to AmB in fungi has been slow to emerge , in spite of over 50 years of use [14] , has underpinned a belief that the fitness costs associated with any resistance might protect against the problem [15] . However , there are increasing reports of AmB resistance in fungi [16–18] . Moreover , several reports of AmB treatment failure have been reported in leishmaniasis patients in India [19 , 20] and in immunocompromised patients in France [21] and resistance to the drug has been reported to occur in at least one field isolate already [19] . Resistance in that case was proposed to relate to several phenotypic changes to the parasite , notably a change in sterol metabolism [19] and increase in defence against oxidative stress [22] . In common with several reports in L . mexicana [23] and L . donovani [19 , 24] , selection of resistance was associated with the replacement of ergostane-type sterols with cholestane-type sterols , the latter being less avid binders of AmB [3] . Other studies into changes occurring in selected AmB resistance in Leishmania point to alterations in enzymes of cellular thiol [24 , 25] and ascorbate [22] metabolism leading to an enhanced resistance to oxidative stress being associated with selection . Although changes to C24-Δ-sterol methyl transferase gene expression suggested a possible genetic marker for resistance [19 , 26] direct corroboration is lacking , and no specific gene mutations have yet been described that correlate unequivocally with resistance . Understanding molecular mechanisms of drug resistance provides potential biomarkers to assess the spread of resistance and can also offer routes to slow the emergence of resistance or even bypass the problem . Due to the lack of economic incentivisation for new drug development , it is essential to retain existing drugs for neglected tropical diseases , such as the leishmaniases , if we are to achieve aims of bringing the disease under control . Here we use the complementary high throughput data approaches of metabolomics and whole genome sequencing to reveal a gene whose mutation causes resistance to AmB in Leishmania mexicana .
Promastigotes of L . mexicana strain MNYC/BZ/62/M379 were cultured in Homem ( GIBCO ) medium [27] supplemented with 10% foetal bovine serum—Gold ( FBS ) ( PAA Laboratories GmbH ) starting at a density of 1 x 105 cells/ml and maintained at 27°C , passaging once every 72 hours . The cells were selected for AmB resistance by increasing concentrations of the drug , initially exposing cells to 0 . 0135 μM of AmB ( Sigma-Aldrich ) with stepwise doubling of the drug concentration to a final concentration of 0 . 27 μM . Cells able to grow in the presence of the drug were cloned under drug pressure by limiting dilution to 1 cell/ml in 20 ml of growth medium and plated out into 96-well plates . Susceptibility of the cells to various drugs was determined using an adaptation of the Alamar Blue assay [28] . A starting density of 1 × 106 cells/ml were incubated at 27°C in the presence of various drug concentrations for 72 hours in a 96-well microtiter plate . Resazurin ( Sigma-Aldrich ) in 1× phosphate-buffered saline ( PBS ) ( Sigma-Aldrich ) pH 7 . 4 solution was added to a concentration of 44 . 6 μM and cells incubated for a further 48 hours . The fluorescence of the reacted dye was measured on a FLUOstar OPTIMA ( BMG LabTech , Germany ) spectrometer set at excitation and emission wavelengths of 530 nm and 590 nm , respectively . The drugs used in the susceptibility assays , unless stated otherwise , were bought from Sigma-Aldrich . To assess the sensitivity to H2O2 , wild-type and derived AmB resistant cells at a starting density of 2 × 106 cells/ml were exposed to 20 μM , 200 μM , 500 μM and 1 mM of H2O2 [29] in growth medium in 6-well plates . The response by the two cell lines to H2O2 were compared by observation under a light microscope at different time points over 72 hours . An alternative test for H2O2 sensitivity involved glucose oxidase as described previously [30] . Briefly , 180 μl of cells at 5 x 105cells/ml were plated into a 96-well plate and 20 μl of glucose oxidase solution ( Sigma-Aldrich ) were added in varying concentrations then tested using the AlamarBlue assay described above . The cell body length of L . mexicana promastigotes was determined using the SoftWoRx 5 . 5 software on a DeltaVision Applied Precision Olympus IX71 microscope . Smears of cells from late log phase culture were spread onto a microscope slide . The cells were fixed in absolute methanol overnight at -20°C then rehydrated with 1 ml of 1 × PBS for 10 minutes . 50 μl of PBS containing 1 μg/ml 4 , 6-diamidino-2-phenylindole ( DAPI ) ( Sigma-Aldrich ) and 1% 1 , 4-Diazobicyclo- ( 2 , 2 , 2 ) octane ( DABCO ) ( Sigma-Aldrich ) were added to stain the cells . The cell length was measured from the anterior end to the posterior end of the cell body . Cells were grown to mid-log phase and 1 × 108 cells for each sample collected and metabolism was quenched rapidly by cooling them to 4°C in a dry ice/ethanol bath while mixing vigorously to avoid freezing and possible cell lysis [31] . Cells were separated from medium by centrifugation at 1 , 250g for 10 minutes at 4°C and 5 μl of supernatant was used for spent medium analysis . Metabolites were extracted from the cell pellet by addition of 200 μl of chloroform-methanol-water ( 1:3:1 ) solution and mixed vigorously at 4°C for 1 hour . The metabolites were separated from the cell debris by centrifugation at 13 , 000g for 5 minutes at 4°C and the samples were stored under argon gas at -80°C until analysis . Separation and mass detection of the metabolites was performed according to [32] , using the DionexUltiMate3000 Liquid chromatography system using a SeQuant ZIC-HILIC column coupled to the Orbitrap Exactive mass spectrometer at Glasgow Polyomics , University of Glasgow . Raw data was processed and analyzed using the mzMatch [33] and IDEOM [34] software platforms . Metabolite identifications were given at Level 2 according to the Metabolomics Standards initiative ( MSI ) where accurate masses and predicted retention times were used to yield putative annotations but when retention times of authentic standards were available , the identification should be considered as Level 1 [35] . Metadata to support the identification of each metabolite is available in the IDEOM file for each study ( S1 Table ) . It is important to note that many of the metabolite names given in the IDEOM file are generated automatically as the software provides a best match to database entries of the given mass and formula . In the absence of additional information these must be considered as putatively annotated hits; the confidence score in the column adjacent to that hit serves as a guide to this . Clearly it is beyond the scope of any study to provide authenticated annotations to many hundreds of detected compounds , but the full datasets are included in the spirit of open access data . Mid log phase cells were taken and washed in PBS before 1 . 5 ml 25% KOH in 60% ethanol was added to each 100 mg cells in glass tubes . Samples were incubated at 85°C for one hour then an equal volume of n-heptane was added . Samples were vortexed then incubated at room temperature for 10 min . The top layer containing the sterols was transferred to a new glass vial for analysis . One microlitre of heptane extract sample was injected into a Split/Splitless ( SSL ) injector at 270°C using splitless injection ( 1 minute ) into Trace 1310 gas chromatograph ( Thermo Scientific ) . Helium carrier gas at a flow rate of 1 . 2 ml/min was used for separation on a TraceGOLD TG-5SILMS 30 m length with 5 m safeguard × 0 . 25 mm inner diameter × 0 . 25 μm film thickness column ( Thermo Scientific ) . The initial oven temperature was held at 50°C for 2 min . Separation of sterols was performed using a gradient of 20°C/min from 50 to 325°C with an 8 . 5 minutes final temperature hold at 325°C . Eluting peaks were transferred through an auxiliary transfer temperature of 275°C into a Q-Exactive GC mass spectrometer ( Thermo Scientific ) . Electron ionisation ( EI ) was at 70 eV energy , with an emission current of 50 μA and an ion source of 230°C . A filament delay of 3 . 5 minutes was used to prevent excess reagents from being ionised . Full scan accurate mass EI spectrum at 60 , 000 resolution were acquired for the mass range 50 to 750 m/z . Peak detection used the Xcalibur software ( Thermo Scientific ) . Masses were compared to those in the NIST/EPA/NIH Mass Spectral Library ( EI ) . Cells for genomic DNA extraction were grown to mid-log phase in a 10 ml culture and harvested by centrifugation at 1 , 250g for 10 minutes and washed once in 1 × PBS . The cells were re-suspended in 500 μl NTE buffer ( 10 mM Tris-HCl pH 8 . 0; 100 mM NaCl; 5 mM EDTA ) to which 25 μl of 10% SDS and 50 μl of 10 mg/ml RNase A ( Sigma-Aldrich ) were added and warmed to 37°C . The solution was mixed by inverting and incubated at 37°C for 30 minutes . After addition of 25 μl of 20 mg/ml pronase ( Sigma-Aldrich ) the lysates were incubated at 37°C overnight . The samples were then extracted twice with phenol:chloroform:isoamyl alcohol ( 25:24:1 ) ( Sigma-Aldrich ) and chloroform , with mixing for 5 minutes between extraction steps . The aqueous phase was obtained after centrifugation at 16 , 000g for 10 minutes and the DNA was precipitated with absolute ethanol and washed once with 70% ethanol . The DNA was dried in the fume hood and after being dissolved in water the concentration was determined using a NANODROP 1000 spectrophotometer ( Thermo Scientific ) . Paired-end samples of the genomic DNA for the progenitor wild-type and derived AmB resistant cells were sequenced using Illumina GAIIx next generation DNA sequencing platform and analysed at Glasgow Polyomics , University of Glasgow . All DNA sequence information is deposited at the European Nucleotide Archive ( ENA ) under project number PRJEB10872 . The expression vector pNUS-HnN for Crithidia fasciculata and Leishmania [36] was used to express the WT sterol 14α-demethylase gene ( LmxM . 11 . 1100 ) fused to the His-tag at the N-terminus in AmB resistant L . mexicana . The vector pNUS-GFPcN was used for expression of both WT and N176I CYP51 with the Green Fluorescent Protein ( GFP ) tag at the C-terminus in both resistant and WT L . mexicana . The genes were amplified by PCR using Phusion High-Fidelity DNA polymerase ( New England Biolabs ) . Primers for pNUS-HnN incorporating NdeI and XhoI restriction sites ( underlined ) for WT CYP51 were forward 5' GCATATGATGATCGGCGAGCTTCTCC3' and reverse 5'CTCGAGCTAAGCCGCCGCCTTCT3' . For expression of the WT and N176I CYP51 in pNUS-GFPcN , the forward and reverse primers were 5’CATATGATGATCGGCGAGCTTCTCCT3’ and 5’AGATCTAGCCGCCGCCTTCTTC3’ , respectively , with NdeI and BglII restriction sites . Sterol C14-reductase ( LmxM . 31 . 2320 ) was expressed in pNUS-GFPcN using forward 5’CATATGATGGCAAAACGCAGAGGTACTG3’ and reverse 5’AGATCTGTATATGTACGGGAACAGCC3’ primers , respectively . The genes were initially sub-cloned into pGEM-T Easy vector ( Promega ) and multiplied in XL1 Blue E . coli competent cells ( Promega ) prior to cloning into the pNUS vectors . The presence of the gene fragments was confirmed by their PCR amplification using vector-specific primers designed from the vector sequences on http://www . ibgc . u-bordeaux2 . fr/pNUS/index . html . Thus , presence of WT CYP51 in the pNUS-HnN was verified with forward 5'CATCATCATCATCACAGCAGC3' and reverse 5'GTCGAAGGAGCTCTTAAAACG3' primers , while the presence of both the WT and N176I CYP51 in pNUS-GFPcN was verified with the forward 5’TATCTTCCACTTGTCAAGCGAAT3’ and reverse 5’CCCATTCACATCGCCATCCAGTTC3’ primers . Similarly , the presence of these genes was confirmed by PCR in DNA extracted from the transfectants . The presence of mutated chromosomal CYP51 in AmB resistant L . mexicana expressing the WT CYP51 gene was confirmed by PCR amplification using a forward primer ( 5'CGCGAAATAGATATAAAGCACACG3' ) starting from 43 bp upstream of the start codon of CYP51 or 569 bp from the point mutation and a reverse primer ( 5'TCGCGAGCGATGATAATCTCG3' ) starting 213 bp downstream the mutated base resulting in a 788 bp PCR fragment . PCR amplification fragments were sequenced at Eurofins MWG Operon , Germany and aligned using CLC workbench Genomics software . All primers were purchased from Eurofins MWG Operon , Germany . L . mexicana promastigotes were grown to log phase and 1 × 107 cells were harvested and washed then re-suspended in 100 μl transfection buffer ( 90 mM NaPO4 pH7 . 3; 5 mM KCl; 50 mM HEPES pH7 . 3; 0 . 15 mM CaCl2 ) and added to 10 μg of plasmid DNA in a cuvette before transfection with an Amaxa biosystems NucleofectorII ( Lonza ) using program U-033 . The cells were incubated on ice for 10 minutes and then transferred into pre-warmed 10 ml of Homem supplemented with 10% FBS-Gold and left to recover for 18 hours at 27°C . G418 disulfate salt ( Sigma-Aldrich ) at 50 μg/ml was added to select cells carrying the plasmids . Clones of the selected cells were obtained in the presence of 50 μg/ml G418 in growth medium by limiting dilution . Immunofluorescence microscopy was performed with WT and AmB resistant cell lines expressing GFP-CYP51 or tagged sterol reductase ( GFP-SR ) from episomal vectors . 200 μl of mid-log phase cells were collected and washed two times with PBS , fixed in 1% formaldehyde ( methanol-free , Thermo Scientific ) for 30 minutes . Triton X-100 ( Sigma-Aldrich ) was added up to final concentration 0 . 1% and incubated for 10 minutes , afterwards , glycine was added to the final concentration of 0 . 1 M and incubated for an additional 10 minutes . Cells were centrifuged , resuspended in PBS , spread on microscopy slides and left to dry . Slides were washed with PBS and blocked with PBS , 0 . 1% Triton X-100 , 0 . 1% BSA ( Sigma-Aldrich ) for 10 minutes . Primary antibody against the ER specific chaperone BiP [37] , a gift from Professor J . Bangs ( University of Buffalo , New York ) , was applied in dilution of 1:5000 , overnight , at 4°C . Subsequently , slides were washed three times with PBS and incubated with secondary anti-rabbit Alexa Fluor antibody ( Molecular Probes ) . Following 1 hour incubation , slides were washed three times with PBS , dried and mounted with 5 μM 4’ , 6-diamidino-2-phenylindole ( DAPI ) . Microscopy was performed using Axioscope , Volocity software and processed with ImageJ software . For mitochondrial staining , cells were incubated with 100 nM MitoTracker red ( Molecular Probes ) at 25°C for 30 minutes . Subsequently , cells were fixed as described above and mounted with DAPI .
AmB resistant L . mexicana promastigotes were selected by stepwise increase in drug concentration in culture medium over 18 passages stretching over six months . During this period , a 23-fold increase ( P = 0 . 0007 ) in EC50 value above that of the wild-type ( WT ) was observed ( Fig 1 and S1 Fig ) . Sustained growth in a drug concentration above 0 . 27 μM could not be achieved . The acquired AmB resistance was stable over at least 15 passages in drug free medium . There was no appreciable difference in the growth phenotype between the resistant and the WT cells , although during the process of resistance induction , the derived AmB resistant cells required at least five passages of adaptation to a given drug concentration before they would grow at similar rates to WT . The cells showing the highest resistance level had a significantly reduced cell body length compared to the WT cells ( P < 0 . 0001 ) . The late log-phase WT cells and the resistant clone had average cell body lengths of 11 . 16 ± 0 . 19 μm ( n = 126 ) and 9 . 86 ± 0 . 16 μm ( n = 126 ) , respectively . The AmB resistant cells exhibited mild cross-resistance to potassium antimony tartrate ( PAT ) and miltefosine with fold change in EC50 values of 2 . 9 and 3 . 9 representing significant differences ( P = 0 . 0005 and P < 0 . 0001 , respectively ) to the WT ( Table 1 and S2 Fig ) . A marginal 1 . 7-fold increase in the EC50 value ( P = 0 . 0007 ) to ketoconazole ( an inhibitor of sterol synthesis at the sterol 14α-demethylase step ) was also observed in the AmB resistant line . Interestingly , the AmB resistant cells were found to be more susceptible to pentamidine ( P = 0 . 0001 ) with decreases in EC50 values of 13 . 3-fold . We also tested the effect of various reagents causing oxidative stress . Exposure of both cell lines to methylene blue , a stress inducing agent [39] , showed that AmB resistant cells were far more susceptible to this agent with EC50 values of 0 . 117 ± 0 . 001 μM against 4 . 20 ± 0 . 22 μM for WT ( P < 0 . 0001 ) . Addition of 500 μM H2O2 directly to cells induced swelling resulting in their assuming rounded shapes , and sluggish to no movement . Resistant cells recovered from exposure more slowly than WT cells ( as judged by inspection of flagellar motility ) and by 72 hours following exposure reached average densities of 4 × 106 cell/ml whilst WT cells were at 9 × 106 cells/ml . Because H2O2 is labile , we also tested the effect of glucose oxidase in medium . Glucose oxidase produces H2O2 continuously [30] and the concentration of enzyme added to medium can , therefore , act as a surrogate for quantitation of susceptibility to the peroxide . 4 . 5 mU/ml of glucose oxidase were required to inhibit growth of WT cells by 50% whilst the EC50 for the resistant cell line was just 1 . 8 mU/ml , confirming the increased sensitivity to stress of the resistant cell line . Using an untargeted liquid chromatography-mass spectrometry ( LC-MS ) metabolomics approach we compared the two cell lines . Principal components analysis ( PCA ) revealed the WT and resistant lines to have clear differences ( Fig 2A ) . Among the most significant changes were alterations around the sterol metabolic pathway . Previous studies in both L . donovani [19 , 24] and L . mexicana [23] have also identified changes to sterols ( specifically an increase in cholesta-5 , 7 , 24-trien-3β-ol in L . donovani and 4 , 14-dimethyl-cholesta-8 , 24-dienol and other methyl sterols in L . mexicana ) . A metabolite of m/z 394 . 32 putatively identified as ergosta-5 , 7 , 22 , 24 ( 28 ) -tetraen-3β-ol was diminished in resistant cells compared to WT cells ( Fig 2B and S1 Table ) while a metabolite of m/z 410 . 35 putatively identified as 4 , 4-dimethylcholesta-8 , 14 , 24-trien-3β-ol and another with m/z 426 . 35 consistent with 4α-formyl-4β-methylcholesta-8-24-dien-3β-ol were more abundant in resistant cells compared to WT cells ( Fig 2B and S1 Table ) . While the LC-MS approach taken allows comprehensive coverage of the metabolome and is thus ideally suited to initial identification of those areas of metabolism changing in response to biological perturbation , the hydrophilic interaction liquid chromatography ( HILIC ) -based liquid chromatography platform is not suitable for separation and robust identification of individual lipids . Having identified that changes in sterol metabolism were key , we adopted a gas chromatography ( GC ) -MS approach since this methodology had been previously applied to the identification of sterol metabolism in L . mexicana [40] . Fig 3 shows chromatograms obtained from the GC-MS , and identities of the detected peaks are indicated in Table 2 based on matches with the NIST library ( https://www . nist . gov/srd/nist-standard-reference-database-1a-v14 ) . The major difference between WT and AmB resistant cells is depletion of peak 6 representing ergosterol ( the most abundant sterol in WT ) and concomitant increase in peak 5 , corresponding with 14-methylergosta-8 , 24 ( 28 ) -dien-3β-ol in the resistant cell line . Peak 4 , not detected in WT , is abundant in resistant cells , and corresponds to 4 , 4-dimethylcholesta-8 , 14 , 24-trien-3β-ol , a product of the sterol 14α-demethylase reaction ( note , however , that isomers of this compound exist that we cannot distinguish ) . In addition , peaks 3 and 8 are increased 50-fold and 80-fold , respectively , in the AmB resistant cell line , putatively identified as ergosta-5 , 24 ( 28 ) -dien-3β-ol and 4 , 14-dimethylergosta-8 , 24 ( 28 ) -dien-3β-ol . The level of cholesterol was unchanged because it is acquired from the medium rather than synthesised [41] . The detected sterols were mapped onto a pathway based on work by Roberts et al . , [42] and metacyc . org database ( Fig 4 ) . Overall , components from the upper part of the pathway are accumulated whereas intermediates of the downstream steps are decreased . Replacement of ergostane-type sterols in WT cells with cholestane-type sterols in resistant derivatives , are similar to those noted in L . donovani promastigotes selected for resistance [24] and also a field isolate of L . donovani from a refractory patient [19] . In a separate study , L . mexicana selected for resistance had also lost ergosterol , but in this case the sterol species that accumulated was 4 , 14-dimethylcholesta-8 , 24-dienol [23] , indicating that there may be several distinct ways whereby loss of ergosterol synthesis can be achieved , with the accompanying accumulation of other sterols . In principle , the loss of any enzyme in the ergosterol synthetic pathway could lead to loss of production of that sterol and acquisition of resistance to AmB . For example , Pourshafie et al . [26] point to possible mutations in C-24-Δ-sterol-methyltransferase causing the increase in cholesta-5 , 7 , 24-trien-3β-ol they identified . The related parasite Trypanosoma brucei accumulates exogenous cholesterol for membrane biogenesis [43] and L . donovani deficient in a cytochrome P450 enzyme related to sterol 14α-demethylase , termed CYP5122A1 , produce less ergosterol than WT cells and grow less well but recover WT growth rates if medium is supplemented with exogenous ergosterol [44] . We therefore tested whether addition of exogenous ergosterol would accumulate in membranes of our resistant cells and re-sensitise them to AmB . However , addition of exogenous ergosterol ( 7 . 6 μM , as used in reference [44] ) for 5 passages prior to testing drug efficacy , failed to re-sensitise , rather it further reduced sensitivity ( 2 . 84-fold increase in EC50 value for AmB ( P = 0 . 0004 ) ) which could be attributed to the drug binding ergosterol in medium . Whole genome sequencing of the resistant line and its WT progenitor ( passaged in parallel during the course of resistance selection ) resulted in more than 50% of the reads being aligned in both cases to the reference L . mexicana MHOM/GT/2001/U1103 genome ( 17 , 138 , 430 and 15 , 392 , 124 reads out of totals of 32 , 238 , 036 and 27 , 514 , 220 reads which were obtained for the WT and the AmB resistant clone , respectively , were aligned ) . Comparison of read coverage depth between AmB resistant and WT cells showed variations in chromosome copy numbers . An extra copy was observed to have been gained for chromosomes 05 , 19 , 22 , and 27 and there was loss of a copy for chromosomes 12 and 17 of the AmB resistant cells as compared to the parental WT cells . In addition , a total of 5 , 047 single nucleotide polymorphisms ( SNP ) distinguish the WT and resistant lines . Since the metabolomic data indicated changes in sterol metabolism we looked specifically for any genetic alterations in genes encoding enzymes of this pathway in AmB resistant cells . A single homozygous SNP was found among genes encoding the enzymes of the ergosterol pathway , namely in sterol 14α-demethylase ( EC 1 . 14 . 13 . 70 ) , where a non-synonymous mutation from A to T results in an amino acid alteration from asparagine to isoleucine ( N176I ) in this enzyme . Lines of several Candida species resistant to AmB also had mutations to sterol 14α-demethylase ( ERG11 ) [45–47] which lead to a cessation of ergosterol production . These cells , however , were also resistant to azoles that target the demethylase whilst our Leishmania cell line was not . Since the Candida lines also accumulate lanosterol ( the substrate of sterol 14α-demethylase ) whilst our Leishmania cell line accumulated the enzyme’s product , we conclude that the Candida mutants have lost enzyme activity whilst our mutant retains demethylase activity but fails to provide the product into later steps of the ergosterol synthetic pathway . Interestingly , 4 , 4-dimethylcholesta-8 , 14 , 24-trien-3β-ol is the product of the sterol 14α-demethylase reaction and it was increased in the resistant cells , indicating that the enzyme itself was functional , but that its metabolic product accumulates and no longer feeds the remainder of the ergosterol pathway . Modelling of the site of the mutation revealed it to reside on an external loop of the enzyme , some way from the active site ( Fig 5A ) . This is compatible with the enzyme’s retaining activity , but somehow becoming divorced from other features required to progress the product further through the ergosterol pathway . In L . donovani , gene knockout experiments with sterol 14α-demethylase concluded that the enzyme was essential and double knockout only possible in the presence of an expressed episomal version of the gene [48] . By contrast , null mutants were made in L . major and the cells were viable , albeit hyper sensitive to temperature stress [49] . An alignment of the primary sequences of sterol 14α-demethylase from different trypanosomatids , yeast and humans ( Fig 5B ) revealed that the mutated asparagine residue is conserved among trypanosomatid species analysed and not in yeast ( Saccharomyces cerevisiae ) or humans ( Homo sapiens ) . The conservation could indicate important function across the Kinetoplastidae , beyond enzyme activity , for instance it could be important for protein-protein interactions . Connection of ergosterol synthesis with AmB resistance was reported in previous studies , and here we observed substantial changes in the ergosterol biosynthetic pathway including loss of ergosterol , and mutation in sterol 14α-demethylase ( CYP51 ) . We therefore re-expressed the WT allele in resistant cells to see if ergosterol synthesis could be restored and whether reversion to AmB sensitivity occurred . Indeed , LC-MS revealed the restoration of the key marker of ergosterol synthesis ( Fig 6 ) , and concomitant reversion to AmB sensitivity was also associated with expression of WT CYP51 in resistant cells . The line now expressing WT CYP51 was found to be less sensitive to ketoconazole with a fold change in EC50 value of 2 . 6 ( P = 0 . 0072 ) as compared to WT cells indicating a possible over-expression of the demethylase , a known target of ketoconazole , in the re-expressor line ( Table 3 ) . The implication of overexpression would be the presence of more protein , requiring more drug to achieve the same level of inhibition of demethylase activity . The AmB resistant cells expressing the WT gene for sterol 14α-demethylase were found to have lost hypersensitivity to pentamidine by reverting back to the WT EC50 value for this drug and sensitivity to miltefosine was also restored . Since the mutation that affects sterol 14α-demethylase falls outside the active site and the enzyme retains activity as assessed by the ability of cells to convert lanosterol to its product , the mutation in the enzyme must prevent entry of the product into the remainder of the sterol pathway . Altering the subcellular localisation of the enzyme such that the product is divorced from the next enzyme in the pathway would offer a means to allow this . We therefore tested the subcellular localisation of both WT and mutant enzyme by tagging both with green fluorescent protein ( GFP ) at the C-terminus , the N-terminus having been proposed as important to localisation [44 , 50] . A staining of the GFP-tagged CYP51 expressing cells with antibodies to the endoplasmic reticulum ( ER ) specific protein BiP , revealed localisation to the ER . There was no indication that the mutated enzyme localises differently from the WT enzyme at this resolution ( Fig 7 ) hence the mutation did not seem to affect the broad compartmental localisation of the enzyme . We also tested localisation of the next enzyme in the sterol pathway , sterol C14-reductase , by tagging with GFP and it too was found in the endoplasmic reticulum in both WT and AmB resistant cells ( Fig 7 ) . The point mutation in CYP51 therefore has no effect on organellar targeting of that protein , nor the next enzyme in the pathway , although we have not been able to ascertain whether these enzymes are linked in either cell line . Higher resolution microscopy or the use of different tagging system might yield more information on the localisation of the enzymes .
The leishmaniases represent a significant health burden in many parts of the tropical and sub-tropical world . Elimination is a public health priority . Treatment of diagnosed patients is central to elimination plans . AmB has , in recent years , gained favour as a first line treatment for the leishmaniases , particularly in its liposomal formulation , AmBisome , which can be given in lower doses and is substantially less toxic than non-liposomal formulations of the drug . Efficacy is such that a single injection of AmBisome ( 10 mg/kg ) is currently proposed for primary intervention [11] . A single dose regimen carries the public health benefit of assured compliance with no need for prolonged hospitalisation . However , the policy also brings with it a risk of resistance selection . Future plans for sustained therapeutic intervention with combination therapies [9 , 10 , 51 , 52] once the best combination regimens have been chosen , might mitigate against this risk . However , where AmB is part of the combination , selection of resistance during the single shot monotherapy phase of the control programme would be calamitous . The fact that resistance to AmB has not emerged to any great extent in the treatment of fungal infections , in spite of over 50 years of use [14 , 15] , coupled to various laboratory based experiments corroborating the difficulty of selecting stable AmB resistance [15] has led to the perception that the benefits of the single dose AmBisome approach outweigh the risks of resistance . Several laboratory studies , however , have revealed that Leishmania can be selected for resistance to AmB , both as promastigotes and also amastigotes [23 , 24] . Moreover , the first reports of parasites that are of reduced sensitivity to the drug being isolated from patients refractory to AmB are emerging [19 , 20] , in spite of the drug’s use against the leishmaniases having been relatively limited . Genes responsible for resistance have yet to be identified , although a number of common features have been detected in AmB resistant Leishmania cell lines . These include changes in sterol metabolism , where ergosterol , the primary sterol of WT Leishmania cell membranes is reduced or lost and replaced by different cholestane-type sterols . Enhanced ability to resist oxidative stress is also a prominent feature and proteomic analysis [19 , 23 , 24] has demonstrated increases in abundance of stress related proteins . Here we set out to seek genes responsible for resistance by applying a polyomics-based approach , combining data from untargeted metabolomics analysis with whole genome sequencing . We focused on L . mexicana promastigotes which are relatively easy to work with to generate an understanding of how resistance to AmB can occur . Ultimately it will be necessary to test the relevance of these results to L . donovani amastigote forms that are responsible for visceral leishmaniasis , the primary target for AmB therapy . We identified changes in sterol metabolism with a loss of ergosterol and could associate this with a single change to the enzyme sterol 14α-demethylase . PCR analysis of the gene encoding this enzyme from parasites selected after 50 days of drug exposure with low level resistance ( Fig 1 ) had a WT sterol 14α-demethylase gene . By day 162 , after higher level resistance had been selected , the mutant allele had appeared , indicating that lower level resistance was associated with changes other than the alteration is sterol metabolism , but the acquisition of higher level resistance required loss of ergosterol . Sterol 14α-demethylase ( CYP51 ) has been considered an important target for chemotherapy as the azole drugs like ketoconazole and itraconazole inhibit this enzyme and show anti-leishmanial activity , albeit with disappointing results in vivo . Although gene knockout experiments indicated the gene was essential in L . donovani [48] , recently it was shown that a L . major null mutant of sterol 14α-demethylase was viable [49] . These cells accumulated the 14-methyl sterols , 14-methyl fecosterol and 14-methyl zymosterol , and also acquired resistance to AmB , due to loss of ergosterol production . They also acquired resistance to azoles exemplified by itraconazole , which targets the demethylase . Our resistant line retained sensitivity to ketoconazole , which is explained by its having retained the demethylase activity , which also explains why the resistant line accumulates the enzyme’s product 4 , 4-dimethylcholesta-8 , 14 , 24-trien-3β-ol . In yeast , the enzymes of ergosterol biosynthesis have been proposed to exist within a multi-enzyme complex , the ergosome [53] . By analogy , a similar multi-enzymatic complex could exist in Leishmania , although no evidence for such a complex has yet been described . To test whether the mutation we identified in sterol 14α-demethylase led to mislocalisation of the enzyme , we tagged both WT and mutant copies with GFP and followed cellular localisation . In L . mexicana the enzyme is found primarily in the ER as in L . major [49] . This localisation is retained in both WT and resistant lines . No gross change in localisation of the following enzyme , sterol C14-reductase , is apparent when mutated CYP51 N176I is expressed . It seems likely , therefore , that the mutation , instead , prevents an interaction with this or another protein and this alteration may prevent the channelling of the product into the subsequent reactions of ergosterol synthesis leading to accumulation of 4 , 4-dimethylcholesta-8 , 14 , 24-trien-3β-ol and other intermediates . The accumulated sterols are presumably sufficient for key roles of sterols in the resistant cell lines . Leishmania therefore may contain a multi-enzyme ergosome analogous to that described in yeast [53] and direct protein interactions may be essential for the proper function . We plan to investigate the presence and composition of the leishmanial ergosome in future work It was also of note that the AmB resistant line we selected was hypersensitive to oxidative stress ( created by hydrogen peroxide and methylene blue ) and to pentamidine , an anti-leishmanial drug previously indicated to exert its mode of action through induction of oxidative stress [54 , 55] . Hypersensitivity to hydrogen peroxide , methylene blue and pentamidine was reversed along with the AmB resistance phenotype upon expression of the WT demethylase gene . Possible explanations for this increase in sensitivity to oxidative stress include changes to the cell membrane integrity and fluidity after loss of ergosterol , as was observed previously [19 , 24] , or possibly the enhanced capability of ergosterol itself as an agent of protection against oxidative stress [56] . Increases in stress-response proteins have also been reported in other AmB resistant lines [22 , 25] . It is possible that this relates to their selection leading to loss of ergosterol and the concomitant increase in sensitivity to oxidative stress is secondarily compensated by additional changes to enzyme pathways dealing with oxidative stress . It is important to note here that other leishmanicides may lead to selection of enhanced resistance to oxidative stress [57] , hence these stress tolerant parasites might form resistance to AmB relatively readily . Even environmental pressures , such as high levels of arsenic in drinking water , can lead to selection of antimony resistance and reduced oxidative stress sensitivity [58] . The potential of Leishmania strains , pre-adapted in ways that will allow relatively easy selection for AmB resistance is therefore of significant concern . Resistance to AmB in this study therefore relates to changes in the sterol composition of the parasite’s membrane with AmB binding ergostane-type sterols replaced by less avidly binding cholestane-type sterols . This is achieved , in this instance , by mutating an enzyme , sterol 14α-demethylase , of the sterol synthesis pathway in a manner which affects not its active site but its ability to channel its enzymatic product to subsequent steps of the pathway . A survey of the literature describing other AmB resistant Leishmania indicates that loss of ergostane sterols is a common step in development of resistance to the drug [19 , 23 , 24] . However , it appears likely that different mutations to various enzymes in the pathway can contribute . The fact that treatment failures with AmB are reported in India and in at least one case sterol metabolism is changed [19] points to a necessity to contemplate spread of resistance to this drug . The frequency with which changes to sterol composition emerge does point to possible tests to survey for resistance . In addition to seeking different genes whose mutation can cause resistance , simple tests for sterol composition including spectrophotometric discrimination between ergosterol and cholestane-type sterols [59 , 60] offer approaches with which to develop tests for resistance to the drug . Mutations in sterol 14α-demethylase can also be identified , but other mutations too might also provide the same ultimate result of loss of ergosterol synthesis and further analysis of genes associated with resistance lines will enhance understanding in this area . | Antimicrobial resistance threatens to reverse many of the great strides made against pathogens responsible for disease . Understanding the molecular processes underlying resistance is crucial to quantifying and tackling the problem . Here we select resistance in Leishmania parasites to amphotericin B , an antileishmanial drug of increasing importance . We then combine genome sequencing with untargeted and targeted metabolomics analyses to identify a gene , sterol 14α-demethylase , mutation of which drives a change in sterol metabolism and loss of ergosterol , the molecular target of amphotericin B . Accumulation of a downstream intermediate of ergosterol biosynthesis indicated the enzyme itself retains activity , but the pathway to ergosterol is truncated . Expression of wild-type sterol 14α-demethylase in the resistant cells restored amphotericin B sensitivity and normal ergosterol production . | [
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] | 2017 | Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana |
Prion diseases are driven by the strain-specific , template-dependent transconformation of the normal cellular prion protein ( PrPC ) into a disease specific isoform PrPSc . Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals . Using non-replicating cells allows the accumulation of higher levels of PrPSc and , thus , greater amounts of infectivity . Here , we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts . We demonstrate that prion-infected myotubes generate substantial amounts of PrPSc and that the level of infectivity produced in these post-mitotic cells , 105 . 5 L . D . 50/mg of total protein , approaches that observed in vivo . Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents . Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact . We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity .
Prions are the etiological agents responsible for the transmissible spongiform encephalopathies . These neurodegenerative diseases affect mammals , are inevitably fatal , and are always associated with the accumulation of a specific post-translationally modified isoform of a normal host glycoprotein , PrPC . This abnormal conformation , the PrPSc isoform , differs from PrPC structurally , resulting in dramatic functional consequences . While PrPC is readily degraded by proteases , soluble in detergent and rich in alpha-helical structure , PrPSc is typically characterized by resistance to proteinase K ( PK ) digestion , detergent insolubility , amyloid formation and β-sheet structure . In vivo the disease-specific isoform of the prion protein ( PrPSc ) accumulates to high levels , a process that is marked by a progressive neurodegeneration that is always fatal as well as the generation of hundreds of millions of lethal doses of transmissible prions . Experimentally , prion infections are typically performed in rodents: wild type mice , transgenic mice or hamsters . Incubation periods are generally “short” ( compared to prion infections of cervids , sheep or cattle ) , ranging from two months in hamsters and certain transgenic mouse lines to greater than a year for other mouse strains and agent strain combinations . Brain infectivity levels are extraordinarily high at clinical stage , 109 50% lethal doses ( LD50 ) in end stage hamster brain and 108 LD50 in mouse models . In vitro models of prion replication have been established by incubating infectious brain homogenates with various susceptible cell lines , most of neuronal origin , and all expressing PrPC , obligatory as a source for PrPSc generation . Typically , dividing cells are exposed to brain homogenates , derived from infected mice , and the cells are then serially passaged until the inoculum is diluted out . PrPSc accumulates to a steady state determined by the accumulative effect of prion replication , the dilutive effect of cell division and subsequent passaging , prion secretion into the media [1] , [2] and prion degradation [3]–[6] . In vitro replication of prions has been observed in numerous cell types including scrapie mouse brain cells [7]–[9] , fibroblasts [10] , [11] , epithelia [12] , glia [13] , [14] , microglia [15] , PC12 [16] , Schwann cells [17] , hypothalmic neurons [18] and neuron-like cells [19] . By far , however , the most widely used cells for in vitro replication of prions are mouse Neuro-2a ( N2a ) , neuroblastoma cells [13] , [20]–[22] . Prion infection of cell cultures typically results in relatively low levels of PrPSc and infectivity being generated . In the N2a cells , the level of infectivity is very low , ∼3×103 LD50 per 1×107 cells [20] , [21] . When infected neuroblastoma cell lines are subcloned and highly susceptible sublines are isolated however , the infectivity can increase to ∼2×104 LD50 per 1×107 cells [23] . Alternatively , highly susceptible N2a sublines can be isolated and subsequently infected [24] , allowing for cognate uninfected cells to be propagated as controls . One difficulty in generating and maintaining in vitro cultures of prion infection is that the infectivity levels are low and some species and strain combinations do not result in infection or stable infection [24]–[27] . Cell lines that divide rapidly tend not to support prion replication , presumably due to the dilutive effect of cell replication [28] . One potential means of overcoming these effects is the use of post-mitotic , differentiated cells for studies of prion replication . Murine-derived C2C12 myoblast cells [29] provide an intriguing possibility as myoblasts are proliferative but following serum deprivation , terminally differentiate into post-mitotic myotubes , a syncytium of fused myoblasts . Muscle expresses relatively high levels of PrPC [30] , which promotes muscle regeneration in vivo [31] , and can harbour and replicate prion infectivity [32] , [33] . We report that differentiated non-proliferative myotube cultures can replicate prions to surprisingly high levels . Previous work with this cell culture system only observed infection , with 22L prions , when C2C12 cells where co-cultured with susceptible neuroblastoma cell lines [34] . Our studies have focussed on the infection of myotubes , not myoblasts , an approach that may prove useful as the terminally differentiated cells do not divide , removing the dilutive effect of passage in assays of scrapie replication . This system more closely mimics the in vivo situation , where a less dynamic population of cells accumulates PrPSc .
Proliferative myoblasts are capable of undergoing terminal differentiation into muscle fiber-like myotubes ( Figure 1a ) . Spontaneous differentiation occurs at high cell density and after serum withdrawal . Fully differentiated myotubes are multinucleated , contain sarcomeres and can contract . Monolayers of myotubes can remain intact for weeks . Importantly , as a cell culture system for PrPSc replication , myotubes express approximately one fifth as much normal prion protein ( PrPC ) as brain normalized per mg protein and an equivalent amount to N2a neuroblastoma cells ( Figure 1b ) though N2a cells are known to be variable in their characteristics [35] . Slightly higher levels of PrPC are consistently observed in C2C12 myotubes compared to myoblasts . PrPC expressed in myoblasts is predominately di-glycosylated . To examine the replicative potential of prions in a terminally differentiated muscle cell line and contrast them with non-differentiated replicative muscle precursor cells , we exposed both C2C12 myoblast and myotube cultures to RML prions ( Figure 2 ) . C2C12 cells were propagated and expanded as myoblasts , seeded into individual wells or flasks , grown to confluence and differentiated into a layer of myotubes upon serum withdrawal . Myotubes can easily be infected with brain homogenates by diluting infected brain homogenate into the media . Myoblast cultures were infected as sub-confluent proliferating cultures , splitting as necessary to prevent differentiation . By passage nine , fifteen days after infection , accumulation of PrPSc was not detectable in myoblast cultures ( Figure 2b ) consistent with the results of Dlakic et al . 2007 [34] . The infection of differentiated myotubes gave a very different result . We routinely observed accumulation of newly generated proteinase K resistant PrPSc by 10 days post-infection in myotubes ( Figure 2a ) . Levels of PrPSc subsequently increased with time . To determine the titre of myotube-generated PrPSc , aliquots of cell homogenates were used to infect C57Bl/6 mice . To generate material for the bioassay , we infected T75 flasks of confluent myotubes with RML prions and harvested cells at indicated time points ( Figure 3 ) . 30 µL of this lysate was inoculated , intracerebrally , into weanling C57Bl/6 mice . A standard curve for infectivity was performed in parallel with mice being inoculated with serial 10-fold dilutions of RML brain homogenate ( Figure S1 ) . Mice inoculated with the infected C2C12 cell lysate ( 15 days post-infection ) had an incubation period of 181 . 7 days . This suggests a level of infectivity approximate to a 0 . 3% brain homogenate from mice clinically affected with RML . Between 4- and 15- days post infection , the amount of infectivity present in C2C12 myotubes increased ∼10 fold and a statistically significant difference in incubation period was observed ( Figure 3 ) . C2C12 myotubes accumulated 105 . 5 LD50/mg protein ( Figure 3B ) as calculated based upon comparison of incubation periods from C2C12 derived material compared to standard 10-fold dilutions of RML brain homogenate ( Figure 3A ) . 10% RML brain homogenate contained 107 LD50/mg protein ( Figure 3B ) . Cell cultures containing prion-infected and mock-infected myotubes were observed daily . Over the time course of the experiments ( up to 21 days ) , no cell loss was observed in prion-infected plates as compared to mock-infected . To examine the cells for molecular responses to prion infection , gene expression profiling was performed ( Figure 4 ) . Expression profiles from infected and uninfected cells were similar and no genes were up-regulated greater than 2-fold by prion infection . Two genes were downregulated by >2-fold by prion infection , Carbonic anhydrase 3 and 2310046K23Rik , an un-annotated transcript . No genes were differentially regulated to an 85% confidence interval as assessed by T-test and a FDR based approach [36] . Subsequently , we examined those genes which were up-regulated in C2C12 cells infected with prion disease by submitting the “top 20” genes to the Prion Disease Database ( http://prion . systemsbiology . net/ ) [37] , [38] for which annotations were known . The genes were up-regulated in infected tubes between 1 . 36 and 1 . 6 fold . Four of the genes in the “top 20 list” ( Iigp1 , tgfb1 , ifit1 , Aldh1l2 ) were found to be up-regulated in brains of prion-infected mice suggesting that the changes observed might contain a prion-specific signature . To compare PrPSc levels in prion-susceptible mouse cell lines , following exposure to RML agent , total protein ( 100 µg ) collected from infected cell lysates and from infected brain homogenates was subjected to PK digestion ( 50 µg/mL final concentration ) . Strong signals were observed in the prion-infected C2C12 samples and the brain homogenates . Weaker PrPSc levels were observed in the N2a and SMB cells ( Figure 5 ) . To semi-quantify the differences in signal intensity , we digested and loaded ten times more total protein from infected myotubes than RML brain homogenate and ten times more N2a and SMB cell protein than myotube protein . These data indicate the PrPSc generation in C2C12 cells is considerably more robust than the other cell culture systems and within an order of magnitude of RML-infected brain homogenates . To examine whether known inhibitors of PrPSc replication could be used to “cure” prion-infected myotubes , we applied pentosan polysulfate to infected C2C12 myotubes . PPS is a sulfated polyanion previously identified to inhibit prion accumulation in cells [39] , [40] , prevent scrapie following intraperitoneal inoculation [41] and some therapeutic effect has been observed in human prion diseases , reviewed by Rainov et al . , 2007 [42] . In our studies , replicate infected myotube cultures were treated with or without PPS ( 1 µg/mL final concentration ) and harvested at specific time points . Equal amounts of total protein were subjected to PK digestion and immunoblotting ( Figure 6 ) . Without PPS , PrPSc abundance increased with time , whereas in cells treated with PPS , PrPSc accumulation was inhibited and the PrPSc signal substantially diminished by 16 days post-infection . To examine the strain selectivity of the C2C12 myotube system , we infected differentiated myotube cultures with 3 different mouse prion strains ( RML , 22L and ME7 ) . Myotube cultures were infected in parallel and analyzed by immunoblotting for the presence of PrPSc . All strains examined replicated prions in this myotube system ( Figure 7 ) , albeit with apparent strain specific kinetics . Signal at day 4 is carryover from the infection . The absence of signal at day 8 in 22L and ME7 would suggest that these strains replicate slower than RML , and the weak signal at day 14 in 22L would suggest that this strain replicates the slowest in C2C12 myotubes . The characteristic shift in glycosylation pattern to C2C12-like diglycosylated PrP is apparent . In vivo transmission of prions between different species is typically associated with a species barrier characterized by low penetrance and extended incubation period upon first passage . To examine the C2C12 model as an in vitro surrogate for examining interspecies transmission , we exposed the C2C12 myotubes to the Hyper ( HY ) strain of hamster-adapted transmissible mink encephalopathy prions at >109 LD50/mL ( Figure 8 ) . While hamster PrPHY appeared to persist in C2C12 myotube culture for 5 days , no mouse PrPSc was detected as a result of infection with HY prions . Control experiments using RML prions readily established PrPSc accumulation . In similar experiments using cervid prions , no conversion of mouse PrPC was observed ( data not shown ) .
We have demonstrated that non-replicative myotube cultures can readily be infected with mouse prion agent . PrPSc replication occurs in a short period of time , is robust , and levels of infectivity are relatively high . Input mouse brain homogenate-derived PrPSc , evidenced by dominance of the mono-glycosylated band , routinely became undetectable after 5 days post-exposure ( Figures 2 , 5 , 6 , 7 , 8 ) . As media was changed daily , it is remains unclear whether the input PrPSc was actively degraded by the cells or diluted out by media changes . Three lines of evidence suggest that the C2C12 myotubes are replicating prions: 1 ) in scrapie mouse brain homogenate , the inocula used on our cells , the dominant band of PrPSc is monoglycosylated , whereas the C2C12-generated PrPSc is predominantly diglycosylated ( Figures 2 , 5 , 6 , 7 , 8 ) . The shift in the glycosylation pattern allows us to discriminate between input material and de novo generated material ( Figure S2 ) ; 2 ) HY TME prions were only detectable at 5 days post-exposure suggesting agent does not persistence ( Figure 8 ) ; and 3 ) bioassay of cell lysates demonstrated an increase in infectivity over the course of the cell infection ( Figure 3 ) . Therefore , we conclude that the PrPSc observed is due to replication and not persistence . Although this is the first study to establish prion infection in C2C12 myotubes , we are not the first to attempt infection of C2C12 cells . Dlakic et al . established infections of C2C12 cells with 22L prions but only when co-cultured with susceptible N2a cells [34] . We were unsuccessful in infecting myoblasts but consistently infected myotubes directly , i . e . , without co-infection with N2a cells . The successful infection of myotubes with 22L , ME7 and RML suggests that the differences observed are a result of both prion strain used for infection and/or the differentiation state of the C2C12 cells . Infection of myotubes with RML and ME7 was more robust than with 22L and we were only successful in infecting completely differentiated myotubes . Typical proliferative cell cultures are constantly dividing and must be split . This means that , for prion infections to be maintained in culture , PrPSc replication must outpace cell replication and degradation [28] , [43] . This is especially important considering that cell lines replicate at different rates . We observe that C2C12 myoblasts double every 18 hours . Contrast this cellular replication rate with in vivo accumulation ( replication and degradation ) of prions , a process that can take years to fulminate in clinical disease . The replication rate of cell lines may explain the differential susceptibility of different cell lines [11] , [22] , [24] , [44]–[47] or clonally-derived sub-lines [24] to infection with different prion strains . In C2C12 cells , we observed replication of 3 different strains of prions , albeit with different levels of PrPSc at 14 days post-exposure . Another issue with proliferating cell lines is the potential genomic instability of these cells . For example , the median chromosome number of the N2a neuroblastoma cell line is 95 and the range is 59–193 ( ATCC datasheet , CCL-131 ) . By contrast , C2C12 myoblasts are diploid [48] . It is clear that stochastic genetic drift of chromosome number over generations of cell culture could cause changes in cell replication rate and/or PrPC expression , both of which may affect PrPSc accumulation or molecular responses [35] , [49] . Finally , proliferative adherent cell cultures are routinely passaged by trypsinization . Since PrPC is expressed on the cell surface , and cleaved by trypsinization , passaging cells may result in a temporary decrease of PrPC required for conversion . Myotube cultures , by contrast , are relatively static allowing direct comparison between parallel plates where the influence of cell division ( replication rate , changes in chromosomal abnormalities , effect of trypsinization , etc ) is removed . Myotubes can accumulate substantial PrPSc ( Figure 5 ) and associated infectivity ( Figure 3 ) . We routinely observe robust PrPSc signals from infected myotubes when loading only 10 µg of PK-digested total protein equivalent . Our data suggest that ∼10 fold higher levels of PrPSc are being generated in C2C12 myotube culture than in common N2a cells . While we observed substantially higher levels of PrPSc in C2C12 myotubes compared with chronically infected N2a cells , the heterogeneity of N2a cells with respect to PrPSc replication cautions against concluding with respect to PrPSc levels in general . Many different sublines of N2a cells can be isolated some which fail to replicate PrPSc and others which are quite prolific . Subclones of infected N2a cells accumulate 1 LD50 per 158 cells [50] or per 500 cells [21] , suggesting a titre of approximately104 LD50 per 1×107 cells . A T75 flask of C2C12 myotubes at 15 days post infection contains 106 LD50; as C2C12 myotubes form a monolayer of non-proliferative multinucleated myofibers , titre cannot be expressed as LD50 per cell , however , a confluent 75 cm2 flask of non-differentiated C2C12 myoblasts contains ∼1×107 cells . This result is comparable to that observed in differentiated PC12 cells [44] , [51] suggesting a similar conclusion , that differentiated non-replicative cells can accumulate prion infectivity to high levels . Importantly , C2C12 myotubes are not of neuronal origin , implying that the non-replicative differentiated state , and not the neuronal state , is the critical factor for allowing high levels of infectivity to accumulate in both C2C12 and PC12 cells . We did not detect overt effects of prion infection in C2C12 myotubes as assessed by cell morphology or gene expression profiling despite the accumulation of appreciable levels of PrPSc and infectivity . This apparent lack of cell toxicity has been observed in most cell cultures systems [49] for ex-vivo PrPSc replication , excepting GT-1 cells [18] . The accumulation of high levels of infectivity in C2C12 myotubes and PC12 cells absent any observable cytopathic effect [51] suggests that PrPSc itself is generally non-toxic to cultured cells; the cytoxicity appears to be specific to neurons in the central nervous system [52] . This strongly suggests that the apparent lethality of prion disease must be due at least in part to the multiple cell types or complex architecture present in the CNS . To examine the potential of myotubes to serve as a model system for assay of prion inhibition , we tested pentosan polysulfate ( PPS ) , a well-established molecule with known anti-PrPSc replication properties , for its ability to hinder PrPSc accumulation in the myotubes . PPS inhibited PrPSc replication in the myotubes ( Figure 6 ) but its efficacy was less than that observed in proliferative systems where PPS treatment and cell passage can completely remove infectivity [39] , [40] . It is likely that , in cultured replicating cells , the dilutive effect of cell passage coupled with the PPS inhibition of PrPSc replication enhances the apparent effect of PPS . This observation supports the suggestion by Weissmann et al . [6] of that current proliferative cell-based models for assay of inhibition of PrPSc replication are inadequate . In vivo , there is no comparable dilution of PrPSc to that created by the serial passage of proliferative cell cultures . Treatment of infected myotubes with PPS also extended the time that inocula , as indicated by dominance of mono-glycosylated PrPSc , persisted in the culture . A strong signal from carry-over inocula is present at day 4 in PPS treated cells and seems to persist longer ( Figure 6b ) than in myotubes without PPS ( Figure 6a ) . One possibility is that PPS enhances the binding of PrPSc to cells preventing it being washed out by media changes . Alternatively , PPS may interfere with degradation of PrPSc . In vivo , transmission of hamster HY or white-tailed deer CWD prions to mice is not efficacious and is associated with a large species barrier[53] , [54] . We examined our cell-based assay for prion replication fidelity by challenging C2C12 myotubes with hamster ( Figure 8 ) and deer prions and were unable to replicate prions from either species , consistent with the normal host range of these agents . This suggests that C2C12 myotubes may be a good model system with which to probe species barriers and prion adaptation , at least with regard to mouse prion protein . In summary , we have developed a novel , non-proliferative cell culture system that replicates prion infectivity to high levels generating substantial amounts of protease-resistant prion protein . This approach may be useful for probing prion strain and species barrier phenomena . Moreover , this system may allow a better assessment of putative anti-scrapie compounds as it removes the confounding effect of cell-replication from that of prion replication . We are currently adapting the myotube system for use with other prion agents .
This study was carried out in accordance with the recommendations and guidelines of the Canadian Council on Animal Care under protocol 647/10/11C and was approved by the Institutional Animal Care Committee at the University of Alberta . All animal manipulation and care was performed under institutionally approved animal use protocols approved by the University of Alberta Animal Care and Use Committee . Preparations for bioassay were formulated/diluted in sterile water . 30 µL of each preparation was injected into the anterior fontanelle of weanling C57Bl/6 mice . Animals were scored weekly for the onset of clinical disease . Prion preparations were obtained by homogenization of brain tissue in Dulbecco's phosphate buffer or water . Brains were collected from animals afflicted with end-stage clinical prion disease , infection was confirmed by proteinase K digestion and western blotting . A 10% ( w/v ) homogenate prepared from a pool of RML affected brains was used for this work . This pooled homogenate was minimally 109 LD50 per gram of brain as determined by bioassay using the Kärber formula [55] . Infectivity of C2C12 samples was calculated from the regression equation ( Figure S1 ) derived from plotting the incubation period observed from inoculation of standard 10-fold dilutions of RML brain homogenates versus infectivity; time interval assay [56] . 22L and ME7 prions were prepared from clinically-affected tga20 mice [57] and contain approximately 108 LD50 per mL [58] . C2C12 ( CRL-1722 ) myoblast cells were purchased from the American Type Culture Collection , expanded and aliquots stored in liquid nitrogen and expanded as needed . Myoblasts were grown in Dulbecco's Modified Eagle Medium , 10% fetal bovine serum ( FBS ) with penicillin and streptomycin ( PS ) . Myoblasts were seeded onto experimental plates , differentiated when confluent by switching to differentiation medium; DMEM , 10% horse serum ( HS ) , and PS . Three days after myotubes first appeared , infections were initiated by the addition of prion-infected brain homogenates diluted 1∶100 in differentiation media ( DMEM , 1% HS , 1% PS ) . A typical infection experiment involved treatment of ∼1×107 cells with ∼1×107 LD50 infectious prions , 100 µL of 10% brain homogenate per 10 mL of media . See Methods S1 . Media was changed daily , washing cells and removing residual inocula . Cell lysates were prepared by removing media , washing the cells with PBS and then adding RIPA lysis buffer . Total protein concentration was determined by BCA assay . Typically , 100 µg of total protein was digested with 3 . 5 µg of Proteinase K ( PK ) ( Roche ) for 30 minutes at 37°C in a volume of 70 uL ( 50 mg/ml PK final concentration ) . Digestion was terminated by addition of 30 ul of 5X SDS sample buffer . Each sample was loaded ( 10–15 µg based on the pre-digestion concentration ) and resolved on 15-well 12% NuPAGE bis-Tris gels ( Invitrogen ) , transferred to PVDF membrane and probed with anti-PrP antibodies 3F4 ( a kind gift from Richard Rubenstein ) epitope ( 107–112 ) [59] , 3F10 ( a kind gift from Yong-Sun Kim ) epitope ( 137–151 ) [60] and/or SAF-83 ( Cayman Chemical ) epitope ( 126–164 ) [61] . Relative quantification of western blotting was performed by loading dilutions of samples until quasi-equivalent signals were obtained on western blots as determined by image analysis software ( Adobe Photoshop ) . Gene expression profiling was performed as described [62] . Briefly , RNA was purified from cell pellets using the QIAshredder and RNeasy mini kit ( Qiagen , Valencia , CA ) in accordance with the manufacturers' instructions . Total RNA was amplified and labeled in preparation for chemical fragmentation and hybridization with the MessageAmp Premier RNA amplification and labeling kit ( Life Technologies , Grand Island , NY ) . Amplified and labelled cRNAs were hybridized on Affymetrix ( Santa Clara , CA ) mouse genome 430 2 . 0 high density oligonucleotide arrays . Raw data were analyzed with Arraystar 5 . 0 ( DNA Star , Madison , WI ) . Robust multiarray normalization using the quantile approach was used to normalize all microarray data . Data are deposited into the National Center for Biotechnology Information Gene expression omnibus database with accession number GSE44563 . Prion Protein ( MGI:97769 ) , 2310046K23Rik ( MGI:1924218 ) , Carbonic Anhydrase 3 ( MGI:88270 ) , Iigp1 ( MGI:1926259 ) , tgfb1 ( MGI:98725 ) , ifit1 ( MGI:99450 ) , Aldh1l2 ( MGI:2444680 ) | This manuscript describes the generation of a new cell culture system to study the replication of infectious prions . While numerous cell lines exist that can replicate prions , these systems are usually based upon proliferating cells . As mammalian cell cultures double approximately every day , prions established in the culture must also , at least , double to be maintained . This is problematic , however , as prions replicate relatively slowly and cell replication may outpace prion replication . In fact , many cell culture systems do not replicate prions and those that do often do not replicate all strains of prions . Here we describe the use of differentiated non-proliferative muscle cells to replicate prions without the interfering effect of cell division . We observed that prions accumulate to very high levels in this muscle cell culture with infectivity approaching that observed in animals . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2013 | Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes |
Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break ( DSB ) repair is important to understand how mutations arise during cancer development and treatment . To this end , we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining ( alt-NHEJ ) , single-strand annealing ( SSA ) , and homology directed repair ( HDR/GC ) . Considering early steps of repair , we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly , in that repair is suppressed by KU and promoted by CtIP . In contrast , both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs . During later steps of repair , we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA , both HDR/GC and alt-NHEJ are significantly less dependent upon these factors . As well , while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA , inhibition of this factor did not affect alt-NHEJ . These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ , SSA , and HDR/GC . However , at later steps of repair , alt-NHEJ is a mechanistically distinct pathway of DSB repair , and thus may play a unique role in mutagenesis during cancer development and therapy .
Faithful repair of DNA damage is essential to suppress genetic instability and tumorigenesis . Conversely , the efficacy of cancer therapies that utilize DNA damaging agents is likely limited by the ability of cancer cells to repair such damage . One form of DNA damage that is prone to causing mutations is a chromosomal double-strand break ( DSB ) , which can result from DNA replication , reactive oxygen species , radiation therapy , and some types of chemotherapy [1] . Characterizing the factors and pathways of DSB repair is important to understand the process of mutagenesis during cancer development and treatment . Non-homologous end joining ( NHEJ ) is a major pathway of DSB repair , in which the ends are ligated without the use of extensive homology . NHEJ appears to comprise both classical-NHEJ and alternative-NHEJ ( alt-NHEJ ) . Classical-NHEJ requires a number of factors important for V ( D ) J recombination , including the KU70/80 heterodimer ( KU ) , XRCC4 , Ligase IV , and DNA-PKcs [2] , [3] . Also , classical-NHEJ is predicted to result in minimal processing of the DSB during repair [3] , [4] . In contrast , alt-NHEJ appears to be independent of the above factors , and often results in a deletion with microhomology at the repair junction [4]–[12] . Genetic rearrangements consistent with alt-NHEJ have been observed in chromosomal translocations associated with both spontaneous and therapy-related cancer [13] , and in reversion mutations of BRCA2 following DNA damage caused by PARP-inhibition [14] . Thus , alt-NHEJ-derived mutations appear to be associated with cancer development and may result from some cancer therapeutics . In contrast to the NHEJ pathways , homology-directed repair ( HDR/GC ) and single-strand annealing ( SSA ) employ significant degrees of homology [15] . HDR/GC utilizes a homologous template for gene conversion ( GC ) through strand-invasion and nascent DNA synthesis . HDR/GC is most precise when the identical sister chromatid is used as the template for repair . Thus , factors that are important for HDR/GC might be expected to be genome stabilizing . In contrast to HDR/GC , SSA involves annealing of homologous single strands to bridge the ends of the DSB , resulting in a deletion between the repeats . Such deletions have been observed between homologous segments of ALU elements in germ-line mutations of several tumor suppressor genes [16] . It is not clear to what degree alt-NHEJ is mechanistically distinct from SSA or even HDR/GC in mammalian cells . We sought to examine this mechanistic distinction by developing an assay for alt-NHEJ repair of a chromosomal DSB , where the predominant repair product is a 35-nucleotide ( nt ) deletion with 8 nt of microhomology at the repair junction . We have used this assay , along with a novel method for inducible control of the I-SceI endonuclease in stable cell lines , for a comparative genetic analysis of alt-NHEJ , SSA , and HDR/GC . From these studies , we found that alt-NHEJ shares KU/CtIP-mediated regulation of end-processing in common with SSA and HDR/GC , but involves a unique mechanism for completion of repair with regards to the role of ERCC1 , RAD52 , and RAD51 .
We have developed two GFP-based chromosomal reporters to measure NHEJ . The first reporter , EJ5-GFP , detects multiple classes of NHEJ events , and thus can be considered an assay for total-NHEJ . We have presented this reporter mostly to provide context for the other reporter ( EJ2-GFP ) , which is designed to monitor only alt-NHEJ events . EJ5-GFP is modeled after other reporters for NHEJ [4] , [6] , [9] , in that it measures repair between two tandem endonuclease cut sites . Specifically , EJ5-GFP contains a promoter that is separated from a GFP coding cassette by a puro gene that is flanked by two I-SceI sites that are in the same orientation ( Figure 1A ) . Once the puro gene is excised by NHEJ repair of the two I-SceI-induced DSBs , the promoter is joined to the rest of the expression cassette , leading to restoration of the GFP+ gene . Since the two I-SceI-induced DSBs have complementary 3′ overhangs , such NHEJ could potentially restore an I-SceI site . Alternatively , NHEJ could fail to restore the I-SceI site , leading to an I-SceI-resistant site . In addition , a restored I-SceI site could also be re-cleaved and repaired to result in an I-SceI-resistant site . To determine the relative contribution of these different NHEJ products from repair of EJ5-GFP in mammalian cells , we integrated EJ5-GFP into both wild-type mouse embryonic stem ( ES ) cells , as well as transformed human embryonic kidney ( HEK293 ) cells ( see Materials and Methods ) . Following transient expression of I-SceI in these cell lines and sorting GFP+ cells , we amplified the GFP genes and digested the products with I-SceI . From this analysis , we found evidence of I-SceI-restoration in approximately 40% of the total products from both ES and HEK293 cells ( Figure 1B ) . Regarding the other events ( 60% ) , we cloned I-SceI-resistant products from the ES cell line sample and sequenced individual clones ( Table S1 ) . Based on these sequences , the I-SceI-resistant NHEJ products showed deletions between 8–27 nucleotides , where the majority of clones ( 11/12 ) showed 2–4 nucleotides of microhomology at the junctions . Thus , NHEJ repair of the EJ5-GFP reporter results in either restoration of the I-SceI site , or generation of deletion NHEJ events , often with microhomology at the junctions . In previous studies with similar NHEJ reporters , KU-deficient cells showed a defect in restoration of the I-SceI site [4] , [6] . To test this notion further , we integrated EJ5-GFP into Ku70-/- ES cells . We then transfected this line with an I-SceI expression vector , and subsequently measured the frequency of NHEJ events that resulted in a GFP+ gene , and quantified the restoration of the I-SceI site as described above . In these experiments , we found that Ku70-/- ES cells showed approximately equivalent overall frequencies of repair relative to wild-type cells ( 2 . 2% and 2 . 5% respectively , Figure 1C ) . However , PCR analysis of the repair products in the Ku70-/- cells showed only I-SceI-resistant products ( Figure 1B ) . These results suggest that restoration of the I-SceI site during NHEJ repair is absolutely KU-dependent , but that I-SceI-resistant NHEJ events are KU-independent . In summary , EJ5-GFP provides an assessment of total-NHEJ events , which comprises both KU-dependent restoration of an I-SceI site , as well as deletion products with some evidence of microhomology at the junctions . We have chosen to focus on the subset of total-NHEJ events that show evidence of microhomology at the junctions , also called alt-NHEJ events . For this , we developed a novel reporter ( EJ2-GFP ) , which is designed so the GFP+ products would predominantly reflect a discrete alt-NHEJ event . This reporter contains a single expression cassette for an N-terminal tag ( NLS/Zinc-finger , [17] ) fused to GFP , except the coding sequence is disrupted between the tag and GFP by an I-SceI site followed by stop codons in all three reading frames ( Figure 2A ) . As well , the I-SceI site and stop codons are flanked by 8 nts . of microhomology , which if annealed during alt-NHEJ would restore the coding frame between the tag and GFP , and cause a 35 nt deletion . This alt-NHEJ repair product also generates an XCM1 restriction site . We determined the contribution of the XCM1+ product relative to total GFP+ repair products of EJ2-GFP , as integrated in ES and HEK293 cells . For this , we sorted GFP+ cells that resulted from I-SceI expression , amplified the GFP genes by PCR , and digested the amplification products with XCM1 . From these experiments , we found that the XCM1+ product accounts for approximately 85% of the total repair products in both ES and 293 cells ( Figure 2A and 2B ) . In addition to this predominant repair event , GFP+ products derived from EJ2-GFP also include a few minor repair events , which we identified by sequencing of cloned PCR products ( Figure 2A and 2B; see Table S2 ) . For instance , one minor repair event involves a 23 nt deletion with no evidence of microhomology at the junctions ( approximately 10% of total events ) . The final set of events showed larger deletions , which ranged between 140–350 nt and showed microhomology at the repair junctions ( approximately 5% ) . The larger deletion products apparently restore a GFP+ cassette because the GFP start codon was placed proximal to the transcription start site ( unpublished data ) . In summary , while GFP+ products derived from EJ2-GFP can include some minor repair events , the predominant event ( XCM1+ ) involves 8 nt of microhomology and a 35 nt deletion , which is characteristic of alt-NHEJ [4] . From previous studies [4] , [6] , and the above experiments with EJ5-GFP ( Figure 1B and 1C ) , alt-NHEJ appears to be KU-independent . To investigate this notion further , we compared the efficiency of EJ2-GFP repair in wild-type and Ku70-/- ES cells following transfections with an I-SceI expression vector . We found that the Ku70-/- cells exhibited a 4-fold increase in the restoration of the GFP+ gene over wild-type cells , and that this increase was reversed by co-transfection of a KU70 expression vector ( Figure 2C ) . Furthermore , analysis of GFP+ products from Ku70-/- showed a similar pattern as wild-type cells , in that the XCM1+ alt-NHEJ product was predominant ( Figure 2B ) . Thus , the alt-NHEJ repair events measured by EJ2-GFP are not only KU-independent , but also appear to be inhibited by KU . In relation to other pathways , KU also suppresses HDR/GC and SSA , as described previously [18] , and as confirmed in parallel experiments with EJ2-GFP ( Figure 2C , see Figure 3 ) . Given that KU-deficiency can lead to elevated DSB end-processing [19] , [20] , these results raise the possibility that alt-NHEJ , SSA , and HDR/GC share such end-processing as a common intermediate . To continue to test the above hypothesis , we sought to perform siRNA experiments in HEK293 cells with the DSB reporters . However , we first would like to describe a novel technological approach for such siRNA experiments . In general , use of I-SceI-based reporters for such experiments would require transfection of the siRNA followed by a second transfection of the I-SceI-expression vector . Such serial transfections appear to cause increased toxicity , which can lead to variability between experiments ( unpublished observations ) . Thus , we have developed a method for inducible activation of I-SceI in stable mammalian cell lines to bypass the need for a second transfection during siRNA experiments . Specifically , we used a mutant form of the estrogen receptor ligand binding domain , where in the absence of 4-hydroxytamoxifen ( 4OHT ) , this domain ( TAM ) appears to restrict access of fused proteins to chromosomes , while addition of 4OHT releases this restriction [21] . We made a series of expression vectors for fusion proteins between the TAM-domain and I-SceI ( see Figure S1 ) , and we chose to continue with an expression vector for TAM fused to both ends of I-SceI: TAM-I-SceI-TAM ( TST ) . We generated stable cell lines expressing the TST fusion protein using a wild-type ES cell line and an HEK293 cell line , each containing an integrated copy of the DR-GFP reporter ( see Materials and Methods ) . Repair of DR-GFP by the HDR/GC pathway results in the restoration of a GFP gene ( Figure 3A ) , as previously described in mouse embryonic stem ( ES ) cells [18] . Following establishment of the TST-expressing cell lines , we analyzed 4OHT-dependent activation of I-SceI , as measured by GFP+ cells . From these experiments , we found low background levels of GFP+ cells from untreated samples , whereas 4OHT treatment resulted in an approximate 50-fold and 10-fold induction of GFP+ cells in ES cells and HEK293 cells , respectively ( Figure 3B ) . Furthermore , we found that the low background levels were stable for at least 4-6 weeks of continuous culture ( unpublished data ) . To measure not only HDR/GC , but also other repair pathways using this method , we subsequently developed similar TST stable cell lines with HEK293 cells containing the EJ2-GFP , EJ5-GFP , and SA-GFP reporters ( see Materials and Methods ) . The SA-GFP reporter measures SSA ( Figure 3C ) , as previously described in ES cells [18] . As discussed in this previous report , while it is formally possible that HDR/GC associated with crossing-over ( CO ) could also result in a GFP+ product from SA-GFP , two lines of evidence strongly suggest that CO events provide a negligible contribution to this assay . For one , multiple independent analyses have shown that CO during DSB repair in mammalian cells occurs at a frequency of less than 1% of the efficiency of the GFP+ repair events measured by SA-GFP [18] , [22] , [23] . As well , disruption of strand-exchange factors ( BRCA2/RAD51 ) causes a significant increase in the efficiency of GFP+ repair of SA-GFP [18] , which is inconsistent with a CO mechanism . In summary , we have generated HEK293 cell lines with stable expression of an inducible I-SceI ( TST ) and four different reporters to measure alt-NHEJ ( EJ2-GFP ) , total-NHEJ ( EJ5-GFP ) , SSA ( SA-GFP ) , and HDR/GC ( DR-GFP ) . As described above , we sought to examine whether DSB end-processing may be a common mechanistic step in alt-NHEJ , SSA , and HDR/GC . For this , we focused on the factor CtIP [24] , which is important for processing DSBs into ssDNA , detected as RPA-foci in mammalian cells following DNA damage [25] , [26] . Regarding repair pathways , CtIP appears important for HDR/GC in both human cells and S . pombe , but is dispensable for plasmid end joining in S . pombe [25] , [27] . We tested the hypothesis that CtIP in mammalian cells promotes not only HDR/GC , but also alt-NHEJ and SSA . For this , we performed siRNA knock-down of CtIP in the relevant HEK293 cell lines with individual reporters and stable expression of the inducible I-SceI protein ( TST ) . We knocked-down CtIP levels using two different siRNA reagents: a pool of three siRNA duplexes ( siCTIP-p ) , and a previously described single unique siRNA duplex ( siCTIP-1 ) [25] , ( see Materials and Methods and Figure S1D ) . We compared these CtIP-depleted cells to control cells transfected with a non-targeting siRNA ( siCTRL ) . We transfected each set of siRNAs into the HEK293 cell lines 48h prior to induction of I-SceI with 4OHT . The induction with 4OHT continued for 24h , and we assayed repair frequencies ( %GFP+ cells ) 3d after the start of the 4OHT treatment . We confirmed reduction in CtIP mRNA for both siCTIP-p and siCTIP-1 by RT-PCR of RNA isolated from parallel transfections at the onset of 4OHT addition ( see Materials and Methods; unpublished data ) . From these experiments , we observed that HDR/GC , alt-NHEJ , and SSA were all significantly reduced in CtIP-depleted cells treated with either siCTIP-p ( 1 . 9-fold , 1 . 7-fold and 1 . 6-fold , respectively; Figure 3D ) or siCTIP-1 ( 1 . 9-fold for each pathway; Figure 3D ) . In contrast , the absolute level of total-NHEJ was slightly increased in CtIP-depleted cells using either siCTIP-p or siCTIP-1 ( 1 . 3-fold and 1 . 4-fold , respectively; Figure 3D ) . Thus , CtIP appears to promote HDR/GC , alt-NHEJ , and SSA , but is dispensable for total-NHEJ . We suggest that CtIP-mediated DSB end-processing is important to generate ssDNA for the later steps of repair by HDR/GC , alt-NHEJ , and SSA . We also considered how alt-NHEJ might diverge from SSA and HDR/GC at later mechanistic steps . In particular , we addressed how factors important for completion of SSA may influence alt-NHEJ , since both pathways often involve annealing of flanking homology and subsequent processing of non-homologous single-stranded tails . Regarding SSA , these annealing and processing steps appear to be promoted by RAD52 and ERCC1 , since cells deficient in these factors show a decreased level of SSA [18] , and also because these factors possess relevant in vitro activities . RAD52 can function in vitro to directly promote homologous strand annealing , and also to mediate RAD51 function during strand exchange [28] . Though , only the strand annealing activity would be expected to be important for SSA in mammalian cells , since RAD51 appears to inhibit SSA [18] . ERCC1/XPF is a structure-specific endonuclease that catalyzes 5′ excision during nucleotide excision repair [29] . In addition , this complex shows efficient cleavage of 3′ overhangs , which could promote processing of non-homologous single-stranded tails during DSB repair [30] . Furthermore , ERCC1/XPF can form a complex with RAD52 , which may suggest that single-strand tail processing and annealing may be coordinated by this complex during repair [31] . To directly examine the role of RAD52 and ERCC1 in alt-NHEJ , we integrated EJ2-GFP into Rad52-/- and Ercc1-/- ES cells ( see Materials and Methods ) , and determined the fold-change in repair resulting from complementation with the relevant expression vector ( i . e . RAD52 or ERCC1 ) . Specifically , we transfected cells with an I-SceI expression vector along with either the relevant complementation vector or empty vector , and then assayed repair three days later as in Figure 2C . As well , previously described Rad52-/- and Ercc1-/- ES cell lines with DR-GFP and SA-GFP [18] were transfected in parallel . These experiments showed that the efficiency of SSA ( SA-GFP ) increased upon complementation with each of the relevant expression vectors ( 3 . 8-fold for ERCC1 , Figure 4A; 1 . 9-fold for RAD52 , Figure 4B ) . In contrast , the efficiency of alt-NHEJ and HDR/GC only slightly increased by complementation with the expression vector for ERCC1 ( 1 . 5-fold , and 1 . 4-fold , respectively; Figure 4A ) , and mildly decreased by complementation with RAD52 ( 1 . 4-fold reduced , and 2-fold reduced , respectively; Figure 4B ) . These absolute measurements of alt-NHEJ could include any of the products that result in a GFP+ gene ( see Figure 2A ) . Notably , while ERCC1 complementation promotes each pathway to some extent , the effect is significantly greater for SSA , as compared to alt-NHEJ and HDR/GC ( 2 . 5-fold and 2 . 7-fold , respectively ) . These results indicate that alt-NHEJ is mechanistically distinct from SSA , in that this pathway is both less dependent upon ERCC1 and is not promoted by RAD52 . Finally , since the above studies showed several mechanistic similarities between alt-NHEJ and HDR/GC , we next considered a probable mechanistic distinction between these pathways . Namely , we suspected that alt-NHEJ might not require RAD51-mediated strand-exchange . To examine this , we used two dominant negative inhibitors of RAD51: BRC3 and RAD51-K133R . BRC3 is a short peptide derived from BRCA2 that can inhibit RAD51 function [32] . RAD51-K133R is a mutant peptide defective in ATP-hydrolysis that results in hyper-stable strand invasion intermediates [33] . We tested the effect of these peptides on repair of the EJ2-GFP , DR-GFP , and SA-GFP reporters in otherwise wild-type ES cells . For each cell line , we co-transfected the I-SceI expression vector along with vectors expressing either BRC3 or RAD51-K133R , and compared the efficiency of repair relative to cells transfected with I-SceI and empty vector . From these experiments , BRC3 and RAD51-K133R resulted in a 2 . 3-fold and 6-fold decrease in HDR/GC , respectively , and a 1 . 4-fold and 1 . 8-fold increase in SSA , respectively ( Figure 4C ) , which is consistent with previous results [18] . In contrast , from parallel transfections with the EJ2-GFP ES cell line , BRC3 and RAD51-K133R showed no significant effect on alt-NHEJ repair ( Figure 4C ) . Thus , alt-NHEJ is distinct from HDR/GC and SSA , in that it is not affected by disruption of RAD51 function . In summary , alt-NHEJ shows a number of mechanistic distinctions from SSA and HDR/GC during later steps of repair .
Chromosomal DSBs can be repaired by a variety of pathways with distinct mechanistic requirements and potentials for mutagenesis . Given the role of mutagenesis during cancer development and treatment , it will be important to understand the mechanistic overlap of these pathways in detail . To this end , we have identified some mechanistic commonalities and differences between three DSB repair pathways: alt-NHEJ , SSA , and HDR/GC ( Figure 5 ) . To begin with , each of these pathways appears to be similarly affected by factors implicated in the control of DSB end-processing , in that they are each suppressed by KU and promoted by CtIP . Such DSB end-processing probably involves 5′ to 3′ resection , as has been directly observed to extend several kilobases in S . cerevisiae [34]; however , the precise nature and extent of DSB end-processing has yet to be determined in vertebrate cells . For example , it is possible that ssDNA could be formed via chromatin remodeling followed by unwinding by a DNA helicase . In any case , activation of end-processing likely requires bypassing KU-mediated protection of DSB ends [19] , [20] , [35] . Such bypass may be initiated by disrupting the binding of KU with DNA . Alternatively , as KU is removed from DSBs , factors could increase the probability that KU-free ends are then processed , for example , by promoting open chromatin structures [36] , and/or by activating the end-processing machinery . CtIP could function via any of these mechanisms during early steps of repair to promote HDR/GC , alt-NHEJ , and SSA . However , its ability to bind to the MRE11 complex and promote its nuclease activity , suggests it may directly promote the end-processing machinery to generate ssDNA [25] , [26] . Alternatively , since CtIP is also a transcription factor [37] , it could conceivably promote DSB end-processing by opening chromatin or affecting some other upstream process . Notably , CtIP is cell cycle regulated in mammalian cells and in S . pombe , showing its highest levels in S-phase through G2/M [27] , [38] , [39] . Thus , repair pathways that are promoted by CtIP , including alt-NHEJ , might be expected to be more prevalent in these later stages of the cell cycle . In general , further characterization of the nature and mechanism of end-processing in mammalian cells will lead to insight into the role of CtIP in regulating this process during repair . Along these lines , our findings that CtIP promotes repair of both EJ2-GFP and SA-GFP , which involve deletions of 35 nt and 2 . 7 kb , respectively , suggests that CtIP-mediated DSB end-processing can extend over a relatively wide-range of sizes . Following DSB end-processing that results in ssDNA as described above , the mechanisms of alt-NHEJ , SSA , and HDR/GC appear to diverge based on how the ssDNA is utilized during repair . For example , such ssDNA could allow either annealing of flanking homology for alt-NHEJ and SSA , or RAD51-mediated strand exchange during HDR/GC . Consistent with this notion , inhibiting RAD51 function disrupts only HDR/GC , such that RAD51 assembly on ssDNA likely commits repair to HDR/GC versus other pathways of repair . Considering the mechanisms of annealing and 3′ end-processing , we have observed that alt-NHEJ is slightly inhibited by RAD52 , and is only moderately promoted by ERCC1 . In contrast , SSA is significantly promoted by both of these factors . This mechanistic distinction may result from variations in the distance between homologous sequences , the length of the homology , and/or the absolute requirement for homologous annealing . For instance , RAD52 may play a specific role for annealing extensive regions of homology , and hence only promote SSA . This mechanism is supported by in vitro studies of RAD52 , showing that its preferred binding substrate appears to be long stretches of ssDNA , though some binding to small regions of ssDNA can also be observed [40] . Similarly , the specific role for ERCC1 during SSA could reflect a necessity for this factor in cleaving particularly long 3′ single-stranded tails; however , inconsistent with this model , ERCC1/XPF shows significant cleavage activity on short ( 15 nt ) single stranded tails [30] . Then again , alt-NHEJ may only rarely involve processing of 3′ single-stranded tails , and thus may often involve other intermediate structures that could be cleaved by a different nuclease complex . Notably , with regard to each of these mechanistic steps of alt-NHEJ , mammalian cells show both similarities and differences with yeast . For instance , our findings with KU/CtIP in mammalian cells are consistent with experiments in S . cerevisiae that showed KU-independence [6] and SAE2-activation [41] of alt-NHEJ , the latter of which may be relevant to mammalian cells assuming that SAE2 is a homologue of CtIP [25] . Regarding later steps of alt-NHEJ , the XPF homologue ( RAD10 ) in S . cerevisiae is critical for this process [6] , whereas RAD52 appears dispensable [42] . Thus , apart from the increased dependence on ERCC1/XPF for alt-NHEJ in yeast , these findings are similar to our results with EJ2-GFP in mammalian cells . In contrast , an S . pombe study on alt-NHEJ showed the opposite of the S . cerevisiae results , in that XPF ( Rad16 ) appears dispensable , and RAD52 ( Rad22 ) is important [12] . However , these S . pombe experiments were plasmid-based and involved microhomology very close to the end of the DSB . Similarly , a plasmid-based alt-NHEJ assay in S . cerevisiae also showed activation of repair by RAD52 [43] . In general , these distinctions highlight the notion that the mechanism of alt-NHEJ may be distinct between mammalian cells and yeast , but may also be affected by the length of homology , the distance separating the homologous segments , and/or the context of a DSB in a plasmid versus a chromosome . Reflecting such differences , alt-NHEJ pathways have been categorized using multiple names , each of which reflect certain features of a defined set of repair events: micro-SSA , microhomology-mediated end-joining ( MMEJ ) , KU-independent end-joining , and backup-NHEJ ( B-NHEJ ) [4]–[12] . While it may be beneficial to find consensus on a particular term , the diversity of terminology also suggest the presence of multiple subclasses of NHEJ events . The predominant event measured by EJ2-GFP , described here as alt-NHEJ , is most similar to MMEJ , in that this product is KU-independent , shows evidence of microhomology at the junction , and results in a deletion . In contrast , other events could be mechanistically more akin to SSA or micro-SSA , with respect to extent of homology , the distance between homologous sequences , and RAD52/ERCC1-dependence . Furthermore , some repair events , while KU-independent , lack evidence of microhomology [4] , [9] , such that so-called KU-independent NHEJ or B-NHEJ may reflect a larger class of events relative to only MMEJ . Further analysis of the mechanisms of this variety of repair events will continue to clarify the subclasses of NHEJ . Among these different subclasses of NHEJ , alt-NHEJ/MMEJ appears to play a significant role in the etiology of mutations that arise during cancer development and treatment . For instance , a screen for PARP-inhibitor resistant BRCA2-mutant cells revealed a set of reversion mutations that are consistent with alt-NHEJ [14] . Thus , combination of PARP-inhibition and simultaneous disruption of alt-NHEJ may be effective in eliminating PARP-inhibitor resistant cancer cells . As supported by our findings with EJ2-GFP , a target for such therapy may include CtIP [37] , whereas disruption of KU-dependent NHEJ pathways would be predicted to be ineffective . Though , PARP has been shown to play a role in plasmid-based NHEJ assays [11] , such that it would be important to ensure that alt-NHEJ is targeted separately from PARP function . Similar to the BRCA2 example , tumors deficient in ERCC1 would also be predicted to be relatively proficient at repair of DSBs by alt-NHEJ , which is consistent with the notion that DSB-inducing agents may be less effective on these tumors than interstrand crosslinking agents [44] . Finally , since alt-NHEJ appears to play a significant role in therapy-induced oncogenic chromosomal translocations [13] , targeting this pathway , again perhaps via CtIP , may enhance the efficacy of such therapy . In summary , further analysis of the mechanisms and mutagenic potential of individual DSB repair pathways will continue to inform the development of therapeutic approaches to cancer treatment .
The expression vector for the fusion protein of TAM-I-SceI-TAM ( TST ) was generated by PCR amplification of the TAM domain from TAM-CRE [21] , and the I-SceI coding sequence from pCBASce , which were cloned in frame into pCAGGS-BSKX [45] , as shown in Figure S1 . The EJ2SceGFP gene ( EJ2-GFP ) was generated by cloning gcctagggataacagggtaattagatgacaagcc into the XCM1 site of pCAGGS-NZEGFP [46] . EJ2SceGFP was then cloned into pim-DR-GFP [47] , and downstream of pgk-puro to generate pim-EJ2-GFP and EJ2-GFP-Puro , respectively . For EJ5-GFP , first an I-SceI site was cloned between the AgeI and BclI sites of pim-EJ2-GFP ( EJ5sceGFP ) , and also at the HindIII site of pgk-puro ( puroSce ) . Then , an EcoRI/I-SceI fragment of puroSce was cloned into EJ5SceGFP , followed by cloning an I-SceI site into the EcoRI site of this vector . Pim-EJ5-GFP was then completed by replacement of an EcoRI fragment that was lost in the previous step . ES cells were cultured as previously described [45] , and HEK293 cells ( HEK293-A7 , New England Biolabs ) were cultured according to the directions of the supplier , except we used DMEM high-glucose without phenol red containing Hepes buffer ( Invitrogen ) . HEK293 cells were grown on plates treated with 0 . 01% poly-lysine ( Sigma ) . Mouse ES cell lines with DR-GFP and SA-GFP targeted to hprt or Pim1 were described previously [18] , [45]–[47] . Pim-EJ2-GFP was used to target the Pim1 locus of AB2 . 2 wild-type ES cells [48] , and Ku70-/- ES cells [49] , using methods previously described [45] , except targeting was detected by PCR . Pim-DR-GFP , Hprt-SA-GFP , and EJ2-GFP-Puro were randomly integrated into HEK293 cells by electroporation with 1×107 cells suspended in 800 µl PBS in a 0 . 4 cm cuvette , followed by pulsing the cells at 250 V , 950 µF , and selecting single clones with 3 µg/ml puromycin . Similarly , EJ2-GFP-Puro was randomly integrated into Ercc1-/- and Rad52-/- ES cells as above , except using electroporation conditions of 680 V and 10 µF . Integration of an intact copy of each randomly integrated reporter was confirmed in single clones by Southern blot analysis with a GFP fragment as the probe ( data not shown ) . Stable cell lines expressing TST were generated by electroporation as described above , except with voltages varying between 200–250V , with 20–30 µg of TST expression plasmid and a selection plasmid . We used two different selection cassettes , with 10 µg of pgk-bsd ( gift from Dr . Pentao Liu ) for the HEK293 and 5 µg of pmc1neo for the ES cells . Clones were selected in the relevant antibiotic for 6–10d at 400 µg/ml G418 or 5–10mg/ml blasticidin ( Invitrogen ) . Individual selected clones were screened for significant induction of GFP+ cells following 24h treatment with 0 . 3 µM and 3 µM 4-hydroxytamoxifen ( 4OHT , dissolved in ethanol , Sigma ) for ES and HEK293 cells , respectively . To measure the repair by transient transfection , 2 . 5×104 cells/cm2 were plated and transfected the next day with 0 . 8 µg/ml of pCBASce mixed with 3 . 6 µl/ml of Lipofectamine 2000 ( Invitrogen ) along with a variety of other vectors . The KU and RAD52 expression vectors were added at 0 . 8 µg/ml , the ERCC1 vector was added at 0 . 4 µg/ml , the RAD51-K133R vector was added at 0 . 1 µg/ml , and the BRC3 vector was added at 0 . 2 µg/ml . For each experiment , an equivalent amount of empty vector ( pCAGGS-BSKX ) was included in the parallel transfections . Each of these expression vectors have been previously described [18] . GFP positive cells were quantified by flow cytometric analysis ( FACS ) 3d after transfection on a Cyan ADP ( Dako ) . Amplification of PCR products from sorted GFP+ cells , associated restriction digests , and quantification of bands were performed using the primers KNDRF and KNDRR as previously described for analysis of DR-GFP [50] . To measure repair using the inducible I-SceI protein ( TST ) in combination with siRNA-mediated inhibition of CtIP , HEK293 cell lines with each of the reporters and stable expression of TST were first plated on 24 well plates at 105 cells/well . The following day , the wells were transfected with 70nM siRNA duplex mixed with 4ul/ml of Lipofectamine 2000 in Optimem ( Invitrogen ) . After 4 . 5h , transfection complexes were diluted two-fold with media without antibiotics , and 48h after the initiation of transfection , 4OHT was added at 3 µM for 24h . Three days after 4OHT was added , the percentage of GFP+ cells was analyzed by FACS as described above . Knockdown of CtIP levels using the various siRNAs was confirmed by RT-PCR from RNA samples isolated from parallel transfections at the time of 4OHT addition ( data not shown ) . Amplification product was quantified at the threshold cycle by including SYBR green in the PCR reaction and using an iQ5 cycler for real-time analysis at the end of each cycle ( BioRad ) . Products were normalized relative to a primer set directed against actin . Sequences of the siRNAs siCtIP-p ( Santa Cruz Biotechnology ) , and siCtIP-1 [25] , and primers for RT-PCR are shown in Figure S1D . Repair frequencies are the mean of at least three transfections or four 4OHT treatments , and error bars represent the standard deviation from the mean . For some experiments , repair frequencies are shown relative to samples co-transfected with I-SceI and an empty vector ( EV ) . For this calculation , the percentage of GFP+ cells from each sample was divided by the mean value of the EV samples treated in the parallel experiment . Similarly , to calculate the fold-difference in repair between siRNA-treated and control-siRNA treated cells , the percentage of GFP+ cells from each sample was divided by the mean value of control-siRNA samples from the parallel experiment . Statistical analysis was performed using the unpaired t-test . | Changes to the sequence of DNA , or mutations , can disrupt cellular growth control genes , which can lead to cancer development . Such mutations likely arise from damage to DNA that is repaired in a way that fails to restore the original sequence . One type of DNA damage is a chromosomal double-strand break . We have developed assays to measure how these breaks are repaired , and also how such repair can lead to mutations . In particular , we present an assay to measure a pathway of repair that results in deletion mutations , often with evidence of short homologous sequences at the repair junctions ( alt-NHEJ ) . We have compared the genetic requirements of this repair pathway in relation to other pathways of repair that use extensive homology . We find that factors KU and CtIP appear to affect the initial stages of repair of each of these pathways , regardless of the length of homology . However , these pathways appear to diverge at later steps , as relates to the role of the repair factors RAD52 , ERCC1 , and RAD51 . Given that mutations observed in some cancer cells are consistent with alt-NHEJ repair , these mechanistic descriptions provide models for how such mutations could arise in cancer . | [
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] | 2008 | Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair |
Capnocytophaga canimorsus is a usual member of dog's mouths flora that causes rare but dramatic human infections after dog bites . We determined the structure of C . canimorsus lipid A . The main features are that it is penta-acylated and composed of a “hybrid backbone” lacking the 4′ phosphate and having a 1 phosphoethanolamine ( P-Etn ) at 2-amino-2-deoxy-d-glucose ( GlcN ) . C . canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS . Surprisingly , C . canimorsus lipid A was 20 , 000 fold less endotoxic than the C . canimorsus lipid A-core . This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A . The binding to human myeloid differentiation factor 2 ( MD-2 ) was dramatically increased upon presence of the LPS core on the lipid A , explaining the difference in endotoxicity . Interaction of MD-2 , cluster of differentiation antigen 14 ( CD14 ) or LPS-binding protein ( LBP ) with the negative charge in the 3-deoxy-d-manno-oct-2-ulosonic acid ( Kdo ) of the core might be needed to form the MD-2 – lipid A complex in case the 4′ phosphate is not present .
Capnocytophaga canimorsus , a usual member of dog's mouths flora [1] was discovered in 1976 [2] in patients who underwent dramatic infections after having been bitten , scratched or simply licked by a dog . The most common syndrome is sepsis , sometimes accompanied by peripheral intravascular coagulation and septic shock [3] . C . canimorsus is a Gram-negative rod belonging to the family of Flavobacteriaceae in the phylum Bacteroidetes [4] , [5] . Human infections occur , worldwide , with an approximate frequency of one per million inhabitants per year [6] . C . canimorsus are able to escape complement killing and phagocytosis by human polymorphonuclear leukocytes and macrophages [7] , [8] . Whole bacteria are also poor agonists of Toll-like receptor ( TLR ) 4 , which results in a lack of release of pro-inflammatory cytokines by macrophages [9] . In addition to these “passive” features , C . canimorsus have been shown to harvest glycan moieties from glycoproteins at the surface of animal cells , including phagocytes [10] , [11] , [12] , in addition they also deglycosylate human IgG [12] . One of the most pro-inflammatory bacterial compounds is the lipopolysaccharide ( LPS , endotoxin ) [13] , consisting of three domains: lipid A , the core-oligosaccharide and the O-polysaccharide ( O-antigen ) . As a potent activator of the innate immune system , LPS can induce endotoxic shock in patients suffering from septicemia . Recognition of LPS by the host occurs via the TLR4/MD-2/CD14 receptor complex [14] , [15] , [16] , at which two proteins , CD14 and LBP , have been shown to enhance the response to LPS by transporting single LPS molecules [17] , [18] , [19] , [20] . It has been shown that the lipid A moiety of the LPS is sufficient for TLR4 binding and stimulation [21] , [22] . The interaction of lipid A and its receptor was unraveled by x-ray crystallography pioneering studies of complexes between MD-2 and the lipid A analog Eritoran [23] or lipid IVA [24] . The identification of the binding sites of lipid A to MD-2 and also to the Leucine-rich repeat ( LRR ) -domains of TLR4 [21] is a landmark achievement that enables a deeper understanding of the structure-function relationship between LPS/lipid A and its receptors . According to these data , the 1 and 4′ phosphates of the lipid A backbone , which form charge interactions with TLR4 and MD-2 , are the key elements for receptor activation [21] , [25] , even though for some of the interactions conflicting data have been reported [26] . It was further shown that the β-hydroxymyristate chain at position 2 forms hydrogen bonds and hydrophobic interactions with TLR4 . At present , there is no evidence that the LPS-core plays any major role in binding to TLR4; only a 10- to 100-fold difference in endotoxicity of lipid A and LPS has been reported for E . coli , Porphyromonas gingivalis or Proteus mirabilis [27] , [28] , but these small differences have been attributed to changes in solubility , even if solid experimental proof is lacking . The core-oligosaccharide has so far never been shown to alter TLR4/MD-2 binding of a specific lipid A , only slight changes in MD-2 binding have been reported [29] . In this work , we investigated the lipid A structure of C . canimorsus in order to clarify its contribution to the septicemia and shock provoked by these bacteria . Very few lipid A structures have actually been solved in the Cytophaga/Flavobacterium group , with the exception of the lipid A from Elizabethkingia meningoseptica ( former Flavobacterium meningosepticum ) [30] . Already some time ago , the acyl chains present in the LPS of Cytophaga bacteria have been identified as [13-Me-14:0 ( i15:0 ) , 13-Me-14:0 ( 3-OH ) ( i15:0 ( 3-OH ) , 16:0 ( 3-OH ) and 15-Me-16:0 ( 3-OH ) ( i17:0 ( 3-OH ) ] [31] , whereat i15:0 is iso-pentadecanoic acid ( 13-methyltetradecanoic acid , 13Me-14:0 ) , i15:0 ( 3-OH ) represents iso- ( R ) -3-hydroxypentadecanoic acid [ ( R ) -3-hydroxy-13-methyltetradecanoic acid , 13Me-14:0 ( 3-OH ) ]; 16:0 ( 3-OH ) is ( R ) -3-hydroxyhexadecanoic acid and i17:0 ( 3-OH ) represents iso- ( R ) -3-hydroxyheptanoic acid [ ( R ) -3-hydroxy-15-methylhexanoic acid , 15-Me-16:0 ( 3-OH ) ] . Here we show that lipid A of C . canimorsus consists of the penta-acylated hybrid backbone 2 , 3-diamino-2 , 3-dideoxy-d-glucose ( β-d-GlcN3N′ ) linked ( 1′→6 ) to α-d-GlcN where the 4′ phosphate group is missing and the 1 phosphate is linked to an ethanolamine group , forming a P-Etn . Not unexpectedly , this lipid A was of very low endotoxicity but , surprisingly , when bound to the core [lipid A-core ( LA-core ) ] it became 20 , 000 fold more endotoxic . In agreement with this observation , we show that the LPS core promotes the binding of C . canimorsus lipid A to MD-2 . This is the first example of a core-oligosaccharide dramatically changing the endotoxicity of lipid A , in which the carboxy group of Kdo probably takes over the function of ionic binding of the missing 4′ phosphate in the lipid A .
GlcN and GlcN3N were found in a ratio of approx . 2∶1 ( Table 1 ) . Based on the notion that by gas-liquid chromatography ( GLC ) analysis synthetic GlcN3N expressed a response factor of about 50% when compared with GlcN ( or Galactosamine ( GalN ) as internal standard ) , it was inferred that GlcN and GlcN3N are present in equimolar amounts in the lipid A backbone , suggesting the presence of a “hybrid backbone” in C . canimorsus lipid A ( Table 1 ) . Total fatty acid analysis revealed i15:0 , i15:0 ( 3-OH ) , 16:0 ( 3-OH ) , and i17:0 ( 3-OH ) in a molar ratio of approximately 1∶1∶1∶2 in lipid A preparations . Analysis of ester-bound acyl chains indicated the presence of i15:0 and i15:0 ( 3-OH ) in approximately equimolar amounts , indicating that one 16:0 ( 3-OH ) and two i17:0 ( 3-OH ) residues are primary acyl chains N-linked to the lipid A backbone ( Table 1 ) . This result suggests a penta-acylated lipid A species . The reversed phase HPLC profile of the lipid A sample is shown in Fig . S1 . Peak 2 expressed a molecular ion at m/z 1716 . 30 , which is in excellent agreement with a lipid A containing i15:0 , i15:0 ( 3-OH ) , 16:0 ( 3-OH ) , and two moles of i17:0 ( 3-OH ) attached to the lipid A backbone ( GlcN3N-GlcN ) , which also carries one P-Etn residue . The second major fraction ( peak 5 ) at m/z 1594 . 29 was compatible with lipid A lacking the P-Etn . Based on peak intensities ( peaks 2 and 5 ) about 40% of the P-Etn was liberated , most likely from the lipid A under the hydrolysis conditions used ( Fig . S1 ) . All lipid A fractions investigated expressed a certain heterogeneity with respect the chain length of acyl chains ( -CH2- groups ) , as all MS showed peak “clusters” differing by 14 u , thus suggesting acyl chain heterogeneity ( Table 2 , Fig . S2 ) . Combined GLC/mass spectrometry ( GLC-MS ) analysis of the acyl chains revealed that the mass difference of Δm/z = 14 u was not due to the exchange of one single , prominent shorter acyl chain [e . g . 16:0 ( 3-OH ) →i15:0 ( 3-OH ) ] . Instead , the lipid A showed a certain structural “fuzziness” with respect to the size and position of the individual acyl chains , which , according to this finding , appeared to be statistically distributed over all positions with no specific structural variation . The ESI-MS data of the wt strain shown in Table 2 indicated identical mass at m/z 1716 . 30 for peaks 2 and 3 . As these lipid A fractions differed in their retention time , we conclude that they represent different structural isomers as they could be baseline-separated by HPLC . This HPLC analysis in combination with ESI-MS data thus shows that structural heterogeneity might not be solely related to the chain length of one acyl chain , but also to its position within the lipid A backbone . In order to allocate the type of the hybrid lipid A backbone , the acyl chain distribution over the lipid A backbone , and the attachment side of the P-Etn , electrospray-ionization Fourier transform ion-cyclotron resonance ( ESI FT-ICR ) MS/MS in the positive mode was run [32] . The triethylammonium salt of HPLC purified lipid A at m/z 1820 . 40 was selected as precursor ion ( Fig . S3 ) . Infrared multiphoton dissociation ( IRMPD ) -MS/MS generated one abundant characteristic B-fragment oxonium-ion of the non-reducing end at m/z 907 . 77 , which is in excellent agreement with the mass value calculated for GlcN3N with i15:0 , 16:0 ( 3-OH ) , and i17:0 ( 3-OH ) attached ( m/z 907 . 77 ) . This fragmentation pattern also showed that P-Etn is attached at the reducing end - most likely at position C-1 . Thus the lipid A in C . canimorsus is penta-acylated with an acylation pattern of three being attached to the “non-reducing” GlcN3N′ and two to the reducing GlcN sugar ( 3+2 ) in the lipid A hybrid backbone . The lipid A was studied further by high-field NMR spectroscopy using correlation spectroscopy ( COSY ) , total correlation spectroscopy ( TOCSY ) , rotating-frame nuclear Overhauser effect spectroscopy ( ROESY ) , 1H , 13C-heteronuclear single-quantum coherence ( HSQC ) , 1H , 31P-heteronuclear multiple-quantum coherence ( HMQC ) , and 1H , 31P-HMQC-TOCSY experiments . The results are depicted in the supplement ( Table S1 ) . The 1H , 13C-HSQC spectrum ( Fig . 1 ) showed two H-1 , C-1 cross-peaks at δ 4 . 28/103 . 4 and 5 . 29/92 . 8 for GlcN3N′ and GlcN , which were distinguished by correlations between protons at nitrogen-bearing carbons and the corresponding carbons ( C-2′ and C-3′ of GlcN3N′ and C-2 of GlcN , at δ 52 . 9 , 54 . 6 , and 51 . 4 , respectively ) . 3J1 , 2 coupling constants of 8 . 0 and 2 . 9 Hz for the H-1 signals at δ 4 . 28 and 5 . 29 , were determined from the 1H NMR spectrum and showed that GlcN3N is β- and GlcN α-linked . The H-1 signal of α-GlcN was additionally split due to coupling to phosphorus ( 2J1 , P 7 . 9 Hz ) , thus indicating that α-GlcN is phosphorylated with P-Etn and β-GlcN3N′ represents the “non-reducing” end of the lipid A backbone . The β1′→6-linkage between the two amino sugars was evident from strong cross-peaks of H-1′ of GlcN3N′ with protons H-6a′ , 6b′ of GlcN at δ 3 . 64 and 3 . 87 in the ROESY spectrum . The location of the P-Etn residue at position 1 of α-GlcN was further confirmed by 1H , 31P-HMQC and 1H , 31P-HMQC-TOCSY ( Fig . S4 ) as well as ROESY experiments , which showed correlations between H-1 of GlcN at δ 5 . 29 and H-1a , 1b of ethanolamine ( Etn ) at δ 3 . 91 and 3 . 98 . In accordance with the 1′→6 linkage and the position of GlcN3N at the “non-reducing end” , the 13C NMR spectrum ( Table S1 ) displayed a typical down-field displacement by ∼10 ppm for C-6 of the 6-substituted GlcN ( δ 71 . 0; compared with δ 60 . 0 for C-6 of GlcN3N′ , which is non-substituted in the free lipid A ) . The acylation pattern was confirmed by 1H , 13C-HSQC spectroscopy ( Fig . 1 ) , which showed only one characteristic downfield shift due to a deshielding effect for the i17:0[3-O ( i15:0 ) ] R2′ i . e . the H-3/C-3 R2′ cross-peak at δ 4 . 95/70 . 7 . This finding indicated that only the OH-group of i17-0 ( 3-OH ) is acylated giving rise to an acyloxyacyl residue [i17:0-3-O ( i15:0 ) ] showing a 3+2 type acyl chain distribution in the penta-acylated lipid A , which is in good agreement with the MS data ( Figs . S2 and S3 ) . Taking together the data of the chemical studies defines the structure of the lipid A of C . canimorsus shown in Fig . 2 A . The structure of E . coli hexa-acylated lipid A is depicted for comparison ( Fig . 2 B ) . The E . coli lipid A consists of a β- ( 1′→6 ) -linked GlcN disaccharide that is phosphorylated at positions 1 and 4′ and carries four ( R ) -3-hydroxymyristate chains ( at positions 2′ , 3′ , 2 and 3 ) . The R2′ and R3′ 3-hydroxylated acyl groups in GlcN′ are further esterified with laurate and myristate , respectively [22] . The structure of C . canimorsus LA-core is depicted in Fig . 2 C and its structural analysis will be described elsewhere ( Zähringer et al . , manuscript in preparation ) . The C . canimorsus LPS core features only one Kdo , to which a P-Etn is attached in position 4 . Usually , mono-Kdo LPS-core have a phosphate attached to the Kdo at that position . Thus , the only net negative charge in this core oligosaccharide originates from the carboxy-group of the Kdo . The inner core continues with two mannoses ( Man ) to which another P-Etn is attached in position 6 of ManI residue in the core oligosaccharide . The outer core consists of Galactose ( Gal ) and l-Rhamnose ( to which the O-antigen is attached ) . A positively charged Galactosamine ( GalN ) residue is linked to the ( second ) ManII residue in position 6 ( U . Zähringer , unpublished results ) . E . coli lipid A biosynthesis has been unravelled in detail [22] , [33] . Analyzing the genome of C . canimorsus 5 [5] , we identified the genes required for the synthesis of lipid A-Kdo [33] . Only lpxA , lpxA′ , lpxC and lpxD seem to cluster in one operon , the other genes are dispersed ( Fig . 3 A ) . The difference in acylation of the 3′ and 3 position and the hybrid backbone of the lipid A consisting of a β-1′ , 6-linked GlcN3N′-GlcN disaccharide , suggests that two lpxA genes might be present in C . canimorsus and indeed two lpxA genes were identified ( termed lpxA and lpxA′ ) in the C . canimorsus 5 genome ( Fig . 3 A ) . In Acidithiobacillus ferrooxidans GnnA and GnnB are responsible for the biosynthesis of GlcN3N [34] . Based on the sequences of A . ferrooxidans , gnnA and gnnB could be identified in the genome of C . canimorsus ( Fig . 3 A ) . In the biosynthetic pathway of E . coli lipid A , enzyme LpxM adds the acyloxyacyl-residue [14:0-3-O ( 14:0 ) ] representing the 6th acyl chain [22] . In good agreement with the penta-acylation of lipid A in C . canimorsus 5 was our finding that lpxM could not be identified in the genome ( Fig . 3 A ) . C . canimorsus LPS core features only one Kdo , suggesting a mono-functional Kdo transferase ( WaaA/KdtA ) or a Kdo hydrolase two-protein complex ( KdoH1/2 ) as in Helicobacter pylori or Francisella novicida [35] , [36] . Searches with KdoH1/2 did not hit any gene in the C . canimorsus 5 genome . Therefore , C . canimorsus possesses either a mono-functional WaaA or a KdoH1/2 complex without significant sequence similarity to known Kdo hydrolases . We have further investigated the enzymes leading to the addition of an Etn at the 1 phosphate of lipid A . In H . pylori , the addition of a P-Etn at 1 position has been proposed to result from a two-step mechanism [37] . In a first step the 1 phosphate is removed by a phosphatase ( LpxE ) , and subsequently a P-Etn-transferase ( EptA or PmrC , YjdB ) adds a P-Etn to the 1 position of lipid A [37] ( Fig . 3 B ) . In H . pylori lpxE and eptA are encoded by one operon ( Hp0021-Hp0022 ) . C . canimorsus eptA was annotated as Ccan 16950 . Search for a lipid A phosphatase were based on lpxE and/or lpxF sequences from P . gingivalis [38] , F . novicida [39] , Rhizobium etli [40] H . pylori [37] , [41] and on all available Bacteroidetes-group pgpB sequences . Three lpxE/F candidates have been found in the C . canimorsus 5 genome ( Ccan16960 , Ccan14540 and Ccan6070 ) . All candidates were deleted and the mutated bacteria were tested for endotoxicity . Only deletion of Ccan16960 affected endotoxicity ( data not shown ) . Interestingly , Ccan16960 is located within the same operon as eptA and the two genes overlap by 20 bp . Following the operon organisation of H . pylori , Ccan16960 has been annotated as lpxE . The predicted function of lpxE and eptA was validated by KO and analysis of the resulting phenotype ( Ittig et al . , manuscript in preparation ) . The presence of the 4′ kinase LpxK and the absence of a 4′ phosphate leads to the assumption of the presence of a 4′ phosphatase , LpxF . Several candidate genes were identified ( besides lpxE: Ccan 14540 and Ccan6070 ) and deleted but they had to be ruled out , as no deletion did affect the endotoxic activity ( data not shown ) , thus , we lack annotation of lpxF . The proposed complete biosynthesis of C . canimorsus lipid A-Kdo is depicted in Fig . 3 C , starting from UDP-N-acetyl-d-glucosamine and ribulose-5 phosphate . The endotoxic activity of wt C . canimorsus 5 LPS ( S-form ) was compared to the endotoxic activity of E . coli O111 LPS using three different approaches: ( i ) Purified LPS samples were assayed for TLR4 dependent NFκB activation with HEK293 cells overexpressing human TLR4/MD-2/CD14 and a secreted reporter protein ( HEKBlue human TLR4 cell line ) , ( ii ) purified LPS samples were assayed for induction of TNFα release by human THP-1 macrophages , ( iii ) purified LPS samples were tested for stimulation of IL-6 release by canine DH82 macrophages . In the two assays involving human TLR4 ( Fig . 4 A and Fig . 4 C ) C . canimorsus LPS appeared to be about 100 fold less endotoxic than E . coli O111 LPS ( both S-form LPS ) . In contrast to human macrophages , where C . canimorsus LPS was found 10–100 fold less endotoxic than E . coli O111 LPS ( Fig . 4 B ) , for canine macrophages the difference in endotoxicity of the two LPS was around 1000 fold ( Fig . 4 E ) . In addition , lipid IVA seems not to be an agonist of canine TLR4 as is the case for murine TLR4 [42] . Generally , the lipid A part of a LPS is considered as sufficient to trigger full TLR4 activation . Minor differences between lipid A and LPS or LA-core have so far been attributed to differential bioavailability/solubility in water even if solid experimental proof is lacking . We have , therefore , examined the endotoxic activity of C . canimorsus lipid A , LA-core and LPS using the HEKBlue hTLR4 cell line and the TNFα release by human THP-1 macrophages . LPS and LA-core exhibited an endotoxicity in the same range , whereas the LPS was less than 10-fold more endotoxic than the LA-core ( Fig . 4 B and Fig . 4 D ) . In contrast , C . canimorsus lipid A appeared to be absolutely non-stimulatory up to 5 µg/ml ( Fig . 4 B and Fig . 4 D ) , around 20 , 000-fold less active than the LA-core and 200 , 000-fold less active than LPS on a weight basis ( ng/ml ) indicating a even higher difference on a molar basis . As the C . canimorsus LPS and the LA-core showed similar endotoxicity , the increase in endotoxicity in comparison to the lipid A must have been raised by the contribution of the core oligosaccharide . Minor differences in endotoxicity between LPS and LA-core as the 10- to 100-fold difference observed between E . coli lipid A and E . coli O111 LPS ( Fig . 4 B and Fig . 4 D ) might be explained by differential bioavailability/solubility in water/buffer and by a direct contribution of the core-oligosaccharide in TLR4/MD-2 binding as suggested [21] . However , in the case of C . canimorsus LA-core the direct contribution of the core-oligosaccharide might be far more pronounced as in E . coli , since C . canimorsus has a lipid A lacking a net negative charge . A role of the core-oligosaccharide in providing solubility to lipid A was ruled out by the fact that no increase in endotoxicity was observed by the addition of triethylamine ( TEN ) or dimethyl sulfoxide ( DMSO ) to the C . canimorsus lipid A stock solution followed by sonication ( see Fig . 4 F ) . The increase in endotoxicity of the C . canimorsus LA-core in comparison to the lipid A must have been raised by the contribution of the core oligosaccharide ( Fig . 4 ) . The 4′ phosphate of E . coli lipid A is known to interact with Arg264 and Lys362 of TLR4 and Lys58 and Ser118 of MD-2 [21] . C . canimorsus lipid A lacks the 4′ phosphate and features only one net negative charge in the LPS core , namely the carboxylic oxygen of Kdo . Based on the known structure of E . coli LPS bound to TLR4/MD-2 ( 3FXI , [21] ) we measured the interaction distances from the carboxylic oxygen of Kdo to Arg264 and Lys362 of TLR4 and to Lys58 and Ser118 of MD-2 . The carboxylic oxygen of Kdo is within close distance to Arg264 and Lys362 of TLR4 and Lys58 and Ser118 of MD-2 and hence could contribute to binding to MD-2 or TLR4 . To assess the ability of C . canimorsus lipid A or LA-core to interact with human MD-2 , we monitored their ability to compete with the binding of E . coli LPS-Biotin to MD-2 . Culture supernatants of cells producing human MD-2 were incubated with biotinylated E . coli O111 LPS , either alone or in combination with different concentrations of a competitor . As a source of LBP and soluble CD14 , 7 . 5% FCS ( v/v ) was added . After purification of LPS based on biotin , co-purification of MD-2 was monitored by Western blotting . C . canimorsus LA-core abolished the copurification of MD-2 with the E . coli LPS-Biotin at higher concentration than the positive controls , E . coli O111 LPS and lipid IVA but at lower concentration than unbiotinylated E . coli penta-acyl lipid A ( Fig . 5 A and B ) . Lipid IVA is expected to be a very potent competitor , as it has been shown to bind deeper into the MD-2 pocket and thus likely stronger to MD-2 than E . coli lipid A [21] , [24] . These results indicate that C . canimorsus LA-core binds to human MD-2 , likely in the same pocket as E . coli LPS . This experiment does not reflect the antagonistic capacity of C . canimorsus LA-core as even native E . coli O111 LPS could prevent the co-purification of human MD-2 ( Fig . 5 A and B ) . In contrast to the LA-core , C . canimorsus lipid A did not significantly affect the copurification of MD-2 with E . coli LPS-Biotin even at high concentration ( Fig . 5 A and B ) . Thus , C . canimorsus lipid A seems not to bind to human MD-2 at all or to bind to MD-2 only very weakly , in contrast to the LA-core . To rule out a major contribution of the core-oligosaccharide by providing solubility to the lipid A , the same MD-2 binding experiment has been performed with C . canimorsus lipid A pre-treated with DMSO or TEN and sonicated to improve solubility . These C . canimorsus lipid A samples did not significantly affect the copurification of MD-2 with E . coli LPS-Biotin even at high concentration ( Fig . 5 C ) . We conclude from this experiment that the C . canimorsus LPS core promotes the interaction and binding of the lipid A to MD-2 either via direct interaction with MD-2 or via binding to LBP or CD14 . In order to assess the contribution of the C . canimorsus LPS core in binding of the lipid A to MD-2 , we modelled the binding of C . canimorsus lipid A to human MD-2 ( Fig . 6 A ) and compared it to the binding of E . coli lipid A . Some differences between the two complexes could be observed at the level of the lipid chains after just few ns of simulation ( Fig . 6 A ) . In both cases the R3′ and R3 chains ( see Fig . 2 for nomenclature ) were fully stretched and interacted with the same residues . No empty space was left by R3″ ( missing in C . canimorsus ) because the longer R2′ and R2″ chains filled the void . While in E . coli the R2 chain is stretched toward the inner side of the pocket , in C . canimorsus it was projected toward the pocket exterior , due to both i ) its longer size and ii ) to the presence of the bifurcated terminus of the close R2″ . The R2 chain of C . canimorsus lipid A was thus not completely buried inside the MD-2 pocket and it was even more exposed to the surface than the hydroxymyristate chain at position 2 in E . coli . This probably enables the i17:0 ( 3-OH ) chain at position 2 to interact with TLR4 , as has been reported for the R2 chain of hexa-acylated E . coli LPS [21] . It should be mentioned here that penta-acylated E . coli lipid A is endotoxically almost inactive [13] , and the acyl chains might be completely buried inside MD-2 . Thus C . canimorsus penta-acylated lipid A is expected to behave differently from penta-acylated E . coli lipid A due to the extended length of the acyl chains and the bulky iso-groups . Overall the arrangement of the sugar moieties with respect to the MD-2 was similar for both complexes , the only major discrepancies being the orientation of the 1-phosphoryl group ( 1 phosphate in E . coli , 1 P-Etn in C . canimorsus ) . The calculated binding energy for the two complexes was very similar when calculated at both MM-GBSA ( molecular mechanics , the generalized Born model and solvent accessibility ) and MM-PBSA ( molecular mechanics , Poisson-Boltzmann solvent accessible surface area ) level , being in both cases the MD-2 – E . coli lipid A complex slightly more stable ( Fig . 6 C ) . To understand this trend the total binding free energy was fractionated into a list of interaction energies between each residue of MD-2 and each fragment of lipid A ( Fig . 6 B ) , as coded in Fig . 2 . Each pairwise binding free energy value has been further fractioned into its electrostatic , steric ( Van der Waals ) , and solvation ( polar and cavitation ) components . For each term contributions arising from backbone and sidechain have been singled out . In both cases the GlcN′ ( E . coli ) or the GlcN3N′ ( C . canimorsus ) moieties ( 2′ NH group ) interacted with the backbone carbonyl of Ser120 establishing a strong ( about 4–5 kcal/mol ) and persistent interaction . Favorable interactions were also observed between GlcN and residues Phe121 and Lys122 . The side chain of Phe121 established a strong apolar interaction ( Van der Waals , non-polar solvation ) with the extended R3 acyl chain in both complexes . The hydrogen bond between the NH group of Ser120 and the carbonyl of the R3′ chain was found to be strong and persistent in both cases . Neither the 1 phosphate group ( E . coli ) nor the 1 P-Etn ( C . canimorsus ) established favorable interactions with MD-2 , whereas the 4′ phosphate group ( missing in C . canimorsus ) could be accounted for the slightly greater stability of the MD-2 E . coli lipid A complex , due to the strong ( about 7 . 5 kcal/mol ) interaction established with both the backbone and the sidechain of Ser118 ( see Fig . 6 B ) . In summary , we found that in the final complex the arrangement of the sugar moieties with respect to the MD-2 and the calculated binding energy for the two complexes was very similar for E . coli lipid A and C . canimorsus lipid A . C . canimorsus LPS , lipid A or LA-core were further tested for a possible antagonistic activity on the action of E . coli O111 LPS using HEKBlue human TLR4 cells . The cells were preincubated for 3 h with various concentrations of purified C . canimorsus lipid A , LA-core or LPS samples , then stimulated with 5 ng/ml E . coli O111 LPS for further 20–24 h and the TLR4 dependent NFκB activation was measured . C . canimorsus LPS , LA-core and lipid A appeared to be no antagonist of E . coli O111 LPS binding to human TLR4 , in contrast to the tetra-acylated antagonist lipid IVA ( Fig . 7 A and B ) . In a second assay , human THP-1 macrophages were preincubated for 3 h with purified C . canimorsus lipid A , LA-core or LPS samples at the concentration indicated . Then the THP-1 cells were stimulated with 1 ng/ml E . coli O111 LPS for further 20 h and TNFα release was measured . C . canimorsus lipid A exhibited no antagonism to E . coli O111 LPS binding to human TLR4 ( Fig . 7 D ) . Again lipid IVA showed the expected antagonism ( Fig . 7 C and D ) . Dependent on the assay no antagonism or a very weak antagonism of C . canimorsus LPS was observed . This is in agreement with the notion of a partial agonist [43] , which includes a certain degree of antagonism at sub-agonist concentration . All tested lipid A and LA-core fractions exhibited no activity towards human TLR2 , as tested by HEK293 cells overexpressing human TLR2/MD-2 and a secreted reporter ( Fig . S5 ) . This proves that the stimulation of HEKBlue human TLR4 cells with C . canimorsus lipid A-core observed is only due to activation of TLR4 .
We showed here that C . canimorsus has a penta-acylated lipid A , a feature often correlated to low endotoxicity [13] , [25] . In addition , the ester-bound 4′ phosphate is lacking . This structural feature is known to reduce the endotoxic activity by a factor of ∼100 [13] , which can now be better explained based on the recent data obtained with x-ray crystallography on the TLR4/MD-2/LPS complex [21] . In this complex , phosphate groups of lipid A play a crucial role . The 4′ phosphate is thought to bind to positively charged amino acids in the LRR of TLR4 ( Arg264 , Lys362 ) as well as to MD-2 ( Ser118 and Lys58 ) in a well-defined manner . This ionic interaction seems to be critical for the ligand affinity of lipid A , enabling formation of a hexameric ( TLR4/MD-2/LPS ) 2 complex necessary for signalling [21] . In the endotoxic lipid A , there is another negatively charged group , 1 phosphate , which binds to positively charged amino acids in the complex , especially in the LRR of both TLR4 and the counter TLR4 , called TLR4* ( Lys388* of TLR4* , Lys341 , Lys362 of TLR4 ) and also to Arg122 of MD-2 . In contrast to the 4′ phosphate which binds to two proteins ( TLR4 and MD-2 ) , the 1 phosphate is involved in binding to three proteins in the complex ( TLR4 , TLR4* , and MD-2 ) , suggesting that this group might be even more important for the formation of a stable hexameric ( LPS/TLR4/MD-2 ) 2 complex , as has been reported [44] . We showed in this work that the lipid A of C . canimorsus contains a P-Etn group at position 1 , thus neutralizing the negative charge of the 1 phosphate group . Therefore , we propose that such modified phosphorylation may exert a “shielding effect” on the negative charge of the phosphate and , hence , can explain why the lipid A of C . canimorsus is significantly reduced in its endotoxic activity . The lipid A structure of C . canimorsus is similar to that of the closely genetically related E . meningoseptica with respect to the nature and position of the acyl chains [30] . As reported for E . meningoseptica , we also found some heterogeneity with respect to the nature of the amino sugar at the non-reducing end in the lipid A backbone , but it was significantly lower ( 2–5% in C . canimorsus compared to ∼30% in E . meningoseptica ) [30] . It has to be pointed out that this structural modification has no influence on the biological activity of lipid A , as it was shown for Campylobacter jejuni [45] . The Etn substitution at position 1 of C . canimorsus lipid A is however not present in E . meningoseptica [30] . One might thus expect that the lipid A of C . canimorsus is less endotoxic than that of E . meningoseptica . To confirm this suggestion a comparative study of lipid A of both species must be carried out . Since the genus Capnocytophaga belongs to the Bacteroidetes phylum [46] , it is also not surprising that the structure of lipid A from C . canimorsus shares some important traits involved in specific TLR4 and MD-2 binding with the structure of Bacteroides fragilis lipid A , which we determined earlier [47] . In particular , the lipid A from both bacteria are ( 3+2 ) penta-acylated , lack the 4′ phosphate and share iso-branched and linear acyl chains , including i15:0 , 16:0 ( 3-OH ) , and i17:0 ( 3-OH ) . In agreement with its structural specifics , C . canimorsus lipid A was shown here to exhibit a very low activity towards human TLR4 . C . canimorsus LPS and LA-core are 100- respectively 1000- fold less active than E . coli O111 LPS towards human TLR4 , which reminds the activity of the closely related lipid A of E . meningoseptica [30] . The data obtained with human TLR4 may seem to contradict previous findings that whole heat killed C . canimorsus bacteria do not stimulate human TLR4 [9] . However , in that early study , only one concentration of bacterial lysate was used and compared to purified E . coli LPS . From the results presented here , we know that below a certain concentration , pure C . canimorsus LPS is weakly active and the threshold concentration for endotoxicity is higher than that of E . coli LPS . Thus , the C . canimorsus extracts used in previous experiments may have contained LPS in insufficient concentrations . In contrast to what was shown in Capnocytophaga ochracea [48] , C . canimorsus LPS and lipid A were found not to antagonize the action of E . coli LPS on human TLR4 . The endotoxicity of the C . canimorsus LPS is probably reduced to the level , which is tolerable in the dog's mouth . We found C . canimorsus LPS was even slightly less active towards canine than human TLR4 in comparison to E . coli LPS . This reduced inflammatory potential might benefit colonization of the dog's mouth . This reduced endotoxicity may probably as well explain why the disease in humans often begins with mild symptoms [2] , [6] , [49] and finally progresses to severe septicemia with shock and intravascular coagulation . The higher threshold concentration for endotoxicity of C . canimorsus LPS is in line with an initial immune evasion . Nevertheless , at high concentrations it reaches an activation comparable to the highly active E . coli LPS , which might contribute substanitally to the septic shock observed in patients suffering from C . canimorsus infections . Features of the LPS could therefore account for initial evasion of C . canimorsus from the host immune system , while the same LPS might later on induce the endotoxic shock when present at higher concentration . E . coli lipid A and O111 LPS exhibit a 10- to 100-fold difference in endotoxicity and similar findings were made for P . gingivalis or Proteus mirabilis [27] , [28] . The lipid A from E . meningoseptica also shows only minor differences in TLR4 activation to its LPS [30] . In contrast , we found that C . canimorsus lipid A was around 20 , 000 fold less endotoxic than the LA-core , even higher when compared on a molar basis , suggesting an important role of the core-oligosaccharide in TLR4/MD-2 binding and activation . This indicates the importance of the LPS core for TLR4 activation in the case of C . canimorsus , which has a lipid A devoid of a net negative charge . The C . canimorsus LPS core exhibits only one unshielded negative charge , on the carboxylic oxygen of Kdo . The negative charged carboxyl-group of Kdo in the C . canimorsus core could therefore directly participate in TLR4 or MD-2 binding , besides the reported inner core interactions with TLR4/MD-2 [21] . We found that the MD-2 binding ability of C . canimorsus lipid A is strongly reduced compared to the LA-core and we could exclude that changes in solubility were the reason for the differences observed . This finding could explain the difference in endotoxicity , as a lipid A not properly bound to MD-2 cannot activate TLR4 . It seems as if the C . canimorsus LPS core interacts with CD14 , LBP or MD-2 and thus enables the binding to MD-2 . By molecular modeling C . canimorsus lipid A was predicted to bind MD-2 in a very similar way as E . coli lipid A and the calculated binding energy for the two complexes was similar . As the energetic state of the final complex would therefore be stable and favorable in the case of C . canimorsus lipid A , we propose that the interactions of the LPS core with MD-2 ( or LBP/CD14 ) preceed the final lipid A – MD-2 binding , rather than only stabilizing it . In our model , summarized in Fig . 8 , we suggest an intermediate state in which the lipid A in the case of E . coli or the core in the case of C . canimorsus form ionic interactions or hydrogen bonds with MD-2 allowing the lipid A – MD-2 complex to form at all . However , we could not rule out a direct role of the LPS-core in binding to CD14 or LBP . To our knowledge , this is the first reported example of the core-oligosaccharide changing dramatically the endotoxicity of lipid A .
13:0 ( 3-OH ) was purchased from Larodan , Malmö , Sveden and 2 , 3 diamino-2 , 3-dideoxy-d-glucose ( 2× HCl ) from United States Biochemical Corporation , Cleveland , OH , USA . All other chemicals , solvents and reagents were of highest purity commercially available . E . coli O111 LPS was purchased from Sigma-Aldrich , lipidIVA from PeptaNova . E . coli F515 lipid A ( hexa- and penta-acyl ) was purified as described [50] , [51] . The analysis and isolation of C . canimorsus LA-core will be described elsewhere ( Zähringer et al . , manuscript in preparation ) . Purchased reagents were resolved according to manufacturer's instructions . Aliquots of lipid IVA were kept at −80°C . C . canimorsus bacteria were harvested from 600 blood plates in phosphate buffered saline ( PBS ) and washed with distilled water , ethanol ( 300 ml ) and acetone ( 300 ml ) , followed each time by centrifugation at 18 , 000× g for 30 min . Bacteria were air dried and resuspended in PBS containing 1% phenol for killing and storage in the deep freezer prior to LPS extraction . Cells were washed with ethanol , acetone and diethyl ether ( each 1 L ) under stirring ( 1 h , room temperature ) . After centrifugation cells were dried on air to give 11 . 2 g . For the isolation of LPS , C . canimorsus 5 bacteria were extracted by phenol-water [52] . The LPS was identified in the water phase , which also contained a large amount of an unknown glucan polymer separated by repeated ultracentrifugation ( 100 , 000× g , 4 h , 4°C , 3 times ) . The glucan was further analyzed ( U . Zähringer and S . Ittig , manuscript in preparation ) and the LPS identified in the sediment . The crude LPS preparation was further subjected to RNAse/DNAse treatment ( 30 mg , Sigma ) for 24 h at room temperature followed by Proteinase K digestion ( 30 mg , 16 h , room temp . ) and dialysis ( 2 days , 4°C ) , and lyophilization . The yield of enzyme-treated LPS related to bacterial dry mass was 70 mg ( 0 . 6% ) . Lipid A was prepared from C . canimorsus 5 ( 25 mg ) LPS by hydrolysis with 2% AcOH ( 4 ml ) at 100°C until precipitation of lipid A ( 2–8 h ) . The sediment was extracted three times with a water-chloroform mixture ( 10 ml ) and the organic phase was concentrated to dryness under a stream of nitrogen to give 17 . 7 mg of crude lipid A . The lipid A was purified by reversed phase HPLC as described elsewhere [53] with the following modifications: an Abimed-Gilson HPLC system equipped with a Kromasil C18 column ( 5 µm , 100 Å , 10×250 mm , MZ-Analysentechnik ) was used . Crude lipid A samples ( 2–5 mg ) were suspended in 0 . 4 mL solvent A and the mixture was sonicated . A 0 . 1 M EDTA-sodium salt solution ( 100 µl , pH 7 . 0 ) was added forming a bi-phasic mixture which was vortexed and injected directly onto the column . Samples were eluted using a gradient that consisted of methanol-chloroform-water ( 57∶12∶31 , v/v/v ) with 10 mM NaOAc as mobile phase A and chloroform-methanol ( 70 . 2∶29 . 8 , v/v ) with 50 mM NaOAc as mobile phase B . The initial solvent consisted of 2% B which was maintained for 20 min after injection , followed by a linear three step gradient raising from 2 to 17% B ( 20–50 min ) , 17 to 27% B ( 50–85 min ) , and 27 to 100% B ( 85–165 min ) . The solvent was held at 100% B for 12 min and re-equilibrated 10 min with 2% B and hold for additional 20 min before the next injection . The flow rate for preparative runs was 2 ml/min ( ∼80 bar ) using a splitter ( ∼1∶35 ) between the evaporative light-scattering detector ( ELSD ) and fraction collector . The smaller part of the eluate was split to a Sedex model 75C ELSD ( S . E . D . E . R . E . , France ) equipped with a low-flow nebulizer . The major part was collected by a fraction collector in 1 min intervals ( ∼2 ml each ) . Nitrogen ( purity 99 . 996% ) was used as gas to nebulize the post column flow stream at 3 . 5 bar into the detector at 50°C setting the photomultiplier gain to 9 . The detector signal was transferred to the Gilson HPLC Chemstation ( Trilution LC , version 2 . 1 , Gilson ) for detection and integration of the ELSD signal . Sugar and fatty acid derivatives were analysed by gas-liquid chromatography ( GLC ) on a Hewlett-Packard HP 5890 Series chromatograph equipped with a 30-m fused-silica SPB-5 column ( Supelco ) using a temperature gradient 150°C ( 3 min ) →320°C at 5°/min . GLC-MS was performed on a 5975 inert XL Mass Selective Detector ( Agilent Technologies ) equipped with a 30-m HP-5MS column ( Hewlett-Packard ) under the same chromatographic conditions as in GLC . Analyses of lipid A were performed in negative and positive ion modes on a high resolution Fourier transform ion cyclotron resonance mass spectrometer , FT ICR-MS ( Apex Qe , Bruker Daltonics , Billerica , MA , USA ) , equipped with a 7 T superconducting magnet and an Apollo dual electrospray-ionization ( ESI ) /Matrix-assisted laser desorption ionization ( MALDI ) ion source . Data were recorded in broadband mode with 512 K data sampling rate . The mass scale was calibrated externally by using compounds of known structure . For the negative ion mode samples ( ca . 10 ng/µl ) were dissolved in a 50∶50∶0 . 001 ( v/v/v ) mixture of 2-propanol/water/triethylamine ( pH∼8 . 5 ) . For the positive ion mode samples , a 50∶50∶0 . 03 ( v/v/v ) mixture of 2-propanol/water/30 mM ammonium acetate adjusted with acetic acid to pH 4 . 5 was used . The samples were sprayed at a flow rate of 2 µL/min . The capillary entrance voltage was set to 3 . 8 kV and the drying gas temperature to 150°C . The mass numbers given refer to that of the monoisotopic ion peak . For MS/MS in the positive ion-mode small amounts of TEN were added to the sample preparation to obtain the [M+TEN+H]+ adduct ions [32] which were selected for collision induced decay ( CID ) in the collision cell infrared multiphoton dissociation ( IRMPD ) within the ion cycIotron resonance ( ICR ) cell . Lipid A samples ( 1–3 mg ) were exchanged twice with deuterated solvents [chloroform-d1/methanol-d4 1∶1 ( v/v ) , Deutero GmbH , Kastellaun , Germany] and evaporated to dryness under a stream of nitrogen . Samples were dissolved in 180 µl chloroform-d1/methanol-d4/D2O 40∶10∶1 ( v/v/v , 99 . 96% ) and analyzed in 3 mm NMR tubes ( Deutero ) . 1H- , 13C- , and 31P-NMR spectra were recorded at 700 . 7 MHz ( 1H ) on an Avance III spectrometer equipped with a QXI-cryoprobe ( Bruker , Germany ) at 300 K . Determination of NH-proton signals was performed in chloroform-d1 ( 99 . 96% ) /methanol/H2O 40∶10∶1 without exchange in deuterated solvents . Chemical shifts were referenced to internal chloroform ( δH 7 . 260 , δC 77 . 0 ) . 31P NMR spectra were referenced to external aq . 85% H3PO4 ( δP 0 . 0 ) . Bruker software Topspin 3 . 0 was used to acquire and process the NMR data . A mixing time of 100 ms and 200 ms was used in TOCSY and ROESY experiments , respectively . Quantification of GlcN , GalN ( internal standard ) and GlcN3N by GLC and GLC-MS was done after strong acid hydrolysis of 0 . 5 mg lipid A in 4 M HCl ( 16 h , 100°C ) , followed by acetylation ( N-acetylation ) in pyridine/acetic acid anhydride ( 10 min , 85°C ) , reduction ( NaBH4 ) and per-O-acetylation . The response factor of the per-O-acetylated GlcNAc-ol , GalNAc-ol , and GlcNAc3NAc-ol derivatives , necessary for the quantification of GlcN3N by GLC , was determined in addition by external calibration with synthetic reference sugars . Etn , GlcN , GlcN3N and their corresponding phosphates ( GlcN-P and Etn-P ) , were determined from the hydrolysate by reversed phase HPLC using the Pico-tag method and pre-column derivatization with phenylisothiocyanate according to the supplier's instructions ( Waters , USA ) . Quantification of total phosphate was carried out by the ascorbic acid method [54] . For analysis of ester- and amide-linked acyl chains , the lipid A was isolated from LPS ( 1 mg ) by mild acid hydrolysis ( 0 . 5 mL , 1% AcOH , 100°C , 2 h ) , centrifuged and the lipid A sediment was separated into two aliquots and lyophilized . Ester-linked acyl chains were liberated from the first aliquot by treatment with 0 . 05 M NaOMe in water-free methanol ( 0 . 5 mL ) at 37°C for 1 h . The mixture was dried under a stream of nitrogen and acidified ( M HCl ) prior to extraction with chloroform . The free acyl chains were converted into methyl esters by treatment with diazomethane and hydroxylated acyl chains were trimethylsilylated with N , O-bis ( trimethylsilyl ) trifluoroacetamide for 4 h at 65°C [55] . The acyl chain derivatives were quantified by GLC-MS using the corresponding derivatives of 17:0 ( 50 µg ) and 13:0 ( 3-OH ) ( 50 µg , Larodan , Malmö , Sweden ) as internal standards for the calibration of the response factor of non-hydroxylated and hydroxylated acyl chains , respectively . For analysis of total acyl chains , the second aliquot was subjected to a combined acid/alkaline hydrolysis as described [56] . Briefly , acyl chains were liberated from the lipid A by strong acid hydrolysis ( 4 M HCl , 100°C , 21 h ) and extracted three times with water/chloroform ( 0 . 5 mL each ) . The organic phase containing the N- and O-linked acyl chains was treated with diazomethane , trimethylsilylated and quantified as described above . The strains used in this study are listed in Table S2 . E . coli strains were grown in LB broth at 37°C . C . canimorsus 5 [9] was routinely grown on Heart Infusion Agar ( HIA; Difco ) supplemented with 5% sheep blood ( Oxoid ) for 2 days at 37°C in presence of 5% CO2 . Bacteria were harvested by scraping colonies off the agar surface , washed and resuspended in PBS . Selective agents were added at the following concentrations: erythromycin , 10 mg/ml; cefoxitin , 10 mg/ml; gentamicin , 20 mg/ml; ampicillin , 100 mg/ml . HEK293 stably expressing human TLR4 , MD-2 , CD14 and a secreted NFκB dependent reporter were purchased from InvivoGen ( HEKBlue hTLR4 ) . Growth conditions and endotoxicity assay were as recommended by InvivoGen . Briefly , desired amount of LPS or lipid A were placed in a total volume of 20 µl ( diluted in PBS ) an added a flat-bottom 96-well plate ( BD Falcon ) . 25000 HEKBlue hTLR4 cells in 180 µl were then added and the plate was incubated for 20–24 h at 37°C and 5% CO2 . If the antagonistic activity of a compound on the action of E . coli O111 LPS was assayed , the compound was added in a total volume of 10 µl ( diluted in PBS ) , 25000 HEKBlue hTLR4 cells in 180 µl were added and the plate was incubated for 3 h at 37°C and 5% CO2 . Then the cells were stimulated with 5 ng/ml E . coli O111 LPS and the plate was incubated as above . Detection followed the QUANTI-Blue protocol ( InvivoGen ) . 20 µl of challenged cells were incubated with 180 µl detection reagent ( QUANTI-Blue , InvivoGen ) . Plates were incubated at 37°C and 5% CO2 and colour developed was measured at 655 nm using a spectrophotometer ( BioRad ) . If needed the C . canimorsus lipid A stock solution ( 1 mg/ml ) was supplemented with 0 . 1% v/v TEN or 50% v/v DMSO and sonicated for some minutes just before the assay . The TEN containing lipid A stock solution was further diluted in a 0 . 1% TEN solution to keep the TEN concentration constant in all samples . Due to the high concentration of DMSO used , this lipid A stock solution was further diluted with PBS . As a control the same amount of TEN or DMSO has been added to E . coli O111 LPS samples tested in the same assay . DMSO concentration in 50 µg/ml and 5 µg/ml were found to affect physiological test conditions . These data have therefore been excluded from the figure . Human THP-1 monocytes ( ATCC TIB-202 ) were cultured as recommended by the American Type Culture Collection ( RPMI 1640 medium complemented with 10% v/v heat-inactivated fetal bovine serum , 2 mM L-Glutamine ) . Monocytes were seeded at 1 . 5×105 cells/ml in 24 well-plates ( BD Falcon ) in growth medium containing 10−7 M PMA ( Sigma-Aldrich ) . For differentiation and attachment the cells were incubated for 48 h at 37°C and 5% CO2 and then washed with growth medium and fresh PMA-free medium was added . After further incubation for >1 h the cells were challenged with the indicated amount of LPS or lipid A in a total volume of 20 µl ( diluted in PBS ) . After 20 h of incubation the supernatants were harvested and immediately analyzed for TNFα by an ELISA . ELISA was performed in accordance with the manufacturers instructions ( BD OptEIA ) . If an antagonist of E . coli O111 LPS was assayed , the compound was added in a total volume of 10 µl ( diluted in PBS ) to the THP-1 cells and the plates were incubate for 3 h at 37°C and 5% CO2 . Then the cells were stimulated with 1 ng/ml E . coli O111 LPS and the plate was incubated for 20 h at 37°C and 5% CO2 . Canine DH82 macrophages ( ATCC CRL-10389 ) were cultured in DMEM supplemented with 15% v/v heat-inactivated fetal bovine serum , 2 mM L-Glutamine and non-essential amino acids ( Sigma-Aldrich ) in a humidified incubator at 37°C and 5% CO2 . Cells were seeded at 1×105 cells/ml in 24 well-plates ( BD Falcon ) . The cells were incubated for 24 h at 37°C and 5% CO2 , before being challenged with the indicated amount of LPS in a total volume of 10 µl ( diluted in PBS ) . After 14 h of incubation the supernatants were harvested and immediately analyzed for content of IL-6 by an ELISA . ELISA was performed in accordance with the manufacturer's instructions ( R&D Systems , DY1609 ) . Biotinylation of E . coli O111 LPS ( Sigma-Aldrich ) was performed as described previously [57] using biotin-LC-hydrazide ( Pierce , Rockford , IL ) . To verify that the biotinylation did not affect the functionality of the LPS , E . coli LPS-Biotin was assayed for endotoxicity with the HEKBlue human TLR4 cell line ( Data not shown ) . Biotinylation reduced the endotoxic potential at low concentrations , but only slightly at concentrations used in the MD-2 binding assay . MD-2 binding assays was performed as described [57] , [58] . HEK293 cells were transfected using Fugene6 ( Roche , 3∶2 protocol ) with a plasmid ( kind gift of K . Miyake and C . Kirschning ) encoding human MD-2 with a C-terminal Flag-His-tag ( pEFBOS-hMD2-Flag-His ) [15] . The medium was exchanged 3–8 h post transfection with fresh growth medium . The cells were incubated for 48 h and the supernatant was harvested and pooled . Fresh FCS was added to the hMD-2 supernatant ( 7 . 5% v/v ) as a source of CD14 and LBP . For each binding reaction , 4 ml of hMD-2 supernatant were combined with 250 ng , 500 ng , 1 µg , 2 µg , 5 µg or 10 µg of the competitor , incubated at room temperature and gently rocked for 30 min . If needed the C . canimorsus lipid A stock solution ( 1 mg/ml ) was supplemented with 0 . 1% v/v TEN or 50% v/v DMSO and sonicated for some minutes just before addition to the hMD-2 supernatant . 1 µg of biotinylated E . coli O111 LPS was added and the supernatant was further incubated for 3–4 h at room temperature . Biotinylated LPS–hMD-2 complexes or single biotinylated LPS were captured by addition of 120 µl ( total volume ) streptavidin-agarose beads ( IBA ) per sample . The beads were previously prepared by washing them three times with a buffer ( 100 mM Tris , 150 mM NaCl , pH 8 . 0 ) . For binding , the supernatants containing the beads were incubated overnight on a rotator at 4°C . Agarose beads were pelleted by centrifuging for 30 s at 5000× g and 4°C and washed three times with PBS containing 0 . 5% Tween 20 . The beads were finally resuspended in 60 µl SDS-loading dye ( without dithiothreitol ) and boiled for 5 min at 95°C . The protein content in the sample was analyzed by non-reducing , denaturing 4–12% Tris-glycine Polyacrylamide gels ( Invitrogen ) or 4–15% Tris-glycine Polyacrylamide gels ( BioRad ) and then transferred to polyvinylidene fluorid ( PVDF ) membrane ( ImmobilonP , Millipore ) . Membranes were probed using monoclonal anti-Flag antibody ( Sigma-Aldrich ) according to the manufacturer's instructions using ECL-Plus reagent ( GE Healthcare ) . Blast-p search tool [59] against the C . canimorsus 5 genome [5] was used . Search sequences were obtained from the National Center for Biotechnology Information . All available Bacteroidetes-group sequences were used as search if available , but standard E . coli sequences have always been included . The highest scoring subjects over all the searches have been annotated as corresponding enzymes . Difficulties in annotation were only observed for lpxE . lpxE search was based on lpxF and/or lpxE sequences from P . gingivalis [38] , F . novicida [60] , R . etli [40] , H . pylori [37] , [41] and on all available Bacteroidetes-group pgpB sequences . Three lpxE/F candidates have been found in the C . canimorsus 5 genome ( Ccan 16960 , Ccan 14540 and Ccan 6070 ) . All candidates have been deleted and only deletion of Ccan 16960 affected endotoxicity ( data not shown ) . Since this gene is encoded in an operon with the predicted eptA and since the same operon structure ( lpxE-eptA ) has been identified in H . pylori [37] Ccan16960 was annotated as lpxE . The MD-2 - E . coli LPS complex ( PDB code 3FXI ) [21] was used to construct models for the MD-2 - E . coli lipid A and for the MD-2 – C . canimorsus Lipid A . The modeling of the lipid A moieties was performed using the VMD [61] program and the leap module of the AMBER11 [62] suite of programs . To investigate the time-dependent properties of the two MD-2 – lipid A complexes , the constructed systems were subjected to molecular dynamics simulations [63] in the framework of a classical molecular mechanics [64] ( MM ) description . MM parameters from the Glycam06 [65] , [66] force field were adapted to describe the acyl chains and the sugar moieties , while the Amber99SB [67] , [68] force field was employed for the MD-2 protein . Advanced methods based on quantum chemistry were employed to obtain the missing parameters of the ester linkages and hydroxyl groups on the acyl chain C2 atoms , the branching at the bottom of the C . canimorsus acyls , the phosphate/P-Etn groups and the GlcN3N′ moiety . Bonding parameters were obtained by performing relaxed potential energy scans [69] ( bonds , angles , dihedrals ) , while charges were calculated on the optimized geometries of selected capped fragments . All the scan and geometry optimizations were conducted at the RI-MP2/def2-TZVP [70] , [71] , [72] level using the COBRAMM [73] suite of programs efficiently linking the ORCA2 . 8 [74] ( wave-function calculation ) and the GAUSSIAN09 [75] ( optimization/scan driver ) programs . Charges were calculated according to the RESP procedure at the HF/6-31G*//MP2/def2-TZVP . Both MD-2 – lipid A complexes were embedded in a 6 . 5×6 . 5×6 . 5 nm3 box of TIP3P [76] water molecules and the appropriate number of Na+ and Cl− ions were added to neutralize the systems charge . The systems were relaxed ( conjugate gradient geometry optimization ) to remove clashes before stating molecular dynamics simulations . The systems were both heated to 300 K in the NVT ( constant particle number , volume , temperature ) ensemble for 500 ps and then equilibrated in the NPT ( constant particle number , pressure , temperature ) until relevant structural parameters ( density , RMSD on the protein Cα ) were found to be stable ( 1 ns ) . Statistics were then performed on trajectories collected from 10 ns long simulations of the equilibrated systems . All molecular dynamics calculations were performed with the sander module of the AMBER11 package; bonds involving H atoms were constrained using the SHAKE algorithm [77] to allow for using a time step of 2 fs . Pressure was controlled via a simple Berendsen weak coupling approach [78] , while a Langevin thermostat ( collision frequency set to 3 ps−1 ) was used to enforce the desired temperature . Molecular dynamics trajectories were analyzed using the VMD software , the ptraj module of the AMBER11 suite and the ProDy [79] package . A set of 300 snapshots of the equilibrated trajectories was subjected to further analysis to quantify the binding energy between MD-2 and each of the two lipid A moieties . Both the MM-PBSA and MM-GBSA approaches [80] were used to calculate the MD-2 – lipid A binding energy , while a full interaction energy decomposition [81] , [82] was performed using the cheaper MM-GBSA method; the MMPBSA . MPI module of AMBER11 was used to perform the binding free energy calculations , while a locally developed software was used to process , analyze and plot the results . Quantification was performed using MultiGauge software ( Fujifilm ) . | Capnocytophaga canimorsus , a commensal bacterium in dog's mouths , causes rare but dramatic infections in humans that have been bitten by dogs . The disease often begins with mild symptoms but progresses to severe septicemia . The lipopolysaccharide ( LPS ) , composed of lipid A , core and O-antigen , is one of the most pro-inflammatory bacterial compounds . The activity of the LPS has so far been attributed to the lipid A moiety . We present here the structure of C . canimorsus lipid A , which shows several features typical for low-inflammatory lipid A . Surprisingly , this lipid A , when attached to the core-oligosaccharide was far more pro-inflammatory than lipid A alone , indicating that in this case the core-oligosaccharide is able to contribute significantly to endotoxicity . Our further work suggests that a negative charge in the LPS-core can compensate the lack of such a charge in the lipid A and that this charge is needed not for stabilization of the final complex with its receptor but in the process of forming it . Overall the properties of the lipid A-core may explain how this bacterium first escapes the innate immune system , but nevertheless can cause a shock at the septic stage . | [
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] | 2012 | The Lipopolysaccharide from Capnocytophaga canimorsus Reveals an Unexpected Role of the Core-Oligosaccharide in MD-2 Binding |
Modular organization in biological networks has been suggested as a natural mechanism by which a cell coordinates its metabolic strategies for evolving and responding to environmental perturbations . To understand how this occurs , there is a need for developing computational schemes that contribute to integration of genomic-scale information and assist investigators in formulating biological hypotheses in a quantitative and systematic fashion . In this work , we combined metabolome data and constraint-based modeling to elucidate the relationships among structural modules , functional organization , and the optimal metabolic phenotype of Rhizobium etli , a bacterium that fixes nitrogen in symbiosis with Phaseolus vulgaris . To experimentally characterize the metabolic phenotype of this microorganism , we obtained the metabolic profile of 220 metabolites at two physiological stages: under free-living conditions , and during nitrogen fixation with P . vulgaris . By integrating these data into a constraint-based model , we built a refined computational platform with the capability to survey the metabolic activity underlying nitrogen fixation in R . etli . Topological analysis of the metabolic reconstruction led us to identify modular structures with functional activities . Consistent with modular activity in metabolism , we found that most of the metabolites experimentally detected in each module simultaneously increased their relative abundances during nitrogen fixation . In this work , we explore the relationships among topology , biological function , and optimal activity in the metabolism of R . etli through an integrative analysis based on modeling and metabolome data . Our findings suggest that the metabolic activity during nitrogen fixation is supported by interacting structural modules that correlate with three functional classifications: nucleic acids , peptides , and lipids . More fundamentally , we supply evidence that such modular organization during functional nitrogen fixation is a robust property under different environmental conditions .
With the advent of bioinformatics and high-throughput technologies , plentiful sources of information stored in databases are now available to help unveil how a variety of biological entities interact among each other and to elucidate how these interacting networks support phenotypic behaviors in microorganisms . Notably , a variety of studies accomplished with these networks have contributed to elucidation of some fundamental organizing principles by which the cell presumably regulates and coordinates its vital biological functions . Among these organizing properties , structural modularity is a systemic property that has been observed in a variety of biological networks , which range from genetic transcriptional regulation to metabolic activity [1] , [2] , [3] , [4] , [5] . On an even more fundamental level , there is evidence that these modules inferred from network topology can be associated with functional modules; these latter are defined as a set of biological components ( metabolites , proteins , or genes ) that coordinately participate in accomplishing a specific biological function in the cell [6] . On the other hand , the idea that optimization principles guide metabolic activity in microorganisms has been an interesting hypothesis that , in combination with genome-scale metabolic reconstructions , has resulted in a successful framework for a systematic , quantitative , and predictive scheme in systems biology [7] . Briefly , the optimization problem in this context is reduced to identification of the metabolic flux along the network that ensures maximal production of a specific array of metabolites representing a specific phenotypic state in the microorganism . An optimal metabolic phenotype constitutes the basis of constraint-based modeling , and its application domain has been extended to a variety of organisms in the past few decades [8] , [9] . In this context , an immediate and fundamental question emerges: how do these structural modules organize and coordinate among themselves to support an optimal metabolic phenotype in microorganisms ? . Even though this enterprise is far from being solved , some remarkable advances have been reported in the field . For instance , a recent experimental and in silico study on a metabolic reconstruction for Escherichia coli supported the idea that feedback inhibition in metabolic units , called modules , constitutes a mechanism capable of inducing an optimal growth rate [10] . Equally relevant , there are studies that have pointed out that modular organization on a genomic scale may be a natural strategy for coordinating the transcriptional and metabolic activities required for ensuring that cells evolve and efficiently respond to environmental perturbations [1] , [5] . Despite these and other advances , the study of the principles governing the metabolic organization in cells is in its infancy , and additional discoveries are required for surveying how structural modules in a metabolic network link together to efficiently achieve their biological functions [10] . In this work , by using a systems biology description , we supply computational and experimental evidence that suggests that structural and functional modularities are robust properties when the cell operates under its optimal metabolic phenotype at different physiological conditions . To support our conclusions , we carried out an integrative study involving computational modeling and high-throughput sequencing technology for characterizing the metabolic activity of Rhizobium etli CFN42 during symbiotic nitrogen fixation in symbiotic association with Phaseolus vulgaris ( bean plant ) . Here , this organism is our benchmark model , a decision that was favored based on the availability of 1 ) a computational description of its genome-scale metabolic reconstruction , 2 ) an integrative description among high-throughput technologies at nitrogen fixation stages , and 3 ) the valuable physiological knowledge already available that describes the metabolic activity during nitrogen fixation by this organism in symbiotic association with P . vulgaris [11] . Hence , to elucidate the metabolic activity of R . etli during nitrogen fixation , constraint-based modeling was applied on an updated version of the metabolic reconstruction for this organism ( iOR450 ) [11] . At present , the metabolic reconstruction of R . etli is an integrated network of 402 reactions involving the participation of 450 genes and 377 metabolites . Unlike our previous studies , here we used metabolome technology to experimentally support our in silico interpretations of R . etli metabolism under two physiological conditions: when it fixes nitrogen in symbiosis with P . vulgaris , and under free-living conditions with succinate and ammonium as carbon and nitrogen sources , respectively ( see the Materials and Methods section ) . Thus , by applying capillary electrophoresis and mass spectrometry ( CE-MS ) [12] , [13] to R . etli and its products , we report the relative abundances of 220 metabolites under these physiological conditions . To improve the interpretations obtained from the constraint-based modeling , we used metabolome data to identify metabolites with meaningful biological roles in bacterial nitrogen fixation . Consequently , this information was used to guide the reconstruction of a more proper objective function ( OF ) to computationally simulate this biological process . Next , with the results of our integrative analysis carried out between the constraint-based modeling and the metabolome data for R . etli , we concluded that the metabolic activity at optimal nitrogen fixation is supported by structural modules with well-defined functional activities . Furthermore , our in silico study let us show that those modular structures tend to be robust during changes in environmental conditions . Overall , our study supplies evidence that modular organization in metabolic networks is required for promoting an optimal metabolic phenotype in microorganisms .
In addition to transcriptome and proteome approaches , metabolome technology represents a third complementary approach to characterize the phenotypic state of a microorganism , through quantitative and qualitative descriptions of its metabolite concentrations . In order to elucidate the metabolic capabilities and organizing properties of R . etli metabolism , we investigated the metabolome profiles of this bacterium in two physiological situations: during nitrogen fixation with P . vulgaris and under free-living conditions ( see the Materials and Methods section and reference [11] ) . Biological samples from each physiological condition were analyzed in triplicate using CE-MS technologies [12] , [13] , and the output was used to sense the relative abundance levels of metabolites under each physiological condition . Metabolome measurements were carried out through a facility service at Human Metabolome Technology Inc . , Tsuruoka , Japan . Briefly , the samples were prepared following an experimental protocol supplied by Human Metabolome Technology Inc . , and the ionic metabolites were separated through electric fields . Having separated the ionic metabolites by capillary electrophoresis the samples were subjected to spectrometric analysis , and their identities were selectively detected by monitoring ions over a large range of m/z values . Thus , spectrum profiles were compared in consultation with the Human Metabolome Technology database and normalized with respect to internal controls for uncovering and estimating their identities and metabolic abundances in both physiological conditions . The relative peak area observed by spectrometry and the average , standard deviation , and log-ratio obtained for each metabolite are reported in Dataset S1 in the supporting information and visually depicted in Figure 1 . Overall , high-throughput analysis led us to identify and characterize the abundances of 220 ionic metabolites ( 93 cations and 127 anions ) under both physiological conditions . A biological analysis of these results led us to arrive at the following conclusions: Undoubtedly , the above descriptions are valuable for elucidation of the metabolic landscape during bacterial nitrogen fixation in R . etli; however , in order to explore the relationships among network topology , functionality , and optimal metabolic phenotype , it is necessary to move toward an integrative , quantitative , and predictive description . To this end , metabolome data were used for improving and assessing the flux metabolic activity inferred from constraint-based modeling when applied on the genome-scale metabolic reconstruction of R . etli . Thus , from the set of 220 metabolites experimentally detected , we identified 119 of the 377 metabolites that participate in the metabolic reconstruction reported for R . etli OR450 [11] . This metabolic set covered 32 . 75% of the total number of metabolites participating in the metabolic reconstruction and constituted our experimental dataset for improving , supporting , and assessing the results that emerged from the constraint-based modeling . Constraint-based modeling is a paradigm in systems biology for exploring the metabolic phenotype in cells under specific environmental conditions and/or subject to genetic perturbations [16] , [17] , [18] . In particular , Flux Balance Analysis ( FBA ) has proven to be a useful computational tool for surveying the metabolic phenotype capacity in a microorganism and to simultaneously evaluate its coherent description with high-throughput technology [9] , [11] . Briefly , once the genome-scale metabolic reconstruction for an organism has been completed , FBA can be divided into two main steps: 1 ) the reconstruction of an OF that simulates a particular phenotypic state for the microorganism ( for instance , the growth rate or maximal nitrogen fixation , to name two ) , and 2 ) the search for a flux distribution along the entire network that maximizes the selected OF when the system is at steady-state [7] . Among the variety of applications that can be tackled with this systems-level framework , bacterial nitrogen fixation carried out by R . etli is an example of how an integrative study using computational modeling and high-throughput technology can contribute to understanding , predicting , and elucidating the metabolic activity during this biological process [11] . Based on these previous achievements , we selected this organism as our model system for surveying the relationships among structural organization , functionality , and the maximal phenotype during bacterial nitrogen fixation . Before proceeding with our in silico analysis , we took advantage of the metabolome data to refine and improve a previous FBA carried out for this organism [11] . Thus , given the metabolic reconstruction iOR450 , we improved the FBA by suggesting a more proper OF , one capable of modeling bacterial nitrogen fixation in a more accurate and realistic fashion . Reconstruction of an OF is a crucial issue in constraint-based modeling for computationally mimicking the metabolic activity of a microorganism , in this case , bacterial nitrogen fixation . For this reason , an OF is mainly defined in terms of those metabolites whose permanent production is essential for sustaining bacterial nitrogen fixation [11] , [19] . Currently , the OF for modeling bacterial nitrogen fixation has been entirely constructed in terms of a query in the scientific literature [11] , [19]; however , the metabolome data depicted in Figure 1 open the possibility for identifying new potential metabolites that were not included in previous analyses due to a lack of physiological evidence . The issue described above is extremely important because it directly influences the quality of the in silico interpretations and the coherent description based on the combination with high-throughput data . In order to proceed with this improvement , our strategy was based on the argument that metabolites detected at higher concentrations at the symbiotic nitrogen fixation stage , compared with those observed under free-living conditions , may mirror their fundamental participation in sustaining a functional phenotype during the biological process . Hence , under this assumption , we identified those metabolites whose concentrations increased during nitrogen-fixing activity by applying a statistical test on the log-ratio data , as shown in Figure 1 and Dataset S1 in the supporting information . To reduce the probability of including false-positive results , we limited the selection of metabolites to only those that obeyed the following conditions: 1 ) the log-ratio increased by at least by 2-fold during nitrogen fixation , with a p-value<0 . 01 ( see the Materials and Methods section ) ; and 2 ) once the candidate metabolite was included in the OF , there existed at least a solution for the metabolic flux distribution that maximized the OF at steady state . A graphical representation of those metabolites obeying the first criterion is shown in Figure 2 ( A ) ( blue points ) . Consequently , by applying the second criterion over this metabolite subset , we identified nine metabolites that potentially have an important role in driving and supporting bacterial nitrogen fixation . Having identified candidate metabolites , we integrated them into the OF and applied linear optimization to identify the metabolic flux distribution that maximized the metabolism of nitrogen-fixing bacteria ( see the Materials and Methods section ) . The time line for the metabolic components , integrating the previous OF in bacterial nitrogen fixation for R . etli ( ZFix ) , including the one obtained with the criteria described above , is summarized in Figure 2 ( B ) and the Materials and Methods section . As a result , the OF reported in this study shows a significant number of components that were not included in previous versions , and among them are the following: 3-phospho-d-glycerate ( 3pg ) , 2-oxoglutarate ( akg ) , l-arginine ( arg-L ) , l-aspartate ( asp-L ) , citrate ( cit ) , CMP ( cmp ) , fumarate ( fum ) , malate ( mal-L ) , and l-tryptophan ( trp-L ) [see Figure 2 ( B ) ] . To assess the consequences of this improvement and compare the results reported here with those obtained in our previous study , we performed FBA on the metabolic reconstruction for R . etli ( iOR450 ) , taking into account the new OF depicted in Figure 2 ( B ) . To proceed with this comparative analysis , our simulation was carried out under equivalent conditions as those described in reference [11] , which can be summarized as follows: In this context , FBA carried out for iOR450 with the new OF led us to conclude that the consistency coefficients for genes ( ηGenes ) and proteins ( ηEnzymes ) were 0 . 61 and 0 . 71 , respectively [see Figure 2 ( D ) ] . Given that the new OF was based on the experimental metabolome profile , we argue that our in silico analysis moves towards a more accurate and improved description of metabolic activity during bacterial nitrogen fixation . In order to evaluate the metabolic implications of these results and compare our results with previous reports carried out for bacterial nitrogen fixation by R . etli , we report in Table 1 the consistency coefficients and the degrees of coverage obtained in each case . Here , the degree of coverage is defined as the fraction of genes ( proteins ) , from the total number of genes ( proteins ) obtained from constraint-based modeling , that were detected by transcriptome or proteome technology ( see the data reported in reference [11] ) . As Table 1 shows , the values of the consistency coefficients for genes and proteins had slight variations in each case . However , as Table 1 chronologically shows , the OF has systematically improved two variables: the total number of genes ( proteins ) obtained from constraint-based modeling , and the subset of genes ( proteins ) belonging to this group detected by transcriptome or proteome technologies . Thus , the OF reconstructed in this work led us to increase the degree of coverage compared with previous cases: 72 of 101 enzymes predicted in silico were experimentally identified with high-throughput technologies . In agreement with this finding , 187 genes were experimentally identified by high-throughput technologies from the 306 genes predicted in the in silico analysis . In general terms , we conclude that while the consistency coefficients slightly varied among them , the OF constructed here induced the activity of additional reactions for the biosynthesis of new metabolites in bacterial nitrogen fixation . We consider that the latter issue is a crucial step in moving toward a more accurate description for characterizing and understanding the metabolic activity supporting bacterial nitrogen fixation and eventually uncovering their fundamental organizational principles at this biological level . The predominance of a hierarchical organization in a metabolic network seems to have advantages that can be observed on short-term and longer-term time scales , because structural modularity has been suggested as a fundamental network property by which cells evolve and orchestrate their physiological responses [3] , [4] , [20] . In order to elucidate the relation between structural modules and biological functionalities in the metabolic activity of R . etli , we focused first on identification of the structural modules in the genome-scale metabolic reconstruction for iOR450 and , consecutively , analyzed the biological roles of their metabolic components . In terms of the first issue , modular structures were identified by considering a pure topological criterion , which has been suggested as a useful method for surveying the organization in a biological network [4] , [5] . Hence , as we explained in the Materials and Methods section , we defined a metric of closeness for each pair of metabolites in the network by calculating the inverse square of the minimal path length between them . Consequently , by taking into account that the metabolites integrating a module are those whose numerical values of the metric tend to be similar , we classified those metabolites with a similar pattern of closeness through a hierarchical clustering analysis ( see the Materials and Methods section ) . As a result of this analysis , nine topological modules were identified over the entire metabolic reconstruction [see Figure 3 ( A ) and ( B ) ] . Even though the previous finding supplied information about the metabolic organization underlying the reconstruction , it was necessary to carry out a functional analysis to determine how these modules can be linked and support a specific metabolic phenotype . To this end , we proceeded to characterize the biological role of the set of metabolites that comprised each topological module defined in Figure 3 ( B ) . The functional composition of each module was determined by identifying the biological roles of their components , which in turn were defined according to the classification reported in the KEGG database [6] . By considering only those metabolites appearing in the KEGG database classification , we noted that the identified metabolites fell into three main biological groups: nucleic acids , peptides , and lipids [see Figure 3 ( C ) ] . This functional analysis suggested that most of the modules were formed by a heterogeneous composition among the three biological functionalities described above; however , we noted that two modules were characterized by a well-defined functional activity , i . e . , production of nucleic acids and peptide groups [see modules one and seven in Figure 3 ( C ) ] . Intuitively , one can expect that the metabolites integrating a functional module can be characterized by their coordinate response to physiological changes in such a way that the relative concentrations of the metabolites comprising the modules be up or down-regulated in a coherent fashion under both physiological conditions . To qualitatively assess this assumption , we first identified those metabolites that were experimentally detected by high-throughput technology in each modular structure defined in Figure 3 ( B ) . Consequently , in order to estimate the coordinate behavior of metabolites inside modules , we evaluated their relative changes under the two physiological conditions: nitrogen fixation versus free-living conditions . Notably , most of the metabolites experimentally detected suggested that components inside modules up-regulate their concentrations during bacterial nitrogen fixation compared to free-living conditions [see Figure 3 ( D ) ] . This finding supports the intuitive idea that coherent activity occurs inside the metabolic modules reported in Figure 3 ( B ) . However , converse to this global trend , we observed a few metabolites with an opposite behavior [see the black lines in Figure 3 ( D ) ] . These components are potentially central metabolites with a specific regulatory control that is perhaps required for transforming the functional background during bacterial nitrogen fixation , a hypothesis to be evaluated in future studies . A list of metabolites integrating each of the structural modules , those metabolites that were experimentally detected by high-throughput technology , the ratio obtained under the two physiological conditions , and their functional classifications according to the KEGG database are depicted in Dataset S2 in the supporting information . The metabolic profile in Figure 1 represents valuable data for surveying how metabolites are organized during bacterial nitrogen fixation on a network scale . In particular , modular organization in biological systems has been suggested as a common property in biological networks at different biological levels [4] , [5] . Here , as discussed above , we have supplied evidence that structural modules for the metabolic reconstruction of R . etli can be considered a potential organizing principle by which the bacterium orchestrates its physiological response during nitrogen fixation ( see Figure 3 ) . However , these modules were identified through a static description , in which physiological information associated with a specific environment was completely absent . In order to study how the topological modules described in Figure 3 can work together to support an optimal phenotype for R . etli , we applied FBA to simulate the metabolic flux activity during bacterial nitrogen fixation . The aims in this section are 2-fold: 1 ) identification of the metabolic flux profile that acts during bacterial nitrogen fixation and to evaluate to what degree the components of the structural modules participate in supporting maximal nitrogen fixation , and 2 ) evaluation of the extent to which the metabolic array required for reaching a maximal phenotype for bacterial nitrogen fixation is robust under perturbations of the physiological conditions . As we described above , data-driven reconstruction of the OF represents a significant contribution to simultaneously improving the predictive scope of constraint-based modeling and unveiling the structural organization for cell metabolism . Hence , we proceeded to apply FBA to the metabolic reconstruction for R . etli to obtain the flux metabolic distribution that maximizes the metabolome-driven OF described above . The solution was found by solving the linear optimization problem , which was subject to thermodynamic and enzymatic constraints , over the entire set of biochemical reactions in the metabolic reconstruction ( see the Materials and Methods section and Dataset S3 in the supporting information ) . Having applied FBA , we identified those reactions required for optimizing nitrogen fixation and , with them , reconstructed a subnetwork involving only their corresponding substrates and products ( see Materials and Methods ) . To dissect the functional participation of the metabolites integrating this subnetwork , we took into account the modular classification previously defined in Figure 3 ( B ) and Dataset S2 in the supporting information . As expected , we identified the metabolites that potentially participate in support of bacterial nitrogen fixation , their biological roles , and their distributions along the modules ( see Figure 4 ( A ) and the Materials and Methods section ) . The metabolic properties inferred from this subnetwork were such that 47 . 96% ( 47 of 98 ) of the metabolites predicted by constraint-based modeling were experimentally detected inside the bacteroids for R . etli . Even though this percentage of alignment with computational modeling is relatively low , this finding represents a significant advance towards a more realistic method for the study of the metabolic activity of bacterial nitrogen fixation . As far as our knowledge extends , this metabolome study is the first performed for Rhizobiaceas in such a way that it defines a benchmark point for future improvements . According to the functional classification described in Figure 3 ( A ) and in Dataset S2 at supporting information , our in silico analysis suggested that nitrogen fixation requires a variety of metabolites , mostly peptides and lipids , belonging to diverse structural modules . Converse to the idea that all the metabolites in a specific module participate during this biological process , we found that a metabolic heterogeneity—in terms of both the number of components and module classification—is required for optimizing bacterial nitrogen fixation in R . etli . Notably , the subnetwork depicted in Figure 4 ( A ) shows a few metabolites that participate in the synthesis of nucleic acids ( dark green dots ) . These findings are in qualitative agreement with the fact that R . etli bacteroids do not grow during symbiotic nitrogen fixation with P . vulgaris ( bean plant ) . In order to distinguish the modules obtained from pure topological criteria from modules specific for a physiological condition , here after we denote these latter as functional modules . At this stage , a question that immediately emerges is to what extent the functional modules depicted in Figure 4 ( A ) are robust under external environmental perturbations , and also whether this network array can be used as a fingerprint to characterize a functional phenotype in bacterial nitrogen fixation . For the purpose of evaluating whether the metabolic organization shown in Figure 4 ( A ) can be used as a fingerprint to define the optimal phenotype for bacterial nitrogen fixation in R . etli , we explored to what extent this topological structure is robust during changes under external environmental conditions . To this end , we evaluated the robustness of this metabolic array through the systematic reduction of uptake rates of two carbon sources: succinate and inositol . While the plant supplies succinate to the bacteroid as the main carbon source , inositol is an internal metabolite that has been detected at high concentrations inside bacteroids in nodules . As has been experimentally verified , both metabolites are important components in the support of nitrogen fixation in Rhizobiaceas [11] . To explore how the availability of inositol and succinate alter the metabolic phenotype shown in Figure 3 ( A ) , we constructed a phenotype phase plane over these carbon sources [see Figure 4 ( B ) ] . In agreement with the physiological knowledge for Rhizobiaceas , we observed that according to the uptake rate , carbon sources in bacterial metabolism were reduced , and a reduction effect on nitrogen fixation was obtained [see the black region of low nitrogen fixation in Figure 4 ( B ) ] . To evaluate the topological changes at different points in the phenotype phase plane , i . e . , with different succinate and inositol uptake rates , we selected a subset of 20 points along the metabolic phase plane . As depicted by the diagonal line in Figure 4 ( B ) , uptake rates for both carbon sources were selected such that their fluxes simultaneously decreased from 20 to 0 nmol of gDW/hr . Then , for each one of the selected uptake rate conditions , we graphically represented the metabolic subnetwork that resulted from FBA . As described in the Materials and Methods section , these subnetworks were constructed by considering only those metabolites participating in the biochemical reactions required for optimization of the phenotype under defined conditions of succinate and inositol uptake rates [see Figure 4 ( C–F ) ] . To quantify the topological variations that resulted under different external conditions , we defined an overlapping coefficient as the fraction of metabolites that overlap these networks ( see the Materials and Methods section ) . With the purpose of quantifying the differences among all pairs of subnetworks , we constructed an overlapping matrix whose calculated numerical entries indicated the overlapping coefficient for each pair of subnetworks obtained at different succinate and inositol uptake rates . The comparative analysis over the 20 points depicted in Figure 4 ( B ) is shown in Figure 5 . This study led us to conclude that the metabolic profile required for optimizing bacterial nitrogen fixation does not change in a significant way for a wide range of uptake rates for either carbon source [see Figure 5 ( A–B ) ] . Hence , our study supplies evidence that , while limited carbon sources reduce the bacterial phenotype , the metabolic organization supporting bacterial nitrogen fixation tends to be robust at different succinate and inositol uptake rates . In other words , even though there was a significant reduction for a phenotype , the topology of the metabolic network did not change in a significant fashion . In light of these results , we concluded that functional nitrogen fixation , at low or high rates , seems to be achieved through a conservative metabolic profile involving those metabolites required for support of optimal nitrogen fixation under specific environmental conditions ( see Figure 5 ) . Notably , this property opens the possibility for the use of network topology for characterizing cellular phenotypes through a specific pattern of metabolites and potentially distinguishing functional from dysfunctional states associated with a specific biological system . This latter approach is an avenue to be addressed in the future . Converse to the naive idea that a reduced phenotype in bacterial nitrogen fixation is the consequence of a broken modularity in metabolism , our study supplies evidence that a reduced efficiency of a phenotype can be mainly a consequence of a decrement in flux activity along the network , but without a significant rupture of the functional modules defined in Figure 4 ( A ) . In this contextual scheme , modular organization in metabolism seems to be a necessary condition for supporting not only the maximal but a suboptimal functional phenotype in bacterial nitrogen fixation ( see Figure 4 ) .
A description of the integration between high-throughput data and computational modeling is a central issue in systems biology that is required for moving toward a quantitative and predictive analysis of the metabolic activity in microorganisms . This enterprise has relevant implications , not only in solving practical issues in biotechnology but also in supplying schemes that contribute to unraveling the principles and mechanisms that regulate and organize living systems . In this work , we have explored the relationships between three biological concepts in metabolic networks: structural modularity , biological functionality , and optimal ( maximal ) phenotype . Here , the relationships among these concepts were studied at a systems level for metabolism in bacterial nitrogen fixation , an important biological process participating in the balance of nitrogen in the biosphere [19] . Hence , by using constraint-based modeling and measuring metabolome data under two physiological conditions , we explored the metabolic organization in R . etli bacteroids while they fix nitrogen in symbiotic association with P . vulgaris ( bean plants ) . Unlike our previous study [11] , here we present a refined version of the OF used for simulating bacterial nitrogen fixation based on our taking into account metabolome data . When constraint-based modeling was applied in this new context , we concluded that 71% of the metabolic reactions predicted in silico were justified by the proteome and microarray data previously stored in the GEO database [see Figure 2 ( D ) and the Materials and Methods section] . On the other hand , based on the metabolic reconstruction for R . etli , we identified nine structural modules in which some of the metabolites components fell in one of the following biological roles: nucleic acids , peptides or lipids ( see Dataset S2 in supporting information ) . Furthermore , by considering the metabolome profile of 220 metabolites under two physiological conditions , we supplied evidence that most of the metabolites integrating each one of these modules works in a coordinated fashion by increasing their metabolite concentrations at nitrogen fixation stages [see Figure 3 ( D ) ] . This latter finding supports the idea that metabolites inside a module tend to respond in a coordinated fashion . This systems-level description supplies evidence that the diverse metabolites forming the modules participate to support an optimal phenotype in bacterial nitrogen fixation . Even more fundamentally , we note that the network representation for those arrays of metabolites associated with an optimal metabolic performance is robust under different external conditions [see Figures 4 and 5] . In light of this study's findings , the main contributions can be summed up as follows:1 ) we have supplied evidence that a robust modular organization at the metabolic level underlies optimal bacterial nitrogen fixation for R . etli; 2 ) we have proposed how these functional modules interact together for supporting bacterial nitrogen fixation; and 3 ) given the robustness observed for these functional modules when there are physiological changes , we suggest that these can be used as fingerprints to associate the active topological structure in a network with optimal phenotypic behavior . Why the metabolic activity is so robust under environmental changes ? , maybe a common explanation can be found in other complex systems , for instance the social organization in factories , where even though the production of cars can change as a consequence of external factors—as low or high demand—the hierarchical and modular organization among employees is still maintained for covering a specific demand in an efficient way . Finally , even though a variety of avenues should be addressed in future research to enrich this study , we envision that this unified description will contribute to the design of experiments for evaluating the interrelation and the role that these modules have in supporting functional states in bacterial nitrogen fixation . This latter issue is fundamental for uncovering the principles by which the cell organizes its metabolism to support functional states in bacterial nitrogen fixation .
The bacterial strain used was R . etli CFN42 wild type . Culture media and growth conditions for R . etli and plant experiments were performed as previously described by our group in reference [11] . Metabolic flux distribution supporting nitrogen fixation in R . etli was predicted in silico by using constraint-based modeling [19] . Briefly , simulations were carried out by defining a mathematical function , called the OF , for computationally mimicking the metabolism of bacterial nitrogen fixation and identifying the flux distribution that maximizes it at a steady-state behavior . The OF , ZFix , is composed of some key compounds , which are classified in two groups: 1 ) metabolites essential for sustaining nitrogen fixation , and 2 ) metabolites that are imported or exported to the bacteroid and establish the symbiotic relationship between R . etli and the plant ( these are denoted by the index [e] ) . Thus , we write the OF as follows:where glycogen , histidine , lysine , polyhydroxybutyrate , valine , alanine , aspartate , and ammonium are denoted as glycogen , hist[c] , lys , phb[c] , val[c] , ala[e] , asp[e] , and nh4[e] , respectively . Similarly , mal , trp , arg , cit , cmp , fum , 3pg , and akg denote malate , tryptophan , arginine , citrate , CMP , fumarate , 3-phospho-d-glycerate , and 2-oxoglutarate , respectively . All these metabolites are required to support an effective symbiotic nitrogen fixation , and their spatial location in the cytoplasm is indicated by the index [c] . For the purpose of obtaining a computational profile of metabolic fluxes , we assumed that the metabolic state of the bacteroid during nitrogen fixation is one that optimizes the OF , ZFix . This problem was mathematically solved as a linear optimization algorithm subject to enzymatic and thermodynamic constraints , i . e . , where Si , j represents the stoichiometric coefficient of metabolite i participating in the jth reaction . Thermodynamic and enzymatic constraints in each metabolic reaction were characterized through the parameters αj and βj ( for a detailed numerical description of these parameters , see the lower and upper bounds in Dataset S3 in supporting information ) . For the sake of simplicity , all the coefficients ( ci ) were chosen as a unit along all the analyses . Linear optimization programming was carried out using the Tomlab optimization package from Matlab . Metabolome data supply valuable information to survey the metabolic phenotype associated with a biological process , and in turn , elucidate the components integrating the OF used in the constraint-based modeling . With the purpose of carrying out FBA in the metabolic reconstruction for R . etli , a more realistic OF for simulating bacterial nitrogen fixation was obtained by identifying those metabolites with a statistically significant change during bacteroid activity compared to levels under free-living conditions . To this end , we calculated the corresponding intensity log-ratio for each metabolite between nitrogen fixation and free-living conditions . Taking into account the three experimental replicates obtained for each physiological condition , a one-side statistical t-test was applied to determine those metabolites that significantly increased their relative quantity between the two physiological conditions ( see Figure 1 ) . Those metabolites with a p-value lower than 0 . 01 and with a ratio higher than 2 during nitrogen fixation were selected as potential candidates to be included in the OF , see region I in Figure 2 ( A ) . Finally , these sets of metabolites were separately included in the OF and were accepted if their effect on nitrogen fixation activity was not reduced to one-third of the corresponding previous OF . To assess the predictive scope of the computational model , we downloaded transcriptome and proteome data so that we could integrate a set of genes whose presence and upregulation suggested an important role for sustaining nitrogen fixation in R . etli . Earlier characterizations of nodules formed after 18 days of nodulation with root plants under both experimental conditions were carried out by our group and previously stored in public databases . The complete dataset of the transcriptome analysis is freely available at GEO ( http://www . ncbi . nlm . nih . gov/geo ) , with accession numbers GPL10081 for the R . etli platform and GSE21638 for data on free-living and symbiotic forms . In addition , to enrich the set of genes required for model assessment , proteome data obtained for R . etli bacteroids were downloaded from the ProteomeCommons . org Tranche by using the following hash:BY/eCcVjwTWN1+m+2ArvJ0QVnesGx5Ekgd4wUOASACfm/ueNl7YI3iLf4xz0lnGsepV5LkpMWOQOrZtjYExlNpQkIBcAAAAAAAABjA = = . Bacteria were grown in minimal medium and were harvested at the exponential phase . Free-living cells were collected by centrifugation and washed once with double-distilled water , and immediately 2 ml of methanol and the internal standards were added and the mixture was treated in an ultrasonic bath for 30 s . A 1 . 6-ml cell suspension was transferred to centrifuge tubes , 1 . 6 ml of CHCl3 plus 640 µl of milliQ water was added , and the mixture was vortexed and then centrifuged at 2 , 300×gat 4°C for 5 min . A 1 . 5-ml aliquot of the aqueous layer was filtered through a Millipore 5-kDa cutoff filter . Then , the sample was fully dried by using a centrifugal evaporator . The bacteroid metabolome extraction followed the same method , except by day 18 after inoculation with root plant , bacteroids were extracted in a self-Percoll gradient in accordance with methods previously described [21] . Measurements of extracted metabolites were performed by using CE coupled with electrospray ionization–time-of-flight analysis and MS with electrophoresis buffer ( solution ID H3302-1021; Human Metabolome Technologies Inc . , Tsuruoka , Japan ) . To assess agreement between in silico predictions and interpretations of high-throughput data , we defined a consistency coefficient that quantified the fraction of genes predicted to be upregulated in silico and simultaneously detected or induced by proteome or transcriptome technologies ( ηGene ) . Simultaneously , we defined a consistency coefficient that quantified the fraction of active enzymes that were predicted by constraint-based modeling and identified by high-throughput technology ( ηEnzyme ) . To proceed with this evaluation , E jkegg ( G jkegg ) was denoted as the set of enzymes ( genes ) that conformed to the j-esime metabolic pathways in the KEGG database , with j ranging from 1 to 22 . Similarly , the set of enzymes ( genes ) that integrated the i-esime metabolic pathway in the reconstruction and the set of enzymes that were detected by high-throughput data were denoted E jRec ( G jRec ) and E jHT ( G jHT ) , respectively . Finally , the set of enzymes obtained from constraint-based modeling were denoted E jiModel andG jiModel . In order to evaluate and create a proper frame of comparison between in silico predictions and high-throughput data , we defined the consistency coefficient as the fraction of enzymes ( genes ) that were actively predicted in silico and were identified by high-throughput technology as follows:This ratio ranged from 0 to 1 and constituted our central parameter to assess and quantify the agreement between the constraint-based modeling and high-throughput data . To assess the output from FBA , we selected 22 metabolic pathways and evaluated the degree of coherence between the flux activity predicted in silico with data for the proteome and transcriptome previously reported for R . etli during nitrogen fixation ( see reference [11] ) . Topological analysis was accomplished by representing the set of biochemical reactions in the metabolic reconstruction as an undirected network . In this network , nodes represent the metabolites and edges indicate their participation in a specific metabolic reaction [4] . Thus , we linked the metabolites in the reactants to all the products in the biochemical reaction , and this procedure was repeated for all the reactions included in the metabolic reconstruction , to finally obtain the results shown in Figure 3 ( B ) . In the case of the FBA , the subnetworks depicted in Figure 4 ( A ) , ( C–F ) were obtained when we applied these rules only to those metabolic reactions for which the flux obtained from FBA was different from 0 . In order to analyze those metabolites with biological roles , the following metabolites were excluded from the entire network representation: pi[e] , ala-L[e] , accoa[e] , glu-L[e] , pi[c] , fdp[c] , nh4[e] , nh3[c] , nh3[e] , h[e] , ppi[c] , o2[c] , o2[e] , nadph[c] , nadp[c] , nadh[c] , nad[c] , n2[c] , n2[e] , h2o2[c] , h2o[c] , h2o[e] , h[c] , fdred[c] , fdox[c] , fadh2[c] , co2[c] , co2[e] , nh4[c] , and fad[c] . A detailed list of the metabolic reconstruction iOR450 used in this work , including the metabolic reactions and metabolite abbreviations , is provided in Dataset S3 in supporting information . Modular composition along the entire metabolic reconstruction was obtained by a clustering analysis applied on a matrix whose entries represented the inverse squares of minimal path lengths for pairs . Specifically , the clustering analysis was performed by calculating the shortest path length between every pair of genes ( i . e . , dij is the shortest path length between gene i and gene j ) . Next , we calculated the association function , defined as 1/dij2 , for all pairs of metabolites along the metabolic reconstruction . This parameter gives a measure of the closeness among genes , amplifying the parameter for pairs of metabolites with low path lengths and minimizing pairs of metabolites located at remote distances . With the purpose of identifying topological modules along the network , these sets of parameters were used as input to perform a hierarchical clustering [3] , [4] , [5] , [22] , see Figure 2 ( A ) . A detailed description of the modules identified by this algorithm and the corresponding biological roles for some of the components are shown in Dataset S2 . The clustering analysis was performed using a hierarchical agglomerative average-linkage clustering algorithm , considering Kendall'sτ value as the similarity metric . In order to compare the similarities and differences that emerged from the functional modules obtained along the phase plane region depicted in Figure 4 , we define the following coefficient:where ηi and ηj represent the number of metabolites that appeared in only module i or j , respectively , and indicates the number of metabolites in common between modules i and j . Clearly , this coefficient ranges from 1 to 0 , depending on the complete or null analogy between the i and j functional subnetworks . | Biological networks are an inherent concept in systems biology that is useful in elucidating how biological entities—as metabolites or proteins—work together in supporting specific phenotypes in microorganisms . Notably , topological analyses carried out over these networks have shown that modular organization is a ubiquitous property at different levels of biological organization , in such a way that modular organization may serve as an organizing principle governing the metabolic activity in microorganisms . With the aim of elucidating the relationship among functional modules , network topology , and optimal metabolic activity , here we present an integrative study that combines computational modeling and metabolome data for evaluation of the metabolic activity of the soil bacterium Rhizobium etli during symbiotic nitrogen fixation with Phaseolus vulgaris . As a result , we supply experimental and computational evidence supporting the concept that the optimal metabolic activity during this biological process is guided by modular structures in the metabolic network of R . etli . Even more fundamentally , we suggest that these biochemical modules interact among each other to ensure an optimal phenotype during nitrogen fixation . Finally , through the in silico analysis on the genome scale metabolic reconstruction for R . etli , we give some examples that suggest that these modular structures supporting nitrogen fixation are robust to external physiological conditions . | [
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] | 2012 | Functional Modules, Structural Topology, and Optimal Activity in Metabolic Networks |
Novel vaccination approaches are needed to prevent leishmaniasis . Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field . The nonpathogenic to humans lizard protozoan parasite , Leishmania ( L ) tarentolae , has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models . Correspondingly , pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis . Here , we tested the efficacy of a novel combination of established protective parasite antigens expressed by L . tarentolae together with a sand fly salivary antigen as a vaccine strategy against L . major infection . The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L . tarentolae stably expressing cysteine proteinases ( type I and II , CPA/CPB ) and PpSP15 , an immunogenic salivary protein from Phlebotomus papatasi , a natural vector of L . major , were tested both in susceptible BALB/c and resistant C57BL/6 mice . Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection . In both strains of mice , the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L . tarentolae stably expressing cysteine proteinase genes . The present study is the first to use a combination of recombinant L . tarentolae with a sand fly salivary antigen ( PpSP15 ) and represents a novel promising vaccination approach against leishmaniasis .
Leishmaniasis is one of the greatest health challenges in nearly 98 countries , contributing to 2 million new clinical cases per year in tropical and subtropical regions of the globe [1] . The disease is transmitted by sandflies and is manifested in several clinical forms , mainly cutaneous leishmaniasis ( CL ) , mucocutaneous leishmaniasis ( MCL ) , and visceral leishmaniasis ( VL ) [2] . The geographical spread of the various clinical forms depends on vector availability . For instance , over 90% of CL cases occur in Afghanistan , Algeria , Brazil , Iran , Peru , Saudi Arabia and Syria; while , 95% of VL cases are found in Bangladesh , India , Nepal , Sudan , Ethiopia and Brazil [3] . High treatment costs , toxicity of drugs , and the constant emergence of parasite resistance highlight the need for a vaccine . Despite the observation that individuals with a healed primary Leishmania infection are protected against reinfection , no effective vaccine has been developed thus far . Lack of success may be due to our incomplete understanding of the control and regulation of immune responses during infection/reinfection and the mechanisms involved in the development of immune memory . In humans , acquired resistance to L . major infection is mediated primarily by cellular immunity , in particular antigen-specific type 1 T helper ( Th1 ) cells . Similarly , Th1 dependent protection is observed in mouse experimental models of L . major infection . Most efforts for antigen identification have been focused on parasite proteins . More recently , it was shown that immunization with defined sand fly salivary proteins confers protection against leishmaniasis [4] . This suggests that salivary molecules can contribute to protection as a component of an anti-Leishmania vaccine . Live attenuated vaccines are the gold standard for protection against intracellular pathogens . Importantly , there have been some recent attempts using this approach for the development of Leishmania vaccines [5] , [6] . Other approaches manipulate the Leishmania genome to engineer genetically modified parasites by introducing or eliminating particular virulence genes [7] , [8] , [9] . These approaches are powerful alternatives for the development of new generation vaccines against leishmaniasis . Nonpathogenic to humans Leishmania strains are also being assessed as promising vaccine tools [10] . Vaccination with a L . tarentolae recombinant strain expressing select immunogenic components of L . infantum , including the A2 and the cysteine proteinases A and B ( CPA/CPB ) genes as a tri-fusion conferred protection against L . infantum infection [11] . In the present study , we evaluated the efficacy of a new prime-boost vaccine combination consisting of a live recombinant nonpathogenic parasite and a vector salivary protein in eliciting a more powerful protective immunity against L . major infection . For this , we combined a recombinant L . tarentolae expressing the CPA/CPB cysteine proteinases with the immunogenic sand fly salivary molecule PpSP15 delivered as a DNA vaccine . We used different prime-boost regimens and evaluated the immunogenicity and protective effectiveness of this novel vaccine combination against L . major infection in both BALB/c and C57BL/6 mice .
All mouse experiments including maintenance , animals' handling program and blood sample collection were approved by Institutional Animal Care and Research Advisory Committee of Pasteur Institute of Iran ( Research deputy dated October 2010 ) , based on the Specific National Ethical Guidelines for Biochemical Research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education ( MOHM ) of Iran that was issued in 2005 . All solutions were prepared using MilliQ ultrapure ( Milli-QSystem , Millipore , Molsheim , France ) and non-pyrogenic water to avoid surface-active impurities . G418 , and Sodium dodecyl sulfate ( SDS ) were purchased from Sigma-Aldrich ( Sigma , Deisenhofen , Germany ) . The material for PCR , enzymatic digestion and agarose gel electrophoresis were acquired from Roche Applied Sciences ( Mannheim , Germany ) . Cell culture reagents including M199 medium , HEPES , L-glutamine , adenosine , hemin , gentamicin , DMEM and Schneider were purchased from Sigma ( Darmstadt , Germany ) and Gibco ( Gibco , Life Technologies GmbH , Karlsruhe , Germany ) , respectively . Fetal Calf Sera ( FCS ) was purchased from Gibco ( Gibco , Life Technologies GmbH , Karlsruhe , Germany ) . All cytokine kits were purchased from DuoSet R & D kits , ( Minneapolis , USA ) . A 2 . 3 kb fragment content CPA/CPB/EGFP fused genes ( with stop codons at the end of the EGFP ORF ) was digested from pCB6-CPA/CPB/EGFP using SacI and BamHI and then cloned into the corresponding sites of pEGFP-N1 vector ( Clontech , Palo Alto , CA ) to provide the vector referred to as pEGFP-CPA/CPB/EGFP . After confirmation of the tri-fused gene through PCR and sequence determination , the pLEXSY-NEO2 vector ( EGE-233 , Jena bioscience , Germany ) was used as an integrative vector to incorporate the CPA/CPB/EGFP fusion gene into the genome of the parasite . The CPA/CPB/EGFP was digested from pEGFP-CPA/CPB/EGFP using XhoI and XbaI and cloned into NheI and XhoI sites of the pLEXSY vector ( XbaI and NheI are isoschizomers and make compatible sticky ends ) . For integration , the SwaI was used to linearize the vector at the 5′ and 3′ ends . Then the L . tarentolae ( Tar II ATCC 30267 ) was grown in M199 5% inactivated fetal bovine serum ( iFBS ) to an optimal concentration . Parasite density was measured by counting the cells dissolved in Hyman's solution ( HgCl2 0 . 5 g , NaCl 1 g , Na2SO4 , 10H2O 11 . 5 g ) using a hemocytometer . The pellet was resuspended in ice-cold electroporation buffer ( 21 mM HEPES , 137 Mm NaCl , 5 mM KCl , 0 . 7 mM Na2HPO4 , 6 mM glucose; pH 7 . 5 ) to a final density of 108 parasites/ml , as recommended [12] . A total of 4 . 0×107 parasites/300 µl were mixed with 5–10 µg linearized DNA for stable transfection in a 0 . 2 cm electroporation cuvette ( BioRad , USA ) and stored on ice for 10 min . Electroporations were performed twice at 450 V , 500 µF using Bio Rad Gene Pulser Ecell device ( Bio-Rad , USA ) and the cell suspension was immediately put on ice for 10 min . Electroporated parasites were then transferred to 3 ml complete M199 media supplemented with 10% iFBS free of antibiotic and incubated at 26°C for 24 hours . Then , the live parasites were collected by centrifugation at 3000 rpm for 10 min at 4°C . Cells were subsequently transferred onto semi-solid plates of M199 medium containing 50% Noble agar ( Difco , USA ) and 50 µg/ml G418 ( Gibco , Germany ) and incubated at 26°C . The genotype of transfected parasites was confirmed by Southern blotting using the EGFPORF through incorporation of radiolabeled dCTP in a PCR reaction . In addition , genomic DNA obtained from transfected and wild type ( WT ) cells was amplified by PCR with specific primers to the upstream and downstream of the flanking region of 5′SSU . Forward primer ( F3001 ) anneals upstream of the 5′SSU on WT genome and reverse primer ( A1715 ) anneals to the backbone of the vector , downstream of 5′SSU and upstream of CPA/CPB/EGFP gene . The sequences for primer F3001 are: 5′ GAT CTG GTT GAT TCT GCC AGT AG 3′ and for primer A1715: 5′ TAT TCG TTG TCA GAT GGC GCA C 3′ . The expression of CPA/CPB in the recombinant parasites was confirmed by RT-PCR , Western blot as well as flow cytometry . The gene coding for PpSP15 ( NCBI accession number: AF335487 ) from the NH2 terminus to the stop codon was amplified from P . papatasi SP15-specific cDNA by PCR as reported previously [13] and cloned into the TOPO TA cloning vector PCRII ( Invitrogen ) . The plasmid VR1020-SP15 was purified using the Endo Free Plasmid Mega kit according to the manufacturer's instructions ( QIAGEN , Germany ) . Frozen and thawed ( F/T ) L . major and L . tarentolae CPA/CPB/EGFP antigens were prepared from stationary phase promastigotes . Parasites were washed with PBS ( 8 mM Na2HPO4 , 1 . 75 mM KH2PO4 , 0 . 25 mM KCl , 137 mM NaCl ) prior to 10 times exposition to liquid nitrogen and 37°C water bath alternately . The rCPA and rCPB were also prepared as previously reported [14] . Protein concentrations were measured with a BCA kit ( PIERCE , Chemical Co . , Rochford III ) . For preparation of salivary gland homogenate ( SGH ) , P . papatasi females , Israeli strain , were used for dissection of salivary glands 3–7 d after emergence as previously described [15] . Briefly , salivary glands were disrupted by ultra-sonication and centrifuged at 10 , 000 g for 3 min and the resultant supernatant was dried in a Speed Vac ( Thermo Scientific ) and reconstituted before use in the listed experiments . Female BALB/c ( H-2d ) and C57BL6 ( H-2b ) mice ( 6–8 weeks old , weighting 20±5 g ) were purchased from the Pasteur Institute of Iran animal breeding facilities . All animals were housed in plastic cages with free access to tap water and standard rodent pellets in an air-conditioned room under a constant 12∶12 h light-dark cycle at room temperature and 50–60% relative humidity . Six groups of BALB/c or C57BL/6 mice ( n = 20 per group ) were vaccinated in different prime/boost modalities given three weeks apart in the right hind footpad ( Table 1 ) . These included , G1: vaccination with L . tarentolae CPA/CPB/EGFP+ and boosting with L . tarentolae CPA/CPB/EGFP+; G2: vaccination with VR1020-SP15 and boosting with L . tarentolae CPA/CPB/EGFP+ followed by VR1020-SP15 the day after; G3: vaccination with L . tarentolae CPA/CPB/EGFP+ followed by VR1020-SP15 the next day and boosting with L . tarentolae CPA/CPB/EGFP+ followed by VR1020-SP15 the next day; G4: Control group , vaccination with PBS; G5: vaccination and boosting with VR1020-SP15; G6: vaccination and boosting with L . tarentolae EGFP+ . L . major EGFP+ ( MRHO/IR/75/ER ) parasites were used for the infectious challenge and were kept in a virulent state by continuous passage in BALB/c mice . The promastigotes were cultured in M199 medium supplemented with 5% iFBS and 40 mM HEPES , 0 . 1 mM adenosine , 0 . 5 µg/ml hemin , 2 mM L-glutamine and 50 µg/ml gentamicin at 26°C . For mice challenge , a total of 2×105 stationary phase promastigotes were injected subcutaneously in the left hind footpad 3 weeks after the booster immunization . For G2 , G3 , G4 and G5 , 0 . 5 pair of sand fly SGH was mixed with parasites and used for challenge . The profile of cytokine production in the groups vaccinated with L . tarentolae CPA/CPB/EGFP+ ( G1 ) and a combination of L . tarentolae CPA/CPB/EGFP+ and VR1020-SP15 ( G2 , and G3 ) and the PBS-immunized control group G4 in both BALB/c and C57BL/6 mice was measured before challenge and at 3 and 10 weeks post challenge in two independent repeats . Briefly , at each time point , 4 mice from each group were sacrificed . Their spleen was treated with a tissue grinder and red blood cells were lysed for 5 minutes using the ACK lyses buffer ( NH4Cl 0 . 15M , KHCO3 1 mM , Na2EDTA 0 . 1 mM ) . Splenic cells were then washed , put in culture at 3 . 5×106 cells/ml and exposed to recombinant antigens rCPA ( 10 µg/ml ) and rCPB ( 10 µg/ml ) , F/T lysate of L . major ( 15 µg/ml ) , L . tarentolae harboring cysteine proteinase genes of interest ( 25 µg/ml ) , and SGH ( 2pairs/ml ) . Cell culture supernatants were collected after 24 hours for IL-2 and TNF-α assays and 72 hours later for IFN-γ and IL-4 assays . Cytokine measurements were performed by Sandwich ELISA using the DuoSet R & D kits as per the manufacturer's instructions . The minimum detection limit is 2 pg/ml for mouse IFN-γ and IL-4 , 3 pg/ml for IL-2 and 5 pg/ml for TNF-α . All measurements were run in duplicates for two independent experiments . Concanavalin A ( Con A; 5 µg/ml ) was used in all experiments as a positive control . For the groups vaccinated with L . tarentolae CPA/CPB/EGFP+ ( G1 ) and a combination of L . tarentolae CPA/CPB/EGFP+ and VR1020-SP15 ( G2 , and G3 ) and the PBS-immunized control group G4 , mice were bled to obtain serum for determination of antibody responses . The serum sample obtained from each mouse was analyzed by ELISA for specific IgG1 and IgG2a isotype responses three weeks after booster immunization ( against F/T lysate of L . tarentolae CPA/CPB/EGFP+ ( 10 µg/ml ) and SGH ( 2pair/ml ) and at 5 weeks after challenge against F/T lysate of L . major ( 10 µg/ml ) and SGH ( 2pair/ml ) . Briefly , 96-well plates ( Greiner ) were coated with each antigen overnight at 4°C . Plates were blocked with 100 µl of 1% BSA in PBS at 37°C for 2 h to prevent nonspecific binding . Sera ( 1∶100 ) were added and incubated for 2 h at 37°C . After three washes , goat anti-mouse IgG1-HPR ( 1∶10 , 000 , Southern Biotech , Canada ) or goat anti-mouse IgG2a-HPR ( 1∶10 , 000 , Southern Biotech , Canada ) were added and incubated for 2 h at 37°C . After four washes , plates were incubated for 30 min at 37°C with Peroxidase Substrate System ( KPL , ABTS ) as substrate . Reactions were stopped with 1% SDS and the absorbance was measured at 405 nm . The parasite load in different groups of BALB/c and C57BL/6 mice ( G1 , G2 , G3 , G4 , G5 and G6 ) were determined by the limiting dilution assay at 3 and 10 weeks post challenge [16] . Briefly , at each time point 4 mice from each group were taken randomly , sacrificed and the lymph nodes ( LN ) were excised and weighed . After homogenizing , 20 different serial dilutions ( 10−1 to 10−20 ) were prepared in Schneider's Drosophila medium supplemented with 10% iFBS and gentamicin ( 0 . 01% ) . Diluted cells were cultured in 96 well plates in duplicate and investigated 7 and 14 days later for positive wells . The parasite load was calculated using the following formula: −Log10 ( last dilution with live parasites/weight of homogenized LN ) . To demonstrate the in vivo level of infection , the infected footpad ( FP ) was imaged 10 weeks after challenge with the KODAK Image Station 4000 Digital Imaging System . Briefly , six BALB/c mice from each group ( G1 , G2 , G3 , G4 , G5 and G6 ) were treated with a depilatory substance ( Nair ) to remove hair from their FPs to reduce background auto fluorescence . Afterward mice were temporarily anesthetized intraperitoneally with a mixture of xylazine 2% ( 7 . 5 µl ) , Ketamine 10% ( 30 µl ) and saline solution ( 260 µl ) per mouse and then imaged . Pixel counting and measurement of the lesions were performed using the KODAK molecular image software version 5 . 3 . Measurements were reported as “Net intensity” , a quantitative measurement defined as the number of green pixels in a given area multiplied by the average intensity of each pixel minus the background intensity . Statistical analysis was performed using Graph-Pad Prism 5 . 0 for Windows ( San Diego , California ) . Depending on data passing normality tests , ANOVA or Mann-Whitney U tests were computed . P values less than 0 . 05 were considered significant . The specific test employed is indicated in each figure .
Expression of the 2 . 3 kb CPA/CPB/EGFP tri-fusion genes in L . tarentolae is under the regulatory control of the rRNA Pol I promoter . We integrated the CPA/CPB/EGFP fragment flanked by 5′ ( ∼860 bp ) and 3′SSU ( ∼1080 bp ) sequences into the rRNA locus of L . tarentolae ( Figure 1A ) . The recombinant L . tarentolae strain expressing CPA/CPB/EGFP genes displayed a normal morphology ( a drop-like shape ) with a normal length of the flagellum comparable to that of the wild type strain . EGFP expression and intensity were verified by visualization using an epifluorescence microscope . The EGFP was attached to the C-terminal end of CPB and fluorescence is distributed through the whole cytoplasm ( Figure 1B ) . Confirmation of CPA/CPB/EGFP expression at the level of RNA and protein was verified using RT-PCR and western blot , respectively ( data not shown ) . A major requirement of vaccines is to protect the majority of a population that normally displays a high diversity in MHC haplotypes . It is known that L . major causes a non-healing cutaneous infection in susceptible BALB/c mice characterized by progressive skin lesions and visceralization of the parasites to the spleen [17] , [18] . In contrast , C57BL/6 mice are naturally resistant against L . major and the infection normally causes transient symptoms and is self-healing [18] . Therefore , we evaluated the immune response in both BALB/c ( H-2d ) and C57BL/6 ( H-2b ) mice in the groups vaccinated with L . tarentolae CPA/CPB/EGFP+ ( G1 ) , combination of L . tarentolae CPA/CPB/EGFP+ and VR1020-SP15 ( G2 , and G3 ) and the PBS-immunized control group G4 ( Table 1 ) . It has been shown that IFN-γ and TNF-α are important parameters for vaccine evaluation since they synergize their capacity to mediate killing of pathogens . Furthermore , IL-2 also enhances the expansion of T cells , leading to a more efficient effector responses [19] . Since these effector cytokines mediate protection , we evaluated antigen specific immune responses three weeks after booster immunization by measuring the production of IFN-γ , IL-4 , IL-2 and TNF-α in the supernatant of splenocytes in response to rCPA/rCPB or F/T lysate of L . tarentolae CPA/CPB/EGFP . In susceptible BALB/C mice , the levels of IFN-γ production by splenocytes after rCPA/CPB stimulation were significantly higher ( p<0 . 05 ) in the G2 and G3 vaccinated groups compared to the control-immunized group G4 ( Figure 2A ) . No significant difference in the levels of IFN-γ production was observed in any of the vaccinated groups when stimulation was done with L . tarentolae CPA/CPB/EGFP ( Figure 2A ) . We further investigated whether splenocytes from the three different vaccinated regimens secreted the Th2-associated cytokine IL-4 . Upon stimulation with rCPA/CPB , G3 exhibited a small but significantly higher level of IL-4 as compared to control group G4 ( Figure 2A ) . Stimulation of splenocytes with L . tarentolae CPA/CPB/EGFP resulted in significantly higher levels of IL-4 in G1 , G2 andG3 groups as compared to control group G4 ( Figure 2A ) . Furthermore , G1 , G2 and G3 produced significantly higher levels of IL-2 compared to G4 ( p<0 . 05 ) when stimulated with rCPA/CPB or L . tarentolae CPA/CPB/EGFP ( Figure 2A ) . For TNF-α , G1 , G2 and G3 showed significantly higher levels in comparison to control group G4 upon stimulation with rCPA/CPB ( Figure 2A ) but no difference was observed among these groups after L . tarentolae CPA/CPB/EGFP stimulation ( Figure 2A ) . The antibody response of vaccinated BALB/c mice against L . tarentolae CPA/CPB/EGFP for groups G1 , G2 , G3 and G4 , and against sand fly salivary gland homogenate ( SGH ) for groups G2 , G3 and G4 was determined before challenge ( Figure 2B ) . Higher levels of both IgG1 and IG2a antibodies against L . tarentolae CPA/CPB/EGFP was observed in vaccinated groups G1 , G2 and G3 compared to control group G4 ( Figure 2B , p<0 . 05 ) . In both G2 and G3 , the level of IgG2a was higher than IgG1 but the opposite was obtained for G1 ( Figure 2B , p<0 . 05 ) . When SGH was used as antigen , both the G2 and G3 groups produced significantly higher levels of IgG2a as compared to control group G4 ( p<0 . 05 ) while no significant differences were observed for IgG1 levels in all three vaccinated groups ( p>0 . 05 ) . For resistant C57BL/6 mice , splenocytes produced significantly higher levels of IFN-γ and IL-4 in the three vaccinated groups ( G1 , G2 , G3 ) as compared to control group G4 when stimulated with either rCPA/CPB or L . tarentolae CPA/CPB/EGFP ( Figure 3A , p<0 . 05 ) . Nevertheless , the level of IL-4 was lower than that of IFN-γ in all vaccinated groups ( Figure 3A ) . Groups G1 , G2 and G3 produced significantly higher levels of IL-2 compared to G4 ( p<0 . 05 ) when stimulated with rCPA/CPB or L . tarentolae CPA/CPB/EGFP ( Figure 3A ) . As for TNF-α , it was only produced upon stimulation with rCPA/CPB where G1 , G2 and G3 showed significantly higher levels in comparison to G4 ( Figure 3A ) . The three vaccinated groups produced significantly higher levels of IgG2a against L . tarentolae CPA/CPB/EGFP compared to control G4 ( Figure 3B ) . The level of IgG1 was only significantly higher in G3 as compared to control G4 ( p<0 . 05 ) . Furthermore , there were no significant differences between IgG1 and IgG2a levels against SGH in the three tested groups G2 , G3 and G4 ( Figure 3B ) . All six groups of vaccinated and control BALB/c mice ( Table 1 ) were challenged with 2×105 late-stationary phase L . major GFP+ promastigotes in their left footpads in the presence ( G2 , G3 , G4 , G5 ) or absence ( G1 , G6 ) of SGH . Weekly measurements showed a sharp increase in footpad swelling in the control groups G4 and G6 at weeks 8 , 9 and 10 that was significantly larger than that observed in groups G1 , G2 , G3 and G5 ( Figure 4A p<0 . 05 ) . As a main parameter , the parasite burden was measured in the lymph nodes of all six groups at 3 and 10 weeks post challenge using a limiting dilution assay ( Figures 4B ) . Three weeks after challenge ( 3WAC ) , groups G1 , G2 , G3 and G5 showed a significantly lower parasite load than groups G4 and G6 ( Figure 4B ) with G2 and G5 showing the lowest lymph node parasite burden ( Figure 4B ) . At the end of week 10 , the parasite burden of groups G1 , G2 , G3 and G5 remained significantly lower ( p<0 . 05 ) compared to groups G4 and G6 ( Figure 4B ) . In addition , both G2 and G3 has significantly lower parasite load in respect to G1 and G5 ( p<0 . 05 ) . In vivo imaging of fluorescent parasites in the footpad 10 weeks after challenge ( 10WAC ) shows a significant reduction in the level of fluorescence intensity in the footpad of the vaccinated groups G1 , G2 and G3 as compared to the control group G4 ( Figures 4C–D ) . Group G3 had the lowest fluorescence intensity with two mice showing no GFP fluorescence ( Figure 4C–D ) . Moreover , the fluorescence intensity of group G3 was significantly lower in comparison to groups G1 , G4 , G5 and G6 ( p<0 . 05 ) but was not statistically significant from that of group G2 ( Figure 4D ) . For assessment of the immune response after challenge , we focused on the groups vaccinated with L . tarentolae CPA/CPB/EGFP+ ( G1 ) and a combination of L . tarentolae CPA/CPB/EGFP+ and VR1020-SP15 ( G2 , and G3 ) compared to the PBS-immunized control group G4 . Splenocytes stimulated with L . major F/T antigen at 3WAC show that groups G1 , G2 and G3 produced significantly higher levels of IFN-γ compared to control group G4 ( Figure 5A , p<0 . 05 ) . Though group G3 produced higher levels of IFN-γ compared to group G2 , it also produced significantly higher levels of IL-4 ( p<0 . 05 ) as compared to groups G1 , G2 and G4 ( Figure 5B ) . The difference in the levels of these two cytokines became less pronounced at 10WAC ( Figure 5B ) . All vaccinated groups showed a positive IFN-γ/IL-4 ratio and group G2 had the highest IFN-γ/IL-4 ratio at 3WAC indicative of a Th1 response ( Figure 5C ) . At 10WAC , group G3 had the highest ratio of IFN-γ/IL-4 ( p<0 . 05 ) in comparison to G1 , G2 , G4 . With regard to IL-2 production , only G2 produced significantly higher levels of this cytokine as compared to G1 , G3 and G4 at 3WAC and 10WAC ( Figure 5D ) . TNF-α production was similar at in all vaccinated and control groups at 3WAC but it was significantly higher in the three vaccinated groups ( G1 , G2 and G3 ) compared to control group G4 at 10WAC ( Figure 5E , p<0 . 05 ) . The specific antibody response against L . major in BALB/c mice was measured in the above-mentioned groups at 5 weeks after challenge . Groups G2 and G3 displayed the highest level of IgG2a and IgG1 antibodies to Leishmania compared to group G1 , and control group G4 ( p<0 . 05 , Figure 5F ) . The low levels of IgG1 and IgG2a antibodies to Leishmania were similar in groups G1 and G4 ( Figure 5F ) . Regarding anti-sand fly saliva antibodies , the levels of IgG2a antibodies were significantly higher in groups G2 and G3 compared to control group G4 ( Figure 5G ) . Furthermore , the ratio of saliva-specific IgG2a/IgG1 was greater in groups G2 and G3 ( Figure 5G ) . In C57BL/6 mice the increase in footpad swelling was similar between groups G1 , G4 and G6 ( Figure 6A ) . There was a significant decrease ( p<0 . 05 ) in footpad swelling in groups G2 and G3 in comparison to groups G1 , G4 , G5 and G6 ( Figure 6A ) . Measurements of parasite burden from lymph nodes at 3 and 10 weeks post-challenge showed that , group G2 had significantly the lowest parasite burden ( p<0 . 05 ) as compared to groups G1 , G3 , G4 , G5 and G6 ( Figure 6B ) . We also observed at 10WAC a significant decrease in parasite burden in G1 , G3 and G5 as compared to control group G4 as well as G6 . In addition , the level of parasite burden in G6 is also significantly lower than G4 ( p<0 . 05 ) ( Figure 6B ) . Overall , these data demonstrate that in C57BL/6 mice priming with VR1020-SP15 and boosting with a combination of the live vaccine and VR1020-PpSP15 elicited a higher protective efficacy than the two other regimens in controlling footpad swelling and parasite propagation up to 10 weeks post-challenge ( Figure 6A , B ) . Although we were able to detect the swelling in the footpad of all groups , we were unable however , to determine the fluorescent intensity by imaging in the footpad of C57BL/6 mice as it was done for BALB/c . In fact , in C57BL/6 mice resistant strain , the level of parasite propagation in the footpads of all groups was limited ( below the instrument detection limit ) . Similar to BALB/c mice , we focused on the groups vaccinated with L . tarentolae CPA/CPB/EGFP+ ( G1 ) and a combination of L . tarentolae CPA/CPB/EGFP+ and VR1020-SP15 ( G2 , and G3 ) compared to the PBS-immunized control group G4 for assessment of the immune response after challenge . Splenocytes stimulated with L . major F/T antigen at 3WAC show that groups G2 and G3 had higher levels of IFN-γ compared to control group G4 ( Figure 7A ) . At 10WAC , IFN-γ production was similar among the vaccinated groups G1 , G2 and G3 and was significantly higher than control group G4 ( Figure 7A ) . The levels of IL-4 were lower in group G2 as compared to group G3 at 3WAC ( Figure 7B , p<0 . 05 ) , however this cytokine decreased significantly 10WAC in group G3 compared to groups G1 , G2 and G4 . Similar to BALB/c mice , the ratio of IFN-γ/IL-4 was higher in groups G1 , G2 and G3 compared to the control group G4 , particularly at 10WAC ( Figure 7C ) . It is worth to mention that G3 has significantly the highest ratio in compare to G1 and G2 ( p<0 . 05 ) . As for IL-2 , its production was significantly higher ( p<0 . 05 ) in groups G2 and G3 compared to groups G1 and G4 at 3WAC , but no statistical significance was observed among the groups at 10WAC ( Figure 7D ) . Importantly , the induction of TNF-α was significantly higher in Group G2 as compared to control group G4 at 10WAC and was 2-folds higher compared to G1 and G3 ( Figure 7E ) . Antibodies against L . major in vaccinated groups G1 , G2 and G3 showed significantly higher levels of IgG2a in comparison to group G4 ( p<0 . 05 , Figure 7F ) . Furthermore , the levels of IgG1 were significantly lower than IgG2a in these three vaccinated groups ( G1 , G2 and G3 ) in comparison to G4 ( p<0 . 05 , Figure 7F ) . Overall , the antibody response to SGH was low ( Figure 7G ) . With the exception of group G2 that produced significantly higher levels of IgG2a compared to groups G3 and G4 , the antibody response to SGH was mixed ( Figure 7G ) .
Despite substantial progress in fundamental Leishmania research , there are many unanswered questions concerning pathogenesis of the disease and the acquisition of protective immunity against reinfection . In this respect , immunization with live attenuated strains as a vaccine tool to induce a protective immune response in the host has a long tradition [20] . The major drawback of this approach is that under certain circumstances , the strains may gain virulence and become pathogenic again . To overcome this problem , subunit vaccines , instead of the whole organism , emerged as a vaccination strategy [21] . A number of parasite antigens have been tested for their potential to induce anti-Leishmania responses . The most extensively studied antigens using a wide range of adjuvants and delivery systems are GP63 , LACK , CPs , and the poly-antigen Leish111f [22] , [23] , [24] . In an attempt to engage Leishmania infection at an early stage , salivary proteins of the sand fly have also been evaluated for vaccination . Studies in mice , hamsters and dogs showed promising results with the induction of Th1-like responses and long-term protection against both cutaneous and visceral infections using these salivary proteins [4] , [8] . Here , we describe the outcome of a new vaccination strategy with different modalities using a live recombinant nonpathogenic L . tarentolae vaccine expressing CPA/CPB/EGFP combined to a DNA vaccine containing the cDNA for PpSP15 , the predominant 15 kDa salivary protein from the sand fly P . papatasi . Our target parasite antigens are cysteine proteinases , which are conserved among different Leishmania species and are highly immunogenic . L . tarentolae , the lizard protozoan parasite , has been previously introduced by Breton et al . [25] as a candidate vaccine against visceral leishmaniasis . Furthermore , we have demonstrated that a recombinant L . tarentolae strain expressing the L . donovani A2 gene elicited a strong protective immunity against virulent L . infantum challenge [26] . Recently , we have shown that vaccination with L . tarentolae expressing A2/CPA/CPB induced a strong parasite-specific Th1 response and conferred protection against L . infantum challenge in BALB/c mice [11] . As for PpSP15 , it was shown previously to protect vaccinated C57BL/6 mice challenged with parasites plus SGH [15] , [27] . A major requirement of vaccines in general , is that they are able to protect the majority of a population , which normally displays a high diversity in MHC haplotypes . For this reason , we tested the efficacy of the recombinant live L . tarentolae expressing CPA/CPB/EGFP candidate vaccine combined to PpSP15 DNA in eliciting protective immune responses in two different strains of mice . While BALB/c mice develop progressive lesions upon infection with L . major , C57BL/6 mice are naturally resistant and the infection normally causes transient symptoms ( contained lesion development and visceralization ) and is self-healing . In this study , L . major IR75 was used for an infectious challenge because it is more virulent in comparison to L . major 39 and the Friedlin strain ( Modabber F , personal communication ) . Both strains of mice showed the strongest protective effect following immunization with a prime/boost regimen based on PpSP15 DNA and recombinant L . tarentolae ( groups G2 and G3 ) demonstrating an enhanced vaccine efficacy compared to the sole use of L . tarentolae CPA/CPB/EGFP ( G1 ) or PpSP15 DNA ( G5 ) . While group G3 showed a more potent immune response in susceptible BALB/c mice , group G2 showed the strongest immunogenicity in C57BL/6 mice and it was the best group in controlling parasite growth in the lymph nodes of both mice strains . In both strains of mice , immunization with PpSP15 as a DNA vaccine combined to L . tarentolae CPA/CPB/EGFP showed considerable level of protection as demonstrated by footpad thickness measurements and parasite burden . This demonstrated for the first time the effectiveness of co-immunization of a sand fly salivary protein , PpSP15 , with live L . tarentolae CPA/CPB/EGFP in controlling the disease . In the case of BALB/c mice , the effect of Live L . tarentolae CPA/CPB/EGFP is less pronounced although we observed a significantly lower parasite burden in G2 and G3 compared to G1 , G4 , G5 and G6 . Inclusion of PpSP15 DNA as a vaccine may be relevant at two levels: i ) as an inducer of adaptive immunity , thus reducing lesion pathology and parasite propagation and ii ) as an potential enhancer of innate immunity due to the intrinsic properties of this molecule that may contribute to the control of intracellular growth of L . major . Furthermore , there are extensive data showing that live L . major plus CpG DNA prevents lesion development and causes the specific induction of Th17 cells , which enhances the development of a protective cellular immunity against the parasite [28] , [29] . Data presented by Mendez et al . [30] suggest that a vaccine combining live pathogens with immunomodulatory molecules may strikingly modify the natural immune response to infection in an alternative manner to that induced by killed or subunit vaccines . Therefore , it may be possible that PpSP15 working as an immunomodulatory molecule and enhancing the development of a protective cellular immunity against the parasite . Comparing the data obtained in C57BL/6 with BALB/c , the highest level of TNF-α production , indicative of a Th1 response , was seen with group G2 at 10WAC although there were no significant differences in IFN-γ production . Of note , we only checked four key cytokines to demonstrate the immunogenicity of each vaccine modality using the live recombinant L . tarentolae . We acknowledge the need to further investigate the role or contribution of other cytokines when studying live parasite vaccines . Our future efforts should be also focused on the analysis of the immunological memory and the factors that could correlate with the size of the memory pool using these vaccine strategies . One of these aspects is the induction of CD8 T+ cell responses , which remains to be elucidated . The concept of using live vaccination against leishmaniasis is not new . Actually , the inoculation of live parasites to produce a lesion that heals , named leishmanization , has been the only vaccination strategy implemented at a large scale because it provides lifelong protection against the development of additional lesions [31] . However , this approach was discontinued because of raised non-healing or slow healing lesions in several human cases [31] , [32] . During the last few years , several attenuated strains of Leishmania have been developed . As an alternative , various defined genetically modified parasites have been achieved using a gene targeted disruption strategy through homologous recombination . One of the first examples was the in vivo evaluation showed that the dhfr-ts−/−parasites survived but were unable to establish a persistent infection or to cause disease even in the most susceptible mouse strains [33] . Other examples such as LPG2−/− parasites protected highly susceptible BALB/c mice against a L . major virulent challenge even in the absence of a strong Th1 response [34] , [35] . In contrast to L . major mutants , L . mexicana LPG2−/− mutants retained their virulence for macrophages and mice [35] , which suggested that different Leishmania species possess alternative virulence repertoires to interact with their host . Therefore , major safety constrains , such as a possible reversion to virulence or reactivation in immunosuppressed individuals , are still among the limiting factors against the use of such vaccines . In contrast to all of the above-mentioned approaches , L . tarentolae is non-pathogenic to humans and can be used in immunocompromised individuals . As such , recombinant L . tarentolae could offer more advantages for vaccine development not only against Leishmania , but also against other pathogens . A recombinant L . tarentolae expressing HIV-1 Gag protein induced strong cell-mediated immunity in mice and decreased HIV-1 replication in an ex vivo system , suggesting that this species can be applied as a promising live vaccine against intracellular pathogens [10] . Recently , a recombinant L . tarentolae strain expressing HPV-E7 antigen-green fluorescent protein ( GFP ) was developed and showed a potential as a live vaccine against HPV infection [36] . Additionally , modification of , and insertion into , the genome of L . tarentolae can be done easily and there is no insert size limitation making it a versatile tool for vaccine development . Our data clearly demonstrate that group G2 ( prime with PpSP15 DNA and boost with L . tarentolae CPA/CPB/EGFP+PpSp15 DNA ) has the lowest level of parasite propagation at 3WAC in both mice strains and at 10WAC in C57BL/6 mice . Therefore , apart from the specific immunogenicity of PpSP15 , this salivary protein may have an immunomodulatory role that in combination with a live vaccine potentially enhances its efficacy against Leishmania . In summary , the present study suggests that this new approach that combines a prime-boosting strategy using recombinant L . tarentolae with a sand fly salivary protein offers a promising platform for developing a more effective vaccine against leishmaniasis . | More than 98 countries are reported as endemic for leishmaniasis , a vector-borne disease transmitted by sand flies . Drug-resistant forms have emerged and there is an increased need to develop advanced preventive strategies . Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field . The lizard protozoan parasite , L . tarentolae , is nonpathogenic to humans and has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models . Correspondingly , pre-exposure to sand fly saliva or immunization with salivary proteins has been shown to protect mice against cutaneous leishmaniasis . Herein , we used DNA/Live and Live/Live prime-boost vaccination strategies against cutaneous leishmaniasis based on recombinant L . tarentolae stably expressing CPA/CPB genes with and without the sand fly salivary antigen PpSP15 in both resistant and susceptible mice models . Assessment of the immune response and parasite burden in vaccinated mice at different time intervals post-challenge demonstrated that combination of recombinant L . tarentolae CPA/CPB with PpSP15 DNA elicits an enhanced protective immune response against cutaneous leishmaniasis in mice . This parasite- and insect vector-derived antigen combination represents an important step forward in the development of new vaccine strategies against Leishmania infections . | [
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] | 2014 | Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen |
Treatment for clinical schistosomiasis has relied centrally on the broad spectrum anthelmintic praziquantel; however , there is limited information on its mode of action or the molecular response of the parasite . This paper presents a transcriptional and functional approach to defining the molecular responses of schistosomes to praziquantel . Differential gene expression in Schistosoma japonicum was investigated by transcriptome-wide microarray analysis of adult worms perfused from infected mice after 0 . 5 to 24 hours after oral administration of sub-lethal doses of praziquantel . Genes up-regulated initially in male parasites were associated with “Tegument/Muscle Repair” and “Lipid/Ion Regulation” functions and were followed by “Drug Resistance” and “Ion Regulation” associated genes . Prominent responses induced in female worms included up-regulation of “Ca2+ Regulation” and “Drug Resistance” genes and later by transcripts of “Detoxification” and “Pathogen Defense” mechanisms . A subset of highly over-expressed genes , with putative drug resistance/detoxification roles or Ca2+-dependant/modulatory functions , were validated by qPCR . The leading candidate among these was CamKII , a putative calcium/calmodulin-dependent protein kinase type II delta chain . RNA interference was employed to knockdown CamKII in S . japonicum to determine the role of CamKII in the response to praziquantel . After partial-knockdown , schistosomes were analysed using IC50 concentrations ( 50% worm motility ) and quantitative monitoring of parasite movement . When CamKII transcription was reduced by 50–69% in S . japonicum , the subsequent effect of an IC50 dosage of praziquantel was exacerbated , reducing motility from 47% to 27% in female worms and from 61% to 23% in males . These observations indicated that CamKII mitigates the effects of praziquantel , probably through stabilising Ca2+ fluxes within parasite muscles and tegument . Together , these studies comprehensively charted transcriptional changes upon exposure to praziquantel and , notably , identified CamKII as potentially central to the , as yet undefined , mode of action of praziquantel .
At least 200 million people are afflicted by schistosomiasis [1] , where clinical symptoms associated with the disease range from fever , headache and lethargy , to severe fibro-obstructive pathological changes , portal hypertension , ascites and hepatosplenomegaly , with complications that are frequently fatal . Meta-analysis indicates that the disease burden , in terms of morbidity and mortality is far greater than previously estimated [1] . Current public health approaches to control of schistosomiasis is underpinned by mass or targeted drug treatment with the heterocyclic pyrazino-isoquinoline compound , praziquantel ( PZQ ) . Since the 1980s , treatment for schistosomiasis has relied almost exclusively on this broad spectrum anthelmintic , which is safe , effective against all species , is administered orally , has minimal side effects and is inexpensive . Remarkably , however , there is only limited information on the mode of action of PZQ or how schistosome parasites respond to the drug . With the increasing spread of schistosomiasis and the concomitant extensive deployment of PZQ , a menacing spectre of appearance and spread of drug resistant schistosomes is a worrisome concern . Early effects of PZQ on Schistosoma mansoni worms include contraction and paralysis , which may result from membrane depolarisation and the influx of extracellular calcium [2] . These effects are compounded by uncontrolled muscle tension which results in adult worms being flushed from the mesenteric venules back to the liver , where vacuolisation and disintegration of the schistosome surface and leukocyte migration through the tegument can be readily observed [2] . Sex specific sensitivities for in vitro and in vivo PZQ exposure are seen in S . mansoni; males are more sensitive than females to PZQ [3] . PZQ also disrupts Ca2+ homeostasis in schistosomes by an unknown mechanism [4] . Greenberg and colleagues have suggested that PZQ sensitivity in schistosomes is brought about via the beta-subunit variant of the Ca2+channel ( Cavß ) , which results in a massive influx of calcium ions . Competitive binding of Ca2+ channels with cytochalasin D ( an actin disruptor ) interferes with the effects of PZQ , particularly in adult male worms via the disruption of Ca2+ homeostasis , subsequently impacting on the tegument actin cytoskeleton [5] . In addition , expression of ATP-binding cassette ( ABC ) superfamily proteins in schistosomes , including multidrug resistance-associated protein 2 ( SMDR2 ) , is altered in worms exposed to sub-lethal levels of PZQ [6] , [7] . SMDR2 is also expressed at higher levels in parasite isolates with reduced PZQ sensitivity , and this protein interacts directly with PZQ [6] , [7] . Cioli and colleagues [8] hypothesised , however , that calcium influx represents only one component of a complex mechanism which leads to the anti-schistosomal effects of PZQ . Despite these and other documented effects of PZQ , the precise identity and location of the molecular targets of PZQ remain unknown [4] . Genetic crosses of resistant and susceptible strains of S . mansoni has revealed that PZQ insensitivity is a quantitative trait , indicating that there may be more than one major physiological target of the drug [9] . Cioli and colleagues [9] speculated that drug metabolism could be the key feature of resistance , rather than the result of significant structural changes to the drug target itself . This notion is supported by earlier findings that revealed an accumulation of fluorescent substrates within the schistosome and an increase in the expression of several ABC transport proteins following exposure to PZQ [10] . Although resistance might also arise from a mutation or structural change in the drug target , resulting in decreased binding [11] , other features might be relevant . These could include drug accessibility to tegumental and other schistosome cells . Alternatively , PZQ might be cleared through an up-regulation of antioxidant enzymes . Selective advantage of rare alleles encoding these defences could give rise to multi-drug resistance , as has occurred parasitic nematodes and protozoans [12] . Changes in transcriptional levels of the drug target , rather than a direct mutation , have been suggested as a mechanism for pyrantel resistance in Ancylostoma caninum with resistant hookworms down-regulating expression of a nicotinic acetylcholine receptor [13] . Some information is available on transcription of genes associated with calcium homeostasis and putative PZQ resistance mechanisms in S . mansoni . One report described transcriptomic responses to PZQ and included in vitro culture of adult schistosomes and microarray analysis , which identified 607 up-regulated genes , 247 of which were shown to correlate with known oxidative-stress processes and calcium regulation [14] . PZQ displays a bimodal spectrum of activity , in that it is active against newly transformed schistosomules ( <3 days old ) , inactive against immature 21 day-old worms , and full activity against the sexually mature blood flukes [15] . Recently , Hines-Kay and colleagues utilised transcriptomics to address this refractory/susceptible nature of developmental stages of schistosomes in terms of PZQ activity [16] . The study profiled gene expression in adult and juvenile S . mansoni with and without in vitro PZQ exposure . The findings suggested that juveniles , which are refractory to PZQ , display enhanced transcriptomic elasticity in the percentage of differentially expressed genes which the authors hypothesise endows the immature stages of schistosomes with the means to withstand the anthelmintic effects of PZQ . Here we describe the use of a novel approach to examine the transcriptional responses of adult S . japonicum parasites exposed in vivo to a combination of PZQ and the host immune system [17] , [18] .
Microarray gene expression analyses were undertaken to investigate the sub-lethal effects of PZQ on S . japonicum in vivo . Hybridisations were performed on mRNA isolated from male or female adult parasites for each time point to allow the identification of ±≥2-fold differentially expressed genes , relative to controls ( time point 0 ) . For female worms , 264 genes were up-regulated between 30 min and 4 h , increasing to 1 , 009 genes between 12 and 24 h after drug exposure . PZQ had a broader effect on transcription in males with 1 , 508 genes up-regulated at 30 min , increasing to 2 , 718 genes at 24 h post-drug exposure . The number of differentially expressed genes are summarised in Table 1 and notable examples of differentially expressed genes are presented in Table S1 . Distinct transcriptional responses by adult S . japonicum to PZQ were sex-dependent and varied with the duration of PZQ exposure ( Figure 1 ) . A list of all the differentially expressed genes is presented in Table S2 . A comparison of similar gene expression patterns between the genders was performed . Using a 2 fold cut of differential regulation compared to time point 0 controls , this included gene expression common to both male and female parasites as grouped for 30 minute or 4 hour time points as early responses and 12 hour or 24 h points as later responses ( Table S3 ) . Up-regulated genes ( for both sexes ) including 19 early , 60 later , and 7 both early and later . By contrast , down-regulated genes common between the sexes included 380 early , 175 later , and 38 that were modulated consistently during early and later time points . Some novel genes were observed both at early and later intervals to be , consistently differentially expressed in both sexes in response to PZQ exposure . This included up-regulated Contig06312 ( Dual specificity protein kinase CLK1 ) which contains a PKc like superfamily motif , and Contig03692 ( Early growth response protein 1 ) which has zinc and nucleic acid binding functions . Down-regulated genes for both sexes and across the entire time course , were more numerous; these included Contig05338 ( Exportin-2 ) a gene related to cell proliferation , and a number of genes associated with ion transport Contig04920 ( Cation/acetate symporter ) Contig07059 ( Solute carrier family 22 member 3 ) Contig01777 ( Probable cation-transporting P-type ATPase ) Contig07303 ( Uncharacterized symporter ) and Contig03785 ( Uncharacterized sodium-dependent transporter ) . The differential expression of Contigs with KEGG annotation associated with the “Calcium Signalling Pathway” , were mapped to present an overview of how PZQ impacts on the pathway as a whole , as illustrated in Figure 2 . Generally , most genes of the pathway were up-regulated for male S . japonicum but down-regulated for female worms over the 24 h observation period , with notable exceptions , including CamKII ( Contig01285 ) which was up-regulated in both males and females ( Table S4 ) . To validate these findings of differential expression , nine genes were analysed further by quantitative ( q ) PCR . The relative fold change of gene expression obtained by microarray and by quantitative PCR was similar for the majority of data points for all nine genes ( Figure S1 ) . The microarray and quantitative PCR data sets of the nine genes indicated a significant correlation ( alpha = 0 . 05 ) between the two methods ( Spearman's Rho = 0 . 83 , P<0 . 001 , n = 90 ) , providing strong support for the integrity of the microarray findings . Based on microarray data , and then validated by qPCR , we selected five genes that showed a high level of differential expression after exposure to PZQ . The selected candidate genes ( Contig02253 Multidrug resistance protein , Contigs05840/02748 ABC transporter H family member 2 ) or Ca2+-dependant/modulatory functions ( Contig10357 Calretinin , Contig01285 Calcium/calmodulin-dependent protein kinase type II delta chain ) ( Figure S1 ) have also been shown to function in either putative drug resistance or detoxification . In general , these genes exhibited increased up-regulation in adult males of S . japonicum compared with female worms , a result correlating with our hypothesis that transcriptional responses to PZQ in schistosomes are sex-specific . Subsequently , the impact of these five genes on adult worm survivalibity following drug exposure was evaluated by RNAi , all of which resulted in knockdown , which in the case of CamKII ( Ca2+/calmodulin-dependent protein kinase II ) reached ∼60% ( female 69% , 63% , 57%; male 61% , 59% and 50% , in triplicate experiments ) when compared with irrelevant ( luciferase ) controls . The other four candidate genes either did not exhibit marked or as consistent knockdown ( not shown ) compared to CamKII . Gene silenced worms were examined in motility assays to identify phenotypical differences . The IC50 values for PZQ-treated female and male S . japonicum were calculated from motility index analysis using the xCELLigence system ( Figure 3 ) . Male and female worms were exposed to 12 . 3 , 37 , 111 , 333 , 1000 , 3000 ng/ml PZQ in CSM after which motility was monitored for up to 72 h . After 48 h , 20 ng PZQ/ml reduced movement by ∼50% in both male and female worms ( Figure 3A , B ) . To ensure the worms used for PZQ treatment were alive at 48 h after dsRNA electroporation , motility was measured by xCELLigence for 3 h before addition of the drug; the range ( % relative to un-electroporated parasites ) of motility was for females 86–89% and 85–110% for males ( Figure 4 ) . No differences were apparent ( p-value>0 . 05 ) among motility of CamKII and luciferase knockdown groups for both females and males , before addition of PZQ . Only living worms were retained for a further culture in the presence or absence of PZQ and subsequent calculation of the average motility index . Figure 5 shows the changes in the motility of adult males and females with about 60% CamKII knockdown in controls ( no PZQ , Figure 5A , B ) and with the addition of IC50 concentrations of PZQ ( 5C , D ) , over 72 h . Worms exhibited decreased motility immediately upon addition of PZQ and , in males , this was followed by spasmodic contraction during e then five hours ( Figure 5C , Figure 5D ) . Worm motility in the luciferase control groups was consistently maintained at 50–60% in the presence of PZQ , whereas CamKII knockdowns of both male and female parasites displayed further reduction in motility after incubation with IC50 PZQ ( Figure 5 ) . With the addition of IC50 PZQ over 72 h ( Figure 5C , Figure 5D ) , the motility of male worms with CamKII knockdown , relative to luciferase controls , decreased significantly from 61% to 23%; in female parasites motility was reduced from 47% to 27% . Statistical differences of paired treatments at each time point ( p-value≤0 . 05 ) for both male and female parasites , between knockdown groups , was apparent from ∼40 h post PZQ administration , and generally continued until the conclusion of the assay . These results indicated that female and male S . japonicum worms , with reduced CamKII levels , were sensitive to IC50 PZQ treatment in terms of motility , and both sexes demonstrated a CamKII-dependence in mitigating the effects of PZQ .
This study reports transcriptional and functional approaches to defining the molecular responses of schistosomes to PZQ . We demonstrate the functionally utility of the xCELLigence system to provide real time assessment of motility , a key phenotype of adult S . japonicum after PZQ treatment . The findings suggested that specific inhibitors of CamK may increase efficacy of PZQ and that a focus on prospective drug targets in the Calcium Signalling Pathway could facilitate development of improved or alternative anti-schistosomals . Exposure to anti-parasitic compounds can result in diverse outcomes in terms of the modulation of gene expression . For example , with Plasmodium falciparum , chloroquine induces relatively few transcriptional changes ( ∼100 genes ) [19] , whereas exposure to artesunate results in ∼400 regulated gene alterations [20] . A microarray-based study of Trypanosoma brucei [21] revealed that exposure to thiazolidinediones , and the resultant cellular differentiation , could be attributed to up-regulation of the expression site associated gene 8 ( ESAG8 ) . In S . mansoni , genes such as glutathione S-transferase , are up-regulated in response to xenobiotics [22] . Adult worms of S . japonicum respond immediately to exposure to PZQ . A notable feature is rapid disruption of the tegument to expose surface antigens , which has also been linked to perturbation of calcium ion homeostasis [23] . The worms contract , a feature clearly visible to the naked eye , and surface blebbing and other damage is evident by light or electron microscopy [24]–[26] . The concentration of PZQ examined in our study ( 20 µg/ml ) was ∼1000 fold less than that described by Xiao and colleagues [27] who examined tegumental damage in S . japonicum adults exposed in vitro to the much higher concentrations of 10–30 µg/ml . Xiao and colleagues [27] were able to detect the formation of surface blebs in worms using light microscopy; by contrast , this damage was not apparent in any of the worms examined here ( not shown ) and we conclude that tegumental damage by PZQ did not occur in control or CamK knockdown parasites . Our approach to understanding the mechanism of action of PZQ , follows that of others who have investigated the responses of yeast and other microbes in which immediate transcriptional changes occur , reflecting the mode of action of antimicrobial agents [28] . The combination of chemotherapeutic and host immune effects on the schistosomes [17] , [18] provides an unique insight into the complete action of PZQ . Our in vivo model involved the administration of PZQ to schistosome-infected mice followed by transcriptional analysis of the drug-exposed flukes . The gene expression profiles , which have not been reported previously , demonstrate a polarised response between male and female worms ( Table 1 and Table S2 ) . We hypothesise that schistosomes up-regulate genes to compensate for the effect of PZQ . This phenomenon mirrors that of drug-treated human cells which leads to complex responses upon the binding target [29] . It addition it is apparent that known mechanisms occur which produce a positive feedback loop that modulates the expression level of the respective target protein [29] . Key biological functions associated with ensuring schistosome worm integrity were identified in the transcriptome analysis ( Figure 1; Table S1 ) . In adult males , up-regulated genes included , but were not limited to , those associated with tegument and muscle function , lipid and ion regulation , and drug resistance . Whereas fewer genes were up-regulated in females , biological functions related to pathogen defence , general detoxification , drug resistance and Ca2+ regulation were prominent . The schistosome tegument provides structural and functional elements for nutrient uptake and physical and immunological protection , processes for which many components have been identified by other methods including proteomic analysis [30] , [31] . Tegument-associated genes up-regulated in male S . japonicum included annexins ( Contigs00538 and 04019 ) , glucose transporters ( Contig05129 ) , calpain ( Contig05997 ) , tetraspanins ( Contig00678 and 04880 ) and sodium/potassium-transporting ATPase ( Contig05103 ) . Also up-regulated were cytoskeletal components , including tubulin ( α , β and δ , Contigs 00204 , 00027 and 06580 ) , actin ( Contig05360 ) ; talin ( Contig05315 ) and supervillin ( Contig03361 ) , regulatory elements such as microtubule-actin cross-linking factor 1 ( Contig06241 ) and actin cytoskeleton-regulatory complex protein ( Contig02825 ) , and muscle components such as paramyosin ( Contig07435 ) , myosin ( Contig13962 ) and actin ( Contig05360 ) . Components of the basement membrane , another important tegument structure , including laminin ( Contig02502 ) , collagen ( Contig06926 ) and a basement membrane-specific heparin sulfate proteoglycan core protein ( Contig11129 ) , were up-regulated early in the response to PZQ by male worms , possibly reflecting tegument stress . Ultrastructural analysis of S . mansoni worms has shown that damage to the basement membrane is characteristic of schistosomes exposed to sub-optimal dosages of PZQ [32] . Endocytosis across the schistosome tegument , particularly in males , is a major route of nutrient uptake [33] . Dynamin ( Contig01607 ) and myoferlin ( Contig07932 ) are components of endocytosis within both endothelial and muscle cells of mammals [34] . Both genes were up-regulated in male S . japonicum in response to PZQ , suggesting increased tegument activity . This notion was also supported by the over expression of clathrin heavy chain 1 ( Contig04218 ) and phosphatidylinositol-binding clathrin assembly protein ( Contig07474 ) , both of which are structural components of coated pits [35] . Thus , while a sub-lethal dose of PZQ appeared to place some facets of tegumental function under stress , resulting in an increase in cytoskeletal elements , other specific processes such as active vesicle-mediated transport appeared to increase . Ion regulation , drug resistance and immunological defences were phenomena where differential gene expression was prominent for both male and female S . japonicum . The majority of the genes within the KEGG Calcium Signalling Pathway were differentially expressed , generally resulting in an up-regulation in males and down-regulation in females ( Figure 2; Table S4 ) . The effect of PZQ on Ca2+ homeostasis is well documented [3] , [5] , and is confirmed by these findings . However , we show also that the impact on related pathways appears to be sex-specific . Our transcriptional analysis of S . japonicum expands on the findings of Aragon et al . [14] who studied the gene expression of S . mansoni exposed in vitro to PZQ . In contrast , we used a mouse model to analyse in vivo exposure to PZQ in adult S . japonicum . In addition , we performed microarray analyses separately on male and female worms , facilitating description of distinct profiles for each gender . Similarities in the findings of the two studies are , however , evident including the up-regulation of calcium-associated genes ( such as Contigs 03004 , 08226 , 09553 ) in male schistosomes . Additionally , other ion-associated genes , including those for sodium ( Contigs 05931 , 0513 , 03882 ) and potassium ( Contigs 10776 , 10915 , 05103 , 02734 ) , were up-regulated in both schistosome species . The induction of extracellular superoxide dismutase precursors ( Contig04124 , 00246 ) , shown in worm pairs of S . mansoni [14] , was only apparent in female S . japonicum . In the calcium pathway , many Ca2+-mediated events occur when the released Ca2+ binds to and activates the regulatory protein calmodulin ( Contig10880 ) , which was strongly up-regulated in both female ( 93-fold increase ) and male ( 11-fold increase ) S . japonicum at 24 h post-PZQ treatment in vivo . In mammals , calmodulin is thought to activate CamKII ( Contig01285 ) by binding calcium ions [36] . From our findings calmodulin appears as an important component of calcium signaling and in response to PZQ strong up-regulation . However , we did not initially select calmodulin for RNAi since it directly interacts with multiple genes ( at least 7 genes downstream ) within the pathway , and its knockdown may have presented a much more complicated phenotype . We intend to focus on calmodulin , now that we established protocols and have characterised CamK as a basis for future work . Other differentially up-regulated genes of note included those encoding mucins ( Contigs 04112 , 08178 and 07699 ) , a family of proteins which , in schistosomes , may play a role in immune evasion and other host-parasite interactions [37] . Mucins have been shown to be expressed only in the intra-molluscan stages of S . mansoni [37] and in the egg , miracidium and sporocyst stages of S . japonicum [38] . Their up-regulation , in male and female S . japonicum after PZQ exposure is the first indication that mucins are utilised by the mammalian stages as well . The anti-microbial peptide β-defensin ( Contig07230 ) is a key host defence peptide in human neutrophils , and a component of innate immunity , and related peptides perform similar roles for other vertebrates and in invertebrates , fungi and flowering [39] . For example , it serves as anti-microbial role in Caenorhabditis elegans and Ascaris suum [40] . The current findings represent the first report of up-regulation of this gene in adult schistosomes and may reflect a defense response of the tegument to insult . The identification of these two defence responses in schistosomes emphasises the utility of the in vivo PZQ assay presented here . A central feature of the transcriptional changes in S . japonicum subjected to PZQ in vivo was the up-regulation of ABC transporters , putative detoxification and multidrug resistance genes . The up-regulation of the ATP-dependent efflux pump SMDR2 in PZQ-treated S . mansoni in vitro [6] , [7] was mirrored in the S . japonicum homologue Contig02253 ( Multidrug resistance protein 3 ) which , in male parasites , was up-regulated ∼4-fold by four hours after drug treatment . The role of peroxiredoxins ( Contigs01526 and 11579 ) in schistosomes has been linked to detoxification , specifically in restricting oxidative damage [41] . Oxidative stress in S . japonicum described here may result from the indirect action of PZQ or to immune-mediated damage or a combination of the two . Efficacy of anthelmintic action can be unambiguously quantified by ascertaining worm motility [42] , [43] . To ascertain the real time efficacy of PZQ on S . japonicum , we employed the xCELLigence approach recently pioneered by Loukas and colleagues to quantitatively characterize effects of anti-worm drugs by determining worm movements in real time [44] . This system measures conductivity indicative of worm surface contact with the gold electrodes on the surface of the culture plate . The sensitivity of xCELLigence allowed detection of subtle changes in motility in real time for numerous individual worms . Since each worm was contained in a single well , producing its own signal representing motility , this delivered statistically robust observations . To determine a sub-lethal concentration with a 50% reduced motility phenotype , PZQ was used in vitro to establish the IC50 of 20 ng/ml . This IC50 is similar to that used in other studies with schistosomes [6] , [7] . It has been demonstrated that when schistosomes are exposed to PZQ in vitro , they undergo a rapid influx of calcium ions [45] accompanied by intense muscular paralysis in male worms . This expected response was evident using the xCELLigence approach . The extent of Ca2+ overload in mammalian cells is partly mediated by the actions of CamKII , which also participates in regulation of muscle contraction in [46] . Isolated muscle cells from S . mansoni exhibit Ca2+-dependent contractility [47] but the effect that CamKII has on this process , until now , not been determined . Responsive to fluctuations in Ca2+ , CamKII functionally modulates many ion channels and transporters in mammalian cells [48] . CaMKII , which exhibits amino acid sequence similarity to the CaMKII auto-inhibitory domain [49] , phosphorylates the β2a subunit of voltage gated Ca2+ channels to facilitate Ca2+ channels . CamKII is necessary for Ca2+ homeostasis in mammalian cells and it likely has a similar function in schistosomes , a role particularly important after PZQ exposure . PZQ produces a well-documented effect on intracellular Ca2+ levels in adult schistosomes [50] . Contigs 01107 and 01396 , both representing potentially Voltage-dependent calcium channel subunit alpha , were up-regulated in males but down-regulated in female S . japonicum when exposed to PZQ in vivo . The increase in intracellular calcium stimulates activity of the calcium-sensitive proteins , CamKII and protein kinase C ( PKC ) [51] , both of which are involved in the calcium pathway and potential drug targets . PKC ( Contig07198 , Protein kinase C-like 2 ) , which can act by phosphorylation on voltage-gated Ca2+ channel subunits [52] , [53] , was up-regulated in both male and female S . japonicum worms in late response to PZQ . CamKII is known to act on both α and β subunits of voltage-gated Ca2+ channels , resulting in the modulation of ion entry into cells [49] . The increase in transcription of both of these kinases in S . japonicum suggests that these genes act as a response element to increase Ca2+ levels , a known event in PZQ action . It is also apparent that both PKC and CamKII can act on calcium channels themselves . It may be that the interaction with CamKII is needed to restrict the effects of PZQ in schistosomes and when CamKII is reduced , as represented here by RNAi , the motility effects of PZQ are exacerbated . Our data also suggest that a combination of PZQ with CamKII inhibitors such as STO-609 [54] , may be synergistic for anti-schistosomal efficacy . However , S . japonicum CamKII shares similarity with both human and murine homologs ( CAB65123 . 1: 81% identify , 5e-113 evalue , NP_076302 . 1: 81% identify , 1e-112 evalue , as determined by Basic Local Alignment Search Tool BLASTp [55] ) and thus deployment of inhibitors in the clinic would require careful scrutiny . Our results partially supports findings of a recent comparison of gene transcription in S . mansoni worms exposed to PZQ in vitro [16] . Cunningham and co-workers exposed worms to PZQ in a similar time course to that presented here , where their 1 h and 20 h exposures [18] are comparable to the present study's 0 . 5 h and 24 h intervals . In juvenile S . mansoni , 1329 genes at 1 h and 3482 at 20 h were differentially up-regulated , as were 208/ND and 1393/1223 at 1 and 20 h in adult male/female worms [18] . In both developmental stages of S . mansoni there was greater differential expression at the later time point , a similar outcome to the present findings . Furthermore , comparison between adult S . mansoni male and female parasites showed limited overlap between the sexes - only 20% of same genes were regulated in the same direct ( up or down ) for both sexes . Although numerous genes were identified in both studies , including ABC transporters , multi-drug resistance genes , and calcium signaling pathway members , many were observed only in juvenile S . mansoni , in contrast to the current findings where numerous S . japonicum genes in adults were differentially expressed in response to PZQ . RNAi has been used to suppress a number of schistosome genes so as to investigate their function , but many may not be amenable to exogenous RNA interference [56] . This likely relates to developmental and tissue specificity of the genes . Moreover , refractoriness to RNAi may be due to the secondary structure of transcripts , gene dosage and pathway member redundancy . Moreover , as an informative example of the subtlety of RNAi analysis in schistosomes , whereas suppression of the TGF-β homologue SmInAct lead to a modest 40% suppression at the RNA level , eggs produced by SmInAct knockdown females failed to develop [57] . Accordingly , for other targets in addition to CamKII , we plan in future studies to examine alternative approaches including shRNAi [58] , [59] . The data presented here provide new insights into the mechanism of action of PZQ , and the response of schistosomes to the drug . Future investigation will focus on elucidation of the roles of those genes , either as the direct target of PZQ or as member of pathways that are affected by the binding of the drug to its targets . Understanding the emergence of drug resistance in schistosomes requires characterisation of the mode of drug action . If resistance is associated with a mutation of the target ( s ) , identification of other targets within the pathway , demonstrated as critical to parasite survival , would be informative for development of next generation anthelmintics . We re-emphasise that PZQ is the only drug effective against all schistosome species . Should drug-resistance develop , the public health implications would be considerable .
The conducts and procedures involving animal experiments were approved by the Animal Ethics Committee of the Queensland Institute of Medical Research ( project number A0108-054 ) . This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . Forty BALB/c mice ( female , 6 weeks old ) were infected with 30 S . japonicum ( Anhui , China isolate ) cercariae . Six weeks later , 20 of the mice were given a single oral dose of PZQ ( 300 mg/kg in PBS ) ; the other 20 received PBS . Mice ( five per group ) were euthanised at four time points ( 30 min , 4 h , 12 h and 24 h after administration of PZQ or PBS and adult worms recovered by portal perfusion using RPMI 1640 medium at 37°C [38] , [60] . The half-life of PZQ in mice is 1–1 . 5 h [61] , and visual effects on the parasite have been reported after 15 min , post subcutaneous administration [32] . Male and female worms were washed separately in 37°C RPMI 1640 , pooled for each time point , and stored at −80°C prior to RNA extraction . All parasites were motile at time of storage . Male and female adult parasites were separated , pooled ( 8–10 worms per mouse ) , stored in RNAlater ( Ambion ) at −20°C before microarray analysis . Total RNA was isolated from the pooled frozen parasites [62] . RNA quality/quantity was determined using the Bioanalyzer RNA Nano LabChip ( Agilent Technologies ) and NanoDrop ( Thermo Scientific ) . Labelling ( CY3 Agilent One-Color Amp Labeling Kit ) and hybridisation methods ( Agilent One-Color Microarray-Based Gene Expression Analysis Protocol ) were carried using optimised procedures [38] . A 4×44 k feature format microarray was constructed using the S . japonicum ( Anhui , China isolate ) transcriptome [63] by Agilent Technologies custom design and manufacturing pipeline . The array comprises 60-mer oligonucleotide probes for 14 , 171 SjC contiguous target sequences ( Contigs ) laid out in triplicate , in addition to proprietary positive and negative controls as supplied by the eArray software interface with Agilent's control grid . Details of the microarray design are available at www . ncbi . nlm . nih . gov/geo/ Accession No . GPL9759 and in Table S5 Series Accession No . GSE41149 . Applications of the array for studying different aspects of the biology of S . japonicum have been described [38] , [64] . Experiments were conducted using an Agilent one-colour protocol and scanned on an Agilent microarray scanner at 550 nm . Images from the DNA Microarray Scanner were extracted with Feature Extraction ( v10 . 5 ) . Automatic outliner flagging was used and the list filtered on the basis of p-value generated . Feature-extracted data were analysed and visualised using GENESPRING ( version 11; Agilent Technologies ) . Microarray data were normalised using a scenario for ‘Agilent FE one-color’ and ProcessedSignal values were determined using Feature Extraction and GeneSpring microarray software , including signal to noise ratio , spot morphology and homogeneity . ProcessedSignal represents the signal after localised background subtraction and includes corrections for surface trends . Features were deemed ‘Absent’ when the processed signal intensity was less than twice the value of the processed signal error value . Features were deemed ‘Marginal’ when the measured intensity was at a saturated value or if there was a substantial amount of variation in the signal intensity within the pixels of a particular feature . Features that were neither absent nor marginal were deemed ‘Present’ . Data points were included only if they were present or marginal , and probes or Contigs were retained if at least half of the data points were ‘Present’ . Differential probe hybridisation was statistically evaluated as a p-value , and a cut-off value of ≤0 . 05 in at least 4 of 10 conditions used as the confidence level . Samples were normalised to untreated parasites at time point equals 0 , and expressed as a relative fold change on a log2 scale . Microarray data were analysed using GeneSpring and calculated p-values were used to filter data ( ≤0 . 05 ) , carefully considering false-positive results . Multiple testing techniques available via GeneSpring were used including the Benjamini and Hochberg False Discovery Rate [65] , [66] . KEGG ( Kyoto Encyclopedia of Genes and Genomes ) metabolic pathways were considered for the microarray data [67] which have been mapped for S . japonicum [68] . The expression profiles of a subset of genes identified during the analysis were validated by real-time PCR . Total RNA samples were DNase-treated ( Promega , Annandale , Australia ) before complementary DNA ( cDNA ) synthesis [69] . The SuperScriptTM III protocol with p ( dT ) 15 primers was used to synthesise cDNA . Real-time PCR was performed and analysed as described [60] . Primers used are presented in Table S6; each sample was checked for primer dimerisation , contamination or mis-priming through inspection of its dissociation curve . Contig01379 ( DNA double-strand break repair rad50 ATPase ) was used as a reference gene for quantitative PCR analyses as the microarray analysis showed constitutive levels of expression of this gene at all time points for both male and female worms after exposure to PZQ . Two independent experiments ( from cDNA synthesis ) were carried out for the validation of selected genes . Data from the microarray and real time PCR analyses were examined to ascertain if they fitted normal distributions using the D'Agostino and Pearson omnibus and the Shapiro-Wilk normality tests . Statistical analyses were conducted using GraphPad Prism V5 or Microsoft Excel . Further characterisation of gene function was carried out using RNAi , an approach now feasible for schistosomes , in light of recent advances in knocking down schistosome genes [70] , [71] . RNAi was used in conjunction with an in vitro assay where S . japonicum worms were cultured in the presence of PZQ , so as to clarify the role of specific genes associated with drug action or in PZQ resistance mechanisms . BALB/c mice ( females , 6 weeks old ) were challenged with 30 S . japonicum ( Anhui , China isolate ) cercariae . Six weeks post-infection mice were euthanised and adult worms obtained by portal perfusion using 37°C RPMI 1640 medium . Adult worms were incubated in complete schistosome media ( CSM ) containing RPMI 1640 medium , supplemented with 20% ( v/v ) heat-inactivated fetal calf serum , 100 IU/ml penicillin and 100 µg/ml streptomycin , at 37°C in an atmosphere of 5% CO2 in air overnight [72] . dsRNAs were transcribed in vitro from template PCR products using gene-specific primers tailed with the T7 promoter sequence . Briefly , luciferase dsRNA ( dsLUC ) was used as a negative control , as reported in other studies with schistosomes [70] , [71] . dsRNA of CamKII ( contig01285 ) was synthesised from S . japonicum cDNA using gene-targeted primers containing T7 promoter sequences: ( F: 5′-TAATACGACTCACTATAGGGAGAGAAGATGGCTACTTCTGTACTCC-3′; R: 5′-TAATACGACTCACTATAGGGAGATTCCATACGGTTCTTTGCGTAAAA-3′ ) . dsRNA was synthesised and purified using a Megascript RNAi kit ( Ambion ) . Five pairs of worms in 50 µl electroporation buffer [73] , containing 0 . 5 µg/µl long dsRNA , were electroporated in a 4 mm cuvette by applying a square wave with a single 20 ms impulse at 125 v [74] . Following electroporation , parasites were transferred to 150 µl pre-warmed ( 37°C ) CSM . After overnight culture , media were replaced with 300 µl of CSM . Worms were collected at 48 h post-treatment with dsRNA , and male and female worms were separated for total RNA extraction . Gene transcript levels were measured by real time PCR , with NADH- ubiquinone reductase included as the reference gene [38] . Adult worms were perfused from mice with 37°C RPMI , as described above for RNAi . The motility of adult male or female S . japonicum and IC50 values were assessed using the xCELLigence system ( Roche Inc . ) [44] . RTCA controller software ( Roche Inc . ) was used to determine how the information was gathered from the 96 well E-plate ( Roche Inc . ) . For real-time monitoring of parasite motility , individual female or male worms were cultured in vitro in a 96 well E-plate ( one worm per well ) . Each worm was cultured in 180 µl of CSM per well and motility was monitored every 15 seconds for 3 h to obtain a baseline motility ( to identify healthy parasites ) reading prior to the addition of 20 µl of a 10× solution of PZQ ( stock solution in 100% ethanol at 5 mg/ml ) . Before the motility of multiple parasites in a treatment group was combined to produce an average and standard deviation ( SD ) , we manually curate the data to identify any worms that had died or was severely damaged by handling either at the baseline collection or during the subsequent culture . As such the data from that specific e-plate well ( representing the dead parasite ) were removed from further analysis . The motility index of each worm was calculated , for the 3 h prior to the addition of PZQ , as the SD over 150 data points of the cell index ( CI ) difference from the rolling average ( average of the 10 proceeding and preceding CI values - 5 min total ) over 20 data points [44] . For generation of the IC50 of PZQ in vitro , a final working concentration range of 12 . 3–3 , 000 ng/ml PZQ was used ( Figure S2 ) . After addition of PZQ , worms were monitored every 15 seconds for a further 72 h; motility index was calculated as the SD over 800 data points , and the CI difference from the rolling average over 20 data points . We were able to use 800 data points , and thus more accurately determine SD , due to the longer time course of 72 h . Dead worms ( heat killed ) were included as immobile controls and considered to exhibit 0% motility . Positive control worms ( without PZQ ) were cultured in the presence of the ethanol concentration equivalent to that for the highest drug concentration , and represented 100% worm motility . A log10 ( drug concentration ) versus normalised response ( 100%-0% ) formula with variable slope and automatic removal of outliers . Statistical analyses were undertaken using Graphprism 5 . 0 [44] . The Hill Slope and LogIC50 value were used together and compared for significant differences using an extra sum-of squares F-test . For RNA interference , worms were cultured for 48 h after dsRNA treatment; individual female or male worms were transferred to the E-plate in 180 µl medium per well to monitor mobility , as above . Briefly , worms were monitored for 3 h to obtain a baseline motility reading before addition of PZQ ( IC50 concentration of 20 ng/ml ) . After adding the drug , worms were monitored for 72 h . Worms subjected to dsRNA but not PZQ served as knockdown controls , and allowed the differentiation of the separate effects of PZQ incubation and RNAi on motility . The motility index and motility ( % ) of treated or untreated worms were determined as described . For each assay , 6–10 worms were monitored simultaneously and separately for each sex and treatment group ( with or without PZQ ) . A t-test ( two-tailed , two-sample equal variance ) for each time point was undertaken ( in Microsoft Excel ) to evaluate significance of differences in motility between treatment groups . | Schistosome infected mice were treated orally with a single sub-lethal dose of the anthelmintic praziquantel . Parasites were subsequently isolated 0 . 5–24 hours later and analysed by gene expression microarray . The transcriptional pattern was influenced by the sex of the parasite . Males up-regulated genes associated with surface integrity , muscle function , ion regulation and drug resistance . While for female worms , genes associated with calcium regulation , drug resistance , detoxification and pathogen defense were transcriptionally elevated . Based on these findings , genes with putative ion homeostasis or drug metabolism roles were further investigated for their prospective roles praziquantel action , using the gene silencing procedure RNA interference . The leading gene was CamKII ( calcium/calmodulin-dependent protein kinase type II ) , a member of the Calcium Signalling Pathway . A quantitative assay for parasite motility during praziquantel exposure was used to study worms in which the CamKII gene had been partially silenced . When CamKII transcription was reduced , the effects of praziquantel were augmented , visualised by reduced parasite movement . These functional studies indicate a key role for the kinase CamKII in the mode of action of the praziquantel , and suggest that future studies on this enzyme and other calcium signaling pathway components will be constructive in understanding how praziquantel kills schistosomes . | [
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] | 2013 | Transcriptional Responses of In Vivo Praziquantel Exposure in Schistosomes Identifies a Functional Role for Calcium Signalling Pathway Member CamKII |
Toxoplasmic encephalitis in patients with AIDS is a life-threatening disease mostly due to reactivation of Toxoplasma gondii cysts in the brain . The main objective of this study was to evaluate the performance of real-time PCR assay in peripheral blood samples for the diagnosis of toxoplasmic encephalitis in AIDS patients in the French West Indies and Guiana . Adult patients with HIV and suspicion of toxoplasmic encephalitis with start of specific antitoxoplasmic therapy were included in this study during 40 months . The real-time PCR assay targeting the 529 bp repeat region of T . gondii was performed in two different centers for all blood samples . A Neighbor-Joining tree was reconstructed from microsatellite data to examine the relationships between strains from human cases of toxoplasmosis in South America and the Caribbean . A total of 44 cases were validated by a committee of experts , including 36 cases with toxoplasmic encephalitis . The specificity of the PCR assay in blood samples was 100% but the sensitivity was only 25% with moderate agreement between the two centers . Altered level of consciousness and being born in the French West Indies and Guiana were the only two variables that were associated with significantly decreased risk of false negative results with the PCR assay . Our results showed that PCR sensitivity in blood samples increased with severity of toxoplasmic encephalitis in AIDS patients . Geographic origin of patients was likely to influence PCR sensitivity but there was little evidence that it was caused by differences in T . gondii strains . ClinicalTrials . gov NCT00803621
The protozoan Toxoplasma gondii is a cosmopolitan parasite that virtually infects all warm-blooded animals , including humans who become infected postnatally by ingesting tissue cysts from undercooked meat , consuming food contaminated with oocysts , or by accidentally ingesting oocysts from the environment [1] . The genetic diversity of this parasite is limited to a few successful clonal lineages in North America , Europe , Africa and China but is considerably higher in tropical South America [2] . In non-immunocompromised persons , toxoplasmosis is usually asymptomatic or limited to a mild symptomatology except in tropical South America . The prevalence of acquired and congenital ocular toxoplasmosis is much higher in Brazil and Colombia than in another place in the world and the Amazonian toxoplasmosis is a disseminated infection that requires management in intensive care units even in otherwise healthy adults [3 , 4] . There is more and more evidence that the greater severity of toxoplasmosis in South America results from poor host adaptation to the genetically diverse T . gondii strains from this region [5] . Toxoplasmic encephalitis ( TE ) in patients with AIDS is a life-threatening disease mostly due to reactivation of Toxoplasma gondii cysts in the brain . TE can be inaugural of AIDS in patients who are not aware of their HIV seropositivity , but poor compliance with cotrimoxazole prophylaxis in patients with CD4 cell counts <200/μL is the major event leading to TE [6] . The incidence of TE in AIDS patients has greatly decreased since the introduction of HAART , but HIV-associated toxoplasmosis hospitalizations remain substantial , even in the United States [7 , 8] . TE must be treated with specific anti-toxoplasmic therapy as soon as the diagnosis of TE is clinically and radiologically suspected [9] . Cerebral biopsy showing T . gondii tachyzoites is the only way to make a definite diagnosis of TE but is rarely undertaken in AIDS patients at baseline . The clinical and radiological response to specific therapy is still the gold standard for confirming a posteriori the diagnosis of TE in AIDS patients . This presumptive diagnosis has important limitations since up to 40% of AIDS patients with suspected TE and treated with specific therapy could not have in fact TE [10] . Laboratory investigations are considered not helpful in the diagnosis of TE . The majority of patients have positive IgG and negative IgM against T . gondii , simply indicating that they acquired toxoplasmosis in the past , mostly during childhood . However , the negative predictive value of a negative serologic testing for toxoplasmosis is high because it is estimated that < 3% of patients with AIDS have no demonstrable antibodies to T . gondii at the time of diagnosis of TE [11] . The diagnostic performance of PCR tests in various biological samples , mostly CSF and blood , was regularly assessed since the early years of the AIDS pandemic [12] . Blood samples are the only ones that can be easily obtained from the patients without invasive procedures . Although specificity was high , the use of PCR testing in blood samples for the diagnosis of TE has been limited by its poor sensitivity in the studies conducted in Europe [12–16] . A few studies have been conducted in tropical South America and the results of sensitivity were highly controversial [17–20] . Considering that T . gondii strains from tropical South America have substantial genetic and pathogenic differences with those from USA and Europe , it is therefore important to re-evaluate the performance of the PCR assay in blood samples for the diagnosis of TE in AIDS patients from this region . The main objective of this study was to evaluate the performance of real-time PCR assay in peripheral blood samples for the diagnosis of TE in AIDS patients from the French departments of America . The French departments of America ( DFA ) are French tropical overseas departments that include French Guiana in mainland South America and the French West Indies islands of Martinique and Guadeloupe in the Caribbean . The secondary objective was to collect and genotype T . gondii isolates from these patients .
This study was approved by the French Ethics Committee “Comité de Protection des Personnes du Sud-Ouest et Outre-Mer 4” on April 4 , 2008 , with the reference number cpp08-006a . The Toxo-DFA study is registered in the ClinicalTrials . gov database ( Identifier: NCT00803621 ) . All patients included in the study were adult and provided written informed consent . The Toxo-DFA study is an epidemiological , prospective , and multicentric study to validate the real-time PCR assay in peripheral blood samples for the diagnosis of TE in AIDS patients from a tropical area . It was conducted between September 16 , 2008 , and December 30 , 2011 , in four hospital centers of the French departments of America: two in French Guiana ( Cayenne and Saint-Laurent du Maroni ) and two in the French West Indies ( Fort de France in Martinique and Pointe à Pitre in Guadeloupe ) . Study participants had to meet all of the following criteria: age >18 years , informed written consent , positive serologic test for HIV , and clinical and radiological suspicion of TE with start of specific antitoxoplasmic therapy . Patients legally protected or uncovered by social insurance , or with a specific antitoxoplasmic therapy already initiated since 72h or more were excluded from the study . The collection of data was done in an online secured case report form with CS-ONLINE from CAPTURE SYSTEM software . Patients were assessed clinically at baseline , between days 6–8 , 15–21 , and 42–56 . Neuroradiographic scans by computed tomography ( CT ) or magnetic resonance imaging ( MRI ) were performed at baseline , between days 15–21 , and 42–56 . The gold standard for diagnosing TE was based on the clinical and radiological responses to specific antitoxoplasmic therapy after suspicion of cerebral toxoplasmosis . A validation committee of independent experts reviewed and classified the cases in 4 categories . TE was considered definite when there was a complete or significant clinical and radiological response to specific therapy ( with not necessarily disappearance of radiological and clinical lesions ) , and no elements for an alternative diagnosis . TE was considered probable when radiological lesions were compatible but only partial improvement was observed with specific therapy ( due to non-optimal treatment or incomplete follow-up ) , and no elements for an alternative diagnosis . Absence of TE was considered definite when there was no improvement or worsening of lesions with specific therapy or absence of T . gondii in cerebral biopsy samples and presence of elements for an alternative diagnosis . Absence of TE was considered probable when there was no response to specific therapy and no elements for an alternative diagnosis . Two different centers were involved in the laboratory investigations: the Limoges center in metropolitan France and the Cayenne center in French Guiana . A Neighbor-Joining tree was reconstructed from microsatellite data to examine the relationships between strains collected from human cases of toxoplasmosis in South America and the Caribbean . The tree was constructed with Populations 1 . 2 . 32 ( http://bioinformatics . org/populations/ ) based on Cavalli-Sforza and Edwards chord-distance estimator [26] and generated with MEGA 6 . 05 ( http://www . megasoftware . net/history . php ) software . The software used for statistical analyses was SAS 9 . 3 and the significant threshold of the p value was 0 . 05 ( SAS Institute , Cary , USA ) . Median and interquartile intervals were given for quantitative variables while qualitative variables were presented as sample size and percentages . Nonparametric tests were used for comparing variables between patients with TE and those without TE: a Fisher exact test was performed for the qualitative variables and a Mann-Whitney test was used for the quantitative variables . To evaluate the diagnostic performance of the PCR assay in detecting TE in peripheral blood samples from AIDS patients in the French West Indies and Guiana , the reliability and the validity of the test were assessed . For these analyses , cases reviewed by the validation committee with definite or probable TE were coded positive and those with definite or probable absence of TE were coded negative . The reliability of the PCR assay was estimated by the Cohen’s Kappa coefficient of agreement and its 95% confidence interval between results of the test in Cayenne and Limoges centers . The validity of the PCR assay was estimated by the sensitivity and the specificity of the test in detecting definite and probable cases of TE that had been identified by the validation committee . The 95% confidence intervals of sensitivity and specificity were estimated by the exact method . A study of the false negative results was carried out to search explanatory factors according to the sample size of this sub-group by using a logistic regression model with the false negative status as the response variable and the potential associated factors as the explicative variables .
A total of 46 patients were included in this study: 17 in French Guiana ( 8 in Cayenne and 9 in Saint-Laurent du Maroni ) and 29 in the French West Indies ( 23 in Guadeloupe and 6 in Martinique ) . The validation committee reviewed the cases as follows: 2 cases were not assessable because of insufficient data ( both included in the center of Saint Laurent du Maroni in French Guiana ) , 36 cases were classified in the TE group and 8 in the non-TE group . In the TE group , 30 were classified as definite TE and 6 as probable TE . In the non-TE group , 6 were classified as definite absence of TE and 2 as probable absence of TE . The demographic , laboratory and clinical baseline characteristics of the 44 patients classified in TE and non-TE groups by the validation committee are available in Table 2 . There was no statistical difference between patients with TE and those without TE with respect to the variables listed in Table 2 except for the place of birth and the results of T . gondii serology . Two different classifications were used for clustering the 44 patients into two groups according to their place of birth . In the first classification , the first group gathered the 29 patients who were born in the French West Indies and Guiana ( 6 in French Guiana , 17 in Guadeloupe , and 6 in Martinique ) while the 15 patients who were born elsewhere were put together in a second group ( 7 in Haiti , 3 in Brazil , 2 in Suriname , 1 in Dominica , 1 in Dominican Republic , and 1 in Spain ) . Being born in the French West Indies and Guiana was significantly more common in patients without TE than in those with TE ( p = 0 . 04 ) because all patients without TE ( n = 8 ) were born in the French West Indies and Guiana ( Table 2 ) . The second classification was based on geography with 32 patients born in the Caribbean ( 17 in Guadeloupe , 7 in Haiti , 6 in Martinique , 1 in Dominica , and 1 in Dominican Republic ) , 11 in South America ( 6 in French Guiana , 3 in Brazil , 2 in Suriname ) , and 1 in Europe . According to this second classification , being born in the Caribbean was not statistically different between patients with TE and those without TE ( Table 2 ) . Of the 41 patients with available data on T . gondii serology , only 2 had negative test results for IgG and IgM against T . gondii , and both of them had definite absence of TE ( Table 2 ) . The blood samples of the remaining 39 patients tested positive for IgG and negative for IgM , indicating past immunization against T . gondii . At presentation , only 7 patients were receiving systemic antiprotozoal prophylaxis: trimethoprim-sulfamethoxazole ( cotrimoxazole , n = 5 ) , pyrimethamine ( n = 1 ) , and atovaquone ( n = 1 ) . Of the 5 patients with cotrimoxazole prophylaxis , good compliance was reported in only one patient . The choice of specific empirical antitoxoplasmic first-line therapy was based on routine practice of each center: all patients from French Guiana ( n = 15 ) , five out of six patients from Martinique and only one patient from Guadeloupe were treated with trimethoprim-sulfamethoxazole ( cotrimoxazole ) whereas 22 out of 23 patients from Guadeloupe were given a pyrimethamine-based combination with either sulphadiazine ( n = 16 ) or clindamycin ( n = 6 ) . One patient was treated with a combination of pyrimethamine plus atovaquone in Martinique . Five ( 11 . 4% ) patients , all with TE , died within the first 12 weeks after antitoxoplasmic therapy was begun . Of the 44 patients , the PCR assay tested positive in blood samples of 9 patients with TE and tested negative in 35 patients ( 27 in the TE group and 8 in the non-TE group ) . The sensitivity was 25 . 00% ( 95% CI 12 . 12–42 . 20 ) which is low , and the specificity was 100% ( 95%CI 63 . 06–100 . 00 ) which is maximal . Of the 9 blood samples with a positive PCR test , only 4 were detected simultaneously in both Limoges ( Parasites/mL blood: 0 . 01–0 . 13 , 0–0 . 42 , 4 . 67–6 . 49 , 2 . 30–8 . 80 ) and Cayenne ( Parasites/mL blood: 1 . 46–1 . 83 , 0 . 45–0 . 53 , 15 . 38–22 . 25 , 2 . 15–9 . 83 , respectively ) laboratories , whereas 4 were detected in the Cayenne laboratory alone ( Parasites/mL blood: 0 . 02–0 . 24 , 0 . 01–0 . 18 , 0 . 37–0 . 98 , 4 . 80–8 . 30 ) and 1 in the Limoges laboratory alone ( Parasites/mL blood: DNA: 0–1 . 16 ) . The sensitivity was 11 . 11% ( 95% CI 3 . 11–26 . 06 ) , 13 . 89% ( 95% CI 4 . 67–29 . 50 ) , and 22 . 22% ( 95% CI 10 . 12–39 . 15 ) when positive PCR tests were observed in both centers , in the Limoges center , and in the Cayenne Center , respectively . The Cohen's kappa coefficient used to estimate the agreement between results of PCR tests performed in both centers was 0 . 5528 ( 95% CI 0 . 2112–0 . 8945 ) , which indicates moderate agreement [27] . From these data , we can conclude that the majority of PCR results were close to the limits of detection and the difference in detection of T . gondii DNA at the two centers were due to differences in the analytical sensitivity of the PCR assay at each site . The relationship between the risk of having a false negative result with the PCR assay in the blood for the diagnosis of TE and different baseline variables is shown in Table 3 . Altered level of consciousness and being born in the French West Indies and Guiana were the only two variables that were associated with significantly decreased risk of false negative results with the PCR assay according to multivariate logistic regression analysis . Five blood samples that tested positive with the PCR assay in the Limoges laboratory were inoculated into mice and only one strain was isolated . This strain was isolated from a patient who was living in Guadeloupe but born in Haiti . The blood sample of this patient tested positive with the PCR assay in both laboratories of Limoges and Cayenne , and corresponded to the sample with the highest parasite load ( Parasites/mL blood: 4 . 67–6 . 49 and 15 . 38–22 . 25 , respectively ) . This strain was designated HTI01 in this study and cryopreserved at the Toxoplasma BRC with the denomination code TgH40001 . The genotype of this strain with 15 microsatellite markers was compared with the genotypes of the three reference type I , II , and III strains and with those of 43 strains collected in human cases of toxoplasmosis from South America and the Caribbean region ( Table 1 ) . The neighbor-joining analysis clustered the HTI01 strain collected in the present study with the type II reference strain in the unrooted tree ( Fig 1 ) . The 43 strains collected from human cases of toxoplasmosis in the Caribbean and in South America by the French national reference center for toxoplasmosis were clustered as follows: i ) strains collected in patients from the French West Indies were highly structured in only two groups: 2 strains clustered with the HTI01 strain in the type II group and 8 strains were grouped into a separate cluster called Caribbean group; ii ) the anthropized strains from French Guiana were also highly structured into three groups: 2 strains were assembled in the type I group , 2 in the type III group , and 1 in the Caribbean group; iii ) the 18 wild strains from the Amazonian forest of French Guiana were found on separate long branches and were highly divergent from all the other strains , as reported previously [24 , 25]; iv ) of the 10 Brazilian strains , six were structured in one separate group called Brazilian group and four strains were divergent and found on separate long branches like wild strains from French Guiana .
There is a need of treatments and diagnostic tools for TE adapted to AIDS patients in the specific context of tropical areas . For example , the standard therapy of TE is the combination of pyrimethamine and sulfadiazine [9 , 28] . However this treatment has several limitations , such as its cost , the high frequency of adverse reactions in AIDS patients , the absence of intravenous formulation and its frequent unavailability in poor resource settings . For these reasons , cotrimoxazole is often preferred as a first line therapy of TE in AIDS patients in tropical areas because it is efficacious , cheap , better tolerated , with an intravenous formulation and , most of all , is widely available in developing countries [29 , 30] . Diagnosis of TE is not straightforward because the majority of clinicians rely initially on an empiric diagnosis based on clinical and radiographic improvement to specific anti-T . gondii therapy in the absence of a likely alternative diagnosis [28] . In tropical areas , many patients are diagnosed with HIV only after developing opportunistic infections such as TE and the differential diagnosis of focal neurological disease in patients with AIDS can be complex in the context of poor-resource settings [29] . Under-diagnosis is likely to be the consequence of the difficulties with diagnosing TE in tropical areas [31] . In this study , we aimed at evaluating the diagnostic performance of TE with the real-time PCR assay in peripheral blood samples from AIDS patients in a tropical setting . A total of 44 patients , 36 with TE and 8 without TE , from the French West Indies and Guiana in the French departments of America were included in the present study . The standards of healthcare are close to those of mainland France but this region is a crossroads for poor Caribbean and South American people who emigrate there for socio-economic reasons [32] . In this region , the HIV epidemic is a major public health problem and TE is a leading cause of death among HIV-infected adults [33 , 34] . All patients without TE tested negative with the PCR assay in blood samples and all patients with a positive PCR result had TE . The 100% specificity of the PCR assay in blood samples for the diagnosis of TE in patients with AIDS in our study confirms the very high specificity of this test reported in the literature ( median 99% , IQR 93 . 1%–100% ) [12–18 , 20] . However , with a sensitivity of 25% , the capacity of the PCR assay to detect TE in blood samples from patients with AIDS is low in a tropical area like the French departments of America . In fact , the sensitivity of this test to diagnose TE in blood samples from patients with AIDS seems to vary with geography in the literature . According to 5 studies conducted in the 1990s [12–16] , the sensitivity ranged from 13 . 3% to 29 . 5% in Europe ( median 24 . 3% , IQR 17 . 9–25 . 6 ) which is similar to the result of our study although this latter was conducted in a tropical area . In contrast , 3 studies from tropical South America in the 2000s showed contradictory results with a sensitivity of 1 . 2% in north-east Brazil , 18 . 8% in Colombia , and 80% in south-east Brazil [17 , 18 , 20] . It is difficult to compare these studies from Europe and South America with our study in the French departments of America because the volumes of blood samples ( 1 , 5 , or 10 mL ) , DNA extraction protocols ( buffy coat versus whole blood ) , DNA targets ( REP529 , B1 , TgRE1 , and rDNA repetitive gene ) , and even primers for the same DNA target ( B1 or REP529 ) were different . The 6 oldest studies performed a conventional PCR [12–17] whereas real-time PCR was done in the present work and in the most recent studies [18 , 20] . The Brazilian study with the highest PCR sensitivity ( 80% ) used a volume of 10 mL of blood sample and a conventional PCR assay targeting the B1 gene with primers B22 and B23 [17] . The DNA extraction step is essential for detecting T . gondii in blood samples . A recent study in the animal model showed that it was preferable to use buffy coat rather than whole blood , but stressed the importance of the volume of blood sample to increase the sensitivity of PCR assay [35] . The volume of blood sample in most studies that evaluated the performance of the PCR assay in AIDS patients with TE was 5 or 10 mL [12–15 , 17] except in 2 studies that used only 1 mL [18 , 20] . Using a limited volume of blood sample might explain , in part , the poor sensitivity of the PCR assay reported in one Brazilian study [19] . The choice of the DNA target for the PCR assay is also important because studies that tested different targets showed different sensitivity results according to the target [14 , 15] . Experts generally recommend the use of REP529 DNA target and real-time PCR for reaching the highest sensitivity but they stress the importance of the proficiency of the laboratory performing the diagnosis and the need for optimization of PCR conditions [36] . It is therefore better to use a well-optimized PCR assay targeting the B1 gene with a conventional PCR assay in a reference laboratory rather than a non-optimized PCR assay targeting REP529 with a real-time PCR assay in an inexperienced laboratory [37] . The use of different primers for the same target may also lead to different results of sensitivity , as suggested in one study [15] . In our study , the target was REP529 in Limoges and Cayenne laboratories but , because each center independently developed their own laboratory-optimized PCR assay for routine diagnosis of toxoplasmosis , primers were different and the sensitivity in each center was also different ( 13 . 89% and 22 . 22% , respectively ) . However , it is also true that identical primers can give variable results of sensitivity depending on the laboratory , which underlines , once again , the crucial importance of PCR optimization [36] . What makes consensus is the systematic use of uracyl DNA N-glycosylase ( UDG ) to avoid false-positive results caused by carry-over contaminations and an internal positive control ( IPC ) to avoid false-negative results caused by PCR inhibitors of PCR [38] . Such basic precautions were taken in the present study , in 3 studies from Europe , and in 1 study from Colombia [12 , 14 , 15 , 20] . One study in Europe and one in Brazil reported the use of IPC but not UDG with sensitivities of 20% and 1 . 2% , respectively [13 , 18] . The two studies that performed PCR without IPC and UDG reported a sensitivity of 25% in Europe and 80% in Brazil [16 , 17] . Altogether , it seems that the methodological issues raised here cannot entirely explain the huge difference between sensitivities of the PCR assay in blood samples for diagnosing TE in AIDS patients from 4 tropical areas: 1 . 2% in patients from Recife , north-east Brazil [18] , 18 . 8% in Colombia [20] , 25% in the French West Indies and Guiana ( this study ) , and 80% in São Paulo , south-east Brazil [17] . In the present study , the geographic origin of patients was likely to influence the sensitivity of the PCR assay because being born in the French West Indies and Guiana was a variable significantly associated with a decreased risk of false negative results according to multivariate logistic regression analysis . The first hypothesis to explain the link between geography and sensitivity of T . gondii DNA detection in blood samples is the strain hypothesis because the hotspot of T . gondii genetic diversity is in tropical South America and because the genotype of T . gondii strains is strongly linked to the presumed geographical origin of infection in immunocompromised patients [2 , 39] . Most cases of TE result from local reactivation of brain cysts without parasitemia which explains the absence of detection of T . gondii in most blood samples . If some strains are more likely to disseminate in blood flow than others , this would have a strong effect on sensitivity of PCR in blood samples . For example , the sensitivity of PCR in blood samples is very low for diagnosing ocular toxoplasmosis in immunocompetent patients from France and T . gondii DNA is detectable only in ocular fluid samples [40 , 41] . In contrast , T . gondii genotypes involved in ocular toxoplasmosis in south and south-east Brazil were not characterized from ocular fluid samples but from peripheral blood , and this prolonged parasitemia was confirmed by direct microscopic observation of tachyzoites in some blood samples [42–44] . Brazil is a big country with a complex T . gondii population structure and it is possible that such differences also exist at a regional scale in Brazil . If strains from south and south-east Brazil are more likely to disseminate in blood flow than those from the north-east , this could explain the regional variation of PCR sensitivity in blood samples for diagnosing TE in AIDS patients from Brazil [17 , 18] . Another example of disseminating disease is the Amazonian toxoplasmosis whose diagnosis is always confirmed by a positive result of the PCR assay in blood samples despite the fact that the patients are not immunocompromised [4] . Little is known about the genetic background that characterizes disseminating strains but , based on what is known from wild strains of the Amazonian rainforest , the genotypes of these atypical strains are found on separate long branches in neighbor-joining trees and are highly divergent from the genotypes of all other strains , especially from the clonal type II and III strains that are common in Europe [24] . However , in the present study , we found little evidence that the effect of geographic origins on PCR sensitivity in blood samples for the diagnosis of TE in AIDS patients was caused by differences in T . gondii strains . Unfortunately , we isolated only one T . gondii strain in a patient who was not born in the French departments of America but in Haiti . The genotype of this strain was not atypical but rather related to type II which represents >95% of strains in Europe where the sensitivity of PCR assay is low in blood samples . The other patient who was not born in the French West Indies and Guiana and who had a positive PCR result was born in Spain and therefore also likely infected by a type II strain . If the strain hypothesis were true in our study , we would have expected positive PCR results in blood samples of the 3 patients from Brazil but none of them tested positive . In fact , the proportion of positive PCR results was higher in patients born in the French West Indies and Guiana ( 7/29 , 24% ) than in those born elsewhere ( 2/15 , 13% ) . We included in the analysis the genotyping data of 43 T . gondii strains collected by the French national reference center from patients infected in tropical South America and the Caribbean . Strains that infect humans in the French West Indies and anthropized areas of French Guiana were not found on separate long branches in the neighbor-joining tree like wild strains from the Amazonian rainforest or some strains from Brazil but were highly structured like in Europe . Type II and III strains that are common in Europe are also common in the French departments of America . The difference with Europe is the predominance of an endemic lineage called Caribbean group that comprises the Caribbean 1 , 2 and 3 genotypes already described in domestic animals from the anthropized area of French Guiana and in immunocompromised patients from the French West Indies [24 , 25 , 39] . Although we did not isolate strains in patients born in the French departments of America in this study , it is likely that they were also infected by T . gondii strains belonging either to the types II and III lineages or to the Caribbean group but not to highly divergent strains that could have explained the better detection of T . gondii in blood samples for these patients . In the absence of a clear explanation by differences in T . gondii strains , the effect of geography on the sensitivity of PCR in blood samples of AIDS patients remains to be elucidated . The main result of our study is that the sensitivity of PCR in blood samples increases with the severity of TE . The main severity factors of TE in AIDS patients are profound immunodepression and impaired consciousness . In a study conducted in AIDS patients with TE at admission in intensive care units , the factors independently associated with a poor outcome were a Glasgow coma scale ≤8 and a CD4 cell count <25/μL [45] . In our study , a CD4 cell count <25/μL was not associated with a decreased risk of false negative results with the PCR assay . However , altered level of consciousness was the second variable significantly associated with a decreased risk of false negative results according to multivariate logistic regression analysis . Of the 8 patients with altered level of consciousness and TE in our study , 5 ( 62 . 5% ) tested positive in blood samples . All patients ( n = 3 ) with a Glasgow coma scale ≤9 had a positive test result with the PCR assay in blood samples . The high PCR sensitivity of 80% in blood samples of the 64 patients from São Paulo , Brazil , could be explained by a high number of severe TE cases in this study but clinical data were not available [17] . In conclusion , the PCR assay in blood samples is not recommended for diagnosing TE in the tropical setting of the French departments of America areas because of a poor sensitivity . The only interest of PCR would be in the most severe forms of TE with altered consciousness because PCR is more likely to be positive . Even in these cases , it seems difficult to reach a good sensitivity with the PCR assay because the concentration of T . gondii DNA is very low . PCR protocols have to be perfectly optimized because positive PCR results rely on high Ct values that are at the limit of the detection of the method which jeopardizes a good agreement between diagnostic laboratories , as showed in our study . There is no argument that the PCR sensitivity could be influenced by the genetic background of T . gondii strains in this area even if the geographic origin of patients is likely to play a role for unclear reasons . We believe that our results can be expanded in any tropical setting with the exception of other parts of tropical South America , especially Brazil where T . gondii strain diversity is far more complex than in the French West Indies and the anthropized areas of French Guiana . Other studies are needed in Brazil to know whether genetic-based differences in the capacity of hematogenous dissemination of locally acquired T . gondii strains are likely to explain the considerable regional variations of the sensitivity of the PCR assay in blood samples of AIDS patients from this country . | Diagnosis of toxoplasmic encephalitis ( TE ) in patients with AIDS is not straightforward because clinicians rely initially on an empiric diagnosis based on clinical and radiographic improvement to specific anti-Toxoplasma gondii therapy . There is therefore a need for biological tools to improve the diagnosis of TE , especially in tropical areas where this diagnosis is likely to be underestimated . The use of PCR testing in blood samples for the diagnosis of TE has been limited by its poor sensitivity in the studies conducted in Europe . In tropical South America , the results of PCR sensitivity in blood samples were controversial . Considering that T . gondii strains from tropical South America have substantial genetic and pathogenic differences with those from USA and Europe , it is therefore important to re-evaluate the performance of the PCR assay in blood samples for the diagnosis of TE in AIDS patients from this region . Our results showed that the only interest of PCR would be in the most severe forms of TE with altered consciousness because PCR is more likely to be positive . We also provided important genotyping data on T . gondii strains isolated in human cases of toxoplasmosis in the Caribbean and in South America . | [
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] | 2016 | Performance Testing of PCR Assay in Blood Samples for the Diagnosis of Toxoplasmic Encephalitis in AIDS Patients from the French Departments of America and Genetic Diversity of Toxoplasma gondii: A Prospective and Multicentric Study |
Parkinson disease affects more than 1% of the population over 60 y old . The dominant models for Parkinson disease are based on the use of chemical toxins to kill dopamine neurons , but do not address the risk factors that normally increase with age . Forkhead transcription factors are critical regulators of survival and longevity . The forkhead transcription factor , foxa2 , is specifically expressed in adult dopamine neurons and their precursors in the medial floor plate . Gain- and loss-of-function experiments show this gene , foxa2 , is required to generate dopamine neurons during fetal development and from embryonic stem cells . Mice carrying only one copy of the foxa2 gene show abnormalities in motor behavior in old age and an associated progressive loss of dopamine neurons . Manipulating forkhead function may regulate both the birth of dopamine neurons and their spontaneous death , two major goals of regenerative medicine .
Midbrain dopamine neurons play important roles in motor control , reward , addiction , attention , and cognition [1 , 2] . A progressive loss of dopamine neurons is a defining feature of Parkinson disease . This disease will cause increased hardship in many countries , as 30% of the population will be over the age of 65 y around 2025 [3] . Dopamine neurons are normally generated in limited numbers , for a restricted time , in a small region of the embryonic midbrain [4] . To improve access to these neurons , techniques have been developed to derive them from precursors dissected from the fetal midbrain and from pluripotent embryonic stem ( ES ) cell lines [5–11] . Despite this effort , our knowledge of the mechanisms controlling the birth and death of these cells is still limited . Foxa2 is a forkhead transcription factor known to play a critical role in the early development of the endoderm and midline structures , including the notochord and floor plate [12–16] . FOXO transcription factors are closely related to the foxa genes and have a central role in cell survival , cancer , and the longevity of organisms [17–19] . Here , we show that midbrain dopamine neurons are derived from the floor plate and that foxa2 plays a central role in specifying dopamine neurons . Late in life , foxa2 heterozygous mice spontaneously develop significant motor problems and an associated late-onset degeneration of dopamine neurons . The initial deficit is asymmetric and preferentially affects dopamine neurons of the substantia nigra ( SN ) while leaving the ventral tegmental area ( VTA ) intact , a pattern of sensitivity also seen in Parkinson patients [20] .
Dopamine neurons can be identified by expression of tyrosine hydroxylase ( TH ) , the rate-limiting enzyme in dopamine synthesis . In the nervous system , foxa2 expression is restricted to the floor plate , a specialized ventral region that regulates the differentiation of nearby neurons by secreting the morphogenic signal sonic hedgehog ( SHH ) [14 , 15] . The newly generated dopamine neurons form a band at the most ventral edge of the midbrain and express FOXA2 in their nuclei ( Figure 1A and 1B ) . A panel of antibodies against transcription factors was used to define the domains of neuronal precursors in the ventral midbrain ( Figure S1 ) . The expression of the transcription factors LMX1b , NKX2 . 2 , and PHOX2a between embryonic day ( E ) 9 . 5 and 11 . 5 defines three adjacent ventral domains of neural progenitors before the first dopamine neurons are formed ( Figure 1C ) . The transcription factor LMX1b is expressed in the most ventral domain , the medial floor plate , continuously from E9 . 5 through E11 . 5 ( Figure S1C–S1E ) . SHH is coexpressed with LMX1b in the floor plate ( Figure 1D ) . From previous studies , it is unclear whether SHH-expressing cells are the direct precursors to dopamine neurons or if they are derived from a more lateral precursor that is induced by signals from the floor plate [21–23] . Genetic tracing using Cre-recombinase expressed from the shh regulatory sequences ( shh-creR26R mice ) allows the derivatives of these most medial cells to be identified [24] . When shh-cre mice were crossed with the R26R reporter strain , the expression of ß-galactosidase is surprisingly found outside the midline ( Figure 1E and inset ) . At E15 . 5 , when the majority of dopamine neurons have become postmitotic , they express TH and also express ß-galactosidase , definitively demonstrating that midbrain dopamine neurons are derived from the medial floor plate ( Figure 1F–1H ) . For the efficient ex vivo generation of dopamine neurons , it is important to understand the mechanisms that induce dopamine neuron identity . SHH gives increased numbers of dopamine neurons in midbrain primary explants and in neuronal cultures derived from ES cells [8 , 22 , 23] . This effect is thought to reflect the morphogenic induction of the dopaminergic fate . Access to reagents that define distinct precursor types allows the morphogenic effects of SHH in vitro to be reexamined . When dissociated cells from the developing ventral midbrain ( E8 . 5 ) were exposed to increasing concentrations of SHH , the proportion of LMX1b+ cells did not change , but the proportion of the NKX2 . 2+ and NKX6 . 1+ cells increased while the more dorsal PAX7 expression decreased ( Figures 2A and S2 ) . This result shows that , under these conditions , SHH does not induce the precursors of dopamine neurons , whereas the proportion of more lateral precursors is altered in the graded way expected of a morphogenic signal . However , SHH is also a mitogen for neural precursors and this mitogenic effect may account for the increased number of dopamine progenitors ( Figure 2B ) . These results suggest that growth control systems for dopamine neuron precursors are distinct from most other ventral midbrain neuron types . A consequence of these results is that production of dopamine neurons in the laboratory will be limited by the number of floor plate cells . As foxa2 is known to control the generation of the floor plate , we asked whether this gene controls the number of TH-positive cells in vitro . Because foxa2-null embryos do not survive past E10 . 5 , E8 . 5 midbrain explants from foxa2 homozygous mutant ( n = 8 ) and control embryos ( n = 12 ) were placed in culture . Neural progenitors migrate out from the explant and differentiate into Tuj1-positive neurons . In all of the wild-type explants , TH+ neurons were abundant ( Figure 2C ) . In contrast , the mutant explants generated no TH+ neurons ( Figure 2D ) . Using transcription factor expression , the neuron types in the ventral midbrain were distinguished ( Figure S3 ) . The islet1-positive oculomotor neurons were also absent , but more-lateral neurons expressing GATA3 and LIM1/2 were generated in cultures from foxa2−/− embryos ( Figure 2E ) . These data show that the foxa2 gene is specifically required for the generation of dopamine and motor neurons , the two neuron types derived from the floor plate . To ask whether expression of foxa2 would cause an increase in the number of dopamine neurons generated in vitro , a foxa2 expression plasmid was transfected into E10 . 5 midbrain cells . A 4-fold increase in the proportion of TH+ cells was observed ( Figure 2F ) . A mouse embryonic stem ( mES ) cell line engineered to inducibly express a foxa2 transgene generated 7-fold more TH+ cells ( Figure 2G ) . The SHH antagonist , cyclopamine , inhibited the production of TH+ neurons in vitro . This effect was reversed upon induction of foxa2 even in the presence of cyclopamine ( Figure 2H ) . These results are consistent with an early role for shh in dopamine neuron specification demonstrated in mice carrying a conditional mutation in the SHH receptor smoothened [25] . Genetic analysis in zebrafish also demonstrates a role for foxa2 in the specification or patterning of ventral neurons in the midbrain and hindbrain [26] . Our data suggest that foxa2 specifies dopamine neurons in mammals . Dopamine neurons in the adult brain continue to express foxa2 ( Figure 3A ) . A foxa2 null allele was crossed into the C57BL/6 strain because this background is widely used in physiological and behavioral studies . Mice entirely lacking foxa2 die in early development . In all the data reported here , the effect of a single copy of the foxa2 gene , haploinsufficiency , was assessed in a C57BL/6 background . Haploinsufficient mice spontaneously developed major motor abnormalities . These behaviors were first observed at 18 mo of age when mice present with an asymmetric posture associated with a muscular rigidity that progresses from the tail , through the hind limbs , to the trunk . foxa2+/− animals showed a slower speed of movement but reduced horizontal movement and a complete loss of vertical movement ( Figure 3B–3D , foxa2+/+ n = 6 , foxa2+/− n = 4; Video S1 ) . The mice demonstrate a unilateral constriction/torsion of the trunk so the spine becomes curved towards either the left or right side . In a group of affected animals , the spinal curvature was 15° measured by analysis of footprints ( Figure 3E ) . Occasional episodes of high-frequency tremoring have been observed . Rigidity and loss of mobility in the hindlimbs become so severe that the limb is abnormally extended or splayed ( Figure 3G ) . These defects could be caused by deficits in non-dopaminergic systems . A widely used and quantitative behavioral assay of dopaminergic function in rodents is amphetamine-induced rotation [27] . Rats or mice lesioned acutely and unilaterally with 6-OHDA circumambulate ipsilaterally in response to amphetamine , and the extent of rotational movement is directly correlated with the severity of the lesion . We assayed amphetamine-induced rotational behavior in old foxa2+/− and wild-type C57BL/6 mice . A significant increase in rotational movement was observed in foxa2 mutants ( Figure 3F ) . Rotations occurred in the clockwise or counterclockwise direction , depending on the individual mouse , and were ipsilateral to the “kinked” side of the mouse . In the ventral midbrain of 1-y-old heterozygous animals , there is a partial loss of FOXA2 protein , suggesting that a reduced level of the FOXA2 protein leads to a behavioral deficit that spontaneously appears in old age ( Figure 3H ) . The amphetamine-induced rotational behavior suggests an asymmetric loss of dopamine neurons . Immunohistochemistry for TH-positive cells was performed to shed light on potential cellular causes of the motor problems . Affected mice demonstrated an asymmetric loss of TH expression in midbrain dopamine neurons in the SN sparing the VTA ( Figure 4A and 4B ) . No loss of dopamine neurons was seen in old foxa2 mutants without behavioral abnormalities ( Figure 4C and 4D ) . In the SN of affected foxa2+/− animals , there are few neurons recognized by Nissl staining ( Figure 4E ) . Serial sections through the midbrain dopaminergic system of a single animal show an almost total loss of TH+ neurons in the SN on one side of the brain and much less damage to the contralateral SN ( Figure S4 ) . In foxa2+/− animals ( n = 3 ) showing abnormal motor behaviors , compared to foxa2+/+ age-matched controls , there is a specific loss of SN neurons ( Figure 4G ) . This loss of dopamine neurons occurs in one third of animals over 18 mo old , and the asymmetric cellular loss explains the amphetamine induced rotation . Within the SN , there are different types of dopamine neurons . The most ventral neurons specifically express retinaldehyde dehydrogenase-1 ( RALDH1; [28] ) . In foxa2 mutant mice , there is a loss of RALDH1-positive neurons even when substantial numbers of dopamine neurons are still present in the SN . In the animal shown in Figure 4F , the SN on one side of the brain has a severe loss of dopamine neurons . In the contralateral SN , TH-positive cells are present , but there is a selective loss of RALDH1-positive cells ( Figure 4F and 4H ) . Many of the affected animals show an asymmetric loss of dopamine neurons , and the most affected side could be either the right or left ( marked by a star in Figures 4B and 4F ) . These data suggest the degenerative process is progressive , the RALDH1-positive neurons are the most vulnerable cells , and the SN on one side of the brain is affected first and then the disease progresses to the contralateral midbrain . Lewy bodies , cellular inclusions immunoreactive for alpha-synuclein , ubiquitin , and other proteins , are often found in the basal ganglia and cortex of the human parkinsonian brain [29] . Pathological staining for alpha-synuclein was not observed in foxa2 mutant mice , but a small number of TH-expressing dopamine neurons that were highly immunoreactive for ubiquitin were seen in the SN of mutant but not in wild-type C57BL/6 mice . Gliosis is an activation of astrocytes that often occurs in neurodegenerative disease [30] . Accompanying the loss of dopamine neurons in foxa2 mutants , there is an increase in the number of activated glia measured by expression of the glial fibrillary acidic protein ( GFAP ) in the substantia nigra pars compacta , in the substantia nigra pars reticulata , and other regions of the ventral midbrain ( Figure S5A–S5D ) . In contrast to neuron loss , this activation occurs in a symmetric manner .
Rigidity , tremor , reduced and slow movement with asymmetric behavioral features , and dopamine neuron loss are characteristically found in Parkinson patients [31] . The age-dependent motor defects and the late loss of dopamine neurons seen in foxa2+/− mice are similar to symptoms of Parkinson disease . In mammalian cells , survival is controlled by phosphorylation of FOXO proteins that respond to oxidative stress by regulating apoptosis [32 . 33] . Overexpression of pha-4 , the nematode foxa homolog , in Caenorhabditis elegans induces the superoxide dismutase , sod-1 , and increases longevity [34] . These data suggest that FOXA2 regulates the response of dopamine neurons to oxidative stress , but it is clear that a cell autonomous function in dopamine neurons should be explicitly assessed . In this report , we have shown that mice heterozygous for a mutation in foxa2 develop a late-onset condition associated with a significant loss of midbrain dopamine neurons . It will be important in the future to determine how functional interactions with other brain regions are altered as the dopamine neurons degenerate . Here , we show the neurons of the SN are more sensitive than the neurons of the VTA to the pathology in foxa2+/− mice . Asymmetric motor deficits and preferential loss of nigral neurons are characteristic in disorders of the nigrostriatal system [20] . The asymmetric degeneration of nigral dopamine neurons shows that foxa2+/− mice model features of human degenerative disease not previously seen in animal models . Oxidative stress is considered to be a primary cause of Parkinson disease [35 , 36] . Recent studies have identified mutations in a number of genes ( PARK genes ) associated with Parkinson disease . These genes include α-synuclein [37] , parkin [38] , DJ-1 [39] , PINK1 [40] , and most recently , LRRK2 [41 , 42] . Mice have been generated with mutations in many of the PARK genes but , surprisingly , spontaneous degeneration of dopamine neurons and motor function deficits have not been observed in these animals . The forkhead transcription factors are targets of the PI3K/Akt signaling pathway that is an important regulator of cell growth and survival [43] . Growth factors proposed as treatments for Parkinson disease also act through this pathway [44] . Recently , genes associated with human parkinsonism have been shown to be regulators of the PI3K/Akt pathway [45 . 46] . The clear cell loss reported here suggests that FOXA2 is the transcriptional effector of dopamine neuron survival . It will be interesting to analyze potential interactions between FOXA2 , mutant PARK genes , and neurotrophic factors . This spontaneous model is important because it will allow us to understand the dynamics of dopamine neuron degeneration . In the SN of Parkinson patients , the RALDH1-expressing ventral dopamine neurons are particularly vulnerable to the etiology of Parkinson disease [47] . An initial asymmetric motor deficit , an asymmetric dopamine loss measured by positron emission tomography ( PET ) scanning , and dopamine neuron pathology are characteristic features of Parkinson disease [31 , 48] . Most studies on whole animals that address Parkinson disease use specific toxins to kill dopamine neurons [49] . Toxins are valuable , and they can specifically target the ventral tier neurons at risk in Parkinson disease , but they inadequately address the progressive nature of the disease and the increased sensitivity with age [50] . foxa2+/− animals give us access to the normal mechanisms that initiate and sustain loss of neurons in the SN . There would be great clinical benefit if the progression of disease could be slowed when patients are first diagnosed . We present data that address the related questions: how to make dopamine neurons efficiently in the laboratory and how to support their survival in the brain . We present a precise map of the distinct origin of dopamine neurons that is fundamental to solving a major current problem in cell therapies for Parkinson disease , the identity of dopamine neurons in the graft . However , our data have a significance that goes beyond the technology of cell therapy . Stem cell biology is often criticized for the slow transition to explicit clinical benefit . The work presented here illustrates how a developmental approach that is implicit in stem cell biology leads to a focus on the mechanisms that control the origin and survival of the cell of interest . In the case of dopamine neurons , our data suggest a central role for the PI3K/Akt/Fox survival pathway in defining the origin of dopamine neurons and developing an effective response to Parkinson disease .
Timed pregnant CD-1 mice ( Charles River Laboratories ) were obtained and embryos were manually dissected and fixed in 4% paraformaldehyde ( PFA ) in PBS , transferred to 20% sucrose overnight , and embedded in O . C . T compound ( Tissue Tek , Sakra ) . 12-μm sections were cut on a Microm MH 500 OM cryostat and processed for immunohistochemistry . Sections stained with the anti-SHH antibody were treated with 50 mM NH4Cl for 20 min at room temperature and washed twice with PBS prior to incubation with the primary antibody . Adult mice were anesthetized with pentobarbital sodium ( Nembutal ) and perfused with 20 ml of PBS followed by 40 ml of 4% PFA . Brains were dissected out , transferred to 20% sucrose overnight , and 40-μm sections were cut on a cryostat and maintained as floating sections in PBS . Immunostaining was visualized on a confocal microscope . All animal work was carried out in accordance with National Institute of Neurological Disorders and Stroke ( NINDS ) Animal Studies Protocols 1204–05 , 1205–05 , and 1247–05 , as approved by the NINDS Animal Care and Use Committee . Cell counts were performed on every sixth section within the midbrain , directly at the microscope . In cases in which cell density made it difficult to easily discern individual cells , cell bodies stained for TH , RALDH1 , and GFAP were matched to their DAPI-labeled nuclei . To estimate the absolute number of midbrain dopamine neurons , we multiplied the total number of cells by six ( sampling frequency ) and made the Abercrombie correction . We measured the nuclei of 50 midbrain dopamine neurons , both in the SN and VTA , and determined the mean nuclear diameter to be 8 . 69 μm , which we used for the Abercrombie correction . For Nissl staining , 40-μm brain sections were washed in distilled water and mounted on slides . Sections were treated with cresyl violet for 5 min and then washed again in distilled water . Slides were then run through an ethanol series ( 50% , 70% , 90% , 95% , and 100% ) and finally dipped in xylene for 2 min , before mounting in Permount . For immunohistochemistry of mouse embryo sections and fixed cells in vitro , the following antibodies were used: β-galactosidase ( 1:500 , rabbit monoclonal; Biogenesis ) ; BrdU ( 1:200 , rat monoclonal; Axyll ) ; FOXA2 , Islet1 , LIM1/2 , NKX2 . 2 , PAX7 , and SHH ( 1:10 , mouse monoclonal; Developmental Studies Hybridoma Bank , University of Iowa ) ; FOXA2 ( 1:4 , 000 , rabbit polyclonal; gift of Ariel Ruiz I Altaba ) ; FOXA2 ( 1:50 , goat polyclonal; Santa Cruz Biotechnology ) ; GATA3 ( 1:200 , mouse monoclonal; Santa Cruz Biotechnology ) ; GFAP ( 1:500 , rabbit polyclonal; DAKO ) ; LMX1b ( 1:4 , 000 , guinea pig polyclonal; gift of T . Jessell ) ; LMX1b ( 1:500 , rabbit polyclonal; gift of T . Muller ) ; nestin ( 1:500 , rabbit polyclonal; McKay lab ) ; NKX2 . 2 ( 1:400 , guinea pig polyclonal; gift of B . Sosa Pineda ) ; NKX6 . 1 ( 1:1 , 000 , rabbit polyclonal; gift of M . German ) ; PTX3 ( 1:400 , rabbit polyclonal; Upstate ) ; RALDH1 ( 1:100 , rabbit polyclonal; gift of Greg Duester ) ; Phox2a ( 1:800 , rabbit polyclonal; gift of J . -F . Brunet ) ; SOX1 and SOX2 ( 1:500 , rabbit polyclonal . gift of R . Lovell-Badge ) ; and tyrosine hydroxylase ( 1:500 , rabbit and sheep polyclonals; Pel-freez ) . E8 . 5 embryos ( 9–13 somites ) were obtained from timed pregnant CD-1 mice ( Charles River Laboratories ) . Embryos were incubated in N2 medium containing 1% amylase ( Sigma ) for 1 h at 37 °C , to facilitate manual dissection . After removal of ectoderm and head mesenchyme , embryonic midbrains were moved to amylase-free N2 medium . Pooled tissue was washed 3× in PBS and then digested in N2 medium containing 0 . 005% Trypsin-EDTA ( Gibco ) for 5 min at room temperature . Dissociated mesencephalic cells were added to N2 medium containing Trypsin Inhibitor ( Sigma ) , 100 ng/ml FGF2 , 100 ng/ml ( FGF8 ) , and 500 ng/ml Shh ( R&D Systems ) . Three embryo equivalents of mesencephalic cells were plated per well in a 24-well plate ( Costar ) , coated with poly-L-ornithine ( Sigma ) and fibronectin ( R&D Systems ) . Upon cell attachment , the medium was changed to N2 medium ( without trypsin inhibitor ) containing 100 ng/ml FGF8 and shh . Cells were fixed with 4% PFA after 4 d of expansion . To assay cell proliferation , cells were treated with 10 μM BrdU ( Roche ) for 1 h prior to fixation . Four independent experiments were performed in which each condition was analyzed in duplicate wells . CNS explants were dissected from E8 . 5 foxa2−/− embryos and wild-type embryos . Explants were plated directly in N2 medium containing 100 ng/ml FGF8 and 500 ng/ml shh , as described above . After 4 d in culture with mitogens , the explants were grown for four additional days in the absence of exogenous growth factors to promote differentiation . After 4 d of differentiation , the explants were fixed with 4% PFA . For overexpression of foxa2 , E10 . 5 embryonic midbrain cells were harvested and plated in a manner identical to the E8 . 5 experiments , above , with the exception that two embryo equivalents of mesencephalic cells were plated per well of a 24-well plate ( Costar ) . Cells were transfected , using the lipofectamine reagent ( Invitrogen ) , with a CMV-foxa2 vector ( generous gift of R . J . Matusik ) and with a GFP expression vector ( gift of T . Misteli , National Cancer Institute ) . The mouse embryonic stem cell line , F4 , which expresses foxa2 inducibly , under the control of doxycycline , will be described in greater detail elsewhere ( A . Kuzmichev and R . D . G . McKay , unpublished data ) . Briefly , these cells were generated by homologous recombination into the HPRT locus using a vector modified from a previously described vector [51] in which the oct4 transgene was replaced by foxa2 . We differentiated the mES cells to neurons using a method modified from a previously described protocol [52] . Approximately 50 , 000 F4 cells were plated per well of a fibronectin-coated , 24-well plate into N2 medium . The cells were differentiated for 28 d in N2 , in the absence of exogenous growth factors . foxa2 was induced using doxycycline from days 6–23 of culture ( no induction for the first and last 5 d of the differentiation ) . Spontaneous motor activity was analyzed using the Versamax Animal Activity Monitoring System ( AccuScan Instruments ) . Animals were placed into the 16 . 5” by 16 . 5” chamber for 60 min where vertical and horizontal movements detected by infrared beams were analyzed by VersaMax software . When wild-type and mutant mice were compared , there was no simple difference in the time spent in the center of the chamber . For amphetamine-induced rotation , mice were injected with 2 . 5 mg/kg of amphetamine intraperitoneally , and clockwise and counterclockwise rotations were measured using the Rota-count 8 system ( Columbus Instruments ) for 60 min . | The restoration of dopamine neurons is a major focus of stem cell biology and regenerative medicine . The gradual loss of these neurons is a hallmark of Parkinson disease . Dopamine neurons in the midbrain convey important sensory and motor functions to the forebrain . We show that the transcription factor FOXA2 plays a central role in the birth and death of dopamine neurons in the midbrain . By defining their precursors in the ventral midbrain , we show that dopamine neurons are derived from organizer cells in the floor plate ( the ventral cells of the neural tube , the embryonic foundation of the central nervous system ) . We also show that FOXA2 specifies the floor plate and induces the birth of dopamine neurons . Mice with only a single copy of the foxa2 gene acquire motor deficits and a late-onset degeneration of dopamine neurons . This spontaneous cell death preferentially affects neurons associated with Parkinson disease . This work provides new strategies to generate neurons in the laboratory and to block their death in old age . | [
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] | [
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] | 2007 | The foxa2 Gene Controls the Birth and Spontaneous Degeneration of Dopamine Neurons in Old Age |
Genome-wide association studies ( GWAS ) have identified ∼100 loci associated with blood lipid levels , but much of the trait heritability remains unexplained , and at most loci the identities of the trait-influencing variants remain unknown . We conducted a trans-ethnic fine-mapping study at 18 , 22 , and 18 GWAS loci on the Metabochip for their association with triglycerides ( TG ) , high-density lipoprotein cholesterol ( HDL-C ) , and low-density lipoprotein cholesterol ( LDL-C ) , respectively , in individuals of African American ( n = 6 , 832 ) , East Asian ( n = 9 , 449 ) , and European ( n = 10 , 829 ) ancestry . We aimed to identify the variants with strongest association at each locus , identify additional and population-specific signals , refine association signals , and assess the relative significance of previously described functional variants . Among the 58 loci , 33 exhibited evidence of association at P<1×10−4 in at least one ancestry group . Sequential conditional analyses revealed that ten , nine , and four loci in African Americans , Europeans , and East Asians , respectively , exhibited two or more signals . At these loci , accounting for all signals led to a 1 . 3- to 1 . 8-fold increase in the explained phenotypic variance compared to the strongest signals . Distinct signals across ancestry groups were identified at PCSK9 and APOA5 . Trans-ethnic analyses narrowed the signals to smaller sets of variants at GCKR , PPP1R3B , ABO , LCAT , and ABCA1 . Of 27 variants reported previously to have functional effects , 74% exhibited the strongest association at the respective signal . In conclusion , trans-ethnic high-density genotyping and analysis confirm the presence of allelic heterogeneity , allow the identification of population-specific variants , and limit the number of candidate SNPs for functional studies .
Genome-wide association studies ( GWAS ) have identified many common genetic variants associated with human diseases and complex traits ( www . genome . gov/gwastudies ) , including ∼100 loci associated with triglycerides ( TG ) , high-density lipoprotein cholesterol ( HDL-C ) , low-density lipoprotein cholesterol ( LDL-C ) , or total cholesterol [1]–[5] . A majority of the lead SNPs at these loci have shown small effect sizes , leaving much of the trait heritability unexplained . Some of this missing heritability may be due to the incomplete coverage of functional common or rare variants and the poor representation of appropriate proxies on commercial genotyping arrays [6] , [7] . Other missing heritability may result from a failure to detect the full spectrum of causative variants present at GWAS-identified loci . Fine-mapping of GWAS signals should increase the power to detect variants that influence trait variability . Genotyping of additional variants at GWAS loci can identify SNPs with stronger evidence of association than the reported GWAS index SNPs and may help detect or further localize the underlying causal variants [7] , [8] . The Metabochip is a high-density custom genotyping array designed to replicate and fine-map known GWAS signals for metabolic and atherosclerotic/cardiovascular endpoints , and more extensively , to identify all signals around the index SNPs [9] , [10] . The fine-mapping SNPs spanned a wide range of allele frequencies including rare ( minor allele frequency ( MAF ) <0 . 005 ) and less common ( 0 . 005≤MAF<0 . 05 ) SNPs selected from the catalogs of the International HapMap Project and the August 2009 release of the 1000 Genomes Project . SNPs annotated as nonsynonymous , essential splice site or stop codon were included regardless of MAF , design score , or the presence of nearby SNPs [10] . The Metabochip contains densely spaced SNPs at 18 , 22 , and 18 loci previously reported for TG , HDL-C , and LDL-C , respectively . Allelic heterogeneity , in which different variants at the same gene/locus affect the same phenotype , is a frequent characteristic of both single-gene and complex disorders . Recently GWAS have identified more than one independent signal at loci associated with coronary artery disease [11] and type 2 diabetes [12] , [13] . Among a set of 30 lipid loci reported through GWAS , secondary SNPs that exhibited weak to moderate LD with the corresponding index SNPs and displayed little change of association in conditional analyses were detected at seven loci including CETP , LIPC , APOA5 , APOE , LDLR , ABCG8 , and LPL [4] . More than one association signal also was detected at 26 of 95 lipid loci reported by the Global Lipids Genetics Consortium [5] . However , allelic heterogeneity has not been comprehensively evaluated for common traits including lipid traits across ethnically diverse populations , especially in non-European populations such as African Americans and East Asians . Due to divergent evolutionary and migratory histories , patterns of linkage disequilibrium ( LD ) vary across ancestry groups [14] . Greater haplotype diversity in some ancestry groups , especially in African ancestry populations , may facilitate the localization of functional variants that show association signals delimited in part due to weaker LD with neighboring SNPs [14] , [15] . A recent multi-ethnic analysis of lipid associated loci demonstrated that genetic determinants at many lipid loci differed between European Americans and African Americans [16] . For example , in African Americans from the PAGE consortium [9] , [17] , a reported regulatory variant rs12740374 at CELSR2/PSRC1/SORT1 locus [18] was more strongly associated with LDL-C compared to many nearby variants demonstrating similar strength of association in European ancestry individuals [5] . High-density genotyping enables trans-ethnic fine-mapping studies to narrow the set of plausible candidate functional variants at GWAS loci without introducing uncertainty through imputation [19] . In this study , we analyzed high-density genotyped SNPs on the Metabochip for their associations with TG , HDL-C , and LDL-C in 6 , 832 African Americans , 9 , 449 East Asians , and 10 , 829 Europeans at 58 known lipid loci . We sought to ( i ) identify the variants with the strongest evidence of association at each locus in populations with different ancestries and in the combined trans-ethnic samples; ( ii ) investigate allelic heterogeneity and population-specific signals at the established lipid loci; ( iii ) explore whether high-density genotyping in diverse ethnic populations would narrow the sets of plausible candidate functional variants for further study; and ( iv ) assess whether the variants reported to have functional effects on gene expression or protein function during the past 30 years of biological study exhibited the strongest evidence of association at the corresponding GWAS signals .
Descriptions of the collection , phenotyping , and genotyping of study samples for each study site are provided in Table S1 . Given that all 58 loci have a priori genome-wide significant evidence of association with one or more of these three lipid traits , we used a P value threshold of 1×10−4 as an approximate correction for the mean of 451 SNPs tested at each locus in African Americans ( Table S2 ) . An average of 273 SNPs per locus was tested in East Asians and an average of 291 in Europeans , but we applied the same , more conservative , P value threshold of 1×10−4 to these two groups as well . A total of 33 loci ( nine for TG , 14 for HDL-C , and 10 for LDL-C ) exhibited evidence of association at P<1×10−4 in at least one of the three ancestry groups , including 22 loci in African Americans , 17 in East Asians , and 31 in Europeans ( Table S3A–S3C ) . The variants that reached this threshold of significance were common ( MAF≥0 . 05 ) , except at three loci ( PCSK9 and ABO for LDL-C , and APOA5 for HDL-C ) in African Americans and two loci ( PCSK9 and TOP1 , both for LDL-C ) in European ancestry individuals . When individuals of diverse ancestry groups were combined , 11 , 15 , and 12 loci showed evidence of significant association with TG , HDL-C , and LDL-C , respectively ( Table S4A–S4C ) . Among these 38 loci , six loci had not reached the P value threshold of 10−4 within any individual ancestry group , including CETP and NAT for TG , GALNT2 and MMAB for HDL-C , and TRIB1 and TIMD4 for LDL-C . One locus , COBLL1 , was significantly associated with HDL-C in Europeans alone ( P = 8 . 5×10−5 ) , but displayed less evidence of association in the combined trans-ethnic samples ( P = 1 . 6×10−4 ) . To assess the presence of two or more signals at each locus that exhibited evidence of association in at least one ancestry group , we performed sequential conditional analyses by adding the most strongly associated SNP to the regression model as a covariate and testing the association with each of the remaining regional SNPs independently . A set of sequential conditional analyses were followed by inclusion of the strongest SNP in each conditional model until the most strongly associated SNP showed a conditional P value>10−4 and was not annotated as a nonsense or nonsynonymous substitution . We also investigated whether association signals were population-specific , which we defined as association signals with variants that are not variable in the samples from the other two ancestry groups in this study or in the 1000 Genomes Project populations that represent those groups among total European ancestry ( EUR ) , total East Asian ancestry ( ASN ) , or total west African ancestry ( AFR ) . In African Americans , sequential conditional analyses revealed that 10 of the 22 loci with evidence of association exhibited two or more signals at P<10−4 ( Table 1 ) . Two loci ( PCSK9 and the TOMM40-APOE-APOC4 cluster; both for LDL-C ) each had seven signals , four loci ( APOB for LDL-C , LDLR for LDL-C , LCAT for HDL-C , and CETP for HDL-C ) had three signals , and another four loci ( APOB , APOC1 , APOA5 , and LPL; all for TG ) had two signals . Among the 10 loci with two or more signals , all these signals led to an average 1 . 8-fold increase in the amount of phenotypic variance ( R2 ) compared to that explained by the strongest signals alone ( See Method ) in African Americans . Among these 34 signals , 15 were represented by less common ( 0 . 005≤MAF<0 . 05 , n = 11 ) or rare ( MAF<0 . 005 , n = 4 ) variants . In addition , 15 signals at eight loci were African American-specific . If we only include SNPs that meet a locus-specific P-value threshold based on the number of genotyped SNPs ( Table S2 ) , LPL for TG and APOB for both TG and LDL each had one signal , and the seven loci with multiple signals still showed an average of 1 . 8-fold increase in the explained phenotypic variance . The seven signals at PCSK9 in African Americans included six nonsense or nonsynonymous variants previously shown to associate with LDL-C levels and to affect PCSK9 expression or function [20]–[22] , along with an unreported intronic variant ( Table 1 ) . The strongest signals were a nonsense variant rs28362286 ( C679X , Figure 1A ) and a nonsynonymous variant rs28362263 ( A443T , Figure 1B ) , which showed no reduction of association evidence when conditioned on C679X . Conditional analysis on both C679X and A443T yielded a third signal at rs28362261 ( N425S , Figure 1C ) ; and further conditional analyses successively implicated rs67608943 ( Y142X , Figure 1D ) , rs72646508 ( L253F , Figure 1E ) , and an intronic variant rs11800243 ( Figure 1F ) . The seventh signal , which did not reach the Pconditional<10−4 threshold , was represented by the nonsynonymous variant rs11591147 ( R46L , Figure 1G ) that exhibited the strongest and directionally consistent evidence of association with LDL-C in Europeans ( Pinitial = 2 . 8×10−30 , Table 2 ) . The seven signals were weakly correlated with each other in African American individuals , and all pairwise LD r2 values were less than 0 . 02 . Among the seven PCSK9 signals , the top five were African American-specific , and six were either less common or rare in African Americans . The lead SNP C679X accounted for 1 . 3% of the explained LDL-C phenotypic variance and the seven signals together explained 3 . 6% of the phenotypic variance in African Americans . PCSK9 exhibited two signals in Europeans ( R46L and rs2495477 , Table 2 ) , but no SNP reached Pinitial<10−4 in East Asians . At the TOMM40-APOE-APOC4 cluster , the seven signals in African Americans explained 6 . 6% of the LDL-C phenotypic variance compared to 4 . 1% explained by the strongest signal R176C , which had reported functional effects [23] ( Table 1 , Figure S1 ) . These seven signals were not entirely independent of one another . The fourth signal , rs157588 , showed association with LDL-C ( P = 2 . 0×10−7 ) only after conditioning on the top three signals , but not in the original unconditioned association analysis ( P = 0 . 72 ) . The trait-decreasing allele ( G allele: freq = 0 . 176 ) of rs157588 was present on haplotypes containing the trait-increasing allele of the third signal rs1038026 ( A allele: freq = 0 . 351 ) , thus the association of the fourth signal increased in significance after accounting for linkage disequilibrium ( r2/D′ = 0 . 35/0 . 92 ) with the third signal at the same locus . Haplotype analysis revealed that compared to the reference A-A ( increasing-increasing ) haplotype , the G-G ( decreasing-decreasing ) haplotype only displayed modest association with LDL-C ( P = 7 . 5×10−3 ) , but the A–G ( rs1038026 increasing- rs157588 decreasing ) haplotype showed significant association with decreased level of LDL-C ( P = 1 . 5×10−10 ) ( Table S5 ) . In Europeans ( Table 2 ) and East Asians ( Table 3 ) , three and two signals were identified at TOMM40-APOE-APOC4 , respectively . The known functional variant R176C exhibited the strongest evidence of association across the three ancestry groups , with effect sizes of −0 . 536 , −0 . 505 , and −0 . 411 mmol/L in individuals of African American , European , and East Asian ancestry , respectively ( Table 1 ) . However , another APOE variant rs429358 ( C130R ) , that together with R176C , defines the three major isoforms of APOE ( ε2 , ε3 , and ε4 ) [7] , [24] , was not successfully genotyped , therefore the LDL-C association with either C130R or the APOE haplotype was unavailable in this study . In Europeans , 21 signals at nine of the 31 loci exhibited multiple signals for at least one of the three lipid traits at P<10−4 ( Table 2 ) . Three loci ( APOA5 for TG , TOMM40-APOE-APOC4 cluster for LDL-C , and CETP for HDL-C ) each had three signals while another six loci ( PCSK9 for LDL-C , GCKR for TG , LIPC for HDL-C , APOB for LDL-C , and LPL for both TG and HDL-C ) each had two signals . At the nine loci that had two or more signals , all association signals resulted in an average of 1 . 3-fold increase in the explained phenotypic variance compared to the strongest signals alone across loci . At PCSK9 , rs11591147 ( R46L ) exhibited the strongest evidence of association in Europeans . As reported above , R46L also represented the seventh signal in African Americans . R46L accounted for 1 . 2% of the total variation in LDL-C levels in Europeans compared the 0 . 16% in African Americans . This SNP was not variable in the 1000 Genomes Project ASN samples ( East Asian ancestry ) and the >9 , 000 East Asian individuals in this study . In East Asians , we observed three signals at the TG locus APOA5 , and two signals at three loci including TOMM40-APOE-APOC4 cluster for LDL-C , CETP for HDL-C , and ABO for LDL-C ( Table 3 ) . At the four loci that exhibited multiple signals , all the association signals increased the explained phenotypic variance by an average of 1 . 3-fold compared to the strongest signal across loci . The second signal at APOA5 was the nonsynonymous variant G185C previously reported to affect the protein function [25] . Although G185C was not unique to East Asians , the frequency was very low in African Americans ( MAF = 0 . 002 , P = 0 . 028 ) and Europeans ( MAF = 0 . 0003 , P = 0 . 23 ) , and the low allele frequency meant that this study had less than 5% statistical power to detect the association in these groups . At APOA5 , which exhibited multiple signals in all three populations ( Table 1 , Table 2 , Table 3 ) , the strongest TG-associated SNPs differed and were not in high LD ( r2<0 . 8 ) with each other in any of the ancestry groups . In African Americans , the two signals S19W ( MAF = 0 . 058 , P = 8 . 4×10−15 ) and rs79624460 ( MAF = 0 . 083 , P = 4 . 8×10−12 ) , showed no evidence of significant association in East Asians ( Table 1 ) , likely due to the low allele frequency and the limited power ( ∼10% ) to detect the association . The three signals at APOA5 in East Asians were only modestly associated with TG in African Americans ( all P>10−3 , Table 3 ) . The SNP LD r2 values between the African American and East Asian signals were less than 0 . 02 in both populations , suggesting that they represent distinct APOA5 signals in the two ancestry groups . In addition , the APOA5 signal rs3741298 ( P = 9 . 7×10−44 , MAF = 0 . 222 ) in Europeans exhibited evidence of association with TG in African Americans ( P = 9 . 8×10−5 , MAF = 0 . 327 ) and East Asians ( P = 1 . 2×10−20 , MAF = 0 . 357 ) , but the significance levels of the association with rs3741298 were substantially attenuated by conditioning on the strongest signals S19W in African Americans ( P = 0 . 10 ) and rs651821 in East Asians ( P = 0 . 88 ) . In Europeans , the associations with rs3741298 were partially removed when conditioning on S19W and rs651821 ( Pconditional = 1 . 7×10−28 and 3 . 1×10−17 , respectively ) . The European signal rs3741298 was moderately correlated with the African American signal S19W ( LD r2 = 0 . 21 and 0 . 10 in the 1000 Genomes Project EUR samples ( European ancestry ) and in PAGE African American samples , respectively ) , and with the East Asian signal rs651821 ( LD r2 = 0 . 31 and 0 . 28 in 1000 Genomes Project EUR and ASN samples , respectively ) . Notably , the effect sizes of the two reported functional variants S19W [26] and G185C [25] at APOA5 were similar across the three groups ( S19W , African American: 0 . 136; East Asian: 0 . 136; European: 0 . 121 and G185C , African American: 0 . 204; East Asian: 0 . 201; European: 0 . 269 mmol/L in loge scale ) despite the limited power to detect significant evidence of association at low allele frequencies . These findings support the hypothesis that causative variants may have a similar genetic impact on trait variation across populations if not influenced by hidden gene-gene or gene-environment interactions [27] . We also observed that the second European signal rs75919952 exhibited nominal evidence of association ( P initial = 0 . 018 , MAF = 0 . 041 ) , but was not associated with TG in the other two groups ( Table 2 ) . The lack of association may be due to insufficient power ( 15% and 55% in African Americans and East Asians , respectively; assuming α = 0 . 05 ) corresponding to the lower allele frequency ( MAF = 0 . 012 ) in African Americans , the smaller sample sizes in both populations , or underlying interactions . We next examined whether trans-ethnic meta-analysis or comparison across ancestries would refine the association signals by narrowing the genomic regions where functional variants might be expected to reside . The trans-ethnic analysis allowed the refinement of association signals at loci of GCKR , PPP1R3B , ABO , LCAT , and ABCA1 ( Table 4 , Table S3A–S3C ) . The signal at GCKR was localized to the reported functional variant P446L [28] due to the limited LD in African Americans ( Figure S2A–S2D ) . Notably , there were seven and six variants in high LD ( r2>0 . 8 ) with P446L in the 1000 Genomes Project ASN and EUR samples , but no SNP with LD r2>0 . 8 in African American individuals . At the signal ∼200 kb from the PPP1R3B gene for which no functional regulatory variant ( s ) have been reported , the association signal was narrowed from 4 SNPs spanning 36 kb ( P<10−4 ) in Europeans to two highly correlated SNPs located 1 kb apart in African Americans ( rs6601299 , P = 8 . 0×10−8 and rs4841132 , P = 2 . 9×10−7; LD r2>0 . 94 ) ( Figure 2 ) . The lead SNP rs6601299 was in high LD with 11 variants in the 1000 Genomes Project EUR samples but only highly correlated with two and one variant in the 1000 Genomes Project AFR samples ( West African ancestry ) and PAGE African American individuals , respectively . At the ABO locus , trans-ethnic meta-analysis revealed six SNPs exhibiting stronger evidence of association ( P<1 . 1×10−11 ) with LDL-C compared to other variants in the same region ( P>2 . 3×10−7 ) ( Figure S3A–S3D ) . At the locus LCAT for HDL-C , the association signals spanned ∼800 kb , ∼360 kb , and ∼360 kb in Europeans , East Asians , and African Americans , with a ∼50 kb overlapping region . Trans-ethnic meta-analysis of all samples localized the signal to four variants spanning this 50 kb region ( Figure S4A–S4D ) . At HDL-C locus ABCA1 , the reported GWAS index SNP rs1883025 consistently showed the strongest association within each of the three ancestry groups that we examined , but the significance level of the association was similar to those of the nearby SNPs . Trans-ethnic meta-analysis refined the signal by revealing that rs1883025 ( P = 4 . 3×10−17 ) and rs2575876 ( P = 1 . 8×10−15 ) displayed much stronger association than the neighboring SNPs ( P>8 . 4×10−10 ) ( Figure S5A–S5D ) . Among loci associated with at least one lipid trait ( P<10−4 ) , at least 27 variants at 15 loci have been previously reported [18] , [22] , [23] , [25] , [26] , [28]–[47] to functionally influence gene expression or protein function in vitro ( Table 5 ) . Among the 27 variants , 17 are present on the Metabochip and two are well-represented by perfect proxies in complete LD ( r2 = 1 ) based on the 1000 Genomes Project EUR data . Of the 19 reported functional variants , 14 ( 74% ) exhibited the strongest association P-value among all SNPs at that signal in at least one population . In addition , two more reported functional variants ( APOB-rs7575840 , P = 7 . 0×10−17 and LPL-rs328 , P = 2 . 3×10−11 ) were in high LD ( r2>0 . 95 ) with the most strongly associated variants and showed similar evidence of association ( APOB-rs934198 , P = 3 . 7×10−17; LPL-rs1803924 , P = 1 . 1×10−11 ) . If we include these two variants , then 16 of the 19 ( 84% ) reported functional variants displayed the strongest association P-value at the primary , secondary , or successive signals . The remaining three reported functional variants: LDLR-rs688 ( N591N ) , LPL-rs1801177 ( D9N ) , and HMGCR-rs3761740 ( 911C>A ) , were poorly tagged ( LD r2<0 . 2 ) by the strongest variants in our data . Additional functional variants may exist at these loci that have not yet been reported to change gene expression/protein function or that were not identified in our literature search . For example , P2739L and P145S that represented the two signals at APOB ( Table 1 ) were predicted by PolyPhen [48] to be ‘probably damaging’ with a score of ‘1’ , although their functional roles were unclear . Among the 16 reported functional variants and proxies that exhibited the strongest association P-value at a signal ( Table 5 ) , R176C at APOE was strongest in all three populations and GCKR L446P was identified in both African Americans and Europeans . The remaining 14 variants showed the strongest associations in only one of the populations , including 10 in African Americans , three in East Asians , and one in Europeans . Five of the 10 variants in African Americans were at the PCSK9 locus . Furthermore , nine of the 16 variants represented the strongest signal at a given locus , three for a 2nd signal , and four for the 3rd or additional signals . These functional variants covered a wide allele frequency spectrum ( MAF: 0 . 003–0 . 481 ) , including five less common or rare variants observed only in African Americans .
This study evaluated densely spaced SNPs at 58 lipid loci across three ancestrally diverse populations . The results support evidence that allelic heterogeneity is a frequent feature of polygenic traits [5] , [49] and extend the findings to non-European populations , especially to African ancestry populations that have high levels of haplotype diversity . The results also provide strong evidence that fine mapping at GWAS loci can identify population-specific signals . Despite comparable sample sizes , we identified more signals per locus and more signals overall in African Americans ( 34 signals at 10 loci ) compared to Europeans ( 21 signals at nine loci ) and East Asians ( nine signals at four loci ) , and 15 of the 34 signals identified in African Americans were population-specific ( Table 1 , Table 2 , Table 3 ) . These observations may reflect the larger number of SNPs genotyped in African Americans ( Table S2 ) , variation across populations subject to natural selection during human evolution [14] , or genetic drift [50] . Due to the varied number of signals per locus , different associated markers , and different effect sizes , the phenotypic variance explained differs across populations [51]–[53] . Sampling variability , epistasis , and gene-environment interactions may cause over- or under-estimation of the proportion of explained phenotypic variance . In this study , we also observed that many population-specific signals , including those at PCSK9 and APOA5 , are largely confirmatory [20] , [22] , [54]; however , the association evidence at other signals , in particular the additional signals at APOE , LDLR , and APOC1 identified by the conditional analyses , requires replication in future studies . At PCSK9 , the strongest signal C679X identified in African Americans is population-specific and showed substantially stronger evidence of association with LDL-C ( P = 4 . 1×10−22 ) compared to the GWAS index SNP rs2479409 [5] ( P = 0 . 12 ) and the most strongly associated SNP R46L identified via fine-mapping [7] ( P = 2 . 3×10−3 ) , both of which were previously reported in Europeans . The proportion of phenotypic variance explained in African Americans increased from 0 . 16% by the GWAS index SNP to 1 . 3% by the Metabochip signal C679X , and all variants at the locus together explained 3 . 6% of the total variation in LDL-C , providing evidence that heritability at identified loci may be underestimated by GWAS [7] . A limitation of these variance estimates is that calculations included the SNPs based simply on their significant association P values rather than the variants with biological function , which could over-estimate effects due to the winner's curse . Results across the genotyped loci demonstrated that the majority of signals were represented by common variants , yet high-density genotyping also identified less common and rare variants associated with lipid traits . At PCSK9 , the MAFs of six out of the seven signals were <0 . 05 in African Americans . These signals , along with other low frequency variants identified at APOE , LDLR , LCAT , APOB , APOC1 , and LPL provide evidence of the substantial contribution of low frequency genetic variants to the variance of lipid traits [6] . Other variants , some with very low allele frequency , may exist at these loci , suggesting that future sequencing studies may identify additional functional variants that influence lipid variation . Sequential conditional analyses provided further insight into the genetic architecture of the established lipid loci by explaining additional phenotypic variation and revealing complex patterns of association . We observed loci at which signals were not independent of each other , but partially correlated based on moderate LD estimates and changes of association statistics before and after accounting for other signals . For these dependent signals , such as those at TOMM4-APOE-APOC4 , the significance of residual association would increase when trait-increasing alleles were present on opposite haplotypes and decrease when trait-increasing alleles were on the same haplotype . Other signals that appeared to be independent on the basis of low pairwise LD and unchanged association evidence after conditional analysis may still be partially tagging an un-typed , yet influential , variant [55]–[57] . Therefore , deeper sequencing that identifies all variants at a locus will be required to characterize more fully the allelic heterogeneity and the patterns of association . One of the major goals of high-density genotyping is to aid in identification of the functional variants by recognizing the most compelling candidate variants for experimental study . Because of the diverse LD structure across populations , particularly in terms of the limited LD extent in African ancestry populations , trans-ethnic fine-mapping of GWAS loci can narrow the region where functional variants are most likely to reside . This study was able to narrow the association signals at five lipid loci , based on the much smaller subsets of most strongly associated variants located in smaller regions . One signal was localized to a reported causal variant ( GCKR-P446L ) [28] and another to an uncharacterized nonsynonymous variant ( SLC12A4-E4G near LCAT ) . These findings demonstrate that trans-ethnic association analyses can increase the resolution of fine-mapping by enlarging the haplotypic diversity of samples with different ancestries and consequently , narrowing the sets of candidate functional variants [58] , [59] . The previously described functional variants at LCAT [44] and ABCA1 [42] , [43] , which are not present on the Metabochip , were physically located 22 kb and >43 kb away from the narrowed association signals observed in this study ( Table 4 ) . Refining signals by trans-ethnic meta-analysis largely relies not only on the existence of distinct LD patterns across ancestry groups but also on shared functional variants . If functional variants are shared across populations , as observed with GCKR-P446L , performing trans-ethnic meta-analysis and integrating LD information across different populations may refine the signal . On the contrary , if trait variation is influenced by distinct functional variants across populations , as our data suggest for APOA5 ( Figure S6A–S6D ) , the lead SNPs produced by meta-analysis would be influenced by the sample size , magnitude of genetic effects , and allele frequencies . Similarly , in the case of population-specific functional variants , such as those at PCSK9 , the results from meta-analysis would reflect the association in one particular population rather than the combined effect across populations if signals unique to this population drive the results . Therefore , accurate assessment of allelic variability is needed on a population-by-population and locus-by-locus basis . Although genotype imputation has become a standard practice to increase genome coverage in GWAS by predicting the genotypes at SNPs that are not directly genotyped , imputation accuracy tends to be lower for rare variants owing to the lower degree of LD and the more challenging haplotype reconstruction [60] . In addition , African American samples pose a challenge for imputation due to their varying degree of admixture [61] . A major strength of our study is that all variants we tested for association were directly genotyped using the Metabochip , which was designed to provide a high-density coverage for both overall SNPs and low frequency variants concentrated around GWAS-identified loci and/or signals [9] , [10] . This approach increases the reliability of our association results overall , but in particular the variants with low allele frequencies . In conclusion , we performed a large-scale trans-ethnic fine-mapping study to investigate the established lipid loci using the Metabochip high-density genotyping array and focusing on diverse groups including African Americans , East Asians , and Europeans . Our results highlight the value of high-density genotyping in diverse populations to identify a wider spectrum of susceptibility variants at established loci , both in terms of additional signals and in terms of population-specific and/or potentially functional variants . The additional signals revealed through the sequential conditional analyses lead to a 1 . 3- to 1 . 8-fold increase in the explained phenotypic variance across the different populations . In addition , integrating diverse LD patterns across diverse ancestry groups allows for the refinement of association signals . Lastly , our findings that 74% of the reported functional variants exhibited the strongest association at these densely typed signals suggest that at loci and signals where functional variants are unknown , the variants with strongest association may be good candidates for functional assessment .
The 6 , 832 African Americans studied are comprised of individuals from the Atherosclerosis Risk in Communities Study ( ARIC ) [62] , the Multiethnic Cohort Study ( MEC ) [63] , and the Women's Health Initiative ( WHI ) [64] , [65] that are part of Population Architecture using Genomics and Epidemiology ( PAGE ) consortium [66] and from Hypertensive Genetic Epidemiology Network ( HyperGEN ) [67] . The 9 , 449 East Asian samples are comprised of 1 , 716 Filipinos from the Cebu Longitudinal Health and Nutrition Survey ( CLHNS ) [68] and 7 , 733 Chinese from Taiwan-Metabochip Study for Cardiovascular Disease ( TAICHI ) . The 10 , 829 European samples are comprised of Finnish and Norwegian individuals; the Finns are from the Finland-United States Investigation of NIDDM Genetics ( FUSION ) , Dehko 2D 2007 ( D2D2007 ) , Diabetes Prevention Study ( DPS ) , Dose-Responses to Exercise Training ( DR's EXTRA ) , and Metabolic Syndrome in Men ( METSIM ) [69] , [70] , and the Norwegians were from the cohorts of Nord-Trøndelag Health Study ( HUNT 2 ) and the Tromsø Study ( TROMSO ) [71] , [72] . All study protocols were approved by Institutional Review Boards at their respective sites . Brief descriptions of the studies are provided in the Text S1 . General characteristics and measurements of TG , HDL-C , and LDL-C in each cohort are summarized in Table S1 . Values of triglycerides were natural log transformed to approximate normality in each study sample separately . We genotyped all study samples with the Metabochip according to the manufacturer's protocol ( Illumina , San Diego , CA , USA ) . Table S1 summarizes the quality control criteria of genotyping , including call rate , sample success rate , Hardy-Weinberg equilibrium , and MAF that varied across studies . We applied multiple linear regression models and assumed an additive mode of inheritance to test for association between genotypes and HDL-C , LDL-C , or log-transformed triglycerides . We performed each test of association separately in each of the 11 groups ( Table S1 ) prior to meta-analysis . We constructed principal components ( PCs ) using the software EIGENSOFT . We used age and sex as covariates in each individual cohort; other cohort-specific covariates including age2 , enrollment site , socioeconomic status , and principal components varied across studies ( Table S1 ) . The European samples include type 2 diabetes ( T2D ) cases and unaffected controls; to avoid confounding due to T2D status , samples were analyzed separately as Finnish T2D patients , Finnish unaffected individuals , Norwegian T2D patients , and Norwegian unaffected individuals . We first conducted the meta-analysis within the African Americans , East Asians , and Europeans separately . We then performed combined trans-ethnic meta-analyses by combining the statistics of each the 11 participating groups to assess the association with the SNPs at the 58 lipids loci . At loci that exhibited evidence of association at P<10−4 , we next performed a series of sequential conditional analyses by adding the most strongly associated SNP into the regression model as a covariate and testing all remaining regional SNPs for association . We conducted a set of sequential conditional analyses until the strongest SNP showed a conditional P value>10−4 and had no annotation or literature evidence that suggested a functional role . For single SNP analyses , we applied PLINK ( http://pngu . mgh . harvard . edu/~purcell/plink/ ) [73] for population-based studies . We used the R package GWAF [74] for the family-based study of HyperGEN . We applied an inverse variance-weighted fixed-effect meta-analysis implemented in METAL [75] . Unless otherwise noted , linkage disequilibrium estimates were obtained from the 1000 Genomes Project November 2010 release . SNP positions correspond to hg18 . We performed haplotype analysis at LDL-C locus TOMM40-APOE-APOC4 in 5 , 593 unrelated African Americans from the PAGE consortium , using the ‘haplo . stat’ R package . Haplotypes and haplotype frequencies were estimated using the R function ‘haplo . em’ . The association between haplotypes and LDL-C was assessed using the R function ‘haplo . glm’ . An additive model was assumed , in which the regression coefficient β represents the expected change in LDL-C level with each additional copy of the specific haplotype compared with the reference haplotype , which was set as the A-A ( trait increasing-increasing ) haplotype . We created the regional association plots using LocusZoom [76] . To plot the association results in Europeans and East Asians , we used the LocusZoom-implemented LD estimates from the 1000 Genomes Project ( June 2010 ) CEU and CHB+JPT samples , whose LD structures are similar to our samples with European and East Asian ancestries . We applied the user-supplied LD calculated from the genotype data of the PAGE African American samples to plot the regional association in African Americans [9] , because the LD patterns may vary from any pre-computed LD sources implemented in LocusZoom . We evaluated the proportion of variance explained by a single SNP or any given locus by including the SNP or a set of SNPs into a linear regression model with all covariates used in association analysis and calculating the R2 for the full model . We subtracted the variance explained by a basic model in which only covariates were included from the variance we obtained from the full model . We performed these analyses using SAS version 9 . 2 ( SAS Institute , Cary , NC , USA ) . | Lipid traits are heritable , but many of the DNA variants that influence lipid levels remain unknown . In a genomic region , more than one variant may affect gene expression or function , and the frequencies of these variants can differ across populations . Genotyping densely spaced variants in individuals with different ancestries may increase the chance of identifying variants that affect gene expression or function . We analyzed high-density genotyped variants for association with TG , HDL-C , and LDL-C in African Americans , East Asians , and Europeans . At several genomic regions , we provide evidence that two or more variants can influence lipid traits; across loci , these additional signals increase the proportion of trait variation that can be explained by genes . At some association signals shared across populations , combining data from individuals of different ancestries narrowed the set of likely functional variants . At PCSK9 and APOA5 , the data suggest that different variants influence trait levels in different populations . Variants previously reported to alter gene expression or function frequently exhibited the strongest association at those signals . The multiple signals and population-specific characteristics of the loci described here may be shared by genetic loci for other complex traits . | [
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] | 2013 | Trans-Ethnic Fine-Mapping of Lipid Loci Identifies Population-Specific Signals and Allelic Heterogeneity That Increases the Trait Variance Explained |
We present a selection design that couples S-adenosylmethionine–dependent methylation to growth . We demonstrate its use in improving the enzyme activities of not only N-type and O-type methyltransferases by 2-fold but also an acetyltransferase of another enzyme category when linked to a methylation pathway in Escherichia coli using adaptive laboratory evolution . We also demonstrate its application for drug discovery using a catechol O-methyltransferase and its inhibitors entacapone and tolcapone . Implementation of this design in Saccharomyces cerevisiae is also demonstrated .
Methylation is the transfer of a methyl group from one molecule to another . Its importance in gaining bioactivity and acquiring bioavailability of drugs has been recognized by chemists for some time [1] . Chemical methylation ordinarily utilizes noxious reagents and generates toxic waste and often lacks regioselectivity [2] . In contrast , enzymatic methylation is specific , environmentally friendly , and safer to work with . Most methylation in cells takes place by S-adenosylmethionine–dependent methyltransferases ( SAM-dependent Mtases ) , using SAM as the methyl donor . SAM-dependent methylation is involved in many important biological processes , including epigenetics and synthesis of a wide range of secondary metabolites ( e . g . , flavonoids , neurotransmitters , antibiotics ) . In fact , SAM is one of the most commonly used cofactors in cellular metabolism , second only to ATP [3] . SAM-dependent Mtases have become an important enzyme category , used either as biocatalysts , as part of fermentative production pathways in biotechnical and chemical industries [2–4] , or as drug targets in the pharmaceutical industry [5 , 6] . Implementing and engineering functional SAM-dependent Mtases is difficult since all existing assays lack robustness , are not cost effective , and are not generalizable to all types of Mtases or high throughput . Consequently , study and engineering of Mtases or building efficient methylation-dependent pathways is hard to achieve . We designed an in vivo synthetic selection system by coupling SAM-dependent methylation to growth via a homocysteine intermediate of the SAM cycle . This selection system design was first implemented in E . coli by deleting serine acetyltransferase ( cysE ) to prevent endogenous homocysteine and cysteine synthesis . We diverged homocysteine of the SAM cycle to cysteine using heterologously expressed yeast cystathionine-β-synthase ( Cys4 ) and cystathionine-γ-lyase ( Cys3 ) ( Figs 1 and S1 ) . Effectively , this design couples methylation to the conversion of exogenous methionine to the biosynthesis of cysteine , a required amino acid for growth . This growth-coupled design was computationally validated using a genome-scale metabolic model ( Fig 1 ) .
We demonstrate the use of our system to improve enzyme properties . Using adaptive laboratory evolution ( ALE ) with growth selection , we first achieved directed evolution of phenylethanolamine N-methyltransferase ( Pnmt ) using a non-natural substrate , octopamine ( OCT ) . In recent years , ALE has emerged as a productive approach to address a wide range of biological questions [7–9] . We implemented an ALE-driven workflow to demonstrate our selection system because of the operational simplicity of ALE ( serial passages ) , its ultra-high–throughput screening capability ( over 10 million cells per passage ) , its ability to engage phenotype-driven in vivo evolution , and the affordability of DNA resequencing ( Fig 2A ) . Following ALE , isolated strains were characterized and were subjected to full DNA resequencing . Mutations in E . coli cfa , involved in phospholipid synthesis , were identified in all non-growth–coupled isolates . The cfa gene encodes for a SAM-dependent Mtase , suggesting its role as a competing Mtase during ALE . On the other hand , a Pnmt ( F214L ) mutation was present in growth-coupled isolates , and a cell-based characterization showed that it led to approximately 2-fold activity improvement on synephrine ( SYN ) synthesis ( Fig 2B ) . Most often , Mtase substrates may not be readily available in large quantities or even membrane permeable in order to perform directed enzyme evolution in vivo , and it may then only be feasible to engage an active metabolic pathway . We thus applied our selection system to evolve a methylation-dependent pathway . The chosen candidate pathway was a de novo three-step melatonin biosynthesis pathway from 5-hydroxytryptophan ( 5HTP ) . It consisted of three enzymatic steps: decarboxylation ( aromatic-amino-acid decarboxylase [Ddc] ) , acetylation ( aralkylamine N-acetyltransferase [Aanat] ) , and methylation ( acetylserotonine O-methyltransferase [Asmt] ) ( Fig 2C ) . By using ALE , Asmt was evolved in vivo , and three sequence variants ( A258E , G260D , and T272A ) were discovered ( Fig 2D ) . All variants showed improved turnover compared to wild-type Asmt under physiological conditions , with the highest improvement observed in A258E ( approximately 2 . 5-fold ) . It was additionally discovered that high levels of ddc expression from a plasmid caused genetic instability , and mutations in cfa could be seen in non-melatonin–producing cells , affirming its role as an unwanted sink for SAM in E . coli . Upon incorporating Asmt ( A258E ) , a single copy of ddc , and cfa deletion in the background strain , Aanat was further evolved in the next ALE , and the D63G mutation was identified , leading to approximately 2-fold activity improvement ( Fig 2D ) . These results demonstrated the usefulness of this growth selection system for directed evolution of enzymes or metabolic pathways when linked to a methylation reaction . We next demonstrate the use of our system for drug discovery . SAM-dependent Mtases participate in many important cellular functions and are targeted by a number of drug development programs ( such as DNA or histone Mtase inhibitors ) [6] . We applied our selection system on catechol O-methyltransferase ( Comt ) , a known drug target for treating Parkinson's disease [5] . Cells bearing human Comt were evolved to grow at high rates using ALE ( Fig 2E ) . All isolates were growth-coupled to Comt activity . Resequencing results showed the comt gene did not acquire any mutations , while many isolates accumulated mutations on RpoC ( such as A328P , E1146A , or E1146G ) , a subunit of E . coli RNA polymerase , suggesting a host factor effect . The suitability of using evolved cells to screen Comt inhibitors by growth was evaluated next by determining Z-factor in a 96-well format [10] . The Z-prime value was calculated to be between 0 . 87 to 0 . 97 when cells were grown for 3 h or more , indicating a high-throughput-screening ( HTS ) –compatible assay with large separation ( Fig 2E and S1 Table ) . We then tested one evolved isolate with two known Comt inhibitors: entacapone and tolcapone , respectively . Both drugs reduced Comt-dependent cell growth at concentrations as low as 200 nM , with a slightly higher potency observed for tolcapone ( Fig 2E ) . Both inhibitors were highly specific to Comt and showed no observable adverse effects on other cellular proteins ( such as heterologous Cys3 and Cys4 or the essential E . coli proteins ) when homocysteine was additionally supplemented , implying a general suitability of our selection system for in vivo Comt inhibitor screening ( Fig 2E ) . Lastly , we implemented our design in budding yeast S . cerevisiae . S . cerevisiae is an industrially important production host with growing interest for biobased production of value-added methylated products [11] . It is also a well-studied eukaryotic model organism expressing diverse cellular Mtases [12] . In contrast to E . coli , yeast is capable of synthesizing cysteine through reverse transsulfuration from homocysteine because of the natural appearance of the CYS3 and CYS4 genes ( Fig 3A ) [13] . Therefore , blockage of homocysteine biosynthesis from aspartate is required to enable the selection , and this was achieved by deleting the genes encoding homoserine O-acetyltransferase ( MET2 ) and O-acetylhomoserine sulfhydrylase ( MET17 ) . Additional gene deletion of phosphatidylethanolamine methyltransferase ( CHO2 ) and phospholipid methyltransferase ( OPI3 ) required for phosphatidylcholine biosynthesis was performed to remove potential competing native Mtases for SAM [14] . In this quadruple knock-out strain , the heterologous caffeine synthase I gene ( CCS1 ) , encoding an N-Mtase from Coffea arabica acting on theobromine to synthesize caffeine , was introduced . The presence of Ccs1 conferred growth advantage when exogenous theobromine was supplemented compared to nonsupplemented cells , affirming the applicability of the design in yeast ( Fig 3B ) . Control cells without Ccs1 expression showed similar growth regardless of theobromine supplementation ( Fig 3C ) . Acknowledging the large number of native Mtases in yeast [12] , this phenotype might be the result of the activity of remaining native Mtases for homocysteine synthesis required for growth .
In summary , we have designed and validated a methylation-dependent growth selection system for Mtases . Not only did this selection system lead to the discovery of causal mutations that improve enzyme properties of heterologous Mtases and acetyltransferase , but it also demonstrated usefulness in drug discovery and the identification of critical host factors . Further applications of this technology can be used to acquire in-depth understanding of Mtases ( such as their evolutionary path and governing factors for substrate specificity ) or to engineer pathways or host cells of metabolic engineering interests . We have implemented our design in E . coli and S . cerevisiae; however , the conceptual design of this selection is transferable to any organism with an active SAM cycle . Implementing this selection system in higher eukaryotic cells ( such as mammalian cells ) is particularly valuable for drug development since permeability and stability of promising drug candidates can be determined at early stages . Overall , the described selection is robust , generalizable , compatible with HTS , and widely applicable , and it is likely to become a useful and valuable tool for the chemical biology and metabolic engineering communities .
The E . coli BW25113 strain and derivatives were used throughout this study . Genome engineering of E . coli was facilitated by λ-red recombination , P1 transduction , and/or site-specific Tn7 transposon [15 , 16] . S . cerevisiae CEN . PK 102-5B ( MATa ) was used as background yeast strain for the engineered yeast strains . Genome engineering of S . cerevisiae was facilitated by CRISPR/Cas9 [17 , 18] , and transformations were performed with the lithium acetate/single-stranded carrier DNA/PEG method [19] . A list of strains used is shown in S2 Table . Unless stated otherwise , all E . coli strains were maintained at 37°C in LB ( Lennox ) ( Sigma Aldrich , St . Louis , MO , USA ) , 2xYT , or M9 media containing M9 minimal salts ( BD Difco , BD , Franklin Lakes , NJ , USA ) , 2 mM MgSO4 , 100 μM CaCl2 , 500-fold diluted trace minerals ( 10 g/L FeCl3·6H2O , 2 g/L ZnSO4·7H2O , 0 . 4 g/L CuCl2·2H2O , 1 g/L MnSO4·H2O , 0 . 6 g/L CoCl2·6H2O , and 1 . 6 mM EDTA [pH 8 . 0] ) , 1× ATCC Vitamin Supplement ( ATCC MD-VS ) , and 0 . 2% glucose ( w/v ) . Unless stated otherwise , ampicillin and spectinomycin were used at 50 mg/L; chloramphenicol and kanamycin were used at 25 mg/L . Yeast strains were cultured on either rich yeast extract-peptone dextrose ( YPD ) , yeast synthetic drop-out media ( lacking the proper amino acids for selection ) ( Sigma Aldrich ) , or Delft medium [20] with 2% glucose . Delft medium was sterilized by filtration . All Δcho2- and Δopi3-derived strains were maintained on media supplemented with 1 mM choline chloride ( Sigma Aldrich ) , except for YPD medium . Plasmid DNA assemblies were performed by Gibson Assembly , USER cloning , or the EasyCloning method [20–22] . E . coli TOP10 ( Invitrogen , Carlsbad , CA , USA ) and DH5α were used for plasmid propagations . For propagation of yeast plasmids , 100 mg/L ampicillin was used . A list of plasmids used is summarized in S3 Table . All ALE experiments were performed at 37°C using M9 supplemented with 50 mg/L methionine and substrates , unless stated otherwise . Cell passages were performed automatically according to previously described [23] , and six lineages of the initial strain were usually maintained . OCT and PCA at 50 mg/L were supplemented for Pnmt and Comt evolution , respectively . The melatonin pathway was evolved in the presence of 100 mg/L of methionine and 100 mg/L of 5HTP . Antibiotics were not supplied during ALE . The E . coli ECAH2 strain ( ΔcysE ) , derived from JW3582 of the Keio Collection [24] , was used as the host strain to test methylation-dependent growth in the presence of pHM11 . The pHM11 plasmid harbored cys3 and cys4 from S . cerevisiae S288C . Homocysteine and Cys3/Cys4-dependent cysteine synthesis was demonstrated by transforming ECAH2 with pHM11 . The transformed strain , ECAH3 , was allowed to grow on an M9 plate containing 50 mg/L homocysteine at 37°C for 24 h to demonstrate homocysteine-dependent growth . The ECAH6 and ECAH7 strains were used for Pnmt and Comt evolution using ALE . All ΔcysE-derived strains were maintained on LB plates supplemented with 25 mg/L cysteine . The E . coli HMP236 strain was initially used to evolve the melatonin pathway from 5HTP ( S2 Table ) . It contained two plasmids , pHM11 and pHM12 . Its parent strain was HMP221 with the following genome modifications: FolE ( T198I ) , YnbB ( V197A ) , ΔtnaA , ΔcysE , ΔmetE , and ΔmetH . Deletion of tnaA was to prevent 5HTP degradation . Deletion of metE and metH was to prevent a reverse methionine-to-homocysteine synthesis via methionine synthase encoded by both genes , but it was later determined not to be required . The E . coli HMP579 was used for the subsequent melatonin pathway evolution . It carried two plasmids , pHM70 and pHM79 . The pHM70 plasmid was modified from pHM11 with an insertion of Asmt ( A258E ) . The second plasmid , pHM79 , was not required in this study because of 5HTP feeding . Its parent strain was HMP553 , carrying a single chromosomal copy of ddc and aanat . The ddc and aanat genes were introduced to the attTn7 site using pGRG25 . The S . cerevisiae SCAH124 strain , derived from S . cerevisiae CEN . PK102-5B ( MATa ) , was constructed by sequential CRISPR/Cas9-facilitated full ORF markerfree knock-out of MET17 , CHO2 , OPI3 , and MET2 mediated by homology-directed recombination using guide RNA ( gRNA ) -expressing plasmids PL_01_A2 , PL_01_A3 , PL_01_C8 , and PL_01_E1 and two linear DNA fragments homologous to the flanking regions up- and downstream of the targeted ORF , as well as PL_01_A9 with CAS9 . The following gRNA sequences were used for Cas9-targeting of indicated ORF and identified with the webservice CRISPy adapted for the S . cerevisiae CEN . PK genome sequence [25]: MET17 ( 5′-GATACTGTTCAACTACACGC-3′ ) , CHO2 ( 5′-ACCACCTGTAACCCACGATA-3′ ) , OPI3 ( 5′- GCAGAAACAACCAGCCCCGC-3′ ) and MET2 ( 5′- GTAATTTGTCATGCCTTGAC-3′ ) . SCAH134 and SCAH138 , derived from SCAH124 and harboring the Mtase gene CCS1 ( PL_01_D2 ) and an empty vector ( pRS415U ) , respectively , were used for demonstration of selection design in S . cerevisiae . Total DNA was extracted using a PureLink Genomic DNA Kit ( Invitrogen ) and was processed either commercially by Beckman Coulter Genomics ( Danvers , MA , USA ) or in house . When prepared in house , DNA libraries were prepared using a Kapa Hyper Prep Library Prep Kit ( Roche Molecular Systems , Pleasanton , CA , USA ) . DNA samples were sequenced using Illumina MiSeq or NextSeq . Trimmomatic tool ( v0 . 32-v0 . 35 ) was used for quality trimming of raw sequencing data with "CROP:145 HEADCROP:15 SLIDINGWINDOW:4:15 MINLEN:30" parameters [26] . Breseq ( v0 . 27 . 1 ) was employed for variant calling on processed sequencing data with "-j 4 -b 20" parameters [27] . E . coli BW25113 genome sequence with NCBI accession CP009273 was used as reference along with other relevant parts and plasmids . All compounds were purchased from Sigma Aldrich . PCA and its methylated product vanillic acid ( VIII ) were quantified using a Dionex 3000 HPLC system ( Dionex , Sunnyvale , CA , USA ) equipped with a Cortecs UPLC T3 column from Waters ( Milford , MA , USA ) and a guard column from Phenomenex ( Torrance , CA , USA ) . The column temperature was set to 30°C , and the mobile phase consisted of a 0 . 1% formic acid and acetonitrile . Runtime was 11 min , including 4 . 2 min separation without acetonitrile , 1 min washing with 75% acetonitrile , and an additional 4 . 5 min run without acetonitrile . The flow rate was constant at 0 . 3 ml/min , and the injection volume was 1 μl . Both compounds were detected by UV at wavelengths 210 , 240 , and 300 nm as well as a 3D UV scan . HPLC data were processed using Chromeleon 7 . 1 . 3 software ( Thermo Fisher Scientific , Waltham , MA , USA ) , and compound concentrations were calculated using calibration curves . 5HTP , serotonin ( HT ) , acetylserotonin ( AcHT ) , and melatonin were quantified using a Dionex 3000 HPLC system equipped with a Zorbax Eclipse Plus C18 column ( Agilent Technologies , Santa Clara , CA , USA ) and a guard column from Phenomenex . To achieve separation , the column was heated to 30°C , and the mobile phase consisted of a 0 . 05% acetate and a variable amount of acetonitrile . Runtime was 12 min , including 10 min of separation , whereas acetonitrile was reduced from 95% to 38 . 7% in 9 . 4 min . After holding 0 . 6 min , acetonitrile concentration was returned to 95% in 1 min and was held till the end of the run . The flow rate was set to 1 ml/min , and the injection volume was 1 μl . Elution of the compounds was detected by UV at wavelengths 210 nm , 240 nm , 280 nm , and 300 nm as well as a 3D UV scan . HPLC data were processed using Chromeleon 7 . 1 . 3 software ( Thermo Fisher Scientific ) , and compound concentrations were calculated using calibration curves . OCT was quantified using a Dionex 3000 HPLC system equipped with a Cortecs UPLC T3 column from Waters and a guard column from Phenomenex . The column temperature was set to 30°C , and the mobile phase consisted of 0 . 1% formic acid and acetonitrile . Runtime was 9 min , including 2 . 5 min separation without acetonitrile , 0 . 5 min washing with 70% acetonitrile , and an additional 5 . 5-min run without acetonitrile . The flow rate was constant at 0 . 3 ml/min , and the injection volume was 1 μl . OCT was detected by UV at wavelengths 210 , 240 , and 300 nm as well as a 3D UV scan . SYN was detected by LC-MS ( Fusion , Thermo Fisher Scientific ) in the positive full-scan mode using the same separation profile as OCT quantification . SYN was detected as [M + H]+ m/z 168 . 10191 with a mass accuracy of 2 . 2 ppm . Data were processed using Chromeleon 7 . 1 . 3 and X-calibur 4 . 1 from Thermo Fisher Scientific , and compound concentrations were calculated using calibration curves . Growth of E . coli was measured using a Duetz 96-well low well system ( Enzyscreen , Heemstede , The Netherlands ) coupled to a humidified Innova 44 shaker ( 5 cm orbit ) ( New Brunswick Scientific , Edison , NJ , USA ) at 37°C and 300 rpm . Seed cells were grown in 400 μl LB in the presence of appropriate antibiotics for 4–5 h in 96-well deep well plates . When transferred to M9 , 10 μl of cells were added to 400 μl of M9 with 25 mg/L cysteine and antibiotics . After overnight growth , cells were added to 150 μl of fresh M9 with 50 mg/L of methionine and 50 mg/L methylation substrates to approximately 4% in a 96-well low well plate . Changes in optical density at 600 nm ( OD600 ) were recorded using a SynergyMx microplate reader ( BioTek Instruments , Winooski , VT , USA ) . Growth rates were calculated from an average of four independent biological replicates using KaleidaGraph 4 . 1 . 3 . Six biological replicates of S . cerevisiae SCAH134 and SCAH138 were inoculated from seed cultures to similar ODs for sulfur amino acid starvation in Delft medium supplemented with histidine , uracil , and choline chloride and incubated for approximately 24 h . The seed cultures were grown in yeast synthetic complete medium without leucine , supplemented with choline chloride , for approximately 25 h . Both replicate seed cultures and starvation cultures were cultured in a total volume of 500 μl in 96-deep well plates at 30°C/300 rpm . Upon completion of starvation , cells were transferred to Delft medium supplemented with histidine , uracil , choline chloride , 1 mM L-methionine , and with/without 1 mM theobromine ( Sigma Aldrich ) to similar ODs and to a final volume of 150 μl in a flat-bottomed microtiterplate . Two technical replicates were inoculated for each of the six biological replicates . Growth was recorded in a microtiterplate reader ( ELx808 Absorbance Reader , BioTek Instruments ) with continuous shaking at strong setting , at 30°C , and with recording of absorbance at 630 nm every 30 min . The cultures had incubated in the plate reader 30 min prior to the first reading at time 0 h . Growth curves were plotted using mean absorbance of the replicates for 30 h . Blank media values were not subtracted . HL1818 was used to test the inhibition effect of entacapone and tolcapone . Cells were prepared for growth measurement as described above . The test growth medium was M9 with 50 mg/L PCA , 50 mg/L methionine , 1% DMSO , and various amount of inhibitors . The stock concentration of entacapone and tolcapone was 20 g/L dissolved in DMSO . To the positive controls , 50 mg/L homocysteine was additionally included so that effects of the drugs on E . coli cells ( such as those other than Comt ) could be determined . A drug concentration response curve was plotted using average OD600 after 6 h growth from three independent biological replicates . The EC50 values were calculated using OriginPro 2018b ( version b9 . 5 . 5 . 409 ) . Measurements of SYN production were performed using a Duetz 96-well deep well system ( Enzyscreen ) coupled to an Innova 44 shaker ( 5 cm orbit ) ( New Brunswick Scientific ) at 37°C and 300 rpm . HL1815 and HL1816 were used to measure SYN production ( S1 Table ) . Seed cells were grown in LB in the presence of chloramphenicol for 4–5 h and thereafter grew in M9 overnight . Fresh M9 containing 200 mg/L OCT was inoculated with seed culture to 4% . These cells were transferred to a 96-well deep well plate , and each well contained 400 μl . 200 μl samples were withdrawn periodically for exometabolites analysis , while the remaining 200 μl cells were used to determine OD values using a SynergyMx microplate reader ( BioTek ) . In vivo enzyme activity was averaged from four independent biological measurements normalized to dried cell weight . The conversion factor from OD to dried cell weight is 1 ( i . e . , 1 OD = 1 g/L ) for our setup . It was observed that Pnmt activity was biomass dependent . A Duetz 24-well deep well system ( Enzyscreen ) coupled to an Innova 44 shaker ( 5 cm orbit ) ( New Brunswick Scientific ) was used . Physiological Asmt activity was determined by growing HMP231 , HMP416 , HMP416 , HMP417 , and HMP418 in 2 ml M9 supplemented with 100 mg/L AcHT at 37°C with shaking at 300 rpm . Samples were withdrawn periodically for exometabolites analysis using HPLC and OD measurements . Physiological Aanat activity was measured using HMP850 and HMP851 in the presence of 100 mg/L HT . In vivo enzyme activity was averaged from three independent biological measurements normalized to dried cell weight . It was observed that Asmt and Aanat activity was biomass independent . Validation of the selection system design was performed using the most recent E . coli genome-scale metabolic model [28] , which computes the flux states of the entire metabolic network . Following established procedures [29] , the cysE gene was “knocked out” in silico by setting the upper and lower bounds of the metabolic reaction it catalyzes to 0 . Metabolic reactions catalyzed by Cys3 and Cys4 were inserted into the metabolic model . Additionally , a methylation-dependent reaction was inserted into the model ( Pnmt , Comt , or Asmt ) . The metabolic model was then solved for its flux state using linear programming by setting cell growth as the objective . All model simulations were performed using the python package COBRApy 0 . 7 . 0 in Python 2 . 7 [30] . | Many important biological processes require methylation , e . g . , DNA methylation and synthesis of flavoring compounds , neurotransmitters , and antibiotics . Most methylation reactions in cells are catalyzed by S-adenosylmethionine ( SAM ) –dependent methyltransferases ( Mtases ) using SAM as a methyl donor . Thus , SAM-dependent Mtases have become an important enzyme category of biotechnological interests and as healthcare targets . However , functional implementation and engineering of SAM-dependent Mtases remains difficult and is neither cost effective nor high throughput . Here , we are able to address these challenges by establishing a synthetic biology approach , which links Mtase activity to cell growth such that higher Mtase activity ultimately leads to faster cell growth . We show that better-performing variants of the examined Mtases can be readily obtained by growth selection after repetitive cell passages . We also demonstrate the usefulness of our approach for discovery of Mtase-specific drug candidates . We further show our approach is not only applicable in bacteria , exemplified by Escherichia coli , but also in eurkaryotic organisms such as budding yeast Saccharomyces cerevisiae . | [
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] | 2019 | Coupling S-adenosylmethionine–dependent methylation to growth: Design and uses |
The primary cilium , which disassembles before mitotic entry and reassembles after mitosis , organizes many signal transduction pathways that are crucial for cell life and individual development . However , how ciliogenesis is regulated during the cell cycle remains largely unknown . Here we show that GSK3β , Dzip1 , and Rab8 co-regulate ciliogenesis by promoting the assembly of the ciliary membrane after mitosis . Immunofluorescence and super-resolution microscopy showed that Dzip1 was localized to the periciliary diffusion barrier and enriched at the mother centriole . Knockdown of Dzip1 by short hairpin RNAs led to failed ciliary localization of Rab8 , and Rab8 accumulation at the basal body . Dzip1 preferentially bound to Rab8GDP and promoted its dissociation from its inhibitor GDI2 at the pericentriolar region , as demonstrated by sucrose gradient centrifugation of purified basal bodies , immunoprecipitation , and acceptor-bleaching fluorescence resonance energy transfer assays . By means of in vitro phosphorylation , in vivo gel shift , phospho-peptide identification by mass spectrometry , and GST pulldown assays , we demonstrated that Dzip1 was phosphorylated by GSK3β at S520 in G0 phase , which increased its binding to GDI2 to promote the release of Rab8GDP at the cilium base . Moreover , ciliogenesis was inhibited by overexpression of the GSK3β-nonphosphorylatable Dzip1 mutant or by disabling of GSK3β by specific inhibitors or knockout of GSK3β in cells . Collectively , our data reveal a unique cascade consisting of GSK3β , Dzip1 , and Rab8 that regulates ciliogenesis after mitosis .
The primary cilium is an antenna-like organelle projecting from the apical surface of most vertebrate cells and plays pivotal roles in mediating signal transduction for the cell and regulating the balance between cell proliferation and differentiation [1–4] . It consists of a basal body , a microtubule-based axoneme generated from the basal body , and a signaling-receptor-enriched ciliary membrane sheet extending from the cell membrane . Between the ciliary membrane sheet and the cell membrane , there is a periciliary diffusion barrier ( PDB ) , a transition zone that forms a selective barrier to the membrane proteins that are laterally transported on the membranes [5 , 6] . The primary cilium is also gated by the pinwheel-shaped transition fibers that originate from the distal appendages of the basal body and end at the “cilium necklace . ” Bidirectional transport of ciliary proteins between the cytoplasm and the cilium is mediated by a multiprotein complex , the IFT ( intraflagellar transport ) machinery [7] . The primary cilium is structurally dynamic during the cell cycle . It disassembles before the mitotic entry and reassembles at the end of mitosis [8 , 9] . Building a cilium , or ciliogenesis , is a sequentially coordinated process [10 , 11] , during which polarized membrane vesicle trafficking to , and fusion with , the cell membrane—mediated by vesicle-bound Rab GTPases—is of great importance for formation of the ciliary membrane sheet [10 , 12] . Among the Rab GTPases , Rab8 is a core modulator of membrane vesicle trafficking to cilium , and specifically functions at the steps of vesicle docking and fusion with the cell membrane [13] . Rab8 in its GTP-bound form ( Rab8GTP ) is active and can be converted into the inactive form ( Rab8GDP ) by hydrolysis of the GTP molecule , which is mediated by its GTPase-activating protein . Conversely , the conversion of Rab8GDP to Rab8GTP requires several specific factors including GDP-dissociating inhibitor protein ( GDI ) , GDI displacement factor ( GDF ) , and Rab8’s guanine nucleotide exchange factor ( GEF ) , Rabin8 [14–16] . The GTP/GDP-bound status of Rab8 has antagonistic effects on ciliogenesis: overexpression of the Rab8GDP-mimicking mutant Rab8T22N blocks cilium assembly , whereas overexpression of the Rab8GTP-mimicking mutant Rab8Q67L promotes cilium assembly [17] . Both the proper localization and the efficient GTP-GDP cycling of vesicle-bound Rabs are important for vesicle trafficking and ciliogenesis [15] . Dzip1 is a zinc-finger-containing protein that is predominantly expressed in human embryonic stem cells and germ cells [18] . The Dzip1 gene was first identified in zebrafish ( where it is called iguana ) , and its mutation results in failure of ciliogenesis in Kupffer’s vesicle cells [19 , 20] . In cultured mammalian cells , Dzip1 and the Dzip1-like protein ( Dzip1L ) promote primary cilium formation [21 , 22] . The glycogen synthase kinase 3 ( GSK3 ) family contains two structurally similar isoforms in mammals , GSK3α and GSK3β . Beyond its function in regulating glycogen metabolism , as a multifunctional serine/threonine kinase , GSK3 also regulates the Hedgehog signaling transduction pathway [23] . A fraction of GSK3β is localized to the centrosomes , and it has increased kinase activity during the metaphase–anaphase transition [24] and is constantly active in resting cells [25] . GSK3β has also been shown to be a key component of an interlinked signaling pathway that maintains the primary cilium [26] . In this work , we investigated the mechanism of ciliogenesis during the cell cycle and found that GSK3β , Dzip1 , and Rab8 co-regulate ciliogenesis by promoting ciliary membrane assembly .
To investigate the function of Dzip1 , we first analyzed the protein level and the subcellular localization of endogenous Dzip1 . Western blot analysis with a commercial rabbit antibody against a peptide equivalent to amino acids ( aa ) 594–610 of human Dzip1 ( Mid2 ) revealed Dzip1 at ~110 kD both in non-ciliated HeLa and ciliated NIH 3T3 cells ( Fig 1A ) . Based on immunofluorescence with this antibody , Dzip1 was found mainly in the cytoplasm , with a small amount in the nucleus . By super-resolution microscopy , we observed that the pericentriolar matrix ( PCM ) and the mother centriole that acts as the basal body to assemble the primary cilium were strongly stained by this antibody in G0-phase NIH 3T3 cells ( Fig 1B ) . Similar results were obtained using an antibody against a peptide equivalent to aa 373–510 of mouse Dzip1 ( Mid1; S1A and S1B Fig ) . Moreover , we established a stable GFP-Dzip1-expressing NIH 3T3 cell line ( S1C Fig ) . With this cell line , we confirmed that GFP-Dzip1 was also localized to the basal body and the PCM , and found that GFP-Dzip1 was preferentially concentrated at one of the two centrioles ( S1D Fig ) . By immunofluorescence staining the cells expressing GFP-Cep120—a protein that is asymmetrically localized to the daughter centriole [27]—we found that Dzip1 was enriched at the centriole that showed less staining for GFP-Cep120 ( Fig 1C ) , further confirming that endogenous Dzip1 is asymmetrically enriched at the mother centriole . Next , we investigated the precise localization of Dzip1 at the PCM by super-resolution microscopy . We observed that , in addition to its localization at the cilium base , Dzip1 was also partially co-localized with PCM1 ( Fig 1D ) . By expressing the PDB marker YFP-GL-GPI ( glycosylphosphatidylinositol-anchored glycan tagged with YFP ) [5 , 28] , we found that this portion of Dzip1 was localized inside the PDB marker ( Fig 1E ) , indicating that the PCM-localized Dzip1 is localized to the PDB in ciliated cells ( Fig 1F ) . Furthermore , we examined the localization of Dzip1 during the cell cycle . We observed that Dzip1 was localized to the basal body/centrosome and the PCM in interphase , regardless of the presence or absence of a cilium . However , when the duplicated centrosomes were separated , Dzip1 showed a preferential localization on one of the two daughter centrosomes ( Fig 1G ) , and a trace amount of it was partially co-localized with PCM1 at one of the two spindle poles ( S1E Fig ) . This asymmetry was confirmed by GFP-Dzip1 localization in living cells ( S1F Fig ) . In telophase/early G1 phase , Dzip1 was localized to the midbody and re-accumulated at the centrosome with priority in one of the two daughter cells ( Fig 1G ) . By co-staining Dzip1 with the mature/mother centriole marker protein GFP-Cep164 [29 , 30] , we confirmed that the daughter cell that inherited the grandmother centriole recruited Dzip1 earlier than the other daughter cell ( Fig 1G ) , an outcome that is correlated with the asymmetrical ciliogenesis of the two daughter cells [31] and which suggests that Dzip1 contributes to ciliogenesis . To investigate the role of Dzip1 in ciliogenesis , we established two stable Dzip1-knockdown NIH 3T3 cell lines ( 1308–3 and 2172–1 ) by RNA interference ( RNAi ) ( S2A Fig ) . We found that the cilium length was significantly shortened from 6 . 5 ± 3 . 2 μm in the control to 2 . 1 ± 0 . 6 and 2 . 6 ± 0 . 5 μm in the two Dzip1-knockdown cell lines , and that the percentage ciliation ratios were also decreased ( S2B—S2D Fig ) , consistent with the concept that Dzip1 knockdown interferes with cilium assembly [8 , 20 , 21] . We further demonstrated that the defects of ciliogenesis caused by Dzip1 knockdown could be rescued by simultaneously expressing full-length RNAi-resistant Dzip1 ( S2E and S2F Fig ) . We also observed that the localization of IFT88 , which is necessary for cilium assembly [32 , 33] , was not affected in the Dzip1-knockdown 1308–3 cells ( Fig 2A ) , and that no IFT88 or γ-Tubulin were detectable in the GFP-Dzip1 immunoprecipitates ( S3A Fig ) . Nevertheless , we found that ciliary Rab8 was significantly decreased in the Dzip1-knockdown 1308–3 cells ( Fig 2A and 2B ) , although the basal body localization of Rab8 and Rabin8 was unaffected ( Figs 2A and S3B ) . To confirm the role of Dzip1 in the entry of Rab8 into the cilium , we transiently expressed the active-mimicking mutant Rab8Q67L tagged with GFP , and found that it was also unable to enter the cilium in the Dzip1-knockdown cells ( Fig 2C and 2D ) . We further examined the localization of Smo , a membrane protein that is transported to the primary cilium depending on Rab8 [34] . Strikingly , we found that even in cells with comparable cilium lengths , Smo-YFP was localized to the cilium much less in 1308–3 cells than in the control cells ( 25% ± 1% versus 80% ± 2%; Fig 2E and 2F ) . Together , these results suggest that Dzip1 is required for the ciliary localization of Rab8 and for Rab8-mediated cargo transport to the ciliary membrane . Next , we investigated whether Dzip1 interacts with Rab8 . Using immunoprecipitation ( IP ) assays , we found that Dzip1 interacted strongly with Rab8 ( Fig 2G and 2H ) . To assess the binding specificity of Dzip1-Rab8 , we screened seven other Rab proteins that are involved in regulating various aspects of intracellular membrane trafficking , especially ciliogenesis . We found that Myc-Dzip1 also bound to GFP-tagged Rab10 but not Rab5a , 6a , 7a , 9a , 11a , or 27a ( Fig 2G ) . In addition , we found that GFP-Dzip1 and RFP-Rab8 were co-localized at the cilium base in living NIH 3T3 cells ( Fig 2I ) , and that the interaction of Dzip1 with Rab8 mainly took place at the PCM ( S4A Fig ) . To determine whether Dzip1 discriminates between Rab8GTP and Rab8GDP , we co-expressed Dzip1 with Rab8Q67L or Rab8T22N in HEK 293T cells , and found that Dzip1 bound with much more Rab8T22N than Rab8Q67L ( Fig 2J and 2K ) . We also found that Rabin8 , the Rab8 GEF , did not immunoprecipitate with Dzip1 ( S3C Fig ) , suggesting that the Rab8GDP bound by Dzip1 could not be converted to active Rab8GTP by Rabin8 . To map the region of Dzip1 that bound with Rab8 , we expressed truncates of Dzip1 and analyzed their interactions with Rab8 . The results showed that the C-terminus ( from aa 430 to the end ) of Dzip1 interacted with Rab8 , of which aa 430–600 were crucial ( S4B and S4C Fig ) . Taken together , these data demonstrate that Dzip1 preferentially binds Rab8GDP via aa 430–600 at the cilium base . The enzyme cycle of Rab GTPases and their functions are regulated by not only the binding of the GTP molecule but also by GDIs , which stabilize Rab proteins in an inactive state by preventing the release of GDP from Rab proteins [15] . To understand why Dzip1 prefers to bind with Rab8GDP , we investigated whether the association of Rab8 with GDI2 , which promotes the membrane dissociation of Rab8 [35] , is regulated by Dzip1 . We found that Myc-GDI2 was co-immunoprecipitated with GFP-Rab8T22N from cells and that the Rab8-GDI2 interaction was abolished by overexpressing Myc-Dzip1 ( Fig 3A ) . We also confirmed in a cell-free system that GST-GDI2 , but not GST , pulled down endogenous Rab8 , and when increasing amounts of His-Dzip1 aa 373–600 were added , the amount of Rab8 bound by GDI2 progressively decreased ( Fig 3B ) . As a control , adding Myosin Va ( aa 1320–1346 ) , which interacts with Rab8 [36] , had no effect on the Rab8-GDI2 dissociation ( S4D Fig ) . Since proteins with GDF activity are able to displace GDI [16] , we asked whether Dzip1 interacts with GDI2 to free Rab8GDP . We found that endogenous Dzip1 and GDI2 were co-localized and interacted at the basal body and the PCM ( Fig 3C and 3D ) , and that Dzip1 also interacted with GDI2 via aa 430–600 ( S4E Fig ) . Therefore , we concluded that Dzip1 binds with the Rab8GDP-GDI2 complex via its middle region and promotes the dissociation of Rab8GDP from GDI2 . To directly visualize the released free Rab8GDP , we designed an AB-FRET assay by generating a reporter through fusing CFP and YFP tags , respectively , to the N- and C-termini of the RBD ( Rab8-binding domain ) of Rabin8 [14] ( CFP-RBD-YFP; S5A Fig ) . This reporter was co-expressed with Flag-tagged Rab8T22N or Rab8Q67L in cells , and showed strong binding with Flag-Rab8T22N but not Flag-Rab8Q67L ( S5B Fig ) , indicating that CFP-RBD-YFP mimics Rabin8 binding with Rab8GDP . Moreover , like endogenous Rabin8 , CFP-RBD-YFP was localized to the basal body and the PCM ( S5C Fig ) . Presumably , CFP-RBD-YFP alone may fold automatically to produce fluorescence resonance energy transfer ( FRET ) , and the FECFP will increase after photo-bleaching of YFP ( S5A Fig , panels a and b ) . Once bound by Rab8GDP , CFP and YFP will either come closer or separate from each other ( S5A Fig , panels c and d ) , resulting in a change of FECFP after YFP photo-bleaching . Indeed , we found that a fraction of CFP-RBD-YFP was co-localized with endogenous Rab8 or Flag-Rab8T22N but not with Flag-Rab8Q67L in the cytoplasmic aggregates ( S5D Fig ) , and that the FECFP in the aggregates was significantly increased after YFP photo-bleaching ( S5E and S5F Fig ) . We also found that the more free Rab8GDP was available to combine with CFP-RBD-YFP , the stronger the FECFP produced ( S5E and S5F Fig ) . These results demonstrate that the binding of free Rab8GDP with CFP-RBD-YFP separates CFP from YFP , leading to a drop in FRET from CFP to YFP and an enhancement of FECFP ( S5A Fig , panel d ) . With this reporter , CFP-RBD-YFP , we examined control and Dzip1-knockdown cells with comparable CFP-RBD-YFP fluorescence intensity . We found that after YFP photo-bleaching , a high FECFP signal occurred around the cilium base ( FECFP = 9 . 3% ± 8 . 2% ) and scattered from the cilium base to the cytoplasm in control cells , but the FECFP signal at the cilium base was markedly decreased in 1308–3 cells ( FECFP = −3 . 6% ± 6 . 9%; Fig 3E and 3F ) . The heat map showed that the FECFP signal mainly occurred at the PCM but not the centrosome ( Fig 3F ) , indicating that Rab8GDP accumulated at the PCM but was excluded from the centrosome in normal cells ( Figs 3F , S4A and S4F ) . Taken together , these results demonstrate that Dzip1 promotes the release of Rab8GDP from GDI2 at the PCM . Since the PCM localization of Dzip1 during the cell cycle is prior to ciliogenesis and Dzip1 plays a positive role in the regulation of ciliogenesis by promoting the dissociation of Rab8GDP-GDI2 and ciliary entry of Rab8 as shown above , we set up experiments to test how Dzip1-mediated ciliogenesis is regulated after mitosis . By comparing the expression of Dzip1 in NIH 3T3 cells arrested at G0 phase and prometaphase through Western blot analysis , we found that Dzip1 was up-shifted in G0-phase cells and that these up-shifted bands were sensitive to λ phosphatase ( S6A Fig ) , indicating that a portion of Dzip1 is phosphorylated in resting cells . In searching for the kinase ( s ) responsible for the phosphorylation of Dzip1 , we realized that GSK3β might be the best candidate because of its centrosomal localization , its increasing activity from metaphase to anaphase , and its high activity in the absence of growth factors [37–39] ( S6B Fig ) . Indeed , we found that GSK3β was immunoprecipitated by Dzip1 , and vice versa ( Fig 4A and 4B ) , and that the two proteins were co-localized at the basal body ( Fig 4C and 4D ) . To determine whether GSK3β interacts with Dzip1 in a kinase-substrate-binding manner , we assessed the binding intensity of wild-type and mutant GFP-GSK3β with Myc-Dzip1 . We found that the binding of Myc-Dzip1 with the mutant GSK3βR96A—which cannot phosphorylate primed substrates but retains intact kinase activity for the unprimed substrates [37 , 40]—showed no decrease in comparison with wild type , the constantly active mutant GSK3βS9A , or the kinase-dead mutant GSK3βK85R [37] . Furthermore , inhibition of CK2 by CX4945 caused a remarkable reduction in the extent of Dzip1 band up-shifting , whereas inhibition of CK1 by D4476 did not change the up-shift of Dzip1 compared with untreated cells ( Fig 4E ) , indicating that Dzip1 is phosphorylated by CK2 as previously reported [41] . Interestingly , both CK2-phosphorylated and-nonphosphorylated Myc-Dzip1 equally bound to GFP-GSK3β ( Fig 4E ) , suggesting that the phosphorylation of Dzip1 by CK2 does not change the Dzip1-GSK3β interaction . Notably , expression of GSK3βK85R yielded a weaker up-shift of Dzip1 than expression of wild type or other mutants ( Fig 4E ) . Together , these results indicate that Dzip1 is a substrate of GSK3β independent of priming phosphorylation by CK1 and CK2 . Next , we investigated Dzip1 phosphorylation during cell cycle exit and re-entry . We found that depletion of serum over a time period of 48 h caused a mild increase in total GSK3β at the protein level and enhancement of its kinase activity , as indicated by the phosphorylation decrease of GSK3β at S9 [37] ( Fig 4F ) . In contrast , when serum was reintroduced , GSK3β kinase activity decreased ( Fig 4F ) . We also found that the up-shifted bands of Dzip1 became evident after 12 h of serum depletion and more pronounced in the following 36 h . However , the up-shifted bands rapidly disappeared during the G0 phase–G1 phase transition ( Fig 4F ) . Moreover , we assessed the phosphorylation status of Dzip1 in the presence of the GSK3 inhibitors BIO and CHIR99021 or the CK2 inhibitor CX4945 in resting immortalized MEFs . The efficiency of GSK3 inhibition was monitored by de-phosphorylation of its canonical substrate β-Catenin . The up-shifted bands of Dzip1 disappeared in cells treated with the CK2 inhibitor but not in control cells , as previously reported [41] , and weakened under treatment with the GSK3 inhibitors , further supporting that GSK3β phosphorylates Dzip1 ( Fig 4G ) . To demonstrate Dzip1 phosphorylation by GSK3β , an in vitro kinase assay was performed using the kinase His-GSK3β with the wild-type Dzip1 truncates aa 1–378 , aa 373–600 , and aa 594–852 . The results showed that His-GSK3β phosphorylated all three wild-type truncates ( Fig 4H ) . The specificity of the in vitro phosphorylation assay was confirmed by the findings that the truncate aa 373–600 was neither phosphorylated by His-GSK3βK85R nor under the inhibition of GSK3 ( S6C Fig ) , and that neither His-Rab8 nor GST-GDI2 was phosphorylated by GSK3β in a parallel experiment ( S6D Fig ) . To characterize the phosphorylation site ( s ) on Dzip1 by GSK3β , GFP-Dzip1 was immunoprecipitated and subjected to identification of phospho-peptide by mass spectrometry analysis . The results showed that S520 on Dzip1 was phosphorylated ( Fig 4I ) and that this phosphorylation disappeared in the cells treated with the GSK3 inhibitor BIO . This site was also confirmed by the in vitro kinase assay , which showed that the S520A mutant of the truncate aa 373–600 was unable to be phosphorylated by the kinase ( Fig 4H ) . Taken together , these data demonstrate that Dzip1 is phosphorylated at S520 by GSK3β in G0-phase cells . Due to the importance of aa 430–600 of Dzip1 in its binding with Rab8-GDI2 , we tested whether phosphorylation at S520 within this region influences this interaction . We found that inhibition of GSK3 markedly decreased the binding affinity of Dzip1 for GDI2 ( Figs 5A and S6E ) and significantly increased the binding affinity of Rab8GDP for GDI2 ( Fig 5B ) , suggesting that GSK3 is involved in the regulation of Rab8GDP dissociation from GDI2 via regulating the binding of Dzip1 with GDI2 . To further confirm this notion , we performed a semi-quantitative GST-GDI2 pulldown assay . We found that as increasing amounts of GST-GDI2 were added , the pulled-down amounts of endogenous Dzip1 and Rab8 showed different trends: the rate of Dzip1-GDI2 binding was much higher in control cells than in the cells treated with CHIR99021 ( i . e . , GDI2 was much more quickly saturated by Dzip1 in control cells than in CHIR99021-treated cells ) , whereas the rate of Rab8-GDI2 binding was lower in control cells than in CHIR99021-treated cells ( Fig 5C ) . To directly compare the amounts of Rab8GDP released from GDI2 with and without GSK3 regulation , we measured FECFP in cells expressing comparable amounts of CFP-RBD-YFP . We found that the efficiencies of YFP photo-bleaching at the basal body were steadily decreased by ~85% both in control cells and the cells treated with the GSK3 inhibitor . However , the FECFP signal after YFP photo-bleaching was lower in cells treated with BIO ( 3 . 3% ± 2 . 1% ) and CHIR99021 ( 2 . 6% ± 1 . 7% ) than in control cells ( 4 . 8% ± 2 . 8%; Fig 5D and 5E ) . Consistently , the binding of Flag-Rab8T22N to the FECFP reporter CFP-RBD-YFP was decreased under GSK3 inhibition ( S6G Fig ) . To understand whether the decreased Dzip1-GDI2 and increased Rab8-GDI2 binding with GSK3 inhibition was due to the phosphorylation of Dzip1 at S520 , we compared the amounts of Dzip1 mutants and endogenous Rab8 pulled down by GDI2 . We found that the rate of Rab8-GDI2 binding was much lower in cells expressing Dzip1S520D than in those expressing Dzip1S520A , and the rate of Dzip1S520D-GDI2 binding was higher than that of Dzip1S520A–GDI2 ( Fig 5F ) . Coinciding with this , when wild-type or mutant GFP-Dzip1 was co-expressed with Myc-GDI2 in G0-phase HEK 293T cells , we found that the mutant Dzip1S520A showed much less binding affinity for GDI2 than wild-type Dzip1 and the mutant Dzip1S520D ( Fig 5G ) . Taken together , these results demonstrate that the phosphorylation of Dzip1 at S520 by GSK3β promotes the dissociation of Rab8GDP-GDI2 . To understand the function of Dzip1 phosphorylation by GSK3β , we then investigated the effect of GSK3 inhibition on post-mitotic NIH 3T3 cells , and found that the percentage ciliation ratios of the post-mitosis daughter cells were dramatically decreased to 3 . 5% ± 1 . 0% with BIO treatment and to 4 . 5% ± 0 . 8% with CHIR99021 treatment , compared with 36% ± 3 . 1% in control cells at the 71-h time point after cell synchronization ( i . e . , after serum starvation for 4 h ) ( Fig 6A–6C ) . The reduced percentage ciliation ratio with CHIR99021 treatment was not due to cytokinesis delay or failure , since less than 20% of the cells serum-starved for 24 h were ciliated with this treatment , whereas ~70% of the untreated serum-starved control cells were ciliated at the same time point ( S7A and S7B Fig ) . Moreover , the lengths of the assembled cilia in BIO- and CHIR99021-treated cells were markedly shorter ( 2 . 2 ± 1 . 0 μm and 1 . 8 ± 1 . 2 μm , respectively ) than that in control cells ( 4 . 9 ± 1 . 0 μm; S7C Fig ) . As the kinase domains of GSK3α and GSK3β are highly homologous [37] , we wondered whether GSK3α or GSK3β or both are responsible for regulating ciliogenesis . By separately investigating ciliogenesis in immortalized MEFs from wild-type and GSK3α- and GSK3β-knockout mice [25] ( S7D Fig ) , we found that only GSK3β−/− cells exhibited ciliogenesis failure after exit from mitosis ( Fig 6D and 6E ) . Notably , although wild-type GSK3β and its mutants S9A and K85R showed identical basal body localization , only wild type and the mutant S9A , but not K85R , efficiently rescued ciliogenesis failure in post-mitotic and in resting GSK3β−/− cells ( Figs 6F and 6G , S7E and S7F ) , demonstrating that the enzymatic activity of GSK3β is required for ciliogenesis in post-mitotic cells . We also used R96A to rescue ciliogenesis failure in GSK3β−/− cells and found that it could partially rescue ciliation in post-mitotic and in resting cells ( Figs 6F and S7F ) , suggesting that direct phosphorylation of the substrate ( s ) by GSK3β is involved in cilium assembly . Finally , we set up an experiment to determine whether the phosphorylation of Dzip1 by GSK3β regulates ciliogenesis . By expressing the wild-type Dzip1 , the nonphosphorylatable mutant Dzip1S520A ( by GSK3β ) , or the phosphorylation-mimicking mutant Dzip1S520D in GSK3β−/− cells , we found that the expression of Dzip1S520D markedly elevated the ciliogenesis ratio from 3 . 0% ± 0 . 3% to 38% ± 3 . 2% , whereas wild-type Dzip1 and the mutant Dzip1S520A had no ability to rescue ciliogenesis in GSK3β−/− cells ( Fig 6H and 6I ) . Consistently , Dzip1S520D , but not Dzip1S520A , elevated the ratio of cilium-localized Rab8 in GSK3β−/− cells , and rescued the cilium defect in 1308–3 cells ( S7G—S7J Fig ) . Taken together , these results demonstrate that Dzip1 phosphorylation by GSK3β is required for ciliogenesis after exit from mitosis .
Several lines of evidence suggest that the PDB plays an important role in regulating the transport of cilium proteins , but how the PDB proteins execute their functions remains largely unknown . The present work revealed that a PDB-localized protein , Dzip1 , functions as a GDF that promotes Rab8GDP dissociation from GDI2 at the PCM through binding with GDI2 , and that this binding is positively regulated by the activation of GSK3β during ciliogenesis . The Rab proteins bound with GDP bind GDI with very strong affinity , whereas the membrane-bound GDFs promote the release of the GDP-bound Rab proteins from GDI , thereby facilitating the association of Rab proteins with relevant membranes [15] . Given that GEFs for Rab proteins cannot directly bind with RabGDP-GDI complexes [16] , we propose that , in the present case , the Dzip1-mediated Rab8-GDI2 dissociation is a prerequisite for Rab8 activation by its GEF Rabin8 . So far , two proteins besides Dzip1 have been identified as GDFs . One is Yip3/PRA1 , a protein with GDF activity and without GEF activity , which promotes the dissociation of Rab9 from GDI and facilitates Rab9 insertion on the membrane [42 , 43]; the other is SidM/DrrA , which retains both GDF and GEF activities and promotes the recruitment of Rab1 to Legionella-containing vacuoles during host-cell infection [44] . Dzip1 contains a Zn-finger in its N-terminus , like another Zn-finger-containing protein , Mss4/Dss4 , an evolutionarily conserved Rab chaperone factor that promotes nucleotide release from exocytic Rab GTPases at a very low rate [45–47] . However , Dzip1 shows no sequence similarity to Mss4 . Dzip1 specifically bound with Rab8 and Rab10 in our assays , unlike the broad binding of Mss4 with a number of Rab members [48] . Considering that Dzip1 and Rab8 are only co-localized at the centrosome and that Rabin8 could exchange the nucleotide for Rab8 at the centrosome [14 , 17] , we conclude that Dzip1 functions more like a GDF specific for Rab8 than like a GEF at the centrosome . Moreover , it is noteworthy that Dzip1 also interacts with Rab10 , the closest Rab8 paralogue , which is associated with the primary cilium and co-localizes and interacts with members of the exocyst complex in renal epithelial cells [49] . However , whether this interaction is due to the high sequential similarity between Rab8 and Rab10 ( identity 76% ) or the involvement of Dzip1 in regulating Rab10 activity remains so far unclear . Once dissociated from GDI2 , Rab8GDP is ready for a subsequent switch into Rab8GTP under the regulation of Rabin8 . In the absence of Dzip1 , Rab8—as well as its active-mimicking form Rab8Q67L—did not enter cilium , although it was still localized to the basal body . Dzip1 interacts with Cep164 [22] , and loss of Cep164 leads to impaired vesicle docking at the mother centriole and failed ciliary localization of the wild-type Rab8 but not the mutant Rab8Q67L [50]; therefore , we propose that in addition to regulating Rab8 activation , Dzip1 also regulates the translocation of Rab8GTP across the transition zone or PDB downstream of Cep164 . As ciliogenesis occurs first in daughter cells that have inherited the grandmother centriole [31] , which is persistently capped by a primary cilium-derived vesicle during mitosis [30] , our finding that the grandmother-centriole-containing daughter cell recruited Dzip1 to its centrosome earlier than the other daughter cell indicates that Dzip1 plays a crucial role in regulating this asymmetrical ciliogenesis . In the absence of growth factors , the kinase activity of GSK3β is de-repressed . Our findings suggest that once the amount of active GSK3β reaches a threshold after mitosis , GSK3β regulates the release of Rab8GDP from GDI2 at the cilium base by phosphorylating Dzip1 at S520 to promote ciliogenesis . However , when cells are growing in a nutrient-rich environment , GSK3β is inactivated by its inhibitory kinases and is unable to phosphorylate Dzip1 . Therefore , the lack of free Rab8GDP may fail to support the generation of Rab8GTP for ciliogenesis , leading to the disappearance of cilia from proliferating cells . Interestingly , in contrast to the viability of mice lacking GSK3α , knockout of GSK3β is lethal due to either hepatic apoptosis [25] or hypertrophic myopathy induced by cardiomyocyte hyperproliferation [51] . As the primary cilium is proposed as a “brake lever” of the cell cycle [52] , negatively regulating cell cycle re-entry [53 , 54] , the finding that GSK3β knockout induces cardiomyocyte hyperproliferation suggests a link between GSK3β and cilium assembly . Indeed , it has been shown that GSK3β is required for cilium maintenance via regulation of the microtubule-stabilizing protein pVHL [26 , 55] . Our present findings further extend the function of GSK3β in the regulation of ciliogenesis , support a positive role of GSK3β in the regulation of cilium assembly , and may have implications for understanding why rapidly-proliferating cells ( such as cancer cells ) often lose their cilia .
The CK1 inhibitor D4476 ( Tocris Bioscience , 2902 ) , rabbit anti-GSK3β ( Santa Cruz Biotechnology , sc-9166 ) , β-Catenin ( BD , 610154 ) , phosphorylated GSK3β ( S9 , Cell Signaling Technology , #93365 ) , and phosphorylated β-Catenin ( N3 , Cell Signaling Technology , #9561 ) were kind gifts from Dr . Wei Wu ( Tsinghua University ) . The CK2 inhibitor CX4945 ( Sequoia Research Products , SRP04555c ) was kindly provided by Dr . Seong H . Kim ( Korea University of Science and Technology ) . The GSK3 inhibitors BIO ( Tocris Bioscience , 3194 ) and CHIR99021 ( Tocris Bioscience , 4423 ) were from Tocris Bioscience . Rabbit anti-Dzip1 polyclonal antibody ( re-named Mid2 ) was from Abgent ( AP8926c ) . Mouse anti- Dzip1 and anti-GDI2 polyclonal antibodies were produced from mice immunized with aa 370–510 ( named Mid1 ) of mouse Dzip1 and with the full-length of human GDI2 , respectively . Rabbit anti-IFT88 ( Proteintech , 13967-1-AP ) , Rabin8 ( Proteintech , 12321-1-AP ) , PCM1 ( Santa Cruz Biotechnology , sc-67204 ) , GDI2 ( Santa Cruz Biotechnology , sc-133939 ) , histone H3 pS10 ( Abcam , ab47297 ) , mouse anti-GAPDH ( Proteintech , 60004-1-lg ) , GFP ( MBL International , M048-3 ) , Flag ( MBL International , PM020 ) , Myc ( Sigma , M4439-100UL ) , γ-Tubulin ( Sigma , T6557 ) , α-Tubulin ( Sigma , T5168 ) , AcTub ( Sigma , T7451 ) , and Rab8 ( BD , 610844 ) were from the indicated companies and were used according to the standard protocols provided . All animal experiments were performed according to the approved guidelines . Cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum under standard conditions . Wild-type and GSK3α- and GSK3β-knockout MEF cells were kindly provided by Prof . Jim Woodgett ( University of Toronto ) . To obtain mitotic NIH 3T3 cells , the cells were double-treated with thymidine to synchronize them in early S phase , and then released into medium containing nocodazole ( 50 nM ) for 8 h . To arrest NIH 3T3 cells at the mitosis–G0 transition , the round-up cells after nocodazole treatment were shaken off and reseeded onto coverslips in fresh medium containing 0 . 5% serum with or without the GSK3 inhibitors BIO ( 2 μM ) or CHIR99021 ( 10 μM ) . Transient cDNA transfections were carried out on cells using the Megatrans transfection reagent ( Origene , TT200002 ) according to the manufacturer’s instructions . To select cell lines stably expressing GFP-Dzip1 or with Dzip1 knockdown , NIH 3T3 cells were split 24 h after vector transfection and treated with G418 or Puromycin ( Sigma ) for 2 wk until the clones became macroscopic . Stable clones were verified by Western blot assay . Human GSK3β ( BC000251 ) , GDI2 ( BC005145 ) , Rabin8 ( BC059358 ) , and mouse Dzip1 ( BC098211 ) were each cloned from the cDNA libraries of the human B cell and mouse embryo brain ( E14 ) . Mutagenesis was conducted using standard molecular approaches . The DNA fragments for expressing the short hairpin RNAs that target the base pair positions 1308–1327 and 2172–2191 of the mouse Dzip1 gene were generated by annealing the following pairs of oligonucleotides ( only sense sequences are shown ) : 5′-GATCCCCCTGAAAGGGACTCCTTTAATTCAAGAGATTAAAGGAGTCCCTTTCAGTTTTTA-3′ and 5′-GATCCCCCTGACAGGAACCTCCATTATTCAAGAGATAATGGAGGTTCCTGTCAGTTTTTA-3′ , and the annealed oligonucleotides were then ligated into the pSuper-RetroPuro plasmid ( OligoEngine ) . Transfection of these plasmids using Megatrans ( Origene ) was performed according to the manufacturer’s instructions . Basal body purification was carried out as previously described [56] . A 30%–70% sucrose gradient was prepared in PIPES buffer ( 0 . 1% Triton X-100 , 10 mM PIPES [pH 7 . 2] , 0 . 2% EtSH ) . The collected basal bodies were then loaded onto a gradient containing 70% , 50% , 40% , and 30% sucrose from bottom to top , and centrifuged at 120 , 000g for 1 h . Fractions ( ~100 μl each ) were carefully collected from the top , mixed with sample buffer , and analyzed by standard Western blotting procedures . For IP assays , NIH 3T3 or HEK 293T cells transfected with the indicated plasmids were lysed using IP buffer ( 0 . 1% NP-40 , 50 mM HEPES [pH 7 . 00] , 125 mM NaCl , 10% glycol , 0 . 5 mM PMSF ) on ice for 15 min . Lysates were centrifuged at 12 , 000 rpm for 15 min , and the supernatants were incubated with the primary antibody-coated beads for 1 . 5 h at 4°C on a rotator . After six washes with IP buffer , the beads were collected and the bound proteins were analyzed by standard Western blotting [8] . The intensity of the indicated bands was quantified using ImageJ ( US National Institutes of Health ) . Immunofluorescence was performed as previously described [8] . Briefly , cells were fixed in 4% paraformaldehyde/phosphate buffer solution ( PBS ) followed by extraction in 0 . 2% Triton X-100/PBS , or cells were fixed in cold methanol . The cells were sequentially immunostained with the indicated primary and secondary antibodies . DNA was stained with DAPI , and the coverslips were mounted with Mowiol ( Sigma ) . For live-cell imaging of cells co-transfected with GFP-Dzip1 and RFP-Rab8 , the original images were collected at 2-μm thickness for five layers every 20 min . For image acquisition and processing , Zen 2009 Light Edition and Velocity ( 6 . 1 . 1 ) were respectively used for management of the confocal immunofluorescence microscope ( Carl Zeiss , LSM-710NLO and DuoScan ) , and the UltraView Vox spinning disc confocal microscope ( PerkinElmer ) . Super-resolution microscopy was performed with a 3-D structured illumination microscope ( Nikon , N-SIM ) , equipped with 405- , 488- , and 594-nm lasers , electron-multiplying CCD cameras ( iXon DU-897E , 512 × 512 , 16 μm × 16 μm ) , and a CFI SR Apo TRIF 100× ( 1 . 49NA ) oil objective ( Nikon ) . The 3-D images were then reconstructed using the NIS-Elements AR software package ( Nikon ) . The N- and C-termini of the RBD ( Rab8-binding domain ) of Rabin8 were fused with CFP and YFP , respectively . This fusion protein was introduced into G0-phase NIH 3T3 cells . For fixed cells , only those that displayed apparent basal body localization of CFP-RBD-YFP were selected for AB-FRET assay . Regular immunofluorescence images ( all channels ) were taken first to show common features of the selected cell . Before acceptor ( YFP ) bleaching , CFP and YFP channel images were respectively captured three times to calculate the mean fluorescence intensity of ICFP before and IYFP before . At the time point of photo-bleaching , a 514-nm laser at 100% intensity was activated 400 times to bleach the emission of YFP in the selected regions ( boxed ) , after which the fluorescence intensity of YFP ( IYFP after ) steadily dropped over 80% . After photo-bleaching of YFP , at the following seven time points ( with the same intervals ) , the fluorescence intensities of CFP and YFP were each measured to calculate the means of ICFP after and IYFP after . The fluorescence efficiency of FECFP was calculated as IFRET = ( ICFP after − ICFP before ) /ICFP before . Heat maps were automatically generated according to the manual of image processing ( Carl Zeiss , LSM-710NLO and DuoScan ) . His-tagged Dzip1 fragments , His-GSK3β , or GST-GDI2 were expressed and purified from Escherichia coli . BL21 with extraction buffer ( 50 mM NaH2PO4 , 300 mM NaCl , 10 mM imidazole [pH 8 . 0] ) or PBS . Briefly , the bacterial pellet was ultrasonically lysed , and the supernatant was incubated with His60 Ni Superflow resin or GST resin ( Clontech ) at 4°C for 1 h . Nonspecific binding to the resin was eliminated by three washes with the wash buffer ( 50 mM NaH2PO4 , 300 mM NaCl , 20 mM imidazole [pH 8 . 0] ) or PBS , and the indicated proteins were eluted with elution buffer ( 50 mM NaH2PO4 , 300 mM NaCl , 250 mM imidazole [pH 8 . 0] , for His-tagged proteins; 10 mM reduced glutathione in 50 mM Tris-HCl [pH 8 . 0] , for GST-tagged proteins ) and substituted by PBS buffer . For purification of the aa 1–378 fragment of Dzip1 from inclusion bodies , 8 M urea denaturation and graded re-naturation with a dialysis bag ( 8 M–4 M–3 M–2 M–1 M in PBS with 3 mM reduced glutathione , 0 . 3 mM glutathione oxide , and 5% glycerol ) was performed . The same amounts of His-GSK3β and Dzip1 fragments were mixed in kinase buffer ( 20 mM Tris-HCl , 10 mM MgCl2 , 5 mM DTT , 200 μM ATP with 10 μCi γ32P ATP [pH 7 . 5] ) , and incubated for 30 min at 30°C . Then , loading buffer was added to each sample to stop the reaction . After separation by SDS-PAGE electrophoresis , the gel was exposed to X-ray film at 4°C for 6 h or overnight . To examine the Dzip1-mediated dissociation of Rab8GDP from GDI2 , GST or GST-GDI2 was incubated with GST affinity binding beads in PBS buffer at 4°C for 1 h . Then , the cell lysate was added to the system for a further 1-h incubation at 4°C . The Rab8-GDI2-coated beads were collected and gently washed three times with PBS , and re-suspended in PBS buffer . The indicated amounts of His-Dzip1 ( aa 373–600 ) or His-Myosin Va ( aa 1320–1346 ) were each added to the system , followed by incubation at 4°C for 2 h . To semi-quantify the role of Dzip1 phosphorylation by GSK3β in regulating the dissociation of Rab8GDP from GDI2 , 4 , 8 , 12 , 16 , and 20 μg of GST-GDI2 were each preloaded onto the GST affinity binding beads in PBS buffer at 4°C for 1 h , and each group of GDI2-coated beads was further equally divided into four groups ( i . e . , 1 , 2 , 3 , 4 , and 5 μg of GST-GDI2 coated onto beads ) for pulldown assays . Each lysate from control , CHIR99021-treated , or S520A- or S520AD-expressing cells was equally divided into five groups and added into the system for a further 1 . 5-h incubation at 4°C . Finally , the beads were collected and washed four times with IP buffer and analyzed by standard Western blotting . All analyses were performed on an LTQ Orbitrap XL mass spectrometer ( Thermo Scientific ) at a resolution of 60 , 000 . For nano-liquid chromatography , Eksigent nanoLC 1D plus systems were equipped with 2-mm ReproSil-Pur C18-AQ ( Dr . Maisch ) trapping columns ( packed in house; i . d . , 1 , 150 μm; resin , 5 μm ) and 200-mm ReproSil-Pur C18-AQ ( Dr . Maisch ) analytical columns ( packed in house; i . d . , 75 μm; resin , 3 μm ) . The solvents used were 0 . 5% formic acid-water solution ( buffer A ) and 0 . 5% formic acid-acetonitrile solution ( buffer B ) . Trapping was performed at 2 μl/min buffer A for 15 min , and elution was achieved with a gradient of 0%–32% buffer B over 80 min , 32%–50% buffer B over 6 min , and 80% buffer B over 6 min at a flow rate of 300 nl/min . Eluting peptide cations were converted to gas-phase ions by a Nanospray Flex ( Thermo Scientific ) ion source at 2 . 0 kV . The mass spectrometer was operated in the data-dependent mode to automatically switch between mass spectrometry ( MS ) and tandem mass spectrometry ( MS/MS ) . Survey full-scan MS spectra were acquired from m/z 300 to m/z 1 , 800 , and the ten most intense ions with a charge state above 2 and above an intensity threshold of 500 were fragmented in the linear ion trap using a normalized collision energy of 35% . For the orbitrap , the AGC target value was set at 1 × 106 , and the maximum fill time for full MS was set at 500 ms . Fragment ion spectra were acquired in the LTQ Orbitrap XL with an AGC target value of 3 × 104 and a maximum fill time of 150 ms . Dynamic exclusion for selected precursor ions was set at 90 s . The lock mass option was enabled for the 462 . 14658 ion . The raw data were processed using Proteome Discoverer ( version 1 . 4 . 0 . 288 , Thermo Fisher Scientific ) . MS2 spectra were searched with the Sequest HT engine against the UniProt mouse complete proteome database ( release 2013_06 , 50 , 790 protein sequences ) . The database was searched with the following parameters: precursor mass tolerance , 20 ppm; MS/MS mass tolerance , 0 . 6 Da; two missed cleavages for tryptic peptides; dynamic modification oxidation ( M ) , phosphorylation ( STY ) ; static modification carbamidomethylation ( C ) . Peptide spectral matches were validated by a targeted decoy database search at 1% false discovery rate . With Proteome Discoverer , peptide identifications were grouped into proteins according to the law of parsimony . Statistical analysis was carried out using Microsoft Office Excel 2007 . p-Values were calculated using the paired t-test from the mean values of the indicated data . Significant differences in figures are marked with asterisks ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001 ) . | The primary cilium is an antenna-like organelle that projects out from the surface of cells and is present in almost all vertebrate cells , playing crucial roles in many cellular processes , including chemical sensation , signal transduction , and control of cell growth . The primary cilium assembles via a dynamic process called ciliogenesis that is regulated during the cell cycle: it assembles after mitosis and disassembles again before entering the next mitotic cycle . Here we investigate the regulatory mechanisms underlying this process . We show that Dzip1—a protein known to promote ciliogenesis—is preferentially recruited to the centrosome of the daughter cell that contains the grandmother centriole . Once in the centrosome , Dzip1 promotes release of Rab8GDP—a small GTPase that regulates membrane vesicular trafficking to the cilium—from its inhibitor GDI2 at the pericentriolar region , thereby facilitating ciliogenesis . This process is further regulated by an enzyme , GSK3β , whose increased kinase activity during the M- to G0-phase transition of the cell cycle results in phosphorylation of Dzip1 , promoting the ability of Dzip1 to release Rab8GDP . Our findings identify the molecular mechanism underlying the GSK3β-Dzip1-Rab8 signaling cascade , shedding light on how ciliogenesis is coordinated with mitotic exit . They also provide an understanding of why ciliogenesis always takes place earlier in one of the two daughter cells . | [
"Abstract",
"Introduction",
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"Methods"
] | [] | 2015 | GSK3β-Dzip1-Rab8 Cascade Regulates Ciliogenesis after Mitosis |
The Drosophila glucoside xylosyltransferase Shams xylosylates Notch and inhibits Notch signaling in specific contexts including wing vein development . However , the molecular mechanisms underlying context-specificity of the shams phenotype is not known . Considering the role of Delta-Notch signaling in wing vein formation , we hypothesized that Shams might affect Delta-mediated Notch signaling in Drosophila . Using genetic interaction studies , we find that altering the gene dosage of Delta affects the wing vein and head bristle phenotypes caused by loss of Shams or by mutations in the Notch xylosylation sites . Clonal analysis suggests that loss of shams promotes Delta-mediated Notch activation . Further , Notch trans-activation by ectopically overexpressed Delta shows a dramatic increase upon loss of shams . In agreement with the above in vivo observations , cell aggregation and ligand-receptor binding assays show that shams knock-down in Notch-expressing cells enhances the binding between Notch and trans-Delta without affecting the binding between Notch and trans-Serrate and cell surface levels of Notch . Loss of Shams does not impair the cis-inhibition of Notch by ectopic overexpression of ligands in vivo or the interaction of Notch and cis-ligands in S2 cells . Nevertheless , removing one copy of endogenous ligands mimics the effects of loss shams on Notch trans-activation by ectopic Delta . This favors the notion that trans-activation of Notch by Delta overcomes the cis-inhibition of Notch by endogenous ligands upon loss of shams . Taken together , our data suggest that xylosylation selectively impedes the binding of Notch with trans-Delta without affecting its binding with cis-ligands and thereby assists in determining the balance of Notch receptor’s response to cis-ligands vs . trans-Delta during Drosophila development .
Notch signaling is a juxtacrine signaling pathway broadly used during animal development and tissue homeostasis [1] . Both Notch and its ligands are type I transmembrane proteins containing multiple epidermal growth factor-like ( EGF ) repeats , which are involved in Notch-ligand interactions and are the sites of several O-linked sugar modifications [2 , 3] . Interaction of the Delta/Serrate/Lag-2 ( DSL ) ligands on the surface of the signal-sending cell and the Notch receptor on the signal-receiving cell initiates Notch trans-activation and results in the induction of Notch downstream targets [4 , 5] . In contrast , binding of Notch and ligands expressed in the same cell can result in cis-inhibition of the pathway [6–10] . The balance between the cis and trans interactions of Notch with ligands is thought to determine whether each cell assumes a signal-sending or a signal-receiving role with regard to a given ligand [11 , 12] . The Notch ligands Delta and Serrate function redundantly in several contexts during fly development [13] . However , there are developmental processes in which Delta and Serrate show non-redundant roles [14–17] . For example , although Serrate plays a minor , fully redundant role during wing vein formation , Delta is the ligand primarily involved in wing vein development [13 , 15 , 18] . In this context , both trans-activatory and cis-inhibitory interactions between Delta and Notch are important for demarcation of the boundary between vein and intervein tissues [15 , 18] . Specifically , high levels of Delta in the central provein cells are thought to cis-inhibit Notch and allow the cells to assume the wing vein fate . Delta from central provein cells also trans-activates Notch in neighboring cells to prevent the adoption of vein fate and to establish the wing-intervein boundary . The haploinsufficient wing vein phenotypes exhibited by Notch+/–and Delta+/–animals and their mutual suppression in Notch+/–; Delta+/–double-heterozygous flies [19] further indicate that both cis-inhibition and trans-activation of Notch by Delta are required for proper wing vein formation , and that altering the relative expression levels of these two proteins can tip the balance between cis- and trans-interactions between them . However , it remains to be determined whether other mechanisms exist to regulate this balance . Sugar modifications of the Notch receptors are known to affect Notch pathway activation at various steps , including folding , trafficking , ligand binding and potentially cleavage [20–22] . For instance , addition of N-acetylglucosamine ( GlcNAc ) to O-fucose residues on Notch EGF repeats by Fringe proteins differentially regulates the response of Notch to its ligands by regulating Notch-ligand interactions [23–26] . It is noteworthy that Fringe proteins promote the binding of Notch receptors to Delta ligands both in cis and in trans configurations [11] and as such , are not likely to be involved in regulating the balance between these opposing activities of Delta ligands . Another type of Notch sugar modification is the addition of O-glucose onto Notch EGF repeats by the protein O-glucosyltransferase 1 ( Poglut1 ) . In Drosophila , Poglut1 is encoded by rumi [27] and promotes Notch activation [27–30] . O-glucosylated EGF repeats also serve as a substrate for the addition of xylose by glucoside xylosyltransferase ( GXYLT ) enzymes [31 , 32] . Functional studies of the fly GXYLT , Shams , indicate that addition of xylose onto a specific subset of Notch EGF repeats ( EGF16-20 ) negatively regulates Notch signaling in specific contexts , i . e . wing veins and head bristles [32] . Loss of xylose from Notch results in increased cell surface expression of Notch in the pupal wing but not in third instar larval wing discs [32] , suggesting that additional mechanisms underlie the context-specificity of the shams loss-of-function phenotype . Here , we provide evidence that Notch xylosylation by Shams decreases Delta-mediated trans-activation of Notch by reducing the binding of Notch to trans-Delta without affecting the binding of Notch to cis-ligands . The effect of loss of Shams on trans-activation of Notch by overexpressed Delta can be mimicked by decreasing the level of endogenous cis-ligands , suggesting that upon loss of Notch xylosylation , Delta trans-activation overcomes Notch cis-inhibition by ligands . Altogether , our observations indicate that Shams regulates the balance between trans-activation and cis-inhibition of Notch by Delta to ensure optimal Notch activation in several contexts during Drosophila development .
To assess the role of Delta in the wing vein loss phenotype observed in shams mutants , we performed gene-dosage experiments using Delta genomic rescue transgenes [11] . Providing two additional genomic copies ( 4X ) of Delta in a wild-type background does not generate any adult wing phenotypes at 30°C ( Fig 1A and 1B ) or at room temperature [11] . The absence of phenotype is likely due to a simultaneous increase in the level of cis-inhibition and trans-activation of Notch upon increasing ligand levels . As previously reported , loss of shams results in a temperature-sensitive loss of distal part of adult wing veins L4 and L5 and a partial loss of the posterior cross-vein ( Fig 1D ) [32] . In a shamsΔ34/Df null background , providing one additional copy of Delta results in a fully penetrant , partial loss of wing vein L2 in addition to L4 , L5 and posterior cross vein ( Fig 1E ) , suggesting that shams mutants are sensitive to increased Delta levels compared to control animals . We performed similar genetic interaction experiments in flies harboring wild-type Notchgt-wt or xylosylation-deficient Notchgt-16_20 genomic transgenes [28] . Increasing the gene dosage of Delta does not result in wing vein loss in N+/+; Ngt-wt/+ animals , which have three copies of the wild-type Notch ( Fig 1G and 1H ) . However , providing an additional copy of Delta in N+/+; Ngt-16_20/+ animals results in a partially penetrant loss of the distal wing vein L5 ( Fig 1J and 1K ) , which resembles the shams mutant phenotype at 25°C [32] . Together , these data indicate that Notch signaling in shams mutants is sensitive to Delta levels and support the hypothesis that lack of Notch xylosylation affects Delta-mediated signaling . We also examined the effects of a Serrate transgene in similar experiments . Providing two additional copies of Serrate does not generate any wing vein loss in a wild-type background ( Fig 1C ) [11] . Moreover , increasing Serrate gene dosage does not enhance the wing vein loss phenotype in a shamsΔ34/Df null background ( Fig 1F ) . Finally , N+/+; Ngt-wt/+ and N+/+; Ngt-16_20/+ animals do not exhibit wing vein loss upon addition of an extra copy of Serrate ( Fig 1I and 1L ) . These results indicate that in the context of wing vein formation , lack of xylosylation does not render Notch sensitive to Serrate levels . Genetic interaction experiments were performed to examine the effect of decreasing Delta levels on the shams mutant phenotypes . Loss of one copy of Delta in Delta9P/+ ( Dl9P/+ ) animals results is extra wing vein material ( Fig 2A ) [33] . When one copy of Delta is removed in a shamsΔ34/Δ34 background , the shams mutant wing vein loss is completely suppressed , and the extra wing vein phenotype of Dl9P/+ is partially suppressed ( Fig 2B and 2C ) . We have previously reported that loss of shams also results in the loss of post-vertical ( PV ) and ocellar ( OC ) bristles in the adult head [32] . Genetic interaction studies indicate that removing one copy of Delta in shams mutants rescues the loss of head bristles ( Fig 2F ) similar to the wing vein loss phenotype . Together , these observations support the notion that the shams loss-of-function phenotypes are due to increased Delta-mediated signaling . We also examined the effect of decreasing Serrate levels on the above-mentioned phenotypes ( loss of wing vein and head bristles ) . Removing one copy of Serrate does not affect the loss of wing vein and head bristles in shams mutants ( Fig 2D–2F ) . These observations indicate that altered Serrate-mediated signaling is not likely to contribute to shams loss-of-function phenotypes . Surprisingly , removing one copy of Serrate in shamsΔ34/Δ34 mutant animals results in wing margin loss in some animals ( S1A Fig; 21% penetrant , n = 73 ) , which resembles the loss-of-function mutants of Notch and Serrate . Adding one copy of a Serrate genomic rescue transgene [11] fully rescues the wing margin phenotype without affecting the wing vein loss caused by the loss of shams ( S1B Fig ) , indicating that the observed wing margin loss is indeed due to decreased Serrate levels in a shams mutant background . This indicates that in the wing margin , shams might play a redundant role which is revealed when one copy of Serrate is removed . Thus , a role for Shams in Serrate-mediated Notch signaling remains a distinct possibility in the context of wing margin formation . To examine the effects of loss of shams on the Notch activity in the wing imaginal disc , we performed clonal analysis in the 3rd instar larval wing discs by using the MARCM system [34] . We expressed Flippase with an Ultrabithorax enhancer ( Ubx-FLP ) , which induces FRT-mediated recombination in the wing imaginal discs with a high efficiency [27 , 35] . We first examined the effect of loss of shams on Notch activity by staining for Notch targets Cut and Wingless ( Wg ) in shamsΔ34 mutants clones . These mutant clones do not exhibit clear abnormalities in the expression of Cut and Wg when compared to adjacent wild-type or heterozygous cells ( Fig 3A–3B’ ) . However , slight broadening observed in Cut and Wg expression in shamsΔ34 mutants clones supports a mild increase in Notch activation ( Fig 3A–3B’ ) . To further assess the effect of loss of shams on ligand-mediated Notch activity , we examined Cut expression in single-mutant clones for ligands ( Serrev6-1 and DlRevF10 ) and double-mutant clones for ligands and shams ( shamsΔ34 Serrev6-1 and DlRevF10 shamsΔ34 ) . To analyze the effects of loss of shams on each ligand without interference from the other ligand , we focused on mutant clones which cross the dorsal-ventral ( DV ) boundary . Serrev6-1 clones show a highly penetrant loss of Cut , except for the cells that directly abut the wild-type tissue ( Fig 3C , 3C’ and 3G; n = 40 ) . This observation indicates that when cells around the DV boundary lack Serrate , they fail to activate the Notch target Cut despite the presence of Delta in the clones . In contrast , Cut-positive cells non-adjacent to the clone boundary are present in 42% of shamsΔ34 Serrev6-1 double-mutant clones which cross the DV boundary ( Fig 3D , 3D’ and 3G; n = 31 ) . Since Delta is the only ligand remaining in these clones , these observations are in agreement with the conclusion that loss of Shams promotes Delta-mediated Notch activation . Next , we performed a similar analysis on Delta single-mutant and Delta shams double-mutant clones , where Serrate is the only remaining ligand . DlRevF10 single-mutant clones crossing the boundary showed a ~65% penetrant loss of Cut expression ( Fig 3E , 3E’ and 3G; n = 29 ) . Similarly , DlRevF10 shamsΔ34 double-mutant clones which cross the DV boundary exhibited a ~64% penetrant loss of Cut expression ( Fig 3F , 3F’ and 3G; n = 34 ) . The comparable loss of Cut expression in DlRevF10 and DlRevF10 shamsΔ34 clones suggests that loss of shams does not alter the activation of Notch by Serrate , which is the only remaining ligand inside these clones . In addition to wing disc stainings , we examined the effects of shamsΔ34 , Serrev6-1 and DlRevF10 single-mutant and shamsΔ34 Serrev6-1 and DlRevF10 shamsΔ34 double-mutant clones on the adult wing size . Although the clones in these adult wings are not marked , inspection of 3rd instar wing imaginal discs indicates that Ubx-FLP induces sizeable clones in 100% of the discs . Adult wings harboring shamsΔ34 clones do not exhibit any wing margin phenotypes , although some animals show wing vein loss ( Fig 3I ) . Adult wings harboring clones of the Serrev6-1 allele show severe wing scalloping ( Fig 3J ) , in agreement with previous reports on this allele and other Serrate null alleles [36–38] . However , the degree of scalloping in adult wings harboring shamsΔ34 Serrev6-1 double-mutant clones is significantly less than that of the wings harboring Ser rev6-1 clones ( Fig 3K ) . Indeed , the average area of the wings harboring Serrate clones was significantly less than that of the wings harboring Serrate shams double-mutant clones ( Fig 3N; P<0 . 05; n = 13 for each genotype ) . In contrast , the average area of wings harboring DlRevF10 clones was not significantly different from the wings harboring DlRevF10 shamsΔ34 double mutant clones ( Fig 3L–3N; P>0 . 05; n = 13 for each genotype ) . These results are in agreement with the data obtained from clonal analysis in larval wing discs and further support the notion that loss of shams partially suppresses the phenotypes caused by the loss of Serrate , likely by enhancing the Delta-mediated Notch activation in the developing wing . In some cell types , the decision whether Notch signaling is activated or inhibited is determined based on the relative levels of Notch trans-activation and cis-inhibition by its ligands [6–8] . Given the complex interplay between trans and cis functions of ligands and potential feedback mechanisms , we used the GAL4-UAS system [39] to overexpress Delta or Serrate along the anterior-posterior boundary of the developing 3rd instar wing imaginal discs and assessed the effects of loss of shams on the cis and trans effects of each ligand on Notch . When raised at 25°C , animals overexpressing Delta within the Dpp expression domain ( dpp>Dl ) exhibit two phenotypes in wing imaginal discs: loss of the Cut-positive cells and disruption of the endogenous wing margin within the Dpp expression domain due to cis-inhibition ( Fig 4A and 4A’; arrowhead ) , and ectopic Cut-positive cells that flank the Dpp expression domain in the dorsal-posterior quadrant due to trans-activation ( Fig 4A and 4A’; arrows ) . In a shamsΔ34/Δ34 mutant background , there is no obvious effect on cis-inhibition within the Delta-expressing stripe ( Fig 4B and 4B’ ) . However , we observe broad ectopic activation of Cut in a significant portion of the dorsal-anterior quadrant ( Fig 4B and 4B’ ) . These observations support the notion that loss of Shams promotes Delta-mediated trans-activation of Notch without affecting cis-inhibition . Of note , in late 2nd instar wing discs , the dpp-GAL4 driver shows a much broader anterior expression domain compared to 3rd instar wing discs ( Fig 4C ) , providing a likely explanation for the wide Cut expression domain observed in the dorsal-anterior quadrant of dpp>Dl shamsΔ34/Δ34 3rd instar discs . To determine the effects of loss of Notch xylosylation on Serrate-mediated trans-activation and cis-inhibition of Notch , we performed similar overexpression experiments using a Tomato-tagged Serrate transgene ( UAS-SerTom ) [40] . As expected , overexpression of SerTom results in cis-inhibition of Notch at the endogenous wing margin region and trans-activation of Notch outside of the Dpp expression domain in the ventral compartment ( Fig 4D and 4D’ ) . In contrast to dpp>Dl , no changes in Cut activation in or outside of the Dpp expression domain was observed between dpp>SerTom and dpp>SerTom shamsΔ34/Δ34 animals ( Fig 4D–4E’ ) . These data indicate that in the developing wing disc , loss of Shams does not affect the trans-activation and cis-inhibition of Notch mediated by overexpressed Serrate . Given the dramatic increase in the ability of dpp>Dl to trans-activate Notch upon loss of Shams , we decided to further explore the mechanism that prevents dpp>Dl from activating Notch in a shams+/+ background . In the sensory bristle lineage , decreasing the endogenous levels of Delta enhances the trans-activation of Notch by overexpressed Delta [9] . Accordingly , we examined the effects of removing one copy of endogenous Delta and/or Serrate on the expression of Cut in dpp>Dl larvae . In a DlRevF10 or Serrev2-11 heterozygous background , dpp>Dl induces moderate levels of Notch trans-activation in the dorsal-anterior quadrant ( Fig 5A–5B’ , compare to Fig 4A and 4A’ ) . Moreover , in a DlRevF10/+ SerRX82/+ double-heterozygous background , dpp>Dl results in massive induction of Cut in the dorsal-anterior quadrant , broadening of the Cut expression domain in the dorsal-posterior quadrant , and appearance of Cut-expressing cells in the ventral-posterior compartment ( Fig 5C and 5C’ ) . In addition , Delta-mediated cis-inhibition is significantly decreased , because many cells in the Dpp expression domain express Cut and the endogenous wing margin is restored ( Fig 5C and 5C’; arrow ) . These observations indicate that cis-inhibition of Notch by endogenous ligands potently limits the ability of overexpressed Delta to trans-activate Notch outside of the Dpp domain and cooperates with overexpressed Delta to cis-inhibit Notch inside of the expression domain , in agreement with the previous report by Jacobsen et al [9] . Therefore , loss of shams and decreasing endogenous ligands both result in a similar increase in the ability of dpp>Dl to trans-activate Notch in 3rd instar wing discs , suggesting that in shams mutants , Delta mediated trans-activation of Notch is able to overcome the cis-inhibitory effect of endogenous ligands . To determine whether the effects of Shams on Delta-mediated signaling can be explained at the level of Notch-ligand binding , we performed S2 co-culture assays and assessed the effects of shams knock-down ( KD ) on the rate of aggregation formation and size of the aggregates formed between S2-Notch ( S2-N ) and S2-Delta ( S2-Dl ) or S2-SerrateTom ( S2-SerTom ) cells , which are indications for Notch-ligand binding [40 , 41] . For control experiments , we used plain S2 cells , which do not express Notch and Delta [41] , and EGFP dsRNA . When co-cultured with plain S2 cells treated with shams dsRNA , S2-Dl cells form small aggregates which do not increase with time ( Fig 6A ) . Co-culture of S2-Dl and control S2-N cells for one minute resulted in the formation of small aggregates , which increased in number and size as time elapsed ( Fig 6A ) . Co-culture of S2-Dl with Shams KD S2-N cells formed much larger cell aggregates at 5 and 15 minutes ( Fig 6A ) . Quantification of cell aggregates containing more than 6 cells at 5 minutes of co-culture showed that the number of aggregates was significantly higher when S2-N cells were incubated with shams dsRNA ( Fig 6B; P<0 . 01 ) . qRT-PCR experiments indicate that shams dsRNA decreases shams mRNA levels greater than 80% in S2 cells ( Fig 6C ) . These observations suggest that Shams decreases the binding between Notch and Delta in trans . Of note , co-culture experiments between S2-SerTom and S2-N cells showed that the size and the number of the S2-SerTom/S2-N aggregates did not change when shams levels were decreased ( Fig 6A and 6B ) . These observations suggest that Shams KD does not affect the binding between Notch and trans-Serrate . To more directly assess the effects of Shams KD on Notch-ligand binding , we used a quantitative receptor-ligand binding assay [23 , 42] . We incubated control and Shams KD S2-N cells with various concentrations of alkaline phosphatase ( AP ) -tagged ligand extracellular domains ( Delta-AP or Serrate-AP ) and asked whether Shams KD alters the binding of each ligand to S2-N cells in terms of the AP activity . Binding of each ligand-AP to plain S2 cells , which do not express Notch [41] , was used as control . In agreement with the aggregation assays , treating S2-N cells with shams dsRNA significantly increased the binding of Delta-AP to these cells compared to EGFP dsRNA treated S2-N cells ( Fig 6D ) . However , the amount of Serrate-AP bound to Shams KD and control S2-N cells was comparable at all four Serrate-AP concentrations tested ( Fig 6D ) . These observations indicate that Shams negatively regulates Notch-Delta interaction but does not affect Notch-Serrate interaction . Binding of Notch with trans-ligands can be affected by cell surface levels of Notch . Recent work has shown that combined loss of O-fucose and xylose residues from Notch alters its trafficking in 3rd instar wing discs , but loss of xylose by itself does not [43] . We have previously reported that loss of Shams or mutations that prevent the addition of xylose-glucose-O glycans to Notch EGF16-20 did not affect cell-surface expression of Notch in the 3rd instar wing discs , but enhanced the cell surface levels of Notch in the developing pupal wing disc [32] . Accordingly , we examined whether shams KD in S2 cells affects the cell surface expression of Notch . Detergent-free immunofluorescent staining of control and shams KD S2-N cells with an antibody against the Notch extracellular domain did not show any significant changes in the surface levels of Notch when shams is decreased ( S2A and S2B Fig; n = 40 cells for each groups ) . Thus , the increased aggregation in Shams KD S2-N/S2-Dl co-cultures is not caused by an increase in Notch surface expression . Although Serrate does not contain any predicted O-glucosylation sites , one of the Delta EGF repeats harbors the consensus O-glucosylation motif [44] . However , adding shams dsRNA to S2-Dl cells did not alter the size or the number of aggregates formed when cultured with S2-N cells ( S3A and S3B Fig ) . We conclude that shams KD in Delta-expressing cells does not affect Notch-Delta trans-binding and that Shams functions in Notch-expressing cells . We also examined the effect of shams KD on surface distribution of Delta in S2-Dl cells using cell surface immunostaining with an antibody against the extracellular domain of Delta . Shams dsRNA treated S2-Dl cells did not show any significant change in surface level of Delta as compared to EGFP dsRNA treated S2-Dl cells ( S3C and S3D Fig; n = 40 cells per group ) . This observation indicates that shams KD does not affect the surface level of Delta in S2 cells . We also examined whether loss of shams affects surface expression of Delta in vivo . Detergent-free staining of 3rd instar wing imaginal discs did not show any significant difference in surface expression of Delta between shamsΔ34 mutant clones and the neighboring wild-type and heterozygous cells ( S3E Fig; n = 12 clones ) , indicating that Shams does not regulate the surface levels of Delta in 3rd instar wing imaginal discs . To determine whether Shams modulates binding between Notch and cis-ligands , we asked whether shams KD affects the ability of ligands co-expressed with Notch to decrease the aggregation between the Notch-expressing cells and S2-Dl cells . To this end , we performed aggregation assays between S2-Dl cells and S2 cells transiently transfected with a Notch expression vector as control ( S2-Ntransient ) or equal amounts of Notch and cis-ligand expression vectors ( S2-N&Dltransient or S2-N&Sertransient ) . The relative aggregation between S2-Dl and S2-Ntransient cells in the presence and absence of cis-ligands was used as an indication for the degree of cis-inhibitory effect of each ligand . For example , if the number of aggregates between S2-Dl and S2-N&Sertransient cells is 25% of the number of aggregates between S2-Dl and S2-Ntransient cells ( i . e . relative aggregation 25% ) , we would conclude that cis-Serrate was able to block the interaction between Notch and trans-Delta by 75% . Co-culture of S2-Dl and control S2-Ntransient cells formed small aggregates , which grew in size and number with time , similar to the co-culture between S2-Dl and stable S2-N cells ( Figs 7A and 8A , compare to Fig 6A ) . The aggregation was dramatically decreased when S2-Dl cells were co-cultured with S2-N&Dltransient or S2-N&Sertransient cells ( Figs 7A , 7B , 8A and 8B ) . This is most likely due to cis-inhibition of Notch by ligands expressed in the same cell , as shown previously in Drosophila and mammalian cell-culture assays [11 , 40] . Co-culture of S2-Dl cells and shams KD S2-Ntransient cells formed aggregates more quickly and resulted in larger aggregates , recapitulating the results seen with stable S2-N cells ( Figs 7A and 8A ) . Addition of cis-ligands to these cells ( shams KD S2-N&Dltransient or S2-N&Sertransient ) also decreased their aggregation with S2-Dl cells ( Figs 7A , 7B , 8A and 8B ) . Importantly , quantification of aggregates after five minutes of co-culture showed that the magnitude of this cis-inhibition was comparable to the cis-inhibition observed in co-culture between S2-Dl and control S2-N&Dltransient or S2-N&Sertransient cells , which were incubated with EGFP dsRNA instead of shams dsRNA ( Figs 7C and 8C ) . These observations suggest that shams KD does not diminish the ability of cis-ligands to oppose the binding between Notch and trans-Delta . One caveat of this experiment is that the relative level of cis-ligands expressed in these cells might be too high and therefore be able to inhibit trans-Delta/Notch binding irrespective of shams levels . To address this concern , we performed three additional sets of aggregation assays for each cis-ligand and successively decreased the ratio of cis-ligand expression plasmid to the Notch expression plasmid used in each set ( 0 . 5:1 , 0 . 2:1 , 0 . 1:1 ) . To ensure similar baseline levels of trans-Dl/Notch binding , we used the same amount of Notch expression plasmid in all experiments and only changed the amount of cis-ligand expression plasmids . Aggregation between S2-Dl and S2-Ntransient cells was once again inhibited by expression of cis-ligands ( both cis-Delta and cis-Serrate ) even at lowest levels . Quantitation of the number of cell aggregates greater than six cells at five minutes of co-culture showed that the magnitude of inhibition by cis-ligands was concentration-dependent: the lower the ratio of cis-ligand to Notch expression plasmid , the lower the ability of cis-ligands to decrease the number of aggregates between S2-Dl and S2-Ntransient cells ( Figs 7C and 8C ) . These data indicate that the level of cis-ligands used in our assays are not saturating . Under each cis-ligand/Notch ratio , the cis-inhibitory effect of each ligand on S2-Dl/S2-Ntransient aggregation was almost identical in cells treated with EGFP dsRNA and shams dsRNA ( Figs 7C and 8C ) . These observations indicate that shams KD does not decrease the ability of cis-ligands to block the interaction between trans-Delta and Notch , and suggest that Shams does not affect the binding between Notch and cis-ligands . Taken together , these cell culture data support the conclusion that loss of Shams specifically enhances the binding of Notch with trans-Delta without affecting its binding with cis-ligands .
Modifications of the extracellular domain of Notch receptor by glycosylation influence its activity in different contexts [20–22] . Xylosylation of the Notch receptor by the glycosyltransferase enzyme Shams has been reported to negatively regulate Notch signaling in Drosophila [32] . Loss of Notch xylosylation is associated with increased Notch expression at the cell surface in pupal wing disc but not in the larval wing imaginal discs [32] . However , the mechanisms underlying the tissue-specific phenotypes of shams and the effects of xylosylation on other steps of Notch signaling such as ligand binding and cis-inhibition are not known . In the present study , we provide several lines of evidence suggesting that the wing vein loss phenotype observed upon loss of Notch xylosylation results from a specific increase in Delta-mediated Notch activation . First , adding one copy of Delta enhances wing vein loss phenotype in shams mutant animals . Second , a Notch transgene with mutations in functional xylosylation sites results in wing vein loss when combined with an extra copy of Delta . Third , removing one copy of Delta suppresses the wing vein phenotype in shams mutant animals . Last , Shams KD in S2-N cells enhances their binding with AP-tagged Delta and their aggregation with S2-Dl cells without affecting cell surface levels of Notch . Since functional xylose residues reside in EGF16-20 of the Notch receptor [32] , our work identifies the glycosylation of this domain as a novel mechanism for the modulation of Delta-mediated Notch activation in Drosophila . Although Notch EGF11-12 are required for binding of Notch to both ligands [2] , a mutation in Notch EGF8 affects Notch-ligand binding and signaling in a ligand-selective manner [42] . Together with this report [42] , our data suggest that distinct EGF repeats other than EGF11-12 are involved in preferential or exclusive modulation of the response of Notch to its ligands . Moreover , the differential effects of decreasing Notch xylosylation on the binding and response of Notch to trans-Delta versus cis-ligands suggest different domains or structural conformations of Notch might be involved in binding to trans- versus cis-ligands . The increased Delta-mediated signaling upon loss of shams can theoretically be due to increased trans-activation , decreased cis-inhibition , or both . If the primary mechanism for activation of Notch signaling and loss of wing vein in shams mutants were decreased cis-inhibition by Delta , removing one copy of Delta should have enhanced the wing vein loss phenotype in shams mutants . On the contrary , the shams wing vein loss was suppressed upon Delta heterozygosity . Moreover , loss of shams dramatically enhances Notch trans-activation but does not affect Notch cis-inhibition by Delta in dpp>Dl animals , and shams KD promotes S2 cell aggregation mediated by Notch and trans-Delta without decreasing the inhibitory effect of cis-Delta on Notch in these assays . Therefore , although it is still possible that Shams plays a minor role in the interaction of Notch with cis-ligands in certain contexts , our data strongly suggest that the primary mechanism for the wing vein loss in shams mutants is an increase in Notch trans-activation by Delta , not a decrease in Notch cis-inhibition by Delta . In line with a previous report in the bristle lineage [9] , we find that removing one copy of Delta and/or Serrate in dpp>Dl animals results in ectopic activation of Notch in the dorsal-anterior quadrant , indicating that cis-inhibition by endogenous ligands normally opposes the trans-activation of Notch by ectopic Delta in this region . These observations are in agreement with quantitative analyses indicating that the balance between the activity of trans- and cis-ligands determines whether a given cell assumes a signal-receiving state or not [11 , 12] . A similar increase in Delta-mediated trans-activation of Notch is seen in dpp>Dl animals upon loss of Shams despite the presence of endogenous ligands . Accordingly , we propose that Shams functions to regulate the balance between trans-activation of Notch by Delta and cis-inhibition of Notch by ligands , and that in the absence of shams , trans-activation of Notch by Delta overcomes the cis-inhibitory effects of ligands . Our cell aggregation assays suggest that Shams mediates this role by impeding the ability of Notch to bind Delta in trans , without altering the binding of Notch to cis-ligands . As shown in Fig 1 , increasing the gene dosage of Delta by itself or combined with an additional copy of wild-type Notch does not result in wing vein loss , likely because the balance between the Notch and Delta levels and also the balance between the trans and cis activities of Delta are preserved . Based on our model , loss of xylose residues on Notch due to loss of Shams or mutations in biologically-relevant Shams target sites on Notch tips the balance between trans- and cis- activities of Delta in favor of trans-Delta , as evidenced by the net gain of Notch signaling and loss of wing vein in animals with three copies of Delta and simultaneous loss of Notch xylosylation . Both Shams and Fringe regulate Notch signaling by adding carbohydrate residues to O-linked monosaccharides on Notch EGF repeats and generating disaccharides: xylose-glucose-O in the case of Shams , and GlcNAc-fucose-O in the case of Fringe [23 , 25 , 32 , 45] . Moreover , as shown here for Shams and previously for Fringe [23 , 46] , both proteins regulate Notch-ligand interactions . However , the effects of these enzymes on the binding and response of Notch to Delta versus Serrate and to trans-Delta versus cis-Delta seem to be distinct from each other . Fringe promotes Delta-mediated trans-activation and simultaneously decreases Serrate-mediated trans-activation of Notch [26] . Moreover , it has recently been shown that Fringe proteins affect the trans and cis interaction of Notch with each ligand in the same direction , i . e . , they promote Notch-Delta interactions both in cis and in trans , and inhibit Notch-Serrate interaction both in cis and in trans [11] . In contrast , our data indicate that Shams decreases the binding of Notch to and its activation by trans-Delta without affecting its interactions with cis-ligands . Further , our aggregation assays and most of our in vivo observations indicate that binding of Notch to and its activation by trans-Serrate is not significantly affected by Shams , although the appearance of a low-penetrance wing margin loss phenotype in shams–/–Serrate+/–animals suggests that Shams might play a redundant role in Serrate-induced Notch signaling in some contexts . The functional differences between Shams and Fringe likely explain their distinct mutant phenotypes in the wing , i . e . loss of wing vein in the case of shams and wing vein thickening and loss of wing margin in the case of fringe mutants [47] . The distinct roles of Fringe and Shams in regulating Notch signaling along with the differentially distributed EGF repeats with Shams ( xylose ) versus Fringe ( GlcNAc ) elongation across the Notch extracellular domain [32 , 48] suggest that the combined function of the sugar modifications mediated by these enzymes ensures optimal level of Notch pathway activity in several contexts during fly development .
The following strains were used in this study: y w , y w; D/TM6 , Tb1 , w; nocSco/CyO , w; nocSco/CyO; TM3 , Sb1/TM6 , Tb1 , dpp-GAL4 , Df ( 3R ) BSC494/TM6C , Sb1 , Dl9P/TM6 , Tb1 , DlRevF10 SerRX82/TM6 , Tb1 , UAS-CD8::GFP ( Bloomington Drosophila Stock Center ) , shamsΔ34/TM6 , Tb1 [32] , PBac{Ngt-wt}VK22 , PBac{Ngt-16_20}VK22 , [28] , P{Dlgt-wt}attP2 , PBac{Dlgt-wt}VK37 , PBac{Sergt-wt}VK37 [11] , y w Ubx-FLP tub-GAL4 UAS-GFPnls-6X-Myc; FRT82B y+ tub-GAL80/TM6 , Ubx [27] , w; UAS-Dl ( Gary Struhl ) , DlRevF10/TM6 , Tb1 [49] , Serrev6-1/TM6 , Tb1 [50] , Serrev2-11/TM6 , Tb1 [50] , SerRX106 [51] , UAS-Serwt-Tomato ( UAS-SerTom ) [40] , FRT82B shamsΔ34 Serrev6-1/TM6 , Tb1 , Dl9P shamsΔ34/TM6 , Tb1 , FRT82B shamsΔ34 DlRevF10/TM6 , Tb1 , FRT82B DlRevF10/TM6 , Tb1 , dpp-GAL4 shamsΔ34/TM6 , Tb1 , UAS-Dl; shamsΔ34/TM6b , Tb1 , UAS-SerTom/CyO; shamsΔ34/TM6b , Tb1 , y w Ubx-FLP/FM7; FRT82B Sb63 y+/TM6 , Ubx ( this study ) . All crosses were performed on standard media . All crosses were incubated at 25°C except for adult wings listed at 30°C , which were incubated at 25°C until late larval stage and shifted to 30°C during pupal stages . To generate MARCM clones [52] in 3rd instar wing discs , y w Ubx-FLP tub-GAL4 UAS-GFPnls-6X-Myc; FRT82B y+ tub-GAL80/TM6 , Ubx females were crossed to FRT82B mutant/TM6 , Tb1 males , wherein mutant stands for shamsΔ34 , Serrev6-1 , DeltaRevF10 , DeltaRevF10 shamsΔ34 , or shamsΔ34 Serrev6-1 . Crosses were kept at 25°C and mosaic Tb+ 3rd instar larvae were selected for dissection . To generate clones in adult wings , FRT82B shamsΔ34/TM6 , Tb1 , FRT82B Serrev6-1/TM6 , Tb1 , FRT82B shamsΔ34 Serrev6-1/TM6 , Tb1 , FRT82B DeltaRevF10/TM6 , Tb1 , and FRT82B DeltaRevF10 shamsΔ34/TM6 , Tb1 males were crossed to y w Ubx-FLP/FM7; FRT82B Sb63 y+/TM6 , Ubx females and raised at 25°C . Sb63 , Tb+ flies were selected for scoring the wings . All of the scored flies had regions of Sb+ microchaete on the thorax , confirming the generation of mutant clones in wing imaginal discs . dpp>Dl shamsΔ34/Δ34 animals were generated by crossing animals harboring a dpp-GAL4 shamsΔ34 recombinant chromosome to UAS-Dl; shamsΔ34/TM6b , Tb1 animals . Tb+ 3rd instar larvae were selected for dissection . To generate dpp>SerTom shamsΔ34/Δ34 animals , dpp-GAL4 shamsΔ34 animals were crossed to UAS-SerTom/CyO; shamsΔ34/TM6b , Tb1 animals . Tb+ 3rd instar larvae expressing Tomato were selected for dissection . To examine the expression pattern of cDNAs driven by dpp-GAL4 in larval wing imaginal disc , dpp-GAL4/TM6 , Tb1 males were crossed to UAS-CD8::GFP females . Late 2nd and late 3rd instar Tb+ larvae were selected and dissected . Dissection and staining were performed by using standard methods . For surface staining , S2 cells were incubated with antibodies against the Notch extracellular domain ( NECD ) or the Delta extracellular domain ( Dl-ECD ) in the absence of detergent . A similar detergent-free protocol was used for Delta surface staining of wing imaginal discs . Antibodies used were mouse α-Cut ( 2B10 ) 1:500 , mouse anti-Wg ( 4D4 ) 1:100 , mouse anti-NECD ( C458 . 2H ) 1:100 ( all from DSHB ) , guinea pig anti-Dl-ECD 1:3000 ( Gift from M . Muskavitch ) [15] , goat α-mouse-Cy3 1:500 , donkey α-mouse-Cy5 1:500 and donkey α-Guinea Pig-Cy3 1:500 ( Jackson ImmunoResearch Laboratories ) . Adult wings were imaged using Zeiss Axioscope-A1 and Nikon Ci-L upright microscopes . Wing areas were measured in term of square pixels using ImageJ 1 . 47 . Confocal images were scanned using a Leica TCS-SP5 microscope and processed with Amira5 . 2 . 2 . Images were processed with Adobe Photoshop CS5; Figures were assembled in Adobe Illustrator CS5 . S2 cells ( Invitrogen ) were cultured in Schneider's Drosophila Medium ( Lonza ) supplemented with 10% fetal bovine serum and penicillin-streptomycin ( 100 U/mL ) . For S2-N and S2-Dl stable cell lines ( DGRC , Bloomington , USA ) , 200 nM methotrexate ( Sigma-Aldrich ) was added . S2-SerTom stable cells [40] were cultured in M3 medium ( Gibco ) supplemented with 10% fetal bovine serum and 100 μg/mL of hygromycin B . For knock-down , 7 . 5 μg of either EGFP dsRNA ( control ) or shams dsRNA was added to the culture medium of S2 , S2-N or S2-Dl cells , and the cells were cultured for 24 hours at 25°C prior to induction with CuS04 ( 0 . 7 mM ) . Ligand-receptor binding assays were performed as described previously [42] with minor modifications . In brief , S2 cells ( 2 x 106 cells/well ) were transiently transfected with the constructs expressing the extracellular domain of Delta or Serrate fused to alkaline phosphatase ( 6 . 0 μg of either pMT-Delta-AP or pMT-Serrate-AP per well ) . After 24 hours , the transfected cells were induced by adding 0 . 7mM CuSO4 to the media . After three days of induction , conditioned media were collected and the amount of AP was determined by quantifying AP activity in each medium by following the manufacturer’s instructions ( Phospha-Light System , Applied Biosystems ) and using FLUOstar OPTIMA ( BMG Labtech ) . In parallel , plain S2 or S2-N cells ( 2 x 106 cells/well ) were induced by 0 . 7mM CuSO4 after 24 hours of treatment with EGFP dsRNA or shams dsRNA . After three days of induction , cells ( S2 or S2-N cells ) were collected and incubated with conditioned media ( containing defined concentrations of Delta-AP or Serrate-AP ) for 90 minutes on rotary shaker . After three times washing and cell lysis , the endogenous AP activity was heat inactivated ( 60°C for 10 min ) . The amount of AP-tagged ligand bound to S2-N cells was assayed as per the manufacturer’s instructions ( Phospha-Light System , Applied Biosystems ) and using FLUOstar OPTIMA ( BMG Labtech ) . Binding of each ligand ( Delta-AP or Serrate-AP ) with plain S2 cells ( which lack endogenous Notch protein [41] ) was used as control . Experiments were performed in triplicate and repeated three times . Cells were incubated with CuSO4 for 1–2 days and then used in aggregation assays . For each aggregation assay , 2 . 5 x 105 of the dsRNA-treated cells ( S2 , S2-N , S2-Dl ) were mixed with 5 x 105 S2-Dl , S2-SerTom or S2-N cells ( induced with 0 . 7 mM CuSO4 for 3 hours prior to co-culture ) in a total volume of 200 μl medium in a 24-well plate . For co-culture assays expressing cis-ligands , after 24 hours of treatment with either EGFP or shams dsRNA , S2 cells were transiently transfected with 2 μg total of either pBluescript ( control ) , pBluescript and pMT-Notch ( S2-Ntransient ) , pMT-Notch and pMT-Delta ( S2-N&Dltransient ) , or pMT-Notch and pMT-Serrate ( S2-N&Sertransient ) using 3 μL of FuGENE HD ( Promega ) . One μg of pMT-Notch was used in all assays . The remaining 1 μg was either pBluescript alone , pMT-Delta alone , pMT-Serrate alone , or a mixture of 0 . 5 μg , 0 . 2 μg or 0 . 1 μg of pMT-Delta or pMT-Serrate and pBluescript ( Figs 7C and 8C ) . For aggregation assays , 2 . 5 x 105 dsRNA treated cells were mixed with 5 x 105 S2-Dl cells ( induced for 3 hours prior to co-culture with CuSO4 ) in a total volume of 200μL in a 24-well plate . Co-cultured cells were gently shaken at 150 rpm to allow aggregation . Images of aggregate formation were taken at reported time points . The number of cell aggregates was quantified using a hemocytometer after 5 minutes of co-culture . Each assay was repeated at least three times . P-values were determined either by Student’s t-test or by One-Way ANOVA with Tukey’s multiple comparisons test . Template DNAs were generated by PCR amplification from pAct-EGFP plasmid DNA or y w genomic DNA . Purified PCR products were used as template for in vitro transcription reactions using the T7 MEGAscript kit ( Ambion ) . Double-stranded RNA was purified with RNeasy Mini kit ( Qiagen ) . shams and Rp49 ( RpL32 ) mRNA expression in S2 cells treated with either EGFP or shams dsRNA were assayed by qRT-PCR using TaqMan One-Step RT-PCR Master Mix and TaqMan primers/probe sets ( Life Technologies Dm02144576_g1 and Dm02151827_g ) . Relative shams mRNA levels were compared using the 2–ΔΔCT method . P-values were determined by Student’s t-test . | One of the key mechanisms used by neighboring cells to communicate with each other in animals is signaling through an evolutionarily conserved receptor family called Notch . Binding of Notch to ligands of the Delta/DLL and Serrate/JAG families from neighboring cells leads to activation of the signaling pathway and is called trans-activation . In contrast , interaction of Notch with ligands expressed in the same cell has an inhibitory effect on signaling and is known as cis-inhibition . The balance between trans- and cis- interactions ensures optimum Notch signaling in some contexts during animal development . We have used the model organism Drosophila ( fruit fly ) to decipher the mechanism through which a carbohydrate residue , xylose , regulates Notch signaling in specific contexts . We provide evidence that addition of xylose residues to the Notch receptor decreases its interaction with trans-Delta ligand without affecting its interaction with cis-ligands . Thereby , xylose tunes the Notch pathway by modulating the balance between Notch trans-activation by Delta and Notch cis-inhibition by same-cell ligands . Misregulation of Notch signaling causes a number of human diseases including cancer and developmental disorders . Therefore , understanding the role of xylosylation in Notch signaling can potentially establish a new framework for therapeutic targeting of this pathway . | [
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] | 2017 | Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila |
During bacterial exponential growth , the morphogenetic actin-like MreB proteins form membrane-associated assemblies that move processively following trajectories perpendicular to the long axis of the cell . Such MreB structures are thought to scaffold and restrict the movement of peptidoglycan synthesizing machineries , thereby coordinating sidewall elongation . In Bacillus subtilis , this function is performed by the redundant action of three MreB isoforms , namely MreB , Mbl and MreBH . mreB and mbl are highly transcribed from vegetative promoters . We have found that their expression is maximal at the end of exponential phase , and rapidly decreases to a low basal level upon entering stationary phase . However , in cells developing genetic competence , a stationary phase physiological adaptation , expression of mreB was specifically reactivated by the central competence regulator ComK . In competent cells , MreB was found in complex with several competence proteins by in vitro pull-down assays . In addition , it co-localized with the polar clusters formed by the late competence peripheral protein ComGA , in a ComGA-dependent manner . ComGA has been shown to be essential for the inhibition of cell elongation characteristic of cells escaping the competence state . We show here that the pathway controlling this elongation inhibition also involves MreB . Our findings suggest that ComGA sequesters MreB to prevent cell elongation and therefore the escape from competence .
In response to nutritional deprivation and high population density , the rod-shaped model Gram-positive bacterium Bacillus subtilis enters stationary phase and develops diverse environmental adaptations , namely competence for genetic transformation , sporulation , cannibalism or biofilm formation [1] . These developmental programs are exquisitely regulated in order to anticipate starvation and optimize the survival of at least a fraction of the population . During the development of these adaptations , cells initiate a large reorganization of gene expression [2 , 3] , protein localization [4 , 5] and cell shape [5] . In the case of genetic competence , the central regulator ComK activates the expression of more than a hundred genes [2 , 6 , 7] . Competence development in B . subtilis is a well-known bistable system [1] . Only a small fraction of a population ( 2 to 10% ) expresses the ComK-dependent genes , and thus the large majority of the population remains in the non-competent state [8 , 9] . Within the ComK regulon , twenty-eight genes are essential for genetic transformation [10] , a process defined as the genetic alteration of a competent cell by incorporation of foreign DNA in its genome . The remaining genes upregulated in the presence of ComK may be involved in functions other than transformation . Accordingly , it was proposed to rename the ComK-determined physiological state the K-state , a more neutral term than genetic competence [2] . For instance , it has been shown that growth is inhibited during the escape from competence . When the environmental conditions improve ( e . g . upon dilution into fresh medium ) , non-competent cells rapidly resume growth whereas competent cells remain in a growth-limited state during which both cell elongation and cell division remain inhibited for more than 90 minutes before they start to grow again [11 , 12] . This delay relative to non-competent cells is thought to constitute a tightly regulated checkpoint to allow the repair of the chromosome following homologous recombination of the transforming DNA , before replication initiation [11 , 12] . Growth inhibition during the escape from competence is controlled at two levels: cell elongation is inhibited through the late competence peripheral protein ComGA [11] and cell division is inhibited by ComGA and the highly conserved protein Maf [11 , 12] . The ComGA-mediated mechanism that inhibits cell elongation during outgrowth remains unknown . After exhibiting a diffuse localization in the cytoplasm , ComGA accumulates preferentially at polar clusters where it co-localizes with other competence proteins to form the transformation machinery [4 , 13] . Upon dilution into fresh medium , ComGA stays at the poles for 120 minutes before delocalizing , presumably through degradation or inactivation , ultimately reversing elongation inhibition [12] . Among the different classes of proteins regulating bacterial cell elongation , the bacterial actin-like MreB proteins have been the most studied over the past fifteen years . MreB proteins ( Mre , for Murein cluster e ) are essential for cell morphogenesis in most non-spherical bacteria [14 , 15] . In exponentially growing rod-shaped cells , MreB proteins localize in membrane-associated assemblies that rotate perpendicularly to the long axis of the cell [16–21] . These MreB structures are thought to control cell elongation by directing the assembly and movement of macromolecular complexes that effect synthesis of the sidewalls ( cell cylinder ) during growth [14 , 16 , 17] . In B . subtilis , sidewall elongation during vegetative growth is controlled by the redundant action of three MreB isoforms: MreB , Mbl and MreBH [22] . mreB and mbl are essential under normal growth conditions [23 , 24] , while mreBH is essential only under certain adverse conditions [22 , 25] . The mreB gene is found in the third position of an operon composed of seven genes; immediately upstream the mreCD morphogenes and the minCD division-related genes , and downstream maf , involved in division inhibition during competence [12] , and radC , of yet unknown function . It has been shown that several promoters are located within or upstream the mreB operon [12 , 26 , 27] . mbl is found immediately downstream spoIIID , a gene encoding a sporulation-specific transcriptional regulator [28] and usd , a gene located upstream spoIIID and necessary for its translation [29] . A sigma-E dependent promoter , activating the mbl expression during sporulation , is located upstream usd and spoIIID [26 , 30] . However , it has been shown that expression of mbl during vegetative growth is ensured by a sigma-A dependent promoter located between spoIIID and mbl [26] . Finally , mreBH forms an operon with a small gene of unknown function , ykpC [26] . Transcription of the mreBH operon is driven by the alternative sigma factor sigma-I , which is induced during heat shock [31] . The specific expression of the three mreB isoforms , from different promoters depending on different sigma factors , is in agreement with their partial functional redundancy upon various stress conditions [22] . Interestingly , mreB and mbl were identified as competence-induced genes in a transcriptomic study [2] . However , a detailed profile of expression of these two genes throughout growth and stationary phase remained to be characterized , and a possible role of MreB-like proteins in stationary phase adaptations was not investigated so far . Here , we report a new role associated to MreB during genetic competence in B . subtilis . We show that mreB ( but not mbl ) belongs to the ComK regulon , and that in competent cells MreB forms a complex with several competence proteins . Additionally , MreB co-localizes with ComGA in polar clusters . We finally show that ComGA-dependent growth inhibition displayed by cells escaping the K-state also involves MreB . We propose a model in which ComGA sequesters MreB in order to prevent cell elongation during outgrowth and therefore the escape from competence .
In previous transcriptional profiling studies of B . subtilis grown to competence , all the genes of the mreB operon were found to be down-regulated in comK mutant cells relative to wild-type cells [2] . mbl was also down-regulated but only when mecA , which codes for the adaptor protein that targets ComK for proteolysis [32] , was knocked-out to increase the percentage of competent cells [33] . It was proposed that expression of both mreB and mbl was ComK-dependent and thus induced during competence , although it could not be excluded that mbl expression was affected by ComK only in the pleiotropic mecA background [2] . We examined whether transcription of mreB , mbl and/or mreBH was specifically induced during competence . Fragments of different sizes ( 500 to 2300 bp ) upstream the open-reading frames of mreB ( Fig 1A ) , mbl ( S2A Fig ) and mreBH ( S2B Fig ) containing several promoters were fused to the firefly luciferase ( luc ) coding sequence . In the case of mreB , three promoters were previously identified: P1 , upstream the maf-radC-mreBCD-minCD operon [12 , 26]; P2 , inside maf [27] and P3 , between radC and mreB [26 , 27] ( Fig 1A ) . P1 and P3 are dependent on the major housekeeping sigma factor sigma-A , while P2 is dependent on extracytoplasmic sigma factors [26 , 27 , 34] . P1 also contains ComK binding boxes ( Fig 1A ) and was shown to drive expression of maf during competence [12] . We measured the transcription rate from three fragments upstream mreB: PmreB123 , containing the three promoters; PmreB23 , containing promoters P2 and P3 , and PmreB3 , containing P3 ( Fig 1A ) during growth ( measured by OD600 , S1 Fig ) in competence medium ( CM ) . During exponential growth , expression of luc fused to PmreB123 , PmreB23 and PmreB3 was virtually identical ( Fig 1B ) . The transcription rate progressively increased to reach a maximum during the transition from exponential growth to stationary phase , which marks the beginning of competence ( T0 ) . This indicated that in exponentially growing cells expression of mreB comes from P3 . No transcript generated from P1 and P2 could be detected , even if P1 has been shown to drive the low , basal expression of maf during exponential growth [12] . Upon entering stationary phase ( after T0 ) , the transcription rate from all three fragments rapidly decreased ( Fig 1B ) . Transcription from PmreB23 ( P2 + P3 ) and PmreB3 ( P3 alone ) exhibited a relatively sharp and progressive decrease , reaching a low basal level approximately 3 h after T0 . However , expression from PmreB123 ( P1 + P2 + P3 ) was significantly higher and exhibited a prominent burst about 2 hours after T0 ( T2 , which corresponds to the time of maximal competence [35] ) . Thus , in stationary phase there was a substantial ( 4–6 fold , see inset in Fig 1B ) increase in mreB transcription that came from P1 , the promoter in front of the operon . This was consistent with the recent finding that maf is expressed during competence from P1 , regulated by the master regulator ComK [12] . As expected , when we monitored mreB transcription in a comK mutant , the transcriptional burst observed at T2 was abolished and the transcription rate from PmreB123 was comparable to that from the two shorter fragments PmreB23 and PmreB3 ( Fig 1C ) . Two promoters were previously identified for mbl: P1 , a sigma-E-dependent promoter located upstream the usd gene and P2 , a sigma-A-dependent promoter right upstream mbl ( S2A Fig ) [26 , 30 , 36] . Like mreB , mbl was transcribed predominantly during exponential growth , and maximum of expression was reached right before T0 ( S2C Fig ) . Expression of mbl in exponentially growing cells came exclusively from P2 . In contrast to mreB , however , expression of mbl was not reactivated in stationary phase and was not affected by ComK ( S2C Fig ) , even though a small peak can be observed around T2 . mbl was previously reported to be over-expressed in comK mutant cells only when mecA was also knocked-out [2] . Since mecA mutants are very pleiotropic [37 , 38] , our results indicate that activation of mbl transcription in the comK- mecA- background was indirect , resulting from secondary effects of the absence of mecA . For mreBH , only a sigma-I-dependent promoter , induced during heat shock , has been identified [31] . Consistently , no transcription of mreBH was detected during growth in competence medium ( S2D Fig ) . Taken together , our findings indicate that mreB , but not mbl and mreBH , is a competence-induced gene , regulated by ComK . To provide insight into a possible role of MreB in competent cells , we sought to identify MreB binding partners during competence . To this end , MreB was fused to the sequential peptide affinity ( SPA ) tag [39] . Unlike cells lacking MreB , cells containing spa-mreB as only copy of mreB in their genome displayed normal morphology in both exponential and stationary phase ( S3 Fig ) indicating that the SPA-MreB fusion was functional . The strain expressing the SPA-MreB fusion was grown to T2 in CM at 37°C , and MreB-associated proteins were purified and identified by mass spectrometry . Strains expressing no SPA-tagged protein and a SPA fusion to PerR , a non-related protein of B . subtilis , were used as negative controls . Interestingly , several competence proteins ( ComGA , Maf , ComEB , ComC and ComFA ) were specifically and reproductively detected in the MreB pull-down complexes ( Table 1 ) . Among these proteins , ComGA was the most abundant in the complex based on the Protein Abundance Index ( PAI , established according to [40] ) . ComGA was co-purified with SPA-MreB well above the contaminant value found in the control strains ( Table 1 ) , indicating that their co-purification was specific . ComEB , ComC and ComFA were specifically co-purified , and Maf was greatly enriched in the SPA-MreB eluate relative to the control strains ( Table 1 ) . Taken together , these results indicated that MreB is associated with several competence proteins in B . subtilis . Next , we determined whether MreB displays a specific localization in the subpopulation of competent cells . We have shown that expression of mreB is complex , driven from three different promoters ( Fig 1 ) . To avoid a possible artifact of overexpression and/or misregulation , we replaced mreB by gfp-mreB at the native locus expressed under control of the native mreB regulatory sequences ( Pnativegfp-mreB ) , without leaving any scar or resistance cassette in the vicinity ( see Methods for details , S4A Fig and S1 Movie ) . We then analyzed nativeGFP-MreB localization in cells that natively expressed a functional ComGA-RFP fusion ( PnativecomGA-rfp ) as a marker for competence . Strikingly , in stationary phase cells ( T2 ) , most nativeGFP-MreB signal disappeared from the membrane and became diffuse in the cytoplasm ( ‘b’ cells in Figs 2A and S4C ) . In competence-expressing cells at T2 , in addition of exhibiting a diffuse signal , nativeGFP-MreB formed clusters at one or both cell poles ( ‘a’ cells in Figs 2A and S4C ) . All polar MreB clusters ( n > 200 ) were found to co-localize with ComGA polar clusters , while no nativeGFP-MreB signal was found in 15% ( n > 200 ) of ComGA polar assemblies ( Figs 2A and S4C ) . In the absence of comGA , MreB polar clusters were never observed and nativeGFP-MreB fluorescence signal was diffuse in all cells ( n > 200 ) ( Fig 2B ) . Control experiments showed that this co-localization was not due to bleed-through of the bright ComGA-RFP signal into the GFP channel ( S4E Fig ) . In a given field of view , the integrated fluorescence signal of nativeGFP-MreB per cell was more than 3 times higher in competent ( AU = 67 . 2 ±17 . 6 , n = 102 ) than in non-competent ( AU = 20 . 8 ±11 . 6 , n = 103 ) cells , indicating that ComK-dependent expression of mreB ( Fig 1 ) leads to increased levels of MreB protein in competent cells . In exponential growth , a functional nativeMbl-GFP fusion displayed the characteristic ‘motile patches’ localization ( S4B Fig and S2 Movie ) . However , in contrast to nativeGFP-MreB , at T2 a nativeMbl-GFP fusion was still localized in membrane-associated patches ( albeit no longer motile , S4 Movie ) along the sidewalls , which did not co-localize with ComGA polar clusters ( S4D Fig ) . Thus , in competent cells MreB , but not Mbl , relocalizes into polar clusters that colocalize with , and are dependent on , the multi-functional competence protein ComGA . ComGA was first described for its essential role in natural genetic transformation [2] . We then tested if MreB could play a role during this process . Strikingly , transformation efficiency of in-frame mreB null mutants was increased about a hundred fold relative to the wild-type strain , while mbl and mreBH mutants had transformation efficiencies comparable to that of the wild-type ( Table 2 ) . However , in cells lacking mreB , both the percentage of competent cells and the timing of competence development were not affected ( S5A and S6A Figs respectively ) . High concentrations of magnesium ( Mg2+ ) rescue the viability and shape defects of mreBs and other mutants involved in different aspects of cell wall synthesis by a yet unknown mechanism [14] . It has been proposed that Mg2+ may stiffen the cell wall , compensating for structural defects associated to the absence of mreB [41] . CM is traditionally supplemented with 5 mM Mg2+ [35] . Remarkably , increasing Mg2+ concentrations in CM progressively rescued the mreB transformation phenotype ( Table 2 ) . At 25 mM Mg2+ , the transformation efficiency of mreB mutant cells was down to wild-type levels ( Table 2 ) . Taken together , these results suggested that the effect of MreB in transformation is indirect . They also raised the interesting possibility that specific cell wall defects could promote transformation in B . subtilis . One hypothesis was that the assembly or localization of the transformation apparatus across the cell wall was affected in the absence of mreB . To investigate this , we compared the localization of the transformation machinery in the wild-type and mreB mutant backgrounds using our nativeComGA-RFP fusion . The dynamic localization of ComGA during competence has been extensively described [4] . In wild-type cells developing competence ComGA first appears diffuse in the cytoplasm ( S7B Fig ) . Then , ComGA forms clusters associated to the inner face of the membrane with an important bias for the regions near the poles ( S7C and S7D Figs ) , where it co-localizes with other main competence proteins to form the transformation machinery [4] . The number of ComGA focus per wild-type competent cell varies from one to nine , but the large majority of wild-type competent cells ( 41% , n>1500 ) display a single ComGA polar cluster ( S5C and S5D Figs ) . The percentage of competent cells that displayed nativeComGA-RFP clusters at T2 ( S5B Fig ) and among these the number of ComGA clusters ( S5C Fig ) were significantly higher in cells lacking mreB relative to wild-type cells . More specifically , the majority of mreB mutant competent cells ( 37% , n>1500 ) displayed three foci ( S5C and S5E Figs ) . Expression of the comGA gene ( S6B and S6D Figs ) and ComGA protein levels ( S6E Fig ) were nevertheless unaffected in the mreB mutant . At high Mg2+ concentrations , mirroring the recovery of wild-type transformation efficiency of the mreB mutant , the distribution of the number of ComGA clusters per competent mreB mutant cell shifted back to wild-type levels ( S5B Fig ) . We concluded that MreB is not directly required for natural transformation and that ComGA localization might be impacted by the cell wall integrity . It was shown that ComGA is also required for inhibition of cell elongation in cells exiting competence [11] , while MreB directs cell elongation in exponentially growing cells [14 , 16 , 17] . We hypothesized that MreB could be involved in inhibition of cell elongation during competence escape through its association with ComGA . To test this , we performed outgrowth experiments using a ComK-GFP construct to distinguish competent from non-competent cells , as previously described [11] . Wild-type , ΔcomGA , ΔmreB and ΔmreB ΔcomGA mutant strains were grown to T2 , when maximal competence is achieved , and diluted 20-fold into fresh medium . Samples were taken prior to dilution ( T2 ) and 90 minutes after dilution ( T2+90 ) for size and morphology characterization . At T2 competent and non-competent cells were indistinguishable in length for all strains ( S3 Table ) [12] . At T2+90 , non-competent cells had resumed growth and division [11 , 12] . Competent cells of the wild-type strain ( Fig 3A ) were only slightly longer than at T2 ( S3 Table ) , confirming the previously reported growth limitation imposed during the escape from competence [11] . ΔmreB competent cells also remained in a growth-limited state after 90 minutes of outgrowth and were significantly shorter than wild-type competent cells ( Fig 3C and 3F and S3 Table ) as previously reported for exponentially growing mreB mutant cells [41] . In contrast , ΔcomGA competent cells were filamentous and often bent ( Fig 3B and 3F and S3 Table ) , indicating that ComGA directly or indirectly inhibits cell elongation during the escape of competence [11] . However , when mreB was knocked out in the ΔcomGA mutant strain , the filamentous phenotype of ΔcomGA competent cells was rescued , and the average cell length of the ΔmreB ΔcomGA mutant was similar to that of wild-type competent cells at T2+90 ( Fig 3D and 3F and S3 Table ) . Wild-type rod shape is restored in mreB and mbl null mutants by addition of 25 mM Mg2+ to the growth medium , while addition of 2 . 5 mM Mg2+ is sufficient to restore wild-type growth rate of mreB mutants [41 , 42] . Consistently , ΔmreB mutant cells were viable and displayed wild-type growth and moderate cell shape defects in classic CM ( i . e . 5mM Mg2+ ) ( S8 Fig and S5 Movie ) . To exclude an indirect effect due to the inability of mreB-like mutants to elongate properly at low Mg2+ concentrations , we repeated the outgrowth experiments in CM containing 25 mM Mg2+ . At T2+90 , the length of ΔcomGA ΔmreB competent cells was similar in conventional CM ( 5 mM Mg2+ ) and in CM with 25 mM Mg2+ ( Fig 3F and S3 Table ) . In contrast , deletion of mbl ameliorated but did not rescue the ΔcomGA filamentous phenotype , and in the presence of 25 mM Mg2+ ΔcomGA Δmbl competent cells filamented like ΔcomGA competent cells ( Fig 3F ) . Taken together , these results indicated that MreB plays a direct role in the growth limitation imposed during the escape from competence . However , it was still plausible that in the absence of MreB , competent ΔcomGA cells did filament but started dividing during the 90 minutes of outgrowth , as previously shown for Δmaf ΔcomGA mutant cells [12] . If this was true , the average length of ΔmreB ΔcomGA double mutant cells would first increase ( during filamentation ) and then decrease ( upon initiation of cell division ) between T2 and T2+90 . Measurement of the length of competent cells at different times during the outgrowth experiment showed that ΔmreB ΔcomGA cells length slightly but progressively increased their average length from T2 to T2+90 ( Fig 3G ) , excluding that they had filamented and then divided . These findings suggested that ComGA-mediated inhibition of cell elongation during the escape from competence also involves MreB . One possibility is that ComGA directly or indirectly sequesters MreB in competent cells to delay the initiation of cell elongation upon outgrowth . According to this prediction , over-production of MreB could totally or partially bypass the ComGA checkpoint and thus promote elongation of competent cells during outgrowth . Consistently , when native levels of MreB were increased by expressing the functional spa-mreB fusion ( S3 Fig ) in the presence of the endogenous copy of mreB , competent cells filamented in a manner similar to ΔcomGA cells after 90 minutes of outgrowth . The mean length of cells overproducing SPA-MreB was almost twice the mean length of wild-type cells and cell length distribution was much broader , with cells exceeding 20 µm in length ( Fig 3F ) . We found that at the time of maximum competence ( T2 ) MreB forms polar clusters that co-localize with ComGA polar clusters and are dependent on the presence of ComGA ( Fig 2 ) . In addition , our findings suggest that MreB is involved , alongside ComGA , in the inhibition of cell elongation during outgrowth . We then verified if the localization of MreB and ComGA was still correlated during the escape from competence . After 90 minutes of outgrowth , MreB polar clusters were still present and co-localized with ComGA clusters in wild-type competent cells ( Fig 3H ) . However , in the filamentous ΔcomGA competent cells , MreB had already re-localized into motile patches along the sidewalls ( Fig 3I ) . These results suggested a direct correlation between ComGA-dependent polar localization of MreB and the absence of elongation during the escape from competence . Our findings above suggest a model in which ComGA would directly or indirectly sequester MreB in competent cells . Unfortunately , difficulties to purify active recombinant MreB proteins currently unable biochemical work with MreB proteins of B . subtilis [14] and thus the direct interaction between MreB and ComGA could not be tested in vitro . No direct protein-protein interaction between MreB and ComGA was detected in pairwise yeast two-hybrid assays using full-length proteins ( S9 Fig ) . False negatives are nevertheless frequent in two-hybrid assays [43] , and thus the absence of interaction in yeast did not exclude a true protein interaction . Alternatively , we analyzed the effect of expression of comGA during exponential growth . In wild-type cells , comGA is exclusively expressed during competence [12] . In the same background , unnatural expression of comGA during exponential phase from an inducible promoter , was reported to have no effect on growth [11] . However , we reasoned that defects due to the sequestration of MreB by ComGA could be masked by the partial functional overlap between the three MreB isoforms [22] . Thus , we analyzed the effect of over-expression of comGA from the very strong ( although poorly repressed ) hyperspank promoter ( Phs ) in both wild-type and mbl mutant cells growing in rich ( LB ) medium . The mbl mutant strain grew almost like the wild-type strain in LB ( Fig 4A ) , and a low percentage of cells ( 11% , n = 400 at OD = 0 . 15 ) displayed mild morphological defects ( arrows in Fig 4D ) . Expression of comGA in the wild-type background had virtually no effect on growth ( Fig 4A ) [11] and morphology ( Fig 4B and 4C ) . However , growth of the mbl mutant carrying the Phs-comGA-rfp construct , was significantly affected both in the absence and ( to a bigger extent ) in the presence of inducer ( Fig 4A ) . This result clearly indicated that over-expression of comGA is toxic in the absence of Mbl . Furthermore , the majority ( 65% , n = 400 at OD = 0 . 15 ) of comGA-overexpressing mbl mutant cells showed progressive bulging and aberrant morphologies including Y-shaped cells and polar bulges characteristic of mreB ( but not mbl ) mutant cells [22 , 44] ( Fig 4E ) , indicating impairment of cell morphogenesis and explaining the lethal effects on growth . We concluded that when comGA is expressed in exponentially growing cells , MreB cannot fully compensate for the absence of Mbl . These findings were consistent with the hypothesis that ComGA sequesters MreB to prevent cell elongation and limit growth . We could not test the effect of expression of comGA on the localization of MreB in the mbl mutant background because GFP fusions to MreB do not support growth in a Δmbl ΔmreB background .
In bacterial cells , like in their eukaryotic counterparts , proteins localize to specific locations , often in a dynamic manner , during growth . Spatiotemporal localization of proteins is critical for their function and orchestrates cellular processes . In exponentially growing B . subtilis cells , the mreB gene is highly expressed and MreB assembles into membrane-associated patches that move processively around the cell to control sidewall elongation [16–20] . Here , we show that when B . subtilis cells enter stationary phase in competence medium , expression of mreB drastically decreases and MreB delocalizes from the membrane exhibiting a largely diffuse localization in the cytoplasm . Such transcriptional regulation of mreB and the disassembly of MreB patches from the membrane may inhibit deposition of peptidoglycan along the sidewalls during stationary phase . Additionally , we show that expression of mreB is reactivated in cells that develop competence . In competent cells , MreB relocalizes in polar clusters together with the late competence protein ComGA . Co-localization of MreB and ComGA at the cell poles persists for at least 90 minutes of outgrowth into fresh media . MreB subsequently relocalizes as motile patches along the sidewalls to reinitiate elongation . Altogether , these findings underline the importance of dynamic regulation of gene expression and protein localization for bacteria to adapt to changing environmental conditions . In cells lacking mreB , transformation efficiency was increased a hundredfold and the number of membrane-associated ComGA clusters was significantly higher than in wild-type cells . Both phenotypes were however rescued by high Mg2+ concentrations , suggesting that ( i ) MreB is not directly required for natural transformation in B . subtilis , and ( ii ) assembly of the transformation apparatus might be affected by structural features of the cell wall , as Mg2+ has been proposed to rigidify weakened cell-walls [41] . The transformation apparatus , which includes a type IV pilus-like structure that traverses the thick cell wall and is required for binding and importing the transforming DNA [45] , preferentially localizes near the poles at the junction between the cylinder and the polar caps [4] . This region represents the interface between the sidewalls , which are intensively reshaped during growth , and the almost inert cell wall at the poles . Interestingly , this region is also chosen by phage SPP1 to bind and inject its DNA into the cytoplasm of B . subtilis [46] . During infection , SPP1 has to irreversibly bind to its receptor , YueB , encoded by a putative type VII secretion system gene cluster in B . subtilis [47 , 48] . YueB extends across the cell wall and also localizes at the junction between the cylinder and the polar caps [46] . Thus , this structurally differentiated region of the cell wall may contain positional information for the assembly of structures that need to cross the cell envelope . Initial assembly of the transformation apparatus pilus-like structure at these sites could then direct the localization of cytoplasmic competence-induced proteins such as ComGA at the inner leaflet of the cytoplasmic membrane . Specific defects in the structure or the organization of the cell wall of mreB mutant cells may favor the assembly of additional transformation apparatus at ectopic sites . Consistently , it has been shown that the absence of mreB induced the apparition of multiple sites containing polar material in E . coli cells [49 , 50] . Furthermore , inactivation of MreB in Pseudomonas aeruginosa led to the mislocalization of a normally polar type IV pilus [51] . We show here that in competent cells mreB is specifically transcribed from the same promoter than maf and that MreB protein levels are increased relative to non-competent cells . Competent comGA mutant cells filament upon dilution into fresh medium [11] . These long comGA mutant cells are unable to divide because Maf is still present and inhibits cell division [12] . When mreB was deleted in a ΔcomGA background , competent cells did not filament during the early stages of competence escape . When mbl was deleted , the average length of ΔcomGA cells exiting competence was also slightly reduced . High Mg2+ concentrations fully rescued ΔcomGA cells elongation in the absence of mbl but not in the absence of mreB . Taken together , these findings indicate that elongation of cells escaping competence primarily depends on MreB and cannot be rescued by the redundant action of Mbl and/or MreBH . Mbl could nevertheless play a mild secondary role in this process . Consistently , a low level of transcription of mbl was detected during stationary phase at T2 , while expression of mreBH was completely switched off . When mreB was overexpressed in a wild-type background , cells escaping competence exhibited a filamentous phenotype , like ΔcomGA cells . We hypothesize that in this condition excess of MreB can bypass the ComGA-mediated inhibition of elongation and activate cell wall synthesis . Finally , confirming the implication of the two proteins in order to limit cell elongation , MreB was found ( i ) in the same complex than several competence proteins and ( ii ) co-localizing with ComGA polar clusters at T2 and throughout the 90 minutes following dilution into fresh medium . In the light of our results , we propose the model presented in Fig 5 , in which ComGA inhibits cell elongation during the escape from competence by sequestering MreB , either directly or indirectly . No direct interaction between MreB and ComGA was detected in yeast two-hybrid assays and such interaction cannot be tested in vitro because active recombinant MreB of B . subitlis is currently not available for biochemical work [14] . However , we show here that expression of comGA in exponentially growing mbl mutant cells induces growth and morphological defects similar to those of mreB mutants . This result suggests that ComGA may be able to sequester MreB during exponential phase too . Therefore , if ComGA and MreB do not interact directly , then the potential protein ( s ) mediating their interaction during competence is ( are ) also expressed during vegetative growth . However , to date , all proteins found to co-localize with ComGA at the poles of competent cells are specifically over-produced during competence [4 , 52] . How the ComGA-MreB interaction is mediated remains an important question for future work . General principles governing protein localization include capture by a cellular factor ( e . g . interacting protein , DNA binding site , membrane domain or substrate ) and self-assembly , where polymerization/depolymerization dictate the location of a protein at a given time [53] . Polymerization may also be regulated by binding proteins , like in the case of eukaryotic actin , where a myriad of actin-binding proteins ( ABPs , [54 , 55] ) regulate actin activity and dynamics . ABPs can nucleate , cross-link , bundle , anchor and regulate the state of polymerization of polymeric , filamentous actin ( F-actin ) , and they can cap and stabilize the monomeric , globular actin ( G-actin ) pool in the cytoplasm . Here , we show that in cells entering stationary phase , MreB dissociates from the sidewalls and becomes diffuse in the cytoplasm . Interestingly , it has been recently shown that the concentration of lipid-linked peptidoglycan precursors regulates the association of MreB to the membrane [56] . When precursors are depleted , MreB filaments disassemble into the cytoplasm . During the entry into stationary phase peptidoglycan precursor depletion probably occurs [57] , as the metabolism slows down and the need of cell wall synthesis decreases , potentially explaining MreB relocalization . However , the details of the mechanism regulating the dynamic localization of MreB remain unknown . Numerous studies have identified a number of proteins that modulate FtsZ ring formation in B . subtilis [58–62] while the first ABP-like protein regulating MreB has yet to been found . It is plausible that one or several ABP-like protein ( s ) , sensing the peptidoglycan precursor’s availability , promote MreB depolymerisation and/or stabilize the monomeric form of MreB in the cytoplasm . In addition , we propose a model in which ComGA would sequester MreB in competent cells to prevent its localization to the sidewalls and therefore cell elongation . ComGA could then be considered as a new cellular regulator of the actin-like protein MreB . Only one protein that spatially regulates the MreB proteins has been reported in bacteria [63] . Indeed , the progressive depletion of RodZ leads to the misassembly of MreB into non-spiral structures before inducing a total loss of shape in Escherichia coli [63] . While RodZ can be considered as a positive regulator favoring the assembly of MreB at the right sites , ComGA could be classified as a negative regulator preventing the canonical MreB localization along cylindrical sidewalls . We suggest that sequestration by ComGA spatially regulates MreB during competence in B . subtilis . When ComGA is eventually degraded or inactivated allowing competent cells to resume growth [11] , excess of MreB relative to non-competent cells would be free to rapidly form membrane-associated patches and initiate fast elongation . ComK-dependent induction of mreB expression during competence would therefore compensate for the timing disadvantage imposed by genetic transformation . The two levels of regulation ( i . e . gene expression and protein localization ) might generate and orchestrate the pathway controlling simultaneously a delay in growth and a way to compensate for it . It has been shown that MreB , Mbl and MreBH display partial functional redundancy in B . subtilis [22] . Overexpression of any one of the isoforms is sufficient to sustain lateral peptidoglycan synthesis and maintain cell shape in normal growth conditions . However , no single MreB isoform could support growth in various stress conditions , suggesting that multiplicity of MreB isoforms may become essential in specific environmental conditions [22] . Here we show that unlike mreB , mbl and mreBH are not specifically expressed during genetic competence . Consistently mbl and mreBH mutants displayed no competence-associated phenotypes . This specialization of MreB in competence further suggests that each isoform could be essential for specific environmental adaptations . A sigma-E sporulation specific promoter has been detected upstream mbl [26 , 30 , 36] , while mreBH is part of the SigI regulon induced during heat stress [31 , 64] . Similarly to MreB in the context of competence , the localization and/or activity of Mbl and MreBH could be modulated by a regulator specifically produced during their respective adaptation . Future studies will reveal whether Mbl plays a role in sporulation and MreBH in stress response .
Bacillus subtilis strains were constructed by natural genetic transformation with selection for the appropriate antibiotic resistance marker . For transformation , competent cultures were prepared and incubated in competence medium ( CM ) with transforming DNA ( ~1 µg/ml ) for 30 minutes at 37°C [35] . When needed , B . subtilis chromosomal DNA was prepared as detailed in [65] . Transformants were selected using 100 µg/ml spectinomycin , 10 µg/ml kanamycin , 5 µg/ml chloramphenicol , 16 µg/ml phleomycin and 1 µg/ml erythromycin . All the plates used to select transformants contained 25 mM of Mg2+ . The details of all the new constructs in this publication are presented below . All new constructs were sequenced after introduction in the B . subtilis chromosome . B . Subtilis strains were grown in CM or LB media . When needed , the CM Mg2+ final concentration was increased to 25 mM . Strains are listed in S1 Table . Because some of our genes of interest are in the middle of operons , we decided to clone our constructs ( promoter + RBS + luciferase ) at the ectopic amyE locus . Fragments of different lengths upstream the genes of interest ( mreB , mbl and mreBH ) and ending right before the genes RBS were amplified by PCR from the B . subtilis chromosome . To amplify the fragments PmreB123 , PmreB23 , PmreB3 ( Fig 1A ) , Pmbl12 , Pmbl2 ( S2A Fig ) and PmreBH1 ( S2B Fig ) we used the primers MCS-PmreB1-F and RBS-PmreB-R , MCS-PmreB2-F and RBS-PmreB-R , MCS-PmreB3-F and RBS-PmreB-R , MCS-Pmbl1-F and RBS-Pmbl-R , MCS-Pmbl2-F and RBS-Pmbl-R and MCS-PmreBH1-F and RBS-PmreBH-R respectively . In parallel , we amplified by PCR the upstream ( amy-Front and choramphenicol cassette ) and downstream ( amy-Back and luciferase gene ) amyE fragments from the plasmid pUC18cm-luc [66] using primers amyF-F and MCS-R and primers amyR-R and MCS-F respectively . Finally , using the Gibson method based on isothermal assembly [67] , we joined the three fragments to obtain the PCR product “amy-F–Cm–Promoter–RBS–Luc- amy-R” . The final PCR product was used to transform strain NC57 by selection for cloramphenicol resistance . Luciferase experiments were performed as we previously described in [68] . All primers are listed in S2 Table . A method developed to construct scar-less and marker-less deletions in the genome of B . subtilis [69] , was adapted to insert the gfp directly upstream mreB , at the native locus . The first step was to delete , in the recipient strain ( NC101 , NeoR ) , the radC gene which is positioned right before mreB , by inserting a deletion cassette ( PhleoR ) . The cassette was first amplified by PCR from plasmid pUC19-K7-010 [69] using the primers K7PH-F and K7PH-R . Then , the regions upstream ( radC front ) and downstream ( radC back ) the radC gene were amplified using the primers HindIII-Pmaf-F and Phleo-radC-R or Phleo-radC-F and HindIII-mreB-R , respectively . Finally , the three fragments were joined using the Gibson method [67] to obtain the following PCR product n°1:“radC front–Phleo cassette–radC back” . Transformation of the recipient strain ( 168 Δupp ) with this PCR product generated strain NC102 which is NeoS and PhleoR . Then , the deletion cassette was replaced by a fragment that re-introduced the radC gene and inserted gfp in front of mreB . This fragment was constituted by two blocks , namely Pmaf-maf-radC ( block 1 ) and gfp-mreB ( block 2 ) . These blocks were amplified by PCR using the following primers: Pmaf-F and GFP-radC-R ( for block 1 ) and RBS-mreB-GFP-F and mreB-R ( for block 2 ) . The gfp-mreB block was amplified from chromosomal DNA of strain 3723 [41] . The two blocks were then joined using the Gibson method [67] to generate the PCR product n°2: Pmaf-maf-radC-gfp-mreB . This final PCR product was used to transform the strain NC102 to obtain strain NC103 ( NeoR and PhleoS ) , which now contains gfp right in front of mreB inside its own operon . Cells of this strain ( NC103 ) and its derivatives , in which Pnative-gfp-mreB is expressed as the only copy of mreB in the genome , were viable and displayed almost wild-type growth and morphology , indicating that the fusion is virtually functional ( S4A Fig and S1 Movie ) . All primers used are listed in S2 Table . We decided to express the comGA-rfp fusion under the control of the native comGA promoter ( PcomGA ) from the thrC locus . The Gibson method [67] was used to join four PCR fragments corresponding to the upstream ( thrC front ) and downstream ( thrC back ) regions of the thrC gene , the comGA promoter and orf , and the mrfpruby gene . These fragments were amplified using the primers hom-F and pDG1664-MCS-R ( thrC front ) , pDG1664-MCS-R and thrB-R ( thrC back ) , pDG1664-MCS-PcomGA-F and RFP-comGA-R ( PcomGA-comGA ) and comGA-RFP-F and pDG1664-MCS-RFP-R ( mrfpruby ) . The four fragments were joined to produce the final PCR product “thrC front–PcomGA−comGA–mrfpruby- thrC back” . The thrC front and thrC back ( which also contains an erythromycin resistance cassette ) fragments were amplified from plasmid pDG1664 [70] . The mrfpruby gene was amplified from chromosomal DNA of strain RWSB5 [16] . The final PCR product was used to transform the NC57strain to generate strain NC118 . In this strain and its derivatives , ComGA-mRFPruby displays the expected dynamic of localization during competence [4] , indicating that the fusion is virtually functional ( S5A–S5D Fig ) . All primers are listed in S2 Table . The method was comparable to the construction of the natively expressed comGA-rfp fusion described above . The Gibson method [67] was used to join four PCR fragments corresponding to the upstream ( thrC front ) and downstream ( thrC back ) regions of the thrC gene , the comGA gene and the mrfpruby gene . The Phyperspank promoter was introduced through the thrC front fragment . The four fragments were amplified using the primers hom-F and pDG1664-MCS-R ( thrC front ) , pDG1664-MCS-R and thrB-R ( thrC back ) , pDG1664-MCS-comGA-F and RFP-comGA-R ( comGA ) and comGA-RFP-F and pDG1664-MCS-RFP-R ( mrfpruby ) . The four fragments were joined to produce the final PCR product “thrC front–Phyperspank−comGA–mrfpruby- thrC back” . The thrC front ( that contains the Phyperspank promoter ) and thrC back ( that also contains an erythromycin resistance cassette ) fragments were amplified from the pDP150 plasmid [71] . The mrfpruby gene was amplified from chromosomal DNA of strain RWSB5 [16] . The final PCR product was used to transform the wild type strain ( 168 ) to generating strain NC208 . All primers are listed in S2 Table . We decided to clone the rfp gene under the control of the comK promoter at the amyE locus . The Gibson method [67] was used to join four fragments corresponding to the upstream ( amy front ) and downstream ( amy back ) regions of the amyE gene , the comK promoter and the mrfpruby gene . These fragments were amplified using the primers amy-F and PcomK-amy-R ( amy front ) , RFP-amyR-F and amyR-R ( amy back ) , amyF-PcomK-F and RFP-PcomK-R ( PcomK ) and PcomK-RFP-F and amyR-RFP-R ( mrfpruby ) respectively . The amy front and back ( which also contains a spectinomycin resistance cassette ) fragments were amplified from plasmid pDG1730 [70] . The mrfpruby gene was amplified from chromosomal DNA of strain RWSB5 [16] . The four fragments were joined to produce the PCR product “amy front–PcomK−mrfpruby- amy back” . The final PCR product was used to transform the wild type strain ( 168 ) , inserting the PcomK- mrfpruby construct , at the amyE locus and selecting for chloramphenicol resistance . All primers are listed in S2 Table . Translational fusion between the SPA-encoding ( Sequential Peptide Affinity ) and mreB open reading frame was cloned at the ectopic amyE locus under control of the xylose-inducible promoter Pxyl ( pSG-SPA-Nter ) . pSG-Spa-Nter was generated by replacing the GFP contained in pSG1729 [72] by affinity purification tags ( Sequential Peptide Affinity , or SPA ) [39] , right downstream from the Pxyl promoter . However to generate a N-ter fusion , the tags were inverted in comparison to the original SPA construct ( i . e . Flag-TEV site-CBD ) . The inverted SPA tag was synthesized by Genscript . Then , the mreB open reading frame was PCR-amplified using primers ac-983/ac984 , and cloned into the pSG-Spa-Nter vector , using the XhoI and EcoRI restriction sites . The resulting pAC637 plasmid ( pSG-Pxyl-spa-mreB ) was transformed into B . subtilis strain 4281 ( ΔmreB::cm ) and selected for resistance to spectinomycin , to obtain strain ABS1370 . Finally , we used chromosomal DNA of strain ABS1370 to transfer by natural transformation the amyE::Pxyl-spa-mreB ( Spc ) construct in strain NC60 to obtain strain NC66 . Chromosomal DNA from strain Bas013 [73] was used to transform the wild-type strain ( 168 ) and transfer the Pxyl-perR-spa fusion . Chromosomal DNA of strain NC60 was then used to sequentially incorporate by natural transformation the mcComS and ComK-GFP constructs to generate the final strain NC135 . Experiments were carried out as previously described [66 , 68] . In brief , the high instability of the luciferase , used as transcriptional reporter in B . subtilis , allows us to approach the measurement of a rate of expression [66] , with a relatively small contribution from the cumulative effect of transcription . This particular characteristic of luciferase is in stark contrast with the behavior of other reporters , e . g . β-galactosidase . All the strains used in the luciferase experiments carried a multi-copy plasmid , mcComS [74] , in order to increase the percentage of competent cells ( from 2% to 35% in the wild-type background in the conditions used here , see Fig 4A ) . For detection of luciferase activity , strains were first grown in LB medium to an optical density at 600 nm ( OD600nm ) of 2 . Cells were then pelleted and resuspended in fresh competence medium , adjusting all the cultures to an OD600nm of 2 . These pre-cultures were then diluted 20 fold in fresh competence medium and 200 µl was distributed in each of two wells in a 96-well black plate ( PerkinElmer ) . 10 µl of luciferin ( PerkinElmer ) was added to each well to reach a final concentration of 1 . 5 mg/ml ( 4 . 7 mM ) . The cultures were incubated at 37°C with agitation in a PerkinElmer Envision 2104 Multilabel Reader equipped with an enhanced sensitivity photomultiplier for luminometry . The temperature of the clear plastic lid was maintained at 38°C to avoid condensation . Relative Luminescence Units ( RLU ) and OD600nm were measured at 2 minutes intervals . The data were plotted as RLU/OD ( luminescence readings corrected for the OD ) versus time from inoculation . B . subtilis strains were transformed using chromosomal DNA of strain BD4893 carrying a spectinomycin marker [35] . The number of transformants was evaluated by plating the transformed cultures on LB agar plates containing spectinomycin . Each transformation culture was also plated on non-selective LB agar in dilution series to establish the viable cell count . Transformation efficiency was calculated by dividing the number of transformants by the viable count of each strain . The strains containing the SPA fusions were grown to T2 in competence medium supplemented with 0 . 4% xylose ( to induce the SPA fusions ) . The cultures were then centrifuged and promptly frozen in liquid nitrogen . The xylose concentration used was chosen in order to optimize the SPA fusions production and minimize the shape and growth phenotypes associated to the over-expression of MreB . The frozen cells pellets were then disrupted by cryogenic grinding ( 4 cycles of 2 minutes , always maintaining the cupules and the pellets in liquid nitrogen ) . The powder recovered from the grinding was resuspended in buffer A ( Tris-HCl pH7 , 5 10 mM , NaCl 150 mM , EDTA 0 , 2 mM , Triton 0 , 1 mM and proteases inhibitors ) and centrifuged to eliminate cell debris . SPA-MreB , PerR-SPA and No-SPA containing protein complexes were then isolated and analyzed as described in [75] . Cultures were grown in competence medium at 37°C from single freshly isolated colonies on plates containing the appropriate antibiotic selection . Samples for microscopic observation were taken at T2 ( 2 hours after the beginning of competence development ) and T2+90 ( 90 minutes after dilution of a T2 culture in fresh competence medium ) and immobilized on 1% agarose-coated microscope slides . Bacteria were imaged with an inverted microscope ( Nikon Ti-E ) equipped with a 100× oil immersion objective and an environmental chamber maintained at 37°C . Conventional epifluorescence Images were recorded on phase-contrast and fluorescence channels ( 472/30-nm excitation filter and 520/35-nm emission filter for GFP , 562/40-nm excitation filter and 641/75-nm emission filter for RFP ) with an ORCA-R2 camera ( Hamamatsu ) . Images were processed with NIS-Elements ( Nikon ) software . Exposure time was set up to 200 ms for nativeGFP-MreB and 500 ms for nativeComGA-RFP . All TIRFM images were acquired on the same inverted microscope with a diode-pumped solid-state laser ( Cobolt Calypso , 50mW , 491nm ) and an Apo TIRF 100x oil objective ( Nikon , NA 1 . 49 ) . All images were collected with an electron-multiplying charge-coupled device ( EMCCD ) camera ( iXON3 DU-897 , Andor ) with a gain of 300 . Incidence angles and z-position were adjusted individually for all channels to obtain comparable evanescent wave penetration depth and focus position . In order to follow B . subtilis growth over time , we used a microfluidic flow chamber technique ( CellAsic part of EMD Millipore ) . The technology is divided in two parts: a perfusion control system and a microfluidic plate ( specific for bacteria , B04A ) that keeps cells in a single focal plan and allow us to induce and follow events during many generations . The day before the experiment , strains were grown on selective plates . The next day , cells were resuspended in competence medium to OD = 1 . 1µl of this resuspension was used to inoculate 1mL of fresh competence medium . Once the cultures reached early exponential phase , cells were injected in the chamber and incubated under a continuous flow ( 5µl/hour ) of medium at 37°C . In order to characterize ComGA-RFP foci at the single cell level , phase contrast and fluorescence images were taken simultaneously for cells grown to stationary phase ( T2 ) in competence media . Fields of view of both images were used to generate sub-images displaying individual cells by applying a two-step algorithm . First , each single cell was detected by applying segmentation to phase-contrast images , resulting sub-images of individual cells with cell contours . Next , diffraction-limited comGA foci in each cell were identified in fluorescence images . Examples of individual cells are presented in S5 Fig Custom image processing codes ( S10 Fig ) were implemented in Matlab ( Mathworks ) . Kymograph analysis was applied to obtain the rotation speed of MreB patches as we previously described [16] . In brief , a series of parallel lines were created from one cell pole to the other ( every other pixel ) , all perpendicular to the cell midline . Next , kymographs were generated , corresponding to movement of MreB patches at all positions along the cell longer axis . Finally , angles of the clear MreB traces on the kymographs were used to calculate the rotation speed . Length of competent cells was measured using the Metamorph software ( Molecular Devices ) . Phase contrast images were used and the distance from one pole to the other was evaluated . Length of competent cells during the outgrowth experiment is shown as boxplots ( refers to Fig 3F ) . The blue box edges indicate the first and third quartile while the red line indicates the median of the data set . In addition , the whiskers indicate the 5th and 95th percentiles and individual red points indicate outliers . All values with means , standard deviations ( SD ) and sample sizes are listed in S3 Table . Boxplots were plotted using Matlab 2013 . The statistical significance of the differences observed is presented in S3 Table . Whole cell extracts were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer’s protocol ( Bio-Rad ) . After incubation with 5% nonfat milk in TBST ( 10 mM Tris , pH 8 . 0 , 150 mM NaCl , 0 . 05% Tween 20 ) for 60 minutes , membranes were incubated with antibodies against GFP ( 1:10000 ) overnight at room temperature . Membranes were washed 3 times for 10 minutes with TBST and incubated with a 1:10000 dilution of anti-rabbit antibodies for 2h . Blots were washed with TBST three times and developed with the “ECL Prime” kit ( Amersham ) according to the manufacturer’s protocols . The Chemidoc system ( Bio-Rad ) was used to reveal the membrane and the Image Lab™ software ( Bio-Rad ) to analyze the intensity of the bands . Saccharomyces cerevisiae cells expressing B . subtilis selected proteins as GAL4 BD fusions were mated with cells expressing either the same or another protein as GAL4 AD fusions as presented in [76] . For each fusion , two independent yeast clones were used . Binary interactions were revealed by growth of diploid cells after 5 days at 30°C on synthetic complete medium lacking leucine , uracil and histidine ( to select for expression of the HIS3 interaction reporter , annotated-H ) . Specific interactions were reproduced independently at least three times . | In bacterial cells , like in their eukaryotic counterparts , precise spatiotemporal localization of proteins is critical for their cellular function . This study shows that the expression and the localization of the bacterial actin-like MreB protein are growth phase-dependent . During exponential growth , we previously showed that MreB , together with other morphogenetic factors , forms discrete assemblies that move in a directed manner along peripheral tracks . Here , we demonstrate that in cells that develop genetic competence during stationary phase , transcription of mreB is specifically activated and MreB relocalizes to the cell poles . Our findings suggest a model in which MreB sequestration by the late competence protein ComGA prevents cell elongation during the escape from competence . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [] | 2015 | MreB-Dependent Inhibition of Cell Elongation during the Escape from Competence in Bacillus subtilis |
Kaposi’s sarcoma-associated herpesvirus ( KSHV ) is a herpesvirus that is linked to Kaposi’s sarcoma ( KS ) , primary effusion lymphoma ( PEL ) and multicentric Castleman’s disease ( MCD ) . KSHV establishes persistent latent infection in the human host . KSHV undergoes periods of spontaneous reactivation where it can enter the lytic replication phase of its lifecycle . During KSHV reactivation , host innate immune responses are activated to restrict viral replication . Here , we report that NLRX1 , a negative regulator of the type I interferon response , is important for optimal KSHV reactivation from latency . Depletion of NLRX1 in either iSLK . 219 or BCBL-1 cells significantly suppressed global viral transcription levels compared to the control group . Concomitantly , fewer viral particles were present in either cells or supernatant from NLRX1 depleted cells . Further analysis revealed that upon NLRX1 depletion , higher IFNβ transcription levels were observed , which was also associated with a transcriptional upregulation of JAK/STAT pathway related genes in both cell lines . To investigate whether IFNβ contributes to NLRX1’s role in KSHV reactivation , we treated control and NLRX1 depleted cells with a TBK1 inhibitor ( BX795 ) or TBK1 siRNA to block IFNβ production . Upon BX795 or TBK1 siRNA treatment , NLRX1 depletion exhibited less inhibitory effects on reactivation and infectious virion production , suggesting that NLRX1 facilitates KSHV lytic replication by negatively regulating IFNβ responses . Our data suggests that NLRX1 plays a positive role in KSHV lytic replication by suppressing the IFNβ response during the process of KSHV reactivation , which might serve as a potential target for restricting KSHV replication and transmission .
Kaposi’s sarcoma-associated herpesvirus ( KSHV ) , also known as human herpesvirus 8 ( HHV-8 ) , is a linear double-stranded DNA virus . It is causally linked with Kaposi’s sarcoma ( KS ) , primary effusion lymphoma ( PEL ) and multicentric Castleman’s disease ( MCD ) [1 , 2] . In the majority of KS lesion cells or PEL cells , KSHV maintains latent infection , where KSHV lytic gene expression can only be detected in a small subset of cells [3] . While latent infection is important for maintaining the viral reservoir , reactivation of KSHV from latent infection is important for viral particle production and transmission of KSHV [4 , 5] . The balance between lytic and latent infection is critical for KSHV pathogenicity . During reactivation , many cellular signaling pathways are activated and regulated by both host and viral proteins . For example , KSHV infection has been shown to trigger innate immune responses through pattern recognition receptors ( PRRs ) that recognize pathogen-associated molecular patterns ( PAMPs ) , which results in interferon and pro-inflammatory cytokine production ( reviewed in [6] ) . KSHV encodes multiple proteins that modulate host immune responses to facilitate viral replication [7–11] . However , a better understanding of the role of cellular genes in viral reactivation is also critical for understanding the pathogenesis associated with KSHV . NLRX1 ( also known as CLR11 . 3 and NOD9 ) is a member of nucleotide-binding domain , leucine-rich repeat-containing protein family ( also known as NOD-like receptors , NLRs ) [12–14] . NLRs were originally widely studied as sensors or receptors of inflammasome signaling , but were recently reported to regulate type I interferon production as well ( reviewed in [15 , 16] ) . NLRX1 was identified as a negative regulator of RIG-I like receptor ( RLR ) dependent type I interferon production [12] . NLRX1 localizes to the mitochondria and associates with MAVS ( also known as Cardif , VISA and IPS-1 ) , a mitochondria-localized adaptor downstream of RIG-I , to disrupt cytosolic RNA induced type I interferon responses . Depletion of NLRX1 led to enhanced antiviral responses [12] . Notably , NLRX1 was also identified to facilitate HIV-1 primary infection through negative regulation of the cytosolic DNA sensing pathway . NLRX1 was shown to sequester the DNA-sensing adaptor STING from interaction with TBK-1 , and therefore block sufficient type I interferon production . In a similar vein , NLRX1 deficient mice also exhibited stronger innate immune responses and thus reduced HSV1 replication [17] . In correlation with these findings , in SIV infection of rhesus monkeys , expression of NLRX1 was negatively correlated with a type I interferon signature [18] . Therefore , in the context of viral infection , NLRX1 might be a key factor in determining the battle between host and virus . KSHV reactivation has been previously reported to activate MAVS-dependent cytosolic RNA sensing pathways . Blocking MAVS was shown to downregulate type I interferon and thus facilitate KSHV lytic replication [19] . Therefore , as a MAVS negative regulator , it is plausible that NLRX1 plays an important role in KSHV reactivation , and functions as a potential target for restricting KSHV transmission .
To investigate the role of NLRX1 as a potential restriction factor against KSHV lytic reactivation from latency , we utilized KSHV latently infected epithelial cells , iSLK . 219 cells [20] . We used control siRNA or siRNA against NLRX1 to specifically deplete NLRX1 in iSLK . 219 cells before reactivation . The iSLK . 219 cells harbor a latent BAC16-KSHV with a constitutive GFP marker , a doxycycline ( Dox ) -inducible RTA , which is necessary and sufficient to induce lytic reactivation , and a RFP marker driven by the promoter of the KSHV lytic gene , PAN , which is activated during KSHV lytic replication . Therefore , GFP serves as a marker for infected cells while RFP serves as a marker for KSHV lytic replication . As shown in Fig 1A , while Dox successfully induced KSHV reactivation and lytic replication in both non-specific ( NS ) siRNA and NLRX1 siRNA transfected cells at each time point , there were lower percentages of RFP positive cells in the NLRX1 depleted samples . Moreover , while we did not observe significant GFP variation , RFP intensity quantitation showed significant inhibition of RFP intensity in the NLRX1 siRNA transfected samples compared to the control NS siRNA transfected samples . This suggests that KSHV lytic replication is suppressed in NLRX1 depleted cells ( Fig 1B and 1C ) . We also tested a second NLRX1 siRNA ( siNLRX1 #2 ) and examined its effect on viral reactivation by investigating RFP fluorescence . The results with the second NLRX1 siRNA was similar to the first siRNA ( S2A and S2B Fig ) . We next monitored KSHV viral particles in the supernatant as well as in infected iSLK . 219 cells from each group harvested at 24 , 48 and 72 hours post reactivation . Consistent with RFP expression levels , we detected significantly fewer KSHV genomes in the NLRX1 siRNA transfected samples compared to control NS siRNA transfected samples as determined by KSHV genome copy number ( Fig 1D and 1E ) . NLRX1 knockdown efficiency was checked by qRT-PCR ( Fig 2A ) . To determine the impact of NLRX1 on viral genes , we examined KSHV lytic gene expression . Knockdown efficiency of NLRX1 was monitored by qRT-PCR ( Fig 2A , S2E Fig ) . NLRX1 deficient cells showed a reduced ability to induce lytic gene transcription ( such as ORF57 , vIRF1 and K8 . 1 ) than the control NS siRNA transfected cells ( Fig 2B and 2C; S2C and S2D Fig ) . Both ORF45 and K8alpha lytic proteins were downregulated at 24 , 48 and 72 hours post reactivation in the NLRX1 siRNA group ( Fig 2D ) . Knockdown efficiency of NLRX1 was also monitored by immunoblot analysis ( Fig 2D ) . To broadly profile KSHV viral gene expression during reactivation in control versus NLRX1 depleted samples , we also performed KSHV whole genome transcriptional profiling . As seen in Fig 2E , Dox treatment successfully induced KSHV gene expression , and depletion of NLRX1 led to a suppression and delay of viral genome transcription at each time point that we tested , which correlates well with our qRT-PCR data ( Fig 2E ) . We also monitored viral gene transcription at later time points , and we observed a smaller difference between the siNS and siNLRX1 groups at later time points ( S3A–S3C Fig ) . We next probed for the mechanism by which NLRX1 depletion restricts KSHV lytic reactivation from latency . In order to rule out the possibility that siNLRX1 affects the induction of RTA , we tested if siNLRX1 would affect RTA mRNA transcription induced by Dox . Because RTA transcription in iSLK . 219 cells can be both affected by Dox and KSHV reactivation , we used iSLK . RTA cells without KSHV infection to test if siNLRX1 would affect Dox induced RTA . As shown in S3D and S3E Fig , knockdown of NLRX1 did not attenuate RTA mRNA transcription . Since NLRX1 has been reported to negatively regulate type I interferon , we then investigated if NLRX1 altered type I interferon responses upon KSHV reactivation . As seen in Fig 3A , loss of NLRX1 did result in stronger ifnb transcriptional activity . IFNβ is known to activate the JAK/STAT pathway to induce ISGs for effective antiviral responses against viruses . To investigate this further , we next tested whether the upregulated ifnb transcription level in NLRX1 deficient cells led to the activation of the JAK/STAT pathway . We performed microarray analysis of genes in the JAK/STAT pathway using KSHV infected cells that were transfected with either control or NLRX1 siRNAs at timepoints of 0 hour , 24 hours and 48 hours post reactivation . We compared JAK/STAT responsive genes that were activated in cells transfected with NLRX1 and NS siRNA at each time point . At 0 , 24 and 48 hours , many JAK/STAT pathway genes were induced at least 2 fold higher in NLRX1 depleted cells compared to NS siRNA transfected cells , indicating a higher potential for JAK/STAT pathway upregulation upon NLRX1 depletion ( Fig 3B–3D ) . Moreover , as shown in Fig 3E and 3F , at 24 hours post Dox treatment , 16 genes were upregulated by 2 fold in the NS siRNA control samples while 48 genes were upregulated at least 2 fold in the in NLRX1 siRNA transfected samples . At 48 hours post Dox treatment , 20 genes were upregulated by more than 2 fold in the NS siRNA control samples while 46 genes were upregulated at least 2 fold in the NLRX1 siRNA transfected samples ( Fig 3G and 3H ) . In summary , NLRX1 deficient cells induce a much wider variety of JAK/STAT pathway related genes at each time point tested in KSHV reactivated cells compared to the control cells . Fig 3I and S1 Table depicts the overall gene expression of all 84 JAK/STAT related genes tested . As shown , most genes exhibit a higher expression level in NLRX1 deficient cells compared to control cells at each time point tested . We noticed that many JAK/STAT related genes were upregulated in the absence of KSHV lytic reactivation . To eliminate that this is not due to an off target effect of the NLRX1 siRNA , we also tested an alternative NLRX1 siRNA ( siNLRX1 #2 ) to monitor the activation of the JAK/STAT pathway before KSHV was reactivated . As shown in S4A–S4D Fig , we have also observed many upregulated genes . Specifically , of 57 upregulated genes in siNLRX1 #2 group , 54 of the genes were also upregulated in siNLRX1 group , compared to the siNS group . We also transiently overexpressed NLRX1 in iSLK . 219 cells to explore the role of NLRX1 in KSHV reactivation . Consistent with the siRNA experiments , we detected attenuated levels of IFNβ mRNA and increased levels of viral ORF57 mRNA ( S1A and S1B Fig ) . NLRX1 mRNA levels were monitored by qRT-PCR ( S1C Fig ) . NLRX1 was identified as a negative regulator of MAVS . Therefore , in order to further detail the mechanism by which NLRX1 facilitates KSHV reactivation , we looked at IFNβ regulation by MAVS . MAVS has previously been shown to play a role in limiting KSHV reactivation . Therefore , we hypothesized that NLRX1 blocks MAVS to attenuate type I interferon responses in KSHV-infected cells , and thus facilitates KSHV reactivation . To prove this , we first tested the integrity and functionality of the MAVS signaling pathway . As shown in Fig 4A , we transfected poly I:C into KSHV infected iSLK . 219 cells and successfully triggered MAVS dependent IFNβ induction , which is indicative of a functional RLR pathway . Moreover , NLRX1 depletion in these cells resulted in higher induction of IFNβ , suggesting NLRX1 blocks MAVS signaling in KSHV-infected iSLK . 219 cells without reactivation . NLRX1 knockdown efficiency was monitored by qRT-PCR ( Fig 4B ) . We also tested if poly I:C , an activator of the MAVS pathway , could mimic the effect of NLRX1 knockdown to inhibit KSHV reactivation . As shown in S5A–S5F Fig , poly I:C induced IFNβ during reactivation , and this correlated with inhibition of KSHV reactivation as determined by RFP fluorescence and viral lytic gene expression . To further test if NLRX1 blocks MAVS signaling in KSHV-infected cells upon reactivation , we used BX795 , an inhibitor of TBK1 , which acts directly downstream of MAVS . As shown in Fig 4C , when we treated cells with NLRX1 siRNA and BX795 , we observed significant inhibition of IFNβ compared to the NLRX1 siRNA and vehicle only treated group at 24 and 48 hours post reactivation . IFNβ levels in the NLRX1 siRNA and BX795 treated group were similar to that of the NS siRNA treated group . NLRX1 knockdown efficiency was monitored by qRT-PCR ( Fig 4D ) . To eliminate the possibility that NLRX1 directly acts as a negative regulator of IRF3 , we tested whether NLRX1 could modulate IFNβ promoter luciferase activation by an IRF3 super activator ( SA ) . As shown in S1D–S1F Fig , NLRX1 overexpression inhibited dRIG-I- or MAVS- dependent activation of the IFNβ promoter , but the IRF3 ( SA ) activated IFNβ promoter activation was not affected . These data suggest that NLRX1 inhibits MAVS in KSHV-infected cells and supports KSHV lytic replication . We next investigated whether BX795 inhibition of the MAVS-TBK1 node would rescue the effect of NLRX1 depletion on KSHV reactivation . As shown in Fig 5A , while NLRX1 depletion resulted in less RFP positive cells than the control NS siRNA group , BX795 treatment partially rescued the block of lytic replication . RFP intensity quantitation also showed significant inhibition of RFP intensity in NLRX1 siRNA treated samples compared to the control group , and a subsequent increase of RFP intensity when the NLRX1 siRNA group was treated with BX795 ( Fig 5C ) . No significant variations were observed in GFP intensity among groups ( Fig 5B ) . We then monitored KSHV viral particles from the reactivated cells from each group harvested at 24 , 48 hours and 72 hours post reactivation . We detected significantly fewer KSHV virions in cells transfected with NLRX1 siRNA compared to control NS siRNA samples , and a partial rescue of KSHV viral genomes upon BX795 treatment ( Fig 5D ) . Similar patterns were observed when we monitored KSHV ORF57 gene transcription levels in reactivated iSLK . 219 cells ( Fig 5E ) . NLRX1 depletion led to a significant inhibition of ORF57 gene transcription , but this was partially rescued by BX795 treatment ( Fig 5E ) . To further corroborate our experimental data obtained with BX795 treatment , we also utilized TBK1 specific siRNA . As shown in Fig 6A , while NLRX1 depletion resulted in fewer RFP positive cells than the control NS siRNA sample , siNLRX1+siTBK1 treatment rescued the block to KSHV lytic reactivation and replication . RFP intensity quantitation also showed significant inhibition of RFP intensity in NLRX1 siRNA treated samples compared to the control samples and a recovery of RFP intensity when the NLRX1 siRNA group was co-transfected with siNLRX1 and siTBK1 ( Fig 6C ) . No significant variations were observed in GFP intensity among groups ( Fig 6B ) . We also monitored multiple KSHV viral gene transcripts in reactivated iSLK . 219 cells . As shown in Fig 6D–6G , NLRX1 depletion led to significant inhibition of ORF57 , K8 . 1 and vIRF1 gene transcription , but they were all rescued when the cells were co-transfected with TBK1 siRNA . NLRX1 knockdown efficiency was monitored by qRT-PCR as shown in Fig 6D . We have also tested if TBK1 knockdown alone promoted KSHV replication by examining viral lytic gene transcription by qRT-PCR . As shown in S6A–S6D Fig , TBK1 knockdown resulted in elevated transcription of viral genes , such as orf57 , virf1 and k8 . 1 . We also investigated NLRX1’s role in KSHV infected PEL cells . BCBL-1 is a KSHV-infected B lymphoma cell line . We transfected NS or NLRX1 siRNA into BCBL-1 cells , and then induced lytic replication of KSHV by addition of TPA and sodium butyrate ( NaB ) as previously described [11] . The cells and supernatant were harvested at 0 hour , 24 hours and 48 hours post reactivation , and KSHV genome copy number was determined by qRT-PCR . As shown in Fig 7A and 7B , NLRX1 depletion resulted in significant inhibition of KSHV viral replication both in the cells and in the supernatant , as determined by the genome copy number . We also tested KSHV gene transcription levels in reactivated BCBL-1 cells transfected with NLRX1 siRNA or NS siRNA . NLRX1 depletion resulted in significant inhibition of lytic gene transcription such as ORF57 ( Immediate early ) , ORF36 ( early ) , and K8 . 1 ( late ) than NS siRNA transfected cells ( Fig 7D–7F ) . NLRX1 knockdown efficiency was monitored by qRT-PCR as shown in Fig 7C . Furthermore , we also introduced another siRNA ( siNLRX1 #2 ) and performed qRT-PCR assay to monitor the status of KSHV lytic genes in reactivated BCBL-1 cells . As shown in S7A–S7D Fig , we observed that both NLRX1 siRNAs modulated KSHV reactivation similarly in BCBL-1 cells . Because NLRX1 is a negative regulator of IFNβ , we tested if NLRX1 blocked type I interferon responses upon KSHV reactivation . As seen in Fig 8A , NLRX1 depletion enhanced ifnb transcriptional activity compared to the NS siRNA group , confirming NLRX1’s role in restricting IFNβ . We then performed microarray analysis of the JAK/STAT pathway in BCBL-1 cells to explore NLRX1’s effect on KSHV reactivation . We compared genes that were activated in cells treated with NLRX1 siRNA or NS siRNA at 0 , 24 and 48 hours post reactivation . At 0 , 24 and 48 hours , a significant number of genes were induced at least 2 fold higher in NLRX1 siRNA transfected cells compared to NS siRNA transfected cells , indicating a higher potential of JAK/STAT pathway upregulation when NLRX1 is depleted in BCBL-1 cells ( Fig 8B–8D ) . Moreover , at 24 hours post reactivation , 21 genes were upregulated by more than 2 fold in the siNS group while 41 genes in the siNLRX1 group were upregulated by 2 fold . At 48 hours post reactivation , 29 genes were upregulated by more than 2 fold in the siNS group while 49 genes in the siNLRX1 group were upregulated by more than 2 fold . These data suggest that NLRX1 deficient cells exhibited induction of a much wider variety of JAK/STAT pathway related genes at each time point tested compared to the control groups ( Fig 8E–8H ) . Fig 8I and S2 Table summarizes the overall gene expression of all 84 JAK/STAT related genes tested . As shown , most genes exhibit higher expression levels in NLRX1 depleted cells compared to control cells at each time point tested .
KSHV reactivation from latency is a complex process for both virus and host , which is tightly regulated by a variety of signaling pathways . Efficient lytic replication of KSHV requires disruption of restrictive signaling pathways that keep the virus latent . This can be achieved by the action of either viral proteins or host proteins . For example , upon KSHV reactivation , cytosolic RNA and DNA dependent pathways were reported to be activated and type I interferon was produced to suppress viral replication [8 , 19] . Previously , we have reported that in order to facilitate viral lytic replication , KSHV encodes multiple proteins to inhibit type I interferon production , such as vIRF1 . vIRF1 knockdown in the context of viral reactivation can result in enhanced IFNβ production and insufficient reactivation [8] . While viral inhibitors of type I interferon are important for KSHV lytic replication , in this study , we focused on exploring the role of a host type I interferon inhibitor , NLRX1 , during KSHV reactivation . We demonstrated that NLRX1 is required for optimal KSHV reactivation . NLRX1 deficiency in iSLK . 219 cells led to enhanced type I interferon production , as well as global suppression of KSHV genome transcription activity , decreased level of lytic proteins , and attenuated virion production . A similar phenotype was observed in BCBL-1 cells as well , suggesting NLRX1 is critical for KSHV reactivation and subsequent replication in multiple cell lines . We noticed that viral genes clustered together based on their expression patterns following NLRX1 depletion . A future goal will be to further explore the differences among these different clusters to better understand the regulation of KSHV reactivation . NLRX1 was previously reported to suppress the RIG-I-MAVS signaling pathway , which is triggered by cytosolic RNA [12] . It has also been previously reported that KSHV reactivation generates dsRNA intermediates that can trigger RIG-I-MAVS signaling in KSHV-infected cells [19] . As shown in our study , we demonstrate the functionality of the MAVS-dependent pathway in both KSHV-infected iSLK . 219 and BCBL-1 cells . More importantly , NLRX1 negatively regulated MAVS-dependent IFNβ transcription before and after KSHV reactivation in both iSLK . 219 and BCBL-1 cells , indicating that NLRX1 regulates KSHV reactivation through a MAVS-dependent pathway . Inhibition of type I interferon induction by the TBK1 antagonist , BX795 , mitigated the effect of NLRX1 deficiency on KSHV lytic replication . We have also explored TBK1’s effect on KSHV lytic reactivation . Although this is the first time that TBK1 was reported to be a negative regulator of KSHV lytic reactivation , TBK1 was previously reported as a restriction factor of RNA viruses , such as Newcastle Disease virus ( NDV ) and Sendai virus ( SeV ) , by acting downstream of MAVS and positively regulating IFN responses [21 , 22] . This correlates with our previously published results that MAVS play a negative role in KSHV reactivation [19] . Moreover , TBK1 also inhibits replication of HSV-1 , an alpha herpesvirus [23] . Although NLRX1 plays an important role in KSHV reactivation , we did not observe significant upregulation or downregulation of NLRX1 at either the transcript or protein level during KSHV reactivation . Therefore , it is possible that NLRX1 serves as a steady-state negative regulator of type I interferon to facilitate KSHV reactivation . However , it is also plausible that NLRX1 may undergo some type of post translational modifications upon KSHV reactivation , which might further benefit KSHV lytic replication . A third possibility is that during lytic reactivation , a KSHV encoded protein ( s ) might also bind directly or indirectly to the NLRX1 signaling complex to regulate its function . In sum , we report for the first time that NLRX1 plays a pivotal role in modulating KSHV reactivation from latency .
iSLK . 219 ( doxycycline-inducible SLK cells harboring latent rKSHV . 219 ) ( a kind gift from D . Ganem ) were maintained in DMEM ( Corning ) supplemented with 10% FBS ( Sigma ) , 1% penicillin and streptomycin ( Corning ) , G418 ( 250 μg/ml ) ( Sigma ) , hygromycin ( 400 μg/ml ) ( Corning ) , and puromycin ( 10 μg/ml ) ( Corning ) . BCBL-1 cells ( a kind gift from D . Ganem ) were maintained in RPMI ( Corning ) medium supplemented with 20% FBS , 1% penicillin and streptomycin ( Corning ) , 1% L-glutamine ( Corning ) , and 0 . 05 mM β-mercaptoethanol ( Sigma ) . All cells were maintained at 37°C in a 5% CO2 laboratory incubator subject to routine cleaning and decontamination . poly ( I:C ) was purchased from Invivogen . Antibodies were obtained from the following sources: mouse anti-NLRX1 ( Jenny Ting laboratory ) , KSHV ORF45 ( MA5-14769 ) ( Thermo Scientific ) , Goat anti β-actin-HRP ( 1615 ) ( Santa Cruz ) , KSHV K8 . 1alpha ( SC-57889; Santa Cruz ) . The NLRX1 antibody was previously reported [12] . The pCIG2-Puro and pCIG2-NLRX1-FLAG plasmids were previously described [17] . The dRIG-I and MAVS expression plasmids were previously described [12] . pRL-CMV renilla vector was obtained from Promega . IFNβ promoter luciferase was a generous gift from Zhijian Chen , University of Texas Southwestern , Dallas . pUNO-IRF3sa was obtained from Invivogen . iSLK . 219 cells were maintained as described above and were transfected using Lipofectamine RNAiMAX ( Life Technologies ) according to the manufacturer’s instructions . At 48 hours post-transfection , the medium was changed to DMEM containing 1% Pen-Strep , 10% FBS , and 0 . 2 μg/ml of doxycycline for reactivation [20] . BCBL-1 siRNA transfections were performed using Lonza nucleofector V kit according to the manufacturer’s recommendations ( Lonza ) . BCBL-1 PEL cells were reactivated with 1 mM sodium butyrate ( Sigma ) and 25 ng/ml 12-O-tetradecanoyl-phorbol 13-acetate ( TPA ) ( Sigma ) where indicated . At 0 , 24 , 48 or 72 hours post-reactivation , cells and supernatant were collected . RNA was harvested from cells via the RNeasy Plus mini kit ( Qiagen ) for analysis of levels of viral transcripts . DNA was harvested from both cells and supernatant via DNeasy mini kit ( Qiagen ) for analysis of genome copy numbers . Protein from cells was harvested for WB analysis . Chemically synthesized siRNA duplexes were obtained from Dharmacon GE . ON-TARGETplus Non-targeting Control siRNAs #1 ( D-001810-01 ) ; ON-TARGETplus NLRX1 siRNA ( J-012926-10 ) Sequence: UCGUCAACCUGGUGCGCAA; ON-TARGETplus NLRX1 siRNA #2 ( J-012926-12 ) Sequence: GUGCUGGGUUUGCGCAAGA; ON-TARGETplus TBK1 ( 29110 ) siRNA SMARTpool ( L-003788-00 ) Sequences: ( J-003788-08 ) AGAAGGCACUCAUCCGAAA; ( J-003788-09 ) GAACGUAGAUUAGCUUAUA; ( J-03788-10 ) UGACAGCUCAUAAGAUUUA; ( J-003788-11 ) GGAUAUCGACAGCAGAUUA Total RNA was isolated by using RNeasy RNA extraction kit ( Qiagen ) and cDNA synthesis was performed using iScript cDNA Synthesis Kit ( Bio-rad ) according to manufacture protocols . Real-time PCR was performed using a ViiA 6 Real-Time PCR System . A SYBR green assay from Bio-rad was used for human ifnb as well as KSHV ORFs detection . Primers used for SYBR green qRT-PCR were: KSHV orf57 F: 5’-TGGACATTATGAAGGGCATCCTA-3’; R: 5’-CGGGTTCGGACAATTGCT-3’ . KSHV orf36 F: 5’-TGCGTCCTCTTCCAGTGTTA-3’; R: 5’-GTCAGCAGAGTGTAGCCCAA-3’ . KSHV virf1 F: 5’-CGTGTCCTTTGGTGAAACTG-3’; R: 5’-TCGGCATTATTTCGAGTACG-3’ . KSHV k8 . 1 F: 5’-AAAGCGTCCAGGCCACCACAGA-3’; R: 5’-GGCAGAAAATGGCACACGGTTAC-3’ . Human actin F: 5’-AAGACCTGTACGCCAACACA-3’; R: 5’-AGTACTTGCGCTCAGGAGGA-3’ . Human ifnb F: 5’-AGTAGGGCGACACTGTTCGTG-3’; R: 5’-GAAGCACAACAGGAGAGCAA-3’ . Human nlrx1 F: 5’-CCTCTGCTCTTCAACCTGATC-3’; R: 5’-CCTCTCGAAACATCTCCAGC-3’ . Human tbk1 F: 5’- CCTCCCTAAAGTACATCCACG-3’; R: 5’- CAATCAGCCATCGTATCCCC-3’ . The relative amount of IFNβ , ORF57 , ORF36 and K8 . 1 mRNA was normalized to actin RNA level in each sample and the fold difference between the treated and mock samples was calculated . Total RNA was isolated from iSLK . 219 or BCBL-1 cells by using RNeasy RNA extraction kit ( Qiagen ) and cDNA synthesis was performed using iScript™ cDNA Synthesis Kit ( Bio-rad ) according to the manufacturer’s protocols . Human JAK/STAT Signaling Primer Library was purchased from Realtimeprimers . com ( Cat #: HJAK-I ) , which contains 88 primer sets directed against human JAK/STAT related genes and 8 housekeeping gene primer sets . Fold-Change ( 2^ ( - Delta Delta Ct ) ) is the normalized gene expression ( 2^ ( - Delta Ct ) ) in the Test Sample divided the normalized gene expression ( 2^ ( - Delta Ct ) ) in the Control Sample . Data were analyzed by GENE-E software ( https://software . broadinstitute . org/GENE-E/ ) . We used a real-time qPCR array to quantify all KSHV mRNAs . Briefly , 192 primer pairs were included to target multiple regions towards the 3’ end of each annotated ORF . Multiple reference genes for cellular transcripts were included for normalization . The array results in amplification reactions with similar efficiencies and annealing temperatures and thus allows us to directly compare the expression levels among different mRNAs . qPCR was plated in 384-well plates using the Tecan Freedom Evo liquid handling robot and cycled using Roche LightCycler 480 , as previously described . A detailed , step-by-step protocol is available at http://www . med . unc . edu/vironomics/protocols . Statistical significance of differences in cytokine levels , mRNA levels , viral titers , and luciferase intensity in reporter assay were determined using Student’s t-test . * indicates P<0 . 05 . ** indicates P<0 . 01 . | Kaposi’s sarcoma-associated herpesvirus ( KSHV ) is linked to a number of different human cancers , including Kaposi’s sarcoma ( KS ) , primary effusion lymphoma ( PEL ) and multicentric Castleman’s disease ( MCD ) . KSHV predominantly establishes life-long latency in the infected host . Lytic reactivation from latency is critical for KSHV survival and replication . During KSHV reactivation from latency , host innate immune responses are activated to restrict viral replication . Here , we report that NLRX1 , a negative regulator of the type I interferon response , is important for optimal KSHV reactivation and subsequent lytic replication . | [
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] | 2017 | NLRX1 negatively modulates type I IFN to facilitate KSHV reactivation from latency |
Significant selection pressure has been exerted on the genomes of human populations exposed to Plasmodium falciparum infection , resulting in the acquisition of mechanisms of resistance against severe malarial disease . Many host genetic factors , including sickle cell trait , have been associated with reduced risk of developing severe malaria , but do not account for all of the observed phenotypic variation . Identification of novel inherited risk factors relies upon high-resolution genome-wide association studies ( GWAS ) . We present findings of a GWAS of severe malaria performed in a Tanzanian population ( n = 914 , 15 . 2 million SNPs ) . Beyond the expected association with the sickle cell HbS variant , we identify protective associations within two interleukin receptors ( IL-23R and IL-12RBR2 ) and the kelch-like protein KLHL3 ( all P<10−6 ) , as well as near significant effects for Major Histocompatibility Complex ( MHC ) haplotypes . Complementary analyses , based on detecting extended haplotype homozygosity , identified SYNJ2BP , GCLC and MHC as potential loci under recent positive selection . Through whole genome sequencing of an independent Tanzanian cohort ( parent-child trios n = 247 ) , we confirm the allele frequencies of common polymorphisms underlying associations and selection , as well as the presence of multiple structural variants that could be in linkage with these SNPs . Imputation of structural variants in a region encompassing the glycophorin genes on chromosome 4 , led to the characterisation of more than 50 rare variants , and individually no strong evidence of associations with severe malaria in our primary dataset ( P>0 . 3 ) . Our approach demonstrates the potential of a joint genotyping-sequencing strategy to identify as-yet unknown susceptibility loci in an African population with well-characterised malaria phenotypes . The regions encompassing these loci are potential targets for the design of much needed interventions for preventing or treating malarial disease .
Sub-Saharan Africa bears a disproportionately high share of the global Plasmodium falciparum malaria burden , with 90% of the estimated 212 million annual cases and 92% of 429 , 000 annual deaths , mostly in children under five years of age [1] . Whilst the majority of cases of Plasmodium falciparum infection are asymptomatic or cause only mild to moderate clinical symptoms , a subset of affected individuals present with severe manifestations such as severe malarial anaemia and cerebral malaria . Risk factors for severe malaria and its various clinical subtypes are poorly understood , although host and parasite genotype , age and immune status have all been established as playing a significant role in individual host susceptibility [2] . Plasmodium falciparum has also exerted significant selection pressure upon the human genome , as evidenced by the geographical concurrence of malaria parasite prevalence with sickle cell trait ( HbAS ) and other haemoglobinopathies , such as the thalassemias and glucose-6-phosphate dehydrogenase ( G6PD ) deficiency . Recent studies , set in a region of high malaria transmission in north-eastern Tanzania , estimated that host genetic factors account for approximately 22% of the total variation in severe malaria risk [3] , consistent with previous findings in a Kenyan family-based study [2] . Less than half of this variation can be explained by erythrocyte-associated polymorphisms [4] , including HbS ( sickle cell trait ) , alpha-thalassaemia , ABO blood group [5] and G6PD deficiency [4] . Novel polymorphisms in or around USP38 , FREM3 [3] , glycophorins gypA/B/E [6 , 7] , DDC [8] , MARVELD3 and ATP2B4 [9] account for additional variation but , in sum , are less protective than heterozygous carriage of HbS [3] . Moreover , the effects of some of these loci are subtype- , location- , or population-specific [3 , 6 , 7 , 9] , reinforcing the need for targeted genome-wide association studies ( GWAS ) in different African populations . Utilising such an approach with robust malaria phenotypes in parallel with whole genome sequencing of study populations is crucial to unravelling host genetic factors that could lead to a greater understanding of protective immunity and development of new tools for disease prevention . To identify novel loci associated with severe malaria in north-eastern Tanzania , we applied genome-wide association and haplotype-based selection methods to a case-control dataset with extensive phenotypic data for 914 participants and 15 . 2 million SNPs . In addition to the expected HbS ( sickle cell ) association , our analyses reveal multiple novel loci under association or selection . Association analysis highlighted significant SNPs clusters within IL-23R , IL-12RB2 , LINC00944 , and KLHL3 whilst lone SNP associations were also present within TREML4 and ZNF536 . Further , we reveal loci under recent positive selection including GCLC and loci within the Major Histocompatibility Complex ( MHC ) . These analyses were supported by whole genome sequencing of an independent dataset consisting of 247 Tanzanian individuals within parent-child trios , which was used to confirm the allele frequencies of putative associations and determine if there are any linked common structural variants in chromosome regions encoding important polymorphisms .
All severe malaria cases ( n = 449 ) and controls ( n = 465 ) came from the Tanga region of North-Eastern Tanzania . Severe malaria cases presented with varying combinations of hyperlactataemia ( 57 . 0% ) , severe malarial anaemia ( 49 . 2% ) , respiratory distress ( 27 . 6% ) and cerebral malaria ( 26 . 7% ) ( Table 1 ) . Compared to controls , malaria cases were younger ( t test P<2 . 2x10-16 ) and marginally more likely to be male ( Chi squared P = 0 . 012 ) ( Table 1 ) . DNA from all samples ( n = 914 ) was genotyped on the Illumina Omni 2 . 5 million SNP chip , and imputed against the 1000 Genomes reference panel ( Phase 3 ) [10] and a Tanzanian parent-child trio panel ( see below ) , using Beagle 4 . 1 [11] , leading to 15 . 2 high quality SNPs . These markers were complemented by 180 SNPs within malaria candidate genes , including HBB ( encoding HbS ) [3 , 4 , 5] on the same cases and controls . DNA from a validation cohort of 78 healthy parent and child trios and 13 independent individuals ( “Trios dataset” , n = 247 ) were whole genome sequenced using Illumina HiSeq2500 technology . For the GWAS samples , a principal component analysis ( PCA ) using all genome-wide SNPs revealed a low degree of stratification by ethnicity and case-control status ( S1 Fig ) and potential cryptic relatedness due to familial clustering . A similar analysis revealed that GWAS and Trio sample clusters overlap , and there is some separation from the other 1000 Genome African populations , including Yoruba ( Nigeria ) and Luhya ( Kenya ) ( S1 Fig ) . GWAS analysis was undertaken with EMMAX mixed model regression [12] , controlling for age as a fixed effect and relatedness ( represented in a kinship matrix ) as a random effect to account for the cryptic population clustering . Separate models of association were fitted for each SNP ( additive , heterozygous , dominant , recessive ) , with their respective genomic inflation factors all being close to one ( see S1 Fig for the heterozygous results ) , consistent with reliable adjustment for stratification . A total of 53 SNPs ( in 16 genomic regions ) were identified with a significance level below our threshold ( P<1x10-6 ) ( Fig 1 , Table 2 , S1 Table ) . Relaxing the stringency would lead to 258 SNPs with a p-value below 1x10-5 and 2 , 322 below a threshold of 1x10-4 . As expected , the most significant association was with the sickle cell locus , rs334 ( P = 8 . 59x10-13 , heterozygous odds ratio = 0 . 07 ) ( Table 2 ) . Controlling for HbS status through a complementary conditional GWAS demonstrated our top associations as robust against linkage with rs334 ( Table 2 , S1 Table ) . Novel associations of note also include SNPs within the KLHL3-MYOT region ( 13 SNPs , Min P = 5 . 85x10-7 , Additive OR = 0 . 590 ) , the IL23R-IL12RB2 region ( 7 SNPs , Min P = 7 . 98x10-7 , Recessive OR = 0 . 479 ) , FAM155A ( 6 SNPs , Min P = 6 . 24x10-7 , Additive OR = 0 . 207 ) , and CSMD1 ( 5 SNPs , Min P = 7 . 98x10-7 , Additive OR = 4 . 795 ) . ( Fig 2 ) . Three significant SNPs are also found within both LINC00943/4 and lincRNA AF146191 . 4–004 . Lone SNP associations are present within proximity of TREML4 ( P = 1 . 21x10-7 , Heterozygous OR = 4 . 087 ) , zinc finger-containing ZNF536 ( P = 8 . 69x10-7 , Recessive OR = 0 . 507 ) , C4orf17 ( P = 3 . 75x10-7 , Recessive OR = 0 . 289 ) , and near LINC00670 ( P = 2 . 15x10-7 , Additive OR = 3 . 867 ) . And finally , three intergenic regions display clusters of significance , most notably a region within chromosome 5 ( 43 , 892 , 232–43 , 964 , 366bp; Min P = 2 . 17x10-7 , Heterozygous OR = 0 . 354 ) , as well as regions within chromosome 7 and 11 . As expected , allele frequencies of the putative polymorphisms within the Trios dataset are generally equivalent to frequencies in our case and control groups , whilst there were some differences from the 1000 Genomes populations , including within the HBB locus ( Table 2 ) . Using the Trios dataset , we sought to identify structural variants that could confound the association analysis or be putative hits . We identified no structural variants within HBB , IL-12RBR2 or LINC00943/4 , one deletion ( 2 , 904bp ) within IL23R , and 152 deletions within KLHL3 ( 63 distinct variants , all singletons except for one 1 , 325bp deletion in 91 individuals ) ( S2 Table ) . None of the common variants are in linkage disequilibrium with the putative GWAS hits , and eight putative regions had structural variants in the Tanzanian trios , but were absent in the 1000 Genomes populations ( S2 Table ) . Subtype specific association analyses were undertaken for those SNPs found to be significantly associated with severe malaria in the primary GWAS ( Table 2 ) . The majority of significant associations are with the hyperlactataemia subtype , a phenotype that includes 57 . 0% of cases , with variants within FAM155A , and the HBB and KLHL3/MYOT regions exhibiting associations exceeding our 1x10-6 significance threshold . In contrast , variants within IL-23R , IL-12RB2 , CSMD1 , ZNF536 and TREML4 appear to be most significantly associated with severe malarial anaemia , who comprised 49 . 2% of cases . Candidate SNPs identified in previous studies , with the same individuals , were included to provide appropriate context for novel findings . ABO blood group , USP38 , FREM3 and alpha-thalassemia have previously been putatively associated with severe malaria in a Tanzanian population [3 , 5] , but these associations are no longer statistically significant ( P>10−4 ) at a more stringent GWAS significance threshold ( S3 Table ) . We also performed targeted imputation of HLA haplotypes within the MHC , finding the most significant SNP to be rs1264362 , which demonstrated a marginal association with hyperlactatemia ( additive model P = 2 . 33x10-5 ) . For the analysis of structural variation within candidate regions in the Trios dataset , we identified 28 distinct deletions within FREM3 , of which all but one are present in only one individual , and six distinct deletions in GYPB , for which copy number variation has previously been identified [6] . Nine distinct variants were identified in ABO , including six duplications , one deletion , one insertion and one inversion . All such ABO variants are present in single individuals , though 18 individuals have a 23bp insertion . In contrast to a diversity of structural variation present within HLA and the wider MHC region , minor frequency variants were identified in ATP2B4 ( 25 deletions across 25 samples ) , MARVELD3 ( five deletions across five samples ) , HBA2 ( 3 deletions across three samples ) , and HBA1 ( one sample with one deletion ) . No structural variants were found in HBB or USP38 ( S2 Table ) . We imputed structural variants within the wider region of human glycophorin genes ( gypA , gypB , gypE ) on chromosome four , using 55 distinct large polymorphisms identified in 59 individuals within our Trios dataset ( S2 Table ) . The glycophorin region is structurally highly diverse , and specific individual variants are of low frequency ( mean frequency: Case Control dataset = 0 . 098 , Trios dataset = 0 . 022 ) , consistent with observations in other African populations [7] . Whilst these large variants could be potentially protective against severe malaria , we identified no significant associations looking at each individually ( P ≥ 0 . 301 ) . Grouping these variants into forms based on genomic location and function may enhance signals within this region , but could also introduce experimenter bias . Further , there exists a multitude of potential variant combinations analysis of which , without specific hypotheses , could risk so-called ‘P hacking’ . A full and in-depth analysis of this region is required but beyond the scope of this study . Two approaches were applied to identify regions under recent positive selection within the Tanzanian GWAS population as a whole ( Integrated Haplotype Score , iHS ) [13] , or between the cases and controls ( Cross-Population Extended Haplotype Homozygosity , XP-EHH ) [14] . A common genome-wide absolute score threshold of 4 ( equivalent to P = 6 . 3x10-5 ) was established for both approaches . At this threshold iHS identified 244 loci , 116 ( 47 . 5% ) within chromosome 6 . Ninety-four of these significant signals are within the MHC , with three loci within HLA-DOA having an absolute score greater than 6 . Other MHC genes with significant signals include the immunophilin FKBP5 ( 35732-37931bp , 9 SNPs , iHS: 4 . 00–4 . 84 ) , SAMD3 ( 12490-13053bp , 6 SNPs , iHS: 4 . 00–4 . 68 ) and the exocytosis regulator RIMS1 ( 72805811-72828559bp , 3 SNPs , iHS: 4 . 17–4 . 62 ) ( S4 Table ) . Most notably , two regions within chromosome 17 ( 3496105-3689132bp , 6 SNPs , 8 genes including integrin ITGAE ) and chromosome 19 ( 38743962-38900106bp , 14 SNPs , 10 genes including two transmembrane channels ) represent regions with a high density of selection signals , akin to those within the MHC . Further signals of note include the transcription factor ZFHX3 ( ATBF1 ) ( chr . 16 , 16326-73133bp , 3 SNPs , iHS: 4 . 27–4 . 91 ) , ABHD5 ( chr . 3 , 43794949bp , 1 SNP , iHS: 4 . 75 ) , DUSP19 & NUP35 ( chr . 2 , 99180–18528 , 3 SNPs , iHS: 4 . 30–4 . 66 ) , surface tyrosine-kinase receptor ERBB4 ( chr . 2: iHS: 4 . 31–4 . 54 ) , transcription-associated RORC ( chr . 1 , 151792842-151817543bp , 2 SNPs , iHS: 4 . 31–4 . 66 ) . No structural variation was identified in ABHD5 or DUSP19 , whilst variants were present but rare for the remaining iHS hits ( S2 Table ) . In total , two deletions were identified in RORC , three in ZFHX3 , NUP35 , and ITGAE , one of which is an 86bp deletion found in seven individuals , and 14 deletions and a 31bp insertion in RIMS1 . Particularly variable are ERBB4 and FKBP5 for which we identify 49 and 75 distinct variants respectively . ERBB4 consists of 44 deletions , four insertions , and one inversion , whilst FKBP5 consists of 70 deletions , three duplications , one insertion , and one inversion . The between case-control XP-EHH approach identifies 10 significant SNPs across six genetic regions ( S5 Table ) . Relative selection for the control population lies within three regions , including POM121L12 ( XP-EHH: 4 . 33 ) , SYNJ2BP , ADAM21 , ADAM20 ( XP-EHH: 4 . 12 to 5 . 57 ) and ERG ( XP-EHH: 4 . 04 ) , whilst three regions are under relative selection in the case population , including MCUR1 ( XP-EHH: -4 . 26 ) , GCLC ( XP-EHH: -4 . 69 ) and the MHC ( XP-EHH: -4 . 02 ) ( S5 Table ) . We identify no structural variants within POM121L12 , ADAM21 , or ADAM20 , but a singleton 75bp deletion in SYNJ2BP , two deletions within MCUR1 , three deletions in GCLC , and 20 distinct deletions within ERG , of which 8 individuals share a 106bp deletion and 6 share a 325bp deletion ( S2 Table ) .
As expected , the most significant SNP association is the heterozygous protective rs334 effect ( P = 2 . 61x10-13 ) , with thirty-nine further SNPs within HBB also being significantly associated with resistance to severe disease . SNPs in other candidate genes , including FREM3 , GYPA , GYPB and USP38 [3] , did not exceed a significance threshold of 1x10-6 , and their p-values were different ( greater ) to those previously published because of the use of the more conservative EMMAX mixed model regression [12] . Marginal evidence for a role of HLA association with severe malaria was also identified , and is broadly consistent with previous work in a West African population that demonstrated that carriers of HLA Class I Bw53 and HLA class II DRB1*1302-DQB1*0501 were protected against severe malaria [15] . Note that our targeted imputation of HLA utilised a Caucasian reference panel and may therefore overlook further true associations within the HLA locus . Further , we identified signals of positive selection within the MHC region , this being consistent with malaria as a driver of MHC polymorphism in the human population [16 , 17] . Of the novel SNP associations identified here , two of the top candidates are located between the interleukin receptors IL-23R and IL-12RB2 , a region that has been identified in GWAS of other inflammatory and immune-linked diseases [18] . IL-12 and IL-23 are related pro-inflammatory cytokines that share both the p40 subunit and the IL-12Rβ1 receptor subunit . IL-12 signals through a receptor comprising IL-12Rβ1 and IL-12Rβ2 and is a potent inducer of IFN-γ which mediates both clearance of infection and immunopathology in infections with Plasmodium parasites . IL-23 signalling ( through its receptor , comprising IL-12Rβ1 and IL-23R ) promotes transcription of RORC which encodes RORγ , a transcription factor involved in generation of IL-17 . RORC was found to be under recent positive selection in our analysis , further supporting the importance of the pathway . Decreased IL-12 levels have been associated with progression from uncomplicated malaria to severe disease , specifically an increased risk of severe malarial anaemia in children [19 , 20] . Variants in IL-12B have been linked to P . falciparum parasite density and associated with protection against cerebral malaria in children whilst , variants in the related IL-12A and IL-12RB1 loci have been associated with protection against severe malarial anaemia among children in western Kenya [19] . Conversely , the IL-23/IL-17 immune pathway has been implicated in the development of inflammatory reactions in children that develop severe malarial anaemia [21] , in multi-organ dysfunction and acute renal failure in adult P . falciparum cases from India [22] and with the risk of cerebral malaria in Africa [23] . IL-23R haplotypes have also been associated with increased susceptibility to severe malarial anaemia in Kenya [24] . Three significantly associated SNPs are present within LINC00944 , with one being 80bp from a known CTCF binding site [25] . Structurally , although the LINC00943/4 region is a known deletion site [25] , we identified no such deletions within the region in our ‘Trios’ dataset . Broader functionality of this long intergenic non-coding RNA is unclear , given limited experimental characterisation , making it difficult to determine a role for these SNP variants . A strong association peak was also identified within KLHL3 , kelch-like protein 3 , being a region known to contain an enhancer and various deletions [25] . Correspondingly , we identify 152 such deletions within our Trios reference panel , of which 62 distinct variants are present in only one individual and one 1 , 325bp deletion is present in 91 individuals . This frequent deletion is located within an open chromatin-containing region between 137 , 022 , 562 and 137 , 023 , 887bp . Mutations of KLHL3 have previously been linked with hypertension and metabolic acidosis [26] suggesting that these novel SNP associations and deletions may prime individuals to have a greater risk for severe malarial acidosis ( hyperlactataemia ) . A number of the most significantly associated SNPs are present as lone , or paired , associations rather than “stacks” . This includes SNPs within or very near to TREML4 and ZNF536 . Whilst this may demonstrate false positive outliers , the existence of these SNPs and their minor frequencies are confirmed in our Trios reference panel . The broad picture of whole population iHS selection is unsurprising , with the MHC region demonstrating the most striking evidence for recent selective sweeps . Our results are also consistent with a number of previously identified iHS signals , such as those for loci containing the alcohol dehydrogenase ADH7 , cadherin PCDH15 , synaptotagmin SYT1 , the nociception receptor TRPV1 , and the transmembrane protein SPINT2 [27] . It should also be emphasised that our iHS signals reflect selection within our case-control dataset and therefore oversample , relative to a general Tanzanian population , for those signals associated with susceptibility to severe malaria . Recent differential selection between the case and control groups , as determined by XP-EHH , identified very few significant signals . There is likely to be limited differential selection between subsets of a closely related population , despite malaria infection being a strong selector . We identified the MHC , GCLC , MCUR1 , POM121L12 and the SYNJ2BP-ADAM21-ADAM20 region . The strongest of these signals covers ADAM20 and ADAM21 , both members of a larger family of disintegrins and metalloproteinases that are believed to be exclusively expressed in the testis [28]; this association might simply reflect differences in the gender ratio between the cases and controls , for which XP-EHH does not control . Selection for this region is more likely driven by a variant of SYNJ2BP , a Synaptojanin-2 binding protein with potential roles in membrane trafficking and signalling [29] . Our previous work has demonstrated that novel associations with potentially significant roles in malaria susceptibility remain to be uncovered [3] , and here we show that an integrated approach that identifies signals of association , selection and structural variation can empower such studies . However , with only 914 individuals in this study , sample size is a notable limitation for interpretation . Initial approaches to account for this were pursued through robust contextualisation of novel variants within the secondary ‘Trios’ dataset , and the wider 1000 Genomes project . More generally , it remains vital that further validation , through larger scale studies , be undertaken to better characterise the SNP and structural variants uncovered . This is particularly true for structural variation such as within KLHL3 , which may impact gene expression and would therefore benefit from incorporation of transcriptomic data . Distributions of human genetic variants with putative roles in P . falciparum malaria susceptibility are diverse . The HbS sickle cell polymorphism is present across most regions of sub-Saharan Africa but is known to have arisen multiple times leading to a number of distinct haplotypic backgrounds [30] . Similarly , other variants , such as G6PD polymorphism and glycophorin structural variants vary both in frequency across populations and in their direction of association , leading in some cases to allelic heterogeneity that may be subtype specific . Many protective variants identified within our study , such as IL-23R and KLHL3 , were found at similar frequencies within the ‘Trios’ dataset but differed from the global 1000 Genomes panel , and may therefore represent examples of Tanzanian- or regional-specific associations . Such variants are informative to our understanding of human-parasite interactions , yet risk being overlooked in inadequately designed studies . Ultimately , human GWAS in parallel with whole genome sequencing of host and parasites in large study populations across Africa will be crucial to unravelling host genetic and parasite interactions that could lead to novel malaria control measures such as vaccines .
All DNA samples were collected and genotyped following signed and informed written consent from a parent or guardian . Ethics approval for all procedures was obtained from both LSHTM ( #2087 ) and the Tanzanian National Institute of Medical Research ( NIMR/HQ/R . 8a/Vol . IX/392 ) . All participants were from the Tanga region of North-Eastern Tanzania , as described previously [3] . Briefly , severe malaria cases ( n = 449 ) were recruited in the Teule district hospital and surrounding villages in Muheza district , Tanga region , Tanzania between June 2006 and May 2007 . The controls ( n = 465 ) were recruited , matched on ward of residence , ethnicity and age ( given in months ) , during August 2008 from individuals without a recorded history of severe malaria [3] . Four severe malaria subtypes were identified within case individuals including hyperlactatemia ( Blood lactate > 5 mmol/L , n = 256 ) , severe malarial anaemia ( Hemocue Hb < 5g/dL , n = 221 ) , respiratory distress ( n = 124 ) and cerebral malaria ( Blantyre coma score <5 , n = 120 ) ( Table 1 ) . Parasite infection was initially assessed by rapid diagnostic test ( HRP-2 –Parascreen Pan/Pf ) and confirmed by double read Geimsa-stained thick blood films . A further 247 anonymously sampled individuals , consisting of 78 healthy parent and child trios ( 156 parents , 78 children , 13 singletons; 80 ( 32 . 4% ) Chagga , 77 ( 31 . 2% ) Pare , 90 ( 36 . 4% ) Wasambaa ) , were collected between 2007 and 2008 . These individuals are those that had no current illness or no history of malaria . The samples were collected from highland , medium and lowland villages near the Kilimanjaro , Pare and West Usambara mountains in the Tanga region of Tanzania . This is a region that experiences low to medium to high levels of malaria transmission . This dataset was used to confirm allele frequencies and identify candidate region structural variation within the general Tanzanian population , as well as to impute variants onto the case-control set . DNA was extracted from processed blood samples , as described previously [3 , 5] . The DNA was genotyped on the Illumina Omni 2 . 5 million SNP chip and SNP genotypes called by the MalariaGEN Resource Centre at the Sanger Institute and the Wellcome Trust Centre for Human Genetics , using previously described methods [6 , 7] . These data were complemented by Iplex genotyping assays that included 180 single nucleotide polymorphisms ( SNP ) across 50 loci on the same individuals [3] . 107 additional candidate SNPs , including the HbS SNP rs334 , were included from previous candidate genotyping of the same case-control individuals; their collection having been described previously [3] . DNA for the individuals in the Trio dataset ( n = 247 ) was sequenced using Illumina HiSeq2500 technology at the Sanger Institute , and aligned to the GRCh37 build of the human genome [7] . The minimum genome-wide coverage across the samples was 22-fold . SNPs were called from the alignments using the standard samtools-bcftools pipeline [31] . This process led to 2 , 788 , 671 high quality SNPs with quality scores of at least 30 ( 1 error per 1000bp ) and perfect trio-consistent genotype calls . Haplotypes were phased from genotypes using SHAPEIT ( www . shapeit . fr; default settings ) . Structural variants , including duplications , deletions , insertions and inversions , were identified within the secondary ‘Trios’ dataset for candidate regions using DELLY version v0 . 7 . 3 [32] . This software was applied using default settings , and its use in pipelines has been shown to reliably uncover structural variants from the 1000 Genomes Project , and validation experiments of randomly selected deletion loci show a high specificity [32] . Structural variants greater than 100 , 000 basepairs in length were removed to conservatively exclude false positives . To increase genome-wide SNP resolution , our initial case-control dataset was imputed using a combined reference panel of the Phase 3 1000 Genomes project [10] and children within the trio dataset , using Beagle 4 . 1 [11] . This allowed for the inclusion of 13 . 5 million additional high quality SNPs , to a total of 15 . 2 million SNPs . A total of 621 , 019 SNPs were removed from the pre-imputation dataset due to evidence of: ( i ) deviations in genotypic frequencies from Hardy-Weinberg equilibrium ( HWE ) as assessed using a chi-square test ( >0 . 0001 ) ; ( ii ) high genotype call missingness ( >10% ) ; or ( iii ) low minor allele frequency ( <0 . 01 ) . 51 individuals were removed due to: ( i ) genotypic missingness ( >0 . 1 ) ; ( ii ) abnormal PCA clustering or ( iii ) missing malaria phenotype data . 849 , 134 strand flips were identified with snpflip , with these being corrected pre-imputation with Plink v1 . 07 . Raw hybridisation plots were manually verified for all top non-imputed GWAS associations , excluding rs334 for which the data was unavailable . Linkage disequilibrium between SNPs in close genomic distance was calculated using Plink v1 . 07 [33] . Targeted imputation was performed for HLA haplotypes within the major histocompatibility complex using 9 , 785 high quality SNPs within the region; for this we utilised SNP2HLA software ( version 1 . 0 . 3 ) and the default Caucasian reference panel [34] . Association tests for this targeted analysis were performed through the pipeline described above . Similarly , 1 , 202 structural variants ( 698 deletions , 311 duplications , 19 insertions , 174 inversions ) within chromosome four were imputed into our primary ‘case-control’ dataset using IMPUTE2 with default parameters , akin to standard SNP imputation . This approach allowed us to perform association analysis on those structural variants using EMMAX mixed model regression [12] . Trio parental SNP data was also used to provide additional context for our case-control SNPs within the wider Tanzanian population , as seen in S1 Table . Case-Control association analysis of SNPs was undertaken with EMMAX mixed model regression [12] , controlling for age as a fixed effect and relatedness ( represented by a kinship matrix ) as a random effect ( to reduce associations relating to familial clustering ) . Several genotypic models were implemented separately , including additive , heterozygous , dominant and recessive . Minimum P values from each model were utilised for top hit identification . Odds ratios were estimated with Plink v1 . 07 [33] . Our complementary conditional GWAS shared the pipeline for the main GWAS , but with HbS status added as an additional covariate . To evaluate the statistical potential of our GWAS study , we performed a retrospective power calculation ( using http://zzz . bwh . harvard . edu/gpc/cc2 . html ) . A study of 460 cases and 460 controls can detect odds ratios of at least 2 for a high risk allele minor allele frequency of 5% with a statistical power of 85% ( and type I error of 10−6 ) . A significance threshold of 10−6 was established using a permutation approach [35] . In particular , both the case-control status of the chromosomes were randomly permuted 10 , 000 times . From each of the 10 , 000 random experiments , we determined the maximum chi-square statistics ( across the four genotypic tests ) over all SNPs genotyped . We ordered these statistics and then calculated the 95 percentile . This was the estimate of the 0 . 05 significance level for the experiment performed , assuming inference is taken with respect to maximum chi-square statistic observed over all genotyped SNPs , and accounts for the linkage disequilibrium between SNPs and correlation between the results from applying the 4 genotypic tests . Whole population Integrated Haplotype Scores ( iHS ) [13] and case-control Cross-Population Extended Haplotype Homozygosity ( XP-EHH ) [14] were calculated and normalised over the whole genome using selscan and norm [36] . Core SNPs with a minor allele frequency below 0 . 01 were excluded from this analysis . In this context , high iHS values indicate a whole population selection signal whilst positive XP-EHH values indicate relative selection within the control population and negative XP-EHH values indicate relative selection within the case population . We looked for structural variants in regions with SNP-based signals of positive selection , as it possible that selection may actually be driven by structural variants ( see [37] for an example ) . | Malaria , caused by Plasmodium falciparum parasites , is a major cause of mortality and morbidity in endemic countries of sub-Saharan Africa , including Tanzania . Some gene mutations in the human genome , including sickle cell trait , have been associated with reduced risk of developing severe malaria , and have increased in frequency through natural selection over generations . However , new genetic mutations remain to be discovered , and recent advances in human genome research technologies such as genome-wide association studies ( GWAS ) and fine-scale molecular genotyping tools , are facilitating their identification . Here , we present findings of a GWAS of severe malaria performed in a well characterised Tanzanian population ( n = 914 ) . We confirm the expected association with the sickle cell trait , but also identify new gene targets in immunological pathways , some under natural selection . Our approach demonstrates the potential of using GWAS to identify as-yet unknown susceptibility genes in endemic populations with well-characterised malaria phenotypes . The genetic mutations are likely to form potential targets for the design of much needed interventions for preventing or treating malarial disease . | [
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] | 2018 | Novel genetic polymorphisms associated with severe malaria and under selective pressure in North-eastern Tanzania |
Plant gas exchange is regulated by guard cells that form stomatal pores . Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2 for photosynthesis , loss of water , and entrance of air pollutants such as ozone . We mapped ozone hypersensitivity , more open stomata , and stomatal CO2-insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in MITOGEN-ACTIVATED PROTEIN ( MAP ) KINASE 12 ( MPK12 ) . In parallel , we showed that stomatal CO2-insensitivity phenotypes of a mutant cis ( CO2-insensitive ) were caused by a deletion of MPK12 . Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels . We demonstrated that MPK12 interacted with the protein kinase HIGH LEAF TEMPERATURE 1 ( HT1 ) —a central node in guard cell CO2 signaling—and that MPK12 functions as an inhibitor of HT1 . These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2 signaling controls plant water management .
Human activities have increased the concentrations of CO2 and harmful air pollutants such as ozone in the troposphere . During the last 200 y , the CO2 concentration has increased from 280 to 400 ppm , and it is predicted to double relative to the preindustrial level by 2050 [1] . Elevated CO2 is likely to have complex effects on plant productivity , since CO2 is not only a driver of climate change but also the main substrate for photosynthesis . Altered atmospheric chemistry is not limited to CO2; the concentration of tropospheric ozone has more than doubled within the past 100 y [2] . Ozone is a notorious air pollutant causing severe damage to crops; present day global yield reductions caused by ozone range from 8 . 5%–14% for soybean , 3 . 9%–15% for wheat , and 2 . 2%–5 . 5% for maize [3] . Both CO2 and ozone enter the plant through stomata—small pores on the surfaces of plants that are formed by pairs of guard cells . Guard cells also regulate plant water balance since plants with more open stomata allow faster water evaporation . Water availability is the most limiting factor for agricultural production , and insufficient water supply can cause large reductions in crop yields [4] . Thus , plants are constantly facing a dilemma; assimilation of CO2 requires stomatal opening but also opens the gates for entrance of harmful air pollutants and leads to excessive water loss . A consequence of increased atmospheric CO2 concentration can be higher biomass production [5] , but at the same time , plants adjust to elevated CO2 by partial closure of stomata [5 , 6] and expressing an altered developmental program that leads to reduced stomatal number [7] . CO2-induced stomatal closure reduces water loss; hence , it can directly modify plant water use efficiency ( WUE ) —carbon assimilated through photosynthesis versus water lost through stomata . Natural variation among Arabidopsis thaliana accessions provides a rich genetic resource for addressing plant function and adaptation to diverse environmental conditions . The Arabidopsis accession Cvi-0 from the Cape Verde Islands has impaired CO2 responses , more open stomata than Col-0 , and is extremely sensitive to ozone treatment [8 , 9] . A single amino acid change in Cvi-0 MITOGEN-ACTIVATED PROTEIN KINASE 12 ( MPK12 ) was recently shown to affect water use efficiency as well as stomatal size and to impair abscisic acid ( ABA ) -induced inhibition of stomatal opening [10] . MPK12 also regulates auxin signaling in roots [11] . However , the involvement of MPK12 in the CO2 signaling pathway in guard cells has not been addressed thus far . Among the important components of A . thaliana guard cell CO2 signaling are carbonic anhydrases ( βCA1 and βCA4 ) that catalyze the conversion of CO2 to bicarbonate and the protein kinase HIGH LEAF TEMPERATURE 1 ( HT1 ) that has been suggested to function as a negative regulator of CO2-induced stomatal movements [12 , 13] . Ultimately , for stomata to close , a signal from the bicarbonate has to activate protein kinases such as OPEN STOMATA 1 ( OST1 ) that in turn activate plasma membrane anion channels , including SLOW ANION CHANNEL 1 ( SLAC1 ) , followed by extrusion of ions and water that causes stomatal closure [14–17] . Isolation of a dominant HT1 allele , ht1-8D , revealed that HT1 may directly inhibit OST1- and GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1 ( GHR1 ) -induced activation of SLAC1 [18] . Bicarbonate-induced activation of SLAC1 has been reconstituted in Xenopus laevis oocytes [19 , 20] . The pathway was shown to consist of RESISTANT TO HIGH CARBON DIOXIDE 1 ( RHC1 ) , HT1 , OST1 , and SLAC1 [19] , while more recently the importance of CARBONIC ANHYDRASE 4 ( βCA4 ) , aquaporin PIP2;1 , OST1 , and SLAC1 was demonstrated [20] . Although guard cells are perhaps the best characterized single cell signaling system in the plant kingdom , there are still large gaps in our understanding of how CO2 signaling in guard cells is regulated and by which mechanism CO2 might regulate plant water management and WUE [5 , 21 , 22] . Here , we present the results of quantitative trait loci ( QTL ) mapping and sequencing of near-isogenic lines ( NILs ) of Cvi-0 ozone sensitivity . In a parallel approach , we mapped more open stomata and CO2-insensitivity phenotypes of a mutant cis ( CO2 insensitive ) . A single amino acid change ( G53R ) in MPK12 and complete deletion of MPK12 are the causes of more open stomata and altered CO2 responses of Cvi-0 and cis , respectively . Based on kinase activity assays , we conclude that MPK12 acts as an inhibitor of the HT1 kinase , which represents a crucial step in the regulation of plant stomatal CO2 responses .
Our initial QTL mapping of ozone sensitivity in Cvi-0 placed the two major contributing loci on the lower ends of chromosomes 2 and 3 [8] . To identify the causative loci related to the extreme ozone sensitivity and more open stomata of Cvi-0 , we created a NIL termed Col-S ( for Col-0 ozone sensitive ) through eight generations of backcrossing of Cvi-0 with Col-0 ( Fig 1A , S1A Fig , and S1 Video ) . In parallel , ozone tolerance from Col-0 was introgressed to Cvi-0 by six generations of backcrossing , which generated the ozone-tolerant Cvi-T ( S1 Video ) . Using these accessions , NILs , and recombinant inbred lines ( RILs ) , we mapped the causative ozone QTLs to a region of 90 kb on chromosome 2 and 17 . 70–18 . 18 Mbp on chromosome 3 ( S1B Fig ) . We have previously shown that the QTL on chromosome 2 also controls plant water loss and stomatal function [8] . We isolated both QTLs by backcrossing Col-S with Col-0 and obtained the NILs Col-S2 and Col-S3 . Both of these were less sensitive to ozone than Col-S ( S1A Fig ) , indicating that these QTLs act additively to regulate ozone sensitivity . Col-S2 ( but not Col-S3 ) showed much higher daytime stomatal conductance than Col-0 ( Fig 1B ) . The mapping resolution on chromosome 3 was not sufficient to identify the causative gene . Hence , we focused on Col-S2 and its role in stomatal function . Within the 90-kb mapping region on chromosome 2 , one gene , At2g46070 , encoding a MAP kinase MPK12 , shows strong preferential guard cell expression [23] . A single point mutation was found in Cvi-0 MPK12 , leading to a glycine to arginine substitution at position 53 of the protein . CAS ( calcium-sensing receptor ) is a chloroplast-localized protein important for proper stomatal responses to external Ca2+ [24 , 25] . While testing stomatal phenotypes in cas mutants , we observed phenotypic discrepancy between different alleles of cas . Whereas the cas-2 ( GABI-665G12 ) line had more open stomata and impaired CO2 responses , this was neither observed in cas-1 nor in cas-3 ( Fig 1C and S1C Fig ) . Further experiments showed that the T-DNA insert in the CAS gene was not linked to the CO2-insensitive phenotype of cas-2 . In a backcross with Col-0 , the T-DNA insert in cas-2 was removed , thereby generating the mutant cis ( CO2 insensitive ) . Both cis and Col-S2 had impaired responses to high CO2 ( 800 ppm ) , leading to longer half-response times , but a residual CO2 response could still be observed ( Fig 1D and S1E Fig ) . In order to identify the causative mutation in cis , mapping and whole genome sequencing of cis × C24 population was performed , which revealed a complete deletion of the MPK12 gene and its neighbor BYPASS2 in cis ( Fig 1E and S1D Fig ) . Thus , cis was renamed mpk12-4 . A second mutant ( gdsl3-1 ) from the GABI-Kat collection ( GABI-492D11 ) contained an identical deletion of BYPASS2 and MPK12 ( S2 Fig ) . We also identified a line with a T-DNA insert in exon 2 of MPK12 from the SAIL collection ( Fig 1E ) , which was recently named mpk12-3 [26] . No full-length transcript was found in mpk12-3 ( S3 Fig ) . SALK T-DNA insertion lines of MPK12 were previously described as lethal [11 , 23]; similarly , we were unable to retrieve homozygous plants of the same alleles , possibly indicating the presence of an additional T-DNA in an essential gene . The new mpk12 deletion , SAIL T-DNA insertion , and Col-S2 point mutation alleles allowed a detailed characterization of the role of MPK12 in stomatal regulation . Stomatal conductance was higher throughout the day in all three lines ( Col-S2 , mpk12-3 , and mpk12-4 ) ( Fig 2A ) , suggesting that the amino acid substitution in Cvi-0 MPK12 leads to loss of function . Furthermore , Col-0 transformed with MPK12 from Cvi-0 showed stomatal conductance similar to Col-0 , which excludes the option that the G53R substitution in MPK12 would lead to gain of function ( S1F Fig ) . Moreover , the wild-type ( Col-0 ) stomatal phenotype was observed in heterozygous F1 plants from a cross of Col-S2 and Col-gl1 , in which the gl1 mutation that gives a trichome-less phenotype was used as a noninvasive method for selecting successfully crossed plants in the first generation ( S1G Fig ) . Increased stomatal conductance may result from an increased number of stomata , larger stomata , or more open stomata . However , the stomatal index , length , and density did not differ between the lines , indicating that MPK12 regulates a function related to the stomatal aperture ( S4 Fig ) . Because of the higher degree of stomatal opening , the instantaneous WUE was lower in mpk12-3 , mpk12-4 , and Col-S2 ( Fig 2B ) . Altered WUE was previously also seen in mpk12-1 and a NIL with Cvi-0 MPK12 in Ler [10] . Cvi-0 and Col-S2 were complemented by expression of MPK12 from Col-0 ( Fig 2C and 2D ) . Similarly , mpk12-4 was complemented by expression of Col-0 MPK12 but not by Cvi-0 MPK12 ( Fig 2E ) . We conclude that MPK12 is a crucial regulator of stomatal conductance , and a single amino acid substitution ( G53R ) in Cvi-0 leads to loss of function of MPK12 . Reduction of CO2 levels inside the leaf [27] is a signal that indicates a shortage of substrate for photosynthesis and triggers stomatal opening . The rate of stomatal opening in response to low CO2 was severely impaired in mpk12 and Col-S2 ( Fig 3A and S5A Fig ) . Another signal for stomatal opening is light; this response was intact in plants with impaired or absence of MPK12 ( S5B and S5C Fig ) . The hormone ABA has dual roles in stomatal regulation; it induces stomatal closure but also inhibits light-induced stomatal opening . The latter response was impaired in mpk12 mutants and Col-S2 ( Fig 3B and S5C Fig ) . Stomata close in response to several signals , including darkness , reduced air humidity , ozone pulse , elevated CO2 , and ABA . Of these , only the response to elevated CO2 was impaired in mpk12 and Col-S2 ( Fig 3C and 3D , S5D–S5H and S6 Figs ) . CO2 signaling is impaired in the carbonic anhydrase double mutant ca1 ca4 [13] , and the product of carbonic anhydrase , bicarbonate , activates S-type anion currents [15] . In Col-S2 and mpk12-4 , bicarbonate-induced S-type anion currents were strongly impaired ( Fig 3E ) . Collectively , these data indicated that MPK12 has an important role in the regulation of CO2-induced stomatal movements in Arabidopsis . Only a few regulators of stomatal CO2 signaling in Arabidopsis have been identified . These include the protein kinases HT1 and OST1 [12 , 15 , 16] . To find the interaction partners of MPK12 , we conducted pairwise split-ubiquitin yeast two-hybrid ( Y2H ) assays against several kinases and phosphatases involved in stomatal signaling ( Fig 4A and 4B and S7A and S7B Fig ) . A strong interaction was observed between MPK12 and HT1 in yeast . The MPK12–HT1 interaction was also confirmed in Nicotiana benthamiana with bimolecular fluorescence complementation ( BiFC ) ( Fig 4C–4E ) and split luciferase complementation assays ( S7C Fig ) . Strong interaction between MPK12 and HT1 was observed in the cell periphery ( Fig 4C ) . Recently , HT1 was shown to be a plasma membrane–associated protein [28] . In contrast , Col-0 and Cvi-0 MPK12-YFP were located inside the cell ( S8A–S8D Fig ) . Hence , it is likely that the interaction with HT1 brings MPK12 to the plasma membrane . HT1 interacted with both the Col-0 and Cvi-0 versions of MPK12 , but the interaction with Cvi-0 MPK12 ( G53R ) was weaker both in quantitative BiFC and Y2H assays ( Fig 4B and 4D ) . MPK11 , an MPK from the same group as MPK12 [29] , did not interact with HT1 ( Fig 4C ) . INDOLE-3-BUTYRIC ACID RESPONSE 5 ( IBR5 ) is a MPK phosphatase that regulates auxin signaling in roots and has been shown to interact with and regulate the activity of MPK12 [11] . We confirmed the interaction between MPK12 and IBR5 ( S7A Fig ) . However , the ibr5-1 mutant exhibited wild-type stomatal phenotypes in response to CO2 changes ( Fig 3A and 3C , and S5A and S5E Fig ) , suggesting that IBR5 is not required in stomatal CO2 signaling . The function of MPK12 in ABA and CO2 signaling was further explored through genetic analysis . A strong loss-of-function allele , ht1-2 , that has low stomatal conductance [12] was used to evaluate the relationship between mpk12 and ht1-2 . The Col-S2 ht1-2 and mpk12-4 ht1-2 double mutants had a more closed stomata phenotype similar to ht1-2 ( Fig 4F ) , suggesting that HT1 is epistatic to MPK12 . The strong impairment of stomatal function in abi1-1 ( ABA insensitive1-1 ) was additive to Col-S2 in the double mutant Col-S2 abi1-1 ( Fig 4F ) . Hence , signaling through MPK12 seems to act—at least to some extent—independently of the core ABA signaling pathway . Taken together , the MPK12-HT1 interaction ( Fig 4A–4E ) and the epistasis between ht1-2 and mpk12-4 ( Fig 4F ) suggest that MPK12 functions upstream of HT1 and could regulate the activity of HT1 . To test this directly , we performed in vitro kinase assays with casein as the substrate for HT1 ( Fig 5A ) . HT1 displayed strong autophosphorylation and phosphorylated casein efficiently . Addition of the Col-0 version of MPK12 and a hyperactive version ( MPK12 Y122C ) efficiently inhibited HT1 activity ( Fig 5A and quantified in Fig 5B ) . A point-mutated version ( MPK12 K70R ) designed to remove the kinase activity of MPK12 also inhibited the autophosphorylation activity of HT1 and phosphorylation of casein by HT1 , although it was less efficient than the wild-type ( Fig 5B ) . Importantly , the Cvi-0 version of MPK12 ( G53R ) displayed strongly suppressed inhibition of HT1 activity ( Fig 5A and 5B ) . MPK12 did not phosphorylate the kinase-dead version of HT1 ( K113M ) ( Fig 5C ) . The kinase-dead version of HT1 ( K113M ) was used as a substrate , since the strong autophosphorylation activity of HT1 would otherwise have obscured the result . Wild-type MPK12 and hyperactive MPK12 ( Y122C ) displayed autophosphorylation , whereas MPK12 ( G53R ) as well as MPK12 ( K70R ) had lost their autophosphorylation activity , indicating that the G53R substitution in Cvi-0 MPK12 disrupts the kinase activity of the protein ( Fig 5C ) . The inhibition of HT1 by MPK12 was specific , as MPK11 , which belongs to the same group as MPK12 , was not able to affect HT1 kinase activity ( S9 Fig ) . We conclude that the stomatal phenotypes of mpk12 mutants and Cvi-0 can be explained by a lack of inhibition of HT1 activity by MPK12 , which leads to more open stomata and impaired CO2 responses ( Figs 2A , 3 and 5A and 5C ) . MPK12 belongs to the same group of MPKs as MPK4 , a crucial regulator of pathogen and stress responses [29] . In tobacco , the silencing of MPK4 impaired CO2-induced stomatal closure [30] . Since Arabidopsis MPK4 and MPK12 are highly similar [31] , it is possible that both MPK4 and MPK12 could regulate stomatal CO2 responses . Indeed , in an Y2H screen to identify HT1 interacting proteins , one prominent interactor was MPK4 [18] . The Arabidopsis mpk4 mutant is severely dwarfed , and measurements of accurate stomatal conductance with these plants are not feasible [32] . However , the impaired stomatal response to CO2 in mpk12-4 ( Fig 3 ) was further enhanced by guard cell–specific silencing of MPK4 [18]; hence , in guard cells MPK4 is acting redundantly with MPK12 in stomatal CO2 signaling . Furthermore , MPK4 could also inhibit HT1 kinase activity [18] . The G53 residue in MPK12 is conserved in all Arabidopsis MPKs [10] . Since the G53R mutation blocked MPK12 function , we tested whether a similar mutation would impair MPK4 function . This experiment showed that MPK4-induced inhibition of HT1 activity was blocked by the introduction of a G55R mutation in MPK4; this mutation corresponds to G53R in Cvi-0 MPK12 ( Fig 6A ) . Since MPK11 did not inhibit HT1 activity ( S9 Fig ) , the function of MPKs as kinase inhibitors in Arabidopsis may be restricted to MPK12 and its closest homologue MPK4 . The Arabidopsis MPK6 crystal structure [33] was used to model the structure of MPK4 and MPK12 and to address the role of the G55R and G53R mutations that were shown to be crucial for the function of these proteins ( Fig 6B and 6C ) . The mutation of Gly to Arg in position 53 in MPK12 caused the protrusion of the arginine sidechain on the surface of the protein , which could affect its binding affinity for other proteins in addition to an altered structure of the loop region . Similarly , the Arg in position 55 of MPK4 protruded from the surface as compared to the wild-type . Thus , the MPK12 G53R and MPK4 G55R amino acid substitutions may alter protein binding affinities of these MPKs to other proteins . Collectively , the presented experiments suggest that the CO2 signal leading to stomatal movements is transmitted through MPK12 and MPK4 , leading to inhibition of HT1 , and this enables SLAC1 activation by its activators , including OST1 and GHR1 . Neither MPK12 G53R from Cvi-0 nor MPK4 G55R can fully inhibit HT1 ( Fig 7 ) .
Natural variation within a species holds great potential to identify regulatory mechanisms that are not easily uncovered through mutant screens . The Cvi-0 accession originates from the southern border of the Arabidopsis distribution area , the Cape Verde Islands . The Ler × Cvi RIL population was one of the first RILs produced , and it has been phenotyped for multiple traits [34] . Despite this , only a few QTLs from Cvi-0 have been identified at the molecular level . Our earlier research identified a locus related to ozone sensitivity and more open stomata phenotype of Cvi-0 in chromosome 2 [8] . Recently , the G53R substitution in MPK12 that affects plant water use efficiency was identified by using the Ler × Cvi populations , but the biochemical function of MPK12 in stomatal regulation was not further investigated [10] . Here , we generated NILs by backcrossing Cvi-0 eight times to Col-0 and show that the same natural mutation in Cvi-0 and lack of MPK12 in cis are the causes of ozone sensitivity , more open stomata , and altered CO2 responses of Arabidopsis plants . Furthermore , we showed that MPK12 regulates the activity of the protein kinase HT1 , a major component of the CO2 signaling pathway in guard cells . The regulators of HT1 have remained largely unknown , despite the exceptionally strong CO2-insensitivity phenotype of plants with impaired HT1 function [12 , 15] . Our findings provide the first evidence for the role of MPK12 in guard cell CO2 signaling and provide a mechanistic insight for the MPK12 function in the regulation of plant water management . The role of MPKs in Arabidopsis guard cell signaling has concentrated on MPK9 and MPK12 , which are preferentially expressed in guard cells . Plants with point mutations in MPK9 ( mpk9-1 , L295F ) and MPK12 ( mpk12-1 , T220I ) had wild-type ABA responses , but mpk12-1 has decreased WUE [10] . The mpk9-1 , mpk12-1 , and mpk12-2 alleles are Tilling ( Targeting Induced Local Lesions IN Genomes ) lines in the Col-erecta background and the previously characterized MPK12-Cvi NIL is in the Ler background [10 , 23] . Mutations in ERECTA modify transpiration efficiency and stomatal density , which may have influenced some of the previously described mpk12-1 phenotypes [10 , 35] . In contrast , the full knockout alleles described here , mpk12-3 and mpk12-4 , are in Col-0 and imply a major function for MPK12 in CO2 signaling . Additional roles for MPK12 in stomatal responses have been inferred through the use of the double mutant mpk9-1 mpk12-1 that has impaired stomatal closure responses to ABA and H2O2 treatment and has impaired S-type anion channel activation in response to ABA and Ca2+ [23] . It is also highly susceptible to Pseudomonas syringae infection and impaired in yeast elicitor- , chitosan- , and methyl jasmonate–induced stomatal closure [36] . Since the mpk9 mpk12 double mutant appears to be more severely impaired in abiotic and biotic stomatal responses and S-type anion channel activation than the loss of function MPK12 alleles ( Fig 3 ) , it is possible that MPK12 together with MPK9 regulates stomatal aperture in response to various signals . MPK12 also regulates auxin responses in the root [11 , 26] . However , beyond the observation that plants with impaired MPK12 are hypersensitive to auxin inhibition of root growth , no details about the targets of MPK12 in roots are known . HT1 was the first component shown to be specifically associated with stomatal CO2 signaling , and the ht1-2 mutant has more closed stomata displaying constitutive high CO2 response at ambient CO2 levels ( Fig 4D [12] ) . The opposite phenotypes of mpk12 and ht1-2 allowed us to use genetic analysis to position MPK12 in the guard cell signaling network . The stomata of mpk12 ht1-2 were more closed , thus positioning MPK12 upstream of HT1 and possibly as a direct regulator of HT1 ( Fig 4D ) . CO2 signaling in guard cells is initiated through the production of bicarbonate by carbonic anhydrases , and bicarbonate initiates signaling leading to activation of S-type anion channels [13 , 15] . In mpk12 , the bicarbonate-dependent activation of S-type anion channels was impaired , as was previously found for the plants with impaired OST1 and SLAC1 ( Fig 3E ) [15] . The combined evidence from mpk12 phenotypes , genetic analysis , and measurements of S-type anion currents all pointed towards MPK12 as a crucial regulator of CO2 signaling acting through HT1 . Indeed , HT1 kinase activity was inhibited in the presence of Col-0 MPK12 but not by the Cvi-0 version of MPK12 ( Fig 5A ) . Thus , the inhibitory function of MPK12 was impaired by the G53R amino acid substitution , probably by its weaker interaction with HT1 ( Figs 4 and 5A ) . This explains the similar phenotypes of the NIL Col-S2 , mpk12-3 , and mpk12-4; they all display lack of inhibition of the negative regulator HT1 , leading to higher stomatal conductance . Further support for the regulatory interplay between HT1 and MPK12 is provided by the isolation of a dominant mutation in HT1 , ht1-8D , which in contrast to ht1-2 has constitutively more open stomata and is biochemically resistant to inhibition by MPK12 [18] . Cvi-0 has altered phenotypes in many traits , including drought and pathogen resistance [34 , 37 , 38] . All of these traits are regulated through stomatal function; thus , the MPK12-HT1 regulatory module identified here may influence many of the previously observed phenotypes of Cvi-0 . Recently , two independent studies used X . laevis oocytes as a heterologous expression system to reconstitute bicarbonate-induced activation of the SLAC1 anion channel [19 , 20] . Tian et al . [19] reported that a multidrug and toxic compound extrusion ( MATE ) -type transporter RHC1 functions as a bicarbonate-sensing component that inactivates HT1 and promotes SLAC1 activation by OST1 . More recently , it was demonstrated that expression of RHC1 alone was sufficient to activate ion currents in oocytes; these currents were independent of bicarbonate , calling into question the role of RHC1 as a bicarbonate sensor [20] . Furthermore , it was shown that SLAC1 activation can be reconstituted by extracellular bicarbonate in the presence of aquaporin PIP2;1 , carbonic anhydrase CA4 , and the protein kinases OST1 , CPK6 , and CPK23 [20] . However , in the guard cell , any proposed CO2 signaling pathway should include HT1 , since plants with mutations in HT1 completely lack CO2-induced stomatal responses [12 , 18 , 28] . We showed that bicarbonate-induced S-type anion currents were strongly impaired in guard cell protoplasts , which lacked functional MPK12 ( Fig 3E ) . Thus , MPK12 , MPK4 , and possibly other MPKs that are expressed in guard cells play a role in controlling the activity of HT1 , and future research should identify the signaling pathway upstream of MPK12 ( Fig 7 ) . Dissection of different domains in SLAC1 revealed that the CO2 signal may involve the transmembrane region of SLAC1 , whereas ABA activation of SLAC1 requires an intact N- and C-terminus [39] . Hence , ABA and CO2 regulation of SLAC1 could use different signaling pathways , and this may explain the lack of strong ABA phenotypes in plants with mutations in MPK12 . We propose that stomatal movements triggered by changes in CO2 concentration are regulated by MPK12- and MPK4-induced inhibition of HT1 activity ( Fig 7 ) . The MPK12 glycine 53 is conserved in all Arabidopsis MPKs [10] and is located on the protein surface in the glycine-rich loop that coordinates the gamma-phosphate of ATP ( Fig 6B and 6C ) . Thus , this glycine may also be important for the function of other Arabidopsis MPKs . Further studies into the mechanisms controlling activation of MPKs in guard cells will help to identify molecular switches that function in plant acclimation to environmental stress and modulate the overall plant water use efficiency . Such information may allow the designing of molecular targets that can be used for breeding crops with improved water management .
Col-0 , Col-gl , Cvi-0 , gdsl3-1 ( GABI-492D11; CS447183 ) , cas-1 ( SALK_070416 ) , cas-2 ( GABI-665G12 ) , and cas-3 ( SAIL_1157_C10 ) were from the European Arabidopsis Stock Centre ( www . arabidopsis . info ) . Seeds of ht1-2 were a gift from Dr . Koh Iba . Col-0 × Cvi-0 RILs were obtained from INRA Versailles . The abi1-1 allele used was in Col-0 accession . Double mutants and other crosses were made through standard techniques and genotyped with PCR-based markers ( S1 Table ) . For ozone screening , seeds were sown at high density on a 1:1 v/v mixture of vermiculite and peat ( type B2 , Kekkilä , Finland ) , and kept for 2 d at 4°C for stratification . The plants were grown in controlled growth chambers ( Bio 1300 , Weiss Umwelttechnik , Germany ) under a 12 h photoperiod , with a 23°C/19°C day/night temperature and a 70%/90% relative humidity or in growth rooms with equivalent growth conditions . The average photosynthetic photon flux density during the light period was 200 μmol m-2 s-1 . When seedlings were 1 wk old , they were transplanted into 8 × 8 cm pots at a density of five plants per pot . Three-week-old plants were exposed to ozone in growth chambers under the same conditions as they were grown until the experiments . Ozone exposure was acute ( 300–350 ppb for 6 h ) and started 2 h after light was switched on . Ozone damage was visualized with trypan blue stain or quantified as electrolyte leakage . NILs were created by crossing Col-0 with Cvi-0 and selecting the most ozone-sensitive plant in F2 and backcrossing to Col-0 for eight generations ( generating Col-S ) or selecting the most tolerant plant and backcrossing to Cvi-0 for six generations ( generating Cvi-T ) . The genomes of Cvi-0 and Cvi-T were sequenced at BGI Tech Solutions ( Hong Kong ) with Illumina technology , and the genomes of Col-S and Cvi-T were sequenced at the DNA Sequencing and Genomics lab of the University of Helsinki with SOLiD technology . Genome sequence data is available from the NCBI BioProject database with the accession number PRJNA345097 . The 90-bp-long Illumina paired end sequencing library reads were mapped onto the Col-0 reference genome ( TAIR10 ) with using the Bowtie2 aligner ( version 2 . 0 . 0-beta7; [40] ) in “end-to-end” alignment mode , yielding an average genomic sequence coverage of 45-fold . Variation calling and haplotype phasing was performed with the help of samtools ( tools for alignments in the SAM format , Version: 0 . 1 . 18; [41] ) . Based on the aligned sequences , various PCR-based markers ( S1 Table ) were designed to genotype Cvi-0 versus Col-0 in the NILs and informative RILs from the INRA Versailles Col-0 × Cvi-0 RIL population . The markers were also used to genotype ozone-sensitive individuals from segregating F2 populations . Mapping population was created by crossing cis ( Col-0 ) and C24 as an Arabidopsis accession with low stomatal conductance . High water loss from excised leaves and decreased responses to high CO2 were used as a selective trait . Rough mapping with 22 markers using 59 F2 samples showed linkage to the bottom of chromosome 2 , at the marker UPSC_2–18415 at 18 . 4 Mbp . Pooled genomic DNA from 66 selected F3 plants was used for sequencing . Whole genome sequencing was conducted with Illumina HiSeq 2000 , and the reads were mapped against Col-0 genome ( release TAIR10 ) by BGI Tech Solutions ( Hong Kong ) . Genome sequence data is available from the NCBI BioProject database with the accession number PRJNA345097 and PRJNA343292 . For mapping the genomic area of the mutation , the Next Generation Mapping tool was used [42] , which positioned the mutation on chromosome 2 between 18 , 703 , 644 –19 , 136 , 098 bp . The deletion mutation in cis was verified by PCR to be 4 , 770 bp ( at the position 18 , 945 , 427–18 , 950 , 196 bp ) . MPK12 and its promoter were amplified from Col-0 or Cvi-0 genomic DNA using Phusion ( Thermo Fisher Scientific ) and Gateway ( Invitrogen ) cloned into entry vector pDONR-Zeo . Subsequently , the genes were cloned into pGWB13 and pMCD100 . Plants were transformed with floral dipping [43] . Total DNAs from different genotyping plants were extracted by CTAB method , and 12 micrograms of total DNA was digested by HindIII or EcoRI . The DNAs were running on the gel and transformed onto Nylon membrane . Hybridization was performed with digoxigenin-labeled specific genomic DNA amplified by primers F3 and R4 for 12 h . The membrane was washed several times by washing buffer and Maleic acid buffer . The membrane was blocked by blocking solution for 1 h at room temperature and washed and incubated with anti-DIG-AP for 30 min . Detection was performed using substrate DIG CSPD . Seeds were planted on a soil mixture consisting of 2:1 ( v:v ) peat:vermiculite and grown through a hole in a glass plate covering the pot as described previously [44] . Plants were grown in growth chambers ( MCA1600 , Snijders Scientific , Drogenbos , Belgium ) at 12 h/12 h day/night cycle , 23°C/20°C temperature , 100 μmol m-2 s-1 light , and 70% relative humidity ( RH ) . For gas exchange experiments , 24- to 30-d-old plants were used . Stomatal conductance of intact plants was measured using a rapid-response gas exchange measurement device consisting of eight through-flow whole-rosette cuvettes [44] . The unit of stomatal conductance mmol m-2 s-1 reflects the amount of H2O moles that exits the plant through stomata per one m2 of leaf area per second . Prior to the experiment , plants were acclimated in the measurement cuvettes in ambient CO2 concentration ( ~400 ppm ) , 100 μmol m-2 s-1 light ( if not stated otherwise ) , and ambient humidity ( RH 65%–80% ) for at least 1 h or until stomatal conductance was stable . Thereafter , the following stimuli were applied: decrease or increase in CO2 concentration , darkness , reduced air humidity , and ozone . CO2 concentration was decreased to 100 ppm by filtering air through a column of granular potassium hydroxide . In CO2 enrichment experiments , CO2 was increased by adding it to the air inlet to achieve a concentration of 800 ppm . Darkness was applied by covering the measurement cuvettes . In blue light experiments , dark-adapted plants were exposed to blue light ( 50 μmol m-2 s-1 ) from an LED light source ( B42180 , Seoul Semiconductor , Ansan , South Korea ) . The decreased or increased CO2 concentration , darkness , and blue light were applied for 58 min . In the long-term elevated CO2 experiment ( Fig 1D and S1E Fig ) , CO2 concentration was increased from 400 ppm to 800 ppm for 2 . 5 h . To calculate stomatal half-response time , the whole 2 . 5-h stomatal response to elevated CO2 was scaled to a range from 0% to 100% , and the time when 50% of stomatal closure had occurred was calculated . Humidity was decreased by a thermostat system to 30%–40% RH , and stomatal conductance was monitored for another 56 min . In ozone experiments , the plants were exposed to 350–450 ppb of ozone for 3 min and stomatal conductance was measured for 60 min after the start of the exposure . In ABA-induced stomatal closure experiments , 5 μM ABA solution was applied by spraying as described in [45] . At time point 0 , plants were removed from cuvettes and sprayed with either 5 μM ABA solution ( 5 μM ABA , 0 . 012% Silwet L-77 [PhytoTechnology Laboratories] , and 0 . 05% ethanol ) or control solution ( 0 . 012% Silwet L-77 and 0 . 05% ethanol ) . Thereafter , plants were returned to the cuvettes and stomatal conductance was monitored for 56 min . In ABA-induced inhibition of stomatal opening experiments , plants were acclimated in measurement cuvettes in darkness . At time point 0 , plants were removed from cuvettes and sprayed with 2 . 5 μM ABA solution ( 2 . 5 μM ABA , 0 . 012% Silwet L-77 [PhytoTechnology Laboratories] , and 0 . 05% ethanol ) or control solution ( 0 . 012% Silwet L-77 and 0 . 05% ethanol ) . Thereafter , plants were returned to the cuvettes , dark covers were removed , and stomatal conductance was monitored in light for 56 min . Prior to the measurement of the diurnal pattern of stomatal conductance , plants were preincubated in the measurement cuvette for at least 12 h in respective light and humidity conditions . Plants were measured in 16-min intervals . WUE was calculated based on the data of diurnal experiments as an average of daytime light period ( from 9:00 to 17:00 ) . CO2-induced stomatal conductance in S2 Fig was measured as following . Five-week-old healthy plants growing in a growth chamber with 70% humidity and a 16 h light/8 h dark condition were used for stomatal conductance analyses at different CO2 concentrations by a LiCOR-6400XT , as previously described [13] . Relative stomatal conductance values were normalized relative to the last data point preceding the [CO2] transitions ( 400 to 800 or 1 , 000 ppm ) . The MPK12 deletion mutant mpk12-4 and wild-type plants were grown in a growth chamber at 70% humidity , 75 μmolm-2 s-1 light intensity , 21°C , and 16 h light/8 h dark regime . Leaf epidermal layers from 2-wk-old plants of both genotypes were preincubated in an opening buffer ( 10 mM MES , 10 mM KCl , and 50 mM CaCl2 at pH 6 . 15 ) for 2 h , and stomata were individually imaged and tracked for measurement before treatment . After that , the leaf epidermal layers were incubated with buffers containing 10 μM ABA for 30 min and the individually tracked stomata were imaged . Stomatal apertures were measured by ImageJ software and genotype-blind analyses were used . The data presented are means and SEM n = 3 experiments , with 30 stomata per experiment and condition . Plants at the age of 28–30 d were used for stomatal index and density measurements . Rosette leaves of equal size were excised , and the abaxial side was covered with the dental resin ( Xantopren M mucosa , Heraeus Kulzer , Germany ) . Transparent nail varnish was applied onto the dried impressions after the removal of the leaves . The hardened nail varnish imprints were attached onto a microscope glass slide with a transparent tape and imaged under a Zeiss SteREO Discovery . V20 stereomicroscope . For quantification , an image with the coverage of 0 . 12 mm2 was taken from the middle of the leaf , next to the middle vein . In total , 81–84 plants per line from two independent biological repeats were analyzed—one leaf from each plant , one image from each leaf . Stomatal index was calculated with the following formula: SI = Stomatal density / ( Density of other epidermal cells + Stomatal density ) . For the stomatal complex length measurements , plants at the age of 28–35 d were used . Whole leaves were preincubated for 4 h abaxial side down in the buffer ( 10 mM MES , 5 mM KCl , 50 μM CaCl2 , pH 6 . 15 [with TRIS] ) in the light . Four to six plants per genotype and one leaf per plant were analyzed , and altogether 84–126 stomatal complexes per genotype were measured . Interactions between MPK12 and selected protein kinases and phosphatases were tested in pairwise split-ubiquitin Y2H assays using the DUALhunter and DUALmembrane 3 kits ( Dualsystems Biotech ) . For bait construction , the coding sequences of MPK12 were PCR-amplified from total cDNAs from Col-0 and Cvi-0 . Other MPK12 variants with point mutations ( K70R , Y122C , and D196G+E200A ) were created by two-step overlap PCR using the Col-0 MPK12 as a template . HT1 was also PCR-amplified from Col-0 cDNA . All MPK12s and HT1 were digested with SfiI and cloned to the corresponding site in pDHB1 , which contained the Cub-LexA-VP16 fusion . For prey constructs , coding sequences of each selected gene were amplified from total Col-0 cDNAs , digested with SfiI , and cloned into either pPR3-N ( HT1 , OST1 , BLUS1 , IBR5 , MKP2 , MPK12 , MPK12G53R , MPK11 ) or pPR3-STE ( SnRK2 . 2 , SnRK3 . 11 , ABI1 , ABI2 , HAB1 , HAB2 ) , which contained a mutated NubG . All primers used are listed in Table S1 . The pAI-Alg5 with a native NubI was used as a positive prey control , whereas the pDL2-Alg5 containing NubG served as a negative control . For pairwise Y2H assays , the yeast strain NMY51 was cotransformed with bait and prey plasmids and grown on SD-Leu-Trp plates to select for presence of both plasmids . At least ten colonies from each transformation were pooled and resuspended in water to an OD600 of 0 . 5 , from which 100 , 1 , 000 , and 10 , 000x serial dilutions were prepared and spotted on SD-Leu-Trp and SD-Leu-Trp-His-Ade plates . SD-Leu-Trp plates were incubated at 30°C for 2 d , photographed , and used for β-galatosidase overlay assays . SD-Leu-Trp-His-Ade plates were incubated for 2–4 d and photographed . The quantitative β-galactosidase assay was performed with three pools of ten independent colonies from each pairwise combination using the Yeast β-galactosidase assay kit ( Thermo Fisher Scientific ) by the nonstop quantitative method . Binary constructs containing split YFPs were designed and generated for cloning genes of interest by the ligation independent cloning ( LIC ) method as described in [18] . Each gene of interest was amplified by two consecutive PCR reactions: first with gene-specific primers and later with a pair of universal primers designed specifically for the LIC method . All primers used are listed in S1 Table . To prepare vectors for LIC , plasmids of 35S:YFPn and 35S:YFPc were linearized by PmlI digestion , followed by T4 DNA polymerase treatment with dGTP to create 15–16 nucleotide 5ʹ-overhangs . For insert preparation , the final PCR products of target genes were incubated with T4 DNA polymerase in the presence of dCTP to create the complementary overhangs with the vectors . Both vector and insert were mixed at room temperature and proceeded with Escherichia coli transformation after 5 min . The final constructs were sequence verified and transformed to Agrobacterium tumefaciens GV3101 for agro-infiltration experiments . For the ratiometric BiFC assays , four different agrobacterial clones—each harboring a YFPn fusion , a YFPc fusion , the SLAC1-CFP internal control , or the gene silencing suppressor P19—were co-infiltrated to the leaves of N . benthamiana at an OD600 of 0 . 02 for each clone in the infiltration buffer ( 10mM MES , 10mM MgCl2 , 200 μM acetosyringone ) . Images were acquired at 3 dpi with a Zeiss LSM710 confocal microscope using a 63x objective ( for high magnification images ) or a 20x objective ( for fluorescence quantification ) . The YFP signals were excited by a 514 nm laser , and emission between 518–564 nm was collected . The CFP signals were excited by a 405 nm laser , and emission at 460–530 was collected . Z-stack images of approximately 15 μm thickness were collected , and all images were acquired at the 16-bit depth for a higher dynamic range . The fluorescence intensity was measured by the ImageJ software . The leaf samples used for imaging were collected and used for protein extraction followed by western blot analysis . The leaf samples ( 30–40 mg ) were ground under liquid nitrogen and boiled for 10 min in 100 μL of 6X Laemmli buffer . 12 μL of each sample were separated on 10% SDS polyacrylamide gel . After SDS-PAGE , proteins were transferred onto nitrocellulose membrane . Immunodetection of HA-tagged proteins was performed with a monoclonal anti-HA antibody . The MPK12 cDNA was cloned into a vector containing the N-terminal half of luciferase ( nLUC ) and HT1 was cloned into the cLUC . The constructs in the A . tumefaciens strain GV3101 were co-infiltrated into N . benthamiana leaves with P19 at an OD600 of 0 . 8 . The infiltrated leaves after 3 d of infiltration were harvested for bioluminescence detection . Images were captured with a CCD camera . Arabidopsis guard cell protoplasts were isolated as described previously [46] . Guard cell protoplasts were washed twice with a washing solution containing 1 mM MgCl2 , 1 mM CaCl2 , 5 mM MES , and 500 mM D-sorbitol ( pH 5 . 6 with Tris ) . During patch clamp recordings of S-type anion currents , the membrane voltage started at +35 to –145 mV for 7 s with –30 mV decrements , and the holding potential was +30 mV . The bath solutions contained 30 mM CsCl , 2 mM MgCl2 , 10 mM MES ( Tris , pH 5 . 6 ) , and 1 mM CaCl2 , with an osmolality of 485 mmol/kg . The pipette solutions contained 5 . 86 mM CaCl2 , 6 . 7 mM EGTA , 2 mM MgCl2 , 10 mM Hepes-Tris ( pH 7 . 1 ) , and 150 mM CsCl , with an osmolality of 500 mmol/kg . The free calcium concentration was 2 μM . The final osmolalities in both bath and pipette solutions were adjusted with D-sorbitol . Mg-ATP ( 5 mM ) was added to the pipette solution before use . 13 . 5 mM CsHCO3 ( 11 . 5 mM free [HCO3-] and 2 mM free [CO2] ) was freshly dissolved in the pipette solution before patch clamp experiments . The concentrations of free bicarbonate and free CO2 were calculated using the Henderson–Hasselbalch equation ( pH = pK1 + log [HCO3-] / [CO2] ) . pK1 = 6 . 352 was used for the calculation . [HCO3-] represents the free bicarbonate concentration and [CO2] represents the free CO2 concentration . For in vitro kinase assays , the respective sequences of HT1 , HT1 K113M , MPK11 , MPK12 , MPK12 G53R , MPK12 K70R , and MPK12 Y122C were cloned into a pET28a vector ( Novagen , Merck Millipore ) using primers listed in S1 Table . Point mutations corresponding to K113M in HT1 , K70R in MPK12 , and Y122C in MPK12 were created with two-step PCR using primers listed in S1 Table . MPK4 was cloned as previously described [18] . 6xHis-HT1WT , 6xHis-HT1 K113M , 6xHis-MPK12 , 6xHis-MPK12 G53R , 6xHis-MPK12 K70R , 6xHis-MPK12 Y122C , 6xHis-MPK11 , 6xHis-MPK4 WT , and 6xHis-MPK4 G55R were expressed in E . coli BL21 ( DE3 ) cells . A 2 mL aliquot of an overnight culture was transferred to a fresh 1 L 2xYT medium and grown at 37°C to an absorbance of ~0 . 6 at OD600 . The cultures were chilled to 16°C and recombinant protein expression was induced by 0 . 3 mM isopropyl b-D-thiogalactopyranoside for 16 h . The cells were harvested by centrifugation ( 5 , 000 rpm , 10 min , 4°C ) and stored at –80°C until use . All purification procedures were carried out at 4°C . The cells were resuspended in 30 mL of lysis buffer ( 50 mM Tris-HCl [pH 7 . 4] , 300 mM NaCl , 5% [v/v] glycerol , 1% [v/v] Triton X-100 , 1 mM PMSF , 1 μg/ml aprotinin , 1 μg/ml pepstatin A , 1 μg/ml leupeptin ) and lysed using an Emulsiflex C3 Homogenizer . Cell debris was removed by centrifugation at 20 , 000 rpm for 30 min . The protein-containing supernatant was mixed for 1 h at 4°C with 0 . 20 mL of Chelating Sepharose Fast Flow resin ( GE Healthcare ) , charged with 200 mM NiSO4 and pre-equilibrated in the lysis buffer . The protein–resin complex was packed into a column , and the beads were washed with 5x10 column volumes ( CV ) of a wash buffer I ( 50 mM Tris-HCl [pH 7 . 4] , 600 mM NaCl , 5% [v/v] glycerol , 1% [v/v] Triton X-100 ) , 5x10 CV of a wash buffer II ( 50 mM Tris-HCl [pH 7 . 4] , 300 mM NaCl , 5% [v/v] glycerol , 0 . 1% [v/v] NP-40 ) , and 2x10 CV of a wash buffer III ( 50 mM Tris-HCl [pH 7 . 4] , 150 mM NaCl , 5% [v/v] glycerol , 0 . 1% [v/v] NP-40 ) . The protein was eluted by incubating the beads for 5 min at room temperature with an imidazole-containing elution buffer ( 50 mM Tris-HCl , 150 mM NaCl , 5% [v/v] glycerol , 0 . 1% [v/v] NP-40 , 300 mM imidazole ) . MPK12 proteins were concentrated and imidazole was removed by Millipore Amicon Ultra-0 . 5 Centrifugal Filter Concentrators ( NMWL 3000 ) . Glycerol was added to a final concentration of 20% ( v/v ) , and 20 μL aliquots of the eluted protein were snap-frozen in liquid nitrogen and stored at –80°C . Protein concentrations were estimated on 10% SDS-polyacrylamide gel using BSA as a standard . HT1 kinase activity assay was performed by incubating a constant amount of purified recombinant HT1 and 0–30 μM MPK12 , 0–20 μM MPK4 , or 0–10 μM MPK11 in a reaction buffer ( 50 mM Tris-HCl [pH 7 . 4] , 150 mM NaCl , 20 mM MgCl2 , 60 mM imidazole , 1 mM DTT , 0 . 2 mg/ml insulin ) at room temperature for 10 min . Then , casein ( 1 mg/ml ) , 500 μM ATP , and 100 μCi/ml 32P-γ-ATP were added and reaction aliquots were taken at the 30 min time point . Reactions were stopped by the addition of SDS loading buffer . Proteins were separated on a 10% SDS-polyacrylamide gel and visualized by Coomassie brilliant blue R-250 ( Sigma ) staining . HT1 activity was determined by autoradiography and quantified by ImageQuant TL Software . Sequence searches and alignments were conducted with SWISS-MODEL [47] . The crystal structure with the best sequence identity and resolution was selected for building homology models . Arabidopsis MPK12 and MPK4 have sequence identity to the 3 Ångstrom resolution Arabidopsis MPK6 structure ( 5CI6; [33] ) of 64 . 61% and 70 . 67% , respectively . This structure was then used to construct models for the wild-type and mutant structures . The RMSD from aligning the structures for MPK12 and MPK12 G53R was 0 . 324 Ångstroms ( i . e . , a close structural similarity ) . Structures were checked for clashes and with quality controls and were then superposed . Statistical analyses were performed with Statistica , version 7 . 1 ( StatSoft Inc . , Tulsa , Oklahoma , United States ) . All effects were considered significant at p < 0 . 05 . | Human activities have increased the concentrations of CO2 and harmful air pollutants such as ozone in the troposphere . These changes can have detrimental consequences for agricultural productivity . Guard cells , which form stomatal pores on leaves , regulate plant gas exchange . To maintain photosynthesis , stomata open to allow CO2 uptake , but at the same time , open stomata lead to loss of water and allow the entrance of ozone . Elevated atmospheric CO2 levels reduce stomatal apertures , which can improve plant water balance but also increases leaf temperature . Using genetic approaches—in which we exploit natural variation and mutant analysis of thale cress ( Arabidopsis thaliana ) —we find that MITOGEN-ACTIVATED PROTEIN KINASE 12 ( MPK12 ) and its inhibitory interaction with another kinase , HIGH LEAF TEMPERATURE 1 ( HT1 ) ( involved in guard cell CO2 signaling ) , play a key role in this regulatory process . We have therefore identified a mechanism in which guard cell CO2 signaling regulates how efficiently plants use water and cope with the air pollutant ozone . | [
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] | 2016 | Natural Variation in Arabidopsis Cvi-0 Accession Reveals an Important Role of MPK12 in Guard Cell CO2 Signaling |
The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea , and the oxygen-resistant spores that it forms have a central role in the infectious cycle . The late stages of sporulation require the mother cell regulatory protein σK . In Bacillus subtilis , the onset of σK activity requires both excision of a prophage-like element ( skinBs ) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence . Importantly , the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell . In C . difficile , σK lacks a pro-sequence but a skinCd element is present . The product of the skinCd gene CD1231 shares similarity with large serine recombinases . We show that CD1231 is necessary for sporulation and skinCd excision . However , contrary to B . subtilis , expression of CD1231 is observed in vegetative cells and in both sporangial compartments . Nevertheless , we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID . We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234 , and that this gene is required for sporulation and skinCd excision . CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration . Thus , CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell . Finally , while the skinCd element is not essential for sporulation , deletion of skinCd results in premature activity of σK and in spores with altered surface layers . Thus , skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development .
Endosporulation is an ancient bacterial cell differentiation process allowing the conversion of a vegetative cell into a mature spore through a series of morphological steps [1 , 2] . Many bacilli , clostridia and related organisms form bacterial spores . The spores have the ability to withstand extreme physical and chemical conditions and their resistance properties allow them to survive for long periods in a variety of environments . Spores serve as the infectious vehicle for several pathogens such as Bacillus anthracis , Bacillus cereus and Clostridium difficile [3 , 4] . C . difficile is the main cause of antibiotic-associated diarrhea . Disruption of the intestinal flora caused by antibiotherapy increases the risk to develop a C . difficile infection . After ingestion , C . difficile spores germinate in the intestine in the presence of specific bile salts [5] . Then , vegetative forms multiply and produce two toxins , TcdA and TcdB , which are the main virulence factors [6] . These toxins cause enterocyte lysis and inflammation leading to diarrhea , colitis , pseudomembranous colitis or more severe symptoms including bowel perforation , sepsis and death . During the infection process , C . difficile also forms spores in the gut that are essential for transmission of this strict anaerobe and contribute to the establishment of reservoirs in the environment including the host and hospital settings [7 , 8] . Despite the importance of spores in the infectious cycle , our knowledge of the molecular mechanisms underlying spore development in C . difficile is still scarce . Sporulation has been extensively studied in the model organism Bacillus subtilis [9 , 10] . At the onset of sporulation , an asymmetric division forms a forespore and a mother cell . A key developmental transition is when the mother cell finishes engulfing the forespore , which becomes fully surrounded by the mother cell . The mother cell maintains metabolic potential in the forespore and contributes to assembly of the spore protective structures and to the release of mature spores . The developmental program of sporulation is mainly governed by the sequential appearance of four cell type-specific sigma factors: σF in the forespore and σE in the mother cell control early stages of development , prior to engulfment completion , and are replaced by σG and σK following engulfment completion . The main morphological stages of sporulation are conserved among spore-formers , which also share a core of sporulation genes [11 , 12] . Nevertheless , recent work has highlighted important differences in the genetic control of sporulation between the aerobic bacilli and the anaerobic clostridia [13–15] . In C . difficile , the main functions and periods of activity of the sporulation σ factors are largely conserved relative to B . subtilis [16–18] . In B . subtilis , several mechanisms including signaling pathways between the two compartments and the architecture of the mother cell- and forespore-specific lines of gene expression , formed by interlocked feed-forward loops ( FFLs ) , converge for the timely activation of the σ factors at specific developmental stages [9 , 19] . However , in C . difficile , the communication between the forespore and the mother cell appears less effective , contributing for a weaker connection between morphogenesis and gene expression [16–18] . Indeed , the activation of the σE regulon in the mother cell just after asymmetric division , is rigorously dependent on σF in B . subtilis , but is partially independent of σF in C . difficile . Likewise , the synthesis of the forespore signaling protein SpoIIR , essential for pro-σE processing , is strictly dependent on σF in B . subtilis but partially independent of σF in C . difficile [18] . Furthermore , in B . subtilis , the onset of σG activity coincides with engulfment completion and requires the activity of σE , while in C . difficile σG activity is detected in pre-engulfment sporangia and this early activity is independent of σE [20 , 21] . Finally , several levels of regulation ensure that the activity of σK in B . subtilis is restricted to the mother cell following engulfment completion . Firstly , the sigK gene is interrupted by an intervening prophage-like element , skinBs . Secondly , expression of sigK and of spoIVCA encoding a member of the large serine recombinases ( LSRs ) superfamily [22] responsible for skinBs excision is under the control of σE and requires the transcriptional regulator SpoIIID [19 , 23] . Expression of spoIIID is also controlled by σE , but since SpoIIID is auto-regulated [24] , a coherent FFL delays expression of the spoIVCA and sigK genes towards the end of engulfment [19 , 25] . Moreover , σK activity depends on the cleavage of an inhibitory pro-sequence , a step controlled by σG . Finally , σK directs expression of an anti-sigma factor , CsfB that inhibits σE , thereby promoting transition from σE- to σK-controlled stages in the mother cell [26] . σK is required for assembly of the spore cortex and the more external coat , the main spore surface structures , as well as for mother cell lysis . The segregation of σK activity to post-engulfment sporangia in B . subtilis is explained by multi-level regulation of σK synthesis and activation . Redundancy ensures fail-safe solutions and increases robustness of the developmental process . In C . difficile , σK is dispensable for cortex biogenesis but is required for the assembly of the spore coat and of the exosporium and for mother cell lysis [17] . sigK is interrupted by a skinCd element , which is excised during sporulation [27] . The skin elements of B . subtilis and C . difficile have different sizes and gene content and are inserted at different sites and in opposite orientation indicating that integration into sigK has occurred independently during evolution [27] . Previous studies have also shown that some transcription of sigK takes place during engulfment [17] . However , σK of C . difficile lacks a pro-sequence [27] and accordingly , σG is dispensable for σK activation [16–18] . In the absence of this cleavage at the end of engulfment , skinCd excision is likely a crucial element in the regulation of σK activity in C . difficile . Importantly , given the role of σK in the assembly of the spore surface layers together with the observation that some strains of C . difficile lack skinCd [27 , 28] , it is imperative to better understand excision of the element in this organism . The CD1231 gene , located within skinCd , codes for a protein similar to the SpoIVCA recombinase of skinBs [27 , 29] . Surprisingly , σE does not control CD1231 expression in C . difficile [18 , 30] , which , as we now show , is expressed constitutively . In this work , we studied the role of CD1231 in sporulation and in skinCd excision . We demonstrated that another factor present in the skinCd element , CD1234 encoded by a gene under σE and SpoIIID control is required for skinCd excision but not integration . Thus , CD1234 is a recombination directionality factor that restricts skinCd excision to the mother cell . Importantly , we showed that skinCd is a key element in controlling the onset of σK activity , which in turn is important for proper spore morphogenesis and function .
The skinCd element of strain 630Δerm inserted into the sigK gene contains 19 genes ( Fig 1A ) . CD1231 , located immediately upstream of the 3´-moiety of sigK ( CD1230 ) , is the unique gene within the skinCd element coding for a protein with similarity to large serine recombinases ( LSRs ) superfamily [22] . The first 300 amino acid residues of CD1231 share 24% identity with SpoIVCA , the recombinase encoded by skinBs [31] , and 30% identity over its entire length to the SprA protein , responsible for the excision of the B . subtilis SPβ prophage [32] . CD1231 is also similar to other C . difficile recombinases associated with conjugative transposons . A domain analysis of CD1231 ( Fig 1B ) identifies the three main structural domains of LSRs and the motifs that connect them: the N-terminal resolvase domain ( LSR-NTD ) bearing the conserved catalytic nucleophile ( Ser at position 10 ) and additional catalytic residues , followed by a recombinase domain ( RD ) , a zinc β-ribbon domain ( ZD ) and a coiled coil ( CC ) motif ( Figs 1B and S1 ) . The RD , the ZD and the CC form the C-terminal domain ( LSR-CTD ) ; the NTD and CTD are linked by a long α-helix ( αE ) while a short linker connects the RD to the ZD [22] . In CD1231 as in other LSRs , the CTD is followed by an extension of variable length , which is mostly α-helical ( S1 Fig ) [22] . In B . subtilis , the excision of the skinBs element occurs in the mother cell , and creates an intact sigK gene , essential for sporulation . Since σK is required for sporulation in C . difficile [15] , inactivation of CD1231 , which is probably involved in skinCd excision , would cause a block in the process . We constructed a CD1231 mutant ( CDIP526 ) using the Clostron system ( S2 Fig ) as well as a complemented strain ( CDIP533 ) carrying CD1231 under the control of its promoter ( pMTL84121-CD1231; see below ) . We then examined the morphology of the strains by phase contrast and fluorescence microscopy after 24 h of growth in sporulation medium ( SM ) , and we tested the efficiency of heat-resistant spore formation at 72 h . The 630Δerm strain produced 2 x106 heat-resistant CFU/ml and phase bright spores , either free or still inside the mother cell , were seen ( Fig 1C ) . In contrast , less than 10 heat resistant CFU/ ml were detected for the CD1231 mutant . While some phase gray spores were seen in cultures of the mutant at 24 h , free spores were not detected ( Fig 1C ) . Complementation of the CD1231 mutation restored the wild-type phenotype ( Fig 1C ) . Therefore , inactivation of the CD1231 gene severely impaired sporulation . The phenotype caused by the CD1231 mutation phenocopied that imposed by a sigK mutation in that phase gray , heat-sensitive spores were formed that often were seen in a angle relative to the long axis of the cell ( Fig 1C ) [16 , 17] . Moreover , as found for a sigK mutant , formation of the phase gray spores was not accompanied by loss of viability of the mother cell , as is the case for the wild-type strain [17] . These observations strongly suggest that σK is inactive in this mutant . Expression of the spoIVCA gene in B . subtilis is under the dual control of the mother cell proteins σE and SpoIIID leading to the restriction of skinBs excision to this compartment [19 , 33] . However , previous transcriptome studies suggested that σE or SpoIIID does not control CD1231 expression in C . difficile [18 , 30] . Moreover , qRT-PCR using RNA extracted from SM cultures of the strain 630Δerm , a sigE mutant and a spoIIID mutant did not show variations in the level of CD1231 expression in the sigE mutant and only a slight increase in the spoIIID mutant relative to the wild-type strain ( Fig 2B ) . In our genome-wide mapping of promoters in strain 630Δerm [34] , a transcriptional start site ( TSS ) was found 21 bp upstream of the CD1231 start codon and -35 ( TTTAAA ) and -10 ( TATAAT ) sequences for σA-dependent promoters are present upstream of this TSS while no consensus for σE is found ( Fig 2A ) . This suggests that expression of CD1231 is under the control of σA and therefore probably not confined to the mother cell . To test this possibility , we constructed a PCD1231-SNAPCd fusion and this fusion was transferred to the 630Δerm strain . Samples of cultures expressing PCD1231- SNAPCd were collected at 24 h of growth in SM , and the cells doubly labeled with the SNAP substrate TMR-Star and the membrane dye MTG . Expression of PCD1231- SNAPCd was detected in 93% of the vegetative cells scored , consistent with the presence of a σA-type promoter . However , expression of PCD1231-SNAPCd was also detected in sporulating cells in both the forespore and the mother cell ( 78% of the sporangia ) ( Fig 2C ) . Thus , in agreement with the absence of a requirement for σE and SpoIIID for its expression as determined by qRT-PCR , CD1231 is not a mother cell-specific gene . Previous work indicated that the skinCd element is excised from the chromosome only during sporulation [27] . This suggests that a factor is required in addition to CD1231 to trigger excision during sporulation . In a first step to search for this factor , we wanted to establish the requirements for sigK transcription and σK activity . Previous work has shown that SpoIIID is required for sporulation and for the transcription of the sigK gene in C . difficile [16 , 18 , 30] . Importantly , expression of a skinCd-less version of the sigK gene from a SpoIIID-independent promoter largely bypasses the requirement for SpoIIID for sporulation [30] . While showing that a critical function of SpoIIID in sporulation is to ensure efficient sigK expression , this result does not discard a possible role of SpoIIID in skinCd excision . Here , we examined the effect of a spoIIID mutation on sigK transcription using a PsigK-SNAPCd fusion and on the activity of σK using a fusion of the σK-controlled PcotE to SNAPCd [17] . Two TSSs have been mapped in the sigK promoter region ( Fig 3A ) [18] . The upstream promoter ( P1 ) matches the consensus for σE recognition , whereas the downstream promoter ( P2 ) matches the consensus for σK recognition [18] . Using the consensus of SpoIIID of B . subtilis [19] , a possible SpoIIID binding site is found upstream of P2 and downstream of P1 ( Fig 3A ) . Transcription of sigK was detected in the mother cell of the wild-type strain soon after septation and during engulfment [17] , but increased following engulfment completion , when it was detected in 88% of the sporangia ( Fig 3B ) . In contrast , transcription of sigK was detected in only 67% of the spoIIID mutant sporangia in which engulfment was completed ( Fig 3B ) . Moreover , quantification of the fluorescence signal from PsigK-SNAPCd in those cells revealed a reduction of the signal per sporangia , from 1 . 1±0 . 8 arbitrary units ( A . U . ) in the wild-type strain , to 0 . 7±0 . 3 A . U . in the spoIIID mutant ( Fig 3C ) . The arrangement of the sigK promoter region suggests that the low level of transcription during engulfment may arise from P1 whereas the main period of sigK transcription could involve utilization of P2 possibly by σE first , then by σK , with the assistance of SpoIIID . P2 may also be involved in the late , positive auto-regulation of σK in cells carrying phase bright spores [17 , 18] ( see also the Discussion ) . Thus , following engulfment completion , transcription of sigK is reduced , but not abolished , in the absence of SpoIIID both in terms of the number of cells in which transcription is activated and , although a less pronounced effect , in the level of expression per cell . The reduction in sigK transcription in the spoIIID mutant is in line with earlier results [18 , 30] . As a measure of σK activity , PcotE-driven production of SNAPCd was detected in the mother cell in 6–13% of the wild-type sporangia during engulfment but increased to 68% just after engulfment completion and was seen in 86% of the sporangia when phase bright spores became visible ( Fig 3D ) . Expression of PcotE-SNAPCd was detected in only 40% of the sigK mutant sporangia that reached late stages of morphogenesis to form partially refractile spores , and the average fluorescence signal per sporangia decreased to 0 . 6±0 . 1 A . U . ( Fig 3E ) . Importantly , disruption of spoIIID or CD1231 reduced expression of PcotE-SNAPCd to only 21% and 29% of the sporangia that reached late stages of morphogenesis . Moreover , the average fluorescence intensity per cell was of 0 . 6±0 . 2 A . U . and 0 . 4±0 . 1 A . U . in the spoIIID or CD1231 mutant , respectively as compared to 1 . 1±0 . 8 A . U . for the wild-type strain ( Fig 3D and 3E ) . Thus , disruption of sigK , spoIIID , or CD1231 reduced expression of the PcotE-SNAPCd fusion approximately to the same extent . In any event , the increase in σK activity following engulfment completion is not seen in CD1231 and spoIIID mutants , compatible with a role for CD1231 and SpoIIID in the control of σK activity . This strongly suggests that SpoIIID may play a role in skinCd excision . To examine the time and requirements of skinCd excision , we devised an assay to monitor reconstitution of a functional sigK gene in C . difficile . We previously described a plasmid , pFT38 , carrying a sigK gene disrupted by a mini-skinCd element bearing a deletion of all the skinCd genes except CD1231 [17] . We modified this plasmid in order to create a translational fusion between the C-terminal moiety of σK and SNAPCd and to remove the 5´-end of CD1231 ( i . e . , only the chromosomal CD1231 is functional ) ( Fig 4A ) . This plasmid , pFT74 , was introduced in strain 630Δerm and in the spoIIID mutant . Excision through recombination involving sequences at the ends of the mini-skinCd element reconstitutes the sigK gene ( Fig 4A ) . The recombined sigK in pFT74 named pFT74R was first detected by PCR using an oligonucleotide hybridizing to the 5’ moiety of the sigK gene and a second in the SNAPCd gene: a fragment of 1500 bp is expected upon mini-skinCd excision instead of 1977 bp for pFT74 ( Fig 4A ) . The 1500 bp PCR fragment was detected in strain 630Δerm but not in the spoIIID mutant ( Fig 4B ) indicating that SpoIIID is necessary for mini-skinCd excision . This is also the case for the chromosomal copy of the skinCd element as described below . To gain further insight into the time of excision relative to the course of spore morphogenesis , we monitored production of the σK-SNAPCd translational fusion formed after mini-skinCd excision at the single cell level by fluorescence microscopy ( Fig 4C ) . Production of σK-SNAPCd was detected only in the mother cell in 58% of the wild-type sporangia in which spores were not yet discernible in the mother cell by phase contrast microscopy but that were close to or just after engulfment completion as judged from the MTG staining pattern ( membranes almost fused or fused ) ( Fig 4C ) . However , σK-SNAPCd was detected in 91% of the sporangia in which partially phase bright or phase bright spores were visible by phase contrast microscopy ( Fig 4C ) . This parallels the pattern of sigK transcription and σK activity [17] and suggests that σK is active as soon as it is produced after skinCd excision . In contrast , no accumulation of σK-SNAPCd was detected in the spoIIID mutant ( Fig 4C ) even if the sigK gene remains expressed in this mutant ( Fig 3A ) . σK-SNAPCd ( 47 kDa ) was detected by Western blotting using anti-SNAP antibodies and by fluorimaging in crude extracts of strain 630Δerm but not in extracts prepared from the spoIIID mutant ( Fig 4D ) . These results strongly suggest that SpoIIID is required for skinCd excision in C . difficile as also observed for B . subtilis . Moreover , since the main period of sigK transcription , σK accumulation and σK activity appear to coincide during the course of morphogenesis , skinCd excision and sigK transcription seem to concur to delay the onset of σK activity . Given that skinCd excision did not take place in vegetative cells or in the forespore in spite of CD1231 expression in these cells and the suspected role of SpoIIID in controlling σK activity via skinCd excision , we inferred that a factor probably encoded within skinCd and produced under the joint control of σE and SpoIIID could modulate CD1231 synthesis and activity . Among the 19 skinCd genes , only CD1234 ( Fig 5A ) was down-regulated in a sigE and in a spoIIID mutant in transcriptome analyses [18 , 30] . CD1234 codes for a small protein of 72 amino acids , with a predicted pI of 5 . 5 and no significant similarity to proteins found in databases . We confirmed by qRT-PCR using RNA extracted from SM cultures that CD1234 expression decreased 25-fold and 40-fold in a sigE and in a spoIIID mutant , respectively as compared to the wild-type strain ( Fig 5B ) . We mapped a TSS 14 bp upstream of the start codon of CD1234 , and a consensus sequence for σE recognition was detected upstream of this TSS [18] . Using the SpoIIID consensus sequence of B . subtilis [19] , we also identified a putative SpoIIID binding motif ( TGTAACAAT ) centered 46 bp upstream of the CD1234 TSS ( Fig 5A ) in agreement with the positive control of CD1234 expression by SpoIIID . To examine the compartment and time of CD1234 expression during sporulation , we constructed a PCD1234-SNAPCd transcriptional fusion . This fusion was transferred by conjugation into the 630Δerm strain and the sigE or spoIIID mutant . In the wild-type strain , SNAP production confined to the mother cell , was detected in 48% and 72% of the cells just after asymmetric division and during engulfment , respectively and persisted until late stages in development , when phase bright spores were seen ( Fig 5C ) . SNAPCd production was eliminated by mutation of sigE ( Fig 5C ) while the average intensity of the SNAPCd-TMR signal decreased from 1 . 4±1 in the 630Δerm strain to 0 . 5±0 . 1 in a spoIIID mutant ( Fig 5D ) . Together , these results indicate that CD1234 is a mother cell-specific gene expressed under the joint control of σE and SpoIIID . To investigate the role of CD1234 in sporulation , we constructed a CD1234 mutant , CDIP396 , using the Clostron system ( S2 Fig ) . A complemented strain , CDIP397 , carrying the CD1234 gene expressed under the control of its native promoter was also constructed . We examined the morphology of the CD1234 mutant using phase contrast and fluorescence microscopy . Some phase bright or partially phase bright spores were present in cultures of the CD1234 mutant but free spores were not detected . The mutant formed only 1 . 5 x 102 heat-resistant spores/ml of culture at 72 h of growth in SM ( i . e . 104 less than the wild-type ) ( Fig 1C ) . Importantly , the wild-type phenotype was restored in the complemented strain ( Fig 1C ) . Thus , inactivation of the CD1234 gene strongly impaired sporulation . Nevertheless , the CD1234 mutant like the spoIIID mutant was not as severely affected in sporulation as the sigE and sigK mutants [16 , 17] or CD1231 mutant ( Fig 1C ) . Since both CD1231 and CD1234 are likely required for skinCd excision , a prerequisite for σK activation , we tested the impact of CD1231 and CD1234 inactivation on the expression of σK or σE targets using qRT-PCR ( Table 1 ) . We extracted RNA from strain 630Δerm , the CD1231 and CD1234 mutants and the complemented strains after 24 h of growth in SM , a time where σK target genes are highly expressed [18] . As expected , expression of CD1231 and CD1234 was strongly reduced in the CD1231 and CD1234 mutants , respectively ( Table 1 ) . The expression of three σE targets ( spoIIIAA , spoIVA , spoIIID ) was not significantly altered in the CD1231 and CD1234 mutants as compared to the wild-type strain . In sharp contrast , expression of six σK target genes strongly decreased in the CD1231 or CD1234 mutant compared to the wild-type strain ( Table 1 ) , as observed previously for a sigK mutant [18] . CD1231 and CD1234 were about 10-fold more expressed in the CD1231 ( pMTL84121-CD1231 ) and CD1234 ( pMTL84121-CD1234 ) strains than in 630Δerm ( Table 1 ) , and expression of the σK target genes was fully or partially restored in these strains . Accordingly , expression of a PcotE-SNAPCd fusion decreased in a CD1231 mutant compared to strain 630Δerm as described above and was reduced in the CD1234 mutant . Only 33% of the sporangia that reached late stages of development expressed the fusion when CD1234 is inactivated compared to 86% for the wild-type strain ( Fig 3D ) . Moreover , the average intensity of the fluorescence signal decreased from 1 . 1±0 . 8 A . U . ( WT ) to 0 . 5±0 . 1 ( CD1234 ) , similar to the intensity seen for the CD1231 mutant ( Fig 3E ) . In conclusion , expression of σK-dependent but not of σE-dependent genes requires CD1231 and CD1234 as expected for proteins involved in sigK reconstruction through skinCd excision . The temporal control of skinCd excision and its confinement to the terminal mother cell may thus require the σE- and SpoIIID-controlled gene , CD1234 . We tested excision of the chromosomal skinCd in strain 630Δerm and in the CD1231 , CD1234 , sigE and spoIIID mutants ( Fig 6 ) . In B . subtilis , skinBs excision occurs within two 5 bp inverted repeats that flank an imperfect 21 bp repeat [35] . In C . difficile , excision is expected to occur by means of a recombination event involving attL and attR ( by analogy with the sequences involved in phage excision ) at the left and right ends of skinCd ( Fig 6A ) [27] . As in B . subtilis , attL and attR consist of two half-sites formed by a 5 bp inverted repeat external to a longer 22 bp imperfect inverted repeat ( Fig 6A ) and of two conserved 12 bp motifs , one in attL and one in attR , within which recombination take place ( in green in Fig 6A ) . The intervening DNA is excised as a circular molecule carrying attP ( by analogy with sequences responsible for phage integration ) , leaving behind a chromosomal attB site ( analogous to prophage insertion sequences in the bacterial chromosome ) ( Fig 6A ) . The excised circular element obtained after excision of skinCd can be monitored by PCR using primers annealing upstream and downstream of attP ( Fig 6A ) . During sporulation , skinCd excision was detected in strain 630Δerm ( Fig 6B , lane 1 ) , but not in the CD1231 ( lane 2 ) , CD1234 ( lane 4 ) and sigE ( lane 6 ) mutants . A very faint band corresponding to the excised skinCd was detected in the spoIIID mutant ( lane 8 , red arrow and red dot ) . Plasmids bearing the disrupted chromosomal genes restored skinCd excision to all mutants ( Fig 6B , lane 3 , 5 , 7 , 9 ) . Thus , skinCd excision during sporulation requires both CD1231 and CD1234 . Moreover , the results confirm the key role of SpoIIID and σE in skin excision likely through their control of the cell type-specific production of CD1234 . To test whether expression of CD1234 could result in skinCd excision during vegetative growth , we constructed a plasmid carrying CD1234 under the control of an ATc-inducible promoter ( pDIA6103-PtetCD1234 ) . This plasmid or the empty vector pDIA6103 were introduced into strain 630Δerm and the sigE , spoIIID , CD1231 and CD1234 mutants . The resulting strains were grown for 4 h in TY medium . Following induction of CD1234 expression , the cells were either plated onto BHI or harvested for DNA extraction . qPCR was then performed with 2 primer pairs , one corresponding to DNApolIII as a control and the second to sigK on both sides of attB for the detection of skinCd excision . The ΔCt ( CtsigK-CtpolIII ) was determined for each strain carrying either pDIA6103 or pDIA6103-PtetCD1234 . The ΔCt was >10 for all strains containing pDIA6103 . Interestingly , the ΔCt was reduced to < 1 for all strains containing pDIA6103-Ptet-CD1234 with the exception of the CD1231 mutant where a ΔCt of 18 was observed , as was also the case for the strain CD1231 ( pDIA6103 ) ( Table 2 ) . In parallel , chromosomal DNA was extracted from 8 independent clones obtained after seeding BHI plates with samples from the cultures of the different strains carrying pDIA6103-PtetCD1234 . For each clone , we tested the presence of skinCd in the chromosome by PCR amplification of the 5’ junction of the skin ( attL ) using one oligonucleotide located in the 3’ part of CD1231 and the second in sigK ( Fig 6A ) . While the skinCd/chromosome junction was detected in only 1 out of 8 clones tested for strains 630Δerm , sigE , spoIIID or CD1234 , this junction was amplified for the 8 clones of the CD1231 mutant ( Table 2 ) . This confirmed that skinCd was excised after induction of CD1234 expression during growth of the wild-type strain and of the sigE , spoIIID and CD1234 mutants . In contrast , skinCd remained integrated in the CD1231 mutant under similar conditions . In conclusion , these results indicate that during C . difficile growth: i ) excision occurs if and only if CD1234 is produced; ii ) under these conditions skinCd excision is independent of σE and SpoIIID; and iii ) excision is absolutely dependent on the skinCd CD1231 recombinase , even if CD1234 is induced . Together , these results indicate that both CD1231 and CD1234 are necessary for skinCd excision in C . difficile . Since CD1231 was not specifically transcribed during sporulation and its expression was not altered in a CD1234 background ( Table 1 ) , we reasoned that CD1234 could post-transcriptionally control the synthesis or activity of CD1231 . The integration reaction catalyzed by the LSRs is unidirectional , in that excision often requires an additional recombination directionality factor ( RDF ) that modulates the LSR activity by direct protein-protein interactions [22 , 36 , 37] . We therefore tested whether CD1231 and CD1234 could interact using pull-down assays . Whole cell extracts prepared from E . coli BL21 ( DE3 ) strains producing separately CD1234-His6 and CD1231-Strep or co-producing the two proteins under the control of PT7lac were prepared . None of the proteins was detected by Coomassie staining , but they were detected by immunoblotting with antibodies to their C-terminal tags ( S4A and S4B Fig ) . The extracts were then incubated with Ni2+-NTA agarose beads , and following washing and elution , the bound proteins were identified by immunoblotting . While a protein of about 30 kDa recognized by the Strep-tag antibody seems to bind non-specifically to the beads , the full length CD1231-Strep as well as two probable degradation products , of about 37 and 40 kDa , were only detected in the presence of CD1234-His6 ( S4B Fig ) . The two likely degradation fragments of CD1231-Strep may contain the CTD domain followed by the C-terminal extension ( residues 405–505 ) consistent with the existence of a protease-sensitive site just downstream of the NTD in the recombinases from phages C31 and Bxb1 and from transposon TnpX [22 , 38] ( S4D Fig ) . These fragments may be retained by the Ni2+ column because they bind to the full-length CD1231-Strep recombinase or to CD1234-His6 ( S4D Fig ) . In a different set of experiments , full-length CD1231-Strep was retained by the beads when these were pre-incubated with extracts prepared from BL21 ( DE3 ) cells producing CD1234-His6 and not Tgl-His6 , an unrelated spore-associated protein from B . subtilis [39 , 40] , which accumulated to much higher levels than CD1234-His6 ( S4C Fig ) . Together , these results indicate that CD1234 and CD1231 were part of a complex that formed in E . coli and suggest that CD1234 might control the activity of CD1231 by direct interaction . To test whether CD1234 and CD1231 were sufficient for skinCd excision , we used a heterologous host E . coli . The plasmid pFT74 carrying a mini skinCd element integrated into sigK was introduced in E . coli carrying plasmids for expression of CD1231 , CD1234 or the co-expression of both genes . The cells were first induced to produce CD1231 , CD1234 or both . The plasmid pFT74 was then purified and examined for the recombination reaction by digestion with BamHI and NotI ( Fig 7A ) . Digestion of the resulting recombined plasmid obtained after mini skinCd excision , termed pFT74R , with BamHI and NotI should produce a fragment of 1000 bp as compared to a fragment of 1288 bp for the parental plasmid carrying the mini-skin ( pFT74 ) . A fragment of 1000 bp was not isolated from cells producing neither CD1234 alone nor CD1231 alone or a mutant allele of CD1231 in which the putative catalytic serine in the NTD was changed to an alanine ( S10A ) ( Figs 1B , 7B and S5 ) . By contrast , pFT74R was detected in cells in which both proteins were produced ( Fig 7B ) . Together , these results show that CD1231 requires CD1234 as an auxiliary factor for the recombination reaction between attL and attR that results in skinCd excision . While both CD1231 and CD1234 are required for skinCd excision in E . coli , we also wanted to test the involvement of these proteins in integration reactions . With that purpose , the attP site of the prophage-like element was inserted into pFT74R , which already contains the integration sequence attB as the result of the excision reaction ( Fig 7A ) . Two plasmids were constructed , one with attP and attB in the same orientation ( pMS511; Fig 7C ) and one with attB and attP in opposite orientation ( attP´ in pMS510; Fig 7E ) . These plasmids were introduced in E . coli cells carrying the plasmids for expression of CD1231 , CD1234 or both . Cells were induced to produce CD1231 , CD1234 or both and then the plasmids were purified and examined for recombination events between attP and attB by digestion with BamHI and NotI . Two types of recombination events serve as readouts for the ability of CD1231 to catalyze DNA integration . A recombination event between attP and attB should result in the removal of the intervening DNA for pMS511 ( Fig 7C ) or in the inversion of the intervening DNA in the case of pMS510 ( Fig 7E ) . We showed that CD1231 is necessary and sufficient for both types of recombination events involving attP and attB ( Fig 7D and 7F ) . No recombined products of pMS510 or pMS511 were retrieved when the catalytically inactive CD1231 bearing the S10A substitution was produced , alone or together with CD1234 ( S5 Fig ) . These results showed that CD1231 is sufficient for the integration event that results from the recombination between attP and attB , but requires CD1234 for the excision event that results from the recombination reaction involving attL and attR . Thus , CD1234 is a recombination directionality factor ( RDF ) assisting CD1231 in skinCd excision . While the regulated excision of skinCd has been suggested as a critical mechanism for efficient sporulation in C . difficile [17 , 27] , a more recent study suggests that skinCd is not essential for the formation of heat-resistant spores in this organism [30] . However , skinCd excision controls the onset of σK activity . The deletion of the skin in a pro-less sigK strain in B . subtilis imposes changes in the mother cell line of gene expression leading to altered spore structure and functional properties , while the final titer of spores formed is reduced compared to the wild-type [41 , 42] . To analyze more precisely the involvement of skinCd in sporulation , we took advantage of the 630Δerm , which expressed CD1234 under Ptet control , to obtain a congenic derivative of strain 630Δerm lacking skinCd ( summarized in S6A Fig ) . Addition of ATc during growth led to skinCd excision and after plating of the cells , DNA was extracted from isolated colonies . We identified several clones that carried a reconstructed sigK gene ( S6B Fig , lane 1 ) but lacked the 5’ junction of skinCd in the chromosome ( S6B Fig , lane 2 ) and the excised form of skinCd that was lost after cellular division ( S6B Fig , lane 3 ) . In a second step , a clone carrying an intact sigK gene was cured of the pDIA6103-PtetCD1234 plasmid by successive cycles of growth and dilution in TY medium . After plating , Tm-sensitive clones that had lost pDIA6103-PtetCD1234 were isolated . One clone was named 630Δerm ΔskinCd . Phase contrast microscopy experiments revealed the presence of free spores in both the 630Δerm and 630Δerm ΔskinCd strains ( Fig 1C ) and the titer of heat resistant spores measured 48 h and 72 h after inoculation in SM medium was almost identical for both strains ( Fig 1C ) . Moreover , the percentage of sporulation measured for the two strains at 12 h ( 0 . 4% for 630Δerm and 0 . 3% for 630Δerm Δskin ) , 18 h ( 1 . 6% and 1 . 1% ) and 24 h ( 6 . 4% and 5 . 4% ) following inoculation into SM also did not differ significantly . Thus , in agreement with the previous results using a skinCd-less sigK gene expressed from a SpoIIID-independent promoter [30] , deletion of skinCd did not appear to affect the final titer of spores and the kinetics of sporulation . We then analyzed transcription of sigK and of σK target genes by qRT-PCR in the 630Δerm and 630Δerm ΔskinCd strains . We first harvested the cultures between 10 h and 24 h of growth in SM . After RNA extraction , we tested the expression of sigK using oligonucleotides located on both sides of the skinCd insertion into sigK and of σK target genes . The results showed that the expression of sigK and of several sigK targets ( cotE , cotBC , sleC , cdeC , bclA1 and bclA3 ) was higher in the Δskin strain than in the 630Δerm strain ( S2 Table ) . Lastly , we monitored the activity of σK at the single cell level using a PcotE-SNAPCd transcriptional fusion in the wild-type and ΔskinCd background . Fluorescence microscopy revealed that the signal intensity from the accumulation of TMR-Star-labeled SNAPCd did not differ significantly between wild-type and ΔskinCd sporangia , before or after engulfment completion . Strikingly , however , the ΔskinCd mutation increased the fraction of cells that showed PcotE-SNAPCd expression and hence σK activity , prior to engulfment completion , from 20% ( WT ) to 60% ( ΔskinCd ) ( Fig 8A ) . Thus , expression of a skinCd-less sigK gene from its native promoter results in premature σK activity . Activation of σK in B . subtilis is tightly linked to engulfment completion , and mutations that cause its premature activation result in alterations in the properties of the resulting spores [41 , 42] . σK plays an important role in the assembly of the spore coat [16 , 17] and because σK was active prior to engulfment completion in a larger fraction of the 630Δerm ΔskinCd sporangia as compared to the 630Δerm strain , we examined ultrastructure and the polypeptide composition of the spore surface layers in the two strains . Spores were density gradient purified from cultures of the 630Δerm and 630Δerm ΔskinCd strains , and processed for electron microscopy . Under our conditions , spores of the 630Δerm strain showed a more internal lamellar coat ( Fig 8B , yellow arrows in panels a to c ) , covered by a more external electrondense layer ( red arrows ) ; this layer had a uniform and compact appearance around the entire spore ( Figs 8B and S7; see also [17] ) . It may correspond to an exosporium-like layer , which in C . difficile seems to be closely apposed to the underlying coat ( [3]; see also below ) . In contrast , the electrondense outer layer was less compacted in spores of the ΔskinCd strain , and absent in some sections ( Fig 8B , black arrows in panels d to i ) . The disorganization of the outer layer presumably allowed the visualization of a thin electrondense layer forming the edge of the lamellar coat and in close apposition to it ( Fig 8B , red arrows in panels d to i ) . However , this thin layer was missing in some sections and material from the inner lamellar layer appeared to peel off the spore in those sections ( Fig 8B , black arrows in panels d and f ) . Overall , the lamellar coat layer also appeared to be less dense , making its structural organization more apparent ( Fig 8B , d to i and S7 Fig ) . Spore surface proteins from both the coat and exosporium layers were extracted from spores of the 630Δerm and ΔskinCd strains and resolved by SDS-PAGE . Several proteins showed increased extractability from spores of the ΔskinCd strain relative to the wild-type strain ( Fig 8C , black arrows , bands a through d and e ) , whereas a protein of about 12 kDa ( red arrow , band f ) showed reduced extractability . Mass spectrometry analysis indicates that band a ( size close to 200 kDa ) contains CotB , whereas band b ( size around 80 kDa ) contains CdeC . Because the predicted sizes of CotB and CdeC are 34 . 9 kDa and 44 . 7 kDa , respectively , these two species may represent cross-linked products of these proteins . CotB and CdeC are likely critical determinants for the assembly of the spore exosporium [3] . Rubrerythrin ( Rbr ) with a predicted size of 20 . 6 kDa and CysK , with a predicted size of 32 . 6 kDa , are detected in bands c and d , at about 37 kDa ( Fig 8C ) . At least Rbr , found in a band of about twice its predicted molecular weight , may form cross-linked homodimers , or be cross-linked to a protein of about 20 kDa . In contrast , a likely proteolytic fragment of the 19 . 2 kDa-exosporium protein CdeM , shows decreased representation or extractability in the mutant ( Fig 8C , band f ) . The alterations in the assembly of CotB , CdeC and CdeM may explain at least in part the morphology of the outer spore layers in the ΔskinCd strain ( Fig 8B ) ; possibly , the Δskin deletion affects mainly the assembly of the exosporium-like layer that in C . difficile seems to be juxtaposed to the coat , while the morphological alterations seem at the level of the coat may be in part a consequence of a misassembled exosporium-like layer . The increased representation of SspB ( a forespore-specific protein ) [18] may indicate that the spores of the mutant are more permeable or more sensitive to the extraction procedure ( Fig 8C , band e ) . In toto , we conclude that deletion of skinCd affects the assembly of the C . difficile spore surface layers . The alterations in the assembly of the spore coat and of a more external possible exosporium-like layer are most likely due to the premature activity of σK in 630Δerm ΔskinCd sporangia .
The mother cell-specific excision of the skin element , in either B . subtilis or C . difficile , is essential for the production of a functional sigK gene . In B . subtilis , expression of the gene coding for SpoIVCA , the LSR responsible for skinBs excision is under the joint control of σE and SpoIIID [33] . Hence , skinBs excision is restricted to the terminal mother cell . Excision of the skinCd element is absolutely dependent on CD1231 encoding a SpoIVCA homologue , but expression of this gene is not restricted to the mother cell . Rather , CD1231 is transcribed from a σA-dependent promoter and is expressed in vegetative cells and during sporulation in both compartments . An important finding of the present investigation is that excision of skinCd requires , in addition to CD1231 , the product of a gene , CD1234 , whose expression is under the control of both σE and SpoIIID ( Fig 9 ) . It is the requirement for CD1234 that restricts skinCd excision to the mother cell . However , the inactivation of CD1234 or SpoIIID does not reduce sporulation to the level observed for the CD1231 or sigK gene disruption . CD1234 and SpoIIID thus appear less crucial for sporulation than σK or the catalytic function of the LSR , CD1231 . It seems possible that CD1231 occasionally performs excision of skinCd without CD1234 . Nevertheless , the normal requirement of CD1234 for skinCd excision in C . difficile is paralleled by its requirement for the CD1231-dependent excision of a mini-skinCd in a heterologous host , E . coli but conversely , CD1234 is not required for the CD1231-dependent skinCd integration . Thus , our recombination assays show that CD1234 is a RDF [22 , 32 , 37] . RDFs likely stimulate formation of the synapse complex between attL and attR by competing for inhibitory interactions involving the CC motifs of LSRs or inhibit formation or otherwise destabilize the synaptic complex formed between attB and attP possibly by stabilizing the auto-inhibitory activity of the CC motifs in this complex [22 , 37] . Accordingly , the RDFs interact with the recombinase in solution , in line with our observation that CD1231 and CD1234 are part of a complex that formed in E . coli . Both structural studies and the isolation of mutations close or within the CC motif of the ϕC31 LSR that allows it to recombine attL and attR in the absence of the cognate RDF , suggest binding of the CC motifs by the RDF [22 , 37] . More studies are needed to unravel the mechanism by which CD1234 cooperates with the CD1231 LSR to control skinCd excision . RDFs have been identified for several LSRs . However , they do not share significant sequence similarity and most of them appear to be small , basic proteins . In contrast , CD1234 is acidic like SprB , the RDF of phage SPβ [32] . SprB is required for the SprA recombinase-mediated excision of phage SPβ . A RDF assisting SpoIVCA in the excision of skinBs has not been identified , but the function of the skinBs-encoded genes has not been inspected individually . Recent studies have examined in detail the sporulation program of C . difficile and provided evidence that the temporal compartmentalization of sigma factor activity is less tightly regulated in C . difficile as compared to B . subtilis [16–18] . A difference between the sporulation programs in the two organisms may be the existence of a higher degree of redundancy in the control of gene expression at key stages in morphogenesis in B . subtilis . The control over spoIVCA and sigK transcription , skinBs excision , and proteolytical removal of the inhibitory pro-sequence ensures that σK is only active following engulfment completion in B . subtilis [41–43] . Since σK in C . difficile lacks a pro-sequence , the main period of σK activity may be mainly dictated by the time of CD1234 production and skinCd excision ( Fig 9 ) . In B . subtilis , both the sigK and spoIVCA genes are controlled by a coherent FFL involving σE and SpoIIID [19] . Thus , both the SpoIVCA-mediated skinBs excision and the transcription of sigK are delayed towards the end of the engulfment sequence , when the forespore signal that leads to pro-σK processing results in “just-in-time” activation of σK . In C . difficile , transcription of CD1234 and sigK decreased in a spoIIID mutant . We detected a common binding motif in the promoter region of these genes suggesting a direct effect of SpoIIID on their transcription . This motif is also present upstream of spoIIID itself allowing us to propose an auto-regulation for SpoIIID and to define a first consensus for the SpoIIID binding site of C . difficile ( KRTAACARK ) ( S3A and S3B Fig ) sharing partial similarity with the SpoIIID consensus of B . subtilis ( S3C Fig ) [19] . Our results show that expression of CD1234 in C . difficile relies on a coherent FFL involving σE and SpoIIID ( Fig 9B ) [19] . Although transcription of CD1234 is detected in the mother cell soon after asymmetric division and does not increase during or following engulfment completion , it is possible that the CD1234 protein only reaches a threshold level following engulfment completion . The effect of a spoIIID deletion on the transcription of sigK as assessed here using SNAPCd labeling and scoring of cells in which engulfment had been completed is less pronounced than previously reported on the basis of a more sensitive qRT-PCR analysis [18] . It seems possible that binding of SpoIIID to the sigK regulatory region represses the σE-dependent P1 promoter towards the end of the engulfment process , while allowing activation of transcription from P2 ( Fig 3A ) . If so , deletion of spoIIID would allow prolonged utilization of P1 by σE , possibly explaining the reduced effect of the mutation following engulfment completion as seen here ( Fig 3B ) . We do not presently know whether P2 is initially utilized by σE with the help of SpoIIID , and later by σK . Further work is needed to test these possibilities . In any event , positive auto-regulation of sigK transcription at P2 may then increase production of active σK locking the cells in a late-mode of gene expression [16–18] ( Fig 9B ) . This genetic architecture may allow delaying sigK transcription and skinCd excision to ensure that the main period of σK activity follows engulfment completion [17] . Thus , although the pro-sequence is absent from σK , some level of redundancy also appears to be embedded in the activation of σK in C . difficile , with delayed transcription and rearrangement contributing to its timely activation . As we show here , the rearrangement is important , since expression of a skinCd-less sigK gene increases σK activity during engulfment . In this regard , we comment on the possible function of sigK P1 . If indeed this promoter is repressed through binding of SpoIIID to the downstream box , P1 would be subject to a type I incoherent FFL , which could result in a pulse of sigK transcription ( Fig 9B ) [44] . Provided skinCd excision also occurs , a pulse in sigK transcription may result in σK activation during engulfment in some cells in a population ( Fig 9B ) . Given that a skinCd-less sigK allele leads to alteration in the spore surface structure , we speculate that this regulatory scheme could produce spores with different structure and functional properties in a fraction of the population . The presence of a skin element remains an exception in clostridia . Although the still more familiar designation of C . difficile was used herein , the organism differs significantly from typical Clostridial species , and was recently placed in the Peptostreptococcaceae family and renamed Peptoclostridium difficile [45] . Nevertheless , a prophage integrated into sigK is found in Clostridium tetani [46] , in some strains of C . botulinum and in a strain of C . perfringens [47] . Absence of a skin element may be related to the involvement of σK in functions in addition to its role as a late mother cell-specific transcription factor . For instance , in C . acetobutylicum and C . botulinum , σK is required for entry into sporulation and for solventogenesis in the former organism and for cold and salt stress in the latter , while in C . perfringens σK is also involved in enterotoxin production [48] . It thus seems likely that skinCd together with the compartmentalized expression of CD1234 helps preventing ectopic and heterochronic activity of σK . This suggestion is in agreement with the observation that bypassing both the recombination and pro-protein processing levels of control leads to some activity of σK during stationary phase under conditions that do not support efficient sporulation by B . subtilis [42] . This inappropriate activity of σK is most likely the result of auto-regulation , and shows that even in B . subtilis , transcriptional control alone is not sufficient to restrict the activity of σK to the mother cell during late stages of spore development but also for proper sporulation as a pro-less skin-less strain of B . subtilis shows reduced sporulation [41 , 42] . In contrast , the early activity of σK in the ΔskinCd strain of C . difficile , while causing alterations in coat assembly , does not affect the final titer of heat resistant spores . It is possible that the sporulation program in C . difficile is more permissive to changes in the proper timing of morphogenetic events [16–18] . It is also possible , however , that an additional as yet unidentified mechanism , controls the activity of σK in C . difficile . Why the expression of CD1231 is not restricted to the mother cell is intriguing and might suggest the possible existence of other functions than skin excision for this recombinase . CD1231 may also function occasionally in the forespore or in vegetative cells , in the absence of CD1234 , resulting in permanent elimination of skinCd , as it is known that some strains of C . difficile including epidemic strains lack this element [27 , 28 , 49] . This possibility also raises the question of whether in those strains σK is recruited to additional functions , by analogy with C . perfringens , C . botulinum and C . acetobutylicum . Importantly , as we show that the premature activity of σK during sporulation in C . difficile imposes alterations to the assembly of the spore surface layers , we speculate that spores of the skinCd-less epidemic strains may have alterations in structural and/or functional properties important for spore persistence and infection . Reminiscent of the situation with skin , whose deletion alters assembly and function of the spore surface layers , it is noteworthy that the B . subtilis SPβ prophage is inserted into the spsM gene , required for the glycosylation of proteins at the spore surface , and that SPβ excision during sporulation depends on the mother cell-specific expression of the SPβ gene coding for the SprB RDF . Remarkably , SPβexcision during sporulation does not result in the assembly of phage particles , most likely because the prophage genes lack mother cell-specific promoters [32] . In different spore-formers , several other sporulation genes such as spoVFB encoding the dipicolinate synthase and spoVR involved in cortex formation are interrupted by phage-like elements that are able to excise during sporulation [32 , 50] . Moreover , developmentally-controlled , recombinase-dependent excision of phage-like intervening elements also takes place in cyanobacteria where these elements , interrupting genes required for nitrogen fixation , are excised during heterocyst differentiation [51] . Insertion of phages or phage like elements in genes that are specifically expressed in terminal cell lines ensures their vertical transmission and minimizes cost for the host . However , regulated prophage excision may also occur in non-terminally differentiated cells . The Listeria monocytogenes comK gene , coding for the competence master regulatory protein , is interrupted by a prophage [52] . The DNA-uptake complex formed by the Com proteins is required for L . monocytogenes to escape from macrophage phagosomes and remarkably , excision of the comK intervening sequence is specifically induced during intracellular growth within phagosomes [52] . Also remarkably , prophage excision from comK as observed for SPβ excision in B . subtilis sporangia does not result in the production of phage particles [52] . Clearly , whether or not in terminally differentiated cells , host “domestication” of prophage excision , may lead to additional levels of genetic control over important cellular functions [22 , 53] .
C . difficile strains and plasmids used in this study are presented in Table 3 . C . difficile strains were grown anaerobically ( 5% H2 , 5% CO2 , and 90% N2 ) in Brain Heart Infusion ( BHI , Difco ) , which was used for selection of conjugants , in TY ( Bacto tryptone 30 g . L-1 , yeast extract 20 g . L-1 , pH 7 . 4 ) or in sporulation medium ( SM ) [54] , which was used for sporulation assays . SM medium contained per liter: 90 g Bacto tryptone , 5 g Bacto peptone , 1 g ( NH4 ) 2SO4 , 1 . 5 g Tris Base . When necessary , cefoxitin ( Cfx; 25 μg/ml ) , thiamphenicol ( Tm; 15 μg . ml-1 ) or erythromycin ( Erm; 2 . 5 μg . ml-1 ) was added to C . difficile cultures . E . coli strains were grown in LB broth . When indicated , ampicillin ( 100 μg . ml-1 ) or chloramphenicol ( 15 μg . ml-1 ) was added to the culture medium . The antibiotic analog anhydrotetracycline ( ATc; 100 ng . ml-1 ) was used for induction of the Ptet promoter present in derivatives of the C . difficile pDIA6103 vector [34] . Sporulation assays were performed as follows . After 12 h , 18 h , 24 h , 48 h or 72 h of growth in SM medium , 1 ml of culture was divided into two samples . To determine the total number of cells , the first sample was serially diluted and plated on BHI with 0 . 1% taurocholate ( Sigma-Aldrich ) to ensure efficient spore germination [5] . To determine the number of spores , the vegetative bacteria of the second sample were heat killed by incubation for 30 min at 65°C prior to plating on BHI with 0 . 1% taurocholate . The rate of sporulation was determined as the ratio between the number of spores/ml and the total number of bacteria/ml ( x100 ) . The ClosTron gene knockout system [55] was used to inactivate the CD1231 and CD1234 genes in the strain 630Δerm , to produce strains CDIP526 ( CD1231::erm ) and CDIP396 ( CD1234::erm ) ( Table 3 ) . Primers to retarget the group II intron of pMTL007 to these genes ( S1 Table ) were designed using the Targetron design software ( http://www . sigmaaldrich . com ) . The PCR primer sets were used with the EBS universal primer and intron template DNA to generate , by overlap extension PCR , a 353-bp product that would facilitate intron retargeting to CD1231 or CD1234 . The PCR products were cloned between the HindIII and BsrGI sites of pMTL007 to yield pDIA6346 ( pMTL007::Cdi-CD1231-206a ) and pDIA6314 ( pMTL007::Cdi-CD1234-89a ) . pDIA6346 and pDIA6314 were introduced into E . coli HB101 ( RP4 ) and the resulting strains subsequently mated with C . difficile 630Δerm . C . difficile transconjugants were selected on BHI agar containing Tm and Cfx and then plated on BHI agar containing Erm . Chromosomal DNA of transconjugants was purified using the Instagene kit ( Biorad ) . The Erm resistance phenotype was due to the splicing of the group I intron from the group II intron following integration , as shown by PCR using the ErmRAM primers ( ErmF and ErmR ) . To verify integration of the Ll . LtrB intron into the right gene targets , we used PCR with two primers flanking the insertion site in CD1231 ( IMV736/IMV737 ) or CD1234 ( IMV695/IMV696 ) , the intron primer EBSu and the CD1231- ( IMV736 ) or CD1234-specific primers ( IMV696 ) ( S2 Fig ) . The Southern blot probe was generated by PCR using pMTL007 as a template and the primer pair OBD522 and OBD523 ( S1 Table ) , yielding a 374 bp PCR product that hybridized with the group II intron . Southern blot analyses were performed as previously described [17 , 18] . For complementation studies , the CD1231 gene with its promoter ( positions -192 to +1569 from the translational start site ) and the CD1234 gene with its promoter ( -168 to +343 from the translational start site ) were amplified by PCR using primers IMV728 and IMV729 or IMV695 and IMV696 , respectively ( S1 Table ) . The PCR fragments were cloned between the XhoI and BamHI sites of pMTL84121 [56] to produce pDIA6307 ( CD1234 ) and pDIA6348 ( CD1231 ) . These plasmids were introduced into E . coli HB101 ( RP4 ) and then transferred by conjugation into CDIP526 ( CD1231::erm ) to produce CDIP533 , and into CDIP396 ( CD1234::erm ) to produce CDIP397 ( Table 3 ) . To construct transcriptional SNAPCd fusions to the CD1231 and CD1234 promoters , 485 and 139 bp DNA fragments containing the promoter region of each gene were PCR-amplified using genomic DNA from strain 630Δerm and primer pairs CDsigK3’ Fw/CDsigK3’_EcoRI Rev or IMV677/IMV678 , respectively . These fragments were cloned into pFT47 [17] to create pMS469 and pDIA6192 ( Table 3 ) . Plasmid pDIA6192 was transferred to 630Δerm , 630Δerm spoIIID::erm or 630Δerm sigE::erm and pMS469 was transferred to 630Δerm by conjugation from derivatives of E . coli HB101 ( RP4 ) ( Table 3 ) . Primers PCDsigK Fw and CDsigK5’Rev ( S1 Table ) were used to amplify a fragment from the sigK gene using pFT38 [17] as the template . The resulting 819 bp fragment encompasses 415 bp of the sigK regulatory region and the 5´-end of the interrupted coding region , 404 bp downstream of the sigK translational start site [18] . This fragment was cleaved with NotI and EcoRI . In a second PCR , the region containing the 3´-end of the coding sequence of sigK , and 180 bp upstream of this position , was PCR amplified using primers RecFP-Fw and SigK-SNAP Rev , yielding a 445 bp fragment . This fragment was cleaved with EcoRI and BamHI . The two fragments were inserted between the NotI and BamHI sites of pFT58 to produce pFT74 ( Fig 4A ) . pFT74 was transferred by conjugation into C . difficile 630Δerm and 630Δerm spoIIID::erm ( Table 3 ) . To express the CD1234 gene under the control of a Ptet inducible promoter , the CD1234 gene with its ribosome-binding site ( positions -19 to + 343 from the translational start codon ) was amplified using primers IMV720 and IMV696 ( S1 Table ) . The resulting PCR product was digested with StuI and BamHI and cloned into pDIA6103 , a derivative of pRPF185 lacking the gusA gene [34] . Using the E . coli HB101 ( RP4 ) strain containing either pDIA6103 or pDIA6353 ( pDIA6103-CD1234 ) , these plasmids were transferred by conjugation into C . difficile 630Δerm , 630Δerm spoIIID::erm , 630Δerm sigE::erm , 630Δerm CD1234::erm or 630Δerm CD1231::erm . The various strains of C . difficile containing either pDIA6103 or pDIA6103-CD1234 ( Table 3 ) were grown overnight in TY containing 0 . 025% of taurocholate . The pre-cultures were diluted 100-fold in TY medium and the resulting cultures incubated at 37°C for 4 h . To induce expression of CD1234 , ATc ( 100 ng/ml ) was then added to the medium . After 2 h of incubation , the cells were harvested by centrifugation and the chromosomal DNA was extracted from each strain using the Puregene Yeast/Bact kit ( QIAGEN ) . To quantify the reconstruction of sigK associated to skinCd excision , quantitative PCR ( qPCR ) was performed using primers upstream ( QRTBD325 ) and downstream ( QRTBD326 ) of the skinCd insertion site . A qPCR of the DNA-PolIII gene was used as a control . For each strain , a dilution of the culture was also plated on BHI . Eight clones per strain were re-isolated on BHI plates , and DNA was extracted from each using the Instagene kit ( Biorad ) . To detect the presence of skinCd into the chromosome , the 5’ junction of the skinCd element was PCR-amplified using primers OBD0742 ( in CD1231 ) and QRTBD326 ( in sigK ) . A derivative of strain 630Δerm lacking the skinCd was obtained as follows . Strain 630Δerm ( pDIA6103-CD1234 ) was grown 4 h in TY . After inducing expression of CD1234 expression , the cells were serially diluted , plated on BHI and DNA extracted for several clones . To verify skinCd excision , PCR was performed with primers flanking the site of skinCd insertion ( QRTBD326 and IMV833 ) or the 5’ junction of the skinCd element ( OBD0742 in CD1231 and QRTBD326 in sigK ) . One clone in which sigK was amplified and lacking the 5’ junction of skinCd skin was selected . To cure the selected clone of pDIA6103-CD1234 , cells were diluted 6-fold in TY medium before plating on BHI . About 70% of the clones obtained after plating were TmS and had lost pDIA6103-CD1234 as checked by PCR . Total RNA was isolated from strains 630Δerm and 630Δerm Δskin , from the CD1231 and CD1234 mutants and from the complementation strains . These strains were grown in SM medium , the cells were collected by centrifugation , resuspended in RNApro™ solution and RNA extracted using the FastRNA Pro Blue Kit , according to the manufacturer’s instructions ( MP Biomedicals ) . Quantitative real-time PCR ( qRT-PCR ) analysis was performed as previously described [57] . The primers used for each marker are listed in S1 Table . In each sample , the quantity of cDNAs of a gene was normalized to the quantity of cDNAs of the DNApolIII gene . The relative change in gene expression was recorded as the ratio of normalized target concentrations ( ΔΔCt ) [58] . The CD1234 coding region was PCR-amplified using primers CD1234-Fw and -Rev . The resulting 252 bp DNA fragment was cleaved with NdeI and XhoI and cloned between the same sites of pET33b ( Novagen ) , creating pMS484 . CD1234 fused to the His6-tag was PCR-amplified from pMS484 with primers CD1234 Fw and T7ter , cleaved with NdeI and cloned between the NdeI and EcoRV sites of pETDuet-1 , to create pMS494 . The CD1231 coding region was amplified using primers CD1231-Fw and -Rev . The resulting 1518 bp fragment was digested with NcoI and SalI and ligated to pFN127 ( a pET16b derivative carrying a Strep-tag ) to yield pMS486 . CD1231 fused to the Strep-tag was removed from pMS486 by digestion with NcoI and XhoI and cloned into pETDuet-1 producing pMS499 , or in pMS494 to give pMS500 . We used pMS499 and pMS500 ( see above ) and CD1231-specific primers to convert the serine codon at position 10 to an alanine codon producing pMS512 and pMS513 , respectively . Derivatives of BL21 ( DE3 ) were constructed bearing pFT74 and pMS494 and either pMS499 , pMS500 , pMS512 , or pMS513 . The resulting strains were grown to an OD600nm of about 0 . 6 , induced with 1 mM isopropyl-D-thiogalactopyranoside ( IPTG ) , and incubated for 3 h before the cells were harvested . Plasmid DNA was extracted and analyzed by cleavage with NotI and BamHI . pFT74R results from miniskinCd excision , by recombination , from pFT74 . The attP site was obtained by PCR using IMV825 and IMV824 and DNA extracted from 630Δerm after 24 h of growth in SM . The PCR product was first cloned into pGEM-T-easy ( Promega ) and the attP site was then released with NotI and inserted into pFT74R , in both directions , to produced pMS510 and pMS511 . Derivatives of BL21 ( DE3 ) were constructed , bearing pMS510 or pMS511 and either pMS494 , pMS499 , pMS500 , pMS512 , or pMS513 . The various strains were grown to an OD600nm of about 0 . 6 , induced with 1 mM IPTG , and incubated for 3 h before the cells were harvested . Plasmid DNA was extracted and cleaved with BamHI and NotI for the detection of recombination events . Derivatives of BL21 ( DE3 ) bearing plasmids for the production of CD1231-Strep , CD1234-His6 or Tgl-His6 , were grown to mid-log phase ( OD600nm≈0 . 6 ) in LB , and induced with 1 mM IPTG for 3 hours before the cells were harvested by centrifugation . The cell sediment was resuspended in 1-ml portions of buffer A [100 mM NaCl , 10 mM Tris-HCl ( pH 8 . 0 ) , 10% glycerol] per 50 ml of induced culture and lysed in a French pressure cell ( 18 , 000 lb/in2 ) . In one set of assays , the lysates containing CD1231-Strep , CD1234-His6 , or both , were cleared by centrifugation and 1 ml of each lysate was independently incubated with 50 μl of a 50% slurry of Ni2+-NTA agarose ( Qiagen ) at room temperature for 30 min . The Ni2+-NTA agarose was washed three times in buffer B ( same as A but with 200 mM NaCl ) . In another set of assays , lysates containing either CD1234-His6 or Tgl-His6 were incubated with the Ni2+-NTA beads as above , and then incubated with a CD1231-Strep-containing extract for a further 30 min at room temperature . The beads were washed as described above . In all cases , the washed beads with bound proteins were resuspended in a final volume of 30 μl . The samples were resolved on 15% SDS-PAGE and subject to immunoblotting . Anti-His6 or anti-Strep antibodies were used at a 1:1000 dilution , and a rabbit secondary antibody conjugated to horseradish peroxidase ( from Sigma ) was used at a 1:10000 dilution . The immunoblots were developed with enhanced chemiluminescence reagents ( Amersham Pharmacia Biotech ) . For spore production , 5 ml of BHI was inoculated with of C . difficile and grown for 12 h at 37°C in anaerobic conditions . Fresh BHI ( 125 ml ) was then inoculated with 1 . 25 ml of the pre-inoculum and the cultures incubated at 37°C under anaerobic conditions for 7 days . Cells were collected by centrifugation ( for 10 min at 4800xg ) , resuspended in cold water and stored overnight at 4°C . Spores were then purified with a 42% Gastrografin ( Schering ) step gradient , as previously described [59] . The pellet was washed 10 times with cold water , and stored at -20°C . To analyze the profile of spore coat proteins , the spores were resuspended in extraction buffer ( 0 . 125 mM Tris-HCl , 5% β-mercaptoetanol , 2% SDS , 0 . 025% bromophenol blue , 0 . 5 mM DTT , 5% glycerol , pH 6 . 8 ) to a final OD580 of 4 , and boiled . The extracted proteins were resolved by 15% SDS-PAGE and visualized by Coomassie brilliant blue R-250 staining . For identification , protein bands were excised and digested with trypsin , before analysis by matrix-assisted laser desorption ionization mass spectrometry . Samples ( 1 ml ) were withdrawn from SM cultures at the desired times following inoculation , and the cells collected by centrifugation ( 4000 xg , for 10 min , at 4°C ) . The cells were washed with 1ml of PBS and resuspended in 0 . 1 ml of PBS supplemented with the membrane dye Mitotracker Green ( MTG ) at 0 . 5 μg . ml-1 and the DNA stain DAPI ( 4' , 6-diamidino-2-phenylindole; 50 μg . ml-1 ) ( Invitrogen ) . For SNAP staining , culture samples of 1 ml were stained for 30 min with 250 nM SNAP-Cell TMR-Star ( New England Biolabs ) as described before [17] . Cells were washed four times by centrifugation ( 4000 g , 5 min ) and ressupended in 1ml of PBS . The cells were resuspended in 1 ml of PBS containing 0 . 5 μg . ml-1 of MTG . Cells were mounted on 1 . 7% agarose coated glass slides and imaged as previously described [60] . Images were analyzed using the Metamorph software suite version 5 . 8 ( Universal Imaging ) . For quantification of the SNAPCd-TMR Star signal , 6x6 pixel regions were defined in the desired cell and the average pixel intensity was calculated , and corrected by subtracting the average pixel intensity of the background . Small fluctuations of fluorescence among different fields were corrected by normalizing to the average pixel intensity obtained for the intrinsic autofluorescence of C . difficile cells [61] . For thin sectioning transmission electron microscopy ( TEM ) analysis , C . difficile were purified by density gradient centrifugation as described above . Samples were processed for TEM as described previously [62] . | Clostridium difficile , a major cause of antibiotic-associated diarrhea , produces resistant spores that facilitate its persistence in the environment including hospitals . C . difficile transmission is mediated by contamination of gut by spores . Understanding how this complex developmental process is regulated is fundamental to decipher the C . difficile transmission and pathogenesis . A less tight connection between the forespore and mother cell lines of gene expression is observed in C . difficile compared to Bacillus subtilis especially at the level of the late sigma factor , σK . In C . difficile , the sigK gene is interrupted in most of the strains by a prophage-like intervening sequence , skinCd , which is excised during sporulation . Contrary to B . subtilis , CD1231 encoding the large serine recombinase required for skinCd excision , is constitutively expressed and a recombination directionality factor , whose synthesis is detected only in the mother cell , restricts skinCd excision to this terminal cell . These two proteins are necessary and sufficient to trigger skinCd excision promoting the timely appearance of σK , which in turn switches-on late sporulation events . While several strains of C . difficile lack a skin element , we show that deletion of skinCd results in premature σK activity and in spores with altered surface layers , a property that might be important for host colonization . | [
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] | 2016 | A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile |
Giardia duodenalis is highly endemic in East Africa but its effects on child health , particularly of submicroscopic infections , i . e . , those below the threshold of microscopy , and of genetic subgroups ( assemblages ) , are not well understood . We aimed at addressing these questions and at examining epidemiological characteristics of G . duodenalis in southern highland Rwanda . In 583 children <5 years of age from communities and health facilities , intestinal parasites were assessed by triplicate light microscopy and by PCR assays , and G . duodenalis assemblages were genotyped . Cluster effects of villages were taken into account in statistical analysis . The prevalence of G . duodenalis as detected by microscopy was 19 . 8% but 60 . 1% including PCR results . Prevalence differed with residence , increased with age , and was reduced by breastfeeding . In 492 community children without , with submicroscopic and with microscopic infection , underweight ( weight-for-age z-score <−2 standard deviations ) was observed in 19 . 7% , 22 . 1% , and 33 . 1% , respectively , and clinically assessed severe malnutrition in 4 . 5% , 9 . 5% , and 16 . 7% . Multivariate analysis identified microscopically detectable G . duodenalis infection as an independent predictor of underweight and clinically assessed severe malnutrition . Submicroscopic infection showed respective trends . Overall , G . duodenalis was not associated with gastrointestinal symptoms but assemblages A parasites ( proportion , 13% ) were increased among children with vomiting and abdominal pain . The prevalence of G . duodenalis in high-endemicity areas may be greatly underestimated by light microscopy , particularly when only single stool samples are analysed . Children with submicroscopic infections show limited overt manifestation , but constitute unrecognized reservoirs of transmission . The predominance of assemblage B in Rwanda may be involved in the seemingly unimposing manifestation of G . duodenalis infection . However , the association with impaired child growth points to its actual relevance . Longitudinal studies considering abundant submicroscopic infections are needed to clarify the actual contribution of G . duodenalis to morbidity in areas of high endemicity .
Giardia duodenalis ( syn . G . intestinalis , G . lamblia ) is among the most common intestinal protozoa and is the most frequent parasitic agent of gastroenteritis worldwide . The regional prevalences of infection differ enormously and may be >30% in children in the African and the Eastern Mediterranean region [1] . Although G . duodenalis is known for causing gastrointestinal symptoms , such as acute or chronic diarrhoea , bloating , and stomach cramps , asymptomatic infections may occur , particularly in highly endemic areas , and there is also evidence for protection against acute diarrhoea in infected individuals [2]–[4] . Chronic ( or recurrent ) infection has been associated with malnutrition , wasting and stunting , most likely due to malabsorption caused by the parasites , and with reduced cognitive functions at later age [5] . The pathogenetic determinants are poorly understood but may involve both host and parasite factors [2] . As for the latter , eight major genetic groups of G . duodenalis have been revealed , i . e . , assemblages A1 , A2 , and B–H with A1 , A2 , and B being considered pathogenic in humans [6] , [7] . Assemblages have inconsistently been linked with symptoms: assemblage A parasites have been associated with more severe clinical symptoms as compared to assemblage B parasites in Australia , Bangladesh , Peru , Spain , and Great Britain [8]–[13] , but the opposite has been reported from the Netherlands and Ethiopia [14] , [15] , and no association in Brazil [16] . Assemblage-associated differences in the pathogenesis of giardiasis have been observed in murine studies where the infection with clone GS ( assemblage B ) , but no with clone WB ( assemblage A ) , caused disaccharide deficiency in infected animals [17] . Diagnosis of G . duodenalis is traditionally based on the detection of the parasites by light microscopy in direct stool smears or following concentration techniques , e . g . , by formalin-ethyl acetate centrifugation . Multiple rather than single sample testing is recommended to improve sensitivity but this is often difficult to implement . Immunoassays are of superior sensitivity but also more expensive [18] , [19] . Similarly , highly sensitive PCR-based methods have been developed but rarely applied in developing countries so far [20] . Limited evidence shows that prevalence estimates based on PCR may exceed those derived by microscopy , e . g . , recently in western Uganda where up to two thirds of people in rural areas were found to be ( asymptomatically ) infected [21] . No data are available , however , regarding the epidemiology and clinical significance of submicroscopic G . duodenalis infections , i . e . , infections detected by PCR but not by light microscopy , in highly endemic areas . East Africa is considered to be among the most endemic regions for G . duodenalis surpassed only by the Indian subcontinent [22] although recent community-based figures are rare . In the present study from southern Rwanda , we aimed at assessing prevalences and epidemiological features of G . duodenalis infection among children in communities and health facilities applying both microscopy and PCR for diagnosis . Also , we aimed at examining the influence of G . duodenalis infection , particularly of submicroscopic infection and different assemblages , on the children's clinical status .
All children's parents were thoroughly informed on the purpose and procedures of the study , and recruitment was preceded by HIV pre-counseling and obtaining informed written consent from the children's parents . The study was reviewed and approved by the National Ethics Committee , Republic of Rwanda . The study was conducted from January to March 2010 , i . e . during a delayed rainy season , in Butare and the neighbouring rural Huye subdistrict . Butare ( population approximately 100 , 000; altitude 1 , 768 m ) is the capital of Huye district , southern province of Rwanda . Located on the country's central plateau ( average altitude , 1 , 700 m; yearly rainfall , 1 , 200 mm; mean temperature , 19°C ) , the city is surrounded by densely populated farmland hills . Governmental health services in the area are provided by several primary health centres , Kabutare district hospital and the University Teaching Hospital of Butare ( CHUB , Centre Hospitalier Universitaire de Butare ) . Due to Rwanda's mandatory health insurance system , treatment of common diseases is basically free of charge including utilization of district and provincial hospitals provided there is adherence to a strict referral system [23] . The study was designed as a cross-sectional survey to assess the prevalences of malaria , HIV , and intestinal pathogens in children under five years of age in the CHUB catchment area , i . e . , at the levels of community , health center , and district hospital . The present report focuses on G . duodenalis . Details of the study among a total of 749 children have been described previously [24] . Briefly , for the community level and based on most recent census data , each 25 households were randomly chosen by bottle-spin in a total of 24 randomly selected villages ( out of 45 ) in the rural Huye subdistrict ( population approximately 20 , 000; Figure 1 ) . Community health workers randomly selected one child per family , and asked the child to be presented to the study team located at Sovu health center ( or a non-permanently staffed branch ) on a scheduled ( usually next ) day ( n = 545 ) . In parallel and during 16 and 11 days , respectively , 103 and 101 pediatric health facility attendants aged five years or less and presenting with a health problem at the primary Sovu health center and at the referral Kabutare district hospital , respectively , i . e . the health facilities serving this population , were consecutively recruited . Leading primary diagnoses in the health facility attendants were respiratory tract infection , gastro-intestinal tract affection , and malaria . Clinical details are reported elsewhere [24] . Brief questionnaires were filled in on socio-economic aspects of the children's families including household assets . Age , sex , weight , and fever ( axillary temperature ≥37 . 5°C ) were documented . Breastfeeding was documented as any vs . none . Using standardized forms , children were examined by a physician and a medical history was obtained . For the latter , parents were specifically asked about the presence of gastrointestinal and other symptoms during the preceding two weeks , e . g . , vomiting , diarrhoea , and abdominal pain . For this study , we used two categories to describe impaired child growth and/or malnutrition , i . e . underweight and clinically assessed severe malnutrition . Underweight was defined as a weight-for-age z ( WAZ ) -score of <−2 standard deviations ( SD ) following calculation of WAZ-scores using WHO Anthro [25] . Clinically assessed severe malnutrition was evaluated by the study physician ( CSh ) based on clinical signs , growth percentiles and/or mid-upper-arm circumference . Malaria [24] , urinary tract infection ( Multistix 10SG , Bayer , Germany ) , and other diseases were treated according to Rwanda health authority guidelines [26] . In the communities , stool collection containers were handed to the parents who were instructed to bring a fresh sample of the selected child at the scheduled day of examination . In the health facilities , fresh samples were collected . During presentation of the children , intestinal pathogens were screened for by direct wet mount microscopic stool examination . A second microscopic examination was performed later on the examination day following the ether-based concentration technique [27] . Aliquots of stool were conserved in merthiolate-iodine-formaldehyde ( MIF ) , transported to Berlin and once more examined microscopically after ether-based concentration . Triplicate microscopy of identical samples was performed to identify potential flaws in the diagnostic procedures and to reduce inter-observer variability . Of the 749 children , 86 , 1% ( 645 ) provided a stool sample: 95 . 4% of the community children ( 520/545 ) but only 61 . 3% ( 125/204 ) of health facility attendants . Overall , 622 stool samples could be assessed by all three microscopic examinations . Quantification of pathogens was omitted . Samples were considered microscopically positive for a given parasite if it was detected in at least one of the three assays , and negative if negative in all three assays . For molecular assays , aliquots of stool were stored at −70°C and transported on dry ice to Berlin . Of the 622 microscopically assessed samples , 583 were available for PCR analysis . DNA was extracted by commercial kits ( Qiamp DNA Stool Mini Kit , Qiagen ) . For each sample , phocine herpesvirus 1 ( PhHV-1 ) , kindly provided by Dr . Martin Schutten , Department of Virology , Erasmus MC , Rotterdam , The Netherlands , was added to the isolation lysis buffer to serve as an internal control for the extraction process [28] . Infections with Necator americanus , Ancylostoma duodenalis , or Ascaris lumbricoides were detected by real-time PCR assays [29] , [30] . A multiplex real-time PCR assay with pathogen-specific fluorescent detection probes was performed to identify G . duodenalis , Entamoeba histolytica ( or E . dispar ) , and Cryptosporidium parvum [20] . Real-time PCR assays were run on a Lightcycler 480 ( Roche Diagnostics ) including controls from microscopically confirmed , positive G . duodenalis samples as well as negative controls . Cycle threshold ( Ct ) values of >36 were considered to reflect limited reproducibility due to low copy numbers , and all respective assays were repeated . For G . duodenalis , the real-time PCR was repeated for 62 samples with an initial Ct-value of 36 . 01–45 . 1 ( median , 37 . 7 ) , of which 47 were reproducibly positive ( Ct-value , range , 21 . 6–38 . 3; median , 35 . 0 ) while 15 were negative . No sample had to be excluded because of evidence of fecal inhibitory factors ( Ct-value for the internal control PhHV-1 >36 ) . For G . duodenalis genotyping , stool DNA samples were analysed by a previously described method with slight modifications [31] . A DNA fragment of the Giardia triosephosphate isomerase ( TPI ) gene was amplified by nested PCR using primer TPI-f1 , AAATYATGCCTGCTCGTCG and TPI-r1 , CAAACCTTYTCCGCAAACC for the first PCR reaction and primer TPI-f2 , CCCTTCATCGGNGGTAACTT and TPI-r2 GTGGCCACCACNCCCGTGCC for the second PCR reaction . The PCR products were purified ( QIAquick , Quiagen , Hilden , Germany ) and unidirectional sequencing reactions were performed with the ABI Big dye 3 . 1 Terminator kit using TPI-f2 primer and send for in-house sequencing service at the Robert Koch-Institute . Sequences were analyzed with Geneious Pro software tool ( Biomatters Ltd . , Auckland , New Zealand ) . To determine Giardia genotypes , a sequence fragment from base 610 to 1052 of a reference sequence ( Genbank accession number L02120 ) was compared to previously defined TPI references [32] . The further subdivision of assemblage B isolates into subtypes based on analyzing the tpi locus was not attempted because of the known heterozygosity at this locus [32] , which we also observed in the majority of isolates typed here . Complete data sets were available for 583 children . Data analysis was performed using SPSS for Windows ( release 18 ) and SAS for Windows ( release 9 . 2 ) ( SAS Institute Inc . ) . Descriptive analyses include absolute frequencies and percentages or means , medians , and ranges . Evaluation of determinants of G . duodenalis infection and of clinical symptoms was performed by simple and multiple logistic regression analysis , and odds ratios were reported . Confirmatory analyses ( means , percentages , P-values , significance tests , 95% confidence intervals ( 95% CIs ) ) were adjusted for intracluster correlations ( cluster = villages ) by use of Generalized Estimating Equations ( GEE; [33] , exchangeable correlation structure for subjects within identical villages ) . Of note , this leads to discrepancies between “naively” computed percentages from absolute frequencies and reported percentages . Therefore , prevalences reported herein are weighed with respect to differing cluster sizes and do not necessarily correspond to the additionally given absolute numbers . A P-value≤0 . 05 was considered statistically significant .
Gastrointestinal parasites were assessed in all children ( Table 2 ) . G . duodenalis was the most prevalent parasite identified , detected by microscopy in 19 . 8% ( 114/583 ) and by PCR in 59 . 7% ( 366/583 ) ( Table 2 ) . All but two of the microscopically positive samples were also detected by PCR ( i . e . , total positivity , 60 . 1% ( 368/583 ) ) . In 51 . 3% ( 254/469 ) of samples diagnosed as negative for G . duodenalis by microscopy , PCR yielded a positive result . Setting G . duodenalis detected by any means as reference , the sensitivity of microscopy in detecting the parasite was 30 . 8% ( 95%CI , 25 . 3–36 . 8% ) and that of PCR was 99 . 5% ( 95%CI , 97 . 9–99 . 9% ) . Of 208 successfully typed isolates , 85 . 9% ( 179/208 ) were assemblage B and 12 . 7% ( 27/208 ) were assemblage A2 , in addition to one assemblage A1 and one mixed assemblage A+B . Infections with assemblage B isolates were more frequently submicroscopic ( 53 . 2% , 95/179 ) than infections caused by assemblage A ( 28 . 0% , 7/28; P = 0 . 02 ) . A . lumbricoides was the second most common intestinal pathogen affecting almost one third of the children . Other intestinal pathogens were comparatively rare ( Table 2 ) . The presence of G . duodenalis was positively associated with the apathogenic species E . coli , E . dispar , and I . bütschlii , and negatively with C . parvum and E . histolytica ( each , P<0 . 05 ) . Overall , 18 . 0% , 33 . 3% , 25 . 9% , 13 . 2% , and 9 . 6% of the children harbored 0 , 1 , 2 , 3 or ≥4 , respectively , of the parasites listed in Table 2 . The most common constellations were G . duodenalis mono infections ( 22 . 1% ) , followed by co-infestations with G . duodenalis and A . lumbricoides ( 9 . 8% ) or with E . coli ( 5 . 7% ) , and triple infestations with G . duodenalis , A . lumbricoides , and E . coli ( 3 . 9% ) . Assemblages A and B did not associate with any other intestinal parasite species ( data not shown ) . Table S1 displays the distribution of intestinal parasites separated for community children and health facility attendants . Factors potentially associated with the presence of G . duodenalis were analyzed including available socio-economic data . G . duodenalis was more common in community children ( 65 . 2% ) than in health facility attendants ( 46 . 4%; P = 0 . 001 ) ( Table 3 ) , and varied with residence ( P = 0 . 002 ) . Among community children , prevalences in the villages ranged from 26 . 7% to 87 . 0% ( P<0 . 0001 ) but no geographic focus was discernible ( Figure 1 ) . Moreover , age had a prominent impact in community children: the prevalence of G . duodenalis increased from 34 . 1% ( 14/44 ) in infants to 81 . 1% ( 79/90 ) in children ≥4 years of age ( P<0 . 0001 ) . This was not observed in health facility attendants ( P = 0 . 10 ) . Also , the proportion of microscopic among all G . duodenalis infections in community children tended to increase with age . While only 7 . 4% ( 1/15 ) of infections in infants were microscopically detectable , this was the case in 30 . 8% ( 21/68 ) , 30 . 3% ( 26/88 ) , 43 . 3% ( 36/82 ) , and 29 . 3% ( 21/73 ) , in the age groups 1<2 , 2<3 , 3<4 , and 4<5 years , respectively ( P = 0 . 06 ) . Interestingly , breastfeeding reduced the odds of being infected with G . duodenalis ( P = 0 . 04 ) while a high number of siblings showed the opposite effect . In multivariate analysis , G . duodenalis remained positively associated with increasing age many siblings , and negatively with health facility attendance and breastfeeding ( Table 3 ) . Separating into microscopic and submicroscopic infections among community children , only age remained significantly associated in each case ( data not shown ) . Several factors expected to be associated with G . duodenalis were actually not , including parents' education , household income , household assets , the number of individuals living in the household , availability of piped water , and household cattle possession ( data not shown ) . Only 11 children ( 1 . 8% ) had reportedly taken drugs with anti-giardial effects within the preceding two weeks ( metronidazole , 2; mebendazole , 9 ) . In these , G . duodenalis was found in 45 . 5% ( 5/11 ) as compared to 60 . 5% ( 361/581 ) of respectively untreated children ( P = 0 . 27 ) . For assemblages B and A , no differences with respect to the above mentioned factors were discernible . In particular , similar proportions of G . duodenalis were of assemblage A in community children ( 14 . 3% , 25/186 ) and health facility attendants ( 16 . 0% , 3/21; P = 0 . 99 ) , and in households with ( 22 . 0% , 6/35 ) and without cattle possession ( 13 . 5% , 22/171; P = 0 . 50 ) . The age ( median months , range ) of infected children with assemblage A or assemblage B parasites did not differ ( 38 . 0 ( 22–59 ) vs . 37 . 0 ( 8–60 ) , P = 0 . 24 ) . A notable exception was residence: in the community children's villages , the proportion of assemblage A among detected G . duodenalis ranged between 0% and 50% ( P = 0 . 02 ) but again no focus was discernible . We focused the analysis of the clinical significance of G . duodenalis infection on community children because of their large sample size and because of the diverse additional morbidity of health facility children [24] . Most community children were asymptomatic: fever and a history of fever were recorded in 3 . 5% ( 17/490 ) and 8 . 5% ( 41/452 ) , respectively , and malaria in 2 . 6% ( 13/492 ) . Also , gastrointestinal symptoms were rare . Frequencies of loss of appetite , diarrhoea , vomiting , and abdominal pain did not differ between children with and without G . duodenalis ( Table 4 ) . Defining giardiasis as at least one of the before mentioned gastrointestinal symptoms in the presence of G . duodenalis , 69 of 492 community children were affected ( based on microscopic diagnosis only , 21 of 492 ) . Abdominal distension tended to be increased in infected children [OR , 2 . 94 ( 95%CI , 0 . 94–9 . 20 ) , P = 0 . 07] . Underweight ( WAZ score <−2 SD ) was observed in 23 . 7% ( 117/490 ) of community children . Of note , G . duodenalis infected children more frequently exhibited clinically assessed severe malnutrition [OR , 2 . 15 ( 1 . 39–3 . 35 ) , P = 0 . 0006] than uninfected children . This was paralleled by slightly decreased WAZ scores ( P = 0 . 14 ) and an increased proportion with underweight ( P = 0 . 14 ) among infected children ( Table 4 ) . Looking separately at submicroscopic and microscopic infections , children with microscopic infections exhibited significantly increased odds of clinically assessed severe malnutrition [OR , 3 . 71 ( 2 . 06–6 . 70 ) , P<0 . 0001] and of underweight [OR , 1 . 93 ( 1 . 23–3 . 04 ) , P = 0 . 004] as well as reduced WAZ-scores ( P = 0 . 01 ) , as compared to uninfected children . Children with submicroscopic infections had an intermediate prevalence of clinically assessed malnutrition [OR , 1 . 80 ( 1 . 14–2 . 86 ) , P = 0 . 01] but WAZ scores were not significantly reduced ( P = 0 . 47 ) . In multivariate analysis , G . duodenalis was independently associated with increased odds of clinically assessed severe malnutrition [adjusted OR ( aOR ) , 2 . 06 ( 95%CI , 1 . 26–3 . 39 ) ; P = 0 . 005] , adjusted for lacking paternal education [aOR , 2 . 57 ( 1 . 21–5 . 46 ) ] , and number of siblings [aOR , 1 . 17 ( 1 . 02–1 . 34 ) ] . All other covariates ( age , absence of any household possession , sex , maternal education , household income , possession of cattle , number of additional intestinal parasites ) were not significant . In this model , the adjusted odds of clinically assessed severe malnutrition in microscopic G . duodenalis infection was 3 . 34 ( 1 . 85–6 . 42 , P<0 . 0001 ) while submicroscopic infection showed a smaller effect [aOR , 1 . 76 ( 1 . 00–3 . 09 ) , P = 0 . 05] . An identical approach identified microscopic G . duodenalis infection to be an independent predictor of underweight [aOR , 1 . 83 ( 1 . 12–2 . 99 ) , P = 0 . 02] but not submicroscopic infection [aOR , 1 . 09 ( 0 . 66–1 . 79 ) ] or G . duodenalis infection per se [aOR , 1 . 32 ( 0 . 84–2 . 08 ) ] , adjusted for maternal education , and absent household possessions . Lastly , we examined the clinical relevance of assemblages A and B ( Table 4 ) . The increases in underweight and clinically assessed malnutrition observed for G . duodenalis infection per se were pronounced for assemblage B but less obvious for the small group of children infected with assemblage A parasites . The latter , however , were more frequently affected by abdominal pain ( P = 0 . 02 ) and vomiting ( P = 0 . 03 ) than uninfected children . This was not the case for children with assemblage B isolates . Compared to them , children with assemblage A parasites more frequently reported abdominal pain ( P = 0 . 004 ) and vomiting ( P = 0 . 04 ) .
In rural communities in southern highland Rwanda , G . duodenalis infects two of three predominantly asymptomatic children , is underestimated by conventional microscopy of single stool samples , and contributes to underweight and clinically assessed malnutrition . This suggests an underrated but considerable burden of disease due to G . duodenalis . The prevalence of G . duodenalis of 60% in Rwandan children – who mainly were randomly selected from communities – considerably exceeds figures reported from other parts of the world [1] . The superior sensitivity of PCR in detecting G . duodenalis has recently been shown in Danish patients [34] . In contrast , PCR and microscopy had similar sensitivities in a Dutch study [35] . In our study , light microscopy , which commonly is the only method available in resource-poor areas , failed to detect more than two-thirds of actually present G . duodenalis infections . Repeated microscopic examinations ( which are difficult to implement ) or the use of immunoassays could have reduced this discrepancy . In addition , chronically and/or repeatedly infected individuals in highly endemic settings might shed low cyst numbers . Thus , microscopy-based prevalence figures from high-endemicity areas may be underestimations , and analyses comparing individuals categorized by microscopy into Giardia infected and non-infected may be confounded by a significant number of false negatives . While some false negatives may persist even when applying PCR assays , that risk cannot be determined accurately in the absence of a well defined gold standard . As to the clinical significance of G . duodenalis among community children , we herein describe associations of symptoms with the presence of the parasite . G . duodenalis infection is known to vary widely in clinical manifestation including acute , chronic , and asymptomatic courses [2] . Therefore , we did not attempt to a priori define clinical giardiasis in our study . Nevertheless , no evidence for causation of gastrointestinal symptoms was seen , with the potential exception of abdominal distension . The association with malnutrition , however , was impressive: approximately every third and every sixth child with microscopic infection had underweight and clinically assessed severe malnutrition , respectively , although causation cannot be clarified in a cross-sectional study . Association does not mean causality , and reduced sample sizes in subgroup analyses increases the chance of type 1 statistical errors . Selection bias during recruitment at home , e . g . , due to preferential presentation by the parents of children with ( a recent history of ) sickness cannot completely be excluded . However , recruitment teams were instructed to select children from households randomly . Also , confounding cannot completely be excluded but our results remained stable in multivariate analyses adjusting for socio-economic parameters and for the number of other intestinal pathogens serving as a proxy parameter for polyparasitism ( data not shown ) . Notably in this regard , A . lumbricoides infections found in one third of the children had no clear-cut effect on malnutrition ( data not shown ) , similar to findings from Brazil and Iran [36] , [37] . Clinically assessed severe malnutrition in this study reflects the subjective evaluation of children's status which , nevertheless , was supported by comparison with growth percentiles und MUAC . In interpreting our results , it consequently needs to be kept in mind that this parameter is not standardized . Though , the stronger association with G . duodenalis of clinically assessed severe malnutrition as compared to underweight may also indicate a greater selectivity of the former . Unfortunately , we were unable to analyze the influence of G . duodenalis on stunting and wasting , i . e . , categories based on children's height , because this parameter was inconsistently measured in the field study , had implausible values , and respective Z-scored were consequently omitted . However , our interpretation of malnutrition as a consequence of giardiasis is in line with other studies [37]–[42] . Children with submicroscopic G . duodenalis infection had a prevalence of clinically assessed severe malnutrition , which was intermediate between children without and with microscopic infection . In multivariate analysis , submicroscopic infections still tended to be associated with clinically assessed severe malnutrition but showed no link with underweight . Longitudinal observations and immunological studies may help to understand the relevance of this type of infection and the underlying mechanisms . G . duodenalis assemblage B parasites clearly predominated ( 86% ) , which is in accordance with most larger studies ( recently reviewed in [6] ) . Interestingly , we detected only one co-infection by both assemblage A and B parasites . By random distribution , a much higher proportion would have been expected possibly reflecting cross-assemblage competition or protection . Unfortunately , we were unable to type all Giardia infections . Although the typing rate was higher than in other studies ( e . g . , [21] ) this is most likely due to the fact that typing relied on the single copy tpi gene while diagnosis was based on the amplification of the multicopy rDNA . However , this is unlikely to have resulted in a significant bias in the determination of the prevalence of A and B genotypes in the study population . The success of typing correlated with the ct values in the diagnostic PCR , i . e . the content of target DNA in the samples and this was not significantly different for typed samples independent of their A or B status ( not shown ) . In our study , children infected with assemblage A isolates showed increased proportions of abdominal pain and vomiting while assemblage B infections associated with underweight and clinically assessed severe malnutrition . Much of the available data on the role of assemblages in clinical disease is inconsistent [8]–[16] . Our data are in line , however , with findings from Western Australia where assemblage A parasites were more likely to be found in symptomatic children with diarrhoea while assemblage B parasites were more prevalent in asymptomatic children [8] . Likewise , against high prevalences of assemblage B parasites in Bangladesh , assemblage A isolates were found to be associated with symptomatic disease , i . e . , diarrhoea [9] , [10] . In contrast , abdominal pain in children was associated with assemblage B parasites in Argentina [43] . Further investigations in our study population will help elucidating whether - in contrast to possibly endemic assemblage B parasites - the genetically distinct assemblage A parasites may appear epidemically and thus are more correlated with acute abdominal symptoms . Socio-economic factors had no great influence on the presence of G . duodenalis in the present study . One reason involved may be a limited selectivity of the questionnaires used in this generally poor agricultural and rural population . Also , we were unable to identify hot spots , let alone their characteristics , potentially underlying the pronounced spatial differences in infection prevalence . In multivariate analysis , only the increases of G . duodenalis with children's age and with a high number of siblings as well as the reduction with breastfeeding remained statistically significant . Increasing prevalence with increasing number of siblings supports the role of transmission within households . Protection from G . duodenalis infection by breast milk has previously been reported [44] . Considering the high endemicity of G . duodenalis in the study area it seems plausible that protective antibodies [45] are transmitted by the breast-milk of seropositive mothers . Soluble IgA has also been shown to contribute to the clearance of Giardia spp . in murine studies [46] . In addition , lactoferrin and leukocytes in breast milk could be involved in protection [47] , [48] . The association with impaired child growth as observed in the present and other studies [5] , [37]–[42] suggests control of G . duodenalis infection to be potentially rewarding , e . g . by preventive chemotherapy . Yet , the influence of repeated treatment of G . duodenalis infection on anthropometric development is unclear [49]: evidence for a beneficial impact has been observed in Brazilian children [50] but not in Bangladeshi infants [51] . Beyond that , G . duodenalis infection in highly endemic areas has been associated with protection from acute diarrhea [3] , [4] . Considering the importance of diarrhoea as a leading cause of childhood mortality , such a protective effect might outweigh impaired child growth due to G . duodenalis infection . To date , there is insufficient data to balance the potential effects of anti-Giardia treatment against each other . Lastly , food supplementation in areas highly endemic for G . duodenalis also appears to be complex . G . duodenalis associated protection from diarrhea has recently been reported to be lost in Tanzanian children with multi-nutrient supplementation [4] while vitamin A plus zinc supplementation reduced the incidence of giardiasis in Mexican children [52] . Thus , large-scale longitudinal studies are needed to disentangle the role of G . duodenalis among the interacting factors contributing to malnutrition and diarrhea in high-endemicity regions and to estimate the potential impact of control measures . In conclusion , our data provide evidence of a very high prevalence of G . duodenalis assemblage B without causing diarrhoea but associated with underweight and clinically assessed severe malnutrition in Rwandan children . The underestimation of G . duodenalis by light microscopy suggests that prevalences and consequences have previously been underrated and asks for the implementation of more sensitive diagnostic , yet simple diagnostic tools . The clarification of the clinical significance of G . duodenalis in high endemicity areas needs to take account of an abundance of submicroscopic infections . | Giardia duodenalis is a protozoan parasite causing gastroenteritis . Although the parasite occurs worldwide , its regional prevalence varies considerably . Using PCR as a highly sensitive molecular diagnostic tool , we detected G . duodenalis in 60% of 583 children younger than five years in southern Rwanda . It was by far the most frequent intestinal parasite detected in this population . Importantly , two out of three infections would have been undetected if only the commonly used light microscopy had been applied . Genotyping revealed the presence of two distinct types of parasites , and only the infrequent subtype showed a weak association with gastrointestinal symptoms . However , G . duodenalis infection was associated with underweight and clinically assessed severe malnutrition . The data call for the establishment of more sensitive than light microscopy , yet simple diagnostic tools to identify infected children as well as for the consideration of abundant submicroscopic infections in evaluating the significance of G . duodenalis in high endemicity areas . | [
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] | 2012 | High Prevalence of Giardia duodenalis Assemblage B Infection and Association with Underweight in Rwandan Children |
Correct segregation of meiotic chromosomes depends on DNA crossovers ( COs ) between homologs that culminate into visible physical linkages called chiasmata . COs emerge from a larger population of joint molecules ( JM ) , the remainder of which are repaired as noncrossovers ( NCOs ) to restore genomic integrity . We present evidence that the RNF212-like C . elegans protein ZHP-4 cooperates with its paralog ZHP-3 to enforce crossover formation at distinct steps during meiotic prophase: in the formation of early JMs and in transition of late CO intermediates into chiasmata . ZHP-3/4 localize to the synaptonemal complex ( SC ) co-dependently followed by their restriction to sites of designated COs . RING domain mutants revealed a critical function for ZHP-4 in localization of both proteins to the SC and for CO formation . While recombination initiates in zhp-4 mutants , they fail to appropriately acquire pro-crossover factors at abundant early JMs , indicating a function for ZHP-4 in an early step of the CO/NCO decision . At late pachytene stages , hypomorphic mutants exhibit significant levels of crossing over that are accompanied by defects in localization of pro-crossover RMH-1 , MSH-5 and COSA-1 to designated crossover sites , and by the appearance of bivalents defective in chromosome remodelling required for segregation . These results reveal a ZHP-4 function at designated CO sites where it is required to stabilize pro-crossover factors at the late crossover intermediate , which in turn are required for the transition to a chiasma that is required for bivalent remodelling . Our study reveals an essential requirement for ZHP-4 in negotiating both the formation of COs and their ability to transition to structures capable of directing accurate chromosome segregation . We propose that ZHP-4 acts in concert with ZHP-3 to propel interhomolog JMs along the crossover pathway by stabilizing pro-CO factors that associate with early and late intermediates , thereby protecting designated crossovers as they transition into the chiasmata required for disjunction .
During the two specialized divisions of meiosis , a single round of DNA replication is followed by two rounds of segregation that ultimately produce gametes with half the parental number of chromosomes . Central to chromosome segregation accuracy is the formation of chiasmata between paired homologous chromosomes that are the visible product of genetic crossing over . The critical series of events leading to the formation of these linkages occurs during meiotic prophase when programmed meiotic DNA double-strand breaks ( DSBs ) are repaired using homologous recombination ( HR ) . An early step in this process is resection of a DSB end to form a single-stranded stretch of DNA that can recruit Rad51 , an event that initiates invasion of the homologous chromosome and the formation of a joint molecule ( JM ) intermediate to link the homologs ( reviewed in [1] ) . Resolution of these JMs can proceed through a crossover ( CO ) or noncrossover ( NCO ) pathway and the route chosen at any given site is carefully monitored . To ensure crossover formation , the number of induced DSBs is in excess of the final number of COs ( reviewed in [2] ) , however , the number of crossovers is in turn strictly regulated in any given organism ( e . g . [3 , 4] ) . Consequently , a decision must be made to stabilize certain JM intermediates for entry into the CO pathway , while the remaining events are repaired as NCOs [5] . These events are particularly tightly regulated in Caenorhabditis elegans , where an estimated 5–12 DSBs along a chromosome pair must be processed to yield a single exchange event [6–8] that serves to both physically link the homologous chromosomes and asymmetrically reconfigure the bivalent in preparation for interaction with the segregation machinery ( reviewed in [9] ) . C . elegans meiotic chromosomes exhibit robust interference that effectively limits each homolog pair to a single crossover [10–11] . As in other organisms , CO formation in the nematode is promoted by conserved players that act to stabilize and protect JM intermediates in the CO pathway , including the scaffolding protein RMH-1 ( RM1 ) , MSH-4/5 ( MutSγ ) , and cyclin-like COSA-1 ( CNTD ) [11–14] . In addition to these factors , a family of proteins resembling SUMO E3-like ligases has emerged as pivotal regulators of the decision to transform JM recombination intermediates into crossovers [15–17] . The canonical budding yeast Zip3p [18] exhibits E3 SUMO activity in vitro [19] and orthologs have subsequently been identified in mammals , plants , nematodes , other fungi , and Drosophila [20–29] . Members of the Zip3 E3-ligase related family are required for CO formation and share similar protein structures: an N-terminal RING finger domain , followed by a coiled-coil domain and a C-terminal domain enriched in serine residues [25] . In these organisms , the Zip3-like proteins diverge into two possible clades , one defined by Zip3 , the vertebrate RNF212 and nematode ZHP-3/ZHP-4 , and the other represented by HEI10 and its orthologs [21 , 23 , 29] . Budding yeast possesses a single member of the Zip3/RNF212 group [18] , while plants and the filamentous fungus Sordaria appear to carry a single ortholog of the HEI10 subgroup [23 , 24 , 26] , and C . elegans and mammals possess members from both subgroups [20 , 21 , 25 , 27 , 29] . However , all members of both groups share a similar pattern of localization by appearing as numerous foci or stretches along the synaptonemal complex ( SC ) and eventually persisting at the few obligate CO sites . In C . elegans , for example , a predicted ZHP-3/ZHP-4 heterodimer is required for crossing over , localizes to the SC and finally restricts to the six late CO intermediates typically observed in each meiotic nucleus at late pachytene stages [22 , 29 , this study] . The pattern of ZHP-3/4 localization is reminiscent of other pro-crossover factors ( RMH-1 , MSH-5 , and COSA-1 ) , which similarly begin with abundant early localization that is then confined to the sites of the obligate crossovers at late pachytene stages , and finally disappears as chromosomes desynapse and chiasmata emerge [11 , 12] . An elusive question in the study of meiosis is how the well-studied molecular events of DNA strand exchange that lead to CO formation transform into the microscopically evident chiasmata required for chromosome segregation . Early microscopy studies of these events revealed a physical connection ( chiasma ) between chromatids [30] and their correlation in number with the frequency of genetic exchange [31] . Consequently , while it is widely accepted that chiasmata originate with the formation of crossovers , the question of how HR at the DNA level becomes a cytologically evident chiasma capable of supporting chromosome segregation remains largely unexplored . In the case of C . elegans , the emergence of chiasmata is coupled to remodelling of the bivalent in preparation for interaction with the spindle machinery and regulated cohesion loss [32 , 33] . In this study , we have shown that zhp-3/4 are required at distinct stages in this transition . First , we show that at early pachytene stages , zhp-3/4 are required to promote the formation of an RMH-1-mediated JM competent intermediate to recruit pro-CO factors . Second , we show that zhp-3/4 are required at late pachytene exit stages for the transition from the late crossover intermediate ( likely the double Holliday Junction , dHJ [34] ) to chiasmata . In fact , zhp-4 is required to stabilize RMH-1 at early JMs and is necessary for recruitment/stabilization of MSH-5 and signalling the end to meiotic DSB induction . Furthermore , genetic crossovers that occur in zhp-4 mutants and are not marked by the pro-crossover factors ( RMH-1 , MSH-5 , COSA-1 ) are unable to form chiasmata capable of triggering the bivalent remodelling required for accurate chromosome segregation at meiosis I . Together , our data suggest that the ZHP-3/4 complex is recruited to the SC as it forms [22] to convene the complex in proximity to early recombination intermediates where it stabilizes pro-crossover factors that first promote JM resolution along the crossover pathway and finally resolve crossover-designated sites into chiasmata .
An EMS screen for recessive nondisjunction mutants ( Materials and Methods ) isolated a mutation ( vv96 ) in Y39B6A . 16 ( ZHP-4 ) , a gene with significant predicted protein sequence similarity to ZHP-3 ( 13% identity and 23% similarity ) . Since ZHP-4 and ZHP-3 share the structural features of the C3HC4-type RING finger domain characteristic of known SUMO E3 ligases ( Fig 1A; reviewed by [35] ) , we investigated its role in meiosis and its relationship to ZHP-3 . The zhp-4 ( vv96 ) mutation results in a premature translation termination codon at amino acid ( a . a . ) 160 that is predicted to produce a truncated protein with an intact RING finger domain ( Fig 1A ) and is compatible with other data presented below that it represents a severely hypomorphic allele . To determine the null phenotype , CRISPR-Cas9 mutagenesis ( Materials and Methods ) was used to generate the deletion allele zhp-4 ( vv103 ) , a frameshift mutation that creates a premature stop codon before the last two cysteines in the predicted RING-finger domain ( a . a . 56; Fig 1A ) . The germlines of both zhp-4 mutants displayed no overt defects in nuclear morphology as assessed by DAPI staining ( S1A Fig ) . The broods of zhp-4 ( vv96 ) and zhp-4 ( vv103 ) mutant homozygotes were marked by statistically similar levels of high embryonic lethality and incidence of XO males amongst the surviving progeny , phenotypic hallmarks of autosomal and X-chromosome nondisjunction ( [36] Fig 1B and 1C ) . Previous studies observed that ZHP-3 first localizes to synapsed chromosomes in an SC-dependent manner , and is then restricted at late pachytene stages to foci that correspond to sites of crossing over [21 , 22] . To investigate if ZHP-4 functions with its paralog ZHP-3 in CO formation , we first examined the localization of the proteins during meiotic prophase and tested their codependency in recruitment to chromosomes ( Fig 2 ) . In the case of ZHP-4 , antibodies raised against the C-terminal 123 a . a . and a CRISPR-generated HA tag ( Materials and Methods ) both revealed that ZHP-4 localizes to the SC from earliest pachytene and is similarly restricted to the 5–7 late pachytene foci reported for ZHP-3 ( Figs 2A and S2A; [21 , 22] ) . Like ZHP-3 , ZHP-4 recruitment to chromosomes is SC dependent ( S2C Fig ) , and the protein colocalizes with ZHP-3 at the SC and at the CO sites that emerge at late pachytene ( Fig 2A ) . ZHP-3 localization was reduced to weak background levels throughout meiotic prophase in the absence of ZHP-4 ( Fig 2B ) , and conversely ZHP-4 localization was similarly abrogated in zhp-3 ( jf61 ) mutant germlines ( Fig 2B ) , indicating that the two paralogs are co-dependent in their recruitment to meiotic chromosomes . Our observations that ZHP-3/4 colocalize in a co-dependent manner to the same chromosome features and the results of a recent characterization of the ZHP-3 family [29] are most simply reconciled with a model in which the two paralogs physically cooperate to mediate the formation of crossovers . Consistent with the crossover-specific function of ZHP-3 [21] , ZHP-4 is similarly not required for pairing , synapsis ( S1B and S1C Fig ) , or for meiotic DSB induction [29] as evidenced by the formation of foci of the strand exchange RAD-51 protein [37 , 38] . While wild-type diakinesis oocytes stained with DAPI invariably contained 6 bivalents ( representing the 12 chromosomes linked by chiasmata ) , zhp-4 ( vv103 ) and zhp-4 ( vv96 ) mutants respectively exhibited an average of 11 . 4 and 8 . 2 DAPI-stained figures ( p < 0 . 001 compared to WT , Fig 3A and 3B ) , instead of the 12 predicted in the event of complete loss of crossover potential [39] . To directly assess the effect of loss of ZHP-4 function on crossing over , we genetically measured the frequency of genetic exchange between visible markers ( Fig 3D; Materials and Methods ) in large genetic intervals comprising ~ 1/3 of the chromosome III ( 12 m . u . ) and ~3/4 of the X chromosome ( 38 m . u . ) . We were unable to measure crossing over in zhp-4 ( vv103 ) null mutants , as the mutation in combination with several visible markers tested was near inviable ( could not be maintained as a strain ) and the embryonic lethality and aneuploidy phenotypes made it impossible to attain data sets for statistical comparisons; however , rare recombinants ( <1/100 wild-type progeny ) that segregated progeny of the recombinant phenotype were recovered in both intervals . The frequency of isolation of the rare recombinants in vv103 mutants is similar to the rare events previously reported for zhp-3 null mutants ( 2/93 ) [21] . In contrast , zhp-4 ( vv96 ) mutants attained 33% of wild-type crossover levels on the X chromosome ( p < 0 . 001 ) and 77% of wild-type crossover levels on chromosome III ( p > 0 . 05 ) , indicating that vv96 mutants remained competent for significant levels of genetic exchange . ZHP-3/4 contain a conserved C3HC4-type RING finger domain required for the catalytic activities of E3 ubiquitin and SUMO ligases ( reviewed by [35 , 40] ) . We investigated its contribution to ZHP-3/ZHP-4 function by targeting conserved histidine residues at positions known to be essential for RING finger function during meiosis in other organisms ( S3 Fig ) ; for example , S . cerevisiae zip3H74A and zip3H80A mutants exhibit defects in SC assembly and sporulation efficiency [19] , while Sordaria hei10H30A mutants are defective in crossing over and chiasma formation [26] . While zhp-4 ( H26A ) mutants exhibited high embryonic lethality and males amongst surviving progeny ( p < 0 . 01 compared to WT ) , zhp-3 ( H25A ) mutants produced only 3% dead embryos and 3% male progeny ( p > 0 . 05 in comparison to WT , Fig 1B and 1C ) , indicating that the RING domain of ZHP-3 is largely expendable for its function . The severity of the embryonic lethality defects observed in the two mutants was also reflected at diakinesis where only 5% of zhp-4 ( H26A ) nuclei exhibited the 6 DAPI bodies observed in wild-type nuclei ( average of 8 . 0 , p < 0 . 001 ) , while 95% of zhp-3 ( H25A ) diakinesis nuclei ( average of 5 . 9 , p > 0 . 05 ) did so ( Fig 3C ) . Since neither zhp-3 ( H25A ) nor zhp-4 ( H26A ) single mutants replicated the phenotypes of the respective null mutant , we addressed the possibility of RING domain redundancy by examining the consequence of loss of both RING domains . zhp-3 ( H25A ) ; zhp-4 ( H26A ) double mutants exhibited phenotypes that were more severe than those of zhp-4 ( H26A ) single mutants and not different from those observed for the null mutant; homozygotes segregated 95% dead embryos and 43% male progeny ( Fig 1B and 1C , p > 0 . 05 ) and 80% of diakinesis nuclei showed 12 univalents ( average of 11 . 7 p < 0 . 001; Fig 3C ) . In zhp-3 ( H25A ) ; zhp-4 ( H26A ) double mutants , ZHP-4 was not detectably recruited to synapsed chromosomes at any stage , and no enriched nuclear localization could be detected ( Fig 4B ) . We could not detect ZHP-3H25A localization in the double RING mutant using α-ZHP-3 antibodies ( the aliquot did not provide a reliable signal even in wild-type controls ) ; however , its localization is likely to be equally abrogated given the co-dependent co-localization of the proteins and the relative fertility of zhp-3 ( H25A ) . In the case of zhp-3 ( H25A ) single mutants , ZHP-4 adopted the wild-type pattern of SC localization throughout pachytene ( Fig 4B ) to finally restrict to ~ 6 foci marking putative crossover/chiasma sites that correlate with the lack of meiotic defects in this mutant . In zhp-4 ( H26A ) single mutants , however , ZHP-4H26A localization to the SC was reduced , discontinuous , and evident only as punctate foci of varying intensities ( Fig 4B ) , indicating that an intact ZHP-4 RING domain is required for the contiguous pachytene pattern of ZHP-3/4 association with the SC that is observed in wild-type . Despite this disrupted localization during early/mid-pachytene stages , 1–3 bright ZHP-4H26A foci/nucleus emerged at late pachytene ( Fig 4C ) ; this in combination with the detection of significant levels genetic crossing over ( 57% and 40% of wild-type chromosome X and III frequencies; Fig 3C ) and the presence of bivalents at diakinesis ( Fig 3C ) indicates that zhp-4 ( H26A ) mutants can support reduced levels of crossover formation and localization of the protein to those sites . Our results are collectively consistent with a model in which 1 ) the RING domain of ZHP-4 is critical for the localization of the heterodimer to the SC where it cooperates with ZHP-3 to promote crossover formation and 2 ) the ZHP-3 RING domain can partially compensate for loss of ZHP-4 RING domain activity to foster the formation of a severely reduced number of crossovers/chiasmata . In crossover-defective mutant backgrounds , RAD-51-marked recombination intermediates typically appear on time and at wild-type levels in early pachytene , however , they accumulate and persist into late pachytene stages [38] . We measured RAD-51 foci in nuclei of the mitotic ( zone 1 and 2 ) , leptotene/zygotene ( referred to as the transition zone ) /pachytene entry ( zone 3 ) , early pachytene ( zone 4 ) , mid-pachytene ( zone 5 ) , and late pachytene stages ( zone 6 ) ( Fig 5 ) . In both zhp-4 ( vv103 ) and zhp-4 ( vv96 ) mutants , RAD-51 foci appeared , peaked in number , and disappeared with wild-type like timing as previously reported for zhp-3 mutants [21] , suggesting appropriate recombination initiation and timely DSB processing and repair . However , the levels of RAD-51 foci in both zhp-4 mutants were dramatically elevated at every stage until their disappearance at late pachytene ( Fig 5 ) . In particular , meiotic RAD-51 foci first emerge in zone 3 where 70% of the wild-type nuclei have no foci ( 30% have 1–6 foci ) while only 4% of vv96 and 14% of vv103 mutant nuclei lack any foci; at this same stage , 34% of vv96 and 17% of vv103 mutant nuclei have >7 RAD-51 foci , a category that does not appear in wild-type germlines until zone 4 . This initial increase of RAD-51 foci in zhp-4 mutants persists into later stages , but with wild-type-like dynamics; their average number per nucleus peaks in zone 4 ( 4 . 2 in wild-type , 10 . 5 and 11 . 1 in vv96 and vv103 mutants , respectively . p < 0 . 001 ) , and appropriately disappears in the last zone ( 1 . 0 in wild-type , 2 . 0 and 1 . 4 in vv96 and vv103 mutants , respectively . p < 0 . 01 for vv96 and p > 0 . 05 for vv103 ) . While the elevated numbers of RAD-51 foci observed in zhp-4 mutants could originate in their impaired turnover , RAD-51 foci kinetics followed the wild-type pattern and did not exhibit the accumulation observed in other CO-defective mutants [38] . This suggests that the initial elevation that appears in concert with meiotic DSB formation is not a reflection of an impairment in processing RAD-51-marked early recombination intermediates but reflects a requirement for ZHP-4 in down regulating DSB formation . To test this possibility , we examined the levels of RAD-51 foci in a rad-54 ( RNAi ) background where DSB repair is blocked and RAD-51 foci are not removed , thereby permitting quantitation of the total number of DSBs formed [41] . Hermaphrodites injected with rad-54 RNA did not show the sterility previously reported for rad-54 deletion mutants or rad-54 ( RNAi ) [41] . However , cytological analysis at 2 days post-injection ( 3 days post L4 stage ) revealed an increase in RAD-51 foci until early/mid-pachytene stages ( zone 4 ) at levels comparable with those previously reported [41] . We observed that the levels of RAD-51 foci decreased in mid-pachytene ( zone 5 ) and disappeared in late pachytene stages ( zone 6 ) , in contrast to rad-54 mutants in which the levels remain high until the end of pachytene . This suggests a partial effect of the RNAi treatment , and consequently we measured RAD-51 foci levels up to zone 4 in zhp-4 ( vv103 ) ; rad-54 ( RNAi ) germlines and compared them to the levels observed in the same zone in rad-54 ( RNAi ) controls ( S4 Fig ) . We observed a significant increase of RAD-51 foci in zone 4 in the double mutant in comparison to rad-54 ( RNAi ) alone ( average of 13 . 5 versus 9 . 6 for rad-54 ( RNAi ) , p < 0 . 00001 ) . This result is consistent with the interpretation that the higher levels of RAD-51 foci observed in vv103 germlines are the consequence of increased DSB formation rather than defective DSB repair . This suggests a role for ZHP-4 in negatively regulating recombination initiation , possibly by being required for the formation of a crossover intermediate that can feedback to negatively regulate DSB formation ( reviewed in [42] ) . Since zhp-4 mutants robustly initiated recombination but failed to resolve these events into crossovers , we used known markers of crossing over to probe the origin of this defect . We first characterized the dynamics of RMH-1 , a conserved scaffolding component thought to co-operate with Bloom’s helicase ( BLM; nematode HIM-6 ) during an early step in crossover designation [43] and the resolution of recombination intermediates into crossovers or noncrossovers [12] . In C . elegans , GFP::RMH-1 is first recruited to synapsed chromosomes at early pachytene in numbers exceeding the number of obligate COs , suggesting that it marks both COs and non COs at this stage; by late pachytene , the number of RMH-1 foci decreased to ~6 per nucleus putatively marking the obligate crossover sites [12] . We observed a similar kinetics of appearance and disappearance of RMH-1 foci in wild-type germlines ( Fig 6A–6F ) as previously reported [12] . Both vv103 null mutants and vv96 hypomorphs acquired low levels of RMH-1 foci in very early pachytene stages in numbers not significantly different from wild-type ( p > 0 . 05 , Fig 6A ) , indicating that ZHP-4 mutants are competent for formation of these early RMH-1-marked recombination intermediates with appropriate timing and levels . In zhp-4 ( vv103 ) mutants , the number of RMH-1 foci did not significantly increase throughout pachytene ( p < 0 . 001 ) , and the 2–3 foci that did form disappeared at the approach of pachytene exit ( Fig 6E and 6F ) , similar to the timing and kinetics observed for RMH-1 foci in zhp-3 ( jf61 ) mutants [12] . In contrast to vv103 null mutants , hypomorphic zhp-4 ( vv96 ) mutants steadily accumulated RMH-1 foci as pachytene progressed , reaching levels not different than those observed in wild types at mid-late pachytene stages ( median of 10 , p > 0 . 05; Fig 6D , 6E and 6G ) . However , the accumulation of these foci was delayed with respect to the kinetics observed in wild types germlines ( Fig 6B and 6C ) ; for example , while the number of RMH-1 foci in wild-type peaked at mid-pachytene with a median of 16 RMH-1 foci , vv96 mutants exhibited 8 foci and displayed a peak later at mid-late pachytene with 13 foci ( Fig 6D ) . Although zhp-4 ( vv96 ) mutants appeared competent in the formation of early RMH-1 marked recombination , they proved to be defective in presenting the bright RMH-1 foci at very late pachytene stages in which RMH-1 marks the sites of the obligate crossovers ( Fig 6F and 6H ) . While in wild types the excess early RMH-1 foci disappeared to a final median population of 6 , zhp-4 ( vv96 ) mutant nuclei exhibited no detectable foci at the stage approaching pachytene exit ( Fig 6F and 6H; p < 0 . 001 in comparison to wild types ) , despite their abundant presence earlier ( Fig 6B–6E ) . Collectively , these data support dual roles for ZHP-4 in RMH-1 dynamics . Our data suggest that wild-type levels of early RMH-1 foci can form in the absence of ZHP-4 , however , ZHP-4 is then required to stabilize or localize RMH-1 at early JMs that will become either COs or NCOs , and later at designated crossover sites where the final intermediate will be resolved as a crossover . In addition to RMH-1 , the crossover-designated sites at mid-late pachytene stages also colocalize with ZHP-3 , MSH-5 , and COSA-1 [11 , 12] . zhp-4 ( vv96 ) mutants displayed punctate staining of ZHP-3 on chromosome tracks at late pachytene ( Fig 4A ) , and a zhp-4 ( ha::vv96 ) -tagged variant that phenocopies the genetic mutant ( Materials and Methods ) adopted a pattern similar to the ZHP-3 localization in the zhp-4 ( vv96 ) background ( S5 Fig ) . Given that zhp-4 ( vv96 ) mutants exhibit evidence of significant levels of bivalent formation and crossing over ( Fig 3 ) , these data suggest that the ZHP-4vv96 mutant protein can associate with crossover pathway intermediates in the absence of its contiguous recruitment to the SC to facilitate crossing over . We next examined the localization of MSH-5 , a component of the pro-crossover MutSγ complex , that is required for formation of the obligate crossovers from a population of earlier recombination intermediates . Abundant early MSH-5 foci form during mid-pachytene where they colocalize with a subset of RMH-1 foci and finally mark the six crossover sites in late pachytene stages [11 , 12] . In the absence of ZHP-4 , only a few sporadic small MSH-5 foci that did not colocalize with chromosome tracks could be detected ( Fig 7A ) . Given the correlated deficit of RMH-1-marked recombination intermediates in zhp-4 ( vv103 ) mutant germlines , these data suggest that MSH-5 recruitment to a potential crossover intermediate may require an RMH-1 processed JM and/or that the two proteins also co-operate in one anothers’ localization/stabilization at these sites . In zhp-4 ( vv96 ) hypomorphs , abundant MSH-5 foci formed in mid-late pachytene , however , these foci were of varying sizes and intensities that precluded accurate scoring and ultimately did not reduce to the ~6 bright foci observed in wild types . Given that RMH-1-marked recombination intermediates are fewer or more unstable in vv96 mutants , a likely consequence is that their loss translates to impaired MSH-5 recruitment/stabilization [12] , in turn leading to the reduced crossing over and chiasma defects observed in these mutants . Furthermore , no bright wild-type-like COSA-1 foci were detected on chromosome tracks in the germlines of vv96 and vv103 mutants ( Fig 7B ) . Its localization was instead reduced to a faint and diffuse signal punctuated by numerous foci of variable sizes and intensities that occasionally overlapped with the synapsed chromosomes , but were often extranuclear . This localization differed from the few faint foci observed in spo-11 mutants that do not initiate meiotic recombination and may reflect protein interactions outside of the context of crossover formation . Based on these results we conclude that COSA-1-mediated steps in crossover designation require ZHP-4 function . The pro-crossover factors that showed disrupted localization to late crossover intermediates in vv96 mutants participate in the resolution of recombination intermediates into class I crossovers that show interference ( the reduced probability that a second crossover forms in the vicinity of the first ) . An alternative class II pathway that includes the MUS-81 structure-specific endonuclease is required for a small subset of meiotic crossovers that are noninterfering [44] and we investigated whether the bivalents that formed in vv96 mutants depended on the activity of MUS-81 or RMH-1 ( Fig 8 ) . The diakinesis nuclei of zhp-4 ( vv96 ) ; mus-81 ( tm1937 ) double mutants did not show significantly different distributions of DAPI figures in comparison to vv96 single mutants ( p > 0 . 05 ) , indicating that the residual chiasmata observed in zhp-4 ( vv96 ) mutants do not required mus-81 . In contrast , an average of 12 DAPI figures and no bivalents were observed at diakinesis in zhp-4 ( vv96 ) ; rmh-1 ( jf92 ) double mutants ( Fig 8B and 8C ) , indicating that bivalent formation in vv96 mutants is dependent on RMH-1 . Furthermore , the residual chiasmata observed in rmh-1 single mutants was in turn dependent on zhp-4 , suggesting possible co-dependent functions in crossover formation . In summary , our collective results suggest that ZHP-4 is first required for the stabilization of RMH-1 at early recombination intermediates; this event either generates a crossover intermediate recognized by pro-crossover factors or stabilizes those factors at the site ( or both ) , leading to resolution of the intermediate into a crossover at pachytene exit and its transition into a chiasma . Characterization of the severe hypomorph zhp-4 ( vv96 ) revealed a functional paradox: although the levels of embryonic lethality and frequency of males in the self progeny did not differ from the null allele ( p > 0 . 05 , Fig 1B and 1C ) , zhp-4 ( vv96 ) mutants exhibited surprisingly substantial levels of both bivalent structures and of crossovers as measured by genetic exchange ( Fig 3A , 3B and 3D ) despite the defects in acquiring late-pachytene stage RMH-1 , MSH-5 and COSA-1 foci that mark sites of the obligate crossovers in wild-type . Close examination of chromosomes in the diakinesis nuclei of zhp-4 ( vv96 ) mutants revealed an unexpected phenotype; the occasional appearance of well-condensed bivalent-like structures that had separated chromosomes and were tethered to one another by a chromatin mass , with the axial element HTP-3 often congregated within ( Fig 9A ) . While abnormal bivalent morphology in which chromosomes linked by chromatin bridges has been observed in mutants that disrupt Holliday junction resolution and crossover intermediate processing , the chromatin linkages in these cases appear more thread-like and do not contain axis components [12 , 45] . The anomalous diakinesis structures observed in zhp-4 ( vv96 ) mutants appeared in a SPO-11-dependent manner , indicating that they were the outcome of a meiotically-programmed DSB intermediate ( Fig 3A and 3B ) , and are for simplicity referred to as “tethered bivalents” . To characterize this disruption , we probed the bivalents observed in vv96 mutants for evidence of the remodelling associated with chiasma formation ( Fig 9 ) , including restriction of the AIR-2 kinase ( aurora B kinase; [46 , 47] ) and SC component SYP-1 to the short arms of the bivalent [32] , and the meiotic sister chromatid cohesin regulator HTP-1 to the long arms [33] . In vv96 mutant oocytes at diakinesis , bivalents ( as assessed by size and cruciform staining of the axis marker HTP-3 ) failed to appropriately remodel ( Fig 9A–9C ) and instead exhibited SYP-1 ( 13/13 bivalents ) and HTP-1 ( 11/12 bivalents ) along the axes of both the long and short arm . zhp-4 ( vv96 ) mutants similarly displayed disruptions to AIR-2 localization that interfered with quantitation; these included failure to localize or its appearance in chromatin masses between the tethered bivalents . Taken together , these results indicate that the crossovers forming in vv96 mutants are defective in triggering the associated remodelling of factors implicated in chromosome segregation , or that this triggering is not executed in vv96 mutants . To further investigate the functional implications of this localization defect , we next examined the localization of the single C . elegans SUMO ortholog whose conjugation to target proteins promotes chromosome alignment at metaphase I [48] . SMO-1 localizes to the chromatin of germline nuclei and then to the axes of the bivalent short arms in late diakinesis stage oocytes ( [48]; S6 Fig ) ; this dynamic localization is recombination dependent , since SMO-1 remains diffusely associated with chromatin rather than restricted to HTP-3-marked chromosome axes in spo-11 ( ok79 ) mutants ( Fig 9D ) . In both zhp-4 ( vv103 ) null and zhp-4 ( vv96 ) hypomorphic mutants , SMO-1 localizes appropriately to chromatin of mitotic and prophase chromosomes and remains associated with the chromatin of univalents present in late-stage diakinesis nuclei , similar to the localization observed in spo-11 mutants ( Figs 9D and S6 ) . In contrast to wild types , however , SMO-1 remained associated with the chromatin of diakinesis bivalents in vv96 mutant nuclei and with the chromatin of a rare bivalent observed in a vv103 mutant nuclei ( Fig 9D . 2-3 ) . In the case of vv96 mutants , SMO-1 also occasionally appeared enriched in the chromatin at the ends of tethered bivalents ( Fig 9D . 4 ) . Consequently , we conclude that the crossovers that form in vv96 or vv103 mutants can give rise to bivalent-like figures , however , these exhibit structural anomalies and are defective in the chromosome remodelling that normally accompanies crossover formation and chiasma emergence . In contrast , the bivalents that form in the diakinesis nuclei of zhp-4 ( H26A ) mutants show appropriate localization of SYP-1 , HTP-1 , and SMO-1 and no tethered bivalent phenotypes ( Fig 9B–9D; no tethered bivalents observed in 86 diakinesis nuclei ) , suggesting that the reduced number of crossovers that form are competent to trigger chromosome remodelling , correlated with improved embryonic survival ( Fig 1B ) . To investigate the possible functional consequences of the formation of aberrant bivalent structures in zhp-4 ( vv96 ) mutants , we examined chromosome congression and segregation at the metaphase plate in meiosis I . In wild-type , bivalents align at the metaphase plate I between overlapping microtubule bundles that form channels through which the chromosomes move during segregation [49 , 50] . At metaphase I , zhp-4 ( vv96 ) oocytes displayed a spectrum of phenotypes from occasional wild-type spindle organization to stray chromosomes and abnormal spindle morphology as assessed by disorganized microtubule channels ( S7 Fig ) . These structural defects in spindle assembly correlated with aberrant segregation behaviour at anaphase I; while in wild-type oocytes two distinct masses of chromatin were separated by the microtubule channels , the chromosomes of some zhp-4 ( vv96 ) mutant oocytes appeared unresolved and tangled while being pulled to the poles ( S7 Fig ) . Given the presence of bivalents in zhp-4 ( vv96 ) mutants , we favour the interpretation that the crossovers that form in the absence of full ZHP-4 function do not trigger the bivalent remodelling associated with preparation for segregation and consequently lead to the same level of embryonic lethality and X chromosome nondisjunction as observed in zhp-4 ( vv103 ) null mutants . These results are consistent with a model in which crossover and chiasmata formation are genetically separable events that require ZHP-4 for the physical transformation of crossovers into the chiasmata capable of directing chromosome segregation at the meiotic spindle . zhp-4 ( H26A ) mutants shared several similar phenotypic features with zhp-4 ( vv96 ) mutants , including faint and punctate SC localization and significant levels of genetic crossing over . In contrast to zhp-4 ( H26A ) mutants , however , zhp-4 ( vv96 ) mutants 1 ) did not form ZHP-3/4 late pachytene foci , despite the presence of genetic crossovers , 2 ) contained aberrant tethered bivalents in diakinesis nuclei which were not observed in zhp-4 ( H26A ) mutants , and 3 ) exhibited significantly higher levels of embryonic lethality ( Fig 1B ) . Given that the zhp-4 ( vv96 ) mutant defects correlated with disrupted RMH-1 stabilization at recombination intermediates and failure to retain/recruit markers of designated crossovers/chiasmata , we investigated these processes in ZHP-4 RING domain mutants . zhp-4 ( H26A ) mutants exhibited early RMH-1 foci dynamics that resembled those observed in zhp-4 ( vv96 ) mutants ( Fig 6A–6E , p > 0 . 05 for all except p < 0 . 05 for C ) , including: 1 ) their appropriate appearance at very early pachytene , 2 ) a delay in their accumulation , and 3 ) the appearance of wild-type levels of RMH-1-marked recombination intermediates at mid-late pachytene stages ( Fig 6D , p > 0 . 05 vs . WT ) . However , in nuclei entering the pachytene exit stage in which designated crossover/chiasma markers emerge , the majority of zhp-4 ( H26A ) nuclei exhibited 1–3 RMH-1 foci/nucleus ( consistent with the appearance of 1–3 ZHP-4 foci in the RING mutant ) , while none were detected in zhp-4 ( vv96 ) mutants ( Fig 6F–6H , p < 0 . 05 compared to vv96 mutants ) . The presence of these RMH-1 foci at this late stage suggests that the RING domain mutant is competent to form a reduced number of the crossover intermediates that are observed in wild types . To investigate this possibility , we next examined zhp-4 ( H26A ) pachytene exit stage nuclei for the appearance of COSA-1 marked-crossover intermediates , which could not be detected on chromosomes above background levels in zhp-4 ( vv96 ) mutants ( Fig 7B ) . zhp-4 ( H26A ) mutants exhibited levels of COSA-1 foci formation similar to that observed for RMH-1 focus formation at late pachytene ( median of 2; Fig 7C , p < 0 . 001 in comparison to wild types ) , and in both cases the bright COSA-1/RMH-1 foci appeared on HTP-3-marked synapsed axes as observed in wild types . Furthermore , well-formed bivalents with respect to DNA condensation and the localization of SYP-1/SMO-1 to the short arm and HTP-1 to the long arm appeared in zhp-4 ( H26A ) mutants at late diakinesis , indicating that the genetic crossovers detected ( Fig 3D ) correlate with the presence of designated crossover markers and appropriate remodelling of the bivalent ( Fig 9B–9D , white arrow ) . The association of late pro-crossover factors with the designated crossover site correlated not only with bivalent remodelling , but also with the ability of the chiasmata to direct segregation as evidenced by the lower embryonic lethality observed in zhp-4 ( H26A ) mutants in comparison to zhp-4 ( vv96 ) mutants ( Fig 1B ) . These results strongly suggest that the crossover intermediates that do acquire RMH-1 , ZHP-4 and COSA-1 at very late pachytene stages in zhp-4 ( H26A ) mutants define crossovers competent to form chiasmata and trigger the bivalent remodelling that is required to ensure accurate chromosome segregation . Consistent with this interpretation , zhp-4 ( vv96 ) mutants are competent for the formation of RMH-1/MSH-5 foci in mid-pachytene stages and reduced levels of crossing over; however , the crossovers that do form are not cytologically visible as foci containing the chiasmata markers , and correlate with defects in chromosome remodelling and spindle function . We propose that ZHP-4 acts in concert with ZHP-3 to stabilize RMH-1 at early recombination intermediates to foster the formation of an early crossover intermediate competent for negatively regulating meiotic DSB induction and association with other pro-crossover factors like MSH-5 and COSA-1 . Our analysis suggests that the ZHP-4-mediated stabilization/recruitment of the pro-crossover complex at designated crossover sites by late pachytene is required to convert the DNA exchange events into the chiasmata solely capable of triggering bivalent remodelling in preparation for meiotic spindle assembly and chromosome segregation .
A further evolution in our understanding of E3 ligases has been the observation that some can function as heterodimers , including the Ub ligases BRCA1-BARD1 [51–53] and SUMO-directed Ub ligases Slx5-Slx8 , [54–57]; however , no such examples have yet emerged for SUMO E3 ligases . Here , we have shown that ZHP-3/4 colocalize throughout meiotic prophase and that their localization to the SC and to designated CO sites is interdependent , suggesting a cooperative activity . Although the ZHP-3 RING finger is competent to support the formation of rare COs , the RING finger domain of ZHP-4 is the preferred contestant in localizing the complex to the SC since ZHP-3 RING activity can only be detected in its absence . This difference in terms of requirement between the two RING finger domains mirrors other examples of heterodimeric Ub E3 ligase complexes: in the case of BRCA1 and BARD1 , BRCA1 is the ‘active’ partner while BARD1 , the ‘inactive’ partner , stabilizes the complex in vivo ( reviewed by [40] ) . Although ZHP-3/4 has been suggested to act as a heterodimeric E3 SUMO ligase [29] , such a function is not supported by the phenotype of mutants in the single nematode SUMO gene , which form bivalents rather than the predicted univalents [22 , 58] . An outstanding question is whether ZHP-3/4 is a ubiquitin ligase , and if so , if it could perform a structural role at crossover sites that becomes a catalytic role in restructuring the resulting bivalent . The family of RNF212-like orthologs have differences and similarities in terms of localization and their relationship with DSB formation and SC assembly that largely reflects the relationship between recombination initiation and SC initiation in each organism . Recent studies have revealed that S . cerevisiae Zip3 and mouse RNF212 recruitment to meiotic chromosomes occurs in two distinct modes , one being DSB-dependent foci , and the other requiring SC formation for localization [18 , 25 , 59] , indicating a DSB-independent mechanism of a CO-promoting factor . In the divergent world of plants and filamentous fungi , the single Zip3-like protein ( HEI10 ) similarly localizes in pachytene to the SC central regions and is then restricted to detectable foci at late stages that correspond to sites of both COs and chiasmata [23 , 24 , 26] . Although SC initiation is independent of DSB formation in C . elegans , ZHP-3/4 exhibit two patterns of localization that grossly reflect the localization of RNF212 in mice: 1 ) ZHP-3/4 initially localize along the SC in an SC-dependent ( and SPO-11-independent ) manner , and 2 ) are restricted to a few sites of crossovers ( ~ 6 foci ) that at late pachytene stages are dependent on both the SC and SPO-11 ( S2C Fig; [21] ) . While in Drosophila , the RNF212-like ortholog Vilya is required for DSB formation to occur [28] , our study indicates that ZHP-3/4 are not required for the initiation of meiotic DSB formation ( DSBs form in the absence of the proteins ) and SC assembly ( Figs 5 and S1C; [21] ) . Instead , ZHP-3/4 are required to foster the transition of a limited number of crossover intermediates into bona fide crossover entities , a function which includes negatively regulating meiotic DSB induction once crossover intermediates have been formed . Overall , a common and recurring feature of all RNF212-like orthologs is their localization to the SC either as continuous linear stretches , or as a population of small foci that later emerge as larger discrete foci that mark crossing over sites . Given these localization dynamics across species with differential requirements for synapsis initiation ( DSB dependent or independent ) , our results are consistent with an intimate relationship between RNF212 family members and the SC from the earliest stages of recombination initiation that supports relatively rare events to go forward as the crossovers that will support chromosome segregation . In this study , we have shown that zhp-3/4 are required at distinct stages in the transition of JMs to chiasmata; at mid-pachytene stages where they promote the formation of an RMH-1-mediated JM competent to recruit pro-CO factors and at pachytene exit stages where they are required for the transition from crossover-designated sites to chiasmata . Since DSB formation , as visualized by RAD-51 , loading initiates on time and at robust levels in zhp-4 mutants ( see below ) , the deficit of chiasmata observed in the diakinesis oocytes indicates a defect in post-initiation/strand exchange process . At mid-pachytene stages RMH-1 cooperates with BLM ( nematode HIM-6 ) to promote crossover outcomes at JMs and repair the remaining recombination intermediates as noncrossovers [12 , 34] . We observed that the first population of RMH-1 appears on time and at appropriate levels in zhp-4 mutants , indicating that recruitment of RMH-1 to early recombination intermediates does not require ZHP-4; at early/mid-pachytene , however , both null and hypomorphic mutants exhibited severe defects in presenting wild-type numbers of RMH-1 foci , collectively consistent with the interpretation that ZHP-4 is required for stabilization of RMH-1 at JM rather than recruitment per se . The failure to stabilize RMH-1 at the early-mid-pachytene stage in zhp-4 mutants correlates with altered dynamics of other markers of recombination progression . First , RAD-51-marked recombination intermediates showed dramatically increased levels from the transition zone/earliest pachytene stages that followed wild-type-like kinetics of appearance and disappearance , suggesting an overall increase in meiotic recombination initiation rather than a defect in RAD-51 turnover . Second , the mid-pachytene MSH-5 foci that colocalize with RMH-1 at JMs in wild-type fail to form in the absence of zhp-4 and in vv96 hypomorphs appear with delayed timing and altered morphologies . Both phenomena are most parsimoniously explained as outcomes of a single event; zhp-3/4 is required for stabilization of RMH-1 at early/mid-pachytene stages to produce a JM intermediate that can signal the end to meiotic DSB initiation , leading to stabilization of interhomolog intermediates , CO designation , and crossover formation . Consequently , we favour the interpretation that the loss or reduction of crossing over observed in zhp-4 mutants originates in the failure to form a stable RMH-1-associated JM intermediate that can progress into the crossover pathway , and is instead repaired as an NCO . In addition to the early role of zhp-3/4 in the crossover pathway , our analysis of the zhp-4 mutants revealed a genetically separable role for ZHP-4 at pachytene exit stages in the designation of crossover intermediates destined to become chiasmata . zhp-4 ( vv96 ) and zhp-4 ( H26A ) RING mutants share a similar early/mid-pachytene phenotypic profile: in both cases , ZHP-3/4 do not show localization along the SC , RMH-1 appears with delayed kinetics that ultimately reaches wild-type levels , detectable genetic crossing over occurs , and similar distributions of bivalent structures appear at diakinesis . At late pachytene stages when pro-crossover markers are restricted to CO sites , zhp-4 ( H26A ) RING mutants exhibit 1–3 bright foci appropriately marked by RMH-1 , ZHP-4 , and COSA-1 , suggesting that the earlier problems in RMH-1 dynamics are overcome to generate a reduced number of wild-type crossovers . In zhp-3 ( H25A ) ; zhp-4 ( H26A ) double RING mutants , these ZHP-4-marked foci fail to form , a phenotype which is accompanied by loss of bivalent formation ( Figs 3C and 4B ) . This suggests that crossing over in ZHP-4 RING mutants is dependent on ZHP-3 and likely reflects a scenario in which the ZHP-3 RING domain is sufficient to support highly reduced ZHP-3/4 localization to chromosomes where its function is unaltered . In the case of vv96 mutants , however , crossing over and bivalent formation are not accompanied by the appearance of crossover-designated sites as defined by RMH-1/COSA-1 focus formation , indicating that CO designation and formation are separable events . The failure to form late RMH-1/COSA-1 foci in vv96 mutants correlates with the appearance of anomalous bivalent structures unique to zhp-4 ( vv96 ) mutants and well-formed bivalents that fail to exhibit the CO-directed remodelling associated with preparation for segregation . These chromosomes often show gross defects in alignment and congression at the metaphase I spindle and remain entangled at anaphase I , consistent with the chromosome segregation defects and high embryonic lethality observed in zhp-4 ( vv96 ) mutants . Similarly , zhp-3::gfp mutants ( the gfp construct does not fully rescue the null mutant phenotype at standard culture temperatures [22] ) display a competency for crossover formation that is nevertheless accompanied by unexpectedly high levels of embryonic lethality and X-chromosome nondisjunction , suggesting that the significant levels of crossing over in the presence of altered ZHP-3 function does not always guarantee chiasmata formation and bivalent remodelling [22] . In many organisms , the correct placement of crossovers on the chromosomes has been proven to be pivotal for promoting segregation; COs at the centromeres or ends of chromosomes are less effective at ensuring disjunction ( reviewed by [3] ) . In the case of zhp-4 mutants , an argument can be made that the inability of the COs to ensure accurate segregation is a consequence of their displacement to disjunction-ineffective regions . However , the nematode rec-1 mutant redistributes a wild-type number of COs in a pattern reflecting the physical map without compromising chromosome segregation [60] , indicating that CO redistribution per se is not sufficient to provoke nondisjunction . The early prophase localization of the ZHP-3/4 complex presents an elegant solution to the requirement for zhp-3/4; they function in promoting CO intermediate formation , while being dispensable for initial pairing or meiotic DSB induction [39 , 61] , The SC is required for crossing over and recruitment of the complex to the structure and concentrates its activities in proximity to nascent HR intermediates from the earliest time point that JMs can enter the crossover pathway . An outstanding question that remains is the function of ZHP-3/4 at the crossover-designated sites that appear at late pachytene stages . The behaviour of vv96 mutants suggests that lost or disrupted retention of late pro-crossover markers at designated sites does not necessarily abolish crossing over , but does disrupt some aspect of CO formation that has functional consequences for the resulting bivalent during chromosome segregation . Recent work on the architecture of nematode recombination complexes and their relationship to the SC during meiotic prophase has observed that CO/NCO outcomes are visibly manifested at late pachytene stages [34] . HR repair proteins are lost from NCO sites ( presumably indicative of completed repair ) and pro-crossover MSH-5 , COSA-1 , and BLM ( RMH-1-interacting nematode protein HIM-6; [12] ) appear at CO-designated sites in the context of central region components of the SC that envelop them in a bubble-like structure . Although the function of this structure is not known , an intriguing possibility is that it reflects an enzymatic caging which can concentrate the pro-CO activities within and protect the CO intermediate from the NCO activities taking place outside . Since ZHP-3/4 are dependent on central region SC components for their localization , it is possible that their function in this compartment is to stabilize the pro-crossover factors until desynapsis at diplotene frees the double Holliday junction ( dHJ ) precursor for resolution into a crossover . In this context , the consequences of the inability of vv96 mutants to form these late pro-crossover factor-enriched sites may result in premature exposure of the dHJ to resolvases that temporally uncouple crossover formation from chiasma emergence and regulated swapping or remodelling of the axes to which the involved DNA is tethered [62] . Such a function may explain the fact that zhp-4 ( vv96 ) mutants are in part marked by the appearance of diakinesis bivalents that are tethered by chromatin masses engaged with axis components , in addition to other bivalent structural anomalies that are suggestive of perturbed coordination between dHJ resolution and CO-triggered chromosome morphogenesis . We speculate that the failure to coordinate these events may distinguish a genetic crossover at the DNA level from a chiasma competent to direct chromosome segregation by disrupting axis exchange and/or patterning of sister chromatid cohesion .
C . elegans strains were cultured under the conditions described by Brenner [63] and all experiments were conducted at 20°C . The N2 var . Bristol strain was used as a wild-type reference and the following mutations and rearrangements were used: zhp-3 ( jf61::unc-119+ ) /hT2 I . meIs8 [Ppie-1::gfp::cosa-1 + unc-119 ( + ) ] II; cosa-1 ( tm3298 ) III . jfsi38 [gfp::rmh-1 cb-unc-119+] II . dpy-18 ( e364 ) unc-25 ( e156 ) III . spo-11 ( ok79 ) /nT1 IV . spo-11 ( me44 ) /nT1 IV . dpy-3 ( e27 ) unc-3 ( e151 ) X . rmh-1 ( jf92[M01E11 . 3::unc-119+] ) I . mus-81 ( tm1937 ) I . syp-2 ( ok307 ) V . msh-5::gfp IV . Hermaphrodites were singled at L4 stage and transferred daily to fresh plates for three consecutive days . The number of eggs of each hermaphrodites was recorded immediately after each transfer; in the last plate , the number of eggs was recorded 24 hours after transfer . The number of hermaphroditic and male progeny were scored three days later . Embryonic lethality rate was calculated as the total number of surviving progeny divided by the total number of eggs . Incidence of males was calculated as the number of males divided by the total number of surviving progeny . Recombination was assayed using visible markers by crossing zhp-4 ( vv96 ) /+ males with zhp-4 ( vv96 ) V; dpy-3 ( e27 ) unc-3 ( e151 ) X and zhp-4 ( vv96 ) V; dpy-18 ( e364 ) unc-25 ( e156 ) III hermaphrodites . Similarly , zhp-4 ( H26A ) males were crossed with zhp-4 ( H26A ) V; dpy-3 ( e27 ) unc-3 ( e151 ) hermaphrodites . NonUnc , nonDpy F1 cross progeny were picked and allowed for self-fertilize . F1s that were homozygous for zhp-4 ( vv96 ) and zhp-4 ( H26A ) respectively were identified by embryonic lethality ( Emb ) and high incidences of males ( Him ) in the F2 progeny . For wild-type , 1973 ( 1334 wild-type and 639 recombinants ) F2 progeny were scored from 10 dpy-3 unc-3/+ + and 3451 ( 2966 wild-type and 485 recombinants ) F2 progeny were scored from 15 dpy-18 unc-25/+ + heterozygotes . For zhp-4 ( vv96 ) mutants , 507 ( 437 wild-type and 70 recombinants ) F2 progeny were scored from 37 dpy-3 unc-3/+ +; zhp-4 ( vv96 ) /zhp-4 ( vv96 ) and 815 ( 710 wild-type and 105 recombinants ) F2 progeny were scored from 67 dpy-18 unc-25/+ +; zhp-4 ( vv96 ) /zhp-4 ( vv96 ) . For zhp-4 ( H26A ) mutants , 1165 ( 913 wild-type and 252 recombinants ) F2 progeny were scored from 57 dpy-3 unc-3/+ +; zhp-4 ( H26A ) /zhp-4 ( H26A ) heterozygotes and 558 ( 524 wild-type and 34 recombinants ) F2 progeny were scored from 35 dpy-18 unc-25/+ +; zhp-4 ( H26A ) /zhp-4 ( H26A ) heterozygotes . Recombination frequencies were calculated as previously described [61] , where the frequency ( p ) between two markers was calculated using the formula p = 1 - ( 1 - 2R ) 1/2 , where R is the number of visible recombinant individuals divided by the number of total progeny . The number of total progeny for the hermaphrodite was calculated as 4/3 X ( number of Wts + one recombinant class ) to compensate for the inviability of the double homozygote class . Both classes of recombinants were used in the calculations . The zhp-4 ( vv96 ) allele was recovered from a “Green Egg” mutagenesis screen ( 50 mM EMS ) that isolated mutants with X-chromosome segregation defects [14] . Cloning of vv96 revealed a C to T substitution at the 160th codon , which changes the glutamine residue ( Q ) into a premature stop codon in the coding sequence Y39B6A . 16 , predicted to be a paralog of ZHP-3 and named ZHP-4 [29] . The wild-type tagged line of zhp-4 ( vv117[zhp-4::ha] ) was generated by Shaolin Li ( Gene Editing Services ) . All the other alleles were generated by directed mutagenesis using CRISPR-Cas9 protocol previously described [64] with the only difference that Cas9 protein was purchased from PNA Bio ( CP01-200 ) . For a list of sgRNAs and repair templates refer to S1 Table . In the case of zhp-4 ( vv103 ) an indel mutation was introduced in the RING-finger domain in front of the last two cysteine residues , which resulted in a frameshift and eventually a premature stop codon . The wild-type sequence is ATTATGTCATCCACCGGAAG-AAG while the mutant sequence is ATTATGTC——CGGAAGAAAG . The mutant zhp-4 ( vv96::ha ) has been created by the positioning of the tag right before the stop codon introduced by vv96 mutation . This strain perfectly mimics the zhp-4 ( vv96 ) untagged worms allowing us to use either of them according to necessity . The ring mutants zhp-3 ( vv137[H25A] ) and zhp-4 ( vv138[H26A] ) harbour the following mutations respectively: CAC-to-GCC and CAT-to-GCC , both substituting a highly conserved His to an Ala . To raise antibodies against ZHP-4 and avoid cross-reactivity with other RING domain containing proteins , a fragment of 372 base pairs corresponding to the C-terminus of ZHP-4 was cloned into two bacterial expression vectors: pGEX-6p-2 , containing the GST tag at the N-terminus ( GE Healthcare ) and pET28a ( Qiagen ) , to generate an N-terminal 6xHis-fusion protein . Recombinant proteins were purified under native conditions using anti-GST beads ( GE Healthcare ) and Ni-NTA matrix ( Qiagen ) respectively following the manufacturer’s instructions . GST::ZHP-4 was used for antibody production in rat and 6xHis::ZHP-4 was used for sera purification ( Medimab ) . ZHP-4 antibody was purified using activated supports according to the manufacturers’ protocols ( Affi-Gel 10 , BioRad ) . For whole embryo staining , thirty-forty of 24-26h post-L4-staged adults were dissected in 1xPBS , followed by freeze crack using liquid nitrogen and fixation in methanol -20°C for 30 minutes . For whole germline staining , gonads of 24-26-h post-L4 staged adults were dissected in 1XPBS and fixed by 1% paraformaldehyde for 5 minutes , followed by freeze crack and fixation in 100% methanol for 5 minutes at -20°C . After fixation in methanol , slides were washed with PBS-T ( 0 . 1% Tween-20 ) for 5 minutes 3 times . Gonads were then blocked with 1% BSA in PBS-T for an hour and incubated with primary antibodies overnight at 4°C . The following day , slides were washed for 3 times 15-minutes each and then incubated with secondary antibodies for two hours . Afterwards , they were washed 3 times , 15 minutes each . 1μg/μL of DAPI in anti-fading agent ( Vectashield ) was added onto the slides . Images consisting of 15–20 stacks ( of 0 . 2μm increments ) , were acquired and processed using a Delta Vision Deconvolution system equipped with an Olympus 1X70 microscope or a Spinning-disc confocal microscope ( Leica DMI 6000B inverted microscope equipped with a Quorum WaveFX spinning Disc and EM CCD camera ) . The following antibodies were used in this study: guinea pig and rabbit α-HTP-3 ( 1:500–1:750 ) , goat α-SYP-1 ( 1:1000 , gift from M . Colaiacovo ) , rabbit α-HIM-8 ( 1:200 , Novus Biological , 41980002 ) , mouse α-GFP ( 1:200 , AbCAM ab290 ) , rabbit α-RAD-51 ( 1:1125 ) , guinea pig α-ZHP-3 ( 1:750 ) , rabbit α-AIR-2 ( 1:200 ) , rabbit α-HTP-1 ( 1:400 ) rat α-ZHP-4 ( 1:200 ) , mouse α-HA ( 1:100 , BioLegend , 901513 ) , mouse monoclonal α-SMO-1 6F2 ( 1:10 , DSHB ) , tubulin-FITC conjugate ( 1:500 , Sigma F-2168 ) , guinea pig α-SUN-1 S8Pi ( 1:700 ) . For specificity of anti-HA staining see S2B Fig . Secondary antibodies used in this study were: AlexaFLuor 555 goat α-guinea pig ( Molecular Probes , A21435 ) and α-rabbit ( Invitrogen , A21429 ) , AlexaFluor 488 goat α-rabbit ( Molecular Probes , A11034 ) , AlexaFLuor 488 donkey α-guinea pig , AlexaFluor 555 donkey α-goat ( Abcam 150130 ) , AlexaFluor 488 goat α-rat and AlexaFluor 488 goat α-mouse ( Jackson ImmunoResearch , 106498 ) , all of which were used in 1:1000 dilution . RMH-1 and COSA-1 are both fused with GFP and , following anti-GFP staining , all the foci were scored in each nucleus in the entire pachytene region of each gonad . For representation of the data , the pachytene region was divided into six equal zones and labelled as: early , early-mid , mid , mid-late , late pachytene , and pachytene exit; three gonads were scored for each genotype . For RAD-51 an anti RAD-51 antibody was used for the staining , and foci were scored along the whole gonad of each genotype untill the end of pachytene and then divided into six equal zones ( three gonads per genotype were scored ) . RNA interference experiments were performed as described previously [65] . In brief , dsRNA was generated using PCR amplification of 946 bp of rad-54 gene using the primers TTCAGGACGAACGGAGGAAC and TTCCACTGTCCACTGGCATC , followed by in vitro transcription with T7 RNA polymerase ( Ambion ) . At 6–8 hours post-L4 , very young hermaphrodites were injected and after 2 days processed for cytological analyses . The efficacy of the RNAi was never complete since the injected animals never showed sterility as expected by complete knockdown of rad-54 [41] . All the eggs laid by injected animals hatched , and their progeny were not sterile; however , cytological analysis of two day post injection animals showed an increase of RAD-51 foci in their germlines until mid-pachytene stage ( S4B Fig ) . Scoring of these foci revealed levels of RAD-51 foci that were comparable to those previously reported [41] . Therefore , our analysis was based on scoring RAD-51 foci in zones 1 through 4 which were affected by rad-54 ( RNAi ) . Distributions of DAPI-stained bodies , RMH-1 foci and COSA-1 foci were statistically tested by Kruskal-Wallis test followed by Dunn’s multiple pairwise coparison tests . The significance of RAD-51 foci scoring was tested by Mann-Whitney U test while embryonic lethality and incidence of males by ANOVA followed by multiple pairwise comparison tests . All calculations were performed with Prism 5 ( GraphPad ) and p < 0 . 05 was considered significant . | The creation of a viable individual from the fusion of egg and sperm requires that they each contain the correct number of chromosomes . This is ensured through the meiotic divisions , which initially fasten identical chromosomes through DNA linkages that hold them together until the cell is ready to separate them . To make these linkages , called crossovers , the cell breaks the DNA in many places , and must repair them to create a crossover , or a noncrossover . We investigate here the role of ZHP-4 , and its partner ZHP-3 which form a complex that associates along paired chromosomes and finally with crossover sites . ZHP-3/4 are conserved proteins found in many organisms that function in recruiting proteins required to decide which DNA event will become a crossover and how this DNA event is coordinated with changes in chromosome structure . Using mutations that reduce the function of ZHP-4 , we show that the complex cannot localize normally to meiotic chromosome and that crossing over fails . Our results suggest that ZHP-3/4 work at early and late steps in the process to stabilize other factors required for crossover formation . | [
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] | 2018 | C. elegans ZHP-4 is required at multiple distinct steps in the formation of crossovers and their transition to segregation competent chiasmata |
Broadly-neutralizing monoclonal antibodies ( bNAbs ) may guide vaccine development for highly variable viruses including hepatitis C virus ( HCV ) , since they target conserved viral epitopes that could serve as vaccine antigens . However , HCV resistance to bNAbs could reduce the efficacy of a vaccine . HC33 . 4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date , binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope ( E2 ) protein , respectively . Given their distinct epitopes , it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates , suggesting that some viral polymorphisms may confer resistance to both bNAbs . To investigate this resistance , we developed a large , diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins ( E1 and E2 ) . By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb , we identified E1E2 variants with resistance to one or both bNAbs , despite 100% conservation of the AR4A binding epitope across the panel . We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor , scavenger receptor B1 ( SR-B1 ) . This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation , highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes .
Hepatitis C virus ( HCV ) infects over 170 million people worldwide [1] and kills more people in the United States annually than HIV [2] . Appalachian regions of the United States saw a >350% increase in the number of new HCV infections from 2009–2012 [3] and recent outbreaks in the United States have been attributed to the rapid increase in injection drug use [4] . While direct-acting antiviral ( DAA ) therapy has revolutionized care for patients with HCV , control of the HCV pandemic remains challenging due to frequent reinfection in high-risk individuals who have achieved a sustained virologic response after DAA therapy [5] , transmission of NS5A inhibitor-resistant HCV variants from individuals failing DAA therapy [6] , and the high proportion ( ~50% ) of infected individuals who are unaware asymptomatic carriers [7] . A major goal for the development of a prophylactic vaccine against HCV is stimulation of an immune response that is protective against a wide range of naturally occurring viral variants [8 , 9] , which is a daunting challenge given the enormous genetic diversity of HCV [10–18] . Broadly neutralizing antibodies ( bNAbs ) are a useful guide for vaccine development , since they bind to relatively conserved viral epitopes , prevent successful entry of diverse HCV isolates , and have been associated with spontaneous clearance of HCV [19] . Despite the relative conservation of bNAb epitopes , polymorphisms conferring resistance to various bNAbs have been identified [20–24] , and increasing evidence has shown that polymorphisms distant from bNAb binding sites can modulate E1E2 resistance [20 , 22 , 24] . BNAb resistance polymorphisms have been identified by various methods , including alanine-scanning mutagenesis , mapping of longitudinal sequence evolution in infected humans [22] , and passage of replication competent virus ( HCVcc ) in vitro in the presence of bNAbs [21 , 23] , but an efficient method to identify common naturally-occurring resistance polymorphisms in circulating E1E2 variants has not been available . Recently , we and others have observed significant variation in sensitivity of natural E1E2 variants to a diverse panel of monoclonal bNAbs and HCV-infected sera [24 , 25] . When we compared the rank orders of neutralization of a diverse array of 19 genotype 1 HCVpp by individual bNAbs , distinct relationships between antibodies were observed , allowing grouping of all bNAbs into three distinct clusters of functionally-related antibodies , and suggesting that common E1E2 determinants of neutralization sensitivity are shared between bNAbs within each cluster [24] . In that study , we identified E2 polymorphisms conferring resistance to most antibodies falling in a group we called neutralization cluster 1 , which included bNAbs that are known to target the CD81-binding site of E2 . We were previously unable to explain the functional relationship between antibodies in a second group that we called neutralization cluster 2 . Surprisingly , this cluster includes the potent human bNAbs HC33 . 4 and AR4A , although their described binding epitopes are at opposite termini of the E2 protein [26 , 27] . HC33 . 4 is a human monoclonal antibody that binds to a continuous epitope near the N-terminus of E2 , at amino acids ( aa ) 412–423 , commonly known as ‘epitope I’ [28] . Recently , aa 408 was also shown to be a HC33 . 4 binding residue [29] . AR4A , also a human monoclonal antibody , binds to a conformational epitope including the C-terminal , membrane proximal region of E2 as well as residues on E1 . BNAbs like HC33 . 4 targeting ‘epitope I’ have been shown to neutralize HCV by blocking E2 interaction with CD81 [30–35] , while AR4A does not appear to interfere with this interaction [26] . Despite the clearly distinct binding epitopes of these two bNAbs , and possibly differing mechanisms of neutralization , we hypothesized that shared E1E2 resistance polymorphisms to these antibodies would explain the unexpected correlation between neutralization profiles of HC33 . 4 and AR4A . No obvious polymorphisms mediating this effect were identified in a small panel of E1E2 variants with varying HC33 . 4 and AR4A sensitivities , so we developed a larger panel of more than 100 E1E2 variants as well as a statistical approach to identify natural polymorphisms that were associated with resistance to each bNAb . Using these tools , we identified polymorphisms conferring resistance to HC33 . 4 and AR4A individually , as well as polymorphisms outside of either binding epitope that confer resistance to both bNAbs by modulating binding to the HCV co-receptor , scavenger receptor B1 ( SR-B1 ) .
To construct a library of E1E2 genes to predict relationships between amino acid sequence and neutralization sensitivity , we cloned more than 700 naturally occurring HCV genotype 1 E1E2 genes . Of these cloned E1E2s , 113 produced HCV pseudoparticles ( HCVpp ) that were functional in repeated tests when co-transfected with an HIV NL4 . 3Δenv-Luc reporter genome , as previously described [24] . The resulting library includes 71 subtype 1a and 42 subtype 1b E1E2 variants isolated from a total of 27 unique donors . It is not known why the majority of cloned E1E2 variants were nonfunctional in HCVpp , but this has also been observed in other studies [36] . We analyzed genetic variation in our functional E1E2 panel to confirm that it is representative of circulating strains . Loci of greatest amino acid variation of the panel across E1E2 mirror those of a reference panel of 643 genotype 1 HCV isolates from GenBank , with the greatest amino acid diversity observed in hypervariable region 1 ( HVR1 ) of E2 ( Fig 1A ) . As many as 9 possible amino acids are represented at some E1E2 loci . Overall , this neutralization panel of functional E1E2 variants contains 97% of amino acid polymorphisms present at ≥5% frequency in the Genbank genotype 1 reference panel . We first assessed variation at the known binding epitopes of the two bNAbs across the HCVpp panel . The HC33 . 4 epitope varied at position 408 , while the AR4A epitope was 100% conserved across the panel at all known binding residues ( Fig 1B ) . We then quantitated the fraction unaffected ( Fu ) of each HCVpp in the presence of each bNAb by measuring hepatoma cell entry of HCVpp in the presence of 10 μg/mL HC33 . 4 or AR4A relative to entry in the presence of nonspecific human IgG ( Fig 1C ) . Neutralization was assessed at a single concentration of bNAb rather than with serial bNAb dilutions in order to increase the throughput of the assay and to minimize the quantity of bNAb required . We have previously shown that Fu values measured by this method are reliably quantitative , as they correlate significantly with IC50 values calculated from neutralization curves of the same HCVpp/bNAb combinations as well as with IC50 values calculated from neutralization curves of replication competent virus ( HCVcc ) [24] . Another recent study also confirmed strong concordance between neutralization results obtained using HCVpp or HCVcc [36] . Each bNAb showed a more than 100-fold variation in neutralization across the HCVpp panel , which was surprising given the conservation of the bNAb binding epitopes . HC33 . 4 and AR4A were associated with a median ( min-max ) Fu of 0 . 22 ( 0 . 003–1 . 1 ) and 0 . 17 ( 0 . 01–1 . 15 ) , respectively . Using a cutoff of Fu<0 . 5 , which is roughly equivalent to an IC50 cutoff of 10 μg/mL , HC33 . 4 and AR4A neutralized 88% and 85 . 8% of the HCVpp panel , respectively . As we previously observed using a panel of 19 HCVpp [24] , there was a significant positive correlation between the rank order of sensitivity of the 113 HCVpp in this panel to HC33 . 4 and AR4A ( r = 0 . 44 p = 7e-7 ) ( Fig 2A ) . We identified E1E2 variants with exquisite sensitivity to both bNAbs ( Fu < 0 . 02 with either bNAb ) as well as variants with high level resistance to both bNAbs ( Fu >1 ) , with other E1E2 variants distributed between those extremes . This correlation between neutralization profiles of the two bNAbs was surprising , given that they do not share binding residues ( Fig 1B ) . To further confirm that the two bNAbs bind to distinct epitopes , we performed binding competition assays between the two bNAbs . E1E2 protein-coated ELISA wells were pre-incubated with a high concentration of either HC33 . 4 or AR4A ( blocking bNAbs ) , followed by biotinylated HC33 . 4 or AR4A at a concentration selected to give 50% of maximal binding ( EC50 ) , with binding of the biotinylated bNAb detected using streptavidin-horseradish peroxidase . A ratio of binding of each biotinylated bNAb in the presence of blocking bNAb divided by binding in the absence of blocking bNAb was calculated ( Fig 2B ) . As expected , each bNAb competed for binding with itself , but HC33 . 4 and AR4A showed minimal competition for E1E2 binding with each other , confirming that the bNAbs bind to distinct epitopes . In spite of their shared resistance pattern , HC33 . 4 more potently neutralized subtype 1a than subtype 1b HCVpp ( Fu median = 0 . 17 for 1a vs . 0 . 27 for 1b , p = 0 . 002 ) , while AR4A displayed minimal difference in subtype neutralization ( Fig 3A ) . The significant positive correlation between HC33 . 4 and AR4A neutralization profiles observed with the full HCVpp panel was also observed on analysis of the subtype 1a-only ( r = 0 . 39 , p = 7e-4 ) and subtype 1b-only ( r = 0 . 69 , p = 1e-6 ) subsets of the panel ( S1 Fig ) We used novel ( SNAPR ) and established ( LASSO ) methods to identify E1E2 sequence determinants of resistance to HC33 . 4 and AR4A . An E1E2 amino acid alignment was generated including sequences of each of the 113 variants in the panel . For the SNAPR method , due to the higher degree of similarity among E1E2 variants originating from the same HCV-infected donor , and a variable number of E1E2 variants contributed by each donor , neutralization and sequence data from variants from underrepresented donors were randomly selected for replication until the number of data points in the analysis representing each individual donor was identical . For each amino acid position across E1E2 , all HCVpp were divided into groups according to the amino acid occupying that position , and the amino acid associated with lowest median Fu ( greatest neutralization sensitivity ) for each bNAb was identified . For each bNAb , at each position , a nonparametric ( Wilcoxon rank-sum ) test was used to compare the neutralization values of all HCVpp with E1E2 carrying the amino acid associated with the lowest mean Fu with the neutralization values of all of the other variants , generating a “SNAPR value” for each position ( Fig 3B ) . While the replication of some sequence and neutralization data reduces the effect of over-representation of some donors , the method also artificially inflates the sample size with data that are not independent , so calculated p-values are artificially low . Therefore , the p-values themselves cannot be used to determine whether variation in neutralization sensitivity at an individual position is statistically significant . Rather , these "SNAPR-values" provide a metric for comparison between effects at different polyprotein positions . Because of the potential for subtype differences to dominate findings , grouped genotype ( S2 Fig ) and subtype 1a only analyses were performed separately for further focused investigation . SNAPR-values spanned approximately 10 and 20 orders of magnitude for HC33 . 4 and AR4A , respectively ( Fig 3C ) . We also re-analyzed the neutralization and sequence data using a method that considers multiple residues in combination—LASSO , without the replication of data required for the SNAPR analysis [37] . Of the 20 most likely resistance polymorphism position predictions from SNAPR for HC33 . 4 and AR4A ( Table 1 ) 5 positions ( for HC33 . 4 ) and 5 positions ( for AR4A ) were also among the 20 and 18 most likely LASSO predictions , respectively . Of note , position 408 was predicted by SNAPR to modulate resistance to HC33 . 4 but not AR4A , which supports sensitivity and specificity of the SNAPR analysis since lysine ( K ) 408 is a known binding residue for HC33 . 4 but not AR4A , and it is the only known binding residue for either bNAb that varies significantly across the neutralization panel . Position 408 was not among the top 20 LASSO predictions for HC33 . 4 . Given the distinct binding epitopes of HC33 . 4 and AR4A ( Figs 1B and 3C ) , and the imperfect correlation between neutralization profiles of the bNAbs ( Fig 2A ) , it is not surprising that many of the loci predicted to modulate sensitivity to HC33 . 4 and AR4A are not shared . Interestingly , SNAPR predicted positions 242 , 403 , and 438 as determinants of neutralization sensitivity for both HC33 . 4 and AR4A . Positions 242 and 403 were also predicted by LASSO to be determinants of sensitivity for both bNAbs ( Table 1 ) . To further investigate the 8 most likely SNAPR predictions for HC33 . 4 and AR4A , we compared Fu values of HCVpp grouped by the amino acid present at each position , without the replicated neutralization data included in the SNAPR analysis ( Fig 4 ) . In the analysis for HC33 . 4 , E1E2 variants with methionine ( M ) vs . valine ( V ) at position 242 showed significant differences in neutralization sensitivity ( median Fu 0 . 10 vs . 0 . 25 , p = 0 . 02 ) ( Fig 4A ) . For AR4A , M vs . V at position 242 were also associated with significant differences in neutralization sensitivity ( median Fu 0 . 12 vs . 0 . 27 , p = 0 . 02 ) ( Fig 4B ) . Variation at the 242 position resulted in the 13th and 8th most extreme LASSO coefficients of any position in E1E2 for HC33 . 4 and AR4A , respectively ( Table 1 ) . Variants with leucine ( L ) vs . phenylalanine ( F ) at position 403 showed significant differences in neutralization sensitivity to HC33 . 4 ( median Fu 0 . 042 vs . 0 . 22 , p = 1E-3 ) . ( Fig 4A ) . For AR4A , L vs . F at position 403 were also associated with significant differences in neutralization sensitivity ( median Fu 0 . 08 vs . 0 . 20 , p = 0 . 01 ) ( Fig 4B ) . Variation at the 403 position resulted in the most extreme LASSO coefficients of any position in E1E2 for HC33 . 4 and AR4A ( Table 1 ) . For HC33 . 4 , E1E2 variants with leucine ( L ) vs . valine ( V ) at position 438 also showed significant differences in neutralization sensitivity ( median Fu 0 . 096 vs . 0 . 39 , p = 3E-3 ) . ( Fig 4A ) . For AR4A , L vs . V at position 438 were also associated with significant differences in neutralization sensitivity ( median Fu 0 . 11 vs . 0 . 44 , p = 0 . 01 ) ( Fig 4B ) . To test SNAPR predictions , putative resistance polymorphisms at the 8 positions with the lowest SNAPR-values for each bNAb were introduced individually by site directed mutagenesis into 2–3 distinct wild type ( WT ) E1E2 variants in which they were not naturally present . These WT variants were each from subtype 1a and differed from each other prior to mutagenesis by an average of 42 amino acids ( 7% ) . Mutated E1E2 and corresponding WT variants were used to produce HCVpp , which were tested for neutralization by HC33 . 4 ( Fig 5A ) or AR4A ( Fig 5B ) . To control for experimental variation between HCVpp neutralization experiments , neutralization of each mutated and WT HCVpp pair was tested in at least 2 independent experiments . Experiments were considered independent only if independently produced HCVpp preparations ( transfections ) were used and independent neutralization assays were performed . M242V , L403F and L438V were predicted to modulate resistance to both HC33 . 4 and AR4A , so these mutations were tested for effect on each bNAb . Four of the 8 polymorphisms predicted by SNAPR to confer resistance to HC33 . 4 showed statistically significant effects after introduction by site directed mutagenesis . Notably , mutation of lysine ( K ) 408 to methionine ( M ) led to an increase in resistance ( WT mean Fu of 0 . 09 vs . K408M mean Fu of 0 . 72 , p<0 . 0001 ) , which was expected since K408 was recently identified by alanine scanning as a binding residue for HC33 . 4 [29] . Mutation of leucine ( L ) 403 to phenylalanine ( F ) also led to a significant increase in resistance to HC33 . 4 . Curiously , mutation of L438 to valine ( V ) , which was predicted by SNAPR to confer resistance to HC33 . 4 , instead conferred significantly increased sensitivity to the bNAb ( WT mean Fu 0 . 22 vs . L438V mean Fu 0 . 04 , p = 0 . 004 ) . Four of the 8 polymorphisms predicted to confer resistance to AR4A also showed statistically significant effects after introduction by site directed mutagenesis . As with HC33 . 4 , L403F conferred significant resistance to AR4A neutralization ( WT mean Fu 0 . 08 vs . L403F mean Fu 0 . 23 , p = 0 . 007 ) , and L438V conferred increased AR4A sensitivity ( WT mean Fu 0 . 20 vs . L438V mean Fu 0 . 06 , p = 0 . 03 ) . Notably , mutation of serine ( S ) 686 to threonine ( T ) and valine ( V ) 720 to isoleucine ( I ) also conferred significant resistance to AR4A . Neither S686 nor V720 fall at a known binding residue for AR4A , but they are 6 and 22 amino acids from known AR4A binding residues , respectively . Though polymorphisms at 242 were also predicted by SNAPR and LASSO to be determinants of resistance for AR4A and HC33 . 4 ( Fig 3C; Table 1 ) , mutagenesis at this position did not confer a significant change in sensitivity to either antibody . Taken together , these results confirm that L403F and L438V modulate sensitivity to neutralization by both HC33 . 4 and AR4A . All resistance polymorphisms except V720I that had been validated by introduction into neutralization sensitive E1E2 variants were reverted in 2–5 distinct E1E2 variants where they were naturally present ( Fig 6 ) . Mutated E1E2 variants and corresponding WT variants were used to produce HCVpp , which were tested for neutralization by HC33 . 4 ( Fig 6A ) or AR4A ( Fig 6B ) . Three of four polymorphisms that showed an effect on HC33 . 4 when introduced into neutralization sensitive E1E2 variants also showed a significant effect when reverted in E1E2 variants where they were already naturally present . Mutation of M408 to the known HC33 . 4 binding residue , K , resulted in significantly increased sensitivity to HC33 . 4 neutralization ( Wild type mean Fu 0 . 75 vs . M408K Fu 0 . 10 , p = 0 . 001 ) . Mutation of F403 to L also increased sensitivity to HC33 . 4 . Mutation of V438 to L also conferred a small but significant increase in HC33 . 4 sensitivity ( Wild type mean Fu 0 . 43 vs . V438L Fu 0 . 38 , p = 0 . 02 ) . This was unexpected because in other E1E2 variants , the reciprocal mutation of L438 to V had also conferred increased neutralization sensitivity , but the magnitude of the effect of V438L was very small ( mean Fu fold change of 0 . 9 ) relative to the magnitude of the effect of L438V ( mean Fu fold change of 0 . 2 ) Two of three polymorphisms that showed an effect on AR4A when introduced into neutralization sensitive E1E2 variants also showed a significant effect when reverted in E1E2 variants where they were already naturally present . Most notably , mutation of F403 to L and mutation of V438 to L conferred increased sensitivity to AR4A , just as they had for HC33 . 4 . Taken together , these results show that mutation of L403 to F and mutation of F403 to L confer reciprocal neutralization resistance and sensitivity effects on both HC33 . 4 and AR4A , while mutation of L438 to V in some E1E2 variants and V438 to L in others confers increased sensitivity to neutralization by both HC33 . 4 and AR4A . To measure the magnitude of the effect of L403F and L438V mutations on neutralization sensitivity , we measured neutralization of wild type 1a154 ( H77 ) , 1a154_L438V , and 1a154_L403F HCVpp by serial dilutions of HC33 . 4 and AR4A ( Fig 7A ) . As expected , the 1a154_L438V HCVpp variant was most sensitive to neutralization . Wild type 1a154 HCVpp showed a 3-fold increase in IC50 relative to 1a154_L438V for both antibodies , and 1a154_L403F HCVpp showed a 24-fold increase in IC50 relative to 1a154_L438V HCVpp for HC33 . 4 and a 90-fold increase in IC50 for AR4A . We also confirmed the resistance phenotypes of the mutations using replication competent cell culture virus ( HCVcc ) ( Fig 7B ) . Wild type 1a154 , 1a154_L438V , or 1a154_L403F E1E2 genes were cloned into a J6/JFH-1 HCVcc genome lacking E1E2 [38] , and replication competent virus was produced from each chimeric strain . HCVcc neutralization results mirrored those observed with HCVpp very closely , with 1a154_L438V most sensitive to neutralization by each bNAb , wild type 1a154 8-fold more resistant to HC33 . 4 and 7-fold more resistant to AR4A , and 1a154_L403F 40-fold more resistant to HC33 . 4 and 24-fold more resistant to AR4A . To understand whether these changes in neutralization sensitivity were mediated by changes in binding of the bNAbs to E1E2 , we performed an ELISA to measure binding of serial dilutions of the bNAbs to 1a154_L438V , 1a154 , and 1a154_L403F E1E2 proteins ( Fig 7C ) . No significant difference in binding of either bNAb to the E1E2 variants was observed , suggesting that differences in bNAb binding to E1E2 are likely not the mechanism by which L403F and L438V modulate resistance to neutralization by HC33 . 4 and AR4A . Using Chinese hamster ovary ( CHO ) cells stably expressing either human CD81 or human SR-B1 [33] , we investigated relative binding of wild type 1a154 ( H77 ) , 1a154_L403F , and 1a154_L438V E2 proteins to these HCV receptors . We used previously described methods to clone these variants without E1 and with replacement of their transmembrane domain with a histidine tag , allowing their expression as soluble E2 ( sE2 ) [39] . Serial dilutions of these soluble proteins were incubated with CD81-CHO , SR-B1-CHO , or wild type CHO cells , then labeled with anti-HIS and fluorescent secondary antibodies to allow detection of binding of sE2 on the cell surface . We were able to quantitate dose-dependent binding of sE2 to both CD81 and SR-B1 using this technique . Fig 8A shows flow cytometry histogram plots of binding of serial dilutions of 1a154 sE2 to CD81-CHO cells , relative to background binding to wild type CHO cells without CD81 or SR-B1 . After normalizing for total sE2 protein input ( S3 Fig ) , we compared binding of serial dilutions of 1a154 , 1a154_L403F , and 1a154_L438V sE2 proteins to SR-B1 and CD81 ( Fig 8B ) . Remarkably , we saw a clear increase in binding of 1a154_L403F to SR-B1 relative to binding of wild type 1a154 , and we saw a decrease in SR-B1 binding of 1a154_L438V , matching the hierarchy of neutralization resistance of these E2 variants . In comparing binding of the same variants to CD81 , we observed a small decrease in binding of 1a154_L403F relative to 1a154 , and a large decrease in binding of 1a154_L438V , confirming that the differences between 1a154 and 1a154_L403F binding to SR-B1 are not likely due to differences in protein input . We next compared binding of a matched , fixed concentration of 1a154 and 1a154_L403F sE2 to SR-B1 and CD81 in the presence of increasing concentrations of nonspecific IgG or HC33 . 4 ( Fig 8C ) . The 1a154_L438V sE2 variant did not have high enough baseline binding to allow accurate measurement of percent inhibition by HC33 . 4 , and we were also not able to study AR4A in this manner because it requires both E1 and E2 for binding . HC33 . 4 reduced binding of 1a154 and 1a154_L403F variants to both SR-B1 and CD81 in a dose-dependent manner . It is not surprising that HC33 . 4 inhibits both SR-B1 and CD81 binding , since a prior study of HC33 . 4-like antibodies showed that some could block binding to both receptors [33] . The concentrations of HC33 . 4 inhibiting 50% of binding to SR-B1 or CD81 ( IC50 values ) of 1a154 and 1a154_L403F sE2 were nearly identical , suggesting that the differing affinities of these proteins for SR-B1 and CD81 did not alter the percent binding inhibition of each by equivalent concentrations of mAb . Unlike HCVpp in neutralization assays , the sE2 variants are normalized for protein concentration , so it is also informative to consider absolute sE2 binding in the presence of mAb . To determine whether modulation of SR-B1 binding could mediate mAb neutralization resistance , we analyzed absolute SR-B1 binding of a fixed concentration of sE2 in the presence of varying concentrations of HC33 . 4 ( Fig 8D ) , and binding of varying concentrations of sE2 in the presence of a fixed concentration of HC33 . 4 ( Fig 8E ) . Comparison of sE2 binding of 1a154 and 1a154_L403F in the presence of increasing concentrations of HC33 . 4 ( Fig 8D ) , showed that 1a154_L403F sE2 bound more SR-B1 than an equivalent concentration of 1a154 sE2 at inhibitory but non-saturating concentrations of HC33 . 4 . We also measured binding of serial dilutions of 1a154 , 1a154_L403F , and 1a154_L438V sE2 proteins to SR-B1 after preincubation with a fixed concentration of HC33 . 4 ( 40 μg/mL ) ( Fig 8E ) . As observed in the absence of antibody , multiple concentrations of 1a154_L403F sE2 incubated with a high concentration of HC33 . 4 showed greater binding to SR-B1 relative to 1a154 sE2 , and 1a154_L438V sE2 showed consistently less binding . Together , these results suggest that , in the presence of inhibitory but non-saturating concentrations of HC33 . 4 , 1a154_L403F sE2 binds more SR-B1 than an equivalent concentration of 1a154 sE2 , and 1a154_L438V sE2 binds less , providing a likely mechanism by which these polymorphisms could confer increased resistance or sensitivity , respectively , to mAbs whose mechanism of neutralization is interference with the E2-SR-B1 interaction .
We have developed a high-throughput platform for measurement of neutralizing antibody breadth and prediction of HCV neutralizing antibody resistance polymorphisms . Despite the relative conservation of HC33 . 4 and AR4A binding epitopes , with 100% conservation of known AR4A binding residues across the panel , we identified E1E2 variants with resistance to one or both bNAbs . We also identified amino acid polymorphisms in E2 conferring resistance to each bNAb individually , as well as polymorphisms outside of both binding epitopes that modulate resistance to both bNAbs . We determined that two of these polymorphisms , L403F and L438V , modulate resistance of both HCVpp and HCVcc to both HC33 . 4 and AR4A . These mutations increase or reduce E2 binding to SR-B1 , identifying a novel mechanism of broad bNAb resistance . It is interesting that HC33 . 4 IC50 values calculated from inhibition of binding of 1a154 and 1a154_L403F to SR-B1 were nearly equivalent , despite the apparent differences in affinity of the two sE2 variants for SR-B1 ( Fig 8C ) . This could be a limitation in the sensitivity of the binding assay , or alternatively could suggest that HC33 . 4 binding affinity for sE2 is significantly higher than the affinity of even the 1a154_L403F-SR-B1 interaction . These binding inhibition IC50 values were higher than the neutralization IC50 values measured for the same variants , likely due to differences in the assays , such as the amount of E2-receptor interaction necessary to generate a detectable signal above background . We show that , despite the equivalent binding inhibition IC50 values of 1a154 and 1a154_L403F , differences in sE2 binding to SR-B1 are a likely mechanism of neutralization resistance , since the neutralization resistant variant , 1a154_L403F , binds more SR-B1 than the same amount of 1a154 sE2 in the presence of non-saturating concentrations of HC33 . 4 ( Fig 8D and 8E ) . As our ability to query larger sets of naturally occurring HCV isolates for their sensitivity to bNAbs increases , so does our understanding of determinants of bNAb resistance—a key barrier to developing an effective prophylactic vaccine against HCV . In a previous report , we found that bNAbs cluster into functional groups with respect to the HCV variants that they neutralize most and least potently . This clustering of bNAbs is determined at least in part by shared or overlapping binding epitopes , but we and others have shown that polymorphisms distant from known binding epitopes can also confer bNAb resistance [20 , 22 , 24] . This study provides evidence that these extra-epitopic polymorphisms play an important role in neutralization resistance of natural E1E2 isolates . Mutations arising within mAb binding epitopes tend to be an antibody-specific resistance mechanism , and cannot confer resistance to bNAbs with epitopes that are 100% conserved . Here we describe a novel mechanism that can confer resistance to multiple anti-HCV bNAbs , even if the bNAb binding epitopes are completely intact , by modulating E2 binding to SR-B1 . To our knowledge , L403F and L438V are the first examples of a naturally-occurring mutations that confer resistance or sensitivity to bNAbs by this mechanism . L438 falls near the CD81 binding site of E2 [40] , which is consistent with our finding that mutation at this site reduced sE2 binding to CD81 , possibly also contributing to the increased bNAb sensitivity of L438V mutants . Introduction of L438V significantly decreased E1E2 fitness to mediate HCVpp or HCVcc entry into hepatoma cells ( S5 Fig ) , which is consistent with the observed reduction in binding of 1a154_L438V sE2 to CD81 and SR-B1 . We found that L403F increased binding to SR-B1 but decreased binding to CD81 . Notably , despite these opposing binding effects , we observed a net increase in E1E2 resistance to neutralization by HC33 . 4 and AR4A after introducing this mutation . The effect of L403F on SR-B1 binding may be dominant over the CD81-binding effect because the interaction of E2 with SR-B1 most likely occurs before binding of CD81 during HCV entry [41 , 42] , or because of differences in relative expression of SR-B1 and CD81 on the surface of hepatocytes . This warrants further study , as it has potentially interesting implications for strategies to inhibit HCV entry with antibodies or small molecules . The binding epitope of HC33 . 4 has been mapped in prior studies , and L403 and L438 were not found to be binding residues [28] . The binding epitope of AR4A is less clearly defined , but L403 and L438 were also not among probable AR4A binding residues [26] . Notably , L438 was identified as a contact residue for mAb AR3C in the crystal structure of AR3C/strain H77 E2 described by Kong et al [39] , and another study showed that AR3C and AR4A do not compete for binding to E2 [26] , Together , these data are all consistent with our finding that L403 and L438 are extra-epitopic for HC33 . 4 and AR4A . Recent crystallization of the E2 protein core in complex with a bNAb has been informative [39] . However , large deletions in E2 to facilitate crystallization preclude analysis of many bNAb epitopes , including the HC33 . 4 and AR4A epitopes . Given the difficulty and limitations of co-crystallizing HCV bNAbs with HCV E2 , much of what we know about bNAb-E1E2 interactions will need to be inferred by a comprehensive approach including binding studies with alanine-scanning mutants as well as binding competition assays . This study shows the utility of an additional , complementary approach that can be used to measure neutralizing breadth , group functionally similar bNAbs , and identify bNAb resistance polymorphisms that may fall within or outside of known binding epitopes . These data are particularly relevant given studies in animal models suggesting that combinations of bNAbs may be necessary to provide sterilizing protection against HCV infection [43 , 44] . Based on their distinct binding epitopes , it would have been reasonable to assume that neutralizing breadth of bNAbs like HC33 . 4 and AR4A would be greater if they were used in combination . That may still be true , but this study shows that an unexpectedly high proportion of HCV variants with resistance to one bNAb may also have resistance to the other , which could reduce the efficacy of this bNAb combination . While we were able to identify polymorphisms modulating resistance to multiple bNAbs , there were limitations to the study design and approach . We only sampled 97% of the naturally occurring polymorphisms that exist at a ≥5% threshold in a large set of Genbank HCV genotype 1 sequences . When the frequency threshold for polymorphism prevalence is reduced to ≥1% , the coverage is reduced to 78% . While SNAPR correctly predicted the 438 locus as a modulator of HC33 . 4 and AR4A resistance , it incorrectly predicted that L438V would confer bNAb resistance , when in fact this mutation confers increased sensitivity to both bNAbs . The error may have arisen due to genetic linkage between the 438 locus and other resistance-determining loci in E1E2 , since the LASSO algorithm , which adjusts for linkage , did not predict that the 438 locus is a determinant of neutralization sensitivity . Notably , the SNAPR algorithm accurately predicted position 408 , a known binding residue , as a mediator of HC33 . 4 resistance , while LASSO did not . Further testing would be necessary to more clearly determine whether SNAPR , LASSO , or a combination of the two methods would be best suited to predict resistance polymorphisms in HCV E1E2 . While the effects of the L403F and L438V polymorphisms are significant , they are small in magnitude relative to the large variation in neutralization sensitivity observed between natural isolates , suggesting that combinations of polymorphisms likely play an important role in bNAb resistance . Even larger , more diverse E1E2 panels are required to reduce confounding from genetic linkage , to probe rarely occurring natural polymorphisms , and to better define the influence of combinations of polymorphisms on neutralization resistance . In conclusion , we have developed a large , diverse HCV neutralization panel and a statistical approach using amino acid sequence variation and neutralization sensitivity to identify bNAb resistance polymorphisms in E1E2 . Despite conservation of HC33 . 4 and AR4A binding epitopes across the E1E2 panel , we discovered variants with resistance to both bNAbs , identifying polymorphisms conferring resistance to each bNAb individually , as well as polymorphisms outside of either binding epitope that modulate resistance to both bNAbs . We determined that two of these polymorphisms , L403F and L438V , modulate resistance to HC33 . 4 by increasing or decreasing E2 binding to SR-B1 , which is a novel mechanism of bNAb resistance . This study highlights the important contribution of extra-epitopic polymorphisms to bNAb resistance , presenting a potential mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes . This diverse viral panel and novel computational pipeline are broadly applicable to future studies to define neutralizing antibody breadth , identify functionally-related bNAbs , and define mechanisms of bNAb resistance .
HC33 . 4 [29] was a gift of Steven Foung ( Stanford University School of Medicine , Stanford , California . AR4A [27] was a gift from Mansun Law ( The Scripps Research Institute , La Jolla , California , USA ) . Plasma samples obtained from HCV infected subjects in the BBAASH cohort [15 , 16 , 45] , Irish Anti-D cohort [46] , and Swan Project [47] were used to construct a library of genotype 1 E1E2-expressing lentiviral pseudoparticles using a high-throughput production and screening approach . The E1E2 region was PCR amplified from cDNA reverse transcribed from viral RNA purified from subject plasma and cloned into the expression vector pcDNA3 . 2/V5/Dest ( Invitrogen ) using Gateway technology in a one-tube BP/LR reaction , as previously described [19] . HCVpp were produced by lipofectamine-mediated transfection of HCV E1E2 and pNL4-3 . Luc . R-E- plasmids into HEK293T cells ( ATCC ) in 96-well plates as previously described [19] . Hep3B cells were exposed to transfected 293T supernatants in order to test for the presence of infectious HCVpp , as previously described [19] . HCVpp were considered infectious in the initial screen if infection of Hep3B cells ( ATCC ) in a 96 well format resulted in greater than 200 , 000 RLU of luciferase activity , which is >10X typical values obtained from infection with mock pseudoparticles . E1E2 variants included in the panel differed by at least one amino acid from every other clone contained in the library . Envelopes that displayed enhanced infection in the presence of neutralizing bNAbs ( Fu >1 . 2 with either bNAb ) were not included in the analysis or in the description of library meta-data as these values most often resulted from HCVpp with poor infectivity . 18 of the 113 E1E2 variants in the final panel were previously described: 1a38 , 1a53 , 1a72 , 1a80 , 1a114 , 1a123 , 1a129 , 1a142 , 1a154 , 1a157 , 1b09 , 1b14 , 1b20 , 1b34 , 1b38 , 1b52 [19] and 1a116 , 1b21 [24] . Sequences of the remaining 95 E1E2 clones have been submitted to GenBank accession numbers KY565136—KY565230 . Sanger sequencing of the entire length of the cloned E1E2 region was performed . Amino acid sequences from a nucleic acid MUSCLE alignment [48] were used to build a phylogenetic tree . Initial tree ( s ) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model , and then selecting the topology with superior log likelihood value [49] . All trees are drawn to scale , with branch lengths measured in the number of substitutions per site , and all positions containing gaps and missing data were eliminated . Evolutionary analyses were conducted in MEGA6 [50] . Sequence logos were generated using VisSPAv1 . 6 ( http://sray . med . som . jhmi . edu/SCRoftware/VisSPA/ ) . Polymorphisms associated with bNAb resistance or sensitivity were introduced into at least two independent E1E2 clones . Mutants were created using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit ( Agilent ) and Sanger sequencing was performed to verify that all mutants differed from parent clones at only the desired locus . For infectivity and neutralization testing of the panel of 113 HCVpp , 2 , 000 Hep3B cells per well were plated in 384-well white flat bottom tissue culture plates . For infectivity and neutralization testing of site-directed mutants 8 , 000 Hep3B cells per well were plated in flat bottom 96-well tissue culture plates and incubated overnight in a humidified CO2 incubator at 37°C . Media was removed from the cells the following day and replaced with 50μL of culture supernatant containing HCVpp ( 96-well plates ) or 25μL of HCVpp supernatant ( 384 well plates ) . The plates were placed in a CO2 incubator at 37°C for 5 hours , after which the HCVpp were removed and replaced with 100μL of phenol-free Hep3B media ( 96-well plates ) or 50μL of phenol-free Hep3B media ( 384-well plates ) and incubated for 72 hours at 37°C . For 96-well plate infections , media was removed from the cells and 50 μL of 1x Cell Culture Lysis Reagent ( Promega ) added and left to incubate for >5 minutes then 45μL from each well were then transferred to a white , low-luminescence 96-well plate ( Berthold ) and read in a Berthold Luminometer ( Berthold Technologies Centro LB960 ) . Each sample was tested in duplicate . For 384-well plate infections , cells were lysed directly in the culture plate with 25μL of lysis buffer and luciferase activity measured using a BMG Labtech Fluostar Omega luminometer . A mock pseudoparticle ( no envelope ) was used as a negative control . The same procedure used to measure infectivity was employed , except that HCVpp were incubated with 10μg/mL of bNAb or serial dilutions of bNAb at 37°C for 1 hour prior to addition to the Hep3B target cells . Infections were performed in duplicate with the test antibody and nonspecific human IgG , the negative control . Murine Leukemia Virus ( MLV ) was used as a control for nonspecific neutralization . Fraction Unaffected ( Fu ) was calculated as RLU in the presence of test antibody/RLU in the presence of nonspecific human IgG . Each replicate RLUmAb value was divided by the average of two replicate RLUIgG values . % Neutralization was calculated as ( 1-Fu ) x100% . To estimate the precision of Fu neutralization measurements , we compared Fu neutralization values of HCVpp measured in independent replicate experiments and observed a highly significant correlation ( S6 Fig ) Amino acid alignments were assembled as described in the phylogenetic analysis . To account for the uneven number of the infectious clones per human donor , neutralization values and corresponding E1E2 sequences were selected at random from each human donor and added to the initial data set until all donors were represented by an equal number of isolates . For each position in the alignment , HCVpp were grouped according to the amino acid encoded at that locus . The amino acid at each position associated with greatest bNAb sensitivity was identified by comparing median Fu values of the HCVpp in each amino acid group . Fu values for HCVpp in the most sensitive amino acid group were then compared to the Fu values of HCVpp with any other amino acid at the same position using a Wilcoxon rank-sum test . The resulting p-value is the SNAPR-value associated with that locus . Analysis was implemented using code developed for R , which is freely available upon request . The LASSO combines a prediction error term ( the least squares error ) with a model complexity penalty , which regularizes the model coefficients to perform variable selection and prevent over-fitting [37] . The two replicate fraction unaffected values were square root transformed , and the mean of these used as the outcome variable , which the model aims to explain using the amino acid sequence . The amino acids at each site were our explanatory variables , and these were encoded as indicator variables . Leave-one-out cross validation was used to select the optimal LASSO penalty ( with the lowest mean-squared error ) , which gives the coefficients for each amino acid at each site , which were used as the LASSO results throughout . This was performed in R using the Lasso implementation from the package "glmnet" ( https://cran . r-project . org/web/packages/glmnet/ ) . BNAb binding to E1E2 was quantitated using an enzyme-linked immuosorbent assay ( ELISA ) as previously described [51] . 293T cells were transfected with E1E2 expression constructs . 48 hours post-transfection cell lysates were harvested . Plates were coated with 500ng Galanthus nivalis ( GNA ) lectin ( Sigma-Aldrich ) and blocked with phosphate-buffered saline containing 0 . 5% Tween 20 , 1% non-fat dry milk , and 1% goat serum . E1E2 cell lysates were added . BNAbs were assayed in duplicate 2 . 5-fold serial dilutions , starting at 10 μg/ml . Binding was detected using HRP-conjuagated anti-human IgG secondary antibody ( BD Pharmingen no . 555788 ) . For binding competitions ELISA , E1E2 protein-coated ELISA wells were pre-incubated with 20 μg/ml of either HC33 . 4 or AR4A ( blocking bNAbs ) , followed by biotinylated HC33 . 4 or AR4A at a concentration selected to give 50% of maximal binding ( EC50 ) , with binding of the biotinylated bNAb detected using streptavidin-horseradish peroxidase . A ratio of binding of each biotinylated bNAb in the presence of blocking bNAb divided by binding in the absence of blocking bNAb was calculated . HCVcc chimeras were generated as previously described [38 , 52] . Briefly , after digestion of the HCVcc backbone with AfeI ( New England Biolabs ) , 1a154 ( H77 strain ) , 1a154_L438V , and 1a154_L403F E1E2 genes amplified from library plasmids were inserted in frame using In-Fusion cloning ( Clontech ) . 2μg of plasmid DNA was linearized using XbaI ( New England Biolabs ) then used for in vitro RNA transcription using the T7 MEGAscript kit ( Ambion ) . RNA clean-up was performed using RNeasy mini kit ( Qiagen ) , quantified using a NanoDrop 1000 spectrophotometer ( Thermo Scientific ) , and stored at –80°C . 10μg of RNA was transfected into 1 . 8e6 Huh7 . 5 . 1 cells ( a gift of Charles Rice , The Rockefeller University , New York City , New York , USA ) using Nucleofector Kit T ( Amaxa ) and plated in a 6-cm plate . Transfection supernatants were collected 4–11 days later and stored at -80°C . Supernatants were titered by serial dilution and infection of Huh7 . 5 . 1 cells . HCVcc neutralization assays were performed in triplicate as described elsewhere [38 , 52] . Briefly , human hepatoma Huh7 . 5 . 1 cells were maintained in DMEM supplemented with 10% fetal bovine serum and nonessential amino acids . 10 , 000 Huh7 . 5 . 1 cells per well were plated in flat bottom 96 well tissue culture plates and incubated overnight at 37°C . The following day , HCVcc were mixed with mAb ( 3-fold dilutions started at 50μg/mL ) then incubated at 37°C for 1 hour . Media was removed from the cells and replaced with 50 μL of HCVcc/antibody mixture . The plates were placed in a CO2 incubator at 37°C overnight , after which the HCVcc were removed and replaced with 100μL of Huh7 . 5 . 1 media and incubated for 48 hours at 37°C . Medium was then removed and cells were fixed with 4% formaldehyde then stained for HCV NS5A using primary anti-NS5A antibody 9E10 ( a gift of Charles Rice , The Rockefeller University , New York City , New York , USA ) at 1:2 , 000 dilution for 1 hour at room temperature . Cells were washed twice with PBS and stained using secondary antibody Alexa Daylight 488–conjugated goat anti-mouse IgG ( Life Technologies ) at 1:500 dilution for 1 hour at room temperature . Cells were washed twice in PBS and then stored in 100μl PBS at 4°C . Images were acquired and spot forming units were counted for infection in the presence of mAb ( HCVccSFUtest ) or PBS alone ( HCVccSFUcontrol ) using an AID iSpot Reader Spectrum operating AID ELISpot Reader version 7 . 0 . Percent neutralization was calculated as 100% x [1- ( HCVccSFUtest /HCVccSFUcontrol ) ] . A truncated , soluble form of the 1a154 ( H77 ) strain E2 ectodomain ( sE2 ) that retains antigenticity and function as previously described [39] , encompassing residues 384–645 , was cloned into a mammalian expression vector ( phCMV3_Ig Kappa_HIS , a gift of Leopold Kong , The Scripps Research Institute , La Jolla , California , USA ) from plasmids containing H77 structural proteins . The vector allows expression of E2 protein with a C-terminal His tag as well as an N-terminal murine Ig Kappa leader signal for efficient protein secretion . H77 mutants , L403F and L438V , were created as described above and verified by Sanger sequencing . Each E2 construct was co-transfected with pAdvantage ( Promega ) into HEK293T cells and incubated for 72 hours at 37°C . Supernatant was collected at 48 and 72 hours , passed through a 0 . 2μm filter , and concentrated using a regenerated cellulose centrifugal filter with a 10kDa cutoff ( Amicon ) . Serial 6 . 25 fold dilutions of each sE2 supernatant beginning with a 1 to 40 dilution were immobilized onto ELISA wells pre-coated with 500 ng Galanthus nivalis lectin ( Sigma-Aldrich ) and blocked with PBS containing 0 . 5% Tween 20 , 1% nonfat dry milk , and 1% goat serum . Wells were probed with 0 . 5 μg of a mouse monoclonal anti-6x His-tag antibody ( ab18184 , Abcam ) and quantified using a HRP-conjugated goat anti-mouse IgG secondary antibody ( ab97265 , Abcam ) . The EC50 for each sE2 construct was calculated by nonlinear regression analysis and fold differences in EC50 used to normalize sE2 concentration in subsequent experiments . CHO-CD81 and CHO-SR-B1 binding experiments were carried out as previously described [33] . CHO cells expressing recombinant human CD81 or SR-B1 ( a gift from Dr . Matthew Evans , Icahn School of Medicine , Mount Sinai , New York ) were detached using PBS supplemented with 4mM EDTA and 10% FBS and washed in PBS containing 1% BSA . Cells ( 2E+05 ) were pelleted in a 96-well u-bottom plate and re-suspended in 2 fold serial dilutions of each sE2 construct ( H77 , L403F , and L438V ) . Following 30 minutes incubation on ice the cells were washed twice and incubated with 0 . 5 ug of anti-6x His-tag antibody for another 20 minutes on ice . The cells were then washed again , re-suspended in an Alexa fluor 647-labeled goat anti-mouse IgG secondary antibody , and incubated on ice for 15 minutes . After a final wash , the cells were fixed with 1% paraformaldehyde and analyzed on a LSRII ( Becton-Dickinson ) using FloJo software ( Tree Star ) . For mAb binding-inhibition experiments , sE2 was normalized for protein concentration , then diluted 1:32 for SR-B1 binding , 1:16 for CD81 binding . sE2 was preincubated with serial dilutions of HC33 . 4 or nonspecific human IgG , then used to stain CHO cells as above . | Generation of an immune response that is protective against a wide variety of naturally occurring isolates is necessary for vaccines against highly variable viruses like hepatitis C virus ( HCV ) . Two broadly neutralizing human monoclonal antibodies , HC33 . 4 and AR4A , neutralize multiple highly divergent HCV isolates , raising hope that a vaccine against HCV is possible . Previous reports have defined the distinct , highly conserved sites on the viral envelope proteins where these antibodies bind . However , little is known about naturally occurring variation in sensitivity of different HCV isolates to these antibodies . We developed a high throughput assay and computational algorithm to evaluate over 100 naturally occurring HCV variants for their sensitivity to these two antibodies , identifying several resistance polymorphisms to each antibody which do not fall within their mapped binding sites . Furthermore , two of these polymorphisms modulate resistance to both antibodies by enhancing or reducing envelope protein binding to HCV co-receptor scavenger receptor B1 ( SR-B1 ) . By developing this broadly applicable platform , we have shown the important neutralization resistance conferred by changes distant from antibody binding sites , presenting a potential mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved sites . | [
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] | 2017 | Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1 |
The cellular prion protein , designated PrPC , is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in other tissues . Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases . However , the physiological functions of PrPC remain largely unknown , particularly in non-neuronal tissues . Here , we show that PrPC is expressed in lung epithelial cells , including alveolar type 1 and 2 cells and bronchiolar Clara cells . Compared with wild-type ( WT ) mice , PrPC-null mice ( Prnp0/0 ) were highly susceptible to influenza A viruses ( IAVs ) , with higher mortality . Infected Prnp0/0 lungs were severely injured , with higher inflammation and higher apoptosis of epithelial cells , and contained higher reactive oxygen species ( ROS ) than control WT lungs . Treatment with a ROS scavenger or an inhibitor of xanthine oxidase ( XO ) , a major ROS-generating enzyme in IAV-infected lungs , rescued Prnp0/0 mice from the lethal infection with IAV . Moreover , Prnp0/0 mice transgenic for PrP with a deletion of the Cu-binding octapeptide repeat ( OR ) region , Tg ( PrPΔOR ) /Prnp0/0 mice , were also highly susceptible to IAV infection . These results indicate that PrPC has a protective role against lethal infection with IAVs through the Cu-binding OR region by reducing ROS in infected lungs . Cu content and the activity of anti-oxidant enzyme Cu/Zn-dependent superoxide dismutase , SOD1 , were lower in Prnp0/0 and Tg ( PrPΔOR ) /Prnp0/0 lungs than in WT lungs . It is thus conceivable that PrPC functions to maintain Cu content and regulate SOD1 through the OR region in lungs , thereby reducing ROS in IAV-infected lungs and eventually protecting them from lethal infection with IAVs . Our current results highlight the role of PrPC in protection against IAV infection , and suggest that PrPC might be a novel target molecule for anti-influenza therapeutics .
The normal cellular prion protein , designated PrPC , is a membrane glycoprotein tethered to the outer cell membrane via a glycosylphosphatidylinositol anchor moiety and expressed most abundantly in brains , particularly by neurons , and to a lesser extent in non-neuronal tissues including hearts , kidneys , and lungs [1 , 2] . Conformational conversion of PrPC into the abnormally folded , amyloidogenic isoform is a pivotal pathogenic event in prion diseases , a group of neurodegenerative disorders including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals [2] . In brains , glial cells including microglia , astrocytes , and oligodendrocytes also express PrPC [3–7] . PrPC expression has also been reported in non-cardiomyocytes in hearts [8] , in glomeruli , proximal convoluted tubules and collecting ducts in kidneys [8 , 9] , activated hepatic stellate cells in livers [10] , in lymphoid nodules [11] and perilymphoid zones of the red pulp [8] in spleens , and in neuronal cells in the lamina propia and parasympathetic ganglions [8] , some epithelial cells [12] , Peyer’s patches [12] and enteric glial cells [13] in intestines . In lungs , alveolar walls were reported to be positive for PrPC expression [8] . However , the exact function of PrPC remains to be clarified . Neuroprotective function has been suggested for PrPC . Mice devoid of PrPC ( Prnp0/0 ) have been reported to be vulnerable to ischemic brain injury , with enhanced neuronal cell apoptosis in the injured brains [14–16] . PrP lacking the octapeptide repeat ( OR ) region failed to rescue Prnp0/0 mice from ischemic brain injury [17] . These results suggest that PrPC might exert an anti-apoptotic activity through the OR region , thereby protecting neurons from ischemic damage . It was recently reported that the hearts and kidneys of Prnp0/0 mice were also vulnerable to ischemic injury [18 , 19] , indicating that PrPC could have a protective function even in non-neuronal tissues . However , the exact mechanism underlying the protective function of PrPC remains elusive . The OR region binds to Cu ions via histidine residues [20–22] . Some investigators showed that Cu content was reduced and the enzymatic activity of anti-oxidant enzyme Cu/Zn-dependent superoxide dismutase , SOD1 , was lower in the brains of Prnp0/0 mice [20 , 23 , 24] , suggesting that PrPC might function to maintain Cu levels , thereby regulating SOD1 activity to exert anti-oxidative activity and eventually protecting neurons from apoptosis . However , others reported normal levels of Cu content and SOD1 activity in the brains of Prnp0/0 mice [25] . Thus , the role of PrPC in maintenance of Cu content and regulation of SOD1 in terms of its protective activity remains to be determined . Several groups have investigated the role of PrPC in virus infection in mice [26–28] . Nasu-Nishimura et al . reported that PrPC could have a protective role against infection with encephalomyocarditis virus B variant by reducing neuronal apoptosis in the brains of infected mice without affecting viral replication [27] . It was also reported that human immunodeficiency virus type 1 ( HIV-1 ) production was strongly inhibited by expression of PrPC in cultured cells transfected with an infectious HIV-1 molecular clone [28] . On the other hand , PrPC overexpression was shown to enhance acute infection of herpes simplex virus type 1 ( SC16 ) in the central and peripheral neuronal tissues , causing higher mortality in mice , although latent infection of the virus in these tissues was inhibited by overexpression of PrPC [26] . Influenza A virus ( IAV ) is an enveloped , negative sense , single-stranded RNA virus , causing seasonal epidemic outbreaks of influenza [29] . High morbidity and mortality are observed in infected people , particularly in the young and elderly and those with underlying chronic diseases in lung or cardiovascular systems [29] . Several lines of evidence indicate that reactive oxygen species ( ROS ) play a pivotal role in IAV infection-induced lung injury , by causing apoptosis in infected lung epithelial cells [30–33] . However , the role of PrPC in IAV infection remains unknown . In the present study , we show that Prnp0/0 mice were highly susceptible to IAV infection , with higher mortality , compared to wild-type ( WT ) mice . PrP lacking the Cu-binding OR region failed to rescue Prnp0/0 mice from lethal infection with IAV . Infected Prnp0/0 lungs were severely injured , with higher epithelial cell apoptosis and higher ROS levels than control WT lungs . Treatment with anti-oxidants rescued Prnp0/0 mice from lethal infection with IAV . SOD1 activity and Cu ion content were lower in Prnp0/0 lungs than in WT lungs . These results suggest that PrPC could have a protective role against lethal infection with IAVs through the OR region probably by exerting an anti-oxidative activity by maintaining Cu content and regulating SOD1 in lungs .
We first investigated expression of PrPC in lung tissues of C57BL/6 WT mice on Western blotting . PrPC was detectable in various tissues , with highest expression in brains ( Fig 1A ) . Lower but considerably high levels of PrPC were detected in lungs , followed by that in spleens and intestines ( Fig 1A ) . Only very low level of PrPC was detectable in hearts and livers ( Fig 1A ) . These results are consistent with PrPC being expressed most abundantly in brains and , to lesser extents , in other non-neuronal tissues [1 , 2] . Weak signals were observed in Prnp0/0 lungs ( Fig 1A ) . However , no signals for PrPC were detectable in the brains of Prnp0/0 mice ( Fig 1A ) , clearly indicating that PrPC expression is absent in Prnp0/0 mice . Therefore , the signals observed in Prnp0/0 lungs are not specific for PrPC . PrPC is a glycoprotein with two glycosylation sites , therefore di- , mono- , and un-glycosylated forms of PrPC are being expressed and detected as a broad band on Western blotting . We then performed immunofluorescent staining for PrPC in lungs . No specific signals were detected on Prnp0/0 lung slices ( Fig 1B ) . In contrast , bronchiolar and alveolar epithelial cells on WT lung slices showed positive staining ( Fig 1B ) . Double staining with anti-podoplanin , anti-surfactant protein C ( SP-C ) , or anti-Clara cell 10-kDa protein ( CC10 ) antibodies , which specifically detect alveolar type 1 and 2 epithelial cells ( AT1 and AT2 cells ) and bronchiolar Clara epithelial cells , respectively , revealed expression of PrPC in these lung epithelial cells ( Fig 1C ) . To investigate the role of PrPC in IAV infection , we intranasally infected Prnp0/0 and C57BL/6 WT mice with 50 infectious units ( IFU ) of influenza virus strain A/Puerto Rico/8/34 ( H1N1 ) ( hereafter referred to as IAV/PR8 ) . Compared to control WT mice , male Prnp0/0 mice showed higher sensitivity to IAV/PR8 , with markedly elevated mortality ( Fig 2A ) . At 14 days post-infection ( dpi ) , only about 7% of male Prnp0/0 mice survived the infection while more than 70% of male WT mice were still alive . Higher mortality was also observed in infected female Prnp0/0 mice , compared to control female WT mice ( Fig 2A ) . Viral titers were higher in infected Prnp0/0 lungs than in control WT lungs ( Fig 2A ) . Western blotting showed similar expression of PrPC in male and female WT lungs ( S1 Fig ) . We also intranasally infected male Prnp0/0 and WT mice with increasing infectious doses ( 100 IFU ) of IAV/PR8 . None of Prnp0/0 mice survived the infection by 14 dpi ( Fig 2B ) . However , about 40% of WT mice remained alive ( Fig 2B ) . Viral titers were higher in infected Prnp0/0 lungs than in control WT lungs ( Fig 2B ) . According to the Reed and Muench method [34] , a 50% mouse lethal dose ( MLD50 ) for IAV/PR8 was calculated as 66 IFU in WT mice and less than 50 IFU in Prnp0/0 mice . We also used Prnp0/0 and Prnp+/+ littermates for intranasal infection with 50 IFU of IAV/PR8 . Male and female Prnp+/+ mice showed a mortality rate of about 40% at 14 dpi ( Fig 2C ) . However , more than 80% of male and female Prnp0/0 mice died by 14 dpi ( Fig 2C ) . Higher viral titers were observed in infected Prnp0/0 lungs compared to control Prnp+/+ lungs ( Fig 2C ) . We also tested other IAV strains , A/Aichi/2/68 ( H3N2 ) and A/WSN/33 ( H1N1 ) ( hereafter referred to as IAV/Aichi and IAV/WSN , respectively ) , for their pathogenicity in Prnp0/0 mice . Prnp0/0 and WT mice were intranasally infected with 500 IFU of IAV/Aichi and 3 , 000 IFU of IAV/WSN . IAV/WSN belong to the same H1N1 subtype family as IAV/PR8 . However , IAV/WSN was established by passages in mouse brains , thus being neurotropic , while IAV/PR8 is highly pathogenic to lungs [35] . Therefore , higher virus titers were used for intranasal infection with IAV/WSN . No male Prnp0/0 mice were alive by 14 dpi with IAV/Aichi and IAV/WSN ( Fig 2D and 2E ) . However , about 90% and 75% of male WT mice survived at 14 dpi with IAV/Aichi and IAV/WSN , respectively ( Fig 2D and 2E ) . Virus titers were higher in Prnp0/0 lungs than in WT lungs after infection with IAV/Aichi and IAV/WSN ( Fig 2D and 2E ) . Higher mortality was also observed in female Prnp0/0 mice infected with IAV/Aichi and IAV/WSN , compared to control WT mice ( Fig 2D and 2E ) . Taken together , these results indicate that Prnp0/0 mice are highly susceptible to IAV infection , with higher mortality and higher virus loads in the lungs compared to WT mice , suggesting that PrPC could have a protective role against lethal infection with IAVs . To confirm that lack of PrPC is responsible for the higher susceptibility of Prnp0/0 mice to IAV infection , 50 IFU of IAV/PR8 were intranasally infected into Tg ( MoPrP ) /Prnp0/0 mice , in which multiple copies of the transgene encoding mouse PrPC are expressed on the Prnp0/0 background [36] . Western blotting showed higher expression of PrPC in the lungs and brains of Tg ( MoPrP ) /Prnp0/0 mice than in WT mice ( Fig 3A ) . Mortality was markedly reduced in male Tg ( MoPrP ) /Prnp0/0 mice compared to male Prnp0/0 littermates after infection ( Fig 3B ) . More than 90% of male Tg ( MoPrP ) /Prnp0/0 mice survived the infection while only less than 10% of male Prnp0/0 littermates remained alive at 14 dpi . Virus titers were also reduced in Tg ( MoPrP ) /Prnp0/0 lungs compared to Prnp0/0 lungs ( Fig 3B ) . A higher survival rate was also observed in female Tg ( MoPrP ) /Prnp0/0 mice after infection , compared to control female Prnp0/0 littermates ( Fig 3B ) . These results confirm that the higher susceptibility of Prnp0/0 mice to IAV infection could result from the lack of PrPC . We then investigated whether the OR region might be involved in the protective role of PrPC against lethal infection with IAVs , by intranasal infection with 100 IFU of IAV/PR8 into Tg ( PrPΔOR ) /Prnp0/0 mice and their Prnp0/0 littermates . Tg ( PrPΔOR ) /Prnp0/0 mice express transgenic mouse PrP with a deletion of the OR region alone on the Prnp0/0 background [37] . Western blotting with 6D11 anti-PrP antibody , which recognizes residues 93–109 of mouse PrP , revealed expression of PrPΔOR in Tg ( PrPΔOR ) /Prnp0/0 lungs and PrPC in WT lungs ( Fig 4A and 4B ) . SAF32 anti-PrP antibody , which recognizes the OR region , did not detect PrPΔOR in Tg ( PrPΔOR ) /Prnp0/0 lungs ( Fig 4A and 4B ) , confirming lack of the OR region in PrPΔOR . We increased the dose of IAV/PR8 for infection into Tg ( PrPΔOR ) /Prnp0/0 mice to 100 IFU since they were highly resistant to 50 IFU of IAV/PR8 . IAV/PR8 infection caused similar mortality in Tg ( PrPΔOR ) /Prnp0/0 and Prnp0/0 mice ( Fig 4C ) . However , mortality in these mice was significantly higher than that in control WT mice ( Fig 4C ) . Only 10% of Tg ( PrPΔOR ) /Prnp0/0 mice and no Prnp0/0 mice survived the infection while 50% of WT mice were alive at 14 dpi ( Fig 4C ) . Tg ( PrPΔOR ) /Prnp0/0 and Prnp0/0 lungs showed similar virus titers , but they were higher than those in WT lungs ( Fig 4C ) . These results suggest that the OR region could play an important role for PrPC to protect against lethal infection with IAVs in mice . To gain insights into the protective role of PrPC against lethal infection with IAVs , we investigated the pathology of IAV/PR8 ( 50 IFU ) -infected Prnp0/0 and WT lungs . No macroscopic lesions were observed on the lung surface of control saline-administrated Prnp0/0 and WT mice ( Fig 5A ) . In contrast , reddish lesions were evident on the surface of infected WT and Prnp0/0 lungs at 5 and 8 dpi , with larger size and higher number of the lesions in the Prnp0/0 lungs than in the WT lungs ( Fig 5A ) . Prnp0/0 and WT lungs had increased wet weights after infection , with the Prnp0/0 lungs being significantly heavier than the WT lungs ( Fig 5B ) , suggesting higher exudates in Prnp0/0 lungs than in WT lungs after infection . Microscopic examinations showed higher infiltration of inflammatory cells in Prnp0/0 lungs than in WT lungs after infection ( Fig 5C ) . Immunofluorescent staining also showed viral nucleocapsid protein NP accumulated in the inflammatory regions ( Fig 5C ) . Atelectatic areas were therefore larger in Prnp0/0 lungs than in WT lungs after infection ( Fig 5D ) . We also investigated levels of inflammatory cytokines , including interleukin-6 ( IL-6 ) , tumor necrosis factor-α ( TNF-α ) , and interferon-γ ( IFN-γ ) , in these infected lungs . All the cytokines examined had higher levels in Prnp0/0 lungs than in WT lungs ( Fig 5E ) . We also investigated viral proteins in these infected lungs . Western blotting showed that viral proteins , including NP , NS1 nonstructural protein , and M2 matrix protein , became detectable in Prnp0/0 and WT lungs at 3 dpi , reached a peak level at 5 dpi , and decreased at 8 dpi ( Fig 5F ) , with slightly but not significantly higher levels in the Prnp0/0 lungs than in the WT lungs ( Fig 5G ) . We evaluated innate and adaptive immune responses against IAV infection in Prnp0/0 mice . To this end , we first investigated expression of the innate immunity-related genes , including those for retinoic acid-inducible gene I ( RIG-I ) , melanoma differentiation-associated protein 5 ( MDA5 ) , TNF-α , IFN-α and IFN-γ , in IAV/PR8 ( 50 IFU ) -infected WT and Prnp0/0 lungs . Reverse transcriptase-polymerase chain reaction ( RT-PCR ) showed upregulated expression of these genes in infected WT and Prnp0/0 lungs ( S2A Fig ) . However , expression levels of these genes except for the MDA5 gene were higher in infected Prnp0/0 lungs than in control WT lungs ( S2A Fig ) . Expression of the viral NP gene was also higher in infected Prnp0/0 lungs than in control WT lungs ( S2A Fig ) , suggesting that the higher expression of the innate immune-related genes in infected Prnp0/0 lungs might be due to the higher viral loads in the lungs . We then investigated antibody responses against IAV/PR8 infection in WT and Prnp0/0 mice . Plasma levels of IAV/PR8-specific IgG and IgM antibodies were similarly elevated in infected WT and Prnp0/0 mice ( S2B Fig ) . Enzyme-linked immunoSpot ( ELISPOT ) assay also showed that the spot number of TNF-α- or IFN-γ-secreting cells was the same in the lungs and spleens of infected Prnp0/0 and WT mice ( S2C Fig ) . These results indicate that Prnp0/0 mice could activate innate and adaptive immune responses against IAV infection . To understand the protective mechanism of PrPC against lethal infection with IAVs , we investigated apoptotic cell death in IAV/PR8 ( 50 IFU ) -infected Prnp0/0 and WT lungs , by performing Western blotting for the cleaved fragments of the apoptotic marker caspase 3 . Prnp0/0 and WT lungs showed increased the fragments after infection ( Fig 6A ) . However , the fragments were higher in infected Prnp0/0 lungs than in control WT lungs ( Fig 6A ) . Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling ( TUNEL ) staining also showed more abundant apoptotic cells in alveolar and bronchiolar epithelial areas in Prnp0/0 lungs than in WT lungs ( Fig 6B ) . We also performed Western blotting with anti-podoplanin , anti-SP-C , and anti-CC10 antibodies . Podoplanin levels were unaffected in Prnp0/0 and WT lungs after infection ( Fig 6C ) . This is consistent with IAV/PR8 infection not damaging AT1 cells in C57BL/6 mice [38] . In contrast , SP-C and CC10 were markedly decreased in Prnp0/0 and WT lungs after infection , with their levels significantly lower in infected Prnp0/0 lungs than in control WT lungs ( Fig 6C ) . Consistently , immunofluorescence staining showed that SP-C-positive AT2 cells and CC10-positive Clara cells were less in infected Prnp0/0 lungs than in control WT lungs , while podoplanin-positive AT1 cells were similarly observed in infected these lungs ( Fig 6D ) . These results suggest that AT2 and Clara cells in Prnp0/0 lungs could be more vulnerable to apoptosis than those in WT lungs after infection with IAVs , and that PrPC could exert an anti-apoptotic activity in AT2 and Clara cells after infection with IAVs . To investigate the role of ROS in the higher mortality of IAVs-infected Prnp0/0 mice , we addressed whether Prnp0/0 mice could be rescued from lethal infection with IAV/PR8 by treatment with a ROS scavenger . To this end , we first measured ROS in the lungs of Prnp0/0 and WT mice intranasally infected with IAV/PR8 ( 100 IFU ) . The virus dose used was higher than 1 MLD50 since it is assumed that the effect of a ROS scavenger on the survival rate of infected mice , if any , would be evaluated more easily for the mice developing mortality more than 50% after infection than those with less than 50% mortality after infection . No difference in ROS levels was detected between uninfected Prnp0/0 and WT lungs ( Fig 7A ) . IAV/PR8 infection at 5 dpi increased ROS levels in Prnp0/0 and WT lungs ( Fig 7A ) . However , ROS levels were higher in Prnp0/0 lungs than in control WT lungs ( Fig 7A ) . ROS levels were also higher in infected Tg ( PrPΔOR ) /Prnp0/0 lungs than in control WT lungs at 5 dpi ( Fig 7A ) . These results suggest that PrPC might exert an anti-oxidative activity to reduce ROS levels through the OR region in IAV-infected lungs . We then examined the anti-oxidative effect of butylated hydroxyanisole ( BHA ) , a ROS scavenger , on ROS levels in the lungs of IAV/PR8 ( 100 IFU ) -infected WT mice . Treatment with BHA for 4 days starting at 2 dpi effectively reduced ROS in infected WT lungs at 5 dpi ( Fig 7B ) . We then similarly treated Prnp0/0 and WT mice with BHA after intranasal infection with IAV/PR8 ( 100 IFU ) . The treatment decreased the mortality of infected WT mice ( Fig 7C ) . This could be consistent with that ROS could be a major player in IAV infection-induced lung injury [30–33] . The mortality of infected Prnp0/0 mice was also decreased to that of control WT mice after treatment with BHA ( Fig 7C ) . Viral titers were also decreased in Prnp0/0 lungs to those in WT lungs after treatment with BHA ( Fig 7C ) . These results suggest that the higher ROS levels in Prnp0/0 lungs could be involved in the higher mortality of Prnp0/0 mice after infection with IAVs . To investigate the role of XO , a major ROS-generating enzyme in IAV-infected lungs [30] , in IAV-infected Prnp0/0 lungs , we first perform Western blotting of IAV/PR8 ( 100 IFU ) -infected Prnp0/0 and WT lungs for XO . Expression of XO was increased in these infected lungs ( Fig 8A ) . However , the expression levels of XO were higher in infected Prnp0/0 lungs than in control WT lungs ( Fig 8A ) . We then treated Prnp0/0 and WT mice with the XO inhibitor allopurinol after intranasal infection with IAV/PR8 at 100 IFU , a dose higher than 1 MLD50 in WT mice . Allopurinol treatment starting from one day before intranasal infection with IAV/PR8 to 14 dpi reduced the mortality of Prnp0/0 and WT mice to a similar rate ( Fig 8B ) . Viral titers were also decreased in Prnp0/0 lungs compared to those in WT lungs after treatment with allopurinol ( Fig 8B ) . These results suggest that XO could be a key ROS-generating enzyme in IAV-infected Prnp0/0 and WT lungs , and that the higher expression of XO in IAV-infected Prnp0/0 lungs could be involved in the higher mortality of IAVs-infected Prnp0/0 mice probably through producing higher levels of ROS . We also investigated infected Prnp0/0 and WT lungs for the enzymatic activity of SOD , an anti-oxidative enzyme in IAV-infected lungs [31] . Western blotting revealed similar expression of SOD1 and SOD2 between uninfected and infected Prnp0/0 or WT lungs ( Fig 8C ) . However , the total SOD activity was significantly lower in uninfected Prnp0/0 lungs than in uninfected WT lungs ( Fig 8D ) . The SOD1-specific inhibitor diethyl-dithio-carbamate ( DDC ) reduced SOD activity in both uninfected Prnp0/0 and WT lungs to the same levels ( Fig 8D ) , suggesting that SOD1 activity might be impaired in Prnp0/0 lungs . IAV/PR8 infection increased the total SOD activity in both WT and Prnp0/0 lungs ( Fig 8D ) . However , the activity was lower in infected Prnp0/0 lungs than in control WT lungs ( Fig 8D ) . DDC decreased the SOD activity in infected Prnp0/0 and WT lungs to the levels in uninfected Prnp0/0 lungs ( Fig 8D ) . These results suggest that SOD1 might not be fully activated in Prnp0/0 lungs after IAV infection . Lower SOD1 activity was also detected in infected and uninfected Tg ( PrPΔOR ) /Prnp0/0 lungs than in control WT lungs ( Fig 8D ) . Cu ions , which are important for SOD1 activity , were lower in uninfected Prnp0/0 and Tg ( PrPΔOR ) /Prnp0/0 lungs than in control WT lungs ( Fig 8E ) . These results suggest that PrPC might function to maintain Cu levels and thereby might regulate SOD1 activity through the Cu-binding OR region in lungs . To further gain insights into the protective role of PrPC in IAV infection , we infected primary lung cells from WT , Prnp0/0 and Tg ( MoPrP ) /Prnp0/0 mice with IAV/PR8 at 1 . 0 multiplicity of infection ( MOI ) . Prnp0/0 cells were more vulnerable to the infection than WT cells ( S3A Fig ) . In contrast , Tg ( MoPrP ) /Prnp0/0 cells were highly resistant to the infection ( S3A Fig ) . Higher expression of viral proteins NP , HA and M2 was observed in Prnp0/0 cells than in WT cells at 2 dpi ( S3B Fig ) . In contrast , their expression was lower in Tg ( MoPrP ) /Prnp0/0 cells than in WT cells ( S3B Fig ) . Higher activation of caspase 3 was detected in infected Prnp0/0 cells than control WT cells ( S3B Fig ) . In contrast , activation of caspase 3 was lower in infected Tg ( MoPrP ) /Prnp0/0 cells than in control WT cells ( S3B Fig ) . siRNA-mediated knockdown of PrPC in the A549 human lung epithelial cells also caused higher expression of NP , HA and M2 and higher activation of caspase 3 after infection with IAV/PR8 ( S3C Fig ) . ROS levels were also higher in infected Prnp0/0 cells and lower in infected Tg ( MoPrP ) /Prnp0/0 cells compared to those in control WT cells ( S3D Fig ) . In contrast , SOD activity was lower in Prnp0/0 cells and higher in Tg ( MoPrP ) /Prnp0/0 cells after infection with IAV/PR8 compared to that in infected WT cells ( S3E Fig ) . These results are consistent with those from the in vivo experiments , suggesting that PrPC might exert a protective activity against IAV infection in a cell-autonomous way . Intranasal administration of LPS is known to cause lung injuries in mice [39] . To investigate whether PrPC might be also protective against LPS-induced lung injuries , we intranasally administrated LPS into Prnp0/0 and WT mice . No Prnp0/0 and WT mice died from the administration ( S4A Fig ) . The body weight of Prnp0/0 and WT mice was similarly reduced by 3 days after administration and thereafter increased ( S4A Fig ) . Caspase 3 was similarly activated in WT and Prnp0/0 lungs 24 h after administration with LPS ( S4B Fig ) . ROS and inflammatory cytokines , including TNF-α and IFN-γ , were also similarly elevated between Prnp0/0 and WT lungs 24 h after administration with LPS ( S4C Fig ) . These results suggest that PrPC might have no protective activity against LPS-induced lung injuries .
In the present study , we showed that Prnp0/0 mice were highly susceptible to infection with IAVs , with markedly higher mortality , compared to control WT mice . Pathological changes were more severe , inflammatory cytokines including IL-6 , TNF-α , and IFN-γ were higher , and viral loads were higher in IAV/PR8-infected Prnp0/0 lungs . We confirmed that the higher mortality of infected Prnp0/0 mice is due to lack of PrPC , by demonstrating that transgenic expression of mouse PrPC rescued Prnp0/0 mice from lethal infection with IAV/PR8 . We also showed that mouse PrP lacking the OR region failed to protect Prnp0/0 mice from the lethal infection with IAV/PR8 . These results suggest that PrPC could have a protective role against lethal infection with IAVs through the OR region in mice . Prnp0/0 mice activated innate and adaptive immune responses against IAV infection . These results rule out the possibility that lack of PrPC might cause defective immune responses against IAV infection , therefore Prnp0/0 mice being highly susceptible to IAV infection . PrPC was expressed in alveolar AT1 and 2 epithelial cells and bronchiolar Clara epithelial cells in lungs . Other investigators also reported expression of PrPC in alveolar walls and Clara cells [8 , 40] . Consistent with the previous report showing that AT1 cells were resistant to infection with IAV/PR8 in WT mice [38] , AT1 cells were unaffected by infection with IAV/PR8 not only in WT lungs but also in Prnp0/0 lungs . In contrast , infection with IAV/PR8 markedly damaged AT2 and Clara cells in Prnp0/0 and WT lungs . However , these epithelial cells in Prnp0/0 lungs were more susceptible to the infection than those in WT lungs . Caspase 3 was activated more robustly in Prnp0/0 lungs than in WT lungs after infection with IAV/PR8 . TUNEL staining also displayed more abundant apoptotic cells in the alveolar and bronchiolar epithelial areas of infected Prnp0/0 lungs than in control WT lungs . Primary Prnp0/0 lung culture cells were also vulnerable to IAV/PR8 infection-induced apoptosis compared to control WT lung cells . These results suggest that AT2 and Clara epithelial cells in Prnp0/0 lungs might be more vulnerable to IAV infection-induced apoptosis than those in WT lungs , and that PrPC might play an anti-apoptotic role in these lung epithelial cells in a cell-autonomous way . However , intranasal administration with LPS , which induces lung injuries through binding to Toll-like receptor 4 ( TLR4 ) [41] , similarly activated caspase 3 in Prnp0/0 and WT lungs , suggesting that the anti-apoptotic activity of PrPC has no effect on the LPS/TLR4-induced apoptosis in lungs . Viral loads were significantly but only slightly higher in Prnp0/0 lungs than in WT lungs after infection with IAV/PR8 . It has been shown that caspase 3 activation induces efficient replication of IAV in cells [42] . It is thus possible that the slightly higher viral loads in IAV/PR8-infected Prnp0/0 lungs might be associated with the higher activation of caspase 3 observed in the lungs . Thus , PrPC might exert its protective activity against IAV infection through its anti-apoptotic activity in lung epithelial cells , not through directly affecting viral replication efficiency in lungs . However , the possibility remains unanswered if PrPC could directly affect IAV replication in the lungs , thereby reducing viral loads and eventually repressing caspase 3 activation in the lungs . AT2 cells are small cuboidal cells covering about 2–5% of the alveolar surface area and secreting surfactant proteins , which are important to reduce alveolar surface tension [43 , 44] . Clara cells are the predominant cell type in bronchioles and known as important progenitor cells for the repair of bronchiolar epithelia [45] . Recently , it was reported that Clara cells are also major progenitor cells for alveolar epithelial regeneration through differentiation to AT1 and 2 alveolar cells after IAV infection [46 , 47] . AT2 and Clara cells were more severely damaged in Prnp0/0 lungs than in WT lungs after infection with IAV/PR8 . It is thus possible that the AT2 cells-mediated regulation of alveolar surface tension and the Clara cells-mediated alveolar and bronchiolar epithelia regeneration after IAV infection might be disturbed more severely in Prnp0/0 lungs than in WT lungs after infection with IAVs , eventually causing higher mortality in Prnp0/0 mice infected with IAVs . We showed that ROS levels were higher in IAV/PR8-infected Prnp0/0 lungs than in control WT lungs . We also showed that the ROS scavenger BHA rescued Prnp0/0 mice from lethal infection with IAV/PR8 , reducing mortality to the levels in IAV/PR8-infected , BHA-treated control WT mice , suggesting that the higher ROS levels in infected Prnp0/0 lungs could be responsible for the higher mortality of Prnp0/0 mice infected with IAVs . It has been shown that Prnp0/0 cells were more susceptible to treatment with agents inducing oxidative stress , readily succumbing to apoptosis , compared with WT cells [23 , 48] , suggesting that PrPC could have a protective role against oxidative stress-induced apoptosis . Therefore , PrPC might play an anti-oxidative role in lungs after infection with IAVs , thereby reducing ROS levels and protecting lung epithelial cells from IAV infection-induced apoptosis . We also demonstrated higher ROS levels in Tg ( PrPΔOR ) /Prnp0/0 lungs than in WT lungs after infection with IAV/PR8 , suggesting that the OR region could be important for PrPC to exert the anti-oxidative activity in lungs after infection with IAVs . XO was shown to be a major ROS-generating enzyme in IAV-infected lungs [30] . We showed that XO expression was elevated in infected Prnp0/0 lungs compared to control WT lungs . We also showed that the XO inhibitor allopurinol rescued Prnp0/0 mice from lethal infection with IAV/PR8 . These results suggest that the XO up-regulation observed in infected Prnp0/0 lungs might be responsible for the higher mortality in Prnp0/0 mice infected with IAVs . It has been shown that inflammatory cytokines such as TNF-α and IFN-γ up-regulate the expression of XO [49–51] . Higher levels of these cytokines were detected in infected Prnp0/0 lungs than in control WT lungs , suggesting that the higher expression of XO in infected Prnp0/0 lungs might be induced by the higher levels of these cytokines in the lungs . We also showed that IAV/PR8 infection increased SOD1 activity in Prnp0/0 and WT lungs . However , SOD1 was not fully activated in infected Prnp0/0 lungs compared to control WT lungs , suggesting that SOD1 activation might be disturbed in Prnp0/0 lungs infected with IAVs . Together with the reported results that administration of pyran polymer-conjugated SOD1 successfully reduced the mortality of WT mice infected with IAV [31] , it is suggested that the lower activity of SOD1 in infected Prnp0/0 lungs might be responsible for the higher mortality of Prnp0/0 mice infected with IAVs . Cu ions are important for the SOD1 enzymatic activity . We found that Cu ion content were lower in Prnp0/0 lungs than in WT lungs , suggesting that the lower activity of SOD1 in Prnp0/0 lungs might be due to the lower Cu content in the lungs . Reduced SOD1 activity and lower Cu content were also detected in Tg ( PrPΔOR ) /Prnp0/0 lungs . It is thus possible that PrPC might have a role to maintain Cu levels in lungs through the OR region , thereby regulating SOD1 activity and eventually exerting an anti-oxidative activity in lungs . PrPC is known to bind to Cu ions via the OR region , suggesting that PrPC might transfer the bound Cu ions to and activate SOD1 [23 , 24] . However , the exact mechanism of how PrPC is involved in the activation of SOD1 remains to be determined . It has been also proposed that PrPC itself could have SOD activity [52] . However , other investigators failed to confirm this proposed SOD activity in PrPC [53 , 54] . Elucidation of the mechanism underlying the anti-oxidative function of PrPC could be helpful for further understanding the pathogenesis of IAV infection and for development of anti-influenza therapeutics based on the PrPC-mediated protective mechanism . Anti-oxidative therapeutics against IAV infection , by targeting the ROS-generating enzymes or by administrating anti-oxidants or anti-oxidant enzymes , has been shown to successfully protect mice from lethal infection with IAVs [30–33] . Our current results showing that PrPC could have a protective role against lethal infection with IAVs in mice possible by exerting ant-oxidative activity , suggest PrPC to be a new target molecule for anti-oxidative therapeutics against IAV infection . It has been reported that PrPC elicited a protective signal against anisomycin-induced apoptosis in neurons via interaction with stress-inducible protein 1 ( STI1 ) , a STI1-derived peptide , or anti-PrP antibodies [55 , 56] , and that the interaction with STI1 could be involved in PrPC-dependent activation of SOD [57] . It is thus interesting to investigate whether these ligands could elicit the protective activity of PrPC against IAV infection .
All animal experiments were conducted in compliance with Japanese legislation ( Act on Welfare and Management of Animals ) . The Ethics Committee of Animal Care and Experimentation of Tokushima University approved the animal experiments in this study ( approval number T27-86 ) . Animals were cared for in accordance with The Guiding Principle for Animal Care and Experimentation of Tokushima University and guidelines under the jurisdiction of the Ministry of Education , Culture , Sports , Science and Technology , Japan . C57BL/6 mice were purchased from Japan SLC Inc . ( Shizuoka , Japan ) . Prnp0/0 mice used in this study had been obtained elsewhere by at least more than 9 time-backcrosses to C57BL/6 mice with Prnp0/0 mice , which originally carry a mixed background of C57BL/6×129Sv×FVB mice [37 , 58] . The backcrossed Prnp0/0 mice were maintained by intercrossing the backcrossed Prnp0/0 mouse pairs and used in this study . Prnp0/0 and Prnp+/+ littermates used in this study were produced by intercross of the backcrossed Prnp0/+ mouse pairs , which were produced by intercrossing the backcrossed Prnp0/0 mice with C57BL/6 mice . Tg ( MoPrP ) /Prnp0/0 mice were obtained elsewhere by intercross between the backcrossed Prnp0/0 mice and Tg ( MoPrP ) mice with a FVB background [36] . Prnp0/0 and Tg ( MoPrP ) /Prnp0/0 littermates used in this study were produced by intercross between the resulting Tg ( MoPrP ) /Prnp0/0 mice and the backcrossed Prnp0/0 mice . Tg ( PrPΔOR ) /Prnp0/0 mice with the C57BL/6 background were produced elsewhere [37] . Tg ( PrPΔOR ) /Prnp0/0 mice were maintained by intercrossing the backcrossed Prnp0/0 mice and Tg ( PrPΔOR ) /Prnp0/0 mice and used in this study . The Tg allele was detected by PCR using a primer pairs ( SH3UT-S , 5’-tcggacgacaagagacaatc-3’; SHPA-A , 5’-taggggccacacagaaaaca-3’ ) , which specifically amplifies the 3’ UTR region of the hamster PrP gene used in the Tg construct . The knockout allele was detected by PCR for the neomycin resistant gene using primer pairs ( Neo163S , 5’-ggtgccctgaatgaactgca-3’; Neo390A , 5’-ggtagccggatcaagcgtat-3’ ) . The PrP allele was detected by PCR using primer pairs ( PrP-23aa-S , 5’-aaaaagcggccaaagcctgga-3’; PrP-231aa-AS , 5’-gctggatcttctcccgtcgtaataggcctg-3’ ) . IAV A/PR/8/34 ( H1N1 ) , A/Aichi/2/68 ( H3N2 ) , and A/WSN/33 ( H1N1 ) were injected into the allantoic sac of 11-day-old chicken embryos in eggs and incubated at 36°C for 48 h . The eggs were chilled at 4°C for at least for 4 h prior to harvesting the allantoic fluids . Cellular debris in the allantoic fluids was removed by centrifugation at 2 , 380×g at 4°C for 30 min . The clarified allantoic fluids were layered over a 20% sucrose cushion and centrifuged at 25 , 000×g at 4°C for 120 min . The pellet containing viruses was suspended in phosphate-buffered saline ( PBS ) , and stored in multiple aliquots at -80°C until used . Male and female mice aged 5 weeks were intranasally inoculated with IAVs in a total volume of 20 μL ( 10 μL in each nasal cavity ) , and monitored for survival and weight loss for 14 days . The IAV stock aliquot was thawed and diluted in saline before used . Sixty micrograms of LPS ( 026:B6 , Sigma-Aldrich , St Louis , MO ) in 20 μL PBS were intranasally administered into a mouse using a micropipette ( 10 μL in each nasal cavity ) . PBS was similarly administered as a control . BHA and allopurinol were purchased from Sigma-Aldrich . BHA was dissolved in dimethyl sulfoxide ( DMSO ) and stored at -20°C until use . BHA was orally administered at 200 mg/kg/day using a sonde needle from 2 to 5 dpi . According to the Material Safety Data Sheet ( MSDS ) ( ScienceLab . com , Inc . Dickinson , Texas ) , the oral LD50 of BHA is 1 , 100 mg/kg in mice . Allopurinol was prepared in PBS and then stored at -20°C until use . Allopurinol was orally administered at 2 mg/kg/day using a sonde needle from -1 to 14 dpi . According to the MSDS ( Sigma-Aldrich ) , the oral LD50 of allopurinol is 78 mg/kg in mice . Tissues were homogenized using a Polytron homogenizer ( PT 2100 , Brinkman Instruments , Inc . , Westbury , NY ) in 2 mL of PBS for measurement of cytokines and Cu ions , and in a lysis buffer ( 0 . 5% Triton X-100 , 0 . 5% sodium deoxycholate , 150 mM NaCl , 50 mM Tris-HCl , pH 7 . 4 , 1 mM EDTA ) containing protease inhibitor cocktail ( Nakalai Tesque , Kyoto , Japan ) for Western blotting and for measurement of ROS levels and SOD activity . Cells were homogenized in a protease inhibitor cocktail ( Nakalai Tesque ) -containing lysis buffer and subjected to Western blotting and measurement of ROS levels and SOD activity . The homogenates were clarified by centrifugation at 1 , 000×g for 2 min at 4°C . Protein concentration of the homogenates was measured using the BCA method ( Thermo Scientific , Rockford , IL ) . Virus titers were expressed as IFU/mL . IFU/mL was determined using Madin-Darby canine kidney ( MDCK ) cells as follows . MDCK monolayer cells were incubated with 10-fold serial dilutions of each sample of interest for 14 h at 37°C . The cells were then fixed with 4% paraformaldehyde , permeabilized with 0 . 3% Triton-X100 in PBS , and immunostained with anti-NP monoclonal antibodies ( GeneTex , Irvine , CA ) . Signals were visualized using horseradish peroxidase ( HRP ) -conjugated anti-rabbit IgG antibodies ( GE Healthcare , Waukesha , WI ) and True Blue Peroxidase Substrate ( KPL , Gaithersburg , MD ) . IFU/mL was defined as the number of the cells positive for the anti-NP signals in 1 mL of each sample . After euthanasia of mice , lungs were quickly removed , fixed with 4% paraformaldehyde , dehydrated , embedded in paraffin , and sliced into 5 μm-thick tissue sections . The sections were deparaffinized , rehydrated , and stained with hematoxylin for 5 min and eosin for 30 sec . The atelectatic lung area was evaluated using Photoshop software ( Adobe , San Jose , CA ) and ImageJ software ( NIH , Bethesda , MD ) . Briefly , the original RGB color images of H-E stained lung sections were converted to black-on-white images using Photoshop software and saved in TIFF format . The binary images in TIFF format were again converted into a white-on-black image using the ImageJ application . Atelectatic lung area was expressed as the area of white pixels , which represent solid areas , against total lung area ( white and black pixels ) . Five micrometer-thick tissue sections on glass slides ( Matsunami , Tokyo , Japan ) coated with poly-L-lysin ( Wako Pure Chemicals , Osaka , Japan ) were deparaffinized and rehydrated , and treated with proteinase K ( Wako Pure Chemicals , 20 mg/ml in 10 mM Tris HCl , pH 7 . 6 ) at 37°C for 30 min . After washed with PBS , the sections were incubated with primary antibodies against PrP ( IBL-N , Immuno Biological Laboratories , Gunma , Japan ) , podoplanin ( MBL , Nagoya , Japan ) , SP-C ( Santa Cruz Biotechnology ) , CC10 ( Santa Cruz Biotechnology ) , and NP virus protein ( GeneTex ) overnight at 4°C , and stained with Alexa Fluor 594 goat anti-rabbit IgG ( Invitrogen ) for IBL-N anti-PrP antibodies , Texas Red-X goat anti-rat IgG ( Invitrogen ) for anti-podoplanin antibody , Alexa Fluor 488 donkey anti-goat IgG ( Invitrogen ) for anti-SP-C and anti-CC10 antibody , and Alexa Fluor 488 goat anti-rabbit IgG ( Invitrogen ) for anti-NP antibody for 2 h at room temperature . The sections were mounted with CC/Mount ( Diagnostic BioSystems , Pleasanton , CA ) containing DAPI ( Dojindo Laboratories , Kumamoto , Japan ) . Fluorescent images were visualized using BIOREVO BZ-9000 ( Keyence , Osaka , Japan ) . TUNEL staining was performed using the in situ cell death detection kit and fluorescein ( Roche Diagnostics , Mannheim , Germany ) in accordance with the manufacturer’s protocol . In brief , the deparaffinized tissue sections were treated with 20 μg/mL proteinase K in 10 mM Tris-HCl for 30 min at room temperature and incubated in the TUNEL reaction mixture for 1 h at 37°C in a humidified dark chamber . The sections were washed with PBS for 5 min 3 times and signals were then detected using BIOREVO BZ-9000 ( Keyence ) . Proteins in each sample were denatured by boiling for 5 min in Laemmli’s sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Proteins were electrically transferred onto Immobilon-PVDF membranes ( Millipore , Bedford , MA ) , and membranes were blocked for 2 h with 5% non-fat dry milk-containing TBST ( 0 . 1% Tween-20 , 100 mM NaCl , 10 mM Tris-HCl , ph 7 . 6 ) . Primary antibodies against PrP ( 6D11 , COVANCE , Dedham , MA; SAF32 , Cayman Chemical Company , Ann Arbor , MI; 3F4 , BioLegend , San Diego , CA ) , pro-caspase 3 ( Cell Signaling , Beverly , MA ) , the cleaved caspase 3 ( Cell Signaling ) , NP ( GeneTex ) , NS1 ( GeneTex ) , HA ( GeneTex ) , M2 ( GeneTex ) , podoplanin ( MBL ) , SP-C ( Santa Cruz Biotechnology ) , CC10 ( Santa Cruz Biotechnology ) , XO ( Santa Cruz Biotechnology ) , SOD1 ( Abcam , Cambridge , UK ) , SOD2 ( Abcam ) and β-actin ( Sigma-Aldrich ) were incubated with the membrane overnight at 4°C . Signals were visualized using HRP-conjugated anti-mouse IgG antibodies ( GE Healthcare ) , anti-rabbit IgG antibodies ( GE Healthcare ) , anti-rat IgG antibodies ( GE Healthcare ) , or anti-goat IgG antibodies ( R&D systems , Minneapolis , MN ) , and detected using a chemiluminescence image analyzer LAS-4000 mini ( Fujifilm Co . , Tokyo , Japan ) . Signal intensities were measured using ImageJ 64 . IL-6 , TNF-α , and IFN-γ levels in samples were determined using a Quantikine ELISA kit ( R&D systems ) according to the respective protocols provided by the manufacturer . In brief , the samples were diluted 1:1 with the assay diluent provided in the kit and added to the ELISA microplate wells . The protein standards for IL-6 , TNF-α , and IFN-γ were also added to other wells . The plates were then left for 2 h at room temperature , and the wells were washed with wash buffer 5 times and mouse IL-6 , TNF-α , or IFN-γ conjugate added followed by incubation for 2 h . The wells were then washed with the wash buffer and the substrate reagent added followed by incubation for 30 min . The reaction was stopped by addition of the stop solution . The optical density of each well was measured at 450 nm in an automated microplate reader ( Thermo LabSystems , MA , USA ) . The amounts of IL-6 , TNF-α , or IFN-γ in each sample were determined using the standard curve for the amounts of IL-6 , TNF-α , or IFN-γ . ROS concentration in samples was measured using an OxiSelect Intracellular ROS Assay Kit ( Cell Biolabs , San Diego , CA ) . The assay uses 2’ , 7’-dichlorodihydrofluorescin diacetate ( DCFH-DA ) , which is deacetylated to non-fluorescent 2’ , 7’-dichlorodihydrofluorescin and then oxidized by ROS to highly fluorescent 2’ , 7’-dichlorofluorescin ( DCF ) . Each of the samples were mixed with 1×DCFH-DA solution in a 96-well black plate and incubated at 37°C for 48 h . ROS concentration in the samples was measured by determining the fluorescence intensities of DCF at 480 nm using Spectra Max Gemini EM ( Molecular devices , Sunnyvale , CA ) . SOD activity in samples was determined using an OxiSelect Superoxide dismutase activity assay kit ( Cell Biolabs ) . This assay uses a xanthine/XO system to produce superoxide anions , which reduce chromagen to produce a formazan dye , which is colorimetrically detectable at 490 nm . SOD activity in the samples was determined as the inhibition of formazan dye production . Each of the samples was mixed with 1× XO solution in a 96-well black plate and incubated at 37°C for 60 min and the formazan dye produced was colorimetrically detected at 490 nm using Spectra Max Plus ( Molecular devices ) . SOD1 inhibition was achieved by the addition of DDC ( Sigma-Aldrich ) to a final concentration of 1 mM into the mixture as described elsewhere [59–61] . Total copper levels in samples were assessed using the Metallo assay low copper LS kit ( Metallogenics , Chiba , Japan ) according to the manufacturer’s instructions . The pH of the samples was adjusted to 3 . 0 by adding a small amount of 0 . 25 mM HCl . Color reagent was then added and incubated at room temperature for 10 min . The copper concentration in the samples was calculated by measuring the absorbance at 580 nm using Spectra Max Plus ( Molecular devices ) . After euthanasia of mice , whole lungs were removed after perfusion of the mice with saline . The lungs were then cut into pieces and sieved through a 40 μm nylon cell strainer ( BD Falcon , Franklin Lakes , NJ ) with PBS . Lung cells were then collected by centrifugation at 1 , 000×g at 4°C for 2 min . The collected cells were suspended in Ham’s F-12K medium ( Life Technologies , Grand Island , NY ) supplemented with 15% FBS and cultured for 24 h . The cells were then cultured in F-12K medium without FBS in a 96-well plate at a density of 5 . 0×104 cells/well for another 24 h , and infected with IAV/PR8 at a 1 MOI in the presence of 0 . 05% trypsin ( Invitrogen ) . Cell viability was assessed using a Cell Counting Kit-8 ( Dojindo ) . Human lung epithelial A549 cells were maintained in Dulbecco’s modified Eagle’s medium ( DMEM , Wako Pure Chemicals ) with 10% FBS and transfected with non-targeting control siRNA ( cat: D-001210-01-05 , Thermo Scientific ) and human PrP-specific siRNA ( cat: D-011101-02 , Thermo Scientific ) . Briefly , 6 . 25 μL of RNAiMAX transfection reagent ( Invitrogen ) was mixed with 125 μL of Opti-MEM ( Life Technologies ) and incubated for 5 min at room temperature . In a separate tube , siRNA was added to 125 μL of Opti-MEM at a final concentration of 150 nM and the solution was then mixed with the RNAiMAX mixture for 20 min at room temperature . The siRNA/RNAiMAX mixture was then added to A549 cells in a 6-well plate . At 24 h after transfection , cells were washed with PBS and infected with IAV/PR8 at 1 MOI in 10% FBS-containing DMEM . Cells were collected , lysed , and subjected to Western blot analysis 24 h after infection . After euthanasia of mice , whole spleens were removed and sieved through a 40 μm nylon cell strainer ( BD Falcon ) with PBS . Splenocytes were then harvested by centrifugation at 1 , 000×g for 2 min at 4°C . The resulting pellet was suspended in ACK buffer ( 0 . 15 M NH4Cl , 1 . 0 mM KHCO3 , 0 . 1 mM Na2EDTA , pH 7 . 2 ) at room temperature for 2 min to disrupt red blood cells and centrifuged at 1 , 000×g for 2 min at 4°C . The collected splenocytes were adjusted to a concentration of 2 . 5×106 cells/mL in RPMI 1640 medium ( Sigma-Aldrich ) supplemented with 10% FBS , 1% L-glutamine ( Sigma-Aldrich ) , 2 μM L-glutamate ( Sigma-Aldrich ) , non-essential amino acids ( Sigma-Aldrich ) , 10 mM HEPES , and 1 mM sodium pyruvate . RT-PCR was performed using OneStep RT-PCR Kit ( QIAGEN , Hilden , Germany ) according to manufacturer’s recommendations . Total RNA was first extracted from tissues using RNeasy Mini Kit ( QIAGEN ) . Tissue homogenates in buffer RLT were transferred to a QIAshredder spin column ( QIAGEN ) . The flow-through was mixed with 1 volume of 70% ethanol and then transfer to an RNeasy spin column ( QIAGEN ) . Total RNA bound to the membrane was washed with buffer RW1 and then with buffer RPE , and eluted with RNase-free water . Eight ng of total RNA was then mixed with primers , dNTPs and OneStep RT-PCR enzyme mix . The mixture was incubated at 50°C for 30 min at RT and then subjected to PCR reaction ( Initial PCR activation step at 95°C for 15 min; 3-step cycling: Denaturation at 94°C for 30 sec , Annealing at 56°C for 30 sec , Extension at 72°C for 1 min; Final extension at 72°C for 10 min ) . Sequences of the primers used and the number of PCR cycles used for each gene examined are given in S1 Table . The products were analyzed by 2% agarose gel electrophoresis . IAV/PR8-specific IgG and IgM titers in plasma were determined by ELISA . Each well of a 96 well immunoplate ( Thermo Fisher Scientific , Roskilde , Denmark ) were coated with the already prepared split IAV/PR8 vaccine [62] in PBS overnight at 4°C . The wells were then washed with PBS 3 times and blocked with PBS containing 4% Block Ace ( Megmilk Snow Brand Co . , Ltd . , Hokkaido , Japan ) for 1 h at 37°C . Mouse plasma samples were first diluted to 1:16 and subsequently 1:2 in PBS and added to the wells at 37°C for 4 h . The wells were washed with PBS containing 0 . 05% Tween-20 3 times , and immune complexes were detected using HRP-conjugated goat anti-mouse IgM or IgG antibodies ( Bethyl Laboratories , Inc . , Montgomery , TX ) and 1-Step Ultra TMB-ELISA ( Thermo Scientific ) . The signals were detected spectrophotometrically at 450 nm using Spectra Max Plus ( Molecular devices ) . Antibody titers are defined as the reciprocal of the highest dilution of samples for which the optical density was at least twice of that of the negative control samples , and are expressed as reciprocal log2 titers . ELISPOT assay for TNF-α- and IFN-γ-secreting cells in lungs and spleens was performed using Mouse TNF-α ELISpotBASIC ( HRP ) kit ( Mabtech Inc . , Cincinnati , OH ) and Mouse IFN-γ ELISpotBASIC ( HRP ) kit ( Mabtech Inc . ) , respectively , according to the manufacturer’s recommendations . In brief , each well of the 96-well nitrocellulosebottomed Millititer HA plate ( Millipore ) was coated with capture MT1C8/23C9 anti-TNF-α antibody ( Mabtech Inc . ) and capture AN18 anti-IFN-γ antibody ( Mabtech Inc . ) in PBS overnight at 4°C . Primary lung cells ( 4×104 cells/well ) and splenocytes ( 2 . 5×105 cells/well ) from mice at 14 dpi were added to the wells and stimulated with 5 nM of the IAV peptide PA224-233 ( Anaspec Inc . , Fremont , CA ) for 3 days at 37°C in a humidified chamber with 5% CO2 in air . The wells were then washed with PBS 5 times and immune complexes were detected with biotin-conjugated detection antibodies ( Mabtech Inc . , MT11B10-biotin for TNF-α and R4-6A2-biotin for IFN-γ ) , Streptavidin-HRP ( Mabtech Inc . ) , and 1-Step Ultra TMB-ELISA ( Thermo Scientific ) . The number of the spots in the wells was quantified using a computerized ELISPOT reader ( CTL-Immunospot S6 analyzer , Cellular Technology Ltd . , Cleveland , OH ) . Survival rates were analyzed using the log-rank test . All other data were analyzed using the Student’s t-test . | Influenza A virus ( IAV ) is an enveloped , negative sense , single-stranded RNA virus , causing seasonal epidemic outbreaks of influenza . Anti-influenza agents targeting viral molecules , such as neuraminidase inhibitors , are currently available . However , these agents have accelerated emergence of mutant IAVs that are resistant to these agents among human populations . Development of new types of anti-influenza agents is awaited . We show that the cellular prion protein PrPC has a protective role against lethal infection with IAVs through the octapeptide repeat ( OR ) region by abrogating lung epithelial cell apoptosis induced by reactive oxygen species ( ROS ) in infected lungs . We also show that PrPC could reduce ROS in IAV-infected lungs through the OR region by maintaining Cu ion homeostasis and thereby activating Cu/Zn-dependent superoxide dismutase , SOD1 . These results highlight the protective role of PrPC in IAV infection . Elucidation of the exact mechanism underlying the PrPC-mediated protection against IAV infection would be important for further understanding the pathogenesis of IAV infection and could be useful for development of new types of anti-influenza therapeutics . | [
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] | 2018 | Prion protein protects mice from lethal infection with influenza A viruses |
The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break ( DSB ) sites where they induce histone acetylation , thereby influencing the chromatin structure and DNA repair process . Whether p300/CBP at DSB sites also acetylate non-histone proteins , and how their acetylation affects DSB repair , remain unknown . Here we show that p300/CBP acetylate RAD52 , a human homologous recombination ( HR ) DNA repair protein , at DSB sites . Using in vitro acetylated RAD52 , we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis . An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation , and that non-acetylated RAD52 initially accumulates at DSB sites , but dissociates prematurely from them . In the absence of RAD52 acetylation , RAD51 , which plays a central role in HR , also dissociates prematurely from DSB sites , and hence HR is impaired . Furthermore , inhibition of ataxia telangiectasia mutated ( ATM ) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity , through the formation of RAD52 , p300/CBP , SIRT2 , and SIRT3 foci at DSB sites . Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism .
Ionizing radiation ( IR ) induces deleterious DNA lesions , such as DNA double-strand breaks ( DSB ) . In response to DSBs , DNA damage response ( DDR ) signaling is induced . Ataxia telangiectasia mutated ( ATM ) protein kinase is one of the central players for phosphorylation-mediated DDR signaling , which is activated at DSB sites and phosphorylates numerous proteins , including the histone variant H2AX , and cell cycle checkpoint and DNA repair proteins [1] . Homologous recombination ( HR ) is an important mechanism for the repair of DSBs [2] . HR repairs DSBs through DNA strand invasion and exchange , in which the damaged DNA strand retrieves genetic information from an undamaged homologous DNA strand . After DSB formation , HR is initiated by a 5' to 3' end resection generating 3' single-stranded ( ss ) DNA overhangs . In mammalian cells , DSB end resection is mediated by the MRE11-RAD50-NBS1 ( MRN ) -CtIP complex and the EXO1 protein [3 , 4 , 5] . Afterwards , replication protein A ( RPA ) rapidly coats the 3'-overhang ssDNA regions , thereby removing secondary structures that form on the ssDNA region . Subsequently , the RPA coating the ssDNA regions is displaced by the RAD51 recombinase , to form a right-handed nucleoprotein filament . The RAD51 nucleoprotein filament then catalyzes DNA strand invasion and exchange between ssDNA and the homologous sequence within double-stranded ( ds ) DNA . The replacement of RPA with RAD51 requires additional proteins , such as recombination mediators , because prior binding of RPA to ssDNA inhibits the nucleation of RAD51 on ssDNA . Biochemical studies using recombinant proteins demonstrated that the yeast Rad52 protein stimulates the Rad51-mediated displacement of RPA from ssDNA regions [6] . In the mouse , the targeted inactivation of RAD52 reduces HR and may be involved in certain types of DSB repair processes [7] . However , this mediator function of human RAD52 for the loading of RAD51 onto the RPA-coated ssDNA region has never been demonstrated , despite extensive biochemical analyses [2 , 8] . Instead , biochemical studies have revealed that the human BRCA2 protein , which does not have a yeast homologue , promotes the RAD51 nucleoprotein filament formation on RPA-covered ssDNA in vitro [9 , 10] . Therefore , instead of RAD52 , BRCA2 is thought to mediate RAD51-dependent HR in human cells . This is supported by the fact that a knockdown of BRCA2 in human cells decreases the efficiency of IR-induced RAD51 foci formation . Interestingly , a RAD52 knockdown in BRCA2-knockdown or BRCA2-deficient cells almost completely inhibits IR-induced RAD51 foci formation [11] , which suggests that human RAD52 could act as a RAD51 mediator or complement the RAD51-dependent pathway in HR . Since most previous biochemical studies of RAD52 have utilized an unmodified , recombinant RAD52 protein expressed in Escherichia coli , it is possible that a recombination mediator activity of RAD52 might only be revealed upon post-translational modifications , as discussed by San Filippo et al . [2] . RAD52 preferentially binds ssDNA [12] rapidly and tightly , by wrapping the ssDNA around itself [13] . In contrast , RAD52 binds dsDNA slowly and weakly , but changes the dsDNA mechanics probably by intercalating into the DNA helix [13] . RAD52 also interacts with RPA and RAD51 [14 , 15] . Both yeast and human RAD52 exhibit ssDNA annealing activity [12 , 16] , which may be required in the steps following strand invasion mediated by RAD51 [17 , 18] , as well as in the RAD51-independent single-strand annealing ( SSA ) pathway [2] . Human RAD52 also has a D-loop formation activity [19] . Both human and yeast RAD52 are multimeric proteins [6 , 20] . Three-dimensional reconstitution from electron microscopy images revealed that full-length human RAD52 exists as a heptameric ring [21] . The crystal structure of the amino ( N ) -terminal half of RAD52 revealed an undecameric ring with a highly positively-charged groove outside the ring [22 , 23] . The N-terminal half of human RAD52 encompasses the catalytic domain for homologous pairing . Structure-based alanine scan mutagenesis of the N-terminal half of RAD52 revealed that several lysine ( K ) residues within the positively-charged groove are essential for DNA binding [22 , 24] . The carboxyl ( C ) -terminal region of human RAD52 contains domains that interact with RAD51 and RPA . Post-translational modifications , such as phosphorylation , ubiquitylation , small ubiquitin-like modifier ( SUMO ) ylation , and acetylation , regulate biological processes by controlling a wide variety of protein functions . Previously , some post-translational modifications of Rad52 were identified . Yeast Rad52 is modified by SUMO at the K10 , K11 , and K220 sites , and the SUMOylation is induced by a treatment with DNA-damaging agents [25] . SUMOylation of yeast Rad52 protects it from proteasomal degradation . Human RAD52 is also modified by SUMO , but the SUMOylation sites of human RAD52 differ from those of yeast Rad52 . The in vitro SUMOylation sites of human RAD52 are K411 , K412 , and K414 , which are located within the putative nuclear localization signal near the C-terminus ( Saito et al . , 2010 ) . SUMOylation does not affect the biochemical activities of human RAD52 , but mutations at SUMOylation sites inhibit RAD52 nuclear localization [26] . Nuclear phosphatase and tensin homolog on chromosome 10 ( PTEN ) was recently found to be involved in regulating RAD52 SUMOylation [27] . PTEN is also modified by SUMOylation , which is involved in the exclusion of the protein from the nucleus [28] . The function of the SUMOylation of human RAD52 , however , remains poorly understood . Human RAD52 is also phosphorylated at tyrosine ( Y ) 104 by c-ABL tyrosine kinase upon exposure to IR , and the phosphorylation deficiency inhibits the IR-induced foci formation of RAD52 [29] . Phosphorylation at Y104 enhances the ssDNA annealing activity of RAD52 by increasing the binding specificity for ssDNA [30] . No other post-translational modifications of RAD52 have yet been identified . Among the several post-translational modifications , acetylation occurs on specific lysine residues and is catalyzed by histone acetyltransferases ( HATs ) . Histones are well-known target proteins for acetylation . Histone acetylation influences chromatin structure , thereby regulating a wide variety of DNA transaction processes , such as transcription [31] , DNA replication [32] , DNA recombination [33] , and DNA repair [34] . HATs can also acetylate non-histone proteins , including some DNA repair proteins [35 , 36] . During HR , CtIP is deacetylated by SIRT6 histone deacetylase ( HDAC ) , and deacetylation is required for DNA end-resection , although the specific acetyltransferase for CtIP has not yet been identified [37] . The HATs , p300 and CBP , accumulate at laser microirradiation- or I-SceI-induced DSB sites , and promote histone acetylation at DSB sites [34] . Whether the accumulated p300 and CBP at DSB sites also induce the acetylation of non-histone proteins involved in DSB repair , however , is unclear . Here , we provide evidence for human RAD52 acetylation by p300/CBP upon DSB induction , and its involvement in RAD51 localization at DSB sites during HR repair . We show how HR is regulated via RAD52 acetylation and reveal the link between the acetylation event and the ATM-dependent phosphorylation .
The acetylation of non-histone DNA repair proteins has attracted recent attention [35 , 36 , 37] . We searched for new HAT substrates among human DNA repair proteins , and found that human RAD52 interacted with CBP , one of the well-known HATs ( Fig 1A ) . FLAG-tagged CBP coimmunoprecipitated with RAD52 , but not with the glutathione S-transferase ( GST ) control . RAD52 also specifically interacted with p300 , which is structurally and functionally similar to CBP ( S1A Fig ) . An in vitro acetylation assay was performed to examine whether RAD52 is acetylated by either CBP or p300 . DNA polymerase β was used as a positive acetylation control substrate [35] . Strikingly , the incubation of human RAD52 with p300 or CBP , in the presence of acetyl CoA , promoted RAD52 acetylation , which was detected by immunoblotting using an anti-acetyl lysine antibody ( Fig 1B ) . RAD52 acetylation was also confirmed by an in vitro acetylation assay using 14C-labeled acetyl CoA; the 14C-labeled acetyl group was transferred onto the ε-amino group of the lysine residue . Acetylation of RAD52 was specifically detected when RAD52 was incubated with CBP ( Fig 1C; lane 6 ) or p300 ( S1B Fig; lane 6 ) in the presence of 14C-labeled acetyl CoA . Notably , RAD52 was more efficiently acetylated than the control substrate , DNA polymerase β ( lane 3 in Fig 1C and S1B Fig ) . By contrast , neither RAD51 nor DNA polymerase κ [38] , which are key factors in homologous recombination , was acetylated by CBP or p300 in vitro ( S1C–S1E Fig ) . To map the acetylated sites in RAD52 , we performed an in vitro acetylation assay with the N- or C-terminal half of RAD52 ( Fig 1D and S1F Fig ) . Both RAD52 fragments were acetylated by CBP or p300 , although the C-terminal fragment of RAD52 ( 209–418 ) was more efficiently acetylated . To identify the acetylated residues , we performed a liquid chromatography mass spectrometry ( LC-MS ) analysis using in vitro acetylated full-length , N-terminal , and C-terminal RAD52 fragments . We identified 11 acetylation sites in RAD52 ( FL ) , and two additional acetylation sites ( K133 , K177 ) in the C-terminally truncated RAD52 ( N ) ( S1–S4 Tables and Fig 2A ) . We next purified two full-length RAD52 mutants , one containing arginine substitutions at the 11 acetylation sites ( 11xR ) , and the other containing arginine substitutions at all 13 identified acetylation sites ( 13xR ) . Using 11xR and 13xR , we confirmed that the p300/CBP-mediated acetylation of RAD52 was diminished when 11 or all 13 of the identified lysine residues were mutated to arginine ( Fig 2B ) . The identified sites are well conserved among different species ( S2 Fig ) . Lysines 411 , 412 , and 414 were previously identified as SUMOylation sites , and overlap with the nuclear localization signal ( NLS ) [26] . Mutations at these SUMOylation sites inhibited RAD52 nuclear localization . Another notable acetylation site is lysine 133 , which is an important site for DNA binding [24] . To study the RAD52 acetylation in detail , we used the acetylation-deficient , lysine-to-arginine substituted mutants in in vivo studies ( S3 Fig ) . The 11xR mutant and the unmodified RAD52 ( Wt ) displayed similar ssDNA binding activities , suggesting that the multiple lysine to arginine substitutions do not affect the RAD52 activity ( S4A and S4B Fig ) . To examine whether RAD52 is acetylated in human cells , we expressed an N-terminally FLAG-tagged RAD52 in human embryonic kidney 293 ( HEK293 ) cells , and immunoprecipitated RAD52 using anti-FLAG antibody-conjugated agarose . Overexpression of CBP induced RAD52 acetylation , based on immunoblotting using an anti-acetyl lysine antibody ( S5A Fig ) . To evaluate the acetylation status and localization of RAD52 in vivo , we produced anti-acetyl RAD52 antibodies against the acetylated lysine residues 274 or 323 . The antibody specificity for acetylated RAD52 was confirmed by comparison with the in vitro acetylated or non-acetylated RAD52 protein ( Fig 3A and 3B ) . Immunoblotting with each antibody revealed a positive reaction to the acetylated RAD52 protein ( Fig 3A and 3B ) . We then used these anti-acetyl RAD52 antibodies to examine whether the induction of DSBs changes the acetylation status of RAD52 in cells . We used the chemical DSB inducer doxorubicin , because it constitutively produces DSBs in the cells , and thus we thought it may induce a stronger DSB signal . Doxorubicin induced RAD52 acetylation in repair-proficient mesenchymal stem cells ( MSCs; Fig 3C ) [39] . A band shift of in vivo acetylated RAD52 was observed following doxorubicin treatment ( Fig 3D ) . The migration distance of the in vitro acetylated RAD52 was indistinguishable from that of the non-acetylated RAD52 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE ) ( Fig 1B ) . This was not the case for the acetylated RAD52 produced in vivo ( Fig 3A ) , raising the possibility that the in vivo acetylation induces additional modifications of RAD52 . Furthermore , we expressed a RAD52 construct in which 10 of the acetylation sites , except for lysine 411 , 412 , and 414 involved in the nuclear localization of RAD52 [26] , were substituted with arginine ( 10xR; Fig 3E and S3 Fig ) , and performed immunoblotting analyses to examine whether the mutations affect the interaction between RAD52 and the anti-acetyl RAD52 antibody . We did not achieve a positive signal with the anti-acetyl RAD52 antibody ( K323 ) in the presence of the 10xR mutant ( Fig 3E ) or in the absence of RAD52-HA expression by the vector ( S6 Fig ) . These findings indicate that the anti-acetyl RAD52 antibody ( K323 ) specifically detects and precisely evaluates the cellular acetylation status of RAD52 . The anti-acetyl RAD52 antibody ( K274 ) also specifically detected the acetylation of RAD52 , but the specificity of the anti-acetyl RAD52 antibody ( K323 ) against acetylated RAD52 was superior to that of the anti-acetyl RAD52 antibody ( K274 ) ( Fig 3C and 3E ) . To examine the RAD52 acetylation at DSB sites , MSCs expressing RAD52 ( Wt ) or the RAD52 ( 10xR ) mutant were irradiated with 8 Gy γ-rays and examined by an immunofluorescence approach . IR treatment transiently induces DSBs in cells , and thus IR is better suited for examining the level of RAD52 acetylation as a function of time . RAD52 foci , which colocalize with phosphorylated H2AX ( γH2AX ) , were detected with both wild-type and mutant RAD52 ( Fig 3F ) . In contrast , radiation-induced acetylated RAD52 ( assessed using the anti-acetyl RAD52 antibody ) was only detected in cells expressing RAD52 ( Wt ) protein at 1 h after IR . Acetylation of RAD52 ( Wt ) was not detected at 6 h after IR , suggesting that deacetylation of RAD52 occurs between 1–6 h at DSB sites . Significant acetylated RAD52 signals were not detected in cells expressing the RAD52 ( 10xR ) protein throughout the time period studied , indicating that the anti-acetyl RAD52 antibody specifically recognized acetylated RAD52 by immunofluorescence . We also investigated the distribution of p300 and CBP in these irradiated cells , and observed p300 and CBP foci , which colocalized with γH2AX , at 1 h and 6 h after IR in human fetal lung fibroblast ( MRC5 ) cells ( S5B and S5C Fig ) . This finding is consistent with a previous report that describes the recruitment of p300/CBP to laser-induced DSB sites [34] . The p300 and CBP foci also colocalized with the RAD52 foci , as observed at 1 h or 6 h after IR in HEK293 cells ( S5D and S5E Fig ) . In addition , the specific interaction of p300 or CBP with RAD52 , but not GST , was detected by dithiobis succinimidyl propionate ( DSP ) -mediated in vivo cross-linking experiments in exogenously p300- or CBP-overexpressing HEK293 cells ( S5F and S5G Fig ) . The interaction of p300 with RAD52 was increased upon doxorubicin treatment ( S5F Fig ) . Finally , the knockdown of both p300 and CBP inhibited the DSB-induced acetylation of RAD52 ( Fig 3G and 3H; S5H and S5I Fig ) . Taken together , our findings indicate that IR induces p300/CBP foci formation and acetylation of RAD52 at DSB sites . By analogy with yeast Rad52 , human RAD52 is reported to bind to RPA-coated ssDNA followig resection . RAD52 is also thought to bind to both ssDNA and dsDNA during the DNA strand exchange reaction . The N-terminal half of RAD52 contains a DNA binding region , whereas the C-terminal half contains an RPA binding region . Therefore , using an in vitro acetylation assay , we examined whether RAD52 binding to DNA or RPA affects the level of its acetylation . The addition of linear ( L ) ssDNA to the reaction mixture for the in vitro acetylation of RAD52 ( FL ) decreased the level of RAD52 acetylation ( Fig 4A ) . The addition of circular ( C ) or linear dsDNA also decreased the acetylation of RAD52 ( FL ) , but less than that of the linear ssDNA . Auto-acetylation of CBP was not affected by the addition of these DNA substrates . The N-terminal half of RAD52 , RAD52 ( 1–212 ) , contains the DNA-binding domain ( Fig 2A ) . The acetylation of RAD52 ( 1–212 ) was completely inhibited by the addition of any one of the DNA substrates ( Fig 4B ) , whereas the acetylation of the C-terminal half of RAD52 , RAD52 ( 209–418 ) , was not affected by the addition of the DNA substrates ( Fig 4C ) . RAD52 ( 209–418 ) contains the RPA-binding domain ( Fig 2A ) , and RAD52 ( 209–418 ) acetylation was inhibited by the addition of RPA in a dose-dependent manner ( Fig 4D ) . Therefore , these results suggest that at least some of the acetylation sites of RAD52 are located at the DNA and RPA interacting surfaces . HATs and HDACs regulate protein acetylation levels . Therefore , we next examined which HDACs are involved in the deacetylation of acetylated RAD52 . After RAD52 acetylation by CBP in vitro , linear ssDNA was added to the reaction mixture to inhibit further RAD52 acetylation . The reaction mixture was divided , and each sample was subjected to an in vitro deacetylation assay using recombinant HDAC proteins . Among the HDAC proteins examined , the recombinant HDAC3/NCOR2 complex , SIRT2 , and SIRT3 proteins deacetylated the acetylated RAD52 ( Fig 5 ) . Since the acetylated RAD52 appeared to be deacetylated at DSB sites after IR ( Fig 3F ) , we examined whether the identified HDACs for RAD52 were recruited to DSB sites after IR . We observed that SIRT2 and SIRT3 , but not HDAC3 , colocalized with γH2AX and/or RAD52 at 1 h after IR ( Fig 6A–6F ) . The SIRT2 and SIRT3 foci colocalized with γH2AX and RAD52 even at 6 h after IR ( Fig 6A , 6B , 6E and 6F ) , suggesting that SIRT2 and/or SIRT3 are involved in the deacetylation of acetylated RAD52 at DSB sites . Therefore , we examined whether the knockdown of Sirt2 or Sirt3 affects the decrease of RAD52 acetylation observed at 6 h after IR . The expression of SIRT2 and SIRT3 was effectively reduced by a small interfering ( si ) RNA treatment ( Fig 7A and 7B , S7A and S7B Fig ) . Importantly , acetylated RAD52 was maintained at DSB sites even at 6 h after IR following SIRT2 or SIRT3 depletion ( Fig 7C ) . Therefore , our results strongly suggest that SIRT2 and SIRT3 deacetylate RAD52 at DSB sites . The RAD52 protein localized to DSB sites after IR ( Figs 3F and 8A ) . Therefore , we examined whether lysine to arginine substitutions at the acetylation sites influence the accumulation of RAD52 at DSB sites . The RAD52 ( 13xR ) mutant ( with arginine substituted for lysine at the 13 identified acetylated lysine sites , including 3 substitutions in the C-terminal NLS sequence; S3 Fig ) localized to the cytoplasm rather than the nucleus , independently of IR ( Fig 8B ) . Interestingly , the RAD52 ( 13xR ) foci colocalized with γ-tubulin , indicating that RAD52 can localize to the centrosome . To examine the acetylation-deficient effect of the RAD52 ( 13xR ) mutant in the nucleus , we constructed a RAD52 ( 13xR ) mutant with an N-terminally fused NLS ( NLS-RAD52 [13xR]; S3 Fig ) . NLS-RAD52 ( 13xR ) was detected in the nucleus , but did not colocalize with γH2AX in MRC5 cells at 6 h after IR ( Fig 8C ) . We next examined the kinetics of the colocalization of RAD52 ( Wt ) and RAD52 acetylation-deficient mutants with γH2AX in irradiated MSCs . The control NLS-RAD52 ( Wt ) foci colocalized with γH2AX for 6 h after IR . In contrast , the NLS-RAD52 ( 13xR ) foci initially colocalized with γH2AX , but then dissociated from γH2AX within 2 h after IR ( Fig 9A and 9C ) . The RAD52 C-terminal region , including the acetylation sites K411 , 412 , and 414 , is known to be essential for the nuclear localization of RAD52 . Therefore , we next used the RAD52 ( 10xR ) mutant harboring a normal NLS sequence for analyzing the acetylation effects of RAD52 on IR-induced foci-formation at DSB sites in cells . The RAD52 ( 10xR ) mutant foci similarly showed an initial colocalization with γH2AX , followed by an early dissociation ( Fig 9B and 9D ) . Strikingly , during the first 30 min or 1 h after γ-irradiation , both NLS-RAD52 ( 13xR ) and RAD52 ( 10xR ) showed clear colocalization with γH2AX ( Fig 9A–9D ) . In order to confirm the effect of the 10xR mutation on the cellular localization of RAD52 expressed by the native promoter , we generated RAD52 ( Wt or 10xR ) knock-in cells by using CRISPR/Cas9-mediated genome editing . The DNA donor plasmid shown in S9 Fig was constructed by the Multisite Gateway method , and was co-transfected with CRISPR Nuclease Vector plasmids into HeLa pDR-GFP or HEK293 cells . The expression of the HA-tagged RAD52 proteins from the native promoter was confirmed by an immunoblotting analysis ( S9F and S9G Fig ) . We then used the knock-in cells for an immunostaining analysis . In order to deplete the RAD52 protein expressed from the untargeted allele , the knock-in cells were treated with an siRNA targeted to the 3'UTR region of RAD52 ( S7C Fig ) . Consistent with the results described above , the colocalization of RAD52 with γH2AX foci was decreased in the 10xR mutant at 6h after irradiation , whereas both the Wt and 10xR RAD52 proteins colocalized with γH2AX at 1h after irradiation ( S10A and S10B Fig ) . To clarify the critical acetylation sites involved in this colocalization defect , we further examined various RAD52 proteins mutated in several functional domains , such as the highly conserved region ( K133R , K133/K177R ) , the RPA binding region ( K262R ) , and the RAD51 binding region ( K323R ) , and also mutated residues outside the domains ( 190/192R ) . We also used the RAD52 ( 8xR ) mutant containing multiple mutations , except in the highly conserved region and the C-terminal NLS region ( S3 Fig ) . In contrast to the results obtained with the NLS-RAD52 ( 13xR ) and RAD52 ( 10xR ) mutants , the colocalization of these RAD52 mutants ( K133R , K133/177R , K190/192R , K262R , K323R , and 8xR ) with γH2AX was not affected after γ-irradiation ( Fig 10A and 10B ) , indicating that this defect is associated with all of the acetylation sites except for those in the C-terminal NLS region . These findings demonstrate that RAD52 acetylation is required for its sustained retention at DSB sites . ATM is a pivotal mediator of signal transduction in response to DSBs . As RAD52 acetylation was induced by DSBs , we examined whether its acetylation was triggered by ATM . KU55933 is a potent and specific ATM kinase inhibitor ( ATMi ) . To verify the functionality of the inhibitor , we observed that the ATM-mediated phosphorylation of CHK2 on threonine 68 ( T68 ) was inhibited in the KU55933-treated cells used in our present study . We found that the doxorubicin-induced acetylation of RAD52 was inhibited by the KU55933 ( ATMi ) treatment ( Fig 11A ) . Consistent with this result , the knockdown of ATM by siRNA treatment decreased the doxorubicin-induced acetylation of RAD52 ( Fig 11B , 11C and 11D ) . The IR-induced acetylation of RAD52 was also inhibited by the treatment with KU55933 ( ATMi ) ( Fig 11E ) . Notably , the ATM inhibition with KU55933 ( ATMi ) did not completely diminish the γH2AX foci formation , consistent with the previous findings that the IR-induced phosphorylation of H2AX can be redundantly affected by ATM or DNA-PK [40] . These γH2AX foci did not colocalize with the RAD52 foci , indicating that the treatment with KU55933 ( ATMi ) inhibited the accumulation of RAD52 at DSB sites ( Fig 11E and 11F ) . The IR-induced accumulation of both p300 and CBP at DSB sites was inhibited by the treatment with KU55933 ( ATMi ) in MRC5 cells ( Fig 12A–12D ) . ATM is involved in the DNA damage-induced phosphorylation of p300 at serine 106 ( S106 ) [41] . We used p300 mutant proteins in which S106 was substituted with alanine ( S106A ) or aspartic acid ( S106D ) , which mimics the phosphorylated state ( Fig 12E ) . Both mutant proteins colocalized with γH2AX after IR , suggesting that the ATM-mediated phosphorylation of p300 at the S106 site is not involved in its colocalization at DSB sites . The colocalization of these two mutant proteins with γH2AX was inhibited by KU55933 ( ATMi ) . Interestingly , the radiation-induced accumulation of SIRT2 and SIRT3 at DSB sites was also inhibited by the treatment with KU55933 ( ATMi ) ( Fig 13A–13E ) . These ATM-associated events were not caused by non-specific effects , because the colocalization of 53BP1 with γH2AX was not inhibited by KU55933 ( ATMi ) ( Fig 13F ) . The number of foci of RAD52 , p300 , CBP , SIRT2 , and SIRT3 decreased in the presence of KU55933 ( ATMi ) ( Figs 11F , 12C , 12D , 13C and 13D ) , whereas the cellular levels of these proteins did not significantly change ( S8A–S8E Fig ) . Thus , ATM inhibition prevents the accumulation of RAD52 , p300/CBP , and SIRT2/SIRT3 at DSB sites , thereby causing the intra-cellular diffusion of these proteins , even after IR treatment . Therefore , our results suggest that the interaction of RAD52 with p300 and CBP will be reduced by ATM inhibition , thereby decreasing the acetylation of RAD52 . Taken together , our results indicate that the DSB-induced acetylation of RAD52 occurs in the vicinity of DSB sites in an ATM-associated manner . We next examined whether RAD52 acetylation influences the accumulation of the RAD52-associated proteins , RPA and RAD51 , at DSB sites . Expression of the RAD52 ( 10xR ) mutant protein disrupted the IR-induced colocalization of RAD51 with γH2AX at 6 h in MSCs ( Fig 14A and 14B ) and at 4 h in HEK293 cells ( Fig 14C and 14D ) . Furthermore , RAD52 ( 10xR ) mutant expression in MSCs did not affect RAD51 foci formation or colocalization with γH2AX from 0 . 5 to 2 h after IR ( Fig 14E ) . A time course experiment revealed that RAD51 colocalized with γH2AX at 2 h after irradiation in both RAD52 ( Wt ) and RAD52 ( 10xR ) -expressing HEK293 knock-in cells , with no significant difference ( Fig 15A and 15B ) . Thereafter , at 4 and 6 h after irradiation , the colocalization decreased only in the RAD52 ( 10xR ) -expressing cells ( Fig 15A and 15B ) . These findings suggest that RAD52 acetylation is dispensable for the initial loading of RAD51 at DSB sites , but is required for the sustained retention of RAD51 at DSB sites . If the DNA resection is affected by RAD52 acetylation , then RPA foci formation should also be affected . However , the expression of the RAD52 ( 10xR ) mutant protein did not affect the colocalization of RPA with γH2AX ( Fig 16A ) . Therefore , our result suggests that DNA resection is not affected by RAD52 acetylation . Previously , BRCA1 was demonstrated to function in the loading of RAD51 at DSB sites via the PALB2-mediated interaction with BRCA2 [42] . IR-induced phosphorylated BRCA1 foci were observed at DSB sites in RAD52 ( 10xR ) and NLS-RAD52 ( 13xR ) -expressing cells ( Fig 16B ) . These findings suggest that the non-acetylated RAD52 protein disturbs the colocalization of RAD51 at DSB sites , but does not influence BRCA1 foci formation . These results are consistent with the aforementioned finding that the initial loading of RAD51 at DSB sites was not affected by RAD52 acetylation . RAD52 depletion is reportedly synthetically lethal with the BRCA2 deficiency , and inhibits cell growth in BRCA2-deficient cells [11] . Therefore , we examined whether BRCA2 depletion also inhibits cell growth in RAD52 ( 10xR ) -expressing cells . BRCA2 depletion inhibited cell growth in RAD52 ( 10xR ) -expressing cells , but not in RAD52 ( Wt ) -expressing cells ( Fig 17A ) . Expression of the RAD52 ( 10xR ) mutant did not affect the γH2AX foci formation after irradiation ( Fig 9B and 9E ) . This result might be due to the existence of a backup DSB repair system by the NHEJ pathway , and is consistent with the previous report showing that the inactivation of mouse RAD52 reduces HR , but does not affect the resistance to ionizing radiation [7] . However , the NHEJ pathway is dispensable for the repair of cross-linking DNA damage , but the HR pathway is required for its repair with the Fanconi anemia DNA repair pathway [44] . In the survival assay of cells treated with the DNA cross-linker cisplatin , the RAD52 ( 10xR ) -expressing cells were more sensitive to cisplatin than the RAD52 ( Wt ) -expressing cells ( Fig 17B ) . These results suggest that the acetylation of RAD52 is involved in HR repair . Therefore , we next examined whether the expression of the RAD52 ( 10xR ) mutant affects the HR efficacy . We quantified IR-induced sister chromatid exchanges in HEK293 cells expressing an empty vector or a vector encoding RAD52 ( Wt ) or RAD52 ( 10xR ) . Expression of RAD52 ( Wt ) did not influence the IR-induced sister chromatid exchanges , whereas the RAD52 ( 10xR ) mutant expression decreased the frequency of sister chromatid exchanges ( Fig 17C ) . In order to confirm the requirement of RAD52 acetylation for HR repair , we used a reporter assay with a cell line bearing a direct repeat green fluorescent protein ( DR-GFP ) reporter cassette [43 , 45 , 46 , 47] . The DR-GFP reporter cassette comprises two inactive GFP genes in a direct repeat orientation . One of the genes , SceGFP , contains an I-SceI cleavage site that is absent in the human genome . The other gene , iGFP , comprises the internal GFP fragment . HR repair between SceGFP and iGFP is induced when a specific DSB at the I-SceI site is introduced by the expression of I-SceI endonuclease . Since HR repair generates an intact GFP gene , the HR repair efficiency can be monitored as the frequency of GFP-positive cells ( Fig 18A ) . Using this HR assay system , we first examined the impact of the depletion of RAD52 , RAD51 , and BRCA2 proteins . These proteins were efficiently depleted by siRNA treatment ( S7C–S7G Fig ) . The depletion of RAD51 almost completely inhibited I-SceI-induced HR repair , as expected ( Fig 18B ) . The HR repair efficiency was reduced more by the BRCA2 depletion than by the RAD52 depletion ( S11 Fig ) , which is consistent with the notion that BRCA2 and RAD52 function in different pathways of RAD51-dependent HR repair [11] . Then , we examined the impact of the RAD52 acetylation-deficient mutation on HR repair ( Fig 18C and 18D ) . We used two types of HeLa pDR-GFP cells , expressing either the HA-tagged RAD52 ( Wt ) or RAD52 ( 10xR ) protein . The RAD52 ( Wt ) and RAD52 ( 10xR ) proteins were expressed by the native and CMV promoters , respectively , and the expression levels of the proteins were almost the same in both types of cells ( Fig 18E and S9F Fig ) . The endogenous untagged RAD52 protein was depleted by a treatment with an siRNA targeted to the 3'UTR region of RAD52 ( S7C Fig ) . In both cell lines , the expression of the RAD52 ( 10xR ) protein inhibited HR repair ( Fig 18C and 18D ) . Collectively , these findings demonstrate that the acetylation of RAD52 is required for HR repair . The interaction of human RAD52 with human RAD51 was previously detected in a yeast two-hybrid analysis [15] . The interactions of yeast Rad52 with yeast Rpa1 , Rpa2 , Rpa3 and Rad51 have also been detected [14] . Therefore , we analyzed the interactions between the human acetylation mimic RAD52 ( 10xQ ) and its target-proteins , using a yeast two-hybrid analysis . Glutamine ( Q ) is widely used to mimic acetylated lysine ( K ) , because the effect of the lysine-to-glutamine substitution is similar to the effect of the acetylation of lysine [48] . The yeast cells lacked p300 and CBP . Therefore , we constructed the plasmids expressing either GAL4-DBD or the NLS-GA4-AD-fused RAD52 ( 10xQ ) mutant for a yeast two-hybrid analysis ( S3 Fig ) . When the NLS-GAL4-activation domain ( AD ) -fused protein interacts with the GAL4-DNA binding domain ( DBD ) -fused protein , the reporter gene , His3 , is expressed , and the yeast cells show growth on an SC-Leu-Trp-His agar plate containing 25 mM 3-Amino-1 , 2 , 4-Triazole ( 3AT ) . Interactions of the GAL4-DBD-fused human RAD52 ( Wt ) with the NLS-GA4-AD-fused human RAD52 ( Wt ) , RAD51 , RPA1 , RPA2 or RPA3 were observed in the yeast two-hybrid analysis ( S12 Fig ) . Another reporter gene , lacZ , can also be used in this yeast two-hybrid analysis system . Therefore , we quantitatively examined the protein-protein interactions of RAD52 , by using a liquid β-galactosidase assay ( Fig 19A–19E ) . Both RAD52 ( Wt ) and RAD52 ( 10xQ ) were expressed almost equally in yeast cells ( Fig 19F ) . The self-interaction of RAD52 was increased 1 . 7-fold by the 10xQ mutation ( Fig 19A ) . The interactions of RAD52 with RAD51 , RPA1 , RPA2 and RPA3 were remarkably increased by the 10xQ mutation ( Fig 19B–19E ) . These results suggest that the interactions of RAD52 with these proteins are enhanced by its acetylation .
Based on our findings , we propose the following working model ( Fig 20 ) . Following DSB formation , human RAD52 and p300/CBP are recruited to DSB sites , and interact with each other near the DSB sites , thereby inducing RAD52 acetylation . Although 13 lysine residues can be acetylated , the acetylation efficiency of each site is probably different . SIRT2 and SIRT3 are also recruited to DSB sites shortly after DSB induction , and deacetylate RAD52 . During HR , RAD52 interacts with RPA or DNA . These interactions may prevent the ongoing acetylation of RAD52 by p300/CBP . As a result , the acetylation level of RAD52 is diminished by SIRT2 and SIRT3 . The acetylation sites of RAD52 are located in the DNA binding region ( K133 ) , and also within regions involved in protein-protein interactions . Indeed , the acetylation-mimicking RAD52 showed increased interactions with RPA and RAD51 in yeast cells ( Fig 19B–19E ) , and acetylated RAD52 binds ssDNA more robustly than non-acetylated RAD52 in vitro ( S13 Fig ) . These findings suggested that acetylated RAD52 plays a critical role in the maintenance of RAD51 recruited to DSB sites . Non-acetylated RAD52 dissociates prematurely from the DSB sites , and thus impairs the retention of RAD51 at the DSB site and prevents the completion of HR . The change in the levels and sites of acetylation during HR might control several activities of the multifunctional RAD52 protein . HR is a multistep process mediated by the concerted actions of many proteins . Regulation of each protein in both positive and negative manners is probably required to achieve the multistep and multi-enzyme processes . At present , the precise molecular mechanisms by which RAD52 exerts its functions in HR have not been elucidated . It is possible , however , that the strength of the RAD52 interactions with DNA and proteins could change during HR , which would enable RAD52 to change interaction partners and act as a multifunctional protein . Acetylation may provide important contributions to this regulation . Feng et al . demonstrated that the depletion of human RAD52 has small effects on decreasing HR and IR-induced RAD51 foci formation , as compared to the depletion of human BRCA2 [11] . They also demonstrated , however , that the depletion of human RAD52 in a BRCA2-depleted background further impairs both HR and RAD51 foci formation [11] . Based on these observations , they proposed that two pathways lead to RAD51-dependent HR: One is a dominant pathway in which BRCA2 mediates the recruitment of RAD51 to DSB sites , and the other is a pathway in which RAD52 alternatively mediates RAD51 recruitment [11] . The latter pathway becomes evident when the dominant BRCA2-mediated pathway is disrupted . Importantly , in the present study , non-acetylatable RAD52 impaired the localization of RAD51 foci at DSB sites , even in the presence of the dominant BRCA2 pathway , suggesting that the non-acetylatable RAD52 may competitively interfere with the BRCA2-mediated pathway . With regard to the reduced co-localization of RAD51 with γH2AX , the acetylation-defective RAD52 10xR mutant may have a dominant-negative effect by binding to RAD51 and preventing the localization of RAD51 at DSB sites . In the presently determined kinetics of RAD51 foci , the RAD52 10xR mutant does not inhibit the initial step of RAD51 recruitment to DSB sites in the presence of BRCA2 . We suppose that RAD51 transiently accumulates and interacts with RAD52 at DSB sites; however , RAD51 might subsequently dissociate from the DSB sites along with RAD52 in this mutant . Here we have described the novel post-translational acetylation of RAD52 , and demonstrated that the failure to acetylate RAD52 critically impacts HR . Our results suggest that an inhibitor of RAD52 acetylation could be exploited for anticancer therapy . Our results demonstrated that DSB-induced RAD52 acetylation requires ATM kinase ( Fig 20 ) . We also found that inhibiting the function of ATM caused a decrease in the accumulation of RAD52 and p300/CBP at DSB sites . From these results , we drew the following hypothesis: In the absence of DSBs , p300 , CBP , and RAD52 are diffused in the nucleus . After DSB-induction , these proteins accumulate at DSB sites , which will promote the interaction between RAD52 and p300 or CBP , thereby inducing the acetylation of RAD52 . Accordingly , in the absence of functional ATM , the interaction of RAD52 with p300 and CBP will not be promoted , leading to the loss of acetylated RAD52 . Our findings revealed that the accumulation of SIRT2 and SIRT3 at DSB sites is also triggered by ATM . Similar results were reported for the other HDAC , SIRT1 , with ATM being required for its accumulation at DSB sites [49] . Human RAD52 is phosphorylated by c-ABL , and this phosphorylation is required for the IR-induced foci formation of RAD52 [29] . The activation of c-ABL is induced in response to various types of DNA damage , including DSB , and the activation depends on ATM . Therefore , ATM might be required for the IR-induced foci formation of RAD52 at DSB sites through the c-ABL-mediated phosphorylation of RAD52 . ATM functions in the DNA damage-induced phosphorylation of p300 [41] . Therefore , we examined whether ATM-mediated phosphorylation is required for the accumulation of p300 at DSB sites , and concluded that p300 phosphorylation is dispensable for its accumulation at DSB sites . To our knowledge , there are no reports that these HATs and HDACs possess DNA binding activity . Therefore , it is unclear how they accumulate near DSB sites . One possibility is that they are recruited to DSB sites via interactions with a protein that exists at DSB sites . ATM phosphorylates several proteins at DSB sites . Taken together , our results suggest that such an ATM-phosphorylated protein at DSB sites interacts specifically with several HATs and HDACs , thereby recruiting them to DSB sites . In contrast to the requirement for ATM in the accumulation of several HATs and HDACs at DSB sites , SIRT1 is required for the accumulation of ATM at DSB sites [49] . Furthermore , ATM is also acetylated by another HAT , Tip60 [50] . ATM acetylation enhances its activation as a protein kinase in response to DNA damage . Thus , there is interplay between acetylation and phosphorylation in response to DNA damage .
The roles of HATs and HDACs are important topics in many fields of biology and medicine . In this paper , we found the novel roles of HATs ( p300/CBP ) and HDACs ( SIRT2 and SIRT3 ) in the HR process through the acetylation of human RAD52 , indicating that human RAD52 is required for HR , depending on its acetylation status . We further demonstrated that ATM protein kinase is required for DSB-induced RAD52 acetylation , as well as for the accumulation of RAD52 , p300/CBP , SIRT2 , and SIRT3 at DSB sites . These findings indicate the presence of crosstalk between acetylation and phosphorylation . Therefore , the ATM kinase activation/RAD52 acetylation axis may be important for HR repair . At DSB sites , several HATs and HDACs regulate histone acetylation , which is required for DNA repair . In addition to histone acetylation , we have demonstrated that HATs ( p300/CBP ) and HDACs ( SIRT2 and SIRT3 ) directly regulate the acetylation of the non-histone protein , RAD52 . We speculate that HATs and HDACs target more DSB repair proteins at DSB sites . These findings provide important information for future studies using in vitro reconstitution systems in the context of chromatin , to clarify the molecular mechanisms of DSB repair .
Confluent cells were exposed to X-rays at a dose rate of 1 Gy/min at room temperature . The cells were immediately subcultured in media with bromodeoxyuridine ( 3 μg/ml ) . After adding colcemid for 6 h , the cells were harvested and treated with a hypotonic KCl solution ( 75 mM ) at 37°C for 20 min , followed by methanol:acetic acid ( 3:1 ) fixation . After three rounds of fixation , the cells were dropped onto slides . The slides were treated with Hoechst 33258 ( 10 μg/ml ) for 20 min and exposed to a black light for 30 min at 55°C . Finally , the slides were treated with 2XSSC ( saline sodium citrate ) solution for 20 min at 65°C . The slides were stained using 5% filtered Giemsa solution mixed in Gurr . Images of metaphase cells were obtained using a Zeiss Axioplan microscope ( Carl Zeiss ) equipped with a QImaging Exi Aqua Cooled CCD camera ( QImaging , Surrey , Canada ) . Sister chromatid exchanges were counted per chromosome . | DNA double strand breaks ( DSBs ) are the most dangerous type of DNA damage in cells . Homologous recombination ( HR ) is a DSB repair system in which a central player , RAD51 , functions with several proteins , including RAD52 . DSBs activate the DNA damage response signaling network , in which the ataxia telangiectasia mutated ( ATM ) protein plays a chief role , by phosphorylating numerous target proteins . As compared to phosphorylated proteins , relatively few acetylated proteins have been functionally characterized in DNA repair . In addition , beyond the roles in phosphorylation signaling , much less is known about whether ATM functions are linked with other protein modifications , such as acetylation . Here , we found that RAD52 at DSB sites is acetylated by p300/CBP acetyltransferases and then deacetylated by SIRT2/SIRT3 deacetylases . RAD52 acetylation is required for sustained RAD51 colocalization at DSB sites , and is therefore essential in HR . ATM is required for the recruitment of RAD52 , p300/CBP and SIRT2/SIRT3 to DSB sites , and therefore is essential for RAD52 acetylation . Thus , the RAD52-acetylation state is critical for HR , and its regulation is linked to ATM signaling . Our work demonstrates the importance of the regulation of RAD52 acetylation in HR and its underlying mechanism . | [
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] | 2018 | Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites |
Empirical evidence suggests that the prevalence of soil-transmitted helminth ( STH ) infections in remote and poor rural areas is still high among children , the most vulnerable to infection . There is concern that STH infections may detrimentally affect children’s healthy development , including their cognitive ability , nutritional status , and school performance . Medical studies have not yet identified the exact nature of the impact STH infections have on children . The objective of this study is to examine the relationship between STH infections and developmental outcomes among a primary school-aged population in rural China . We conducted a large-scale survey in Guizhou province in southwest China in May 2013 . A total of 2 , 179 children aged 9-11 years living in seven nationally-designated poverty counties in rural China served as our study sample . Overall , 42 percent of the sample’s elementary school-aged children were infected with one or more of the three types of STH—Ascaris lumbricoides ( ascaris ) , Trichuris trichuria ( whipworm ) and the hookworms Ancylostoma duodenale or Necator americanus . After controlling for socioeconomic status , we observed that infection with one or more STHs is associated with worse cognitive ability , worse nutritional status , and worse school performance than no infection . This study also presents evidence that children with Trichuris infection , either infection with Trichuris only or co-infected with Trichuris and Ascaris , experience worse cognitive , nutritional and schooling outcomes than their uninfected peers or children infected with only Ascaris . We find that STH infection still poses a significant health challenge among children living in poor , rural , ethnic areas of southwest China . Given the important linkages we find between STH infection and a number of important child health and educational outcomes , we believe that our results will contribute positively to the debate surrounding the recent Cochrane report .
A recent Cochrane Report [1] has raised questions about the nature of the relationship between infection with soil-transmitted helminths ( STHs ) and children’s healthy development . In a meta-analysis of 42 papers , the authors of the report found that there was no clear , consistent relationship between deworming and improvements in children’s cognitive ability , nutritional indicators , or school performance . The report ended with a call for more concerted research that would help clarify the nature of the relationship between STH infection and these outcomes in children . Health policymakers depend on this type of information when deciding how to allocate resources to different disease types , in general , and how to allocate for STH control and treatment , in particular . Because of high STH prevalence , China is an especially suitable setting in which to conduct such additional research . According to Wang et al . [2] , 40 percent of school-aged children in rural areas of Guizhou province are infected with one or more types of three STHs: Ascaris lumbricoides ( ascaris ) , Ancylostoma duodenale ( hookworm ) , and Trichuris trichuria ( whipworm ) . In some villages , the prevalence is as high as 80 percent . Similarly high prevalence has also been reported elsewhere in China , such as in rural Guangxi and Hainan provinces [3] . The uncertainty in the international literature about the link between STH infections and child outcomes is reflected in the China-specific literature . Of six total China-based studies , five measured the link between STH infection and child health ( either physical development or hemoglobin levels ) . Three of these [2–4] found a significant negative correlation between STH infection and children’s health , while two found no correlation [5–6] . None of the six studies measured school performance , although three attempted to measure the relationship between STH infections and cognitive ability using formal tests; however , the sample sizes in these studies were small , ranging from 140 to 200 children in 2 to 11 communities or clusters . With such small sample sizes , it is statistically improbable to produce meaningful results . In short , in the context of China—as in the rest of the world—there is uncertainty about the relationship between STH infections and child outcomes . In this paper we will answer questions raised by the Cochrane report [1] and build evidence on the relationship between STH infections and health outcomes in children in rural China . To achieve this goal , we have three objectives . First , we will document the prevalence of STHs in the study areas , thus better defining the severity of the STH problem in poor areas of rural China . Second , we will document the levels of cognitive ability , nutritional indicators and school performance among our sample children in order to assess how children in poor rural areas fare in terms of these measured outcomes . Finally , we will examine the links between STH infection and cognitive ability , nutritional indicators , and school performance .
We collected the data used in May of 2013 as part of a large-scale survey of elementary school-aged children in Guizhou province . Our study was conducted in seven rural counties in Qiandongnan prefecture . We chose our sample to include regions that were poor and populated by ethnic minorities , the subpopulations that are at higher risk for STH infection . Fig 1 depicts the sample selection process . Based on rural per capita income levels reported in Guizhou Statistical Yearbook [7] , the research team randomly selected a total of seven rural counties from the poorest half of the counties ( 8 out of 16 ) in Qiangdongnan . According to national statistics , at 4 , 625 yuan , the average rural individual in our sample areas has a per capita income in the bottom quartile of China’s rural income distribution [8] . Once we chose the sample counties , we selected the sample townships and villages . In each county , we included all townships except for the township which houses the county government . We did not include the township that housed the county government because such townships are almost always wealthier and more urban than a typical rural township . A total of 112 townships were selected . Then sample villages within each township were selected . Since our survey would take place in schools , we obtained a list of all the 9–11 year old children attending the central primary school in each township . We classified all 9–11 year old children by their home village , and then randomly selected 20 sample children from the home village with the largest number of children at that school . We excluded villages that housed the local township government , since ( as discussed above in the context of towns/counties ) these villages are typically wealthier and more urban than a typical village . If the home village had fewer than 20 children in our age group attending the school , we randomly selected children from the next-largest village to fill in the gap . Overall , a total of 20 school children were randomly chosen from either one or two villages in each township . A total of 2 , 240 children from 146 villages and 112 townships in the 7 poor rural counties were chosen as sample students . The survey team collected four types of information: data from a socioeconomic survey; scores on a test of cognitive ability; measures of child health ( including STH infection status , height , weight , and hemoglobin levels ) ; and school absence and performance ( as measured by absenteeism and performance on a standardized math test ) . This study received ethical approval from the Stanford University Institutional Review Board ( IRB ) ( Protocol ID 25027 ) , and from the Sichuan University Ethical Review Board ( Protocol ID 2013005–02 ) . All participating children gave their assent for their involvement in the study , and the children’s legal guardians gave their written consent for both their own and their children’s involvement . Children who were found to have severe anemia were referred to the local hospital for treatment . Anemia status was determined based on finger prick blood analysis for hemoglobin ( Hb ) . Following internationally accepted standards , anemia was defined as Hb<115 g/L [20] . Physical indicators of height and weight were used to construct height-for-age z-scores ( HAZ ) and Body Mass Index ( BMI ) -for-age z-scores using WHO AnthroPlus , a software application of the WHO Reference 2007 for children aged 5–19 years that is used to monitor the growth of school-aged children and adolescents [21] . Weight-for-age z-scores ( WAZ ) were calculated using a SAS program for the 2000 CDC growth chart for children aged 0–20 years [22] . We followed internationally recognized cutoffs [23] to consider children whose HAZ , WAZ , or BMI-for-age z-score to fall more than two standard deviations below the international mean to be stunted , underweight , or malnourished , respectively . Raw scores obtained from the four core subtests of the WISC-IV were converted to age-scaled index scores using tables of norms in the Mandarin version of the WISC-IV administration and scoring manual . Two index scores are considered for analysis: Working Memory Index ( WMI ) and Processing Speed Index ( PSI ) . Scores are divided into internationally-recognized ranges . A score of 90–110 is considered “average”; a score of 80–89 is considered “low average”; a score of 70–79 is considered “borderline”; and a score of below 70 is considered “extremely low” and at risk for intellectual disabilities or mental retardation . All statistical analyses were performed using STATA 12 . 0 . P-values below 0 . 05 were considered statistically significant . All P-values were adjusted for multiple hypotheses testing by the Bonferroni method . The statistical significance of differences in all outcomes by subgroup populations was assessed using student’s t-test in STATA . STATA’s multiple linear regression model was used in the multivariate analysis for those continuous outcome variables: WMI , PSI , Hb , HAZ , WAZ , BmiAZ , as well as standardized math test scores . Meanwhile , STATA’s logistic regression model was used in the multivariate analysis for those binary outcome variables: anemic ( yes/no ) and school absence ( yes/no ) . We included the following independent variables as potential confounders in the multivariate analysis: gender , age , boarding status , minority status , sanitation behaviors , and household characteristics . Definitions of key variables to be used in the rest of the paper are presented in Table B in S1 Text .
Of the 2 , 179 children who provided fecal samples , participated in the socioeconomic survey , cognitive testing , health examination as well as school absence and performance tests , 42 percent were infected with one or more of the three types of STH ( Table 2 ) . The most prevalent type of STH in the survey areas is Ascaris ( 31 percent ) , followed by Trichuris ( 22 percent ) , and finally by hookworm ( 1 percent ) . The prevalence of Ascaris only and Trichuris only was 19 percent and 11 percent , respectively . Eleven percent of sample children were co-infected with Ascaris and Trichuris . In contrast , the prevalences of infection with hookworm only , co-infection with Ascaris and hookworm , co-infection with Trichuris and hookworm , or co-infection with Ascaris , Trichuris and hookworm were negligible . A total of 63 percent of children had a Working Memory Index ( WMI ) that was either “extremely low” ( <70 ) or “borderline” ( 70–79 ) . The breakdown shows that 13 percent of children scored “extremely low” on the WMI portion of the test , and 50 percent of children scored in the “borderline” range . A total of 36 percent of children had a Processing Speed Index ( PSI ) that was either “extremely low” ( <70 ) or “borderline” ( 70–79 ) . The breakdown shows that 10 percent of children scored “extremely low” on the PSI portion of the test , and 26 percent of children scored in the “borderline” range . We find that 16 percent of our sample children are anemic ( Table 2 ) , 28 percent are stunted ( HAZ < -2 ) , 6 percent are malnourished ( BMI-for-age < -2 ) , and 26 percent are underweight ( WAZ < -2 ) . Around 13 percent of children in our sample had been absent from school at least once during the most recent semester . The average child in our sample earned a failing score on the TIMSS test ( score < 60 ) , scoring an average of 52 . 6 out of 100 on the TIMSS test ( Table 2 ) . There are significant differences in children’s cognitive ability between infected children and uninfected children ( Fig 2 and Table 2 ) . Our data show that the mean WMI of infected children is 76 . 6 , significantly lower than that of the uninfected group ( 80 . 0 , p < 0 . 005 ) . Moreover , 71 percent of infected children had an “extremely low” or “borderline” WMI , significantly higher than that of uninfected children ( 57 percent , p < 0 . 005 ) . These differences remain statistically significant after controlling for confounding factors ( Table 3 ) . Our data show that the mean PSI of infected children is 83 . 4 , significantly lower than that of the uninfected group ( 88 . 1 , p < 0 . 005 ) . Moreover , 45 percent of infected children in the infected group had an “extremely low” or “borderline” PSI , significantly higher than that of uninfected children ( 29 percent , p < 0 . 005 ) . These differences remain statistically significant after controlling for confounding factors ( Table 3 ) . There are no significant differences between children with Ascaris only and children with no infection of any of the three types of STHs in terms of WMI and PSI ( See Panel A , Table C in S1 Text ) . The same is true when we compare children with both Ascaris and Trichuris against children with Trichuris only ( Panel D2 ) . However , compared with children with no infection of any of the three types of STHs , children with Trichuris only infection had significantly lower WMI ( p<0 . 001 ) and PSI ( p<0 . 001 ) after controlling for confounding factors ( Panel B ) . The same strong relationship remains when we compare children with both Ascaris and Trichuris against children with no infection with any of the three types of STHs ( Panel C ) . Our data also show that children with a co-infection of both Ascaris and Trichuris had significantly lower PSI ( p<0 . 001 ) than children with Ascaris only infection , after controlling for confounding factors . However , there is no significant difference in WMI between these two groups of children ( Panel D1 ) . There are significant differences in terms of both mean hemoglobin level and mean anemia rate between infected and uninfected children ( Table 2 ) . The hemoglobin level among infected children is 126 . 8 g/L , significantly higher than that among the uninfected group ( 125 . 4 g/L , p < 0 . 01 ) . Meanwhile , the anemia rate among infected children is 19 percent , significantly higher than that among the uninfected group ( 15 percent , p < 0 . 05 ) . However , after controlling for confounding factors , the differences between infected children and their uninfected peers were found to be statistically insignificant ( Table 3 ) . There are significant group differences in height-for-age z-scores ( HAZ ) between infected and uninfected children ( Table 2 ) . The mean HAZ among infected children is -1 . 59 , compared with -1 . 25 among uninfected children ( p < 0 . 005 ) . An average of 34 percent of infected children are stunted , compared with 23 percent of uninfected children ( p < 0 . 005 ) . After controlling for confounding factors , the difference in HAZ remains statistically significant ( Table 3 , p < 0 . 001 ) . There are significant group differences in weight-for-age z-scores ( WAZ ) between infected and uninfected children ( Table 2 ) . The mean WAZ among infected children is -1 . 55 , compared with -1 . 27 among uninfected children ( p < 0 . 005 ) . An average of 31 percent of infected children are underweight , compared with 23 percent of uninfected children ( p < 0 . 005 ) . After controlling for confounding factors , the difference in WAZ remains statistically significant ( Table 3 , p < 0 . 005 ) . The mean BMI-for-age z-score among infected children is -0 . 64 , compared with -0 . 55 among uninfected children ( p < 0 . 05 ) . The proportion of being malnourished does not vary significantly between infected and uninfected children ( Table 2 ) . Similar to the cases of hemoglobin level and anemia rate , after controlling for confounding factors , the difference in BMI-for-age z-scores becomes statistically insignificant ( Table 3 ) . There are no significant differences between children with Ascaris only and children with no infection of any of the three types of STHs in terms of mean hemoglobin level , mean anemia rate , HAZ , WAZ and BmiAZ ( Panle A , Appendix Table 3 ) . The same is true when we compare children with both Ascaris and Trichuris against children with Trichuris only ( Panel D2 ) . However , compared with children with no infection of any of the three types of STHs , children with Trichuris only infection had significantly lower Hb ( p<0 . 001 ) , lower HAZ ( p<0 . 001 ) and lower WAZ ( p<0 . 001 ) after controlling for confounding factors although their mean anemia rate and BmiAZ are not statistically different ( Panel B ) . Our data also show that children with a co-infection of both Ascaris and Trichuris had significantly lower HAZ ( p<0 . 001 ) and lower WAZ ( p<0 . 001 ) than children with no infection of any of the three types of STHs although their mean hemoglobin level , mean anemia rate and BmiAZ are not statistically different ( Panel B ) . Finally , compared with children with Ascaris only infection , children co-infected with both Ascaris and Trichuris also had significantly lower WAZ ( p<0 . 001 ) , but there are no significant differences in terms of mean hemoglobin level , mean anemia rate , HAZ or BmiAZ ( Panel D1 ) . The absence rate among infected children is 16 percent , compared to 11 percent among uninfected children ( p < 0 . 005 ) ( Table 2 ) . This difference becomes statistically insignificant after controlling for confounding factors ( Table 3 ) . The mean TIMSS score among infected children is 47 . 5 , compared with 56 . 4 among uninfected children ( p < 0 . 005 ) . A total of 70 percent of infected children failed the TIMSS test ( scores < 60 ) , compared with 56 percent of uninfected children ( p < 0 . 005 ) ( Table 2 ) . These differences remain statistically significant after controlling for confounding factors ( Table 3 , p < 0 . 001 ) . Compared to children without infection of any of the three types of STHs , children with Ascaris only infection see no difference in the incidence of school absence ( See Panel A , Table C in S1 Text ) . The same is true when we compare children with Trichuris only ( Panel B ) or children with a co-infection with both Ascaris and Trichuris against children with no infection of any of the three types of STHs ( Panel C ) . Similarly , our data show no significant difference in incidence of school absence between children with a co-infection with both Ascaris and Trichuris with children with Ascaris only ( Panel D1 ) or Trichuris only ( Panel D2 ) . Our data also show that there is no difference in standardized test scores between children with Ascaris only infection and children with no infection of any of the three types of STHs , after controlling for confounding factors ( See Panel A , Table C in S1 Text ) . The same is true when we compare children with a co-infection of both Ascaris and Trichuris to children with Trichuris only infection ( Panel D2 ) . However , compared with children with no infection of any of the three types of STHs , children with Trichuris only infection or children had lower standardized test scores ( p<0 . 001 ) , after controlling for confounding factors ( Panel B ) . The same strong correlation holds when we compare children with a co-infection of both Ascaris and Trichuris against children with no infection of any of the three types of STHs ( Panel C ) . Our data also show that children with a co-infection of both Ascaris and Trichuris had lower standardized test scores ( p<0 . 001 ) than children with Ascaris only infection ( Panel D1 ) .
In this paper we document the prevalence of STHs using results from stool sampling and socioeconomic testing of 2 , 179 school children living in seven nationally-designated poverty counties in Qiandongnan prefecture in Guizhou province . We observed that 42 percent of the sample children were infected with one or more of the three types of STH—Ascaris , Trichuris , and hookworm . This prevalence is consistent with previous , smaller-scale studies in China [2 , 3] , but is more than twice the observed STH prevalence from the National Survey on Current Status of the Important Parasitic Diseases in Human Population in 2004 [25] . According to the WHO treatment guidelines , the prevalence we document warrants mass treatment . We also document children’s cognitive ability , nutritional indicators , school absence and performance . Our data show that sample children are lagging far behind the international standard in terms of each of these measured outcomes . We further found that after controlling for a set of socioeconomic confounders , infection with one or more STHs is associated with worse cognitive ability ( in terms of WMI and PSI ) , worse nutritional status ( in terms of HAZ and WAZ ) , and worse school performance ( in terms of standardized math test scores ) . Without implying there is any causal link per se , this study also presents evidence that infection with Trichuris , either infection with Trichuris only or co-infected with Trichuris and Ascaris , makes children experience worse cognitive , nutritional and schooling outcomes than their uninfected peers or children infected with only Ascaris . These results are consistent with findings from other epidemiological studies and randomized controlled trials examining the relationship between STH infections and cognitive ability [3 , 10–14 , 26] , nutritional indicators [2 , 3 , 13 , 27–32] , and school performance [33–34] . In a study of children between the ages of 5 and 14 years in China , Shang reported that STH infection was associated with high incidence of malnourishment , stunting and anemia . The Shang study also found infections were correlated with worse performance on WMI and PSI [3] . These observations support our findings that infections with one or more STHs are associated with worse cognitive ability , worse nutritional status and worse school performance than their uninfected peers . A randomized controlled trial of STH infected children who were treated with albendazole demonstrated less school absenteeism than children in the comparison group who were not treated with albendazole [35] . However , we do not find any strong correlation between STH infection with school absenteeism . A previous cross-sectional study among schoolchildren in Brazil suggested that polyparasitised children experience worse cognitive outcomes than children with only one helminth infection [14] . We attempted to assess the effects of polyparasitism on child development outcomes , specifically cognitive ability , nutritional indicators , school absence and performance . To do so , a method was developed whereby development outcomes were compared between children with co-infections of STHs and children with only one STH infection . The findings suggest that infection with Trichuris , either infection with Trichuris only or co-infected with Trichuris and Ascaris , makes children experience worse cognitive , nutritional and schooling outcomes than their uninfected peers or children infected with only Ascaris . Our study has several limitations . First , due to budgetary constraints , we collected two stool samples per child ( on consecutive days ) , rather than three samples per child . While we believe that two samples adequately allows for the cyclical nature of roundworms’ egg laying patterns , three samples may have allowed for even greater sensitivity in the detection of the true prevalence of infection . Second , while we made every effort to keep samples refrigerated for as long as possible between sample production and laboratory testing , the samples were not produced on site , and therefore children may have waited up to several hours before delivering their samples to the nearest refrigeration facilities ( either at the village clinic or at the school ) . This waiting period was outside of our control , but may have contributed to the degradation of hookworm eggs . Since both of these limitations may have resulted in an underestimate of total STH prevalence , the estimates presented here should be considered to be a lower bound . A third study limitation is that we were unable to collect data on the intensity of infection in the sample areas . Our study shows that STH infection still poses a significant health challenge among children living in poor , rural , ethnic areas of southwest China . Given the important linkages we find between STH infection and a number of important child health and educational outcomes , we hope that our results will contribute positively to the debate surrounding the recent Cochrane report [1] . Although our results are correlational , we believe that the strength of the correlations is striking , and indicates a need for more rigorous research on the impacts of STH treatment on child outcomes . Due to the cross-sectional nature of our data , we are unable to identify the precise reasons behind the linkages we observe through this study . However , we can speculate that one possible explanation might be that STH infections lead to nutritional deficits , which in turn contribute to cognitive impairments . Another possible explanation might be that children from disadvantaged households are both more likely to have poor sanitation practices that contribute to chronic STH infection and are also more likely to have poor nutritional intake that may lead to worse cognitive performance . | Empirical evidence suggests that the prevalence of soil-transmitted helminth ( STH ) infections in remote and poor rural areas is still high among children , the most vulnerable to infection . There is concern that STH infections may detrimentally affect children’s healthy development , including their cognitive ability , nutritional status , and school performance . Medical studies have not yet identified the exact nature of the impact STH infections have on children . The objective of this study is to examine the relationship between STH infections and developmental outcomes among a primary school-aged population in rural China . We conducted a large-scale survey in Guizhou province in southwest China in May , 2013 . Overall , 42 percent of elementary school-aged children were infected with one or more of the three types of STH—Ascaris lumbricoides ( ascaris ) , Trichuris trichuria ( whipworm ) and the hookworms Ancylostoma duodenale or Necator americanus . After controlling for socioeconomic status , we observed that children infected with one or more STHs have worse cognitive ability , worse nutritional status , and worse school performance than their uninfected peers . While not causal , this study also presents evidence that children with Trichuris infection , either infection with Trichuris only or co-infected with Trichuris and Ascaris , have worse cognitive , nutritional and schooling outcomes than their uninfected peers or children infected with only Ascaris . Given these important linkages , we hope that our results will contribute positively to the debate surrounding the recent Cochrane report . | [
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"Discussion"
] | [] | 2015 | Soil-Transmitted Helminths in Southwestern China: A Cross-Sectional Study of Links to Cognitive Ability, Nutrition, and School Performance among Children |
The global-scale epidemiology and genome-wide evolutionary dynamics of influenza B remain poorly understood compared with influenza A viruses . We compiled a spatio-temporally comprehensive dataset of influenza B viruses , comprising over 2 , 500 genomes sampled worldwide between 1987 and 2015 , including 382 newly-sequenced genomes that fill substantial gaps in previous molecular surveillance studies . Our contributed data increase the number of available influenza B virus genomes in Europe , Africa and Central Asia , improving the global context to study influenza B viruses . We reveal Yamagata-lineage diversity results from co-circulation of two antigenically-distinct groups that also segregate genetically across the entire genome , without evidence of intra-lineage reassortment . In contrast , Victoria-lineage diversity stems from geographic segregation of different genetic clades , with variability in the degree of geographic spread among clades . Differences between the lineages are reflected in their antigenic dynamics , as Yamagata-lineage viruses show alternating dominance between antigenic groups , while Victoria-lineage viruses show antigenic drift of a single lineage . Structural mapping of amino acid substitutions on trunk branches of influenza B gene phylogenies further supports these antigenic differences and highlights two potential mechanisms of adaptation for polymerase activity . Our study provides new insights into the epidemiological and molecular processes shaping influenza B virus evolution globally .
Influenza viruses cause significant morbidity and mortality worldwide and present major challenges for public health . Two types of influenza virus circulate widely in human populations: influenza A and influenza B viruses . While rates of hospitalization and mortality attributed to influenza B are lower than for influenza A subtype A ( H3N2 ) , they were higher than the less virulent seasonal A ( H1N1 ) subtype of influenza A viruses [1] . Influenza B viruses cause epidemics worldwide each year , contributing approximately one third of the global influenza disease burden [2] , and are associated particularly with severe disease in children [1 , 3] . Despite the significance of influenza B viruses to public health , their epidemiological characteristics and their global evolutionary and antigenic dynamics are poorly understood compared to influenza A viruses [4 , 5] . Influenza B viruses are classified into two co-circulating phylogenetically- and antigenically-distinct lineages , named after viruses B/Yamagata/16/88 ( Yamagata-lineage ) and B/Victoria/2/87 ( Victoria-lineage ) that diverged in the 1970s [6 , 7] . The Yamagata- and Victoria-lineages have had a complex epidemiological history since their divergence , co-circulating globally since at least 2002 and often alternating in regional dominance [8] . Disparities from antigenic mismatches between the predominant circulating influenza B virus lineage in a given year and that year’s seasonal influenza trivalent vaccine ( which contains representatives of A ( H1N1 ) , A ( H3N2 ) plus one of the two influenza B virus lineages ) have occurred . Consequently , updated quadrivalent vaccines that contain representative Yamagata-lineage and Victoria-lineage viruses have been recommended [9] . A number of studies have reported the genetic and epidemiological characteristics of influenza B viruses in specific geographic regions [2 , 10–15] yet few have investigated the large-scale evolutionary dynamics of influenza B viruses at the genome-wide level or global scale [16–19] . Nevertheless , existing insights into the evolutionary dynamics of influenza B viruses show they undergo slower antigenic evolution than influenza A viruses [19 , 20] , with genetic changes including nucleotide insertions , nucleotide deletions , and frequent reassortment events between and within lineages , contributing to their continued diversification [16 , 17 , 21 , 22] . Recent analyses have revealed that the polymerase basic 1 and 2 ( PB1 , PB2 ) and hemagglutinin ( HA ) genes of Victoria- and Yamagata-lineage viruses remain as distinct lineages despite high levels of overall reassortment , likely through genomic incompatibility among viral genome segments [17 , 23] . Other differences between the two lineages have been observed; Victoria-lineage viruses appear to undergo more rapid lineage turnover and antigenic drift [18] and persist for longer in local geographic regions before wider dissemination [19] . Despite these advances , there remain substantial unanswered questions about the genomic evolution of influenza B viruses on a global scale , including whether the genetic differentiation observed in HA is mirrored in other less-studied gene segments and the influence of geography on genome-wide viral genetic diversity . Until recently , efforts to address these issues have been hampered by the paucity of globally sampled influenza B virus hemagglutination inhibition ( HI ) data and full-length genome sequences available , particularly from Europe , Africa , Central Asia , and South America . To address this , we used samples from multiple locations worldwide to generate 382 new complete influenza B virus genome sequences . We further compiled the largest and most spatio-temporally-representative dataset of influenza B virus whole genome sequences to date . This dataset included 2 , 651 complete genomes ( 1 , 265 Yamagata- and 1 , 386 Victoria-lineage HA viruses ) sampled worldwide between 1987 and 2015 . We used antigenic cartography and phylogenetic approaches to identify patterns of reassortment , compare the dynamics of antigenic evolution among lineages , and characterize genome-wide demographic histories in geographic regions . We identify substitutions along the trunk branches of the phylogenies for each gene and structurally map changes in the HA and polymerase complex that may contribute to molecular adaptation . Our study shows how the global phylodynamics and epidemiologic interactions of influenza B viruses are shaped by reassortment , genomic compatibility , and differing patterns of antigenic change .
For this study , we sequenced and assembled 382 new , full-length genomes of influenza B viruses collected globally from 2007 to 2013 ( Fig 1 ) . In total , we analyzed all available gene sequence data from over 10 , 000 distinct influenza B viruses sampled from 1987 to 2015 , of which 2 , 651 were complete genomes . Our sequencing efforts increased the total number of complete influenza B genomes by 17% , with the new genomes representing a 44% increase in the number of genomes for the years 2008–2013 ( Fig 1B ) . Crucially , our genomes were sampled from geographic regions under-represented by previous influenza B virus molecular surveillance . Specifically , we increased the number of genomes from Europe ( 20 to 243 genomes ) , Africa ( 11 to 89 genomes ) , Central Asia ( 10 to 37 genomes ) , and South America ( 21 to 31 genomes ) . Our sequencing has therefore substantially improved the global context of influenza B genomic diversity ( Fig 1A ) . One region that remains deficient in influenza B genome sequences is the Indian subcontinent , as assessed by lack of submission to sequence databases , which was previously shown to be an important source of influenza A and B virus diversity [19] . Despite this , our study encompasses the most comprehensive dataset of influenza B complete genomes to date . The Yamagata-lineage has been separated previously into two major antigenically distinct clades ( clade 2 , the B/Massachusetts/02/2012 clade , and clade 3 , the B/Wisconsin/1/2010 clade ) , based on phylogenetic analysis of its HA and neuraminidase ( NA ) genes [24 , 25] . However , it was unknown whether this separation also extended to the other genes . Our analysis demonstrates that this phylogenetic divergence is indeed present across all genes , resulting in each Yamagata-lineage clade comprising a distinct ‘whole genome’ genotype ( Fig 2 , S1 Fig ) . Using molecular clock phylogenetic analysis , we estimated that this whole genome split occurred progressively over a period of approximately 10 years , beginning with the PB1 segment around 1993 ( 95% highest posterior density ( HPD ) 1992–1994 ) ( S2 Fig ) , followed by polymerase acidic protein ( PA ) in 1996 ( 95% HPD 1995–1997 ) , then nucleoprotein ( NP ) , PB2 , HA , NA , non-structural protein 1 ( NS1 ) , and matrix protein 1 ( M1 ) in 2002–2003 ( 95% HPD 2001–2004 ) ( Table 1 ) . While several Yamagata-Victoria inter-lineage reassortment events were apparent after the genome-wide split of Yamagata-lineage viruses into clades 2 and 3 , especially for NA , we observe that after the split of Yamagata-lineage viruses , there is little evidence of substantial reassortment between the Yamagata-lineage clades , with them maintaining their unique genomic constellations for over 12 years ( Fig 2 , S2 Fig ) . In contrast , Victoria-lineage influenza B viruses show evidence of continued reassortment between clades within the Victoria-lineage over time . As a result , we observed multiple co-circulating Victoria clades that do not maintain distinct genome constellations ( Fig 3 , S1 and S3 Figs ) . In particular , we noted considerable inter-clade reassortment between recently circulating B/Brisbane/60/2008 ( clade 1A ) , B/Odessa/3886/2010 ( clade 1B ) , and B/Malaysia/2506/2004 clade viruses . The abovementioned differences in the genome-wide evolutionary patterns between Yamagata- and Victoria-lineage viruses led us to investigate if the genetic differences also extended to the antigenic properties of the viruses , as measured by hemagglutination inhibition ( HI ) data . We compiled available HI measurements and associated HA gene sequences for influenza B viruses sampled between 1987–2013 . We then removed known egg-adapted viruses , resulting in a dataset of 309 Victoria- and 308 Yamagata-lineage viruses with both genetic and antigenic data . We integrated these data under a Bayesian framework [20] to jointly infer the antigenic and genetic relationships of influenza B viruses in two antigenic dimensions ( Fig 4 , S4 Fig ) . Under a Bayesian multidimensional scaling ( BMDS ) model that does not account for variations in virus avidities and serum potencies in the HI assays ( ‘fixed effects , ’ model 7 in [20] ) , the two extant Yamagata-lineage clades appear to experience little antigenic change over time ( S4 and S5 Figs ) with an estimated drift rate slower than the Victoria-lineage , in line with previous observations by Vijaykrishna et al . [18] . However , using a more comprehensive model that does consider these experimental variations ( ‘full model , ’ model 10 in [20] ) , we found no significant difference in antigenic drift rate between the Victoria-lineage and the Yamagata-lineage ( Table 2 ) , in agreement with Bedford et al . [20] . Previous model performance testing indicated that the latter model provided the greatest predictive power and least test error for HI titers [20] , providing further support for influenza B virus lineages experiencing antigenic drift at similar rates . Despite comparable rates of antigenic drift , we observed notable differences in the dynamics of antigenic evolution between the Victoria- and Yamagata- lineages . Around 2005 , the genetically-distinct clades 1A and 1B of the Victoria-lineage emerged , replacing the previously-circulating lineages and subsequently dominating the Victoria-lineage virus population ( Fig 3 ) . While the HA genes of these Victoria-lineage clades are clearly different ( Fig 3 ) , antigenic mapping showed they are not antigenically distinct ( Fig 4A ) . Conversely , the genetically-divergent Yamagata-lineage clade 2 and 3 viruses do exhibit measurable antigenic divergence ( Fig 4B ) . In contrast to the serial replacement of novel antigenic types in the Victoria-lineage viruses ( Fig 4A ) , the two antigenically-distinct clades of the Yamagata-lineage co-circulate globally , alternating in dominance ( nextflu . org/yam/12y/ ) ( S6 Fig ) . However , despite the divergence and counter-cyclical maintenance of Yamagata-lineage clades 2 and 3 over 10 years , recent reports indicate that the incidence of clade 2 viruses has decreased substantially ( https://www . crick . ac . uk/research/worldwide-influenza-centre/annual-and-interim-reports/ ) . Other long-lived Yamagata-lineage clades previously became extinct . In particular , B/Yamanashi/166/98 clade viruses emerged in 1993 ( 95% HPD: 1992–1994 ) , and constituted the predominant circulating Yamagata-lineage clade worldwide until 2002 , when they were replaced by B/Harbin/7/94-like Yamagata-lineage viruses ( Fig 2 ) . Although these two Yamagata-lineage clades were genetically distinct , the B/Harbin/7/94 clade was antigenically similar to the B/Yamanashi/166/98 clade ( Fig 4B ) . Our whole-genome phylogenetic analysis showed that in 2000–2001 ( 95% HPD: April 2000-April 2001 ) the B/Yamanashi/166/98 clade provided the NA gene that became incorporated into the Victoria-lineage ( S3 Fig ) . Subsequently , the global incidence of Victoria-lineage viruses increased dramatically , while the B/Yamanashi/166/98 clade went extinct . This suggests that factors involving other gene segments or differing patterns of reassortment may have influenced influenza B lineage dynamics on a global scale . However , we were unable to investigate this further due to limited availability of genome sequences covering this time period . Given the observed influenza B virus inter-lineage differences in the phylodynamics and patterns of antigenic evolution , we sought to compare levels of natural selection acting on Victoria- and Yamagata-lineage viruses . As selective sweeps are difficult to detect by dN/dS methods , we used ancestral sequence reconstruction to quantify the accumulation of potentially adaptive substitutions in all the major influenza B virus genes ( Fig 5 , S1 Fig , S1 Table ) . We focused on amino acid changes occurring on the ‘trunk’ of the phylogenies , which are less sensitive to varying sampling densities over time that occur due to differences in sequence availability . Substitutions along the trunk represent changes that have fixed in the virus population and are at least neutral or could confer selective advantages that are swept to fixation . We first compared trunk substitutions in Victoria- and Yamagata- lineage HA phylogenies ( S7 Fig ) . Fewer nonsynonymous changes were found along the trunk of the Victoria-lineage HA phylogeny ( mean: 0 . 81; 95% HPD: 0 . 76–0 . 86 nonsynonymous substitutions/year ) than the Yamagata-lineage phylogeny ( mean: 1 . 06; 95% HPD: 0 . 93–1 . 17 nonsynonymous substitutions/year ) ( Figs 2 and 3 ) . Structural mapping of these trunk mutations showed that , in both lineages , the majority of changes were in solvent-accessible residues on the globular head region of HA ( S7 and S8 Figs ) . As expected , these substitutions predominantly occurred within predicted antigenic epitopes in the Yamagata- and Victoria-lineages [26 , 27] ( S7 and S8 Figs ) . Since 2002 and the global re-emergence of the Victoria-lineage [16] , both lineages have experienced trunk substitutions in three residues located in HA1 antigenic epitopes; Yamagata-lineage ( amino acid changes N116K , S150I , N202S ) and Victoria-lineage ( amino acid changes K129N , I146V , N165K ) ( Figs 2 and 3 , S7 Fig ) . Previous experimental work has shown that transitions between influenza antigenic clusters are predominantly associated with substitutions at sites near the receptor-binding site ( RBS ) [28] . We identified four trunk substitutions adjacent to the RBS: V137I , which fixed early in Victoria-lineage HA prior to 1995 ( Fig 3 ) ; residues N150S ( and S150N ) , R162K , and N202S in Yamagata-lineage HA . We identified a smaller number of trunk substitutions in structurally ‘buried’ residues , namely P108S , V179I and V25M1 in Yamagata-lineage HA , with P108A notably a clade 2-defining substitution that fixed early in the Yamagata-lineage clade 2/3 divergence ( Fig 2 ) . Ancestral sequence reconstruction along the Victoria- and Yamagata-lineage HA phylogenies also revealed residues that experienced multiple amino acid replacement and therefore temporary fixation over time ( Figs 2 and 3; S2 Table ) . For Victoria-lineage viruses , two such positions ( T75N/N75K , T129K/K129N ) were solvent-accessible ( exposed ) residues of known antigenic epitopes . For Yamagata-lineage viruses , residue N150S/S150I was in a partially-exposed position within a major antigenic epitope , adjacent to the RBS . Additionally , we observed a number of residues that experienced amino acid substitutions that subsequently reverted back to their ancestral state . Three of these HA reversions ( K48R/R48K in Yamagata-lineage , V146I/I146V and T121I/I121T in Victoria-lineage ) occurred in known antigenic epitopes , while two other reversion residues ( 172 in Victoria- and 179 in Yamagata-lineages ) were not located in or near predicted epitopes ( S2 Table ) . Furthermore , we observed identical substitutions in major antigenic epitopes ( N116K , R149K ) that emerged and became independently fixed in different Yamagata-lineage clades . Both substitutions occurred in the B/Yamanashi/166/98 clade that went extinct around 2002 , and around a similar time , R149K also arose in B/Harbin/7/94 viruses . More recently , N116K became fixed in clade 3 viruses . Finally we observed changes to a given residue that were different depending on the Yamagata-lineage clade . In particular , around the year 2005 , changes at residue 229 were independently fixed in Yamagata-lineage clade 3 ( as G229D ) and clade 1 ( B/Florida/4/2006 clade: G229S ) ; clade 2 Yamagata-lineage viruses however , retained the ancestral amino acid ( 229G ) at this site . Consequently , from 2005–2010 , the Yamagata-lineage comprised three co-circulating populations that varied at position 229 in HA . Applying the same rationale , we estimated the time of emergence of trunk substitutions across the entire genome of Victoria- and Yamagata-lineage viruses ( Fig 5 ) . Over the 20-year period , only one amino acid change , R105K present in contemporary Yamagata-lineage viruses of both clades , fixed in matrix protein ( M1 ) in the global influenza B virus population ( Fig 5B , S1B Fig ) . There was potential co-emergence of substitutions in some gene segments , for example emergence of trunk substitutions in NS1 appeared to coincide with the emergence of substitutions in NA . There was also evidence of temporal ordering of Yamagata-lineage ‘clade-defining’ mutations , which first accumulated in PB1 , followed by PA , and then the rest of the genes ( Fig 5C and 5D ) . To determine whether these early trunk substitutions had potential functional consequences contributing to the clade 2/3 divergence of the Yamagata-lineage , we mapped them onto an influenza B virus polymerase complex structure ( Fig 6 ) . Yamagata-lineage clade 2 and clade 3 viruses accumulated changes in sites where PB1 and PA interact or where polymerase contacts viral RNA ( vRNA ) , respectively . PB1-I357V and PA-I617V substitutions fixed in clade 2 viruses; both residues are positioned at the PB1-PA interface , with PA-617 at a known interaction site with the N-terminus of PB1 critical for PB1-PA binding [29] ( Fig 6A and 6B ) . Differently , PB1-K652R and PB1-H38Y substitutions fixed in clade 3 viruses , both potentially interact with vRNA bound in the polymerase structure [30] ( Fig 6C and 6D ) . Additional substitutions occurred in sites of the polymerase structure not at these interfaces . Around the same time ( 1996 , 95% HPD 1996–1997 ( S1 Table ) ) , K390R and K391R PB1 substitutions emerged in Yamagata-lineage clade 2 and clade 3 viruses , respectively , which are located beside each other and are exposed on the polymerase structure ( S9 Fig ) . Further , three ‘clade-defining’ substitutions that emerged later appeared to be ‘buried’ in the polymerase subunits: PB2-L555M in clade 2 viruses , and PA-V485I and PB1-V66I in clade 3 viruses ( S9 Fig ) . Finally , we sought to determine whether the differences in the molecular evolutionary dynamics of Victoria- and Yamagata-lineage viruses that we observed at the global level were also present at regional scales . Previous studies have focused either on the circulation of influenza B viruses in a specific geographic region [18 , 31] , or have analyzed the global circulation of the HA segment only . Unlike influenza A ( H3N2 ) HA , influenza B HA lineages circulate independently in China , India , and Southeast Asia for long periods of time before spreading elsewhere in the world [19] . Here new data , especially from Europe , enables us to combine these two approaches and analyze whole virus genomes within specific geographic regions: Europe , the United States ( USA ) , Australia and New Zealand ( Oceania ) , and Southern China and Southeast Asia ( SC/SEA ) . Until 2011 , Victoria-lineage viruses experienced selective sweeps across all segments simultaneously in different regions of the world ( Fig 7 ) . However , after 2011 regional differences became apparent , with only viruses in the USA and Europe maintaining this genome segment linkage ( Fig 7A and 7B ) whilst acquisition of the Victoria-lineage NA by Yamagata-lineage viruses in Oceania resulted in NA disassociating from the rest of the Victoria-lineage genome ( Figs 7D and 8D , S10 Fig ) . Regional phylogenies also highlight the persistence of a Victoria-lineage NA gene ( B/Malaysia/2506/2004 clade ) that circulated almost exclusively within SC/SEA since 2003 ( Fig 8C , S10 Fig ) . Throughout this period , viruses from this lineage were sporadically observed in other regions ( Fig 8A , 8B and 8D ) , but did not persist outside of SC/SEA . Victoria-lineage viruses in SC/SEA show greater levels of inter- and intra-lineage reassortment , maintaining genetic diversity in NA , M1 , and HA ( Fig 7C ) . Unlike Victoria-lineage viruses , no major regional differences in the dynamics of genomic diversity were observed for the Yamagata-lineage ( Fig 7E , 7F , 7G and 7H ) . Rather , the accumulation of diversity was associated with the split of the Yamagata-lineage into clades 2 and 3 , with PB1 and PA showing greater accumulation of genetic diversity over time than other genes . Although influenza B virus sampling was more limited , these patterns of Victoria-lineage and Yamagata-lineage virus diversity were also observed for the geographic regions of Africa and the Eastern Mediterranean ( S12 and S13 Figs ) . Whole genome analysis of Victoria-lineage B/Malaysia/2506/2004 clade viruses revealed that they maintained a distinct genomic constellation until 2008–2009 , when they underwent extensive reassortment of all segments except the NA gene ( Fig 3 , S3 Fig ) . The first reassortment event involved replacement of the HA , PB2 , PB1 , PA , and NP genes ( 95% HPD: March 2008-May 2009 ) with those from a globally co-circulating Victoria-lineage clade , the B/Odessa/3886/2010 1B clade . Following this , a subset of clade 3 viruses of the Yamagata-lineage that circulated in multiple geographic regions acquired the Victoria-lineage B/Malaysia/2506/2004 clade NA ( 95% HPD: June 2011-March 2012 ) ( Fig 2 ) . In a separate reassortment event , other viruses of the same Yamagata-lineage clade acquired the NA of the Victoria-lineage B/Brisbane/60/2008 1A clade , indicating a propensity for this Yamagata-lineage clade to replace its NA gene . However , despite this extensive reassortment , viruses containing the B/Malaysia/2506/2004-like NA gene are rarely detected outside of SC/SEA . Conversely , a B/Dakar/10/2012-like NA clade reassortant was observed in many regions of the world , but not in SC/SEA . As influenza A viruses are known to exhibit different dynamics of lineage turnover among regions of the world [32] , we decided to compare lineage turnover of influenza B viruses circulating in different geographic regions . To evaluate lineage turnover , we estimated the average time to most recent common ancestor ( TMRCA ) of contemporaneous viruses at yearly time intervals across the time-scaled phylogenies , which provides a measure of the maximum co-circulating genetic diversity in each year . For Victoria-lineage viruses from 2002–2015 , the average estimated TMRCA is comparable in temperate regions with 4 . 1 years ( 3 . 6–4 . 7 years ) in the USA , 4 . 1 years ( 3 . 7–4 . 8 years ) in Europe , and 3 . 9 years ( 3 . 4–4 . 4 years ) in Oceania . In comparison , the equivalent value for A ( H3N2 ) in the USA and Oceania is approximately 1–2 years [32] , indicating that Victoria-lineage viruses have slower lineage turnover than A ( H3N2 ) viruses . In contrast to the Northern and Southern temperate regions , the genetic diversity of Victoria-lineage viruses in SC/SEA is more constant , with multiple co-circulating clades in this region ( Fig 8C , S10C and S11C Figs ) . These SC/SEA clades of Victoria-lineage are longer-lasting , with an average TMRCA of 5 . 1 years ( 4 . 7–5 . 7 years ) . In contrast , the average TMRCA estimates for Yamagata-lineage viruses are similar at 6 . 5 ( 5 . 9–7 . 1 ) in the USA , 7 . 2 ( 6 . 5–7 . 8 ) years in Europe , 6 . 3 ( 5 . 7–6 . 9 ) in SC/SEA , and 6 . 7 ( 6 . 1–7 . 3 ) years in Oceania , highlighting that a similar level of diversity of Yamagata-lineage viruses exists throughout the world due to the co-existence of the two extant Yamagata-lineage clades .
Here we report the global full-genome molecular epidemiology , antigenic evolution , and phylodynamics of influenza B viruses , putting this important human pathogen into a similar context as in analysis of influenza A viruses . Results were obtained from viruses collected between 1987–2015 , including the complete genomes of 2 , 651 unique viruses . Full virus genome analysis show that in contrast to influenza B Victoria-lineage viruses that undergo reassortment between clades , Yamagata-lineage viruses form two persisting co-circulating clades that genetically diverge across the whole virus genome . Yamagata-lineage clade 2 and clade 3 virus populations have a prolonged absence of intra-Yamagata-lineage reassortment , resulting in the long-term maintenance of separate genome constellations . Moreover , estimated timings of this split reveal that the divergence of Yamagata-lineage viruses began much earlier than previously suggested by analysis of HA and NA phylogenies alone . Evolutionary divergence into two distinct genetic clades began with PB1 over twenty years ago , followed by PA and then the remaining genes . Similar observations were made regarding the maintenance of distinct Yamagata- and Victoria-lineages in PB2 , PB1 , and HA genes , potentially driven by “reassortment incompatibility” [17 , 33] . This idea has been tested and supported recently by in vitro studies [23] . However , unlike the separation between Yamagata- and Victoria-lineage viruses , which is currently restricted to a PB2-PB1-HA complex , the differentiation between the clades of the Yamagata-lineage is maintained across all genes . Interestingly , we observed greater Yamagata/Victoria inter-lineage reassortment for NA and NP than Yamagata intra-lineage reassortment . However , as there are fewer whole-genome sequences than individual HA and NA genes , it is possible that reassortment events between Yamagata-lineage clades remain undetected at low frequencies or in poorly sampled regions of the world . The co-divergence of the Yamagata-lineage genes relates to experimental studies that suggest that coevolution of PB1 with other influenza genes is important for virus fitness for influenza A viruses [34 , 35] . Specifically , evidence suggests that optimal PB1-PA interaction is important for efficient polymerase activity and is essential for in vitro influenza A virus viability [34] . This is underpinned by an influenza A polymerase model proposing that initial binding between PB1 and PA is necessary for efficient transport to the nucleus and subsequent interaction with PB2 to assemble the polymerase complex [36 , 37] . PB1 has also been associated with co-selection of virus-matched HA and NA glycoproteins , with reduced virus growth and antigen yield being observed when miss-matched in vitro [33 , 35 , 38] . Here we observe mutations fixed on the Yamagata-lineage PB1 and PA phylogeny trunk branches at two amino acids ( PB1-I357V and PA-I617V ) in contact areas of PB1 and PA for Yamagata-lineage clade 2 viruses , one of which was previously functionally characterized [29] , and two amino acids ( PB1-K652R and PB1-H38Y ) associated with PB1/vRNA interaction for Yamagata-lineage clade 3 viruses . The functional significance of these requires testing; however , these data suggest that adaptation of influenza B virus fitness through polymerase activity can occur by at least two mechanisms . Work here also highlights the importance of model selection for antigenic drift analyses and supports the view that Victoria and Yamagata lineages have comparable rates of antigenic drift [20] in contrast to differences in estimated Influenza B virus antigenic drift rates from previous reports [18] . Detecting selection in influenza viruses is challenging when using traditional statistical tests based on dN/dS ratios , as such ratios are sensitive to recurrent selection at individual sites [39] . Further , adaptations that arise from egg [40 , 41] and cell-culture [42 , 43] passaging often appear as recurring mutations , also confounding analyses , whereas analyzing the phylogenetic distribution of mutations can assist in the detection of positive selection . Characterizing amino acid substitutions that occur along the trunk of Yamagata- and Victoria-lineage gene phylogenies , identifies changes that become fixed in the virus population across seasons [44 , 45] , and are thus less likely to be passage artefacts . Notably , we did not detect trunk substitutions at HA residues 196/197 or 198/199 , which are known to be highly variable and associated with adaptation to propagation in eggs [40 , 41] . The HA gene ( and encoded glycoprotein ) has been the focus of much influenza research , owing to its role in immune escape . A recent study on the global circulation patterns of influenza HA genes noted the persistence of influenza B virus clades , particularly Victoria-lineage clades , which circulated exclusively in China and India for longer periods of time before migrating to other regions [19] . Our whole-genome analysis indicates that geographical constraint extends to other genes of Victoria-lineage viruses , notably with greater levels of genetic diversity for NA , M1 , and NS1 detected in SC/SEA compared to other geographic regions . It remains unclear how the spatial structure of Victoria-lineage diversity is maintained or why Yamagata-lineage viruses do not also show this spatial pattern . Based on the incomplete availability of influenza B virus genome sequences , particularly from the Indian subcontinent , the existence of other Yamagata- or Victoria-lineage clades may have gone undetected in our analysis . Further , we cannot exclude the possibility that seemingly geographically-constrained virus populations have gone undetected in other regions , for example in Europe outside of our sampling window . Nevertheless , high levels of intra- and inter-lineage reassortment in the Victoria-lineage are seen and considerably affect genetic diversity , with multiple distinct genotypes generated through reassortment events . In particular , introductions of the SC/SEA Victoria-lineage NA into other geographic regions was associated with reassortant viruses containing the Yamagata-lineage HA and genes ( Fig 2 ) . As Yamagata-lineage viruses have been associated with a slightly older age of infection [10 , 13 , 18] and associated with more frequent air travel [19] , this may contribute to the global migration of these reassortant viruses . Analysis of Victoria- and Yamagata-lineage viruses shows differences in modes of antigenic evolution . Structural mapping of amino acid changes in HA confirmed the genetic drift estimates , as the accumulation of adaptations in antigenically-relevant sites in each lineage was comparable . The majority of phylogeny trunk substitutions in influenza B HA appear in the globular head and do not map to the stalk region of HA . Whereas Victoria-lineage viruses experience antigenic drift and turnover of antigenically-distinct viruses , the genetic and antigenic bifurcation of Yamagata-lineage viruses has enabled these viruses to alternate between two antigenic types over time . This provides a mechanism for generating antigenic novelty , as previously proposed [46] . This model is supported by the amino acid reconstruction analysis here , as two substitutions at residues located near the RBS ( sites 150 and 202 ) accumulated along the trunk of Yamagata-lineage clade 3 , but not in clade 2 , potentially affecting antigenicity . The emergence and co-existence of two major antigenic Yamagata-lineage clades in a region has implications for the epidemiological dynamics of influenza B viruses . For example , Yamagata-lineage viruses dominated influenza B viruses in Malaysia in 2013 after a Victoria-lineage dominated season in 2012–2013 . However , in 2014 the Yamagata-lineage continued to dominate in the influenza B virus population , through a shift from clade 2 to clade 3 viruses [13] . This shift in patterns of dominance supports the idea that essentially three ‘lineages’ of influenza B virus co-circulated , with distinct genotypes and antigenicity . Consequently , the persistence of two antigenically-distinct Yamagata-lineage clades may complicate vaccine virus selection . In contrast , we found that Victoria-lineage clade 1a and clade 1b not only genetically reassort , but also occupy the same antigenic dimensions in antigenic map-space , suggesting the WHO-proposed distinction of contemporary Victoria-lineage viruses may not be antigenically relevant . The future coupling of influenza B virus whole genome sequencing and antigenic mapping may well help in global vaccine selection and development of new immunization strategies . The additional whole-genome sequencing data and measurements of antigenic properties of HA presented here , particularly from under-sampled geographic regions , contributes to ongoing public health surveillance of influenza viruses . Our findings provide a better understanding of the interplay of epidemiological , immune-driven , and molecular factors driving the evolution and spread of influenza B viruses worldwide .
Samples ( specimens , clinical samples , or virus isolates ) were received by the WHO Collaborating Centre ( WHO CC ) in London ( The Crick Institute , formerly the MRC National Institute for Medical Research ) from WHO National Influenza Centers ( NICs ) and taken with informed consent obtained in each country as laboratories within the WHO Global Influenza Surveillance and Response System ( GISRS ) for the purposes of global surveillance of influenza under the WHO Global Influenza Program . Samples were anonymized prior to sharing with the WHO CC for influenza B genomic RNA extraction and Institutional Review Board review was not applicable . Samples were collected between 2007 and 2013 from 55 countries across Europe , Africa , the Middle East , Asia , and South America . Samples for extraction were chosen based on lack of recovery of virus ( clinical specimens ) and unusual profiles emerging from HI assays with a panel of post-infection ferret antisera , along with a representative number of viruses showing ‘normal’ HI profiles . Amplification was performed using the SuperScriptIII One-Step RT-PCR system with Platinum Taq DNA High Fidelity polymerase ( Invitrogen ) in two reactions . Each reaction contained 25μl Reaction Mix ( 2x ) , 17μl DNase/RNase-free water , 1μl of each primer ( 10μM ) , 1μl SuperScriptIII RT/Platinum Taq High Fidelity and 5μl of the template RNA . Primers used for the HA , NP , NA , MP , and NS genes were: FluB-S1-F ( 5’ GCC GGA GCT CTG CAG ATA TCA GCA GAA GCA 3’ ) and FluB-S1-R ( GCC GGA GCT CTG CAG ATA TCA GTA GWA RYA A 3’ ) . Primers used for the polymerase complex genes ( PB2 , PB1 , PA ) were: FluB ( 555 ) -L1-F ( 5’ CTG AGT CCG AAC ATT GAG AGC AGA AGC G 3’ ) and FluB ( 555 ) -L1-R ( 5’ CTG AGT CCG AAC ATT GAG AGT AGA AAC AC 3’ ) [47] . The cycling conditions were 42°C for 15 min , 55°C for 15 min , 60°C for 5 min , 96°C for 2 min , and then 5 cycles ( 94°C for 30 s , 45°C for 30 s , slow ramp ( 0 . 5°C /sec from 45°C to 66°C ) and 68°C for 3 min ) , followed by 35 cycles ( 96°C for 30 s , 66°C for 30 s , and 68°C for 3 min ) and finally 68°C for 5 min with subsequent examination of amplicons by agarose gel electrophoresis . Amplicons were pooled and sequenced on Illumina MiSeq or HiSeq 2000 platforms using the paired-end 150bp technology . The resultant reads were quality-controlled using QUASR version 7 . 01 [48] to remove primer sequences , trim low-quality bases from the 3’-ends of reads until the median Phred-scaled quality was 35 and filter reads shorter than 145bp . All raw sequencing reads are available in the European Nucleotide Archive ( ENA ) under study accessions PRJEB19198 and PRJEB2261 . Genomes were generated using de novo assembly and reference-based mapping methods . In brief , quality-controlled reads were de novo assembled using the SPAdes genome assembler version 2 . 4 . 0 [49] with kmer size 127 and minimum contiguous sequence ( contig ) size of 300 . Resulting contigs were arranged by genomic segment and filtered to retain those covering at least 70% of the open reading frame for each segment . In the case where multiple contigs were assembled for a segment , a custom Python script was used to estimate the relative abundance of each contig in the reads ( i . e . to determine composition of variants ) and retain the majority variant . For reference-based mapping , unique references were selected for each sample by performing a BLAST search on a subset of the reads and retaining the best match for each segment . Reads were mapped against the reference sequences using SMALT version 0 . 5 . 0 [50] , and consensus sequences generated using SAMtools version 0 . 1 . 8 [51] and QUASR version 7 . 01 [48] . Sequences generated in this study are available in GISAID under accession numbers listed in S3 Table . All available influenza B virus gene segment sequences , excluding artificial recombinant and laboratory-generated variants , were downloaded from the NCBI Influenza Virus Resource ( IVR ) [52] and GISAID ( http://gisaid . org ) repositories on 28 August 2015 . Acknowledgement of the sources of the GISAID sequences is given in S4 Table and accession numbers of GenBank sequences are listed in S5 Table . After duplicate samples and sequences containing less than 70% of the segment coding sequence were removed , the downloaded sequences were combined with the 413 genome sequences generated for this study ( representing 382 unique viruses ) , resulting in a dataset containing 2651 unique , complete genome sequences , ( from 2992 PB1 , 3090 PB2 , 3012 PA , 9167 HA , 3178 NP , 6608 NA , 3403 MP , and 5159 NS sequences ) sampled worldwide between 1987 and 2015 . Separate alignments were constructed for the longest coding region of each segment ( PB2 , PB1 , PA , HA , NP , NA , M1 , NS1 ) in AliView version 1 . 17 . 1 [53] . To reduce sampling bias from over-represented regions in the time-resolved phylogenetic reconstructions , we downsampled epidemiologically-linked isolates while maintaining phylogenetic structure , temporal range and spatial distribution . Maximum likelihood ( ML ) phylogenies for each segment were estimated using RAxML version 7 . 8 . 6 [54] under a general-time reversible ( GTR ) nucleotide substitution model with gamma-distributed rates to represent among-site heterogeneity . Clade confidence was estimated by bootstrapping with 1 , 000 pseudo-replicates . Trees were visualized , rooted to the oldest virus and colour-coded by lineage and clade using FigTree version 1 . 4 . 2 ( http://tree . bio . ed . ac . uk/software/figtree/ ) . The resulting phylogenetic trees were inspected by linear regression and residual analysis using TempEst v1 . 4 [55] to identify incorrectly dated or anomalous sequences , which were subsequently removed from the alignments . Molecular clock phylogenies were inferred for each gene segment using the Markov chain Monte Carlo ( MCMC ) method implemented in BEAST version 1 . 8 . 0 [56] . Separate Victoria- and Yamagata-lineage phylogenies were inferred for the PB2 , PB1 , and HA genes . For all runs , the SRD06 nucleotide substitution model [57] was used , along with a strict molecular clock , as suggested by the linear regression analysis , and a Bayesian Skyride coalescent prior [58] . At least two MCMC chains were run for 200 million states , and combined with a 10% burn-in and sampling every 40 , 000 states . Mean pairwise diversity measures and 95% highest posterior densities across 9 , 000 trees were inferred for viruses from each major geographic region in yearly time intervals using PACT ( http://bedford . io/projects/PACT/ ) . Amino acid substitutions along the HA phylogenies were inferred using ‘renaissance’ counting ancestral reconstruction methods [59 , 60] . The ‘trunk’ branches of each phylogenetic tree were defined by tracing from the most recent contemporaneous samples back to the oldest . Nonsynonymous substitutions along the trunk lineage were calculated in year time intervals to determine the mean nonsynonymous substitutions/year count and 95% highest posterior densities across a posterior set of 1000 trees . Viruses were categorized into major Yamagata- and Victoria-clades , as previously reported in WHO influenza centre reports for HA and NA genes ( https://www . crick . ac . uk/research/worldwide-influenza-centre/annual-and-interim-reports ) , from the ML and time-resolved phylogenies where viruses grouped together in well-supported clades ( bootstrap value >60% and/or posterior probability >0 . 6 ) . Each gene was assigned to one of the defined clades to generate a complete genotype for each sample . Phylogenetics trees were annotated with resulting genotypes and visualized in R using the ggtree package [61] . Data analysis and visualization scripts are available in Github repository https://github . com/pclangat/global-fluB-genomes . We compiled HI measurements and HA sequence data , which were previously published [20] or collected by the WHO Collaborating Centre ( WHO CC ) in London . Known egg-adapted viruses were removed , resulting in a final HI dataset of 309 Victoria- and 308 Yamagata-lineage viruses isolated from 1988 to 2013 . We implemented a Bayesian multidimensional scaling ( BMDS ) cartographic model to jointly infer antigenic and phylogenetic relationships of the viruses as previously described [20 , 62] . Briefly , MCMC was used to sample virus and serum locations in two antigenic dimensions , as well as virus avidities , serum potencies , MDS precision , and virus and serum location precisions , using an empirical tree distribution of 1 , 000 posterior trees inferred for HA sequences separately ( as detailed above ) . Two antigenic dimensions were specified based on previous findings that two-dimensional models provide the best predictive power for antigenic mapping of influenza B virus [20] . MCMC chains were run for 500 million states with sampling every 200 , 000 states with a 10% burn-in , and checked for convergence in Tracer v1 . 6 ( http://tree . bio . ed . ac . uk/software/tracer/ ) . We obtained a total of 2 , 000 trees from which the maximum clade credibility tree was summarized in TreeAnnotator v1 . 8 . 2 . We estimated the rate of antigenic drift for each lineage , by calculating the mean Euclidean distance in antigenic units ( AU ) of all antigenic map locations at yearly time intervals from the inferred phylogenetic root . From this time series of Euclidean distances , we estimated the rates of antigenic drift ( in AU/year ) using linear regression . 95% highest posterior density ( HPD ) estimates were used to measure the statistical uncertainty in these drift rate inferences from the posterior sample of trees . Source data , including BEAST input XML files , HI tables , and output trees are available in Dryad repository https://doi:10 . 5061/dryad . s1d37 [64] . Amino acid substitutions occurring along the trunk of each lineage were visualized on the crystal structures of the HA trimers for viruses of the Yamagata-lineage B/Yamanashi/166/98 ( PDB ID: 4M40 , [63] ) and Victoria-lineage B/Brisbane/60/2008 ( PDB ID: 4FQM , [27] ) , and influenza B virus PB2-PB1-PA polymerase complex bound to viral RNA ( PDB ID: 5MSG , [30] ) using PyMOL Molecular Graphics System , Version 1 . 7 . 6 . 0 , Schrödinger , LLC . Structural features were mapped as described in S8 Fig . | Influenza B viruses cause roughly one third of the global influenza disease burden . However , many important questions regarding the global-scale molecular epidemiology and evolutionary dynamics of influenza B virus have yet to be comprehensively addressed compared to influenza A virus . This is in part due to limited globally-sampled genomic data . We improved the availability of influenza B virus data by sequencing over 350 full genomes , fillings gaps from under-sampled regions by as much as 12-fold . Using a dataset of over 2 , 500 influenza B virus genomes , we show major differences in the genome-wide evolution , molecular adaptation , and geographic spread between the two major influenza B lineages . These findings have implications for vaccine design and improve our understanding of influenza virus evolution . | [
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] | 2017 | Genome-wide evolutionary dynamics of influenza B viruses on a global scale |
Chikungunya virus ( CHIKV ) has been detected sporadically since the 1950s and includes three distinct co-circulating genotypes . In late 2013 , the Asian genotype of CHIKV was responsible for the Caribbean outbreak ( CO ) that rapidly became an epidemic throughout the Americas . There is a limited understanding of the molecular evolution of CHIKV in the Americas during this epidemic . We sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014–2015 Caribbean/Americas epidemic . Our comprehensive phylogenetic analyses estimated the epidemic history of the Asian genotype and the recent Caribbean outbreak ( CO ) clade , revealed considerable genetic diversity within the CO clade , and described different epidemiological dynamics of CHIKV in the Americas . Specifically , we identified multiple introductions in both Nicaragua and Florida , with rapid local spread of viruses in Nicaragua but limited autochthonous transmission in Florida in the US . Our phylogenetic analysis also showed phylogeographic clustering of the CO clade . In addition , we identified the significant amino acid substitutions that were observed across the entire Asian genotype during its evolution and examined amino acid changes that were specific to the CO clade . Deep sequencing analysis identified specific minor variants present in clinical specimens below-consensus levels . Finally , we investigated the association between viral phylogeny and geographic/clinical metadata in Nicaragua . To date , this study represents the largest single collection of CHIKV complete genomes during the Caribbean/Americas epidemic and significantly expands our understanding of the emergence and evolution of CHIKV CO clade in the Americas .
Chikungunya virus ( CHIKV ) belongs to the Alphavirus genus in the Togaviridae family and is an arthropod-borne virus spread by Aedes mosquitoes . CHIKV has a positive-sense single-stranded RNA genome that is 11 . 8 kb in length and encodes both a nonstructural and a structural polyprotein that are separated by a subgenomic promoter . Although CHIKV causes an acute febrile illness with clinical manifestations similar to other co-circulating mosquito-borne viral diseases , such as dengue , CHIKV infection may be followed by months or years of sequelae including rheumatic disease and debilitating joint pain [1] . The first reported outbreak of chikungunya occurred in eastern Africa during 1952–1953 [2–4] . CHIKV was detected sporadically in limited regions in Africa and Asia until the early 2000s [5–8] . Three distinct genotypes of CHIKV have been identified: West African , Asian , and East/Central/South African ( ECSA ) [9] . In 2005 , the CHIKV ECSA genotype reemerged and caused multiple explosive epidemics across Africa , Asia , and the Indian Ocean , which then subsided [10–13] . In late 2013 , the CHIKV Asian genotype was introduced into the Americas and rapidly spread through numerous Caribbean , South American , and Central American countries via Aedes aegypti mosquitoes [14–16] . There is a growing interest in understanding the epidemiology and molecular evolution of CHIKV during the 2014–2015 epidemic in different regions of the Americas . In Nicaragua , the first imported case was reported in July 2014 , and autochthonous cases were reported since September 2014 [17] . The introduction of CHIKV into Nicaragua was followed by a small epidemic wave in 2014–2015 and a much larger second wave in 2015–2016 . In North America , chikungunya infections were reported in travelers returning from Latin America and the Caribbean , with limited autochthonous transmission of CHIKV reported in Florida in July 2014 [18] . Following our recent effort of complete genome sequencing of CHIKV from the Americas [19] , we strategically collected and sequenced another 185 complete genomes using next-generation sequencing methods representing multiple geographic locations , especially Nicaragua in Central America and Florida in the United States , during the 2014–2015 Caribbean/Americas epidemic . Here we reconstructed the evolutionary history of the CHIKV Asian genotype and described the different epidemiological dynamics of CHIKV in Nicaragua and Florida . We also investigated the association between CHIKV sequence variation with clinical features of the infected patients . Furthermore , we determined the statistically significant amino acid substitutions of CHIKV from the Caribbean/Americas epidemic , as well as assessed minor variants and intra-host diversity that were observed in these strains through deep-sequencing . To our knowledge , this dataset represents the largest single collection of CHIKV complete genomes from the Caribbean/Americas epidemic and augments our understanding of the emergence and evolution of CHIKV in the Americas .
This study was based on three collections of CHIKV samples: Viral RNA , which was shipped to the JCVI from each of the participating institutions , was subjected to sequence-independent single-primer amplification ( SISPA ) [20] . In cases where sequence-independent amplification failed , a sequence-specific overlapping primer amplification strategy was used to generate the entire genome of CHIKV . The primer sequences used to amplify the required regions in each sample are reported in the respective GenBank sequence records . Barcoded libraries were pooled and adaptors were ligated following the manufacturer's instructions ( Illumina ) . Libraries were sequenced on the Illumina MiSeq ( 2x300 bp Paired End ( PE ) ) instrument . After sequencing , barcoded reads from each sample were deconvoluted and trimmed to eliminate low-quality regions , SISPA hexamer primers and barcode sequences . Trimmed reads were subjected to de novo assembly with the clc_novo_assemble program in the CLC Bio software suite . The resulting contigs were queried against a custom full-length CHIKV reference database to determine the closest reference . Sequences were then mapped to the selected reference for each sample using the clc_ref_assemble_long program in the CLC Bio suite . A final mapping of all next-generation sequences to the updated reference sequences was performed with CLC Bio’s clc_ref_assemble_long program . When sufficient read data were available , reference sequences were extended to capture as much of the untranslated regions ( UTRs ) as possible . Assemblies were manually validated and curated prior to annotation with VIGOR [21] . VIGOR was used to predict genes and detect any potential sequencing errors . The annotation was subjected to manual inspection and quality control before submission to GenBank . All sequences generated as part of this study were submitted to GenBank as part of Bioproject PRJNA294670 . We combined the newly-determined 185 complete CHIKV genome sequences together with 271 CHIKV genome sequences available in GenBank ( http://www . ncbi . nlm . nih . gov/genbank/ , as of October 1 , 2016 ) and generated a dataset with a total of 456 complete genome sequences of CHIKV . We excluded vaccine strains , synthetic recombinant strains , and other non-wildtype sequences . Because of the large number of insertion/deletion regions in the UTRs , these regions were excluded from the phylogenetic analyses ( 456_CDS ) . Sequences were aligned using the MUSCLE program in MEGA 6 . 0 with manual adjustment [22] . Potential recombination in the 456_CDS dataset was screened for using seven methods ( RDP , GENECONV , Chimaera , MaxChi , SiScan , 3Seq , and BootScan ) implemented in the Recombination Detection Program version 4 . 46 ( RDP4 ) [23] . Sequences having p-values less than 1×10−4 in at least two methods were reported as evidence of recombination , and the one predicted recombinant strain ( KJ679578 ) was removed from further phylogenetic analyses . Nucleotide and amino acid similarities across complete CDS and in each gene were also calculated . The individual amino acid sites under diversifying selection were detected by three different codon-based maximum likelihood methods: single most likely ancestral reconstruction ( SLAC ) , fixed effects likelihood ( FEL ) , and IFEL ( similar to FEL , but selection is only tested for along internal branches of the phylogeny ) using the Datamonkey webserver ( www . Datamonkey . org ) [24–26] . A significance cutoff of p<0 . 05 was used for all methods . A maximum likelihood ( ML ) phylogenetic tree of complete coding sequences of CHIKV was inferred in MEGA 6 . 0 [27] . A general time reversal ( GTR ) nucleotide substitution model with a gamma distribution of among-site rate variation and a proportion of invariable sites ( GTR+Γ+I ) was selected as the best-fit model by Modeltest in MEGA 6 . 0 and used in the tree inference . The robustness of the ML tree was assessed by bootstrap analyses of 1000 pseudo-replicates , and the phylogenetic tree was midpoint rooted . Due to the long branches of three genotypes ( historical West African , ECSA , and Asian ) suggested by our previous ML phylogeny and the very limited number of sequences in the West African genotype ( n = 11 ) , the phylogenetic trees of the ECSA and Asian genotypes were separately reconstructed using the Bayesian Markov Chain Monte Carlo ( BMCMC ) method available in the BEAST V1 . 8 . 2 package ( http://tree . bio . ed . ac . uk/software/BEAST ) [28] . Two sequences from the ECSA genotype , STMWG01/2011 and Angola/m2022/1962 , were excluded from the BEAST analysis because of potential laboratory contamination or mutations that occurred during passaging [19] . Maximum likelihood phylogenies for the ECSA dataset and Asian dataset were also constructed using the methods described above and rooted by the oldest sequence in the dataset . To incorporate temporal information , each sequence in both datasets was assigned a date of origin that corresponded to the time of collection . To better estimate the spatial diffusion of the viruses , sequences were grouped by the different regions where they were collected . For 442 ( out of 456 ) sequences with detailed travel history information , we assigned the regions based on the country of origin ( Table 1 ) . In the discrete phylogeographic analyses , the GTR+Γ+I substitution model selected by Modeltest in MEGA 6 . 0 was used under a relaxed molecular clock ( uncorrelated lognormal , UCLN ) . The demographic model , Gaussian Markov Random Fields ( GMRF ) Bayesian skyride coalescent tree prior was employed in the BEAST runs . In each BEAST run , to ensure convergence of effective sampling size parameters ( ESS ) over 200 , a Markov chain Monte Carlo ( MCMC ) was run for 500 million generations , and a 10% burn-in was removed . The results were assessed using the Tracer program v1 . 6 . 0 ( http://tree . bio . ed . ac . uk/software/tracer ) to ensure that convergence was achieved . The posterior distribution of BMCMC trees was summarized as the maximum clade credibility ( MCC ) tree and generated using TreeAnnotator v1 . 8 . 2 ( available in BEAST v1 . 8 . 2 package ) , with the first 10% of trees removed as burn-in . MCC trees were visualized using FigTree v1 . 4 . 3 ( http://tree . bio . ed . ac . uk/software/FigTree ) . Host demographic information was available for all Nicaraguan samples , and clinical information was available for 95 of 108 of the Nicaraguan samples . To determine whether there was phylogenetic clustering by age or sex of the patient , virus sequences were grouped into 4 classes according to host age ( <5 years , 5–10 years , 11–18 years , and >18 years old ) or into 2 classes by sex respectively . Similarly , each patient was classified as either positive or negative for each clinical symptom individually . The clinical symptoms analyzed included history of: dehydration , headache , vomiting , myalgia , arthralgia , fever , rash , abdominal pain , and retro-orbital pain . The strength of association between the phenotypic features described above and the CHIKV phylogeny was determined using two phylogeny-trait association statistics , the parsimony score ( PS ) and the association index ( AI ) tests , both of which were implemented in the Bayesian tip association significance testing ( BaTS ) program [29] . A significance cutoff level of p<0 . 05 was used in both statistics . A null distribution of these statistics was determined using the posterior distribution of Nicaragua-only phylogenetic trees obtained from the BEAST package described above . The posterior distribution of Caribbean-only phylogenetic trees generated by BEAST was also used to assess the strength of geographic clustering in the data by BaTS . Each sequence in the Caribbean clade was therefore assigned to a character state by its sampling country . For the sequences collected in North America with detailed travel history information , we assigned the states based on the travel history . The coding regions for the nonstructural and structural polyprotein coding regions were extracted from the same nucleotide-based multiple sequence alignments that were used for the phylogenetic tree reconstruction . Each of the coding regions was then translated into amino acid sequences using standard codon translation tables . Strains were annotated with either their respective clade assignments from the phylogenetic tree or their clinical metadata and analyzed separately . An iterative version of the Metadata-driven Comparative Analysis Tool for Sequences ( Meta-CATS ) algorithm was then applied to identify amino acid positions showing statistically-significant skewing in the residue distributions either between phylogenetic clades or clinical metadata [30] . The resulting amino acid positions in the nonstructural and structural polyproteins were converted to mature peptide positions using the ViPR database ( www . viprbrc . org ) [31] . The publicly available E1-E2 heterodimeric structure for the CHIKV E1-E2 proteins was downloaded from the RCSB ( PDB: 3J2W ) [32 , 33] . UCSF Chimera software was used to navigate and visualize the three-dimensional protein structure ( PDB: 3J2W ) [34] . Deep sequencing analysis was performed on all sequences to identify the minor variants in the sequence population . This involved generating a consensus sequence from all sequence reads for each sample using the CLC mapping assembly program ( clc_ref_assemble_long ) and then mapping all high-quality trimmed reads sequenced from the sample against its own consensus sequence . The annotated consensus was used to provide biological context and to determine whether the minor variation resulted in a synonymous or a nonsynonymous change , and whether any nonsynonymous changes were categorized as either conservative or non-conservative . To find statistically-significant variations in the population , all forward and reverse reads covering each position were checked and a statistical model using a binomial distribution was generated to ensure that coverage at each base position was above a specific threshold ( 3% ) with a 95% confidence interval , followed by Bonferroni correction for multiple-hypothesis testing . Positions where insufficient coverage existed to provide 95% confidence of a minor allele resulted in the position not being reported in the output . The significant variations existing in the sequencing reads were then included in an automated report against the consensus sequence of the same strain to obtain the percentage of minor alleles in the population .
Samples for this study from the three collections are described in Materials and Methods . For the 108 samples obtained from studies in Nicaragua , final assemblies resulted in an average of 20 , 953 reads per sample , with an average coverage of 348X ( range: 60X-1 , 079X ) . Final assembly of 67 complete genomes from the strains provided by the Florida BPHL indicated an average of 15 , 662 reads per sample , with an average coverage of 271X ( range: 43X-745X ) . From the NYSDOH , sequencing of 10 strains resulted in an average of 14 , 559 reads per sample , with an average coverage of 254X ( range: 180X-513X ) . Together , these new complete genome sequences substantially increase the number of complete genome sequences available in GenBank from the Asian genotype and allow for more in-depth sequence analyses . In order to maximize the reliability of downstream phylogenetic analyses , our first step was to identify and remove any sequences that displayed evidence of recombination . RDP results suggested that one of 456 coding sequences , CHIKV_STMWG02 , displayed evidence of potential recombination and was consequently excluded from further phylogenetic analysis . The CHIKV genome was highly conserved across all samples , with an average nucleotide similarity of 96 . 20% and an average amino acid similarity of 98 . 24% . Nonstructural genes ( NSP1 , NSP2 , NSP3 , and NSP4 ) showed similar levels of heterogeneity as structural genes ( Capsid , E3 , E2 , 6K , and E1 ) , with the average nucleotide similarity ranging from 95 . 66% ( NSP3 ) to 96 . 74% ( NSP2 ) , and the average amino acid similarity ranging from 95 . 51% ( 6K ) to 99 . 18% ( NSP2 ) . NSP2 was the most conserved gene across the genome as a whole . We then wanted to gain a better understanding of the role that selection pressure played in the sequence diversity within this collection . As such , we investigated the aligned sequences for individual amino acid sites under diversifying selection ( positive selection ) by three methods ( see Materials and Methods ) , using a significance level of p<0 . 05 . Nonstructural protein NSP1 site 148 was identified as being under diversifying selection by all three methods , whereas no codons in the structural polyprotein were detected to be under diversifying selection by more than one method . To gain a better understanding of the evolutionary relationships between our collection of CHIKV sequences and those that had previously been generated , we constructed a maximum likelihood phylogenetic tree of all available sequences ( Fig 1 ) . This global CHIKV phylogeny revealed the presence of the three major genotypes of CHIKV West African , ECSA , and Asian genotypes that have spread around the globe , consistent with previous studies [19 , 35 , 36] . The recent Indian Ocean outbreak ( IO ) and Caribbean outbreak ( CO ) form monophyletic clades that are descendants of the ECSA and Asian genotypes , respectively . Our newly-sequenced strains collected from patients in Nicaragua , Florida ( with recent travel history to the Caribbean ) , and New York after 2014 ( also with recent travel history to the Caribbean ) fell into the CO clade . Four historical strains collected from New York during 2006–2008 ( with recent travel history to India , La Reunion , and Sri Lanka ) fell into the IO clade , while one strain collected from a patient in New York in 1995 ( with travel history to Central Africa Republic ) belongs to the ECSA genotype but not the IO clade ( Fig 1 ) . The long branch lengths of the ECSA and Asian genotypes in the CHIKV global phylogeny suggest that they have been circulating in Africa and Asia for many years prior to being detected or sampled . We analyzed the Asian ( Fig 2 ) and ECSA genotypes ( S1 Fig ) separately to better understand their evolutionary dynamics and to more accurately reconstruct their epidemic history . We then reconstructed a time-scaled MCC phylogenetic tree focusing on the Asian subtree to provide a finer level of granularity regarding the relationships between historical strains within the Asian genotype and the sequences isolated during the Caribbean/Americas epidemic ( Fig 2 ) . This tree provides estimates of temporal signal , with the CO clade being well-supported by a Bayesian posterior possibility ( BPP ) of 1 and a bootstrap value of 96% in a maximum likelihood tree . The mean evolutionary rate of the entire Asian genotype was estimated to be 4 . 33E-4 substitutions/site/year , with a 95% highest posterior density ( HPD ) between 3 . 54E-4 and 5 . 15E-4 substitutions/site/year . The time to the most recent common ancestor ( tMRCA ) of the Asian genotype was estimated as November 1957 , with a 95% HPD between July 1956 and December 1958 . Our analysis suggests that the Asian genotype has circulated in mainland southeast Asia since the 1950s and then migrated to the surrounding islands around 1980 ( state probabilities over 0 . 9 , marked by asterisks in Fig 2 ) . These viruses continued to spread across the Pacific ( state probabilities over 0 . 5 but less than 0 . 9 ) and Caribbean islands ( state probabilities over 0 . 9 , marked by asterisks ) from 2008–2009 prior to causing the epidemic in the Western hemisphere in 2014–2015 . Since 2013 , CHIKV continued to spread from the Caribbean islands to Florida in North America; Brazil , Colombia , and Guyana in South America; as well as Nicaragua and other Central American nations ( Table 1 , Fig 2 , Fig 3 ) . However , the limited number of non-CO strains prohibited us from accurately modeling the virus movements between southeast Asia , the Pacific islands , and the Caribbean . With the rapid explosion of strains that contributed to the Caribbean/Americas epidemic , we did not observe significant subclades within the CO clade in the maximum likelihood phylogenetic tree . However , we observed considerable genetic diversity in the BMCMC phylogenetic tree , which enabled the separation of two large subclades ( CO1 and CO2 , BPP>0 . 9 , Fig 3 ) , a few small subclades ( CO3-CO6 , BPP>0 . 9 , Fig 3 ) , and some monophyletic groups within the CO1 and CO2 subclades ( CO1 . 1-CO1 . 12 and CO2 . 1-CO2 . 3 , Fig 3 ) . Sequences from Nicaragua and Florida showed different epidemiological dynamics . Nicaraguan sequences tended to cluster together in the upper portion of the CO clade but did not form a single subclade . The exception was a small group of Nicaraguan sequences ( marked by the red pound sign in Fig 2 ) that were phylogenetically distinct from the other Nicaraguan sequences ( Fig 2 and Fig 3 ) . This suggests several introductions and rapid local spread of the viruses in Nicaragua during the 2014–2015 epidemic . In contrast , the CHIKV epidemic in Florida was characterized by multiple introductions of the viruses from travelers ( imported cases ) but very limited autochthonous transmission . Sequences from Florida are located in the lower portion of the CO clade and did not form a single subclade either . With the travel information that was available , the Florida sequences were found to be phylogenetically proximal to the sequences from the regions in Caribbean to which the cases reported travel ( see the following section , Fig 3 ) . We next wanted to determine how the topology of the phylogenetic tree consisting of sequences collected during the Caribbean/Americas epidemic ( CO clade ) was influenced by the geographical location of the patient . The availability of recent travel history for each patient in the majority of the newly-generated sequences from the United States enabled us to trace the origin of the infection . To determine the phylogeographic structure of the CHIKV CO clade , we performed phylogeny-trait association ( BaTS ) tests on the phylogenies of the CO clade alone . This analysis revealed significant phylogenetic clustering by geographical location ( p-values for AI and PS < 0 . 001 ) , suggesting stronger spatial clustering of the virus by either sampling country or country of recent travel than expected by chance alone . To enable a more in-depth view of how geographical location of isolation correlated with tree topology , the CO clade from the Bayesian MCC tree of Asian genotype sequences was annotated with the country of origin ( Fig 3 ) . Superimposing the geographical location information on the tree for the CO strains revealed strong evidence that the strains primarily cluster together by the geographical origin of the infection ( Fig 3 ) . The branches of the tree were colored based on the regions where the samples were isolated , while the ovals represent the probable origins of infections . In general , strains isolated from the same place tended to cluster together ( Fig 3 ) . Our newly sequenced strains from New York and Florida with travel history cluster with sequences of strains that were originally isolated from the same geographical location ( Fig 3 ) . Five Florida strains with no travel history ( black ovals in Fig 3 ) show the local transmission of CHIKV in Florida . Almost all Nicaraguan strains fall into the CO1 subclade , with the exception of a monophyletic group of five sequences in the CO3 subclade ( Fig 3 , left lower panel ) , and Nicaraguan strains in the CO1 clade clustered with strains from other countries in Central America ( e . g . , El Salvador , Guatemala , and Honduras , highlighted with red concentric circles in Fig 3 , right panel ) . The rest of the CO1 strains are from countries in the Caribbean Greater Antilles ( Puerto Rico , Haiti , and Jamaica ) , Caribbean Lesser Antilles ( Trinidad and Tobago ) , and North America ( Florida and Mexico ) , indicating the viral movements between the Caribbean and North America . Although Puerto Rico and the Dominican Republic within the Caribbean Greater Antilles were the most common sources of the origin of infection for sequences belonging to the CO2 subclade ( Fig 3 , left upper panel ) , limited strains were isolated from Caribbean Lesser Antilles ( US Virgin Islands ) , Central America ( Honduras and Panama ) , South America ( Colombia , Venezuela , and Brazil ) , and North America ( Florida ) . The rest of the non-CO1/CO2 sequences ( Fig 3 , left lower panel ) have more diverse origins than the CO1 and CO2 sequences with representative sequences from North America ( Mexico ) , South America ( Guyana , Brazil , Colombia ) , Caribbean Greater Antilles ( Puerto Rico , Jamaica ) , Caribbean Lesser Antilles ( Trinidad and Tobago , US Virgin Islands , Saint Barthelemy , Saint Lucia , Martinique , British Virgin Islands ) , and Central America ( Nicaragua ) . Overall , our phylogeographic analysis across multiple collections suggests numerous international transmission events . Amino acid substitutions that have become fixed into the evolutionary history of the CHIKV Asian genotype were mapped to the branches of maximum likelihood phylogeny ( Fig 4 ) . The CO clade is characterized by amino acid substitutions V3167A and L3242M ( Fig 4 , internal branch 5 ) , relative to the recent Asian strains ( after the year 2000 ) , which have all the amino acid changes on internal branches 1 to 4 ( Fig 4 ) . Additional amino acid substitutions for small monophyletic groups within the CO clade were also mapped to the maximum likelihood tree ( Fig 4 ) . Given that the topology of the BMCMC phylogeny showed subclades and monophyletic groups within the CO clade ( Fig 3 ) , we performed statistical comparisons of the amino acid residues that significantly differed between these subclades and monophyletic groups using Meta-CATS . Specifically , we compared sequences belonging to CO1 vs . CO2 , CO2 . 1 vs . CO2 . 2 , and CO1 . 1 vs . CO1 . 4 vs . CO1 . 6 vs . CO1 . 7 ( Fig 3 ) . Clades CO1 . 2 , CO1 . 3 , and CO1 . 5 were excluded from these statistical comparisons because of insufficient numbers of sequences . We then compared the results from the maximum likelihood phylogeny to those from the BMCMC phylogeny . Significant amino acid substitutions suggested by the Meta-CATS analysis for BMCMC phylogeny were consistent with those suggested by the maximum likelihood phylogeny ( Table 2 , Fig 4 ) . We identified 2 positions that showed strict residue separation between the CO clades , based on the consensus sequence . Aligned position 1381 in the nonstructural polyprotein , corresponding to amino acid position 48 in the NSP3 mature peptide , consists solely of serine residues in the CO1 and CO2 . 2 clades; however , this serine is replaced by a cysteine residue in all 13 strains belonging to clade CO2 . 1 . Similarly , at aligned position 3438 in the structural polyprotein , located at position 155 of the E1 mature protein , only threonine residues are observed in clades CO1 and CO2 . 1 , whereas CO2 . 2 strains code exclusively for an isoleucine . In addition , significant variation was detected at nonstructural polyprotein positions 1157 , 1720 , 1827 , and 2452 of CO1 . 4 strains when compared to CO1 . 1 , CO1 . 6 , and CO1 . 7 . Specifically , 3 of 9 residues differ in the CO1 . 4 strains when compared to the other 3 clades . Interestingly , these 3 strains ( NIC . 7101 , NIC . 1497 , and NIC . 1835 ) were all isolated from Nicaragua in 2015 and belong to a sub-clade of CO1 . 4 ( Fig 3 and Fig 4 ) . We subsequently visualized the amino acid differences identified by Meta-CATS that were located in the structural E1 and E2 proteins onto an existing three-dimensional protein structure ( 3J2W ) to better understand the contribution of the significant amino acid variation that was observed . This structure includes substantial regions of the E1 and E2 proteins that have been co-crystallized in a conformation that has been modeled into a complete virion structure . We focused specifically on the residues that differed in the E1 and E2 regions , since in general they have a higher likelihood of affecting the host B-cell immune response and entry into target cells . While residues 368 and 371 in the E2 protein were found to significantly differ between the CO and previously existing Asian strains , these residues were not resolved in the available crystal structure [33] . However , threonine 155 in E1 and serine 185 in E2 were both present in visible regions of the structure ( Fig 5 ) . Threonine 155 in E1 is located within a pocket that is formed from the quaternary structures of E1 and E2 . In contrast , serine 185 in E2 is located at the tip of a small globular domain that extends outward from the surface of the virion . With an average sequencing depth of at least 250X across all three CHIKV collections , we re-examined the read information to identify minor variants that were present as intra-host quasispecies within each clinical sample . Deep sequencing analysis of the nonstructural polyprotein region identified 250 minor variants that met our filtering criteria in strains belonging to at least one of the three collections and 56 minor variants that were observed across strains belonging to 2 or more collections . In the structural polyprotein , there were 156 minor variants present in strains belonging to at least one collection , with 30 of these variants detected across at least 2 collections ( S1–S3 Tables ) . We identified 20 codons in the nonstructural polyprotein that contained statistically significant minor variation across all three virus collections including: 25 , 26 , 29 , 30 , 158 , 163 , 164 , 235 , 320 , 376 , 377 , 1059 , 1060 , 1234 , 1358 , 1853 , 1976 , 2173 , 2204 , and 2293 . Codon numbering was used to facilitate cross-referencing between minor variants that affect amino acid sequence and other analyses . To determine whether any of the detected minor variants were present within nonsynonymous codons identified by Meta-CATS as significantly differing between subclades , we cross-referenced the 2 sets of results . Codon 1381 in the nonstructural polyprotein had detectable minor variation only within the New York collection . Interestingly , the consensus sequence in this codon codes for serine in members of the CO1 subclade but codes for either serine or cysteine in the CO2 subclade , with minor variants coding for either amino acid being detected . Similarly , 12 codons in the structural polyprotein were found to have minor variants across all three collections at positions: 20 , 22 , 24 , 510 , 511 , 769 , 805 , 810 , 889 , 944 , 948 , and 1135 . These findings suggest that dominant variants have emerged within the viral genome while genetic diversity is still being maintained at the population level as quasispecies that contain multiple minor variants . Clinical symptoms , disease severity , and related metadata fields were collected as part of the Nicaraguan study . Specifically , host demographic information was available for all 108 Nicaraguan samples , and clinical information was available for 95 of these samples . We performed two phylogeny-trait association statistics ( AI and PS ) to identify whether Nicaraguan CHIKV strains might exhibit some phylogenetic clustering by host demographics and/or disease severity ( Table 3 ) . We found no significant association of age distribution and sex ratio with viral genetic variation ( i . e . , phylogenetic topology ) in Nicaraguan strains . Hence , phylogenetic clustering by age and sex was not greater than that expected by chance alone . However , the analysis revealed significant phylogenetic clustering of Nicaraguan strains by the presence of dehydration and myalgia separately , based on both clustering statistics , AI and PS ( p-value ≤ 0 . 004 ) ( Table 3 ) . Additionally , significant phylogenetic clustering of Nicaraguan strains by only one statistical method was found for rash ( AI p-value = 0 . 036 ) and arthralgia ( PS p-value = 0 . 013 ) ( Table 3 ) . Based on the tree topology , we hypothesized that samples from particular geographic areas within Nicaragua could be enriched in virus strains assigned to different phylogenetic subclades . To test this hypothesis , we applied a hypergeometric statistical test . While the majority of the sequenced isolates were collected in Managua , statistically significant results were observed for CO1 . 1 strains in Masaya ( 3/3 virus samples in this subclade and location; p-value 0 . 041 ) , and subclade CO1 . 4 in 2 separate locations including Managua ( 5/8 virus samples in this subclade; p-value = 0 . 047 ) and Nueva Segovia ( 2/8 virus samples in this subclade; p-value = 0 . 014 ) .
This work presents a large number of newly-acquired coding-complete CHIKV sequences collected from patients in the United States and Nicaragua during the 2014–2015 Caribbean and Latin American epidemic . The new data significantly increase the number of publicly-available CHIKV sequences , especially of the Asian genotype , and consequently allowed a comprehensive study of the evolution and genetic diversity of the CHIKV Asian genotype in the Americas . We reconstructed the epidemic history of the Asian genotype and estimated the mean evolutionary rate as 4 . 33E-4 substitutions/site/year ( 95% HPD: 3 . 54E-4–5 . 15E-4 substitutions/site/year ) . Our phylogenetic analyses revealed considerable genetic diversity and phylogeographic clustering within the CO clade and described different epidemiological dynamics of CHIKV in the Americas . Specifically , the Nicaragua epidemic was characterized by several introductions and rapid local spread of the virus , while the Florida epidemic was characterized by multiple introductions but limited autochthonous transmission . Arbovirus transmission by Aedes sp . mosquitoes in Florida has been well documented [37] , especially related to DENV transmission [38–40] . We also identified the significant amino acid substitutions within the entire Asian genotype as well as within the CO clade . Additionally , our deep sequencing minor variants analysis investigated intra-host diversity of CHIKV strains in the CO clade , especially regarding the nonsynonymous codons identified previously by phylogenetic reconstructions . Finally , we found significant phylogenetic clustering according to the presence of dehydration and myalgia ( separately ) in the Nicaraguan sequences . The genetic analyses suggest that CHIKV is a conserved virus , with over 95% nucleotide/amino acid similarity across the genome . Previous studies based on partial genomes and individual genes yielded similar results [35] . Codon 148 in the nsp1 coding region was identified as undergoing positive selection pressure . Given that this position is located in a nonstructural protein and is not present in any currently known B-cell epitopes , it is possible that this residue plays a role in modulating intracellular interactions and/or the host T-cell immune response . Insertion/deletion regions in the CHIKV 3’UTR are associated with genotype/subtype classification and most likely occurred as a founder effect due to their adaptation to mosquitoes [41] . Since insertion/deletion does not count as “substitution” in viral evolution , we did not include 3’UTR region in our BEAST analysis . The substitution rate of CHIKV was estimated to range from 4E-4 ( Asian genotype ) to 6E-4 ( ECSA genotype ) substitutions/site/year by our temporal phylogenetic analysis , consistent with previous studies based on fewer numbers of CHIKV genome sequences [19 , 42] . The substitution rate is lower than human influenza A virus ( 3E-3 substitutions/site/year ) and Zika virus ( 1E-3 substitutions/site/year ) [43 , 44] , but similar to other RNA viruses , such as human respiratory syncytial virus A ( 5E-4 substitutions/site/year ) [45] , West Nile virus ( 5E-4 substitutions/site/year ) [46] , and Ebola virus ( 8E-4 substitutions/site/year ) [47] . Epidemic history of the CHIKV Asian genotype reconstructed by phylogenetic analyses showed a significant temporal-spatial evolutionary pattern , especially among non-CO strains , and indicated that the CHIKV Asian genotype diverged about 50–60 years ago . Long internal branches of non-CO sequences in the phylogenetic trees indicated extensive genetic diversity and suggested that the non-CO viruses have been circulating for many years prior to being detected or sampled . Significant geographical clustering of non-CO strains reflected limited viral transmission between regions , with a previous study showing similar results [19] . In contrast , due to the rapid spread and explosive transmission of the CO viruses , the CO clade showed distinct bush-like patterns with several polytomies . Furthermore , there was more viral transmission between different regions in the CO clade than in non-CO strains . A previous study suggested an intertwined transmission network among different countries and regions [19] . However , with the large number of newly-acquired sequences during the 2014–2015 Caribbean/Americas epidemic , the CO clade presents significant phylogeographic clustering as well . In particular , our Florida sequences with recent travel history available enabled robust interpretations regarding distinct virus introductions and co-circulating virus strains in different regions . However , a lack of such metadata for many previously-published sequences may prevent us from gaining a complete picture of international virus transmission . In Nicaragua , the geographic enrichment analysis revealed a higher-than-expected presence of strains belonging to the CO1 . 4 subclade in both Managua and Nueva Segovia , which are located relatively far apart ( ~300 km = 186 miles ) . This finding could indicate either co-circulation or separate introductions of this virus subclade into two geographic regions . The large sampling of sequences from Nicaragua and Florida in this study allowed us to investigate the epidemiological dynamics of the CHIKV Asian genotype in these two regions during the 2014–2015 epidemic . Different strains of the viruses were introduced to the Americas , including Nicaragua , and were the cause of most of the outbreaks in different regions . However , although there were multiple introductions of the virus in Florida , there was limited autochthonous transmission of CHIKV during the 2014–2015 Caribbean/Americas epidemic . Florida has a large population of residents that travel to countries with ongoing transmission and a high number of visitors that were prior residents of these countries . A few possible reasons for the lack of sustained transmission in Florida could exist . First , the climate of Florida and the mosquito vector population may not be optimal to create the same environment as in other countries in the Caribbean to sustain transmission for a long period of time . Second , the U . S . and Florida have better arbovirus surveillance systems , prevention methods ( for example , home window screens , air conditioning , regular garbage collection services , use of repellent , promotion of dumping standing water around the home , and promotion of covering mosquito breeding sites such as boats , etc . ) , and mosquito control ( through the use of larvicide and adulticide ) . These factors likely contribute to the lack of ongoing CHIKV transmission in Florida so far . Next , we investigated the significant amino acid substitutions in the Asian genotype phylogenies and discovered two CO-clade-specific amino acid substitutions , V3167A ( E2 V368A ) and L3242M ( 6K L20M ) , and other amino acid changes within CO clade . A recent comparison of the nonsynonymous amino acid changes that differentiate American and Asian genotype strains also identified these two sites [42] . The amino acid differences that we identified as being significantly different between Asian pre-CO and Asian CO sequences are of interest and could merit additional experimentation to determine whether they are accompanied by a change in phenotype . While we expect that a subset of these positions are consistent among intra-clade strains due to the evolutionary relatedness of these strains , we also expect a small number of these positions to confer some evolutionary advantage , which would explain their persistence in the virus population . Although high specificity of clade-specific residues was not observed at all significantly different positions , such comparisons may identify regions of viral proteins where amino acid changes are tolerated and may play a role in viral replication , fitness , host interaction , or the immune response . In addition , deep sequencing analysis , which was previously performed by Stapleford et al . on a collection of CHIKV strains , identified a large number of minor variants that met the established criteria of having a significant p-value and being present in at least 20 percent of reads for a given position [36] . Interestingly , when we compared the positions on our list to those reported in the previous study [36] , we found five positions that matched previous results . Specifically , codons 134 in nsp1 , 613 in nsp2 , and 116 , 345 , and 441 in nsp4 overlapped between our study and the prior publication . This finding provides independent verification of quasispecies circulating during the Caribbean outbreak that contained these minor variants , and may indicate convergent evolution among these viruses as they follow a similar , yet independent , evolutionary “path” . Additional laboratory work is needed to determine whether the circulating quasispecies that include minor variants modulate viral replication , host-pathogen interactions , or overall fitness and whether genetic influences , such as covariation , play a role in their dominance . The location of the two clade-specific variations on the E1-E2 three-dimensional protein structure also provides additional knowledge . The region surrounding threonine 155 is not likely exposed to the host immune system since it is buried in the tertiary and quaternary protein structure and is not present in or near any reported B cell epitopes from the Immune Epitope Database ( www . iedb . org ) [48] . Conversely , the globular domain surrounding serine 185 contains a linear 18-mer B cell epitope that is located immediately adjacent to S185 [49 , 50] and may also play a role in binding to host proteins . Additional CHIKV sequence data are critical to understand the genetic diversity of the virus and to successfully monitor the epidemic potential of CHIKV in the Americas . Similarly , metadata describing the virus isolation , patient characteristics , and clinical information are also useful in efforts to identify how viral evolution may contribute to changes in transmission , virulence , and/or disease severity . An example that illustrates this point comes from previous CHIKV studies of the 2005–2006 outbreak in La Reunion , which identified a single mutation in the E1 glycoprotein ( A226V of the ECSA genotype ) [51 , 52] . This mutation enhances the fitness of CHIKV in its mosquito vector Aedes albopictus , which is more prevalent than Aedes aegypti in the southeastern United States and other regions . In our study , while analyzing whether significant phylogenetic clustering of multiple clinical signs and symptoms was present , we observed that both dehydration and myalgia are well-supported by two statistical methods . Conversely , a high degree of confidence in the significant findings for both rash and arthralgia are less warranted since they were each classified as significant by only one statistical method . Even so , the ability to identify significant clustering of these clinical signs and symptoms was only made possible by the extensive collection of clinical metadata that accompanied patient specimens . The identification of two amino acid substitutions that were associated with headache and the one amino acid difference that was associated with dehydration were found to be significant , although the expected residue specificity was lower than expected . Additional experimentation with these positions is needed to better understand how the virus sequence affects the host response to infection . In summary , our study reported a large number of CHIKV complete genome sequences collected from multiple regions in the Americas and revealed the epidemiological and evolutionary dynamics of the CHIKV Asian genotype ( particularly the CO clade ) during the recent CHIKV Caribbean/Latin American epidemic . These results provide additional insight into the evolution of CHIKV during a geographically-diverse epidemic and may have important implications for the control and prevention of other mosquito-borne viruses in the Americas , such as Zika , dengue , and West Nile viruses . | Chikungunya is an arboviral disease that causes fever and acute viral febrile illness , which may be followed by months or years of debilitating joint pain in humans . There are currently no vaccines or anti-viral drugs to prevent or treat chikungunya virus ( CHIKV ) infection . In late 2013 , CHIKV was introduced into the Caribbean and spread rapidly throughout the Americas . CHIKV infections in the United States were primarily reported in Florida , with sporadic cases of traveler-associated infection reported in other states . However , little is known about the molecular evolution of the virus during this epidemic . Here , we sequenced a large number of CHIKV strains from Nicaragua , Florida and New York . Despite multiple introductions , limited local transmission was documented in Florida; in contrast , in Nicaragua , rapid local dissemination was observed . This study greatly increases the number of publicly-available CHIKV complete genome sequences from the Americas and provides a more comprehensive insight into the evolution of CHIKV during a geographically-diverse epidemic . This may have important implications for the control and prevention of other mosquito-borne viruses in the Americas , such as Zika , dengue , and West Nile viruses . | [
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] | 2018 | Differing epidemiological dynamics of Chikungunya virus in the Americas during the 2014-2015 epidemic |
Leptospirosis is a zoonotic infection with significant morbidity and mortality . The clinical presentation of leptospirosis is known to mimic the clinical profile of other prevalent tropical fevers . Laboratory confirmation of leptospirosis is based on the reference standard microscopic agglutination test ( MAT ) , direct demonstration of the organism , and isolation by culture and DNA detection by polymerase chain reaction ( PCR ) amplification . However these methods of confirmation are not widely available in resource limited settings where the infection is prevalent , and reliance is placed on clinical features for provisional diagnosis . In this prospective study , we attempted to develop a model for diagnosis of leptospirosis , based on clinical features and standard laboratory test results . The diagnostic score was developed based on data from a prospective multicentre study in two hospitals in the Western Province of Sri Lanka . All patients presenting to these hospitals with a suspected diagnosis of leptospirosis , based on the WHO surveillance criteria , were recruited . Confirmed disease was defined as positive genus specific MAT ( Leptospira biflexa ) . A derivation cohort and a validation cohort were randomly selected from available data . Clinical and laboratory manifestations associated with confirmed leptospirosis in the derivation cohort were selected for construction of a multivariate regression model with correlation matrices , and adjusted odds ratios were extracted for significant variables . The odds ratios thus derived were subsequently utilized in the criteria model , and sensitivity and specificity examined with ROC curves . A total of 592 patients were included in the final analysis with 450 ( 180 confirmed leptospirosis ) in the derivation cohort and 142 ( 52 confirmed leptospirosis ) in the validation cohort . The variables in the final model were: history of exposure to a possible source of leptospirosis ( adjusted OR = 2 . 827; 95% CI = 1 . 517–5 . 435; p = 0 . 001 ) serum creatinine > 150 micromol/l ( adjusted OR = 2 . 735; 95% CI = 1 . 374–4 . 901; p = 0 . 001 ) , neutrophil differential percentage > 80 . 0% of total white blood cell count ( adjusted OR 2 . 163; 95% CI = 1 . 309–3 . 847; p = 0 . 032 ) , serum bilirubin > 30 micromol/l ( adjusted OR = 1 . 717; 95% CI 0 . 938–3 . 456; p = 0 . 049 ) and platelet count < 85 , 000/mm3 ( adjusted OR = 2 . 350; 95% CI = 1 . 481–4 . 513; p = 0 . 006 ) . Hosmer-Lemeshow test for goodness of fit was 0 . 931 . The Nagelkerke R2 was 0 . 622 . The area under the curve ( AUC ) was noted as 0 . 762 . A score value of 14 reflected a sensitivity of 0 . 803 , specificity of 0 . 602 , a PPV of 0 . 54 , NPV of 0 . 84 , a positive LR of 2 . 01 and a negative LR of 0 . 32 . The above diagnostic model for diagnosis of leptospirosis is suggested for use in clinical settings . It should be further validated in clinical practice .
Leptospirosis is a zoonotic infection caused by spirochaetes of the genus Leptospira , with humans being affected as incidental hosts . Infection occurs when water or soil contaminated with urine of infected animals ( commonly rodents ) comes into contact with abraded human skin or mucous membrane . Clinically , leptospirosis infection has a range of manifestations , from a mild febrile illness to a severe and potentially fatal disease with acute kidney injury , liver dysfunction , pulmonary haemorrhage and acute respiratory distress syndrome , bleeding , and cardiac involvement . The burden of leptospirosis is high; the WHO Leptospirosis Burden Epidemiology Reference Group ( LERG ) estimates 873 , 000 annual cases and 48 , 000 deaths due to leptospirosis[1] . Leptospirosis is endemic in Sri Lanka . Data from the Epidemiology Unit of the Ministry of Health , Sri Lanka , suggests that leptospirosis has emerged as a key infection over the last 20 years . From 167 cases in 1991 , the numbers have increased to 4545 cases in 2010[2] . The clinical manifestations of leptospirosis mimic those of several other tropical diseases including dengue , Hanta-virus infection , rickettsial infection , as well as bacterial sepsis . In many areas where leptospirosis is common , there is also a high incidence of viral infections , including haemorrhagic fevers and infections which result in organ dysfunction . There is often confusion in differentiating leptospirosis from other infections , particularly dengue , during high incidence periods[3 , 4] . Differentiating leptospirosis from these other diseases is often a challenge to the clinician . The laboratory confirmation of leptospirosis is often based on the microscopic agglutination test ( MAT ) , isolation of the organism , or demonstration of leptospiral DNA by means of PCR . While these tests are useful for epidemiological purposes , they are not available freely , or available in a timely manner , to clinicians treating cases of acute febrile illness . For example , in Sri Lanka , where the incidence of leptospirosis , dengue , and rickettsial infections are high , reliance on specific diagnostics is impractical due to lack of availability of tests , and also because of possible cross-reacting antibodies . MAT is performed only in one reference laboratory in the country . PCR is not widely available , and rapid immunodiagnostics are expensive and not widely validated . Clinicians often depend on clinical features to diagnose and treat these conditions , based on available guidelines and clinical experience . In this study , we prospectively evaluated patients presenting to hospital with a suspected diagnosis of leptospirosis , in order to determine the clinical and investigation characteristics which may help differentiate leptospirosis from other infections presenting with a similar clinical picture , and attempted to develop a model for diagnosis of leptospirosis .
Data were collected as a part of prospective study carried out in two hospitals in the Western province of Sri Lanka , which is one of the high prevalence areas for leptospirosis in Sri Lanka . An analysis of hospital based sentinel surveillance data of leptospirosis over 4 years in Sri Lanka has confirmed that , of nearly 4000 suspected cases , 47% were from this province[5] . The selected hospitals were the National Hospital of Sri Lanka ( NHSL ) and the Homagama Base Hospital . The NHSL is the apex tertiary care teaching hospital in Sri Lanka where severely ill patients including those with complicated leptospirosis are transferred for further treatment . The Homagama base hospital is located in an area highly endemic for leptospirosis . Data collection was performed over a period of 24 months starting from June 2012 . All patients aged over 12 years with a suspected diagnosis of leptospirosis , based on the WHO surveillance criteria[6] , were enrolled . The clinical criteria for enrolment were: acute febrile illness with headache , myalgia and prostration , with any of the following—conjunctival suffusion , jaundice , oliguria/haematuria , cardiac arrhythmia or failure , cough , haemoptysis & breathlessness , bleeding , features of meningeal irritation , skin rash , history of exposure to potentially contaminated water or soil . Patients with a definite alternative diagnosis available at the time of admission were excluded . Data collection was by research assistants independent of the primary treating teams . Clinical features and investigation findings were recorded on a daily basis until the point of discharge or death . A total of 600 patients were included , with random allocation of 450 patients into a derivation cohort and 150 patients into a validation cohort . Microscopic Agglutination Test ( MAT ) was performed at the Medical Research Institute , Colombo , for laboratory confirmation of the leptospirosis . A positive result in MAT was considered under three circumstances; i . e . , a ) MAT titer of 400 or greater in single or paired samples , b ) a four-fold increase in MAT titer between acute and convalescent serum samples , or c ) seroconversion to a MAT titer greater than 200 between paired sampling[6] . Patients were classified as leptospirosis or non-leptospirosis fever ( NLF ) based on laboratory confirmation , retrospectively . The research assistants collecting data were blinded to the results of the confirmatory tests . The MAT used was based on the genus specific Leptospira biflexa serovar Patoc strain Patoc-1 . A derivation cohort was selected from the available data using a random sample of 450 out of the total 592 patients . Univariate analysis was performed on the derivation cohort with formulation of clinical , demographic , epidemiological and laboratory criteria associated with MAT positivity . All numerical data for clinical and laboratory values were plotted and tested for normality with histograms and superimposed curves . The mean values were selected for dichotomization and cut-off points for the data conforming to a normal distribution . The median values were selected in the case of non-normal data distributions . These cut-off points were subsequently refined and rounded to the closest integer with clinical application . All clinical and laboratory data extracted for analysis and subsequent model construction were the first available parameters on admission . Day three of illness was the mean day of admission . A backward multivariate logistic regression model was applied to derive the variables for the final diagnostic predictive model . All variables with p < 0 . 2 were utilized in the multivariate model . Significant independent predictors from this model were identified and their coefficients were examined . The goodness of fit of the model was analysed using the Hosmer-Lemeshow statistic . Furthermore , the Nagelkerke R2 was calculated . In order to obtain a practical scoring model , all coefficients were divided by the smallest coefficient and rounded to the closest integer ( fractions of 0 . 5 and above were rounded to the higher number , while those below 0 . 5 were rounded down ) . The scoring model was validated in the remaining patients ( i . e . , those not selected randomly for the derivation cohort ) . ROC curves were generated to examine the sensitivity and specificity of the model with MAT positivity and confirmation of the disease denoted as the gold standard . Ethics clearance was obtained from the Ethics Review Committee of the Faculty of Medicine , University of Colombo ( EC-12-056 ) and the Ethics Review Committee of the NHSL . Informed written consent was obtained from all the participants prior to recruitment to the study .
The results of the univariate analysis of associations of MAT positive , confirmed cases of leptospirosis are depicted in Table 3 . The following were positively associated with confirmed leptospirosis at a significance level of p < 0 . 05: history of exposure , myalgia , conjunctival suffusion , oliguria , acute kidney injury , urea > 18 mmol/l ( normal range [NR] 2 . 9–8 . 2 ) , serum creatinine > 150 micromol/l ( NR 60–120 ) , bilirubin concentration > 30 micromol/l ( NR 5–21 ) , serum sodium concentration < 130 mEq/l ( NR 135–148 ) , total white blood cell ( WBC ) count > 11500/mm3 ( 4000–7000 ) with neutrophil percentage > 80% , haemoglobin concentration < 10 . 5g/dL ( NR 11–12 ) and packed cell volume < 30% and platelet count < 85000/mm3 ( NR 150 , 000–450 , 000 ) . Table 3 . Comparison of clinical and laboratory features in leptospirosis and NLF patients in the derivation cohort ( univariate analysis ) The selection criteria of independent variables for model construction were described under methodology . The presence of conjunctival suffusion , jaundice , exposure history , muscle tenderness , total WBC count > 11 , 500mm3 , neutrophil percentage > 80 . 0% , serum creatinine >150micromol/l , bilirubin > 30 micromol/l , hemoglobin < 10 . 5g/dL , serum sodium < 130 mEq/L , ALT > 70 IU/L ( NR– 10–35 ) , microscopic hematuria , and serum potassium > 5 . 0 ( NR– 3 . 5–5 . 3 ) were entered into the initial model . Correlation matrices were used to adjust for co-dependence between the independent variables . Contact history was adjusted for sex and age , serum creatinine was adjusted for age , and haemoglobin for sex . The variables in the final step model were: history of exposure to possible source of leptospirosis ( adjusted OR = 2 . 827; 95% CI = 1 . 517–5 . 435; p = 0 . 001 ) , serum creatinine > 150 micromol/l ( adjusted OR = 2 . 735; 95% CI = 1 . 374–4 . 901; p = 0 . 001 ) , neutrophil differential percentage > 80 . 0% of total WBC count ( adjusted OR 2 . 163; 95% CI = 1 . 309–3 . 847; p = 0 . 032 ) , serum bilirubin > 30 micromol/l ( adjusted OR = 1 . 717; 95% CI 0 . 938–3 . 456; p = 0 . 049 ) and platelet count < 85 , 000/mm3 ( adjusted OR = 2 . 350; 95% CI = 1 . 481–4 . 513; p = 0 . 006 ) . Hosmer-Lemeshow test for goodness of fit was 0 . 931 . The Nagelkerke R2 was 0 . 622 The diagnostic score derived from the above is shown in Table 4 . The beta coefficients were divided by the smallest coefficient and then multiplied by a factor of 4 to create a more robust and practical scoring system . Receiver operating characteristic ( ROC ) curves were generated separately for serum creatinine , neutrophil differential percentage , serum bilirubin , and platelet count . Furthermore , we generated an ROC curve utilizing the scoring system applied to the validation cohort to differentiate leptospirosis from NLF ( Fig 2 ) . The sensitivity , specificity , positive predictive value ( PPV ) , negative predictive value ( NPV ) and likelihood ratios ( LR ) are presented . The area under the curve ( AUC ) was noted as 0 . 762 . A score value of 14 reflected a sensitivity of 0 . 803 , specificity of 0 . 602 , a PPV of 0 . 54 , NPV of 0 . 84 , a positive LR of 2 . 01 and a negative LR of 0 . 32 . The diagnostic model performance parameters for various cut-off points are presented in Table 5 . All coordinates in the ROC curve with relevant sensitivity and specificity are presented in S1 Table . The dataset is presented as S1 Dataset .
The incidence of confirmed leptospirosis is high among patients admitted to hospital with a suggestive clinical picture . However nearly 52% of patients with suspected leptospirosis were negative on confirmatory testing . Nonetheless , since confirmatory test results are often delayed , the majority of them were treated with appropriate antibiotics to cover leptospirosis; in our study , this was nearly 100% in those with a contact history of leptospirosis . Our results indicates that a diagnostic model with inclusion of serum creatinine , neutrophil percentage , elevated serum bilirubin and platelet count has reasonably good sensitivity and specificity for the diagnosis of leptospirosis . Notably , apart from exposure , none of the clinical features looked for could reliably distinguish leptospirosis from NLF . This reinforces the need for accurate and readily available tests to confirm the diagnosis . History of exposure was noted to have the strongest positive association . Thus , exploring the different sources of exposure is one of the most useful components of the clinical history . An open ended question asking whether there is exposure to muddy water maybe inadequate , and we suggest developing a list of potential exposures that should be asked for at the time of admission . Specific organ involvement , i . e . , kidney and liver , appear to differentiate leptospirosis from NLF . Haematological parameters are of particular use , since a full blood count is often the first investigation to become available . A low platelet count , while often the hallmark of dengue , also appears to be a feature of leptospirosis . The absolute neutrophil percentage appears to be a more useful indicator of leptospirosis rather than the total leucocyte count . Faine’s criteria [7] for the diagnosis of leptospirosis have been suggested for the diagnosis of leptospirosis , with various subsequent modifications[8] . Faine’s criteria essentially use clinical , epidemiological and microbiological features to score the likelihood of leptospirosis . These criteria , with modifications , have been evaluated in various studies , giving varying degrees of specificity and sensitivity [8 , 9] . The use of clinical criteria alone was found to have high negative predictive value but relatively low positive predictive value; however studies have been small [9 , 10] . The numbers included in our study were greater , and a large panel of clinical and laboratory characteristics were evaluated in our diagnostic model . The patients utilized in derivation of this diagnostic model were hospitalized patients and the data may not be universally applicable to patients with milder disease and outpatients presenting with acute febrile illness . Our study has certain limitations . First , the final diagnosis of patients in the NLF category was not available in all cases . This would have created better characterization between other leptospirosis mimics . Secondly , with the high awareness of dengue and the wide availability of Dengue NS1 antigen testing , many patients admitted with a similar clinical picture present to hospital with positive results of dengue NS1 antigen , and would thus have been excluded from the study . Thirdly , the MAT methodology available up to 2015 in Sri Lanka was limited to the Patoc serovar analysis . While cross reactivity between pathogenic serovars and the saprophytic serovars occurs , it is possible that there were false negatives on MAT testing . Further analysis of the study population with a broader panel of pathogenic serovars has been initiated . Finally , the use of MAT as a gold standard has been questioned in prior studies [11] where estimated sensitivity of MAT and MAT + culture is noted to be less than 50% . Nonetheless , this scoring system applies to the optimum currently available laboratory standards in resource limited settings such as Sri Lanka . We suggest the use of the above diagnostic model for the diagnosis of leptospirosis in clinical settings . This model should be further validated in clinical practice , and with a broader panel of serovars . | Leptospirosis is a bacterial disease which is common in tropical countries , and spreads via contaminated rat urine . It has a potential for mortality and can cause failure of multiple body systems . The features of the disease mimics several other tropical diseases prevalent in the region . The modalities for laboratory confirmation of the disease are not readily available in resource limited settings . This study utilizes prospective data and proposes diagnostic criteria using simple clinical presentations and easily accessible laboratory data . | [
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] | 2016 | A Diagnostic Scoring Model for Leptospirosis in Resource Limited Settings |
The rok gene of Bacillus subtilis was identified as a negative regulator of competence development . It also controls expression of several genes not related to competence . We found that Rok binds to extended regions of the B . subtilis genome . These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer . Some of the Rok binding regions are in known mobile genetic elements . A deletion of rok resulted in higher excision of one such element , ICEBs1 , a conjugative transposon found integrated in the B . subtilis genome . When expressed in the Gram negative E . coli , Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association . Together with previous work , our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes , many of which appear to have been acquired by horizontal gene transfer . In these ways , Rok appears to be functionally analogous to H-NS , a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria .
In bacteria , horizontal gene transfer typically occurs through natural transformation , conjugation , and transduction and contributes to rapid evolution and the acquisition of new traits [1] , [2] . For example , genes needed for pathogenesis , symbiosis , resistance to antibiotics , and metabolism of various compounds are often acquired through horizontal gene transfer . Even though horizontal gene transfer can confer potential benefits on a recipient cell , there are potentially lethal costs . For example , the acquisition and expression of genes for restriction enzymes ( often carried on phage ) is potentially lethal . The acquisition of transcriptional regulators could lead to inappropriate rerouting of gene regulatory networks , and the introduction and expression of enzymes could lead to altered metabolic flux , resulting in loss of fitness under some conditions . In addition , the introduction and expression of homologues of essential genes may be detrimental to the cell if the gene products interfere with essential cellular components . Many organisms have mechanisms to inhibit acquisition and expression of foreign DNA . These mechanisms can help to mitigate some of the potentially deleterious effects of acquisition and expression of foreign genes . For example , the H-NS protein of Gram-negative bacteria is a widely studied nucleoid-associated protein that modulates gene expression and is a negative regulator of some genes and mobile elements acquired by horizontal gene transfer [3]–[5] . The presence of H-NS appears to be confined to proteobacteria . A functional analogue has been recently described from the high-G+C Gram positive actinomycete Mycobacterium tuberculosis [6] , [7] , but no analogous proteins have been described in low-G+C Gram positive organisms to date . We found that the transcription factor Rok of the low-G+C ( 43 . 5% ) Gram positive Bacillus subtilis binds to A+T-rich DNA and helps repress the activity of at least one mobile genetic element . Rok is relatively small ( 20 . 7 kD ) and is found in several Bacillus species closely related to B . subtilis [8] . Rok was previously identified as a negative regulator of natural genetic competence in B . subtilis [9] . It binds to and represses the promoter of comK [9] . comK encodes a transcriptional activator that is required for expression of the B . subtilis competence machinery needed for uptake of exogenous native and foreign DNA [10] , [11] . In addition to repressing transcription of comK , Rok represses transcription of several other genes , including many that encode extracellular functions [8] . Rok binds specifically to the promoters of at least a subset of these genes , although a consensus recognition sequence has not been identified [8] . To better understand the function of Rok in gene regulation and cell physiology , we characterized the association of Rok with chromosomal DNA in vivo . We expected to find Rok binding largely limited to chromosomal regions of genes known to be transcriptionally regulated by Rok [8] . In addition , we found extensive binding of Rok to genomic regions characterized by a high A+T , many of which are believed to have been acquired via horizontal gene transfer . We also found that Rok binds to and represses endogenous excision of the mobile genetic element ICEBs1 , an integrative and conjugative element ( conjugative transposon ) found integrated in the 3′-end of a tRNA gene in B . subtilis [12] , [13] . Our results indicate that Rok is a nucleoid-associated protein , is bound to chromosomal regions containing A+T-rich sequences , and inhibits the activity of at least one mobile genetic element . In these ways , Rok helps to repress several genes and elements known or thought to have been acquired by horizontal gene transfer .
Rok is a DNA binding protein and appears to have some sequence specificity , although no consensus binding sequence has been identified [8] . We used fluorescence microscopy to visualize the location of Rok in living cells ( Figure 1A ) . We fused rok to yfp , ( encoding yellow fluorescent protein , IYFP [14] such that rok-yfp was expressed from its native promoter and was the only source of Rok in the cell . Strains expressing the fusion had normal transformation frequencies , indicating that Rok-YFP was functional ( data not shown ) . As expected for a DNA-binding protein , Rok-YFP ( Figure 1A ) appeared to be associated with the nucleoid , as visualized with 4′ , 6-diamidino-2-phenylindole ( DAPI ) ( Figure 1B ) . However , Rok-YFP was not uniformly associated with the nucleoid ( Figure 1 ) . In contrast , the nucleoid binding protein HBsu , the B . subtilis homologue of HU from Gram negative organisms [15] , appeared to be associated with the nucleoid in a relatively uniform manner ( Figure 1C , 1D ) . We fused HBsu ( hbs ) to mCherry [16] such that hbs-mcherry was expressed from the endogenous hbs promoter and the fusion was the only source of HBsu in the cell . hbs is essential for cell growth [17] , and the hbs-mcherry fusion strain appeared to grow normally , indicating that the fusion was functional . Since HBsu-mCherry appeared to be associated with most or all of the nucleoid , and Rok-YFP was not uniformly associated with the nucleoid ( Figure 1 ) , we inferred that the DNA binding preferences of these two proteins are likely to be different . This was further explored in chromatin immunoprecipitation experiments with HBsu and Rok described below . We determined the genome wide binding profile of Rok ( Figure 2A ) and HBsu ( Figure 2B ) in vivo using formaldehyde-mediated crosslinking and immunoprecipitation ( ChIP ) and hybridization of immunoprecipitated DNA to DNA microarrays ( ChIP-chip ) . We fused a cMyc epitope tag to the 3′ end of rok ( rok-myc ) and hbs ( hbs-myc ) such that each fusion is expressed from its native promoter and is the only copy of the gene in the cell . Like Rok-YFP and HBsu-mcherry , both myc-tagged proteins appeared to be functional; rok-myc strains had normal levels of competence and hbs-myc strains were viable ( data not shown ) . We used monoclonal anti-myc antibodies to immunoprecipitate the myc-tagged proteins and detected DNA that was co-precipitated using DNA microarrays containing >99% of the annotated B . subtilis open reading frames as well as a subset of intergenic regions [18] . We found that Rok was associated with several genomic regions ( Figure 2A ) , many of which correlated with the locations of genetic elements known or thought to have been acquired by horizontal transfer [19] , [20] . Since horizontally acquired sequences often have a different G+C content than endogenous genes , we compared the Rok binding profile to the G+C content of the genome ( Figure 2A ) . We calculated the average G+C content in windows of 3 , 000 bp with a step-size of 1 , 000 bp ( Materials and Methods ) . The average % G+C across the entire genome is ∼43 . 5% [19] . Many of the chromosomal regions associated with Rok were strikingly lower in % G+C ( higher % A+T ) than the rest of the genome ( Figure 2A ) . The observed binding profile was specific for Rok , and not the myc-epitope tag or nucleoid binding proteins in general as the binding profile of HBsu-myc ( Figure 2B ) was quite different from that of Rok-myc ( Figure 2A ) . In contrast to Rok , none of the chromosomal regions had >2 . 5-fold enrichment for HBsu-myc ( Figure 2B ) , consistent with the function of HBsu as a general nucleoid binding protein [21] . Our results indicate that in vivo , HBsu is a relatively non-specific DNA binding protein and Rok binds preferentially to A+T-rich regions of the chromosome . Analysis of the ChIP data for smaller chromosomal intervals highlights the difference between Rok and HBsu binding ( Figure 3 ) . Some of the chromosomal regions have genes characteristic of prophages , or defective prophages and thus appear to be or once have been functional mobile genetic elements likely acquired by horizontal gene transfer . These regions were designated as prophage regions and given a number [19] . Prophage regions 4 , 5 , and 6 have regions of G+C content significantly less than 40% ( using a sliding window of 500 bp and a step size of 100 bp ) and these regions are preferentially bound by Rok , but not HBsu ( Figure 3A–3C ) . Similarly , the defective prophage skin [22] ) and the prophage regions 1 and 7 also preferentially bind Rok , but not HBsu ( Figure 2 and data not shown ) . Regions of the prophage SPβ [23] were bound by both Rok and HBsu , although binding of Rok appeared to be much greater ( Figure 2 ) . The sdp operon ( Figure 3D ) , yybN operon ( Figure 3E ) , and the yefC-yezA region ( Figure 3F ) are postulated to have been acquired by horizontal gene transfer [20] . Rok binds these regions in vivo , and binding correlates well with the regions of low G+C content ( Figure 3D–3F ) . Rok does not bind to all horizontally acquired DNA . The defective prophage PBSX [24] is a mobile genetic element likely acquired by horizontal gene transfer and Rok was not significantly bound to this chromosomal region ( Figure 3G ) . However , the nucleotide composition of the PBSX region is not significantly different from the average of the B . subtilis genome ( Figure 3G ) , consistent with the findings that Rok binds preferentially to regions of relatively low G+C content . Similarly , H-NS in Salmonella species does not significantly associate with mobile genetic elements that have G+C content similar to that of the host [25] , [26] . Previously , Rok was found to control expression of at least 20 different transcription units , either directly or indirectly [8] , [9] . At least nine of these appear to be directly regulated by Rok since Rok binds to the promoter regions in vitro [8] . The ChIP-chip data indicate that Rok is also bound to ( or near ) almost all of these in vivo ( Table 1 ) , consistent with a role for Rok in directly repressing their expression . Several of the previously identified Rok targets are located in genomic regions that have extensive binding of Rok in vivo . For instance , prophage 4 contains yjcN , a gene previously identified as a direct target of Rok [8] . Rok binding extends well beyond a simple regulatory region for yjcN ( Figure 3A ) . Similarly , the sdp operon and yybN genomic region were previously identified as direct targets of Rok [8] . Rok binding to these regions in vivo extends greater than 5 kb ( Figure 3D and 3E ) . Rok also appears to bind to some regions that do not have significantly lower G+C content than the average for the B . subtilis genome . For example , the comK region has normal G+C content and Rok binds to this region in vivo ( Figure 3H ) and in vitro [9] . This also seemed to be the case for Rok binding to its own promoter region , although there is also some Rok binding in an A+T-rich region of rok ( Figure 3I ) . These results might reflect recruitment of Rok by other DNA binding proteins . Alternatively , they might indicate that Rok has some DNA sequence-specific binding beyond the recognition of low G+C content , and that binding specificity could be obscured by the less specific and widespread association with low G+C content DNA . We found that when expressed in Escherichia coli , Rok also bound preferentially to A+T-rich DNA . We introduced a plasmid expressing full length Rok with a C-terminal myc-tag ( Rok-myc ) into E . coli and confirmed by Western blotting that the protein accumulated ( data not shown ) . We used ChIP followed by quantitative real time PCR ( ChIP-qPCR ) to compare the association of Rok-myc with six different chromosomal regions in E . coli , three that are A+T-rich and three that are G+C-rich ( Table 2 ) . There was significantly greater association of Rok-myc with the A+T-rich sequences than the G+C-rich sequences ( Table 2; Figure 4A ) . This preferential binding of Rok-myc to A+T-rich sequences in E . coli is consistent with the binding of Rok to AT-rich sequences in B . subtilis as determined by ChIP-chip ( Figure 2 ) . It is not known which region of Rok is needed for DNA binding [8] , [9] , [27] . Using various tools for sequence analyses combined with rok deletions and ChIP-qPCR , we found that the DNA binding activity of Rok is contained in the C-terminal region of the protein . We used a ClustalW2 alignment {http://www . ebi . ac . uk/clustalw/} of primary amino acid sequences of Rok homologs from B . subtilis , B . amyloliquefaciens , B . pumilus , B . licheniformis , B . coagulans and B . pseudomycoides to define conserved regions of the protein ( Figure S1 ) . The analysis revealed three distinct regions: ( I ) a highly conserved N-terminal region; ( II ) a moderately conserved central region; and ( III ) a highly conserved C-terminal region ( Figure 4B and Figure S1 ) . On the basis of the presence of many positively charged residues , we used the third putative domain to query the I-TASSER server for protein structure prediction [28] . The in silico analysis predicted that the C-terminal region might have some structural relatedness ( Tm-score 0 . 6145 ) to winged helix DNA-binding domains of proteins such as FurB from Mycobacterium tuberculosis ( PDB 2o03_A ) {Lucarelli 2007 , FurB} . The C-terminal region of Rok was also classified as containing a possible DNA/RNA-binding 3-helical bundle from the Winged Helix Superfamily , with an estimated precision of 20% by the PHYRE Protein Fold Recognition server [29] . Based on the in silico sequence analysis , we tested for the ability of the three regions of Rok to bind DNA . We fused each of these regions to a myc-epitope tag ( Figure 4B ) and used ChIP-qPCR to measure the ability of each fusion to associate with a region of E . coli DNA with high or low A+T content ( as described above ) . Each of the fusion proteins accumulated to similar levels in E . coli and was detectable with anti-myc antibodies ( data not shown ) . We found that any variant that contained the C-terminal region ( region III ) of Rok was able to bind DNA , and there was a preference for DNA with high A+T content ( Figure 4A ) . In contrast , variants that contained region I , region II , or both , did not appear to have significant DNA binding activity ( Figure 4A , 4B ) . However , association of full length Rok with A+T-rich DNA was greater than that of region III alone ( Figure 4A ) . Based on these results , we conclude that region III of Rok is the DNA binding region and that this region alone has some preference for A+T-rich DNA . We suspect that region I , either alone or in combination with region II , likely contributes to DNA binding and the specificity for A+T-rich DNA , perhaps by affecting dimerization and/or multimerization of Rok and potentially contributing to cooperativity . This would be similar to H-NS and H-NS-like proteins where the N-terminal region affects multimerization [30] . Rok binds to regions of the mobile element ICEBs1 , and the regions with the most binding , at the left and right ends of ICEBs1 , have the lowest G+C content ( Figure 5A ) . The genes encoding the ICEBs1 site-specific recombinase ( int ) and the excisionase ( xis ) that allow excision from the genome are in the left end , and the recombination reaction occurs between 17 bp sequences found at the ends of the integrated element ( attL and attR ) [12] . During normal exponential growth , ICEBs1 gene expression is repressed by the element's repressor ImmR and there is very little excision of ICEBs1 [31] . Upon production of active RapI , a cell sensory protein , or during the RecA-dependent SOS response , ImmR is inactivated and ICEBs1 gene expression is derepressed . This leads to rapid production of excisionase and efficient excision of ICEBs1 from the chromosome [31] , [32] . Because Rok was associated with genes and sequences at the ends of ICEBs1 , we determined the effects of a rok null mutation on the stability of ICEBs1 . We found that endogenous excision of ICEBs1 increased approximately 4-fold in a rok null mutant ( Figure 5B ) . Excision of ICEBs1 by site-specific recombination between the left and right attachment sites ( attL and attR ) leaves behind an empty bacterial attachment site , attB , that is readily detected by PCR using appropriate primers [12] . The occurrence of attB in a population , compared to a nearby chromosomal locus ydbT , is a measure of the frequency of excision of ICEBs1 [33] , [34] . During exponential growth , the excision frequency of ICEBs1 in rok+ cells was ∼4×10−5 ( Figure 4B ) , consistent with previous measurements [33] , [34] . In a rok null mutant , this frequency increased to ∼1 . 6×10−4 ( Figure 5B ) . The increased excision frequency of ICEBs1 in the rok null mutant could be due to an increase in integrase- and excisionase-mediated site-specific recombination , or possibly homologous recombination between the 60 bp direct repeats that mark the ends of ICEBs1 . We found that the increased excision in the rok null mutant was dependent on the ICEBs1 excisionase . An ICEBs1 xis ( excisionase ) null mutation reduced the excision frequency to ∼1-2×10−6 ( Figure 5C ) , consistent with previous findings [33] . This frequency did not significantly change in a rok null mutant ( Figure 5C ) , indicating that the increased spontaneous excision observed in the rok mutant requires excisionase and is not due to increased homologous recombination between the 60 bp direct repeats at the ends of ICEBs1 . Based on these results , we conclude that Rok contributes to keeping ICEBs1 quiescent in the genome .
Rok is a negative regulator of genes involved in horizontal gene transfer in at least two ways: 1 ) as a repressor of competence development , and 2 ) as an inhibitor of expression of genes in A+T-rich chromosomal regions . rok was initially identified as a repressor of of comK [9] . The comK gene product is a transcriptional activator and the master regulator of competence development and the K-state in B . subtilis [10] , [11] , [35] . By virtue of repressing comK , Rok also indirectly represses expression of the genes activated by ComK and is a therefore a strong negative regulator of competence development , thus inhibiting the ability of cells to acquire foreign DNA . Rok also binds to chromosomal regions that are known or thought to have been acquired by horizontal gene transfer and are characterized by a high A+T content . One of these regions contains ICEBs1 , an integrative and conjugative element integrated in the B . subtlis chromosome [12] . Rok binds to ICEBs1 and helps prevent spontaneous excision . Many other horizontally acquired genes are bound by Rok in vivo and expression of some of these were previously found to be de-repressed in a rok null mutant [8] . Interestingly , rok itself appears to have been recently acquired in the B . subtilis - B . amyloliquefaciens - B . licheniformis clade as it is inserted in and interrupts an otherwise conserved genomic arrangement [8] . rok is auto-regulated , repressing its own expression . That is , transcription of rok increases in the absence of functional Rok protein . Thus , when the concentration of Rok decreases at its own promoter , its expression will increase , perhaps allowing cells to adjust the levels of Rok in response to acquisition of new DNA that has a high A+T content . A null mutation in rok causes increased expression of at least 20 transcription units , either directly or indirectly [8] , separate from genes that are activated by ComK . In vitro , Rok binds to sequences upstream of at least a subset of these genes [8] . We found that in vivo Rok is associated with most of these genes , consistent with the notion that Rok directly represses their transcription . Our results indicate that the number of chromosomal genes bound by Rok is s significantly greater than the number of genes whose expression is detectably altered in a rok null mutant [8] . There are two ways to explain this difference . First , many genes bound by Rok are not expected to be expressed under the conditions used for analysis of mRNA levels [8] . This is particularly true for genes that might be controlled by other regulators , and for regions containing the mobile genetic elements that are strongly repressed during normal growth . Second , Rok may be bound to regions but not properly positioned to have an effect on transcription . There is a general correlation between chromosomal regions with low G+C content and Rok binding . However , it is notable that at least a few of the chromosomal regions bound by Rok appear to have a G+C content much closer to or greater than the norm , including the comK and rok regulatory regions . Rok might be recruited to these regions by other DNA binding proteins . Alternatively , Rok might be capable of some sequence-specific binding somewhat different from binding to A+T-rich DNA , and these possibilities are not mutually exclusive . A discriminative MEME motif search [36] using the few regions of Rok binding that are close to the average G+C content identified a motif that appears to be overrepresented ( Figure S2 ) . This motif could be a binding site for another protein that possibly interacts with Rok , or could indicate a specific binding sequence for Rok . Further genetic and functional dissection of Rok and this motif should help determine how Rok is associated with this DNA . Binding of H-NS to DNA also appears to be complex . H-NS can interact with or bind to regions bound by other DNA binding proteins , might have some site-specific binding , and can switch from stimulating DNA bridging to causing DNA stiffening {e . g . , [5] , [37]–[40]} . All bacteria appear to have nucleoid-associated proteins ( NAPs ) [41] that are abundant , bind relatively non-specifically to extended regions of the chromosome , and often cause changes in DNA topology . The most highly conserved nucleoid-associated protein is the “heat-unstable” protein HU ( and the related integration host factor IHF ) found in both Gram negative and Gram positive organisms . In contrast , the heat-stable nucleoid restructuring protein H-NS is found in several Gram negative bacteria , but there are no obvious homologues in Gram positive bacteria ( reviewed in [42] ) . Despite the lack of sequence similarity , the recently characterized Lsr2 protein from Mycobacerium and related high G+C Gram positive bacteria is a functional analogue of H-NS [6] . Like H-NS , Lsr2 binds A+T-rich DNA , including regions acquired by horizontal gene transfer [7] , [43] , can repress transcription [43] , is capable of bridging DNA [6] , [44] , and can partly substitute for H-NS in E . coli [6] . Neither H-NS nor Lsr2 homologues have been found in low G+C content Gram positive species . There are several functional similarities between Rok of B . subtilis and H-NS ( and Lsr2 ) . Both Rok and H-NS act as negative regulators of transcription and bind extended chromosomal regions with high A+T content . H-NS causes significant changes in DNA topology , and this has also been postulated for Rok [27] . Most notably , both Rok and H-NS help silence foreign DNA with a high A+T content . Transcriptional repression exerted by H-NS can be reversed by certain anti-silencing mechanisms {reviewed in [45]} . Likewise , auto-activation of comK transcription is accomplished by preventing Rok-mediated repression [27] . Rok and H-NS are both relatively small ( 20 . 7 kDa and 15 . 4 kDa , respectively ) , although H-NS appears to be more abundant than Rok . There are approximately 20 , 000 molecules per cell of H-NS in growing cells [4] . In contrast , we estimate that there are approximately 1 , 000–3 , 000 molecules of Rok per genome of exponentially growing B . subtilis cells ( see Materials and Methods ) . Based on the several similarities in function , we propose that Rok of B . subtilis and its close relatives is functionally analogous to H-NS of Gram negative bacteria and Lsr2 of Mycobacterium and related high G+C actinomycetes . We suspect that other organisms have H-NS analogues that are not readily recognized by sequence similarities .
For routine growth and manipulations , E . coli and B . subtilis cells were grown in LB medium . For most experiments , B . subtilis cells were grown in the MOPS buffered S750 defined minimal medium [46] with 0 . 1% glutamate , supplemented with required amino acids ( typically tryptophan and phenylalanine ) , 1% glucose or arabinose as a carbon source , and 1 mM IPTG or 1% xylose as inducer as necessary . Strains with plasmids integrated into the chromosome by single crossover were grown with appropriate antibiotic to maintain selection for the integrated plasmid . B . subtilis strains used are listed in Table 3 . PCR Primers used in strain constructions are listed in Table S1 . B . subtilis strains were constructed by transformation using standard procedures [35] , [47] Previously described alleles affecting ICEBs1 include Δxis190 , a deletion of the excisionase gene of ICEBs1 [33] , and Pxyl-rapI [48] , used to induce efficient ICEBs1 gene expression . Cells were grown in defined minimal medium , placed on agarose ( 1 . 5% ) pads containing Spizizen minimal salts [47] . DAPI was added to a final concentration of ∼80 ng/ml five minutes prior to visualization . Images were acquired using Nikon Ti-E inverted microscope under a 100× phase oil objective . Fluorescence images were acquired using Nikon Intensilight mercury illuminator and appropriate sets of excitation and emission filters ( 49008 for mCherry , 49003 for YFP and 49000 for DAPI , Chroma ) . Images were recorded using a CoolSNAP HQ camera ( Photometrics ) and processed using NIS-Elements Advanced Research 3 . 10 Software . TIFF images were processed in Adobe Photoshop CS3 and Figure 1 was prepared in Adobe Illustrator CS3 . Chromatin immunoprecipitation of DNA bound to the various proteins in B . subtilis was done essentially as described [53] , except that DNA was precipitated in the presence of glycogen ( 20 µg ) as a carrier . For ChIP experiments in E . coli , crosslinking was done at room temperature for 20 minutes . Myc-tagged proteins were immunoprecipitated using monoclonal anti-cMyc antibodies ( Zymed ) . Both Rok-myc and HBsu-myc were functional in B . subtilis ( see results ) and both were detected in Western blots ( data not shown ) . Preliminary comparisons between Rok-myc and HBsu-myc in Western blots with anti-myc monoclonal antibodies indicated that there is about 20-50-fold more HBsu in the cell than Rok . Since the cellular concentration of HBsu is about 50 , 000 molecules per genome [54] , we estimate that there are approximately 1 , 000–3 , 000 molecules of Rok per genome . For Rok-myc and HBsu-myc , we verified that the anti-myc antibodies actually immunoprecipitated the protein by depleting it from an extract ( data not shown ) . Even though there was little or no significant enrichment of specific chromosomal regions in the HBsu-myc ChIP experiments , the protein appeared to crosslink to DNA as the signals on the microarrays were significantly above background . ChIP-chip analyses were performed as described previously using printed DNA microarrays with PCR products corresponding to most open reading frames and many intergenic regions [55] . The microarray data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [56] and are accessible through GEO Series accession number GSE23199 ( http://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ? acc=GSE23199 ) . qPCR was performed on a Roche LightCycler 480 II . Samples ( 2 µl ) of immunoprecipitated DNA were analyzed in duplicate in a 20 µl reaction volume that contained Sybr green . Signals were analyzed using the LightCycler 480 SW 1 . 5 software ( Roche ) , according to the manufacturer ( Absolute quantification; 2nd derivative of Max ) . Signals were normalized against a 12-point standard curve obtained from a dilution series of total chromosomal DNA of B . subtilis JMA222 [34] or BL21 ( DE3 ) [52] . Primers used for real time PCRs are listed in Table S1 . For all conditions at least three independent biological replicates were analyzed and data shown represent the average of these replicates . Excision of ICEBs1 was monitored by quantitative real time PCR , as described previously [34] using primers CLO261 , CLO262 , CLO283 , and CLO284 ( Table S1 ) . Conserved regions of Rok from B . subtilis 168 ( NP_389307 . 1 ) , B . amyloliquefaciens FZB42 ( YP_001420994 . 1 ) , B . pumilus ATCC7061 ( ZP_03052836 . 1 ) and SAFR-032 ( YP_001486564 . 1 ) , B . licheniformis ATCC14580 ( YP_078814 . 1 and YP_079175 . 1 ) , B . psuedomycoides DSM12442 ( ZP_04153718 . 1 ) and B . coagulans 36D1 ( ZP_04433348 . 1 ) were identified using ClustalW2 ( http://www . ebi . ac . uk/Tools/clustalw2/index . html ) . Amino acids 96–191 of B . subtilis Rok were used to query the I-Tasser server ( http://zhanglab . ccmb . med . umich . edu/I-TASSER ) [57] and the PHYRE Protein Fold Recognition Server ( http://www . sbg . bio . ic . ac . uk/~phyre/ ) [29] for secondary structure predictions . Sliding window analyses were performed using SWAAP 1 . 0 . 3 ( http://asiago . stanford . edu/SWAAP/SwaapPage . htm ) [58] on genome sequences of Bacillus subtilis ( accession number AL009126 ) and E . coli BL21 ( DE3 ) ( accession number CP001665 ) retrieved from GenBank ( ftp://ftp . ncbi . nih . gov/genbank/genomes/Bacteria/ ) . A discriminative MEME motif search [36] was used to detect overrepresented motifs in selected regions that show Rok binding in vivo . | There are several mechanisms by which bacteria acquire exogenous DNA . Sometimes this genetic material is advantageous for bacterial cells , for example , by making them resistant to antibiotics . Other times , foreign DNA has genes that are deleterious to the new host . Bacteria have mechanisms for helping to silence exogenously ( horizontally ) acquired genes . Many horizontally acquired genes are A+T-rich , a feature which can be important in distinguishing these loci from the host genes . We found that the transcriptional regulator Rok in the bacterium Bacillus subtilis preferentially binds to A+T-rich DNA . Together with previous work , our findings indicate that Rok helps repress expression of A+T-rich genes , many of which are likely to have been acquired by horizontal gene transfer . In these ways , Rok appears to be a functional analogue of the H-NS protein found in Gram negative bacteria ( e . g . , E . coli ) and Lsr2 found in the high G+C Mycobacterium tuberculosis . | [
"Abstract",
"Introduction",
"Results",
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"Methods"
] | [
"genetics",
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] | 2010 | The Transcriptional Regulator Rok Binds A+T-Rich DNA and Is Involved in Repression of a Mobile Genetic Element in Bacillus subtilis |
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