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The regulation of cell migration is essential to animal development and physiology . Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses . Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains , which are synthesized by glycosyltransferases of the exostosin ( EXT ) family . Abnormal HS chain synthesis results in pleiotropic consequences , including abnormal development and tumor formation . In humans , mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses . Complete loss of any of the exostosin glycosyltransferases in mouse , fish , flies and worms leads to drastic morphogenetic defects and embryonic lethality . Here we identify and study previously unavailable viable hypomorphic mutations in the two C . elegans exostosin glycosyltransferases genes , rib-1 and rib-2 . These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects , including abnormal cell and axonal migrations . We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis , and is sustained throughout life in specific cell types , consistent with HSPGs playing both developmental and post-developmental roles . Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance . Our findings provide insights into general principles underlying HSPG function in development .
Cell migration is key to animal development and physiology . To reach their targets , migrating cells rely on guidance factors and morphogens , which can be regulated by heparan sulfate proteoglycans ( HSPGs ) [1] . HSPGs are cell-surface or extracellular proteins characterized by the attachment of heparan sulfate ( HS ) polysaccharide chains to the extracellular domain of their core protein [2] . HSPGs interact with molecules at the cell surface and in the extracellular matrix via both their HS chains and core proteins , and can function as co-factors that regulate the distribution of morphogens and that modulate the interactions between extracellular ligands and their receptors [1 , 3] . HSPGs have been shown to be part of multiple signaling pathways across species and to be key to multiple developmental events , including those elicited by guidance cues such as Slit and Netrin , and morphogens such as Hhg , FGF , Sonic Hedgehog , Wnts , and BMPs [1 , 2 , 4 , 5] . The importance of HSPGs during animal development has been extensively studied using mutations that disrupt individual HSPG core proteins . A number of HSPGs have been characterized in C . elegans using mutations that affect specific core proteins , such as mutations in sdn-1/syndecan , lon-2/glypican , cle-1/collagen type XVIII , unc-52/perlecan , gpn-1/glypican , and agr-1/agrin [6–26] . These studies have uncovered precise roles of individual HSPGs in morphogenesis and nervous system development . For instance , loss of cle-1/collagen type XVIII leads to synaptic defects at neuromuscular junctions , as well as specific neuronal cell and axon guidance defects [6 , 7]; unc-52/perlecan promotes ectopic presynaptic bouton growth and affects the 4° dendritic branching of the neuron PVD [24 , 25]; sdn-1/syndecan mutants exhibit a number of neuronal cell and axon guidance defects [12–14 , 21]; and lon-2/glypican is important for cell and axon guidance [8 , 12 , 17 , 21] , particularly for netrin-mediated guidance events [8 , 13] . Moreover , studies where two or three HSPG core proteins have been simultaneously mutated in double and triple mutants demonstrated that the combined actions of precise HSPGs ensure proper guidance of neurons and axons during development [8 , 12 , 13 , 21] . All these analyses of mutations affecting specific HSPG core proteins have been instrumental to address the roles of HSPGs in development . However , mutations that remove particular HSPG core proteins inevitably also remove the HS chains that would have been attached to the missing core proteins . Therefore , the phenotypic consequences of such mutations in HSPG core proteins can be due to the absence of either the core protein or the HS chains , or both , making it difficult to extract the functional contribution of the HS chains per se with such analysis of HSPG core protein mutants . To address the roles of the HS chains that are attached to HSPG core proteins , various mutations that affect HS chain biosynthesis have been analyzed [1] . HS chains are linear glycosaminoglycan ( GAG ) polysaccharides composed of alternating repeats of D-glucuronic acid ( GlcA ) and N-acetylglucosamine ( GlcNAc ) [27] . HS chain biosynthesis in the Golgi apparatus can be divided into three phases: ( 1 ) initiation , ( 2 ) elongation , and ( 3 ) chemical modifications . First , a HS chain is initiated by the addition of a tetrasaccharide linker ( synthesized by the step-wise addition of a xylose residue , a galactose residue , a galactose residue , and a GlcA residue ) on a specific Ser residue of the HSPG core protein . This initiation step is catalyzed by a set of four initiation enzymes encoded by the glycosyltransferases genes sqv-6 , sqv-3 , sqv-2 , and sqv-8 in C . elegans [28–30] . Mutations in these four initiation genes have been characterized , revealing important morphogenetic roles in embryogenesis and vulva development [28 , 29 , 31] . However , these four initiation enzymes add the same tetrasaccharide linker also to the core proteins of chondroitin sulfate proteoglycans ( CSPGs ) , as they also catalyze the initiation of chondroitin sulfate ( CS ) chains . Thus , specific roles for HS chains cannot be addressed in these mutants in which the initiation of both HS and CS chains is affected , with phenotypes resulting from the combined disruption of both HSPGs and CSPGs . Once initiated , the second phase of HS biosynthesis is the elongation of HS chains . HS chain elongation is catalyzed by the HS copolymerase , a heterodimer composed of two glycosyltransferases of the EXT family . HS chain elongation has been shown to be crucial to animal development across metazoans , as its dysfunction results in pleiotropic consequences including abnormal morphogenesis and tumor growth . In C . elegans , null mutations in the exostosin glycosyltransferases rib-1 and rib-2 are embryonic lethal , indicating that HS elongation is essential for morphogenesis [32–34] . In Drosophila , null mutations in the exostosin genes tout-velu , brother of tout-velu and sister of tout-velu are lethal , and loss of their function leads to severe patterning defects with abnormal morphogen signaling in many developmental contexts [35–39] . For example , tout-velu mutants exhibit a lack of diffusion of Hh in the wing imaginal disc [35] . In zebrafish , mutations in EXT family members ext2 ( dackel ) and extl3 ( boxer ) are also lethal [40] . In mice , complete loss of function of EXT1 induces defective gastrulation and embryonic lethality [41] . Mutations in glycosyltransferases genes EXT1 and EXT2 in humans result in a dominant disorder called hereditary multiple exostoses , characterized by cartilage-capped skeletal tumors known as osteochondromas , which results in skeletal abnormalities and short stature [42–47] . Although the osteochondromas are most often benign tumors , malignant transformation into chondrosarcomas or osteosarcomas occurs in ~2% of HME patients [48] . However , their exact roles in bone development and homeostasis are not well understood . Given the lethality associated with the complete loss of HS elongation across species , genetic analysis of such mutations has been possible for early developmental roles or for conditions where the gene function is only partially lost . For instance , conditional knockouts have been used to study later developmental roles of the HS copolymerase in mice [49 , 50] , and partially maternally rescued mutants ( displaying partial phenotypes ) have been studied in zebrafish [40] and in C . elegans [21 , 32–34] . Indeed , C . elegans deletion mutations in the HS copolymerase genes rib-1 and rib-2 , namely rib-1 ( tm516 or ok556 ) and rib-2 ( tm710 or qa4900 ) die as embryos: homozygous mutant progeny from a homozygous mutant mother ( animals genotypically m-/- z-/- , where “m” and “z” indicate the maternal and zygotic genotypes , respectively ) , all die as embryos . In contrast , first generation homozygous mutant animals from a heterozygous mother ( that is to say animals that are genotypically rib-1m+/- z-/- or rib-2m+/- z-/- ) are “maternally rescued”; they complete development and become adults , due to wild-type gene product inherited from their heterozygous mothers ( Table 1 ) [32–34] . Such adult rib-1 ( tm516 ) m+/- z-/- and rib-2 ( qa4900 ) m+/- z-/- animals are largely but not completely maternally rescued , as they display locomotion defects and cannot lay eggs normally , becoming filled with their dead m-/- z-/- progeny [32–34] . It has been shown that one quarter of rib-1 ( tm516 ) m+/- z-/- maternally rescued animals exhibit axon guidance defects for the neuron HSN , while no HSN defect was seen in rib-2 ( tm710 ) m+/- z-/- maternally rescued animals [21 , 32] . Thus , a thorough phenotypic analysis of rib-1 and rib-2 deletion mutants has been limited by both ( a ) the early embryonic lethality of rib-1m-/- z-/- and rib-2m-/- z-/- mutants , where later developmental stages cannot be examined , and ( b ) the presence of wild-type maternal product in rib-1m+/- z-/- and rib-2m+/- z-/- , which profoundly rescues development , masking HS functional requirements [21 , 32–34] . The function of HS elongation in C . elegans has also been examined by RNAi knockdown of rib-1 and rib-2 , where the HSN axon guidance defects were more penetrant than in maternally rescued animals [17] . Yet , developmental defects are likely partially penetrant as there is no phenocopy of embryonic lethality by RNAi knockdown of rib-1 and rib-2 [17] . Thus , the availability of partial loss-of-function mutations for the genes rib-1 and rib-2 would allow , if viable , a systematic study of developmental roles for HS elongation during the development of the nervous system in C . elegans . After HS chain elongation , the third phase of HS biosynthesis is the chemical modification of HS chains by modifying enzymes such as epimerases and sulfotransferases [1] . Research in C . elegans has elegantly addressed the requirements for HS chain modifications in nervous system development . Specific roles in the guidance of neuronal cell and axon migrations , including the modulation of distinct guidance cues , have been uncovered using mutations in the HS modifying enzymes epimerase hse-5 and in the sulfotransferases hst-2 , hst-3 . 1 , hst-3 . 2 , and hst-6 [9–12 , 14 , 15 , 18–22 , 26] . For instance , in the contexts of PVQ axon guidance and D-type motoraxon guidance , slt-1/Slit signaling acts in the same pathway as hse-5 and hst-6 , suggesting HSPGs modified by hse-5 and hst-6 may function with slt-1/Slit to guide the axons of both PVQ and motorneurons [10] . In addition , ectopic hypodermal expression of hst-6 disrupts the guidance of the axon of the motorneuron DB7 , and is dependent upon both lon-2/glypican and slt-1/Slit , suggesting that ectopic hypodermal 6O-sulfated LON-2/glypican may disrupt axon guidance through impacting slt-1/Slit signaling [11] . Also , interactions between HS modification enzymes and ephrin and integrin signaling have also been investigated in the contexts of PVQ and motoraxon guidance [10] . In contrast , whereas the core protein LON-2/glypican has been shown to function in netrin-mediated guidance [8] , it remains to be determined which specific HS chain modifications may be important for the unc-6/Netrin signaling pathway . Collectively , all of this remarkable prior work on the roles of HSPGs for nervous system development in C . elegans , targeting either core proteins or HS chain chemical modifications , has yielded a view in which sets of specific HSPGs , with distinct HS chemical modification patterns , interact with specific guidance pathways and contribute to context-dependent guidance decisions during the nervous system assembly . However , a general outlook on the functions of HSPGs in nervous system development by specifically disrupting the presence of HS chains across all HSPGs has been unavailable . Here we report the identification of viable partial loss-of-function mutations in the two HS copolymerase glycosyltransferase genes rib-1 and rib-2 of C . elegans . We show that these mutations reduce HS levels and affect cell and axonal migration during nervous system development . We find that the HS copolymerase is expressed dynamically during morphogenesis , and that expression is sustained throughout life in specific cell types , consistent with HSPGs playing both developmental and post-developmental roles . Our findings indicate that proper axon guidance during the development of the nervous system requires coordinated HS chain elongation in both the migrating neuron itself and adjacent cells that secrete the extracellular matrix along which the growth cone extends . Our analysis highlights the functional importance of HSPGs during animal development .
The analysis of uncoordinated mutants of C . elegans has uncovered key genes underlying nervous system development and function over the past decades [51–53] . In order to identify new genes required for neuronal development , a genetic screen searching for maternally-rescued uncoordinated mutants was carried out by Hekimi et al . [54] . Briefly , an F2 clonal screen was performed , where P0 animals were mutagenized , from which individual F1 hermaphrodites were isolated and allowed to self-fertilize , followed by the selection of individual wild-type F2 hermaphrodites that were allowed to self-fertilize , to finally screen for and select broods where all the F3 animals were uncoordinated/abnormal [54] . Such a scheme allowed for the isolation of maternally rescued mutants , where F2 animals appear phenotypically normal even though they are genotypically homozygous mutant ( m+/- z-/- ) , due to wild-type gene product provided by the heterozygous F1 mother . Only in the next generation of homozygous mutants derived from a homozygous mutant mother , namely F3 m-/- z-/- animals , does the abnormal phenotype manifest . Several maternally rescued uncoordinated mutants were isolated in this screen [54–56] , two of which , qm32 and qm46 , had similar phenotypes and were mutations in genes mum-1 and mum-3 , respectively ( mum stands for maternal-effect uncoordinated and morphologically abnormal ) [54] . mum-1 ( qm32 ) and mum-3 ( qm46 ) homozygous mutant animals from a homozygous mutant mother have severe defects: 32% of mum-1 ( qm32 ) m-/- z-/- and 15% of mum-3 ( qm46 ) m-/- z-/- die as embryos , and of the embryos that hatch , 80% of mum-1 ( qm32 ) m-/- z-/- larvae and 26% of mum-3 ( qm46 ) m-/- z-/- larvae die before reaching adulthood and display morphological abnormalities ( [54]; summarized in Table 1 ) . Fortunately , a proportion of mum-1 ( qm32 ) m-/- z-/- and mum-3 ( qm46 ) m-/- z-/- mutant animals are viable and complete development to become fertile adults [54] , which allows the easy propagation of the homozygous mutant strains . In fact , 14% of mum-1 ( qm32 ) m-/- z-/- animals and 63% of mum-3 ( qm46 ) m-/- z-/- animals reach adulthood , all of which are uncoordinated and egg-laying defective [54] . Importantly , mum-1 ( qm32 ) and mum-3 ( qm46 ) mutations are recessive: animals genotypically heterozygous ( z+/- ) exhibit a wild-type phenotype , irrespective of the maternal genotype ( m-/- or m+/- ) [54] . Moreover , mum-1 ( qm32 ) m+/- z-/- and mum-3 ( qm46 ) m+/- z-/- animals are fully maternally rescued: m+/- z-/- animals develop normally , display no lethality or morphological abnormalities , and become adults that are indistinguishable from the wild type , locomoting and laying eggs normally [54] . Worth noting , this maternal rescue effect is incomplete when both mum-1 ( qm32 ) and mum-3 ( qm46 ) mutations are combined . Indeed , first generation double homozygous mutant animals from doubly heterozygous mothers , i . e . mum-1m+/- z-/-; mum-3m+/- z-/- , develop into adults that are uncoordinated and egg-laying defective , becoming bloated with a brood of dead embryos that are mum-1m-/- z-/-; mum-3m-/- z-/- double homozygous mutants . Thus , the double mutant mum-1 ( qm32 ) ; mum-3 ( qm46 ) could not be generated ( we attempted to build it by several crossing schemes ) , as 100% of the animals die as embryos when there is no wild-type maternal contribution . Finally , the phenotype of both mum-1 ( qm32 ) and mum-3 ( qm46 ) over a deficiency is lethal , suggesting that the null phenotype of these two genes is lethal [54] . To uncover what genes were disrupted in the mutants qm32 and qm46 we determined the molecular identity of the lesions in these two mutants . We found that qm32 and qm46 are alleles of the genes rib-1 and rib-2 , respectively , as we demonstrate here . First , we narrowed down the genetic position of mum-1 ( qm32 ) by genetic mapping and then assayed cosmids corresponding to the genetic position of mum-1 ( qm32 ) for transformation rescue ( Fig 1B ) . We found that cosmid F12F6 fully rescued the mum-1 ( qm32 ) mutants for uncoordination , egg laying defects , and abnormal larval morphology and lethality ( Fig 1A and 1B ) . We tested PCR products corresponding to each of the genes located on this cosmid and found that a 9 kb PCR product containing the gene F12F6 . 3/rib-1 fully rescued all of the above mutant phenotypes of mum-1 ( qm32 ) ( Fig 1A and 1B ) . In addition , construct Prib-1::rib-1 ( + ) completely rescued the cell and axon guidance defects of the neuron AVM , the axon guidance defects of the neurons PVQ in mum-1 ( qm32 ) mutants ( Fig 1C ) , and their behavioral defects . We verified the predicted exon structure of the gene by sequencing cDNA clone yk187a9 . We sequenced the genomic region of rib-1 in mum-1 ( qm32 ) mutants and found that the qm32 molecular lesion is a T to A base pair change at position 39528 of cosmid F12F6 , which converts the Stop codon of rib-1 into a Lys residue ( Fig 1B ) . Thus , mum-1 ( qm32 ) corresponds to the gene previously known in the literature as rib-1 , and we will refer to mum-1 ( qm32 ) as rib-1 ( qm32 ) from now on . The gene rib-1 is homologous to exostosin family members mammalian EXT1 and Drosophila tout-velu , and thus encodes one of the two glycosyltransferases that compose the C . elegans HS copolymerase , responsible for HS chain elongation ( see below , Fig 1D ) . The rib-1 ( qm32 ) mutation does not affect the transcript levels of rib-1 as assayed by RT-PCR ( S1B Fig ) . Based on the sequence , it may result in the translation of an open reading frame present in the 3’UTR , which would possibly extend RIB-1 by 114 aa residues until the next in-frame Stop codon . The activity of the mutant RIB-1 protein in rib-1 ( qm32 ) , or of the complex in which it functions ( see below ) , is affected by the mutation . The rib-1 ( qm32 ) mutation is fully recessive , fully maternally rescued , and is completely rescued by expression of wild-type transgenic copies of the gene [54] ( this study ) , suggesting that the predicted protein extension diminishes RIB-1 activity in rib-1 ( qm32 ) mutants , rather than being neomorphic . Consistent with the notion that qm32 is a partial loss-of-function mutation , the phenotype of qm32 over a deficiency is more severe ( i . e . , lethal [54] ) , and the phenotype of null deletion alleles rib-1 ( tm516 ) and rib-1 ( ok556 ) is also more severe than that of rib-1 ( qm32 ) , as 100% of rib-1 ( tm516 ) m-/- z-/- and rib-1 ( ok566 ) m-/- z-/- animals die as embryos [32 , 33] , compared to 32% embryonic lethality in rib-1 ( qm32 ) m-/- z-/- [54] . In sum , these data indicate that the mutation qm32 is a hypomorphic mutation of the gene rib-1 , where residual function allows 14% of the rib-1 ( qm32 ) m-/- z-/- mutants to be viable and become uncoordinated and egg-laying defective adults . The second C . elegans HS glycosyltransferase and subunit of the HS copolymerase that catalyzes HS chain elongation is encoded by the gene rib-2 . Given the phenotypic similarities between the mum-1/rib-1 ( qm32 ) and mum-3 ( qm46 ) mutants and that the genetic mapping position of mum-3 ( qm46 ) corresponded to a chromosomal interval containing the gene rib-2 , we determined whether mum-3 ( qm46 ) was an allele of rib-2 . We tested a 5 . 6 kb PCR product containing rib-2 ( + ) for rescue of mum-3 ( qm46 ) mutants and found that their defects in larval development , locomotion , and egg laying were fully rescued by this transgene ( Fig 2A and 2B ) . In addition , construct Prib-2::rib-2 ( + ) completely rescued the cell and axon guidance defects of the neuron AVM and axon guidance defects of the neuron PVQ in mum-3 ( qm46 ) mutants ( Fig 2C ) , as well as their developmental and behavioral defects . We verified the predicted exon structure of the gene by sequencing cDNA clone yk3c1 . We sequenced the genomic region of the gene rib-2 in the mum-3 ( qm46 ) mutants and found that the qm46 molecular lesion changes a G to A at position 4366 of cosmid K01G5 . The qm46 mutation results in an Arg to Gln amino acid substitution at conserved residue 434 , which is near the exostosin domain in the 814 amino acid long RIB-2 protein ( Fig 2D ) . Thus , mum-3 ( qm46 ) corresponds to the gene previously known in the literature as rib-2 , and we will refer to mum-3 ( qm46 ) as rib-2 ( qm46 ) from now on . The gene rib-2 encodes the second glycosyltransferase subunit of the HS copolymerase and is most homologous to exostosin family members mammalian EXTL3 and Drosophila brother of tout-velu , and also fills the functional roles of EXT2 and Drosophila sister of tout-velu in C . elegans ( Figs 2D and 3A ) . Consistent with qm46 being a missense mutation , the levels of rib-2 transcript are comparable to wild type ( S1B Fig ) . The rib-2 ( qm46 ) mutation is fully recessive , fully maternally rescued , and is completely rescued by expression of wild-type transgenic copies of the gene ( [54]; this study ) , consistent with rib-2 ( qm46 ) being a partial loss-of-function mutation . Moreover , the phenotype of qm46 over a deficiency is more severe ( i . e . , lethal [54] ) , and the phenotype of null deletion alleles rib-2 ( tm710 ) m-/- z-/- and rib-2 ( qa4900 ) m-/- z-/-animals is also more severe as all embryos die [32–34] . In contrast , only 15% of rib-2 ( qm46 ) m-/- z-/- animals die as embryos [54] . Taken together , qm46 is a viable hypomorphic mutation in the gene rib-2 , where residual function allows 63% of the rib-2 ( qm46 ) m-/- z-/- mutants to be viable and become adults that are uncoordinated and egg-laying defective . The genes rib-1 and rib-2 each encode one of the two C . elegans HS glycosyltransferases that elongate HS chains ( Fig 3A ) [32 , 57] , which are composed of alternating GlcA and GlcNAc residues . After a tetrasaccharide linker has been synthesized on the HSPG core protein , the first step for HS chain elongation is the addition of a GlcNAc residue ( Fig 3A ) [57] . The addition of the first GlcNAc residue is catalyzed by RIB-2 in C . elegans , as demonstrated biochemically [57] , similar to EXTL3 in mammals [58] , and Brother of tout-velu in Drosophila [59] . HS chain elongation then proceeds by the repeated addition of disaccharide units of GlcA and GlcNAc ( Fig 3A ) [32] . This alternating addition of GlcA and GlcNAc is catalyzed by a heterodimer of RIB-1 and RIB-2 , as demonstrated biochemically [32 , 57] , similar to EXT1 and EXT2 in mammals [60–63] , and Tout-velu ( Ttv ) and the Sister of tout-velu in Drosophila [59] . Given the biochemically demonstrated roles of RIB-1 and RIB-2 [32 , 57] , the rib-1 ( qm32 ) and rib-2 ( qm46 ) mutations are expected to reduce HS chain elongation and result in decreased levels of HS in these mutants . To directly determine total HS levels in the rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants , we performed Western blot analysis . For this , we extracted proteins from wild-type ( N2 ) animals , rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- single mutants . We treated the protein extracts from these strains with a mix of heparinases I and III , and performed Western blot analysis using an antibody that specifically recognizes heparinase-digested HS chains ( 3G10 , [64] ) . As expected , no signal above background was detected in untreated control samples ( three left lanes , Fig 3B ) , compared to heparinase-digested samples ( three right lanes , Fig 3B ) . Indeed , among the heparinase-digested samples , we found that compared to wild type , the HS content was severely reduced in rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants ( Fig 3B ) , confirming that the rib-1 ( qm32 ) and rib-2 ( qm46 ) mutations decrease HS biosynthesis , as predicted from their known biochemical functions [32 , 57] . We examined whether the HS level reduction observed in these mutants could be rescued by transgenic expression of rib-1 ( + ) and rib-2 ( + ) , respectively . For this , we extracted proteins from a strain of rib-1 ( qm32 ) m-/- z-/- mutants carrying a rib-1 ( + ) -containing extrachromosomal array , and from a strain of rib-2 ( qm46 ) m-/- z-/- mutants carrying a rib-2 ( + ) -containing extrachromosomal array . We found that transgenic expression of rib-1 ( + ) and rib-2 ( + ) re-elevates HS levels in the rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants , respectively ( right most lanes in Fig 3B ) . HS levels rescue appears incomplete likely due to the fact that by the time that the worms populations from these strains were collected , only ~10–20% of the animals actually carried the rescuing transgene ( extrachromosomal arrays are lost at some frequency during cell divisions and over the course of generations [65] ) . Nevertheless , our results clearly indicate that the alleles of rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- strongly reduce the levels of HS compared to the wild type and that copies of the wild-type transgene re-elevate the HS levels . These results support that the rib-1 ( qm32 ) and rib-2 ( qm46 ) mutations reduce the function of the genes which are important for HS elongation , consistent with prior biochemical demonstration of their function [32 , 57] . Having examined how rib-1 ( qm32 ) and rib-2 ( qm46 ) mutations impact global HS levels , we further examined their consequences on two specific HSPGs , LON-2/Glypican and SDN-1/Syndecan . In these experiments , to detect LON-2/Glypican , we expressed green fluorescent protein ( GFP ) -tagged LON-2 ( LON-2::GFP , [66] ) . We carried out Western blot analysis using anti-GFP antibodies as a probe . Whereas two high molecular weight bands corresponding to LON-2::GFP and HS-modified LON-2::GFP are detected in wild-type lysates , only one of the bands is detected in lysates of rib-1m-/- z-/- and rib-2m-/- z-/- mutants ( Fig 3C ) , indicating that HS synthesis onto LON-2/Glypican is affected by loss of rib-1 or rib-2 function . Consistent with this interpretation , wild-type worms expressing a mutant version of LON-2 in which the HS attachment sites are mutated ( LON-2ΔGAG::GFP , [67] ) displayed a single high molecular weight band that migrated to the same molecular weight as LON-2::GFP when expressed in rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) mutantsm-/- z-/- ( Fig 3C ) . We next analyzed HSPG SDN-1/Syndecan using a similar strategy . We expressed GFP-tagged SDN-1/Syndecan ( SDN-1::GFP , [14] ) in wild-type and rib-1 ( qm32 ) m-/- z-/- mutant worms , and probed for GFP in lysates of these worms . In wild-type lysates , we detected two high molecular weight bands corresponding to SDN-1::GFP and HS-modified SDN-1::GFP , but only detected a single band in lysates of rib-1 ( qm32 ) m-/- z-/- mutants ( Fig 3D ) , indicating that loss of rib-1 impairs HS synthesis onto SDN-1/Syndecan . Thus , our results provide compelling evidence that rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutations drastically reduce HS content , consistent with these mutations impairing HS chain elongation . As a note , using fluorescence microscopy , we observed that the expression of LON-2::GFP in rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants was similar to wild type , and that SDN-1::GFP was similar to wild type in rib-1 ( qm32 ) m-/- z-/- mutants ( S2 Fig ) . rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants are uncoordinated and egg-laying defective . To gain insight into the impact of HS chain elongation on neuronal development , we set out to characterize the neuroanatomy of rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/-mutants . For this , we built strains of the rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 m-/- z-/- mutants carrying integrated transgenes to drive the expression of fluorescent proteins and allow the visualization of specific neurons ( see S9 Table ) . We examined the nervous system of rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- adult viable mutants and found that the overall organization of the nervous system is grossly normal , as neuronal ganglia , axon fascicles and isolated neurons were generally well laid out . Examination with single-cell resolution revealed that numerous neuronal migrations are affected in both mutants . For instance , the CAN neuron cell body , which migrates from the head region towards the midbody region in wild-type animals , is frequently positioned too anteriorly or too posteriorly in both mutants ( Fig 4A ) . Also , the HSN neuron cell body , which migrates from the tail region to the midbody region in wild type , is often located too posteriorly in both rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants ( Fig 4B ) . Moreover , the AVM neuron cell body is frequently located in the posterior of the body instead of being anterior to the vulva ( Fig 4C ) . The penetrance and expressivity of these defects is similar in both mutants . Thus , loss of function of the genes rib-1 or rib-2 impairs the guidance of diverse neurons that undergo long-range migrations during development . We also found that axonal projections are defective in rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- single mutants . For example , the axon of the interneuron PVQ , which projects into the ipsilateral fascicle of the ventral nerve cord in the wild type , frequently projects in the contralateral fascicle or even laterally in rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants ( Fig 4D ) . Similarly , the axon of the motorneuron HSN , which projects ventrally and into the ipsilateral fascicle of the ventral nerve cord in the wild type , is misguided in the rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants as it projects into the contralateral fascicle or laterally in these mutants ( Fig 4B ) . These defects in HSN axon guidance are consistent with those reported for RNAi knockdown of rib-1 and rib-2 , as well as in maternally rescued rib-1 animals [17 , 32] . Another example is the axon of the mechanosensory neuron AVM , which extends ventrally towards the ventral nerve cord in the wild type , projects laterally in the mutants ( Fig 4C ) . The axons of cholinergic and GABAergic motorneurons are also misguided in rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants: contrary to the wild type , where most motorneuron axons exit the ventral midline on the right side to migrate along on the right side of the worm’s body wall , many motorneuron axons abnormally project to the left side in rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants ( Fig 5A ) . Finally , the dorsal nerve cord , which is composed of several motoraxons that run as a single fascicle in the wild type , is frequently defasciculated into several bundles in rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants ( Fig 5B ) . Thus , the guidance of numerous axons is disrupted upon loss of function of the genes rib-1 and rib-2 . In a similar way , the migration of mesodermal cells , which share guidance mechanisms with neurons [68] , is defective in rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- single mutants . For instance , the canals of the excretory cell ( two anterior and two posterior canals ) run laterally in the wild type but are frequently too short or extend along the ventral or dorsal aspect of the body in rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants ( Fig 6A ) . Another example of misguided mesodermal cells in the rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants is that of the distal tip cell ( DTC ) , whose path determines the shape of the gonad . In wild-type animals , the anterior gonad arm is located on the right side of the animal , as the anterior DTC migrates along the right side , first anteriorly , then turning dorsally , and migrating back posteriorly towards the midbody region . Similarly , the posterior gonad arm is located on the left side , as the posterior DTC migrates posteriorly , turns dorsally and migrates back towards the midbody region . In rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants , the sidedness of the gonad arms is often abnormal , with the anterior arm of the gonad on the left side of the animal and the posterior arm on the right side , or even having both gonad arms on the opposite side of the animal ( Fig 6B ) . Lastly , rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants display abnormal positioning of the excretory glands ( Fig 6C ) . Thus , loss of function of the genes rib-1 or rib-2 disrupts the guidance of migrations of neuronal and mesodermal cells during development . To gain insight into the roles of HSPGs during development , we determined the expression pattern of the HS copolymerase . For this , we designed a transcriptional fusion , Prib-1::gfp , between the upstream regulatory region of rib-1 and gfp . Since rib-1 is the second gene in a two-gene operon [69] , we included the region upstream of the first gene in the operon , as well as the intergenic region of the operon that lies immediately upstream of rib-1 ( see Materials and Methods ) . Second , we constructed a translational fusion for rib-1 using the same upstream regulatory region as for Prib-1::gfp , and fusing the coding region of rib-1 with venus , a gfp variant that fluoresces in acidic cellular environments [70] , to make the translational fusion Prib-1::rib-1::venus . We generated at least five transgenic lines for each of these two rib-1 reporters and examined transgenic animals by fluorescence microscopy . We observed that the GFP signal from the transcriptional fusion Prib-1::gfp fills the cytoplasm of expressing cells , whereas the VENUS signal displays a punctate cytoplasmic pattern in cells expressing the translational fusion Prib-1::rib-1::venus , consistent with RIB-1 being localized to the Golgi apparatus ( Fig 7A ) . Moreover , we found that both the transcriptional and translational fusions have a very similar spatial and temporal expression pattern during development: expression was visible in neurons , hypodermal cells , muscles of the digestive system , and reproductive tissues ( Fig 7A ) . Importantly , we found that the translational fusion Prib-1::rib-1::venus is functional , as it fully rescues the defective locomotion , egg-laying , morphology , and axon guidance of rib-1 ( qm32 ) m-/- z-/- mutants . Our observations indicate that the observed expression pattern of the translational reporter and of the very similarly expressed transcriptional reporter are functionally relevant and largely reflect sites of endogenous rib-1 expression . Since the transcriptional and translational rib-1 fusions have similar expression patterns , we used Prib-1::gfp , which has a stronger expression level , to characterize the expression pattern of rib-1 in more detail . We found that Prib-1::gfp is broadly expressed in ectodermal and mesodermal cells during embryogenesis . A salient feature of the rib-1 expression pattern is that it is very dynamic in hypodermal cells during development . In embryogenesis , Prib-1::gfp is detected along the entire layer of hypodermoblasts that surrounds the gastrulating embryo at about 200 minutes after fertilization . By the early comma stage of embryogenesis , Prib-1::gfp is expressed at high levels in hypodermal cells of the elongating embryo ( Fig 7C ) , including hypodermal cells extending ventrally during ventral closure and in the two rows of dorsal hypodermal cells undergoing dorsal intercalation . Following these embryonic morphogenetic events , expression of Prib-1::gfp in the hypodermal cells of the body wall is no longer visible during larval and adult stages , except for seam cells undergoing fusion during larval development . Also , hypodermal cells of the developing vulva express Prib-1::gfp ( Fig 7D ) , at a low expression level in L3 larvae and at a stronger level in L4 larvae and just molted young adults , and vanishing in vulval cells in the adult . These dynamic expression patterns in cells undergoing dramatic changes during morphogenesis suggest a potential for rapidly changing needs for particular HSPGs in specific tissues at different time points during development . The nervous and digestive systems express Prib-1::gfp stably and continuously from embryogenesis throughout adulthood . Strong and sustained expression is seen in motorneurons , interneurons , sensory neurons ( including AVM ) , neurons in the head and tail ganglia , with the GFP signal filling axons running along the ventral and dorsal nerve cords , commissures , and sublaterals . Expression in neurons of the ventral nerve cord and of the head ganglia is visible in 1 . 5- , 2- , and 3-fold embryos , and persists into adulthood ( Fig 7A and 7B ) . Strong expression of Prib-1::gfp is also observed in the pharynx from the 2-fold stage of embryogenesis onwards and remained strong in adults ( procorpus , metacorpus , terminal bulb , grinder , and pharyngeal-intestinal valve ) . The anal depressor , the anal sphincter , the two enteric muscles , the spermathecae and the uterine muscles maintain expression in adults ( Fig 7B ) . The continued expression of rib-1 in the nervous , digestive and reproductive systems suggests that HSPGs play not only developmental , but also post-developmental roles in these cell types . A prominent site of expression of the HS copolymerase is the nervous system , including during axon migration in embryonic and larval development ( Fig 7 ) , and disruption of the HS copolymerase in rib-1 ( qm32 ) m-/- z-/- or rib-2 ( qm46 ) m-/- z-/- mutants leads to numerous axon guidance defects ( Fig 4 and Fig 5 ) . To determine in which cells HS production is required for axon guidance , we provided rib-1 ( qm32 ) m-/- z-/- mutants with wild-type copies of rib-1 ( + ) in subsets of cells and assessed rescue of the PVQ axon guidance defects . The axon of PVQ extends along the ventral nerve cord during embryogenesis , following the path of other axons , and is in proximity with the hypodermis and body wall muscles . We built constructs to express rib-1 ( + ) in neurons including PVQ ( using the heterologous promoter Prgef-1 ) , in the hypodermis ( using the heterologous promoter Pdpy-7 ) , or in body wall muscles ( using the heterologous promoter Pmyo-3 ) . We then generated transgenic rib-1 ( qm32 ) worms expressing rib-1 ( + ) in these tissues and analyzed PVQ axon guidance . As a control , we determined that expression of rib-1 ( + ) under its own promoter completely rescued the guidance defects of the PVQ axon ( Fig 8A ) . Targeted expression of rib-1 ( + ) only in neurons , only in the hypodermis , or only in body wall muscles did not rescue the rib-1 mutant PVQ axon guidance defects . However , co-expression of rib-1 ( + ) simultaneously in the hypodermis , neurons , and body wall muscles led to a significant rescue of these defects ( Fig 8A ) , suggesting that HSPGs derived from specific cell types together contribute to proper PVQ axon guidance . The rescue of the PVQ axon was strong but incomplete , possibly due to the inappropriate rib-1 expression level or timing under these heterologous promoters . Nonetheless , expressing rib-1 simultaneously in these three tissues yielded a significant rescue of the PVQ defects , indicating a simultaneous functional requirement for HSPGs in distinct cell types to regulate the guidance of the PVQ axon . We next turned to elucidating the spatial requirements of HS biosynthesis for guidance of the mechanosensory neuron AVM . During the first larval stage , the AVM axon pioneers its own ventral migration through a basement membrane along the body wall , sandwiched between the hypodermis and body wall muscles . We expressed rib-1 ( + ) in the hypodermis ( using the heterologous promoter Pdpy-7 ) , in body wall muscles ( using the heterologous promoter Pmyo-3 ) , or in AVM itself ( using the heterologous promoter Pmec-7 ) in rib-1 ( qm32 ) m-/- z-/- mutants , and analyzed AVM axon guidance . As a control , we determined that expression of rib-1 ( + ) under its own promoter completely rescued the guidance defects of the AVM axon ( Fig 8B ) . We found that the AVM axon guidance defects of rib-1 mutants were rescued by expression of rib-1 ( + ) in AVM itself , or by expressing rib-1 ( + ) in the hypodermis ( Fig 8B ) . These results suggest that HSPGs derived from both AVM and the hypodermis crucially impact AVM axon guidance . Taken together , our results support the notion that HSPGs synthesized in distinct cell types coordinate guided axonal migration during development . Prior studies have addressed roles of specific HSPG core proteins and HS chain modifications in Netrin- and Slit-mediated axon guidance in worms [8 , 10–15 , 18 , 71] , flies [72 , 73] , and mice [49] . HS chain elongation per se has been implicated in Netrin- and Slit-mediated axon guidance using in vitro spinal cord and retinal explant assays [74 , 75] . However , an in vivo study of the impact of HS chain elongation in Netrin- and Slit-mediated guidance events has been lacking . To address how globally disrupting HS chain elongation affects guidance events that require the unc-6/Netrin or slt-1/Slit pathways , we studied the guidance of the AVM axon ( Fig 9A ) . Two complementary and highly conserved guidance pathways guide the AVM axon: attraction mediated by the UNC-40/DCC receptor towards ventral UNC-6/Netrin , and repulsion mediated by the SAX-3/Robo receptor away from dorsal SLT-1/Slit ( Fig 9B ) [68 , 76–81] . Simultaneous complete loss of both unc-6/Netrin and slt-1/Slit function leads to fully penetrant AVM axon guidance defects , where ~95% of AVM axons fail to extend ventrally , as demonstrated in [77] ( also reproduced by [8] ) . We found that rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants are defective in AVM ventral axon guidance ( Fig 4C ) , a result that is consistent with the notion that loss of HS chain elongation may affect signaling through either unc-6/Netrin signaling , slt-1/Slit signaling , or both . It is also possible that an unidentified pathway , also involving HS chains , may help guide AVM ventrally . To test whether HS elongation contributes to both Netrin and Slit signaling pathways , we constructed double mutants of rib-1 ( qm32 ) and rib-2 ( qm46 ) with mutations in unc-6/Netrin and slt-1/Slit . Because rib-1 and rib-2 null alleles are lethal , we used the hypomorphic alleles rib-1 ( qm32 ) and rib-2 ( qm46 ) . For unc-6 , we used the hypomorphic allele e78 [82] , as we found that double mutants with the null allele unc-6 ( ev400 ) [83] rib-1 ( qm32 ) ;unc-6 ( ev400 ) and rib-2 ( qm46 ) ;unc-6 ( ev400 ) died as embryos . For slt-1 , we used the presumptive null allele eh15 [77] , as well as a condition of altered slt-1/Slit signaling , where misexpressing slt-1/Slit in all body wall muscles ( using Pmyo-3::slt-1 ) , leads to AVM axon guidance defects [84] . We found that all four double mutants ( a ) rib-1 ( qm32 ) m-/- z-/-;unc-6 ( e78 ) , ( b ) rib-2 ( qm46 ) m-/- z-/-;unc-6 ( e78 ) , ( c ) rib-1 ( qm32 ) m-/- z-/-;slt-1 ( eh15 ) , and ( d ) rib-2 ( qm46 ) m-/- z-/-;slt-1 ( eh15 ) , have AVM axon guidance defects that are more pronounced than the respective single mutants ( Fig 9B ) . Similarly , animals expressing Pmyo-3::slt-1 in the rib-1 ( qm32 ) m-/- z-/- or rib-2 ( qm46 ) m-/- z-/- mutant backgrounds have severe AVM guidance defects compared to the rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- single mutants or to animals misexpressing Pmyo-3::slt-1 in the wild-type background ( Fig 9B ) . While hypomorphic alleles complicate the interpretation of results , the finding that disruption of HS chain elongation alters axonal guidance in an additive manner to dysfunctional unc-6/Netrin and slt-1/Slit signaling , highlights the importance of HS chain elongation in axon guidance and is consistent with a role of HS chain elongation in unc-6/Netrin and slt-1/Slit signaling to guide AVM . To directly test the functional importance of HS chain elongation in unc-6/Netrin signaling , we used a gain-of-function approach that specifically assays a unc-6/Netrin-dependent guidance event . Similar to AVM , the PVM axon is attracted ventrally towards secreted UNC-6/Netrin via the UNC-40/DCC receptor . However , misexpression of the repulsive UNC-6/Netrin receptor , UNC-5/UNC5 , using the transgene Pmec-7::unc-5 ( [85] , Fig 9C ) in PVM , results in an unc-6/Netrin- and unc-40/DCC-dependent abnormal extension of the PVM axon towards the dorsal side of the animal [86] . As controls , dorsal extension of the PVM axon is never observed in wild type or mutants in the unc-6/Netrin or slt-1/Slit signaling pathways ( Fig 9C ) . We focused on the PVM axon in this assay as both the AVM and ALMR axons extend dorsally in Pmec-7::unc-5 transgenic animals rendering AVM indistinguishable from ALMR . We generated rib-1 ( qm32 ) m-/- z-/- or rib-2 ( qm46 ) m-/- z-/- mutant strains carrying the Pmec-7::unc-5 transgene [85] to misexpress unc-5 in PVM . If loss of rib-1 and rib-2 functionally disrupts unc-6/Netrin signaling , we would expect to see a decrease in the unc-5-mediated dorsal migration of PVM . Indeed , we found that rib-1 ( qm32 ) and rib-2 ( qm46 ) loss of function significantly suppressed the unc-6/Netrin-dependent unc-5-mediated dorsal migration of PVM ( Fig 9C , S8 Table ) , indicating that unc-6/Netrin signaling requires HS chain elongation . This suppression of the dorsal extension of the PVM axon by mutations in rib-1 and rib-2 is specific , as individual loss of function of other genes required for guidance , such as sdn-1/Syndecan , slt-1/Slit and sax-3/Robo , did not suppress these abnormal dorsal extensions ( Fig 9C ) . Taken together our observations support the notion that HS chain elongation plays a critical role in unc-6/Netrin-mediated guidance . Furthermore , if unc-6/Netrin and slt-1/Slit signaling pathways were indeed the sole two key pathways guiding the AVM axon ventrally , as the fully penetrant defects of unc-6 slt-1 double null mutants suggest [8 , 77] , then our results would support that HS chain elongation is important for both unc-6/Netrin and slt-1/Slit signaling . This notion is consistent with prior work implicating the HSPG syndecan in slt-1/Slit-mediated guidance [8 , 14 , 72 , 73] , the HSPG glypican in unc-6/Netrin-mediated guidance [8] , and HS modifications in slt-1/Slit signaling [10 , 11] . Once synthesized , HS chains become extensively modified by epimerases and sulfotransferases ( reviewed in [1] , Fig 3A ) . In C . elegans , key modifying enzymes have been studied , including glucuronyl C5-epimerase encoded by hse-5 , 2O-sulfotransferase encoded by hst-2 , and 6O-sulfotransferase encoded by hst-6 [9–11 , 15 , 87] . These HS modifying enzymes are required for axon guidance as mutations disrupting their function impair this process in a number of developmental contexts [1 , 9–15 , 21] . However , the roles of these HS modifying enzymes in the guidance of the AVM axon , which is mediated by unc-6/Netrin- and slt-1/Slit , are unknown . To determine the functional importance of specific HS modifications in AVM axon guidance , we first analyzed single , double , and triple null hse-5 , hst-2 and hst-6 mutants , and found that loss of each single HS modifying enzyme led to minimal AVM axon guidance defects ( Fig 9D ) , as has previously been reported [10] . However , hse-5; hst-6 and hst-2 hst-6 double mutants , in which the 6O-sulfotransferase and either the 2O-sulfotransferase or the C5-epimerase are mutant , display significant AVM guidance defects ( Fig 9D ) . The defects of these two double mutants are not further enhanced by the loss of the third key HS modifying enzyme in hse-5; hst-2 hst-6 triple mutants ( Fig 9D ) . These observations indicate some level of compensation between HS chain modifying enzymes , which has been observed at the biochemical level [87] , and suggest that combinations of types of HS chain modifications impact the guidance of AVM , which relies on unc-6/Netrin- and slt-1/Slit-signaling . Our results add to prior work showing that specific HS chain modifications regulate precise cell and axon migration events in several other contexts , including of migration events that are slt-1/Slit- and unc-6/Netrin-dependent , and through interactions with the slt-1/Slit signaling pathway [10–15 , 18] . Next , we analyzed AVM ventral axon guidance in double mutant animals lacking just one of the HS modifying enzymes , hse-5 , hst-2 , and hst-6 , and unc-6/Netrin or slt-1/Slit . We found that loss of function of any of the HS modifying enzymes enhanced the AVM guidance defects of unc-6/Netrin null mutants . Similarly , loss of any of the HS modifying enzymes hse-5 , hst-2 , or hst-6 enhanced the AVM guidance defects of presumptive null mutants for slt-1/Slit [77] ( Fig 9D ) . These results show that HS chain sulfations and epimerizations carried out by hse-5 , hst-2 , or hst-6 enzymes participate in AVM axon guidance likely through the two key signaling pathways unc-6/Netrin- and slt-1/Slit . These findings are in agreement with prior studies that demonstrated the importance HS chain modifications to neural development in other contexts [9–15 , 18 , 21] .
In this study we have molecularly identified and characterized two mutations that were previously isolated in a forward genetic screen for maternally rescued uncoordinated mutants [54–56] . We show that mum-1 ( qm32 ) and mum-3 ( qm46 ) are partial loss-of-function mutations in the genes rib-1 and rib-2 , respectively , which encode the two exostosin glycosyltransferases that compose the HS copolymerase in C . elegans . The enzymatic activities predicted by sequence homology have been corroborated using bacterially expressed RIB-1 and RIB-2 [32 , 34] . RIB-2 functions as an alpha1 , 4-N-acetylglucosaminyltransferase that has both GlcNAc transferase I and II activities and is involved in the addition of the first GlcNAc residue onto the tetrasaccharide linker , as well as in the elongation of HS chains [34 , 57] . RIB-1 and RIB-2 glycosyltransferases together form a functional heterodimer that catalyzes HS chain elongation [32] . Moreover , HS levels have been shown to be reduced in maternally rescued worms of rib-1 ( tm516 ) m+/- z-/- and rib-2 ( qa4900 ) m+/- z-/- null mutations [32 , 34] . Here we show that homozygous hypomorphic single mutants rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- have profoundly disrupted HS levels: we found that the global HS levels are severely reduced in these single mutants , and that high molecular species of LON-2/Glypican and SDN-1/Syndecan , likely corresponding to the core protein with HS chains attached , are undetectable in the rib-1 ( qm32 ) and rib-2 ( qm46 ) single mutants . These findings indicate that rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- result in a loss of function of the genes rib-1 and rib-2 . It is noteworthy that rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- single mutants each display severe mutant phenotypes , indicating that rib-1 and rib-2 cannot substitute for each other , consistent with their specific biochemical roles in HS chain elongation . In sum , we provide further evidence that the function of RIB-1 and RIB-2 is required for HS biosynthesis in C . elegans . Several observations indicate that rib-1 ( qm32 ) and rib-2 ( qm46 ) are partial loss-of-function mutations: ( a ) their phenotype is less severe than deletion alleles; ( b ) the phenotype of rib-1 ( qm32 ) and rib-2 ( qm46 ) over a deficiency is more severe that in homozygous mutants [54]; and ( c ) simultaneously disrupting both genes results in complete embryonic lethality . Thus , despite the severe reduction in HS in rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- single mutants , residual HS copolymerase activity appears to be sufficient for viability in the single mutants . However , that disrupting both genes in rib-1 ( qm32 ) m-/- z-/-; rib-2 ( qm46 ) m-/- z-/- leads to a more severe phenotype is likely because the HS copolymerase , a heterodimer of RIB-1 and RIB-2 proteins , is more drastically impaired in the double mutants . We propose that HS copolymerase dimers composed of one mutant protein and one wild-type protein might be stabilized by the presence of one normal protein in the complex in single rib-1 ( qm32 ) and rib-2 ( qm46 ) hypomorphic mutants ( i . e . mutant RIB-1 and wild-type RIB-2 in rib-1 ( qm32 ) , and mutant RIB-2 and wild-type RIB-1 in rib-2 ( qm46 ) ) . In contrast to the single mutants rib-1 ( qm32 ) m+/- z-/- or rib-2 ( qm46 ) m+/- z-/- , which are completely maternally rescued [54] , double mutant animals rib-1 ( qm32 ) m+/- z-/-;rib-2 ( qm46 ) m+/- z-/- are not , and instead become severely uncoordinated and egg-laying defective adults ( Table 1 ) . Thus , maternal product deposited in the oocyte is sufficient to support HS copolymerase activity and allow for normal development and behavior in single hypomorphic mutants rib-1 ( qm32 ) m+/- z-/- and rib-2 ( qm46 ) m+/- z-/- , but is insufficient for double hypomorphic mutants . Incomplete maternal rescue effect is observed for single null mutants rib-1 ( tm516 ) m+/- z-/- and rib-2 ( qa4900 ) m+/- z-/- [32 , 34] . High levels of rib-1 and rib-2 transcripts are detected in the germline of C . elegans ( http://nematode . lab . nig . ac . jp/ ) . These observations highlight the importance of HS copolymerase activity , and therefore HSPGs , from the earliest stages of development . RIB-1 and RIB-2 are not expected to affect the biosynthesis of glycosaminoglycans other than HS . In C . elegans , both HS and CS , but not hyaluronate nor dermatan sulfate , have been detected [88 , 89] . Whereas HS and CS chains share the same tetrasaccharide linker to couple the HS or CS chain to their respective core proteins , the elongation of HS and CS chains are carried out by different enzymes . The elongation of CS chains , a polymer of alternating GlcA and N-acetylgalactosamine ( GalNAc ) residues , is catalyzed by a bifunctional glycosyltransferase encoded by the sqv-5 gene [90] . Thus , rib-1 ( qm32 ) and rib-2 ( qm46 ) mutations facilitate the study of the consequences of globally and specifically disrupting HS elongation in live animals . Complete disruption of HS chain elongation in the deletion alleles of rib-1 ( tm516 ) m-/- z-/- and rib-2 ( qa4900 ) m-/- z-/- affects the mutant organism in a pleiotropic fashion , leading to fully penetrant embryonic lethality [32 , 34] , which had limited the systematic study of the impact of HS chain elongation in later developmental processes . We found that a basal level of the required enzymatic activities in the hypomorphic mutants rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- is sufficient to bypass major pleiotropic effects , allowing a proportion of the animals to fully develop and reach adulthood [54] . In these animals that complete development , major morphogenetic movements , such as gastrulation , ventral closure and organogenesis , occurred normally , and their anatomy , including the specification of neuronal identities and the layout of ganglia and major axon fascicles , was grossly normal . This hypomorphic condition allowed us to study the influence of HS chain elongation on the guidance of cell and axon migration in viable animals . We found that disrupting HS chain elongation affects the migrations of neurons and axons , including migrations that occur during embryonic and post-embryonic development , and along both body axes ( antero-posterior and dorso-ventral ) [78] . It is worth noting that the motility per se of migrating cells is not lost in rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants as soma and axons often overshoot their targets . Rather , it is the guidance of migrations during development that is disrupted by the loss of function of rib-1 and rib-2 . The critical role of HS elongation in axon guidance during nervous system development is evolutionarily conserved , as disruption of HS elongation in mice and fish leads to defective axonal guidance [40 , 49] . Previous analyses of the consequences of disrupting HS chain elongation on neuronal development reported weaker defects than those we describe here using rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- . This is not surprising since only maternally rescued animals rib-1 ( tm516 ) m+/- z-/- and rib-2 ( tm710 ) m+/- z-/- , or partial knock down of gene activities with rib-1 ( RNAi ) and rib-2 ( RNAi ) , could be studied before . For example , HSN soma migration is fully normal in maternally rescued rib-2 ( tm710 ) m+/- z-/- [21] and 27% of maternally rescued rib-1 ( tm516 ) m+/- z-/- animals exhibit HSN axon guidance defects [32] . Similarly , rib-1 ( RNAi ) and rib-2 ( RNAi ) lead to a 40–45% penetrance of combined HSN cell and axon guidance events [17] . In contrast , 77% and 74% of rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutant animals exhibit abnormal HSN soma migration , and 100% and 83% of rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutant animals display defects in the guidance of the HSN axon , respectively . This highlights that the newly identified hypomorphic rib-1 ( qm32 ) and rib-2 ( qm46 ) mutants reported here enable the study of HS chain elongation-dependent biological processes . The guidance of multiple migrating neurons and axons is altered in rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants , suggesting that disruption of HS chain elongation impacts several guidance pathways . In particular , our analysis of the ventral axon guidance of AVM shows that HS chain elongation and HS chain modifications , are important for signaling via the unc-6/Netrin and the slt-1/Slit signaling pathways , and perhaps a yet to be identified HS-dependent pathway . That loss of function of rib-1 or rib-2 was able to suppress the unc-6/Netrin-dependent dorsalization of PVM upon ectopic expression of unc-5/UNC5 supports the model that unc-6/Netrin signaling requires HS chains . We have previously shown that HSPG lon-2/Glypican functions with , and is required for , unc-6/Netrin signaling in axon guidance [8] . Interestingly , the core protein of LON-2/glypican , but not its HS chains , functions in the unc-6/Netrin pathway . In fact , two versions of LON-2/Glypican lacking the HS chains are able to function in unc-6/Netrin-mediated axon guidance ( one where the HS attachment sites were deleted [67] , which indeed prevents the addition of HS onto LON-2/Glypican ( Fig 3C ) , and one where LON-2/Glypican is truncated in a way that removes all HS attachment sites , [8] ) . Taken together , the observation that the core protein of LON-2/Glypican , but not its HS chains , functions in unc-6/Netrin signaling , and that HS elongation is required for unc-6/Netrin signaling ( loss of rib-1 or rib-2 suppresses the unc-6/Netrin-dependent effect of unc-5/UNC5 ectopic expression ) , raises the possibility that an additional unidentified HSPG functions in unc-6/Netrin signaling . One HSPG that has a role in unc-6/Netrin guidance in other contexts is unc-52/Perlecan , which affects the guidance timing defects of distal tip cells upon ectopic early expression of unc-5/UNC5 [16] . However , loss of unc-52 alone does not affect AVM axon guidance and does not enhance defects of sdn-1/Syndecan mutants [8] , indicating that unc-52 likely does not participate in AVM guidance . Thus , another unidentified HSPG may function in unc-6/Netrin signaling through its HS chains . Also , the notion that HS chains are important for slt-1/Slit signaling is consistent with prior reports that ( 1 ) HS chain elongation is required for retinal explant axon outgrowth in vitro [74] , ( 2 ) the HSPG gene sdn-1/Syndecan functions in slt-1/Slit signaling to guide the AVM axon [8] ( Fig 10B ) and other axons ( PVQ , [14] ) , and ( 3 ) HSPG Syndecan is key to Slit signaling and distribution in flies [72 , 73] . Furthermore , studies have demonstrated roles for HS chains in slt-1/Slit-mediated guidance in other contexts [10 , 11] , through the study of HS modifying enzymes mutants , however whether it is the modifications of the HS chains on SDN-1/Syndecan specifically that are required for guidance is not known . We analyzed the spatial requirements for the HS copolymerase by focusing on two specific migrating neurons , namely the embryonically migrating PVQ axon and the AVM axon that extends during the first larval stage . In both cases we found that rib-1 expression in several cell types restored function during development of rib-1 mutants . For the guidance of the migrating PVQ axon , combined HS copolymerase expression in the hypodermis , neurons , and body wall muscles of rib-1 mutants was required to rescue PVQ axon guidance defects . This observation indicates that HS chains synthesized onto HSPGs from multiple tissue types cooperate to properly pattern the ventral midline and suggests that distinct HSPGs from specific tissues may contribute to properly guide the PVQ axons at the ventral midline ( Fig 10A ) . Interestingly , the combined loss of two HSPGs , a glypican and a syndecan , in the lon-2 sdn-1 double mutant leads to a penetrance of defects in PVQ guidance similar to rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants [12] , suggesting that PVQ axon guidance defects observed in rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants may reflect a disruption of HS chains onto LON-2/Glypican in the hypodermis and SDN-1/Syndecan in the PVQ neurons . Consistent with this interpretation , lon-2/Glypican has been found to function non-cell autonomously in the hypodermis to guide the axon of AVM [8] , and sdn-1/Syndecan has been shown to function cell autonomously in the migrating neuron in a variety of contexts , such as AVM axon guidance [8] , PVQ axons , HSN soma , and ALM soma [14] . Furthermore , PVQ axon guidance likely requires that the specific HS chains on core HSPGs not only be synthesized but also modified , as the combined loss of HS modifying enzymes also leads to PVQ axon guidance defects [10] . Indeed , loss of the C5-epimerase hse-5 and the 6-O-sulfotransferase hst-6 in hse-5; hst-6 double mutants , or loss of the 2-O-sulfotransferase hst-2 in double mutants hst-2 hst-6 leads to PVQ axon guidance defects [10] with a similar penetrance to that of rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- mutants . Together , our studies within the context of the literature suggest that the coordinated action of specific HS chains synthesized and modified in different tissues onto distinct HSPG core proteins function to properly guide the PVQ axons at the ventral midline . Similarly , the defective guidance of the axon of the AVM neuron is strongly rescued by expression of the HS copolymerase in the AVM neuron itself , but expression in the underlying hypodermis also contributes to normal AVM development . In this case too , HS chains synthesized onto core HSPGs functioning to guide AVM may be sdn-1/Syndecan in AVM and lon-2/Glypican in the hypodermis , as previously identified by analysis of core protein mutants in the context of AVM axon guidance [8] ( Fig 10B ) . Given that HSPGs decorate most cells in metazoans and are implicated in numerous cellular processes at the cell surface , including cell-matrix , cell-cell , and ligand-receptor interactions during development and tissue homeostasis , it could be expected that the HS copolymerase may be expressed ubiquitously . Functional rib-1::gfp was detected in virtually all cell types at some point during development . Interestingly , the HS copolymerase expression pattern was found to be dynamic , with levels changing and differing across tissues and developmental stages . Expression is strong and transient in hypodermal cells of the embryo , the larva , and the developing vulva , likely reflecting the developmental requirements for cell migration during the formation of complex tissue shapes during morphogenesis . Our observations support a model in which high expression of the HS copolymerase in cells that secrete a basement membrane , such as hypodermal cells , is pivotal for cell migration along this basement membrane . In addition , it is quite likely that migrating cells themselves might dynamically regulate surface HSPGs to modulate their adhesion properties and regulate the signaling of guidance cues . This is reflected by HS copolymerase expression peaking during periods of cell migration during embryogenesis and vulva formation . In addition to this dynamic expression pattern , the HS copolymerase shows sustained expression in a number of structures throughout the life of the animal , including the pharynx , the pharyngeal-intestinal valve , the anal depressor , sphincter , and enteric muscles , as well as the nervous system . These are morphologically complex cells that are under continuous mechanical stress . For instance , the pharynx is constantly pumping bacteria , thus exerting variable pressure on the pharynx itself and the pharyngeal-intestinal valve . Similarly , the enteric muscles , the anal depressor , and the sphincter , all contract to expel waste , and , as the animal moves , the relatively long neuronal axons within the nerve cords are constantly subjected to stretch and relaxation . Other cell types expressing the HS copolymerase are the spermatheca , which stretches to welcome oocytes to be fertilized and contracts to expel the zygotes , and the uterine muscles , which contract to lay embryos in reproducing adults . Our observations point to a role for HSPGs in maintaining the integrity of tissues , possibly by regulating the attachment of cells that undergo considerable mechanical stress from repeated body contractions , and thus contribute to tissue homeostasis . That rib-1 expression persists post-developmentally is consistent with studies in other model systems that describe post-developmental roles for HS and HSPGs [4 , 50] . The rib-1 expression pattern overlaps with known expression patterns of specific HSPG core proteins . For example , membrane bound SDN-1/syndecan is expressed in neurons , hypodermis , and pharynx [14] , GPI-linked LON-2/glypican shows expression in the intestine and hypodermis [66] , and UNC-52/perlecan , a secreted HSPG , is expressed in body wall muscles , digestive system muscles , and pharynx [91 , 92] . Overlap between expression of HS biosynthetic machinery , such as RIB-1 , and the localization of specific HSPGs , suggests that HSPGs may remain near cells where HS synthesis occurs . This may have functional relevance , as glypicans in fibroblast cells were shown to be internalized through endocytosis , returned to the Golgi , and then transported back to the membrane with HS chains altered both in length and modification pattern [93 , 94] . Whether an internal recycling of HSPGs back to the HS biosynthetic machinery in the Golgi occurs in C . elegans , or whether it has functional relevance to guidance , remains to be determined . In conclusion , our studies have identified viable mutations in each of the two subunits of the HS copolymerase in C . elegans , which severely disrupt HS biosynthesis , leading to profound developmental defects . Our findings offer a model system to dissect the functions of HSPGs in C . elegans and uncover general principles of their roles during development and tissue homeostasis .
Nematode cultures were maintained at 20°C on NGM plates seeded with OP50 bacteria as described [51] . mum-1/rib-1 ( qm32 ) and mum-3/rib-2 ( qm46 ) alleles were outcrossed five times before building strains with reporters . 14% of mum-1/rib-1 ( qm32 ) m-/- z-/- animals reach adulthood , as 68% of the embryos hatch into larvae and 20% of larvae reach adulthood; and 63% of mum-3/rib-2 ( qm46 ) m-/- z-/- animals reach adulthood , as 85% of the embryos hatch into larvae , and 74% of larvae reach adulthood [54] . Alleles used in this study are listed in S1 Table . Strains were constructed using standard genetic procedures and are listed in S9 Table . When needed , genotypes were confirmed by genotyping PCR or sequencing , using primers listed in S10 Table . Neuroanatomy was examined in animals of rib-1 ( qm32 ) m-/- z-/- and rib-2 ( qm46 ) m-/- z-/- that are had completed development to L4 larvae and adults ( 14% and 63% of the respective populations ) using specific reporters . Animals were mounted on agarose pads , anaesthetized with 100 mM sodium azide , and examined under a Zeiss Axio Scope . A1 or a Zeiss Axioskop 2 Plus . For mapping mum-1 , a three-point mapping experiment was carried out by picking Unc-non-Dpy and Dpy-non-Unc recombinants from heterozygous mothers of the genotype mum-1/unc-24 dpy-20 , and the presence of mum-1 was assessed among the progeny of the homozygosed recombinants . Two-point mapping was carried out by picking Dpy worms from mum-1 dpy-20/++ heterozygous mothers , and the presence of mum-1 was assessed in the next generation . Also , Lin-non-Dpy recombinants were picked from heterozygous mum-1/lin-3 dpy-20 . As the rib-1 and rib-2 mutants are severely morphologically abnormal , cosmids , constructs , and PCR products were injected into strains carrying the mum-1/rib-1 ( qm32 ) or mum-3/rib-2 ( qm46 ) mutations in a heterozygous state , balanced by flanking visible markers . For rib-1 , we used rib-1 ( qm32 ) /unc-24 ( e138 ) dpy-20 ( e1282ts ) and for rib-2 , we used rib-2 ( qm46 ) /unc-32 ( e189 ) dpy-18 ( e364 ) ( see S9 Table ) . Transgenic F1s were isolated and lines homozygous for rib-1 or rib-2 were established . Transgenic animals were generated by standard microinjection techniques [95] . Each construct or PCR amplicon was injected at 5 to 25 ng/μl with one or two coinjection markers which included pRF4-rol-6 ( su1006d ) ( 100–150 ng/μL ) , Pttx-3::mCherry ( 50 ng/μL ) , Pceh-22::gfp ( 50 ng/μL ) , pCB101 . 1 Prgef-1::DsRed2 ( 50 ng/μL ) , and Punc-122::rfp ( 50 ng/μL ) . pBSK+ ( 90–100 ng/μL ) used to increase total DNA concentration if needed . For coinjection markers used for each rescued transgenic line , see S9 Table . The gene rib-1/F12F6 . 3 is downstream of the gene srgp-1/F12F6 . 5 in an operon of two genes . The nearest gene upstream of the operon is transcribed in the opposite direction . The genomic region between the operon of srgp-1 and rib-1 , and the upstream neighboring gene is 4352 bp , corresponding to coordinates 22290–26642 on cosmid F12F6 . Prib-1::rib-1 ( PCR product ) : A PCR product containing bases 34593–39595 of cosmid F12F6 of the rib-1 locus was amplified with Pfu polymerase . Prib-2::rib-2 ( PCR product ) : A PCR product containing bases 581 to 6196 of cosmid K01G5 of the rib-2 locus was amplified with Phusion polymerase . Prib-1::gfp ( pCB78 ) : A PCR generated piece containing bases 23701 to 26662 of cosmid F12F6 corresponding to the promoter region of the rib-1 operon , as well as the initial 7 codons of srgp-1 , was cloned upstream of gfp in the pPD95 . 77 vector using enzymes PstI and XbaI . Prib-1::rib-1::Venus ( pCB221 ) : The rib-1 promoter region containing bases 23701–26580 of cosmid F12F6 was PCR amplified and cloned upstream of gfp in the pPD95 . 77 vector using enzymes SphI and PstI . A PCR generated piece containing bases 34452–39527 of cosmid F12F6 corresponding to the intergenic sequence between the genes rib-1 and srgp-1 , as well as the rib-1 coding sequence , was cloned downstream of the rib-1 promoter and upstream of gfp using enzymes PstI and AvrII . Then , gfp was replaced with a PCR amplified Venus and cloned in frame with rib-1 using enzymes MscI and ApaI . As a note , for rib-2 , we constructed several transcriptional Prib-2::gfp and translational Prib-2::RIB-2::Venus reporters with different sizes of promoter region , injected at a range of concentrations ( 10–150 ng/μL ) . At least three transgenic lines were examined for each condition , but gave no or a very weak expression level in transgenic worms carrying these constructs . A very faint level of Prib-2::gfp was broadly detected in comma-stage embryos , and in the head and vulva area at later developmental stages . Pdpy-7::rib-1 cDNA ( pCB186 ) : The rib-1 cDNA was amplified from yk1228g12 and ligated into a Pdpy-7 vector with a pPD95 . 75 backbone using enzymes XmaI–NcoI . Pmyo-3::rib-1 cDNA ( pCB196 ) : A Pmyo-3 HindIII–XbaI fragment was ligated upstream of the rib-1 cDNA in a pCB186 HindIII–XbaI fragment in place of Pdpy-7 . Pmec-7::rib-1 cDNA ( pCB204 ) : The rib-1 cDNA was amplified from yk1228g12 and cloned into the pPD96 . 41 vector using enzymes AgeI–BglII . Prib-1::rib-1 cDNA ( pCB225 ) : The rib-1 cDNA was ligated downstream of the rib-1 promoter ( bases 23 , 701 to 26 , 580 of cosmid F12F6 ) using enzymes XmaI–ApaI in the pPD95 . 77 backbone . Prgef-1::rib-1 cDNA ( pCB199 ) : The rib-1 cDNA was ligated downstream of Prgef-1 in place of DsRed2 using enzymes XmaI–ApaI in the pCB101 . 1 vector . All inserts of finalized clones were verified by sequencing . The genomic regions of mum-1/rib-1 and mum-3/rib-2 were PCR amplified using Pfu polymerase and sequenced on two independent PCR products amplified from genomic DNA of qm32 and qm46 , respectively , using primers to cover the entire genomic region . Primers listed in S10 Table sequence over the mutation in each of the two mutants . Mixed-stage wild type ( N2 ) , SDN-1::GFP ( opIs171 ) , rib-1; SDN::GFP ( rib-1; opIs171 ) and rib-1 GFP control ( rib-1; lqIs4 ) worms were collected from plates devoid of bacteria in buffer and protease inhibitors ( Roche ) . Worm pellets were subjected to repeated freeze-thaw cycles . Protein concentration was measured using the Pierce 660 nm Protein Assay on a Nanodrop . 80 μg of samples mixed with 2x Laemmli sample buffer ( Bio-Rad ) were frozen in liquid nitrogen , then boiled , separated by SDS-PAGE on a 4–20% Mini-Protean TGX gel ( Bio-Rad ) , and transferred to PVDF membrane . Membranes were incubated in 1:3000 rabbit anti-GFP primary antibody ( Millipore #AB3080 ) and 1:9000 goat anti-rabbit HRP secondary antibody ( Bio-Rad #166-2408EDU ) . For the loading control , membranes were incubated in 1:5000 rabbit anti-HSP90 antibody ( CST #4874 ) and 1:10000 goat anti-rabbit HRP secondary antibody ( Bio-Rad #166-2408EDU ) . Signal was revealed using Clarity Western ECL Substrate ( Bio-Rad ) , and imaged using film ( LabScientific ) . Mixed-stage wild type ( N2 ) , GFP control ( lqIs4 ) , LON-2::GFP ( TLG257 ) , LON-2ΔGAG::GFP ( TLG199 ) , rib-1; LON-2::GFP ( VQ525 ) , rib-2; LON-2::GFP ( VQ528 ) , rib-1 GFP control ( rib-1; lqIs4 ) and rib-2 GFP control ( rib-2; lqIs4 ) worms were collected from plates devoid of bacteria in buffer and protease inhibitors ( Roche ) , mixed with 2x Laemmli sample buffer ( Bio-Rad ) , and frozen in liquid nitrogen . Samples were boiled and spun down , separated by SDS-PAGE on a 4–20% Mini-Protean TGX gel ( Bio-Rad ) , and transferred to PVDF membrane . Membranes were incubated in 1:3000 rabbit anti-GFP primary antibody ( Millipore #AB3080 ) and 1:9000 goat anti-rabbit HRP secondary antibody ( Bio-Rad #166-2408EDU ) . For the loading control , membranes were incubated in 1:5000 rabbit anti-HSP90 antibody ( CST #4874 ) and 1:10000 goat anti-rabbit HRP secondary antibody ( Bio-Rad #166-2408EDU ) . Signal was revealed using Clarity Western ECL Substrate ( Bio-Rad ) , and imaged using film ( LabScientific ) . Worm RNA was extracted using Trizol ( Invitrogen ) according to manufacturer’s instructions . RNA ( 500 ng ) was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit ( Applied Biosystems ) and random primers . PCR reactions were carried out with cDNA template , and 0 . 25 μM of each primer in 10 mM Tris pH 8 . 3 , 1 . 5 mM MgCl2 , 50 mM KCl , 0 . 2 mM deoxynucleotides , and 1 U Phusion DNA polymerase for 30 cycles of 94°C for 10 seconds , 55°C for 20 seconds , and 72°C for 45 seconds . Primers used to detect rib-1 transcript: oCB1533 ( TGGAATCGACACAACGGATCG ) , oCB1534 ( CAAGCAGTTCGTCGTATTCCC ) , oCB1535 ( GAATACGACGAACTGCTTGCC ) , oCB1536 ( TCCAGCTCAATCTTGTTGTCG ) and oCB1537 ( AGATGTGATGAGGGGAGAACG ) . Primers used to detect rib-2 transcript: oCB1538 ( CAGTTCGTTTGGAATTGACGG ) , oCB1539 ( CTGCTATATGATTGACATCCACAGG ) , oCB1540 ( CACGTCATCACGCCAGATACG ) , and oCB1541 ( TGATTCTGTGGGAGACGCGTC ) . The transcript for Y45F10D . 4 was used as control using the primers oCB992 ( TCGCTTCAAATCAGTTCAGC ) and oCB993 ( GCGAGCATTGAACAGTGAAG ) . | During animal development , cells and neurons navigate long distances to reach their final target destinations . Migrating cells are guided by extracellular molecular cues , and cellular responses to these cues are regulated by heparan sulfate proteoglycans . Heparan sulfate proteoglycans are proteins with long heparan sulfate polysaccharide chains attached . Here we identify and study previously unavailable viable mutants that disrupt the elongation of the heparan sulfate chains in the nematode C . elegans . Our analysis shows that these HS-chain-elongation mutations affect the development of the nervous system as they result in misguided migrations of neurons and axons . Furthermore , we find that heparan sulfate chain elongation occurs in numerous cell types during development and that the coordinated production of heparan sulfate proteoglycans , in both the migrating cell and neighboring tissues , ensures proper migration . Our findings highlight the critical roles of heparan sulfate proteoglycans in nervous system development and the evolutionary conservation of the molecular mechanisms driving guided migrations . | [
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] | 2017 | Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans |
Stem rust ( Puccinia graminis f . sp . tritici; Pgt ) is a devastating fungal disease of wheat and barley . Pgt race TTKSK ( isolate Ug99 ) is a serious threat to these Triticeae grain crops because resistance is rare . In barley , the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race . Segregation for resistance observed on seedlings of the Q21861 × SM89010 ( QSM ) doubled-haploid ( DH ) population was found to be predominantly qualitative , with little of the remaining variance explained by loci other than Rpg-TTKSK . In contrast , analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro , Kenya , revealed several additional quantitative trait loci that contribute to resistance . To molecularly characterize these loci , Barley1 GeneChips were used to measure the expression of 22 , 792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation . Comparison of expression Quantitative Trait Loci ( eQTL ) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 ( within the Rpg-TTKSK locus ) as a candidate susceptibility gene . In parallel , we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated , adult plant resistance discovered through the Njoro field trials . Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK , but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H . It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses , where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome . Hence , the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement of R-gene mediated resistance .
Plants respond to invading pathogens with several forms of defense , ranging from the generation of toxic chemicals to programmed cell death [1] . These defense strategies do not occur coincidentally , but rather by successive rounds of chemical , physical , and enzymatic barriers introduced to impede pathogen progression . Initially , pathogen-associated molecular patterns ( PAMPs ) are recognized by pattern recognition receptors ( PRR ) , which in turn , trigger non-specific defense cascades , also known as PAMP-triggered immunity ( PTI ) [1] , [2] . Generally , these non-specific defense mechanisms successfully block pathogen entry . When these primary impediments fail , a more extreme form of defense known as gene-for-gene resistance , or effector triggered immunity ( ETI ) , may occur if the plant encodes an appropriate resistance ( R ) protein that recognizes , either directly or indirectly , its corresponding pathogen effector [1] , [3] . Though extreme , ETI-mediated programmed cell death restricts pathogen ingress , essentially destroying the nutrient source required for colonization of biotrophic fungi . The translocation of several host R proteins into the nucleus after recognition of cognate pathogen effectors has implicated the regulation of gene expression in ETI [4] . Stem rust , caused by the obligate fungal biotroph Puccinia graminis , has been a serious problem wherever wheat and barley are grown [5]–[8] . Urediniospores of P . graminis germinate within 4 to 8 hours after inoculation ( HAI ) during nights with dew formation or rainfall [9] . After germ tube extension and recognition of stomatal openings , appressoria form around 12 HAI . Growth continues , with the generation of a penetration peg that initiates sub-stomatal invagination of host tissue , development of infection hyphae , and differentiation of haustorial mother cells . In barley , penetration into the sub-stomatal space coincides with activation of the defense response ( 12–24 HAI ) [10] , [11] . Recognition of the pathogen will occur in the presence of Rpg ( Resistance to P . graminis ) genes , which mediate resistance to particular formae speciales of P . graminis by [12] . To date , eight Rpg genes have been identified , with five specifying resistance to races of P . graminis f . sp . tritici ( Pgt ) and three to P . graminis f . sp . secalis ( Pgs ) [12] . The identification of a new highly virulent race of Pgt known as TTKSK , ( commonly referred to as Ug99 ) , initiated a major collaboration to identify resistance genes in germplasm repositories of wheat and barley ( www . globalrust . org ) [13]-[15] . In a search for loci that mediate resistance to Pgt race TTKSK , Steffenson and colleagues identified the Rpg-TTKSK locus on the long arm of chromosome 5H , contributed by the barley cv . Q21861 [12] . This locus had previously been implicated in stem rust resistance by the fine mapping and cloning of rpg4 and Rpg5 , respectively [16] . The recessive resistance gene rpg4 confers immunity to Pgt race QCCJ , while Rpg5 provides dominant/semi-dominant resistance to Pgs isolate 92-MN-90 . Sequencing of the genomic region in cv . Morex ( genotype = Rpg4; rpg5 ) found five candidate genes encoding two nucleotide-binding site ( NBS ) , leucine-rich repeat ( LRR ) proteins , two actin depolymerizing factors ( ADF2 , ADF3 ) , and a protein phosphatase 2C protein ( PP2C ) [16] . Rpg5 co-segregated with the two NBS-LRR , ADF3 , and PP2C encoding genes in the susceptible cv . Morex [16] . Sequencing of resistant cv . Q21861 identified major structural polymorphisms in one of the NBS-LRRs , such that it encoded a unique combination of NBS and LRR domains coupled to a serine/threonine kinase ( S/TPK ) domain [16] . Virus-induced gene silencing and allele sequencing implicated this NBS-LRR-S/TPK as Rpg5 . The recessive resistance gene rpg4 has been associated with Adf2 by allele and recombinant sequencing [16] . Interestingly , resistance to Pgt race QCCJ in the informative recombinants indicates that both Rpg5 and rpg4 may be required to mediate an effective resistance response [17] . It is unknown which gene underlies Rpg-TTKSK mediated resistance to Pgt race TTKSK , but it is hypothesized that both Rpg5 and rpg4 are required [12] . Recently , several studies have exploited natural variation combined with expression profiling to decipher complex regulatory pathways , and in some cases phenotypic consequences [18] . This approach is referred to as genetical genomics or expression quantitative trait locus ( eQTL ) analysis [19] , [20] . Invariant to the organism studied , two types of heritable variation have been identified for gene expression in segregating populations; the most predominant form being linked to local variation near the physical position of genes ( cis-eQTL ) and the weaker distant regulation generated by genes that impact the transcriptional status of other genes ( trans-eQTL ) [21]–[23] . Although trans-eQTL tend to have more moderate effects than cis-eQTL , the functional polymorphisms that permit their discovery often affect more than one gene . For example , if a polymorphism altered activity of a transcription factor or hormone signaling gene , eQTL analysis may trace the regulation of many to hundreds of genes to the locus harboring this polymorphism , which would then be termed a trans-eQTL hotspot [24] . One such trans-eQTL hotspot affecting secondary metabolism has been associated with the AOP ( ALKENYL HYDROXALKYL PRODUCING ) locus , where cis-eQTL in genes involved in glucosinolate biosynthesis lead to the altered transcriptional and metabolic status of Arabidopsis [25] . To gain insight into the regulatory functions of the Rpg-TTKSK locus and the polymorphism ( s ) responsible for its existence , we analyzed the mRNA abundance of 22 , 792 host genes in each member of the Q21861 × SM89010 ( QSM ) doubled-haploid mapping population subjected to Pgt race TTKSK-inoculation ( INOC ) and mock-inoculation ( MOCK ) . By integrating the dynamics of eQTL hotspot formation , inoculation-responsive gene expression , and alternative control of eQTL between INOC and MOCK treatments , we describe two forms of transcriptional regulation that are associated with the resistance response . First , we provide evidence for Adf3 ( within the Rpg-TTKSK locus ) as a candidate susceptibility gene based on a strong cis-eQTL that has its effect magnified by inoculation with Pgt race TTKSK . Second , we report the identification of an inoculation-dependent , trans-eQTL hotspot that governs the expression of hundreds of genes , which under normal conditions , are controlled by additional modular regulators . The position of this chromosome 2H trans-eQTL hotspot is coincident with a quantitative resistance factor that acts as an enhancer of Rpg-TTKSK-mediated resistance in adult plants . Notably , the alleles across this shared genomic position that enhance Rpg-TTKSK-mediated resistance , lead to transcriptional suppression of numerous genes associated with disease defense .
The parents of the QSM population represent resistant and susceptible selections of barley against Pgt race TTKSK , with Q21861 and SM89010 exhibiting seedling infection types ( IT ) of 0; and 213− to 3 , respectively [12] , [26] . As illustrated in Figure 1A , these modified Stakman IT reflect the size of lesions by scoring on a scale from 0 to 3+ ( “;” denotes necrotic flecks ) and are ordered by their observed frequency [12] , [27] , [28] . The variability of IT on SM89010 is a classic example of the mesothetic response , a phenotype frequently observed on barley when challenged with P . graminis [29] . This mixture of responses in SM89010 is in direct contrast to the complete resistance observed in Q21861 . To identify additional loci that contribute quantitatively to resistance , we normalized the Stakman IT from the QSM population described by Steffenson and associates [12] using weighted counting of the ordered IT to generate infection frequencies , IF0 , IF1 , IF2 , and IF3 ( see Materials and Methods; Figure S1 ) [30] . Additionally , these infection frequencies were decomposed using principal components analysis , a method used previously to identify residual phenotypic variability in the barley Steptoe × Morex ( SxM ) population in response to Pgt race MCCF [30] . Principal component 1 ( PC1 ) explained 74 . 4% of the phenotypic variance , with PC2 , PC3 , and PC4 explaining 15 . 1% , 8 . 7% , and 1 . 9% of the remaining variance , respectively . We used a QSM genetic map generated from transcript-derived markers ( TDMs ) ( see Materials and Methods , Dataset S1 ) to perform composite interval mapping ( CIM ) with infection frequencies and principal components [12] , [27] , [28] , [30] . The Rpg-TTKSK locus on chromosome 5H at bin 49 ( 5H . 49 ) was the major qualitative locus for all infection frequencies and PC1 ( Table 1 ) . In addition , several minor effect QTL were detected for IF3 , PC3 , and PC4 at 7H . 7 , 1H . 35 , and 2H . 60 , respectively . Although significant , all three QTL contributed very little to resistance as compared to the Rpg-TTKSK locus . Further analysis of sub-populations fixed for resistance ( Rpg-TTKSK ) or susceptibility ( rpg-TTKSK ) did not identify any other loci that substantially explained residual variation ( Tables S1 and S2 ) . In parallel with the seedling experiments , infection phenotyping was performed in Njoro , Kenya during the 2008 season using natural inoculum of Pgt race TTKSK and isolates in this lineage . Reactions of QSM progeny were assessed three times in October and November by estimating the severity of rust infection ( SEV; scale from 0 to 100% ) on stem and leaf sheath tissue and also lesion size on a semi-quantitative scale ( LES; scale from 0 . 25 to 1 . 00 ) ( Figure 1B ) . An additional trait termed the ‘infection coefficient’ ( IC ) , was generated by multiplying percent rust infection by the numeric code for uredinia size ( SEV × LES ) . As shown in Table 2 and Figure 2 , resistance was predominantly mediated by Rpg-TTKSK ( contributed by the Q21861 allele ) , with negative additive effect estimates ( AEE ) of SEV by 7 . 7% , LES by 0 . 1 units , and IC by 8 . 4% ( i . e . , a reduction in disease ) . The second most prevalent QTL identified among the set of temporal observations was located on chromosome 2H at bin 16 ( 2H . 16 ) . This 2H . 16 locus ( contributed by the SM89010 allele ) had negative AEE of SEV by 6 . 7% , LES by 0 . 1 units , and IC by 8 . 5% . QTL analysis using resistant and susceptible QSM sub-populations ( based on their Rpg-TTKSK allele ) revealed that resistance mediated by 2H . 16 was only detectable in the resistant sub-population . A two-way ANOVA test also revealed a significant interaction term between 5H . 49 and 2H . 16 , further implicating 2H . 16 as an enhancer of Rpg-TTKSK . QTL analysis of both seedling and adult progeny of the QSM population revealed that the most significant locus contributing to resistance was Rpg-TTKSK . In seedlings , greater than 50% of the phenotypic variance for IF0 , IF1 , IF3 , and PC1 was attributed to Rpg-TTKSK , whereas , Rpg-TTKSK explained 11 . 5% to 35 . 2% of the phenotypic variance in adult plants surveyed under field conditions , depending on the trait and date of data collection . In the experiments involving adult plants , the weaker relative contribution of Rpg-TTKSK suggests that it may act so strongly in seedlings that the effect of loci such as 2H . 16 may be masked ( Table 1 ) . We hypothesized that the signaling components associated with Rpg-TTKSK-mediated defense response could be identified by characterizing the regulation of host gene expression in seedlings of the QSM population inoculated and mock-inoculated with Pgt race TTKSK . To maximize the variability among experimental genotypes and treatments , we considered Pgt infection kinetics as well as barley-Pgt time-course expression profiling data [31] . We selected 24 hours after inoculation ( HAI ) , just after formation of Pgt haustoria and during intracellular hyphal growth in seedlings inoculated and mock-inoculated with Pgt race TTKSK [10] , [11] . Furthermore , this time point should capture a mixture of PTI and ETI responses , providing opportunities to assess their potential overlap in barley-stem rust interactions . Variation in gene expression between the parental lines Q21861 and SM89010 was estimated by using four biological replicates that were randomized among the 75 doubled-haploid progeny of the QSM population ( see Materials and Methods ) . Plants were inoculated with Pgt race TTKSK urediniospores suspended in a light-weight mineral oil or mock-inoculated with spore-free mineral oil . For each genotype , pools of 5 first seedling leaves were harvested at 24 HAI , mRNA extracted , and hybridized to individual Barley1 GeneChips , which contain probe sets representing 22 , 792 genes [32] . A two-way ANOVA using genotype ( Q21861 and SM89010 ) and treatment ( INOC and MOCK ) was performed using the natural log normalized expression data to determine the number of differentially expressed genes between genotypes , treatments , and their interaction . Histogram-based estimation for false discovery rate ( FDR ) [33] revealed 6 , 957 , 1 , 902 , and 48 significant differences for genotype , treatment , and genotype × treatment effects when controlled for an FDR of 5% ( Figure 3 ) . Thus , effects of polymorphisms between Q21861 and SM89010 that are independent of treatment account for the differential expression of ∼25% of the genes on the Barley1 GeneChip , or ∼78% of the total number of differentially expressed genes ( Figure S2 ) . The majority of genes with significant differences with respect to genotype had fold change less than 2 ( 71 . 6% ) . For the treatment effect , 1 , 902 genes were differentially expressed , of these , 995 were induced and 907 were suppressed . For those genes that were induced , 362 ( 36 . 4% ) displayed a fold change greater than 2 , while only 107 ( 11 . 8% ) suppressed genes met the same 2-fold change threshold . Concordantly , the relatively small number of genes ( i . e . , 48 ) with an interaction between genotype and treatment was expected , as most variation in gene expression attributed to inoculation is insensitive to the genotype assayed [34] , [35] . These results are similar to other plant-fungal interactions examined , where modulation of gene expression is a robust response [36] , [37] . In short , if a gene is induced , it is almost always induced , or conversely , if a gene is suppressed , it is almost always suppressed in response to pathogen challenge . Observation of differentially expressed genes in the parents alone sheds light on only a small fraction of the diverse genetic responses associated with defense . By contrast , the use of a segregating population provides a biallelic sampling that incorporates genotypic variability when detecting differences between treatments ( INOC and MOCK ) . In addition , the greater number of individuals allows for a precise estimation of differential expression regardless of the allele used in our experiment [38] . Two approaches were used to estimate the difference in expression levels between INOC and MOCK in the QSM segregating population; first , by performing an ANOVA between all QSM lines in INOC versus MOCK , and second , using a paired t-test with respect to QSM line between INOC and MOCK . As summarized in Figure 4 , controlling the FDR at 0 . 1% , 5 , 997 and 5 , 614 genes were differentially expressed among progeny lines using the ANOVA and paired t-test approaches , respectively , with an overlap of 5 , 325 . This suggested that the difference found between INOC and MOCK either by pooling lines ( ANOVA ) or between paired lines ( paired t-test ) was consistently detected as responsive to inoculation . A significant overlap was found between genes that were differentially expressed in the progeny ( 5 , 325 ) and those that were differentially expressed in the parents ( 1 , 902 ) , with the intersection consisting of 1 , 476 genes ( Figure 4 ) . Though highly overlapping , these two gene lists had a considerable number of genes not shared in the intersection ( 3 , 849 for the progeny; 426 for the parents ) . We found that this was mainly accounted for by the higher sensitivity to declare differential expression when using progeny as compared to parents , as the correlation of log-fold change of genes differentially expressed in the parents or DH progeny but not both was r2 = 0 . 83 . These results indicate that a considerable proportion of the barley transcriptome is reprogrammed by 24 HAI in response to Pgt inoculation . The substantial genotypic variability between Q21861 and SM89010 , paired with the strong gene expression response to inoculation with Pgt race TTKSK , suggests that this population is ideal for identifying the regulatory components that reprogram the defense transcriptome of barley . We identified eQTL using composite interval mapping in both INOC and MOCK experiments using natural log normalized expression data from the QSM population [39] , [40] . Individual experiment-wise thresholds ( EWT ) were determined for each expression trait in both experiments by permuting expression values 1 , 000 times; the CIM analyses were performed with reselection of background markers on each permuted data set , which is the more stringent implementation of this approach [41] , [42] . When controlled at α = 0 . 05 , EWT for INOC and MOCK exhibited mean LOD EWT of 3 . 138 and 3 . 142 , respectively . These are slightly lower than global LOD EWT estimated from 1 , 000 random probe sets ( INOC: 3 . 168 and MOCK: 3 . 167 ) [43] , where a single threshold is used for all genes within a tissue/treatment . As shown in Table 3 , at least one eQTL was detected for 13 , 919 and 15 , 468 expression traits in INOC and MOCK , respectively , with an intersection of 10 , 127 traits ( Datasets S2 and S3 ) . Estimates of FDR among expression traits that exceeded their EWTs at one or more genomic locations were 8 . 2% ( 0 . 05×22 , 792/13 , 919 ) and 7 . 4% ( 0 . 05×22 , 792/15 , 468 ) for INOC and MOCK , respectively . The frequency of eQTL that met the EWT for both treatments is shown across the genetic map in Figure 5 . eQTL were found to be unevenly distributed across the genetic map , with several regions appearing to contain either an excess or shortage of eQTL ( hotspots and coldspots , respectively ) . Hotspots may coincide with a greater density of genes ( e . g . , a genomic region with little recombination , common in pericentromeric regions [44] ) or even by the occurrence of a regulator of steady-state mRNA levels with strong allelic variation . Sequencing of the 5-Gb barley genome is still underway [45] , thus , we could not directly compare all eQTL to their physical position or the specific number of genes within each bin . As an alternative , TDMs have been applied as surrogates for the physical positions of genes as a means to estimate the number of genes located within a chromosomal region [46] . Regions over and under-saturated with eQTL can be determined by using a contingency χ2 test on the ratio of TDM:eQTL as compared to the entire experiment . As shown in Figure 5 and Table 4 , we identified five non-overlapping regions in MOCK and five non-overlapping regions in INOC oversaturated with eQTL with p<0 . 001 . Except for the shared hotspot 6H . 40 , all of these are distinct , indicating that Pgt race TTKSK elicits extensive remodeling of transcriptional regulation . We hypothesized that an eQTL hotspot would form at the Rpg-TTKSK locus . However , this region was significantly under-saturated for eQTL in INOC ( -log10 ( p ) = 14 . 62 ) . Alternately , the Rpg-TTKSK locus could impart resistance by modulating the expression of a small set of genes at 24 HAI . We identified 88 genes with Pgt race TTKSK-specific regulation at the Rpg-TTKSK locus ( 5H . 48/49/50 ) , with several genes known to function in PTI , ABA signaling , and reorganization of the actin cytoskeleton ( Table S3 ) . In parallel to de novo regulation , regulatory perturbation at Rpg-TTKSK may manifest itself in the strengthening or weakening of basal expression after inoculation with Pgt race TTKSK . We found seven such cases among the 46 genes with eQTL at the Rpg-TTKSK locus ( Table S4 ) ; for Contig4389_at , Contig4391_at , Contig7092_s_at , Contig7641_at , Contig9278_at , Contig26405_at , and rbah27g12_at , the AEE for the eQTL differed by more than 0 . 10 when the strengths of the effects were compared between INOC and MOCK . Of particular interest was the probe set Contig7092_s_at , as it hybridizes to Adf3 [17] , a gene that was previously implicated in INOC-specific regulation at the Rpg-TTKSK locus with the probe set Contig7093_at . Contig7093_at has AEE of 0 . 90 contributed by the SM allele in INOC , whereas Contig7092_s_at has AEE of 1 . 51 contributed by the SM allele in MOCK as compared to an AEE of 1 . 80 in INOC . This strong allele-dependent expression of Adf3 in INOC was confirmed by probe level analysis . We sequenced Adf3 in Q21861 , SM89010 , and 12 DH progeny ( 6 Q allele and 6 SM allele ) . This analysis revealed that probes 1–5 of Contig7092_s_at and probes 1–3 of Contig7093_at contained no SNPs between the probe source sequence ( cv . Morex ) and all other lines examined above . Analysis of these monomorphic probes verified the true expression level polymorphism as opposed to sequence-dependent hybridization efficiency . Note: Since the Rpg5 sequence was not identified prior to chip design , we were therefore unable to assay its expression for this analysis . Phenotypic QTL analysis of seedling and adult progeny implicated several resistance factors distinct from Rpg-TTKSK . These loci may represent additional basal defense regulators , such as PRR-mediated recognition of PAMPs that alter the expression of genes involved in non-specific resistance [47] . To assess evidence for this type of regulation , we identified regions that were oversaturated for genes that are differentially expressed between Pgt race TTKSK and mock-inoculation . We used the 5 , 997 genes identified as differentially expressed between INOC and MOCK treatments of the QSM progeny ( Figure 4 ) and applied a contingency χ2 test on the ratio of genes with eQTL that are differentially expressed as compared to the total number of genes with eQTL at each bin ( 5 , 997:22 , 792 gene; 26 . 31% ) . As shown in Figure 5 , five non-overlapping regions in both MOCK and INOC were significantly oversaturated for genes with eQTL that are also differentially expressed , inoculation-responsive genes ( p<0 . 01; Table 4 ) . Of particular interest were regions 2H . 28/29 in MOCK and 2H . 16/17/18 , 2H . 21/22 , and 6H . 40 in INOC , as they also were oversaturated with eQTL within their respective experiments . Hundreds of genes come under new regulation at the 2H . 16 trans-eQTL hotspot as a result of inoculation with Pgt race TTKSK . By comparing the positions of the most significant eQTL for each gene in the INOC and MOCK experiments , we asked whether genes regulated at this locus are specific to pathogen-induction , or alternatively , if they are regulated by different loci between MOCK and INOC . We then determined if an overlap between two loci was significant between INOC and MOCK by generating a bootstrap p-value based on the number of genes shared under a random distribution and excluding comparisons between cis-chromosomal positions . As shown in Figure 6 , 5 , 538 of 10 , 127 genes ( 54 . 7% ) have their most significant eQTL on different chromosomes between INOC and MOCK experiments . The altered regulation of eQTL between INOC and MOCK was not evenly spread across chromosomes , but instead was saturated at several vertical and horizontal positions in the map , relative to MOCK and INOC , respectively . Several of the saturated regions with p<0 . 001 ( dark red circles in Figure 6A ) coincided with eQTL hotspots , either with loci that were significantly over-saturated with eQTL in MOCK but not INOC ( 2H . 28 , 2H . 51 , 3H . 25/26 , and 6H . 40 ) , the reverse ( 2H . 21 , 6H . 28 , 6H . 40 ) , or loci not associated with a saturation in eQTL . Many of these genes have alternative regulation at the sites of eQTL hotspots , but others are distributed throughout the genome . The only eQTL hotspot to be shared between treatments was 6H . 40 . However , the composition of 6H . 40 is significantly altered , suggesting a reprioritization in the genes regulated by this locus . The eQTL hotspots at 3H . 43/44 and 6H . 28 in INOC form after inoculation without any apparent saturation from MOCK loci . In addition , some loci ( e . g . , 2H . 59/60 , 7H . 56/57 ) were found to regulate a significant number of genes as a result of inoculation , but were not found to be oversaturated with eQTL in INOC . All the phenomena described above demonstrate the complexity introduced by challenge with Pgt race TTKSK , where transcriptional reprogramming is modulated by the activation , deactivation , or reprioritization of regulatory loci that affect transcription . The barley transcriptome undergoes significant reprogramming in response to environmental stimuli , such as cold [48] , salinity [49] , drought [50] , or pathogen stress [36] , [51] . A marked example of this reprogramming is the transfer of regulatory control from several distinct loci in MOCK to the 2H . 16 locus in INOC , wherein a total of 368 genes come under alternate regulation after challenge by Pgt-TTKSK ( Table S5 ) . As shown in Table 5 , significant loci that met the bootstrap p-value cutoff of 0 . 05 were 6H . 40 , 6H . 36/37 , 1H . 1/2/3/4 , 3H . 27 , and 7H . 37/38 , transferring regulation of 10 , 10 , 3 , 5 , and 3 genes to the 2H . 16 locus after inoculation with Pgt race TTKSK , respectively . Exclusion of eQTL on the same chromosome may have removed loci that are genetically distinct; therefore we used both manual identification and cis-chromosome bootstrap p-values to identify 2H . 28/29 as one additional MOCK locus where regulatory control was transferred to 2H . 16 in INOC . Furthermore , we found that the overall extent of eQTL migration was underestimated , as our analysis of alternative regulation between INOC and MOCK focused on maximal eQTL ( Table 5 ) . As such , 42 , 25 , 23 , 10 , 3 , and 3 genes are regulated at the MOCK loci 2H . 28/29 , 6H . 40 , 6H . 36/37 , 3H . 27 , 1H . 1/2/3/4 , and 7H . 37 , respectively , with the inclusion of non-maximal eQTL . Of the six loci that significantly contribute to the INOC 2H . 16 locus , 1H . 1/2/3/4 , and 7H . 37 lacked an increase in eQTL when including non-maximal eQTL and were excluded from further analysis . In addition to the genes regulated in MOCK by these six loci , the INOC 2H . 16 locus regulates the expression of an additional 199 genes with MOCK eQTL distributed across the genetic map and 73 genes that did not have a detectable eQTL in MOCK . Alternate transcriptional control in the INOC and MOCK experiments suggested that one or more regulator ( s ) at 2H . 16 in INOC override the regulation exerted by MOCK loci 2H . 28/29 , 3H . 27 , 6H . 36/37 , 6H . 40 , as well as at additional loci distributed across the genome . This coalescence of regulation in INOC suggests that each of the loci identified in MOCK may itself coordinate the expression of a distinct regulon . If this were true , one might expect the functional polymorphisms that underlie these eQTL to act pleiotropically on their respective targets in similar ways . To test this , we compared AEE for eQTL between INOC 2H . 16 and the four MOCK loci listed in Table 5 . As illustrated in Figure 7 , the parent that increased gene expression was either conserved ( 2H . 28/29 ) or reversed ( 3H . 27 , 6H . 36/37 , 6H . 40 ) for each MOCK locus as compared to the INOC 2H . 16 , with correlations of r2 = 0 . 97 , −0 . 86 , −0 . 97 , and −0 . 98 , respectively . This predictive power between MOCK and INOC regulatory loci suggests that these genes represent four inoculation-dependent regulons . These regulons were not entirely distinct , as seven genes were shared between 2H . 28/29 and 6H . 40 , and one gene was shared between 2H . 28/51 and 6H . 36/37 , 2H . 28/29 and 3H . 37 , and 3H . 27 and 6H . 36/37 . The overlap in control among regulons is correlated with the number of genes regulated by each locus and is likely an underestimate due to population size . Thus , coordinated reprogramming from multiple loci in MOCK to the single INOC locus suggests that these genes belong to a buffered regulatory complex in mock-inoculated leaves that is consolidated by a master switch in response to pathogen infection . Coupled with the dependence on alternative regulation after challenge with Pgt race TTKSK , over-saturation of inoculation-responsive genes at the 2H . 16 locus implies that this locus largely determines the extent to which a gene is differentially expressed between INOC and MOCK . When we considered the AEE in INOC eQTL at the 2H . 16 locus , we found that it was highly predictive of the direction of differential expression between INOC and MOCK , with 78 of 90 genes affirming this association ( Figure 8 ) . Additionally , significant correlation was observed between the AEE in INOC and the log-fold change of differential expression ( r2 = 0 . 71; 90 genes ) ( Figure 8 ) . Selecting only those genes differentially expressed in the comparison of INOC versus MOCK strengthened the correlation , r2 = 0 . 85 ( 48 genes ) . In contrast , genes not declared differentially expressed between treatments had a considerably weaker correlation of r2 = 0 . 51 ( 42 genes ) . These results indicate that the regulation in INOC from the 2H . 16 locus is either the principal source or major component of gene expression changes due to challenge by Pgt race TTKSK . Our final analyses aimed to characterize shared aspects of the genes regulated by the 2H . 16 trans-eQTL hotspot following challenge by Pgt race TTKSK . First we examined the AEEs of these genes and observed a bias in the directionality of effects . Specifically , of the 229 genes with eQTL that have positive AEE ( Q21861 allele ) , 162 are induced and 67 are suppressed . The opposite is seen for the 139 genes with eQTL that have negative AEE ( SM89010 allele ) , with 39 induced and 100 suppressed . Thus , for the 368 genes regulated , the SM89010 allele at 2H . 16 attenuates expression levels for 262 of them ( ∼71% ) . Since we previously established that the genes regulated by the 2H . 16 trans-eQTL hotspot are oversaturated for differential expression in response to stem rust ( Figure 5 and Figure 6 ) , we wished to consider any additional evidence for their functional involvement in defense . To address this , we performed gene ontology ( GO ) enrichment analysis using the suite of analysis tools from agriGO to identify functional conservation [52] . Singular enrichment analysis ( SEA ) of genes having an eQTL where the SM89010 allele contributes the greater allelic effect identified over-representation of genes targeted to the plastid ( 37 of 111 annotated genes; p = 2 . 0e-6; q = 1 . 2e-4 ) . In contrast , no significant GO terms were identified using SEA for eQTL with Q21861 allele contributing the greater allelic effect . Although SEA directly tests for enrichment of GO terms , it does not take into account the magnitude of differential expression or allelic effects . To address this , we used a parametric analysis of gene set enrichment ( PAGE ) to incorporate these effects . In agreement with the results from SEA , we found that localization to the plastid was significant for genes down-regulated after challenge by Pgt race TTKSK ( p = 1 . 7e-3; q = 3 . 3e-2 ) . We also found that genes with negative allelic estimates , where the SM89010 allele results in lower expression , were moderately over-represented among the set of genes predicted to be localized to the plastid ( p = 6 . 3e-3; q = 0 . 12 ) . Additional statistically significant GO terms were identified with PAGE that were associated with either up-regulation or down-regulation after inoculation with Pgt race TTKSK . GO terms associated with lower expression in INOC ( down-regulation ) included: biosynthetic processes ( GO:0009058 ) , nitrogen compound metabolism ( GO:0006807 ) , nucleobase , nucleoside , nucleotide and nucleic acid metabolism ( GO:0006139 ) , and cellular biosynthetic processes ( GO:0044249 ) . In contrast , protein metabolism ( GO:0019538 ) , protein modification ( GO:0006464 ) , macromolecule modification ( GO:0043412 ) , cellular protein metabolism ( GO:0044267 ) , and localization to the membrane ( GO:0016020 ) are associated with up-regulated genes ( greater expression in INOC ) . The relative enrichments of GO terms observed are diagnostic of plant-pathogen interactions where genes involved in protein metabolism , modification , and membrane localization are typically up-regulated , while those genes targeted to the plastid are down-regulated [53]–[55] . Together with the directionalities of allelic effects , we have shown that the prototypical pattern of gene expression associated with defense is attenuated by the SM89010 allele at the 2H . 16 trans-eQTL hotspot . This result is paradoxical , since presence of SM89010 alleles across this same locus also enhances Rpg-TTKSK-mediated resistance ( Table 2 ) .
The presence of trans-eQTL hotspots at the site of major regulators has become a common theme in the control of gene expression [18] , [61] , [62] . Interestingly , a hotspot was not identified at the Rpg-TTKSK locus , but several models can account for its absence . First , our selection of 24 HAI may represent a very early ( or late ) time point in the activation of resistance signaling , such that only the primary targets of Rpg-TTKSK-specified resistance would be differentially regulated at this time . Second , if the primary transcriptional targets of Rpg-TTKSK-mediated resistance had considerable structural variation in their promoters , then the regulatory contribution from the Rpg-TTKSK locus may be effectively masked by strong cis-eQTL effects , or be too small to be detected with this approach . In this scenario , trans-eQTL hotspots composed of secondary targets will form at the genetic positions of primary transcriptional targets of R-gene signaling , rather than the R gene itself . These two hypotheses overlap , as the selection of time points would determine whether primary , secondary , or more general responses are detected . Lastly , the primary resistance response mediated by Rpg-TTKSK may not include gene expression as a causal component in defense . The absence of a trans-eQTL hotspot refocused our efforts to identify cis-eQTL that are biologically relevant at the Rpg-TTKSK locus , an approach used previously to dissect partial resistance to barley leaf rust [51] . ADF proteins are involved in the reorganization of the actin cytoskeleton by altering the rate of actin dissociation from the pointed ends of actin filaments [63] and are known to play a role in basal defense and mlo-mediated resistance in barley-powdery mildew interactions [64] . Notably , Adf3 is located within 5H . 49 and is proximal to the Rpg5 resistance gene . Previously , this gene was excluded as a candidate for Rpg5 , which mediates resistance to Pgs isolate 92-MN-90 , because the ADF3 amino acid sequence was identical between resistant and susceptible cultivars [17] . Our results suggest that after inoculation with Pgt race TTKSK , Adf3 has enhanced regulation ( AEEINOC > AEEMOCK ) at 5H . 49 , with lower expression in lines carrying Rpg-TTKSK . Therefore , resistance is associated with the suppression of Adf3 expression , a hypothesis suggested for Adf2 by Brueggeman and colleagues [17] . Although Adf3 was not considered a candidate for Rpg5 based on the failure to observe non-synonymous variation between Q21861 and susceptible alleles of the coding region [16] , our data suggest that Adf3 may be a factor contributing to Rpg-TTKSK-mediated resistance based on its strong expression polymorphism . Several hypotheses have been put forth for the biological role of ADF in plant-pathogen interactions [16] , [30] . Here , the enhanced expression of Adf3 in plants carrying the susceptible ( SM89010 ) allele implicates its functional role as a susceptibility factor induced by Pgt race TTKSK . Significant structural variation does occur in the promoter of Adf3 [16] , although additional functional analysis will be required to establish the requirement of Adf3 induction in compatibility . However , it has been shown that an intact host actin cytoskeleton is required for successful colonization in several plant-fungal pathosystems [65] , therefore Adf3 may be a potential target of pathogen-derived effectors . Even in the absence of a trans-eQTL hotspot at Rpg-TTKSK , extensive transcriptome reprogramming due to invasion by Pgt race TTKSK revealed key regulators that were altered between INOC and MOCK treatments . Most significant was the trans-eQTL hotspot at 2H . 16 , where saturation in both the number of eQTL and inoculation-responsive genes suggests that regulator ( s ) at this locus usurp the steady-state regulatory machinery to actively remodel gene expression . Dissection of this hotspot revealed a transcriptional hierarchy of several distinct loci in MOCK that converge on 2H . 16 after challenge with Pgt race TTKSK , with a predictive allelic effect between INOC and MOCK conditions . The co-localization of the hotspot with an enhancer of adult plant , R gene-mediated defense may implicate a causal relationship between gene expression and enhanced resistance . There exists some difficulty in making this connection , as the cloning of both classical and expression QTL can be confounded by the presence of tightly linked genes contributing to the phenotype . In mouse , dissection of the Qrr1 ( QTL rich region on chromosome 1 ) region found that multiple genes likely regulate different subsets of trans-eQTL that had previously been grouped together [66] . With the use of informative recombinants and multiple populations , Mozhui and colleagues [66] separated the Qrr1 region into proximal and distal portions , and found the distal region specifically regulated RNA metabolism and protein synthesis . In doing so , they were able to focus the eQTL candidate gene list that underlies a classical QTL associated with seizure susceptibility . This case study provides a model for how a single locus associated with an abundance of both QTLs and trans-eQTL was broken into two regions that are associated with entirely different pathways . Similarly , it is possible that the 2H . 16 region may be comprised of several regulators . At present , our strongest evidence against this hypothesis is the predictive power in the allelic effects between the MOCK loci ( 2H . 28/29 , 3H . 27 , 6H . 36/37 , and 6H . 40 ) and the INOC 2H . 16 locus , suggesting a single regulator or family of regulators that control these regulons after challenge with Pgt race TTKSK . In our investigation of eQTL regulation after treatment with Pgt race TTKSK , we focused on the dynamics in response to pathogen invasion . This is one approach for identifying genes that act as nodes in regulatory networks , but several other methodologies may be similarly powerful . For example , candidate genes can be identified from unrelated eQTL experiments by using additional information such as physical map position , functional annotation , expression polymorphisms , and correlation [18] , [51] , [67] . Druka and colleagues [30] provided a case study for the eQTL candidate gene selection of the cloned resistance gene Rpg1 [68] by using correlation and tissue-specific expression to associate the causal gene , albeit from unrelated tissue ( grain ) [30] . Similarly , they extended this approach to identify candidate genes for several minor effect stem rust resistance QTLs from the SxM population . They leveraged expression profiling of rpr1 [69] , a gene required for Rpg1-mediated resistance , and physical map position to identify a sensory transduction histidine protein kinase ( represented by probe set Contig13680_s_at ) that was strongly down-regulated in non-inoculated rpr1 plants and physically mapped near the QPgt . StMx-2H QTLs ( IF2 and PC2 ) [30] . In light of the results from this previous study and the importance of both phenotypic QTL and trans-eQTL hotspots on chromosome 2H in our work , we linked the QSM and SxM genetic maps via the conserved TDMs used to generate both maps ( Figure 9 ) . Based on shared TDMs , it appears that the QPgt . StMx-2H QTLs detected in the SxM population inoculated with Pgt race MCCF and the QTL identified in adult QSM progeny in Njoro , Kenya are largely distinct . Both QTLs have broad 2-LOD support intervals that overlap , but the 1-LOD support intervals are separate . Though the histidine protein kinase does have an overlapping 1-LOD support interval with QPgt . StMx-2H , we found that the peak of the QPgt . StMx-2H co-localized precisely with the 2H . 21 and 2H . 22 regions , shown by our results to be a trans-eQTL hotspot and over-saturated with differentially expressed genes after inoculation with Pgt race TTKSK . The histidine protein kinase exhibits a strong expression level polymorphism in the QSM population similar to the SxM population . An eQTL for the gene co-localizes with the 2H . 28/29 trans-eQTL hotspot and is detected in both INOC and MOCK , having a LOD of 16 . 71 ( 15 . 24 ) and AEE of 0 . 67 ( 0 . 66 ) , with greater expression contributed by SM89010 allele in INOC ( MOCK ) . Thus , three tightly linked loci , 2H . 16 , 2H . 21/22 , and 2H . 28/29 control the QTLs to Pgt race TTKSK , Pgt race MCCF , and the regulation of a steady-state trans-eQTL hotspot , respectively . It is of interest that both QTLs detected in the SxM and QSM populations against Pgt races TTKSK and MCCF , respectively , enhance the effect of their respective R genes , Rpg-TTKSK and Rpg1 . As Druka and colleagues used tissue derived from germinating embryos , it is unclear whether a trans-eQTL hotspot underlies the histidine protein kinase in leaf tissue in the SxM population [30] . Clearly , this region of chromosome 2H is a hotbed of phenotypic and expression QTLs that are involved in resistance to stem rust and points to an interconnected set of regulatory loci that link these genetic loci with resistance . It is interesting that the trans-eQTL hotspot at 2H . 16 regulates genes that are both induced and suppressed in response to Pgt race TTKSK invasion . Overall , the allelic effects for trans-eQTL at this locus were biased for greater expression when carrying the Q21861 allele ( 304 Q21861 vs . 219 SM89010 ) . This effect was mutually predictive with up-regulation in response to Pgt race TTKSK associated with the Q21861 allele . In contrast , enhancement of Rpg-TTKSK-mediated adult plant resistance was associated with the SM89010 allele . This is especially relevant , as Q21861 contributes Rpg-TTKSK and SM89010 is susceptible to Pgt race TTKSK . Taken together , these results suggest that transcriptional suppression was correlated with resistance , where enhancement would have been expected . This model of host-mediated gene suppression may be a defense mechanism against pathogen-mediated gene activation , which has been observed in several phytopathosystems as a method to distract or enhance accessibility of the host . The bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 produces the jasmonic acid-mimic coronatine that induces jasmonic acid/ethylene-associated pathways that compete with bacterial defense pathways dependent on salicylic acid signaling [70]–[72] . In contrast , direct binding to host promoters by TAL effectors in Xanthomonas spp . activate genes involved in host susceptibility [73]–[76] . Though counter-intuitive , several systems have shown that this mechanism is a bona fide approach for manipulating the host and enhancing virulence . Therefore this locus may provide a degree of insensitivity to effector-dependent manipulation of the host . It is important to recognize that genes regulated at 2H . 16 in INOC were not dependent on Rpg-TTKSK . Therefore the enhanced resistance conferred by 2H . 16 in the presence of Rpg-TTKSK suggests that the manipulation of gene expression may only impact the interaction of barley and Pgt under the appropriate conditions . Here , regulation at 2H . 16 in INOC overrides the control of several steady-state regulators in MOCK . Thus , these MOCK regulators may be prone to manipulation by Pgt , whereas 2H . 16 is non-responsive . Alternatively , the 2H . 16 locus may control the precise timing of gene expression , such that the full impact of these genes is maximized to strengthen Rpg-TTKSK-mediated resistance . Ultimately , increased resolution in the 2H . 16 region will be required to dissect the causal polymorphisms that enhance R gene-dependent adult plant resistance and the regulator ( s ) that generate the trans-eQTL hotspot .
The barley QSM doubled-haploid mapping population was generated from a single Q21861 × SM89010 F1 plant [26] , [77] . All data used for infection type analysis are derived from Steffenson et al . [12] . Briefly , three to five seeds of each doubled-haploid line or parent were planted in plastic cones and placed in flats in a completely randomized design . Plants were placed in the greenhouse at 22°C with supplemental lighting by 1 , 000-W sodium vapor lamps for 14 hours per day at the USDA-ARS Cereal Disease Lab , University of Minnesota , St . Paul . Pgt race TTKSK isolate 04KEN156/04 was initially increased on a susceptible wheat host , collected , desiccated , and stored in tubes at −80°C . Nine days after sowing ( PO:0007094 - first leaf unfolded ) , flats were inoculated with a low density of Pgt race TTKSK urediniospores ( 0 . 004 mg/plant ) suspended in a lightweight mineral oil carrier using the inoculation protocols described by Sun and Steffenson [29] . After inoculation , the plants were placed in a mist chamber for 16 hours in the dark , followed by light for 5 hours , and then moved to the greenhouse using the previously described conditions . Plants were phenotyped at 14 to 17 days after inoculation . The full experiment was repeated twice , with the evaluation of three to five plants per replicate . Field trials were carried out at the Kenya Agricultural Research Institute in Njoro , Kenya during the 2008 growing season . QSM DH lines were planted in 0 . 3 m rows ( 20–35 seed per row ) in a completely randomized design with one replicate . Parents were included at random in the planting plan in three replicates . Infection phenotypes were scored on 7 October 2008 , 17 October 2008 , and 10 November 2008 . The majority of lines were at the mid-dough stage of development ( Zadoks scale 8 . 5; Feeke's scale 11 . 2 ) at the first scoring date [78] , [79] . Other lines with later maturity reached the mid-dough stage by 17 October and 10 November . The bulk of the natural inoculum found in the field was typed to Pgt races TTKSK ( used in seedling resistance assays ) and TTKST ( same virulence pattern as TTKSK with the addition of virulence for wheat stem rust resistance gene Sr24 ) . Pgt race TTTSK ( same virulence pattern as TTSKS with the addition of virulence for Sr36 ) may have been present at a low frequency . Stakman infection types ( ITs ) for seedling plants were normalized using a modified approach that weights the counts of ordered ITs ( Figure 1A and Figure S1 , Table S6 ) [30] . Weights given were 1 . 0 , 0 . 65 , 0 . 25 , and 0 . 1 for the 1st , 2nd , 3rd , and 4th ordered ITs , respectively . IFs were determined by averaging weights for two replicates , where full weight is given to ITs of 0 , 1 , 2 , and 3 or partial weights for ITs of ‘0;’ , ‘1−’ , ‘1+’ , ‘2−’ , ‘2+’ , and ‘3−’ . For partial weights , 70% is given to the IT shown ( 0 , 1 , 2 , or 3 ) and 30% to the modified IT ( ‘+’ to the greater IT , ‘−’ to the lower IT ) . In the unique case of ‘3+’ , a weight of 1 . 3 was given to IT 3 . For adult plants , LES was quantified on a scale from 0 . 25 to 1 . 0 based on resistance or full susceptibility , where resistant is equal to 0 . 25 , moderately resistant is equal to 0 . 5 , and a fully susceptible LES score is 1 . 0 ( Figure 1B ) . The IC was determined by multiplying the SEV by LES . Principal components analysis for seedling and adult phenotypic data was performed using R ( www . r-project . org ) ( Table S7 ) . Composite interval mapping ( Zmapqtl; model 6 ) was performed with QTL Cartographer v1 . 17j , with a walking speed of 2 cM , window size of 10 cM , and five background markers ( SRmapqtl ) [39] . EWT were computed using permuted data ( Prune ) with reselection of background markers ( SRmapqtl ) , where each iteration maximum LOD scores were stored and after 1 , 000 runs the 95th quantile ( α = 0 . 05 ) was selected as the EWT [41] , [42] . QTLs that exceeded the EWT were extracted using Eqtl . Two flats ( each flat contained 75 doubled-haploid lines + 4 replicates of each parent = 81 cones/flat ) were grown in a completely randomized design at the USDA-ARS Cereal Disease Lab , University of Minnesota , St . Paul . For the INOC flat , a higher density of Pgt race TTKSK urediniospores ( 0 . 25 mg/plant ) was used as compared to the seedling phenotypic assay . For the MOCK flat , spore-free mineral oil was used . After inoculation , both flats were placed in the same mist chamber for 16 hours in the dark , exposed to light for 5 hours , and then moved to the greenhouse for 2 hours . Five seedlings were harvested , pooled , and placed in liquid nitrogen for each line in the population within a 1 . 5 hour period at 24 HAI . RNA was extracted using a hot acid-phenol protocol and RNAeasy columns ( Qiagen ) were used for further purification of the isolated RNA . Labeling , hybridization , washing , and scanning were performed according to standard Affymetrix protocols using the Barley1 GeneChip which contains probe sets representing 22 , 792 ( 21 , 439 non-redundant ) genes [32] at the ISU GeneChip Facility ( www . biotech . iastate . edu/facilities/genechip/Genechip . htm ) . At any single genetic locus , each of the 75 doubled haploid lines carries two copies of either the Q21861 or the SM89010 allele . These two genotypes can be distinguished by differential success in hybridizing RNA to Barley1 arrays , providing robust genetic markers . Transcript-derived markers were generated as described by Potokina and colleagues [46] , using an implementation in Python ( www . python . org ) . This technique identifies single feature polymorphisms ( SFPs ) by using individual probes on the Affymetrix Barley1 GeneChip as quantifiable measures of probe hybridization efficiency . After background correction and quantile normalization using R/Bioconductor ( www . bioconductor . org ) , individual probe signals were separated into two distinct groups with k-means clustering . Goodness-of-fit using a Z-statistic found over 2 , 500 quality markers for the QSM genetic map . This analysis was performed separately with the INOC and MOCK data sets , and only those markers conserved between these data sets ( 1 , 503 markers ) that had three ( of 75 ) or fewer data points missing were included . A scaffold of 294 markers shared with the SxM doubled-haploid mapping population was used to place the remaining 1 , 200 markers [46] . Available information for the genetic positions of genes represented on the Affymetrix Barley1 GeneChip was used to confirm marker order; this included data from a recently developed SNP-derived genetic map [44] . Manual curation of marker positions was assisted with visualization of two-point marker linkages using MadMapper ( Figure S3; http://cgpdb . ucdavis . edu/XLinkage/MadMapper ) [46] , [80] . The final map has a total of 378 unique markers ( bins ) with a genetic length of 1 , 259 cM , with an average of approximately 3 . 3 recombination events between bins ( Figure S4 , Dataset S1 ) . ANOVA was performed with SAS v9 . 1 ( SAS Institute Inc . , Cary , North Carolina ) . All comparisons between these data sets were generated using Python scripts . q-values were determined using histogram-based estimation proposed by Mosig and colleagues [81] , using the implementation by Nettleton and associates [33] . All microarray data for eQTL analysis were normalized with the Bioconductor implementation of the MAS5 . 0 algorithm ( www . bioconductor . org ) . Composite interval mapping was performed with QTL Cartographer v1 . 17j , using a walking speed of 2 cM , window size of 10 cM , and five background markers [39] . eQTL that exceeded individual EWT were extracted using a Python script , such that two peaks within close proximity were declared different eQTL if the distance between peaks was greater than 2 LOD [46] . Individual EWT were computed using a combination of Python scripts , bash shell scripts , and QTL Cartographer . Briefly , composite interval mapping was performed using the same criteria in the eQTL analysis except the data were permuted ( Prune ) with reselection of background markers ( SRmapqtl ) a total of 1 , 000 times [41] , [42] . Maximum LOD scores were stored and the 95th quantile ( α = 0 . 05 ) was selected as the individual EWT . The over and under-saturation of eQTL were identified using a contingency χ2 test on the ratio of TDM:eQTL for a region as compared to the entire experiment . To fulfill the requirements of this contingency χ2 test , we merged successive bins in the genetic map until the sum of observed eQTL and TDMs was greater than 73 for INOC and 85 for MOCK for each set of bins . The same bins were used to analyze both experiments by incorporating the distribution of eQTL in MOCK and INOC in parallel . A bootstrap approach was used to estimate the significance associated with alternate regulation in the INOC and MOCK data sets for genes with eQTL in both data sets , using the maximum LOD eQTL . Genetic regions were compared with non-overlapping merged bins ( superbins ) that were generated with a greedy approach . This approach required a minimum number of TDMs and eQTL in INOC ( 73 TDM and eQTL ) and MOCK ( 85 TDM and eQTL ) to be placed within a superbin . Hence , the same bins in each experiment were collected into a single superbin . This approach is similar to that used for the identification of trans-eQTL hotspots and regions for over-saturation of differentially expressed genes ( see Results ) . Two strategies were used to account for ( 1 ) alternate regulation on the same chromosome and ( 2 ) regulation on different chromosomes between INOC and MOCK . For both , genes were redistributed from MOCK using probabilities determined by the distribution of eQTL in INOC based on the eQTL histogram . This was repeated 1 , 000 times for the maximum LOD eQTL in the overlap between INOC and MOCK . Probabilities were generated differently for the first and second strategies by including all genes with eQTL ( 10 , 127 genes ) in INOC and MOCK or only those genes with maximum LOD eQTL on a different chromosome between data sets ( 5 , 538 genes ) . Bootstrap p-values were determined by comparing the observed overlap versus the 1 , 000 bootstrapped samples . Gene ontology enrichment analysis was carried out using agriGO v1 . 0β ( http://bioinfo . cau . edu . cn/agriGO ) [52] . Singular enrichment analysis ( SEA ) was performed using the default parameters , Fisher test , the Yekutieli multi-test adjustment method , a significance level of 0 . 05 , and a minimum number of five mapped entries using the complete set of gene ontology terms . Parametric analysis of gene set enrichment ( PAGE ) was used with SEA default parameters , with a difference in requiring a minimum of ten mapped entries and FDR cutoff at 0 . 1 . All MIAME-compliant GeneChip profiling data are available as accession BB64 at the PLEXdb expression resource for plants and plant pathogens ( www . plexdb . org ) , accession GSE20416 at NCBI-GEO , as well as accessions GN235 , GN236 , GN237 , GN238 at GeneNetwork ( www . genenetwork . org ) [82] . | An important step in molecular plant pathology is the identification of the biologically relevant events that are directly involved in mediating resistance to pathogens . Historically , it is known that de novo and modulated gene expression are important components of the immune response . And yet , how exactly regulatory cascades orchestrate transcriptional responses to influence immunity remains unexplored . Several molecular tools have enabled the dissection of the defense transcriptome . One such technique , expression Quantitative Trait Locus ( eQTL ) analysis , provides the opportunity to identify genes involved in transcriptional regulation and simultaneously identifying their downstream targets . This paper describes an eQTL analysis of a barley population segregating for qualitative and quantitative immunity to stem rust ( Puccinia graminis f . sp . tritici ) , a devastating pathogen of Triticeae grain crops . Analysis of treatment-specific effects identified several regulatory loci that alter the expression of many inoculation responsive genes . On chromosome 2H , a trans-eQTL hotspot coincides with an enhancer of adult plant resistance . Notably , the resistance allele for this locus is associated with suppressing the transcription for hundreds of genes , with some of these having been previously associated with plant disease defense . In this respect , conventional wisdom is challenged by these findings . | [
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] | 2011 | Quantitative and Qualitative Stem Rust Resistance Factors in Barley Are Associated with Transcriptional Suppression of Defense Regulons |
Sequencing technologies are becoming cheap enough to apply to large numbers of study participants and promise to provide new insights into human phenotypes by bringing to light rare and previously unknown genetic variants . We develop a new framework for the analysis of sequence data that incorporates all of the major features of previously proposed approaches , including those focused on allele counts and allele burden , but is both more general and more powerful . We harness population genetic theory to provide prior information on effect sizes and to create a pooling strategy for information from rare variants . Our method , EMMPAT ( Evolutionary Mixed Model for Pooled Association Testing ) , generates a single test per gene ( substantially reducing multiple testing concerns ) , facilitates graphical summaries , and improves the interpretation of results by allowing calculation of attributable variance . Simulations show that , relative to previously used approaches , our method increases the power to detect genes that affect phenotype when natural selection has kept alleles with large effect sizes rare . We demonstrate our approach on a population-based re-sequencing study of association between serum triglycerides and variation in ANGPTL4 .
Several authors have reviewed the potential contribution of low frequency alleles to variation in phenotypes [2]–[7] . Absent a change in the properties of new mutations during recent history , which we find implausible , systematic differences between SNPs of varying frequencies must be mediated by natural selection . Since the early 20th century , much work has explicated the evolutionary dynamics of quantitative traits , reviewed by Barton and Johnson [22] , [23] . Below we will posit a model of pleiotropic selection whereby the trait under study or a trait with a correlated genetic basis is under purifying selection . More detailed connection and contrast to the existing work on the genetic basis of quantitative traits is found in Text S1 . In Figure 1 , we illustrate direct and apparent selection scenarios which give rise to a correlation between fitness effects and phenotype effects . In Figure 1A , the phenotype itself is under selective pressure; for example , disease leading to propensity to childhood mortality . Figure 1B shows apparent selection by pleiotropy; variants which disrupt an unconstrained role of a gene also tend to disrupt another role which is under selection; for example , variation which increases Alzheimer's Disease risk after reproductive age may relate to other brain function which is relevant for individuals still reproducing . Hartl and Clark [24] carefully constructs and interprets the concept of fitness-effects in classical population genetics . Briefly , in an idealized population , the relative reproductive advantage of an individual is the product of the fitness effects of each variant that person carries , an additive approximation with no dominance or epistasis . We parameterize the problem in terms of the log of multiplicative fitness effects . That is , the fitness of the person is given by where the fitness effect of the variant is denoted and is the unphased genotype at that locus . The fitness effect of a new mutation determines several of its properties , such as average sojourn time before either going extinct or fixing at 100% prevalence and average frequency when sampled at a point in time [24] . Rather than assume that all variants in the region have the same , we assume that the of new mutations are sampled from a distribution of fitness effects ( DFE ) . Just as a fixed would determine properties of the sampled genotype data for a SNP , a DFE along with mutation , recombination , and demographic parameters induces a distribution on the observed frequency spectrum and polymorphism - divergence ratios in sampled data . Several authors have attempted to fit a parameterized DFE from genomic data [25]–[34] . Boyko et al [33] found that a combination of a point mass at neutrality ( not under selection ) combined with a gamma distribution for deleterious differences from neutrality to be a good fit for the DFE of non-synonymous mutations . With these facts in mind , in what follows we will use fitness effects to operationalize the construct of functional status for each SNP . Whereas Johnson and Barton [23] worked directly with the joint distribution of fitness and phenotype effects , we will use an existing DFE estimate [33] as a marginal distribution for fitness effects and construct the conditional distribution of phenotype effects . Since we do not know the true fitness effects of SNPs , we will estimate them with observed SNP frequency , which is statistically ancillary to phenotype-SNP correlation , using a simulation methodology described below .
Assume the context of a simple random cross-sectional sample of individuals ( indexed by ) studying a quantitative trait measured once per individual . Assume that these individuals also possess vectors of covariates and genotypes at each locus inside a sequenced candidate gene or region . The genotypes are coded such that “0” represents homozygous possession of the ancestral allele , “1” heterozygosity , and “2” homozygous possession of the derived allele at the locus . That is , represents the fourth sampled person possessing two derived alleles at the third locus in the sequenced region . We can write a regression model for person 's phenotype in terms of deviation from an average level predicted by covariate effects and additive genotype effects , ( 1 ) Using standard least-squares regression to estimate such a model will pose several problems . First , because there will be many rare variants , will contain many poorly estimated coefficients . The large number of rare variants will give model ( 1 ) a large number of degrees of freedom , decreasing its power to detect association with the candidate gene . Some of the variation uncovered may be perfectly correlated in the sample , meaning that those coefficients are not separately estimable in least-squares regression . Additionally , as the amount of the genome sequenced becomes large , there will be more variants than participants , making the entire model unidentified . To overcome these problems , we need to make more assumptions and model the coefficients . We adopt a model where we view the effects of SNPs in the study as a sample from a wider population of SNP effects , and characterize that entire population using only three parameters . To fix ideas , assume for now that we knew the fitness effect of each SNP . If fitness was perfectly correlated to effect on phenotype , we would use that as a summary for all alleles , , where the parameter relates the scales of the two measures . As the fitness effect is not perfectly correlated to effect on phenotype , we add a mean and an error term acknowledging those limitations to obtain ( 2 ) In applied problems , is not known a-priori , so we will construct a prediction based on the observed frequency . We denote for that estimate , and for its prediction error we write . We plug those estimates in to ( 2 ) to obtain ( 3 ) and combine the two uncorrelated error terms to yield ( 4 ) where ( 5 ) ( 6 ) The first term in ( 4 ) , , allows derived alleles to on average increase or decrease the phenotype . The second term is an unscaled correlation between phenotype effects and expected fitness effects . The error term is the deviation in SNP j's effect on phenotype from the average of SNPs with the same observed frequency . The variance of in ( 6 ) therefore has two components , first corresponds to prediction error of , and second is the variance of phenotype effects for SNPs at the same level of true fitness burden . The function allows that as average burden changes the variability might also change . Although one could imagine “bad” alleles being more variable in their effects than relatively neutral alleles , implying non-constant , we propose constant as a reasonable modeling start . This will still allow for the variance of effect sizes to change with observed frequency because of non-uniformity of with frequency . Equation ( 4 ) asserts that phenotype-effect and fitness-effect are linearly related; that seems correct for the scenario in Figure 1A and a good starting place for the other possibilities . In future work we will be able to empirically examine this assumption by graphical diagnostics and comparing fits using other functional forms . Further discussion of nonlinear relationships is found in Text S1 , and we will demonstrate the impact of an incorrect assumption of linearity in our simulation studies . Our model is quite general in that existing methods correspond to submodels of ( 4 ) . An allele count method tests the model with only allowed to vary; rare alleles below an arbitrary threshold are summarized by an average effect which does not change with frequency , so and all are set to zero , and alleles above that threshold are regarded as free parameters . Similarly a weighted-burden method corresponds to the model with a particular implementation of , such as in Madsen et al [21] where , and forces all in the rare alleles to be zero . Our model will not involve an arbitrary threshold for “rare alleles” and will adaptively pool variant effects in a flexible way . As shown in the results , this will create substantial power gains in a variety of settings . When and in ( 4 ) are zero , our model reduces to a standard random-effect model identical to that of Kwee et al [19] with all variants given the same weight . That is , regardless of frequency all SNPs have the same likelihood of having large effect sizes , and regardless of frequency SNP effects have zero mean . As a result , our method will be robust to the case that fitness and phenotype effects are unrelated by estimating and retaining the flexibility of the method of Kwee et al . The major difference between the above and our method is the use of population genetics to suggest the structure of the variance of SNP effects , including a fallback should fitness and phenotype effect not be related . Kwee's method is developed in the context of tag SNPs and suggests an arbitrary variance of SNP effects given as either a constant , , or any prior-information based form . A related method is that of Hoggart et al [18] . Their approach corresponds to and set to zero ( they assume a mean-zero distribution ) and a different set of restrictions on the distribution of . Their assumptions about the distribution of were chosen to yield estimates with most variants having zero effect , a feature called model selection which eliminates small effects and correlated variables . In contrast , our model will tend to reign in large effect sizes and split effect size between variants in high linkage disequilibrium , but does not eliminate SNPs from the fit . We prefer our choice for resequencing for several reasons . First , there may well be many effects of small size which are cumulatively important , and we want to retain those small effects in the model . Second , we want an estimate of the effect size of each variant for graphical and diagnostic purposes . Third , we accomplish a similar goal of reducing the model size by rejecting the null on a small number of genes . That is , we want to identify a small number of disease relevant genes with our efficient test; doing so will exclude most SNPs without further model selection procedures . Fourth , by smoothly grouping rare SNPs and summarizing them with only a few parameters , we already greatly reduce the multiple testing burden . The specification of equations ( 1 ) , ( 4 ) , and ( 6 ) yields a natural interpretation to the fitted model . After estimating the population parameters of phenotype effects , we will be able to jointly estimate individual SNP effects and their impact on the phenotype of each person in our sample . By calculating , we obtain the expected difference between participant i's phenotype and what we would expect were there no effects of this gene . As a result we can empirically estimate the overall phenotypic variability due to observed genetic variants , over study participants . We can similarly estimate the variability dues to rare alleles by including only rare SNPs in the above calculation . The overall effect is an average change in phenotype per derived allele , perhaps due to inadequate purifying selection . In the variance expression ( 6 ) , is the variability of allelic effects for a given level of true fitness . As will be shown below in Figure 2 , when using the genome-wide distribution of fitness effects for non-synonymous SNPs , common variation is nearly neutral so can also be thought of as the variability of effects of common alleles . represents the correlation between fitness burden and phenotypic burden . This parameter's interpretation relies on accurately estimating the scale of fitness effects and has awkward units , but we can avoid this difficulty by noting that ( 4 ) can be decomposed into a fitness related portion and a fitness unrelated portion which are independent ( 7 ) By calculating we can ascribe a proportion of total variation in phenotype to selection-phenotype correlation without worrying about having gotten the scale of correct . Calculations for separating these variance components are found in Text S1 . We can use the same technique to compare classes of SNPs , for example non-coding vs missense , by jointly fitting separate and comparing the attributable variance for each class of SNPs . We will illustrate this idea in our real data example . This decomposition also shows why it is not crucial for our estimates of fitness to be perfect . The model can fall back by setting to zero and use only to recover a working model which does not pool information across rare alleles . Doing so will mean that the opportunity to gain information by recognizing structure in phenotype effects will not be realized , but the remaining estimation method is still valid . An important consideration is how to interpret the results when multiple ethnic groups are analyzed simultaneously . Because some genetic variation is fixed between ethnic groups in the sample , the average effect of single-population variation will be absorbed into the fitted mean for that group . As a result , the interpretation for “total explained variation” is actually “total explained within-ethnic-group variation;” genetic variation may explain some of the phenotypic difference between groups , but we do not include it in our estimate because of confounding between environmental exposures and ethnic background . Another point requiring clarification is the assumption that genotype effects are independent . In the context of GWAS , nearby SNPs often are thought to have correlated effects because they mutually tag a functional variant . Additionally , estimates of SNP effects will be correlated due to LD making their true separate effects difficult or impossible to identify . However , in the underlying data generating mechanism true genotype effects are independent . Because sequencing identifies all the variation within the region and eliminates much of the correlation due to untyped alleles , we believe that the independence assumption is a useful approximation in this case . Non-independence of the true effects could be accommodated by imposing a covariance structure on SNP effects , for example using their spatial distance in the genome or folded protein . Alternatively , the phylogenetic approach of TreeLD [35] estimates the degree of probable overlap of untyped SNPs . Model ( 4 ) relies on a prediction of the fitness effect of each variant as well as an estimate of the error of that prediction . We use the following procedure to calculate such estimates . To reduce computational requirements , steps 2 and 3 above can be replaced by simulating a smaller number of large populations and calculating the expected mean and variance of fitness using simple random sampling . Figure 2 depicts the relationship of and to frequency when using a genome-wide fitted DFE [33] . Because much of the variation discovered in our multi-ethnic example dataset is confined to one ethnicity , we use the ethnicity-specific frequency and pseudo-data . Because of admixture in our sample , we use the highest observed frequency ( the most skeptical about its being rare ) to assign an ethnicity of origin to SNPs appearing in multiple groups . An advantage of this method is that because it refers to a feature of genetic history rather than a phenotype , it need only be done once for any trait under study on the same cohort . While the fitness - phenotype relationship will be different for all traits , that is modeled by the fitted parameter rather than modification of . If the impact of LD structure on the prediction does not vary too much between genes , the calculation can be recycled for multiple genes under study . In some experiments , we found the impact of LD to be minimal ( data not shown ) . Discussion of taking the DFE as known versus estimating or using some other flexible function of frequency it is included in Text S1 . Discussion of the quality of the existing DFE estimates are also included in Text S1 . We have used the observed frequency to estimate the fitness effect , but there are many other potential predictors of functional status . Discussion of including them in our model is found in Text S1 .
We propose a novel method , EMMPAT , for association between sequenced genes and phenotype which utilizes population genetic theory to pool information among rare variants . Our method generalizes allele-count and allele-burden techniques , and presents several advantages . Of greatest importance to the practicing scientist will be increased power and interpretability . As shown above , our method allows us to leverage allele frequency as auxiliary data related to SNP effects and to substantially increase power to detect association in many scenarios . The availability of a well motivated pooling strategy allows an omnibus test which incorporates common and rare variation simultaneously . Our approach provides clear interpretations for the fitted model , such as the attributable variance in phenotype due to all polymorphisms observed in a gene , particular types of SNPs , or only the rare variation . Furthermore it facilitates tests of meaningful parameters ( such as mean derived allele burden ) and group differences ( such as non-synonymous versus non-coding ) . The regression toolbox allows model checking and exploration , such as in Figure 3 which presents the data in an informative format . Additional model checking proceeds as usual in linear mixed models , and posterior predictive checks are similarly possible . A relevant question is how important our method will be for diseases which have not been strongly selected against . There are three answers to consider . First , when selection and disease effect are completely independent , common SNPs will tend to have just as large effect sizes as rare SNPs and explain much of the heritable variation in phenotype [2] , [3] . We believe that most investigators conducting resequencing studies assume rare variation to have larger effect sizes , since that is the best-justified scenario for the expense of sequencing . Second , our method allows for this possibility in the form of estimating to be zero and non-zero . As demonstrated in our simulations , the loss of power in adding a single unnecessary parameter to describe many SNPs is small . Third , as discussed in the Introduction and Text S1 , direct selection against disease is not a necessary condition for correlation between fitness and phenotype; as long as the disease related gene is under selective pressure in any of its functions , we expect a correlation . We have planned several extensions to this method . In addition to improved techniques of estimating fitness effects , we need to incorporate evidence for adaptive selection . Signatures of positive selection [47]–[49] can be used to prioritize genes for study which may have been more important in differentiating humans from our ancestors and hence contribute to modern phenotypes . We expect positively selected variants to have very different phenotype effects from neutral alleles , but it is not clear a-priori what that relationship should be or if it will be possible to reliably identify positively selected SNPs [50] , [51] . Second , for mathematical and numerical convenience we have developed this method in the context of a prospective probability sample measuring a quantitative trait . Both these assumptions need to be relaxed for the setting of most resequencing projects . Disease phenotypes are frequently non-normal , binary , or censored such as time-to-event from clinical trials , requiring a generalized linear mixed model . The prospective sampling assumption will also require work to relax . Retrospective sampling such as in case-control designs and extreme-phenotype-based sampling [13] , [52] is well known to distort random effect distributions [53] . Third , in our example and simulations , we assume that are independent of one another , but one need not do this . One could add spatial covariance structures between to relax the independence assumption , which would correspond to allowing that variants nearby each other in the genome or folded protein tend to have similar effects . Especially in exome-only resequencing studies , consideration of unobserved linked markers with techniques similar to TreeLD [35] will be important . Our model has not included dominance or epistasis between SNPs or genes , the structure of which is probably not simple , although progress has been made on determining the impact of these features to quantitative traits [54] , [55] . Finally , because our example dataset comes from high-quality Sanger sequencing , we have ignored nonrandom missing data issues . Future work involving second generation sequencing or beyond must address the complex nature of library coverage , alignment error , and genotyping error inherent in those technologies . | Studies correlating genetic variation to disease and other human traits have examined mostly common mutations , partly because of technological restrictions . However , recent advances have resulted in dramatically declining costs of obtaining genomic sequence data , which provides the opportunity to detect rare genetic variation . Existing methods of analysis designed for an earlier era of technology are not optimal for discovering links to rare mutations . We take advantage of 1 ) the advanced theoretical understanding of evolutionary mechanics and 2 ) genome-wide evidence about evolutionary forces on the human genome to suggest a framework for understanding observed correlations between rare genetic variation and modern traits . The model leads to a powerful test for genetic association and to an improved interpretation of results . We demonstrate the new method on previously confirmed results in a gene related to high blood cholesterol levels . | [
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] | 2010 | An Evolutionary Framework for Association Testing in Resequencing Studies |
Polysaccharide intercellular adhesin ( PIA ) , also known as poly-N-acetyl-β- ( 1–6 ) -glucosamine ( PIA/PNAG ) is an important component of Staphylococcus aureus biofilms and also contributes to resistance to phagocytosis . The proteins IcaA , IcaD , IcaB , and IcaC are encoded within the intercellular adhesin ( ica ) operon and synthesize PIA/PNAG . We discovered a mechanism of phase variation in PIA/PNAG expression that appears to involve slipped-strand mispairing . The process is reversible and RecA-independent , and involves the expansion and contraction of a simple tetranucleotide tandem repeat within icaC . Inactivation of IcaC results in a PIA/PNAG-negative phenotype . A PIA/PNAG-hyperproducing strain gained a fitness advantage in vitro following the icaC mutation and loss of PIA/PNAG production . The mutation was also detected in two clinical isolates , suggesting that under certain conditions , loss of PIA/PNAG production may be advantageous during infection . There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenic icaADBC deletion mutant . Together , these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression , that high-level production of PIA/PNAG carries a fitness cost , and that icaADB may contribute to bacterial fitness , by an unknown mechanism , in the absence of an intact icaC gene and PIA/PNAG production .
Phase variation functions as a reversible on/off switch for the expression of a particular gene . The result is commonly an alteration in the expression of some cell surface-expressed antigen . Slipped-strand mispairing is one mechanism that can lead to the production of a phase variant . Slipped-strand mispairing occurs during DNA replication when there is mispairing between mother and daughter DNA strands in regions of DNA that contain simple 1–10 nucleotide repeats [1] . This results in the addition or subtraction of one or more repeats that can bring about a change in transcriptional efficiency or shift the reading frame to alter or halt translation . Staphylococcus aureus infections are responsible for an enormous loss of life; deaths from methicillin resistant S . aureus ( MRSA ) alone exceed 18 , 000 yearly in the United States , making it the leading cause of death by a single infectious agent [2] . Antibiotic resistance is a mounting problem and an effective vaccine is not yet available . Biofilm formation plays an important role , particularly in device-related infections , and it contributes to antibiotic failure and resistance of the bacteria to host immune defenses . Biofilm formation is the aggregation of bacteria on a solid surface within a self-produced extracellular polymeric matrix . Formation of a biofilm confers several survival advantages to the resident bacteria . The biofilm provides protection from adverse environmental conditions such as heat , shear force , and UV damage; as well as protection from the host immune system and antibiotic challenge [3] . Biofilm bacteria are resistant to antibiotic levels up to 1 , 000-fold higher than planktonic bacteria that are genetically identical [4] , [5] . A major component of the S . aureus biofilm extracellular matrix is the polysaccharide poly-N-acetylglucosamine . The staphylococcal polysaccharide intracellular adhesin ( PIA ) is a high molecular weight polymer of β-1-6-linked N-acetyl-glucosamine ( PNAG ) [6] , [7] . In addition to its role in intercellular adhesion and biofilm formation , PIA/PNAG also plays a role in immune evasion [8] , [9] . Evidence suggests that antibodies against PIA/PNAG often recognize secreted PIA/PNAG rather than the surface-associated form , resulting in an ineffective immune response [8] . In contrast , an effective immune response against surface-associated PIA/PNAG , which can be directed by a conjugate vaccine , can successfully eradicate an infection [10] . Thus , PIA/PNAG protects the bacteria from immune defenses but under certain circumstances could actually be the target of an effective immune response . PIA/PNAG is synthesized by the proteins encoded in the icaADBC intercellular adhesin locus [11] , [12] . IcaA is a transmembrane glucosyltransferase that , together with IcaD , produces short PIA/PNAG oligomers [13] . IcaC is an integral membrane protein that is necessary for linking the short oligomers into longer polymer chains , and is thought to be involved in translocation of these chains to the cell surface [13] . Once there , IcaB is responsible for partial deacetylation of the PIA/PNAG molecule , which is required for retention at the cell surface [14] . A number of regulators modulate icaADBC transcription , including IcaR and CodY , which are repressors , and SarA and GraRS , which are positive regulators [15] , [16] , [17] , [18] . Other regulatory mechanisms have been implicated as well and are described in a recent review [19] . We found previously that a 5-nucleotide deletion mutation within the icaADBC promoter was sufficient to induce constitutive transcription of the icaADBC genes and high-level PIA/PNAG production , resulting in a mucoid phenotype and strong biofilm production [15] . In the present study , we noted that growth of the mucoid strain in liquid culture resulted in the rapid accumulation of non-mucoid variants . We investigated this phenomenon and found that the most frequent mutation leading to the PIA/PNAG-off phenotype was a change in the number of a specific tandem repeat in icaC . This mutation was reversible and was found in clinical isolates as well . The PIA/PNAG-negative variants had a growth advantage over the PIA/PNAG-overproducing parent and rapidly predominated the cultures . This represents a newly recognized mechanism of PIA/PNAG regulation in S . aureus .
S . aureus strain MN8m is a spontaneous PIA/PNAG-overproducing mutant of strain MN8 [15] , [20] . A 5 bp deletion in the icaADBC promoter region of MN8m is responsible for constitutive icaADBC transcription and the constitutive hyper-production of PIA/PNAG that gives the strain its mucoid appearance [15] . It is highly aggregative in liquid culture whereas non-mucoid strains are dispersed , making the culture turbid ( Fig . 1A ) . We found that MN8m cultures frequently exhibited an appearance that was somewhere between that of a turbid , non-mucoid strain and an autoaggregative mucoid strain ( Fig . 1A ) . When these cultures were plated on Congo red agar ( CRA ) , both mucoid colonies , which appear dry with irregular edges , and non-mucoid colonies , which are slick , circular , and occasionally surrounded by a transparent red perimeter , were observed ( Fig . 1B ) . We isolated 15 of the variant colonies and sequenced the icaADBC locus ( Fig . 2 ) . All of the isolates still exhibited the 5 bp deletion that leads to constitutive icaADBC transcription . The sequence of the icaADBC locus of four of the isolates was identical to that of MN8m , suggesting a mutation had occurred elsewhere in the chromosome . One of the isolates had a single point mutation within icaB . As icaB has been shown previously to be dispensable , it is likely that a secondary mutation outside of the icaADBC locus was present in this strain as well . One of the isolates had a nonsense mutation at the 5′-end of the icaC gene . The remaining nine isolates all shared the loss of a “ttta” tetranucleotide repeat within the icaC gene . The insertion led to a shift in the translational reading frame and truncation of the protein; reducing IcaC from 350 amino acids to 303 . Because this mutation was the most common amongst the variants , we chose to study it further . We focused on isolate JB12 , a tetranucleotide insertion variant . The icaADBC genes are co-transcribed , so to determine levels of the full-length transcript in JB12 , we quantified levels of the 3′-most transcript , icaC , by realtime RT-PCR . As shown in Fig . 3A , icaADBC transcript levels were more than 300-fold greater in MN8m than in the non-mucoid parent strain MN8 and the level remained elevated in the non-mucoid variant . To determine whether or not PIA/PNAG was produced by the JB12 variants , we performed slot-blot analysis using PIA/PNAG-specific rabbit polyclonal antiserum . As depicted in Fig . 3B , no PIA/PNAG was detected on the cell surface or in the spent media of JB12 cultures . Therefore , the mutation in the icaC gene resulted in the complete loss of detectable levels of PIA/PNAG . To confirm that the nucleotide insertion was responsible for loss of the mucoid phenotype , we complemented the icaC gene in trans . Expression of the intact icaC gene in strain JB12 from the IPTG-inducible plasmid pCL15 , lead to restoration of the mucoid colony morphology on CRA plates ( Fig . 4A ) , PIA/PNAG synthesis ( Fig . 4B ) , and biofilm formation ( Fig . 4C ) . Introduction of the empty vector into the variant had no effect . Phase variation is , by definition , a reversible on/off switch . Therefore , if the tetranucleotide repeat insertion was an example of phase variation , then we would expect to isolate variants of JB12 in which the mucoid phenotype was restored . To determine whether or not the phenotype was reversible we plated cultures of JB12 onto CRA . The reversion back to the mucoid phenotype was a rare event and only 1 variant was detected per approximately 45 , 000 cell divisions . We sequenced 6 mucoid variants from separate JB12 cultures , and all 6 variants sequenced had lost the 4 bp repeat unit that was gained in JB12 meaning that they had reverted back to the MN8m genotype . To determine whether the mutation in icaC was RecA-dependent , we disrupted the recA gene in MN8m with the bursa aurealis mariner transposon . PIA/PNAG-negative phenotypic revertants were still isolated from the recA mutant and of these revertants , the prevalence of the icaC repeat mutation was equivalent ( 6 out of 10 revertants ) . These results indicate that the mutation was RecA-independent and strongly suggest that the mutation occurred through slipped-strand mispairing . To determine whether or not this tetranucleotide repeat indel occurred outside of the laboratory , we examined 51 fully sequenced genomes available in NCBI and 52 sequenced genomes within the NARSA repository . Of these , 9 clinical isolates ( ∼9% ) contained a variation in the tandem repeat region in icaC described in this study , demonstrating that the phase variants do occur in vivo ( Table 1 ) . Sequences from two representative clinical MRSA strains with deletion or expansion of the repeat units are illustrated in Fig . 5A . The isolates with altered repeat number were PIA/PNAG-negative Fig . 5B . We also analyzed a fully sequenced genome using the online server Burrows-Wheeler Tandem Repeat Searcher ( BWtrs ) to determine whether tetranucleotide repeat indels occur within other genes [21] . Strain MN8m has not been fully sequenced so we chose a strain with an icaC tetranucleotide expansion , strain Bmb9393 . The genome harbored 59 regions with 3 or more direct tandem tetranucleotide repeats . Out of these 59 regions , 13 exhibited indels when the region was compared to 48 completed S . aureus genomes and 438 scaffolds or contigs in the NCBI database . Nine of these indel regions were within intergenic regions , but 4 of them occurred within open reading frames . Two of these indels were in putative phage anti-repressor proteins ( SABB_01096 and SABB_02531 ) , one was in a hypothetical phage protein that was not annotated in Bmb9393 , and the fourth was in icaC . The frequency of reversion from non-mucoid ( JB12 ) to mucoid was very low . We hypothesized that the higher frequency with which nonmucoid variants were isolated from MN8m cultures was due to a fitness cost imparted by high-level PIA/PNAG production . To determine if there was a fitness cost associated with constitutive PIA/PNAG synthesis , we inoculated competitive co-cultures with equivalent numbers of MN8m and JB12 and examined shifts in the population over time by assessing colony morphology on CRA . We observed that there does indeed appear to be significant growth advantage in the PIA/PNAG-negative variant JB12 , and that by 12 hours , more than 95% of the culture was non-mucoid ( Fig . 6 ) . Direct calculation of the fitness cost of PIA/PNAG over-production versus PIA/PNAG loss resulted in fitt ( relative bacterial fitness ) values of +1 . 401 at 6 hours , and +1 . 386 at 12 hours , with a value greater than 1 indicating a significant fitness advantage of the JB12 PIA/PNAG-negative phenotype over the mucoid MN8m . We calculated the generation time for strains with differing levels of PIA/PNAG production . The generation time for MN8 , a low-PIA/PNAG-producing strain , was 48 minutes , while that of MN8m , the overproducing strain , was 67 minutes . Interestingly , while the PIA/PNAG-negative strain JB12 had a generation time of 54 minutes , a strain lacking the entire ica locus , MN8ΔicaADBC::erm had a longer generation time of 59 minutes . It stood to reason that the difference in generation time was largely responsible for the frequent isolation of non-mucoid variants from MN8m cultures and that the actual frequency of the repeat insertion was low , similar to the rate of repeat deletion in JB12 . To minimize the contribution of growth rate , we inoculated liquid medium from single MN8m colonies , plated half of the suspension immediately on CRA to ensure that the starter culture was free from variants , and incubated the culture for only 67 minutes , enough time for a single round of cell division before plating the remainder . We did not detect any variants in 45 , 000 cell divisions , suggesting that the frequency is less than 1 in 45 , 000 divisions . In the absence of a fitness advantage to select for PIA/PNAG-off mutants , we would not expect a high prevalence of mutants in culture . Unlike strain MN8m , strain MN8 produces a more typical , moderate amount of PIA/PNAG . Therefore , to determine whether the icaC mutation was only selected for when the parent strain was a PIA/PNAG-overproducer or whether the mutation would occur within an average PIA/PNAG-producing strain , we used high throughput sequencing to determine whether the mutation occurred in MN8 . We amplified a 344-bp region of the icaC gene that included the tandem repeats and sequenced the product . Out of ∼88 , 000 reads , ∼140 contained a 4-nt expansion or contraction of the repeat region confirming that the mutation occurs in an average PIA/PNAG-producing strain in vitro . We also investigated whether the clinical MRSA isolates with icaC-off mutations , NRS63 and NRS264 , were derived from average PIA/PNAG-producing strains or hyper-producing strains . We performed realtime RT-PCR and found that , unlike strain MN8m , icaC transcript levels were moderate and comparable to strain MN8 , not strain MN8m ( data not shown ) . We selected 48 colonies with a darker phenotype on CRA from each strain , performed slot-blot analysis using the PIA/PNAG-specific antiserum , and sequenced the ica loci . Revertants in which the icaC gene reverted to the “on” genotype produced only modest levels of PIA/PNAG ( data not shown ) . Together these data suggest that the “icaC-off” strains NRS63 and NRS264 arose from moderate PIA/PNAG producing strains . We did not directly measure function to confirm that the IcaA , IcaD , and/or IcaB proteins were functional in JB12; however , complementation of the mucoid phenotype in trans by a copy of the icaC gene alone suggests that only the function of IcaC is altered in JB12 and that IcaA , IcaD , and IcaB are still present and functional . We hypothesized that one or more of these proteins may have alternative roles within the cell that contribute to bacterial fitness and that this was the reason why icaC was the target for phase variation . Under starvation conditions , when the bacteria were switched to minimal media containing no carbon source , JB12 survived significantly longer than MN8Δica::tet ( data not shown ) . To confirm that this difference was not due to a secondary mutation introduced during strain passage , three isogenic strains that differed only at the ica locus were made . We performed allelic exchange in strain MN8Δica::tet to replace the tetracycline resistance cassette and the interrupted ica locus with the entire MN8m ( MN8icaC_On ) or the entire JB12 ( MN8icaC_Off ) ica locus on the chromosome . The allelic exchange mutants exhibited survival profiles similar to MN8m and JB12 and both strains survived longer in the minimal media than MN8Δica::tet ( Fig . 7 ) . Production of intact , and presumably functional IcaA , IcaD , and IcaB appeared to significantly increase survival of JB12 under these growth-limiting conditions .
The polysaccharide PIA/PNAG plays an important role in virulence both through its contribution to biofilm formation and immune evasion . In fact , the benefits of PIA/PNAG to survival appear to have resulted in its ubiquitous production by a wide variety of pathogens [22] . It is clear however , that it is not always necessary for survival during infection as PIA/PNAG-negative S . aureus and S . epidermidis strains have been isolated [23] , [24] . In S . epidermidis , and in a minority of S . aureus strains , PIA/PNAG production can be switched off by the insertion of an IS256 element in the icaC gene , however , prior to this study , phase variation of PIA/PNAG expression in S . aureus isolates that lack this insertion element was not clear [25] , [26] . We noted that it was very difficult to maintain pure mucoid cultures of the PIA/PNAG-overproducing strain MN8m . Over time , MN8m cultures appear to contain a mixture of both mucoid and non-mucoid bacteria . In this study , we investigated the molecular basis for this phenomenon . The most common mutation resulting in the PIA/PNAG-negative phenotype was an expansion of a 4-nt repeat within the icaC gene . The rapid increase in the proportion of PIA/PNAG-negative bacteria in these cultures was due to their increased fitness relative to the PIA/PNAG-overproducing parent strain . Of note , we grew our cultures in conical tubes rather than Erlenmeyer flasks , and the depth of the medium likely resulted in microaerobic conditions . The low levels of oxygen during growth may have contributed to PIA/PNAG production and to the relatively low endpoints ( OD600nm ∼3 . 0 ) in our growth curves [27] and may have also contributed to the growth advantage of the PIA/PNAG-negative mutants . The mutation frequency was very low but the growth advantage allowed non-mucoid variants to take over liquid cultures in time . This resulted in a wide variability in the number of variants present in different cultures depending on the point at which the first variants arose . Therefore , the final number of variants present at the end of the culture period would depend upon the timing of the first mutation event and would be stochastic . The mucoid strain , MN8m produces approximately 1 , 000-fold more PIA/PNAG than most clinical isolates . The fitness advantage of the PIA/PNAG-negative variants isolated upon in vitro culture , such as JB12 , may be due to the metabolic cost of hyperproduction of PIA/PNAG . During infection , PIA/PNAG plays an important role in immune evasion , and PIA/PNAG-negative variants would likely be more susceptible to neutrophil-mediated killing . Therefore , despite the metabolic cost , selection pressures that favor the PIA/PNAG-negative phase variants are likely less pronounced in vivo . However , skin colonization and ocular infections appear to favor the PIA/PNAG-negative phenotype in S . epidermidis and ica-negative clinical isolates of S . aureus have been detected as well [23] , [24] , [28] . Furthermore , as PCR amplification of the ica genes is often used to demonstrate the capacity to produce PIA/PNAG [29] , [30] , our finding that a 4-nt indel mutation can shut off PIA/PNAG production suggests that PIA/PNAG negative clinical isolates may be more prevalent than previously appreciated . When we analyzed 103 S . aureus genomic sequences , we found that 9 ( ∼9% ) contained a slipped strand mutation in the tandem repeat region in icaC . We were able to obtain 2 of these isolates , NRS63 and NRS264 , and both were PIA/PNAG-negative . When we isolated “IcaC-on” revertants from NRS63 and NRS264 we found moderate PIA/PNAG production , suggesting that PIA/PNAG hyper-production is not a prerequisite for selection of “IcaC-off” mutants . An immune response against PIA/PNAG could also serve as a selective pressure against PIA/PNAG production . Antibodies against deacetylated PIA/PNAG effectively mediate opsonophagocytosis [31] . Therefore , in the event that the host mounts an effective antibody response , the capacity to switch PIA/PNAG production off could benefit the bacteria in vivo . It could also benefit the bacteria in conditions that favor the planktonic mode of growth . The 5 bp deletion in the MN8m ica promoter leads to constitutive icaADBC transcription and the accumulation of at least 300-fold more transcript than the clinical isolate parent strain MN8 . Such high-level transcript production and protein synthesis would seem to be metabolically costly even in the absence of PIA/PNAG production . We therefore found it somewhat surprising that the most common mutation ( 9 out of 15 variants ) occurred within the last gene in the operon ( icaC ) and that icaADBC transcript was still being produced at MN8m levels . Furthermore , the insertion sequence IS256 has been shown to turn PIA/PNAG synthesis off through insertional inactivation of icaC [26] suggesting again that mutation of icaC is the preferred “off switch” for PIA/PNAG production . We tested our hypothesis that there might be some advantage to continuing synthesis of IcaA , IcaD , and IcaB even though PIA/PNAG is not produced in the absence of IcaC . Our results indicate that under nutrient-limiting conditions , possession of functional icaADB genes was advantageous for survival . The basis for this survival advantage is unclear at this time . Further work is necessary to conclusively determine whether or not the IcaA , IcaD , and IcaB proteins function in some capacity in addition to PIA/PNAG synthesis . In conclusion , we found that the RecA-independent expansion or contraction of a 4-nt tandem “ttta” repeat shifts the reading frame of icaC , leading to a premature stop codon , truncating the protein at 303 amino acids; 47 amino acids shorter than full-length protein . Structural prediction indicates that the mutation disrupts a transmembrane domain of IcaC , and we found that the mutation resulted in the complete abrogation of PIA/PNAG production . We found that the mutation frequency was low , but that in vitro , elevated production of PIA/PNAG carried a fitness cost and consequently , PIA/PNAG-negative phase variants quickly increased in number relative to PIA/PNAG over-expressers . Of note , IcaC appears to be the chosen target for phase variation in PIA/PNAG production and loss of this protein appears to confer a survival advantage under nutrient-poor conditions relative to loss of the entire operon suggesting that the other proteins encoded within the ica locus could have some other function . Alternatively , PIA/PNAG precursors could accumulate within the bacterial cells in the absence of IcaC , and affect growth . Studies to determine the effect of the IcaADB proteins on growth are underway .
Staphylococcus aureus strain MN8 is a clinical isolate from a case of toxic shock syndrome [32] . Strain MN8m was a spontaneous mutant isolated from a chemostat culture of strain MN8 [20] . Strain SA113Δica was provided by Dr . Sarah Cramton [12] and the mutation was transduced to strain MN8 by phage 80α to produce MN8Δica::tet . Strain MN8Δica::tet was complemented by allelic exchange with the ica loci from the entire MN8m and JB12 strains to produce strains MN8icaC_On and MN8icaC_Off , respectively . To this end , the ica loci were amplified by primers SA11 and SA12 as previously described [12] cloned into the pMAD vector , and allelic exchange was performed as previously described [33] . Mutants were tetracycline sensitive and allelic exchange was confirmed by sequencing the ica loci . A recA mutant of strain MN8m was produced by transducing the bursa aurealis transposon from strain NE805 ( NARSA repository ) using phage 80α . NRS264 and NRS63 are sequenced clinical isolates obtained from the NARSA repository . NRS264 was associated with bacteremia and abscess and NRS63 was a bacteremia isolate . All strains were grown aerobically at 37°C on tryptic soy agar ( TSA ) plates containing the appropriate antibiotic . Liquid cultures were in tryptic soy broth containing 1% glucose ( TSBG ) , incubated in air at 37°C , 200 rpm in 5 mL in 50 mL conical tubes ( microaerobic conditions ) . Congo red agar was composed of brain heart infusion ( BHI ) agar +3 . 6% sucrose +0 . 5% glucose +0 . 08% congo red . Plasmid purifications were performed using the QIAprep Spin Miniprep kit ( Qiagen , Valencia , CA ) according to the manufacturer's instructions . Primers were custom synthesized by Integrated DNA Technologies ( Coralville , IA ) . Restriction enzymes were purchased from New England Biolabs ( Beverly , MA ) . The icaC gene was cloned into the isopropyl-β-d-thiogalactopyranoside ( IPTG ) -inducible vector pCL15 ( kindly provided by Dr Chia Lee , University of Arkansas ) [34] . The gene was PCR amplified from strain MN8m genomic DNA with the primer set , icaCSphIFwd ( 5′-CCGCGCATGCCAAAAATGGCAGAGAGGAAGA-3′ ) and icaCKpnIRev ( 5′-CCGCGGTACCCCGCGTGTTTTTAACATAGC-3′ ) . Initial cloning was performed in E . coli using the pCR4TOPO vector ( Invitrogen , Grand Island , NY ) according to manufacturer's instructions . The genes were digested from the cloning vector with the appropriate restriction enzymes , purified after gel electrophoresis using the QIAquick Gel Extraction kit , and ligated into pCL15 using Ready-To-Go T4 DNA ligase ( GE Healthcare , Piscataway , NJ ) . After passage through E . coli , all plasmid constructs were transformed into the restriction-deficient S . aureus strain RN4220 according to the method of Lee [35] . Constructs were transferred to other strains of S . aureus by transduction with phage 80α . To generate a bacterial growth curve for use in calculating strain generation time , TSBG was inoculated with individual bacterial colonies and gently sonicated for 30 seconds to break apart clusters . Each culture was diluted to OD600nm of 0 . 1 and used to inoculate fresh TSBG 1∶100 , with separate tubes for each time point . The cultures were incubated at 37°C , 200 rpm . At each time point the cultures were gently sonicated , the OD600 nm measured , and plated on CRA plates to monitor population mucoid/non-mucoid phenotype over time and ensure that variants did not arise to influence observed growth rates . Logarithmic growth was determined to occur between 180 and 360 minutes for each of the strains . For this time period , the A600 measurements were converted into log2 values , and the generation time was calculated as the inverse of the slope of the line of best fit . For the competitive fitness assay , cultures were grown overnight in TSBG , and gently sonicated for 30 seconds . Each culture was diluted to a concentration of 103 cells and mixed 1∶1 with MN8m + JB12 , with separate tubes for each time point . The cultures were incubated at 37°C , 200 rpm . At each time point the cultures were gently sonicated , serially diluted and plated in triplicate on CRA plates for CFU counting . Calculation of the difference in fitness was determined using the function derived from Sander et al . [36] LN ( ( ( nmt/mt ) / ( nmt-1/mt-1 ) ) ∧ ( 1/gen ) ) where nmt and mt represent the non-mucoid and mucoid cells , respectively at a given time t . While nmt-1 and mt-1 denote the quantity of non-mucoid and mucoid cells at the preceding timepoint . The quotient of the ratios was standardized with the exponent 1/generation , with the assumption that cell numbers determined at 24 hours represents approximately 17 generations . The relative bacterial fitness for a given time was calculated as fitt = 1+St . The fitness value is equal to 1 if there is no difference in fitness between the competing strains , less than 1 if the non-mucoid phenotype reduces fitness , or greater than 1 if the non-mucoid phenotype increases bacterial fitness . Microtiter plate assays for biofilm formation were performed essentially as described previously by Christensen et al . [37] with minor modifications . Cultures were grown overnight in 4 ml of TSBG or TSBG +10 µg ml−1 chloramphenicol , diluted 1∶200 in the same media or media with 1mM IPTG added for plasmid induction , and aliquoted into 96-well polystyrene flat-bottom microtiter plates ( Greiner Bio-One , Monroe , N . Carolina ) . After 24 hours at 37°C , the wells were emptied and washed once with phosphate-buffered saline ( PBS ) . The plates were dried at room temperature , stained with 200 µl safranin for 1 minute , washed gently with water , and allowed to dry . The biofilms were assessed qualitatively by visual inspection and images were taken using a digital scanner . The safranin was then resuspended in 200 µl 33% acetic acid and the wells were analyzed by spectrophotometry at OD562 nm using a 96-well plate spectrophotometer . RNA was isolated from exponentially growing bacteria , following induction with 1 mM IPTG for 2 hours if pCL15 was present , using the Qiagen RNeasy kit ( Qiagen , Valencia , Calif . ) according to the manufacturer's instructions . Contaminating DNA was digested with Turbo DNAse ( Ambion , Austin , Texas ) , and the mRNA transcript levels were measured by quantitative reverse transcriptase ( RT ) -PCR . Reverse transcription of 1 µg of RNA was performed using the Tetro cDNA synthesis kit ( Bioline , Taunton , Mass . ) according to manufacturer's instruction , and 10 pmol icaCRTRev ( 5′-CGTTCCAATAGTCTCCATTTGC-3′ ) , and 16SRTRev ( 5′- TATGCATCGTTGCCTTGGTA-3′ ) . Controls for DNA contamination contained no reverse transcriptase . SensiMix SYBR & Fluorescein mix ( Bioline , Taunton , Mass . ) was used for the quantitative real-time PCR with the primer sets: icaCRTFwd ( 5′-CGAACAACACAGCGTTTCAC-3′ ) and icaCRTRev , or 16SRTFwd ( 5′-GAACCGCATGGTTCAAAAGT-3′ ) and 16SRTRev . PIA/PNAG slot blots were performed essentially as described previously by Cramton et al . [27] with minor modifications . Bacteria were grown overnight in TSBG or TSBG +10 µg ml−1 chloramphenicol +1 mM IPTG . For cell surface extracts 109 cells were collected by centrifugation , washed once with PBS , and resuspended in 250 µl of 0 . 5 M ethylenediaminetetraacetic acid ( EDTA ) . To analyze secreted PIA/PNAG , 250 µl of spent media was retained . All samples were incubated in boiling water for 5 minutes , cooled , and incubated at 65°C for 1 hour with 20 µl proteinase K . Samples were boiled for an additional 5 minutes to inactivate the protease , diluted in PBS , and immobilized on nitrocellulose with a vacuum manifold . Blots were blocked overnight at 4°C in 5% bovine serum albumin , probed with 1∶5 , 000-diluted rabbit antiserum specific for PIA/PNAG ( kindly provided Dr . Gerald B . Pier ) [7] for 2 hours at 21°C , washed , and probed with 1∶10 , 000-diluted goat anti-rabbit immunoglobulin-horseradish peroxidase conjugate for 1 hour at 21°C . Bands were visualized with the ECL Plus western blotting detection system ( GE Healthcare ) . MN8 was cultured for 4 hr at 37°C . DNA was purified using the DNeasy Blood and Tissue kit ( Qiagen ) and amplified by PCR using SSSeqFWD ( 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAACGTTACCAGCTTTTCATATTC-3′ ) and SSSeqREV ( 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCACCGCGTGTTTTTAACATAGC-3′ ) . The PCR product was sequenced at the Nucleic Acids Research Facilities at VCU using Illumina MiSeq . Sequencing yielded ∼88 , 000 paired end reads , which were compared to the MN8 parent sequence using JBrowse to detect indels [38] . Liquid cultures were initially grown shaking aerobically in TSBG for 24 hours . Bacteria were collected by centrifugation , and equal numbers of bacteria for each sample were resuspended in equal volumes of MOPS minimal media ( Teknova , Hollister , Ca ) with no glucose , or other carbon source . The cultures were incubated at 37°C while shaking . At each time point the cultures were serially diluted and plated in triplicate on TSA plates for CFU enumeration . Each strain was analyzed in technical triplicate and biologic replicates . | Staphylococcal polysaccharide intercellular adhesin ( PIA ) , also known as β-1-6-linked N-acetylglucosamine ( PNAG ) plays a role in immune evasion and biofilm formation . Evidence suggests that under certain circumstances PIA/PNAG production is beneficial , whereas at times , it may be advantageous for the bacteria to turn production off . In S . epidermidis , PIA/PNAG can be switched off when an insertion sequence recombines into the intercellular adhesin locus ( ica ) . In this study , we have found a short tandem repeat sequence in the ica locus of S . aureus that can undergo expansion and contraction . The addition or subtraction of non-multiples of three of this repeat shifts the reading frame of the icaC gene , resulting in the complete loss of PIA/PNAG production . We hypothesize that certain conditions that make the PIA/PNAG-negative phenotype advantageous during infection , such as the development of an effective immune response to PIA/PNAG on the bacterial surface , would select for repeat mutants . In support of this hypothesis , we found clinical isolates with expansion and deletion of the repeat . These findings reveal a new on-off switch for the expression of PIA/PNAG . | [
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] | 2014 | Phase Variation of Poly-N-Acetylglucosamine Expression in Staphylococcus aureus |
Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites . Rabies virus ( RABV ) circulates through two different epidemiological cycles: terrestrial and aerial , where dogs , foxes or skunks and bats , respectively , act as the most relevant reservoirs and/or vectors . It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory . We studied the positivity rate of RABV detection in different areas of the country , and the antigenic and genetic diversity of 99 rabies virus ( RABV ) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008 . Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance , the positivity rate of RABV in insectivorous bats ranged from 3 . 1 to 5 . 4% , depending on the geographic location . The findings were distributed among an extensive area of the Argentinean territory . The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis , Myotis spp , Eptesicus spp , Histiotus montanus , Lasiurus blosseviilli and Lasiurus cinereus . Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries . Finally , inter-species transmission , mostly related with Lasiurus species , was demonstrated in 11 . 8% of the samples . This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina . This information is relevant to identify potential areas at risk for human and animal infection .
Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites . Rabies virus ( RABV ) circulates through two different epidemiological cycles: terrestrial and aerial , where dogs , foxs or skunks and bats , respectively , act as most relevant reservoirs and/or vectors . In Argentina , successful vaccination and control of canine rabies in the 1980s revealed the importance of bats in RABV transmission . Cases associated with the hematophagous vampire bat Desmodus rotundus are common in endemic areas of Argentina [1] . Two human rabies cases were associated with this species in 1997 and 2001 [2] . No cases have yet been associated with insectivorous bats , a stark contrast to the United States and Canada where these bats are the most common source of indigenously acquired human rabies infections [3] . Thus , it is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory [4] , [5] , [6] . Monoclonal antibodies ( N-Mabs ) directed against the viral nucleoprotein ( produced by the CDC , USA ) have allowed for identification of antigenic variants ( V ) associated with insectivorous bats circulating in Argentina . Two variants were identified: V4 and V6 , associated with Tadarida brasiliensis and Lasiurus cinereus , respectively [7] , [8] . Additionally , although a limited number of specimens were analyzed , partial sequencing of the viral nucleoprotein has revealed at least four genetic variants or lineages associated with other insectivorous bat species [8] . Here we present an extensive study of the geographical distribution of disease associated with insectivorous bats , to infer which species are involved in the maintenance and transmission cycle of RABV in Argentina and to identify possible interspecies transmission patterns .
The Argentinean National Rabies Network consists of ten regional laboratories distributed in northern and central regions of the country . The south of Argentina is considered free of terrestrial rabies . RABV detection is mainly performed using direct immunofluorescence detection in brain samples and mouse inoculation test . Later , RABV isolates are sent to two National Reference Laboratories for antigenic characterization ( DILACOT , SENASA or Instituto de Zoonosis “Dr Luis Pasteur” ) . Taxonomic characterization of the bats is performed by local specialists at the collection sites . A total of 99 rabies samples were obtained from insectivorous bats and bat related cases between 1991 and 2008 from throughout Argentina . All viruses were isolated by intracerebral inoculation in mice as described previously [9] . Eight other RABV isolates from Argentinean insectivorous bats were also analyzed [8] . The host species and geographic location of rabies isolates are shown in Table S1 . Samples were identified as follows: The first letters indicate abbreviation for the bat species follow by the number for each sample in the virus repository at SENASA or Instituto Pasteur and the year of collection . In addition , four terrestrial specimens ( Saldg126 , Saldg146 , Chadg120 , Chafx119 ) and six vampire bat related cases ( Ctebv01 , Chabv90 , Ctebv011 , Ctehm82 , Chabv72 , Chabv129 ) were included in this study for comparison . In the comparator group , we included 93 sequences corresponding to historical samples available in GenBank , which represent the major bat RABV clades reported in the United States , Canada , Mexico and South America . Antigenic characterization was performed by indirect immunofluorescence using a panel of eight monoclonal antibodies directed against the viral nucleoprotein ( C1 , C4 , C9 , C10 , C12 , C15 , C18 , C19 ) kindly provided by the Centers for Disease Control and Prevention , ( Atlanta , GA , USA ) . Positivity reactivity results were analyzed using previously described antigenic variant patterns [7] . Viral RNA was extracted from isolates using TRIzol® ( Invitrogen , Carlsbad , CA , USA ) . Reverse transcription and PCR amplification were achieved with primers 10 g and 304 , as previously described [10] . The amplified product was sequenced using a BigDye Terminator v3 . 1 cycle sequencing kit according to the manufacturer's protocol with the ABI PRISM® 310 Genetic Analyzer ( Applied Biosystems Inc . Foster City , California , USA ) . A 264-bp region corresponding to the nucleoprotein gene located between nucleotides 1157 and 1420 and amino acids 363 to 450 ( numbered according to strain SAD B19 ) was analyzed [11] . Raw sequence data were first edited using CHROMAS software ( version1 . 3 , Mc Carthy 1996 , Griffith University , Queensland , Australia ) . Complete alignment was performed with Clustal X 1 . 8 [12] . The alignment was analyzed using Kimura 2 parameters as a method of substitution and Neighbor-Joining model to reconstruct the phylogenetic tree ( MEGA version 4 . 1 ) [13] . The statistical significance of the phylogenies constructed was estimated by bootstrap analysis with 1000 pseudoreplicate data sets [14] , [15] . Partial nucleoprotein gene sequences described in this study were deposited in the GenBank database under the following accession numbers JF738250–JF738348 .
Between 1995 and 2007 , public health authorities notified 1096 cases of animal rabies: dogs ( 33 . 6% ) , cattle ( 45 . 9% ) or insectivorous bats ( 15 . 4% ) . The success of vaccination programs meant that of 200 rabies cases reported for 2008–09 , canine rabies only represented 14 . 5% , while rabies transmitted by insectivorous bats increased to 38% , and cattle remained constant ( 45 . 5% ) [16] . Geographic distribution of rabies in Argentina between 2008 and 2009 is shown in Table 1 . Importantly , Northeastern ( NE ) and Northwestern ( NW ) regions show a predominance of rabies by vampires while in Central and South regions , almost all reported cases are associated with insectivorous bats . From 1991 to 2008 , Laboratories of National Rabies Network tested 4536 insectivorous bats for RABV from Buenos Aires city ( CABA ) and 23 from provinces of Argentina . Of these , 207 were found to be positive . Three provinces accounted for 82 . 2% of all reported cases of rabies in bats: Buenos Aires province , 70 cases ( 33 . 7% ) ; CABA , 63 cases ( 30 . 4% ) ; and Santa Fe province , 38 cases ( 18 . 4% ) . National positivity rate of rabies caused by insectivorous bats could not be calculated since the total number of bat specimens received from all provinces for rabies investigation was unavailable . However , local positivity rate could be determined in CABA , 3 . 5% ( 63/1792 ) , Buenos Aires province 5 . 4% ( 70/1307 ) and Santa Fe province 3 . 1% ( 38/1113 ) . Of the bats infected with rabies virus , 65 . 4% of RABV-positive cases of T . brasiliensis were detected in CABA , while 77 . 8% of L . cinereus were detected in Buenos Aires province , and 60 . 0% of Myotis genus and 87 . 5% of Eptesicus genus were found by Santa Fe province . Great diversity was observed in Santa Fe ( ten species ) and Buenos Aires ( nine species ) . T . brasiliensis accounted for 92 . 7% of the total in CABA ( four species ) . Antigenic typing was performed on 103 isolates ( Table S1 ) . Antigenic variant 4 ( AgV4 ) was identified in 57 bats: 53 ( 92 . 9% ) in T . brasiliensis; the rest ( 7 . 1% ) in M . molossus ( n = 2 ) , Eumops patagonicus ( n = 1 ) and unidentified bat ( n = 1 ) . This variant showed the greatest distribution , scattered throughout the provinces . Antigenic variant 6 ( AgV6 ) was identified in 25 specimens: 11 L . cinereus ( 44 . 0% ) , 4 L . ega ( 16 . 0% ) , and the remaining 10 ( 40 . 0% ) in Eptesicus spp ( n = 1 ) , M . molossus ( n = 1 ) , M . levis ( n = 1 ) , M . nigricans ( n = 1 ) , one Myotis spp ( n = 1 ) , four unidentified bats , and one dog . This variant mainly circulated in central provinces . Finally , 22 isolates exhibited 11 different atypical reaction pattern ( ARP ) ( Table 2 ) . These isolates were from Myotis ( n = 7 ) , Eptesicus ( n = 9 ) , Histiotus genus ( n = 3 ) , T . brasiliensis ( n = 1 ) , L . blossevillii ( n = 1 ) bats and one from a cat . A total of 107 insectivorous bat-related sequences segregated into six distinct lineages . This was well supported by significant bootstrap values and clearly differentiated from those related to rabies in terrestrial animals ( Figure 1 ) . The first lineage ( TB ) included 51 samples from T . brasiliensis , two from M . molossus , one from Eumops patagonicus and one from an unidentified bat . All isolates were typed as V4 . This lineage exhibited a high nucleotide and amino acid similarity ( 99 . 0% and 100 . 0% , respectively ) . It showed the amino acidic residue N394 in the nucleoprotein that is characteristic of this genetic variant ( Table 3 ) . Rabies isolates obtained from Myotis , Eptesicus and Histiotus bats grouped in three highly diverse lineages ( nucleotide intra-lineage distance of 6 . 7% , 2 . 0% and 3 . 4% , respectively ) . All these isolates resulted in several atypical reaction patterns with N-Mabs ( ARP ) . Myotis lineage ( MY ) was subsequently divided in five sublineages ( MY1–5 ) less-well supported , scattered throughout central provinces ( Figure 2 ) . The Eptesicus lineage ( EP ) was also divided in three sublineages ( named EP1–3 ) . The three EP sublineages resulted in different ARP circulating in Santa Fe , Entre Rios and Buenos Aires . Both lineages MY and EP presented a similar coding signature containing the amino acids A385 and L419 . Lastly , the Histiotus montanus lineage ( HM ) included three isolates from southern provinces ( Santa Cruz and Chubut ) . It was further divided in two sublineages ( HM1 and HM2 ) in correspondence with their antigenic and nucleotide sequence clustering with nucleotide and amino acid distance of 4 . 3% and 2 . 3%; amino acid difference at position 369 ( Q369K ) . Furthermore , they showed different reaction with C12 N-Mab ( Table 3 ) . Finally , the last two lineages ( named LB and LC ) , were detected in three different species of Lasiurus bats . LB included a single sample of L . blossevilli , antigenically typed as ARP . LC included 30 samples , obtained from 13 L . cinereus ( 43 . 3% ) , four L . ega ( 13 . 3% ) and others species: one Eptesicus spp . , one E . bonariensis , one M . molossus , three Myotis spp . , one dog , and four samples from unclassified bats . LC is one of the less divergent lineages ( 0 . 3% ) and was formed from 30 samples . Virus of the lineage LC were mainly typed as V6 ( n = 25 , 83 , 3% ) ; ARP ( n = 4 , 13 , 3% ) and V4 ( n = 1; 3 , 4% ) Deduced amino acid sequences revealed a characteristic amino acid S414 in both LC and LB lineages . Moreover , a change at position 419 ( S419T ) was identified between LC and LB lineages . Analysis of nucleoprotein RABV sequences associated with insectivorous bats in Argentina and the Americas revealed several monophyletic clusters associated with specific bat species . This was consistent with previous analyses [17] , [18] , [19] ( Figure 3 ) . RABV samples obtained from T . brasiliensis bats from Argentina , Brazil , Chile and Uruguay segregated into a monophyletic cluster . RABV sequences associated with Myotis species occurring in South America were divided in two heterogeneous lineages formed by samples from Argentina , Chile and Uruguay or Brazil . Instead , RABV sequences associated with Myotis species occurring in North America ( USA , Canada ) formed a different cluster . Similarly , RABV samples from Eptesicus bats of Argentina and Brazil , represented primarily by furinalis subspecies , were grouped in two clusters . These clusters did not show relationships with the genetic clusters associated with fuscus subspecies from USA and Canada . Strains that were recovered in Argentina and Chile from Histiotus bats circulated in both countries as at least two sublineages less-well supported . Finally , RABV sequences obtained from different species of Lasiurus from the Americas were divided in three monophyletic clusters associated with subspecies blossevillii , borealis and cinereus/ega . An Argentinean sample obtained from L . blossevillii ( Lb658-BA03 ) grouped with two Brazilian L . blossevillii-related RABV strains ( BR-BAT27 and BR-BAT13 ) . RABV isolates obtained from L . cinereus and L . ega from Argentina , Brazil , Chile , Uruguay , USA , Mexico and Canada grouped in a cluster with the highest nucleotide homogeneity ( 99 . 5% ) .
The current understanding of the epidemiology of rabies in Argentina states that the vast majority of cases in the Northern region are associated with hematophagous bats . Very few cases of terrestrial rabies were also detected in the Northwestern ( Salta and Jujuy ) and Northeastern provinces ( Formosa and Chaco ) during 2010 . The rest of the country has been considered terrestrial rabies-free since the early 1980s . This epidemiological situation makes it difficult to estimate the real impact of the insectivorous bat rabies in our country . In the north , very few cases have been detected probably masked by endemic bovine rabies . While in the center and south , the absence of terrestrial rabies has led to low level of awareness among general public , public health officials and health administrators . As a result , rabies associated with insectivorous bats and its potential consequent implications in public and animal health have been largely neglected in Argentina . Our study reveals that the positivity rate of rabies in insectivorous bats received in the laboratory for analysis ranges from 3 . 1 to 5 . 4% . This proportion is comparable to other countries such as the United States ( 9–10% ) where insectivorous bats are the only cause of concern for RABV surveillance systems , and other South American countries ( Brazil ( 1 . 3% ) and Chile ( 4 . 2% ) [20] , [21] , [22] . Fortunately , <1% of natural bat population have been shown to be infected [23] . Thus , the risk of contracting rabies from insectivorous bats is low . However , evidence indicates that many of the human cases of rabies resulted from exposures to bats that were not recognized or reported [3] . Consequently , prevention of human infection with bat rabies virus variants remains an important public health concern . On the other hand , emergence of rabies in terrestrial hosts after spillover from chiropteran reservoirs has been described but does not typically result in sustained transmission . However , if host switching of rabies virus variants occur , once established could be become enzootic in new reservoir species [24] . Therefore , special attention should be paid to unusual epidemiological patterns of terrestrial rabies transmission in new geographic areas . Antigenic characterization utilizing the eight monoclonal antibodies developed by the CDC is widely used in Latin America for RABV surveillance . However , in some instances , antigenic analysis is unable to identify RABV isolates obtained from several insectivorous bat species because these isolates produced atypical reaction patterns ( unrelated to previously described virus reservoirs ) [8] . In those cases , partial genetic analysis of the viral nucleoprotein sequence allowed further characterization [25] allowing the identification of lineages or genetic variants maintained by insectivorous bat species in an independent enzootic cycle . Indeed , in our work , we identified six RABV lineages that were specifically associated with specific bat species . Moreover , genetic analysis allowed us to differentiate some of the previously accepted antigenic variants in independent sublineages that appear to be related with different geographical or ecological niche behaviors . Tadarida brasiliensis maintains circulation of its own antigenic ( AgV4 ) with high degree of nucleotide and amino acid homogeneity in Argentina , Chile , Brazil and Uruguay . In contrast , analysis of RABV isolates recovered from Myotis and Eptesicus species showed a high antigenic diversity that could be related to the gregarious and non-migratory habits of these species [17] , [18] . The elevated antigenic diversity of RABV sustained by Eptesicus or Myotis species can complicate definitive strain typing . This could be the case of the only Myotis associated strain from Chile , which was assigned to an apparent AgV3 using a reduced panel of eight N-MAbs by Yung et al . [26] , but our genetic characterization revealed its real clustering into Myotis group . All Argentinean isolates obtained from Histiotus montanus , clustered in a single genetic group along with strains from Chile confirming that this species is its own the viral variant reservoir . There is little information about this species other than it lives a solitary life and migrates seasonally between Argentina and Chile [27] . Although both countries are separated by the Andes Chain , an important natural barrier , low-lying passages of the mountain allow different species of bats and terrestrial mammals to move between the countries and thus spreading this viral variant [28] . Members of the genus Lasiurus typically are solitary and migratory bats . In our study , rabies samples from L . blossevillii , cinereus and ega were analyzed . A RABV isolate from Argentinean L . blossevillii bat was distant to others obtained from the Lasiurus genus but clustered with others from the same bat species from Brazil . Indeed , it yields a previously unrecognized genetic lineage circulating in Argentina . On the other hand , antigenic and molecular analysis of rabies isolates from Lasiurus cinereus confirmed that this species maintained its own antigenic ( V6 ) and genetic variant as previously reported [29] . Despite showing a wide geographical distribution ( Canada to Argentina ) , RABV isolates from this species exhibited a high degree of nucleotide and amino acid homogeneity , which could be explained by its ability to transport its own specific strain during its long migration pattern . Two rabies samples of Chilean Lasiurus have been assigned to AgV4 ( T . brasiliensis reservoir ) by Yung et al . [26] , but our phylogenetic analysis showed that they grouped with Lasiurus strains ( AgV6 ) . This apparent discrepancy could be explained by the inadequate use of the N-Mabs panel which could lead to confusion between AgVs . The difference between both variants falls only in the reactivity with monoclonal C1 . According to the N-Mabs manufacturer's instructions , all negative or diminished reactions should be confirmed by furthers tests of the samples with a 10-fold less-dilute antibody [30] . Molecular characterization of RABV isolates revealed that inter-species transmission is a relatively common event . Eleven ( 11 . 8% ) of the 93 bat samples tested showed to be infected by a variant supported by another bat species . Cross-species transmission is facilitated by several types of species life-history traits and perhaps environmental variables structuring communities [31] . In the case of Lasiurus , these seem to have an important role in these events , since we have identified this variant nearly in all bat species studied . Although , L . cinereus generally roosts in isolation , it has been observed occasional aggressive encounters at share roosts or during flight , which could promote viral transmission . The findings of this study demonstrate the presence of rabies in several species of insectivorous bats throughout Argentina . Phylogenetic analysis of an extensive collection of rabies strains obtained from 14 species over a 17-year period shows complex epidemiological patterns characterized by the presence of multiple endemic cycles and relatively frequent inter-species transmission that are affected by several ecological aspects such as migration patterns , roosting and habitat . The establishment of viral variants associated with specific bat species can assist in the epidemiological investigation of cases of human rabies associated with bats and potential events spread to terrestrial mammals . | In Argentina , successful vaccination and control of terrestrial rabies in the 1980s revealed the importance of the aerial route in RABV transmission . Current distribution of cases shows a predominance of rabies by hematophagous bats in the Northern regions where rabies is a major public health concern; in contrast , in Central and Southern regions where rabies is not a major public health concern , little surveillance is performed . Based on the analysis of insectivorous bats received for RABV analysis by the National Rabies system of surveillance , the positivity rate of RABV in insectivorous bats in these regions ranged from 3 . 1 to 5 . 4% . This rate is comparable to other nations such as the United States ( 9–10% ) where insectivorous bats are an important cause of concern for RABV surveillance systems . Antigenic and genetic analysis of a wide collection of rabies strains shows the presence of multiple endemic cycles associated with six bat insectivorous species distributed among an extensive area of the Argentinean territory and several countries of the Americas . Finally , inter-species transmission , mostly related with Lasiurus species , was demonstrated in 11 . 8% of the samples . Increased public education about the relationship between insectivorous bats and rabies are essential to avoid human cases and potential spread to terrestrial mammals . | [
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] | 2012 | High Diversity of Rabies Viruses Associated with Insectivorous Bats in Argentina: Presence of Several Independent Enzootics |
The transcription factor nuclear factor kappa-B ( NFκB ) is a key regulator of pro-inflammatory and pro-proliferative processes . Accordingly , uncontrolled NFκB activity may contribute to the development of severe diseases when the regulatory system is impaired . Since NFκB can be triggered by a huge variety of inflammatory , pro-and anti-apoptotic stimuli , its activation underlies a complex and tightly regulated signaling network that also includes multi-layered negative feedback mechanisms . Detailed understanding of this complex signaling network is mandatory to identify sensitive parameters that may serve as targets for therapeutic interventions . While many details about canonical and non-canonical NFκB activation have been investigated , less is known about cellular IκBα pools that may tune the cellular NFκB levels . IκBα has so far exclusively been described to exist in two different forms within the cell: stably bound to NFκB or , very transiently , as unbound protein . We created a detailed mathematical model to quantitatively capture and analyze the time-resolved network behavior . By iterative refinement with numerous biological experiments , we yielded a highly identifiable model with superior predictive power which led to the hypothesis of an NFκB-lacking IκBα complex that contains stabilizing IKK subunits . We provide evidence that other but canonical pathways exist that may affect the cellular IκBα status . This additional IκBα:IKKγ complex revealed may serve as storage for the inhibitor to antagonize undesired NFκB activation under physiological and pathophysiological conditions .
The nuclear transcription factor κB ( NFκB ) family consists of five DNA-binding proteins ( p65 , p50 , p52 , cRel , RelB ) that differentially modulate gene transcription . Since NFκB activation is involved in many cellular processes including inflammation , proliferation , angiogenesis and anti-apoptosis , only transient expression of the responsive genes ensures proper function of living cells [1] . Impairment of the regulatory system may contribute to malignant transformation , invasion and metastasis [2] . Consequently , NFκB controls its own activity by initiating negative feedback mechanisms including transcriptional up-regulation of cellular inhibitors [3] , [4] . Two independent pathways have been described to induce NFκB activation . While the non-canonical pathway determines the activity of p50:p52 heterodimers , the more prominent canonical pathway controls activation of p65:p50 subunits . In un-stimulated cells NFκB ( p65:p50 ) resides inactively within the cytosol , bound to its inhibitor IκBα , which covers its nuclear localization signal [5] . Following canonical signal transduction , NFκB activation is triggered via the IκB kinase complex ( IKK ) , which consists of two catalytic subunits , IKKα and IKKβ , as well as the regulatory subunit IKKγ . Site specific phosphorylation of IKKβ mediates downstream phosphorylation of IκBα at two Ser residues , serving as a signal for poly-ubiquitination and proteasomal degradation of the inhibitor . Liberated NFκB subsequently translocates into the nucleus to serve its function as a transcription factor which also – and most importantly – includes induction of negative feedback regulation via IκBα re-synthesis [6] . The network of interaction , however , leading to activation , inhibition or post-activational attenuation of NFκB is very complex and can be influenced by changes in signal transduction as well as through specific modifications of the molecules involved . Since dysregulation of NFκB plays a major role in the development of various diseases , a number of mathematical models have been implemented to analyze the non-linear dynamical behavior of this regulatory network [7] , [8] . These models contributed to the analysis of the role of negative feedback loops [9]–[11] , the description of the overall input-output behavior of the pathways [12]–[14] , the understanding of the integration from multiple input signals [14] , [15] , as well as to the identification of sensitive parameters [16] . However , the pattern of NFκB-induced gene expression may significantly change depending on the cell type , the intracellular protein-protein interaction and the physiological context . Following this line , previous studies revealed canonical NFκB responses to dramatically change in cells being exposed to DNA-damaging agents , including ultraviolet-B ( UVB ) radiation . In particular , Interleukin-1 ( IL-1 ) stimulation was shown to protect epithelial cells from death ligand-induced apoptosis via NFκB-dependent up-regulation of anti-apoptotic genes . When co-irradiated with UVB , instead , IL-1 driven and NFκB-dependent repression of anti-apoptotic genes caused enhancement of UVB-induced apoptosis [17] . As a prerequisite , nuclear persistence of NFκB was shown to be facilitated via UVB-induced inactivation of the catalytical subunit of Ser/Thr phosphatase PP2A , causing chronic IKKβ activation and subsequent phosphorylation and proteasomal degradation of resynthesized IκBα . Both modifications in concert were shown to drive the pro-apoptotic properties of a classical non-apoptotic protein [3] , [18] . Using a systems biological approach we could show that not only PP2A inactivation but also global translational inhibition appeared to be involved in preventing IκBα recurrence . Above this , translational inhibition was shown to induce IκBα depletion in cells irradiated with UVB alone , indicating both mechanisms to individually mediate UVB-dependent responses [15] , [19] . Similar to UVB , co-stimulation of cells with IL-1 and the tyrosine phosphatase inhibitor orthovanadate ( OVA ) induced tyrosine kinase cSrc-mediated inactivation of PP2A . Again here , chronic phosphorylation of IKKβ and consequently inhibition of IκBα recurrence provided for sustained NFκB activation [18] . In the present study we aimed to understand which additional regulatory mechanisms may exist to prevent unwanted NFκB activation under physiological conditions . We therefore analyzed OVA-dependent IκBα depletion as a tool to identify additional IκBα sources within the cell by iterative model refinement in combination with model inspired experimentation . The extended model predicted alternative non-canonical IκBα-degradation to occur without affecting NFκB activity . Respective experimental design finally revealed a yet unknown IκBα:IKKγ complex to exist , which might serve as a backup for negative feedback regulation of NFκB .
Stimulation of cells from the epithelial cell line KB with IL-1 caused canonical degradation of the NFκB inhibitor IκBα via phosphorylation of the upstream kinase IKKβ at Ser177/181 . Perfectly in line with the phosphorylation pattern of IKKβ , IκBα was completely degraded 30 min after IL-1 stimulation and became resynthesized after 2 h when phosphorylation of IKKβ started to descent ( Fig . 1A ) . Co-treatment of cells with IL-1+OVA instead accelerated phosphorylation of IKKβ causing early phosphorylation and degradation of IκBα ( Fig . 1B , 15 min ) . Strikingly , re-accumulation of IκBα was completely inhibited under these conditions even though the phosphorylation pattern of IKKβ at later time points ( 2 h; 4 h ) remained largely unchanged compared to IL-1 treated cells ( Fig . 1A ) . This strongly indicated that other OVA-driven mechanisms may superimpose IL-1-mediated canonical IκBα degradation at later time points . In accordance with this assumption we could detect partial , almost linear IκBα depletion at 4–8 h after OVA only treatment , following a much slower kinetics than canonical IL-1-driven IκBα degradation ( Fig . 1C ) . Since IKKβ phosphorylation did not seem to play a major role in delayed OVA-dependent IκBα depletion we next examined whether transcriptional or translational inhibition might be involved . Performing RT-PCR analysis we revealed the IκBα mRNA level to remain completely unchanged , even up to 8 h after OVA treatment , while being up-regulated 1 h after canonical IL-1 treatment , as a positive control ( Fig . 2A ) . Application of the transcription inhibitor actinomycin D ( ActD ) strongly implied transcriptional alterations not to be involved in OVA-induced IκBα depletion . While OVA treatment alone induced a moderate and incomplete reduction of the IκBα protein ( Fig . 2B ) but not the respective mRNA ( Fig . 2C ) over time , transcriptional inhibition by ActD caused pronounced inhibition of both the mRNA and the protein level of IκBα , respectively . Of note , co-application of OVA and ActD further enhanced depletion of IκBα protein without additively affecting the transcription level ( compare Fig . 2B + 2C ) . Results indicated that OVA-induced IκBα depletion is facilitated at the protein level , independent of transcriptional regulation . An analogous IκBα protein pattern was obtained when inhibiting translation by addition of cycloheximide ( CHX ) . While individual treatment with either CHX or OVA caused IκBα reduction over time , co-application of both substances additively enhanced loss of IκBα ( Fig . 2D ) . These data strongly support the concept that OVA-mediated IκBα depletion is independent of translational inhibition but might be caused by activation of upstream signaling pathways apart from canonical NFκB signal transduction . Canonical NFκB activation is well known to involve Ser177/181 phosphorylation of IKKβ , followed by Ser32/36 phosphorylation of IκBα as a prerequisite for its proteasomal degradation . Stepwise documentation of canonical NFκB activation by Western-blot analysis and electro mobility shift assay ( EMSA ) revealed that OVA-induced IκBα depletion does not follow the canonical pattern . While IL-1-induced total IκBα degradation occurred as a fast process being completed after 15 min , OVA-induced subtotal IκBα depletion followed a much slower kinetics , and did not involve classical IKKβ phosphorylation ( Fig . 3A ) . Illustrating canonical IκBα degradation by addition of the proteasome inhibitor MG132 , which stabilizes phosphorylated IκBα , revealed only canonical IL-1 stimulation to cause an IκBα shift , while OVA treatment did not , but still caused IκBα depletion over time ( Fig . 3B ) . Correspondingly , OVA depleted not only IκBα-wt but also the Ser32/36Ala mutant which lacks the IKKβ-dependent phosphorylation sites and can therefore not be degraded in the canonical fashion ( Fig . 3C ) . Interestingly , EMSA revealed no significant nuclear translocation of NFκB to occur upon delayed OVA-induced IκBα depletion ( Fig . 3D ) , indicating other than NFκB-bound IκBα pools to exist within un-stimulated cells . In order to identify this additional IκBα pool we integrated all experimental data into detailed dynamical modeling using ordinary differential equations . Based on our previous NFκB signaling model [15] we generated an extended mathematical model ( variant M-1 , Fig . 4A ) . Since OVA-mediated IκBα depletion appeared to be independent of both , transcriptional/translational inhibition and proteasomal degradation , we assumed proteases to be involved in this process . It is known that free as well as NFκB-bound IκBα can be degraded by proteases in a proteasome- as well as in a lysosome-independent manner [20] , [21] . To monitor the role of OVA within the entire NFκB signaling network , we also included OVA-dependent Src phosphorylation - which subsequently leads to inactivation of PP2A and chronic IKKβ activation - into the model . This part of the signaling pathway , however , exclusively plays a role at early time points during IL-1 dependent canonical IκBα degradation [18] . Successful parameter fitting was performed based on a huge variety of experimental data comprising multiple stimulations: OVA− , ActD− , ActD+OVA , CHX+OVA-stimulation ( Fig . 1–3 ) , IL-1 + OVA-stimulation [18] , as well as data describing IκBα depletion upon IL-1− , UVB− , IL-1+UVB− , CHX− , IL-1+UVB+MG132-stimulation collected before [15] , [19] . All experimental data could be fitted well ( Fig . S1 ) . In particular , the good reproducibility of OVA experiments indicated that addition of an IκBα-degrading protease is sufficient to reproduce OVA-induced IκBα depletion without activating NFκB . The model thereby predicted a pool of free IκBα which is eliminated by a putatively OVA-activated protease , but leaves NFκB-bound IκBα almost unaffected . In the best fit scenario of knock down simulation studies depletion of free IκBα showed major relevance while the depletion of NFκB bound IκBα is negligible ( Fig . 4B ) . Of note , OVA-induced PP2A inactivation is only important in IL-1 stimulated cells and is therefore also irrelevant for OVA only stimulation following a much slower kinetics . Still , the model predictions had to deal with two obstacles: Firstly , due to its immense instability only a very small pool of free IκBα seems to exist within the cell at all [22] . Secondly , proteolytic cleavage of IκBα at least by three major groups of proteases: caspases , calpains and cathepsins could be excluded by the use of specific inhibitors ( Fig . 4C ) . To follow up the model based hypothesis of a stably existing IκBα form that is not bound to NFκB , we immuno-precipitated the NFκB p65 subunit from whole cell lysates and checked the levels of co-precipitated IκBα in un-stimulated versus OVA-treated cells . The amount of IκBα remained largely unchanged , whereas IκBα was absent in cells stimulated with IL-1 , due to complete canonical degradation ( Fig . 4D ) . These data strongly supported the assumption that only IκBα which is not bound to NFκB is depleted in an OVA-dependent fashion . To investigate whether depleted IκBα in fact is free or bound to other cellular components but NFκB , we conducted size exclusion chromatography . Indeed , IκBα appeared to exist in at least three different forms in un-stimulated cells . While only a minimal fraction seemed to refer to unbound IκBα eluting at a size of 44 kDa , surprisingly no complex exclusively consisting of IκBα and NFκB ( p65:p50 ) seemed to exist . Instead , complexes of higher molecular weight containing IκBα , p65 and p50 ( NFκB ) showed up at sizes ranging between 300 kDa and 490 kDa , indicating to incorporate other proteins as well . In addition , different aggregates ranging between 100 kDa and 300 kDa in size appeared which did not contain any NFκB components , implying IκBα to also form complexes with other proteins ( Fig . 4E ) . To investigate whether IKKs might be involved in stabilizing IκBα we knocked down the two catalytic subunits IKKα and IKKβ by RNA interference . While IKKα knock down had no effect on IκBα depletion , knock down of IKKβ seemed to enhance loss of IκBα , implying that binding to IKKβ stabilizes IκBα which is not bound to NFκB ( Fig . 4F ) . The fact that model variant M-1 , and a simple extension by adding a second ( competing ) IκBα complex formation ( variant M-2 , Fig . S3 ) , respectively , could not fully explain the IKK knock down data ( Fig . S2 and S4 ) argues for an additional IκBα-IKK interaction and calls for a more in depth analysis of the IκBα complexes . To stress whether IKK components play a role in IκBα complex formation we first generated model variant M-3 assuming that IKK is permanently bound to IκBα which lacks NFκB ( Fig . 5A ) . According to the chosen model structure , binding of IKK to IκBα results in the formation of an IκBα:IKK as well as an NFκB:IκBα:IKK complex . As phosphorylated IKK ( IKKp ) initiates degradation of IκBα , the IKKp containing complexes are highly unstable and dissociate into free IKKp and free NFκB which translocates into the nucleus . The simulation results of this model variant revealed a very good reproducibility of all experimental data including the OVA+IKK knock down experiment ( Fig . 5B ) . When exploring the model predicted steady state levels in the un-stimulated IKK knock down setting ( Fig . S5 ) , very low IKK yielded decreased formation of both IKK containing complexes IκBα:IKK and NFκB:IκBα:IKK . As a consequence elevated levels of free and NFκB-bound IκBα ( NFκB:IκBα ) were predicted . Consequently , constitutive degradation of NFκB:IκBα results in enhanced NFκB activation and further increased levels of free IκBα ( Fig . S5A ) . Starting from these changed steady state levels in the IKK knock down setting - especially taking the increased level of free IκBα into account - the OVA-mediated degradation of IκBα could be reproduced accurately ( Fig . S6 ) . In this final model OVA mediated the degradation of an NFκB-free IκBα complex ( IκBα:IKK ) , thus preventing activation of NFκB and IκBα mRNA synthesis ( Fig . 5C and Fig . 5D ) . In summary , the mathematical model based analysis clearly proposes the existence of IKK containing IκBα complexes lacking NFκB , supporting the hypothesis that IKKβ stabilizes “unbound” IκBα . Size exclusion chromatography clearly revealed that the high molecular weight complexes ( 300–490 kDa ) in un-stimulated cells consisted of at least IκBα , NFκB ( p65:p50 ) , and all three IKK components IKKα , IKKβ and IKKγ . Most importantly , IKKγ eluted together with IκBα at 100 kDa , perfectly matching the size of a heterodimeric complex ( ±94 kDa ) . Of note , additional complexes containing exclusively IKKβ and IKKγ ( ±140 kDa ) but not IKKα appeared to be formed ( Fig . 5E ) . Treatment with OVA for 8 h did not significantly change the composition of the high molecular weight complexes . In contrast , it caused pronounced depletion of IκBα from the IκBα:IKKγ complex , while IKKγ seems to randomly distribute over numerous fractions . This clearly indicates dissociation of this heterodimeric IκBα:IKKγ complex to precede IκBα depletion ( Fig . 5F ) . The existence of an NFκB-lacking IκBα:IKKγ complex as well as the specific depletion of IκBα from this particular complex following OVA treatment was confirmed by co-immunoprecipitation . While in unstimulated cells reasonable amounts of IκBα but no NFκB were shown to bind to IKKγ , the level of IκBα decreased significantly upon treatment of cells with OVA for 8 h . No IκBα was found bound to IKKγ in IL-1 stimulated cells , due to complete canonical degradation ( Fig . 5G ) . This is perfectly in line with simulation results derived from the final model variant M-3 suggesting that degradation of NFκB-free but IKK-bound IκBα is responsible for the partial IκBα depletion in response to OVA . To assess the functional relevance under physiological conditions , we investigated IκBα depletion in response to stimuli being capable to induce both , NFκB activation and apoptotic cell death [23] , [24] . Treatment with the death ligands TRAIL and FasL , respectively , exclusively caused canonical IκBα degradation , being indicative by sparing the IκBα super-repressor variant from depletion . In contrast , irradiation of cells with UVB additionally resulted in IκBα depletion independent of the canonical pathway – represented by degradation of both , the endogenous and the super-repressor variant ( Fig . 5H ) . These data strongly support the notion that other but canonical pathways exist that may affect the status of IκBα within the cell . Thus , we have uncovered an additional IκBα complex to exist in un-stimulated cells that might serve as storage to antagonize random or undesired NFκB activation and consequently ensures proper cellular function .
In the past decades activation of NFκB has exclusively been attributed to either canonical or non-canonical signal transduction pathways . Both pathways are tightly regulated by a series of phosphorylation and ubiquitination events that cause nuclear translocation of distinct NFκB family members . Canonical signal transduction basically accumulates at two well described protein complexes , namely IKK and NFκB:IκBα . Accordingly , stimulation of cells with the pro-inflammatory cytokine IL-1 causes downstream activation of the IKK complex , in particular the catalytic subunit IKKβ , to mark IκBα for proteasomal degradation . Released NFκB in turn triggers resynthesis of its inhibitor in a negative regulatory feedback loop . Most recently , a number of ubiquitin ligases including TRAF molecules and the lubac complex have additionally been implemented in canonical NFκB activation [25] , [26] , while de-ubiquitinases like A20 and CYLD serve a well-known function in negative feedback regulation [27] , [28] . In the present study we provide evidence that besides the well-known components of canonical NFκB signaling , alternative IκBα containing complexes exist within the cell that might indirectly contribute to NFκB regulation . Short term inhibition of IκBα resynthesis following IL-1+UVB and IL-1+OVA treatment , respectively , is due to abrogation of canonical negative feedback regulation via inhibition of PP2A leading to chronic IKKβ activation [3] , [19] . At later time points , however , canonical feedback regulation seems to be superimposed by an IKKβ-independent OVA- driven mechanism that follows a slower kinetics and only causes partial IκBα depletion . A similar observation could previously be made in UVB-irradiated cells , showing slow and subtotal IκBα depletion resulting in only moderate and delayed NFκB activation [3] . In the present study OVA-induced delayed IκBα depletion appeared to follow a pattern different from canonical NFκB activation , because super-repressor variants of IκBα could be depleted as well . This implies IκBα depletion which does not follow the canonical pattern to serve an important , yet unknown function . Although a number of alternative ways to proteolytically cleave IκBα have been described in the literature [20] , [21] , [29] , [30] inhibition of the major cellular protease families , could be ruled out . More recently an alternative proteasome independent mechanism called PIR has been described to enhance IκBα turnover in B-cells , however , PIR resulted in constitutive p50:cRel activation in those cells and may therefore play a different role [21] , [31] . Still , integration of an OVA-activated protease into all our mathematical models that is able to deplete IκBα- different from the canonical mechanism - was shown to nicely reproduce the OVA induced IκBα degradation with slow activation kinetics determined by a small rate constant ( M-3: kprot = 3 . 76e−7 s−1 , Table S1 ) . Accordingly , significant degradation of IκBα occurs at later time points ( 4 h–8 h ) and may involve PIR-like mechanisms . Reproducing OVA-induced IκBα depletion without NFκB activation in model variant M-1 predicted high levels , 31% , of free IκBα ( 0 . 041 µM out of 0 . 135 µM ) to exist within the cell ( Table S2 ) , whereas only 10% to 15% of free IκBα is supposed to exist [32] , [33] . Remarkably , inclusion of putative NFκB-lacking IκBα complexes nicely scaled down the level of free IκBα to 13% ( M-2 ) and 8% ( M-3 ) respectively which now is in very good accordance to the literature values , matches mathematical models of other groups [11] , [22] , [34] , and could also be verified by size exclusion chromatography . According to previous studies that revealed IKKα and IKKβ to form high molecular complexes that contain IκBα as well as NFκB components [35] , [36] model variant M-3 predicted IκBα to form both , stable IκBα:IKK as well as NFκB:IκBα:IKK complexes . With this final model the entire data of numerous experiments could be reproduced with a good fit quality . Finally formation of an IκBα:IKKγ complex could be verified experimentally by gel filtration analysis as well as co-immunoprecipitation . Expanding the scope of our previous model [15] by iterative model refinement revealed new insights into NFκB regulation and allowed to analyze the effect of an in silico knock down of the IKK complex on the OVA-mediated IκBα depletion . The model predicts that knocking down IKKβ enhances the level of an IκBα compound that can be degraded by the proposed protease . In vitro neither IκBα from the high molecular complex nor IKKβ:IKKγ bound IκBα seems to be depleted after OVA treatment . Thus knocking down IKKβ could result in decreased levels of NFκB:IκBα:IKK as well as IκBα:IKKβ:IKKγ and an elevated concentration of the IκBα:IKKγ complex . Due to larger amounts of IκBα:IKKγ , OVA-mediated degradation of this IκBα component would explain why IKKβ knock down enhances OVA-induced IκBα depletion . Additionally , a constant basic IKKβ-mediated IκBα turnover exists in unstimulated cells [19] , yielding in continuous NFκB-dependent resynthesis of IκBα . In case of IKKβ knock down , this process is completely abrogated consequently causing subtle loss of IκBα over time exclusively dependent on the individual half life in treated versus untreated cells ( see also Fig . 4F ) . Combining experimental methods and detailed dynamical modeling we could provide strong evidence for the existence of an NFκB-free , IKKγ containing IκBα complex presumably acting as a cellular backup pool to capture randomly released NFκB . Above this , our model introduces an IκBα degradation pathway that is independent from canonical processes but is likely to influence the cellular status of IκBα . Our final mathematical model ( M-3 ) created here is able to reproduce a huge number of datasets comprising various intracellular proteins and a wide range of stimulation experiments to extensively reflect on the whole NFκB signaling network above individual top-down signaling pathways . Thus , our model can be used for further modeling approaches regarding NFκB regulation and may provide predictive potential for sensitive parameters that may serve as therapeutic targets in the future .
The human epithelial carcinoma cell line KB ( ATCC ) was cultured in RPMI 1640 , 10% FCS . Recombinant human IL-1β ( R&D Systems , Wiesbaden , Germany ) was applied at 10 ng/ml and Na-Orthovanadate ( Sigma , Munich , Germany ) at 1 mM . Actinomycin D and cycloheximide ( Sigma ) were added to cells at 5 µg/ml , respectively . Specific protease inhibitors ( Calbiochem , Darmstadt , Germany ) were applied at 50 µM for the cathepsin inhibitor CATI-1 , 1 µM for calpastatin and 20 µM for the pan caspase inhibitor zVAD . Proteasomal inhibition was achieved by addition of 25 µM MG132 ( Calbiochem ) . For Fas/CD95 receptor activation 0 . 5 µg/ml of an agonistic antibody ( Immunotech , Monrovia , CA , USA ) was used . Recombinant human iz-TRAIL protein , N-terminally fused to an isoleucine-zipper motif in order to constitutively build the trimerized active form [37] was kindly provided by Dr . Henning Walczak , Centre for Cell Death , Cancer and Inflammation , UCL , London and added at 100 ng/ml . UVB irradiation ( 300 J/m2 ) was performed with TL12 fluorescent bulbs ( 290–320 nm , Philips ) . Cells were lysed in lysis buffer ( 50 mM Hepes , pH 7 . 5; 150 mM NaCl; 10% glycerol; 1% Triton-X-100; 1 . 5 mM MgCl2; 1 mM EGTA; 100 mM NaF; 10 mM pyrophosphate , 0 . 01% NaN3 and Complete protease inhibitor cocktail; Roche , Mannheim , Germany ) for 20 min on ice . Endogenous NFκB ( p65 ) or IKKγ were immune-precipitated using specific antibodies ( sc-372; sc-8330 Santa Cruz , Heidelberg , Germany ) and A/G-plus agarose ( Santa Cruz ) over night . Precipitates were analyzed by Western-blotting using antibodies against NFκB ( F6 , sc-8008 , Santa Cruz ) , IκBα ( L35A5 , Cell Signalling , Beverley , MA , USA ) and IKKγ ( IMG-324A , Imgenex , San Diego , CA , USA ) . For WB analysis cells were lysed by addition of hot ( 95°C ) Laemmli buffer . 80 µg protein extracts were subjected to SDS-PAGE and Western-blot analyses using antibodies against IκBα , P-IKKβ-Ser177/181 , IKKβ , p50 ( L35A5 , 16A6 , 2C8 , #3035 , Cell Signaling ) , p65 ( sc-8008 , Santa Cruz ) , IKKα ( 556532 , BD Biosciences ) , IKKγ ( IMG-324A Imgenex , San Diego , CA , USA ) , and α-tubulin ( DM1A , Neomarkers , Fremont , CA , USA ) , using West-Pico or West-Dura ( Pierce , Thermo Scientific , Rockford , IL , USA ) chemiluminescent substrates . Total RNA was extracted from cells using GIT-buffer ( 4 M guanidinthiocyanate , pH 4 . 8; 0 . 3 M NaOAc; 1% N-lauroylsarcosine; 0 . 2% β-mercaptoethanol ) followed by phenol/chloroform extraction utilizing Phase Lock Heavy tubes ( Eppendorf AG , Hamburg , Germany ) . Six µg of total RNA was reverse transcribed with an AMV Reverse Transcriptase kit ( Promega , Mannheim , Germany ) . The following primers were used in a 20 µl reaction utilizing the RedTaq polymerase system ( Sigma ) : GAPDH: F: 5′-GCCTCCTGCACCACCAACTGC-3′; R: 5′-CCCTCCGACGCCTGCTTCAC-3′ IκBα: F: 5′-ACAGGAATTACAGGGTGCAGG-3′; R: 5′-GAGAAACTCCCTGCGATGAG-3′ For ectopic expression of IκBα -S32/36A 6 , 5×106 cells were transfected with 25 µg of the respective pcDNA3 . 1-based construct by electroporation at 1200 µF and 250 V ( EasyjecT-plus , Peqlab , Erlangen , Germany ) in ice cold RPMI medium w/o FCS . Transfection efficacy ranged between 70 and 80% . 1×105 cells were transfected with 0 . 5 pmol/µl siRNA knocking down IKKα: GCAGAAGAUUAUUGAUCUATT or IKKβ: UUCAGAGCUUCGAGAAGAATT ( Eurofins MWG , Ebersberg , Germany ) using Lipofectamin 2000 ( Life Technologies , Darmstadt , Germany ) according to the protocol . Cells were analyzed after 72 h . Following stimulation cells were harvested and nuclear proteins extracted as described before [38] . The NFκB consensus oligo nucleotide ( sc-2505; Santa Cruz ) was end-labeled using [γ32P] ATP and T4 polynucleotide kinase ( MBI Fermentas , Ontario , Canada ) , followed by column-purification ( QIAquick Nucleotide Removal Kit , Qiagen , Hilden , Germany ) . Binding reactions were carried out in a 20 µl volume containing 8 µg nuclear protein extract in 5× binding buffer ( 20 mM HEPES , pH 7 . 5; 50 mM KCl; 2 . 5 mM MgCl2; 20% ( w/v ) ficoll; 1 mM DTT ) , containing 1 µg poly[dIdC]; 2 µg BSA , and 70 . 000 cpm of 32P-labeled NFκB consensus oligo nucleotide for 20 min at RT . Samples were separated on a 4% native PAGE at 150 V for 2 . 5 h and detected by autoradiography . Cells were harvested in PBS+0 . 01% sodium acide and disrupted by sonication . 10 mg protein extract was fractionated on an agarose bead column ( GE , Healthcare , Frankfurt , Germany ) . Fractionated proteins were precipitated in 50% TCA , washed twice with acetone and applied to SDS-PAGE and subsequent Western-Blot analysis . Proteins of known size ( thyroglobulin: 670 kDa , γ-globulin: 158 kDa , ovalbumin: 44 kDa , myoglobin: 17 kDa , cobalamin: 1 . 3 kDa ) were used as a standard . Western-Blots analyses are presented as mean ± SD of 3 independently performed experiments . Blots were evaluated densitometrically and the results normalized to the maximal measured value . A minimal relative standard deviation of 15% was always assumed . For statistical analysis student's t-test was performed . The mathematical models created in this study are based on our previous ODE model [15] comprising the following components: IL-1-receptor ( IL-1R ) , IL-1-receptor ligand complex ( ILRc ) , free IκBα ( IκBα ) , NFκB bound IκBα ( NFκB:IκBα ) , nuclear IκBα ( IκBαn ) , nuclear NFκB ( NFκBn ) , IκBα mRNA ( IκBαt ) , IKKβ ( IKK ) , phosphorylated IKKβ ( IKKp ) , Protein phosphatase 2A ( PP2A ) . In our recent models IKK ( and IKKp ) is perceived as the IKK complex consisting of IKKα , IKKβ and IKKγ instead of IKKβ alone . First of all we extended the basic model [15] by our previously proposed mechanism of OVA prolonging the IL-1 mediated activity of NFκB [18] . OVA treatment causes phosphorylation of the tyrosine kinase cSrc which in turn phosphorylates and thereby inactivates PP2A: Terms adopted from the model of Witt et al . [15] are highlighted in bold font . PP2Ap represents phosphorylated PP2A . Without loss of generality the total amount of the kinase cSrc ( SRC ) can be assumed to be 1 ( cf . an analogous reasoning for IKK in [19] ) . Considering mass conservation , ( 1-SRCp ( t ) ) then represents the Src kinase in its inactive ( un-phosphorylated ) state . The stimulus OVA is represented by the step function ova ( t ) having a value of 0 ( absent ) or 1 ( present ) . The initial concentration of PP2A is set to 1 whereas PP2Ap is set to zero . In order to reach steady state conditions we started stimulation from steady state established after 240 hours of simulation of the un-stimulated system . Based on our recent results we assumed for the observed OVA mediated IκBα degradation the involvement of a protease prot that is activated by OVA . This assumption is included in all of our model variants by adding the following term: In this equation prot represents the active form of the assumed protease and ( 1-prot ( t ) ) the inactive state considering mass conservation . The total amount of prot is assumed to be 1 without loss of generality . In model variant M-1 this protease is able to degrade free and NFκB bound IκBα: Degradation of NFκB bound IκBα by the protease in model variant M-1 causes translocation of NFκB into the nucleus followed by the initiation of IκBα mRNA synthesis: The factor kv is used to compensate the different volumes of cytosol and nucleus ( see Witt et al . [15] ) . ActD ( t ) represents a step function of the inhibitor of transcription actinomycin D ( ActD ) with a value of 1 ( present ) or 0 ( absent ) . RNA polymerase II and basal transcription factors are known to be essential for transcription of genes [39] , [40] whereas specific transcription factors function as enhancer or repressor . Thus we assumed in M-1 and M-2 a constitutive transcription rate for IκBα mRNA synthesis ( ctransc ) that is independent of NFκB and is also included in the NFκB model of Hoffmann et al . [9] . Model variant M-1 was fitted to time courses of NFκB , IKKβ -P , IκBα mRNA and/or IκBα respectively gained from the following stimulation experiments: The in silico knock down of IKK is realized by adding a factor named siIKK to the model variant and setting its value to 0 . 93 which reduces the initial concentration of IKK to the experimentally determined 7% of IKKβ concentration measured in untreated cells: In all stimulation experiments distinct from the IKKβ knock down experiment the value of siIKK is set to zero . In model variant M-2 we included the proposed IκBα complex IκBα:Comp , consisting of IκBα and components Comp distinct from NFκB: In this model variant , the activated protease prot additionally degrades IκBα from the IκBα complex , IκBα:Comp . Since this complex is part of the measured cellular IκBα concentration the overall IκBα concentration in the model , IκBαobs , becomes: The final model , variant M-3 , ( Fig . 5A ) was designed to investigate the possibility of IKK being part of the proposed IκBα:Comp complex . We therefore extended model variant M-2 by binding reactions of IκBα and IKK or IKKp and removed the component IκBα:Comp . In contrast to M-1 and M-2 we fitted the start concentration of IKK by adding the parameter IKKstart: In addition we assumed that IKK as well as phosphorylated IKK is able to bind IκBα in its free and bound form . In contrast to variants M-1 and M-2 a constitutive IκBα mRNA synthesis ( ctransc ) in M-3 is not necessarily required to reproduce all experimental data since fitting M-3 without ctransc to experimental data results only in a slight decrease of the fit quality ( overall χ2 = 100 . 6 compared to 98 . 6 ) . Thus parameter ctransc is not included in our final model M-3 . This is in line with the models of Lipniacki et al . and Ashall et al . assuming that only nuclear NFκB initiates IκBα mRNA synthesis [11] , [41] . Likewise , we did not integrate a constitutive degradation of IκBα in the NFκB:IκBα:IKK complex analogous to constitutive degradation of IκBα in NFκB:IκBα ( c6a ) since inclusion of this reaction did not improve fit quality . The entire ODE system of each model variant is shown in Text S1 , S2 , S3 . For parameter estimation as well as for solving and analyzing the ordinary differential equation system we used the MATLAB ( Mathworks ) toolbox PottersWheel [42] . For optimization the χ2 value of the following objective function was minimized by using a trust region approach: The χ2-value depends on the estimated parameter values . Variable y represents the i-th measured value whereas f states the simulated state value at time point i and is dependent on the parameter values p . The factor σi represents the standard deviation . Besides the newly introduced parameters the fitted parameters of the previous model were also included in the parameter estimation with the same parameter boundaries ( Table S1 ) . Scaling parameters were included to take the lack of absolute values of the experimental data into account . Fit sequences were applied in the estimation process: the starting value of each parameter in a sequence was thereby calculated as The Factor p represents the fitted parameter value of the currently best fit of the fit sequence ( PottersWheel F3 routine ) and the variable ε determines the strength of disturbance where ε∼N ( 0 , n ) . Four subsequent runs were performed with 100 fits each using n = 4 , 1 , 0 . 1 , 0 . 01 , respectively . In addition we performed an identifiability analysis of the final model M-3 , using the top 10% of 300 fits with random starting conditions . Many parameters are well identifiable with a low relative standard Deviation ( Table S1 ) . Furthermore for some of the less identifiable parameters , linear or non-linear correlations with other parameters exist which indicate that combinations of these parameters are identifiable ( Table S1 ) . All models are provided as PottersWheel model datasets in the supplemental data . | In unstimulated cells , the transcription factor NFκB resides in the cytosol bound to its inhibitor IκBα . Canonical activation of NFκB by numerous stimuli leads to proteasomal depletion of IκBα , thereby liberating NFκB to translocate into the nucleus to induce transcription of genes leading to proliferation , angiogenesis , metastasis , or chronic inflammation . Consequently , only transient activity needs to be warranted by immediate NFκB-dependent induction of negative regulatory mechanisms , including up-regulation of its inhibitor IκBα . Resynthesized IκBα consequently terminates NFκB activity by binding to its nuclear localization sequence . However , under physiological or pathophysiological conditions , random NFκB activation may occur , which needs to be avoided in order to guarantee proper cellular function . Using detailed dynamical modeling , we have now identified an additional IκBα containing complex to exist in un-stimulated cells which lacks NFκB but includes IKKγ ( IκBα:IKKγ complex ) . This additional IκBα is not depleted from cells in the canonical fashion and may therefore serve as a cellular backup to avoid random NFκB activation . | [
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] | 2014 | Identification of New IκBα Complexes by an Iterative Experimental and Mathematical Modeling Approach |
Studies have suggested that Epithelial–Mesenchymal Transition ( EMT ) and transformation is an important step in progression to cancer . Par3 ( partitioning-defective protein ) is a crucial factor in regulating epithelial cell polarity . However , the mechanism by which the latency associated nuclear antigen ( LANA ) encoded by Kaposi's Sarcoma associated herpesvirus ( KSHV ) regulates Par3 and EMTs markers ( Epithelial-Mesenchymal Transition ) during viral-mediated B-cell oncogenesis has not been fully explored . Moreover , several studies have demonstrated a crucial role for EMT markers during B-cell malignancies . In this study , we demonstrate that Par3 is significantly up-regulated in KSHV-infected primary B-cells . Further , Par3 interacted with LANA in KSHV positive and LANA expressing cells which led to translocation of Par3 from the cell periphery to a predominantly nuclear signal . Par3 knockdown led to reduced cell proliferation and increased apoptotic induction . Levels of SNAIL was elevated , and E-cadherin was reduced in the presence of LANA or Par3 . Interestingly , KSHV infection in primary B-cells led to enhancement of SNAIL and down-regulation of E-cadherin in a temporal manner . Importantly , knockdown of SNAIL , a major EMT regulator , in KSHV cells resulted in reduced expression of LANA , Par3 , and enhanced E-cadherin . Also , SNAIL bound to the promoter region of p21 and can regulate its activity . Further a SNAIL inhibitor diminished NF-kB signaling through upregulation of Caspase3 in KSHV positive cells in vitro . This was also supported by upregulation of SNAIL and Par3 in BC-3 transplanted NOD-SCID mice which has potential as a therapeutic target for KSHV-associated B-cell lymphomas .
In 1994 , Kaposi's sarcoma-associated herpesvirus ( KSHV ) was first identified [1] . Studies have shown that KSHV is predominantly involved in two kinds of cancers , originated through B-cells and endothelial cells [2 , 3] . In B-cells , it contributes to primary effusion lymphoma ( PEL ) as well as multicentric Castleman disease a rare form of lymphoproliferative disorder [4 , 5] . Kaposi sarcoma ( KS ) is derived from the lymphatic endothelial cell ( LEC ) lineage [6] , and usually presents as colored reddish brown lesions or patches on the skin , and may spread to internal organs [7] . Additionally , other disease phenotypes like distal metastasis or pulmonary KS can be observed in AIDS-KS patients and causes symptoms like diffused lung disease [8] . B-cell neoplasias are also known to migrate into body cavities or internal organs [9] . Therefore KSHV infection is likely to be important for inducing cell migration , invasion and disease progression [10] . However , the mechanism by which cell migration , and cell invasion markers contribute to KSHV-mediated B-cell infection is still mostly unexplored . It is known that KSHV lytic infection of endothelial cells leads to down-regulation of VE-cadherin protein levels [11] . Interestingly , KSHV induced degradation of VE-cadherin correlated with internalized virus particles [11] . Also , KSHV infection modulates the production of multiple MMPs to amplify cell invasiveness and thus adds to pathogenesis of KSHV-induced malignancies [12] . A fundamental process for unicellular and multicellular organisms is polarization , which is required for proper differentiation , proliferation and morphogenesis [13] . Formation of mesenchymal cells from epithelia was defined as Epithelial-Mesenchymal Transition ( EMT ) . A hallmark of EMT is the loss or attenuation of epithelial polarity , which mainly occurs during metastasis and cancer progression [14 , 15] . In this process junction proteins localize differently in proliferating ( mesenchyma ) or ( TJ ) tight junction -containing epithelial cells , which may be suggestive of their specific functions [16] . EMT allows polarized epithelial cells to interact with the basement membrane through its basal surface , leading to biochemical changes that facilitate the mesenchymal cell phenotype . This enhances the migratory capacity , invasiveness , elevated resistance to apoptosis , and increased production of the ECM components [17 , 18] . Additionally , EMT is illustrated by disruption of epithelial junctions , altering of actin cytoskeleton , loss of cell polarity , variation of cell–matrix adhesion , and amplified cell motility [19] . EMT progresses in a step wise fashion , which starts with disruption of cell junctions and suppression of E-cadherin expression [20] . Recently , Lemma et al . , showed the EMT like structure in B-cell lymphomas [21] . Also , Tilló et al . , demonstrated that the EMT activator promotes tumor growth in mantle cell lymphoma [22] . Hence to study EMT markers in KSHV-induced B-cell lymphoma is crucial to explore . Partitioning-defective ( PARs ) proteins represent a component of the body defense system , and aggressively participates in the inflammatory response [23] . PAR proteins were discovered in C . elegans [23] . More specifically , Par3 plays a crucial role in establishment and progression of epithelial cell polarity [24] . However , only specific stimuli are able to initiate the differentiation of epithelial cells to mesenchymal through genetic re-programming to form mesenchymal-like cells [25] . In another study , using cultured epithelial cells the Par3 complex supports the creation of epithelial cells tight junctions thereby adding significantly to the establishment and maintenance of apical–basal polarity [26] . In many cancer cell lines , SNAIL-1 and SNAIL-2 ( Slug ) are considered strong repressors of E-cadherin expression [27] . SNAIL-1 expression is enhanced in bladder cancer [28] . However , there were no significant relationship of SNAIL-1 to E-cadherin expression [29] . Further , another group demonstrated a direct association between SNAIL-1 and Cadherins [29] . Recently , Shin et al demonstrated that over-expression of SNAIL-1 significantly enhanced tumor progression , lymphovascular invasion , lymph node metastases and perineural invasion [30] . Earlier studies by Gottwein et al showed that Herpesviruses can inhibit p21 expression and attenuates p21-mediated cell cycle arrest [31] . Furthermore , a study from Takahashi et al also suggested that SNAIL represses p21 expression in the process of cellular differentiation [32] . Previous studies have also suggested that NF-kB signaling is important in KSHV-mediated oncogenesis [33 , 34] and the family of matrix metalloproteinase ( MMPs ) ( zinc-dependent photolytic enzymes ) are involved in many physiological and pathological events associated with the virus [35] . It is also known that numerous modulatory processes are regulated by MMPs to drive malignant progression of cancers . These include induction of cell invasion , release of growth factors , remodeling of ECM , promotion of angiogenesis , or modulation of the local immune responses [36] . Importantly MMP9 a well-studied MMP that induces cell invasion and metastasis in various cancers [37] , was shown to be induced by the EBV oncoprotein , LMP1 [38] . Therefore understanding how these EMT markers are influenced in KSHV-mediated oncogenesis , and specifically regulated through one of the essential viral-encoded latent antigen LANA will provide important clues as to their contribution to viral-associated pathologies .
Earlier studies from our group investigating KSHV infection of primary blood mononuclear cells ( PBMCs ) identified a number of genes related to DNA damage and regulators of virus infection [39] . One gene is particular was dramatically changed after KSHV infection . Surprisingly this enhancement was distinctly different from other previously reported genes shown to be important for KSHV-induced oncogenesis . More specifically , we found that infection with KSHV led to an increase of Par3 levels at day 2 and 6 in KSHV infected primary B-cells as seen at the transcript as well as protein levels ( Fig 1A ) . Additionally , we also obtained a similar pattern for Par3 expression when we looked at KSHV positive cells compared to KSHV negative cells at both the transcript and protein levels ( Fig 1B ) . We further compared mock HEK-293 cells with stable HEK-293-BAC-KSHV cells selected with hygromycin . As expected , we found consistent up-regulation of Par3 in the presence of KSHV ( Fig 1C ) . These results strongly suggest that Par3 is strongly up-regulated by KSHV and may likely play a key role in viral-mediated cell transformation . Earlier studies have shown the importance of LANA in KSHV infection [23 , 40 , 41 , 42 , 43 , 44] . To identify the KSHV-encoded antigen responsible for upregulation of Par3 , we analyzed the knock-down of LANA in KSHV-positive BC-3 and JSC-1 PEL cell lines . Transcription analysis by quantitative Real-Time PCR showed a consistent drop of greater than 50% in Par3 transcript levels in BC-3 and JSC-1 cell lines stably knocked down for LANA using a Lentivirus shRNA to target LANA ( sh-LANA ) ( Fig 1D ) . To investigate whether the regulation of Par3 in KSHV-positive cells is directly linked to LANA expression , we asked whether LANA can associate in a complex with Par3 . In HEK-293 cells using ectopic expression , we demonstrated that Par3 was present in a complex with LANA ( Fig 2A ) . A number of earlier studies showed that LANA is a major transcription factor which is an important contributor to KSHV-mediated oncogenesis [40 , 41 , 43 , 44 , 45] . We validated our immunoprecipitation results by exploring this with an in-vitro approach . We used GST pull down assays and in support of the results above , we found that an N-terminal domain of LANA had a stronger binding activity when compared to a C-terminal domain ( Fig 2B ) . To further support our interaction results between LANA with Par3 , we performed endogenous immunoprecipitation assays using Par3 specific polyclonal antibody with the appropriate isotype control in two KSHV-positive PEL cell lines BC-3 and BCBL1 ( Fig 2C ) . BC-3 and BCBL1 cells showed a stronger signal for Par3 which was also able to specifically immunoprecipitate LANA in Par3 pull down complexes from whole cell lysates . To more closely examine the domain of Par3 responsible for interaction with LANA we generated truncations of Par3 described earlier ( Fig 2D ) [46] , and performed immunoprecipitation assays ( Fig 2E ) . Here we show that residues 373 to 653 of Par3 which contains PDZ domains 2 and 3 bound very strongly with LANA when compared to other Par3 regions ( Fig 2E ) . To further support our association studies between LANA and Par3 , we used confocal microscopy to determine co-localization of these two proteins in the B-cell line Ramos ( Fig 3C ) . The results showed that LANA and Par3 colocalized greater than 60% in these assays in the nuclear compartment ( Fig 3C ) . We have also seen that Par3 localization was predominantly in the periphery on the outside of the cytoplasm and cell membrane in the absence of LANA ( S1 Fig ) . Some signals were seen in the nucleus at a lesser extent ( S1 Fig ) . Previous studies localized Par3 signals along the periphery of the cell membrane [41] . However , some Par3 signals were also seen in the nucleus suggesting signaling activities that modulate specific cellular processes at the cell membrane as well as in the nucleus [47] . In this study we were interested in determining the localization of Par3 during KSHV infection and more specifically in the presence of LANA . Par3 staining was observed in KSHV negative cells ( BJAB and Ramos ) to be mostly at the periphery of the cells compared to a shift of the majority of Par3 signals to the nuclear compartment in KSHV-positive cells ( BC-3 , BCBL1 and JSC-1 ) ( Fig 3A and 3B ) . Moreover , changes in localization of Par3 in the presence of KSHV were consistent across different cell lines although these cell lines may have different genetic backgrounds . Interestingly , around 60% of nuclear Par3 signals was colocalized with LANA although most of the signals were nuclear ( Fig 3B ) . Additionally , in PBMCs infected with KSHV the localization pattern of Par3 changed predominantly to nuclear compared to control PBMCs on day six ( Fig 3D ) . Importantly , the expression of Par3 was also significantly enhanced on KSHV infection when compared to uninfected PBMCs ( Fig 3D ) . Identifying the strong association of LANA with Par3 indicates a possible functional role for this complex . Therefore we evaluated whether LANA contributed to increased Par3 levels , as well as determining whether an increasing amount of LANA had any significant effects on the expression or stability of Par3 . Here HEK-293-BAC-KSHV cells were transfected with sh-LANA to knockdown LANA and a control knockdown vector containing a scrambled sequence as control ( Fig 4A ) . The results demonstrated that LANA played a role as a crucial regulator of Par3 and may be important for KSHV-induced oncogenesis . Further we evaluated the stability of Par3 in the absence or presence of LANA ( Fig 4B and 4C ) . Interestingly , we observed that LANA significantly stabilizes Par3 and was consistent in HEK-293 as well as BJAB cells , a B-cell line ( Fig 4B and 4C ) . However , the mechanism utilized by LANA for the stabilization of Par3 was not previously explored . To address this question we treated cells with the proteasome inhibitor MG132 in the presence or absence of LANA compared to a DMSO control in BJAB cells . The results showed that as expected , Par3 was stabilized in the presence of LANA ( Fig 4D ) . We further performed cyclohexamide experiments in BC-3-shControl and BC-3-shLANA cell lines to block de novo protein synthesis . A more rapid reduction in Par3 levels were seen in LANA-negative sh-LANA BC-3 cells compared to BC-3 with the sh-RNA control ( Fig 4E ) . To further confirm our results in LANA knockdown of BC-3 , we utilized MG132 in BC-3 sh-Control and sh-LANA with pull down of Par3 . As expected , we observed greater degradation of Par3 in LANA knockdown compared to the control BC-3 cell background ( Fig 4F ) . This strongly suggested that in KSHV positive cells LANA is a major contributor to stabilization of Par3 . Our studies so far showed that Par3 was up-regulated and stabilized by LANA . Further , Par3 interacted and was predominantly translocated to the nucleus by LANA . Furthermore , we were interested in determining the contribution of Par3 to cell proliferation . Here we used cell growth assays ( Fig 5A and 5C ) , colony formation assays ( Fig 5B ) and cell number determination to evaluate cell proliferation ( Fig 5D ) . We used Par3 knockdown cells compared to the scrambled controls in HEK-293 and BC-3 , BCBL1 cells ( Fig 5A and 5C , S2A Fig respectively ) . The results showed a moderate inhibition of cell growth and proliferation with knockdown of Par3 when compared to the vector control cell lines ( Fig 5A and 5C , S2A Fig ) . Interestingly , we observed that in the presence of LANA no induction in cell proliferation in the Par3 knockdown cells was observed when compared to control cells ( Fig 5A ) . To quantify the proliferation activity we performed cell counting assays for up to 6 days . Cells were selected with puromycin after 24 hour post transfection and plotted for cell density ( S2B Fig ) . Par3 knockdown cells showed a significant drop in growth capabilities compared to the corresponding control cell lines ( S2B Fig ) . We also showed that the efficiency of Par3 knockdown achieved at the transcript level was greater than 80% ( S2C Fig ) . Importantly , Par3 knockdown led to retarded growth patterns in HEK-293 as well as BC-3 and BCBL1 which ranged from 30–75% ( Fig 5B and 5D , S2A and S2B Fig respectively ) . In the colony formation assays , we demonstrated that Par3 knockdown resulted in more than a 80% drop in colonies compared to controls ( Fig 5B ) . Similar to our cell growth assays , the colony formation assays showed a significantly higher number of colonies with LANA in the Par3 knockdown cells ( Fig 5B ) . In our cell growth assays we measured cell density in BC-3 and BCBL1 . There were no dramatic changes . This is likely due to the inclusion of dead cell density in the total cell density as they were not washed off before image processing . However , when counting live cells with Trypan blue the changes were dramatic and Par3 knockdown led to slower cell proliferation ( Fig 5C and 5D ) . This strongly suggested an oncogenic property of Par3 which is directly regulated by LANA and contributes to progression of KSHV-induced cell proliferation . Additionally , de-regulation of apoptosis by Par3 may also be an important contributor to the control of cell growth and proliferation . Previous studies have demonstrated that genes with oncogenic properties also have anti-apoptotic activity [48 , 49 , 50] . Here we also observed that knockdown of Par3 led to increased apoptotic activity when monitored by serum starvation or etoposide treatment ( S2D and S2E Fig ) . Induction of apoptosis was much higher ( 15 fold ) in Par3 knockdown compared to vector control ( 3 fold ) in serum starve cells ( S2F Fig ) . Similarly , etoposide treatment led to an increase by 13 fold in cell death for Par3 knockdown cells compared to control vector ( S2F Fig ) . These results strongly demonstrated that knockdown of Par3 enhanced the apoptotic activities induced by serum starvation or etoposide as seen by an increase in cell death ( S2A–S2F Fig ) . This strengthens our hypothesis that Par3 has an oncogenic role in KSHV-infected cells . Par3 knockdown and exogenous expression of LANA were confirmed through quantitative real time PCR ( S3A–S3C Fig ) . How KSHV-induces EMT markers in B-cell lymphoma have not been previously explored . Par3 was previously described as an important factor for cells to transition from epithelial to mesenchymal [51] . However , this has not been previously explored in B-cell lymphoma . To examine its role in KSHV infected PBMCs , we screened a number of epithelial ( E-cadherin , Zo-1 , DSP ) and mesenchymal ( SNAIL , Lef1 , B-catenin , Cdh2 ) markers in KSHV infected PBMCs at day 2 and 7 ( Fig 6A ) . Surprisingly , we showed that SNAIL expression was higher compared to other EMT markers ( Fig 6A ) . Here LANA was used as a positive control for KSHV infection ( Fig 6A ) . Further we wanted to explore the interference of Par3 in the presence of KSHV infection . Hence , we evaluated EMT markers in HEK293 stably expressing the BAC-KSHV with shControl , -shPar3 ( Fig 6B ) . Interestingly , we showed that Par3 knockdown led to a significant downregulation of SNAIL ( Fig 6B ) . Additionally , we observed that E-cadherin and Zo-1 levels were elevated in Par3 knockdown cells ( Fig 6B ) . Moreover , Par3 knockdown in BJAB cells led to SNAIL downregulation and E-cadherin upregulation albeit moderately ( Fig 6C ) . These results indicated that SNAIL was positively regulated in KSHV infection through Par3 , and that E-cadherin was negatively regulated when compared to the other EMT markers ( Fig 6A–6C ) . Based on the above data we monitored the localization or expression pattern of E-cadherin and SNAIL in HEK-293 cells and BAC-KSHV infected cells ( Fig 6A–6C ) . Surprisingly in the presence of KSHV , these EMTs markers were translocated predominantly to the nucleus from the periphery of the infected cells ( Fig 7A and 7B ) . These results clearly indicate a potential mechanism by which these EMTs markers may be regulated by KSHV encoded antigens after infection . We also observed enhanced signals for E-cadherin in HEK-293 compared to BAC-KSHV infected cells and conversely , SNAIL signals were much stronger in BAC-KSHV infected cells compared to HEK-293 control cells . To corroborate our findings , we performed Western blot analysis for E-cadherin , SNAIL , and MMP9 as well as LANA and Par3 . Importantly , the levels of MMP9 , SNAIL and Par3 were up-regulated in BAC-KSHV infected cells compared to control cells ( Fig 7F ) . These results establish that Par3 , E-cadherin , SNAIL and MMP9 are contributors to KSHV-mediated activities in the B-cell background . Previous studies suggested that SNAIL and E-cadherin had opposing roles during EMTs leading to metastasis [29] . In follow-up to this result , we determined the transcript levels of SNAIL and E-cadherin in the B-cell line Ramos expressing increasing amounts of LANA . Here we also found that SNAIL was up-regulated and E-cadherin was down-regulated as the amount of LANA increased ( Fig 7C ) . In the above studies we demonstrated that KSHV infection of PBMCs at day 2 and 7 modulated EMT markers . To more closely monitor the expression patterns in KSHV-infected PBMCs , we examined these changes at days 1 , 2 , 4 and 7 ( Fig 7D ) specifically for SNAIL and E-cadherin transcripts ( Fig 7D ) . As expected , we showed that E-cadherin expression was significantly induced at day 1 followed by down-regulation through day 7 . Notably , SNAIL expression was consistently upregulated from day 1 to 7 ( Fig 7D ) , which strengthens our hypothesis that SNAIL and E-cadherin plays an important role in KSHV infection . To determine whether these markers are directly linked to LANA expression , we evaluated the expression of SNAIL , Par3 , E-cadherin and MMP9 in LANA knockdown KSHV-positive BC-3 and JSC-1 cell lines ( Fig 7E ) . Importantly , SNAIL levels was reduced in LANA knockdown KSHV positive cells and so provided another clue as to the mechanism by which SNAIL can be regulated by LANA during KSHV infection ( Fig 7E ) . The levels of E-cadherin was strongly upregulated in the LANA knockdown cell lines and the level of increase inversely correlated with the degree of LANA knockdown ( Fig 7E ) . The degree of SNAIL suppression was also directly related to the level of LANA knockdown ( Fig 7E ) . Importantly , MMP-9 another EMT marker , was also reduced in LANA knockdown cells ( Fig 7E ) . Similarly , changes in levels of MMP-9 , E-cadherin and SNAIL were seen with infection of HEK-293 cells with BAC-KSHV infection ( Fig 7F ) . Moreover , knockdown of SNAIL in BC-3 compared to a control shRNA showed a dramatic reduction of LANA and Par3 while E-cadherin was upregulated in SNAIL knockdown cells ( Fig 7G ) . These results strongly supported a positive association of LANA with Par3 and SNAIL as well as a dramatic loss of E-cadherin expression in KSHV infected cells . To determine if our in vitro findings for Par3 and SNAIL expression correlated with that seen in vivo , we generated tumors in a NOD/SCID mouse xenograft model with KSHV positive BC-3 and KSHV negative BJAB cells ( Fig 8A ) . Ten million cells were injected intraperitoneally . Mice were sacrificed approximately 5 weeks after injection with BJAB and BC-3 cells ( Fig 8B ) . Further , the tumors were harvested and subjected to transcript and Western blot analyses ( Fig 8C and 8D ) . SNAIL transcripts were substantially upregulated in BC-3 generated tumors compared to BJAB . Similarly , Par3 protein expression was observed more prominently in BC-3 generated tumors compared to BJAB . We also performed IHC in these tumors with primary antibodies against LANA , E-cadherin , Par3 , and DAPI for nuclear staining ( Fig 8E ) . Here we saw enhanced staining for Par3 and LANA in BC-3 induced tumors . However , E-cadherin staining was prominent in BJAB induced tumors but not in KSHV positive BC-3 cells ( Fig 8E ) . H and E staining was shown for a similar group of tumor tissues capitalize to Fig 8F . Overall , these results clearly showed enhanced expression of Par3 and SNAIL in KSHV positive tumors compared to KSHV negative tumors . For a more detailed understanding of the targets of SNAIL in KSHV induced tumors , we investigated the transcription activity of SNAIL on selected promoter regulatory elements . Further , we identified a number of transcriptional target genes of SNAIL along with SNAIL binding sequences in the upstream regulatory regions of genes including p21 , pTEN , TGFβ3 and the upstream region of SNAIL itself ( Fig 9A–9D ) . Earlier studies identified the "CACCTG" signature sequence as the DNA binding sequence of SNAIL [52] , and also reported the importance of SNAIL as a transcription factor for regulation of cellular genes p21 , pTEN and TGFB3 [53 , 54 , 55] . Therefore , we analyzed the upstream region of these genes to identify the binding sequence of SNAIL in KSHV positive cells . We designed multiple primers within the promoter regions for these genes based on their binding residues ( Table 1 ) . This study was investigated using two physiologically relevant approaches . First , we infected PBMCs with BAC-KSHV purified virus and performed ChIP using SNAIL antibody on day 2 and 5 post-infection ( Fig 9A–9D ) . In our second approach , we performed ChIP for SNAIL in BC-3 cells ( Fig 9A–9D ) . Surprisingly , anti-SNAIL antibody was enriched for DNA within p21 regulatory binding regions 3 and 4 along with SNAIL regulating binding regions 4 and 5 , respectively ( Fig 9A and 9B ) . These regions were found to be enriched for SNAIL binding sites in KSHV infected cells compared to uninfected PBMCs . These results suggest that SNAIL can regulate p21 expression in infected B-cells and can contribute to KSHV induced B-cell oncogenesis . Interestingly , pTEN and TGFB3 did not show any detectable differences in KSHV induced B-cell lymphoma when compared to KSHV infected PBMCs ( Fig 9C and 9D ) . Additionally , we probed the promoter activity of p21 in BJAB cells ( Fig 9E ) . As expected SNAIL knockdown led to upregulation of the p21 promoter activity compared to control vector ( Fig 9E ) . This result clearly emphasized the effect of SNAIL binding at the promoter region of p21 important for its regulation . Knockdown of SNAIL was confirmed through Western blot ( Fig 9F ) . GAPDH was used as a protein loading control ( Fig 9F ) . SNAIL can regulate itself and deregulates p21 in KSHV positive cells . It was important to explore other downstream targets which may be important for oncogenesis . Additionally , previous studies demonstrated the importance of NF-kB in KSHV-induced tumors [56] . Additional studies showed that p21 down-regulation was found to be critical for NF-kB-mediated oncogenesis [57] . This prompted us to follow-up on our current data above to more closely understand the link between p21 and NF-kB in KSHV positive cells . Interestingly , in other studies we also observed that small molecules inhibitors targeted EBV and KSHV lymphoma cells through the NF-kB pathway [58] . Here we analyzed BC-3 cells in the presence of a SNAIL inhibitor , as a KSHV positive cell line compared to the KSHV negative cell line BJAB ( Fig 10A and 10B ) . Interestingly , we observed that in the presence of a SNAIL inhibitor the levels of NF-kB ( p65 and p50 ) in BC-3 cells was significantly diminished . However , this reduction was significantly less in KSHV negative BJAB cells ( Fig 10A and 10B ) . Similarly , NF-kB ( p65 ) levels were substantially down in sh-LANA , compared to vector control in BC-3 cell background ( Fig 10C ) . Earlier , several studies suggested that NF-kB can regulate SNAIL at the transcriptional and post-translational levels [59] . To investigate if this holds up in KSHV positive cells we utilized the proteosome inhibitor MG132 and DMSO control in BC-3 sh-Control and sh-NF-kB cells ( Fig 10D ) . Interestingly , our results revealed that in KSHV positive cells NF-kB knockdown led to increased ubiquitination of SNAIL , which may explain the post-translational regulation of SNAIL by NF-kB in a KSHV positive background . A similar pattern was observed in sh-Control when compared to the NF-kB knockdown cells in DMSO treated cells , suggesting a role for NF-kB in SNAIL degradation ( Fig 10D , compare lane 1 and 2 with 3 and 4 ) . These results strongly demonstrated the importance of NF-kB in SNAIL regulation in KSHV-infected cells . We also showed that LANA knockdown reduced the impact of the SNAIL inhibitor when compared to vector control which strongly suggests a critical role for LANA in SNAIL-mediated gene regulation during KSHV infection ( Fig 10A–10D ) . Furthermore , we also monitored Caspase3 cleavage in sh-Control and sh-Par3 in the presence and absence of LANA , and a SNAIL inhibitor ( Fig 10E ) . Surprisingly , the presence of LANA reduced Caspase3 cleavage in BJAB cells ( Fig 10E ) . However , the SNAIL inhibitor induced Caspase 3 cleavage in the presence as well as the absence of LANA ( Fig 10E ) . These results suggested that NF-kB is positively regulated by SNAIL whereas Caspase 3 is negatively modulated by SNAIL in KSHV positive cells . We have also seen the minimal variation of v-Flip and v-Cyclin in LANA knockdown BC-3 and JSC1 cells ( S4A and S4B Fig ) . Previous results demonstrated that KSHV infection led to up-regulation of SNAIL and down-regulation of E-Cadherin [29] . Moreover , SNAIL inhibition in KSHV positive BC-3 led to E-Cadherin upregulation providing a direct link between SNAIL and E-Cadherin regulation in KSHV positive cells ( Fig 11B ) . Earlier , we showed that knockdown of SNAIL led to downregulation of Par3 and upregulation of E-cadherin . Here we showed that by using a SNAIL inhibitor in vitro the expression of Par3 in BC-3 cells was dramatically reduced as seen by immunofluorescence ( Fig 11A ) . Additionally , E-cadherin signals by immunofluorescence was increased supporting our earlier studies above ( Fig 11B ) . Using cell fractionation assays , Par3 was selectively present in the nuclear compartment with LANA . However LANA knockdown in BC-3 cells lads to transfer of some of the Par3 signals in to the cytoplasmic fraction ( Fig 11C ) . Here GAPDH was used as a loading control for cytoplasmic fractions and H2A was used for the nuclear fraction ( Fig 11C ) . LANA was shown as a positive control for KSHV positive BC-3 cells .
Kaposi Sarcoma ( KS ) is an endothelial tumor typically infected with HHV-8 [60] . Immune-suppression is tightly associated with KS development and remains the second most frequent tumor seen in acquired immune deficiency syndrome patients [61] . Earlier , it was suggested that Par3 functions as a potential “gatekeeper” to sustain the phosphoinositide concentration gradient in polarized cells [62] . Importantly , polarization is a crucial step towards progression of epithelial cells to mesenchymal cells [24] . We evaluated several DNA damage markers known to be critical for viral induced oncogenesis . Surprisingly , Par3 which was shown to be important for cell polarization , was significantly upregulated in the presence of KSHV . We also found significant changes in Par3 levels with KSHV infection of primary B-cells at the transcript and protein levels . This expression increased as the infection progressed . Earlier , we established that KSHV lytic genes are essential for virus infection and increased from day 2 to 6 [63] . Further , in KSHV infected B-cell lines ( BC-3 and BCBL1 ) and with BAC-KSHV infected cells a similar trend was observed compared to control . Interestingly , knockdown of LANA directly inhibited the basal expression levels of Par3 in KSHV positive cells . A prominent association was demonstrated between the 373–653aa residues of the LANA-N-terminal and Par3 when compared to C-terminal ( 611–1266aa ) which had a much lower capacity to bind LANA . Furthermore , we showed a high degree of co-localization between LANA and Par3 in B-cells . These results suggest a possible functional regulation of Par3 by LANA in KSHV-infected cells . Earlier studies from our lab and others demonstrated that LANA can regulate host proteins through protein stabilization or degradation during KSHV infection [64 , 65 , 66 , 67] . To examine Par3 modulation in the presence of LANA we performed stabilization and dose-dependent assays . We observed that Par3 degradation was significantly delayed in the presence of LANA when compared to controls . Earlier studies showed that many cellular proteins are stabilized by LANA through a similar mechanism [68] . Furthermore , localization of proteins are directly linked to their functional roles and their pattern of expression are typically linked to their functional relevance [68] . Interestingly , Par3 is a large protein ( 180 kDa ) , which needs an active process to be transported to the nucleus for signaling [69] . Our results suggested that LANA induced Par3 translocation to the nucleus in KSHV-infected cells . We found a localization pattern in endogenously infected B-cells , where Par3 signals were peripheral in KSHV negative cells , compared to a predominantly nuclear signal in KSHV positive cell lines . This result was validated in KSHV infected PBMCs . Furthermore , Par3 knockdown led to arrest of cell proliferation and enhanced apoptosis . These results are similar to that observed for other well known oncoproteins strongly supporting a role for Par3 as an oncoprotein which contributes to KSHV-induced cell transformation [48] . Pathogenic viruses have evolved mechanisms to disrupt adherence junctions , important for maintenance of the integrity of the endothelium [70] . This disruption of junctional barriers ensures that the viruses can gain entry through the open receptors in the basolateral membranes and so traffics to other tissues through the lymphatic and vascular networks [71] . Qian et al , demonstrated that KSHV infection induces vascular permeability by down-regulating VE-cadherin expression to allow virus entry into cells [11] . Moreover , KSHV-mediated aberrant vascular permeability likely stimulates virus replication , inflammation and angiogenesis , which enhances pathogenesis of KSHV-induced malignancies . Furthermore , loss of E-cadherin increases expression of MMPs , one of the hallmark for EMT progression [72] . However , how these EMT markers are modulated and functionally involved in B-cell associated lymphomas was not extensively explored . Recently , two separate studies from Lemma et al . , and Tillo et al demonstrated the importance of EMT markers in B-cell lymphoma and mantle cell lymphoma respectively [21 , 22] . Therefore , evaluation of these EMT markers as crucial indicators of ECM stability and strength is an important component of the many cellular processes which contribute to KSHV-induced oncogenesis . ECM signaling modulated by LANA expressed from BAC-KSHV after infection showed that expression of E-cadherin was down-regulated in BAC-KSHV infected cells , whereas expression of SNAIL was enhanced . These results suggested a direct association between KSHV infection and EMTs . Additionally , we screened a series of EMT markers in Par3 knockdown B-cells . Surprisingly , the majority of epithelial markers were enhanced whereas mesenchymal markers were down-regulated . Moreover , SNAIL was dramatically modulated in Par3 knockdown , and virus infected B-cells . These results emphasizes the importance of SNAIL in KSHV-induced B-cell oncogenesis . We further corroborated the importance of SNAIL in KSHV infection in primary B cells by demonstrating that SNAIL is directly regulated by Par3 and LANA and can also deregulate E-cadherin for establishment of KSHV latent infection . During EMT , MMPs-mediated E-cadherin disruption is a determining process where E-cadherin is essential for transmitting signals to epithelial cells through catenin , SNAIL and Slug [73] . Furthermore , in gastric cancer SNAIL was shown to affect the invasiveness and migratory ability of cancer cells during metastasis [30] . SNAIL is a zinc-finger transcriptional repressor , which directly represses E-cadherin through regulation of the E-cadherin promoter [74] . It is also recognized as an important regulator in various cancer including ovarian , non-small cell lung , gastric , hepatocellular , and urothelial [75 , 76] . Moreover , SNAIL has been shown to induce VEGF and MMPs , well-established tumor invasion and metastasis markers [30] . In this study we found that SNAIL knockdown and its specific inhibitor diminished the expression of LANA suggesting a level of regulation also at the viral genome , as well as Par3 , while E-cadherin was moderately enhanced in B-cells . We also demonstrated the importance of SNAIL as a transcription factor where it binds its own sequence within its promoter , as well as the promoter region of p21 . Earlier , p21 down-regulation was shown to be important in Herpesvirus infection [31] . Although , NF-kB a well-known signaling molecule is enhanced in KSHV-induction , it is also reduced significantly in the presence of a SNAIL inhibitor in KSHV positive cells compared to KSHV or LANA negative cells , robustly supporting our hypothesis . In this study we showed that LANA regulates Par3 activity and EMT markers important for metastasis of KSHV-infected cells . This is similar to other viral oncogenes like E6 of human papilloma virus ( HPV ) which also targets the cell-polarity proteins Dlg and Scrib for proteolytic degradation [77] . However , a study by McCaffrey et al demonstrated that in breast cancer Par3 was shown to be tumor suppressor [78] . In KSHV infection , LANA can therefore functions as a master protein which regulates both transcription and cell growth during metastasis of KSHV-infected cancer cells and can also modulate gene expression during KSHV latent infection [79] . Further , a large number of cellular and viral promoters are regulated by LANA through its repressing or activating activities [45 , 48 , 64] . Overall , KSHV-infection results in expression of LANA which leads to induction of Par3 and SNAIL expression in B-cells . This enhances MMP9 expression and reduces E-cadherin levels ( Fig 12 ) . Further , SNAIL binds to p21 and up-regulates NF-κB activities through induction of Caspase3 cleavage and suggests a potential mechanism through which LANA can regulate the EMT markers important for transition and invasion of KSHV-infected B-cells through the extracellular matrix ( Fig 12 ) .
PBMC were obtained from University of Pennsylvania Human Immunology Core ( HIC ) and donated by the healthy donors . This study was approved by University of Pennsylvania Human Immunology Core which maintains IRB protocol . In this IRB approved protocol declarations of Helsinki protocols were followed and each donor gave written , informed consent [48] . The mice studies were conducted under the project entitled "therapeutic small molecule inhibitors of EBV and KSHV" . All six-week-old male NOD/SCID mice were purchased from ( Jackson Labs , Bar harbor , ME , USA ) . The office of Laboratory Animal Welfare ( OLAW ) guidelines provided by National Institute of Health ( NIH ) USA , are strictly followed by the University of Pennsylvania Institutional Animal Care and Use Committee ( IACUC ) . Our protocol number for this study was 804617 and we have adhered with all OLAW guidelines . Par3 was cloned from human PBMCs . Primers with specific restriction enzyme sites for cloning in pA3HA vector was used for amplification of specific product for cloning . The pA3F-LANA expresses FLAG-tagged LANA . pA3M-LANA , pA3M-N-LANA and pA3M-GFP-C-LANA express Myc-tagged , full length LANA , N-terminal of LANA ( 1–340 AA ) and C-terminal of LANA ( 930–1162 ) with GFP-tag respectively , and have been described previously [64] . For expression of GST fusion protein , cDNA from PBMCs was amplified using primers specific for Par3 amplicon and cloned in pGEX2T vector . An sh-SNAIL and control construct was provided by Dr . Gerhard Christofori ( University of Basel , Basel , Switzerland ) . Sequences of all the constructs were verified by DNA sequencing ( DNA sequencing facility , University of Pennsylvania ) . HEK-293 ( human embryonic kidney cell line ) was obtained from Jon Aster ( Brigham and Woman's Hospital , Boston , MA ) . KSHV-negative Burkitt’s lymphoma cells Ramos and BJAB were kindly provided by Elliot Kieff ( Harvard Medical School , Boston , MA ) . BC3 and BCBL1 are KSHV-positive , lymphoma-derived cell lines obtained from the American Type Culture Collection ( ATCC ) . JSC-1 cells is a latently infected KSHV B-lymphoma cell line , kindly provided by Richard F . Ambinder ( John Hopkins , Baltimore , Maryland ) . Wild-type KSHV BACmid was a kind of gift from Shou-Jiang Gao ( University of Texas , San Antonio , TX ) . KSHV-negative cell line Ramos , BJAB , and the KSHV-positive cell lines BC-3 , BCBL1 and JSC-1 were cultured in RPMI 1640 medium with 10% bovine growth serum ( BGS ) with additional supplements as described previously [64] . Human embryonic kidney 293 ( HEK-293 ) and HEK-293-BACKSHV cell lines were cultured in Dulbecco's modified Eagle's medium ( DMEM ) supplemented with 5% BGS , penicillin-streptomycin ( 5U/ml and 5μg/ml , respectively ) , and 2 mM L-glutamine . Primary antibodies for SNAIL ( H-130 ) , MMP9 ( SC-1609 ) , E-cadherin ( H-108 ) , NF-kBp65 ( SC-109 ) NF-kBp50 ( SC-114 ) , and Caspase3 ( SC-7272 ) were purchased from Santa Cruz Inc . ( Santa Cruz , CA , USA ) . Anti-Rabbit polyclonal Par3 ( 07–330 ) antibodies were purchased from Millipore ( Massachusetts , USA ) . Hybridoma culture supernatants were used as sources of anti-Myc ( 9E10 ) and anti-LANA ( LANA1 ) . Mouse anti-FLAG monoclonal antibody ( M2 ) was purchased from Sigma-Aldrich Corp . , ( St . Louis , MO ) . Induction of KSHV from HEK-293-BACKSHV procedure were followed as described earlier [48] . The KSHV virus was collected by centrifugation at 23 , 500 rpm for 1 . 5 to 2 hr at 4°C by ultracentrifugation . KSHV-infection was performed by incubation of primary cells ( PBMCs ) with virus using 4 μg/ml polybrene in RPMI 1640 medium ( 10% BGS ) and 5% CO2 at 37°C for 3–4 hr . KSHV-infection was assessed by evaluating the expression of GFP using fluorescence microscopy . Total RNA was isolated from experimental cells using TRIzol reagent Invitrogen Inc . ( Carlsbad , CA ) as per manufacturer's instructions . All procedures were followed as described earlier [39] . Step-one Real-time PCR Cycler was used with the default program settings . A melt curve analysis was also performed to ensure the specificity of amplified products . Relative quantitation was carried out by the threshold cycle method . All reactions were set up in triplicates . Primers used in these studies are listed in Table 1 . For expression of GST and GST-fusion proteins , bacterial cultures were induced with 1 mM IPTG at log phase ( OD600 = 0 . 6 ) and incubated with shaking at 37°C for 4 hr . E . coli BL21-DE3 cells were used for all transformation in this experiment . Bacterial lysis and purification of proteins with Glutathione-Sepharose beads were carried out as described earlier [39] . In brief , the cultures were pelleted at 3000 RPM for 10 minute and the pellets washed with STE buffer and resuspended in NETN buffer ( 100 mM NaCl , 20 mM Tris-Cl ( pH 8 . 0 ) , 0 . 5 mM EDTA 0 . 5% ( v/v ) , Nonidet P-40 ) . Dithiothreitol ( DTT ) and 10% Sarkosyl in STE buffer ( 100mM NaCl , 10 mM Tris , 1 mM EDTA ) were mixed into the solution , and the sample was sonicated to solubilized the proteins . The lysates were collected by centrifugation and the supernatant transferred and followed by addition of Triton X-100 in STE and Glutathione-Sepharose with rotation . GST-fusion proteins , bound to Glutathione-Sepharose beads were incubated with cell lysates to allow binding of GST-fusion protein with the target protein . After stringent washes the beads were boiled in SDS-sample loading buffer to allow denaturation and dissociation of bound proteins . Samples were centrifuged and the supernatant were subjected to SDS-PAGE fractionation followed by western blotting . 25 million cells were used for immunoprecipitation ( IP ) of exogenously expressed proteins using specific mono or polyclonal antibodies as described earlier [39] . In brief , the cell lysates from experimental groups were incubated similar to controls with species specific IgG and beads as well as gene specific primary antibodies and beads . A mixture of Protein A/G-Sepharose beads ( 1:1 ) was used in these assays . For removal of non-specific binding , stringent washes were carried out with RIPA buffer . Further the conjugated beads were boiled in 4x SDS-sample loading buffer and subjected to SDS-PAGE gel for western-blot experiments . Western blot protocols and quantitation of bands were performed as described earlier [39] . The membrane was blocked with 5% skim milk in phosphate-buffered saline ( PBS ) for 1 hour , followed by incubation with the appropriate dilution of primary antibodies ( in PBS ) for 2 hour at RT . IR dye-tagged secondary antibodies were used to detect the binding of primary antibodies , and the membrane was scanned using an Odyssey imager ( LiCor Inc . , Lincoln , NE ) . Quantitation of the protein bands was carried out using Odyssey scanning software . IF experiments were essentially carried out as described earlier [45] . In brief , experimental cells were washed two times with ice cold PBS . Four percent paraformaldehyde containing 0 . 1% TritonX-100 were used to fix and permeabilize the cells for next steps . Again cells were washed with ice cold PBS and blocked using freshly made 5% skim milk for 1 hr . For LANA and Par3 co-localization experiments , we used Ramos , HEK-293 , BJAB , BCBL1 , BC-3 , JSC-1 , BC-3-shControl , BC-3-shLANA and 293BAC-KSHV cells . HEK-293 and 293BAC-KSHV cells were used to evaluate the localization pattern for Par3 , E-cadherin , SNAIL , MMP9 and LANA . All primary antibodies as mentioned earlier were used for specific staining in cells . Evaluation of nuclear staining with different sets of proteins were carried out with DAPI ( 4 , 6-diamidino-2-phenylindole ) . After being washed with PBS , coverslips were mounted on glass slides using mounting medium . An Olympus Fluoview 300 confocal microscope was used in all experiments . pGIPZ vector was used for gene specific shRNA cloning . This vector was purchased from Open Biosystems , Inc . ( Huntsville , AL ) . Oligos for shPar3 clone was designed against a unique region of Par3: 5’ CAACAAGAAGACGCGAATC 3’ and LANA: 5' GCTAGGCCACAACACATCT 3' . A shRNA sequence with no significant homology with other human mRNA was used as a sh-control and was described earlier [80] . To monitor the knockdown effect of SNAIL , we used shSNAIL and control shRNA as described previously [81] . HEK-293 , BJAB control and BJAB stably expression with LANA cells were used for this assay . In HEK-293 , 36 hours post-transfection , cells were exposed to 40 μg/ml cyclohexamide ( CalBiochem , Gibbstown , NJ ) at given time points . For BJAB cells , 25 million cells were incubated with 100μg/ml cyclohexamide in normal serum medium . 20μM/ml MG132 concentration was used for treating the BJAB control and BJAB with stable expression for LANA . Subsequently , proteins were prepared at given time points and analyzed by immunoblotting with suitable antibodies . Odyssey Imager ( LiCor Inc . , Lincoln , NE ) was used to measure band intensities . All procedures for IHC were followed as described earlier [48] . Paraffin-embedded tissues were sectioned at the New Bolton Center , Animal Pathology Service , University of Pennsylvania School of Veterinary Medicine . Slides were deparafinized in Xylene . Treatment with H2O2 was followed by antigen retrieval in sodium citrate buffer ( pH 6 . 0 ) , and samples were blocked with 10% normal rabbit/goat serum prior to incubation with primary antibodies O/N at 4°C . Further procedures were followed as described earlier [48] . In brief , evaluation of nuclear staining was carried out with DAPI ( 4 , 6-diamidino-2-phenylindole ) in mix with secondary antibodies for 1 hour at RT . After being washed with PBS , coverslips were mounted on glass slides using mounting medium . An Olympus Fluoview 300 confocal microscope was used in all experiments . HEK-293 cells with pGIPz-shControl , pGIPz-shPar3 and vector control or LANA plasmids were transfected and placed in petri dishes and grown in culture medium containing puromycin and G418 antibiotic selection ( 2μg/ml ) . All procedures were followed as reported earlier [39] . Culture medium with antibiotics were replaced every alternate day . After two weeks in culture , cells were washed with PBS and fixed in 4% PFA for 30 minutes at room temperature in the dark . Cells were stained with 0 . 1% crystal violet ( Sigma-Aldrich Corp . , St . Louis , MO ) . Plates were washed to remove non-specific color and photographed using an Odyssey scanner ( LiCor Inc . , Lincoln , NE ) . The number of colonies are representated as a percentage using Image J software ( National Institutes of Health , Bethesda , MD ) . HEK-293 cells with pGIPz-shControl , pGIPz-shPar3 and vector control or LANA plasmids were transfected and placed in petri dishes and grown in culture medium . BC-3 and BCBL1 cells with pGIPz-shControl and pGIPz-shPar3 were transfected and placed in T25 flask and grown in culture medium in 6 well plates . 48 hour post transfection 50 , 000 cells were plated for day1 , day2 and day 3 analysis . According to time points , cells were washed with PBS and fixed in 4% PFA for 30 minutes at room temperature in the dark . Cells were stained with 0 . 1% crystal violet ( Sigma-Aldrich Corp . , St . Louis , MO ) . Plates were washed to remove non-specific color and photographed using an Odyssey scanner ( LiCor Inc . , Lincoln , NE ) . B-cells were subjected to scan directly after completion of time points . The number of colonies are representated as a percentage using Image J software ( National Institutes of Health , Bethesda , MD ) . Stable cell line for sh-control and sh-Par3 in HEK-293 cells were used in this study . Before harvesting an equal number of cells were kept with 0 . 1% serum with culture medium for 12 hr . Further cells were harvested by trypsinization , washed three times with PBS and fixed in 1:1 ratio of methanol: acetone for 12 hr at 4°C . Cells were incubated with 200 μg/ml of RNase A and kept in the -20°C freezer for 3 hr . Cells were then stained with propidium iodide ( PI ) 40 μg/ml ( Sigma , St Louis , MO ) and dissolved in PBS containing at least 1 hr at 4°C in the dark . In etoposide treated experiment , cells were incubated with etoposide ( 20 μM/mL ) for 12 hr and a similar protocol followed as described for serum starved cells . Cells in different cell cycle phases with appropriate controls were differentiated through FACS Calibur ( BD Biosciences , San Joe , CA ) and the results were assessed by the FlowJo software ( Tree star , Ashland , OR ) . HEK-293 cells were transfected using Ca3 ( PO4 ) 2 method as described [82] . Twenty four hour post transfection , cells were selected using puromycin ( 2μg/ml ) added to the culture media . Approximately 0 . 1 million cells from both sets of samples were plated into each well of the 6-well plates and cultured for 6 days . All procedures were followed as described earlier [80] . BC-3 and BCBL1 cells with pGIPz-shControl and pGIPz-shPar3 were transfected and placed in T25 flask and grown in culture medium . 48 hour post transfection cells were counted with Trypan blue dye on days dependent manner . Reporter assays were essentially performed as described previously [49] . In brief , plasmid mixes were prepared according to experiments , and control plasmids were added as needed to normalize the amount of total DNA . Following transient transfection , cells were incubated for 48 hours and lysed in reporter lysis buffer ( Promega Inc . , Madison , WI ) . Aliquots of the lysates were transferred to 96-well plates , an luciferase activity monitored as described earlier [49] . Thirty million BC-3-shControl and BC-3-shLANA cells were immunoprecipitated with 1 μg of anti-Par3 antibody and Western blotted with anti-ubiquitination , Par3 , LANA , and GAPDH antibodies . Cells were incubated for 12 h with 50 μM MG132 ( Santa Cruz , Inc . , Dallas , Texas ) . All procedures were followed as described earlier [49] . Thirty million of BC-3-shControl and Bc-3-shLANA were immunoprecipitated with 1 μg of anti-SNAIL antibody and Western blotted with anti-ubiquitination , SNAIL , LANA , and GAPDH antibodies . Six-week-old male NOD/SCID mice ( Jackson Labs , Bar harbor , ME , USA ) were acclimatized for one week and divided into two groups ( 5 mice per group ) . Representative pictures of results are shown in Fig 7 . Five mice were injected with BC-3 ( KSHV positive cells ) and other five mice were injected with BJAB ( KSHV negative cells ) cells intraperitoneally . Prior to injection , each mouse was anesthetized by intraperitoneal administration of ketamine ( 80mg/kg ) and xylazine ( 5mg/kg ) mixture . Five weeks later , mice from all groups were euthanized when the tumor size grew to more than 15 mm in size . All mice were necropsied to determine gross metastases . Tumors were preserved in 10% formalin for histopathology , mRNA and protein extraction and antigen detection . Chromatin immunoprecipitation ( ChIP ) was carried out as has been described earlier [49] . Briefly , after the cells were fixed with formaldehyde , cells were washed with cell lysis buffer . Followed by nuclei isolation and sonication in the nuclear lysis buffer to an average DNA length of 300–700 bp , as confirmed by agarose gel electrophoresis . Samples were precleared with salmon sperm DNA-protein A-Sepharose slurry for 1 hr at 4°C with rotation . Twenty percent of the total supernatant was saved for input control , and the remaining 80% was divided into two fractions and incubated with ( i ) control antibody ( Sigma , Inc . , St . Louis , MO ) or ( ii ) Poly-clonal anti-Rabbit Par3 antibody . The precipitated immune complex was washed for stringency , reverse cross-linked , and purified using a phenol: chloroform extraction method . A p53-Snail binding Inhibitor , GN25 from CalBiochem ( Merck K Darmstadt , Germany ) was purchased and used in this study . SNAIL inhibition study was followed as described in an earlier study [83] . It has been demonstrated that p53 is suppressed and eliminated from cells by direct binding with oncogenic K-Ras-induced SNAIL [82] . Further they generated specific inhibitors against p53-Snail binding ( GN25 ) . This chemicals can induce p53 expression . Moreover , GN25 can selectively activate wild-type p53 in p53WT/MT cancer cells . In vivo xenograft test also supports the antitumor effect of GN25 . | Par3 ( partitioning-defective protein ) is characterized as a cell polarity protein with a crucial role in regulating EMT progression in KSHV-infected cells . Notably , SNAIL , a major EMT regulator is targeted through expression of Par3 and Latency associated nuclear antigen ( LANA ) during KSHV infection . In this study we show that LANA positively regulated Par3 and SNAIL while E-cadherin was inversely regulated during KSHV infection of B-cells . Furthermore , SNAIL binding with the potent kinase inhibitor p21 in KSHV positive cells enhanced NF-kB signaling . These results demonstrate the importance of Par3 and SNAIL in development of KSHV-induced B-cells cancers through regulation of EMT related antigens , and may be critical for driving KSHV-associated primary effusion lymphoma . | [
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] | 2016 | KSHV-Mediated Regulation of Par3 and SNAIL Contributes to B-Cell Proliferation |
Human cytomegalovirus ( CMV ) exerts diverse and complex effects on the immune system , not all of which have been attributed to viral genes . Acute CMV infection results in transient restrictions in T cell proliferative ability , which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems . In a search for new immunomodulatory proteins , we investigated the UL11 protein , a member of the CMV RL11 family . This protein family is defined by the RL11 domain , which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins . We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes . This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45 , identified by mass spectrometry analysis of pUL11-associated proteins . CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells , indicating that pUL11 is a specific CD45 ligand . CD45 has a pivotal function regulating T cell signaling thresholds; in its absence , the Src family kinase Lck is inactive and signaling through the T cell receptor ( TCR ) is therefore shut off . In the presence of pUL11 , several CD45-mediated functions were inhibited . The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired . We therefore conclude that pUL11 has immunosuppressive properties , and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV .
Infection of immunocompetent individuals with human cytomegalovirus ( CMV ) rarely results in symptomatic disease . Following primary infection children and even adults often shed the virus in saliva or urine for weeks or months [1] , suggesting that clearance of CMV by the immune system is a complex process . Cellular immunity , in particular Natural Killer ( NK ) cells and CD8 T cells , has been found to be pivotal in controlling CMV [2] , [3] . Yet , despite the induction of strong cellular immune responses and neutralizing antibodies , CMV is able to establish a latent infection , and reactivation as well as reinfection with multiple CMV strains seems to be quite frequent [4]–[6] . These properties of CMV have been ascribed to the expression of a series of viral immunomodulatory proteins [3] , [7] . In individuals with weakened or immature immune systems the balance between host immune control and viral immunomodulation can easily be shifted in favor of viral replication , resulting in viremia and end-organ disease associated with morbidity and even mortality in CMV-infected transplant recipients , AIDS patients or children congenitally infected with CMV [8] . It is a long standing observation that T lymphocytes in patients with acute CMV infection display reduced proliferation capacity [9]–[13] that may result in transient immunosuppression associated with an increased risk of secondary infection [14] , [15] . A number of mechanisms have been proposed by which CMV may interfere with the priming of T cells as well as with their effector functions . The inhibition of MHC class I antigen presentation pathways by CMV is well established; limiting the recognition and lysis of infected cells by cytolytic T lymphocytes [7] , [16] . Another strategy that acts on the ability of T cells to proliferate is the secretion of host and virally encoded suppressive factors from CMV-infected cells; the virus induces enhanced secretion of transforming growth factor β1 and soluble CD83 , and itself encodes an interleukin-10 homologue that suppresses T cell proliferation [17]–[20] . Other suppressive functions require direct contact between infected cells and T cells [12] . An example is the upregulation of pro-apoptotic ligands on the surface of CMV-infected dendritic cells that can induce apoptosis in activated T cells [21] . The observation that the fraction of T cells that is not driven into apoptosis is also unable to proliferate normally after contact with CMV-infected cells implies the existence of additional suppressive mechanisms [21] . One possibility could be the interaction of CMV-encoded surface proteins with regulatory or inhibitory receptors on T cells . Cellular proteins and also immunomodulatory proteins of various viruses that mediate the interaction with surface proteins of immune cells often contain immunoglobulin-like or MHC-like domains [22]–[24] . The CMV genome encodes a number of putative transmembrane proteins with such a property [25] , the most prominent being the RL11 family that includes 14 largely uncharacterized proteins . The defining motif of this family is the RL11 domain , which has limited sequence homology to immunoglobulin domains and to the immunomodulatory E3 proteins of adenoviruses [26] . The only member of the RL11 family that has been studied more thoroughly , the TRL11/IRL11 protein , encodes an Fc-receptor that binds human immunoglobulins , presumably mediating escape from recognition by anti-viral immunoglobulins [27] , [28] . In this study we focused on another member of the RL11 family , the UL11 protein that has previously been reported to be expressed on the cell surface of CMV-infected cells [29] and therefore has the potential to interact with T cell receptor molecules . The restricted proliferative capacity of T cells from CMV-infected patients has been linked with defects in T cell receptor ( TCR ) signaling [30] . There are only a few transmembrane proteins on T cells that may exert negative regulatory effects on TCR signaling , the most prominent being the receptor tyrosine phosphatase CD45 . The CD45 protein is an essential regulator of the TCR signaling pathway that determines the sensitivity of T cells to TCR mediated stimulation . The absence of CD45 leads to a severe combined immunodeficiency ( SCID ) phenotype in humans [31]–[33] and mice [34]–[36] . The key substrate of the CD45 phosphatase in TCR signaling is the Src family kinase Lck [37] , which is in close proximity to the TCR and , upon activation by an incoming stimulatory signal , phosphorylates immunoreceptor tyrosine-based activation motifs ( ITAMs ) in subunits of the CD3-TCR complex [38] , [39] . In common with other Src family kinases , Lck is regulated via the phosphorylation status of one inhibitory and one activating tyrosine residue; Y505 and Y394 in Lck [40] . When phosphorylated , the negative regulatory tyrosine , Y505 , maintains an intramolecular interaction holding Lck in a closed , inactive conformation [41]–[43] . CD45 dephosphorylates Y505 and releases Lck into an open , primed form , ready to receive a signal from the CD3-TCR complex that results in phosphorylation of Y394 and thereby activates the kinase activity of Lck [44]–[46] . CD45 also has a less favored inhibitory function , to dephosphorylate Y394 [47]–[49] . For TCR signaling to occur , a pool of primed Lck must be available . CD45 is the only phosphatase known to dephosphorylate the inhibitory tyrosine of Lck , and the action of CD45 is therefore essential in setting the threshold at which incoming stimulating signals can be transduced into effects [50] . In this study we show that the CMV UL11 protein interacts with the CD45 receptor phosphatase on the surface of T cells , inhibiting signaling and restricting T cell proliferation .
The UL11 protein is predicted to be a type I transmembrane protein ( Figure 1A ) and has previously been reported to be expressed on the surface of human fibroblasts infected with the highly passaged CMV laboratory strain AD169 [29] . Expression of the protein from more physiologically relevant strains of the virus and in other cell types has not been analyzed , but relatively low levels of UL11 transcription from the Merlin strain of CMV have been observed ( Andrew Davison , personal communication ) . To allow us to work with conveniently detectable levels of pUL11 , we therefore used an adenovirus expression system [51] and constructed a recombinant adenovirus ( rAdV UL11 ) expressing pUL11 from the TB40/E strain of CMV [52] and GFP to allow the identification of transduced cells . Using a polyclonal antiserum specific for the predicted N-terminal extracellular domain of UL11 ( Figure S1 ) we detected pUL11 on the surface of A549 lung epithelial cells and human foreskin fibroblasts ( HFF ) transduced with rAdV UL11 , but not with a control adenovirus lacking UL11 ( rAdV GFP ) by flow cytometry ( Figure 1B ) and confocal microscopy ( Figure 1C ) . These results confirmed that pUL11 is expressed at the cell surface and furthermore indicated that surface expression of pUL11 does not require the presence of other CMV proteins . To characterize the UL11 protein , we transduced A549 cells with rAdV UL11 and performed immunoblots of the cell lysates using an antibody specific for the V5 epitope added at the C-terminus of the UL11 protein . A prominent band of 50 kDa and several faint bands with apparent molecular masses ranging from 60 to 72 kDa were detected ( Figure 1D ) . This suggested posttranslational modification of the UL11 protein since the predicted molecular mass of pUL11 is 31 kDa . To investigate potential glycosylation of pUL11 , lysates of recombinant adenovirus transduced cells were treated with PNGase F , O-glycosidase , or a combination of the two and immunoblotted ( Figure 1E ) . PNGase F treatment led to a reduction of the molecular mass of pUL11 to approximately the predicted 31 kDa , but treatment with O-glycosidase , either alone or in combination with PNGase F , had no effect . N-linked glycosylation therefore appears to form all or the majority of the post-translational modification of pUL11 . As pUL11 is expressed on the cell surface , its role could potentially be to interact with proteins on the surface of neighboring cells such as infiltrating immune effector cells in infected tissue . The UL11Fc protein consisting of the pUL11 extracellular domain with the Fc domain of human IgG fused at the C-terminus ( Figure S2 ) was used to measure interactions of pUL11 with five different cell types by flow cytometry . Markedly higher binding of UL11Fc than the control Fc domain ( Fc ) was detected to the lymphocyte cell lines BJAB and Jurkat but not to the non-hematopoietic cell lines HeLa , 293T or BJ fibroblasts ( Figure 2A ) . Extension of the study to primary PBMCs from a healthy donor indicated interactions of UL11Fc with CD4 and CD8 T cells , B cells , NK cells , monocytes and neutrophils ( Figure 2B ) . These data suggested that pUL11 binds to a protein that is ubiquitously expressed on cells of hematopoietic origin . To identify interaction partners of pUL11 , proteins were precipitated from Jurkat cell lysates using UL11Fc as bait . A doublet of approximately 200 kDa and a few smaller proteins precipitated from Jurkat cell lysates by UL11Fc were detectable by silver staining ( Figure 3A , lane 2 ) . The 200 kDa bands were not present when the Fc control protein was used as bait ( Figure 3A , lane 1 ) or when proteins were precipitated from 293T cells with UL11Fc ( Figure 3A , lane 4 ) . Since pUL11 interacts with a leukocyte surface protein , we wished to determine which of the proteins precipitated from Jurkat lysates were surface proteins . Jurkat or 293T cells were labeled with membrane impermeable biotin before lysis and precipitation with UL11Fc , the Fc domain alone , or as a positive control with an antibody specific for the CD3 ε-chain . Following blotting and detection using peroxidase-coupled streptavidin ( Figure 3B ) , the 200 kDa doublet produced the strongest signal of the proteins from Jurkat lysates precipitated by UL11Fc ( Figure 3B , lane 2 ) , whereas these proteins were not precipitated by the Fc domain , or the CD3-ε antibody ( Figure 3B , lanes 1 and 6 ) . As expected a 23 kDa protein immunoprecipitated by the CD3-ε antibody from Jurkat cell lysates could be visualized ( Figure 3B , lane 6 ) . These data suggest that the 200 kDa proteins are on the surface of Jurkat cells . To determine the identity of these proteins , the double band was subjected to mass spectrometric analysis . Eight peptides stemming from the receptor tyrosine phosphatase CD45 were detected , and no other peptides corresponding to surface proteins , suggesting that CD45 is the interaction partner of pUL11 . To confirm the interaction of pUL11 with CD45 , the proteins precipitated from Jurkat cell lysates by UL11Fc were analyzed by immunoblotting with an antibody against CD45 ( Figure 4A ) . CD45 was detectable in Jurkat , but not 293T cell lysates ( Figure 4A , upper panel , lanes 3 and 4 ) , and corresponding reactivity was observed with the 200 kDa protein doublet precipitated from Jurkat lysates by UL11Fc but not the control Fc domain ( Figure 4A , upper panel , lanes 1 and 2 ) . This confirms that pUL11 can interact with CD45 from Jurkat cell lysates . As a control for the specificity of the interaction , we also analyzed the precipitated proteins by immunoblotting using an antibody against CD43 . CD43 has no homology to CD45 , but a similarly sized and glycosylated ectodomain to CD45RABC [53] , and the lectin MGL , which interacts with CD45 , also binds to CD43 in Jurkat cells [54] . CD43 could be detected in Jurkat lysates ( Figure 4A , lower panel , lane 4 ) , but not in proteins precipitated by UL11Fc ( Figure 4A , lower panel , lane 1 ) . CD45 is expressed on the surface of all nucleated hematopoietic cells [55] and could therefore be the interaction partner of pUL11 seen upon flow cytometric analysis of leukocytes ( Figure 2B ) . To test this assumption , we analyzed the interaction of pUL11 with T cell lines that do not express CD45 . The J-AS-1 cell line is a Jurkat cell line in which CD45 expression has been selectively disrupted by the stable expression of antisense RNA [56] . The J45 . 01 cell line was independently derived from Jurkat cells by irradiation and selection for loss of CD45 expression [57] . In both of these cell lines , the lack of CD45 expression coincided with the lack of UL11Fc binding ( Figure 4B ) , indicating that CD45 expression is needed for the interaction . To show that CD45 expression is sufficient to induce the interaction of pUL11 , we expressed the R0 and RABC isoforms of CD45 in 293T cells by transient transfection . In both cases , an interaction of UL11Fc with the cells expressing CD45 could be seen ( Figure 4C ) . The amount of UL11Fc that binds to the cell surface increases with higher surface expression of CD45 , indicating that the interaction is concentration dependent ( Figure 4C , top row ) . Cells were also transiently transfected with expression plasmids encoding other surface glycoproteins; murine CD45 and human CD43 . No interactions of UL11Fc were detected with cells expressing these proteins ( Figure 4C , top and middle rows ) . Furthermore , binding of the extracellular domain of another member of the RL11 family , pUL6 ( Figure S2 ) , to 293T cells was not affected by transfection with either of the CD45 isoforms ( Figure 4C , bottom row ) . The presence of CD45 on the cell surface therefore appears to be sufficient to induce a specific interaction with pUL11 and no detectable interactions occur between pUL11 and other T cell surface proteins . To confirm by another method that pUL11 binds to Jurkat cells via CD45 , we incubated Jurkat cells with antibodies directed against CD45 and then analyzed subsequent UL11Fc binding by flow cytometry . The AICD45 . 2 antibody that recognizes all isoforms of CD45 [58] blocked UL11Fc binding in a concentration dependent manner ( Figure 4D ) . A second pan-CD45 antibody , MEM-28 , had a marginal effect , and the UCHL-1 antibody that recognizes only the CD45R0 isoform [59] had no effect at all on UL11Fc binding ( Figure S3 ) . This experiment therefore supports the conclusion that pUL11 binding is CD45 dependent . The UL11 protein shows a high degree of polymorphism between different strains of CMV [25] , [29] and so , in order to determine whether the interaction with CD45 is restricted to pUL11 from the TB40/E strain of CMV , or a more general property of the protein , we investigated pUL11 from two additional CMV strains; Toledo and AD169 . The predicted extracellular domains of the Toledo and AD169 UL11 proteins were also expressed as Fc fusion proteins and the binding of all three variants of UL11Fc to Jurkat and CD45 negative J-AS-1 cells was compared ( Figure 4E ) . All three forms of pUL11 interacted with Jurkat but not with CD45-negative J-AS-1 cells , interestingly with some apparent quantitative differences in binding . This experiment indicated that the interaction of pUL11 with CD45 is not strain-specific . We were interested in whether the complete , surface expressed UL11 protein also interacts with CD45 . To investigate this question , we transduced HFF cells with rAdV UL11 or the control rAdV GFP adenovirus and incubated these presenter cells with PBMCs , Jurkat or J-AS-1 cells lacking CD45 . After washing away the unbound cells , rosetting of PBMCs and Jurkat cells around the pUL11 expressing cells could be clearly seen ( Figure 5A , top two rows ) , and was absent from the control cells . No rosetting of the J-AS-1 cells was seen ( Figure 5A , third row ) , indicating requirements for both CD45 and pUL11 for the interaction to take place . The binding of Jurkat cells to rAdV UL11 transduced fibroblasts could be disrupted following treatment of the transduced cells with UL11-specific antiserum , but not with pre-immune serum ( Figure 5B , middle and bottom rows ) . This indicates that the adhesion properties of the soluble extracellular UL11Fc protein are also representative of full-length transmembrane pUL11 . Five different isoforms of CD45 , generated by variation in splicing , have been detected in human lymphocytes . The expression of these isoforms is tightly controlled , depending on cell type , stimulation and maturation [60] . Naïve T cells typically express high molecular weight isoforms of CD45 containing exon A encoded domains . The RA isoforms are downregulated during activation . The expression of the R0 isoform , due to removal of exons A , B and C by splicing , is the major CD45 protein species characteristic for primed and memory T cells [61] . In individuals with a variant form of the CD45 gene , typified by the C77G polymorphism , the splicing pattern of CD45 is altered , meaning that T cells expressing both long RA and short R0 isoforms of CD45 are present after activation [62] , [63] . In addition to CD45 splicing , the glycosylation of the different isoforms is also affected by cell stimulation , which could potentially affect the interaction with pUL11 [60] . It was of interest to understand whether pUL11 interacts preferentially with forms of CD45 associated with a particular activation state of T cells . Primary T cells from both control ( C77C ) and variant ( C77G ) individuals were therefore stained with antibodies against RA and R0 isoforms of CD45 and co-incubated with UL11Fc ( Figure 6A ) . Binding of UL11Fc could be seen to cells expressing either RA , or R0 or both types of CD45 isoforms . Upon stimulation of the cells with phytohaemagglutinin and interleukin-2 , the CD45 isoform expression pattern changed , but UL11Fc binding to all cell populations was unaltered ( Figure 6B ) . This indicates that pUL11 can interact with both long and short isoforms of CD45 on both naïve and mature T cells . CD45 in T cells functions to prime the tyrosine kinase Lck , enabling TCR dependent signaling leading to activation and proliferation . Stimulation through the TCR-CD3 complex activates a signaling cascade resulting in the increased tyrosine phosphorylation of multiple downstream signaling intermediates [64] . To investigate the effect of pUL11 on this function of CD45 , we stimulated Jurkat T cells with the C305 anti-Jurkat TCR antibody in the presence and absence of UL11Fc and detected induced changes in tyrosine phosphorylation by immunoblotting . In untreated cells and cells preincubated with the Fc control protein , the expected increase in tyrosine phosphorylation was readily detectable upon TCR stimulation ( Figure 7A , left and right panels ) . In cells preincubated with UL11Fc , however , this increase was strongly reduced ( Figure 7A , middle panel ) , indicating an inhibitory effect of pUL11 on T cell signaling . Activation of T cells through TCR signaling leads to their proliferation . To determine whether T cell proliferation is also disrupted by pUL11 treatment , we measured the effects of UL11Fc on the proliferation of primary T cells in response to stimulation via CD3 ( Figure 7B ) . T cells were incubated with the OKT3 CD3 antibody or with the mitogen phytohaemagglutinin , in the presence of UL11Fc or the Fc control protein . After 72 h , proliferation was measured and an inhibitory effect of UL11Fc could be seen . pUL11 therefore affects T cell functions that require active CD45 , resulting in reduced TCR signaling and proliferation .
Immune suppression induced by acute CMV infection can have serious consequences for patients with impaired immune functions , such as an increased incidence of severe secondary bacterial and fungal infections in solid organ transplant recipients [65] . As a starting point to identify new CMV encoded immunosuppressive proteins , we considered the RL11 gene family . The RL11 proteins are largely uncharacterized , but the majority possesses the RL11 domain , a variable region of between 65 and 82 residues that has some sequence homology to the adenovirus CR1 domain and to immunoglobulin domains [26] . Adenovirus proteins containing the CR1 domain include immunomodulatory E3 proteins [26] , [66] , and immunoglobulin domains are commonly required for both cellular and viral protein interactions with cell surface components of the immune system [22] , [23] . Acute CMV infection results in a reduction in the proliferation capacity of lymphocytes , which are not themselves infected by the virus [11] . A similar effect is produced in vitro upon contact between lymphocytes and CMV-infected cells , indicating the potential existence of uncharacterized surface expressed viral proteins with immunomodulatory properties [12] , [21] . As it has previously been proposed that the RL11 domain containing protein pUL11 is expressed on the surface of human fibroblasts infected with the AD169 laboratory strain of CMV [29] , we considered pUL11 to be a good candidate for a novel immunosuppressive protein . The surface staining of CMV-infected cells with a UL11-specific rabbit serum was interpreted by Hitomi et al . [29] as proof of the surface expression of UL11 , however , it might rather have reflected the binding of rabbit immunoglobulins to the virally encoded Fc-receptors , as has previously reported by other authors [67] , [68] . Despite the published description of pUL11 surface expression [29] , we could only observe low levels of UL11 mRNAs in fibroblasts or epithelial cells infected with the AD169 or TB40/E strains of CMV ( data not shown ) , in agreement with data using the Merlin strain of CMV ( Andrew Davison , personal communication ) . Therefore , we first re-evaluated whether the UL11 protein from the TB40/E strain of CMV can be expressed on the surface of fibroblasts and epithelial cells , using a recombinant adenovirus expression system . The surface expression of pUL11 that we detected then led us to search for interactions between pUL11 and surface molecules on different cell types . The predicted extracellular domain of pUL11 was used in flow cytometry binding studies and interacted with leukocyte cell lines and primary leukocytes , but not with control cell lines of non-hematopoietic origin , indicating an interaction with a leukocyte specific receptor . Mass spectrometry analysis of surface proteins pulled down by the pUL11 extracellular domain from Jurkat cell lysates identified the receptor tyrosine phosphatase CD45 as a binding partner of pUL11 . That CD45 is also responsible for the interaction of pUL11 with leukocytes seen in flow cytometry analysis was confirmed using two different CD45 deficient cell lines . No interactions could be seen between pUL11 and the Jurkat derived CD45 negative cell lines J-AS-1 or J45 . 01 [56] , [57] . The interaction of pUL11 with the surface of leukocytes could also be demonstrated using fibroblasts expressing full-length pUL11 , to which PBMCs and CD45 expressing T cells adhered . The interaction of pUL11 with Jurkat cells could be blocked by pretreatment with either an CD45 antibody , or pUL11 antiserum , confirming the specificity of the interaction . As pUL11 shows sequence variation between different strains of CMV [25] , [29] , we purified the predicted extracellular domain of pUL11 from two additional CMV strains , Toledo and AD169 , and showed that these forms of pUL11 also interact specifically with CD45 expressing cell lines , apparently with some differences in affinities , demonstrating that the interaction is not a peculiarity of the TB40/E form of pUL11 . Expression of two different isoforms of CD45 in 293T cells both induced pUL11 binding , indicating that CD45 is sufficient for the interaction . A second member of the RL11 family , pUL6 , was used to investigate whether the interaction with CD45 is a general property of RL11 proteins , or specific to pUL11 . No changes in pUL6 binding were seen in relation to CD45 expression , indicating that the interaction is a particular property of pUL11 . An interaction with pUL11 could not be induced by expression of the mouse CD45 protein , or the human CD43 glycoprotein in 293T cells . In conjunction with the observation that pUL11 binding is abrogated in the CD45 deficient T cell lines , this provides strong evidence that pUL11 interacts with CD45 and that the interaction is specific . CD45 exists as a set of different isoforms , the expression and glycosylation of which is tightly controlled and depends on cell type and maturation state . We demonstrated that the interaction of pUL11 with primary T cells is not dependent upon T cell activation state , as interactions were detectable between pUL11 and both long and short isoforms of CD45 , expressed on both naïve and mature T cells . This implies that the immunomodulatory effects of pUL11 in vivo may be wide ranging , potentially affecting both priming and effector functions of T cells . The interaction of pUL11 with CD45 is markedly different from that of other known CD45 ligands . The other CD45 ligands that have been described are all lectins , which recognize oligosaccharide moieties with specificities determined by the lectin carbohydrate recognition domains [69] . Lectins typically bind to multiple ligands and have pronounced differences in their interactions with the various CD45 isoforms and glycoforms due to their differing glycosylation patterns [60] . The C-type lectin macrophage galactose type lectin ( MGL ) , a pattern recognition receptor on myeloid antigen presenting cells which recognizes N-acetylgalactosamine ( GalNAc ) sugars , for example , binds only to the longer isoforms of CD45 due to their higher GalNAc content , and also to the sialoglycoprotein CD43 [54] . Other lectins are even more specific in their preferences; glucosidase II and serum-mannan binding protein only interact with CD45 glycoforms characteristically found on immature thymocytes; in the case of serum-mannan binding protein only with the hybrid-type N-linked glycans on the R0 isoform [70] , [71] . Lectin ligands for CD45 frequently do not show reduced surface binding to CD45 negative T cell lines , due to the abundance of other suitably glycosylated ligands [54] , [72] , and in contrast to the binding pattern observed for pUL11 . As pUL11 interacts with diverse forms of CD45 and shows no detectable binding to CD45 negative T cells , its interaction with CD45 seems to be of a different nature from those of previously described ligands . CD45 is necessary for T cell functions mediated via signaling through the TCR complex . The Src family kinase Lck is primed by CD45-mediated dephosphorylation of tyrosine 505 , which releases an intramolecular bond holding Lck in a closed , inactive conformation [50] . In the absence of primed Lck , signal transduction through the TCR cannot be initiated [73] and inhibition of TCR mediated signaling functions is therefore characteristic of reduced CD45 function [74] . Pretreatment with pUL11 reduced the cascade of tyrosine phosphorylation triggered by T cell stimulation with an anti-TCR antibody and TCR dependent T cell proliferation was also inhibited . These effects indicate that signal transduction through the TCR is impaired in the presence of pUL11 , and are consistent with a restriction in CD45 function . The critical control of signaling thresholds by CD45 implies that its effects must be tightly regulated . Although this process is not yet fully understood , mechanisms have been proposed , some of which are influenced by interactions of ligands with the extracellular domain of CD45 , and might therefore be applicable to pUL11 function . For CD45 to function correctly , it must have a tightly controlled localization in the plasma membrane , with regulated contact to substrate proteins . Lck is present in lipid rafts [75] , and a fraction of CD45 must have access to Lck to be able to dephosphorylate residue Y505 and generate a pool of primed Lck . This partial localization of CD45 in lipid rafts is dependent on the extracellular domain of CD45 and may require interactions in cis with other raft components [53] , [76] . As CD45 is a potent phosphatase , it is however important that only a fraction is present in lipid rafts . Excessive contact between CD45 and the TCR signaling complex can result in dephosphorylation of the active site tyrosine 394 of Lck and potentially of Lck substrates such as ZAP-70 and the ζ-chain of the CD3-TCR complex , blocking the initiation of signal transduction [48] , [77] , [78] . CD45 is therefore excluded from the signaling complex by lipid raft movements [76] , [79] . The inhibitory effects on CD45 function of the therapeutic anti-CD45RB mAb 6G3 [80] , and also the lectin placental protein 14 [81] are associated with excess movement of CD45 into lipid rafts , allowing a deactivation of Lck . It is conceivable that an interaction with pUL11 could disrupt cis interactions of the extracellular domain of CD45 with lipid raft components , affecting the controlled partitioning of CD45 into lipid rafts and thus generating the observed effects on T cell signal transduction . CD45 activity has also been described to be dependent on dimerisation state; a model has been proposed in which dimerisation results in the formation of an inhibitory structural “wedge” disrupting substrate access to the phosphatase domain [82] , [83] . Reduced dimerisation associated with a prevalence of high molecular weight forms of CD45 has been suggested to underlie excessive CD45 activity resulting in hyperresponsive T cell function [84] , [85] . Increased dimerisation , as seen by forced dimerisation of a EGFR-CD45 hybrid molecule and the interaction of the lectins galectin-1 and placental protein 14 with CD45 decreases CD45 function [81] , [86] , [87] . An analogous role for pUL11 is possible , in which pUL11 binding to the extracellular domain of CD45 increases dimerisation , decreasing CD45 phosphatase activity and therefore restricting TCR signaling . Other mechanistic interpretations , such as allosteric regulation of CD45 phosphatase function upon ligand binding , potentially in conjunction with those suggested here , are of course also possible . Further investigations into the mechanisms of pUL11 function seem likely to lead to new insights into CD45 regulation . The role of pUL11 in the context of CMV infection is intriguing; a transient general suppression of T cell function during viral infection has been demonstrated [9]–[13] and the interaction of pUL11 with CD45 may contribute to this effect . It is also clear that a means for the virus to escape from CMV-specific T cell control could enhance viral replication . The consequences of the interaction with CD45 may also extend beyond effects on T cell function as it is well known that CD45 plays important roles in other classes of leukocytes [50] . The observation that UL11 proteins from three different strains of CMV all bind to CD45 , but apparently with some variation in affinity , is also interesting , as it may point towards strain or host dependent immunosuppressive effects of CMV infection . Before these questions can be addressed , however , the expression profile of pUL11 during CMV infection needs to be understood . We would speculate that the expression of pUL11 may be cell type or state specific , but this remains to be demonstrated . In conclusion , we have identified CMV pUL11 as a novel , specific interaction partner of CD45 . pUL11 limits T cell signaling and proliferation , effects which are consistent with a reduction in CD45 activity . The interaction of pUL11 with CD45 appears to represent a previously unknown pathway by which CMV can induce immunosuppression , with potential therapeutic significance .
Human blood cells were provided by voluntary blood donors in the Institute of Transfusion Medicine , Hannover Medical School . All materials and data were analyzed anonymously . The use of the human blood cells was approved by the ethics committee of Hannover Medical School . Human lung adenocarcinoma epithelial A549 cells and human foreskin fibroblasts ( HFF ) were propagated in DMEM containing 10% FCS , 2 mM glutamine and 1% non-essential amino acids . 293T and 293A cells were maintained in DMEM containing 10% FCS . Jurkat T cells , J45 . 01 cells [57] and J-AS-1 cells [56] , were cultured in RPMI 1640 with 2 mM glutamine and 10% FCS , with the medium for the latter two cell lines supplemented with 20 mM HEPES and for the J-AS-1 cells , also with G418 ( 0 . 5 mg/ml ) . For protein production , retinal pigment epithelium ( RPE ) or 293T cells were maintained in serum free Pro293a-CDM ( LONZA ) , containing 2 mM glutamine . PBMCs were flushed from leukocyte filters used to prepare erythrocytes from healthy voluntary blood donors for transfusion . Where indicated , the individuals were identified as carrying wild type or C77G variant CD45 . PBMCs were isolated by density gradient centrifugation using Biocoll Separating Solution or Ficoll ( both from Biochrom ) and cryopreserved until usage . PBMCs were maintained in RPMI 1640 containing 20 mM HEPES or 1 mM sodium pyruvate , 4 mM glutamine and 10% FCS . The recombinant adenoviruses rAdV UL11 , rAdV GFP and rAdV UL6Fc are based on the pAdZ-CV5 replication deficient adenovirus vector [51] . First , the sequence for the V5 epitope tag ( GKPIPNPLLGLDST ) was added at the 3′-end of the UL11 open reading frame ( ORF ) in the CMV TB40/E genome [52] by homologous recombination in E . coli as previously described [88] . The UL11V5 fragment was amplified using the primers 5′-AGTCGGATCCAATTACCTGTGGTAGAATGC-3′ and 5′-GGCCGGATCCTTACGTAGAATCAAGACCTA-3′ and cloned into the pIRES eGFP vector ( BD Biosciences Clontech ) . The UL11V5 IRES eGFP cassette was then introduced into the AdZ-CV5 vector by homologous recombination in the E . coli SW102 strain as previously described [51] . rAdV GFP , rAdV UL6Fc , rAdV Toledo UL11Fc and rAdV AD169 UL11Fc were constructed by introducing the ORFs for GFP , Toledo UL11Fc , AD169 UL11Fc and for UL6Fc ( see below ) into the pAdZ-CV5 vector . The correct construction of the adenovirus genomes was confirmed by restriction analysis and sequencing . Recombinant adenoviruses were produced and titered in 293A cells . The sequence encoding the predicted extracellular domain of pUL11 was amplified from the genome of the CMV strain TB40/E [52] using the primers 5′-CGGGATCCATCAGCCTCCACGATGCCTG-3′ and 5′-CCGGTCGACTGTAGCCACGTGTTGGTGC-3′ and cloned into a pCR3-based vector containing sequences encoding the mouse IgH signal peptide and the Fc region of human IgG1 [89] . The predicted extracellular domains of pUL11 from CMV strains Toledo and AD169 were amplified from the respective viral genomes [90] , [91] using the following primers and cloned into the same vector . pUL11 Toledo: 5′-CGGGATCCATCAGCCTCCATGATGCCTG-3′ and 5′-CCGGTCGACTGTGGCCACGTGTTGGTGC-3′ . pUL11 AD169: 5′-CGGGATCCATCAGTTTCCACGACCATGC-3′ and 5′-CCGGTCGACTGTCGCCACGTGTTGGTAC-3′ . The sequence encoding the predicted extracellular domain of pUL6 was amplified with the following primers: 5′-CGGGATCCCATGCTAAGATAAACGGGTGG-3′ and 5′-CCGGTCGACGAATGCCAAGTTAGTTATGTTC-3′ and cloned in an analogous manner . The ORF for the TB40/E UL11Fc protein was amplified with the primers 5′-CGGCGGCCGCGCCACCATGAACTTCGGGTTC-3′ and 5′-CGGAATTCTCATTTACCCGGAGACAGGG-3′ and cloned into the pSFbeta91-wpre replication deficient retrovirus vector [92] . The ORF for the Fc domain of human IgG1 was cloned into the pSFbeta91-wpre vector in a similar manner . Retroviruses were generated by transfecting the Phoenix-gp packaging cell line with the pSFbeta91-wpre constructs together with the retroviral gag/pol plasmid M25-DAW [93] and the feline endogenous retrovirus envelope glycoprotein expression plasmid RD114 [94] and used to transduce 293T cells as described [92] . TB40/E UL11Fc , Toledo UL11Fc , AD169 UL11Fc , UL6Fc and Fc control proteins were purified from serum free supernatants of retrovirally transduced 293T cells or adenovirally transduced RPE cells by protein A affinity chromatography using hiTrap rProtein A FF columns ( GE Healthcare , Munich , Germany ) . The TB40/E UL11Fc protein was used to generate a rabbit antiserum directed against the extracellular domain of pUL11 ( Pineda Antikoerper Service , Berlin , Germany ) . Plasmids LCA . 1 and LCA . 6 that express the human CD45R0 and CD45RABC isoforms under control of the SR-alpha promoter [56] were kindly provided by David Rothstein . The CD45R0 ORF was removed from LCA . 1 by treatment with Eco RI and Sal I and replaced by an Mfe I/Sal I treated PCR fragment encoding the transmembrane region and cytoplasmic tail of mouse CD45 , which was amplified with primers 5′-GGCCAATTGACGCGTGCGGCCGCTATATTCCTGGTGTTTCTGA-3′ and 5′-GGCCAATTGCCCGTCGACCGTTATGAACTCTGGGTTGGAGCTG-3′ , using a plasmid carrying the mouse CD45RB cDNA [95] . The resulting plasmid was cut with Not I and a PCR fragment was added encoding the extracellular domain of mouse CD45RB , which was amplified from the mouse CD45RB cDNA using primers 5′-GGCGCGGCCGCACGCGTAGGGGCACAGCTGATCTCCAGAT-3′ and 5′-GGCGCGGCCGCTTTAGCATTAAAATTTGTTGACTCATTTC-3′ , leading to the mCD45 expression vector . A PCR fragment encoding the extracellular domain of human CD43 was generated with primers 5′-CCCGCGGCCGCTGTTTCTTAGGGACACGGC-3′ and 5′-GAGGCGGCCGCGCCTCGTGAGTTCTCATCTGGGTTCC-3′ from a cDNA vector encoding human CD43 ( Open Biosystems ) , and was cloned in an analogous manner , resulting in the CD43 expression vector . 1×106 HEK293T cells were transfected with 4 µg of the expression constructs using the Lipofectamine 2000 reagent , and flow cytometric analysis as described below was performed 48 h later . Detection of protein expression from the transfected cells was using FITC-coupled MEM-28 anti-human CD45 ( Immunotools ) , FITC-coupled MEM-59 anti-human CD43 antibody ( Immunotools ) or FITC-coupled IBL-5/25 anti-mouse CD45 ( Immunotools ) . Cell surface expression of pUL11 was measured in HFF or A549 cells , 72 h after transduction with the rAdV at a multiplicity of infection ( MOI ) of 500 and 300 , respectively . The pUL11-specific antiserum was adsorbed for 8 h on uninfected A549 or HFF cells before use . Cells were incubated with antiserum in blocking solution ( 1% BSA , 0 . 1% gelatine , 2 mM EDTA in PBS ) followed by PE-conjugated goat anti-rabbit antibody ( Open Biosystems ) in blocking solution containing 6% goat serum . All steps were performed at 4°C . For flow cytometry based binding assays , 2 . 5 µg of purified Fc fusion proteins were incubated with 1×106 cells in blocking solution ( 5% mouse serum , 2 mM EDTA in PBS ) . Bound Fc proteins were detected using PE-conjugated anti-human IgG ( Acris ) . To determine the effects of CD45 antibodies on Fc protein binding , cells were incubated with MEM-28 ( Immunotools ) , UCHL-1 [59] ( kindly provided by P . Beverley , University of Oxford , UK ) or AICD45 . 2 [58] ( kindly provided by B . Schraven , University of Magdeburg , Germany ) antibodies in blocking solution for 30 min prior to incubation with UL11Fc fusion protein . Sub-populations of PBMCs were identified using antibodies directed to the following surface markers; T cells: anti-CD3-FITC ( Immunotools ) , anti-CD4-Dy647 ( Acris ) , anti-CD8-PE-Dy590 ( Antibodies-online ) . B cells: anti-CD19-PE-Dy590 ( Antibodies-online ) . NK cells: anti-CD56-APC ( Immunotools ) ; NK cells were identified as CD56 positive and CD3 negative cells , monocytes: anti-CD14-APC ( Immunotools ) and neutrophils: anti-CD15-FITC ( BD ) . Measurements were performed on a Beckmann Coulter Cytomics FC500 cytometer and analyzed using CXP analysis software . FACS based binding assays to stimulated CD4 T cells were performed using CD4 T cells prepared from PBMCs from control or variant ( CD45 C77G ) donors by positive MACS separation ( Miltenyi Biotec ) using the OKT-4 anti-CD4 mAb purified from hybridoma . The purity of the CD4 positive fraction was determined by FACS . Cells were stained and measured immediately or were stimulated with 1 µg/ml PHA ( Murex Diagnostics Ltd . ) for 24 h and then treated for 8 days with 25 U/ml IL-2 ( Roche ) . For staining , the cells were incubated in 50% mouse serum in PBS , followed by Fc fusion protein ( 1 µg ) for 45 min . After washing , cells were incubated with FITC-conjugated anti-CD45RA and APC-conjugated anti-CD45R0 antibodies ( BD ) . Bound Fc fusion proteins were detected using PE-conjugated anti-human IgG ( Acris ) . Measurement was performed using a FACSCalibur cytometer and analysis was performed using WinMDI software , version 2 . 9 . For confocal microscopy A549 or HFF cells infected with rAdVs as described above were incubated with the anti-pUL11 serum in blocking solution ( 1% BSA , 0 . 1% gelatine , PBS ) , followed by Alexa 568 conjugated goat anti-rabbit ( Invitrogen ) . Cells were fixed with 3% paraformaldehyde and observed using a Zeiss LSM 510 Meta Confocal Microscope . To observe leukocyte rosetting , HFF infected 96 h earlier with rAdVs at an MOI of 500 were co-cultured with Jurkat T cells , J-AS-1 T cells or freshly isolated PBMCs at a ratio of 1∶20 for 2 h at 37°C , and washed 8 times with PBS . To determine the effects of antisera on leukocyte rosetting , HFF cells infected with rAdVs as described were incubated for 2 h at 37°C with 400 µl of the rabbit anti-UL11 serum or preimmune serum diluted with 600 µl of DMEM . The serum was then removed and the HFF co-cultured with E6 . 1 Jurkat T cells ( 2×106 Jurkat cells per 1×105 HFF ) for another 2 . 5 h , followed by 15 washing steps with PBS . Images were taken using a Zeiss Axio Observer light/epifluorescence microscope . Glycosylation was investigated using purified Fc proteins or proteins of lysates from A549 cells transduced with rAdV at an MOI of 100 72 h earlier and prepared using NP40 lysis buffer ( 150 mM NaCl , 1% NP40 , 10 mM Tris-HCl pH 7 . 4 , 1 mM EDTA , protease inhibitor cocktail [Calbiochem] ) . Cell lysates or purified proteins were boiled for 5 min in denaturing buffer ( 0 . 5% SDS , 0 . 5% 2-mercaptoethanol ) before being treated with N-glycosidase F ( 4 U ) ( Roche ) or Endo-α-N-acetylgalactosaminidase ( 2 , 000 U ) and neuraminidase ( 100 U ) ( New England Biolabs ) in 500 mM sodium phosphate buffer pH 7 . 6 containing 1% NP40 for 2 h or overnight at 37°C . Cell surface proteins were biotinylated by incubating 2 . 5×107cells/ml in PBS with 2 mM Sulfo-NHS-LC-Biotin ( Thermo Fisher Scientific ) , for 30 min . The cells were washed three times with 100 mM glycine in PBS and then lysed in NP-40 lysis buffer . Proteins were pulled down or immunoprecipitated from cell lysates prepared from 1×108 cells/ml of NP40 lysis buffer . 500 µl of cell lysate precleared by incubation for 20 min with protein A sepharose CL-4B ( GE Healthcare ) was incubated with 10 µg of Fc protein or antibody and 20 µl protein A sepharose CL-4B for 90 min at 4°C . CD3 was immunoprecipitated using OKT3 ( eBioscience ) . The preparative pull-down for mass spectrometric analysis used 2×108 cells lysed in 1 ml of NP40 lysis buffer , incubated with 20 µg of protein and 20 µl of protein A sepharose CL-4B . For silver staining of proteins , SDS-PAGE gels were washed twice in 50% methanol/10% acetic acid for 15 min each , once in 10% ethanol/5% acetic acid for 6 min , and rinsed twice for 9 min in water . Gels were then incubated in sodium hydrosulfite ( 20 ng/ml ) for 9 min , followed by 0 . 1% silver nitrate solution , containing 0 . 75 µl/ml 37% formaldehyde for a further 9 min . Gels were then rinsed for 30 s in water and transferred to a 3% sodium carbonate solution containing 0 . 1% of 37% formaldehyde and 10 ng/ml sodium thiosulfate . Development was halted using stop solution ( 2 . 5% acetic acid , 5% Tris ) . For detection of pUL11 , CD45 , CD43 and phosphotyrosine proteins by immunoblotting a mouse anti-V5 antibody ( Invitrogen ) , the rabbit anti-pUL11 serum , the MEM-28 CD45 antibody ( Immunotools ) , the MEM-59 CD43 antibody ( Immunotools ) and the 4G10 phosphotyrosine antibody ( Millipore ) were used , respectively , followed by incubation with the appropriate HRP-conjugated anti-mouse or anti-rabbit antibodies ( Dako ) . Induction of tyrosine phosphorylation was measured after the incubation of Fc fusion proteins ( 2 . 5 µg ) with 4×105 Jurkat cells in 100 µl of culture medium for 30 minutes at 37°C , followed by stimulation with 100 µl of C305 anti-Jurkat TCR mAb [96] hybridoma supernatant ( kindly provided by B . Schraven , University of Magdeburg , Germany ) . Stimulation was stopped by the addition of 1 ml ice-cold PBS and the cell suspension was immediately centrifuged . The cell pellet was then lysed with NP-40-digitonin lysis buffer ( 1% NP-40 , 1% digitonin , 50 mM Tris-HCl pH 7 . 4 , 150 mM NaCl , 10 mM EDTA , 2 mM sodium vanadate and protease inhibitor cocktail [Calbiochem] ) . To measure proliferation of PBMCs , Fc fusion proteins ( 2 . 5 µg ) and OKT3 ( 1 µg; purified from hybridoma supernatant ) were adsorbed onto Maxi-Sorb 96-well plates . 1×105 PBMCs per well were incubated in 200 µl of culture medium . PHA ( Oxoid , Basingstoke , UK ) was added where indicated at 25 µg/ml . After 48 h , 0 . 4 µCi [3H]-thymidine ( Amersham Biosciences , Braunschweig , Germany ) was added . After 24 h the cells were harvested and incorporated [3H]-thymidine measured in a beta-counter ( Perkin Elmer , Rodgau , Germany ) . Protein bands were excised manually from a preparative , Coomassie-stained gel . After destaining two times with 100 µl of 50% acetonitrile ( ACN ) , 20 mM NH4HCO3 at 37°C for 30 min , bands were dehydrated by adding 100 µl ACN and dried . 20 µl of sequencing grade trypsin ( 10 ng/ml; Promega ) was added and after 30 min incubation on ice remaining trypsin solution was discarded . Digestion was continued at 37°C overnight and stopped by adding 0 . 1% trifluoroacetic acid , 50% ACN . Tryptic peptides were extracted with two times 20 µl 50% ACN , 0 . 1% formic acid ( FA ) for 30 min at 37°C and 10 µl ACN for 30 min at RT . All extracts were combined and dried in a vacuum centrifuge . For LC-iontrap-MS analysis peptide samples were dissolved in 10 µl 10% ACN . Five microliter per peptide sample were injected onto a C18 RP-Column ( Zorbax SB , C18 , 80 Å , 5 µm , 150×0 , 5 mm: Agilent ) using a 1100 Series Agilent HPLC System equipped with an autosampler , coupled online to an Esquire3000+ ion trap mass spectrometer ( Bruker Daltonics ) . Using a two buffer system ( A: 5% ACN , 0 , 1% FA; B: 80% ACN , 0 , 1% FA ) and a flow rate of 5 µl/min , a multi-step gradient was applied after injection: 0–5 min: 0% B; 30 min gradient to 53 . 9% B ( 40% ACN ) ; 5 min gradient to 100% B; increase of flow rate to 10 µl/min in 1 min; 10 min at 100% B; 4 min gradient to 0% B; 15 min at 0% B . The MS method used to select and fragment the eluting peptides was set to trigger fragmentation of the three most intensive peaks from an MS scan at a 40 , 000 ion count threshold and a preference of doubly charged ions . Automated precursor exclusion after one acquired spectrum per precursor for 0 . 3 min was used . The ESI source conditions were set to 10 psi nebulizer gas pressure with dry gas heated to 300°C at a flow rate of 4 . 0 l/min . Mass spectrometrical data were searched against the SwissProt Database with carbamidomethylation of cysteins as static and oxidation of methionines as variable modification . For ion trap-MS 150 ppm mass deviation was tolerated for precursors and 0 . 7 Da for peptide fragments in MS/MS . At least two peptides with a Mascot peptide ion score higher than 25 each were used as a threshold for protein identification . | The human cytomegalovirus ( CMV ) belongs to a class of viruses that interferes with the immune response of its host . Accordingly , infection with CMV is a severe risk for immunologically immature newborns and immunocompromised patients such as transplant recipients . The mechanisms by which CMV affects the immune system are not completely understood . Here we show that a CMV protein , pUL11 , which is expressed on the surface of cells , binds to leukocytes by interacting with the receptor tyrosine phosphatase CD45 . In T cells , CD45 is essential for transmission of activating signals received via the T cell receptor ( TCR ) to downstream effector molecules that ultimately lead to activation and proliferation of these immune cells . Binding of the CMV pUL11 protein to CD45 on T cells prevents signal transduction via the TCR and restricts T cell proliferation . Interestingly , the mechanism by which the activity of CD45 is regulated is a matter of debate and no specific cellular ligand of CD45 has yet been described . The identification of a first viral ligand for CD45 may provide the means to investigate CD45 regulatory mechanisms and also allow the development of therapies to interfere with CMV-mediated immunomodulation . | [
"Abstract",
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"Results",
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] | [
"medicine",
"biochemistry",
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] | 2011 | The Human Cytomegalovirus UL11 Protein Interacts with the Receptor Tyrosine Phosphatase CD45, Resulting in Functional Paralysis of T Cells |
Exposure to man-made electromagnetic fields ( EMFs ) , which increasingly pollute our environment , have consequences for human health about which there is continuing ignorance and debate . Whereas there is considerable ongoing concern about their harmful effects , magnetic fields are at the same time being applied as therapeutic tools in regenerative medicine , oncology , orthopedics , and neurology . This paradox cannot be resolved until the cellular mechanisms underlying such effects are identified . Here , we show by biochemical and imaging experiments that exposure of mammalian cells to weak pulsed electromagnetic fields ( PEMFs ) stimulates rapid accumulation of reactive oxygen species ( ROS ) , a potentially toxic metabolite with multiple roles in stress response and cellular ageing . Following exposure to PEMF , cell growth is slowed , and ROS-responsive genes are induced . These effects require the presence of cryptochrome , a putative magnetosensor that synthesizes ROS . We conclude that modulation of intracellular ROS via cryptochromes represents a general response to weak EMFs , which can account for either therapeutic or pathological effects depending on exposure . Clinically , our findings provide a rationale to optimize low field magnetic stimulation for novel therapeutic applications while warning against the possibility of harmful synergistic effects with environmental agents that further increase intracellular ROS .
Editor’s Note: This Short Report received positive reviews by experts . The Academic Editor has written an accompanying Primer that we are publishing alongside this article ( https://doi . org/10 . 1371/journal . pbio . 3000018 ) . The linked Primer presents a complementary expert perspective; it discusses considerations about the status of knowledge and experimental systems in the field that encourage cautious interpretation .
Weak electromagnetic radiation ( μT-mT ) , which increasingly pollutes our environment , has been associated with dual and seemingly contradictory effects on human health . On the one hand , possibly deleterious public health consequences have elicited considerable debate on safety and exposure limits to electromagnetic field ( EMF ) radiation [1–4] . On the other hand , weak magnetic fields have been applied as therapeutic tools , notably in the form of pulsed electromagnetic fields ( PEMFs ) , which have shown benefits in a broad range of regenerative medicine therapeutics , as well as in the alleviation of depression , reducing symptoms of Parkinson disease , and reducing memory loss [5–10] . Such PEMFs also affect nonexcitable tissues [7 , 9] and are below firing threshold for neurons [11 , 12] consistent with magnetic field effects and thereby activation of a biological magnetoreceptor . The current challenge is therefore to identify these putative magnetosensor ( s ) and to propose a mechanism that may explain the seemingly disparate effects of EMFs in medicine and in public health . A possible class of biological magnetoreceptor [13] are the cryptochromes , which are conserved flavoprotein receptors [14] implicated in magnetosensing in organisms ranging from plants to migratory birds [14–16] . Cryptochrome receptors undergo redox reactions in the course of their activation that lead to the synthesis of reactive oxygen species ( ROS ) [17–19] . ROS are global regulators that are implicated in numerous cellular signaling functions related to response to stress and ageing and are toxic at high concentrations [20–23] . In mammalian cells , cryptochromes are both cytosolic and nuclear proteins that have been characterized for a role as core components of the circadian clock [24 , 25] but that are not known to respond to external magnetic fields . However , recombinant mammalian cryptochromes expressed in a heterologous Drosophila system are reported to confer magnetic sensitivity in behavioral assays in flies [16] , and they were recently proposed to play a role as sensors of low EMFs in the onset of childhood leukemia [26] . This raises the question of whether cryptochromes could be implicated in magnetic sensitivity in humans .
To explore this question , we chose to use PEMF exposure as a source of magnetic stimulation because it has demonstrated therapeutic effects on a wide variety of mammalian cell types [5–10] . To determine whether cryptochromes are implicated in PEMF effects , we first established whether a known magnetosensitive cryptochrome can mediate a response to a PEMF signal in a well-established magnetically sensitive model system . We used the fruitfly Drosophila melanogaster , which display a natural behavioral avoidance response to static magnetic fields [16] . Adult flies were placed on square petri plates to lay eggs for 24 hours and were subsequently removed . The ensuing hatched larva migrated freely over the plate for several days before choosing a location to attach to and form sessile pupa for metamorphosis . These pupae were located randomly around the perimeter of the plate , with preference for the corners ( Fig 1 ) . We tested magnetic sensitivity , with a coil generating continuous PEMF at 10 Hz , with peak amplitude of 1 . 8 mT at the level of the larvae ( S1 and S2 Figs ) , placed underneath one of the 4 corners of the petri plate ( see Materials and methods ) . Fly larvae grown under these conditions avoided the corner of the petri plate above the PEMF device ( Fig 1A ) compared to the other corners . Both Canton S ( WTS ) and Oregon ( WTO ) wild-type fly strains showed this avoidance response ( Fig 1A and 1B ) in blue light ( which activates Drosophila cryptochrome; Fig 1B ) but not in red light ( which does not activate Drosophila cryptochrome; S3 Fig ) . As a control , a 1 . 0 mm mu-metal plate , which blocks static or low-frequency magnetic fields , was inserted between the magnetic coil and the petri plate containing the fly larvae . In these conditions , larvae did not show the avoidance response ( S3 Fig ) . As a further control , we tested a coil in which the wire had been wound in an antiparallel fashion in order to cancel the magnetic field without altering the current in any way ( see Materials and methods ) ; this was also ineffective in causing an avoidance response . We next observed that fly mutants deficient in cryptochrome ( cryb and cry02; [27] ) did not avoid the PEMF , confirming a role for cryptochrome in this response . Finally , we tested transgenic fruitflies expressing the human cryptochrome-1 ( HsCry1 ) protein in Drosophila cryptochrome-deficient strains as described previously [16 , 27] ) . HsCry1 expression indeed restored the behavioral avoidance response to PEMF in flies lacking their endogenous cryptochrome ( Fig 1B ) . These results indicate that PEMF can be detected by insects through the action of either Drosophila ( DmCry ) or human ( HsCry1 ) cryptochrome , consistent with the response to static magnetic fields in this organism [16] . A possible mechanistic basis for this fly avoidance response was suggested by recent observations that ROS are byproducts of cryptochrome activation [17 , 28] linked to signaling [29 , 30] . Furthermore , at high concentrations , ROS are toxic metabolites implicated in oxidative stress and ageing , which damage cell membranes , nucleic acids , and proteins [20] , consistent with the behavioral avoidance response . In contrast , at physiological concentrations , ROS are reported to have beneficial effects [20 , 23] , consistent with the observed therapeutic effects of PEMF [5–10] . To determine whether the PEMF signal stimulates formation of ROS , Spodoptera frugiperda ( Sf21 ) insect cell cultures overexpressing DmCry [28] were stimulated by PEMF in blue light for 15 minutes in the presence of the ROS label , {5- ( and-6 ) -chloromethyl-2’ , 7’-dichlorofluorecein diacetate} ( DCFH-DA ) [17 , 28] . Confocal image analysis revealed a marked increase in fluorescent signal in PEMF-treated cells compared to unstimulated cultures ( Fig 1C ) . In contrast , no visible effect of PEMF stimulation was observed in Sf21 cells lacking DmCry ( S4 Fig ) . These data indicate that PEMF stimulation leads to intracellular accumulation of ROS and that this effect requires Drosophila cryptochrome . Although flavin binding affinity is reportedly poor for vertebrate cryptochromes in vitro [31] , they nevertheless confer light-sensitive phenotypes in expressing transgenic flies [16 , 27] and undergo light-sensitive conformational change in the avian retina [15 , 32] , indicating that flavin is bound in vivo . Moreover , vertebrate-type cryptochromes are shown to undergo photoreduction and flavin radical formation in whole cell cultures , using an EPR spectroscopic approach [33] . These properties are consistent with the capacity to undergo flavin redox state interconversion and to form ROS , as do other cryptochromes [17 , 28 , 34] . We therefore tested for ROS induction following PEMF stimulation of human embryonic kidney 293 ( HEK293 ) cells , grown in darkness for 48 hours in the presence or absence of PEMF ( Fig 2 ) . After incubation , the extracellular media were scored for secreted hydrogen peroxide ( H2O2 ) , a byproduct of ROS formation , using the Amplex Ultra Red fluorescence detection substrate as described [35] . The concentration of ROS was significantly elevated in media from PEMF-treated cell cultures compared to controls ( Fig 2 ) . To evaluate toxicity of prolonged exposure to PEMF , we counted cells at the end of the exposure period ( see Materials and methods ) . A marked decrease in cellular growth was observed in PEMF-exposed HEK293 cultures compared to untreated controls , consistent with the toxicity of accumulated ROS ( Fig 2 ) . To assess a possible effect of cryptochrome on this response , short hairpin RNA ( shRNA ) lines with double HsCry1 and HsCry2 mRNA knockdown were constructed ( see Materials and methods , S5 Fig ) and similarly analyzed . These shRNA lines deficient in both HsCry1 and HsCry2 showed no significant effect of PEMF either on cell growth or on ROS secretion ( Fig 2A and 2B ) , in marked contrast to wild type . Therefore , these magnetic field effects appear to involve cryptochrome function and formation of ROS in human cells . We further analyzed PEMF effects on mammalian cells using fluorescence imaging to detect multiple ROS forms . As observed for the Sf21 insect cell experiments above ( Fig 1C ) , HEK293 cells were incubated in the presence of DCFH-DA at 37 °C for 15 minutes in the presence or absence of PEMF ( Fig 3 ) . Fluorescent ROS labeling increased significantly in PEMF-stimulated cells compared to unstimulated control cell cultures . ROS staining can be seen in both nuclear and cytosolic compartments , with areas of concentration in nuclear speckles ( nucleoli ) and vesicular structures ( E . R and Golgi ) , consistent with subcellular localization of mammalian cryptochromes [36] . To further confirm the involvement of cryptochrome in this response , we examined cells from murine cryptochrome mCry1/mCry2 double knockout mice [37] . Specifically , we analyzed immortalized mouse embryonic fibroblast ( MEF ) cell cultures from wild-type and mCry1/mCry2 double knockout lines , using the ROS fluorescence imaging techniques used for the HEK cell cultures . A marked induction of intracellular ROS after 15 minutes of PEMF stimulation was observed in wild-type MEF cells ( Fig 3 , middle panels ) , equivalent to those observed for the HEK293 human cell cultures ( Fig 3 , upper panels ) . However , mCry1/mCry2 null mutant cell cultures treated in an identical manner ( Fig 3 , lower panels ) showed no visible increase in ROS labelling . Taken together , these data show that cryptochrome is necessary for PEMF-induced ROS formation in mammalian cells . To further define the effects of PEMFs and relate them to therapeutic consequences observed in humans [5–10] , we performed microarray analysis of gene expression in HEK293 cells cultured with or without 3 hours of PEMF stimulation ( S1 and S2 Tables ) . Several hundred genes were up-regulated or down-regulated by PEMF stimulation . Of these transcripts , a significant proportion encoded proteins localized to nuclear , Golgi , and endoplasmic reticulum ( ER ) compartments ( S5 Table ) . Significantly , bioinformatic gene ontology ( GO ) analysis of biochemical function showed enrichment in oxidoreductase function consistent with increased production of ROS ( see Materials and methods , S6 Table ) . Furthermore , promoter analysis of PEMF-induced genes indicated that a majority ( 75% ) contained promoter elements known to interact with ROS-responsive transcription factors . These data are consistent with stimulation of ROS-responsive genes following PEMF exposure ( S7 Table ) . Furthermore , they parallel the imaging data of these HEK293 cells , which showed enhanced localization of ROS to the nuclear , Golgi , and ER compartments , whereas transcription of proteins localized to these compartments are particularly enriched among PEMF-regulated genes ( S5 Table ) . Thus , the induction of ROS by PEMF is indicated by two entirely independent and complementary approaches: imaging and transcriptome analysis .
A widely held paradigm for cryptochrome magnetosensing involves a radical pair-based mechanism , whereby the singlet/triple interconversion rates of unpaired radicals formed in the course of cryptochrome redox chemistry can be altered by static magnetic fields [13] . This provides a mechanism whereby cryptochrome reaction rates and product yields , including of H202 and other ROS formed during the cryptochrome redox cycle [17 , 28] , can be altered by magnetic fields . Recent experiments probing the light dependence of magnetic orientation in birds have pinpointed cryptochrome flavin reoxidation as the likely step for radical pair formation leading to magnetic sensitivity [32 , 38] . Such flavin reoxidation , which occurs independently of light , involves reaction of cryptochrome-bound reduced flavin with molecular oxygen and fulfills the criteria of radical pair formation during magnetoreception [39] . Nonetheless , in the case of both avian and drosophila cryptochromes , the initial formation of reduced flavin requires light ( by the process of flavin photoreduction ) [34 , 38] . This explains the requirement for light in establishing magnetic sensitivity in flies and birds because reduced flavin is required for the magnetically sensitive redox reaction ( reoxidation ) to ensue [38] . By contrast , mammalian-type cryptochromes appear to function independently of light in their role in the circadian clock and as negative regulators of transcription [14 , 24 , 25] . However , mammalian-type cryptochromes reportedly occur in a partially reduced redox state in vivo even in dark-adapted cell cultures [40] . As a consequence , they would retain the characteristics to respond to magnetic fields by a mechanism whereby flavin reoxidation is stimulated , with an ensuing burst of ROS synthesis consistent with our observations . We also note that , although there has been overwhelming evidence for a radical pair-based magnetic sensing mechanism involving vertebrate cryptochromes [13] , the possibility of unrelated cry-dependent magnetosensing mechanisms cannot be excluded . For example , a recently suggested interaction of cryptochrome with the putatively magnetosensitive MagR protein could also be consistent with our data [41] , whereas reported magnetic sensitivity mediated through a C-terminal overexpression construct of Drosophila cryptochrome [42] also suggests alternative magnetosensors impacting on a cry-based magnetosensing mechanism . A mechanism based on regulation of ROS can explain both the beneficial and deleterious effects of magnetic stimulation that have so long puzzled the field . For example , proposed deleterious effects [1–4 , 26] of low-frequency EMFs could result from elevated ROS , which inform about exposure to magnetic fields either in human treatment or in public health . This result is furthermore consistent with past suggestions that the lifetimes and reactivity of 02 and ROS ( both paramagnetic species ) may be affected by magnetic fields in living systems [5] . However , prior speculation has focused exclusively on ROS generated via metabolic pathways of the mitochondrial electron transfer chain or via cell membrane–associated NADPH oxidases . Here , we implicate a flavoprotein receptor and signaling molecule , which is suitably positioned within the nucleus [36] , to induce localized changes in ROS concentration and/or reactivity in close proximity to redox-sensitive and/or ROS-regulated nuclear signaling molecules . We note that the prolonged PEMF signal ( S1 and S2 Figs ) used in the current study has no therapeutic application and is apparently harmful to cell cultures over long periods . However , a range of alternate frequencies and amplitudes of PEMF signal have been empirically derived that provide proven physiological benefits involving cellular repair and healing [5–12] . These beneficial PEMF effects are compatible with modulation of intracellular ROS within a therapeutic range resulting in stimulation of ROS responsive cellular defense and repair mechanisms [20 , 23] . In conclusion , from a public health perspective , our work shows that exposure to even such low levels of magnetic fields as those generated by PEMF devices have definite physiological consequences . It should be noted that peak output at less than 1 . 8 mT is within an order of magnitude of emissions by household electronic devices and of current safety guidelines for exposure to EMF in humans [1–4] . In keeping with our results , it has also been shown that the low-level man-made EMFs emitted from electrical equipment in public buildings can disrupt orientation in birds , a process that has also been linked to both cryptochromes and magnetoreception [43] . Although current epidemiological studies have not provided conclusive evidence of EMF-induced pathology in humans [1–4] , our results raise the possibility of synergistic harmful effects with other environmental or cellular factors that stimulate intracellular ROS [5 , 20] . More refined epidemiological studies taking these factors into consideration are therefore essential for a true assessment of long-term impact of EMFs on public health .
The pulsed magnetic field was generated by a commercially available device ( EC10701; GEM Pty Ltd . , Perth , Western Australia ) used for the treatment of musculoskeletal disorders . During Drosophila behavioral tests , PEMF was applied continuously at a frequency of 10 Hz , with the coil 1 cm below the petri plate . Peak magnetic intensity at the experimental distance was 2 mT . The parameters of the PEMF signal were verified by measurement of the current as presented in S1 and S2 Figs . The coil was 9 × 5 . 5 cm and 200 turns and produced a maximum magnetic field intensity 1 cm above the coil of 1 . 8 mT . Fly strains used were as follows: wild-type Canton S , wild-type Oregon-R , cry02 and cryb ( described in [27] ) . Transgenic strains tim-gal4;cry02 and UAS-Hscry1;cry02 were crossed to generate the heterozygote HsCry1-expressing strain as described in Vieira and colleagues [27] . Light sources and growth conditions on complete media were as previously described [27] . For the larvae migration studies ( pupal distribution ) , adult drosophila were transferred to a square plate ( 12 . 5 cm × 12 . 5 cm ) containing complete rich medium and were allowed to lay eggs . After a period of 24 hours , the adults were discarded and the plates placed under the indicated light conditions ( blue or red light ) for 3 days at 23 °C . Subsequently , a single corner of each plate was exposed to PEMF from underneath for an additional 5 days . Temperature differential between corners was less than 0 . 5 °C . The control condition was established by shielding the plate from the PEMF device with 1 . 0 mm mu-metal sheeting , which was measured to reduce magnetic field signal by 85% . Once pupal development was complete , the distribution of the now nonmotile pupae could be readily scored by counting the number of pupae in a defined area of the plate . As the pupae showed a preference for the corners of the plates , we evaluated 3 . 12 × 3 . 12 cm2 areas over each of the 4 ( PEMF treated versus untreated ) corners and compared the corners that had received no PEMF treatment with those exposed to PEMF . Statistical methods were as follows: for each experimental condition , a total of between 8 and 10 plates were analyzed ( n = 8–10 ) . The number of drosophila counted in each corner was expressed as the percentage of the total number of pupae ± SEM per plate . All statistical tests were carried out using SPSS ( version 20 , IBM Corporation , NY ) . Data were analyzed for normality ( Shapiro-Wilk test , p < 0 . 05 ) , so the differences between the PEMF and mean of the 3 non-PEMF corners per plate were compared using Kruskal-Wallis analysis of variance and Mann-Whitney-U post hoc where appropriate . The α value was set to p < 0 . 05 . Further details of the behavioral experimental setup are presented in S3 Fig as follows: the position of the plate containing drosophila growth media and growing larva under which the PEMF coil was placed ( upper left ) is designated as the position “1 , ” the “test” corner . The PEMF coil was at a distance of 1 cm from the bottom of the test plate containing the drosophila larvae . The temperature at all 4 corners was measured , and the PEMF device did not cause any change in temperature from the other corner positions of the plate . Equivalent-size squares at each of the other corners ( designated positions 2 , 3 , and 4 ) serve as the internal “control” positions to the PEMF stimulated “test” position . The number of pupae that were deposited beneath the PEMF coil was compared to the number of pupae deposited within an equivalent volume at each of the other 3 corner positions . Additional controls to the behavioral experiments are shown in S3B and S3C Fig . The PEMF device was shielded from the test plate using mu-Metal sheeting of 1 . 0 mm thickness . Under these conditions ( S3B Fig ) , no significant avoidance of the PEMF corner position was detected . In addition , response to PEMF was scored in red light , which does not activate insect ( Drosophila ) cryptochrome ( S3C Fig ) . In this case also , no avoidance of the PEMF was observed . Preparation of DmCry-expressing and control ( Spa1 ) -expressing insect cell cultures was performed as described in Arthaut and colleagues [28] . For imaging experiments , Sf21 cells were seeded at a density of 400 , 000 cells in a 3 . 5 cm2 observation chamber . After incubation at RT for 2 hours for cell attachment , Sf21 cells were incubated in 40 mM potassium phosphate buffer ( pH 6 . 4 ) containing 12 . 5 μM DCFH-DA ( Molecular Probes , Life Technologies , Grand Island , NY ) for 15 minutes in the dark , rinsed 2 times in phosphate buffer , and were then exposed to blue light with or without PEMF for 15 minutes and observed with an inverted Leica TCS SP5 confocal microscope using a 40× objectif . Green fluorescence from DCFH-DA and differential interference contrast ( DIC ) were excited at 488 and 561 nm wavelengths , respectively . Emission fluorescence intensities and DIC were detected using a photomultiplier between 498 and 561 nm , and a transmission photomultiplier , respectively . Two channels were recorded sequentially . Z series projections were taken using ImageJ software ( W . S . Rasband , ImageJ ) . As a control for these experiments , control cell cultures that did not express DmCry were used ( S4 Fig ) . These cells did not show induction of ROS in response to PEMF . By using the InvivoGen siRNA Wizard tool , shRNA sequences targeting human CRY1 ( NM_004075 . 4 ) and CRY2 ( NM_021117 . 3 ) were selected , and a pair of complementary ( sense and antisense ) oligonucleotides were designed for each sequence as follows: shCRY1 sense ( 5’GTACCTCGGAACGAGACGCAGCTATTAATCAAGAGTTAATAGCTGCGTCTCGTTCCTTTTTGGAAA 3’ ) ; shCRY1 antisense ( 5’AGCTTTTCCAAAAAGGAACGAGACGCAGCTATTAACTCTTGATTAATAGCTGCGTCTCGTTCCGAG3’ ) ; shCRY2 sense ( 5’ACCTCGTACGTATGTCACCTTCACTATCAAGAGTAGTGAAGGTGACATACGTACTT3’ ) ; shCRY2 antisense ( 5’CAAAAAGTACGTATGTCACCTTCACTACTCTTGATAGTGAAGGTGACATACGTACG3’ ) ( complementary sequences of the hairpin are underlined ) . Complementary oligonucleotide pairs were PAGE-purified , and 25 μM of each were annealed by incubation in 0 . 1 M NaCl at 80 °C ( 2 minutes ) followed by slow ( 1 °C per minute ) cooling to 35 °C . The resulting double-stranded DNA fragments were cloned into the same psiRNA-DUO-GFPzeo plasmid according to the manufacturer’s instructions using a two-step procedure ( InvivoGen ) . Briefly , the psiRNA-DUO plasmid was digested with Acc65I and HindIII restriction enzymes and ligated with the first insert ( shCRY1 annealed oligonucleotide pairs ) . The resulting construct was transformed into Escherichia coli GT115 cells ( InvivoGen ) , and positive colonies were selected using Fast-Media Zeo X-gal ( 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside ) ( InvivoGen ) . The plasmid containing shCRY1 was subsequently digested with BbsI restriction enzyme and ligated with the second insert ( shCRY2 annealed oligonucleotide pairs ) . The resulting construct was transformed into E . coli GT115 cells ( InvivoGen ) , and positive colonies were selected using Fast-Media Zeo 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid , cyclohexylammonium salt ( X-gluc ) ( InvivoGen ) . The obtained psiRNA-Cry1Cry2-GFPzeo expression plasmid was used for transfection of HEK cells , and psiRNA-LucLac-GFPzeo encoding shRNA for the silencing of a prokaryote gene ( InvivoGen ) was used to transfect HEK cells as nonsilencing control . Stable transfectants were selected in complete medium containing 300 μg/ml Zeocin ( InvivoGen ) . Expression of HsCry1 and HsCry2 was verified by qPCR ( S5 Fig ) . HEK293 cells were grown and maintained in Eagle’s Minimum Essential Medium ( EMEM ) , supplemented by 10% fetal bovine serum . The cells were cultured in 75 cm2 flasks to expand cell number . After reaching confluence , the cells were seeded in 12-well plates . The volume of medium totaled 1 mL . Medium was then changed every 2 days . The cultures were incubated in a 5% CO2 atmosphere at 37 °C in the same incubator ( Fisher Scientific; Model 5 ) . The temperature and CO2 levels were monitored daily and were maintained at 37 °C and 5% , respectively . All experiments were conducted in the same incubator . To control for location in the incubator and any associated electromagnetic noise or other spatial variation , the orientation of experimental and control cultures were periodically reversed , and 0 . 3 mm mu-Metal shielding was applied between PEMF-treated and control cell culture dishes within the incubator . Cells were seeded and allowed to rest for 4 hours under the same background conditions , at which time the magnetic exposures began . This time is denoted as t0 . Fluorometric detection of H2O2 production was performed using the horseradish peroxidase-linked Amplex Ultra Red ( Invitrogen ) fluorometric assay . HEK cells were seeded at a concentration of 25 . 0 × 104 cells per well in a 12-well plate and were exposed to PEMFs for the duration of the experiment . Medium was aspirated off , and cells were then washed with PBS and incubated for 2 hours with DMEM containing 2% FBS , 0 . 2 units/ml horseradish peroxidase , and 10 μM Amplex UltraRed ( AUR ) . Resorufin fluorescence was measured by a Varian Cary Eclipse spectrofluorimeter . Cellular number and resorufin fluorescence were measured at the same termination points . H2O2 production was normalized to cell count . H2O2 calibration curves with HRP-AUR in PEMFs did not show any difference compared to control , thus demonstrating that PEMFs do not interact with the detection system . Human HEK and MEF cells were grown in Dulbecco’s Modified Eagle Medium ( DMEM ) supplemented with 10% fetal calf serum ( FCS ) and 2 mM l-glutamine in a 95% air–5% CO2 incubator at 37 °C . For intracellular localization of ROS , living HEK or MEF cells were seeded on cell observation chambers and incubated in 40 mM potassium phosphate buffer ( pH 7 ) containing 12 . 5 μM DCFH-DA ( Molecular Probes ) for 15 minutes in the incubator at 37 °C , during which they were either exposed or not to PEMF . Cells were rinsed for 15 minutes in the potassium phosphate buffer solution and were observed with an inverted Leica TCS SP5 confocal microscope equipped with a 95% air–5% CO2−37 °C thermostatic observation chamber and using a 63× objective . Green fluorescence from DCFH-DA and DIC were detected as previously described ( section 5 ) . For quantitation of intensity , using LEICA TCS software , the region of interest ( ROI ) corresponding to cells were dawn and mean fluorescent intensity ( MFI ) measured in each ROI . Human HEK cells were grown in DMEM supplemented with 10% FCS and 2 mM l-glutamine in a 95% air–5% CO2 incubator at 37 °C . Cells were seeded into multiple 3 . 5 cm2 round cell culture dishes and were grown under identical conditions for 48 hours . Prior to confluence , cell culture dishes were treated with 3 hours of continuous PEMF in the absence of light ( test condition ) . Control cell cultures were harvested prior to application of PEMF . Triplicate PEMF treated and control cell cultures were then harvested into liquid nitrogen , and total RNA was extracted by RNEasy RNA extraction kit ( Promega , Inc . ) and related protocols . Microarray gene expression and analysis using Agilent affymatrix technology was performed by IMGM Laboratories GmbH , Martinsried , Germany . As a control to eliminate the possibility of artifact due to temperature and/or vibrational factors generated by the pulsed field device , we designed and built a modified PEMF coil in which the wire was folded in half before precision winding to achieve an antiparallel current travelling in opposite directions within the same coil during activation . This antiparallel coil had the same wire length and dimensions as the test coil used in our experiments , and it was driven by the same pulsed field generator device and with the identical current—which , because of the antiparallel winding of the coil , ran simultaneously in opposing directions within the coil . The signal measured in S7 Fig shows that , whereas there are residual spikes in the antiparallel field coil ( panel B ) that could not be cancelled , these spikes are less than 0 . 01 seconds in duration in comparison to the magnetic signal in the original PEMF coil , which lasts for 0 . 5 seconds ( panel A ) . The residual spikes are not visible on panel D because they are too short lived for the detection limit for the instrument at this time scale . We conclude that these residual spikes are of negligible duration compared to the signal given out by the intact coil and are demonstrably too brief to trigger a biological response . As a result , we achieved a significant reduction ( cancelling ) of the pulsed magnetic field ( S7 Fig ) while keeping all other parameters ( electric current driven by the pulsed field device ) the same . We tested the effect of the cancelled PEMF field on the behavioral avoidance response of wild type ( Canton S ) fly pupa according to the methods used for Fig 1 . We measured the number of pupa in the corner of square petri plates exposed to the antiparallel ( cancelled PEMF field ) coil compared to the test coil ( generating the PEMF signal ) placed beneath the plate corner ( S8 Fig ) . The flies did not show an avoidance response to the cancelled field ( antiparallel coil ) , indicating that the magnetic field of the PEMF was indeed triggering the response . We next evaluated the effect of the cancelled magnetic field coil on the stimulation of ROS in mammalian cell culture experiments . Using both HEK and MEF cell cultures , we observed that significant stimulation of ROS formation occurred only in response to PEMF but not to the antiparallel , PEMF-cancelled magnetic field ( S9 Fig ) . In conclusion , these data indicate that there was no discernable artifact introduced into our experiments through the operation of the pulsed field device and that positive results required the presence of the magnetic field . | Repetitive low-intensity magnetic stimulation has been used in the treatment of disease for over 50 years . Associated benefits have included alleviation of depression , memory loss , and symptoms of Parkinson disease , as well as accelerated bone and wound healing and the treatment of certain cancers , independently of surgery or drugs . However , the cellular mechanisms underlying these effects remain unclear . Here , we demonstrate that repetitive magnetic field exposure in human cells stimulates production of biological stress response chemicals known as reactive oxygen species ( ROS ) . At moderate doses , we find that reactive oxygen actively stimulates cellular repair and stress response pathways , which might account for the observed therapeutic effects to repetitive magnetic stimulation . We further show that this response requires the function of a well-characterized , evolutionarily conserved flavoprotein receptor known as cryptochrome , which has been implicated in magnetic sensing in organisms ranging from plants to flies , including migratory birds . We conclude that exposure to weak magnetic fields induces the production of ROS in human cells and that this process requires the presence of the cryptochrome receptor . | [
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] | 2018 | Low-intensity electromagnetic fields induce human cryptochrome to modulate intracellular reactive oxygen species |
SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells . Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function , although alternative mechanisms have been proposed recently . Here , we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity , restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers . Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity . SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics . However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion . Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells .
HIV-1 replicates poorly in cells of the myeloid lineage and resting T cells through blocks that occur early in infection [1–3] . However , in HIV-2 and related simian viruses ( SIVs ) the lentiviral accessory protein Vpx overcomes this restriction by targeting the cellular protein SAMHD1 for proteasomal degradation [4–6] . SAMHD1 is a GTP/dGTP-activated deoxynucleotide triphosphohydrolase [7] . It was first identified through association with the rare genetic disorder Aicardi Goutières syndrome ( AGS ) , which mimics congenital viral infection [8] . Human SAMHD1 is a 626 amino acid protein comprising an N-terminal nuclear localisation signal [9] , two major structural domains; a sterile alpha motif ( SAM ) and an HD domain , together with CtD , a C-terminal region required for interaction with Vpx isolated from SIV that infects sooty mangabey ( SIVsmm ) [10 , 11] . SAM domains are present in a wide range of proteins and are generally involved in protein-protein or protein-nucleic acid interactions [12] . The HD domain contains the active site of the protein and in combination with N- and C-terminal sequences also incorporates the two allosteric nucleotide-binding sites , AL1 and AL2 , that regulate the enzyme through combined binding of G-based ( AL1 ) and deoxynucleoside ( AL2 ) triphosphates [7 , 13 , 14] . The CtD contains the residues bound by SIVsmm Vpx that target human SAMHD1 to the ubiquitination machinery and direct its subsequent proteasomal degradation [11] . SAMHD1 is proposed to suppress HIV-1 replication in restrictive cells by inhibiting reverse transcription through depletion of the intracellular dNTP pool . This hypothesis is supported by the inverse relationship between SAMHD1 levels and dNTP concentration ( and consequently HIV-1 infectivity ) in a number of cell lines and primary cells [15–17] . However , there is uncertainty as to the extent to which the different domains of SAMHD1 contribute to restriction of HIV-1 infection . Some data suggest that only the HD domain is required for inhibition of infection [18] and that removing the C-terminal region does not affect restriction [18 , 19] , while other studies suggest that the SAM domain may influence function [20] and that the C-terminal region is required for SAMHD1 activity in cells [21] . Although the HD domain has consistently been found to be necessary for lentiviral restriction , some reports have questioned whether this is due to its role in tetramerisation of the protein rather than the presence of the catalytic site [18 , 22] . Furthermore , SAMHD1 has been reported to have alternative nuclease [20 , 23] and/or RNA binding [24 , 25] activities that , dependent on the cellular circumstances , can also mediate restriction of HIV-1 [23] . In addition , restriction is regulated by Threonine 592 phosphorylation [19 , 22 , 26] and removing this regulation may enable SAMHD1 to inhibit HIV-1 in cycling cells ( Cribier et al . , 2013 ) . These latter observations question the role of triphosphohydrolase activity in restriction . However , disparities in the field may be due to variation in assays used to determine enzyme activity or could represent divergent strategies for retroviral restriction . Therefore , given these controversies and the importance of SAMHD1 both as a potential therapeutic target and a key intracellular regulator of metabolism , we set out to thoroughly investigate the activity and regulation of SAMHD1 through a combined virological , structural and biochemical study of the enzyme , both in cells and in vitro .
To determine the regions of SAMHD1 required for restriction activity , a panel of SAMHD1 proteins comprising N- and C- terminal truncations as well as internal deletions was constructed ( Fig 1A ) . The positions at which domains were defined were informed by structural domain boundaries , as they would be more likely to generate stable and active proteins . Expression of each SAMHD1 deletion was assessed by western blotting and only mutants E and H were expressed at lower levels than the full length protein suggesting that most constructs were equally stable in cells ( S1 Fig ) . Each deletion mutant was assessed for its ability to restrict HIV-1 infection of differentiated U937 cells using a single round , two-colour flow cytometry assay . In this assay , U937 cells were first transduced with a bicistronic IRES vector expressing SAMHD1 and YFP and then differentiated . Three days later , differentiated cells were challenged with HIV-1 virus-like particles ( VLP ) expressing GFP , and after a further three days , the percentage of YFP-expressing cells that were infected was measured by flow cytometry . As expected , mutants B and G , that did not contain the HD domain , were unable to restrict HIV-1 infection similar to a catalytically inactive mutant , 206-7AA ( Fig 1B ) . However , not all mutants containing the HD domain were able to restrict HIV-1 infection . For example , mutant A , containing only the HD domain , and mutants C , D and F , containing the HD domain in combination with the NLS or SAM domains or both respectively , did not restrict HIV-1 infection ( Fig 1B , grey bars ) . Only mutants E , H and I , containing both the HD domain and the C-terminal domain ( CtD , residues 584–626 ) , inhibited infection ( Fig 1B , red bars ) . Therefore , in agreement to previous observations [18 , 21] these data demonstrate that both the HD domain and the CtD are required for restriction but that neither the NLS nor SAM domains are necessary . These results were substantiated in stable U937 lines expressing wild type SAMHD1 or mutants F or H ( S2 Fig ) . Restriction by our panel of deletion mutants was also tested in undifferentiated cycling U937 cells using a two-colour assay ( Fig 1C ) . In agreement with previous reports , wild-type SAMHD1 was not able to restrict HIV-1 replication in cycling cells . However , in contrast to a previous observation [19] , none of the deletion mutants , including those with a CtD deletion , were able to restrict replication in these cells either . To test whether the SAM domain influenced the timing of the block to HIV-1 infection , wild type SAMHD1 or domain mutants F ( lacking the CtD ) or H ( lacking the SAM domain ) were introduced into U937 cells . Following differentiation and challenge with HIV-1 VLP , the accumulation of reverse transcription products was measured by qPCR ( Fig 1D ) . These data show that wild-type SAMHD1 inhibits the accumulation of second strand reverse transcription products by approximately 10-fold . Moreover , mutant H also inhibited the accumulation of reverse transcripts to a similar degree , whereas the inactive mutant F did not affect reverse transcription . Together , these data indicate that the SAM domain has no influence on the timing or the magnitude of the SAMHD1-mediated block to HIV-1 infection . We also tested the ability of SAMHD1 to restrict a panel of HIV-1 VLP carrying mutations in reverse transcriptase ( RT ) that reduce the affinity of the enzyme for dNTPs , and therefore reduce RT processivity [27–29] . None of these mutations had a significant effect on viral infectivity . However , all were found to be more sensitive to SAMHD1 restriction ( Fig 1E ) , implying that the affinity of RT for dNTPs correlates with restriction by SAMHD1 . In addition , whilst introduction of SAMHD1 in U937 cells substantially reduced the levels of cellular dNTPs , mutant F , which lacks the CtD , did so but to a lesser extent ( Fig 1F ) . This suggests that mutant F has reduced triphosphohydrolase activity in cells , supporting the proposed correlation between dNTP levels and restriction . Deletion of the CtD region of SAMHD1 , residues 584–626 , ablated its HIV-1 restriction activity in differentiated U937 cells . This region has been shown to promote tetramerisation of the protein [13 , 14 , 21] . Therefore , to examine the effects of this truncation in vitro we compared the solution oligomeric state of SAMHD1 ( 115–626 ) and SAMHD1 ( 115–583 ) following addition of substrates and allosteric regulators using Size Exclusion Chromatography coupled to Multi-Angle Laser Light Scattering ( SEC-MALLS ) . Addition of the allosteric regulator dGTP to SAMHD1 ( 115–626 ) resulted in formation of a ~225 kD species corresponding to a protein tetramer ( Fig 2A ) . In contrast , addition of dGTP to SAMHD1 ( 115–583 ) failed to produce a tetrameric species that was stable under the conditions of the size exclusion chromatography ( Fig 2A ) . Other G-based nucleotides , GTP or ddGTP , were insufficient to induce tetramerisation of SAMHD1 ( 115–626 ) ( S3A Fig ) . However , when GTP was combined with dNTP substrates that also act as AL2 activators [14 , 30 , 31] , a varying amount of tetramer was formed , dependent on the deoxynucleotide employed ( S3B Fig ) . dATP and dGTP support the greatest degree of tetramer formation , TTP is intermediate and dCTP appears to be ineffective . This same rank order has also been reported for the AL2 dNTP binding preference based on the site occupancy of crystal structures [30] . In addition , the GTP/dATP induced tetramer is highly stable ( Fig 2B ) as even though more than 90% of the available dATP is hydrolysed to deoxyadenosine ( dA ) after 5 minutes incubation ( Fig 2C ) , tetramers still persisted for up to 3 hours before the nucleotide-free monomer-dimer equilibrium was re-established . These observations are consistent with chemical crosslinking studies that showed SAMHD1 tetramers persist for hours after dilution of activating nucleotides [31] . Having demonstrated that residues 584–626 are required for stable tetramer formation , a comparison of the steady-state kinetics of dNTP hydrolysis by SAMHD1 ( 115–626 ) and SAMHD1 ( 115–583 ) was undertaken using quantitative real-time measurements of triphosphohydrolase activity [32] . In these experiments , GTP was employed as an AL1 allosteric activator as it is not hydrolysed by SAMHD1 and TTP was used as both an AL2 allosteric activator and a substrate . The concentration dependency of the enzyme rate was then fitted to a steady-state Michaelis-Menten model , to derive the kinetic parameters . These data ( Fig 2D ) , show that both enzymes readily hydrolyse TTP , although SAMHD1 ( 115–583 ) had ~ 10-fold reduced kcat ( 0 . 15 sec-1 compared to 1 . 3 sec-1 ) . Both enzymes had a similar KM ( ~90 μM ) indicating that the active site is competent and the affinity for substrate/transition state is unchanged . Therefore , although stable tetramer formation is not a prerequisite for SAMHD1 catalysis under steady-state conditions , it does support a higher turnover form of the enzyme and this appears necessary for restriction . To identify residues important for substrate binding , catalysis and tetramerisation we determined the co-crystal structures of SAMHD1 ( 41–583 ) and SAMHD1 ( 115–583 ) with the poorly hydrolysable G-based nucleotide analogue , dideoxyguanosine triphosphate ( ddGTP ) . Details of the data collection , structure solution and refinement are presented in Table 1 . SAMHD1 ( 41–583 ) contains the SAM domain however no electron density for the domain was observed , suggesting a degree of flexibly in the linkage between the SAM and HD domain . The CtD is absent in SAMHD1 ( 41–583 ) and SAMHD1 ( 115–583 ) , and only the AL1 activator ddGTP is present , nevertheless , although not stable under the conditions of our MALLS experiments , both structures are tetrameric , made up from a dimer of dimers ( Fig 3A ) . The primary dimer interface is the same as that observed in other ligand-bound SAMHD1 tetramers [13 , 14] and in the apo-stucture [7] . However , the conformation of the tetramer differs , largely due to how the α13 helices at the dimer-dimer interfaces pack against each other . The SAMHD1 ( 41–583 ) tetramer is symmetrical containing two equivalent dimer-dimer interfaces . In each interface , interactions between R372 , D361 and H364 form a hydrogen-bonding network along the whole length of α13 ( Figs 3B , left , and S4A ) . The same α13-mediated dimer-dimer interaction is also observed in the SAMHD1 ( 115–583 ) but in this case , only in one of the dimer-dimer interfaces . In the second interface , only the first three N-terminal turns of α13 pack against each other ( Figs 3B , right , and S4A ) and the side chain of D361 makes an alternative hydrogen bonding interaction with R333 located on α12' of the opposing monomer . This non-equivalence results in a SAMHD1 ( 115–583 ) tetramer with an asymmetric arrangement and importantly also reveals that SAMHD1 tetramers have a degree of conformational flexibility . In SAMHD1 ( 41–583 ) and SAMHD1 ( 115–583 ) the active site contains bound ddGTP and similar to previously described SAMHD1 structures [7 , 13 , 14] , a tightly bound metal ion is co-ordinated by the HD motifs ( H167 , D311 and H206 , D207 ) and the triphosphate moiety of the bound deoxynucleotide . However , comparison with other SAMHD1 tetramer structures crystallised in the presence of a variety of metals [13 , 14] reveals both differences in the nucleotide position and also in the location of the metal centre ( Figs 3C and S4B ) . Specifically , these differences affect how residues R164 and D207 interact with the triphosphate and also how the α , β and γ phosphates of the nucleotide are positioned with respect to the metal ion . Given these variations in active site configuration , the nature of the bound metal was examined by recording anomalous diffraction data from a SAMHD1 ( 115–583 ) -ddGTP crystal at the Fe absorbance edge and by measuring the X-ray fluorescence emission . Inspection of the anomalous-difference electron density ( S5A Fig ) readily identifies a first series transition metal , attributed to Fe . However , the X-ray fluorescence emission spectrum ( S5B Fig ) contains peaks at the Zn and Fe emission energies indicating the presence of both elements , findings also confirmed using inductively coupled plasma mass spectrometry ( ICP-MS ) ( S5C Fig ) . These data reveal the presence of Fe and Zn , in a variety of different SAMHD1 constructs , albeit at varying ratio but with a total stoichiometry of approximately 1:1 metal to protein indicating that both Zn and Fe can occupy the active site . Taken together , these data demonstrate that the SAMHD1 active site can accommodate different metal ions and that the configuration of bound nucleotides is dependent on the type of metal . Moreover , given that Zn is strongly inhibitory to SAMHD1 triphosphohydrolase activity ( S5D Fig ) this supports the notion that differential metal incorporation by SAMHD1 might be a means to regulate SAMHD1 activity and substrate selectivity . In the tetramer structures , as well as occupying the active site , ddGTP was also present at the four allosteric AL1 sites , located at the conserved dimer interfaces . To ascertain if a differential preference for G-based nucleotides in the allosteric site exists , two further structures were determined , one with dGTP included in the crystallisation of an active site mutant SAMHD1 ( 115–583 , R164A ) and the other with GTP and the nucleotide analogue inhibitor 2′ , 3′-didehydro-2′ , 3′-dideoxythymidine ( d4T ) included in the crystallisation of a SAMHD1 ( 115–626 ) construct . Details of the data collection , structure solution and refinement are presented in Table 1 . Both of these crystal forms also contain SAMHD1 tetramers . The SAMHD1 ( 115–583 , R164A ) structure has the same asymmetric conformation observed in the SAMHD1 ( 115–583 ) -ddGTP structure and has dGTP bound at the AL1 allosteric site . Nucleotides are absent from the active site likely as a result of the introduction of the R164A substrate binding mutation . SAMHD1 ( 115–626 ) was crystallised in the presence of both GTP and d4T . The asymmetric unit again comprises four SAMHD1 ( 115–626 ) molecules arranged as two dimers but with only a minimal contact between one pair of adjacent α13 helices . In this structure , GTP occupies AL1 and again the active site is empty . A comparison of the nucleotide configuration at the AL1 site for ddGTP , dGTP and GTP bound structures is shown in Fig 4A . The electron density that nucleotides were built into is presented in S6 Fig . The G base in all three structures makes the same hydrogen bonds with D137 , Q142 and R145 of one monomer along with a stacking interaction with R451' of the opposing monomer . The basic side chains of K116 and R451' also make equivalent electrostatic interactions with the triphosphate of the bound nucleotide illustrating that the binding mode for each of the guanine nucleotides is broadly conserved . Therefore , the capacity of each G-based nucleotide to support tetramerisation when combined with dATP was assessed using SEC-MALLS ( Fig 4B ) . These data show that all G-based nucleotides facilitate tetramer formation but to differing degrees in a rank order of GTP > dGTP > ddGTP . This supports the notion that in a cellular context , where the concentration of GTP is greater than that of any dNTP counterpart , tetramer formation is not limited by the availability of guanine-based nucleotides to occupy AL1 , but instead by the availability and capacity of dNTPs to productively support tetramer formation through occupancy of AL2 . Based upon our observations in the crystal structures , amino acid substitutions were made to disrupt key residues in the active site of the HD domain , the allosteric site and at the dimer-dimer interface . The SAMHD1 mutant proteins were then tested for inhibition of HIV-1 infection using the two-colour restriction assay ( Fig 5 ) . These data show that when residues that co-ordinate the bound iron in the active site ( H167 , H206 , D207 and D311 ) were replaced with alanine , restriction activity was abolished , confirming that SAMHD1 metal co-ordination in the active site is a requirement for restriction . In addition , alanine substitution of residues that make hydrogen bonds with the phosphates of the bound nucleotide ( R164 , H233 , K321 and Y315 ) also abolished restriction activity highlighting the necessity for dNTP substrate binding for restriction activity ( Fig 5A ) . Further alanine substitutions were introduced at allosteric site residues R143 and R145 that are mutated in AGS patients [8] . R145 is involved in guanine base recognition and R143 projects from the allosteric site to the rear of the active site . These alanine mutations also abolished SAMHD1 restriction activity ( Fig 5B ) , demonstrating that allosteric activation of SAMHD1 is a further requirement for restriction . Residue R372 is located at dimer-dimer interface of the tetramer structures and contributes substantially to the hydrogen-bonding network between the adjacent α13 helices ( Fig 3B ) . Therefore , we examined the effects of a charge reversal mutation , R372D , on SAMHD1 restriction and found that this abolished restriction activity ( Fig 5C ) . Moreover , the R372D mutation completely removed the ability of SAMHD1 to form tetramers ( Fig 5D ) and resulted in a loss of SAMHD1 in vitro triphosphohydrolase activity ( Fig 5E ) supporting the notion that SAMHD1 tetramerisation mediated through allosteric activation is a requirement for both SAMHD1 restriction and dNTP hydrolysis . Phosphorylation by CDK2/cyclin A or substitution of T592 with phosphomimetic residues has been shown to prevent HIV-1 restriction by SAMHD1 in cycling and differentiated cells respectively . However , how this affects enzyme activity and dNTP levels is uncertain [19 , 22 , 26] . Our data show that in differentiated U937 cells , alanine substitution at T592 had only a small effect on restriction but that phosphomimetic substitution by aspartate and glutamate completely eliminated the ability of SAMHD1 to restrict ( Fig 6A ) . However , in contrast to Cribier et al . [19] , preventing phosphorylation in cycling U937 cells , by , in our case introducing a T592A alanine mutation , did not rescue SAMHD1 restriction ( Fig 6B ) , suggesting that under these circumstances , inhibiting phosphorylation is insufficient to gain restriction . Given the requirement of the ( 584–626 ) CtD region of SAMHD1 for stable tetramer formation in vitro and the effects of phosphomimetic and alanine mutations on restriction , the contribution of CDK2/cyclin A phosphorylation on SAMHD1 tetramerisation , and triphosphohydrolase activity were investigated in vitro . For these studies SAMHD1 ( 115–626 ) was first in vitro phosphorylated by incubation with CDK2/cyclin A kinase . After treatment , analysis by tryptic digest and tandem mass-spectrometry found only a single site of phosphorylation at T592 ( S7A Fig ) . In addition , the proportion of phosphorylation was assessed using Phos-tag SDS-PAGE showing that only a single new species was produced and that greater than 90% of the SAMHD1 was modified ( S7B Fig ) demonstrating that specific and near stoichiometric phosphorylation of SAMHD1 ( 115–626 ) at T592 was attained . The capacity for phospho ( p ) SAMHD1 ( 115–626 ) to tetramerise upon addition of GTP and dATP was then assessed using SEC-MALLS . These data ( Fig 6C ) show that phosphorylation also resulted in the inhibition of stable tetramer formation , similar to that observed with SAMHD1 ( 115–583 ) and the dimer interface mutant R372D . Given these observations , we determined the crystal structure of pSAMHD1 ( 115–626 ) . The structure ( Fig 6D ) is also tetrameric with the same dimer of dimers conformation mediated by α13 interactions observed in the SAMHD1 ( 115–583 ) and SAMHD1 ( 41–583 ) structures . GTP and dATP were included in the crystallisation condition and GTP occupies AL1 but the active site is empty , likely because of hydrolysis of the dATP . Similar to that observed in the SAMHD1 ( 115–583 ) tetramer in one of the dimer-dimer interfaces , only the first three N-terminal turns of α13 are in close proximity resulting in a tetramer with an asymmetric arrangement . A structural alignment of pSAMHD1 ( 115–626 ) and SAMHD1 ( 115–583 ) tetramers also reveals that although the conformation of dimers are near identical ( 0 . 58 Å rmsd of all Cα ) a small displacement in the positioning of α13-helices ( ~3 Å ) results in a different rotation of one dimer with respect to other between the two tetramers ( Fig 6E ) . The observation of a further alternative dimer-dimer conformation adopted in pSAMHD1 ( 115–626 ) tetramer strengthens the notion of a conformational plasticity within the dimer-dimer-interfaces of SAMHD1 tetramers . Although the pSAMHD1 ( 115–626 ) tetramer includes the CtD region containing the T592 phosphorylation site , no density is observed for residues C-terminal to D583 suggesting that this region of the protein becomes disordered upon phosphorylation . In addition , the pSAMHD1 ( 115–626 ) structure is much more “open” than the structures of unphosphorylated “closed” tetramers [13 , 14] where residues 582–595 comprise a short CtD helix-turn-helix motif ( CtD-HTH ) . In these closed structures residues Q582 and D585 , located in the first helix ( 582–588 ) of the motif , make hydrogen bonds with R528 and Q536 in the β7-β8 loop region of an opposing SAMHD1 dimer and provide further tetramer-stabilising interactions additional to the α13-α13 interface . T592 is located on the second helix ( 591–595 ) where its hydroxyl side chain is hydrogen bonded to the sidechain of D585 as part of the network of interactions that both stabilise and align the helix-turn-helix motif with the opposing β7-β8 loop . In the pSAMHD1 ( 115–626 ) tetramer , residues C-terminal to D583 are disordered , the CtD-HTH is not formed and so these additional tetramer stabilising interactions do not occur . Moreover , modelling of phosphorylation of T592 into closed structures introduces substantial clashes of the phosphate group with residues in the CtD-HTH , especially with the sidechains of D585 and P593 . This gives rise to the notion that T592 phospho-dependent disruption/disordering of the CtD-HTH results in the loss of the additional tetramer stabilising interactions with the β7-β8 loop and that it is these interactions that are required for stable tetramer formation observed by SEC-MALLS . To examine how phosphorylation affects SAMHD1 triphosphohydrolase activity we initially employed steady-state kinetics to compare the activity of SAMHD1 ( 115–626 ) and pSAMHD1 ( 115–626 ) . Surprisingly , these data ( Fig 7A ) revealed that both phospho- and non-phospho forms have comparable kcat and Km for TTP hydrolysis . However , these experiments were conducted under steady-state conditions at “high” substrate and activator concentration . Therefore , in order to better understand how phosphorylation and tetramer disassembly might regulate restriction , an activator-depletion coupled to an “advantageous” substrate experiment was carried out . Here , SAMHD1 ( 115–626 ) and pSAMHD1 ( 115–626 ) were first incubated with GTP and dATP until all substrate dATP was depleted ( Fig 7B ) . Then ddGTP was added and hydrolysis assessed by IEX-HPLC , Fig 7C . ddGTP cannot induce tetramer formation alone ( S3A Fig ) but unlike other ddNTP substrates can be hydrolysed when other dNTP activators occupy AL2 ( S8 Fig ) . Fig 7C shows that whilst SAMHD1 ( 115–626 ) readily hydrolyses the newly added substrate pSAMHD1 ( 115–626 ) is unable to hydrolyse the ddGTP . These data suggest that the capacity to form a stable tetramer allows dNTPs to be retained in the allosteric sites , maintaining the active form of SAMHD1 even when dNTPs are at very low levels . In contrast , phosphorylation of SAMHD1 destabilises tetramer formation resulting in the loss of activating dNTPs and down-regulation of pSAMHD1 activity in a depleted deoxynucleotide environment .
It was the triphosphohydrolase activity of SAMHD1 that was first described to be responsible for restriction of HIV-1 [7 , 15] . However , more recently , it has been reported that SAMHD1 has nuclease activity and that this putative RNase activity is its key antiviral property [20 , 23] . In addition , there are disputes over which domains of SAMHD1 are required for restriction . Here , we demonstrate that both the HD domain and CtD are required for restriction of HIV-1 in differentiated U937 cells expressing SAMHD1 , but that the SAM domain and N-terminal region containing the NLS are dispensable ( Fig 1 ) . In contrast to a recent report [20] , the presence of the SAM domain does not enhance the magnitude or significantly alter the kinetics of restriction ( Fig 1 ) . At the active site , Fe as well as other metals , including Zn , can be co-ordinated ( S5 Fig ) . However , the metal-type affects the configuration of the bound nucleotide ( Fig 3C ) and Zn actually inhibits triphosphohydrolase activity ( S5D Fig ) , suggesting that differential metal binding at the SAMHD1 active site may have a regulatory function . The importance of nucleotide configuration and metal binding for restriction is also apparent as mutations made at both metal-coordinating residues and those required for nucleotide binding all result in a loss of restriction activity ( Fig 5 ) . At the allosteric site , all G-based nucleotides are accommodated and support SAMHD1 triphosphohydrolase activity . Mutation of the AL1 G-recognition residue R145 to alanine or introduction of the AGS mutation R143C that makes hydrogen bonds to both R145 in AL1 and H210 in the active site also both abolish restriction ( Fig 5 ) . These data suggest that both binding of the G-based nucleotide in AL1 and the linkage of active and allosteric sites through the R143 “bridge” are requirements for restriction . These results indicate that inhibition of HIV-1 by SAMHD1 requires the enzyme to be competent for binding both dNTPs and metal ions in the active site and dNTPs at the allosteric site strongly suggesting that restriction is linked to the capacity of SAMHD1 to hydrolyse dNTPs . Further , our data show that HIV-1 with reverse transcriptase ( RT ) mutations that result in a less processive enzyme or increased KM for deoxynucleotide substrates [27–29] are more sensitive to SAMHD1 ( Fig 1E ) . As these mutants have the same genomic RNA they would be expected to have the same sensitivity to an RNase activity , and so these data further support the notion that the SAMHD1 triphosphohydrolase activity limits dNTP availability for RT and that this inhibits viral infection at the time of reverse transcription . Our data show that the CtD of SAMHD1 is required for restriction activity ( Fig 1 ) . Deletion of the CtD abolishes stable tetramer formation in vitro ( Fig 2A ) , although transient tetramers presumably can form as the crystal structures of SAMHD1 ( 1–583 ) and SAMHD1 ( 41–583 ) are tetrameric ( Fig 3A ) . Preventing any tetramer formation by mutation of R372 , a key residue in the primary α13-α13 tetramer interface , also blocks restriction and severely impairs triphosphohydrolase activity ( Fig 5 ) . These data strongly suggest that , in addition to nucleotide binding and hydrolysis , SAMHD1 stable-tetramerisation is also required for restriction . However , although removal of the CtD reduces triphosphohydrolase activity in vitro , by around 10-fold , it is not completely abolished , and the Km for substrates is not strongly affected ( Fig 2D ) , indicating that stable tetramerisation is not a mechanistic requirement for catalysis . These data are supported by the observation that the amount of stable tetramer formed is highly dependent on the identity of the nucleotide occupying AL2 ( S3B Fig ) but regardless , all dNTPs are still hydrolysed at a comparable rate [30] . Again , this suggests that stable tetramerisation is not an absolute requirement for in vitro SAMHD1 triphosphohydrolase activity making its role in restriction unexplained . Recent studies have identified phosphorylation as a means to regulate SAMHD1 activity in cells [19 , 22 , 26] . However , the mechanism of regulation is unclear , as phosphomimetic mutants display no apparent triphosphohydrolase defect in vivo [22 , 26] . Moreover , in the reported structures of non-phosphorylated SAMHD1 tetramers [13 , 14] , the phosphorylation site was remote to both the active and allosteric site of the enzyme . These observations furthered suggestions that SAMHD1 has an alternative activity to dNTP hydrolysis and/or required a co-factor in cells , in order to restrict retroviral replication [22 , 26] . Given the degree of conformational flexibility we observe at the tetramer interface ( Figs 3 and 6 ) combined with our in vitro tetramerisation data , we questioned whether phosphorylation might regulate tetramer formation . We found that introduction of phosphomimetic mutants at T592 abolishes restriction activity in cells and that phosphorylation of residue T592 in vitro inhibits stable tetramer formation ( Fig 6C ) . Moreover , the structure of pSAMHD1 ( 115–626 ) has an open conformation highly similar to that of the C-terminally deleted SAMHD1 structures . However , steady-state kinetic measurements of pSAMHD1 revealed that although T592 phosphorylation prevents stable tetramer formation and blocks restriction , it has surprisingly little effect on SAMHD1 dNTP turnover in vitro ( Fig 7A ) . This could suggest that triphosphohydrolase activity is not linked to restriction and that tetramerisation is required for an alternative mechanism . SAMHD1 oligomers including tetramers have been observed in cells [21 , 25] . However , as the putative RNase activity does not require tetramerisation [23] , and our other requirements for restriction are all linked to dNTP binding , this seems unlikely . Our experiments in vitro were performed under steady state conditions that are likely very different to those in non-dividing differentiated myeloid cells and resting T-cells that have low dNTP levels [6 , 15–17 , 33] . Therefore , we assessed how SAMHD1 activity was affected under conditions of activator-depletion ( Fig 7B and 7C ) where the enzyme response to the environment may be more important than the steady-state rate of catalysis . By using a ddGTP substrate that is hydrolysed only when a dNTP fills AL2 we were able to separate substrate from activator function ( Fig 7C ) . These data reveal that the inability of pSAMHD1 to form a stable tetramer results in a molecule that is catalytically competent at high nucleotide/activator levels ( Fig 7A ) but that is unable to hydrolyse substrate at low activator ( dNTP ) levels ( Fig 7C ) . We therefore conclude that the CtD of SAMHD1 is required to stabilise the tetrameric , active form of the protein by holding it in a closed , tight tetramer form . Phosphorylation of T592 disrupts the CtD , allowing transient , open active tetramers to form but preventing the stabilisation of these tetramers . Removing the CtD also prevents stable tetramer formation and has a more severe effect on the enzyme kinetics . Disruption of the primary α13-α13 dimer-dimer interface has the most severe effect and leads to inability to tetramerise and abolishes triphosphohydrolase activity . Notably , none of our deletion or phosphomimetic SAMHD1 mutants restricted HIV-1 replication in cycling U937 cells ( Figs 1C and 6B ) . Cribier et al . reported that a 576–626 SAMHD1 deletion that removes the phosphorylation site enables SAMHD1 to restrict HIV-1 in cycling cells [19] . However , as well as preventing phosphorylation , we would predict that a 576–626 deletion should prevent SAMHD1 stable tetramerisation as observed with our 583–626 CtD mutant ( Fig 2A ) and therefore would only be functional at high dNTP levels that do not restrict HIV-1 replication . Given all these observations , a unifying model for SAMHD1 restriction of HIV-1 , can be proposed , Fig 8 . Here , we postulate that in the differentiated cellular environment depleted of activating dNTPs , only SAMHD1 variants that can form long-lived , stable tetramers are functionally active due to their ability to retain activating nucleotides in the allosteric site [31] . These stable tetramers are necessary and sufficient for restriction of retroviral replication because they can reduce and maintain the dNTP pool to very low levels below the threshold that HIV requires for reverse transcription . By contrast , in cycling cells , dNTP levels are generally higher because of dNTP synthesis through ribonucleotide reductase activity . In these cells , transient pSAMHD1 tetramers can still hydrolyse dNTPs , but as the pool depletes , activating nucleotides can be lost from AL2 and so the enzyme dissociates to inactive dimers and the dNTP pool is never reduced to very low levels . Such low dNTP levels are difficult to accurately measure against background noise in most assays , possibly contributing to conflicting reports in the field . Improved understanding of the mechanism of SAMHD1 activity and its regulation is crucial as , in addition to inhibiting exogenous , and potentially endogenous , viral replication SAMHD1 is an important regulator of intracellular dNTP levels [34] . Our findings therefore have wide-ranging implications in these fields and may also inform the mechanistic pathogenesis of AGS .
For SAMHD1-YFP expression plasmids , a full-length codon-optimised SAMHD1 sequence was amplified from pLgatewaySN_SAMHD1 [7] , sub-cloned into pENTR/D/TOPO using the pENTR Directional Cloning Kit and transferred into pLgatewayIRESEYFP using Gateway LR Clonase II Enzyme mix ( Invitrogen ) . Mutants A , B , D , F and I were amplified from pLgateway_SAMHD1IRESYFP using primers listed in the S1 Table . PCR reactions contained 0 . 5 μM forward and reverse primers , 200 μM dNTP , 1X Phusion HF buffer , 10 ng template and 1 Unit Phusion High-Fidelity DNA Polymerase ( NEB ) . Cycling conditions consisted of initial denaturation at 98°C for 30 sec , 98°C for 10 sec , 70°C for 30 sec , 72°C for 1 min ( for 35 cycles ) and final extension of 10 min at 72°C . Inserts were cloned into pLgatewayIRESEYFP as above . Mutants C , G and H were made using Quikchange II Site-directed Mutagenesis Kit ( Stratagene ) according to the manufacturer’s instructions using pLGateway_SAMHD1IRESYFP as a template for G and H , F for C , H for E and the primers listed in the S1 Table . For puromycin resistance vectors , full-length SAMHD1 and mutant F were amplified from pLgatewaySN_SAMHD1 , using primers listed in the S1 Table , and cloned into pCMS28 [35] using restriction digest with XhoI and EcoRI or HpaI , and ligation . Mutant H was created from pCMS28_SAMHD1 as above . Point mutations were created in full-length pLgateway_SAMHD1IRESYFP and Reverse Transcriptase mutations were created in p8 . 91 [36] using the Quikchange II Site-directed Mutagenesis Kit ( Agilent technologies ) . RT mutant 114S was a gift from S . Okura . SAMHD1 expression in transduced cells was analysed by immunoblotting using 1/500 anti-SAMHD1 3295 ( in house ) followed by 1/5000 anti-rabbit IRDYE800 ( Tebu Bio ) , and imaged on an Odyssey Infrared Imager ( Licor ) . 293T and TE671 cells were maintained in DMEM ( Invitrogen ) and U937 cells in RPMI +[L]-Glutamine ( GIBCO ) , each supplemented with 10% heat inactivated foetal calf serum ( Biosera ) and 1% penicillin/streptomycin ( Sigma ) . U937 cells stably expressing SAMHD1 variants were prepared by transduction with MoMLV-based Puromycin-expressing VLPs , followed by selection with puromycin at 10 μg/mL . MoMLV-based YFP-expressing VLPs were made by cotransfecting 293T cells with pVSV-G ( gifted from D . Lindemann ) , pKB4 [37] and pLGateway_SAMHD1IRESYFP ( wild-type or mutants ) , harvesting 48 hr post-transfection . HIV-1GFP was produced by cotransfection of pVSV-G , p8 . 91 and pCSGW [38] . MoMLV-based Puromycin-expressing VLPs were made by cotransfection of pKB4 , pVSV-G and pCMS28_SAMHD1 ( wild-type or mutants ) . VLPs were titred on TE671 or 293T cells for normalisation prior to infection . Viruses with mutations in Reverse Transcriptase were normalised by HIV-1 p24 ELISA ( Perkin Elmer ) . U937 cells ( 1 x 106 ) were transduced by spinoculation in a 24 well plate at 1 , 700 rpm ( Sorvall Legend RT 75006441L ) for 90 min with 0 . 5 mL normalised virus in the presence of 10 μg/mL polybrene ( to give approximately 30% transduction ) . Cells were transferred to a 6 well plate and 1 . 5 mL RPMI added . 3 days later , cells were passaged 1/3 , differentiated with 100 nM Phorbol 12-myristate 13-acetate ( PMA , Sigma ) for 72 hours and then infected with HIV-1GFP ( ~10 ng CA ) in fresh media . For experiments using cycling cells , the differentiation step was omitted . Restriction was assessed after 72 hours by 2-colour flow cytometry using BD LSR II or BDVERSE flow cytometers using single colour controls and automatic compensation [39] . Restriction was calculated by dividing the percentage of transduced ( YFP +ve ) cells that were infected ( GFP +ve ) by the percentage of non-transduced cells that were infected . Positive ( wild-type SAMHD1 ) and negative ( HD206-7AA catalytic site mutant ) controls were included for each experiment . Statistical differences between wild type and mutants were determined using the Mann-Whitney test ( Dunn’s multiple comparisons test for reverse transcriptase mutants ) . U937 cells ( 4 x 106 ) were transduced by spinoculation at 1 , 700 rpm ( Sorvall Legend RT 75006441L ) for 90 min with 0 . 5 mL concentrated virus in the presence of 1 μg/mL polybrene ( to give >80% transduction ) . 3 days later , cells were plated at 3 . 8 x 106 cells per well of a 12 well plate in the presence of 100 nM PMA . After 72 hours cells were chilled at 4°C for 30 min and infected with DNase-treated HIV-GFP ( 10 ng CA ) at 4°C for 30 min and transferred to 37°C ( time 0 ) . Cells were harvested at the indicated times post-infection by removing media , washing with ice-cold PBS , trypsinising , resuspending in ice-cold PBS , pelleting at 2000 xg for 2 min at 4°C and flash-freezing on dry ice . DNA was isolated using the DNeasy Blood & Tissue Kit ( Qiagen ) . Eluted DNA was treated with DpnI for 1 hour and quantitation of strong stop and second strand PCR products was determined by qPCR using the primers detailed in S2 Table . PCR reactions contained 9 μM forward and reverse primers , 2 . 5 μM probe , 1X Taqman Fast Advanced Mastermix and 2 μL DNA . Reactions were carried out in a 7500 Fast Real-Time PCR System ( Applied Biosystems ) using standard reaction conditions . Cellular deoxynucleoside triphosphates were extracted from batches of 4x106 differentiated native and SAMHD1 transduced U937 cells according to [40] . The dNTP levels were quantified by radiolabel incorporation assays performed using oligonucleotide templates detailed in [41] and the procedures described in [42] with the following modifications . Standard curves ranged from 0 . 05 to 4 pmole , 1 unit of KOD polymerase ( Merck Millipore ) was used in place of Thermo-Sequenase ( GE Healthcare ) and 2 . 5 μM of α-32P-dATP was employed as an incorporation label . For expression in E . coli the DNA sequences coding for human SAMHD1 residues M1-M626 , M115-M626 , P26-M626 , M115-D583 and D41-D583 were amplified by PCR and inserted into a pET52b expression vector ( Novagen ) using ligation independent cloning to produce N-terminal StrepII-tag fusion proteins . The M115-D583 ( R164A ) and M1-M626 ( R372D ) mutants were prepared from the parent construct using the Quikchange II kit . All insert sequences were verified by DNA sequencing . Strep-tagged SAMHD1 constructs were expressed in the E . coli strain Rosetta 2 ( DE3 ) grown in Luria broth at 37°C with shaking . Protein expression was induced by addition of 0 . 1 mM IPTG to log phase cultures ( A600 = 0 . 5 ) and the cells incubated for a further 20 h at 18°C . Cells were harvested by centrifugation resuspended in 30 ml lysis buffer ( 50 mM Tris-HCl pH 7 . 8 , 500 mM NaCl , 4 mM MgCl2 , 0 . 5 mM TCEP , 1x EDTA-free mini complete protease inhibitors ( Roche ) , 0 . 1 U/ml Benzonase ( Novagen ) per pellet of 1 L bacterial culture and lysed by disruption in EmulsiFlex-C5 homogeniser ( Avestin ) . The lysate was cleared by centrifugation for 1 h at 48 , 000 xg and 4°C then applied to a 10 mL StrepTactin affinity column ( IBA ) followed by 600 mL of wash buffer ( 50 mM Tris-HCl pH 7 . 8 , 500 mM NaCl , 4 mM MgCl2 , 0 . 5 mM TCEP ) at 4°C . Bound proteins were eluted from the column by circulation of 1 mg of 3C protease ( GE ) in 10 mL of wash buffer over the column in a closed circuit overnight . 3C protease was removed by incubation of the eluent with 500 μL GSH-Sepharose ( GE ) . After centrifugation to remove the resin , the supernatant was concentrated to 5 mL and applied to Superdex 200 16/60 ( GE ) size exclusion column equilibrated with 10 mM Tris-HCl pH 7 . 8 , 150 mM NaCl , 4 mM MgCl2 , 0 . 5 mM TCEP . Peak fractions were concentrated to approximately 20 mg/mL and flash-frozen in liquid nitrogen in small aliquots . Prior to crystallisation , protein samples were diluted to 5 mg/mL with gel filtration buffer , supplemented with 1 mM of the appropriate nucleotide ( Jena Bioscience ) . Crystals were produced by hanging drop vapour diffusion at 18°C using an Oryx crystallisation robot ( Douglas instruments ) and 2 μL droplets containing an equal volume of the protein/nucleotide solution and mother liquor . Crystals of SAMHD1 ( 41–583 ) -ddGTP were obtained from hanging drops containing 160 mM succinic acid , 11% PEG 3350 , pH 7 . 0 . Crystals of SAMHD1 ( 115–583 ) -ddGTP were obtained from drops containing 100 mM Bis Tris propane-HCl , 150 mM Na2SO4 , 13 . 5% PEG 3350 pH 6 . 5 . Crystals of SAMHD1 115–583 R164A-dGTP were obtained from drops containing 0 . 2 M sodium citrate , 0 . 1 M Bis Tris propane-HCl , 20% PEG 3350 , pH 8 . 5 and Crystals of SAMHD1 ( 115-626-d4T/GTP ) were obtained from drops containing 0 . 2 M ammonium sulphate and 20% PEG 3350 . Crystals of pSAMHD1 ( 115–626 ) were obtained from drops containing 0 . 2 M sodium formate and 20% PEG 3350 with 25 mM dATP and GTP included in the crystallisation condition . Details of crystallographic spacegroups , cell dimensions and contents of the asymmetric unit are presented in Table 1 . For data collection , crystals were adjusted to 25% glycerol and flash frozen in liquid nitrogen . Datasets were collected on beamline I04 at the Diamond Light Source , UK at a wavelength of 0 . 97949 Å or 0 . 92 Å with the exception of the SAMHD1 115-583-ddGTP dataset , which was collected at a wavelength of 1 . 735 Å , corresponding to the Fe absorption edge . Data were reduced using the HKL [43] or XDS [44 , 45] software suites . Structures were solved by molecular replacement using the program MOLREP [46] implemented in the CCP4 interface [47] using the structure of apo-SAMHD1 120–626 as search model ( PDB code 3U1N , [7] ) . For the SAMHD1 ( 115–583 ) crystal , iterative model building with the program Coot [48] combined with positional , real-space , individual B-factor and TLS refinement in phenix . refine [49] produced a final model for SAMHD1 residues 115–276 , 282–530 , 538–583 ( chain A ) ; 115–277 , 282–506 , 513–530 , 547–583 ( chain B ) ; 115–276 , 284–532 , 538–583 ( chain C ) ; 115–276 , 284–506 , 512–530 , 547–582 ( chain D ) with R-/Rfree-factors of 16 . 49%/22 . 28% . In the model , 97 . 98% of residues have backbone dihedral angles in the favoured region of the Ramachandran plot , a further 1 . 74% are in the allowed regions and 0 . 28% are outliers . For the SAMHD1 ( 41–583 ) crystal , iterative model building with Coot combined with positional , real-space , grouped B-factor , TLS refinement and inclusion of reference restraints from the higher-resolution SAMHD1 115–583 model in phenix . refine produced a final model for SAMHD1 residues 110–276 , 285–505 , 517–531 , 547–582 ( chain A ) ; 113–277 , 287–463 , 466–506 , 517–529 , 548–583 ( chain B ) with R-/Rfree-factors of 20 . 94%/26 . 71% . In the model , 98 . 48% of residues have backbone dihedral angles in the favoured region of the Ramachandran plot , 1 . 28% fall in the allowed regions and 0 . 23% are outliers . For the SAMHD1 ( 115–583 ) -R164A crystal , iterative model building with Coot combined with positional , real-space , individual B-factor and TLS refinement produced a final model for SAMHD1 residues 115–276 , 284–505 , 515–530 , 547–582 ( chain A ) ; 115–276 , 285–505 , 547–582 ( chain B ) ; 115–276 , 283–483 , 490–507 , 514–530 , 539–542 , 547–582 ( chain C ) ; 115–276 , 285–505 , 547–582 ( chain D ) with R-/Rfree-factors of 15 . 62/23 . 44 . In the model , 97 . 8% of residues have backbone dihedral angles in the favoured region of the Ramachandran plot , 1 . 79% are in the allowed regions and 0 . 42% are outliers . For the SAMHD1 ( 115–626 crystal ) , iterative model building with Coot combined with positional , real-space , grouped B-factor and TLS refinement produced a final model for SAMHD1 residues 115–276 , 285–507 , 513–530 , 547–583 ( chain A ) ; 115–276 , 285–506 , 514–530 , 547–583 ( chain B ) ; 115–276 , 284–506 , 515–530 , 547–583 ( chain C ) ; 115–276 , 284–505 , 514–530 , 547–583 ( chain D ) with R-/Rfree-factors of 17 . 71/24 . 73 . In the model , 97 . 9% of residues have backbone dihedral angles in the favoured region of the Ramachandran plot , 1 . 97% are in the allowed regions and 0 . 12% are outliers . For the pSAMHD1 ( 115–626 ) crystal , iterative model building with Coot combined with positional , real-space , grouped B-factor and TLS refinement in phenix . refine produced a final model for SAMHD1 residues 115–276 , 285–505 , 517–526 , 547–583 ( chain A ) , 115–276 , 285–463 , 468–484 , 491–505 , 516–529 , 547–581 ( chain B ) , 115–276 , 284–394 , 407–460 , 468–479 , 496–505 , 518–522 , 547–580 ( chain C ) , 117–273 , 286–303 , 308–460 , 474–484 , 500–505 , 518–529 , 549–553 , 560–581 ( chain D ) with R-/Rfree-factors of 24 . 39/30 . 82 . In the model , 96% of residues have backbone dihedral angles in the favoured region of the Ramachandran plot , 4% are in the allowed regions and 0% are outliers . Structural alignment and superposition was undertaken using SSM [50] and LSQMAN [51] . Protein structure figures were prepared using PYMOL [52] . The coordinates and structure factors of SAMHD1 ( 41–583 ) -ddGTP SAMHD1 ( 115–583 ) -ddGTP , SAMHD ( 115–583 ) -R164A-dGTP , SAMHD1 ( 115–626 ) -d4T-GTP and pSAMHD1 ( 115–626 ) -GTP have been deposited in the Protein Data Bank under accession numbers 5ao0 , 5ao1 , 5ao2 , 5ao3 and 5ao4 respectively . Size Exclusion Chromatography coupled to Multi-Angle Laser Light Scattering ( SEC-MALLS ) was used to determine the molar mass composition of SAMHD1 samples upon addition of deoxynucleotide/nucleotide substrates and activators . For the time course assessing tetramer stability , samples were incubated at 4°C for the specified time after the addition of nucleotide substrates and activators . Samples ( 100 μl ) were applied to a Superdex 200 10/300 GL column equilibrated in 20 mM Tris-HCl , 150 mM NaCl , 5 mM MgCl2 , 0 . 5 mM TCEP and 3 mM NaN3 , pH 8 . 0 , at a flow rate of 0 . 5 ml/min . The scattered light intensity and protein concentration of the column eluate were recorded using a DAWN-HELEOS II laser photometer and an OPTILAB-TrEX differential refractometer ( dRI ) ( dn/dc = 0 . 186 ) respectively . The weight-averaged molecular mass of material contained in chromatographic peaks was determined using the combined data from both detectors in the ASTRA software version 6 . 1 ( Wyatt Technology Corp . , Santa Barbara , CA ) . Inductively coupled plasma mass spectrometry ( ICP-MS ) was employed to determine the identity of the metal ions bound by SAMHD1 . Samples were prepared for ICP-MS by extensive dialysis against 20 mM Tris pH 8 . 0 , 150 mM NaCl , 5 mM MgCl2 and 2 mM TCEP and the final protein concentration adjusted to 20 μM . Samples ( 100 μl ) were then added to 3 mL of 2% HNO3 and quantitative transition metal analysis carried out using an Agilent 7500cx Inductively Coupled Plasma Mass Spectrometer ( Manchester Analytical Geochemistry Unit , University of Manchester ) . External calibration was accomplished using standards with concentrations of 5 , 10 , 50 , 100 and 200 μg/L . Calibration standards were prepared immediately prior to analysis by dilution of concentrated multi-element stock solutions with 2% sub-distilled HNO3 in 18 MΩ deionised water . 3 μM SAMHD1 was incubated with 0 . 2 mM GTP activator and 0 . 5 mM dATP substrate in either Reaction buffer , 20mM Tris-HCl , 150 mM NaCl , 5 mM MgCl2 , 2 mM TCEP ( pH 7 . 5 ) or Reaction buffer including 10 μM ZnSO4 . Reactions were allowed to proceed and samples withdrawn at timed intervals up to 3 minutes and terminated by 10 fold dilution into 18 . 0% acetonitrile , 25mM Tris-HCl , 1 mM EDTA pH 8 . 0 . For assessment of SAMHD1 hydrolysis of ddNTP substrates 3 μM SAMHD1 was incubated with 0 . 2 mM GTP/dATP activators together with 1 mM ddNTP substrates . In activator-depletion experiments 3 μM SAMHD1 ( 115–626 ) or pSAMHD1 ( 115–626 ) was first incubated with 0 . 05 mM GTP activator and 0 . 2 mM dATP in Reaction buffer until all substrate dATP was depleted ( 5 minutes ) . After the depletion phase 0 . 5 mM ddGTP was added and samples of the time-course of hydrolysis withdrawn and terminated as above . The nucleotide hydrolysis reactions were analysed by anion exchange HPLC using a DNA-PAC100 ( 4 x 50mm ) column ( Dionex ) . The column was equilibrated at 30°C at 1 mL/min in 25mM Tris-HCl , 0 . 5% acetonitrile pH 8 . 0 ( Buffer A ) . Samples of the reaction ( 2 nmole ) were eluted with a five-minute isocratic phase of Buffer A followed by linear gradient of 0 to 240 mM NH4Cl over 12 minutes . Absorbance data from the column eluent was continuously monitored between 200-400nm ( 2 nm interval ) using MD-2010 photodiode array detector ( JASCO ) . Peak integration of the absorbance data recorded at 260 nm was used to quantify the amount of substrate and products at each time point of the reaction . To obtain quantitative , time-resolved information and kinetic parameters for SAMHD1 nucleotide hydrolysis , a coupled assay was employed utilising the biosensor MDCC-PBP [53 , 54] to measure phosphate release from combined SAMHD1 triphosphohydrolase and S . cerevisiae Ppx1 exopolyphosphatase activity in real time , as described previously [32] . In a typical experiment , solutions containing SAMHD1 constructs , Ppx1 , MDCC-PBP and GTP were incubated for 5 min in assay buffer ( 20 mM Tris pH 8 . 0 , 150 mM NaCl , 5 mM MgCl2 and 2 mM TCEP ) at 25°C before the reaction was initiated by the addition of substrate ( TTP ) . The final concentrations were 100 nM SAMHD1 , 10 nM Ppx1 , 40 μM MDCC-PBP , 0 . 2 mM GTP and varying concentration of TTP . The fluorescence intensity was recorded at 430 nm excitation and 465 nm emission over a period of 10–30 min in a Clariostar multiwall plate reader ( BMG ) . Steady state rates were obtained from time courses of Pi formation by linear regression of the data points in the linear phase of the reaction . Rates were divided by the SAMHD1 concentration and plotted versus substrate concentration . Apparent dissociation constants for substrate binding ( KM ) and catalytic constants ( kcat ) were then determined by nonlinear least squares fitting using either a hyperbolic or a Hill-function . All measurements were performed in duplicate or triplicate . SAMHD1 was in vitro phosphorylated using preassembled human CDK2/cyclin A complex [55] . In a phosphorylation reaction SAMHD1 ( 115–626 ) was incubated at a ratio of 75:1 ( w/w ) with CDK2/cyclin A in 10 mM Tris-HCl pH 7 . 8 , 150 mM NaCl , 4 mM MgCl2 , 0 . 5 mM TCEP and 2mM ATP . The reaction was initiated by the addition of SAMHD1 ( 115–626 ) and incubated for 6 hours at 4°C . The target for phosphorylation was threonine 592 and this was confirmed as the sole site of phosphorylation by in-gel trypsin digestion combined with MALDI-TOF mass spectrometry . The level of phosphorylation was also quantified by applying pre and post-phosphorylated samples to 6% ( w/v ) acrylamide SDS PAGE gels supplemented with 50 μM Phos-tag [56] and Mn2+ ( Wako , Japan ) . | SAMHD1 is a restriction factor that blocks infection of certain immune cells by HIV-1 . It was discovered to be an enzyme that catalyses the breakdown of dNTPs , suggesting that it inhibits HIV-1 replication by reducing cellular dNTP pools to such low levels that reverse transcriptase cannot function . However , recently , alternative mechanisms have been proposed . SAMHD1 is also regulated by phosphorylation , although the effects of phosphorylation on protein function are unclear . In order to address these issues , we carried out combined structural and virological studies and have demonstrated that in addition to allosteric activation and triphosphohydrolase activity , restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers . Disrupting the tetramer in various ways always abolished restriction but had differing effects on enzyme activity in vitro . SAMHD1 phosphorylation also prevented restriction and tetramer formation but without affecting enzyme catalysis under steady-state dNTP conditions . However phosphorylated SAMHD1 was unable to catalyse dNTP turnover at very low nucleotide levels that more accurately represent conditions in the cells in which restriction takes place . Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity and substantiate that degradation of dNTPs by SAMHD1 is sufficient to restrict HIV-1 . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [] | 2015 | Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction |
Zoonotic cutaneous leishmaniasis has long been endemic in Israel . In recent years reported incidence of cutaneous leishmaniasis increased and endemic transmission is being observed in a growing number of communities in regions previously considered free of the disease . Here we report the results of an intensive sand fly study carried out in a new endemic focus of Leishmania major . The main objective was to establish a method and to generate a data set to determine the exposure risk , sand fly populations' dynamics and evaluate the efficacy of an attempt to create "cordon sanitaire" devoid of active jird burrows around the residential area . Sand flies were trapped in three fixed reference sites and an additional 52 varying sites . To mark sand flies in the field , sugar solutions containing different food dyes were sprayed on vegetation in five sites . The catch was counted , identified , Leishmania DNA was detected in pooled female samples and the presence of marked specimens was noted . Phlebotomus papatasi , the vector of L . major in the region was the sole Phlebotomus species in the catch . Leishmania major DNA was detected in ~10% of the pooled samples and the highest risk of transmission was in September . Only a few specimens were collected in the residential area while sand fly numbers often exceeded 1 , 000 per catch in the agricultural fields . The maximal travel distance recorded was 1 . 91km for females and 1 . 51km for males . The calculated mean distance traveled ( MDT ) was 0 . 75km . The overall results indicate the presence of dense and mobile sand fly populations in the study area . There seem to be numerous scattered sand fly microsites suitable for development and resting in the agricultural fields . Sand flies apparently moved in all directions , and reached the residential area from the surrounding agricultural fields . The travel distance noted in the current work , supported previous findings that P . papatasi like P . ariasi , can have a relatively long flight range and does not always stay near breeding sites . Following the results , the width of the "cordon sanitaire" in which actions against the reservoir rodents were planned , was extended into the depth of the agricultural fields .
Zoonotic cutaneous leishmaniasis ( ZCL ) has long been endemic in Israel [1] . Two Leishmania species cause ZCL in Israel: Leishmania major , which is transmitted by Phlebotomus ( Phlebotomus ) papatasi ( Scopoli ) , and Leishmania tropica , which is transmitted by Phlebotomus ( Paraphlebotomus ) sergenti Parrot and Phlebotomus ( Adlerius ) arabicus Theodor ( Diptera: Psychodidae: Phlebotominae ) [1–7] . The rodent reservoirs of L . major are sand rats ( Psammomys obesus Cretzschmar ) , jirds ( Gerbillus dasyurus Wagner , Meriones crassus Sundevall and Meriones tristrami ( Thomas ) and perhaps also voles [8–11] , whereas rock hyraxes ( Procavia capensis Pallas ) are considered animal reservoirs of L . tropica in Israel [12] . The reported incidence rates of ZCL in Israel increased from 0 . 4/100 , 000 population in 2001 to 4 . 4/100 , 000 population in 2012 [13] . Endemic transmission has been observed in a growing number of communities in regions previously considered free of the disease [11 , 13—14] . This situation and the lack of effective control methods led the Ministry of Environmental Protection and the Ministry of Health in 2012 , to propose that the Israeli Government implement a pilot experimental intervention program . The Ministerial Committee for Social and Economic Affairs approved the program outline in August 2012 . Funds were allocated to increase public awareness in 50 communities , to carry out experimental environmental intervention activities in 15 communities , to monitor the effectiveness of the intervention activities and for research . Activities under the program started in 2013 . Urim , a collective community ( kibbutz ) located in the Western Negev , was chosen as one of the fifteen communities , following a ZCL outbreak as a result of L . major . The outbreak started in 2010 with annual reported incidence rate of 103 . 1/1000 population and continued in 2011 and 2012 with reported incidence rates of 78 . 4 and 24 . 7/1000 , respectively ( Division of Epidemiology , Ministry of Health , Jerusalem , Israel ) . Many active M . tristrami burrows were observed on a man-made embankment surrounding the residential area . This observation and the prevailing view that sand flies progress in short hops and do not disperse far from breeding sites [15] led to the assumption that the embankment is the main source of Leishmania-bearing P . papatasi females threatening the residents of Urim . Thus , the original intervention plan was to protect the residential area by clearing the embankment from jirds to create a narrow "cordon sanitaire" . Sand fly monitoring was designed to study the spatial and temporal distribution of sand flies and Leishmania infections in sand flies and to assess from which directions and what distance sand flies reach the residential area . The monitoring was also intended to verify the likelihood of success of the planned activities against the rodents . The current work summarizes the sand fly data collected throughout the intensive sand fly surveillance efforts in and around Urim during the summer of 2013 . The marking experiment was designed to study the dispersal distances of sand flies by calculating the middle travel distance and not relying only on maximal travel distances . The results have changed the view on the dispersal of sand flies in the area and led to the expansion of the "cordon sanitaire" in which activities against the reservoir should be taken .
Urim ( 31°30N , 34°52E ) is located in the semi-arid plains of the northwestern Negev of Israel ~25 km west of Be'er Sheva and ~15 km east of the Palestinian Gaza Strip ( Fig 1 , small map ) . The summer ( May to October ) is hot with mean daily temperatures of 32–34°C , often reaching 40°C , and mean night temperatures around 20°C . The coldest months are December to February , with maximum mean daily temperatures of 17°C and mean night temperatures of 7°C . The rainy season is between November and April , mean annual rainfall is around 250 mm . The wind regime in the area is characterized by northwest winds in the late afternoon , replaced by southeast winds during the course of the night [16] . Urim is a small cooperative community ( kibbutz ) with around 500 residents . The residential area ( ~0 . 5km2 ) includes on the west side , small family homes surrounded by communal irrigated gardens ( Fig 1 , purple line ) and on the eastern side public and farm buildings ( Fig 1 , black line ) . A boundary zone ~50-100m wide and ~2 . 6km long ( Fig 1 , green line ) separates the residential area from the surrounding agricultural fields . The boundary zone includes a man-made embankment , ~4m wide at its base and 2-3m high , built to prevent unauthorized movement of vehicles , a dirt road and a high chain-link fence . The agricultural fields around Urim include mainly grain crops such as wheat and barley are watered by rain . Field crops as potatoes , carrots , sunflowers , peanuts and other vegetables are irrigated . Depending on the crop type and crop rotation schemes , some of the plots are not cultivated between the spring harvest and the autumn . A natural recreation area , Eshkol National Park , is located about one km west of Urim . The national park , ~3 . 5km2 , is characterized by loess badlands and plains with a landscape cover of biogenic crust matrix and patchy shrub distribution [17] . In some parts of the park , man-made contour banks called Shikim [18] were used as a water harvesting system to trap surface runoff water to sustain trees sparsely planted for shade . The National Park is bordered in the east by the agricultural fields of Urim and in the west by the bank of HaBesor stream . Active Tristam's jird burrows were common on the embankment in the perimeter of the Kibbutz . In the farmland , jird burrows were abundant in non-plowed plots and in the narrow wayside by the agricultural roads separating the cultivated fields . Eight out of the twelve ( 67% ) M . tristrami trapped near the embankment in 2011 and 3 out of 13 ( 23% ) trapped in 2013 , were positive by PCR to L . major ( Roni King , Israel Nature and Parks Authority , Jerusalem , Israel ) . Additional reservoir animal species especially Psammomys obesus were not present . Sand flies were collected outdoors between May and December 2013 . Modified CDC traps operated without light powered by two 1 . 2 V AA rechargeable batteries and baited with ~1kg of dry ice , were placed overnight in a vertical position . The openings were parallel to the ground and ~10 cm above it , the fan causing updraft airflow with the collection boxes hanging above the body of the trap . The catch was chilled and kept at -20°C until sorting . All males and samples of females were identified to species . The identification was made by examining the morphology of male genitalia , female spermathecae , and pharynges using the keys of Abonnenc 1972 [19] and Lewis 1982 [20] . In the marking experiment trappings , the numbers of sand flies with visible food dye were noted for each trap and food dye color ( Fig 2 ) . The information on the food dyes and the application method is reported in the section "monitoring dispersal distances" . In catches exceeding ∼500 specimens , sand fly numbers were estimated by dividing the catch into sub-samples of 1/4-1/8 and counting the sand fly numbers in the sub-sample . All the females from small catches and 100–400 females from traps that collected more than 500 specimens were pooled in groups of up to 20 specimens for molecular detection of Leishmania DNA . Engorged females were kept individually for future blood-meal identification . All samples were stored at -20°C until testing . DNA was extracted from the pooled intact sand fly females using the PureLink Pro 96 Genomic DNA Kit ( Invitrogen by life technology , Carlsbad , CA ) following the manufacturer's instructions . To grind the samples , 3mm stainless steel beads and 200μl proteinase K digestion solution were introduced to each tube . Plates of 96 tubes were set in the TissueLyser II ( QIAGEN , Valencia , CA ) . Parameters were set at 30 Hz , 5min . DNA extracts were amplified with the previously described internal transcribed spacer 1 gene ( ITS1 ) primers ITS-219 [19] . The PCR reaction was performed in a total volume of 20μl , containing ( 10 μl ) of AccuMeltHRM SuperMix ( Quanta Bioscience , Gaithersburg , USA ) , 0 . 5 μM of each primer , 0 . 1% BSA ( w/v ) , 5% DMSO ( w/v ) and 3μl DNA , using the 7500 Fast or StepOne Real-Time systems ( Applied Biosystems , California , USA ) . The cycling parameters were 95°C for 5 min; 40 cycles of 95°C for 10 sec; 62°C for 45 sec . Melting curves were generated by the RT-PCR machine software . The florescent signal was measured while raising the temperature to 95°C for 10 seconds , 62°C for 1 minute , and 95°C for 15 seconds ( ramp rate , 1% ) . The high resolution melt analysis ( HRM ) method described by Talmi-Frank et al . 2010 [21] differentiates between Leishmania species . The specificity of the method for the direct identification of Leishmania species was further verified in our lab using DNA extracted from Leishmania cultures . The cultures used included L . tropica ( MHOM/IL/1990/P283 ) , L . major ( MHOM/PS/1967/Jericho II ) , L . donovani ( MHOM/SD/1962/1S-CLD2 ) , L . aethiopica ( MHOM/ET/1972/L102 ) and L . arabica ( MPSA/SA/83/JISH220 ) . The specific melt curves for the five Leishmania species are shown in Fig 3 . The HRM analysis does not distinguish between L . infantum and L . donovani that have almost identical DNA sequence over the amplified 265 bp region [19] . Two samples of L . tropica and two of L . major parasites were included in each DNA extraction as standard reference controls . Samples of the positive identifications were validated by sequencing . One to three sand fly collections were conducted each month . Traps were set at 55 different sites ( Fig 1 ) in four categories of land use: residential area ( 8 ) ; boundary zone ( 12 ) ; agricultural fields ( 25 ) ; Eshkol National Park ( 10 ) . Trapping sites were not associated with rodents borrows which were scattered and not clustered . In the residential area the traps were placed in the communal gardens after receiving consent from the Kibbutz Urim Secretariat . We were able to operate up to 16–24 traps each night , thus we were unable to place traps in all sites throughout the season . However , in each trapping night , three traps were set in fixed locations ( reference sites—marked by black circles , Fig 1 ) . To study the flight distances of sand flies we used sugar bait marking , exploiting the tendency of sand flies to feed on sugar from the surface of plants [22] . Of the trapping sites we chose five that yielded large captures , each located in a different direction and at a distance of 0 . 6–1 . 1 km from the center of the kibbutz ( Fig 4 ) . In each site a patch of vegetation ~200 m2 was sprayed with 20l of solution containing 10% sugar and 0 . 5% of one of five different food dyes ( Brilliant Blue E133 , Red Carmosine E122 , Yellow E-102; Orange E110 , Brilliant Green E102&E133 , Stern , Netanya , Israel ) . The vegetation was sprayed till runoff using a backpack sprayer ( 15Lt 425 , Solo , Newport News , VA , US ) . Stubble of wheat ~30cm high dominated the area sprayed with the green sugar solution south-west of the Kibbutz , dead standing annual plants ~30cm high dominated the sites sprayed with the blue ( north ) red ( north-east ) and orange ( west ) sugar solutions , while dead standing annual plants and perennial shrubs of ~50cm high dominated the site sprayed with yellow ( south ) sugar solutions . The design of the experiment included an attempt to increase the number of marked sand flies by placing two containers each with 1 kg of dry ice without traps , in the middle of each of the five sprayed patches . Using this experimental design , we gave up on the possibility of collecting information on the flying distance of sand flies during one night . In the following two nights we trapped sand flies in 23 locations within a 2km radius of the marking sites: five in the center of each marking site , eight in the boundary zone just inside the surrounding fence and an additional ten in the fields ( Fig 4 ) . Marked sand flies in the catch of the 22 distant traps represented the dispersing population . Marked sand flies in the catch of the trap placed in the center of the marking area were considered non-dispersing . The material collected by the five traps placed in the marking sites was used to estimate sand fly densities , male/female ratios , proportions of specimens marked by the color of the site and to calculate the ratios between dispersing sand flies and non-dispersing sand flies . The maximal number of specimens collected in each trapping site was used to show the spatial distribution of sand flies densities . The average size of the catch calculated for each trapping site and category of land use ( residential , boundary , agriculture and Eshkol National Park ) were used to compare the findings from the four land use categories . Collections of up to 10 specimens were considered small; collections of 11–100 moderate , collections of 101–1000 large and collections of more than 1000 specimens very large . The data from the three reference trapping sites ( indicated by black circles in Fig 1 ) , were used to study the seasonal trend . The level of transmission was monitored by screening "pools" of specimens and calculating the minimal Leishmania infection rate ( MIR ) which assumes that a positive pool contains only a single infected insect [23] . The MIR was calculated as the number of positive pools per 100 females tested . To compare the relative risk that a female sand fly will infect a human with L . major we calculated the Estimated Risk of Exposure Index ( EREI ) . Following Kilpatrick et al . 2005 [24] and assuming consistent anthropophilic tendency and vector competence for the one vector species present , we calculated a simplified EREI . The EREI was calculated by multiplying the proportion of infected females ( number of positive pools divided by the total number of females tested ) by the number of female specimens in the relevant catch . The MIR representing the percent of infected females was calculated for each trapping month , trapping site , and land use category . The EREI representing the number of infected sand fly females per trap was calculated for each month and land use category . The EREI representing the relative number of infected sand fly females in the different land use categories , may indicate the relative contribution of each category to the infected sand fly females capable of transmitting Leishmania parasites to people . To calculate the dispersal of sand flies from each of the five marking sites , we used the data of the sand flies marked by the site color from all the traps ( 22 ) , excluding the one located in the center of the marking site . The trapping area surrounding each of the marking sites was divided into four annuli , separated by 0 . 5 km ( ArcMap 10 . 2 , Esri , Redlands , CA , US ) . The mean distance traveled ( MDT ) was calculated following the method developed by Lillie et al . 1981 [25] and White & Morris 1985 [26] . To account for differences in trap densities a correction factor ( CF ) was calculated for each annulus . CF = ( the area of the annulus × No . of traps per annulus ) / area of trapping . The estimated recapture ( ER ) was calculated for each annulus . ER = ( No . of colored sandflies per annulus × CF ) / No . of traps per annulus . The mean traveled distance was calculated using the formula: MDT = Σ ( ER × median distance ( for each annulus ) ) / Total number of ER ) . MDTs were calculated separately to evaluate the dispersal of females and males from each of the marking patches . The General MDT calculation was done separately for females and males . One-way ANOVA and post hoc Tukey's HSD tests were used to analyze the effect of land use category on sand fly densities . A Kruskal-Wallis test was conducted to test the difference in sex ratio between the land use categories . Sand fly numbers and sex ratios were Box-Cox transformed to meet statistical tests assumptions . Correlation analysis was conducted between total catch per trap and male/female ratio . A difference test for proportion was conducted between every two sites to detect differences in the proportion of marked sand flies and the marking of females and males between the five marking sites . Bonferroni corrections were applied to prevent false positive results caused by multiple comparisons . Correlation analysis was conducted between the non-dispersing sand flies and the dispersing sand flies . Chi-square was used to test whether the ratios between dispersing and non-dispersing sand flies differ between males and females . STATISTICA software , version 12 . 6 ( StatSoft Inc . , Tulsa , Oklahoma , USA ) was used for all the statistical analysis .
A total of 122 , 507 specimens were collected ( females comprising 68% ) , during 14 trapping nights , using a total of 189 traps sampling 55 sites . Catch size varied between 0–3755 , male/female sex ratios in catches of n≥5 were between 0–1 . 48 . No correlation was found between the sex ratio and the catch size ( r ( 46 ) = 0 . 27 , p = 0 . 065 ) . The very few Sergentomyia specimens were discarded . Phlebotomus papatasi was the sole Phlebotomus species found in the trappings . All the male specimens had coxite with very small process near the base , long and cylindrical style with three short terminal spines and two basal spines much closer to each other than to the terminal spines and surstyle with two short spines at the end . The females were characterized by pharyngeal armature of scaly teethes arranged into a wide meshed network restricted to the posterior third of pharynx . The spermatheca were cylindrical with 8–12 rings ( apical segment short ) and the relative lengths of the ascoids on antennal segment IV were less than 0 . 6 [27] . Approximately 20% of the females ( 15 , 023 ) were tested for the presence of Leishmania DNA by PCR . Leishmania DNA was detected in 78/807 pools ( ~10% ) , in 30/55 sites sampled . The MIR was 0 . 51 . The melting curves of all the positive samples from Urim were similar to those of the L . major control samples ( Fig 3 ) , while L . tropica and L . infantum melting curves were identified in simultaneously tested samples from other regions of Israel . The seasonal trend observed was generally similar in the three reference traps ( Fig 5A ) . Sand fly numbers were low in May and June , increased in July peaked in August and September reaching between 1 , 500 and 3 , 500 specimens per trap . Numbers started to decline in October and further declined in November . Traps operated in December collected no sand flies . Leishmania major DNA was detected in pooled sand fly females from all the collection nights , except the collection night in June . Leishmania MIR was relatively low in May and July , increased in August and was high in September and November ( Fig 5B ) . The highest estimated risk of exposure EREI , was in September ( Fig 5C ) , EREI was very low in May & June and moderate in July , August , October & November . The maximal number of sand flies collected in each trapping site and the MIR are presented in Fig 6 . The trapping efforts and the sand fly data according to land use categories are summarized in Table 1 . Traps placed in the area used for residence collected only a few specimens ( 0–5 ) and most were females ( 82% ) . In the boundary zone close to the embankment , females comprised 69% . In most sites ( 9/12 ) , the catch was large ( 114–946 specimens ) . A high proportion of females ( 86% ) and large differences between the trapping sites characterized the sand fly catch in the Eshkol National Park . Catches were moderate in the two trapping sites located on the bank of HaBesor stream , large in seven sites , and very large in a site ( marked by a flag in Figs 1 and 6 ) located amid a Shikim system . Catches were generally large to very large in the agricultural fields and females comprised 67% . The differences between the number of specimens trapped in the four land use categories were significantly different ( F ( 3 , 183 ) = 80 . 6 , p<0 . 01 ) . Post hoc analysis showed significant differences between the land use categories , except between the boundary zone and the Eshkol National Park . Sand fly densities were similar in all directions around Urim , there was no direction with notably higher or lower densities ( Fig 6 ) . The male/female sex ratio differed between the land use categories ( H ( 2 , 173 ) = 26 . 63 , p<0 . 01 ) . Significant difference in sex ratio was found between the Eshkol National Park and both agriculture and boundary areas . On the other hand , no significant difference was found between the agricultural and boundary areas ( Table 1 ) . Leishmania major DNA was not detected in females collected in the residential area . Leishmania MIR was similar in females collected in the boundary zone ( 0 . 43 ) and the agricultural fields ( 0 . 5 ) . The MIR was highest ( 1 . 12 ) in females from the Eshkol National Park area . The EREI indicating the relative contribution of infected sand fly females based on densities and MIR was higher in the Eshkol National Park and the agricultural fields in comparison with the boundary zone ( Table 1 ) . The total number of sand flies captured during the two trapping nights following the spraying of sugar solutions containing food dyes was 38 , 958 . The five traps placed in the marking sites collected 1 , 111–5 , 853 specimens; females comprised 60–87% ( Table 2 ) . The proportion of sand flies that contained visible traces of the site's marking solutions were similar in the material collected in four ( green , blue , red and orange ) of the five marking sites . In these collections the marking rates of the males ( 23–30% ) were significantly higher than those of the females ( 8–15% ) ( red Z = -2 . 96 , p = 0 . 0015; green , blue , orange Z = -3 . 896 , p<0 . 00001 ) . Marking rates in the catch from the fifth site ( yellow ) were significantly higher ( Z = -3 . 896 , p<0 . 01 after Bonferroni correction ) . The marking rates of the males ( 52% ) were slightly higher than those of the females ( 46% ) . The difference was not significant ( Z = -2 . 1 , p = 0 . 143 ) ( Table 2 ) . The distribution of marked sand flies captured in each of the trapping sites , and the annuli used for MDT calculations are presented for each marking color in Fig 7 . The data of the marked sand flies collected by the 22 distant traps , the maximal travel distances and the calculated middle travel distances ( MDTs ) for the females and the males are summarized in Table 3 . The number of dispersing sand flies ( Table 3 ) was positively correlated with the number of the corresponding non-dispersing sand flies ( Table 2 ) ( females r ( 3 ) = 0 . 56 , p<0 . 05; males r ( 3 ) = 0 . 35 , p<0 . 05 ) . Dispersing green sand flies were found in 8/22 traps , blue specimens were found in 2/22 , red in 10/22 yellow in 18/22 and orange in 12/22 . Apart from the blue marking , dispersing sand flies were found in traps placed on the other side of the residential area . For all marking sites , the maximal flight distances were longer for females ( 0 . 48–1 . 91 km ) than for males ( 0 . 18–1 . 53 km ) . For each of the marking colors , marked males were not caught in the corresponding most distant trap . Female sand flies marked by the red , yellow and orange food dyes were found in the collection sites furthest away from the corresponding marking site ( 1 . 88km , 1 . 57km and 1 . 91km ) . Females marked by the green color were found at a distance of 1 . 23km while the most distant trap was about 90 m further away ( 1 . 32km ) . The most distant trap from the blue marking site was at 1 . 52km . However , the very few blue marked sand flies retrieved were found in only two very close traps ( Fig 7E ) . Males marked by all food dyes except the blue and females marked by the green and orange food dyes were collected on the first trapping night ( second night after marking ) in the maximal distance . Females marked by the red and yellow food dyes were collected on the first trapping night very close to the maximal distance . The ratios between dispersing to non-dispersing females were greater ( χ2 ( 4 ) = 102 . 64 , p<0 . 001 ) than the ratios between dispersing and non-dispersing males ( Table 4 ) . The calculated MDTs were 0 . 25–0 . 95km for females and 0 . 25–0 . 93 for males ( Table 3 ) . The presence of only one blue male obviated the calculation of male-MDT for the blue marking . The calculated MDTs for females were slightly greater than those of the males for all the marking colors except the orange . The calculated overall MDT for females ( 0 . 74km ) was somewhat smaller than the general overall MDT calculated for the males ( 0 . 82km ) ( Table 3 ) . The overall MDT calculated for the females and males together was 0 . 75km .
The residence area of Urim is a small "island" of homes and cultivated garden surrounded by a "sea" of farmland dominated by annual crops . Only a few P . papatasi , none infected with Leishmania parasites , were captured in traps placed in the residential area between the homes , while sand flies were abundant in traps placed in the surrounding boundary area and the agricultural fields ( Table 1 , Fig 6 ) . Similar results , i . e . small number of sand flies in traps placed in residential areas and larger numbers in traps placed in neighboring fallow and cultivated areas , were reported from the southern Jordan Valley [29] , the Judean Desert [46] and the central Jordan Valley [47 , 11] . Phlebotomus papatasi is endophilic and anthropophilic species [6] . Therefore , even the low densities observed in traps placed in the built-up areas , were sufficient to cause severe biting nuisance to the residents of Urim throughout the warm season . Sand fly catches in the boundary zone were significantly larger than catches in the residential area but smaller than catches in the fields . The larger catches in the surrounding fields and the relatively small size of the boundary zone compared with the size of the field area do not support the hypothesis that the man-made embankment was the main source of Leishmania-bearing sand fly threat to the residents of Urim . The large numbers of sand flies collected in most trapping sites in the agricultural fields , in all directions and at all distances , indicate the existence of many scattered sources of sand flies . The agricultural environment around Urim in the summer and autumn was a complex patchwork of irrigated and non-irrigated plots in various stages of cultivation separated by agricultural roads with non-cultivated narrow waysides . This environment seemed to offer P . papatasi many niches suitable for development and resting . Development could occur in rodent burrows that were abundant in the non-cultivated plots and waysides . In addition , the deep and relatively cool and humid cracks with cut roots and branches between the large soil clods created by plowing might have contributed suitable breeding sites for P . papatasi regardless of rodent burrows . The potential role of a plowed field as a major breeding source for P . papatasi was noted in studies from the Jordan Valley and Dead Sea areas in Israel [30 , 48] . Positive association between agriculture and irrigation and a high abundance of P . papatasi was described in studies from Jordan [42] and Israel [29] . A different situation was reported from Tunisia . While irrigation had a positive effect on the abundance of the vectors of L . infantum , P . papatasi densities were positively correlated with aridity and the species was more abundant in the non-irrigated areas [37 , 49] . In the Eshkol National Park , differences in sand fly catch size between trapping sites were large ( Table 1 , Fig 6 ) . Sand fly catches were very large only in one site the Shikim ( marked by a flag in Figs 1 and 6 ) . Probably , as in the agricultural fields , higher soil moisture can account for the increase carrying capacity of this specific site for sand fly development . Leishmania infection rates were highest in females captured in the Eshkol National Park ( Table 1 ) indicating a strong association between the vectors and the reservoir animals . Strong association between P . papatasi and rodents in arid and semi-arid natural environments was reported in earlier studies [50] . Low soil moisture was considered the major factor limiting P . papatasi development [3 , 40] . In dry environments , sand fly development mainly occurs in rodent burrows , which provide microhabitats suitable for the development of sand flies [3 , 48] . The lower MIR of the females collected from the agricultural fields ( 0 . 5 ) compared with the high MIR in the Eshkol National Park ( 1 . 12 ) indicate a weaker association between sand flies and rodents . Given the great density of P . papatasi in the agricultural fields , even with relatively low MIR , the number of infective females was rather large as indicated by the risk of exposure ( EREI ) . Spraying the vegetation with sugar solutions containing food dyes has the advantage of marking sand flies in their habitat without interrupting their normal behavior by capture , treatment and release [22] . Additional advantages are the ability to mark a large number of individuals when sand fly densities are high , and the ease with which marked specimens can be separated under a stereoscopic microscope ( Fig 2 ) . The proportions of marked males were higher than the proportions of marked females in the marking sites traps . Schlein 1987 [22] reported similar marking rates of males and females . Marking rates were significantly higher in the southern marking site where sugar solution containing yellow food dye was sprayed on dead annual plants and perennial shrubs ( Table 2 ) . The vegetation in the other four marking sites was lower , sparser and did not include perennial shrubs . Higher feeding rates on denser vegetation and plants that possibly attract sand flies [22] could be expected . The dispersal of sand flies from four out of the five marking sites ( green , red , yellow and orange ) had similar characteristics , in terms of travel distances and distribution . Sand flies marked by these food dyes were found in trapping sites on the other side of Urim and in different directions ( Fig 7A–7D ) . The capture of only a small number of "blue" specimens outside the marking site ( Fig 7E , Table 3 ) was exceptional . This result may reflect the behavior of the local sand fly population around the blue marking site north of Urim , but more likely the result is atypical . The movement of sand flies in all directions and to distances greater than 1km indicated that sugar fed sand flies in the agricultural fields around Urim were remarkably mobile . The lack of clear movement along the wind direction axis northwest—southeast ( northwest in the first part of the night and southeast in the second part [16] ) , indicates that the dispersal of P . papatasi was not assisted by wind . No evidence for wind assisted dispersal , and a high mobility of sand flies were reported for P . ariasi in south of France [51] . The maximal dispersal distance recorded in the present study was 1 . 91km for females and 1 . 53km for males . Marked females were found in the furthest trapping site in three cases and at a distance only slightly shorter than the furthest trap , in the fourth case ( Table 3 ) . As the maximal dispersal range recorded for females in our study was limited by the distances between the marking sites and the most distant traps it is safe to assume that the actual flight range of females was longer . Marked males were not found in the furthest traps in any of the cases . Hence , the results seem to reflect the actual maximal travel distances of male P . papatasi . Yuval et al . 1988 [30] found dispersing P . papatasi in a fallow field at a distance of 400m away from the closest Psammomys obesus burrows . The authors concluded that the dispersal was mainly a female activity , and that dispersal of infected females away from the site of infection may result in transmission of leishmaniasis at considerable distance from the sites of vector reservoir contact . Alexander and Young 1992 [52] cited a publication of Streklova and Kruglov from 1985 reporting a flight distance of up to 4 km for P . papatasi in Uzbekistan . Killick-Kendrick et al . 1984 [51] mentioned that researchers in the Soviet Union concluded that sand flies ( probably P . papatasi ) occasionally travel a distance of 1 . 15km . Maximal travel distance of females P . ariasi in the south of France were 2 . 2 km when the record for males was 0 . 6km [51] . Additional studies on the dispersal of sand flies in the Old World found maximal travel distances of 730m for P . orientalis Parrot , 1936 in Sudan [53] and 289 m for P . longipes Parrot and Martin , 1939 in Ethiopia [54] . The maximal travel distance provides information on the distance a sand fly can reach , but do not provide information on the typical travel distance of the sand fly population . Estimation of the distance at which sufficient numbers of the sand fly population travel and the flight range relevant for epidemiology , disease transmission and control , can be derived from the MDT . The MDT value calculated for P . papatasi around Urim was 0 . 75km . The MDT values for the females were slightly larger or similar to the MDT values calculated for the males in three cases ( green , red and yellow ) and somewhat smaller in the fourth case ( orange ) . The overall MDTs for the females ( 0 . 74km ) and for the males ( 0 . 82km ) were similar . The slightly lower overall MDT calculated for the females was affected by the relatively large number of females marked by the orange food dye and probably do not represent a shorter effective dispersal distance of females . The ratios between dispersing and non-dispersing sand flies were used to compare the dispersing tendency of the females and the males . The higher values observed for females for each of the marking colors imply a greater tendency of females to disperse . The results of the maximal travel distance and the ratios between dispersing to non-dispersing sand flies indicate that females tend to disperse more than males and travel longer distances . Nevertheless the similar MDT results for females and males and a maximal distance of more than 1 . 5km recorded for males suggest that males and not only females disperse , and that males , although tend to disperse somewhat less than females , do not necessarily stay close to breeding sites as indicated in the literature [29 , 30 , 42 , 51 , 55–57] . Marking sand flies in the field using sugar solutions containing food dyes and an integrated methodical monitoring scheme , we were able to collect in a short period of several months , epidemiologically meaningful information . The overall results of the present work indicated the existence of dense and mobile sand fly population around Urim . Scattered over large areas in the agricultural fields there seemed to be numerous development sources and suitable resting sites for sand flies . Sand flies apparently moved in all directions to distances typically of at least 0 . 75km . The risk of exposure to Leishmania-infected sand fly female had a distinct seasonal peak in September . Given the current study results , the "cordon sanitaire" , in which actions against the reservoir rodents had to be carried out , was extended to the agricultural fields . Identifying a season-high risk of infection in September could be used to encourage the residents to enhance the use of personal protection measures at the critical time . The travel distance noted in the current work did not fit the widely-held view according to which sand flies stay near breeding sites and have a short limited flight range . It is possible that the favorable environment of the agricultural fields providing many moist microsites suitable for resting , allowed the optimal expression of the dispersal capacity noted in our study . In addition , the good energetic state of the sugar fed sand flies in our experiment might have contributed to the long travel distances observed . Perhaps in natural habitats and/or when conditions are less favorable the full dispersal capacity is not expressed and travel distances are shorter . We hope that the great importance of the dispersal distances of sand flies to epidemiology and control and the simple marking method used , will lead to additional studies and the consolidation of data-driven hypotheses regarding the effective flight range of medically important sand fly species and the factors affecting it . | Cutaneous leishmaniasis is a dermal disease of public health concern in Israel . The typical skin lesions are caused by single celled Leishmania parasites that are transmitted by the bite of an infected female sand fly . In the last years , the disease has been spreading and emerged in Urim , a small cooperative community , surrounded by agricultural fields in southern Israel . In this area the transmission cycle of leishmaniasis involves rodents as reservoir animals . For the purpose of experimental intervention against the reservoir animals , it was necessary to find out from what areas and distances infected sand flies arrived to the residential areas . The research in 2013 was based on integrated methodical monitoring of sand flies using CO2 baited traps and on marking . The results indicated the existence of dense and mobile sand fly population around Urim . The agricultural fields seemed to be the major source for sand flies . Sand flies apparently moved in all directions to distances typically of at least 0 . 75km . The risk of exposure to Leishmania-infected sand fly females had a distinct seasonal peak in September . The information was used to determine the "cordon sanitaire" in which actions against the reservoir had to be carried out . | [
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] | 2016 | Distribution and Dispersal of Phlebotomus papatasi (Diptera: Psychodidae) in a Zoonotic Cutaneous Leishmaniasis Focus, the Northern Negev, Israel |
COP1 ( CONSTITUTIVE PHOTOMORPHOGENIC 1 ) , a ubiquitin E3 ligase , is a central negative regulator of photomorphogenesis . However , how COP1 activity is regulated by post-translational modifications remains largely unknown . Here we show that SUMO ( small ubiquitin-like modifier ) modification enhances COP1 activity . Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype . SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1 . A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta . Consistently , COP1 activity is reduced in siz1 and the level of HY5 , a COP1 target protein , is increased in siz1 . Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1 , but SIZ1-mediated SUMO modification does not affect COP1 dimerization , COP1-HY5 interaction , and nuclear accumulation of COP1 . Interestingly , prolonged light exposure reduces the sumoylation level of COP1 , and COP1 mediates the ubiquitination and degradation of SIZ1 . These regulatory mechanisms may maintain the homeostasis of COP1 activity , ensuing proper photomorphogenic development in changing light environment . Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase , COP1 , in plants .
Sumoylation is a post-translational modification in which SUMO ( small ubiquitin-like modifier ) peptides are covalently attached to a SUMO consensus motif ( ψKxE/D; ψ a large hydrophobic amino acid residue; K , the acceptor lysine; x , any amino acid; E/D , glutamate or aspartate ) in target proteins through a series of biochemical steps involving activation ( E1 ) , conjugation ( E2 ) , and ligation ( E3 ) enzymes [1 , 2] . SUMO conjugation can be reversed by SUMO-specific proteases [3] . In yeast and metazoans , sumoylation has been implicated in several aspects of cellular functions , including chromatin remodeling , DNA repair , nuclear/cytoplasmic transport , transcription , and the cell cycle [4] . The PIAS [Protein inhibitors of activated STATs ( signal transducer and activator of transcription ) ]-type SUMO E3 ligase , SIZ1 [SAP ( scaffold attachment factor , acinus , PIAS ) , and Miz1 ( Msx2-interacting zinc finger ) ] , regulates abiotic stress responses ( i . e . , responses to heat , cold , drought , and salt stresses ) , hormone signaling ( i . e . , abscisic acid , salicylic acid , and auxin pathways ) , nutrient ( i . e . , phosphate , nitrogen , and copper ) homeostasis , and development ( i . e . , flowering time and female gametophyte development ) in Arabidopsis [5 , 6] . Increasing evidence indicates that the SUMO and ubiquitin systems are tightly associated . Sumoylation antagonizes ubiquitination by competing for acceptor K residues or promotes ubiquitination by recruiting SUMO-targeted ubiquitin ligases ( STUbLs ) to sumoylated substrates in yeast , mammals , and plants [7 , 8] . Moreover , the PIAS family of SUMO E3 ligases positively or negatively regulates ubiquitin ligase activity by SUMO modification of the SUMO-regulated ubiquitin ligases ( SRUbL ) in humans [9 , 10] . In Arabidopsis , SUMO modifications regulate the protein stability of DELLA , ICE1 ( inducer of CBF/DREB1 expression ) , ABI5 ( ABA insensitive 5 ) , MYB30 , NIA1/2 ( nitrate reductase ) , SLY1 ( SLEEPY1 ) and SnRK1 ( Snf1-related protein kinase 1 ) likely by antagonizing or promoting ubiquitination [11–17] . SRUbLs may also exist in plants; however , they remain to be identified . Dark-grown seedlings have an elongated hypocotyl , closed cotyledons , and an apical hook ( skotomorphogenesis ) . In the light , Arabidopsis seedlings undergo photomorphogenesis and exhibit short hypocotyls and open cotyledons with no apical hooks [18] . The ubiquitin E3 ligase COP1 ( CONSTITUTIVE PHOTOMORPHOGENIC 1 ) , a central repressor of photomorphogenesis , mediates the ubiquitination and degradation of positive regulators of photomorphogenesis , such as HY5 ( ELONGATED HYPOCOTYL 5 ) , HYH ( HY5 HOMOLOGUE ) , LAF1 ( LONG AFTER FAR-RED LIGHT 1 ) , HFR1 ( LONG HYPOCOTYL IN FAR RED 1 ) , STH3 ( SALT TOLERANCE HOMOLOG 3 ) /BBX2 and PIL1 ( PHYTOCHROME INTERACTING FACTOR 3-LIKE1 ) [19–24] . SPA ( SUPPRESSOR OF PHYA-105 ) and PIFs ( PHYTOCHROME INTERACTING FACTORs ) interact with COP1 , and enhances its E3 ligase activity [21 , 25 , 26] . CSU1 ( COP1 SUPPRESSOR1 ) , a RING-finger E3 ubiquitin ligase , regulates COP1 homeostasis by ubiquitinating and degrading COP1 in darkness [27] . CSU2 and FIN219 interact with COP1 , and negatively regulate its E3 ligase activity and protein level , respectively [28 , 29] . In response to lights , phyA ( phytochrome A ) , phyB , CRY1 ( crytochrome 1 ) and CRY2 , repress COP1 activity through modulating COP1-SPA1 complex [30–34] , Reduced COP1 activity in the lights causes the accumulation of HY5 and the transcriptomic reprogramming of HY5 target genes [35 , 36] . However , post-translational modifications that regulate COP1 activity are largely unknown . Recent study has revealed that SUMO modification of phyB represses red light signaling , at least partly , through inhibiting interaction between phyB and PIFs [37] . In this study , we demonstrate that SIZ1 negatively regulates photomorphogenesis , at least partly , through promoting COP1 ubiquitin E3 ligase activity by SUMO modification , and that COP1 in turn mediates the ubiquitination and degradation of SIZ1 . Our results reveal a novel regulatory mechanism of COP1 and SIZ1 in photomorphogenesis .
The observation that loss-of-function siz1 mutant seedlings showed a short-hypocotyl phenotype under white light prompted us to evaluate the light responsiveness of siz1-2 . The siz1-2 seedlings exhibited a short-hypocotyl phenotype under darkness and red , blue , and far-red light conditions ( Fig 1A and 1B ) . Expression of ProSIZ1:SIZ1-GFP in siz1-2 plants ( complemented lines referred to as SSG ) [38] suppressed the short-hypocotyl phenotype of siz1-2 under these conditions , indicating that mutation of SIZ1 is responsible for the short-hypocotyl phenotype ( Fig 1A and 1B ) . The short-hypocotyl phenotype of siz1-2 was due to a reduction in cell elongation but not in cell number ( S1 Fig ) . The expression of light-inducible genes , CAB ( CHLOROPHYLL A/B BINDING PROTEIN ) and RBCS ( RUBISCO SMALL SUBUNIT ) [39 , 40] , and a light-repressed gene , PORA ( PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A ) [41] , was up- and down-regulated , respectively , in siz1-2 during the transition from darkness to light , indicating that the regulation is stronger in siz1-2 than in wild type ( Fig 1C ) . In addition to the short-hypocotyl phenotype , siz1-2 seedlings had unfolded apical hooks in darkness ( Fig 1D ) and exhibited more opened cotyledons than did wild-type seedlings in dark and light conditions ( Fig 1E ) . These results suggest that SIZ1 negatively regulates photomorphogenesis . To determine if SIZ1-mediated SUMO1/2 modification is involved in the regulation of hypocotyl elongation , we determined the hypocotyl length of sum1 and sum2 double mutant under darkness and red , blue , and far-red light conditions ( Fig 2 ) . SUMO1 and 2 have redundant functions , and the sum1 sum2 double knockout mutant is embryo lethal [42] . Therefore , we used a viable weak allele , sum1-1 amiR-SUM2 , in which SUMO2 expression is down-regulated by RNAi in the sum1-1 knockout mutant background [43] . Similar to siz1-2 , sum1-1 amiR-SUM2 seedlings exhibited a short-hypocotyl phenotype under dark and light conditions , suggesting that SUMO1/2 modification regulates hypocotyl elongation ( Fig 2 ) . Elevated SA levels in siz1-2 cause dwarfism , and the dwarf phenotype is substantially suppressed by expression of NahG , a bacterial salicylate hydroxylase [44] . To determine if the short-hypocotyl phenotype of siz1-2 is due to increased SA levels , the hypocotyl lengths of five-day-old wild-type , siz1-2 , NahG , and NahG siz1-2 seedlings were compared under darkness and red , blue , and far-red light conditions ( Fig 2 ) . The expression of NahG did not affect hypocotyl elongation , but the siz1-2 and NahG siz1-2 seedlings exhibited an identical short-hypocotyl phenotype in all tested conditions , indicating that the elevated SA level in siz1-2 did not contribute to the short-hypocotyl phenotype . Since siz1-2 seedlings exhibit a weak cop1-like phenotype , we examined whether SIZ1 physically interacted with COP1 to regulate its activity . To do so , we performed a bimolecular fluorescence complementation ( BiFC ) assay . YFP fluorescence signals were detected in the nucleus of N . benthamiana cells that coexpressed COP1-YFPN ( fused with the N-terminal half of YFP ) and SIZ1-YFPC ( fused with the C-terminal half of YFP ) under light ( 1 h light exposure ) and dark conditions ( Fig 3A and S2 Fig ) . Consistently , SIZ1-GFP was coimmunoprecipitated with Myc-COP1 ( Fig 3B ) . These results indicate that SIZ1 physically interacts with COP1 in the nucleus . SUMOplot ( http://www . abgent . com/sumoplot ) analysis predicted the presence of three potential sumoylation motifs [45] ( VK14PD , IK193ED , and WK653SD ) in COP1 ( S3 Fig ) , suggesting that COP1 may be a SUMO substrate . To test this possibility , we performed an in vitro sumoylation assay to determine SUMO modification of COP1 as described previously [15] . Anti-FLAG and anti-SUMO1 antibody detected slow migrating multiple bands above original COP1 protein in the reaction containing SUMO E1 ( His6-SAE1b and His6-SAE2 ) , SUMO E2 ( His6-SCE1 ) , and His6-SUMO1-GG , but not in the reaction lacking His6-SUMO1-GG , suggesting that COP1 is a possible SUMO substrate ( Fig 3C ) . To further confirm COP1 is sumoylated in vivo , we performed an in vivo sumoylation analysis as described previously [38] . We co-expressed Myc-COP1 with FLAG-SUMO1 or FLAG-SUMO1AA ( a conjugation-deficient mutant ) in Arabidopsis protoplasts or N . benthamiana leaves . Myc-COP1 was immunoprecipitated with anti-Myc antibody and the immunoprecipitated proteins were detected with anti-FLAG antibody . Higher molecular weight sumoylated COP1 bands were detected when Myc-COP1 and FLAG-SUMO1 were co-expressed , but not in Myc-COP1 and FLAG-SUMO1AA co-expressing cells ( Fig 3D and S4A Fig ) . Moreover , to confirm that sumoylation of COP1 occurs in planta , we generated an Myc-COP1 overexpression transgenic line ( referred to as 35S-Myc-COP1 ) and performed an in vivo sumoylation analysis . Anti-SUMO1 antibody detected higher molecular weight sumoylated COP1 bands ( Fig 3E ) . Furthermore , we evaluated if sumoylation of COP1 changes in response to light . Since light exposure promotes nucleus to cytosol re-localization of COP1 [46] , we monitored the sumoylation status of COP1 in the nucleus under dark ( 0 h ) and light ( 12 h ) conditions ( Fig 3F ) . Under darkness , sumoylated COP1 bands were detected in the Myc-COP1 and FLAG-SUMO1 co-expressing nuclear fraction , but the sumoylation level of COP1 was substantially reduced in response to 12 h of light exposure . Taken together , these data demonstrate that COP1 is a SUMO substrate , and sumoylation level of COP1 is regulated by light . To test if SIZ1 mediates SUMO conjugation of COP1 , we cotransformed Myc-COP1 and FLAG-SUMO1 into Arabidopsis protoplasts isolated from wild-type or siz1-2 plants , and analyzed the sumoylation status of COP1 . Whereas COP1-SUMO1 conjugate was detected in the wild type , substantially lower levels were present in siz1-2 ( Fig 3G ) , indicating that SIZ1 facilitates the sumoylation of COP1 . The lower levels of sumoylated COP1 in siz1-2 may be due to another SUMO E3 ligase ( s ) that facilitates the residual SUMO modification of COP1 . Alternatively , it is also possible that E1 and E2 contribute to basal levels of COP1-SUMO conjugation , since E1 and E2 alone mediate the sumoylation of COP1 in vitro ( Fig 3C ) . K-to-R substitutions in sumoylation motifs block SUMO conjugation [47] . To elucidate the sumoylation motifs of COP1 , SUMO conjugation of Myc-COP1K14R , Myc-COP1K193R , and Myc-COP1K653R were evaluated in Arabidopsis protoplasts . The K193R substitution blocked COP1-SUMO1 conjugation , but K14R or K653R substitutions did not ( Fig 3H ) . Unfortunately , we could not confirm the effect of K193R substitution in vitro , due to anti-FLAG antibody detected long smear bands above the original purified MBP-COP1K193R-FLAG , which would strongly affect subsequent in vitro sumoylation analysis . COP1-SUMO1 conjugation was also blocked by the K193R substitution in N . benthamiana leaves ( S4B Fig ) . These results indicate that SIZ1 mediates sumoylation of COP1 and that K193 is critical for SUMO conjugation . Since SIZ1 mediates SUMO modification of COP1 and siz1-2 seedlings exhibit a weak cop1-like phenotype ( Figs 1 and 3 ) , we hypothesis that SIZ1-mediated SUMO modification may enhances COP1 activity . To test this possibility , we first determined the effect of sumoylation of COP1 in planta . Myc-COP1 and Myc-COP1K193R ( a non-sumoylated form ) overexpressing Arabidopsis transgenic plants were generated , and two independent lines with similar levels of transgene expression for each construct were selected for further phenotypic analysis ( S6 Fig ) . 35S-Myc-COP1 seedlings exhibited longer hypocotyls than did the wild type under white light conditions ( Fig 4A ) , confirming a previous report [48] . Under 30 μmol m-2 s-1 fluence rate of white light conditions , the hypocotyls of 35S-Myc-COP1K193R seedlings were longer than that of the wild type , but shorter than that of 35S-Myc-COP1 ( Fig 4B ) . Under relatively higher fluence rate of white light conditions ( 50 and 80 μmol m-2 s-1 ) , while 35S-Myc-COP1 seedlings still exhibit longer hypocotyls than wild type , the hypocotyls of 35S-Myc-COP1K193R seedlings were of similar length as those of the wild type , suggesting that non-sumoylated Myc-COP1K193R exhibits lower activity than Myc-COP1 ( Fig 4A and 4B ) . Next , total protein extracts isolated from 35S-Myc-COP1 , 35S-Myc-COP1K193R , Col-0 and cop1-4 seedlings were used as E3s to perform an in vitro HY5 ubiquitination assay as described previously [49 , 50] ( Fig 4C ) . Ubiquitination of HY5 was stronger in the reaction containing protein extracts isolated from 35S-Myc-COP1 than in that containing extracts from 35S-Myc-COP1K193R . We did not detected ubiquitination of HY5 in the reaction containing Col-0 protein extracts due to short expose time for the blot ( Fig 4C ) . Next , we overexpressed COP1 in siz1-2 plants by crossing the COP1 overexpression line , COP1 OE , with siz1-2 ( referred to here as COP1 OE siz1-2 ) , and tested if the siz1 mutation reduces COP1 OE effect on hypocotyl elongation . Overexpression of COP1 causes a long-hypocotyl phenotype under light conditions ( Fig 4D and 4E ) [48] . Consistent with our hypothesis , the hypocotyl length of COP1 OE siz1-2 was substantially shorter than that of COP1 OE , suggesting that COP1 activity is reduced in siz1-2 ( Fig 4D and 4E ) . Finally , we compared the COP1 ubiquitin E3 ligase activity of Col-0 and siz1-2 . Total protein extracts from the wild type mediated the ubiquitination of HY5 more efficiently than did those from siz1-2 ( Fig 4F ) . Collectively , these results demonstrate that SIZ1-mediated SUMO conjugation promotes COP1 E3 ubiquitin ligase activity . Furthermore , genetic interaction between COP1 and SIZ1 was analyzed . The hypocotyl length of cop1-4 [51] was shorter than that of the wild type and siz1-2 under dark and light conditions ( Fig 4G and 4H and S7 Fig ) . The cop1-4 siz1-2 double mutant exhibited a short-hypocotyl phenotype similar to cop1-4 in both dark and light conditions , suggesting that SIZ1 regulates hypocotyl elongation partly through COP1 ( Fig 4G and 4H and S7 Fig ) . SIZ1 enhances COP1 activity ( Fig 4 ) and COP1 mediates ubiquitination and degradation of HY5 [19] . Therefore , we tested if the protein level of HY5 is up-regulated by mutation of SIZ1 . Anti-HY5 antibody [52] revealed that endogenous HY5 was more abundant in siz1-2 than in the wild type under light conditions ( Fig 5A ) . Consistent with previous report [19] , higher level of HY5 was accumulated in cop1-4 , and the HY5 level in cop1-4 was similar to that of cop1-4 siz1-2 , suggesting that higher level of HY5 in siz1-2 is possibly due to reduced COP1 activity rather than increased transcription of HY5 in siz1-2 ( Fig 5A ) . Consistent with this hypothesis , the level of HY5 expression was similar in the wild type and siz1-2 ( Fig 5B ) . These results indicate that SIZ1 negatively regulates HY5 protein abundance . Consistent with the increased HY5 level in siz1-2 , the expression level of the cell elongation-related direct target genes of HY5 , i . e . , EXP2 ( EXPANSIN2 ) , EXT3 ( EXTENSIN3 ) , XTR6 ( XYLOGLUCAN ENDOTRANSGLYCOSYLASE6 ) , and XTH17 ( ENDOTRANSGLUCOSYLASE/HYDROLASE17 ) [36 , 53] , was down-regulated in siz1-2 compared to the wild type ( Fig 5C ) . The loss-of-function hy5-215 seedlings exhibited a long-hypocotyl phenotype under light conditions [54] . To identify genetic interactions between HY5 and SIZ1 , we generated the hy5-215 siz1-2 double mutant through genetic crossing . The short-hypocotyl phenotype of siz1-2 was partially suppressed by hy5-215 under various light conditions ( Fig 5D and 5E ) , suggesting that the accumulation of HY5 in siz1-2 at least partly accounts for the short-hypocotyl phenotype under light conditions . In addition to HY5 , COP1 also mediates the ubiquitination and degradation of HYH , LAF1 , HFR1 , STH3/BBX2 and PIL1 [19–24] . Thus , it is possible that the protein levels of these positive regulators of photomorphogenesis are higher in siz1-2 , which may causes a weak cop1-like phenotype of siz1-2 . SIZ1-mediated SUMO modification may regulate COP1 expression , COP1 stability , nuclear accumulation level , interaction with other proteins and/or affects its enzymatic activity . The level of COP1 expression was not significantly affected by the siz1-2 mutation , indicating that SIZ1 does not regulate the transcription of COP1 ( Fig 6A ) . Anti-COP1 antibody [30] revealed that endogenous COP1 protein abundance was similar in the wild type and siz1-2 , indicating that the reduced COP1 activity in siz1-2 was not due to reduced levels of COP1 ( Fig 6B ) . Since COP1 functions as a homodimer [55] , we examined whether SUMO modification affected COP1 dimerization . Immunoprecipitation analysis showed that Myc-COP1 and Myc-COP1K193R ( a non-sumoylated form ) immunoprecipitated the same amount of FLAG-COP1 , indicating that sumoylation of COP1 did not significantly affect COP1 dimerization ( Fig 6C ) . Next , we examined if SUMO modification enhanced the COP1-HY5 interaction using in vitro co-immunoprecipitation assays . Sumoylation of COP1 did not enhance the substrate accessibility of COP1 under dark and light conditions ( Fig 6D ) . Moreover , the siz1 mutation did not affect the level of nuclear COP1 under dark and light conditions ( Fig 6E ) . Finally , we evaluated if SUMO conjugation regulates transubiquitination acitivity of COP1 . In vitro sumoylated and non-sumoylated MBP-COP1-FLAG were used as E3s to perform an in vitro HY5 ubiquitination assay . Anti-GST and anti-ubiquitin antibodies detected higher level of ubiquitinated proteins in the reaction containing sumoylated COP1 than did non-sumoylated COP1 ( Fig 6F ) , suggesting that SIZ1-mediated SUMO modification may enhances transubiquitination activity of COP1 . Although the biological functions of SIZ1 have been extensively characterized , the mechanism that regulates SIZ1 activity is largely unknown . Our finding that COP1 interacted with SIZ1 ( Fig 3A and 3B ) prompted us to determine if COP1 mediates the ubiquitination and degradation of SIZ1 . To determine if COP1 promotes degradation of SIZ1 , we analyzed the decay kinetics of SIZ1-GFP in the presence or absence of Myc-COP1 . The degradation of SIZ1 was more rapid in the presence of Myc-COP1 ( Fig 7A ) . Moreover , the COP1-promoted SIZ1 degradation was inhibited by MG132 , a 26S proteasome inhibitor ( Fig 7B , upper panel ) . In agreement with a previous report [21] , MG132 also repressed the degradation of COP1 ( Fig 7B , middle panel ) . SIZ1 expression was not affected in cop1-4 , indicating that COP1 does not regulate SIZ1 transcription ( Fig 7C ) . Next , we evaluated if COP1 mediates the ubiquitination of SIZ1 . MBP-COP1-FLAG was used as the E3 ligase in an in vitro SIZ1 ubiquitination assay . Anti-Myc and anti-ubiquitin antibody revealed that COP1 facilitates ubiquitination of SIZ1 in vitro ( Fig 7D ) . These results suggest that COP1 negatively regulates SIZ1 protein stability through ubiquitination and subsequent 26S proteasome-dependent degradation .
Loss-of-function siz1-2 and sum1-1 amiR-SUM2 seedlings exhibited a short-hypocotyl phenotype under dark and light conditions ( Figs 1 and 2 ) , suggesting that SIZ1-mediated SUMO1/2 modification negatively regulates photomorphogenesis . SIZ1 physically interacts with COP1 and facilitates its SUMO modification ( Fig 3 ) . COP1 exhibits higher activity compared to that of non-sumoylated COP1 , COP1K193R , and the COP1 activity was attenuated in siz1-2 ( Fig 4 ) . The reduced COP1 activity in siz1 resulted in higher levels of HY5 , stronger down-regulation of cell elongation-related HY5 direct target genes ( Fig 5A and 5C ) . These results strongly suggest that SIZ1-mediated SUMO modification enhances COP1 activity; thus , COP1 is a plant SUMO-regulated ubiquitin ligases ( SRUbL ) . The hypocotyl length of cop1-4 siz1-2 was slightly shorter than that of cop1-4 ( Fig 4G and 4H and S7 Fig ) , suggesting that SIZ1 regulates hypocotyl elongation through at least two pathways , a COP1-dependent and -independent pathway . Recently , it has been shown that SUMO modification of phyB negatively regulate photomorphogenesis under red light [37] . OTS1 ( OVERLY TOLERANT TO SALT1 ) mediate desumoylation of phyB , but whether the SUMO modification is facilitated by SIZ1 remains to be determined . However , it should be noted that cop1-4 is a weak allele , which expresses a partially functional truncated COP1 protein ( 1–282 aa , which contains the K193 sumoylation motif ) [51] . Therefore , it is also possible that the partial COP1 activity in cop1-4 is further attenuated in cop1-4 siz1-2 . It has been demonstrated that COP1 is regulated by different mechanisms: ( 1 ) light , FIN219 and cold induces nuclear-to-cytoplasmic export of COP1 , but ethylene enhances nuclear retention of COP1 in the light [29 , 56–58]; ( 2 ) CSU1 , FIN219 and heat shocks reduce protein abundance of COP1 [27 , 29 , 59]; ( 3 ) SPA positively regulates COP1 activity may through enhancing substrate recruitment [21 , 25]; ( 4 ) phyA , phyB , CRY1 and CRY2 repress COP1 activity by affecting COP1-SPA complex in the lights [30–34]; ( 5 ) PIF1 not only enhances substrate recruitment , but also increases autoubiquitination and transubiquitination activities of COP1 [26] . The present study reveals that SIZ1-mediated sumoylation does not regulate COP1 protein abundance , dimerization of COP1 , or nuclear-to-cytoplasmic translocation ( Fig 6 ) . SUMO modification possibly increased transubiquitination activity of COP1 in vitro ( Fig 6F ) , but did not affect substrate recruitment ( Fig 6D ) . At the molecular level , sumoylation affects protein-protein interactions and target protein conformation [60 , 61] . We speculate that the enhanced transubiquitination activity of COP1 by sumoylation possibly due to conformational change of COP1 or increased COP1-E2 interaction . Recent study has demonstrated that CSU2 negatively regulates COP1 activity through their coiled-coil domains [28] . SUMO modification site , K193 , is located in the coiled-coil domain of COP1 . Thus , it is also possible that the sumoylation inhibits interaction between CSU2 and COP1 in vivo , but remain to be elucidated in the near future . It has been shown that a certain amount of COP1 is present in the nucleus even under prolonged light exposure , and plays a critical role in regulating development [27 , 51 , 58 , 62–64] . Interestingly , prolonged light exposure reduced sumoylation levels of nuclear-localized COP1 ( Fig 3F ) , which results in decreased COP1 activity . Thus , we hypothesize that light-induced reduction of COP1-SUMO levels is required for the maintenance of moderate COP1 activity under light conditions to ensure the tight regulation of photomorphogenesis . The reduced sumoylation level of COP1 under light conditions may be due to decreased SIZ1-mediated sumoylation and/or increased SUMO protease ( s ) -mediated desumoylation , but the details of the mechanisms by which light regulates the balance between sumoylation and desumoylation remain to be elucidated . Collectively , our study demonstrates that SIZ1-mediated sumoylation negatively regulates photomorphogenesis , at least partly , through enhancing COP1 activity . Interestingly , COP1 in turn mediates the ubiquitination and 26S proteasome-dependent degradation of SIZ1 ( Fig 8 ) . This feedback repression of SIZ1 activity by COP1 may reflect the requirement of tightly regulated COP1 activity for proper photomorphogenic development .
Seeds of wild type , siz1-2 ( Salk_065397 ) , cop1-4 , COP1 OE , hy5-215 , sum1-1 amiR-SUM2 [43] , SSG [38] , NahG , and NahG siz1-2 [44] were in the Columbia ecotype . cop1-4 siz1-2 , hy5-215 siz1-2 , and COP1 OE siz1-2 were generated by genetic crossing and the double mutants were identified as described in previous reports [51 , 54] . To generate 35S-Myc-COP1 and 35S-Myc-COP1K193R , pBI121-Myc-COP1 and pBI121-Myc-COP1K193R were transformed into Col-0 by the Agrobacterium-mediated floral dip method [65] , respectively , and homozygous T3 plants were used . After surface sterilization , seeds were sown on Murashige and Skoog growth medium ( 1/4× Murashige and Skoog basal salts , 1% sucrose , and 0 . 75% agar ) . After 3 days of incubation at 4°C in darkness , the seeds were exposed to 6 h of white light to induce germination , and then transferred to light chambers containing red , blue , or far-red light emitting diodes at the indicated fluence rates . White light ( 100 μmol m-2 s-1 unless indicated otherwise ) was provided using white fluorescent lamps . To analyze hypocotyl length , cotyledon angle , and hypocotyl cell length and cell numbers , seedlings were photographed with a camera ( Canon ) or a cool CCD camera coupled to an Olympus BX53 microscope and the images were analyzed with NIH ImageJ software ( http://rsbweb . nih . gov/ij/ ) . Total RNA was extracted from seedlings with TRIZOL reagent ( RNAiso Plus , TaKaRa ) and reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit ( Thermo Scientific ) . qRT-PCR was then performed with SYBR Premix ExTaq ( TaKaRa ) according to the manufacturer’s instructions . Three biological replicates were performed . The relative expression level of each gene was normalized to that of Ubiquitin-Conjugation Enzyme ( UBC ) . Primer sequences are listed in S1 Table . COP1-YFPN and SIZ1-YFPC plasmids together with the proper control plasmids ( empty YFPN or YFPC vector plasmids ) were transformed into Agrobacterium tumefaciens strain GV3101 and infiltrated into Nicotiana benthamiana leaves as previously described [66] . After incubation at 22°C for 3 days under 16 h white light ( 100 μmol m-2 s-1 ) /8 h darkness , YFP fluorescence was detected with a fluorescence microscope ( Olympus BX53 ) under darkness or after 1 h white light ( 100 μmol m-2 s-1 ) exposure . For co-immunoprecipitation of COP1 and SIZ1 , Myc-COP1 and SIZ1-GFP were separately expressed in Col-0 protoplasts to avoid degradation of SIZ1 by COP1 . Immunoprecipitation was carried out using a mixture of COP1 and SIZ1 protein extracts . The mixture of protein extracts was immunoprecipitated with anti-Myc-conjugated agarose beads ( Sigma , F-2426 ) and co-immunoprecipitated SIZ1-GFP proteins were detected with anti-GFP antibody ( Clontech , 632375 ) . To analyze the sumoylation effect in the interaction between HY5 and COP1 , 1 μg of GST-HY5 protein purified from E . coli [52] , as bait , was incubated with the protein extracts isolated from N . benthamiana co-expressing Myc-COP1 with FLAG-SUMO1 or FLAG-SUMO1AA . The mixture of protein extracts was immunoprecipitated with anti-Myc-conjugated agarose beads ( Sigma , F-2426 ) and co-immunoprecipitated GST-HY5 proteins were detected with anti-GST antibody ( Abcam , ab19256 ) . To determine the sumoylation status of COP1 , proteins were extracted in a buffer composed of 50 mM Tris-Cl ( pH 7 . 4 ) , 150 mM NaCl , 1 mM EDTA ( pH 8 . 0 ) , 1 mM DTT , 20 mM NEM , 1% TritonX-100 , and 1× complete protease inhibitor mixture ( Roche , 04693159001 ) . Then , the protein extracts were immunoprecipitated with anti-Myc-conjugated agarose beads ( Sigma , F-2426 ) for 3 h . Next , the beads were washed with protein extraction buffer four times , and the immunoprecipitated proteins were eluted with 2× SDS loading buffer for immunoblot analysis . The sumoylated form of COP1 was identified with anti-FLAG antibody ( Sigma , F3165 ) or anti-SUMO1 antibody ( Abcam , ab5316 ) . An in vitro sumoylation assay was performed as described previously [15] with minor modifications . Briefly , 50 ng of His6-AtSAE1b , 50 ng of His6-AtSAE2 , 50 ng of His6-AtSCE1 , 8 μg of His6-AtSUMO1-GG , and 100 ng of MBP-COP1-FLAG were incubated in 30 μl of reaction buffer ( 20 mM HEPES pH7 . 5 , 5 mM MgCl2 , 2 mM ATP ) for 3 h at 30°C . Sumoylated MBP-COP1-FLAG was detected with anti-FLAG ( Sigma , F3165 ) and anti-SUMO1 antibody ( Abcam , Ab5316 ) . An in vitro ubiquitination assay was performed as described previously [50] with minor modifications . E3s , recombinant wheat E1 , 500 ng purified E2 , 5 μg ubiquitin , and 100 ng substrates were incubated in 30 μl of reaction buffer ( 50 mM Tris-Cl ( pH 7 . 4 ) , 10 mM MgCl2 , 5 mM ATP , and 2 mM DTT ) for 3 h at 30°C . The substrates were detected with anti-His ( Sigma , H9658 ) , anti-GST ( Abcam , ab19256 ) , or anti-Myc ( Sigma , M4439 ) antibody , and the ubiquitination was determined with anti-ubiquitin ( Sigma , U5379 ) antibody . Nuclear protein extraction was carried out with the CelLytic PN Extraction Kit ( Sigma , CELLYTPN1 ) as described previous [62] . To generate pSPYNE-COP1-YFPN , full-length COP1 cDNA without the termination codon was amplified with gene-specific primers COP1-F-SpeI/COP1nt-R-XhoI . The COP1 cDNA was inserted in-frame at the SpeI/XhoI sites of the pSPYNE-35S vector [66] . To generate pSPYCE-SIZ1-YFPc , full-length SIZ1 cDNA without the termination codon was amplified with gene-specific primers SIZ1-F-XmaI/SIZ1-R-SpeI and ligated into the pBluescript vector . pBluescript-SIZ1 was digested with SmaI and SpeI , and the SIZ1 cDNA was inserted in-frame at the HpaI/SpeI sites of the pSPYCE-35S vector [66] . To generate pCambia1302-SIZ1-GFP , pBluescript-SIZ1 was digested with SmaI and SpeI . The pCambia1302 vector was digested with NcoI , to generate blunt ends , and then with SpeI . The insert was then ligated into the pCambia1302 vector . To generate pMAL-C2-MBP-COP1-FLAG , full-length cDNA of COP1 without the termination codon was amplified with gene-specific primers COP1-5’-SmaI/COP1nt-3’-XhoI , and ligated into the pBluescript vector . pBluescript-COP1 was digested with SmaI and XhoI , and inserted in-frame at the SmaI and XhoI sites of the pMAL-C2-MBP-FLAG vector . To generate pMAL-C2-MBP-SIZ1-Myc , full-length cDNA of SIZ1 without the termination codon was amplified with gene-specific primers SIZ1-F-XbaI/SIZ1-R-ClaI , and ligated into the pBluescript vector . pBluescript-SIZ1 was digested with XbaI and ClaI , and inserted in-frame at the XbaI and ClaI sites of the pMAL-C2-MBP-MYC vector . To generate p326-Myc-COP1 , the full-length cDNA of COP1 was amplified with gene-specific primers COP1-F-HindIII/COP1-R-XhoI , and ligated into the pBluescript vector . pBluescript-COP1 was digested with HindIII and XhoI , and inserted in-frame at the HindIII and XhoI sites of the p326-35S-nMyc vector . pBluescript-COP1 was used as template with primer pairs COP1 K14R-F/COP1 K14R-R , COP1 K193R-F/COP1 K193R-R , and COP1 K653R-F/COP1 K653R-R , to generate pBluescript-COP1K14R , pBluscript-COP1K193R , and pBluescript-COP1K653R , respectively . pBluescript-COP1K14R , pBluescript-COP1K193R , and pBluescript-COP1K653R were digested with HindIII and XhoI , and inserted in-frame at the HindIII and XhoI sites of the p326-35S-nMyc vector to generate p326-35S-Myc-COP1K14R , p326-Myc-COP1K193R , and p326-Myc-COP1K653R , respectively . p326-Myc-COP1 and p326-Myc-COP1 K193R were digested with SpeI and XhoI , and the digested products were inserted into the SpeI and XhoI sites of the pBI121-35S-nMyc vector to generate pBI121-Myc-COP1 and pBI121-Myc-COP1K193R , respectively . To generate p326-FLAG-SUMO1 and pBI121-FLAG-SUMO1 , SUMO1 cDNA was amplified with gene-specific primers SUMO-F-XbaI and SUMO-R-XhoI , and inserted into the XbaI and XhoI sites of the p326-35S-nFLAG and pBI121-35S-nFLAG vector . To generate p326-FLAG-SUMO1AA and pBI121-FLAG-SUMO1AA , SUMO1 cDNA was amplified with gene-specific primers SUMO-F-XbaI and SUMOAA-R-XhoI , and inserted into the XbaI and XhoI sites of the p326-35S-nFLAG and pBI121-35S-nFLAG vector . All primer sequences are listed in S1 Table . | In darkness , the ubiquitin E3 ligase COP1 accumulates in the nucleus and mediates ubiquitination and degradation of positive regulators of photomorphogenesis , such as HY5 . In response to light , COP1 activity is reduced to ensure proper photomorphogenic development . However , post-translational modifications that regulate COP1 activity are largely unknown . We have found that the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates photomorphogenesis . Genetic and biochemical lines of evidence demonstrate that SIZ1-mediated SUMO modification of COP1 enhances its E3 ubiquitin ligase activity , which causes increased ubiquitination and degradation of HY5 . In response to the light , sumoylation level of COP1 is decreased , which may also contributes to the reduction of COP1 activity in the light . Moreover , COP1 mediates ubiquitination and 26S proteasome-dependent degradation of SIZ1 and this feedback repression may ensure the moderate levels of COP1 activity . Our study established a post-translational regulatory modular consisting of SIZ1-mediated sumoylation and COP1-mediated ubiquitination that tightly regulate photomorphogenesis . | [
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] | 2016 | An Arabidopsis SUMO E3 Ligase, SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity |
Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites . Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material ( CWM ) consisting of three paralogous cyst wall proteins ( CWP1–3 ) via organelles termed encystation-specific vesicles ( ESVs ) . Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution . However , neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation . Based on this “minimal Golgi” hypothesis we predicted maturation of ESVs to a trans Golgi-like stage , which would manifest as a sorting event before regulated secretion of the CWM . Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions , which are sorted and secreted sequentially with different kinetics . This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall , followed by slow secretion of the remaining components . Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs . Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible . Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter . Nevertheless , partitioning and sequential secretion of two portions of the CWM are unaffected in these cells . Although these cysts have a normal appearance they are not water resistant and therefore not infective . Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions .
Infectious parasite stages transmitted to a new host via the oral route ( cysts , oocysts , eggs ) require a highly resistant extracellular matrix to protect them in the environment and during passage through the stomach . The diplomonad Giardia lamblia ( syn . G . intestinalis , G . duodenalis ) is an intestinal protozoan and a leading cause for parasite-induced diarrheal disease [1] . Trophozoites in the small intestine or in culture undergo stage-differentiation to a cyst form in response to environmental cues , e . g . changes in pH , bile and/or cholesterol concentration [2] , [3] . The members of this phylum have undergone strong reductive evolution resulting in minimization or loss of cellular systems and organelles such as mitochondria , peroxisomes and the Golgi apparatus [4]–[6] , but despite significant advances in phylogenetic analysis their point of divergence during evolution remains elusive [7] . Comparative genomic data suggest that the complexity of cellular organization in the last common eukaryotic ancestor with respect to compartments and membrane transport was considerable [4] . Specifically , the central organelle for maturation and sorting of excretory/secretory proteins , a classical Golgi apparatus likely with a typical stacked configuration of functionally distinct cisternae , appears to have been present in this hypothetical cell . Thus , reductive evolution is the most parsimonious , albeit still unproven , explanation for the absence of a Golgi organelle and Golgi functions in Giardia trophozoites [8] . In Giardia trophozoites , secreted proteins appear to traffic directly from the endoplasmic reticulum ( ER ) to the target organelle or the plasma membrane [9] . In contrast , in cells differentiating to cysts secretory cargo is delayed for many hours in specialized organelles termed encystation-specific vesicles ( ESVs ) which arise de novo [5] , [10] . ESVs contain only presorted cyst wall material ( CWM ) and exclude constitutively secreted proteins even during neogenesis . The CWM rapidly polymerizes upon secretion and forms the protective cyst wall ( CW ) on the parasite surface at 20–24 h post induction ( p . i . ) of differentiation in vitro . The CWM biopolymer has a surprisingly low complexity considering its effectiveness as a biological barrier . It consists of three paralogous cyst wall proteins ( CWP1–3 ) and simple β1–3 GalNAc homopolymer chains [11] . The glycan portion constitutes ∼60% of the CW [12] , but where it is synthesized and how it is exported and incorporated into the cyst wall structure is unknown . Galactosamine synthesis from glucose and its incorporation as a polymer is mediated by pathways whose components are upregulated transcriptionally and allosterically during encystation [10] , [13]–[16] . Synthesis of CWP mRNA peaks at ∼7 h p . i . and protein export from the ER to ESVs is completed after 8–10 h p . i . [17] in parasites encysting in vitro . CWPs are sorted away from constitutively secreted proteins presumably during ER export , thus ESVs contain only presorted cargo [9] . The pulsed synthesis and sorting of the CWPs to ESVs the cargo is delayed by many hours in the newly formed ESV organelle system which is best described as transient Golgi cisterna analogs [18] , even though the compartments have no morphological similarity to a classical Golgi with biochemically distinct cisternae . Previously , we and others have shown transient association of COPI components with ESVs [5] , ESV sensitivity to brefeldin A [9] , [10] , and dependence of ESV genesis and maturation on giardial Sar1 and Arf1 GTPases , respectively [18] . Taken together , there is increasing support for a model depicting ESVs as developmentally regulated , minimized Golgi-like organelles which undergo simultaneous maturation before being consumed during secretion of their cargo [8] . If confirmed , ESVs could be considered the most simply organized Golgi system identified as yet . ESVs arise stage-specifically and lack Golgi glycosyl transferases and typical structural or morphological landmarks which define this organelle in most other eukaryotes . This makes it impossible to test directly whether ESVs are indeed Golgi analogs or whether they arose independently during evolution . Thus , this issue can only be addressed by accumulation of circumstantial evidence and rigorous experimental testing of predictions based on this model . Although few details are known it is reasonable to assume that export of the CWM is delayed in ESVs for several hours to allow for post translational maturation before it is secreted in fluid form to cover the entire cell surface where it eventually polymerizes . Proteolytic processing of CWP2 which has a 121 residue C-terminal extension rich in basic amino acids [19] is the only modification of CWPs described in any detail . Although the evidence clearly implicates a cysteine protease , there is a controversy as to which enzyme is responsible [20] , [21] . In addition to processing , the enzymatic formation of disulfide [17] and isopeptide [22] bonds between CWPs appears to play a major role in the export process . In the present study we address the question whether assembly of the giardial cyst wall requires an additional sorting step . This idea follows from a central prediction of our working model [23] , [24] , namely that ESVs as the only Golgi-like organelles in Giardia ultimately mature to a stage corresponding to the trans Golgi compartment of the classical Golgi whose principal function is sorting of mature cargo into distinct transport intermediates . However , a fundamental difference between ESVs and conventional Golgi cisternae is that the giardial organelles contain only a single type of pre-sorted cargo , the CWM , all of which is believed to be simultaneously secreted to the cell surface . In principle this should make a sorting step at this stage unnecessary except if the CWM were divided into distinct subfractions , for example as a result of post translational processing . This hypothesis is testable by analyzing the fate of all CWPs ( pro-proteins and mature forms ) by ( quantitative ) confocal fluorescence microscopy and Western blot . With this approach we discovered a completely novel cargo sorting function in mature ESVs resulting in partitioning of the proteinaceous CWM into two clearly defined fractions . Using specific antibodies and conditionally expressed epitope-tagged variants of CWPs we show that this processing/sorting mechanism which results in the sequential secretion of the CWM to the cell surface is necessary for the functional integrity of the cyst wall as a protective extracellular matrix .
Previous investigations of transport and secretion of the CWM provided evidence for proteolytic processing of the C-terminal extension of CWP2 [25] . The data suggested cleavage of the entire C-terminal extension of ∼13 kDa . However , the small C-terminal portion of the native or the transgenic CWP2 has never been visualized directly [26] . Processing of CWP2 was found to occur before secretion of the CWM but has not been correlated with expression kinetics or maturation and morphology of ESVs . To determine the temporal and spatial distribution of pro-CWP2 and its mature products we engineered a dually tagged CWP2 variant ( Flag-CWP2-HA ) for conditional expression under the CWP1 promoter ( Figure 1A , B ) [18] . Western analysis showed stage-specific expression of pro- Flag-CWP2-HA and appearance of a large processed form with a MR reduced by ∼5 kDa between 8 h and 10 h post induction ( p . i . ) ( Figure 1A ) . The data are consistent with removal of a short C-terminal portion with the attached HA tag ( ΔC-HA ) from Flag-CWP2-HA which appears to be nearly complete at 12 h p . i . We were unable to resolve ΔC-HA on SDS-PAGE although it is readily detected in immunofluorescence microscopy analysis ( IFA ) ( see below ) . To make a rough determination of the proteolytic cleavage site we expressed two modified Flag-CWP2-HA variants containing deletions from N244 to A272 ( ΔPS ) or A300 to V359 ( ΔPS3 ) in the C-terminal domain ( Figures 1B and S1 ) . Western blot analysis of protein from transgenic cells at 10 h p . i . confirmed processing of ΔPS but not of ΔPS3 . Combined with the apparent mass difference after removal of ΔC , this is consistent with cleavage of Flag-CWP2-HA ∼50–60 amino acids from the C-terminus . During the 20–24 h process of encystation , ESVs arise de novo through export of CWM from the ER and attain their final dimension of ∼500 nm between 8 h and 10 h p . i [9] . As a standard marker to follow this organelle development we and others use a commercially available mAb against CWP1 . The signal observed in optical sections of ESVs generated by confocal IFA in encysting trophozoites at 10–12 h p . i . showed a characteristic ring-like distribution of the anti-CWP1 antibody ( Figure 1C ) compared to an even staining of organelle contents typical for earlier stages ( see also Figure 2A ) . The ring-like distribution of the CWP1 marker was a transient phenomenon and disappeared after a few hours . The simplest explanation for this observation was that the CWM in more mature ESVs became condensed during maturation which limited penetration of the anti-CWP1 antibody into the organelle . This was also consistent with the conspicuous electron density of ESV contents ( Figure 1C ) . However , unlike in secretory granules of other unicellular organisms , e . g . , rhoptries of Toxoplasma gondii [27] or dense core granules of Tetrahymena termophila [28] , no lattice-like structure is detected in transmission EM micrographs of ESVs . Together with the demonstrated exchange of a soluble CWP1::GFP reporter within an ESV organelle network [18] at this stage of the encystation process this would argue against condensation in ESVs . As an alternative explanation we therefore considered that CWP2 and/or CWP3 could be involved in the formation of a putative core which excludes CWP1 . To test this we performed high-resolution confocal IFA in cells expressing the Flag-CWP2-HA reporter under stage-specific control . In differentiating transgenic cells ( Figure 2 ) we labeled developing ESVs using the anti-CWP1 antibody ( red ) and detected the N- or the C-terminus of the CWP2 reporter with the anti-HA or the anti-Flag antibody , respectively ( green ) . At 6 h p . i . the HA and the CWP1 signals overlapped completely in ESVs ( Figure 2A , merged image ) as documented by co-localization analysis based on the three-dimensional reconstruction of all optical sections ( scatter plot ) . Until at least 8 h p . i . the tagged CWP2 reporter is not processed ( Figure 1A ) . Consistent with this , the Flag and the CWP1 signals also overlapped completely in ESVs ( data not shown ) . This was still true in cells at 12 h p . i . although both markers now showed the typical ring-like distribution of the proteins at the periphery of ESVs ( Figure 2B ) . ΔC-HA , on the other hand , had a completely different distribution at this stage and localized inside the ring-like staining pattern of the anti-CWP1 antibody ( Figure 2C ) . This visual assessment was confirmed by quantitative analysis of the confocal image stack which demonstrated significant loss of signal overlap ( scatter plot ) . The same characteristic distribution was found when a CWP2 monoclonal antibody ( mAb ) was used in combination with anti-HA instead of the mAb against CWP1 ( Figure S2 ) . As also shown below the anti-CWP2 mAb reacts with an epitope in the N-terminal portion of CWP2 . The combined data was direct evidence for the physical separation of the ΔC-HA and Flag-N products consistent with proteolytic cleavage of the pro-protein as documented in Figure 1A . Thus , the small ΔC-HA fragment was a first marker localizing to a putative core of ESVs . The observed cargo partitioning was unaffected by swapping of epitope tags on the CWP2 reporter ( data not shown ) . To complete this analysis of CWPs in fixed cells we conditionally expressed a HA-CWP3 reporter cloned in the same vector . Analysis of tagged CWP3 by Western blot indicated that , as for the closely related CWP1 , this protein was not processed by proteolytic cleavage ( data not shown ) . Interestingly , by confocal IFA , HA-CWP3 appeared also clearly segregated from CWP1 and localized to the same central portion of ESVs as did ΔC-HA ( Figure 2D ) . Taken together , this is direct evidence for a partitioning of the CWM inside ESVs into two separate and physically distinct fractions , each containing a CWP2-derived product . Partitioning of CWP1 from ΔC-HA within ESVs , together with the fluid nature of a CWP1::GFP reporter documented previously [18] , strongly suggested that these components of the CWM assumed different physical states . Since HA-CWP3 showed the same distribution as ΔC-HA , this allowed us to directly test the hypothesis that the mechanism for cargo partitioning was indeed formation of a condensed core in ESVs . Fluorescence recovery after photobleaching ( FRAP ) was used to quantify the degree of mobility of a CWP3::GFP reporter in the ESV organelle system in living transgenic cells . In analogy to the experiment with CWP1::GFP [18] , exchange of CWP3::GFP between ESVs was used as a measure of condensation and core formation . We tested this in cells prior to appearance of mature ESVs at 6 h p . i . and found clear evidence for recovery of fluorescence in bleached organelles ( Figure 3A , quantitative analysis ) . Recovery showed similar kinetics as observed previously for CWP1::GFP [18] , which proved that in principle the soluble CWP3::GFP reporter could be transported between ESVs . In contrast , in cells at 12 h p . i . which contained maturing ESVs , recovery of fluorescence was consistently absent ( Figures 3B and S3A , B ) . Note also the higher rate of fluorescence loss in control organelles ( 6 h time point ) due to dilution of the GFP pool within the ESV system during the recovery period ( compare quantitative analyses in Figures 3A and 3B ) . To show that CWP3::GFP is immobilized in ESV cores but CWP1::GFP is not we performed fluorescence loss in photobleaching ( FLIP ) experiments in transgenic cells at 12 h p . i . We used six rapid cycles to bleach fluorescence in all but one ESV and quantified fluorescence loss in this organelle as a measure of diffusion in the ESV system ( Figure S3C , D ) . Consistent with previous observations of CPW1::GFP mobility and the FRAP analysis of CWP3::GFP presented herein , we find rapid diffusion and signal loss in ESVs containing CWP1::GFP compared with the sustained fluorescence of CWP3::GFP in ESVs at this stage of encystation . Taken together , this is direct evidence for virtually complete immobilization of the CWP3 reporter in mature ESVs and consistent with core formation and loss of solubility . Combined with previously reported FRAP data using CWP1::GFP [18] this strongly supports the idea that a hallmark of maturing ESVs is partitioning of the CWM into two fractions with distinct physical properties . Sorting mechanisms based on selective condensation of secretory cargo and formation of condensed cores in the trans Golgi network ( TGN ) and in post Golgi vesicles of mammalian cells have been described by the “sorting by retention model” [29] , [30] . In analogy , condensation of CWM components in maturing ESVs suggested that this cargo is selected for differential secretion . Indeed , in transgenic cells at 14–16 h p . i . dual labeling revealed that cargo partitioning in ESVs gave way to actual sorting of cargo into separate compartments ( Figure 4A , B ) . The fraction consisting of CWP1 and the large N-terminal portion of the processed CWP2 ( collectively termed CWMfl ) , presumably remained in a fluid state throughout , and appeared to be concentrated in small compartments with peripheral localization in the cell . Tagged ΔC and CWP3 proteins , collectively termed CWMco ) , were detected in organelles with a more central localization . Thus , whilst the mechanism for partitioning of the CWM within ESVs ( i . e . physical separation of the two fractions ) is clearly condensation , the cellular machinery for the subsequent sorting of CWMco and CWMfl into distinct organelles remains to be identified , but possibly involves coat protein complexes . This idea is also based on localization studies which show that clathrin ( CLH ) is specifically recruited to membranes of maturing ESV ( Figure S4A ) [8] , [31] . CLH is not upregulated during encystation but the protein appears to re-localize from the membranes of the endosome-lysosome-like peripheral vesicle organelles to ESVs . CLH is most abundant on maturing ESVs with evidence for a condensed core , and appears to lose this association as sorting progresses ( Figure S4B , C ) . Whether clathrin is directly involved in sorting of CWMfl or has another role remains to be determined . Classical clathrin coated pits on ESV membranes , at least , have never been demonstrated by electron microscopy . The significance of this sorting event only became evident when newly formed cysts were analyzed by IFA at 16 h p . i . ( Figure 4C ) . The CWMfl fraction ( represented here by CWP1 ) was secreted quantitatively whereas CWMco ( represented by ΔC-HA ) remained in internal compartments . Correspondingly , CWMfl and CWMco lost colocalization completely as CWMfl was deposited on the outer face of the plasma membrane during morphological differentiation of the trophozoites into cysts ( Figure 4C ) . Yet , if the cysts were allowed to mature longer and were harvested at 24 h p . i . , all cysts showed partial and some even full recovery of marker colocalization at the cyst wall ( Figure 4D , E ) as documented in the quantitative analysis ( scatter plots ) . This suggested that ΔC-HA , as well as CWP3-HA or CWP3::GFP ( Figure S5A–C ) were secreted with clearly different kinetics , suggesting a requirement for sequential deposition of the CWM fractions . Partitioning of CWM , core formation and processing of tagged and endogenous CWP2 all appeared to take place around 10–12 h p . i . Together with an idea presented recently by the Lujan laboratory that CWP2 coordinated export of CWM [25] , the simplest explanation was that this change of physical property was triggered by the release of ΔC . To test this we inhibited processing of the Flag-CWP2-HA reporter as well as endogenous CWP2 by treating encysting cells with the protease inhibitor E64 shown to block giardial cysteine protease 2 ( CP2 ) [20] . The Western analysis of parasites harvested at 12 h p . i . confirmed complete inhibition of processing in these conditions ( Figure 5A , B ) . Surprisingly , using labeled anti-CWP1 antibody as a marker we found that cyst formation was not significantly impaired . More detailed analysis of fixed transgenic cells by IFA showed that in cysts derived from treated cells , unprocessed Flag-CWP2-HA remained in internal vesicles containing the CWMco fraction ( Figure 5C ) . General cargo partitioning and sorting of the CWMfl and CWMco fractions and sequential secretion appeared to be unaffected despite the changed composition . This suggested that in treated cells CWP1 was the only family member which was exported during the formation of the first layer of the CW , followed by the components of CWMco which now included the unprocessed CWP2 ( Figure 5C ) . Altogether , the results indicate that proteolytic cleavage of CWP2 is not necessary to induce core formation . This leaves two possibilities for the role of CWMco components: CWP3 can induce condensation alone , or alternatively , through interaction with the ΔC portion of CWP2 independent of processing . Sequestration of unprocessed Flag-CWP2-HA in the condensed core and in CWMco compartments of E64 treated cells suggests the presence of a dominant sorting signal in the short ΔC domain . An additional conclusion from these experiments was that building of the first layer of the cyst wall whose likely function is to provide structural stability to the morphologically transformed cell appeared to be independent of CWP2 processing and trafficking . Interestingly , the truncated ΔPS3 variant of the Flag-CWP2-HA reporter , which was not processed because it lacks the cleavage site , showed an identical distribution in maturing cysts derived from untreated cells ( Figure S5D ) as the wild type variant in cells treated with E64 . This might also indicate that cleavage has to be very precise for the N-terminal part of CWP2 to be exported with CWMfl , or that cleavage and partitioning are coupled processes . Although two CWM fractions appeared to be secreted sequentially in differentiating cells treated with E64 , we suspected that the viability of these cysts was compromised . We tested water resistance as a quantifiable hallmark of correctly formed cysts by exposing mature cysts derived from E64-treated cells and from untreated controls to cold water for >24 h . Quantification of survival rates ( Figure 5D ) shows that the number of viable cysts from treated cells was reduced by ∼90% after exposure to water although their cyst walls remained apparently intact . This is direct evidence that correct composition of the sequentially secreted CWM , which is achieved by processing of CWP2 and by sorting of the two products in maturing ESVs , is essential for the biological activity of cysts .
Efficient formation of water-resistant cysts of Giardia is a major contributing factor for the world-wide distribution of this extremely successful parasite . The simple organization and genetic tractability of Giardia allow for the study of basic principles of Golgi compartment neogenesis , sorting and regulated secretion in an uncluttered system [8] , [32] . More importantly , by looking for universally conserved paradigms of protein trafficking and organelle organization in the minimal secretory system of Giardia we uncovered a completely unknown mechanism for cyst wall formation . The regulated secretory pathway in Giardia is established from ER-derived transport intermediates [9] . As the only Golgi-like compartments in Giardia , ESVs are exceptional since they contain only CWM and no constitutively secreted proteins [9] , [23] , [33]–[35] . Thus , ESVs constitute a laterally connected network of maturation compartments which is clearly distinguishable from the ER and whose synchronous maturation can be tracked during the entire 20–24 h of the differentiation process in vitro . The exported CWM has a very low complexity: Three paralogous CWPs are very likely the major proteins of the extracellular portion of the giardial cyst wall [26] , [36] , in addition to a simple β1–3 GalNAc homopolymer glycan [11] , [12] , whose manner of integration with CWPs is unknown . A cysteine-rich membrane protein ( HCNCp ) which may localize also to the plasma membrane of cyst forms [37] could function as a possible link between the cyst wall and the cell surface . Thus , the structural and organizational minimization in Giardia provides unique opportunities to investigate basic principles of extracellular matrix formation . Compared to Giardia cysts , environmentally resistant infectious stages of other pathogenic protozoa have more elaborate cyst walls . While the first layer of the giardial CW is secreted rapidly [8] , the Entamoeba invadens CW is built more gradually from soluble secreted material and is anchored by a plasma membrane bound Gal/GalNAc lectin . This in turn binds to seven Jacob glycoproteins [38] , [39] , which cross-link chitin fibrils to establish a structural scaffold . In an elegant study , Chatterjee et al . [40] showed that construction of this structural part of the matrix , which also includes a chitinase [41] , [42] involved in its remodeling , was followed by incorporation of Jessie3 proteins which provide the “mortar” that seals it . Sequential secretion of distinct CWM fractions from different secretory organelles was observed during establishment of the three distinct layers of the Eimeria oocyst wall [43] , [44] . Based on these and other models we postulate that sequential assembly of multi-layered cyst walls from protein and carbohydrate is a universally conserved albeit polyphyletic trait required for full protection of infectious stages . CWP2 with its prominent C-terminal extension ( Figure S1 ) was postulated to act as an escorter for the other CWPs during export [25] . We have used a dually tagged CWP2 reporter ( Flag-CWP2-HA ) to investigate processing and trafficking of CWP2 . In contrast to a previous report which postulated the removal of the entire C-terminal domain which is unique to CWP2 [21] , our Western analysis and the examination of deletion variants indicated removal of only ∼5 kDa . Localization of C-terminally tagged CWP2 in the cyst wall by Sun and coworkers was interpreted as the presence of pro-CWP2 [26] , which could mean that processing may not be required for incorporation into the matrix . Our results showed that both CWM fractions receive a portion of this domain rich in basic amino acids , indicating that it fulfills several functions . We also find evidence for the presence of a dominant sorting signal in the ΔC domain ( see also below ) . Our data suggest that proteolytic cleavage of CWP2 is a discrete process that marks the transition from the ESV genesis to the ESVs maturation phase . Encystation is not completely synchronous in an induced population because parasites need to complete the S-G2 transition of the cell cycle in order to exit the proliferation cycle and differentiate [45] . However , our observations indicate that the transition into the maturation phase starts at ∼10 h post induction in the large majority of cells ( Figure 6 ) and coincides with a marked downregulation of CWP synthesis [17] . Cargo partitioning provides a more sophisticated explanation for the incomplete staining by anti-CWP1 antibodies which was observed in maturing ESVs . Considering that ΔC contains a dominant targeting signal , processing can be interpreted as liberating the large N-terminal domain which remains soluble and can be secreted to the outer cyst wall layer . Since only CWP2 was processed and both its products could be detected in IFA all major players were followed either by epitope tagging or using a specific mAb in the case of CWP1 and CWP2 . Investigation of cargo partitioning by high resolution confocal microscopy yields correspondingly clear cut results showing distinct localizations for these factors in either the center or the periphery of ESVs which can be quantitatively analyzed for co-localization . In addition to providing a sorting mechanism for partitioning of CWM , selective condensation theoretically allows for differential post-translational modification of components in fluid and condensed fractions . This could partially offset the lack of a stacked cisternal organization of this organelle system . Biogenesis of secretory granules is still poorly understood [30] . Formation of immature granules occurs at the TGN in endocrine , exocrine and neuronal cells by sorting granule proteins from constitutively secreted cargo . Condensation of soluble proteins is organized by aggregation factors such as chromogranin A of neuroendocrine cells which drive granule formation independently of coat protein complexes [46] , [47] . Core formation in secretory granule biogenesis is dependent on inherent biophysical properties of cargo components and aided by acidic pH and high Ca2+ in these organelles . Our attempts to disrupt or delay this process in ESVs by inhibiting acidification of organelles using ammonium chloride or the H+-ATPase inhibitor bafilomycin , or by depleting intracellular calcium were not successful ( Konrad and Hehl , unpublished ) . This suggests that an inherent tendency to aggregate is the dominant driving force of CWMco condensation . Alternatively , interaction with an as yet unidentified component could prevent circulating CWMfl components from becoming condensed . Further maturation of secretory granules in higher eukaryotes entails sorting and removal of non-granule proteins by vesicular traffic involving AP1/clathrin [48] . We have observed important recruitment of clathrin to membranes of mature ESVs ( Figure S4A ) [9] but this alone does not prove any involvement in the sorting of CWMfl . Furthermore , because over-expression of a clathrin hub fragment during encystation had no effect on cyst formation ( Stefanic and Hehl , unpublished data ) the role of this coat protein remains to be determined . More interestingly , expression of a dominant-negative Arf1 homolog which also recruits AP1/clathrin prevented secretion of CWP1 from ESVs but not morphological transformation which suggests an essential function in late steps of regulated secretion [18] . One of the principal questions in connection with ESV maturation was whether a condensed core was formed . We were able to address this directly using a CWP3::GFP reporter to compare the physical state of CWP3 as a marker for the core with that of the closely related CWP1 protein whose dynamics was investigated previously [18] . FRAP and FLIP experiments provided the key piece of evidence for the interpretation of the results obtained by fluorescence microscopy which revealed progression from cargo partitioning to sorting and sequential secretion . E64-inhibitable proteolytic processing of pro-CWP2 has been described previously by Touz and coworkers [21] using an antibody which binds to its N-terminal portion . In agreement with these findings we detected an initial retention of unprocessed CWP2 in internal compartments . In contrast , by using an extended experimental approach , i . e . , co-labeling of markers for both CWM fractions , we documented the sequential nature of CWM secretion . Interestingly , our data showed that cyst formation was completed even when processing of endogenous and recombinant CWP2 was blocked ( see Figure 5 ) . Treatment of encysting cells with E64 did not affect stage-differentiation , secretion of CWM , or formation of an extracellular matrix , even though pro-CWP2 was retained in the CWMco fraction . Retention of ΔPS3 , which differs from the mature N-terminal CWP2 fragment by only few amino acids , points to a surprisingly stringent dependence on precise cleavage of pro-CWP2 or on cleavage itself , which contrasts with the overall robustness of the sorting process . Detailed investigation of this step , including identification of the proteolytic cleavage site , will be necessary to resolve the link between processing and sorting of the two pro-CWP2 products . Together with results from previous investigations the sorting data presented herein provide a novel scenario for regulated export of CWM in Giardia ( Figure 6 ) . The complete pathway requires two discrete sorting steps which are both consistent with our Golgi model for ESVs: I ) Sorting of CWM from constitutively secreted proteins at ER exit sites [9] , and concomitant export of CWPs to ESVs . II ) Partitioning and sorting of the mature CWM into two fractions shortly before secretion . Processing of CWP2 coincides with , but is not required for , condensed core formation in ESVs . The subsequent separation and sequential secretion of the physically distinct CWMfl and CWMco fractions is consistent with maturation of ESVs to TGN analogs and a “sorting by retention” mechanism for separating differentially secreted cargo [29] . In addition to being a prerequisite for subsequent sorting , condensation of CWMco may serve to sequester this soluble content cargo from modifying factors . CWMfl components , however , continue to circulate and could theoretically intersect with other compartments such as the ER or PVs . Secretion of CWMfl is completed in only a few minutes simultaneously with loss and/or resorption of flagella , disassembly of the ventral disk and nuclear division [8] , [49] , and most likely provides primarily structural stability to the differentiated cell . Unlike reported previously [50] , we find that encysting trophozoites adhere quite well in vitro , until just prior to secretion of the CWM ( Trepp , Spycher and Hehl , unpublished ) when they lose attachment as the cytoskeleton is reorganized . This allows the newly formed cyst walls to reach full function before cysts are finally shed into the environment . Integration of chitin with early and late secreted proteins in encysting E . invadens is essential for establishing a fully functional cyst wall [40] . How the unique β1–3 GalNAc homopolymer chains which provide the bulk of the CW carbohydrate are integrated into this structure during encystation in Giardia remains unknown . Three factors , i . e . , the fibrillar nature of the polymerized CWM in the outer cyst wall as shown in scanning EM [11] , studies showing that this material is composed of carbohydrate and protein [15] , [51] , and the absence of specialized vesicles containing large amounts of this carbohydrate , raise the question how this material is exported . This still awaits resolution , mainly because no known lectin reacts with the carbohydrate portion of the giardial CW with sufficient specificity . The Phaseolus lunatus lectin ( LBA ) has been reported to bind to the G . muris CW [11] , but reactivity with the G . lamblia CW is poor and inconsistent . More importantly , fluorochrome-conjugated LBA weakly labeled the nuclear envelope but not ESVs or other large organelles in encysting cells which might be involved in export of CW carbohydrate ( Hehl , unpublished ) . Whatever the route of carbohydrate export , evidence for extensive covalent cross-linking [12] , [24] , [52] of CWPs and carbohydrate chains underscore the importance of a structurally resistant CW . The simplest explanation for the sequential secretion of CWM components is that the fibrillar shell of polymerized CWMfl requires sealing to become fully protective and infectious . Based on the high proportion of intermediate stages ( Figure 4D ) found in cyst preparations at 16–24 h p . i . , export of the CWMco fraction appears to be considerably slower than secretion of CWMfl , most likely because the former must be decondensed for secretion . In light of the low complexity of the CWM , investigation of the biochemistry of its reversible condensation and subsequent polymerization is expected to reveal fundamental aspects of biopolymer export and assembly . CWPs and their inherent tendency to aggregate may be the primary driving force for cargo partitioning and ultimately for polymerization on the surface . It is likely that carbohydrates play a much more important role in coordinating sequential secretion than merely providing a means for cross-linking the protein components of the CW .
Trophozoites of the Giardia lamblia strain WBC6 ( ATCC catalog number 50803 ) were grown under anaerobic conditions in 11 ml culture tubes ( Nunc , Roskilde , Denmark ) containing TYI-S-33 medium supplemented with 10% adult bovine serum and bovine bile according to standard protocols [17] . For chemical fixation or protein extraction parasites were harvested by chilling the culture tubes on ice for 30 minutes to detach adherent cells , and collected by centrifugation at 1000×g for 10 minutes . Cells were then resuspended in phosphate-buffered saline ( PBS ) and counted . Encystation was induced using the two-step method as described previously [17] , by cultivating the cells for 44 hours in medium without bile and subsequently in medium with porcine bile and a pH of 7 . 85 . Circular plasmid DNA of expression vectors was linearized at the SwaI restriction site [18] and 15 µg of cut DNA were electroporated into 5·106 freshly harvested trophozoites on ice using the following settings: 350 V , 960 µF , 800Ω . Linearized plasmids were targeted to the Giardia lamblia triose phosphate isomerase ( Gl-TPI ) locus ( see below ) and integration occurred by homologous recombination under selective pressure of the antibiotic puromycin ( Sigma , St . Louis , MO ) for 5 days . Transgenic cell lines were maintained and analyzed without antibiotic . For the inducible expression of tagged proteins in Giardia , a previously described vector pPacV-Integ was used which allows for the expression of fusion proteins with a N-terminal HA-tag under the control of the CWP1 promoter [18] . For the expression of double-tagged CWP2 ( Flag-CWP2-HA ) , a Flag-tag was fused downstream of the stretch coding for the CWP1 signal peptide using oligonucleotide primers 44 and 768 ( Table S1 ) to PCR amplify the CWP1 promoter including the CWP1 signal peptide from genomic DNA . The PCR product was ligated into the XbaI and NsiI sites upstream of the CWP2 coding sequence . The CWP2 open reading frame ( ORF ) without the stretch coding for the signal sequence ( E21 - R362 ) was PCR amplified using oligonucleotide primers 760 and 756 . The latter included the sequence coding for the HA epitope tag . This fragment was ligated in frame using the NsiI and PacI sites of the pPacV-Integ expression cassette to generate the basic Flag-CWP2-HA vector . All constructs were sequenced prior to transfection . CWP2 deletion constructs: To express CWP2 lacking N244–A272 ( ΔPS ) , two DNA fragments ( coding for E21–R243 and H273–R362 ) were amplified with oligonucleotides 760 and 842 , and 844 and 756 , respectively , and ligated via the introduced EcoRI site , and used to replace the original NsiI - PacI fragment in the Flag-CWP2-HA vector . The same strategy was used to generate ΔPS3 lacking A300–V359 of the CWP2 ORF: the sequence coding for the E21–T299 fragment of the CWP2 ORF was PCR amplified using primers 760 and 877 and used to replace the NsiI - PacI fragment in the Flag-CWP2-HA vector . CWP3 constructs: the CWP3::GFP expression construct was made by replacing the CWP1 sequence in a previously used construct CWP1::GFP [18] with the CWP3 ORF and promoter region PCR amplified with primers 936 and 937 . An HA-tagged CWP3 ( HA-CWP3 ) fragment was made by PCR amplifying the region coding for M17-R247 of the CWP3 ORF using primers 856 and 420 and replacing the NsiI-Pac fragment in the pPacV-Integ expression vector . Encystation of trophozoites was induced in the presence of 30 µM E64 or an equal volume of the solvent ( control ) . To determine the number of viable cysts in preparations cells were harvested after 48 h , washed with PBS and incubated in ddH2O for >48 hours at 4°C . For the quantification of cyst viability cells were stained with a mixture of acridin orange ( 4 ug/ml ) and ethidium bromide ( 0 . 1 mg/ml ) in PBS for 10 min at room temperature and washed once in PBS . Cells were mounted on a slide and imaged on a Leica DM-IRBE microscope using a 40× lens ( Leica Microsystems GmbH , Wetzlar , Germany ) . For each condition two sets of 13 randomly selected fields were digitally recorded ( Diagnostic Instruments Inc . , USA ) and processed with the Metaview software package ( Visitron Systems GmbH , Puchheim , Germany ) . Percentage values of replicates were averaged . For the preparation of total cell extracts Giardia parasites were harvested as described above . The cell pellet was dissolved in SDS sample buffer to obtain of 2·105 cells in 50 µl and boiled for 3 minutes . Dithiothreitol ( DTT ) was added to a final concentration of 7 . 75 µg/ml before boiling . SDS-PAGE on 12% polyacrylamide gels and transfer to nitrocellulose membranes was done according to standard techniques . Nitrocellulose membranes were blocked in 5% dry milk/0 . 05% TWEEN-20/PBS and incubated with the primary antibodies ( anti-HA , anti-Flag , anti CWP2 mAb ) at the appropriate dilution in blocking solution . Bound antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse IgG ( Bio-Rad , Hercules , CA ) and developed using Western Lightning Chemiluminescence Reagent ( PerkinElmer Life Sciences , Boston , MA , USA ) . Data collection was done in a MultiImage Light Cabinet with AlphaEase FC software ( Alpha Innotech , San Leonardo , CA , USA ) using the appropriate settings . Immunofluorescence analysis: Chemical fixation and preparation for fluorescence microscopy was performed as described [9] . Briefly , cells were washed with cold PBS after harvesting and fixed with 3% formaldehyde in PBS for 40 min at 20°C , followed by a 5 min incubation with 0 . 1 M glycine in PBS . Cells were permeabilized with 0 . 2% triton X-100 in PBS for 20 min at room temperature and blocked overnight in 2% BSA in PBS . Incubations of all antibodies were done in 2% BSA/0 . 2% Triton X-100 in PBS . Cells were incubated with directly coupled mouse monoclonal antibodies , i . e . , Alexa488-conjugated anti-HA ( Roche Diagnostics GmbH , Manheim , Germany; dilution 1∶30 ) , Cy3 conjugated anti-Flag ( Sigma , St . Louis , MO 1∶30 ) , or Texas Red-conjugated anti-CWP1 ( Waterborne™ , Inc . , New Orleans , LA , USA; dilution 1∶80 ) for 1 h at 4°C . CLH was detected with a Giardia-specific polyclonal antibody [31] . Post incubation washes were done with 0 . 5% BSA/0 . 05% triton X-100 in PBS . Labeled cells were embedded for microscopy with Vectashield ( Vector Laboratories , Inc . , Burlingame , CA , USA ) containing the DNA intercalating agent 4′-6-Diamidino-2-phenylindole ( DAPI ) for detection of nuclear DNA . Immunofluorescence analysis was performed on a Leica SP2 AOBS confocal laser-scanning microscope ( Leica Microsystems , Wetzlar , Germany ) equipped with a glycerol objective ( Leica , HCX PL APO CS 63× 1 . 3 Corr ) . Confocal image stacks were recorded with a pinhole setting of Airy 1 and twofold oversampling . Further processing was done using the Huygens deconvolution software package version 2 . 7 ( Scientific Volume Imaging , Hilversum , NL ) . Three-dimensional reconstructions and quantitative analysis of co-localization were done with the Imaris software suite ( Bitplane , Zurich , Switzerland ) . Alternatively , a standard fluorescence microscope ( Leica DM IRBE ) and MetaVue software ( version: 5 . 0r1 ) was used for data collection . Live cell microscopy , fluorescence recovery after photobleaching ( FRAP ) and fluorescence loss in photobleaching ( FLIP ) analysis: For live cell microscopy , induced cells expressing the CWP3::GFP chimera were harvested at 6 or 12 h p . i . and transferred to 24-well plates at a density of 6·106/ml . After incubation on ice for 5–8 h , oxygenated cells were sealed between microscopy glass slides and warmed to 21°C or 37°C . Under these conditions , the encysting cells were stable and even continued to complete encystation . For FRAP , FLIP and time-lapse series , images were collected on a Leica SP2 AOBS confocal laser-scanning microscope ( Leica Microsystems , Wetzlar , Germany ) using a 63× water immersion objective ( Leica , HCX PL APO CS 63× 1 . 2 W Corr ) . Fluorescence in selected regions of interest was quantified using the corresponding Leica software suite . The pinhole was set to Airy 2 in order to increase the thickness of the optical sections to accommodate an entire ESV in the z-plane . Quantifiable criteria for cell viability were active attachment to substrate and continuous beating of the ventral and anterolateral flagella pairs . FRAP experiments were performed with the same settings as used for the CWP1::GFP reporter [18] with Leica FRAP software module to set bleaching parameters and to quantify fluorescence recovery . Electron microscopy: Encysting parasites were prepared for TEM as described previously [18] . To achieve uniform orientation , ultrathin sections were cut parallel to the sapphire surface , stained with uranyl acetate and lead citrate and examined in a CM12 electron microscope ( Philips ) equipped with a slow-scan CCD camera ( Gatan , Pleasanton , CA , USA ) at an acceleration voltage of 100 kV . Recorded pictures were processed further with the Digital Micrograph 3 . 34 software ( Gatan , Pleasanton , CA , USA ) . GiardiaDB accession numbers are given for the following genes: CWP1 GL50803_5638 , CWP2 GL50803_5435 , CWP3 GL50803_2421 , clathrin heavy chain ( CLH ) GL50803_102108 . | The protozoan Giardia lamblia is the leading cause for parasite-induced diarrhea with significant morbidity in humans and animals world-wide , and is transmitted by water-resistant cysts . Giardia has undergone substantial reductive evolution to a simpler organization than the last common eukaryotic ancestor , which makes it an interesting model to investigate basic cellular mechanisms . Its secretory system lacks a Golgi , but trophozoites induced to differentiate to cysts generate organelles termed encystation-specific vesicles ( ESVs ) . Previous work shows that ESVs are most likely minimal pulsed Golgi-like compartments for exporting pre-sorted cyst wall material . We tested whether the sorting function associated with classical trans Golgi networks was also conserved in these organelles . By tracking immature and processed forms of the three cyst wall proteins during differentiation we discovered a novel sorting function which results in partitioning of ESV cargo and sequential secretion of the cyst wall material . Using live cell imaging we identified reversible formation of condensed cores as a mechanism for cargo partitioning . These observations suggest that the requirement for sequential secretion of extracellular matrix components protecting Giardia during transmission has prevented the complete secondary loss of the machinery to generate Golgi cisterna-like maturation compartments; indeed , the preserved functions have been placed under stage-specific control . | [
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] | 2010 | Selective Condensation Drives Partitioning and Sequential Secretion of Cyst Wall Proteins in Differentiating Giardia lamblia |
Hepatitis B virus ( HBV ) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans . However , there is considerable uncertainty over the time-scale of its origin and evolution . Initial shotgun data from a mid-16th century Italian child mummy , that was previously paleopathologically identified as having been infected with Variola virus ( VARV , the agent of smallpox ) , showed no DNA reads for VARV yet did for hepatitis B virus ( HBV ) . Previously , electron microscopy provided evidence for the presence of VARV in this sample , although similar analyses conducted here did not reveal any VARV particles . We attempted to enrich and sequence for both VARV and HBV DNA . Although we did not recover any reads identified as VARV , we were successful in reconstructing an HBV genome at 163 . 8X coverage . Strikingly , both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments , characteristic of an ancient origin . In contrast , phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains ( of genotype D ) , at first suggesting contamination . In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure . This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far , and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity . If genuine , this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century , and enables comparison of potential pathogenic similarities between modern and ancient HBV . These results have important implications for our understanding of the emergence and evolution of this common viral pathogen .
The comparative analysis of viral genomes provides a wide and informative view of evolutionary patterns and processes . In particular , viruses often evolve with sufficient rapidity to inform on evolutionary processes over the timespan of direct human observation ( weeks and days ) [1–3] . Importantly , recent technical developments in next generation sequencing [4 , 5] and ancient DNA ( aDNA ) recovery [6] have enabled the rigorous study of nucleotide sequences from increasingly older historical , archaeological and paleontological samples . Consequently , aDNA sequences can now facilitate the study of more slowly evolving pathogen populations , from which recent samples display limited sequence diversity , by permitting an expansion of the indirectly observable timespan to that of centuries [7 , 8] . Hence , viral and bacterial genomes recovered from such ancient samples have the potential to reveal the etiological agents associated with past pandemics , as well as important aspects of the long-term patterns and processes of evolutionary change within pathogen populations . To date , investigations of ‘ancient’ viruses have been limited in number and scope . Those focusing on pathogens sampled prior to 1900 have considered four human viruses: variola virus ( VARV , the agent of smallpox ) [8 , 9] , human papillomavirus [10] , human T-cell lymphotropic virus [11] , and hepatitis B virus ( HBV ) [12 , 13] . Similarly , aDNA techniques have been used in studies of major 20th century epidemics of influenza virus and human immunodeficiency virus [14 , 15] . Taken together , these studies have helped to clarify the causative agents of specific outbreaks , whether ancient strains differ markedly from recent ones , and the evolutionary and epidemiological processes that have likely shaped virus diversity . Moreover , they have provided key information on the dynamics of evolutionary change , including the ‘time-dependent’ nature of viral evolution in which estimates of evolutionary rates are routinely elevated toward the present and decline toward the past due to a combination of unpurged transient deleterious mutations in the short-term and site saturation in the long-term [16] . A major challenge for any study of aDNA is the accumulation of post-mortem damage in the genome of interest . This damage includes fragmentation , nucleotide deamination , and polymerase-blocking lesions , such as molecular cross-linking , resulting from enzymatic and chemical reactions [17–19] . Certain environmental conditions , including desiccation of organic material and low ambient temperatures , can inhibit the activity of some endonucleases and environmental microorganisms , although oxidative and hydrolytic processes will continue to occur in all conditions at variable rates depending on the preservational context [20] . However , the predictably recurring forms of damage , such as the tendency for cytosine deamination to occur more often near the 3’ and 5’ ends of fragmented DNA molecules , also provide a means of addressing questions of contamination and inferring the authenticity of a recovered sequence through statistical pattern analysis [21] . For rapidly evolving genomes , such as those from viruses , phylogenetic analysis can provide another means of establishing provenance . In particular , as the rate of evolutionary change is high in many viruses [22] , “ancient” viruses should generally fall closer to the root of a tree than their modern relatives . However , a complicating factor is that any phylogenetic inference that aims to determine evolutionary rates or time-scale is only meaningful when the virus in question exhibits clear temporal structure in the phylogeny , such that it is evolving in an approximately clock-like manner over the time-scale of sampling . Because DNA viruses generally exhibit lower rates of evolutionary change than RNA viruses [22] , such populations tend to require larger sampling time-frames to discern temporal structure and clock-like behavior [16] . Hepatitis B virus ( HBV ) ( family Hepadnaviridae ) presents a compelling case of the complexities of analyzing the evolution of DNA viruses . Despite considerable effort , the evolutionary rate and time of origin of this important human pathogen remain uncertain , even though it is chronically carried by approximately 350 million people globally , with almost one million people dying each year as a result [23] . Particularly puzzling is that although HBV utilizes an error-prone reverse transcriptase ( RT ) for replication , estimates of its evolutionary rate are generally low , yet also highly variable . For instance , mean rate estimates of 2 . 2 × 10−6 nucleotide substitutions per site per year ( subs/site/year ) have been derived from long-term studies utilizing internal node calibrations on phylogenetic trees built using conserved regions of the viral genome [24] , while rates of up to 7 . 72 × 10−4 subs/site/year have been recorded within a single patient [25] and pedigree-based studies have returned mean rates of 7 . 9 × 10−5 subs/site/year [26] . As the highest evolutionary rates are observed in the short-term , this pattern is consistent with both relatively high background mutation rates and a time-dependent pattern of virus evolution in which rates are elevated toward the present due to incomplete purifying selection [16] . HBV is also genetically diverse , comprising ten different genotypes designated A–J , as well as additional subgenotypes within genotypes A–D and F [27] . Intra-genotypic sequence differences average 8% , while subgenotypes differ by an average of 4% [28 , 29] . The HBV genotypes differ in their geographic distributions . Genotype A is most prevalent in northwestern Europe and the United States , while genotypes B and C predominate in Asia , and genotype D in the Mediterranean basin , including Italy , as well as the Middle East and India . Similarly , genotype E is mostly seen in west Africa , genotype F in South and Central America , genotype G in the USA and France , and genotype H in Mexico and South America [30] . The more recently described genotype I has been identified in Vietnam [31] and Laos [32] , while the one example of genotype J was isolated from a Japanese sample [33] . With over half of the nucleotide coding for more than one protein , the physical constraints of the HBV genome are likely to have a major impact on evolutionary dynamics . Partially double-stranded , the relaxed-circular DNA genome averages ~3200 bp in length for the longer strand and ~1700–2800 for the shorter , comprised of four overlapping open reading frames ( ORFs ) . These ORFs encode seven proteins; the pre-core and core proteins , three envelope proteins ( small , medium , and large S ) , the reverse transcriptase ( RT , the polymerase ) , and an X protein that is thought to mediate a variety of virus-host interactions [34 , 35] . As noted above , the replication of HBV involves the use of RT , an enzyme that has no associated proofreading mechanism , such that mutational errors are expected to be frequent [25 , 36] . However , due to the overlapping ORFs , many mutations are likely to be non-synonymous and therefore purged by purifying selection . Considerable uncertainty remains as to when HBV entered human populations and when it differentiated into distinct genotypes . Given its global prevalence and the presence of related viruses in other mammals including non-human primates , it is commonly believed that the virus has existed in human populations for many thousands of years [37] . Further , recent studies have shown that the long-term evolutionary history of the Hepadnaviridae is shaped by a complex mix of long-term virus-host co-divergence and cross-species transmission [38] . Hence , it seems reasonable to conclude that HBV diversified within geographically isolated human populations following long-term continental migrations [24] . Ancient DNA has the potential to provide a new perspective on the evolutionary history of HBV . Notably , HBV has been sequenced from a Korean mummy radiocarbon dated to 330 years BP ( ±70 years ) which translates to ca . 1682 ( with an error range of 1612–1752 ) [12] . Phylogenetic analysis of this sequence ( GenBank accession JN315779 ) placed it within the modern diversity of genotype C , which is common to Asia . This phylogenetic position is compatible with low long-term rates of evolutionary change in HBV such that the virus has existed in human populations for many thousands of years , with genotypes diversifying over this time-scale [24] . However , the authenticity of this sequence , and hence the evolutionary time-scale it infers , remains uncertain , as deamination analysis and read length distribution were not reported as the data were generated using PCR based methodologies . In addition , as genotype C is common in modern Asian populations [37] and the mummy sequence ( JN315779 ) clusters closely with modern sequences , there is no phylogenetic evidence to support the historical authenticity of this sequence . For aDNA to resolve the origins of HBV it is of paramount importance to determine whether ancient samples can be used to calibrate the molecular clock to provide more accurate estimates of the time-scale of HBV origins and evolution . To this end , we report the detailed study of a complete HBV genome sampled from a 16th century Italian mummy .
We sampled the remains of an unidentified child mummy , approximately two years of age , found in the sacristy of the Basilica of Saint Domenico Maggiore in Naples , Italy , and exhumed between 1983 and 1985 [39] ( Fig 1 ) . This mummy is described in previous studies as mummy no . 24 ( NASD24 ) [40] . Radiocarbon ( 14C ) dating indicates that this mummy is 439 years old ( ± 60 years ) , thereby placing it to 1569 CE ± 60 years [39] . Evidence from the funerary context , including the dress style [41 , 42] , particularities of the mummification technique , and the known identities and historical records of other mummies , agree with this time frame [39 , 40] . Shotgun analysis of a suite of mummified remains from this site showed that one of the remains , those of NASD24 , yielded sequence reads mapping closely to viral sequences from the Hepadnaviridae ( Table 1 , S1 Table ) . Prior to the deposition in a coffin on a suspended passageway of the sacristy , the body of mummy NASD24 was eviscerated and embalmed . Records indicate the mummy was left undisturbed from 1594 [40] . Notably , the autopsy identified a diffuse vesiculopustular rash on the arm , body and face [40] . Paleopathological interpretation of this rash identified it as evidence of a possible smallpox infection . Electron microscopic images produced in an earlier study of pustular tissue homogenates from this mummy showed egg-shaped , dense structures , and positive results in immunostaining with protein-A/gold complex of ultrathin sections of pustular skin incubated with human anti-vaccinia-virus antiserum supported the presence of a poxvirus [43] . However , in our study , we also attempted SEM analysis of the tissue samples of NASD24 and did not find evidence of particles resembling either VARV or HBV in their dimensions , though did discover particles consistent with an unknown viral origin ( S1 Fig ) . We are uncertain how mummification ( or later processes involved in preservation and preparation for EM analysis ) may have affected the physical appearance of viral particles . We extracted total DNA from samples of the distal femur , skin attached to a rib , skin attached to the frontoparietal bone , thigh muscle , temporo-maxillary skin , and leg skin of mummy NASD24 ( Table 2 ) using a modified organic phenol-chloroform-isoamyl method [44] in dedicated aDNA facilities at McMaster University , Hamilton , Canada . These extracts were converted into double-stranded ( ds ) Illumina indexed sequencing libraries both with and without uracil DNA glycosylase ( UDG ) treatment and enriched for HBV , VARV , and mitochondrial genomes using in-solution bait sets . All libraries were sequenced on an Illumina HiSeq 1500 platform . We generated a total of 1 , 041 , 774 reads for the UDG-treated LM01 library ( distal femur ) and 3 , 869 , 248 for the non-UDG-treated LM01 library ( Table 2 ) . Of the trimmed and merged reads ( minimum 30 base pairs in length ) , 4 , 338 unique reads mapped to HBV D3 genotype X65257 from the UDG-treated library and 4 , 360 from the non-UDG-treated library . A smaller number of reads mapped to X65257 from other samples ( Table 2 ) . A total of 9610 reads from the distal femur , the skin attached to the rib and the fronto-parietal bone , the thigh muscle , the temporo-maxillary skin and the leg skin were pooled for further analysis using a consensus sequence ( Table 2 , S1 Table ) . We first mapped all next-generation sequencing reads to the HBV reference genome ( GenBank accession number NC_003977 ) using a dedicated aDNA pipeline [8] ( Table 2 , S1 Table ) . Using BLAST , as well as an initial phylogenetic analysis of a sequence data set representing the genotypic diversity of HBV ( Fig 2 , S2 Table ) , the consensus of the draft HBV genome was identified as a subgenotype D3 virus . This subgenotype has a broad global distribution and is common in the Mediterranean region , including Italy [30] . To ensure a proper consensus we remapped all reads to a subgenotype D3 HBV ( GenBank accession number X65257 ) . In addition we mapped all reads to the revised Cambridge reference sequence ( rCRS ) for human mtDNA ( GenBank accession number NC_012920 [45] ) and identified the consensus as haplogroup U5a1b ( S3 Fig ) . Haplogroup U5 is common in European populations and U5a1b is most commonly found in Eastern European populations but is also frequently seen in the Mediterranean region , including in Greek , Italian , Portuguese and Spanish populations [46] . The HBV genome used in all evolutionary analyses was assembled after pooling all the reads from the rib with skin , fronto-parietal bone with skin , thigh muscle , temporo-maxillary skin , leg skin , and the distal femur both with and without UDG treatment . This consensus genome is 3 , 182 nt in length and with a gap in the genome of 177 nt ( positions 1427–1603 ) near the 5’ end of the X ORF and in the region of overlap with the polymerase ORF ( S2 Fig ) . This gap is likely due to low bait coverage because of locally high G/C strand imbalance affecting oligo production , rather than the existence of a true biological gap due to a deletion . The first 5 nucleotides of the sequence mapped to X65257 were marked as ambiguous , as were the last 36 . A damage analysis of the mapped reads ( using the mapDamage 2 . 0 program [47] ) from the subsamples with and without UDG treatment revealed an authentic post-mortem damage pattern , as expected for an ancient sample ( Fig 3 , S4 Fig ) . If deamination had occurred within the lifespan of an infected host ( for instance , as a result of deamination induced by human enzyme APOBEC–known to induce deamination in certain viruses ) , then we would expect to have seen deamination spread more evenly throughout the viral reads . Instead , deamination has preferably occurred at the termini of DNA fragments as seen in the non-UDG treated subsamples ( Fig 3A ) . As nearly identical patterns were observed with the mitochondrial reads , this suggests to us that the HBV DNA is more likely to be of the same age as the mtDNA than it is to be derived from a recent contaminant ( Fig 3B ) . As expected , following treatment with UDG , viral reads do not display the deamination pattern , suggesting that the UDG did indeed remove uracils from the DNA fragments ( S4 Fig ) [48] . As it has been previously been suggested that the rash observed in this mummy is the direct result of a smallpox infection [43] , we enriched for VARV using an in-solution bait set previously published [8] . Importantly , we were unable to find a single significant read mapping to the VARV genome ( S1 Table ) . Although we cannot conclusively exclude the presence of VARV in this sample , this result does not lend credence to the presence of any VARV DNA when compared to the successful enrichment of HBV DNA , unless VARV is far more sensitive to degradation post mortem than HBV is , which does not agree with our recent success at enrichment of VARV from a Lithuanian child mummy [8] . We compiled two primary data sets representing ( a ) the full genotypic diversity of HBV and ( b ) that of D genotype alone ( Table 3 ) . These consisted of publicly available HBV sequence data from GenBank with the additions of the HBV genome newly sequenced here ( NASD24SEQ ) and that previously obtained from a Korean mummy ( JN315779 ) ( S2 Table ) . No evidence of recombination was found within either ancient sequence . While there was evidence for recombination in some modern sequences , equivalent results were found in evolutionary analyses conducted with and without these sequences , indicating that recombination has not had a major impact on the phylogenetic results presented here . Maximum likelihood phylogenetic analysis revealed that the Italian sequence ( NASD24SEQ ) and the previously published Korean sequence ( JN315779 ) fall within the genetic diversity of modern HBV [12] . Specifically , in phylogenetic analysis of HBV sequences representative of all genotypes ( data set a-ii ) , NASD24SEQ grouped with modern HBV sequences of the D3 subgenotype collected between 1985 and 2008 with 100% bootstrap support , falling on the branch separating D3 from the D1 and D2 subtypes ( Fig 4 ) . Similarly , JN315779 fell within the genetic diversity of HBV genotype C , on the branch separating subgenotype C2 ( Fig 4 ) . Importantly , NASD24SEQ occupied similar phylogenetic positions within subgenotype D3 when phylogenies were inferred separately for the overlapping and non-overlapping regions of the HBV genome , as well as for the polymerase ORF alone ( S5 Fig ) . Hence , there is no evidence that the grouping of NASD24SEQ with modern subgenotype D3 sequences is a function of genome overlap . The sequence recovered from this 16th century Italian mummy therefore occupies a paradoxical phylogenetic position: although it exhibits legitimate signs of DNA damage , consistent with both the pattern seen in the mitochondrial reads and an ancient origin , it clusters closely with modern HBV sequences , as might be expected if it were a recent contaminant . If the NASD24SEQ sequence is bona fide , then the only reasonable explanation is that our data set representing the last 450 years of HBV evolution is of insufficient duration to exhibit temporal structure , in turn implying that HBV has a long evolutionary history in humans with ancient diversification times of the different viral subtypes . To test this hypothesis , we performed a detailed analysis of HBV evolutionary dynamics , focused on addressing whether the evolution of this virus presents sufficient temporal structure for molecular clock dating . To help determine the veracity of our ancient HBV sequence , we performed a series of analyses using both root-to-tip regression [49] as well as those within a Bayesian framework [50] . These analyses employed various calibrations , including collection dates for modern samples , radiocarbon-dating estimates of ancient samples , and viral co-divergence with human population migration . Data sets must possess temporal structure for tip-dated analyses to be informative [51] . We first assessed for temporal structure using a regression of root-to-tip genetic distances against year of sampling [49] . Strikingly , neither of the primary data sets ( subsets a and b ) , with or without inclusion of ancient sequences , showed evidence of any temporal structure , with R2 values of 8 . 98 × 10−4 ( a-ii ) and 2 . 78 × 10−2 ( b-i ) , respectively ( Fig 5 ) , as was true of the other genomic data sets ( S6 Fig ) . Similarly , no temporal structure was observed in the D3 subgenotype , both without ( R2 = 2 . 85 × 10−2 ) and with NASD24SEQ ( R2 = 2 . 90 × 10−2 ) ( S7 Fig ) . To further assess the extent of temporal structure , we employed a Bayesian date-randomization test in which the nucleotide substitution rate is estimated using the correct sampling dates ( see below ) , and the analysis then repeated 20 times on data sets in which the sampling dates have been randomized among the sequences [52] . Notably , for both ancient sequences ( NASD24SEQ and JN315779 ) , the 95% higher posterior density interval ( HPD ) of the rate overlapped between the true and randomized data for both the modern and complete ( including ancient ) data sets ( Fig 6 and S8 Fig ) . This indicates that there is insufficient temporal structure in HBV to performed tip-date-based analyses of evolutionary dynamics , even when including sequences that date to the 16th century . Because of the lack of temporal structure from tip-dated calibrations , we next specified an informative prior distribution on the clock rate using a previous estimate of the long-term substitution rate of HBV at 2 . 2 × 10−6 subs/site/year [24] . The aim here was to estimate the ages of NASD24SEQ and JN315779 and determine whether these estimates matched the dates inferred from radiocarbon dating of the mummies and tomb materials . To this end , we tested ( i ) a prior of 2010 on the ages of both modern and ancient samples , such that no true sampling dates were considered , ( ii ) a uniform prior on the age of the ancient samples with a lower bound of 0 . 0 and an upper bound of 10 , 000 years before present , and ( iii ) a normal prior matching the radiocarbon dates . For both the ‘modern’ prior of 2010 and the uniform prior calibrations we expect that the posterior will differ from the prior if the rate calibration and the molecular sequence data are informative about the ages of the samples . Importantly , the inclusion of this long-term substitution rate as a clock calibration resulted in estimated ages for the ancient samples that were very similar to each of the priors tested in each case ( S9A Fig ) . Hence , the molecular sequence data and rate calibration do not have sufficient information to estimate the age of these sequences . Similarly , we attempted to estimate the sampling times of NASD24SEQ and JN315779 under the assumption that HBV has co-diverged with human populations . Internal node calibrations can be more informative than tip calibrations when no temporal structure can be ascertained using sampling dates [51 , 53] . Accordingly , we used the same calibration scheme as Paraskevis et al . 2015 , in which human migration dates were used to specify the prior distributions of ages on nodes from HBV subgenotypes found in endemic populations [24] . Our analysis using subset a-ii yielded a mean rate estimate of 6 . 84 × 10−6 subs/site/year ( 95% HPD: 4 . 46 × 10−6 to 9 . 26 ×10−6 subs/site/year ) , which is considerably lower than some previous estimates [25] . Critically , however , the age estimates for the ancient samples again matched the uniform prior distributions ( S9B Fig ) . Therefore , even with internal node calibrations , the sequence data and calibrations were not sufficiently informative to estimate the age of these viral sequences . Finally , we employed a second set of internal node calibrations in which the node separating the F and H genotypes from the rest of the HBV tree had a normal prior with a mean of 16 , 000 years and a standard deviation of 1000 years . This follows a study that estimated that humans may have entered the Americas about 16 , 000 years ago , a more specific date than that estimated in earlier papers [54] . Using this more precise calibration , our analysis resulted in a mean rate estimate of 4 . 57 ×10−6 subs/site/year ( 95% HPD: 2 . 62 × 10−6 to 6 . 96 ×10−6 subs/site/year ) similar to that obtained from the node calibrations employed above . The mean ages of the ancient samples were accordingly estimated at 214 years for JN315779 ( 95% HPD: 23 to 398 ) and 276 years ( 95% HPD: 29 , 505 ) for NASD24SEQ ( Fig 7 ) . However , due to the very wide uncertainty in these estimates , which again closely resemble the prior distributions , these results do not provide posterior estimates that are conclusive on the age of the HBV samples .
We have enriched and sequenced a complete HBV genome from the remains of a mummified child estimated to have died in 1569 CE ± 60 years [39] . The cytosine deamination patterns occurring preferentially at termini in both the viral and mitochondrial DNA fragments support the ancient authenticity of these sequences . We have also subtyped the mitochondrial DNA from the mummy to haplogroup U5a1b , a common European haplogroup [46] , and the HBV to genotype D , a genotype predominant in the Mediterranean region today [30] . The nearly identical fragment length distributions , deamination patterns and same geographically recovered haplotypes ( mitochondrial and HBV ) argue for the authenticity of the sequences . The identification of consistent HBV reads in multiple ( 5 ) tissue samples ( distal femur , fronto-parietal bone with skin , thigh muscle , temporo-maxillary skin and leg skin; Table 2 ) , suggests that the virus is distributed throughout the mummy and not in one location , as might be expected with contamination . Further , other mummies from the same site , excavated at the same time and processed in the same facilities , did not show any HBV reads in shotgun sequencing data . Thus , if the mummy was contaminated , it was specific to this one sample alone . Although hepadnaviruses like HBV exhibit strong tropism for liver cells ( hepatocytes ) , hepadnaviral DNA has been shown to exist in other somatic cells , including mononuclear cells , which are protected by the hydroxalite matrix of the bone [55–57] . HBV particles produced in the bone marrow and protected by the matrix may explain why we recovered the majority of our viral sequences from a femur sample . DNA isolated from ancient bone matrix has also been shown to be better preserved and less damaged than that recovered from corresponding soft tissue from the same remains [58] . Many reports of ancient epidemics and other disease outbreaks have relied upon historical reporting and paleopathological studies of human remains . Recent advances , including next generation sequencing technology [5] and DNA enrichment methods [6] , now allow recovery of ancient nucleotide sequences from these remains and the genetic verification of the pathogens responsible for disease , as well as the identification of pathogens undetectable by other means . Our study provides a strong argument for this latter approach , as mummy NASD24 was originally reported to have been infected with smallpox [40 , 43]; crucially , however , shotgun sequencing following enrichment for VARV ( S1 Table ) and SEM analysis ( S1 Fig ) revealed no evidence of VARV in this mummy . This is particularly surprising given previous results in which electron microscopy studies and immunostaining indicated the presence of VARV particles in these samples [43] . Given our results , a new interpretation is that the child was not suffering from smallpox at the time of death , but rather Gianotti-Crosti syndrome caused by HBV infection [59] . Gianotti-Crosti syndrome is a rare clinical outcome of HBV that presents as a papular acrodermatitis in children between 2 and 6 years old [59] . This , in turn , illuminates the power of aDNA in providing definitive evidence or clarifying retrospective diagnoses , where etiology may be uncertain and morphology complicated for key type specimens that provide critical time points for the origins or presence of specific pathogens ( e . g . smallpox ) . Despite the multiple streams of evidence supporting an ancient origin of NASD24SEQ , the results of the evolutionary analysis are less straightforward . In particular , our phylogenetic analysis reveals a close relationship between NASD24SEQ and modern D genotype sequences , as would be expected if the sequence were a modern ( 1980s ) contaminant , and we note the same phenomenon with the Korean mummy sequence thought to date from the 17th C [12] . Importantly , however , data sets representing only modern HBV sequences , sampled over 50 years to the present , did not display discernible temporal structure . Clearly , without temporal structure we cannot accurately estimate the age of the ancient sequences using phylogenetic methods . Hence , the apparently paradoxical phylogenetic position of NASD24SEQ cannot automatically be taken to mean that this genome is a modern contaminant . In turn , if NASD24SEQ is indeed from the 16th century , then this phylogenetic pattern indicates that the diversification of the HBV genotypes occurred prior to 1500 and that any subsequent accumulation of diversity was either lost through strong purifying selection or masked by multiple substitutions . Our analyses of both modern and ancient HBV samples returned results consistent with the absence of temporal structure , not only within the full diversity of HBV but also within the D genotype and D3 subgenotype . Given that the genomic structure of HBV is likely to result in strong selective constraints , a likely explanation for our results is that many of the mutations that arise in the short-term , such as within chronically infected hosts or along single chains of transmission , are non-synonymous and eventually removed from the HBV population by purifying selection , yet artificially inflating evolutionary rates over this sampling period [60] . Support for this hypothesis comes from short-term studies of HBV evolution in which rates of evolutionary change are greater than those estimated from longer-term studies [24 , 25] . On balance , our analysis suggests that our HBV sequence is authentically 16th century and that no temporal structure is observable in over 450 years of HBV evolution . As such , these results have a number of important implications for the study of HBV evolution . In particular , such a phylogenetic pattern implies that the currently circulating viral genotypes must have been associated with their specific host populations long before the 16th century , and hence supports a long association of HBV with human populations . In addition , the lack of temporal structure means that it is not possible to use molecular clock methods to reliably date HBV evolution over the time span of genome sequences currently available .
Exploration of the coffin and the autopsy of the unidentified two-year old mummy ( NASD24 ) was completed as part of a larger study , conducted between 1984 and 1987 , of mummies from the sacristy of the Basilica of Saint Domenico Maggiore in Naples , Italy . Autopsies were performed for all mummies by paleopathologists wearing sterile surgical coats , sterile latex gloves , sterile masks , headdresses and overshoes . Details of this initial investigation have been reported previously [40] . The samples used for aDNA sequencing were collected during the initial autopsies , and these samples were stored in sealed , sterile plastic bags . The sample bags were first opened in 1985 for a preliminary paleopathological examination that suggested a viral agent , thought to be smallpox , as the likely cause of an apparent skin rash [40] . The samples were handled again in 1986 for examination by immune-electron microscopy [43] . After this , the samples remained in sterile storage before subsamples were removed and sent to the McMaster Ancient DNA Centre at McMaster University ( Hamilton , Ontario , Canada ) in 2013 . Ethical approval to work on this mummy sample was granted to Dr . Fornaciari by the Supervisor for the Artists and Historians of Campania in 1984 . Eight subsamples of 75–125 mg of organic matter were excised from samples of various body parts of NASD24 in a dedicated cleanroom facility at the McMaster Ancient DNA Centre ( Table 1 ) . Tissue samples were cut into small pieces using a scalpel and bone material crushed into powder . These samples were then demineralized , digested , and extracted according to previously published protocols [44] . In brief , samples were demineralised using 1 . 5 mL of EDTA ( 0 . 5 M , pH 8 . 0 ) before being incubated at room temperature for 24 hours with rotation at 1000 rpm . Samples were then digested using a 1 . 5 mL proteinase K solution and incubated at 55°C for 6 hours with rotation at 1000 rpm . The supernatant from the demineralization and digestion steps was subjected to organic extraction using a modified phenol-chloroform-isoamyl ( PCl ) alcohol protocol . This means that 0 . 75 mL of PCl ( 25:24:1 ) was added to the demineralization/digestion supernatant , which was then vortexed , and spun via centrifuge ( 4000×g ) for 20 minutes . The aqueous phase was transferred to a fresh tube and a further 0 . 75 mL of chloroform added . The solution was mixed and spun via centrifuge ( 4000×g ) for 10 minutes . The aqueous phase was collected and concentrated using an Amicon Ultra 0 . 5 mL 30 kDa filter . This concentrated solution was purified over a MinElute column ( Qiagen , Hilden , Germany ) according to the manufacturer’s instructions , and eluted in 10 μL of 0 . 1 TE with 0 . 05% Tween-20 . Reagent blanks were introduced at each step and processed alongside the samples . A library from the distal femoral sample of NASD24 was prepared according to a previously published protocol [61] that was modified to include an overnight ligation and with an input volume of 5 μL . Double indexing was performed using KAPA SYBR FAST ( Kapa Biosystems ) for 8 cycles of indexing amplification [62] . The library and blanks were enriched using two rounds of in-solution capture baits targeting HBV and the human mitochondrial genome ( in separate reactions ) according to the manufacturer’s instructions ( Mycrorarray , MyBaits ) with recommended aDNA modifications . Baits , of 80nt in length with 4x tiling density ( 10nt flexible spacing ) , were designed based on the sequences of 5 , 230 HBV sequences , representing all major viral subtyptes . Template input was 5 μL for each reaction and bait concentrations were 100 ng per reaction using the in-solution bait mix targeting HBV and 50 ng per reaction for that targeting human mtDNA . Target genetic material was reamplified for 12 cycles both between and after rounds of enrichment . The HBV-enriched library generated 1 , 934 , 624 clusters ( 3 , 869 , 248 raw reads ) and the human mtDNA-enriched library generated 2 , 844 , 500 clusters ( 5 , 689 , 000 raw reads ) on an Illumina HiSeq 1500 at the Farncombe Metagenomics Facility ( McMaster University , Hamilton Ontario , Canada ) . Reads were demultiplexed using CASAVA-1 . 8 . 2 ( Illumina , San Diego , California ) , then adapters were trimmed and reads merged using leeHom [63] with aDNA specific settings ( —ancientdna ) . These processed reads were mapped to an appropriate reference genome ( HBV genotype D3 , GenBank accession X65257; revised Cambridge Reference Sequence for human mtDNA , GenBank accession number , NC_012920 ) using a network-aware version of the Burrows-Wheeler Aligner [64] ( https://bitbucket . org/ustenzel/network-aware-bwa ) with distance , gap and seed parameters as previously described [8] . Duplicates were removed based on 5’ and 3’ positions ( https://bitbucket . org/ustenzel/biohazard ) . Reads shorter than 30 base pairs and with mapping quality less than 30 were removed using Samtools [65] . The resulting BAM files were processed using mapDamage 2 . 0 on default settings with plotting and statistical estimation [47] . Haplogrep v2 . 1 . 0 [66] using PhyloTree Build 17 [67] was used to identify the haplogroup of the mtDNA as U5a1b . The complete genome sequence of NASD24 has been submitted to GenBank and assigned accession number MG585269 . We analyzed the ancient HBV sequenced in this study in the context of modern whole-genomes of HBV . To this end we downloaded all human HBV genomes from GenBank that were over 3 , 000 nt in length and for which the year of sampling was available ( all date information , including month and day , if available , was converted into decimal format ) . This initial GenBank data set comprised 3 , 696 sequences sampled between 1963 and 2015 ( S2 Table ) . Sequences were aligned with the MAFFT v7 program using the FFT-NS-1 routine [68] to visually check for obvious errors in database labeling . For initial genotypic subtyping , one representative of each HBV subtype was selected ( S3 Table ) . This data set included the purportedly ancient HBV sequence previously obtained from a 17th century Korean mummy ( GenBank accession number JN315779; radiocarbon-dated to 1682 with an error range of 1612–1752 [12] ) . The subtype of NASD24SEQ was inferred from maximum likelihood ( ML ) phylogenetic trees estimated using PhyML v3 . 0 [69] with the GTR+Г4 model of nucleotide substitution and employing SPR branch-swapping , with nodal support assessed by conducting 1000 non-parametric bootstrap replicates . Following this , a random subsample of HBV sequences was taken using the Ape package in R [70] . Specifically , we sampled five representatives of each genotype and subtype , or the maximum number available if this was not five . This produced a data set of 135 sequences sampled between 1963–2013 which we refer to as subset a ( Table 3 , S4 Table ) . We then added the ancient Italian HBV sequence NASD24SEQ to subset a to make data set a-i with n = 136 . A third subset was built by adding the ancient Korean HBV sequence JN315779 to a-i to make subset a-ii with n = 137 . We next randomly sampled only D genotype sequences from the initial GenBank data set to form subset b with n = 64 ( S5 Table ) . To this we added NASD24SEQ , generating subset b-i with n = 65 . We aligned the nucleotide sequences in each subset using the L-INS-i routine in MAFFT v7 . The RDP , GENECOV , and MAXCHI methods available within the RDP v4 package [71] , with a window size of 100 nt ( and default parameters ) , were used to analyze each subset for recombination . If at least two methods detected recombinant regions in a sequence , then we removed the region of recombination from the alignment . Following removal of recombinant regions , we inferred phylogenetic trees on each subset ( of a and b ) again using the ML method in PhyML v3 . 0 [69] with the GTR+Γ4 model of nucleotide substitution and employing SPR branch-swapping , and with nodal support assessed by conducting 1000 non-parametric bootstrap replicates . We conducted a range of analyses to assess the extent of temporal structure in the data sets and to estimate the rate and time-scale of HBV evolution . To initially verify the temporal structure in the data we conducted regressions of root-to-tip genetic distance as a function of the sampling time ( year ) using TempEst v0 . 1 [49] . We then conducted a date-randomization test [52] . This involved analyzing the data using the Bayesian method implemented in BEAST v1 . 8 . 3 [50] under a lognormal relaxed clock model [72] and assuming a constant population size , with 20 replicates in which the sampling dates were randomized among the sequences . The HBV data were considered to have temporal structure if the mean rate estimate and 95% HPD intervals were not contained within the 95% HPD of any of estimates resulting from the randomized data sets [52] . All analyses were run with an Markov chain Monte Carlo chain length of 107 steps with samples from the posterior distribution drawn every 2 × 103 steps . After discarding the first 10% of steps as burn-in , we assessed sufficient sampling from the posterior by visually inspecting the trace file and ensuring that the effective sample sizes for all parameters were at least 200 . We also performed more detailed Bayesian estimates of evolutionary dynamics . One method of validating the ages of ancient samples is to specify uninformative prior distributions for these and test if the tip dates or an informative rate give the postulated ages . Specifically , if the sequence data and calibrations are informative , the posterior should consist of a narrow distribution that includes the true sampling time of the ancient samples [73] . However , if the prior and posterior distributions for the ages of the ancient samples are the same , then the data are considered to have insufficient information to estimate the ages of these samples . We conducted these analyses by setting uniform distributions for the two ancient samples between 0 and 107 with a mean of 105 . For the Korean sample we set a uniform prior with upper and lower bounds of 400 and 0 , whereas for the Italian sample we used 507 and 0 , with the maximum values in both cases reflecting their presumed sampling date . We also conducted analyses in which , for the Italian sample , we set a normal truncated prior distribution with the upper and lower values at 507 and 387 , and mean of 447 and standard deviation of 10 , whereas for the Korean sample we used 400 and 260 for the bounds , and 330 and 35 for the mean and standard deviation , respectively . These numbers are based on the radiocarbon dating analysis , with the upper and lower values reflecting the error margins . We employed three calibration strategies for these analyses . ( i ) First , we used the sampling times of the modern samples , but with a uniform prior for the rate bounded between 0 and 1 . ( ii ) Second , we assumed that all the modern samples were contemporaneous , and specified internal node calibrations , corresponding to those used by Paraskevis et al . ( 2015 ) and Llamas et al . ( 2016 ) [36 , 54] and assuming that the spread of HBV corresponds to that of early human populations [24 , 36] . These consisted of setting normal priors for the time of the most recent common ancestor ( tMRCA ) of F and H genotypes at 16 , 000 years , with a standard deviation of 1000 , setting the tMRCA for subgenotype B6 at 3500 years with a standard deviation of 3000 , and setting the tMRCA for subgenotype D4 at 8500 years with a standard deviation of 3500 . ( iii ) Finally , we calibrated the molecular clock by specifying an informative prior for the mean ( long-term ) substitution rate of HBV based on the estimate by Paraskevis et al . ( 2013 ) [24] . Accordingly , the prior was in the form of a normal truncated prior with mean of 2 . 2 × 10−6 sub/site/year , standard deviation of 5 × 10−7 , and lower and upper bound of 1 . 5 × 10−6 and 3 × 10−6 , respectively . We again used the GTR+Γ4 nucleotide substitution model for these analyses , although a codon-partitioned HKY model was also considered within the internally calibrated analysis . | Hepatitis B virus ( HBV ) exerts formidable morbidity and mortality in humans . We used ancient DNA techniques to recover the complete genome sequence of an HBV from the mummified remains of a child discovered in the 16th century from Naples , Italy . Strikingly , our analysis of this specimen resulted in two contrasting findings: while the damage patterns lend credence to this HBV sequence being authentically 16th century , phylogenetic analysis revealed a close relationship to recently sampled viruses as expected if the sequence were a modern contaminant . We reconcile these two observations by showing that HBV evolution over the last ~450 years is characterized by a marked lack of temporal structure that hinders attempts to resolve the evolutionary time-scale of this important human pathogen . | [
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] | 2018 | The paradox of HBV evolution as revealed from a 16th century mummy |
Pituitary tumors are common intracranial neoplasms , yet few germline abnormalities have been implicated in their pathogenesis . Here we show that a single nucleotide germline polymorphism ( SNP ) substituting an arginine ( R ) for glycine ( G ) in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production . Compared with FGFR4-G388 mammosomatotroph cells that support prolactin ( PRL ) production , FGFR4-R388 cells express predominantly growth hormone ( GH ) . Growth promoting effects of FGFR4-R388 as evidenced by enhanced colony formation was ascribed to Src activation and mitochondrial serine phosphorylation of STAT3 ( pS-STAT3 ) . In contrast , diminished pY-STAT3 mediated by FGFR4-R388 relieved GH inhibition leading to hormone excess . Using a knock-in mouse model , we demonstrate the ability of FGFR4-R385 to promote GH pituitary tumorigenesis . In patients with acromegaly , pituitary tumor size correlated with hormone excess in the presence of the FGFR4-R388 but not the FGFR4-G388 allele . Our findings establish a new role for the FGFR4-G388R polymorphism in pituitary oncogenesis , providing a rationale for targeting Src and STAT3 in the personalized treatment of associated disorders .
Pituitary tumors occur in almost 20% of the population [1] and represent nearly 10% of surgically resected intracranial tumors [2]–[3] . They can cause significant health problems due to abnormal hormone production and invasion into surrounding brain structures [2]–[3] . However , the mechanisms underlying the development of sporadic pituitary tumors that rarely involve mutations of classical oncogenes or tumor suppressor genes remain to be clarified [2]–[3] . Indeed , the only consistent molecular event reported thus far is activating mutations of the G-protein coupled Gsα that occurs in a subset of somatotroph adenomas [4]–[5] . Germline genetic abnormalities associated with pituitary tumor pathogenesis include inactivating mutations of menin in patients with Multiple Endocrine Neoplasia type 1 [6]–[7] , loss of function mutations of the aryl hydrocarbon receptor-interacting protein ( AIP ) tumor suppressor gene in patients with familial isolated pituitary adenomas [8] , and activating mutations the Protein kinase A type I regulatory subunit PRKA [9] in patients with Carney complex , however these alterations have not been shown to mediate pituitary neoplastic growth in the more common sporadic neoplasms . Evidence suggests that epigenetically controlled growth signals implicated in pituitary development may be relevant to the tumorigenic processes in this gland [10]–[11] . Of note members of the fibroblast growth factor ( FGF ) and FGF receptor families have been proposed as candidate effectors , given their recognized importance in pituitary organogenesis [12]–[13] . FGF signaling is critical in pituitary development . Deletion of FGF10 or its receptor , the FGFR2 IIIb isoform , leads to failure of pituitary development [13] . Mid-gestational expression of a soluble dominant-negative FGFR results in severe pituitary dysgenesis [14] . FGF ligands are over-expressed in pituitary tumors . FGF-2 , originally described in bovine pituitary folliculostellate cells , regulates multiple pituitary hormones and is over-expressed by human pituitary adenomas tumors [15] . We identified altered FGFR4 expression in pituitary tumors [16] due to expression of an N-terminally deleted isoform , pituitary tumor-derived FGFR4 ( ptd-FGFR4 ) [17] generated by alternative transcription initiation from a cryptic promoter [18]–[19] . Prototypic FGFR4 ( FGFR4-G388 ) is a 110 kD membrane-anchored protein expressed in several endocrine cells including the normal pituitary . In contrast , ptd-FGFR4 is a cytoplasmic protein expressed in pituitary tumors . The invasive tumorigenic potential of ptd-FGFR4 , but not full length FGFR4 , was demonstrated by targeted pituitary expression in transgenic mice [17] . The basis for the contrasting functions between these FGFR4 isoforms relates to their differential ability to associate with neural cell adhesion molecule ( NCAM ) and engage N-cadherin [20] . These studies were all carried out with the prototypic receptor prior to the identification of a single nucleotide polymorphism ( SNP ) that alters the coding region of the transmembrane domain . This germ-line polymorphism substitutes a glycine with an arginine at codon 388 of FGFR4 , resulting in a charged amino acid in the highly conserved and normally hydrophobic transmembrane region of the receptor [21] . This FGFR4-R388 allele has been linked with advanced [21] and treatment-resistant breast cancer [22] , prostate cancer [23] , sarcomas [24] , and head and neck carcinomas [25] . The mechanisms underlying FGFR4-R388 actions remain unclear . In this report we identify distinct signaling and hormone regulatory properties that distinguish FGFR4-R388 from the prototypic FGFR4-G388 form . The data unmask important patho-physiologic consequences of this common SNP with therapeutic implications for related diseases .
To determine if the FGFR4 polymorphic isoforms possess distinct functional properties in hormone-producing pituitary cells , we compared the effects of FGFR4-G388 and FGFR4-R388 on pituitary hormone production in rat GH4 mammosomatotroph cells that co-express prolactin ( PRL ) and growth hormone ( GH ) and in PRL235 cells that express PRL only . These GH4 and PRL235 cells express endogenous FGFR4 ( Figure S1 ) and are homozygous for FGFR4-G385 , the rodent equivalent of the human 388 site . Expression of human FGFR4-G388 or FGFR4-R388 to comparable levels shows that FGFR4-G388 enhances PRL and suppresses GH expression whereas FGFR4-R388 increases GH production with a reciprocal effect on PRL ( Figure 1a , 1b ) . To determine the effects of these FGFR4 isoforms on cell growth , stably transfected cells were plated in soft agar and examined for colony formation . GH4 cells expressing FGFR4-R388 were more efficient at forming colonies in soft agar compared with their FGFR4-G388 counterparts ( Figure 1c; left ) . Enhanced colony formation resulting from FGFR4-R388 compared to FGFR4-G388 was also noted in PRL235 pituitary cells ( Figure 1c; right ) . The FGFR4-R388 substitution does not alter receptor kinase activity [21] and ( data not shown ) . Thus , to examine signaling differences induced by the two FGFR4 isoforms in pituitary cells , we compared the ability of FGF to promote phosphorylation of the immediate FGFR substrate FRS2α . In contrast to FGFR4-G388 which showed ligand-dependent stimulation of this docking protein , FGFR4-R388 cells displayed enhanced FRS2α phosphorylation ( Figure 2a ) . Src phosphorylation at Y416 was also appreciably higher in cells expressing FGFR4-R388 compared to those expressing FGFR4-G388 while Src phosphorylation at Y527 remained unchanged . To examine the functional significance of this finding , we compared the ability of the Src inhibitor , dasatinib , to impede pituitary tumor cell growth and hormone production . Dasatinib effectively diminished Src phosphorylation ( Figure 2b ) and significantly inhibited colony formation in soft agar of cells expressing FGFR4-R388 ( Figure 2c ) . By comparison , the less efficient colony forming FGFR4-G388 cells were relatively insensitive to the Src inhibitor ( Figure 2c ) . In contrast to the impact on cell growth , pharmacologic Src inhibition did not alter GH or PRL hormone expression ( Figure 2b ) . Additionally , siRNA-mediated Src down-regulation did not significantly affect GH ( Figure 2d ) or PRL levels ( data not shown ) . These findings suggested that while Src may play a role in driving FGFR4-R388-mediated cell growth , Src signaling may not be intimately coupled with pituitary hormone regulation in these cells . STAT activation is implicated in mediating the effects of FGFR3 mutations associated with thanatophoric dysplasia [26] . We , therefore , examined the ability of the two FGFR4 isoforms to activate STAT signaling . Figure 2a depicts the differential impact of the FGFR4 isoforms on their ability to phosphorylate STAT3 . While FGFR4-G388 supported ligand-induced tyrosyl phosphorylation of STAT3 , this effect was not shared with FGFR4-R388 , which instead resulted in sustained STAT3 serine phosphorylation at S727 ( Figure 2a ) . STAT1 and STAT5 modifications were not affected by either FGFR4 isoform in GH4 or PRL235 cells ( data not shown ) . In contrast to the nuclear residence of pY-STAT3 , pS-STAT3 translocates to the mitochondria where it has been implicated in cellular metabolism [27] . Thus , we performed immunofluorescence to localize pS-STAT3 . Figure 3a identifies the mitochondrial residence of pS-STAT3 in FGFR4-R388; FGFR4-G388 cells are almost negative ( upper panels ) . As controls , we expressed a constitutively active serine form of STAT3 ( STAT3-S727D ) in GH4 cells , and this also co-localized to the mitochondria ( Figure 3a ) . In contrast , an inactive serine form of STAT3 ( STAT3-S727A ) failed to show a mitochondrial signal ( Figure 3a ) . Subcellular fractionation followed by western blotting supported these findings with prominent mitochondrial expression of STAT3 and pS-STAT3 in FGFR4-R388 and in STAT3-S727D control but not in FGFR4-G388 cells in both GH4 and PRL235 cells ( Figure 3b ) . To examine the impact of pS-STAT3 on mitochondrial function , we measured Cytochrome C oxidase activity in pituitary cells expressing the different FGFR4 isoforms ( Figure 3c ) . FGFR4-R388 cells which displayed higher pS-STAT3 levels also demonstrated higher Cytochrome C oxidase activity than FGFR4-G388 cells . In addition , lactate dehydrogenase ( LDH ) levels in FGFR4-R388 cell lysates were higher ( 2612 IU/ml ) than those of the FGFR4-G388 ( 1197 IU/ml; n = 3 ) . To examine the functional impact of distinct STAT3 modifications on pituitary cells we compared growth in soft agar of cells expressing various STAT3 expression vectors ( Figure S2 ) . Of the STAT3 modifications , the active serine ( STAT3-S727D ) form displayed the greatest positive impact on colony formation ( Figure S2 ) . To determine whether Src and STAT3 serine phosphorylation were inter-dependent , we examined the impact of pharmacologic Src inhibition . To this end , dasatinib-mediated inhibition of Src phosphorylation also reduced pS-STAT3 in FGFR4-R388 GH4 cells ( Figure 2b ) . Moreover , siRNA-mediated Src down-regulation also resulted in diminished pS-STAT3 levels in these cells ( Figure 2d ) . Additionally , treatment with the protein kinase C inhibitor G06983 reduced pS-STAT3 whereas the protein kinase A inhibitor H89 had no effect ( data not shown ) . To determine whether differential STAT3 responses were responsible for altered hormone gene expression , we compared the hormonal responses of FGFR4-G388 and FGFR4-R388 cells to a panel of growth factors . FGFR4-G388 cells with intact pY-STAT3 responses exhibited the expected GH inhibition in the presence of IGF-1 and related growth factors and the expected PRL increase in response to growth factors ( Figure 4a ) . In contrast , and consistent with their attenuated pY-STAT3 responses , FGFR4-R388-expressing cells failed to suppress GH or mount a PRL response ( Figure 4a ) . In complementary experiments we down-regulated STAT3 in FGFR4-G388 cells . This forced STAT3 reduction resulted in increased GH and reduced PRL ( Figure S3 ) . Conversely , forced expression of STAT3 induced PRL and diminished GH expression ( Figure 4b , 4c ) . Moreover , introduction of the dominant negative STAT3-Y705F diminished IGF-1 inhibition of GH ( Figure 4b , 4c ) . The STAT3-Y705F mutant also failed to stimulate PRL expression further underscoring the requirement for pY-STAT3 in mediating PRL induction . In contrast to the impact of pY-STAT3 , introduction of the constitutively active serine STAT3-S727D or the serine inactive STAT3-S727A did not alter GH or PRL responses ( Figure 4b , 4c ) . Taken together , these findings suggest that pY-STAT3 , but not pS-STAT3 , plays a more important role in pituitary GH and PRL regulation . To determine if the observed cellular actions of FGFR4 polymorphism translate into biologically relevant actions on pituitary cell growth and function , we examined mice with knock-in ( KI ) of the mouse homologue of the polymorphism , Fgfr4-R385 . Importantly , introduction of this SNP does not alter Fgfr4 expression levels [28] and ( Figure S1 ) . Systematic examination of the pituitary glands from mice carrying the Fgfr4-R385 allele at different ages identified the presence of pituitary tumors by 12 months of age . This revealed increased cellularity with loss of the reticulin network representing the hallmark of true neoplasia in this gland ( Figure 5a–5d ) . Importantly , unlike the more common prolactinomas which are seen sporadically in aging mice [29] , hormone staining identified GH ( Figure 5e ) but not PRL ( Figure 5f ) production by these tumors . Further , we examined pS-STAT3 in pituitary tissue from the knock-in mouse model . Fgfr4-R385 KI mice displayed strong immunoreactivity for pS-STAT3 ( Figure 5g ) which was not noted in control Fgfr4-G385 mice ( Figure 5h ) . Double staining localized this pS-STAT3 in GH- immunoreactive somatotrophs ( Figure 5i ) but not in other cell types such as FSH-immunoreactive gonadotrophs ( Figure 5j ) . The frequency of these pituitary tumors and their morphologic phenotypes according to Fgfr4 genotype are summarized in Figure 5k . No GH-containing pituitary tumors were detected in control littermates ( Figure 5k ) . To corroborate the pituitary phenotypic abnormalities we compared circulating levels of the GH target growth factor IGF-1 . Shown in Figure 5l is the positive impact of the Fgfr4-R385 allele on circulating IGF-1 levels . In contrast , and consistent with the in vitro data , mice carrying the Fgfr4-R385 allele did not demonstrate high PRL levels , and instead showed a tendency to lower concentrations compared to their Fgfr4-G385 littermates ( Figure 5m ) . Given the ability of FGFR4-R388 to facilitate pituitary tumorigenesis and GH production , we sought to identify evidence linking these two processes in human disease . We first examined STAT3 serine phosphorylation in human pituitary tissue . In the normal gland , immunohistochemistry for pS-STAT3 revealed strong reactivity in vascular endothelium ( Figure 6a ) , providing an internal positive control; adenohypophysial cells and stroma were largely negative or showed only faint staining . All but one of 8 somatotroph adenomas exhibited strong positivity ( Figure 6b ) whereas lactotroph adenomas ( n = 4 ) were negative or showed focal weak positivity ( Figure 6c ) . Four of 6 gonadotroph adenomas and all but one of 7 null cell adenomas were also either negative or weakly positive ( Figure 6d ) . We next compared GH levels and pituitary tumor size in 64 patients with pituitary tumors and acromegaly based on their FGFR4 genotypic status . This examination identified a positive correlation between circulating GH levels ( r = 0 . 622 , p = 0 . 006 ) and pituitary tumor size in patients ( n = 30 ) harboring an FGFR4-R388 allele . In contrast , patients homozygous for FGFR4-G388 ( n = 34 ) showed no relationship ( r = 0 . 23; p = 0 . 468 ) between GH levels and pituitary tumor size . Additionally , there was no relationship between FGFR4 genotype and tumor size in non-functional gonadotroph pituitary tumors ( n = 22 ) or lactotroph adenomas ( n = 13 ) . These data support a selective link between the FGFR4-R388 allele , GH hormone production , and clinical pituitary somatotroph tumor formation .
The FGFR4-R388 SNP is known to promote breast cancer cell motility and invasiveness [21] . It has also been associated with accelerated cancer progression and treatment resistance [21]–[25] . However , the mechanisms underlying these actions remain unclear . We show here that FGFR4-R388 significantly alters pituitary function . Compared with the prototypic form of the receptor ( FGFR4-G388 ) , the polymorphic FGFR4-R388 variant supports distinct signaling to deregulate pituitary growth hormone production and cell growth in vitro and in vivo . In the mouse model we report , Fgfr4 expression levels are not altered [28] , providing relevance to the human situation . Unlike the common sporadic pituitary lactotroph adenomas in rodents or the intermediate lobe corticomelanotroph pituitary tumors associated with several mouse models of cancer [29] , the Fgfr4 SNP knock-in mice develop GH-producing pituitary tumors . The resulting GH/IGF-1 excess in these animals is potentially important in the enhanced breast cancer progression associated with this model [28] . FGFR signaling relies heavily on recruitment of the immediate substrate FRS2α through tyrosyl phosphorylation [30] . Using hormone-producing pituitary cells we show that compared to FGFR4-G388 , FGFR4-R388 is associated with enhanced phosphorylation of FRS2α but not with the anticipated downstream MAPK activation . Instead , FGFR4-R388 signaling is accompanied by enhanced Src and STAT3 activation in pituitary cells . Consistent with this feature , pharmacologic Src inhibition results in greater growth inhibition by pituitary cells expressing FGFR4-R388 . Neither pharmacologic inhibition nor Src knockdown , however , could alter the GH excess associated with FGFR4-R388 . Instead , the FGFR4 SNP variant relies heavily on serine ( S727 ) but not tyrosyl ( Y705 ) phosphorylation of STAT3 . These findings suggested that while Src may play a role in promoting cell growth , the observed hormone dysregulation was not intimately coupled with this putative oncogene . The current study implicates multiple consequences of altered STAT3 modifications in the control of pituitary hormonal balance . As anticipated , FGFR4-G388 supports FGF-induced FRS2 phosphorylation to promote pY705-STAT3 activation . In turn , pY705-STAT3 induces PRL , as has been shown previously [31] . Conversely , the attenuated pY-STAT3 response , likely the result of pS-STAT3 [32] , associated with the FGFR4-R388 SNP relieves GH from inhibition leading to higher expression of this hormone . STAT3 is a well-recognized mediator of cytokine signaling , and is known to regulate GH produced by the pituitary gland [27] . Interestingly , STAT5 which is also implicated in GH regulation [33] is not affected by this FGFR4 SNP in pituitary cells . Pituitary auto-feedback mechanisms are candidate pathways whose interruption has become increasingly well-appreciated [2]–[3] . PRL receptor knockout mice develop pituitary lactotroph tumors [34] . Similarly , a somatic pituitary tumor-associated mutation in the extracellular domain of the GH receptor ( GHR ) disrupts N-terminal glycosylation of the receptor , thereby impairing GHR trafficking to the membrane , limiting ligand binding , and disrupting auto-feedback inhibition through diminished STAT activation [35]–[36] . Insulin and IGF-1 are growth factors that are known to exert negative-feedback at the level of the pituitary to inhibit GH [37] . In our study , insulin/IGF-1 efficiently activated pY-STAT3 to inhibit GH expression . In contrast , FGFR4-R388 failed to activate pY-STAT3 following ligand stimulation . The importance of diminished pY-STAT3 in mediating increased GH expression was further gleaned from knockdown of this STAT . STAT3 down-regulation or introduction of dominant negative STAT3-Y705F resulted in augmented GH production . Taken together , these data support the importance of STAT3 in the feedback inhibition control of GH regulation . In the bi-hormonal mammosomatotroph cell line examined , this altered signaling had the reverse effect on PRL . STAT3 down-regulation reduced PRL , whereas forced expression of STAT3 increased PRL and reduced GH expression by facilitating IGF-1 action . It is noteworthy that mice lacking pS-STAT3 have reduced IGF-1 levels [38] providing a complementary model to the increased IGF-1 noted in our FGFR4-R385 KI mice with pituitary somatotroph gain of pS-STAT3 . In contrast to the impact of FGFR4-G388 on pY-STAT3 , FGFR4-R388 was associated with serine STAT3 phosphorylation ( S727-STAT3 ) . STAT3 is generally regarded as a requirement for Src-mediated cell transformation as shown in many carcinomas [39] . Traditionally , STAT3 oncogenic functions have been regarded to rely on pY-STAT3 and its nuclear translocation . However , more recently the positive impact of pS727-STAT3 on cell transformation has emerged . Unlike the tyrosyl modification , serine phosphorylated STAT3 has been also been described in the mitochondria [40] and negatively modulates tyrosyl phosphorylation [32] . Mitochondrial pS-STAT3 as shown in this study has been implicated in augmented electron transport complex and ATP synthase activity to yield higher lactate dehydrogenase [27] , a critical metabolic requirement for transformed cells . Previous studies have shown that pharmacologic inhibition of wild-type FGFR4 was not effective in arresting pituitary tumor xenografts [41] . Given our newly recognized FGFR4-R388 ability to preferentially activate Src in pituitary cells , we set out to re-examine the potential role of pharmacologic interruption on pituitary tumor-associated parameters . Using the Src inhibitor dasatinib [42] , we demonstrate the ability of this agent to inhibit colony formation by FGFR4-R388 pituitary tumor cells . Given the recognized oncogenic actions of Src [43] , and specifically in pituitary tumorigenesis as shown here , our data provide new insights into how this kinase might be an attractive therapeutic target in patients harboring the FGFR4-R388 SNP . It is equally plausible that inhibitors of STAT3 and those targeting oxidative phosphorylation may be of potential value in modulating pituitary tumors for therapeutic purposes . In summary , we show that the heritable FGFR4-R388 allele yields a receptor variant that signals in a distinct manner from its prototypic FGFR4-G388 form in pituitary cells . Through its preferential ability to activate Src and pS-STAT3 , FGFR4-R388 facilitates pituitary cell transformation . Further , the diminished ability to respond through pY-STAT3 results in attenuated negative feedback inhibition to augment pituitary GH expression . Given the recognized impact of FGFR4-R388 [21]–[25] and of the GH/IGF-I axis on cancer progression [44] , the current findings identify the common FGFR4 polymorphism as an endocrine signal participating in these processes . It also highlights Src and STAT3 as potential targets for the treatment of patients with growth disorders in the context of the FGFR4 transmembrane polymorphism .
As there are no human-derived hormone-producing pituitary cell lines , we used rat pituitary GH4 mammosomatotroph cells which were propagated in Ham F10 medium 12 . 5% horse and 2 . 5% fetal bovine serum ( FBS; Sigma , Oakville , ON ) , 2 mM glutamine , 100 IU/ml penicillin , and 100 µg/ml streptomycin ( 37°C , 95% humidity , 5% CO2 atmosphere incubation ) . Rat pituitary PRL235 lactotroph cells were propagated in DMEM 10% FBS , 2 mM glutamine , 100 IU/ml penicillin and 100 g/ml streptomycin . Plasmids encoding human prototypic FGFR4 ( G388 ) or the polymorphic form FGFR4-R388 were generated and stably transfected into GH4 and PRL235 cells as previously described [17] . Construct fidelity was confirmed by DNA sequencing after introduction into pcDNA3 . 1 . STAT3 expression vector was kindly provided by M . Minden ( University of Toronto ) , dominant negative STAT3-Y705F , inactive STAT3-S727A , or constitutively active STAT3-S727D were kindly provided by J . Chen ( University of Illinois ) [42] . Cells were transfected using Lipofectamine 2000 ( Life Technologies , Rockville , MD ) according to the manufacturer's instructions . Stable clones were selected using neomycin ( G418 ) at a concentration of 0 . 7 µg/ml . A minimum of 3 clones of each isoform were pooled for further analyses in each of the cell types examined . Oligonucleotides complementary to the gene of interest were synthesized by Ambion and introduced by transfection using lipofectamine 2000 . Scrambled sequences of equal length were used as controls . Ligand stimulations were performed on cells grown in 100 mm plate ( 4×106 cells/plate ) , pre-incubated as indicated for 1 hr or 24 hrs in serum-free defined medium ( 3 µg/ml putresine , 10-6 M hydrocortisone , 10-11 M tri-iodothyronine T3 , and 0 . 375% albumin bovine factor V ) , without or with dasatinib ( Sequoia Research Products Ltd , Pangourne , UK , 100 nM ) , H-89 ( Calbiochem , San Diego , CA , 1 µM ) , or G06983 ( Sigma , 1 nM ) . Treatments with IGF-1 ( Sigma , 13 nM ) , insulin ( Eli Lilly , 600 nM ) , FGF-1 ( Sigma , 25 ng/ml ) with heparin ( 10 U/ml ) , and EGF ( R & D systems , 25 ng/ml ) were based on earlier dose and time course studies ranging from 5 min up to 24 hrs . Twenty five hundred cells were plated in 35 mm dishes as a single cell suspension in 0 . 3% agar in Ham F10 medium supplemented with 15% horse serum and 10% CS over under-layer of 0 . 5% agar prepared in Ham F10 as above . For Src inhibition , cells were incubated with the pharmacologic inhibitor dasatinib at concentrations ranging from 10−6 to 10−4 M . Colony formation was monitored daily with a light microscope and colonies photographed 4 weeks later as previously described [43] . Cells were lysed by mechanical homogenization for mitochondrial extraction using Qproteome Mitochondria isolation kit ( Qiagen ) . Isolated fractions were analyzed by Western blotting to detect the MnSOD mitochondrial marker . Effective exclusion of contaminating cytoplasmic or nuclear proteins was confirmed by detection of tubulin and acetylated histone 3 respectively . Cytochrome C oxidase activity was used as a measure of electron transport chain activity according to the manufacturer's ( Sigma ) instructions . Cells were lysed in lysis buffer ( 0 . 5% sodium deoxycholate , 0 . 1% sodium dodecyl sulfate , 1% Nonidet P-40 and 1× PBS ) containing proteinase inhibitors ( 100 µg/ml phenylmethylsulfonyl fluoride ( PMSF ) , 13 . 8 µg/ml aprotinin ( Sigma ) , and 1 mM sodium orthovanadate ( Sigma ) . Total cell lysates were incubated on ice for 30 mins , followed by micro-centrifugation at 10 , 000 g for 10 min at 4°C . Protein concentrations of the supernatants were determined by Bio-Rad method . Equal amounts of protein ( 50 µg ) were mixed with 5× SDS sample buffer , boiled for 5 mins and separated by 8 , 10 , or 12% sodium dodecyl sulfate ( SDS ) -polyacrylamide gel electrophoresis , and transferred onto PVDF membranes ( 0 . 45 µm , Millipore , US ) . Intracellular and secreted hormones were determined using the following antibodies: polyclonal antisera to PRL or GH [donated by the National Hormone and Pituitary Program ( NHPP ) , National Institute of Diabetes and Digestive and Human Development , Bethesdsa , MD] applied at dilutions of 1∶8 , 000 and 1∶50 , 000 , respectively . Blots were incubated with polyclonal affinity–purified rabbit antiserum against the carboxy terminus of FGFR4 ( Santa Cruz , Santa Cruz , CA ) . Immunoblotting was performed using anti-FGFR4 ( Santa Cruz , 1∶1000 ) , a monoclonal antibody to the V5-tag ( Invitrogen , Burlington , ON ) , anti-MnSOD ( Millipore , Billerica , USA ) , anti-FRS2α ( R&D systems , Minneapolis , USA , 1∶1000 ) , anti-Erk1/2 ( Sigma , 1∶10000 ) , anti-pFRS2α ( Y196 , 1∶1000 ) , anti-pErk1/2 ( 1∶1000 ) , anti-pY-STAT3 ( Y705 , 1∶1000 ) , pS-STAT3 ( S727 , 1∶1000 ) , STAT3 ( 1∶2500 ) , anti-pSrc ( Y416 , 1∶1000 ) , Src ( 1∶1000 ) , anti-Tubulin ( 1∶1000 ) , anti-acetylated Histone H3 ( 1∶3000 ) were purchased from Cell Signaling ( Pickering , ON ) . Loading was monitored by detection of actin ( 1∶500 , Sigma ) . Non-specific binding was blocked with 5% nonfat milk in 1× TBST ( Tris-buffered saline with 0 . 1% Tween-20 ) . After washing for 3×10 mins in 1× TBST , blots were exposed to the secondary antibody ( anti-mouse or rabbit IgG-HRP , Santa Cruz ) at a dilution of 1∶2000 and were visualized using ECL chemiluminescence detection system ( Amersham , U . K . ) . Cells were grown in 2 chamber slides and pre-incubated in serum-free define medium for 16 hrs . Cells were incubated with MitoTracker Red CMXRos ( Invitrogen ) at 37°C for 20 minutes , washed twice with PBS , fixed with 4% formaldehyde/PBS for 10 minutes , and washed three times with PBS . Cells were permeabilized for 10 minutes in PBS with 0 . 2% Triton X-100 and blocked for 30 minutes with PBS containing 5% FBS . Cells were first incubated with rabbit anti-STAT3 antibody ( 1∶100 ) or anti- pS-STAT3 antibody ( 1∶100 ) for 30 minutes at room temperature , washed three times with PBS , subsequently incubated with anti-rabbit IgG Alexa Fluor 488 for 30 minutes at room temperature , and washed three times with PBS . Coverslips were mounted in Fluoromount-G purchased from Electron Microscopy Sciences ( Hatfield , PA ) on glass slides . Cells were examined with Two-photon microscope ( Zeiss LSM 510 META NLO ) , equipped with a 63× water-immersion objective lens and filters optimized for double-label experiments . Images were analyzed using the LSM IMAGE browser . Fgfr4-R385 knock-in ( KI ) mouse were generated using standard approaches as described previously [28] . Mice were maintained on a pure C57BL/6 background . Genotyping was performed by PCR of genomic tail-DNA [28] . The care of animals was approved by the Institutional Animal Care facilities . Serum IGF-1 ( Quantikine ELISA kit ) and Prolactin ( Calbiotech ) levels were measured according to the manufacturer's protocols . Tissues were frozen in liquid nitrogen and stored at −70°C , or fixed in formalin and embedded in paraffin for histologic and immunohistochemical analyses . At least 6 animals were included at each time point for experimental measures . Pituitary glands were stained with the Gordon-Sweet silver method to demonstrate the reticulin fiber network . Immunocytochemical stains to localize adenohypophysial hormones were performed as previously reported [17] . Primary polyclonal antisera directed against rat pituitary hormones were used at the specific dilutions: GH , 1∶2500; prolactin , 1∶2500; ß-thyroid-stimulating hormone ( ß-TSH ) , 1∶3000; ß-follicle-stimulating hormone ( ß-FSH ) , 1∶600; ß-luteinizing hormone ( ß-LH ) , 1∶2500 ( National Hormone and Pituitary Program , Rockville , MD ) ; and adrenocorticotropin pre-diluted preparation , which was further diluted 1∶20 ( Dako , Carpinteria , CA ) . Human pituitary tissues were retrieved from the files of the University Health Network with REB approval . All had been fully characterized according to currently accepted criteria [45] . Fasting serum growth hormone levels were obtained from patients with histologically proven GH-producing pituitary adenomas . Pituitary tumor size was based on the maximal diameter noted on magnetic resonance imaging ( MRI ) . FGFR4 germline genotyping was performed on DNA isolated from circulating while blood cells as described [21] . The care of animals was approved by the Institutional Animal Care facilities . Human pituitary tumors were retrieved from the files of the University Health Network with REB approval . Data are presented as mean ± standard deviation ( SD ) . In the experimental models , differences were assessed by the unpaired , two-sided t test . P<0 . 05 was considered statistically significant . The analysis of surgical human tumor specimens applied Fisher's exact test . | Several human cancers have been associated with increased growth hormone levels . Here we show that a frequent single nucleotide polymorphism ( SNP ) associated with increased cancer risk and progression also deregulates pituitary function . Through recruitment of a distinct STAT3 signaling cascade , this polymorphic receptor variant drives pituitary growth hormone cell survival and hormonal output . These findings provide an example of a potentially common genetic program shared between cancer and a hormone that promotes its progression . | [
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"biology"
] | 2011 | The FGFR4-G388R Polymorphism Promotes Mitochondrial STAT3 Serine Phosphorylation to Facilitate Pituitary Growth Hormone Cell Tumorigenesis |
Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche . Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 ( extRD1 ) locus that encodes one such pathway , the early secretory antigenic target 6 ( ESAT-6 ) system 1 ( ESX-1 ) secretion apparatus . ESX-1 is required for virulence and for secretion of the proteins ESAT-6 , culture filtrate protein 10 ( CFP-10 ) , and EspA . Here , we show that both Rv3881c and its M . marinum homolog , Mh3881c , are secreted proteins , and disruption of RD1 in either organism blocks secretion . We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B . Secretion of M . marinum EspB ( EspBM ) requires both the Mh3879c and Mh3871 genes within RD1 , while CFP-10 secretion is not affected by disruption of Mh3879c . In contrast , disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM . Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates . EspBM physically interacts with Mh3879c; the M . tuberculosis homolog , EspBT , physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted . We also found interaction between Rv3879c and Rv3871 , a component of the ESX-1 machine , suggesting a mechanism for the secretion of EspB . The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates .
The cell surface–associated and secreted proteins of pathogenic bacteria promote the uptake of nutrients; facilitate attachment to specific surfaces , cells , or proteins; function in cell wall maintenance and cell division; and offer protection from harsh environmental conditions , including the host immune system . In Mycobacteria , there are at least four pathways to secrete proteins—Sec , SecA2 , twin-arginine translocase , and the early secretory antigenic target 6 ( ESAT-6 ) system 1 ( ESX-1 ) . Much attention has been focused on the ESX-1 pathway because it is required for virulence and for the secretion of ESAT-6 and culture filtrate protein 10 ( CFP-10 ) , two major targets of the immune response in infected individuals . M . tuberculosis ESX-1 is required for virulence in mice , growth in macrophages , and the suppression of macrophage inflammatory and immune responses , including the arrest of phagosome maturation and the reduced expression of IL-12 and TNF-α [1–6] . The homologous M . marinum ESX-1 is required for virulence in zebrafish , growth in macrophages , cytolysis and cytoxicity , and cell-to-cell spread , in addition to ESAT-6 and CFP-10 secretion [7 , 8] . In zebrafish embryo infections , M . marinum ESX-1 is required for macrophage aggregation and granuloma formation [9] . In M . smegmatis , ESX-1 , in addition to being required for secretion of ESAT-6 and CFP-10 , modulates conjugal DNA transfer [10 , 11] . In contrast , most strains of M . ulcerans , which is closely related genetically to M . marinum and M . tuberculosis , but persists in extracellular locations during mammalian infection , lack most of the ESX-1 components as well as orthologs of the genes extending from Rv3879c thru Rv3883c [12 , 13] . Although the ESX-1 secretion machinery ( Rv3870 , Rv3871 , and Rv3877 ) is required for the arrest of phagosome maturation by M . tuberculosis during an infection of macrophages , the known ESX-1 substrates are dispensable [6] . The multiple phenotypes and host responses dictated by the ESX-1 secretory apparatus suggest that there may be additional substrates , components , and regulatory molecules yet to be identified . Recently , a third ESX-1 substrate , EspA ( Rv3616c ) , was identified [14] . Unlike ESAT-6 and CFP-10 , EspA is encoded at a locus distant from the ESX-1 machine , yet this substrate is codependent with both ESAT-6 and CFP-10 for secretion . The mechanism for this interdependence has not been determined , but the interaction between ESAT-6 and CFP-10 in the bacterial cytosol appears to be required for secretion of the heterodimer [15–18] . Presumably , the stable heterodimer is also required for the secretion of EspA . The M . tuberculosis region of difference 1 ( RD1 ) locus ( Rv3871-Rv3879c ) and the neighboring genes encode the ESX-1 substrates ESAT-6 and CFP-10 , as well as core components of the secretion machine [1–3] . These core components include at least two putative SpoIIIE/FtsK ATPase family members ( Rv3870 and Rv3871 ) , a proline-rich predicted chromosome-partitioning ATPase ( Rv3876 ) , and a putative transporter protein with 12 transmembrane domains ( Rv3877 ) . The non-RD1 gene cluster Rv3616c-Rv3614c also is required for secretion of the known substrates [14 , 19] . Additional proteins are likely to be necessary for the assembly of the ESX-1 machinery , because in M . smegmatis , genes extending from homologs of Rv3866 through Rv3883c have been shown to be required for ESX-1–mediated secretion [11]; an M . bovis mutant disrupted for the expression of the genes homologous to Rv3867 through Rv3869 fails to secrete ESAT-6 and CFP-10 [20]; and in M . marinum , the locus required for ESX-1–mediated secretion extends at least from the homolog of Rv3866 ( Mh3866 ) to the homolog of Rv3881c ( Mh3881c ) , which in this work we rename espB ( see below ) [7] . Although these studies have identified multiple genes required for ESX-1 function , the biochemical interactions necessary for assembly of the secretion machine and for transport of substrates are still not understood . A model for CFP-10 secretion is that the carboxyterminus of the CFP-10 substrate is recognized by Rv3871 , which in turn interacts with the integral membrane protein Rv3870 to direct CFP-10 through the secretion pore [15] . The interaction of CFP-10 with Rv3871 is also required for secretion of ESAT-6 , suggesting that this is a requisite step in secretion of the ESAT-6/CFP-10 heterodimer by the ESX-1 machine . Here , we show that Rv3881c and its M . marinum homolog , Mh3881c , are substrates for secretion by ESX-1 . For this reason , we have named the gene product of this locus ESX-1 substrate protein B ( EspB ) . In both species , espB encodes a gylcine-rich protein with a predicted molecular weight of ∼47 kDa , without any region of apparent similarity to the secretion signal of CFP-10 or other known secretion signals . Although a substrate of ESX-1 , we find that the specific genes required for secretion of EspB differ from those required for the secretion of CFP-10 . Biochemical investigation demonstrates that EspB forms a complex with Rv3879c and that Rv3879c interacts with Rv3871 , the same component of ESX-1 that interacts with the ESAT-6/CFP-10 complex during its secretion . These data support a model that different substrates are delivered to the ESX-1 machine by molecularly distinguishable pathways . Moreover , each of these pathways for ESX-1–mediated secretion contributes to mycobacterial virulence .
A previous genetic screen for M . marinum mutants that fail to cause hemolysis led to the isolation of eight mutants in the extended RD1 ( extRD1 ) locus , Mh3866-Mh3881c [7] . Of the eight mutants , espBM::tn ( Mh3881c::tn ) was the most attenuated for virulence to zebrafish , growth in macrophages , and cytotoxicity to J774 cells . Thus , we decided to investigate the espBM–encoded protein ( EspBM ) and its M . tuberculosis homolog ( EspBT ) in detail . The gene , espBM , is the first in a two-gene operon . Using quantitative RT-PCR , we found that the mutation disrupts the expression of both genes in the operon ( unpublished data ) . We then sought to determine the genetic requirements for restoration of intracellular growth to the mutant . Introduction of a non-integrating plasmid , expressing either EspBM from the espBM promoter or EspBT from its native promoter , was sufficient to appreciably restore growth in macrophages to espBM::tn ( Figure 1A ) . The non-integrating plasmids expressing both EspBM and Mh3880c or both EspBT and Rv3880c were not superior in restoration of intracellular growth . Thus , EspB is necessary and sufficient to appreciably complement espBM::tn , and the M . tuberculosis homolog functions equally well in M . marinum , demonstrating conservation of function . While the expression of EspBM from a non-integrating plasmid appreciably restored growth in macrophages to espBM::tn , the complementation was not complete . Among possible explanations are that the transposon insertion exerted a polar effect on the operon upstream , Rv3883c-Rv3882c , which also might have a role in intracellular growth , or that a proper stoichiometry between EspB and ESX-1 is required for complete complementation . Therefore , integrating plasmids encoding either espBM along with the espBM promoter , espBM-Mh3880c along with the espBM promoter , or the entire locus Mh3883c-Mh3880c along with the Mh3883c promoter , were introduced into espBM::tn . The locus Mh3883c-Mh3880c , along with the Mh3883c promoter , was also introduced into espBM::tn on a non-integrating plasmid . Of these constructs , only the integrating plasmids encoding espBM-Mh3880c or Mh3883c-Mh3880c fully complemented the growth defect of espBM::tn ( Figure 1B ) . Similarly , espBM alone appreciably restored a rough colony morphology to the espBM::tn mutant , but espBM-Mh3880c or Mh3883c-Mh3880c fully restored the rough colony morphology to espBM::tn ( Figure S1 ) . These results suggest that Mh3880c can contribute to M . marinum growth in macrophages when it is expressed along with espBM from the bacterial chromosome . In contrast , espBM contributes equally well to bacterial virulence whether expressed episomally or on the chromosome , suggesting that its contribution is more independent of its stoichiometry with respect to other virulence components . As a first step toward understanding the role of EspB in virulence and growth in macrophages , we determined its localization in Mycobacteria grown in broth culture . The cell lysate and culture filtrate fractions of M . tuberculosis H37Rv , wild-type M . marinum , and M . marinum espBM::tn were probed with a mouse polyclonal antibody raised against a 100 amino acid fragment of EspBT extending from amino acid 234 to 333 ( Figure 2A ) . EspB was detected in both the cell lysate and the culture filtrate fractions of M . tuberculosis , as well as in both the cell lysate and the culture filtrate fractions of wild-type M . marinum . EspB was not detected in either fraction of the espBM::tn culture , verifying the specificity of the antibody . GroEL , a non-secreted bacterial cytoplasmic protein , was found exclusively in the cell lysate , demonstrating that EspB did not appear in the culture filtrate as a result of cell lysis . The EspB in the cell lysate had an Mr of 55 kDa on SDS-PAGE , while the EspB in the culture filtrate of both species ran at a slightly lower molecular weight . A lower molecular weight of EspB in the culture filtrate was also observed in a prior proteomic analysis of M . tuberculosis H37Rv proteins [21] , in which EspB in the cell lysate was observed on a 2-D gel as a single spot with an apparent molecular weight of 55 . 6 kDa , while the EspB in the culture filtrate was observed as two spots with apparent molecular weights of 49 . 7 kDa and 48 . 4 kDa . Therefore , EspB might be cleaved either during or after secretion . To test this possibility , a V5 epitope tag was fused to the N-terminus of EspBM and a His6x epitope tag was fused to the C-terminus . The resulting construct , V5-EspBM-His6x , was expressed in the espBM::tn mutant . Like the native protein , V5-tagged EspB was detected in the cell lysate as a single band and as a doublet in the culture filtrate ( Figure 2B ) . In contrast , His-tagged protein was only detected in the cell lysate fraction , suggesting that EspBM in the culture filtrate is C-terminally truncated . To assess which ESX-1 genes are required for EspB secretion , its compartmentalization between cell lysate and culture filtrate was determined for several M . marinum ESX-1 mutants ( Figure 2C ) . Although EspBM was found in both the cell lysate and culture filtrate fractions of most mutants , EspBM was not detected in the culture filtrates of MmΔRD1 , Mh3868::tn , Mh3879c::tn , or Mh3871::tn . The Mh3868::tn mutants failed to accumulate protein in the pellet , suggesting that Mh3868 protein could be involved in EspB synthesis or stability . Thus , of the ESX-1 genes tested , only Mh3879 and Mh3871 were clearly involved in EspBM secretion . In contrast , none of the mutants secreted ESAT-6 [7] , and only Mh3879c::tn and Mh3878c::tn secreted CFP-10 normally . This difference in secretion requirements for ESAT-6 and CFP-10 in M . marinum has been noted previously [7] . Complementation of Mh3879::tn and espB::tn restored EspBM secretion . GroEL was absent from culture filtrates of all strains , and secretion of the fibronectin attachment protein ( FAP ) , a protein secreted in a Sec-dependent manner [22] , was not disturbed in any of the extRD1 mutants . Thus , the product of the espBM gene is a secreted protein that requires Mh3871 , a core component of the ESX-1 secretion machine , for export; we have therefore named it ESX-1 substrate protein B ( EspB ) . However , EspB , ESAT-6 , and CFP-10 differ with respect to the extRD1 genes required for their secretion . EspBM secretion depends on Mh3879c , but is independent of Mh3866 and Mh3867 , while CFP-10 shows the inverse pattern . To demonstrate the importance of the ESX-1 machine in EspB secretion in another strain of M . marinum , we examined the 1218R strain and an isogenic mutant in which the Mh3871 gene had been disrupted . The M strain , used for the previous experiments , is a human isolate , whereas 1218R was originally isolated from an infected fish . Wild-type 1218R secreted EspB , but the Mh3871 mutant did not ( Figure S2 ) , confirming the importance of ESX-1 in the secretion of this protein by M . marinum . Complementation of the mutant with either the M . marinum or M . tuberculosis homolog of Mh3871 restored secretion of EspBM to this mutant , suggesting parallel functions for the genes in the two species . To test directly whether ESX-1 was required for EspB secretion by M . tuberculosis , we examined culture filtrates from M . tuberculosis Erdman and the isogenic mutants Rv3870::tn , Rv3871::tn , and ΔCFP-10 ( Figure 2D ) . Secretion of EspBT by wild-type M . tuberculosis was abrogated in the Rv3870 and Rv3871 mutants , but not in the ΔCFP-10 mutant . Thus , EspB is a secreted protein in both M . marinum and M . tuberculosis , and its secretion requires core ESX-1 components in both species of Mycobacteria . Importantly , EspB is the first ESX-1 substrate in M . tuberculosis whose secretion is not disrupted in the ΔCFP-10 mutant . Of the ten M . marinum extRD1 mutants we examined , MmΔRD1 , espBM::tn , and Mh3871::tn were disrupted for the secretion of all three substrates: ESAT-6 , CFP-10 , and EspBM . In contrast , the Mh3879::tn mutant was disrupted only for the secretion of ESAT-6 and EspBM , while the Mh3866::tn and Mh3867::tn mutants were disrupted only for ESAT-6 and CFP-10 secretion . To assess the importance of the multiple ESX-1 substrates for growth in macrophages , we infected murine bone marrow–derived macrophages ( BMDMs ) with wild-type M . marinum , with strains lacking one secreted effector , or with strains lacking secretion of all the known ESX-1 substrates . As shown in Figure 3 , MmΔRD1 , espBM::tn , and Mh3871::tn , which fail to secrete all substrates , are more attenuated for growth in macrophages than Mh3866::tn , which still secretes EspBM , or Mh3879c::tn , which still secretes CFP-10 . Therefore , we conclude that the various substrates of ESX-1 each contribute to virulence . To learn more about the involvement of ESX-1 in EspB secretion , we tested whether EspB would interact with other ESX-1 genes by bacterial two-hybrid analysis ( Figure 4 ) . An advantage of the bacterial two-hybrid system is that it can allow detection of interactions of membrane-bound proteins [23] . In this assay , potential protein–protein interactions are assessed by determining the ratio of colonies that grow on selective medium to the number grown on non-selective medium . For each of the bait plasmids , co-transformation with an empty target resulted in a ratio of colonies on selective to non-selective medium of less than 0 . 1% , as did co-transformation of the EspBT target with an empty bait . In contrast , the Rv3879c bait and EspBT target resulted in a ratio of 7 . 6% , an increase of more than 75-fold . An Rv3876 bait also showed interaction above background with EspBT , but since the M . marinum Mh3876::tn mutant showed significant EspBM secretion ( Figure 2C ) , any interaction between Rv3876 and EspBT is not likely to be required for EspB secretion and thus was not pursued . To test for an analogous interaction between EspBM and Mh3879c and to confirm the potential interaction between EspBT and Rv3879c suggested by the two-hybrid assay , we performed in vitro pull-down assays . All of the proteins used were expressed in Escherichia coli as GST- or V5-epitope-tagged fusions . Controls for nonspecific interactions included GST alone , as well as GST-syntaxin2 , and GST-Shp1 . As shown in Figure 5A , GST-tagged EspBM , but none of the GST controls , bound specifically to V5-tagged Mh3879c . In the reciprocal experiment , GST-tagged Mh3879c bound specifically to V5-EspBM . Similarly , as shown in Figure 5B , GST-tagged EspBT bound specifically to V5-tagged Rv3879c , and GST-tagged Rv3879c bound specifically to V5-tagged EspBT . These data demonstrate that recombinant EspBT and Rv3879c , as well as their M . marinum homologs , interact in vitro . Since Rv3871 mutants in both M . tuberculosis and M . marinum fail to secrete EspB , we used GST pulldowns to test whether Rv3871 interacts with either EspBT or Rv3879c . GST-tagged Rv3879c bound to V5-tagged Rv3871 , whereas the GST controls and GST-EspBT did not bind to Rv3871 . This suggests that Rv3879c may facilitate EspBT secretion through an interaction with Rv3871 . To identify whether EspB , like CFP-10 , requires its carboxyterminus for secretion , we constructed a series of EspBM deletion mutants with N-terminal V5 tags and expressed them in the espBM::tn mutant strain using the espBM promoter . As shown in Figure 6A , V5-tagged full-length EspBM was secreted . This N-terminally tagged protein , like native EspBM , underwent C-terminal truncation either during or after secretion . EspBM deletion mutant constructs Δ ( 2–31 ) , Δ ( 264–271 ) , and Δ ( 400–454 ) were stably expressed in M . marinum , but only EspBM Δ ( 400–454 ) accumulated in the culture filtrate . The secreted EspBM Δ ( 400–454 ) had a higher apparent molecular weight than the secreted full-length EspBM , presumably because deletion of the C-terminal 55 amino acids inhibits some of the carboxyterminal proteolytic processing . This result demonstrates that the C-terminus of EspBM is dispensable for secretion , but N-terminal and internal amino acids are required . Next , we tested how these EspBM mutants interacted with Mh3879c . Lysates of E . coli that express V5-tagged EspBM mutants were incubated with GST-Mh3879c . While full-length EspBM and EspBM Δ ( 400–454 ) bound to GST-Mh3879c , the stably expressed but non-secreted EspBM Δ ( 2–31 ) and EspBM Δ ( 264–271 ) constructs did not bind to GST-Mh3879c ( Figure 6B ) . These data support a model in which EspB interacts with Rv3879c , which in turn interacts with Rv3871 , to facilitate the secretion of EspB . Because CFP-10 and ESAT-6 are secreted as a heterodimer , we assessed whether Mh3879c and EspB might be secreted similarly . The fusion constructs V5-Mh3879c , Mh3879c-His6x , and V5-Mh3879c-His6x were expressed from the endogenous Mh3879c promoter on non-integrating plasmids in both wild-type M . marinum and in the Mh3879c::tn mutant . Introduction of V5-Mh3879c fully complemented the EspBM secretion defect of the Mh3879c::tn mutant , but Mh3879c-His6x and V5-Mh3879c-His6x failed to complement the secretion defect ( Figure S3A ) . In wild-type M . marinum , V5-Mh3879c and V5-EspBM were expressed at nearly identical levels in the cell lysate , but only V5-EspBM was detected in the culture filtrate ( Figure S3A ) . To determine whether failure of secretion reflected inefficient competition of V5-tagged protein with native protein , the V5-EspBM secretion was also analyzed in the Mh3879::tn mutant . In this strain as well , V5-Mh3879c was found only in the cell lysate . Thus , V5-tagged Mh3879c , while fully competent to mediate EspB secretion , was not itself secreted , suggesting that Mh3879c and EspB are not secreted as a heterodimer . The C-terminally His6x-tagged Mh3879c , which did not restore EspB secretion to the Mh3879::tn mutant , also was detected only in the cell lysate . Since Mh3879c-His6x failed to complement the EspBM secretion defect of the Mh3879c::tn mutant , we hypothesized that the carboxyterminus of Rv3879c might be required for interaction with EspB . To test this hypothesis , lysates of E . coli that express V5-tagged Rv3879 mutants were incubated with GST alone , GST-Rv3871 , or GST-EspBT . While full-length Rv3879 and Rv3879 Δ ( 1–166 ) bound to GST-EspBT , Rv3879 Δ ( 564–729 ) failed to bind to GST-EspBT ( Figure S3B ) . None of the constructs bound to GST alone , and all three constructs bound to GST-Rv3871 . Thus , the carboxyterminal 166 amino acids of Rv3879 are required for EspB secretion , but not for interaction with the ESX-1 machine .
In this study , we identified EspB as a novel substrate of the ESX-1 secretion system and demonstrated a requirement for the Mh3879c and Mh3871 genes in the secretion of EspBM . Further , we showed protein complex formation between EspBM and Mh3879c , as well as identical behavior of their M . tuberculosis homologs . Two mutants of EspBM that were stable after synthesis but failed to bind Mh3879c were not secreted , while a large carboxylterminal deletion did not interfere with either Mh3879c binding or secretion . Additionally , the carboxyterminus of Rv3879c/Mh3879c is required for interaction with and secretion of EspB . These results suggest that the EspB/Mh3879c protein complex is required for EspBM secretion . While complex formation between ESAT-6 and CFP-10 is required for their secretion as a heterodimer by M . tuberculosis , Mh3879c appears not to be secreted . Our data , though , do not exclude the possibility that the aminoterminus of Mh3879c is quantitatively removed during or immediately after secretion , since we do not have and could not probe with antibodies to the native protein . We hypothesize that Mh3879c acts as a cytosolic chaperone to deliver EspBM to the secretion machine . We showed that Rv3879c interacts directly with Rv3871 and that Rv3871 , in addition to being required for the secretion of ESAT-6/CFP-10 , is required for the secretion of EspB . Although our work does not reveal precisely how EspB is delivered to the ESX-1 machine , our data demonstrate that Rv3879c can interact with Rv3871 as well as with EspBT , suggesting that EspB may be targeted to Rv3871 in this way . We propose that the mechanisms of EspB and CFP-10 secretion intersect at binding to Rv3871 ( Figure 7 ) . We also found that disruption of Mh3868 leads to loss of accumulation of EspB in the bacterial cytosol . We previously observed that disruption of Mh3868 prevents bacterial accumulation of ESAT-6 and CFP-10 [7] . Mh3868 and its M . tuberculosis homolog Rv3868 are predicted to be AAA ATPases , which suggests that they may function as chaperones for the translocation of ESX-1 substrates , but little is known about this key protein . We have found that CFP-10 and espBM mRNAs are expressed in the Mh3868::tn mutants ( B . McLaughlin and E . Brown , unpublished data ) , suggesting that the Mh3868 gene product affects either the translation or stability of the ESX-1 substrates . Characterizing the function of Mh3868 will certainly be important to better understand ESX-1–mediated secretion . Like ESAT-6 and CFP-10 , EspA is secreted by the ESX-1 machine . Whether any of the M . marinum genes with sequence similarity to espA are functional orthologs has not yet been determined . Loss of either EspA or EspB inhibits secretion of ESAT-6 and CFP-10 , but the reason for their requirement is unknown . It may be that as substrates reach the final common pathway for secretion , they interact in a manner that leads to cooperative secretion . Clearly , though , the secretion of EspB is quite distinct from that of EspA . While EspA requires CFP-10 for its secretion , EspB secretion is independent of CFP-10 . EspB secretion is not disrupted in the M . marinum mutants Mh3866::tn and Mh3867::tn , neither of which secrete CFP-10 , nor is EspB secretion disrupted in the M . tuberculosis ΔCFP-10 mutant . These data are consistent with the model that EspB , unlike either ESAT-6 or EspA , is targeted to the ESX-1 machine independently of CFP-10 . These studies beg the question of whether it is possible to determine which ESX-1 substrates are most important for virulence . This has been a difficult task because of the apparent codependence of the various substrates on each other for secretion . However , our results allowed a somewhat different approach . We used a set of extRD1 mutants in which some ( Mh3866::tn and Mh3867::tn ) failed to secrete CFP-10 , but did secrete EspB; while another mutant ( Mh3879::tn ) secreted CFP-10 but failed to secrete EspB; while mutants that disrupted the core secretion machinery ( MmΔRD1 and Mh3871::tn ) and espBM::tn itself failed to secrete all substrates . We found that mutants lacking secretion of both substrates had a more marked growth defect in macrophages than the mutants lacking secretion of only one substrate . This suggests that the different substrates make distinct , and potentially additive , contributions to virulence . Although we cannot say that the defects in intracellular growth of the various mutants are caused by the substrates we have identified , our work does support the hypothesis that ESX-1 secretes more than one substrate that contributes to the virulence of Mycobacteria and that different substrates may have independent contributions to bacterial pathogenesis . In summary , this work has identified a novel substrate for ESX-1–dependent secretion and has demonstrated interactions of this substrate with a protein encoded within RD1 , expanding our understanding of how genes within this locus contribute to this novel secretion pathway . Furthermore , we have demonstrated that secretion of distinct ESX-1 substrates follows variable pathways to interaction with the core secretion machinery , and that the different substrates may contribute independently to intracellular survival and growth of the bacteria . These data extend the understanding of a major virulence mechanism of Mycobacteria .
All strains and plasmids used in this study are listed in Table 1 . M . marinum strains were grown as previously described [24] . The designations assigned by the Sanger Institute in the annotation of the M . marinum genome and the corresponding DNA sequences are available at http://www . sanger . ac . uk/Projects/M_marinum/ . The transposon insertion in the mutant espBM::tn lies between the 175th and 176th base pairs of the espBM gene , and the kanamycin gene within the transposon is transcribed opposite to the direction of transcription of the espBM gene . The strains M . marinum M attB::hygr and espBM::tn attB::hygr were constructed by transforming the strains M . marinum M WT and espBM::tn with the plasmid pMV306 . hyg . The strains espBM::tn attB:: espBM hygr and espBM::tn attB:: espBM -Mh3880c hygr were constructed by ligating 250 bp upstream of espBM along with espBM or espBM -Mh3880c into pMV306 . hyg and then transforming the resulting plasmids , pBM264 and pBM262 , into espBM::tn . The strain espBM::tn attB:: Mh3883c-Mh3880c hygr was constructed by ligating 345 bp upstream of Mh3883c along with Mh3883c-Mh3880c into pMV306 . hyg and then transforming the resulting plasmid , pBM263 , into espBM::tn . To construct the plasmids pBM841 , pBM540 , and pBM810 , the genes Rv3871 , Rv3879c , and Rv3881c were PCR amplified from the cosmid RD1-2F9 [25] and ligated into pBM510 , a derivative of pET22b+ in which the N-terminal His tag was replaced with the V5 epitope tag . To construct the plasmids pBM843 and pBM504 , the genes Mh3879c and Mh3881c were PCR amplified from M . marinum M genomic DNA and ligated into pBM510 . To construct the plasmids pBM332 and pBM336 , a series of fragments were ligated into pLYG206 to achieve the following sequence ligated into the NotI and XbaI sites: 250 bp upstream of espBM , then the V5 epitope , then the espBM gene , and finally , in the case of the pBM336 plasmid , the His6x epitope . The plasmids pBM869 , pBM870 , and pBM871 were made in a manner synonymous to that of pBM332 and pBM336 , where the Mh3879 promoter and gene were used . The plasmids pBM367 and pBM400v were constructed by PCR from pBM332 and re-ligation of the truncated gene fragments back into pBM332 , while pBM398 was generated by quick-change mutagenesis ( Stratagene , http://www . stratagene . com/ ) . For pBM589 , pBM398e , and pBM400ve , the espBM gene fragments in the plasmids pBM367 , pBM398 , and pBM400v were cut by restriction digest and ligated into pBM504 . For pBM856 , pBM550 , pBM553 , and pBM551 , the genes Mh3879c , espBM , Rv3879c , and espBT were cut by restriction digest from the plasmids pBM843 , pBM504 , pBM540 , and pBM810 and ligated into the GST expression vector pGex-KG . To construct the plasmid pMh3879 , 250 bp upstream of the gene Mh3879c together with Mh3879c was PCR amplified from M . marinum genomic DNA and inserted into pLYG206 . The plasmids pBM1010 and pBM1013 were constructed by restriction digests of pBM540 to excise portions of Rv3879c , and ligation of 5′ phosphorylated hybridized oligos that restored the frame and created the Rv3879 deletions Δ ( 1–166 ) and Δ ( 564–729 ) . To construct the plasmid p ( Mh3883c-Mh3880c ) , the 345 bp upstream of Mh3883c along with Mh3883c-Mh3880c was cut by restriction digest from the plasmid pBM263 and inserted into pLYG206 . To construct the plasmid p ( espBT-Rv3880c ) , 250 bp upstream of the operon Rv3881c-Rv3880c together with the operon were inserted into pLYG206 . M . marinum strains were grown in 40-mL cultures to 0 . 5 OD600 in 7H9 medium . The cultures were centrifuged and washed three times with 15 mL of PBS before re-suspension in 40 mL of Sauton's medium , supplemented with 0 . 015% Tween-80 . When strains containing non-integrating plasmids for complementation were grown in Sauton's medium , the Sauton's medium was supplemented with Zeocin ( 5 μg/ml; Invitrogen , http://www . invitrogen . com/ ) . After growth for 36 h at 30 °C , 105 rpm , in Sauton's medium , the cells were harvested by centrifugation . Supernatants were filtered through a 0 . 22-μm-pore-size filter with a glass pre-filter and concentrated with an Amicon Ultra-15 ( 5 , 000-molecular-weight cutoff; Millipore , http://www . millipore . com/ ) to 200 μL , which was saved as the culture filtrate ( CF ) fraction . Pelleted cells were washed and resuspended in 1 . 5 mL of PBS with a protease inhibitor cocktail and 1 mM PMSF . Pellets were lysed using glass beads and the mini-bead beater ( BioSpec Products , http://www . biospec . com/ ) with three 40-s pulses at maximum speed and incubations on ice in between each pulse , and then centrifuged at 3 , 000g for 2 min at 4 °C to remove unbroken cells . The resulting supernatant was collected and saved as the cell lysate ( CL ) fraction . M . tuberculosis ( Erdman ) culture filtrate and cell lysate fractions were prepared as previously described [1] . Total protein concentrations were determined by a Bradford assay . Pellet and culture filtrate fractions were separated by SDS/PAGE on 10%–20% gradient polyacrylamide gels for detection of CFP-10; 7 . 5% polyacrylamide gels for detection of EspB , GroEL , or V5-tagged Mh3879; and 12 . 5% polyacrylamide gels for detection of FAP . Proteins were visualized by immunoblotting by using antibodies against EspB at a concentration of 1:500 ( mouse polyclonal to the 100 amino acid fragment of Rv3881c [234–333 aa] , Arizona State University CIM Antibody Core ) , and the blot was developed using ECL reagent West Dura ( Pierce , http://www . piercenet . com/ ) . Anti-CFP-10 ( rabbit polyclonal; Colorado State University , http://www . cvmbs . colostate . edu/microbiology/tb/top . htm ) was used at a concentration of 1:50000 , blots of the culture filtrate fraction were developed using West Pico ( Pierce ) , and blots of the cell lysates were developed using West Dura ( Pierce ) . Anti-GroEL ( rabbit polyclonal , SPA-875 / SPS-875; Stressgen , http://www . assaydesigns . com/ ) was used at a concentration of 1:10000 , and blots were developed using West Pico ( Pierce ) . Anti-FAP [22] for M . marinum samples was a rabbit polyclonal , used at a concentration of 1:10000 and developed using West Pico ( Pierce ) . Anti-FAP for M . tuberculosis Erdman samples was CS-93 ( Colorado State University ) , mouse monoclonal , used at a concentration of 1:20 , and developed using West Pico ( Pierce ) . His6x epitope was detected with a mouse monoclonal ( Novagen , http://www . emdbiosciences . com/html/NVG/home . html ) at a concentration of 1:1500 , and V5 epitope was detected with a mouse monoclonal ( R960–25 , Invitrogen ) , at a concentration of 1:5000 , and these blots were developed using West Pico ( Pierce ) . The genes Rv3614c , Rv3615c , and Rv3616c , which were PCR amplified from genomic DNA , and each of the genes in the region Rv3864 through Rv3883 , which were PCR amplified from the cosmid RD1-2F9 [25] , were cloned into the “bait” vector pBT ( BacterioMatch II; Stratagene ) in frame with cI . Rv3881c was cloned into the “target” vector pTRG in frame with the N-terminal subunit of RNA polymerase according to the manufacturer's instructions . The constructs were co-transformed into the E . coli two-hybrid system reporter validation strain XL1-Blue MRF′ hisB lac [F′ laqIq HIS3 aadA Kanr] and plated onto both the selective ( +5 mM 3AT ) and the non-selective screening medium according to the manufacturer's instructions . The non-selective screening plate is histidine-dropout M9 agar supplemented with 0 . 5 mM IPTG , 12 . 5 μg/ml tetracycline , and 25 μg/ml chloramphenicol . The selective screening plate is histidine-dropout M9 agar supplemented with 0 . 5 mM IPTG , 12 . 5 μg/ml tetracycline , 25 μg/ml chloramphenicol , and 5 mM 3-amino-1 , 2 , 4-triazole . GST fusion proteins , GST alone , and V5-tagged proteins were expressed in the BL21-RP codon plus E . coli strain ( Stratagene ) by addition of 0 . 2 mM IPTG ( 3 h at 30 °C ) . Bacterial cultures were lysed in buffer containing 50 mM HEPES ( pH 7 . 4 ) , 300 mM NaCl , 1% Triton X-100 , 0 . 5 mM EDTA , and protease inhibitor cocktail ( Roche ) . Solubilized proteins were separated by centrifugation at 20 , 000g for 10 min . The GST fusion proteins and GST alone were bound to glutathione agarose beads ( Amersham Biosciences , http://www . gelifesciences . com ) by incubation overnight at 4 °C . The beads were then extensively washed with PBS containing 0 . 1% Triton X-100 . Bacterial lysates containing solubilized V5-tagged proteins , in lysis buffer , were incubated with the GST protein–loaded agarose beads overnight at 4 °C . After washing three times with PBS containing 1% Triton X-100 , bead-bound protein was eluted in Laemmli buffer , seperated by SDS-PAGE , and analyzed by western blot . All macrophages used in these experiments were derived from bone marrow cells of C57BL/6 mice that were differentiated for 6 d in DMEM supplemented with 10% CMG supernatant [26] and 10% fetal bovine serum ( FBS; HyClone , http://www . hyclone . com/ ) . Immediately prior to infection , macrophage monolayers were washed once with FBS-free DMEM . M . marinum strains were each grown to OD600 of 1 . 0 , prepared for infection , and incubated with macrophages as previously described [7] . All infections were performed at a multiplicity of infection of 1 , for 2 h at 32 °C , in a 5% CO2 , humidified environment , in 24-well plates . The time at which M . marinum was added to the well was designated time zero . At the end of the 2 h incubation period ( T = 2 h ) , infected monolayers were washed twice with DMEM and further incubated in DMEM containing 0 . 1% FBS and 200 μg of amikacin/ml for 2 h to kill extracellular bacteria . At the end of the antibiotic treatment , monolayers were washed twice with DMEM and incubated in DMEM containing 0 . 1% FBS at 32 °C and 5% CO2 . Intracellular bacteria were enumerated by lysing macrophage monolayers and diluting and plating bacteria exactly as described [7] . Statistical analysis was performed by calculating the one-way analysis of variance ( ANOVA ) with GraphPad Prism 4 . 0 ( GraphPad Software , http://www . graphpad . com ) .
The GenBank ( http://www . ncbi . nlm . nih . gov/Genbank/index . html ) accession numbers for the gene products discussed in this paper are CFP-10 ( NP_218391 ) , ESAT-6 ( YP_178023 ) , Rv3866 ( NP_218383 ) , Rv3867 ( NP_218384 ) , Rv3868 ( NP_218385 ) , Rv3870 ( NP_218387 ) , Rv3871 ( NP_218388 ) , Rv3876 ( NP_218393 ) , Rv3877 ( NP_218394 ) , Rv3878 ( NP_218395 ) , Rv3879c ( NP_218396 ) , Rv3880c ( NP_218397 ) , and Rv3881c ( NP_218398 ) . | A major mechanism used by pathogenic bacteria for disabling host defenses is secretion of virulence proteins . These effectors are often transported by specialized secretion machines . One such pathway , present in Mycobacterium and other Gram-positive genera , is ESX-1 ( early secretory antigenic target 6 system 1 ) . Although ESX-1 is required for multiple phenotypes related to the pathogenesis of infection , only three substrates of the secretion machine have been identified to date , and the mechanism by which these substrates are exported is not understood . In our efforts to understand this virulence-related secretion mechanism , we identified a novel substrate and found that its delivery to the ESX-1 machine requires different protein interactions than previously identified substrates . Finally , we present data that the various ESX-1 substrates contribute additively to virulence . These data are incorporated into a model of ESX-1 function . | [
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] | 2007 | A Mycobacterium ESX-1–Secreted Virulence Factor with Unique Requirements for Export |
Coral reefs are in global decline , with coral diseases increasing both in prevalence and in space , a situation that is expected only to worsen as future thermal stressors increase . Through intense surveillance , we have collected a unique and highly resolved dataset from the coral reef of Eilat ( Israel , Red Sea ) , that documents the spatiotemporal dynamics of a White Plague Disease ( WPD ) outbreak over the course of a full season . Based on modern statistical methodologies , we develop a novel spatial epidemiological model that uses a maximum-likelihood procedure to fit the data and assess the transmission pattern of WPD . We link the model to sea surface temperature ( SST ) and test the possible effect of increasing temperatures on disease dynamics . Our results reveal that the likelihood of a susceptible coral to become infected is governed both by SST and by its spatial location relative to nearby infected corals . The model shows that the magnitude of WPD epidemics strongly depends on demographic circumstances; under one extreme , when recruitment is free-space regulated and coral density remains relatively constant , even an increase of only 0 . 5°C in SST can cause epidemics to double in magnitude . In reality , however , the spatial nature of transmission can effectively protect the community , restricting the magnitude of annual epidemics . This is because the probability of susceptible corals to become infected is negatively associated with coral density . Based on our findings , we expect that infectious diseases having a significant spatial component , such as Red-Sea WPD , will never lead to a complete destruction of the coral community under increased thermal stress . However , this also implies that signs of recovery of local coral communities may be misleading; indicative more of spatial dynamics than true rehabilitation of these communities . In contrast to earlier generic models , our approach captures dynamics of WPD both in space and time , accounting for the highly seasonal nature of annual WPD outbreaks .
The study site was located at the shallow water reef ( depth of ca . 1 . 5 m ) off the shore of the Interuniversity Institute ( IUI ) in Eilat . The reef is relatively uniform with respect to bathymetry and is situated on a gentle slope ( ca . 3° ) on flat beach-rock . The reef did not appear to exhibit any particular dominant water-flow direction due to the high impact of the erratic and changing wave action . The reef is characterized by relatively high coral density , which allows for a relatively large number of infection cases per unit area . Thus the IUI site is particularly suitable for studying the spatial distribution and the dynamics of coral diseases . A 10×10 m plot was surveyed once a month , from June 2006 until May 2007 providing twelve “snapshots” in total . The size of the plot and the period of time between snapshots were based on a preliminary survey where we roughly assessed the clustering size of infected corals , and the development time of new infections . The four corners of the plot were marked in the field , and a grid made of ropes and elastic bands was placed on the plot dividing the plot to 100 subunits of 1×1 m . Using photography ( photoquadrats ) , all 2 , 747 susceptible corals within this area were mapped and an X-Y coordinate of the coral’s centre within the plot was allocated , following the “center rules” of Zvuloni et al . [59] . Once a month , the grid was placed precisely on the same area and the locations of infected corals were recorded . Corals were classified in the field as infected if they showed typical signs of the disease—a sharp line between apparently healthy tissue and a thin zone of bleached tissue grading into exposed coral skeleton ( Fig 1 , [50] ) and some level of progression ( i . e . , increased severity ) relative to the previous snapshot . The Israel National Monitoring Program of the Gulf of Eilat provided continuous measurements of sea-surface temperature ( SST ) , ca . 20 m away from the plot , as obtained from two temperature probes ( Campbell Scientific , Temperature Probe Model 108; accuracy of ±0 . 1°C within the range of 20–30°C and resolution of 0 . 1°C ) . The 12 spatial snapshots of the reef-section were organized as eleven pairs of sequential snapshots , where in each pair infected corals were partitioned into two groups: Newly-Infected Corals ( NICs ) —those corals that had signs of infection in the current snapshot , but not in the previous one . Previously-Infected Corals ( PICs ) —those corals that were infected in the previous snapshot . Our conjecture was that if inter-colonial transmission is significant for the spread of the disease , NICs should develop in closer proximity to PICs than would be expected at random . To test this hypothesis , we developed a simple , but novel , spatiotemporal index , which is based on Ripley’s K-function [60 , 61] . While the K-function tests the spatial pattern of a single group of events , our spatiotemporal index n ( r ) was designed to test the spatial relations between two groups of events , in this case two groups of infected corals—the NICs and the PICs . This index is defined as the mean number of NICs in a given month within a radius r from a PIC of the previous month , and is calculated as: n ( r ) =1m∑i=1m∑j=1kIr ( dij ) . ( 1 ) Here , m and k are the numbers of PICs and NICs , respectively , in the tested pair of sequential sampling dates , dij is the distance between any PIC i and NIC j . The indicator variable Ir ( dij ) indicates whether or not NIC j is located within radius r from PIC i . Thus , Ir ( dij ) receives a value of 1 if dij < r and zero otherwise . In contrast to the nearest-neighbor approach used by Zvuloni et al . [16] to identify whether NICs form aggregations in the vicinity of PICs , the n ( r ) index also quantifies the spatial scale of aggregation , as it is calculated for a range of distances r ( similarly to Ripley’s K function; see Ripley [60 , 61] ) . Using a null model approach , which bases the null expectation on the spatial distribution of the entire pool of susceptible corals , we ascertained whether the k NICs found in the field were significantly aggregated around the PICs ( see Material and Methods ) . We model disease transmission by using a stochastic spatiotemporal model similar to Zvuloni et al . [16] , but with a new maximum-likelihood fitting procedure to estimate model parameters from the field-data . The analysis that follows is based on the classical Susceptible-Infected-Susceptible ( SIS ) model of epidemiology [62 , 63] . Corals are classified as either susceptible or infected . A susceptible coral can become infected when the disease is transmitted from a ( usually ) neighboring PIC , and an infected coral can return to be susceptible if the disease stops showing clinical signs . The model assumes transmission is via local waterborne infections ( i . e . , susceptible corals are infected by suspended infectious material originating from diseased corals within the study site ) . The assumptions underlying the construction of the model are that: ( i ) there is a higher probability that infection events take place in close proximity to existing infections; and ( ii ) there is a cumulative impact of multiple infections on a single susceptible coral , such that the more infected neighbors a susceptible coral has , the more likely it is to become infected itself . More specifically , the model determines the probability of each susceptible coral being infected and thus becoming a Newly Infected Coral ( NIC ) . The probability of being infected by any Previously Infected Coral ( PIC ) within the study site is assumed to be inversely proportional to the distance ( d ) of the PIC . In addition , a susceptible coral can be infected by any of the PICs present . Thus , we define the probability of a coral i ( from all susceptible corals within the study site ) to become infected during a month t ( 1≤t≤11; in total there are eleven sequential sampling dates ) as: pt ( i ) =ct∑j∈PICt1dijα , ( 2 ) where PICt is the set of all PICs in month t and dij is the Euclidean distance between coral-i and PIC-j . The exponent α characterizes the decay of the transmission probability with distance . In this way , infections are preferentially passed to neighboring susceptible corals . Another special feature of the model is the inclusion of seasonal drivers [64] through the constants ct that characterize the transmission strength of WPD in each month t . These constants presumably depend on environmental factors that change in accordance to the season ( e . g . , seawater temperatures ) , and therefore may link between the spatiotemporal model and these factors . Note that all PICs within the study site influence the probability of any susceptible coral to become infected . The definition ensures the probability is inversely proportional to the coral’s distance from any PIC . In addition , the probability increases with the number of PICs and the increase will be largest for neighboring PICs ( where the distances dij are small ) . Model parameters that need to be estimated are: ( i ) the exponent α that characterizes the decay of the transmission probability with distance , and ( ii ) the constants ct that characterize the transmission strength in each month t . In order to find the best fitting parameters α , c1 , … , c11 , we define a likelihood function and then maximize it with respect to these parameters . Given PICt ( the set of PICs in month t ) , the probability that the set of corals infected during this month is precisely the set NICt of NICs is: p ( NICt|PICt , α , ct ) =∏i∈NICtpt ( i ) ×∏i∉NICt∪PICt ( 1−pt ( i ) ) . ( 3 ) Here , the first term on the RHS is the probability that all the corals in the set NICt are infected , and the second product is the probability that all the corals , which are neither in the set NICt , nor in the set PICt , are not infected . The total probability of obtaining the empirical results given the model , that is the likelihood function , is thus given by: L ( α , c1 , … , c11 ) =∏t=111[∏i∈NICtpt ( i ) ×∏i∉NICt∪PICt ( 1−pt ( i ) ) ] , ( 4 ) and the log-likelihood is given by: LL ( α , c1 , … , c11 ) =∑t=111[ ∑i∈NICtlog ( ct∑j∈PICt1dijα ) +∑i∉NICt∪PICtlog ( 1−ct∑j∈PICt1dijα ) ] ( 5 ) The maximum-likelihood estimate for the parameters is obtained by maximizing the function in Eq 5 . The procedure described below reduces the multi-variable optimization problem to a series of one-dimensional problems . We note that since each of the variables ct appears in only one of the summands , we find that: maxα , c1 , … , c11LL ( α , c1 , … , c11 ) =maxαM ( α ) ( 6 ) where: M ( α ) =∑t=111maxct[∑i∈NICtlog ( ct∑j∈PICt1dijα ) +∑i∉NICt∪PICtlog ( 1−ct∑j∈PICt1dijα ) ] . ( 7 ) The profile likelihood function M ( α ) is the maximum of LL with respect to c1 , … , c11 with a fixed α . In order to maximize LL , we proceed as follows in our numerical algorithm: ( i ) We step α incrementally through a certain interval in small steps . For each of the α values we run over t from 1 to 11 ( the number of pairs of sequential sampling dates ) , and for each of the values of t we numerically find ct = ct ( α ) that maximizes: M˜t ( α , ct ) =∑i∈NICtlog ( ct∑j∈PICt1dijα ) +∑i∉NICt∪PICtlog ( 1−ct∑j∈PICt1dijα ) . ( 8 ) Two approaches were used to test the null hypothesis that the observed data is generated by the SIS epidemic model driven by Eq 2: The number of NICs observed in the field ( k ) in each month was compared to the distribution of the simulated number of NICs generated from 1 , 000 model realizations using the best-fitting parameters α , c1 , … , c11 . The model fit was tested by comparing the spatiotemporal index n ( r ) ( Eq 1 ) calculated for the actual data with that generated by repeated model realizations using Eq 2 . For further details see Material and Methods . We link the model to seawater temperatures and test possible effect of increasing temperatures on disease dynamics . By controlling the temperature we can test different climate change scenarios . Our model differs from the usual mean-field SIS models in which susceptible individuals and infectives mix randomly and in a uniform manner; here an explicit spatial component is incorporated through the use of Eq 2 . For all future projections , we use the last month of the real data as initial conditions . Then , at each monthly time step , the susceptible corals that become infected over the coral network are stochastically determined according to Eq 2 , given the spatial compositions of the sampled community . The computations keep track of which of all the corals become infected and which remain susceptible . Two different demographic assumptions were applied in the simulations— ( i ) constant influx of recruits , and ( ii ) free-space regulation of recruitment ( see Material and Methods ) .
Based on our analysis with the sptiotemporal index n ( r ) ( eq 1 ) , we found that in all cases Newly-Infected Corals ( NICs ) appeared to form aggregations around Previously-Infected Corals ( PICs ) over distance scales of up to 4 . 5 m ( see e . g . , Fig 2A for June-July 2006 , and S2a Fig for all eleven sequential snapshots ) . This is because the index n ( r ) of the observed data sits almost always above the Monte Carlo 95% CI envelope generated by the null test ( see Materials and Methods ) . That is , in all cases the hypothesis that the NICs were infected by a random process of disease transmission , independent of the spatial location of the PICs , was rejected . In S3 Fig we provide spatial illustrations of the disease dynamics over the studied year showing the spatial relation between PICs and NICs . Using the maximum likelihood fitting procedure , the best-fitting exponent α , which in Eq 2 expresses the decay of the transmission probability with distance , was found to be α^ = 1 . 9 ( Fig 3 ) . The maximum-likelihood estimates for the best-fitting parameters ct , constants that express the transmission strength of the disease during month t ( c1 , … , c11 ) and presumably depend on environmental factors , are given in S1 Table . For all pairs of sequential sampling dates , the number of NICs observed in the field ( k ) was within the 95% confidence interval ( CI ) envelope of the simulated number of NICs obtained from the model realizations ( Fig 4 ) . We thus could not reject the hypothesis that the observed NICs were produced according to Eq 2 . ( Note that here we are essentially testing the model’s “goodness of fit” to the data , and thus there is no need to use the first half of the time series to predict the second half . ) Additional support for the validity of the spatiotemporal model is that in nearly all cases the observed n ( r ) was purely within the null expectation of the model for all distance scales r ( e . g . , Fig 2B ) . However , in a few cases the observed n ( r ) was found to be greater than the upper bound of the 95% CI envelope generated by the model realizations for certain distance scales ( see , for example , August-September 2006 in S2b Fig ) . The number of infected corals observed within the study site ranged from a low of 11 infected corals during June 2006 to a peak of up to 36 infected corals in November 2006 ( Fig 5A ) . The disease prevalence lagged ca . 3 months behind the sea surface temperature ( SST ) that reached its seasonal peak of 27 . 7°C at the end of August 2006 . On the other hand , we found a high association between SST and ct ( see Fig 5B; Adjusted r2 = 0 . 88 , goodness of fit is SSE: 3 . 02e-07 , RMSE: 0 . 0001943 ) , which is expressed by the polynomial relationship: ct=p1⋅SST2+p2⋅SST+p3 ( 10 ) having coefficients [with 95% CI]: p1 = 2 . 968e-05 [-6 . 95e-06 , 6 . 631e-05] , p2 = -0 . 001216 [-0 . 002972 , 0 . 0005402] and p3 = 0 . 01267 [-0 . 008197 , 0 . 03353] . We calculated the epidemiological reproductive number R0 [65] for the time period between June and August 2006 , when the cumulative incidence of infections grows approximately exponentially with time ( see Material and Methods ) . The result shows that the development of the disease within the coral community resulted in an epidemic-like growth with R0 = 1 . 2 ( r = 0 . 35; TG = 0 . 53 ) . The unexpected high association found between SST and the transmission strength ct of WPD ( Adj . r2 = 0 . 88; see Fig 5B ) extracted from fitting the spatiotemporal model to the data allows us to assess the potential long term impact of WPD on the local coral community under different climate change scenarios . We first examine model projections assuming that there is no climate change and that the seasonal cycle of SST temperature repeats in exactly the same way from year to year . Projections of the disease 80 years into the future under these conditions ( see Material and Methods ) show seasonally driven annual cycles ( Fig 6A and 6D ) . Indeed , each year the transmission strength of the disease increases as SST rises from March to August , and then rapidly decreases from September to February ( Fig 5B ) . We then considered the impact of a general mean increase in SST assuming a scenario of constant influx of recruits ( “recruitment limited” ) . Fig 6B shows the effects of increasing SST by 0 . 5°C while Fig 6C shows the effect of only a 1°C increase . We find multi-annual cycles , in which severe epidemics take place every few years when the density of susceptibles corals build up to relatively high levels ( Fig 6A , 6B and 6C ) . The intensity of these epidemics increases with increasing SST , but their frequency is still restricted by the rate at which corals are replenished . The same simulations were examined under an assumption that the coral community is governed by space limitation and is thus “free-space regulated , ” or dependent on the level of free substrate available in the local patch . This follows from the hypothesis that space is a limiting resource in many marine benthic populations [66–69] . Fig 6D , 6E and 6F show that under a scenario of free-space regulation of recruitment , a mean increase of only 0 . 5°C can cause epidemics to double in size , while a mean rise of 1°C can cause increases scaling in orders of magnitude . Finally , we point out that these model “forecasts” should not be viewed as accurate predictions of monthly changes but more as qualitative guidelines as to what might be expected should there be a future long-term trend in SST temperatures . This corresponds to the “strategic” approach suggested by May [70] , which “sacrifices precision in an effort to grasp at general principles . Such general models , even though they do not correspond in detail… provide a conceptual framework for discussion and further exploration” .
Our work offers the very first model fit of any coral disease epidemic , over the timescale of the epidemic , to be found in the literature . Other attempts failed to succeed either because they did not have the fine resolution data ( e . g . , 12 monthly sampling points ) over the timespan of the epidemic , and/or because they did not have a modelling formulations to conduct parameter estimates and model fits . At best other modelling attempts have only taken into account the total annual numbers of infected corals , which is the coarsest of descriptors when characterizing epidemic dynamics . In the beginning of the transmission season , the spread of the disease in the local community exhibited epidemic-like growth motivating us to study R0 , the epidemiological reproductive number . R0 was estimated ( see Material and Methods ) for the time period between June and August 2006 ( the development period of the disease within the community ) and was found to be greater than unity ( R0 = 1 . 2; r = 0 . 35 , TG = 0 . 53 ) . This value of R0 was lower than these calculated for BBD for the outbreaks of 2006 and 2007 ( R0 = 1 . 6 and 1 . 7 , respectively; [16] ) . In BBD , both the exponential growth rate ( r ) and the mean generation interval of the epidemic ( TG ) were greater than these calculated for WPD . Although the observed seasonal outbreak generated an epidemic-like growth , the disease did not spread over a large fraction of the susceptible corals ( see Fig 5 ) . Our model simulations suggest that seasonality and low R0 are not the only factors responsible for this restriction in disease spread , and in particular , that the spatial component of the system may also play a significant role . The spatial scale of aggregations of NICs in the vicinity of existing infected corals indicates that small-scale inter-colonial transmission is significant within the community under study ( see Figs 2 and S2 ) . That is , infected corals are ‘hotspots’ of potentially infectious material , being transmitted to nearby susceptible corals on the reef ( see S3 Fig ) . We find that the larger the number of infected corals in proximity to a susceptible coral , and the closer they are , the higher the likelihood of this coral becoming infected itself . Similar results were found in previous studies for WPD in the Florida Keys [50] , for BBD in the Red Sea [16] and for aspergillosis in the Caribbean [71] . These findings are in contrast to a recent study by Muller & van Woesik [72] which suggests that coral diseases in the Caribbean do not follow a contagious-disease model . One possible explanation for the inconsistency in the results between these studies , is that there are differences in the infection process of the two identified pathogens ( i . e . , the causative agents are known to be different between regions ) . In addition , coral communities across the Red Sea are much denser than in the Caribbean; while in the present study 2 , 747 corals susceptible to WPD were recorded within a 10×10 plot , Muller & van Woesik [72] recorded only 78±12 ( mean±SE ) susceptible corals within the same plot size . Hence , the average distance among susceptible corals in the Caribbean is much greater than in Eilat , making the probability of identifying inter-colonial transmission significantly lower than in Eilat . As such , the findings of Muller & van Woesik [72] would not necessary contradict the findings of our transmission model . In a similar spirit , Bruno et al . [13] also argue that high coral cover and/or density increases the occurrence for horizontal transmission of White Syndrome between corals across the Great Barrier Reef in Australia . Testing the goodness of fit of our spatiotemporal model ( Eq 2 ) in two different ways [e . g . , distribution of NICs and clustering index n ( r ) ] reveals that in all cases the model could effectively predict the number of NICs and in nearly all cases it could simulate the actual spatial patterns of new infections . However , in a few cases the observed n ( r ) was found to be greater than the upper bound of the 95% CI envelope generated by the model realizations for certain distance scales . These deviations suggest that there may be mechanisms involved in the transmission process that are not fully captured by our simple model . However , by comparing the results obtained from the random simulated infections ( S2a Fig ) and those obtained from the spatiotemporal model ( alongside with S2b Fig ) , it is clear that the spatiotemporal model always outperformed the random transmission model . The unexpectedly high association found between SST and the transmission strength ct of WPD ( Adj . r2 = 0 . 88; see Fig 5B ) extracted from fitting the model to the data indicates the power of the modeling approach . This association strongly suggests that SST is the seasonal driver behind the WPD dynamics , and might well be explained by the response of the host and/or pathogen to seasonal thermal fluctuations . High seawater temperatures may cause stress to coral hosts and increase their susceptibility to disease infections [73] , while at the same time they may increase the virulence of the pathogen [74] . Previous studies from other locations have also identified clear seasonal patterns of various coral diseases , such as white syndrome [13 , 32] , BBD [15 , 22] , ulcerative white spots [46] , aspergillosis [47] and white pox [48] , related particularly to warm seawater temperatures . In this study , the seasonal patterns of the transmission strength of WPD ( ct ) preceded the seasonal patterns of the disease prevalence by ca . three months ( see Fig 5B vs . Fig 5A , respectively ) . This suggests that the high seawater temperatures may directly affect the susceptibility of the corals and/or the virulence of the pathogen , but indirectly affect the prevalence of WPD . That is , the impact of the disease on the reef might be the lagged response ( ca . three months ) to processes that advance the progression of the disease within and among coral colonies . The strong coupling of the transmission strength of the disease ( measured by ct ) and the seasonal variation in SST , forms the basis for our forecasts of future global warming scenarios . The association suggests that the higher seawater temperatures associated with future global warming will intensify the impacts of WPD on reefs . Our future predictions verify that in a demographic scenario , when recruitment is purely free-space regulated , such that the coral community density is relatively constant in steady-state conditions , a mean increase of only 0 . 5°C can cause epidemics to double in size . Likewise , a mean rise of 1°C can even lead to increases in several orders of magnitude . However , in reality , the influx of recruits is likely to be limited to some extent and located along a continuum between the two extremes ( i . e . , constant influx vs . free-space regulation ) . Thus , it is reasonable to assume that during an intense epidemic , when many susceptible corals will be removed through death , the spatial component of the disease will play a role in the disease dynamics . Indeed , our future predictions confirm that the spatial component of the disease transmission system has , to some extent , a protective effect that restricts the magnitudes of annual epidemics . Under a demographic scenario of constant influx of recruits , the mean coral community densities decrease as the SST increase ( Fig 6A , 6B and 6C ) . In this case the intensity of the disease does not change with increased SST scenarios . We suggest that this is because the decrease in density discounts for the increase in the transmission strength of the disease ( i . e . , each of these parameters work in a different direction ) . In practice , the decrease in coral density increases the mean distance between infected and susceptible corals within the community and thus decreases the potential for disease transmission [13] . Such a positive relationship between host density and disease transmission has been demonstrated in many host-pathogen systems [75–78] , and is considered as an important property of the infectious process [79] . Specifically with infectious coral diseases , high coral density may have similar effects to that of high coral coverage; effectively this reduces the mean distance between neighboring corals , and as with our spatiotemporal epidemic model , increases the likeliness of inter-colonial transmission . Indeed , Bruno et al . [13] demonstrated that for white syndrome outbreaks to occur in the Great Barrier Reef in Australia , in addition to thermal stress , coral coverage must be relatively high ( 50% or higher ) . Our model suggests that an infectious disease , such as WPD in the Red Sea , cannot lead to a complete destruction of the coral community , due to the spatial nature of the disease transmission and its protective effect . However , this also implies that signs of recovery of local coral communities may be misleading , and are not truly indicative of their rehabilitation ( see for example the sharp fluctuations in the disease prevalence in Fig 6C ) . In addition , environmental changes , such as increasing levels of SST , can shift the nature of recruitment on local scales , altering the way in which the spatial component of the system restricts or enhances local disease dynamics . In addition , note that the remarkable transition in disease prevalence , which is observed when recruitment is free-space regulated ( Fig 6D , 6E and 6F ) , may indicate that the interaction of the seasonal driving force and the spatial nature of the system has higher levels of complexity , beyond those described here . These more complex aspects of this system are beyond the scope of the present paper . To summarize , we have addressed some fundamental questions regarding the dynamics of WPD in the Red Sea . Spatiotemporal statistics combined with null hypothesis approaches proved to be effective tools for understanding epizootiological processes in coral reef communities . The new spatiotemporal index , n ( r ) , proved to be specifically tuned to detect the localized transmission dynamics among the infected corals . Previous approaches for modeling coral disease have not used powerful statistical inference methodologies to estimate parameters and for choosing the best model structure . Neither have they attempted to model the epidemic curve as it changes over a single season . In this study , however , a specially formulated maximum-likelihood fitting procedure , enabled us to estimate the most likely parameters in the model ( α and ct ) , based on the disease dynamics in space and time . It also allowed us to link the spatiotemporal dynamics of the disease to seawater temperature ( see ct in Eqs 2 and 10 ) and gave us an opportunity to generate future projections that assess the impact of increasing SST on coral communities . Over any season , the spatial model revealed that as the temperature increases , the spread of WPD on corals looks similar to the spread of forest fires , where dense forests tend to burn completely while less dense forests are relatively resistant because the fire can hardly spread [80 , 81] . Current assessments on the future of these reef-building corals are still relatively uncertain , being hindered by a lack of knowledge and understanding . In this context , our study exposes the critical importance of conducting additional multi-annual surveys on local spatial scales , for deepening our insights into these unique systems , and for supporting our efforts to successfully design effective conservation policies .
Using a null model approach , which bases the null expectation on the spatial distribution of the entire pool of susceptible corals , we ascertained whether the k NICs found in the field were significantly aggregated around the PICs . We used n ( r ) ( Eq 1 ) as a statistical index , defined as the mean number of NICs in a given month within a radius r from a PIC of the previous month . The non-aggregated null distribution of the NICs , and thus n ( r ) , was generated as follows . Infected corals from the first month in each pair of sequential sampling dates defined the m fixed PICs . Then , via computer simulation , a group of k simulated NICs was randomly chosen from the entire pool of susceptible corals without any discrimination as to whether individuals were healthy or infected . n ( r ) was then determined for different radii r . This was repeated 1 , 000 times so that n ( r ) could be calculated for each group of k NICs for any value of r . These results made it possible to generate a 95% confidence interval ( CI ) envelope for n ( r ) under the null hypothesis of no aggregation of the NICs . We then calculated n ( r ) using only the k observed NICs found in the field . If the observed n ( r ) was found within the envelope , then the null hypothesis could not be rejected and the spatial distribution of NICs was considered independent of the spatial distribution of the PICs . Otherwise , if the observed n ( r ) was found outside the 95% CI envelope , the null hypothesis was rejected and the spatial distribution of NICs was considered significantly dependent on that of the PICs at α = 5% level ( that is , the null hypothesis was rejected ) . NICs are considered spatially aggregated around PICs where the observed n ( r ) is greater than the null expectation , indicating inter-colonial ( i . e . local ) infections . On the other hand , NICs are considered over-dispersed in relation to PICs , if n ( r ) is smaller than the null expectation . This test was carried out for all pairs of sequential sampling dates . To test whether the spatiotemporal model describes suitably the transmission pattern of the disease , we simulated the infection process at the studied site based on a given set of PICs for a particular date , using the most likely parameters ( α^ , c1 ( α^ ) , … , c11 ( α^ ) ) . Thus , infected corals from the first month in each pair of sequential sampling dates define the m fixed PICs . Then , for a simulation that required a generation of new infections , we simply chose NICs at random from the entire pool of corals , assuming that coral-i has a probability pt ( i ) of being chosen ( Eq 2 ) . We repeated this process 1 , 000 times . Then , the model was tested for each pair of sequential sampling dates in two different ways: ( i ) the number of NICs observed in the field was compared with the distribution of the number of NICs obtained from the 1 , 000 random realizations; and ( ii ) the spatiotemporal index n ( r ) ( Eq 1 ) that was calculated for the real data was compared with the distributions of n ( r ) that was calculated for any distance scale r , for the 1 , 000 random realizations . We tested whether the observed number of NICs and n ( r ) were significantly different from the null distribution of those simulated under a two-tailed test of 5% significance level . If this occurred it implied that the results found in the field are inconsistent with the proposed null model . In the beginning of the transmission season , the spread of the disease in the local community exhibited epidemic-like growth . The epidemiological reproductive number , R0 [65] , was calculated for the time period between June and August 2006 ( the development period of the disease within the community ) , using the approximate relationship R0≈erTG[82] ( cf . , Zvuloni et al . [16] for black-band disease ( BBD ) ) . The exponential growth rate is governed by the parameter r , which is estimated by fitting an exponential function to the ( cumulative ) incidence of the infective numbers . The parameter TG is the observed mean generation interval , i . e . , the interval between a coral becoming infected and its subsequent infection of another coral ( see Zvuloni et al . [16] ) . R0 measures the epidemic potential of a pathogen and is defined as the mean number of secondary infections caused by a typical single infectious individual in a wholly susceptible coral community . When R0 ≤ 1 , the introduction of an infected individual will fail to result in an outbreak . If , however , R0 > 1 , then the introduction of the disease is likely to result in an epidemic that persists for extended periods . Linking the spatiotemporal model ( Eq 2 ) to seawater temperatures allows us to assess the potential future impact of WPD on the local coral community . We calculated the probability of each susceptible coral to become infected according to Eq 2 , where ct in this equation was determined by fitting a quadratic model to fit SST according to the SST in that month ( Eq 10 ) . We set α = 1 . 9 , which was found to be the best fitting exponent . The use of Eqs 2 and 10 for future predictions ensures that the probability of any susceptible coral to become infected has both spatial and seasonal/environmental components . In accordance with our data , simulations are carried out in discrete time steps from month to month . For all simulated projections , we use the last month of the real data as initial conditions for the future projections , and SST is based on a time series measured between June 2006 and May 2007 , which we assume repeats yearly . In light of global change , there is also obvious interest in trying to assess long-term effects of variations in SST , and we do this by varying the levels of SST in our simulated projections . Each year in the beginning of the infection period we randomly infected one of the corals . This insured that the local population did not stay infection free due to stochastic fadeouts in the previous season . Clearance and death rates were month specific and calculated based on collected data , i . e . , the probability of death , recovery , or remaining infected is determined by the fraction of infections that died , cleared , or stayed infected in the same month in the original data . The locations for new recruits in the 10×10 m plot are randomly chosen anywhere on the plot whenever a recruitment event takes place . This approach sets no spatial restrictions on coral settlement , and as such does not constrain the topological distribution of the corals . We assume that the per capita recruitment is either: ( i ) “recruitment limited”—independent of local community density by assuming a constant influx of recruits per year . Alternatively , we assume recruitment is ( ii ) “free-space regulated”—dependent on the level of free substrate in the local patch; following from the hypothesis that this is a limiting resource in many marine benthic populations [66–69] . Here it is assumed that following a coral’s death , a healthy recruit instantaneously replaces it . In the first scenario ( i ) , due to the spatial component of the model , the coral density may play a significant role in the transmission probability of the disease . On the other hand , in the second scenario ( ii ) , the coral community density remains constant , and the role of the spatial component in the model is also expected to be relatively constant . In reality , coral recruitment is likely to lie somewhere between these two extremes , with variations in the location of different reefs along this continuum ( for further reading on these assumptions , see [66–69] . | Coral reefs are deteriorating at alarming rates , with coral disease outbreaks increasing in prevalence and in space . Anomalously high ocean temperatures are thought to significantly contribute to this problem . We collected a unique and highly resolved dataset of a White Plague Disease ( WPD ) outbreak from the coral reef of Eilat ( Israel , Red Sea ) . By fitting a novel epidemiological model to the data , we characterize the dynamics of WPD , and study the possible effects of future increasing sea-surface temperatures ( SST ) on disease dynamics . In contrast to earlier studies , our approach captures the dynamics of coral disease both in space and time , and accounts for the highly seasonal nature of the annual outbreaks . We also apply a novel combination of spatiotemporal statistics and null hypothesis approaches to study the disease progression . Model forecasts into the future show that for some scenarios even an increase of only 0 . 5°C in SST can cause epidemics to double in magnitude . Since the probability of infection is found to be negatively associated with coral density , this implies that the spatial nature of disease transmission can both enhance and restrict the magnitude of annual epidemics . The results have implications for designing management policies appropriate for coral reef conservation . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2015 | Modeling the Impact of White-Plague Coral Disease in Climate Change Scenarios |
During meiosis , Structural Maintenance of Chromosome ( SMC ) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation . While meiotic functions of the cohesin and condensin complexes have been delineated , the role of the third SMC complex , Smc5/6 , remains enigmatic . Here we identify specific , essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin . We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms , respectively . Smc5/6 also localizes to recombination hotspots , where it promotes normal formation and resolution of a subset of joint-molecule intermediates . In this regard , Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex . Furthermore , we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease , Mus81-Mms4Eme1 . Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence , attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastrophe .
Sexually reproducing organisms reduce their genomic content by half in the gametes such that the normal chromosome copy number is restored in the zygote . To achieve this , homologous chromosomes ( homologs ) have to pair and then segregate to opposite spindle poles at the first division of meiosis . In many organisms , homolog pairing and segregation depends upon the developmental induction of hundreds of double-strand breaks ( DSBs ) throughout the genome ( 150–300 DSBs in yeasts and mammals ) [1] . High levels of DSBs are necessary for homologs to pair efficiently along their entire lengths [2] . Moreover , a subset of DSB repair events lead to crossover formation . These reciprocal exchanges between homologs combine with sister-chromatid cohesion to form chiasmata , the physical connections that aid bi-orientation of homologs on the meiosis I spindle . Homolog separation at anaphase I thus requires the release of sister chromatid cohesion between chromosome arms . However , centromere cohesion is specifically protected to allow biorientation and accurate segregation of sister chromatids on the meiosis-II spindles [3]–[5] . Meiotic recombination is highly regulated and temporally coordinated with the meiotic cell cycle . Crossover-specific joint molecule intermediates ( JMs ) are formed during midprophase I of meiosis ( ‘thick threads’ , pachytene ) , when homologous chromosomes are highly compacted and paired along their entire length by the synaptonemal complex . JMs are resolved into crossovers upon pachytene exit when a dedicated resolving process becomes activated by polo-like kinase [6]–[8] . In contrast , most noncrossovers arise during prophase I , independently of known resolving nucleases via a process termed synthesis-dependent single-strand annealing [8] , [9] . The formation of JMs is guided by the RecQ-family DNA helicase Sgs1/BLM , which limits the formation of aberrant JM structures , such as those that interconnect 3 or 4 chromatids instead of the normal two [10] , [11] . Resolution of aberrant JMs requires the activities of structure-selective nucleases , Mus81-Mms4 , Slx1-Slx4 and Yen1 [11]–[14] . Sgs1 together with type-I topoisomerase , Top3 , and accessory factor , Rmi1 , defines a potent double Holliday junction ( dHJ ) “dissolving” enzyme that specifically promotes noncrossover formation [15] , [16] . At pre-crossover sites , this dissolution activity must be attenuated in order to ensure efficient crossing over . In budding yeast , a majority of crossovers are formed via a dedicated pathway defined by the conserved , meiosis-specific MutS complex , MutSγ ( Msh4–Msh5 ) that is predicted to encircle and thereby stabilize JMs [17]–[20] . From extensive studies , we know that components of the MutSγ pathway promote the formation of stable JMs , Single End Invasions ( SEIs ) and dHJs , and protect them from being dissociated by Sgs1 [10] , [20] , [21] . Subsequent resolution of dHJs into crossovers requires the DNA mismatch repair factors , Exo1 and the predicted endonuclease activity of MutLγ , a complex of the MutL homologs Mlh1 and Mlh3 [22] , [23] . In C . elegans , MutSγ promotes all crossovers [24] . However , other organisms , such as fission yeast and Drosophila , lack MutSγ . In Drosophila , an analogous function in protecting JMs from Sgs1/BLM anti-crossover activity has been inferred for two MCM-like proteins ( mei-MCM ) . JM resolution in Drosophila occurs by the XPF-family endonuclease , MEI9-ERCC1 [25] , [26] . In fission yeast , essentially all crossovers are generated by Mus81-Eme1 , another XPF-family endonuclease [27]–[29] . In budding yeast , plants and mammals MutSγ-MutLγ is the predominant pathway of crossover formation , although Mus81-Eme1 ( Mus81-Mms4 in budding yeast ) also promotes a subset of crossovers [30]–[32] . Although Exo1-MutLγ , Mus81-Mms4 , and Sgs1 are the major JM processing activities during budding yeast meiosis , at least two additional endonucleases can also facilitate resolution in budding yeast and metazoans . Yen1 can act as a backup resolvase in the absence of Mus81-Mms4 [13] , [14] , [33] . Similarly , Slx1–Slx4 is essential for resolution of a subset of JMs , specifically when Sgs1 is absent [13] , [14] , [34]–[36] . Collectively , the JM resolution and dissolution activities establish two essential conditions for efficient homolog disjunction at meiosis I: formation of crossovers to facilitate homolog biorientation and the efficient removal of all JMs that would otherwise impede chromosome separation . Meiotic recombination is coordinated with global changes in chromosome morphology , including sister-chromatid cohesion and condensation . These processes are mediated by Structural Maintenance of Chromosome ( SMC ) complexes , large clamp or ring-like structures that include cohesin , condensin and Smc5/6 . Whereas cohesin and condensin have wide-ranging effects on global chromosome morphology as well as DNA repair [37] , the Smc5/6 complex appears to operate locally to attenuate recombination [38]–[43] . During mitotic growth , the Smc5/6 has been proposed to stabilize stalled replication forks and prevent recombination at the fork [43] , [44] . However , if recombinational repair ensues , Smc5/6 also regulates late steps , promoting the resolution of recombination structures [38] , [45] . The core Smc5/6 complex does not contain any DNA repair activities , raising the question of how it facilitates replication and recombination . One model posits that Smc5/6 regulates effector proteins via an intrinsic SUMO E3 ligase activity , catalysed by the associated non-SMC element Nse2/Mms21 [46]–[48] . This SUMO-mediated process has been inferred for regulation of telomeric and kinetochore proteins , and the establishment of cohesion around DSB sites ( in mitotically cycling cells ) [49]–[52] . However , this emerging paradigm has not been extended to enzymes involved in JM resolution . Genetic or physical interactions between Smc5/6 and JM resolving enzymes have not been established . Based upon the findings that chromosome segregation appeared worse in smc6 mutants that also lacked Sgs1 or Mus81 , the Smc5/6 complex has been suggested to work in parallel with both Sgs1 and Mus81-Mms4 during mitotic DNA repair [40] . However , the severity of smc5/6 mutants in combination with mus81 or sgs1 could equally reflect both separate as well as collaborative functions . The only physical interaction described to date is with the Mph1/FANCM DNA helicase , whose interaction with Smc5/6 does not depend upon sumoylation [53] . Despite the central role of Smc5/6 in orchestrating responses to DNA damage in mitotic cells , the role of Smc5/6 in meiotic recombination remains equivocal . In one study , a critical role for budding yeast Smc5/6 was inferred to occur during premeiotic S-phase , since abolition of meiotic DSBs by mutation of Spo11 did not improve the block to chromosome separation caused by smc5/6 mutation [54] . In fission yeast , deletion of Nse5 or Nse6 is epistatic with the Mus81-Eme1 resolvase with regards to crossover generation suggesting that Smc5/6 regulates Mus81-dependent crossovers [55] . However , Mus81-Eme1 appears to be the sole resolvase acting during meiosis in fission yeast [56] , [57] , so it is unknown whether this paradigm extends to organisms that employ multiple resolvases; or whether Smc5/6 influences all resolution activities via global changes in chromosome structure . In contrast to fission yeast , in C . elegans animals depleted for Smc5/6 , crossover formation appears normal but meiocytes contain excess RAD-51 foci indicative of unrepaired DSBs [58] . From these phenotypes , a specific defect in meiotic DSB-repair between sister-chromatids was inferred [58] . This raises the possibility that the Smc5/6 complex regulates a subset of recombination events and their resolution via specific resolvase activities . A possible explanation for these apparently contradictory phenotypes is the extent to which different organisms employ the different JM resolution/dissolution activities [59] . In this study , we demonstrate that budding yeast Smc5/6 has essential roles during meiotic recombination in regulating the ordered formation of interhomolog joint molecules as well as their resolution . In smc5/6 mutants , intersister dHJs as well as multichromatid joint molecules accumulate and fail to be resolved . For the latter , we show that Smc5/6 regulates Mus81-Mms4 activity in joint molecule resolution and localization to meiotic chromosomes . In contrast , the main resolvase activity during meiosis ( MutLγ ) appears to function independently of the Smc5/6 complex .
Affinity-tagged Smc5-13myc and Nse4-TAP proteins were expressed throughout meiosis ( Figure S1A ) . A subset of Smc5-13myc migrated as a highly molecular weight band that likely corresponds to the sumoylated species ( Figure S1A ) . Smc5-13myc displayed linear or punctate immuno-staining patterns along meiotic chromosomes , during prophase I , that became undetectable at diplonema and metaphase I ( Figure S1B ) . The punctate localization of Smc5-13myc was dependent upon Cdc6 ( which is required for meiotic DNA replication ) and to a lesser extent on the type-II topoisomerase Top2 ( Figure S1 ) . In contrast , chromosomal staining of Smc5/6 did not require Spo11 ( required for DSB formation ) , Rec8 ( cohesion ) , or the type-I topisomerases , Top1 and Top3 ( Figure S1C and data not shown ) . To obtain a higher resolution picture of Smc5/6 association with meiotic chromosomes , we carried out genome-wide ChIP-on-chip localization analysis for Smc5 tagged at its C-terminus with three V5 or 13 myc epitopes . Smc5 binds to many of the same chromosomal axis-associated sites as the meiosis-specific cohesin component , Rec8 , and is similarly enriched at centromeres ( Figure 1A , 1B ) . A similar , perhaps even more pronounced , enrichment at cohesin binding sites was also observed when a tagged Smc5-13myc protein was analyzed ( Pearson's correlation coefficient ( PCC ) for Smc5-3V5 vs . Rec8 = 0 . 22 , p<10−15; Smc5-13myc vs . Rec8 = 0 . 43 , p<10−15; Figure S2 ) . The enrichment of Smc5 at cohesin binding sites , centromeres , and telomeres is similar to the localization pattern previously described for Smc5/6 in vegetative cells [60] . However , in contrast to the mitotic distribution [60] , neither we nor Xaver et al . [61] observed an increased density of Smc5/6 association sites along longer chromosomes during meiosis . To determine whether the association of Smc5 with meiotic chromosomes depended upon DSB formation , we determined the binding profile in the absence of Spo11 ( Figure 1B ) . Aside from a small overall reduction in binding , we observed no gross changes in the Smc5-3V5 distribution either at or between core sites in a spo11Δ strain ( Figure 1B ) . This result is consistent with our observation that Smc5 immuno-staining on individual , spread meiotic nuclei is largely unaffected in the absence of Spo11 ( Figure S1C ) , similar to that seen for Smc6 [54] . Some weaker binding sites also occurred in between the axis association sites defined by Rec8 ( Figure 1A , lower panel ) . DSBs tend to occur in between the Rec8 axis association sites [62] , [63] , and Smc5/6 is recruited to DSBs in mitotic cells [60] , [64] . Thus , we explored the idea that a fraction of Smc5/6 binds meiotic DSB sites . The locations of non-axis Smc5 association sites were determined by normalizing the Smc5 binding signal to the Rec8 signal ( Figure 1C ) . This analysis revealed several additional binding sites along each chromosome ( Figures 1C , S2 ) . These weaker binding sites showed significant overlap with DSB sites ( PCC = 0 . 28 , p<10−15; Figure 1C ) , mapped by single-stranded DNA that accumulates at DSB sites in dmc1Δ mutants [65] , [66] . Thus , Smc5/6 displays both a strong localization to chromosomal core sites and a weaker ( perhaps more transient ) localization to DSB sites . Several proteins involved in the formation and processing of meiotic DSBs localize to DSB hotspots even in the absence of DSB formation . Indeed , the Smc5 pattern , including DSB-correlated sites , is essentially unchanged in a spo11 mutant ( Figure 1C ) . This pattern is reminiscent of the binding profiles of Rec114 and other factors required for DSB formation , which are inferred to result from interaction of the DSB sites with the chromosome axes at the time of DSB formation [62] , [63] . We conclude that Smc5/6 associates with cohesin association sites , centromeres , as well as DSB hotspots , and that this association occurs mostly independently of DSB formation . The strong enrichment of Smc5/6 at centromeres ( the strongest cohesin binding sites in the genome ) as well as DSBs were also observed for the Smc6 subunit in independent experiments by Xaver et al . ( 2013 ) . Using ChIP-seq , they observed a small enrichment at cohesin association sites as well . The differences in the magnitude with which Smc5 ( our study ) or Smc6 ( Xaver et al . ) binds cohesin associated sites is likely due to the affinity tags being placed on different subunits of the complex . These may be differentially accessible to the antibodies and/or local DNA . It is unlikely that the enrichment of Smc5/6 that we observe in the ChIP experiments is non-specific , because the patterns are similar for both Smc5-3V5 and Smc5-13myc , which were immunoprecipitated with different antibodies and resins . Moreover , other DSB factors tagged with 13myc did not show any significant enrichment to cohesion binding sites by ChIP-chip ( data not shown ) . Finally , consistent with a fraction of Smc5/6 binding to chromosomal axes , more than 50% of Smc5-13myc foci localize to the synaptonemal complex ( central element component , Zip1 ) in our experiments ( Figure S1B ) . This makes it highly unlikely that non-specific association of the antibodies with proteins or sequences at cohesin binding sites gives rise to false peaks . Smc5 localization at sites of meiotic DSBs , cohesin binding , and centromeres suggests possible roles for the Smc5/6 complex in meiotic recombination and chromosome morphogenesis . Since Smc5/6 is essential , its meiotic functions were studied by depleting the core component , Smc5 , and the kleisin ( Nse4 ) using the CLB2 promoter , which is strongly repressed in meiosis [67] ( Figure 2A ) . Meiosis-specific depletion circumvents the need for temperature-sensitive conditional alleles that require temperature-shift protocols , which may be complicated by the fact that several chromosomal processes are affected by temperature [20] , [68] . Strains carrying the PCLB2-SMC5 or PCLB2-NSE4 alleles ( hereafter , smc5 and nse4 ) had normal vegetative growth and were not sensitive to DNA damaging agents ( data not shown ) . In meiosis , although bulk DNA replication and spindle pole body separation were essentially normal ( Figure 2B , C ) , nuclear divisions were severely defective ( Figure 2D ) . Time-lapse studies revealed that although nuclear divisions were attempted at both anaphase I and II , as soon as spindles disassembled , DNA bodies retracted into a single mass that subsequently failed to be encapsulated in the spores ( Figure 2F , H; Movie S1 , S2 , S3 ) . None of 30 randomly-selected cells imaged for either the smc5 or nse4 mutant managed to stably separate their DNA at the completion of meiosis I or II ( Figure 2E ) . Micronuclei or fragmented nuclei as well as aberrant chromosomal morphologies were also observed ( Figure 2E , arrows ) . Despite the severe nuclear separation defect , both the smc5 and nse4 mutants went on to complete meiosis and form asci with similar efficiencies to wild type ( Figure 2F , G , ∼90% ) . However , the failure to separate the DNA at meiosis I and II , prevented encapsulation of DNA into the spores ( Figure 2H ) . This “meiotic catastrophe” was more pronounced for the nse4 mutant compared to the smc5 . This is likely due to more efficient depletion of Nse4 , because when Smc5 was further depleted using an auxin-inducible degron fusion ( PCLB2-SMC5-AID , [69] ) , the nuclear separation defect became more severe and analogous to that seen in nse4 cells ( Figure 2F–H ) . We could not determine unequivocally that the PCLB2-SMC5-AID was more depleted than PCLB2-SMC5 , since the depletion by PCLB2-SMC5 alone rendered Smc5 undetectable by Western blot ( Figure 2A , data not shown ) . However , analysis of SMC5-AID ( without CLB2 depletion ) demonstrated that auxin-induced degradation of Smc5 does occur , even when Smc5 is expressed at normal levels from its native promoter ( Figure S3 ) . Together , these experiments support the notion that the less severe meiotic catastrophe seen in the PCLB2-SMC5 cells relative to PCLB2-NSE4 is due to less efficient depletion of Smc5 . However , they do not rule out the possibility that Nse4 has a function distinct from Smc5 , perhaps acting as part of the Nse1-Nse3-Nse4 subcomplex [70] . To determine whether meiotic catastrophe required the initiation of recombination , we abolished the DSB activity of Spo11 , using the catalytically-dead spo11-Y135F allele . This suppressed the nuclear separation defects of both smc5 and nse4 ( Figure 3A ) . To address whether DNA damage or replication intermediates accumulated during pre-meiotic S-phase contribute to the nuclear separation defects of smc5 and nse4 , we converted meiosis I into a single mitosis-like division by de-protecting centromeric cohesin at anaphase I ( spo13Δ ) , while simultaneously inactivating recombination ( spo11Δ ) . No effect of smc5 or nse4 mutation on either dyad formation or spore viability was observed ( Figure 3B , C ) . This experiment rules out the possibility that gross S-phase defects alone are responsible for the meiotic chromosome segregation failure in smc5 and nse4 mutants . Thus , depletion of Smc5/6 causes severe recombination-dependent meiotic catastrophe . This is in sharp contrast to the smc6–9 temperature sensitive allele , which was previously shown to cause meiotic catastrophe independently of Spo11 [54] . To investigate possible roles of Smc5/6 in meiotic DSB repair , we analysed meiotic recombination at the well-characterized HIS4LEU2 recombination hotspot construct using a series of Southern blot assays [71] , [72] ( Figure 4 ) . Restriction site polymorphisms combined with 1D or 2D gel electrophoresis and Southern analysis allow formation of DSBs , crossovers , noncrossovers and several different species of joint molecules to be monitored at HIS4LEU2 . Joint molecules include single-end invasions , double Holliday Junctions ( formed between homologs or between sister chromatids ) and multichromatid joint molecules ( involving 3 or 4 chromatids ) [10] , [71] , [72] . In wild-type cells , joint molecule levels peaked around 4 . 5 hours , at ∼3% of hybridizing DNA , and disappeared by 8 hrs , when the majority of cells had completed the meiotic divisions ( Figure 5A , C ) . In contrast , joint molecules in the smc5 mutant appeared with normal timing but persisted at high levels ( 4 . 7% ) until at least 9 hrs . The nse4 mutant had a much more severe defect in joint molecule resolution , with very high levels of joint molecules ( 10% ) persisting at 13 hrs ( Figure 5C ) , when wild type cells have completed the meiotic divisions ( Figure 2D ) . The level of unresolved joint molecules detected in the nse4 mutant is at least 3-fold higher than any other single mutant analyzed to date and is reminiscent of mutants that simultaneously lack multiple joint molecule resolution or dissolution activities [13] , [14] , [33] . Closer inspection of both the intersister- and interhomolog-dHJ signals revealed additional spots or smears ( Figure 5B ) . In the 1st dimension , these new signals migrated ahead of the main dHJ spots , suggesting a lower molecular weight . In contrast , the signals were retarded in the 2nd dimension relative to the main dHJ spots . It is currently unclear whether these JM species are extreme variants of dHJs ( e . g . with very widely spaced Holliday junctions ) or aberrant structures that are never formed in wild type . Regardless , their existence indicates that JM formation as well as resolution is altered in smc5 and nse4 mutants . In contrast to joint molecules , the appearance , disappearance , and resection of DSBs in smc5 and nse4 mutants occurred with largely wild-type kinetics ( Figure 5D , Figure S4 , S5 ) . These observations suggest that the initiation of recombination occurs without any significant defects and that smc5/6-depleted cells are specifically defective in steps leading to the formation and resolution of joint molecules . Crossover formation was delayed and final levels were reduced by 20–30% in smc5 and nse4 mutants . Crossovers accumulated to 22% of the DNA signal in wild type , while nse4 and smc5 mutants formed , respectively , 15% and 17% ( Figure 5D and E , S4B ) . The double mutant ( smc5 nse4 ) was indistinguishable from the nse4 single mutant ( Figure S4 ) . To understand whether smc5/6 mutants accumulate a specific class of joint molecules , we separately quantified the levels of single-end invasions ( SEIs ) , double Holliday Junctions ( dHJs ) , and multi-chromatid joint molecules ( mcJMs ) using 2D gels ( Figure 5C ) . Compared to the wild type , the smc5 mutant showed slightly elevated levels of all joint molecule species and delayed disappearance . In the nse4 mutant , all classes of joint molecule accumulated to higher levels than wild type and remained elevated throughout the meiotic time course ( Figure 5C ) . We infer that Smc5/6 plays a general role in joint molecule metabolism . Our observations that smc5 and nse4 mutants accumulate unresolved joint molecules while still forming high levels of crossovers raise the possibility that more total joint molecules are made in these mutants . To address this question , we used the resolution-defective ndt80Δ mutant to quantify joint molecule formation independently of changes in the efficiency of resolution [6] , [8] . In both ndt80Δ and ndt80Δ nse4 , total accumulated joint molecules plateaued at similar levels and with essentially identical kinetics ( ∼15% , Figure 5C , lower panel ndt80 ) . However , intersister dHJs and multichromatid JMs were increased at the expense of interhomolog dHJs when compared to the ndt80Δ mutant alone ( Figure 5C; lower panel ndt80 ) . Consistently , the ratio of interhomolog dHJs to intersister dHJs ( “interhomolog bias” ) was decreased from 4∶1 ( 4 . 1±0 . 5 ) in the ndt80Δ strain , to 2∶1 in both mutants ( 1 . 9±0 . 3 and 1 . 7±0 . 2 in smc5 ndt80Δ and nse4 ndt80Δ , respectively; Figure 5C and data shown not ) . Similarly , when NDT80 was present , the IH∶IS dHJs ratio was also decreased from a steady-state ratio of ∼3 . 5±0 . 4 in wild type to 2 . 1±0 . 2 in smc5 and 2 . 1±0 . 2 in nse4 ( P<0 . 01; Figure 5C ) . We conclude that overall JM levels are not significantly altered by depletion of Smc5/6 , but the spectrum of JMs is altered such that intersister and multichromatid joint molecules are increased at the expense of interhomolog dHJs . Similar conclusions have been reached by two other labs [61] , [73] . In budding yeast meiosis , Sgs1 helicase is a central regulator of meiotic recombination intermediates during meiotic prophase [10]–[14] . Similar to smc5 and nse4 strains , sgs1 mutants form more multichromatid and intersister JMs , but fewer interhomolog dHJs [10] . However , unlike smc5 and nse4 , joint molecule resolution and chromosome segregation occur efficiently in sgs1 cells . To examine the relationship between Smc5/6 and Sgs1 , we combined smc5 or nse4 depletion mutants with meiosis-specific depletion of Sgs1 ( PCLB2-3HA-SGS1 , hereafter sgs1 ) . Both crossover and noncrossover formation were synergistically decreased in the smc5 sgs1 and nse4 sgs1 double mutants ( Figure 6A and data not shown ) . On their own , smc5 , nse4 , and sgs1 single mutants exhibited , respectively , 1 . 5% , 13% , and 0 . 6% joint molecules at time points when cells had completed meiosis ( 13 h; Figure 6A and data not shown ) . In both the smc5 sgs1 and nse4 sgs1 double mutants , we observed synergistic increases in all species of joint molecules , which accumulated to 14% and 20% , respectively ( Figure 6A and data not shown ) . This level of accumulation of joint molecules is similar to that seen when both Sgs1 helicase and structure-specific endonucleases ( Mus81-Mms , Slx1–Slx4 , and Yen1 ) are lacking ( ∼20% , [13] , [14] ) . Given that crossover and noncrossover levels are high in the smc5 and nse4 strains ( Figure 5E , 6B ) , we infer that Sgs1 can still function proficiently to promote crossovers and noncrossovers when Smc5/6 is depleted . MutLγ is inferred to be an endonuclease that specifically promotes the resolution of dHJs into crossovers along the MutSγ pathway for crossing over [17] , [22] , [23] , [74] . To test whether the crossovers formed in smc5/6 mutants are formed via this pathway , we deleted MLH3 in the smc5 and nse4 mutants . Although the mlh3Δ mutation alone caused a substantial decrease in crossovers ( compare 18%±0 . 5% in wild type to 8 . 2%±0 . 2% in the mlh3Δ; Figure 6D ) , crossing-over in the double mutants was further decreased ( 4 . 5±0 . 5% for smc5 mlh3Δ and 4 . 4%±0 . 2% for nse4 mlh3Δ; Figure 6D; data not shown for smc5 ) . Importantly , noncrossovers were unaffected , consistent with the notion that MutLγ predominantly yields crossovers [23] , [75] . We infer that MutLγ is active and responsible for most crossovers in smc5/6 mutants . MutLγ promotes crossovers in conjunction with MutSγ , which in turn interacts with and requires Zip3 , for its association with meiotic chromosomes ( reviewed in [76] ) . Zip3 associates in a punctate pattern with meiotic chromosomes at axial association sites , where homolog synapsis initiates and where crossovers will form [2] , [77] . We reasoned that if MutLγ and MutSγ are active in the smc5 and nse4 mutants , then Zip3 localization along meiotic chromosomes as well as synapsis should occur with normal proficiency . To assess whether this was the case , we detected a GFP-tagged Zip3 and co-stained for the synaptonemal complex protein , Zip1 ( Figure 6E ) . In the wild type , we observed ∼30 Zip3-GFP foci in pachytene nuclei; this number was increased 1 . 2–1 . 3-fold in the smc5 and nse4 mutants ( Figure 6F ) . This increase was similar in magnitude to that observed in an Sgs1-depleted strain ( Figure 6F ) [78] . Zip3 promotes the assembly of the synaptonemal complexes ( SC ) . No significant differences were observed in the kinetics of SC assembly and disassembly , including turnover of Zip1 protein , in the smc5 and nse4 when compared to the wild type ( Figure S6 ) . Thus , early steps in MutSγ-dependent crossover formation and initiation of synapsis are not adversely affected by depletion of Smc5/6 . Our results further distinguish phenotypes observed for Smc5/6 from those of Sgs1: Smc5/6 depletion does not suppress the crossover defect of MutLγ , unlike that seen in sgs1 mlh3Δ mutants [10] . These phenotypes could be explained if Smc5/6 has additional roles in joint molecule resolution via the Mus81-Mms4 endonuclease , which becomes essential for resolution in sgs1 mutants [11] , [12] . To determine whether Smc5/6 affects the functions of structure-selective endonucleases during meiosis , we deleted MMS4 ( mms4 ) , the regulatory subunit of Mus81 , and also the two cryptic endonucleases Yen1 and Slx1–Slx4 [79] . Yen1 and Slxl–Slx4 have only minor , if any , roles in joint molecule resolution in otherwise wild-type cells [13] , [14] , [33] . Crossover levels were roughly similar in the mms4 yen1 slx4 mutant ( 11±0 . 4% ) , smc5 ( 12%±0 . 7% ) and nse4 ( 14 . 5±1 . 7% ) mutants ( Figure 7A , B and data not shown ) . The nse4 mms4 yen1 slx4 quadruple mutant had a further reduction in the levels of crossovers ( 7 . 4±0 . 7%; Figure 7A , B ) . Noncrossovers were also further decreased in the nse4 mms4 yen1 slx4 quadruple mutant . In the wild type , the noncrossover signal contributed 2 . 4±0 . 1% , compared to 2 . 1±0 . 1% in the nse4 mutant , 1 . 6±0 . 3% in the mms4 yen1 slx4 mutant and 1 . 3±0 . 1% in nse4 mms4 yen1 slx4 quadruple mutant ( Figure 7A , B ) . At least two reasons could account for the further loss of crossover and noncrossover products in the nse4 mms4 yen1 slx4 quadruple mutant . Smc5/6 could promote joint molecule resolution in parallel with one or more of the three endonucleases . Alternatively , the formation of joint molecules leading to crossovers and noncrossovers could be perturbed . Analysis of joint molecules in the nse4 mms4 yen1 slx4 cells lends support to the latter possibility ( Figure 7C ) . The nse4 mms4 yen1 slx4 mutant displayed a further decrease in the IH∶IS dHJ ratio ( 1∶1 ) compared to the nse4 single and mms4 yen1 slx4 triple mutants ( 2∶1 ) . This indicates that Smc5/6 operates in parallel with the resolvases to promote interhomolog template bias ( Figure 7C ) . Assuming a direct relationship between interhomolog-dHJs and the generation of interhomolog products ( crossover and noncrossover ) , the decreased IH∶IS bias ( 50% ) in the nse4 mms4 yen1 slx4 mutant would be predicted to lead to a loss of half the crossovers ( predicted 7 . 3% crossover products based on the 14 . 5% crossovers seen in the nse4 mutant ) . The observed value of 7 . 4% crossovers ( Figure 7B ) is in good agreement with this . The additive reduction of interhomolog bias in the nse4 and mms4 yen1 slx4 mutants is therefore sufficient to explain the further decreases in crossover and noncrossover levels seen in the nse4 mms4 yen1 slx4 quadruple mutant . To further address which endonuclease was affected by Smc5/6 , we focussed upon analysing the genetic interaction with Mus81-Mms4 ( Figure 7D , E ) . Crossover levels ( Figure 7E ) as well as the IH∶IS dHJ ratios ( Figure 7D ) were similar in the mms4 , nse4 , and nse4 mms4 mutants . These observations show that abolishing Mus81-Mms4 activity has little consequence for joint molecule resolution at least when Smc5/6 is depleted . Moreover , crossover levels were substantially higher in the nse4 mms4 mutant compared to the nse4 mms4 yen1 slx4 quadruple mutant , which suggests that Yen1 , or more likely , Slx1–Slx4 promotes a significant amount of crossing over , presumably via a function that promotes interhomolog bias ( Figure 7C ) . In contrast to the effect of depleting Sgs1 in the nse4 mutant background , the level of unresolved joint molecules did not increase in the nse4 mms4 yen1 slx4 quadruple mutant , but instead decreased ( compare 4 . 3±0 . 6% to 12 . 9±2 . 4% in the nse4 single mutant; Figure 7B ) . This was also the case for the nse4 mms4 mutant ( 6 . 6% unresolved joint molecules; Figure 7E ) . We interpret there results to mean that when Smc5/6 is depleted , the Mus81-Mms4 endonuclease renders a significant proportion of joint molecules non-cleavable by Sgs1 and/or MutLγ . To investigate whether chromosomal localization of Mus81-Mms4 was affected in the smc5 and nse4 mutants , we assessed the ability of Mus81-9myc to form foci on spread , meiotic chromosomes at pachytene , when joint molecules reach their highest levels . Pachytene-stage nuclei were selected by virtue of linear staining of the synaptonemal complex component , Zip1 , and the numbers of Mus81 foci were counted . In the wild type , the majority of pachytene nuclei contained more than 20 distinct foci of Mus81 . In contrast , the majority of nuclei from the smc5 and nse4 mutants had no distinct Mus81 foci ( Figure 7F , G ) . We ruled out that this was due to reduced levels of Mus81-Mms4 protein or failure to hyperactivate Mus81-Mms4 upon exit from pachytene ( Figure S7 ) . These observations imply that the ability of Mus81 to associate with or be stabilized on meiotic chromosomes is diminished when Smc5/6 complexes are depleted . Our observations imply that unresolved joint molecules in the smc5 and nse4 cells cause severe failure of chromosome segregation during anaphase I and II and , ultimately , meiotic catastrophe ( Figure 2 ) . This recombination-dependent meiotic catastrophe hypothesis makes at least two predictions . First , the cell cycle should occur with similar timing in the mutant and wild-type strains and , second , individual meiotic nuclei should show increased DNA damage at anaphase I and anaphase II , when cells are attempting to divide their nuclei . To test these predictions , we monitored markers for early prophase I , exit from prophase I , and entry into meiosis II , which allowed us to calculate and thus compare transit times in the wild type to Smc5/6-depleted cells . Induction of the meiotic DNA damage response ( DDR ) , monitored by the Mec1/ATR-dependent phosphorylation of HORMA-domain protein , Hop1 , and γH2A [80] , [81] occurred with similar timing , 3–4 hours after transfer to sporulation medium ( Figure 8A ) . Spindle pole body separation , a marker for pachytene exit , and indeed spindle formation both occurred with relatively normal timing in the two mutants compared to wild type ( Figure 8A ) . Consistent with this , the timing of Cdc5 and Clb1 expression , both under the regulation of the Ndt80 transcription factor that facilitates pachytene exit [67] , were also similar in all three strains . These results suggest that exit from pachytene occurred with similar timing in the smc5 and nse4 mutants compared to the wild type strain . To follow M-phase events , we assessed steady-state levels of Rec8 and Pds1 , the securin orthologue in budding yeast . Degradation of both occur at the onset of anaphase I and anaphase II . Rec8 and Pds1 degradation occurred around 7 hours in all three strains and the second wave of Pds1 degradation ( anaphase II onset ) was observed in both wild type and smc5 ( Figure 8B ) . The nse4 time course was presumably less synchronous such that the second wave of Pds1 and Rec8 degradation was not detected [82] . To assess meiosis II entry , we used the B-type cyclin , Clb3 . In all three strains , Clb3 expression appeared at similar times ( Figure 8A ) . Collectively , these observations strongly support the notion that the meiotic progression is not significantly delayed or arrested in Smc5/6-depleted cells . The population kinetics of γH2A suggest that smc5 and nse4 mutants undergo meiotic catastrophe with damaged DNA . In the wild-type , γH2A disappeared by 7–8 hours , whereas it remained high in the two mutant strains , even at 12 hours when meiosis was completed ( Figure 8A , and data not shown ) . Consistent with this analysis , immunostaining for γH2A foci in combination with tubulin revealed meiosis I and meiosis II cells that also contained an increased number of γH2A foci ( Figure 8C , D ) . In the wild type , cells with anaphase I spindles showed confluent , low intensity background γH2A staining as well as a few punctate foci ( median: 3 foci ) . In contrast , analogous nuclei from both smc5 and nse4 mutants contained large numbers of γH2A foci , many of which were located off the main body of DNA ( Figure 8C ) , suggestive of perturbed DNA/chromatin structure . Furthermore , in nuclei with meiosis II spindles , 5% of smc5 and 42% of nse4 nuclei ( n = 50 ) contained punctate γH2A staining ( Figure 8D ) . The lower number of γH2A-positive staining anaphase II nuclei in the smc5 mutant presumably reflects the lower level of unresolved joint molecules relative to nse4 ( Figure 5 ) . Collectively , these data indicate that smc5/smc6 mutants progress through the meiotic divisions with elevated levels of γH2A . Finally , we investigated whether smc5 and nse4 mutants are deficient in maintaining the DDR-induced meiotic arrest that occur in mutants , where high levels of single-stranded DNA accumulate ( dmc1Δ , rec8Δ , and hop2Δ ) [83] . Depletion of Smc5 or Nse4 had no effect on the meiotic progression in any of these mutants ( Figure 8E ) . Combining the dmc1Δ nse4 or hop2Δ nse4 mutants with fpr3Δ , which is required for checkpoint maintenance [84] , resulted in high levels of checkpoint bypass ( Figure 8E ) . These data demonstrate that smc5 and nse4 mutants are checkpoint proficient and that the progression into the meiotic nuclear divisions with unresolved joint molecules is unlikely to be caused by defective DDR maintenance . Unresolved joint molecules are inferred to impede chromosome separation in cells undergoing the meiotic divisions [11] , [12] . However , cleavage of cohesin by separase is also essential for chromosome disjunction [85] . Smc5/6 localizes to cohesin-binding sites ( Figure 1 ) and in S . pombe , smc5/6 mutants show increased retention of cohesin during mitosis that contributes to chromosome segregation defects [86] , [87] . These considerations led us to evaluate whether cohesin was mis-regulated in meiosis . To this end , we analysed Rec8-GFP dynamics in time-lapse studies [88] . Using Pds1-tdTomato as a marker for anaphase I entry ( Figure 9A ) , cohesin removal along chromosome arms was completed in 14 . 2 ( ±5 . 7 ) minutes in wild type ( n = 30; Figure 9A & B , Movies S4 , S5 , S6 , S7 ) . There was little or no delay in the smc5 cells and a slight but significant delay in the nse4 mutant ( Figure 9C , Mann-Whitney p<0 . 01 ) . Assessment of retention of cohesin in spread nuclei confirmed that the cohesin was associated with meiotic chromosomes ( Figure S8 ) . Moreover , we also observed smc5 nuclei at anaphase II with significant cohesin staining ( Figure S8B ) . It is likely that this residual cohesin that we detect with antibodies but not live cell imaging in the smc5 mutant , reflect relatively low levels of retained cohesin that cannot be detected due to the decreased sensitivity of live cell imaging . To address whether the delayed removal of cohesin relative to the nuclear divisions contributed towards the severe chromosome segregation defects of the smc5/6 mutants , we engineered a TEV protease cleavage site into Rec8 ( in addition to the two separase cleavage sites ) and expressed TEV protease around anaphase I onset ( Figure 10A–D ) . We observed small improvements in chromosome segregation at anaphase I in both strains , with a more pronounced effect in smc5 ( Figure 10F , G ) . However , the contribution of the persistent cohesin towards the severe meiotic catastrophe is likely relatively small compared to the failure to remove joint molecules prior to the meiotic divisions , especially in the nse4 strain . Finally , we noticed that the retention of centromeric cohesin was severely defective in the two mutants ( Figure 9 , S8A , C ) . This premature loss of centromeric cohesin correlated with the precocious separation of sister centromeres ( Figure 9E ) and indicates that smc5/6 mutants experience problems with the establishment and/or retention of cohesion . We conclude that the mis-regulation of cohesin is two-fold in the Smc5/6-depleted cells: removal of arm cohesin is delayed while the protection of centromeric cohesin is compromised as well .
SMC complexes regulate a vast array of chromosomal processes , including DNA repair , during mitosis and meiosis [37] . In this study , we set out to determine whether the third , highly conserved SMC complex , Smc5/6 , has roles in meiotic recombination . We were particularly interested in determining whether depletion of Smc5/6 leads to general recombination defects , like cohesin or condensin [89] , [90] , or whether specific pathways would be perturbed in its absence ( Figure S9 ) . Despite its central role in mitotic cells in mediating resolution and separation of chromosomes in response to DNA damage , the role of the Smc5/6 complex in meiotic recombination has remained enigmatic . Previous findings suggested that Smc5/6 mediated its critical role during premeiotic S-phase , since deletion of SPO11 did not alleviate the chromosome separation defect of smc6 temperature-sensitive mutants [54] . In this work , we show clearly that the budding yeast Smc5/6 complex is required for chromosome resolution following induction of meiotic recombination ( Figure 3 ) . Similar findings are reported by two independent studies in budding yeast [61] , [73] . Collectively , they firmly support the notion that across a range of species , Smc5/6 has essential functions in mediating chromosome resolution in response to induction of meiotic recombination [55] , [58] , [61] , [73] , [91] . During meiosis , Smc5/6 localizes to centromeres , cohesin-binding sites and sites of meiotic DSBs ( Figure 1 ) . However , the chromosome-length dependent increase in the density of Smc5/6 binding sites reported in vegetative cells [60] is not observed in meiosis . We identified at least three factors that contribute to the general failure of chromosome separation seen in smc5/6 mutants . First , high levels of joint molecules , both between homologs and sister chromatids , remain unresolved , especially in the nse4 mutant ( Figure 5 ) . Second , cells enter the meiotic nuclear divisions without a delay that might otherwise allow time for joint molecules to be resolved ( Figure 8 ) . Third , mis-regulation of cohesin also partly contributes to the delayed chromosome separation at anaphase I , especially in the smc5 mutant ( Figure 10 ) . Moreover , a combination of unresolved joint molecules between sister chromatids and precocious separation of sister kinetochores ( Figure 9 ) could also contribute to chromosomal entanglement ( Figure 11B ) . Time-lapse imaging of single cells delineates the sequence of severe chromosome segregation defects and meiotic catastrophe caused by unresolved joint molecules . Meiotic catastrophe was preceded by failure to separate the nuclear mass ( nse4 ) or by failure to keep the nuclear masses separated upon spindle disassembly ( smc5 ) . Spindle formation and elongation were associated with aberrant chromosome morphology such as micronuclei and chromosome spikes ( Figure 2 ) . It has been suggested that even low levels of unresolved joint molecules may block chromosome separation in meiotic cells [11] , [12] . In the nse4 mutant , the 10% of chromosomes trapped in joint molecules at HIS4LEU2 ( Figure 5 ) translates to 20% of cells with an unresolved joint molecule at this recombination hotspot . Assuming that naturally occurring hotspots display a similar dependency on Smc5/6 , each cell will undergo nuclear divisions with 20% , or roughly 30–40 joint molecules , unresolved ( based on DSB levels of 150–200 per cell [92] ) . In the smc5 mutant , the 1 . 8% unresolved joint molecules at 13 hours would equate to ∼5–7 persistent joint molecules per cell . These considerations raise the possibility that a small number of unresolved joint molecules ( less than one per chromosome ) can cause a pan-nuclear segregation defect . Physical monitoring of joint molecules indicates that Smc5/6 regulates both the formation of recombination intermediates as well as their resolution ( Figure 5 ) [61] . In accompanying studies the hypomorphic smc6–56 allele and the SUMO E3 ligase-dead mms21-11 alleles also accumulate joint molecules [61] , [73] . Therefore , inactivation or depletion of four distinct components of the core budding yeast Smc5/6 complex leads to defective joint molecule metabolism during meiosis . Similarly , in S . pombe , nse5 and nse6 mutants show accumulation of Rec12/Spo11-dependent joint molecules [55] . Thus , Smc5/6 has a critical and conserved role in the completion of meiotic DSB repair in both yeasts by facilitating the removal of joint molecules . We have identified three aberrations in the joint molecules that accumulate in the smc5/6 mutants from which we infer that Smc5/6 is critical for directing not only the removal of joint molecules upon prophase I exit ( ‘late prophase I” , Figure 11 ) , but also their proper formation during DSB repair ( Figure 11 ) . Smc5/6 depletion increases the fraction of joint molecules between sister chromatids that involve three and four chromatids ( multi-chromatid JMs ) , while decreasing the levels of interhomolog dHJs . A similar conclusion is reached by Xaver et al . ( 2013 ) , who analyzed joint molecules at a second hotspot . Since single-end invasions formed relatively normally in smc5/6 mutants ( Figure 5 ) , these observations suggest that Smc5/6 may be important for coordinating the two DSB ends or to limit secondary strand invasions between sister chromatids ( Figure 11A ) . Smc5/6 could also redirect multi-chromatid JMs and intersister dHJs to the interhomolog fate , perhaps via regulation of DNA helicases and/or endonucleases during early prophase I ( Figure 11A ) . Such a redirection process was previously envisioned for Sgs1 [10] . Mus81-Mms4 was previously shown to play a small but significant role in inter-homolog bias , primarily by enhancing formation of inter-homolog dHJs [12] , [30] . Since inactivation of Mus81-Mms4 did not cause a further decrease in inter-homolog bias in the nse4 mutant ( Figure 7 ) , it is possible that Smc5/6 regulates this function of Mus81-Mms4 during the formation of interhomolog dHJs . However , Mus81-Mms4 also somehow increases the final level of unresolved joint molecules in nse4 cells ( Figure 7E ) . Perhaps , in the absence of Smc5/6 function , Mus81-Mms4 creates structures that cannot be resolved . Alternatively , the decreased accumulation of JMs in the nse4 mms4 and nse4 mus81 mutants may suggest functions of Mus81-Mms4 in processing DSB repair intermediates that do not lead to crossovers ( see below ) . Inspection of the JM spots revealed additional spots and smears of the main dHJ molecules , suggestive of altered structure of the JMs that accumulate in smc5/6 mutants ( Figure 5B ) . In S . pombe , JMs that accumulate in mus81 mutants can be resolved in vivo by expression of RusA and by RuvC after extraction from gels . In nse5/6 mutants , however , the JMs appeared partially refractory to both RusA and RuvC treatment [55] , although they migrated in similar spots of JMs in mus81 mutants . Our observations suggest that the JMs that are formed in smc5/6 mutants are not normal and this , together with the mislocalization of Mus81-Mms4 on the meiotic chromosomes , could contribute to the lack of resolution by Mus81-Mms4 , despite its normal activation by Cdc5 . In S . pombe , Mus81-Eme1 promotes most or all crossovers and deletion of Nse5 or Nse6 diminishes crossing over [27] , [28] , [55] , [57] . Our findings show that Smc5/6 may be specifically required for resolution mediated by structure-specific endonucleases such as Mus81-Mms4 ( and possibly also Yen1 and Slx1–Slx4 ) in organisms with alternative resolving pathways . Specifically , we found that crossover levels and inter-homolog bias in nse4 mutant were not further reduced when Mus81-Mms4 was also mutated ( Figure 7D , E ) . In contrast , mutation of Sgs1 or Mlh3 synergistically reduced crossover levels in nse4 cells ( Figure 6 ) . These observations suggest that Smc5/6 coordinates resolution of joint molecules that form independently of the major , MutSγ-dependent pathway . It is possible that Smc5/6 affects resolution of all non-Msh4/5 joint molecules . We infer that it is unlikely that Smc5/6 depletion leads to gross , general chromosomal defects that generally affect recombination , as seen in condensin mutants , where Cdc5/Polo-like kinase fails to associate with meiotic chromosomes and recombination is perturbed [90] , [93] . How might Smc5/6 regulate joint molecule resolution ? In the case of Mus81-Mms4 , hyperphosphorylation and presumably hyperactivation of endonuclease activity still occurs in in the smc5 and nse4 mutants ( Figure S7 ) . However , association of Mus81 with meiotic chromosomes is diminished ( Figure 7F , G ) , even during early prophase I , consistent with observed defects during the formation of joint molecules ( Figure 7D , E ) . Although we do not know whether the Mus81 foci we observe reflect catalytically active Mus81-Mms4 complexes , our data support the idea that Smc5/6 mediates chromosomal association of Mus81-Mms4 . Smc5/6 has been reported to have low affinity interactions with single stranded DNA [94] . It is possible that the complex targets Mus81-Mms4 to substrates containing single-stranded regions . However , no direct interaction between Mus81-Mms4 and the Smc5/6 complex has been reported . Another possibility is that Smc5/6 holds joint molecules ( or their precursors ) in a conformation that ultimately allows resolution by Mus81-Mms4 . In this regard , the novel joint molecule species that we detect in the smc5 and nse4 mutants may represent structures that cannot be resolved by Mus81-Mms4 or other resolving endonucleases . EM studies have revealed aberrant JM structures in sgs1 and mms4 sgs1 mutants that might represent hard-to-resolve structures [12] . Finally , Smc5/6 may also regulate local chromosome structure around a subset of DSBs and this could impact on recombination [86] . For example , mis-regulation of cohesin could indirectly influence inter-homolog bias , as seen in rec8Δ mutants [89] .
Strains are described in Table S1 . They are all derived from SK1 . Diploid cells were grown to saturation in YEPD ( 1% yeast extract , 2% bactopeptone , 2% dextrose , pH 6 . 5 ) , then inoculated at 5×106 cells per ml in SPS ( 0 . 05% yeast extract , 1% peptone , 0 . 17% YNB , 1% potassium acetate , 0 . 5% ammonium sulphate , 0 . 05 M potassium hydrogen pthalate at pH 5 . 5 ) and grown to a cell density of 5×107 cells per ml . To induce meiosis , cells were resuspended in SPM ( pH 7 . 0 ) consisting of 1% potassium acetate , 0 . 02% raffinose , 0 . 02% antifoam ( Sigma , A8311 ) , 2% histidine , 1 . 5% lysine , 2% arginine , 1% leusine and 0 . 2% uracil . Genome-wide Smc5 association was measured as previously published [95] . Briefly , Smc5 crosslinked chromatin was immunoprecipitated with 2 µl anti-myc 9E11 ( Abcam ) or 20 µl anti-V5 beads ( Sigma-Aldrich ) . Immunoprecipitated and input DNA samples were cohybridized to a custom DNA microarray ( Agilent ) and data were normalized as previously described . Every 3 points along the chromosome were averaged to produce the smoothed profiles in Figure 1 . The relative enrichment of Smc5 to Rec8 and Smc5 in spo11 versus SPO11 is the ratio of the values in each of the two datasets indicated . The raw data and log ratios from this study are available from the NCBI Gene Expression Omnibus ( http://www . ncbi . nlm . nih . gov/geo/ ) , accession number GSE44852 . Molecular assays were carried out as described previously [72] , with the modification that we used the Phase Lock Gel for phenol extraction . We analysed three independent diploids for each strain . To measure genome wide DSB signal , chromosome-length DNA captured in agarose plugs [96] was separated by pulsed field gel electrophoresis under the following conditions: 1 . 3% agarose in 0 . 5×TBE; 14°C; 6 V/cm; switch angle 120° , ramped switch time of 15–25 seconds over 30 hours ( Biorad CHEF DRIII ) . Following a denaturing transfer to nylon membrane , a radioactive DNA telomeric probe for the left side of chromosomes III ( CHA1 ) was hybridized to the membrane . Radioactive signal was collected on phospho-screens , imaged using a Fuji FLA5100 and quantified using FujiFilm ImageGauge software . DSB signal was measured as a percentage of the total lane signal [97] . DSB molecules occurring further from the probe are under-estimated due to DSBs occurring closer to the probe on the same molecule . To correct for this , the estimated DSB frequency was calculated using Poisson correction: Percentage broken chromosomes ( Poisson corrected ) = −ln ( 1−measured DSB signal ) . To produce lane profiles , 900 lane slices were exported from ImageGauge and combined from 6–10 hours and each slice plotted as a percent of total lane signal . Cells from meiotic cultures ( OD600 1 . 2–1 . 5 , 2 ml ) were disrupted using glass beads in 200 µl of ice cold 20% TCA . Precipitates were collected by centrifugation and washed in 400 µl of ice cold 5% TCA . Precipitates were resuspended in 100 µl of SDS-PAGE sample buffer ( 4% SDS , 5% β-mercaptoethanol , 0 . 15 M DTT , 20% glycerol , 0 . 01% bromophenol blue ) ; boiled for 5 minutes at 95°C , centrifuged , and the supernatant containing protein was collected . Proteins were separated by SDS-PAGE , transferred to nitrocellulose membranes , and probed with the appropriate antibodies followed by HRP-conjugated secondary antibodies ( DAKO , 1∶2000 ) . HRP activity was detected using Pierce ECL Western Blotting Substrate followed by exposure to Amersham Hyperfilm ECL or using the Image Quant LAS 4000 imaging system . Cdc5 ( Santa Cruz sc-6732 , 1∶2000 ) , HA ( 12CA5 , CRUK , 1∶1000 or Abcam Ab9110 , 1∶1000 ) , γH2A ( J . Downs , 1∶1000 ) , H2A ( 1∶5000 , J . Downs ) , Rad51 ( 1∶2000 , S . Roeder ) , PAP ( Sigma P1291 , 1∶2000 ) , Pgk1 ( Invitrogen 459250 , 1∶200 000 ) , Myc ( 9E10 , CRUK , 1∶2000 ) , V5 ( AbDSerotec MCA1360 , 1∶2000 ) , Zip1 ( Santa Cruz sc-48716 , 1∶2000 ) , Hop1 ( F . Klein , 1∶1000 ) , pHop1-T318 ( Cambridge Research Biochemicals , 1∶500 ) , and Clb3 ( Santa Cruz sc-7167 , 1∶500 ) . Meiotic cultures were arrested at pachynema after 6 hours in SPM . TEV protease and Ndt80 were induced by the addition of 1 µM β-estradiol . Protein synthesis was blocked by the addition of cyclohexamide to meiotic cultures to a final concentration of 200 µg/ml . Cyclohexamide was added to meiotic cultures 1 hour after Ndt80 induction . The PCLB2-SMC5 was C-terminally-tagged with the AID [69] . To induce degradation of Smc5 , we added 150 µl of 500 mM auxin ( 3-indoleacetic acid; Sigma I375-0 ) , resuspended in 1N NaOH , to 50 ml meiotic cell cultures . This was added at 1 hour after transfer to SPM . Addition of auxin at earlier time points resulted in arrest during the preceding mitotic divisions when cells underwent premeiotic growth in pre-sporulation medium ( SPS ) . Nuclear spreading and antibodies have been described elsewhere [98] , [99] , except that we treated cells with both zymolyase 100T and glusulase in order to generate spheroblasts for some strains . Fixation followed by indirect immunofluorescence was carried out by fixing cells in 4% formaldehyde for 15–45 minutes at room temperature . When assessing Mus81-Mms4 foci , we carefully controlled for the extent of spreading , because we noted that even in the wild type , a small proportion of nuclei did not contain Mus81-Mms4Eme1 foci . When we applied more extreme spreading techniques , all Mus81-Mms4Eme1 staining ( but not Zip1 ) was abolished in the wild type ( data not shown ) . This suggests that the Mus81-Mms4Eme1 interaction with meiotic chromosomes is less stable than Zip1 . Cells were initially incubated in sporulation media for 6–8 hours . 20 µl of cells were added to a Y04D CellASIC plate ( CellASIC ONIX microfluidic perfusion system ) and imaged inside an environmental chamber set at 30°C . A flow rate of 8 psi was used to load the cells and a steady-state flow rate of 2 psi was used for the duration of the time course . Time-lapse microscopy was carried out using a Personal DeltaVision ( Applied Precision ) with xenon or solid-state illumination , using associated proprietary software ( SoftWoRx software; version 4 . 0 . 0 , Applied Precision ) . Images were captured using an UPLS Apochromat 1 . 4 numerical aperture , ×100 magnification oil immersion objective ( Olympus ) , auxiliary magnification to prevent undersampling , standard DeltaVision filter sets FITC ( ex 490 , em 525 nm ) and TRIC ( ex 555 , em 605 ) , yielding approximate resolutions ( Rayleigh's d ) of ∼229 nm and 264 nm in the xy , respectively , whereas axial resolutions were approximately 811 and 935 nm . Photon detection was carried out using a Cascade2 1 K EMCCD camera ( Photometrics ) using a gain of 230 and no binning . Images were taken using exposure times of 0 . 025 sec . and 32% transmission ( FITC ) and 32% transmission and 0 . 1 sec . exposure ( TRITC ) . 6–7 z-stacks at 1 µm were collected . Final images for sporulation were carried out with DIC , 32% transmission and 0 . 05 sec . exposure . Images were recorded every 5 minutes for the first 90 minutes , every 20 minutes for the next 80 minutes and then every 45 minutes for the last 90 minutes . Around 12 hours after imaging the sporulation of the cells at each point of imaging was assessed . Only cells that sporulated were included in the analyses . Images were deconvolved using SoftWoRx software ( version 4 . 0 . 0 , Applied Precision ) . Subsequent 3D analysis to measure spindle length was carried out using Imaris ( version 7 . 0 . 0 , Bitplane ) . 3D images are presented as maximum projections , rendered in Softworx or Imaris . Some images were manipulated in Adobe Photoshop CS5 . 1 using the following procedure . Images were converted to . psd files from Softworx files before being opened in Adobe Photoshop . Only the max/min input levels of each channel were adjusted manually to adjust differences in the imaging intensities . Images were cropped preserving the relative ratios , and the size bar copied to a second layer of the image . For aesthetic reasons , a broader bar covering the size and the out-of-focus number was added on top of the original . Analysis of foci numbers was carried out manually and with the ‘Find Peaks algorithm’ ( ImageJ plugin is available from: http://www . sussex . ac . uk/gdsc/intranet/microscopy/imagej/plugins and documentation: http://www . sussex . ac . uk/gdsc/intranet/microscopy/imagej/findpeaks ) . Peaks were identified above a background level using non-maximal suppression . An allowance was made for peak regions covering multiple pixels with the same intensity ( plateau maxima ) . A watershed algorithm was used to assign all non-maxima pixels to the appropriate peak by following the maximum gradient . Peak expansion was restricted using the height above background . Following identification the boundaries between peaks were calculated and the highest boundary point between touching peaks stored as saddles . A peak merge algorithm was used to join insignificant smaller peaks into their neighbour peak defined using the highest saddle point . Peaks were identified as insignificant using height and area criteria . Noisy data were smoothed using a Gaussian blur prior to peak identification . Reported peak statistics always use the intensity values from the original unsmoothed image . The algorithm can be applied to 2D or 3D images and is available as a plugin for ImageJ . The plugin allows setting parameters to control the background identification , search method , merge criteria and the results output . The plugin is scriptable via the ImageJ macro facility and provides a GUI that allows the parameters to be adjusted with real-time results update . The plugin will be published separately elsewhere . We used various statistical tests in R ( www . r-project . org ) , as indicated throughout the text . P-values were adjusted for multiple pair-wise comparisons according to Dunn-Sidak to reflect α<0 . 05 . Standard error bars around proportions were calculated as √[p ( 1−p ) /n] , where p is the proportion of the specific class ( n>100 for each strain ) . For the Pearson product-moment correlation , the cor . test uses the t-statistics to calculate the p-value and the Fisher z transform to generate an asymptotic confidence interval ( 95% ) . | Meiosis is a specialized cell division that exactly halves the number of chromosomes transmitted from each parent to their offspring via gamete cells ( such as sperm and eggs ) . This requires that matching ( ‘homologous’ ) chromosomes associate and then separate into different cells such that each gamete contains exactly one complete set of chromosomes . In many organisms , this sequence of events is facilitated by the induction and repair of chromosome breaks via a process called homologous recombination . As homologous chromosomes engage in recombination , matching DNA strands between broken and intact template chromosomes become intertwined in repair intermediates called Joint Molecules . In this study , we show that a highly conserved protein complex called the Structural Maintenance of Chromosomes 5/6 ( Smc5/6 ) complex is important for regulating the choice of recombination template as well as for the resolution of Joint Molecules that is required for chromosomes to separate . Even though Joint Molecules remain unresolved in mutants that lack normal Smc5/6 function , cells still attempt to separate chromosomes and meiosis becomes catastrophic . Thus , Smc5/6 mutants have a two-fold defect: accumulation of unresolved Joint Molecules and a failure to stall meiosis in order to remove these structures . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2013 | Smc5/6 Coordinates Formation and Resolution of Joint Molecules with Chromosome Morphology to Ensure Meiotic Divisions |
Candida albicans is the most common human fungal pathogen , causing infections that can be lethal in immunocompromised patients . Although Saccharomyces cerevisiae has been used as a model for C . albicans , it lacks C . albicans' diverse morphogenic forms and is primarily non-pathogenic . Comprehensive genetic analyses that have been instrumental for determining gene function in S . cerevisiae are hampered in C . albicans , due in part to limited resources to systematically assay phenotypes of loss-of-function alleles . Here , we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants , using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency . We identified 269 genes that were haploinsufficient in four growth conditions , the majority of which were condition-specific . These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes . We also screened 57 chemically diverse compounds that more potently inhibited growth of C . albicans versus S . cerevisiae . For four of these compounds , we examined the genetic basis of this differential inhibition . Notably , Sec7p was identified as the target of brefeldin A in C . albicans screens , while S . cerevisiae screens with this compound failed to identify this target . We also uncovered a new C . albicans-specific target , Tfp1p , for the synthetic compound 0136-0228 . These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification .
Fungal species of the genus Candida generally live communally on and in the human body , yet Candida infections can become systemic and lethal in up to 60% of immunocompromised patients [1] , [2] . C . albicans alone accounts for over 50% of all fungal infections [3] . Furthermore , drug resistance to current therapies is becoming increasingly prevalent [4] , motivating research efforts to understand the genetic basis of C . albicans' pathogenesis and uncover novel therapeutic targets . The etiology of C . albicans is particularly complex , and our understanding of this pathogen lags with respect to the model organism Saccharomyces cerevisiae , which is often used as a proxy for Candida species despite the fact that S . cerevisiae diverged from C . albicans between 150–800 million years ago [5] , [6] . Notably , S . cerevisiae is rarely pathogenic [7] and lacks the multiple morphogenic forms that define C . albicans pathogenicity . C . albicans also exists as an obligate diploid and lacks a traditional meiotic cycle [8]; as a result , many of the genetic tools developed in S . cerevisiae are not easily applied . Finally , only 58% of predicted C . albicans proteins share an ortholog with S . cerevisiae [9] , [10] , underscoring the need for direct study of C . albicans . An effective approach for such analyses would be to extend high-throughput methodologies developed in S . cerevisiae to C . albicans . For example , experimental multiplexing , in which a genome-wide collection of deletion mutants is pooled and grown competitively to determine the fitness of each mutant in an experimental condition , has been particularly effective in S . cerevisiae ( reviewed in [11] and [12] ) . Strain tracking and quantitation is enabled by the presence of unique DNA sequences , or tags , introduced during the construction of each deletion mutant [13] . To measure strain abundances in pooled growth experiments , these strain-specific tags can be amplified and hybridized to a microarray containing the tag complements , or sequenced directly [14] , [15] . The S . cerevisiae deletion collection and pooled phenotypic profiling have been invaluable for examining gene function [14] , genetic interactions [16] , and for the identification of drug targets and their mechanism of action [17] , [18] , [19] , [20] . Strategies for large-scale mutant screening in C . albicans include two studies using transposon mutagenesis to rapidly generate large numbers of mutants [21] , [22] , and a third study using targeted deletions combined with a regulatable promoter [23] . While each has been used to uncover novel biological insights , several factors have limited their widespread utility to the C . albicans research community . For instance , mutants in one heterozygous disruption collection [22] are largely unsequenced , making predictions regarding its coverage difficult . The homozygous transposon disruption collection [21] , although sequenced and archived as individual mutants , is unavailable as an entire collection and its strains are not tagged; thus experiments must be conducted individually . A proprietary tagged deletion collection has been used to identify novel antifungal targets [23] , [24] , [25] , [26] , but the composition of this collection is limited to 2868 strains that share homology to genes essential in S . cerevisiae , other fungi , and higher eukaryotes . Accordingly , a majority of C . albicans-specific genes are not interrogated . Our goal was to create an unbiased , open-access collection of tagged C . albicans mutants useful for high-throughput experimental multiplexing . We previously reported the creation of a pilot pool of 1290 tagged C . albicans mutants , using universal “TagModules” to label transposons that were subsequently used to simultaneously generate mutants and integrate a pair of DNA tags at the insertion site [27] . Here , we describe the creation and validation of a genome-wide C . albicans tagged transposon mutant collection , using TagModule-based transposon mutagenesis to generate 4252 mutant strains , 3633 of which were detectable by microarray in a pooled growth assay . To demonstrate the utility of this collection , we investigated nutrient-specific and drug-induced haploinsufficiency . By screening four different media conditions and 57 primarily synthetic inhibitory compounds , we 1 ) identified genes functioning in core or essential processes , 2 ) uncovered genes specific to growth in a particular nutrient condition , and 3 ) demonstrated the utility of this collection in antifungal screening to determine mechanism of action of inhibitory compounds . This collection represents a public , archived resource of tagged C . albicans mutants that can be used to examine gene function either individually or multiplexed in a pool .
To circumvent the resource-intensive approach of generating gene-specific deletion cassettes to knock out gene function , we used tagged transposon mutagenesis to generate mutants . To incorporate tags into the transposon , we used 4280 Gateway-compatible TagModules developed in a previous study as a source of sequence-verified tags [27] . Each TagModule contains a pair of DNA tags , an uptag and a downtag , flanked by common priming sites for amplification of the tags to determine strain abundance . These TagModules were transferred to a Gateway-compatible transposon modified to contain the UAU1 selectable marker , which allows heterozygous ( Arg+ ) as well as homozygous disruption mutants ( Arg+ , Ura+ ) to be generated [28] . The design of the tagged transposons is shown in Figure 1A . Following the approach of previous transposon mutagenesis studies [21] , [22] , these tagged transposons were used to mutagenize a C . albicans genomic library in vitro . Following in vitro mutagenesis , unique insertions were recovered in E . coli and sequenced individually to determine the disrupted gene and its linked TagModule ( Figure 1B ) . We recovered a total of 21 , 468 usable insertion events ( see Methods S1 for criteria ) representing 4827 unique genes ( ∼78% of predicted open reading frames ) . 4239 of these were successfully transformed ( for criteria , see Methods ) into C . albicans via homologous recombination to create a uniquely tagged , heterozygous disruption mutant ( Table S2 ) . To examine the quality of our collection , we pooled the 4239 strains , amplified their tags , and hybridized them to an Affymetrix TAG4 array ( Figure 1C ) . At a zero timepoint ( i . e . , with no competitive outgrowth ) , 3633 strains were detected ( tag intensity above 3X median background ) , with 619 strains falling below background ( Figure 2A ) . Failure to detect strains may result from either a failure of the transformant to re-grow , or low abundance prior to pooling . If the latter , these strains could assayed as a separate subpool , or alternatively , detected using high-throughput sequencing , which provides increased sensitivity [15] . After twenty generations of growth in a pooled assay , biological replicates were highly correlated ( Figure 2B; R = 0 . 98 , p<10−16 ) and strains showed low cross-reactivity with other features on the array; the vast majority ( 12292/12686 , or 97% ) of unused tags on the array had signal intensity below our cutoff of 3X background ( Figure S3 ) . Thus , our tagged transposon mutants have robust and reproducible performance in a pooled format . Genes that have a growth defect when reduced in copy number from two to one ( termed haploinsufficiency ) are of interest for their potential as drug targets [12] . We therefore sought to use the pooled growth assay to define genome-wide haploinsufficiency in C . albicans , particularly for C . albicans-specific processes and those required for growth in hyphae-inducing conditions , a determinant of virulence . We screened the C . albicans pool in four different nutrient conditions at 30°C: i ) rich YPD media , a standard laboratory growth condition , ii ) a synthetic media used for the selection of transformants ( SC ) , iii ) minimal media , which has been used to induce formation of pseudohyphae and consists of 2% glucose and yeast nitrogen base ( YNB ) , and iv ) a low-nitrogen minimal media ( synthetic low ammonium dextrose , or SLAD ) , which can induce pseudohyphal/hyphal growth in C . albicans . To assess the effect of each gene disruption on growth in these conditions , we tracked tag abundance for each of the detectable 3633 strains , assaying after five , ten , fifteen , and twenty generations of growth with biological replicates . Following calculation of tag abundance by hybridization of amplified tags to a microarray , we fitted a linear regression to each strain's abundance over a timecourse and used the slope of the regression to measure strain sensitivity . Based on Deutschbauer et al . [29] , we defined a strain as having a growth defect if its slope was <0 , p<0 . 05 ( Table S3 ) . We found that regardless of media condition , similar proportions of strains were haploinsufficient ( Figure 3A ) . Overall , 145 ( 4% ) strains were haploinsufficient in rich YPD , 105 ( 2 . 9% ) in SC , 97 ( 2 . 7% ) in YNB , and 140 ( 3 . 9% ) in the low-nitrogen SLAD , representing 269 ( 7 . 4% ) unique genes . Only 9% ( 25 ) of these genes were haploinsufficient in all conditions; the majority ( 55% ) of these 269 genes were haploinsufficient in a single condition , suggestive of condition-dependent haploinsufficiency ( Figure 3B ) . This observation suggests that a substantial portion of the C . albicans genome may be amenable to haploinsufficiency profiling under certain conditions . Finally , comparing our haploinsufficiency profiling data in rich media to that of S . cerevisiae [29] , we found that while the overall proportion of haploinsufficient strains was similar , we found a number of biological differences ( Figure 3C ) . 48/145 ( 33% ) of C . albicans haploinsufficient genes in YPD had no S . cerevisiae ortholog . Of those C . albicans genes that did have an ortholog , 26 are orthologous to S . cerevisiae genes that were essential or haploinsufficient in YPD , and an additional 22 orthologs exhibited a growth defect as a homozygous deletion in YPD . 49 ( 34% ) orthologs had no phenotype in YPD . Of the 269 haploinsufficient genes in C . albicans , 97 ( 36% ) had no S . cerevisiae ortholog . A second striking difference between the S . cerevisiae and C . albicans haploinsufficient gene sets is the number of transcription factors ( TFs ) haploinsufficient in C . albicans . Few transcription factors ( excluding general regulatory factors ) were haploinsufficient in S . cerevisiae [29] . In contrast , we identified seven TFs as haploinsufficient in YPD ( ZCF9 , HAC1 , LYS142 , IRO1 , SUA71 , BDF1 , RBF1 ) , and a total of 13 in the complete haploinsufficient dataset ( FGR17 , SEF1 , SUA72 , orf19 . 6623 , orf19 . 5368 , MSN4 ) . Their functions range from iron utilization to regulators of filamentous growth or stress response . These findings highlight the need for direct study of C . albicans , as our assay uncovers novel phenotypes for orthologs as well as a large number of C . albicans-specific genes . To search for functional enrichment among the 25 genes haploinsufficient in all conditions ( Table S4 ) , we used the Gene Ontology Term Finder from the Candida Genome Database ( http://www . candidagenome . org/cgi-bin/GO/goTermFinder ) . These genes were significantly enriched for the GO processes “translation” and GO functions “structural constituent of ribosome” and “structural molecule activity” ( Table 1 ) . We also observed genes in this set that function in “core” cellular processes that are likely to be either essential or necessary for growth regardless of condition; for example , POL2 ( DNA polymerase epsilon ) and orf19 . 736 , whose S . cerevisiae ortholog Sc-SRB8 is a subunit of RNA polymerase II required for transcriptional regulation [30] . We also identified a putative permease ( orf19 . 3293 ) , which may play a role in nutrient sensing , two genes whose products protect against oxidative stress ( SOD3 and orf19 . 5553 , based on S . cerevisiae orthology ) , and a putative 5′-monophosphate 5′-nucleotidase , ISN1 , which is a fungal-specific gene whose S . cerevisiae ortholog has been implicated in nucleotide scavenging [31] . Interestingly , the S . cerevisiae orthologs for these four genes are not haploinsufficient , suggesting that S . cerevisiae and C . albicans have diverged with respect to the relative importance of these genes for survival , or that these genes are required at greater than heterozygote gene doses in C . albicans . Because the 25 genes that were haploinsufficient in all conditions ( “4 or more” subset ) were enriched for genes involved in fundamental cellular processes , we propose that the unverified genes in this set ( those which lack annotation , or those whose annotation has not been confirmed experimentally ) are also involved in core or essential processes . As the GO enrichments for genes haploinsufficient in 3 or more conditions ( Table S5 ) were similar to the 4 or more subset ( Table 1 ) , we expanded our list of “core” genes to include these genes as well , for a total of 70 genes . Consistent with our observations in the 4 or more subset , this 3 or more group included additional permeases ( MUP1 and orf19 . 5826 ) and two genes with predicted involvement in oxidative stress response ( TRR1 and POS5 ) . To ask if we could assign functions to this unverified “core” gene set , we selected 17 genes whose S . cerevisiae orthologs ( or if an ortholog was not found , the best BLAST hit was used ) are essential , so we could assess function by complementation testing in S . cerevisiae . Although an imperfect test for C . albicans function ( and susceptible to false negatives ) , positive results strongly suggest functional similarity . We used two approaches to test for complementation . First , we cloned these 17 C . albicans ORFs into a CEN/ARS vector with a constitutive promoter and transformed them into the corresponding S . cerevisiae heterozygous mutants ( YKO ) . Sporulation of the heterozygous deletion strain followed by selection for a haploid knockout should yield no growth unless complemented by the plasmid-borne C . albicans gene . To generate homozygous null S . cerevisiae knockouts , we used the Magic Marker strains [32] , which use sequential selections to generate haploid deletion mutants . 6/15 C . albicans ORFs with available corresponding Magic Marker strains showed complementation , as did 9/13 S . cerevisiae ORFs tested as a control . Complementation was assessed based on a significant increase in the number of colonies on the overexpression plate versus the vector-only plate ( Figure 4A , Figure S5 ) . Second , we confirmed all negative Magic Marker results by tetrad dissection ( Figure 4B , Figure S5 ) and found that a total of 10/17 C . albicans ORFs ( two additional YKOs were available for this test ) and 13/13 S . cerevisiae ORFs complemented their corresponding deletion allele . In instances in which we observed complementation , segregation of spore viability improved to 3∶1 or 4∶0 ( from 2∶2 in controls ) , indicating rescue of one or both of the haploid null spores . 3∶1 segregants likely result from incomplete segregation of the CEN/ARS-based plasmid , which generally exists in one or more copies per cell , to all four spores . Interestingly , we also observed two small-size spores in the case orf19 . 7615/Sc-TRS31 , indicating partial complementation . Based on these results , we conclude that 10/17 C . albicans ORFs have the same ( or very similar ) cellular roles as their S . cerevisiae ortholog and propose the following changes to the description of these ORFs: a change of “Feature Type” from “uncharacterized” to “verified” , and a transfer of function from the S . cerevisiae description ( summarized in Table 2 ) . Finally , to test whether this group of “core” genes tested via complementation also represents essential C . albicans genes , we obtained GRACE strains [33] for 12/17 of the “core” group genes . Testing the GRACE strains , which are conditional heterozygous knockouts that can be converted ( functionally ) into homozygous knockout mutants by repression of the second allele with doxycycline , served two purposes: first , to determine whether these strains are viable as homozygotes , and second , to validate that the phenotypes of our mutants represent true positives by validating growth defects in an alternative strain background . This is particularly relevant because it is possible that synthetic effects with BWP17′s uracil or histidine auxotrophies could contribute to the observed growth phenotypes . We examined the growth of these 12 GRACE strains as well as our 17 transposon-derived mutants in selective media . Overall , after 15–20 generations of growth , the majority of our transposon mutants recapitulated a growth defect as observed in the pooled growth assay ( Figure S4A ) . While the growth phenotypes of the GRACE mutants were generally less severe ( Figure S4B ) , we also observed haploinsufficiency in 11/12 of these strains ( defined as <98% of BWP17′s growth rate using the metric AvgG [34] ) . The difference in the degree of haploinsufficiency could be the result of either a “leakiness” in the tetracycline-regulated promoter of the GRACE strains [33] causing production of additional gene product and thereby alleviating the growth phenotype , or could reflect some contribution of synthetic interactions with the histidine and uridine auxotrophies mentioned above . When the GRACE strains were grown in the presence of 100 µM doxycycline , 10/12 ( FOL3 and TIF34 GRACE mutants excepted ) showed a severe to complete growth defect in all media types , suggesting that they are essential in C . albicans ( Figure S4C ) . The fact that two strains were not essential under the GRACE test but were able to complement their essential S . cerevisiae ortholog could be the result of promoter leakiness , or could reflect that these genes , while capable of performing a similar function as their S . cerevisiae orthologs , are not essential in C . albicans . We noted that haploinsufficiency in C . albicans is primarily condition-dependent; 55% of the 269 haploinsufficient genes had a growth defect in a single condition , and 19% of the 269 were identified in only two conditions . Examining these genes , we found two general categories . The first category , identified in rich media , includes genes involved in oxidative metabolism , such as COX2 , NAD5 , ABC1 , KGD2 , and ( by annotation transfer from S . cerevisiae orthologs ) orf19 . 4204/Sc-PET123 . We also observed haploinsufficiency in the C . albicans-specific alternative oxidases AOX1 and AOX2 ( GO:0016682 , n = 2 , 1 . 4% vs 0% in the genome; hypergeometric p = 0 . 046 ) , which are thought to function in maintaining turnover of the TCA cycle , relieving saturation in oxidative metabolism [35] . In reduced nutrient conditions , we observed a class of haploinsufficient genes related to growth in low nitrogen . Genes in this category , outlined in Table 3 , were involved in 1 ) cell wall maintenance ( FAT1 , SIM1 , UPC2 ) , 2 ) nutrient sensing/acquisition ( PUT4 , JEN2 , GPX2 , MEU1 , FUR1 , SEF1 ) , and 3 ) pseudohyphal growth regulators ( GRR1 , FGR17 , CPP1 ) . orf19 . 1617 , an uncategorized gene in both C . albicans and S . cerevisiae ( Sc-YDR282C ) , has also been shown to have a filamentation defect [22] . Because these categories encompass the roles of nutrient scavenging , initiation of filamentation , and cell wall remodeling necessary to produce hyphae , we asked if filamentation may be one mechanism by which C . albicans thrives in low-nutrient conditions . We investigated if these defects in growth rate correlate with defects in filamentation by testing if the 77 genes necessary for growth in the nutrient-limiting conditions ( Table S6 ) manifested a filamentous defect . Only 3/77 ( BET2 , SHE3 , and RPS18 ) mutants had a distinct filamentous defect on Spider agar ( Figure 5 ) , one condition that elicits a filamentous phenotype . SHE3 and RPS18 had a completely smooth appearance with no peripheral filaments compared to wild-type . BET2 had a smooth appearance with some peripheral filaments . SHE3 has previously been shown to be necessary for filamentous growth on Spider media [36] . The other two genes ( RPS18 , a putative ribosomal protein , and BET2 ) have not previously been implicated in a filamentous phenotype . BET2′s S . cerevisiae ortholog functions in vesicle transport , and in C . albicans BET2 is regulated by Mig1p , a transcriptional repressor that regulates carbon source utilization [37] and is downregulated in hyphal growth [38] but upregulated in biofilms [39] . While we have identified two new genes likely involved in filamentous growth , our results suggest that genes required for growth in low-nutrient conditions , as identified in our SLAD-media screens , are , in general , different from those required for filamentation in Spider agar . We next performed a pooled screen on solid SLAD media . Briefly , ∼500 , 000 colonies/pool was plated onto solid SLAD agar plates and grown for 6 days at 37°C . By quantifying cells that had invaded the agarose ( pellet ) and comparing them to the proportions of cells that had not invaded the agar ( supernatant ) , we were able to determine if particular mutants were defective in invasion . At a cutoff of 2-fold greater abundance in non-invaded fraction ( log2 ( supernatant/pellet ) >2 ) , we observed 341 mutants that were invasion-defective ( Table S8 ) . As we previously observed , there was little overlap ( 5/77 ) between strains that were necessary for full growth in low nitrogen and those that had a defect in agar invasion . However , when we compared the 243 S . cerevisiae orthologs/best hits corresponding to these 341 mutants to invasion-defective mutants of the pseudohyphal S . cerevisiae Sigma 1278B strain ( O . Ryan , personal communication ) , we observed a 26% overlap ( 62/243 ) , underscoring 1 ) the ability of our pooled mutants to identify biologically consistent results , and 2 ) the flexibility of the pooled assay to a solid-media format . Additional experiments on solid media will provide a more detailed genome-wide perspective on this key aspect of C . albicans physiology . Haploinsufficiency profiling was developed in S . cerevisiae to identify drugs that target gene products essential for growth , based on the premise that lowering copy number of the target gene sensitizes the corresponding heterozygous deletion strain to the drug [40] . To select compounds for screening against the tagged C . albicans mutants , we focused on those that inhibited C . albicans growth more potently than S . cerevisiae . We reasoned that these compounds would be more likely to have a different mechanism of action in the two yeasts , e . g . , by having distinct targets , different mechanisms of influx/efflux , or different off-target effects . We screened 1521 compounds ( previously titrated to a concentration that inhibits S . cerevisiae by ∼10% , or IC10 ) against wild-type S . cerevisiae and wild-type C . albicans and measured the ratio of compound-treated growth to that of a control . While we found that the majority of compounds inhibited C . albicans growth at levels similar to S . cerevisiae's , a number of compounds inhibited C . albicans more severely ( Figure 6A ) . For example , the top 40 compounds ( highlighted in red ) produced 20–90% greater inhibition in C . albicans . From this screen , we selected for titration and pooled growth screening 67 readily available compounds that inhibited C . albicans more strongly than S . cerevisiae in our initial screen ( see Methods for criteria ) . Comparing the dose response of these compounds for S . cerevisiae and C . albicans , we observed that the compounds that conferred differential growth inhibition fell into two classes of dose response curves ( representative curves are shown in Figure 6B , Figure S6 ) . The first class included those with parallel dose response curves , suggestive of a compound that has the same target in both yeasts but different cell permeability or residence time . A second category included those in which the dose-response curves had different slopes , suggestive of a different mechanism of action or distinct secondary effects . Overall , these results from screening a library of 1521 compounds against wild-type C . albicans and S . cerevisiae suggest that certain compounds may have distinct mechanism of action ( e . g . , a combination of primary and secondary activities ) in C . albicans . To study the genetic basis of the differential action of certain compounds in C . albicans and S . cerevisiae , we screened 57 of the 67 compounds at approximately an IC20 in the pooled growth assay ( chemical structures are shown in Figure S7; to demonstrate that these 57 represented chemically diverse compounds , the distribution of their pair-wise similarities , based on Tanimoto scores , is shown in Figure S8 ) . We performed a 20-generation endpoint assay ( by analogy to the S . cerevisiae haploinsufficiency profiling ) in which the abundance of each strain in a compound-treated pool was compared to its abundance in a set of DMSO-treated controls . To identify drug-induced haploinsufficiency , a normalized z-score was used to examine the response of each strain to a compound , comparing the performance of a strain ( proxied by tag intensity ) in the compound treatment to its performance in the no-drug controls ( Table S7 ) . Positive z-scores indicate increasing sensitivity to the treatment condition; strains with a high z-score were significantly affected by the drug treatment and may be depleted of genes that encode a cellular target . Of the 57 screens , we focused on 25 that had a small number of significantly sensitive outliers , those most likely to be representative of compounds that act through a single or small number of targets . The remaining 32 compounds exerted either widespread or few fitness defects in the pool . An overview of the z-scores for these 25 compounds is shown in Figure 7A . We found that the GTP exchange factor SEC7 , which activates formation of transport vesicles , was the most sensitive strain in screens with two compounds , brefeldin A and nigericin ( Figure 7B ) . In addition , analysis of the 30 most sensitive strains from the nigericin screen in C . albicans showed GO enrichment of a number of vesicle transport-related processes ( e . g . , Golgi vesicle transport ( GO:0048193 ) , 20 . 7% ( n = 6 ) versus 2 . 4% in the genome , hypergeometric p<0 . 008 ) . As nigericin affects ion gradients across lipid membranes ( as opposed to having a direct protein target [41] ) , we speculate that changes in membrane permeability , and by extension intracompartmental pH ( see below ) , interact synthetically with defects in vesicle transport to cause a fitness defect . Brefeldin A , whose protein target is widely considered to be Sec7p [23] had a less pronounced effect on vesicle transport; brefeldin A-sensitive strains were GO-enriched for function “GTPase regulator activity” ( GO:0005085 , 6 . 7% ( n = 2 ) versus 0 . 4% in the genome , hypergeometric p<0 . 07 ) . Interestingly , neither compound induced SEC7 haploinsufficiency in S . cerevisiae ( Figure 7B ) , even though brefeldin A has been shown to inactivate S . cerevisiae Sec7p complexes in vitro [42] . These results 1 ) suggest that S . cerevisiae has additional targets that become rate-limiting for growth prior to the effects of inhibition of SEC7 , and 2 ) confirm the utility of our pool for validating C . albicans-specific targets . Two synthetic , uncharacterized compounds , 1187–1561 and 0136–0228 , also inhibited vesicle transport-related functions in C . albicans ( Figure 8A ) . We confirmed sensitivity of these strains by growth in individual culture ( Figure 8B ) . The most sensitive strain in a screen with 1187–1561 was orf19 . 2411::Tn5/orf19 . 2411 . The S . cerevisiae ortholog Sc-SYN8 ( non-essential in S . cerevisiae ) is a SNARE protein that functions in vesicle fusion [43] . 0136–0228 induced a strong growth defect in a tfp1 mutant , which has no ortholog in S . cerevisiae , but is computationally predicted to encode a V-type ATPase that regulates intracompartmental pH . Because its best BLAST hit is Sc-TFP1 and because these two genes share a fungal orthogroup [44] , we surmise that they also share function . Consistent with an effect on intracompartmental pH , many of the mutants most sensitive to this compound are also sensitive to nigericin ( vesicle transport related ORFs: TFP1 , BTS1 , orf19 . 2078 , orf19 . 880 , orf19 . 6558 , and others: orf19 . 9 , FAA21 , SPT5 , and orf19 . 6435 ) . As noted above , it is likely that the genes disrupted in nigericin-sensitive strains are interacting synthetically with defects caused by altered intracompartmental pH . We then performed a genetic test to see if 0316–0028 may interact with Tfp1p . We overexpressed C . albicans Tfp1p in a wild-type S . cerevisiae BY4743 and an Sc-Δtfp1 homozygous null mutant background . Because we saw no effect with wild-type S . cerevisiae ( Figure 8C ) , we also tested an Δerg6 null mutant to account for the possibility that the compound was not penetrating the cell . Deletion of ERG6 has been shown to increase compound penetration due to defects in the cell membrane and has been used to sensitize S . cerevisiae to brefeldin A [42] . First , we observed that Tfp1p overexpression ameliorated the growth defect of the Δtfp1 null mutant ( and interestingly , also the Δerg6 null mutant ) even in the absence of drug . This is consistent with results that show that Sc-ERG6 and Sc-TFP1 interact synthetically [45] . Second , when strong growth inhibition was applied with 0136–0228 , we observed partial rescue of the growth defect in both the Δtfp1 and Δerg6 backgrounds , most distinctly in the drug-sensitized Δerg6 background ( Figure 8C ) . That we observed rescue in a heterologous system is strong evidence that this protein is a functional target of this compound , and the fact that 0136–0228 also inhibits the Δtfp1 null suggests that it has additional targets in the cell . We thus propose that Tfp1p is a principal protein target of 0136–0228 , and that the other sensitive strains in its chemogenomic profile appear as a secondary consequence of altered intracompartmental pH ( see Discussion ) . Haploinsufficiency profiling in S . cerevisiae with these compounds would have failed to reveal these gene-compound interactions ( Figure 8A ) .
Experimental multiplexing using DNA tags was one of the essential attributes of the pioneering S . cerevisiae deletion collection , enabling high-throughput genome annotation , genetic analysis , and antifungal discovery [14] , [17] , [46] . A publically available , archived , tagged mutant collection for C . albicans has the potential to similarly accelerate research and drug discovery in an organism directly relevant to public health . Using tagged transposon mutagenesis , we constructed a tagged C . albicans mutant collection that is fully sequenced identified and archived as individual mutants . We note that our collection has some caveats . First , our mutants were created in the –Arg –Ura –His strain BWP17 and so remain auxotrophic for uracil and histidine . Synthetic effects with these auxotrophies could produce false positives in some screens and follow-up assays . In such cases , complementation of HIS1 and URA3 , or validation of the phenotype in an alternative strain background can verify the phenotype . Our results sampling for haploinsufficiency in an alternatively constructed strain both supports that our results in BWP17-derived strains represent true positives and also suggests that there are subtle but detectable synthetic effects contributed by these auxotrophies . A second issue is that because these strains are transposon mutants , they are not likely to represent complete loss of function alleles . This latter case has the advantage that multiple insertion events with different degrees of functional disruption can be interrogated for a particular gene . The other advantage is that this approach is scalable . We report the creation of 4239 uniquely tagged gene disruptions , representing 68% of 6197 predicted ORFs . Additional mutagenesis can be used to create additional mutants , or , given their compatibility , the TagModules can be integrated into deletion cassettes to create the remaining mutants via homologous recombination . There are many potential applications of a C . albicans collection; here we investigated haploinsufficiency , the phenomenon in which a single gene copy in a diploid organism results in a fitness defect . We applied the tagged mutant collection in a competitive growth assay to identify haploinsufficient genes in four different nutrient conditions , identifying 269 haploinsufficient genes across four media conditions . This dataset represents a resource for further study of their involvement in growth , morphogenesis , and potential druggable targets . We found that C . albicans has a unique profile of haploinsufficient genes , highlighting the importance of niche ( or C . albicans ) -specific processes for maintaining wild-type fitness . For example , C . albicans relies more heavily on oxidative metabolism , nutrient sensing ( e . g . , permeases and nutrient scavenging mechanisms ) , and resistance to oxidative stress for optimal growth . Consistent with this observation , the host immune response via neutrophils and macrophages involve superoxide production to kill C . albicans [47] , suggesting that these protective mechanisms may be necessary for full growth . Moreover , dependence on oxidative metabolism is consistent with a requirement for efficient energy production for rapid growth . Because it has evolved within a human host , C . albicans may rely more heavily on oxidative metabolism , because carbon sources in the form of fat or proteins may be more accessible than simple sugars . Metabolites of both fat and proteins are shunted into the tricarboxylic acid ( TCA ) cycle as acetyl-CoA for subsequent breakdown in oxidative phosphorylation . Consistently , we found a predicted fatty acyl-coA synthetase ( FAA21 ) haploinsufficient in 3 conditions . Targeting C . albicans-specific metabolic processes may be a useful approach for identifying novel antifungals . We identified several transcription factors as haploinsufficient in C . albicans , a notable distinction from S . cerevisiae , suggesting that transcriptional regulation may be less flexible in C . albicans with heterozygous alleles manifesting haploinsufficiency . One possible explanation for this observation is that C . albicans is less tolerant of changes in gene dosage as it generally exists in a diploid state . In contrast , S . cerevisiae exists as both a haploid and a diploid , and so these changes in dosage can be tolerated without a reduction in fitness . A second possibility is that because C . albicans is an obligate diploid that lacks a traditional meiotic cycle , two alleles of a transcription factor have diverged such that they are no longer functionally equivalent . This is supported by extensive allelic heterozygosity observed during assembly of the C . albicans genomic sequence [48] . Functional allelic variation has also been observed in C . albicans in a number of small-scale studies , for example in drug pumps [49] , or for HWP1 , in which the two alleles are differentially expressed under biofilm conditions [50] . We next used our dataset to identify which genes function in core cellular processes , and which are haploinsufficient in a specific nutrient condition . Selecting from candidates generated through genome-wide screens , we followed up on a subset of 17 genes from a “core” set of 70 haploinsufficient genes using complementation testing and individual strain growth analysis . Although complementation testing in S . cerevisiae is an imperfect test of function , it is useful for generating hypotheses that can be used to infer function for the remaining uncharacterized genes in the “core” dataset of putative essential genes . We found that 10 of these 17 genes were able to complement their essential S . cerevisiae ortholog , strongly suggestive of functional similarity for these conserved processes . For those genes that did not complement , it is possible that their phenotypes arose from the influence of strain auxotrophies . Alternatively , failure to complement can also result from result of alternative codon usage in C . albicans . Interestingly , we found that both complementing and non-complementing genes contained the alternative CUG codon ( Table 2 ) ) . We also identified a set of genes necessary for growth in nutrient-limiting conditions , and found that while necessary for growth in limited nutrients , these genes were generally not necessary for filamentation . As filamentation in fungi is a well-documented response to low-nitrogen conditions ( presumably an adaptation to improve nutrient acquisition ) [51] , we anticipated that filamentation might play a role in growth in nutrient-limited conditions . Contrary to this expectation , our results suggest a disconnect between growth rate ( which may require optimal nutrient utilization ) and filamentation ( which may require specific nutrient sensing ) under the conditions that we tested . From a biological perspective , filamentation may be the preferred lifestyle for tissue invasion or macrophage evasion in which the ability to grow rapidly is less important . C . albicans cells that are unable to filament in vivo are avirulent , and null mutants of EFG1 , a transcriptional regulator of filamentation , have normal growth [52] . However , whether all mutants that have a filamentous defect display wild-type growth has not been systematically determined . From the standpoint of developing new treatment strategies , identifying both fungicides , which can be identified by growth inhibition screens , and inhibitors of pseudohyphal growth are of value . With the appropriate experimental design , both types of screens can be performed with this collection , as we have exemplified by preliminary results from a SLAD solid media pooled experiment . We also investigated drug-induced haploinsufficiency in C . albicans , screening the pool with compounds that were most likely to produce a differential drug response by selecting compounds that more potently inhibited C . albicans than S . cerevisiae . Interestingly , we observed that the same chemical inhibitor can have different effects in S . cerevisiae and C . albicans . For instance , wild-type S . cerevisiae is much less sensitive to nigericin or brefeldin A than wild-type C . albicans , and mutants with the highest sensitivity in screens with these compounds did not include Sc-SEC7 . This suggests that; 1 ) other targets in S . cerevisiae have a greater impact on S . cerevisiae's sensitivity to brefeldin A and nigericin , 2 ) that the drug is detoxified in S . cerevisiae , or 3 ) that C . albicans has less genetic redundancy for the pathways inhibited by these compounds . We also identified two other synthetic , previously uncharacterized compounds that inhibited vesicle transport in C . albicans , 0136-0228 and 1187–1561 . Interestingly , the sensitivity profile of 0136–0228 overlapped that of nigericin , although their chemical structures show no obvious similarity . This result can be explained if 0136–0228 inhibits Tfp1p , a putative V-type ATPase that regulates intracompartmental pH [53] . Support for this scenario came from our complementation experiments , in which the drug-induced growth defect was rescued by overexpression of Tfp1p in S . cerevisiae . Abrogation of intracompartmental pH regulation via disruption of ion flow across vesicle membranes likely results in growth defects similar to those produced by nigericin , which produces a similar disregulation of intracompartmental pH . Interestingly , no orthologs or proteins of similar function were found in S . cerevisiae screens with this compound , again underscoring the need for direct study of C . albicans to identify novel treatment strategies . The approach of using tagged transposon mutagenesis to generate mutant collections can be applied to a wide range of fungi of medical interest . The in vitro mutagenesis method allows flexibility because organism-specific transposons are not required , although a means for homologous recombination is needed . However , this approach could also be adapted to an in vivo format if the transposon could be electroporated directly into the cell ( e . g . , using commercially available transposome technology [54] ) , or if it could be expressed endogenously . Both of these approaches could bypass the transformation step , although they may be subject to insertion bias . Additionally , the TagModules can be readily adapted to a targeted deletion system for fungi with a compatible recombination system . While model organisms such as S . cerevisiae have been invaluable in initiating research in a range of microorganisms of medical interest , ultimately , it will be most fruitful to identify novel treatment strategies using the pathogen itself , owing to pathogen-specific differences . In summary , we have generated a uniquely tagged , publically available and archived disruption collection in C . albicans that can be used in multiplexed phenotypic assays or in individual experiments to identify potential new biology , therapeutic targets and mechanisms of pathogenesis .
C . albicans genomic DNA was isolated from strain BWP17 ( ura3Δ:: λ imm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG ) [55] and partially digested with one or more of the following enzymes: XbaI , EcoRV , SpeI , XbaI/SpeI , XbaI/EcoRV , or BsrBI . The partially digested DNA fragments were gel purified to approximately 2–8 kb in size and ligated into a library backbone cut with the corresponding enzyme and phosphatase-treated . Backbones used were pCR 8/GW/TOPO + linker for XbaI , EcoRV , and BsrBI-cut genomic DNA , and pUC19 + linker for SpeI , XbaI/EcoRV , and SpeI/XbaI-cut genomic DNA . Construction of these vectors was previously described [27] . Each library contained on average 20 , 000 clones . We also used a commercially available C . albicans genomic library ( Open Biosystems; [56] ) . The transposon destination vectors Tn7-UAU1-A and Tn5-UAU1-C . 1 , modified from Tn7-UAU1 [21] and EZ-Tn5 pMOD-3 ( Epicentre Biotechnologies ) to contain the Gateway recombination sites , were obtained from our previous study [27] . The Gateway-compatible TagModules [27] were pooled and transferred to the Tn7-UAU1-A or the Tn5-UAU1-C . 1 vectors as previously described [27] , and the resulting tagged transposons were used in the mutagenesis of C . albicans genomic libraries as described in the same study . The majority of insertions were generated using the Tn5-UAU1 due to technical difficulties with the Tn7 . Each insertion was sequenced using D2_revcomp ( Tn7-based ) or U1 ( Tn5-based ) to identify the gene disrupted and the tag associated with each disruption . Gene-tag pairs were sorted to maximize i ) the number of unique TagModules , ii ) unique gene mutagenesis events , and iii ) highest % gene disrupted ( # base pairs from transposon junction to gene start ) / ( total gene length ) . Overall genome coverage is represented in Figure S1 . Select insertion plasmids were then plasmid prepped with Seqprep 96 ( Edge Biosystems ) , and the genomic fragment containing the tagged insertion was excised with the appropriate enzyme and chemically transformed into BWP17 in 96-well format . At this step , two pools were created , one with unique uptags and one with unique downtags ( for details , see Figure S2 and Methods S1; for pool characteristics , see Table S1 ) . We separately arrayed transformants for each pool to agar plates and scraped the colonies into SC- Arg + uridine +15% glycerol to create the C . albicans pools , which we then stored as 50 µL aliquots at −80°C . For validation , we grew the pools independently in YPD for 20 generations as described in [34] minus the 10-generation recovery time , and extracted genomic DNA using the YeaStar Genomic DNA Kit ( Zymo Research ) . PCR amplification of the uptags and downtags was performed separately with the common primers U1′ & BTEG-U2′ , and D1′ & BTEG-D2′ , and 30 µL each of uptag and downtag product was hybridized to an Affymetrix TAG4 microarray as described [57] . For haploinsufficiency experiments , YPD , SC – Arg + uridine , YNB + uridine , and SLAD + uridine were made as defined in [58] . Growth assays were performed in duplicate in each of the media conditions , and samples were recovered at 5 , 10 , 15 , and 20 generations of growth . Genomic DNA extraction , tag amplification , and hybridization were performed as described above . In total , we performed a series of eight hybridizations per media condition plus two hybridizations for a common zero timepoint . For the zero timepoint , ∼2 OD600 of frozen cells were used as the cell template for the genomic extraction . For array data pre-processing , we followed the protocol outlined in [57] , [59] . Briefly , outliers were masked and removed , and the average of the unmasked replicates were calculated for each tag . Uptags and downtags were mean-normalized , and low-quality tags ( those below 3X background intensity units ) were removed . To identify strains with reduced fitness in each of these growth conditions , we used a linear regression model to track decreases in tag hybridization intensity as a function of time . Linear regression of the log2 ( tag intensity ) as a function of generations of growth ( 0 , 5 , 10 , 15 , and 20 ) was implemented using the lm ( ) function in the statistical program R . A negative regression slope indicates that the tag signal decreases over time , inferring that the strain has a fitness defect in this condition with respect to the pool as a whole . To correct for multiple testing , the p-value for each regression was adjusted using the false discovery rate ( “fdr” ) option of the R function p . adjust ( ) . For comparisons of regression slope between media types , an F-statistic was calculated and a p-value derived from the distribution . Our criteria for strain sensitivity was slope <0 , p<0 . 05 . This is a slightly less stringent cutoff than reported in Deutschbauer et al . ( 2005 ) as we have observed that the competitive growth assay is capable of detecting even slight defects in growth . For complementation testing , we used several pre-existing S . cerevisiae resources for the strain background . We picked individual strains from the Magic Marker collection ( Open Biosystems , [32] ) of yeast heterozygous deletions containing the selectable matA haploid marker . As the Magic Marker strains have some reversion rate , as evidenced by some colonies observed on the no-vector plates , we confirmed a few of these results with traditional tetrad dissection of the sporulated yeast knockout ( YKO ) strain , which has no Magic Marker cassette . For overexpression , we used as our destination vector the Gateway compatible destination vector pAG416GPD-ccdB ( Addgene , [60] ) , which is a Ura+ CEN plasmid under control of a constitutive promoter . For cloning C . albicans ORFs , we PCR amplified approximately 500 bp up and downstream of the start codon using primers specific to each gene and Platinum PCR SuperMix High Fidelity Primer Solution ( Invitrogen ) . PCR products were TOPO cloned into the Gateway entry vector pCR 8/GW/TOPO ( Invitrogen ) . Individual clones were sequenced using primers GW1 and GW2 . Correct clones were then transferred to the pAG416GPD-ccdB destination vector using the LR clonase reaction ( Invitrogen ) , and resequenced as described . For S . cerevisiae overexpression plasmids , we picked clones from the Molecular Barcoded Yeast ( MoBY ) ORFs ( Open Biosystems , [61] ) . As these were already Gateway compatible , we transferred them to the overexpression plasmids using the LR clonase reaction and sequence verified them using primers GPD_ProF and pBluescriptSK . Overexpression plasmids were chemically transformed into the corresponding Magic Marker or YKO strain , selecting for Ura+ transformants . Individual colonies were then inoculated to SC –Ura and grown overnight . Cultures were then harvested , washed twice with water , and then resuspended in sporulation media ( 2% potassium acetate ) at a density of 1–1 . 5 OD600/mL . After sporulation for 5 days , 4 . 5×10−4 OD600 of sporulated Magic Marker culture was plated to Magic Marker selection media –Ura and grown for 2–3 days at 30°C . YKO-based strains were tetrad dissected and plated onto SC –Ura + G418 . Tetrad dissected plates were then replica plated onto 5-FOA and YPD + G418 agar plates to confirm complementation . For individual growth experiments , we picked mutants from the frozen stock and grew them to saturation in SC –Arg + uridine . Each strain was then diluted to a working OD600 of ∼0 . 6 in water . For 5 generation experiments , each strain was diluted to a final OD600 of 0 . 06 in YPD , SC –Arg + uridine , YNB , or SLAD in 96-well plates . For 20 generation experiments , each strain was diluted to a final OD of 0 . 03 in the media of interest in 48-well plates , and growth was measured every 15 minutes robotically diluting every 5 generations as in the pooled growth assay . To measure growth of the GRACE strains , 12 of 17 of the core haploinsufficient strains identified in our screens were obtained as GRACE alleles ( 5 were not constructed ) , in which heterozygotes were constructed such that the remaining allele is under the control of a tetracycline-repressible promoter [33] . 400 cells of each strain ( as determined by hemocytometry ) were inoculated into 650 µL of media in 48-well plates ( Greiner ) and grown with constant shaking at 30°C as described [57] . In these conditions , individual cultures underwent ∼14 generations of growth without necessitating re-inoculation during the course of the experiment . Each strain was assayed in triplicate in selective SC –Arg + uridine media in the presence or absence of the tetracycline analog doxycycline ( 100 µM ) . AvgG ( a metric of growth ) was calculated as previously described [34] . Individual strains were picked from the frozen stock and grown overnight in SC –Arg + uridine . Strains were then stamped in triplicate to Spider agar media and grown for 5 days at 30°C , and colonies were then examined microscopically for filamentation . Methods for the pooled assay on solid SLAD media are described in Methods S1 . A diversity library was obtained from ChemDiv , Inc . and dissolved in DMSO . Other compounds were obtained from Sigma with the exception of itavastatin ca ( Sequoia Research Products ) and enantio-paf C-16 ( Enzo Life Sciences ) . Each compound had previously been titrated to an inhibitory level of ∼10% in S . cerevisiae in YPD buffered to a pH of 6 . 8 with HEPES [62] . We grew C . albicans strain BWP17 and S . cerevisiae strain HO-1 in the presence of these 1521 distinct compounds under the same conditions , measuring AvgG as previously described [34] . Titration experiments were performed in microtitre plates , growing BWP17 and HO-1 in 100 µL buffered YPD plus either 1% DMSO ( control ) or 2 µL of compound at stock concentration . Growth rate in compound dilutions of up to 1/64th original concentration ( or further , if necessary ) were measured as previously described . AvgG from each microtitre plate was normalized to the 12 controls on each plate . Selected compounds were then chosen for follow-up in dose response and pooled growth assays . First , compounds were ranked in order of greatest difference in inhibition between C . albicans and S . cerevisiae ( Δ ( normalized AvgG ) ) . As a number of the compounds which produced the greatest difference in inhibition were not readily available or obtainable , and some hybridizations in the pooled growth assay failed , we focused on ∼67 available compounds for follow-up . The majority of these produced greater inhibition of C . albicans in the dose response assays ( Figure S6 ) . For pooled growth assays with 57/67 of these compounds , 20-generation pooled growth assays were performed as described [34] , [62] . To determine sensitive strains , the experimental array was compared to a matched control set comprised of 11 no-drug arrays [18] . We then calculated z-scores and associated p-values as a metric of sensitivity as described [18] . | Candida albicans is a normal inhabitant in our bodies , yet it can become pathogenic and cause infections that range from the superficial in healthy individuals to deadly in the immunocompromised . Comprehensive genetic analysis of C . albicans to identify mechanisms of virulence and new treatment strategies has been hampered by limited , publically accessible genomic resources . By combining the principles of Saccharomyces cerevisiae strain tagging with transposon mutagenesis to generate individually tagged mutants , we created the first entirely public resource that allows simultaneous measurement of strain fitness of ∼60% of the genome in a wide range of experimental treatments . By identifying genes that confer a fitness or growth defect when reduced in copy number , we uncovered genes whose protein products represent potential antifungal targets . Moreover , screening this strain collection with chemical compounds allowed us to identify anticandidal chemicals while concurrently gaining insight into their cellular mechanism of action . This resource , combined with straightforward screening methodology , provides powerful tools to generate hypotheses for functional annotation of the genome , and our results highlight the value of direct versus model-based pathogen studies . | [
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] | 2010 | Gene Annotation and Drug Target Discovery in Candida albicans with a Tagged Transposon Mutant Collection |
The nematode intestine is a major organ responsible for nutrient digestion and absorption; it is also involved in many other processes , such as reproduction , innate immunity , stress responses , and aging . The importance of the intestine as a target for the control of parasitic nematodes has been demonstrated . However , the lack of detailed knowledge on the molecular and cellular functions of the intestine and the level of its conservation across nematodes has impeded breakthroughs in this application . As part of an extensive effort to investigate various transcribed genomes from Ascaris suum and Haemonchus contortus , we generated a large collection of intestinal sequences from parasitic nematodes by identifying 3 , 121 A . suum and 1 , 755 H . contortus genes expressed in the adult intestine through the generation of expressed sequence tags . Cross-species comparisons to the intestine of the free-living C . elegans revealed substantial diversification in the adult intestinal transcriptomes among these species , suggesting lineage- or species-specific adaptations during nematode evolution . In contrast , significant conservation of the intestinal gene repertories was also evident , despite the evolutionary distance of ∼350 million years separating them . A group of 241 intestinal protein families ( IntFam-241 ) , each containing members from all three species , was identified based on sequence similarities . These conserved proteins accounted for ∼20% of the sampled intestinal transcriptomes from the three nematodes and are proposed to represent conserved core functions in the nematode intestine . Functional characterizations of the IntFam-241 suggested important roles in molecular functions such as protein kinases and proteases , and biological pathways of carbohydrate metabolism , energy metabolism , and translation . Conservation in the core protein families was further explored by extrapolating observable RNA interference phenotypes in C . elegans to their parasitic counterparts . Our study has provided novel insights into the nematode intestine and lays foundations for further comparative studies on biology , parasitism , and evolution within the phylum Nematoda .
The intestine is one of the major organs in nematodes , creating a key surface at the intestinal apical membrane that interacts with the environment . While specific cellular characteristics of the intestine can be diverse among nematode species , they typically conform to polarized epithelial cells with the apical membrane composed of microvilli lining the digestive tube . In apparent contrast to other surfaces of nematodes , digestive and assimilative functions , as well as various metabolic pathways and cellular trafficking , are expected to be extremely active at the intestinal surface . For example , an adult Caenorhabditis elegans is capable of producing oocytes with about the same total biomass as its own body per day [1] , but the average intestinal residence time for foods was estimated to be less than two minutes in C . elegans [2] , suggesting that the microvillous membrane must have an enormous capacity for nutrient digestion and absorption . In addition , the intestine has to offer innate immunity against invasive pathogens , and adaptations at the apical intestinal membrane may be required to protect parasitic nematodes against host immune systems . Furthermore , the nematode intestine has been suggested to be involved in other biological processes such as stress responses , body size control , and aging [1] . Three lines of evidence indicate that the intestine is an important target for the control of parasitic nematodes . First , intestinal antigens enriched for apical membrane-associated proteins have been successfully used to immunize against Haemonchus contortus , a hematophagous nematode of small ruminants [3]–[7] . Surface-bound nematode proteases are a dominant , but not exclusive , group of proteins that have been implicated in inducing this protection . A prospective mechanism of the immunity involves perturbing nutrient digestion and acquisition at the intestinal surface by the ingested host-derived antibodies capable of neutralizing parasite digestive proteases [7] . Further investigations conducted with hematophagous hookworms also produced similar effects [8] . Second , adult H . contortus intestinal cells are hypersensitive to benzimidazole anthelmintics , apparently through the target protein beta-tubulin isotype 1 [9] , [10] . It was suggested that the drug inhibited vesicle transport in the apical secretory pathway , causing the intracellular release of the digestive enzymes destined for secretion and subsequent cytotoxic effects [9] . Third , parasite control has been demonstrated by inhibition of an intestinal enzyme , cathepsin L cysteine protease , by either RNA interference or a chemical inhibitor in the plant parasitic nematode Meloidogyne incognita [11] . These observations generate great interests to uncover the basic characteristics of the intestinal cells that might be further exploited for the broad control of parasitic nematodes . However , the dearth of relevant experimental systems and molecular information such as gene repertoires for many parasitic species has impeded rapid progress . Five major clades ( I–V ) are currently recognized to comprise the phylum Nematoda [12] , [13] . So far , almost all studies of the intestine at the gene level have focused on the clade V nematodes . A small-scale sampling of expressed sequence tags ( ESTs ) from the dissected intestine from adult H . contortus females identified 51 intestinal genes including cysteine proteases [14] , this list was later expanded via a proteomic approach to include a number of apical intestinal membrane proteases from H . contortus and hookworms [15] . Intestinal EST libraries generated from laser-dissected materials from Necator americanus and Ancylostoma caninum allowed the identification of 544 intestine-expressed genes [16] . Although a more comprehensive dataset with >5 , 000 intestinal genes is available in C . elegans [17]–[19] , it is unclear , given the evolutionary diversity within Nematoda , to what extent the molecular and cellular functions of the intestine can be extrapolated across nematode species . In this study , we sampled the transcribed genomes from several tissues and developmental stages from two parasitic nematodes: the clade III nematode Ascaris suum , which presumably feeds on the semi-digested contents in the host intestine , and the clade V blood-feeding parasite H . contortus . Nearly 10 , 000 and 5 , 000 genes were identified from the two nematodes , respectively . More importantly , given the attention to the intestine , we produced the largest collection of intestinal genes in parasitic nematodes by dissecting adult intestine from each species , a procedure that is not practical for many other nematodes because of their small sizes and the lack of laboratory culturing systems . Extensive cross-species comparisons were made among the adult intestinal genes from the parasites and those expressed in the adult intestine of the free-living bacterivore C . elegans . Both diversification and conservation of intestinal gene repertories were evident among the species investigated . The diversities of intestinal transcriptomes by clade and species may reflect the substantial life style differences among these nematodes . A group of 241 protein families were found conserved in the intestine of all three nematodes , accounting for ∼20% of the intestinal gene repertoires from the three species . These genes may include core intestinal functions that are indispensable among many nematodes . Functional annotations were generated for the intestinal genes . Molecular characteristics of the intestinal genes were further explored to highlight various physiological aspects of the nematode intestine .
Dissection of the adult intestine was carefully performed under microscopy as described previously [14] , [20] , [21] . The samples used in this study had also passed another round of visual inspection microscopically to ensure they did not contain other tissues such as muscle , esophagus , or hypodermis . Detailed information on genetic materials and cDNA library construction are available at www . nematode . net . ESTs were processed and clustered as described before [22]–[25] . EST contig sequences were translated individually by Prot4EST , a 6-tier translation pipeline combining both similarity-based methods and de novo predictions [26] , for downstream analysis . Databases used for sequence comparisons were: i ) Caenorhabditis spp . , all amino acid sequences in the complete genomes of C . elegans ( Wormbase Release v150 ) , C . briggsae ( June , 2006 ) , and C . remanei ( June , 2006 ) , ii ) Other Nematoda , all non-Caenorhabditis nematode nucleic acid sequences in GenBank excluding those from A . suum ( when analyzing A . suum sequences ) or H . contortus ( when querying H . contortus sequences ) ( October 18 , 2006 ) , and iii ) Non-Nematoda , all amino acid sequences in the non-redundant protein database NR excluding those from nematode species ( September 20 , 2006 ) . WU-BLASTP ( wordmask = seg postwe B = 1000 topcomboN = 1 ) was used to query the translated sequences against protein databases , and WU-TBLASTN ( wordmask = seg lcmask B = 1000 topcomboN = 1 ) for searching against nucleotide databases [27] . The E-value cutoff of 1 . 0e−5 was used to accept sequence similarities in all BLAST searches . Each intestinal EST cluster was assigned two counts according to the numbers of times it was sampled from either the intestinal or non-intestinal cDNA libraries , respectively . Similarly , each C . elegans intestinal gene was assigned two counts for the numbers of times it was sampled by SAGE tags from either the glp-4 dissected gut or the glp-4 adult whole worm , respectively . The mutants lack the gonad when raised at 25°C , therefore contamination by other tissues is less likely [17] . The SAGE data was downloaded with sequence quality filter = 0 . 99 , no normalization , duplicate ditags and ambiguous or antisense tags removed ( April 19 , 2006; mapped to Wormbase Release v150 ) [17] . A Poisson-based enrichment test , considering both the total sampling sizes and random variations [28] , was implemented to compute an P-value to represent the likelihood of intestinal enrichment for each EST cluster or C . elegans gene using these two counts . The P-value cutoff of 0 . 001 was chosen to define the putative intestine-enriched genes from the three nematodes . A hidden Markov modeling-based algorithm , Phobius [29] , was used with default setting . Each query sequence was further annotated as TM-only , TM with SP , SP-only , or intracellular based on raw Phobius outputs . For each EST cluster , Phobius annotation was predicted for each contig and summarized at the EST cluster level . A modified Wormbase Release v150 containing only the longest splicing isoform at each gene loci was used as the complete gene set of the C . elegans genome . For tissue-level comparisons made between intestine and gonad , InParanoid [30] was used at default settings to identify a total of 1 , 764 putative orthologous groups between all the A . suum EST clusters and the complete gene set of C . elegans ( the modified Wormbase Release v150 containing only the longest splicing isoform at each gene loci ) . InParanoid-generated main orthologous pairs , which are essentially the mutual-best matches between all the available genes from the two species , were further screened against the 447 , 546 A . suum Genome Survey Sequences ( GSSs ) that were generated recently ( Mitreva , unpublished ) , resulting in the final group of 1 , 652 putative main orthologous pairs in which the C . elegans members do not have better matches in GSSs than the A . suum EST partners assigned by InParanoid . C . elegans gonad-expressed genes were extracted from SAGE data generated from dissected gonad ( March 12 , 2007 ) [17] . An all-against-all WU-BLASTP was performed on all the 9 , 918 translated intestinal genes from the three species ( including sequences for EST contigs from the two parasites and 5 , 056 C . elegans genes ) . Raw BLAST results were fed to a C-language implementation of Markov Cluster ( MCL ) Algorithm ( www . micans . org/mcl ) , a fast and scalable unsupervised cluster algorithm based on simulation of flow in graphs [31] . An Inflation Fact of 1 . 6 was chosen for the MCL clustering . The MCL output was then summarized at the EST cluster level , during which we applied an additional filtering step to remove an EST cluster from a MCL protein family if less than 10% of its total contigs were clustered into that family . These parameters were based on manual inspection of the results on a test set consisting of the putative intestine-enriched genes with 210 parasite EST clusters and 247 C . elegans genes ( false positive rate of 3%; data not shown ) . Default parameters for InterProScan v13 . 1 [32] were used to search against the InterPro database [33] . Raw InterProScan results for the translated EST contigs were summarized at the EST cluster level . Gene ontology ( GO ) terms were further assigned and displayed graphically by the AmiGO browser with default parameters and the ontology data released on March 15 , 2007[34] . Complete GO mappings for the three intestinal transcriptomes are available at www . nematode . net . For each GO term , its enrichment in an IntFam group ( such as the IntFam-241 group ) was measured over the complete set of 9 , 918 translated intestinal genes using a hypergeometric test , the p-value cutoff of 1 . 0e−5 was chosen for enrichment . The less informative ontologies , including those at level 4 or higher for Biological Process or Molecular Function , and those at level 2 or higher for Cellular Component , were removed from the enrichment list . Also removed were redundant ontologies by keeping only the lower level more informative ontology if the same group of genes was mapped to more than one GO term . An empirical mixed approach was used for mapping the novel genes to canonical pathways . The E-value cut-off of 1 . 0e−10 reported by WU-BLASTP against the Genes Database Release 39 . 0 from Kyoto Encyclopedia of Genes and Genomes ( KEGG ) was first used for finding homologous matches . Then the top match and all the matches within a range of 30% of the top BLAST score , if meeting the cut-off , were accepted for valid KEGG associations [35]–[37] . A hypergeometric test , measuring the relative coverage of the KEGG-annotated orthologous groups assigned to a pathway , was implemented to identify the enriched pathways for each intestine [38] . Nucleotide sequences data reported in this paper are available in the GenBank , EMBL and DDBJ databases . The accession numbers for ESTs from A . suum are: BI781215-BI784439 , BM032617-BM034650 , BM280443-BM285290 , BM318846-BM319958 , BM515079-BM518821 , BM566483-BM567588 , BM568416-BM569529 , BM732977-BM734435 , BM964439-BM965448 , BQ094886-BQ096565 , BQ380669-BQ383404 , BQ835081-BQ835723 , BU965907-BU966430 , CA849193-CA850481 , CA953713-CA955182 , CB100077-CB102042 , DV018957-DV019894 , EB186562-EB187079 . The accession numbers for ESTs from H . contortus are: CA033335-CA034379 , CA868595-CA870175 , CA956361-CA959150 , CB018493-CB022024 , CB063882-CB065260 , CB099467-CB100076 , CB190871-CB192419 , CB331948-CB333475 .
We constructed 18 A . suum and 6 H . contortus stage- or tissue-specific cDNA libraries , and sequenced 31 , 416 and 14 , 014 5-prime ESTs from the two species , respectively . These ESTs totaled to 13 . 6 and 6 . 3 million bases for A . suum and H . contortus , accounting for 77% and 63% of the total nucleotides from the two species currently available in public databases ( Table S1 ) . Supplemented by 9 , 354 A . suum and 8 , 146 H . contortus ESTs previously deposited in GenBank ( retrieved in January , 2006 ) , all available ESTs were grouped into 17 , 989 A . suum and 9 , 842 H . contortus EST contigs , each containing ESTs derived from nearly identical transcripts according to overlapping sequences to reduce sequence redundancy [23] , [24] . The contigs were further assembled into 9 , 947 A . suum and 5 , 058 H . contortus EST clusters based on sequence similarities identified among contigs as well as in previously identified genes ( Table S1 ) . Each EST cluster likely represents transcripts derived from a single genomic locus and therefore is approximated as one gene [22]–[24] . Given that C . elegans and C . briggsae each contains ∼19 , 000 protein-coding loci , and between 14 , 500 and 17 , 800 genes were inferred from the Brugia malayi draft genome [39] , we have consequently identified a substantial portion of the complete gene sets from the two parasites . These data will vastly facilitate the genome assembly and annotation in the related nematode genome sequencing projects currently underway . Initial investigation of the identities of these novel genes was performed by comparing the translated sequences with known proteins from other organisms ( Text S1; Figure S1 ) . To study the intestinal transcriptomes , four cDNA libraries ( out of the 18 ) from A . suum and three ( out of the 6 ) from H . contortus were constructed from dissected adult intestine with methods based on either Poly-A [40] or spliced leader sequences [24] . Among all the ESTs we generated , a total of 9 , 586 A . suum and 7 , 068 H . contortus ESTs were derived from these intestinal libraries . These ESTs occurred in 3 , 121 A . suum and 1 , 755 H . contortus EST clusters , accounting for about 30% of the total genes sampled in each nematode . Since these EST clusters contained ESTs sampled from the adult intestine , they were considered to represent adult intestinal genes , making this the largest tissue-level gene discovery in parasitic nematodes thus far ( Table 1 ) . In contrast to the two gastrointestinal parasites , the free-living model nematode C . elegans is a bacterivore obtaining nutrients primarily or exclusively from the consumption of bacteria . Two previous studies reported identification of genes expressed in the adult C . elegans intestine: i ) sequence tags generated by serial analysis of gene expression ( SAGE ) from the dissected adult intestine were mapped to over 4 , 000 C . elegans genes [17] , [18]; ii ) a study using mRNA tagging and microarray gene expression profiling identified ∼1 , 900 intestine-expressed genes [19] . Consolidating the two efforts provided us with a non-redundant set of 5 , 065 intestinal genes from adult C . elegans , covering over 25% of all coding loci in its entire genome ( Table 1 ) . The phylum Nematoda is ancient and diverse . Even though the evolutionary distance between clade III A . suum and clade V C . elegans was estimated to be ∼350 million years [41] , the nematode intestine has maintained high similarity in both tissue morphology and presumably physiology ( i . e . involvement in feeding ) . However , it is unknown how much the intestine is conserved , or diversified , at the molecular level across species . The tissue-level gene sampling in this study offered an opportunity to investigate this question . Differences in the intestinal gene repertoires were obvious among the three nematodes . In total , 39% of A . suum and 19% of H . contortus intestinal genes were found to be novel compared to all known proteins in the public databases ( Figure 1 ) . Such novel intestine-expressed parasite genes contained no match in the complete genome of the free-living C . elegans , thus not in the C . elegans intestine , making them unique by comparison to C . elegans . In addition , for the sampled intestinal genes from both parasites , the non-Caenorhabditis nematodes offered the largest numbers of homologous matches than either the Caenorhabditis species or the non-nematode organisms ( Figure 1 ) . Such differences may suggest the existence of lineage- or species-specific diversification in the nematode intestine . Furthermore , we observed higher levels of diversification in the putative intestine-enriched genes from the three nematodes . Taking into consideration sample size and random sampling fluctuation [28] , we identified 150 A . suum , 60 H . contortus , and 247 C . elegans putative intestine-enriched genes based on the “digital” expression levels revealed in EST and SAGE data ( at the Poisson distribution-based P-value cutoff of 0 . 001 ) ( Table S2; Table S3; Table S4 ) . Many of these predicted enrichments suggested unique intestinal functions for the individual species . For example , the group of 60 genes from the blood-feeding H . contortus includes 2 fibrinogen-related proteins that may function as thrombin inhibitors to prevent clotting of ingested blood . Also included are putative enzymes that may be involved in the digestion of hemoglobin , one of the major food sources of blood-feeding parasites , including a serine-type protease , a metallopeptidase , and 13 different cysteine-type proteases that were reported previously [42] ( Table S3 ) . Interestingly , a significantly higher percentages of these genes ( e . g . 15%-31% higher than all the sampled intestinal genes ) encode proteins predicted as secreted or trans-membrane [29] ( Figure 2 ) , suggesting that they interact with the extracellular environment . However , 64% , 54% , and 69% of them , from the three species respectively , were distinct from members of the protein families conserved in the intestine of all three nematodes ( IntFam-241; see below ) , indicating that a large portion of these putative intestine-enriched genes are specific to the intestine of individual nematode lineages or species . This further underlines the diversification of intestinal transcriptomes in accommodating the different life styles and feeding patterns among nematodes . To evaluate common characteristics of the nematode intestine , we first sought evidence for the molecular conservation of the tissue in the context of phylogeny . We made comparisons among genes expressed in the intestine of A . suum and C . elegans and those expressed in another tissue , namely the gonad . These two species have the largest numbers of sequences available , and they also represent the most distant relationship among the three nematodes investigated . The gonad was chosen because the next largest group of genes was sampled from this tissue in A . suum after the intestine . H . contortus was excluded from this analysis because a gonad-expressed gene set was not available from this nematode . Genes expressed in the intestine and gonad were divided into four putative tissue-specific groups: i ) 2 , 453 A . suum and ii ) 2 , 557 C . elegans genes expressed in the intestine but not in the gonad ( the two intestine groups ) , and iii ) 2 , 690 A . suum and iv ) 2 , 589 C . elegans genes that were found in the gonad but not in the intestine ( the two gonad groups ) . The use of the similar numbers of genes in each group is expected to reduce false results caused by over-representation from any single category . Molecular conservation was first evaluated by comparing the numbers of putative homologous pairs identified among the intestine and gonad gene groups . The number of the putative homologs between the two intestine groups was significantly larger than that between the intestine and gonad groups ( p-value = 2 . 5e−04 at the bit-score cutoff of 100 in a permutation two-tailed Z-test; p-value = 4 . 2e−08 at the bit-score cutoff of 50; Figure S2 ) , suggesting that for genes expressed in the intestine of one nematode , their homologous matches in another species are significantly more likely to be expressed in the intestine than in the gonad of the second nematode . These results provide evidence for the molecular conservation of the intestine across these distantly related nematodes . In contrast , the number of putative homologs between the two gonad groups was not statistically different from that between the gonad and intestine ( Figure S2 ) , indicating that the gonad genes appeared to be less conserved than those expressed in the intestine in this two-tissue comparison . To increase the confidence of analysis , we next focused on the putative orthologous pairs predicted among the intestine and gonad gene groups , which was a smaller data set than the homologous pairs used above but with higher stringency . Among the total of 1 , 652 putative orthologous pairs predicted from A . suum and C . elegans ( see Materials and Methods ) , 289 were paired among genes from the intestine and gonad groups . They were used in a Chi Square statistical test , with random distribution of orthologous pairs as the null hypothesis . Compared to the expected numbers , there was a 31% enrichment of orthologous pairs observed between the A . suum and C . elegans intestine groups ( Figure 3 ) , whereas the enrichment between the two gonad groups was only marginal ( 5% ) , and the observed numbers of orthologous pairs between the gonad and intestine groups were less than expected ( Figure 3 ) . Overall , a significant χ2 value of 11 . 9 rejects the null hypothesis at a confidence level higher than 99% ( p-value <0 . 01 ) [43] , and selective pressure is evident on molecular conservation of the intestinal gene repertories . Although the use of the incomplete transcriptomes and a bias towards relatively abundant transcripts in EST sampling can affect results , analyses of either homologous or orthologous pairs both provide direct support for the molecular conservation of the nematode intestine . With the obvious pattern of diversification in the nematode intestine ( discussed earlier ) , our results indicate that a subset of the intestinal gene repertoires , which likely contribute to the intestinal characteristics conserved across diverse nematode species , remain conserved during the evolution of Nematoda . Interestingly , genes expressed in the gonad appear to be less well conserved based on both analyses . However , these results do not suggest the lack of evidence for the conservation of the gonad . Instead , the two-tissue comparisons indicate that the levels of conservation are lower in the gonad than in the intestine , suggesting that the levels of molecular conservation may differ in different nematode tissues . In fact , the conserved characteristics of the gonad may become more evident with larger sample sizes and/or by comparisons with another tissue with a lower level of conservation than the intestine , when new sequence data becomes available . Similarly , differences at the levels of molecular conservation were observed in different tissues between human and mouse , which diverged only about 25 million years ago [44] . Future comparisons with more complete expression data across multiple tissues in different nematode species should offer additional insights into this aspect of nematode evolution . To compare the intestinal transcriptomes of A . suum , H . contortus , and C . elegans in a single analysis , we built protein families from the complete set of 9 , 918 translated intestinal genes combined from the three nematodes . A total of 5 , 587 intestinal protein families ( IntFam ) were identified conservatively based on sequence similarities by MCL clustering [31] ( Figure 4 ) . Proteins assigned into the same protein family contain putative homologous or orthologous matches among the three species . Both diversification and conservation of the intestinal transcriptomes was obvious at the protein family level in this 3-species comparison . A total of 59% of all the sampled intestinal genes were members of the protein families containing proteins from only one nematode ( Figure 4 ) . Although the assignments for many of these single-species families are likely to change when more complete intestinal gene repertories become available , this group includes the genes contributing to the unique intestinal features in each species . The remaining 41% of the intestinal genes formed 910 multi-species protein families; they are conserved in the intestine of at least two nematodes . Among these multi-species families , 241 had members from all three species , accounting for ∼20% of all the intestinal genes under investigation ( Figure 4 ) . Given the differences in life styles and feeding patterns among the three nematodes , we propose that these 241 intestinal protein families represent an ancestral intestinal transcriptome involved in core cellular and physiological intestinal functions common to the investigated species or even across the Nematoda . Therefore , we referred to them as the “core” IntFam-241 group . The 9 , 918 translated intestinal genes sampled from the three nematodes were annotated and classified using Gene Ontology [34] , [45] . Ontologies were assigned at a higher ratio ( 58% ) to the C . elegans intestinal genes than to those from A . suum ( 31% ) or H . contortus ( 35%; Table 1 ) . In addition , genes in the multi-species IntFam groups , which contained members from at least two nematodes , were annotated at higher ratios ( 47%–74% ) , whereas only 8% of the genes were annotated from the two single-species IntFam groups containing members only from A . suum or H . contortus ( data not shown ) . These data may indicate that novel intestinal genes have independently evolved in relation to the different lineages of parasitism . Complete GO mappings for the three intestinal transcriptomes are presented in the searchable AmiGO browser at www . nematode . net [46] . Furthermore , A hypergeometric test was implemented to identify ontologies that are statistically enriched , thus indicating enriched features , in the core IntFam-241 ( Table 2 ) as well as other IntFam groups ( Text S1; Table S6 ) . Five of the 17 enriched Molecular Function ontologies in IntFam-241 are related to protein kinases ( Table 2; Table 3 ) . Protein kinases are one of the largest and most influential protein families , accounting for about 2% of genes in a variety of eukaryotic genomes including C . elegans and B . malayi . They regulate almost every aspect of cellular activities and may phosphorylate up to 30% of entire proteomes [39] , [47] . Based on GO annotations , protein kinases were enriched by ∼3 . 5 fold in IntFam-241 over the complete set of intestinal genes ( 5 . 3% vs . 1 . 5% of the total genes for each group ) . Both serine/threonine- and tyrosine-types of protein kinases were found to be enriched . Novel protein kinases from the parasites were further classified based on their C . elegans homologs ( Table S5 ) . Interestingly , molecular functions such as adenyl nucleotide binding , ATP binding , and GTP binding were also enriched . The involvement of these functions in protein kinase activities further suggested key roles of cellular signaling in the nematode intestine ( Table 2 ) . The other major Molecular Function terms enriched in IntFam-241 were the proteases ( Table 2; Table 3 ) . All but one of the six subtypes of proteases ( glutamic acid-type proteases as the exception ) had been identified in IntFam-241 ( Table 3 ) , suggesting conservation of essential protease functions , such as nutrient digestion and acquisition , among the three species or even across many species of Nematoda . Because each species feeds on distinct food sources , it is possible that related digestive proteases have evolved within each species to adapt for digestion of the different food types . Given the success of parasite control achieved by immunization with H . contortus and hookworm intestinal protease-type antigens [3] , [8] , these proteases may warrant further investigation in A . suum and other parasites . Analysis of the IntFam groups other than IntFam-241 was also conducted . However , in absence of deeper sampling of the intestinal transcriptomes , it is difficult to interpret the results in relation to broadly conserved or lineage- and species-specific characteristics ( Text S1; Table S6 ) . To identify the biological pathways that are active in the nematode intestine , we mapped the 9 , 918 intestinal translated sequences , and for comparison , the complete C . elegans genes ( Wormbase Release v150 ) , to the reference canonical pathways in Kyoto Encyclopedia of Genes and Genomes [35]–[37] ( Table 4 ) . Complete listing of all KEGG mappings including graphical representation is available for navigation at www . nematode . net [46] . The enrichment of specific major KEGG pathways was evident for each intestine by comparisons to the complete KEGG mappings for all C . elegans genes ( Table 4 ) [38] . Carbohydrate metabolism , energy metabolism , and translation were identified as the statistically enriched pathways in all three intestinal transcriptomes ( at the p-value cutoff of 0 . 05 ) . Interestingly , immune system was an enriched KEGG cellular process in the A . suum intestine; this pathway barely missed the cutoff for enrichment in H . contortus ( with a p-value of 9 . 9e−02 ) , but no enrichment was indicated for the C . elegance intestine ( Table 4 ) . The KEGG immune system was built based on studies in mammalian systems . Many of those from the two parasites were mapped to intracellular proteins of immune cells involved in , for example , intracellular signaling or antigen processing ( Table S7; Table S8 ) . Therefore , the potential for their involvement in interactions with the host are not a primary suggestion here , but it cannot be completely excluded either . RNA interference ( RNAi ) has been developed and successfully applied to genome-wide gene silencing to inhibit gene functions in C . elegans [48]–[51] . C . elegans RNAi information can be further extrapolated in understanding functions of orthologous genes in other nematodes , especially in parasitic nematodes where high-throughput screening is not yet practical [52] . For the 3 , 455 IntFam protein families containing C . elegans genes , observed RNAi phenotypes for their C . elegans members ( Wormbase Release v150 ) were extracted and extrapolated to a total of 45% of these IntFams ( Table 5 ) . Protein families from the IntFam-241 were assigned at a higher ratio ( 73% ) than those from other IntFam groups with C . elegans members ( Table 5 ) . Among the IntFams-241 families with RNAi phenotypes assigned , 74% ( 131/176 ) had severe phenotypes including embryonic , larval , or adult lethal , sterile , sterile progeny , and larval or adult growth arrest ( data not shown ) . Since the IntFam-241 families represent proteins conserved in all the three species , these results further support our hypothesis that the core IntFam-241 protein families likely play key roles in the nematode intestine across many species . We have performed large-scale sampling of the transcribed genomes in A . suum and H . contortus from various tissues or developmental stages , accounting for 77% and 63% of total available bases for the two nematodes , respectively . The identification of 9 , 947 A . suum and 5 , 058 H . contortus genes in this study will vastly facilitate the related genome sequencing projects currently underway . The research has produced the largest samplings of the adult intestinal transcriptomes thus far in parasitic nematodes by identifying 3 , 121 A . suum and 1 , 755 H . contortus intestinal genes , making possible the extensive comparative studies with the adult intestinal transcriptome of the free-living C . elegans . We found that , even with the evolutionary distance of an estimated 350 million years separating clades III and V nematodes [41] , both significant conservation and diversification of gene repertories were evident for the intestine . A group of 241 intestinal protein families , each containing members from all three intestines , were further identified . The IntFam-241 group , containing ∼20% of all intestinal genes sampled from the three species , was proposed to represent an ancient intestinal transcriptome responsible for core cellular and physiological intestinal functions that are conserved in the investigated species or many other nematodes . In addition , various aspects of nematode intestinal physiology were revealed by GO and KEGG classifications of the intestinal transcriptomes , and the examination and extrapolation of available RNAi phenotypes from C . elegans . Overall , this study has contributed to a better understanding of nematode biology , providing central information for the development of novel and more effective parasite control strategies . Finally , the use of the C . elegans model to dissect basic parasite biology has been slow to evolve . Results presented here identified numerous specific areas of research where C . elegans might contribute in this way . | Biological properties of the nematode intestine warrant in-depth investigation , the results of which can be utilized in the control of parasitic nematodes that infect humans , livestock , and plants . Both the importance of intestinal antigens from Haemonchus contortus in immunity and the damage to H . contortus intestine by anthelmintic fenbendazole have highlighted the versatility of the intestine as an emerging target . However , biological information regarding fundamental intestinal cell functions and mechanisms is currently limited . Conserved intestinal genes across nematode pathogens could offer molecular targets for broad parasite control . Furthermore , qualitative and quantitative comparisons on intestinal gene expression among species and lineages can identify basic adaptations relative to a critical selective force , the nutrient acquisition . This study begins to identify intestinal cell characteristics that are conserved across representatives of two clades of nematodes ( V and III ) and further clarifies diversities that likely reflect species- or lineage-specific adaptations . Results consistent with functional data on digestive enzymes from H . contortus and RNAi in Caenorhabditis elegans , as examples , support the potential for the comparative genomics approach to produce practical applications . This study provides a platform on which extensive investigation of intestinal genes and a more comprehensive understanding of the Nematoda can be gained . | [
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] | 2008 | Intestinal Transcriptomes of Nematodes: Comparison of the Parasites Ascaris suum and Haemonchus contortus with the Free-living Caenorhabditis elegans |
Complex chromosomal rearrangements ( CCRs ) are rearrangements involving more than two chromosomes or more than two breakpoints . Whole genome sequencing ( WGS ) allows for outstanding high resolution characterization on the nucleotide level in unique sequences of such rearrangements , but problems remain for mapping breakpoints in repetitive regions of the genome , which are known to be prone to rearrangements . Hence , multiple complementary WGS experiments are sometimes needed to solve the structures of CCRs . We have studied three individuals with CCRs: Case 1 and Case 2 presented with de novo karyotypically balanced , complex interchromosomal rearrangements ( 46 , XX , t ( 2;8;15 ) ( q35;q24 . 1;q22 ) and 46 , XY , t ( 1;10;5 ) ( q32;p12;q31 ) ) , and Case 3 presented with a de novo , extremely complex intrachromosomal rearrangement on chromosome 1 . Molecular cytogenetic investigation revealed cryptic deletions in the breakpoints of chromosome 2 and 8 in Case 1 , and on chromosome 10 in Case 2 , explaining their clinical symptoms . In Case 3 , 26 breakpoints were identified using WGS , disrupting five known disease genes . All rearrangements were subsequently analyzed using optical maps , linked-read WGS , and short-read WGS . In conclusion , we present a case series of three unique de novo CCRs where we by combining the results from the different technologies fully solved the structure of each rearrangement . The power in combining short-read WGS with long-molecule sequencing or optical mapping in these unique de novo CCRs in a clinical setting is demonstrated .
Complex chromosomal rearrangements ( CCRs ) are rearrangements involving more than two chromosomes or more than two breakpoints [1 , 2] . Balanced , de novo , CCRs are extremely rare and more than half are associated with an affected phenotype [3] . Traditionally in a diagnostic laboratory , karyotypically detected complex rearrangements are further analyzed using methods such as array comparative genomic hybridization ( aCGH ) and fluorescence in situ hybridization ( FISH ) . Which method to choose and the number of experiments needed to resolve its genomic structure will be dependent on the characteristics of the rearrangements under scrutiny . These characteristics include the number of potential breakpoints , as well as if the rearrangement includes unbalanced events such as cryptic deletions , which are commonly the cause of the affected phenotype [4 , 5] . Additionally , factors such as cost , patient phenotype , time , availability of tests and interests of the lab ( diagnostic versus research ) will all be considered in deciding which methodology to choose . More recently , whole genome sequencing ( WGS ) has been used to study complex rearrangements . Compared to the standard cytogenetic techniques , WGS offers a wide range of advantages , including high resolution and the ability to detect rearrangements of any size and type in a single experiment , providing important clues for the mechanism of formation [6 , 7] . Furthermore , it may be critical for diagnosis of genetic diseases as well as for genotype-phenotype studies . However , solving the structure of a complex rearrangement is costly and time consuming , mainly due to the sheer amount of breakpoints involved , the large variety in sizes and types of structural variants involved , challenges in interpretation , as well as specific limitation detection of the WGS method of choice . Today , Illumina sequencing technology is the most commonly used second-generation WGS technology [8] . Illumina sequencing is cost efficient and provides high quality data with a wide range of library preparation methods available . Each of these library preparation methods produces data at different cost and with different properties . In particular , the long insert-size mate-pair ( MP ) sequencing protocols have been useful for analyzing structural variation , mainly due to its high span coverage which potentially could bridge regions that are hard to map , such as repetitive regions [7 , 9] . On the other hand , the higher coverage PCR-free paired-end ( PE ) libraries enables the use of split reads for exact breakpoint analysis , and highly precise read depth CNV detection [10 , 11] . These different protocols provide a flexible framework for analyzing different types of variations and answering different questions . However , it is not obvious which method to choose when faced with the problem of solving a complex rearrangement . Even though WGS comes with a range of advantages it has important limitations , including low sensitivity in repetitive regions . Furthermore , haplotyping of long genomic segments is still a challenge for WGS even using single-molecule sequencing technologies . Therefore , phasing multiple rearrangement breakpoints that may have been created concomitantly in complex rearrangements including CCRs and chromothripsis events , can be a daunting task . Moreover , the breakpoints found using WGS commonly needs to be validated using orthogonal techniques , most commonly breakpoint PCR and Sanger sequencing . Hence , even when using WGS , the analysis of complex rearrangements is difficult , and a large number of experiments may be needed . Bionano optical mapping is a technology that enables detection of large structural variants across the entire genome , potentially helpful to resolve long haplotypes including detection of genomic variants in cis . In contrast to the short-read WGS methods commonly used today , Bionano optical mapping utilize long DNA molecules ( >100 kb ) . The usage of long input DNA molecules enables the optical maps to span repetitive and poorly mapped regions of the genome . Further , each optical map may span multiple adjacent breakpoints , providing additional information on how the breakpoints relate to each other . Bionano optical maps show great promise for SV detection and phasing [12] , although optical maps are currently limited by lower resolution compared to sequencing technologies and the availability of the technology itself . Linked-read sequencing is a method provided by 10X Genomics , where linked reads are used to detect structural variation and allows for detection of SVs located within repetitive regions using barcoding of long DNA molecules [13 , 14] but it is also limited by availability of a comprehensive software and high false-positive rate . In this study , we have characterized three unique CCRs combining multiple technologies including standard cytogenetic techniques ( aCGH and FISH ) , followed by short- and long-read approaches in addition to Bionano optical mapping . For short-reads , we used two different sequencing protocols , Illumina 30X PCR-free PE WGS and Illumina Nextera MP WGS , whereas linked-read sequencing using 10X Genomics Chromium was used to obtain synthetic long-reads . By combining next generation sequencing and cytogenetic techniques , we were able to fully characterize the molecular structures of each unique CCR . Lastly , we performed a comprehensive comparison of each methodology applied here concerning the resolution and sensitivity to detect SVs of different sizes throughout the genome , which we discuss in detail .
The t ( 2;8;15 ) complex rearrangement was first indicated by regular karyotyping , however , this analysis only showed an abnormal derivative chromosome 15 and the karyotype was reported out as 46 , XX , del ( 15 ) ( q ? 22 ) ( S1 Fig ) . Further delineating of the chromosomes using FISH and aCGH identified the complex rearrangement involving three chromosomes . Deletions in the breakpoints of chromosomes 2 and 8 were detected using FISH and genome-wide aCGH . Of note , although the 14 . 5 Mb deletion at 8q23 is within the resolution of classical chromosome analysis , it was not detected , likely masked by the presence of multiple segments involved in the complex rearrangement ( S1 Fig ) . WGS confirmed the aforementioned chromosomal rearrangements and determined the chromosome 2 deletion sizes to be 2 . 1 Mb and 2 . 3 Mb , respectively . Additionally , a genomic segment of approximately 970 kb , originally located between the two deleted parts from chromosome 2 , was inserted onto chromosome 15 ( fragment D , Fig 1A ) together with a small inverted 12 kb fragment also originating from chromosome 2 ( fragment C , Fig 1A ) . The first deletion of 2 . 1 Mb removed eight protein-coding genes and the second deletion of 2 . 3 Mb deletion removed three protein-coding genes ( Table 1 ) . The deletion on chromosome 8 was determined to be 14 . 5 Mb and removed 49 protein-coding genes ( Table 1 ) . The translocation breakpoint on chromosome 15 was balanced with only loss of three nucleotides ( Fig 1A , S2 Fig ) . Analyzing the breakpoint junctions on the nucleotide level , it was observed that no microhomology was present in any of the breakpoint junctions ( Table 2 , S2 Fig ) . In one breakpoint junction , there was a four-nucleotide indel at the junction , and a three-nucleotide indel 14 nucleotides upstream , none of them present in the dbSNP database , therefore potentially originated concomitantly to the CCR ( S2 Fig ) . According to the WGS results , the molecular karyotype is t ( 2;8;15 ) ( q34;q23 . 3;q21 . 3 ) seq[GRCh37] g . [chr2:pter_cen_209425831::chr15:55083064_qter] g . [chr8:pter_cen_114508085::chr2:214880375_qter] g . [chr15:pter_cen_55083061::chr2: 211567929_211580844inv::chr2:211580785_212551796::chr8:129040005_qter] . No other rare structural events were detected in the microarray or WGS data . A total of eight breakpoints and five breakpoint junctions were identified in Case 1 . Three out of five of the breakpoint junctions were detected using all four WGS technologies . The 12 kb inversion on chromosome 2 ( fragment C , Fig 1A ) was detected using both short-read sequencing ( PCR-free PE and MP ) technologies and the linked-reads technology with the Supernova pipeline , but not by optical mapping likely due to its small size . One of the translocation junctions between chromosome 2 and chromosome 8 was not detected using the linked-reads technology ( Supernova pipeline ) . None of the breakpoint junctions in Case 1 were found with the linked-reads technology using the Long Ranger pipeline . Finally , eight breakpoints were located within repeats elements . Sequence homology was found in two out of these junctions , however only short stretches of microhomology indicate that no fusion repeat elements were formed ( Table 2 , S2 Fig ) . The t ( 1;5;10 ) complex rearrangement was first identified using regular karyotyping and FISH . A large deletion of 14 . 4 Mb located at chromosome 10p12 was not detected by the initial chromosome analysis , possibly due to the fact that multiple chromosomal segments were involved and the deletion was located in the translocation breakpoint region . Instead , the deletion was identified during characterization of the rearrangement using FISH and later confirmed by aCGH . The molecular cytogenetics data was reported previously [15] . WGS confirmed the cytogenetic findings ( Fig 1B ) , including the deletion ( segment F , Fig 1B ) that involved 75 protein-coding genes ( Table 1 ) . A total of five breakpoints and four breakpoint junctions were identified . Breakpoint junctions revealed 0 to 3 nt of microhomology , and no SNVs or insertions neither in the junctions or flanking the junctions ( Table 2 , S2 Fig ) . Three out of five breakpoints of this complex translocation were located within repeat elements , but no evidence for fusion repeats elements were observed in the breakpoint junctions , meaning that the rearrangement produced a truncated repeat ( S2 Fig ) . The breakpoints of the large deletion on chromosome 10 were both located far from any repeat element ( Table 2 ) . Using the WGS data , the molecular karyotype could be determined to be t ( 1;10;5 ) ( q31 . 3;p12 . 31;q23 . 2 ) seq[GRCh37] g . [chr1:pter_cen_196997343::chr5:124956736_qter] g . [chr5:pter_cen_124956731::chr10: 20816168_pter] g . [chr10:qter_cen_20816166::4689760_19120882del::chr1:196997343_qter] . No other rare structural variants were identified in the microarray or WGS data . All breakpoint junctions indicated by the cytogenetic analysis were found by all four WGS technologies applied , although only PCR-free PE WGS and linked-read sequencing resolved the junctions on the nucleotide level . First , karyotyping identified an unusual banding pattern on the short arm of chromosome 1 . Follow-up analysis using FISH and BAC array identified 14 breakpoint junctions including a 0 . 87 Mb deletion located at 1p36 . 2 , which was previously reported in Lindstrand et al . ( 2008 ) [16] . Combined analysis of the four different WGS technologies confirmed those 14 breakpoint junctions and unveiled 12 additional ones ( Table 3 ) originating from chromosomal segments translocated , inverted , and deleted involving both the short and the long arm of chromosome 1 ( Fig 2 ) . Hence , a total of 33 breakpoints and 26 breakpoint junctions were identified after WGS analysis . The WGS analysis identified seven deletions at the rearrangement breakpoints ( Table 1 ) . In the breakpoint junctions , microhomology was found in 10 junctions ( 2–6 nt ) , and one junction contains a 9 bases long non-templated insertion ( Table 3 , S2 Fig ) . Every breakpoint junction contained at least one repeat region , mostly Alu elements ( Table 3 ) . The junctions that do not involve Alu elements are located within a wide range of distinct types of repeat regions . The second major group of repeats were found to be LINE elements ( 11 breakpoints ) , remaining breakpoints seem to be scattered randomly amongst various repeats including simple repeats , LTR elements , and retroviral elements ( Table 3 ) . In total , 26 breakpoint junctions were detected in Case 3 using combined analysis . Two junctions , junction 1 and junction 2 , were not detected using any short-read ( PCR-free PE and MP ) method , likely because they map within low-copy repeats ( LCRs ) in a 125 kb large intron of TTC34 . Breakpoints located within such regions may require longer reads to be correctly mapped , and indeed only optical mapping and linked-read sequencing were able to detect those junctions ( Fig 3 , Table 4 ) . Of note , all 26 breakpoint junctions were detected using the linked-read WGS technology , although we were required to use both the Long Ranger and Supernova pipelines as they complement each other ( Table 4 ) . PCR-free PE WGS detected 24 out of 26 breakpoint junctions , all of them supported by split reads . MP WGS identified 23 out of 26 breakpoint junctions but none of them was solved on the nucleotide level as expected for MP , whereas optical mapping detected 20 out of 26 breakpoint junctions ( Table 4 ) . Comparing the results of the different technologies , it was found that all technologies tested here could detect the majority of the junctions ( Tables 4 and 5 ) . PCR-free PE WGS and the linked-read technology present the highest detection rate: PE detected all but two junctions whereas linked-reads detected all but one junction . Moreover , both PCR-free PE WGS and linked-read WGS share the highest resolution ( 1 bp ) . MP WGS present a resolution of ~400bp with a false-negative rate of 9% ( fails to report three junctions out of the total 35 junctions ) ( Table 5 ) . Lastly , optical mapping has the lowest detection rate since it fails to report seven junctions of the total 35 junctions ( 20% ) and the lowest resolution of ~ 7 kb ( Table 5 ) . The three CCRs presented here were previously identified using chromosome analysis , therefore the WGS analysis was focused on delineating the structure of the derivative chromosomes . However , to simulate a “WGS first” scenario and evaluate the utility of each of the techniques applied here for SV detection without a prior hypothesis , we ran FindSV developed for PCR-free PE and MP WGS data . The FindSV analysis pipeline is described in the WGS methods section . Generated calls were thereafter ranked based on i ) frequency in the SweGen SV database [17] ii ) amount of discordant read pairs and iii ) size in base pairs , or chromosomal position . Based on these filtering criteria , calls pinpointing the breakpoints of the complex rearrangements were ranked high in the generated lists of rare SVs in all cases ( S1 Table ) . The FindSV pipeline generated 162 calls from the PCR-free PE data in Case 3 from which 34 well-supported interchromosomal calls were ranked above the intrachromosomal rearrangement on chromosome 1 ( S1 Table ) . These calls are most likely due to mobile elements [18] , therefore we concluded that filtering of PCR-free PE data needs to be optimized to minimize false-positive interchromosomal calls due to repetitive elements and favor ranking of potentially false-negative intrachromosomal rearrangements . Lastly , not all breakpoint junctions will be detected in the FindSV output data . FindSV may fail to detect breakpoint junctions that are located in repetitive regions as well as regions that are poorly covered due to various technical artifacts or low DNA quality . All highly ranked calls are manually inspected in IGV , which allows for detection of additional breakpoints and small aberrations in the junctions . Analysis of the linked-read and optical map pipelines in a simulated “WGS-first” scenario was more challenging because both technologies produce a large number of calls ( >1000 ) . In addition , there is still a lack of frequency databases for these technologies , which makes filtering and ranking based on public database frequencies currently not possible . We concluded that using optical maps or linked-read sequencing technologies as the initial screening techniques for CCRs might not be feasible with current pipelines until more frequency data is available . Furthermore , optical mapping has a lower resolution limitation which hamper its use to detect smaller and more complex junctions , including the small 12 kb inversion of fragment C in Case 1 ( Fig 3A–3C ) . Importantly , however , we found that optical mapping and linked-read WGS performs better than short-read WGS in highly repetitive regions or paralogous regions ( such as Junctions 1 and 2 , Case 3 , located within LCRs ) . Example of the junctions that could not be detected using any of the short-read WGS protocols is shown in Fig 3D and Fig 3E . To compare the utility of all WGS technologies used here , we also performed a comparison of all polymorphic CNVs present in the three probands of the present study detected with aCGH ( S2 Table ) . In brief , CNV calls obtained by WGS were compared to those from two different aCGH platforms: a custom designed 400K genome-wide array with ~ 2000 targeted high-resolution genes and a commercial 1M medical exon array from OGT with ~5000 targeted high-resolution genes . The array calls that were also found with at least one of the WGS methods were considered confirmed and hence more likely to be true variants . The sensitivity for detecting such confirmed CNVs in each case and method is given in S2 Table and the summarized sensitivity per method in Table 6 . The data suggest that PCR-free PE WGS has a high detection rate on both small ( <0 . 1 kb ) and large ( >10 kb ) SVs and offers the highest overall detection rate . Linked-read WGS technology has the second highest detection rate , mainly because of the Supernova pipeline and the Long Ranger small deletions algorithm . Lastly , MP WGS and optical mapping perform similarly: both technologies perform well on the larger CNVs , but fail to detect a large number of smaller variants ( S2 Table ) . Overall , it is clear that the high resolution of linked-reads WGS and PCR-free PE WGS allows for the detection of small SVs that MP WGS and optical mapping does not have the required resolution to detect . Next , we used the Case 2 WGS data , which were of the best quality among the three cases , to compare the sensitivity , number of calls , reported variant sizes , and reported variant types of the WGS technologies to one-another . Using SVDB ( https://github . com/J35P312/SVDB ) [10] , we found that the technologies differ regarding the overlap of the variant calls ( Table 7 ) . In particular , optical mapping and the Illumina based technologies detect only a few hundred ( 6 . 6% ) overlapping calls ( Table 7 ) . In contrast , MP and PCR-free PE WGS produce the largest fraction of overlapping calls with nearly 71% of the MP calls also detected by PCR-free PE WGS . The overlap between PCR-free PE WGS and linked-reads WGS was 42% ( Table 7 ) . There are many reasons why the number of overlapping calls may differ between technologies , including artifacts and features specific to each technology and calling pipeline that can influence the output ( S2 Table ) . Bias towards variant types is also observed , for instance optical maps report no duplications and 6733 insertions while PE WGS reports 454 duplications but no insertions ( Table 8 ) . Moreover , due to distinct resolutions , each methodology also present a bias towards the SV sizes it detects . For instance , linked-read WGS and PCR-free PE WGS produce a large fraction of small variant calls ( <0 . 1 kb ) , in contrast , most of the optical mapping and MP calls are sized 10–100 kb ( Fig 4 ) . Next , we compared the overlap between the variant calls and three public datasets: the Database of Genomic Variants ( DGV ) [19] , the HG002 integrated call-set compiled by the Genome In A Bottle consortium ( GIAB ) ( https://github . com/genome-in-a-bottle ) [20] , as well as the CNV list published in a previous paper by Conrad et al . ( 2010 ) [21] ( Table 9 ) . Notably , the performance between the technologies differs depending on which database they are compared to . Comparing the methodologies used here to the CNVs reported in Conrad et al . [21] , we found that Bionano and PCR-free PE WGS achieve similar numbers . MP WGS reports fewer variants matching the CNVs listed in Conrad et al , and linked-read WGS detect only a few of those CNVs ( Table 9 ) . In contrast , linked-read WGS performs better on the DGV database , and detects the largest number of deletions ( Table 9 ) . Despite the high detection rate of these deletions , linked-read WGS performs relatively poor on all other variant types . Similar to the comparison of the Conrad et al . dataset [21] , optical maps and PCR-free PE WGS report nearly the same amount of variants . However , optical maps detect a greater number of insertions , while PCR-free PE WGS detects a greater number of deletions . MP WGS detects the smallest number of variants , however , most of the MP variant calls do match a DGV variant indicating that the precision of MP WGS is high . In contrast , a smaller fraction of optical maps and Long Ranger calls are similar to DGV variants , indicating a lower precision of these technologies . Lastly , the technologies were compared to the HG002 ( GIAB ) integrated call-set , which consists of mainly small SVs ( <0 . 1 kb ) ( S4 Table ) detected using both long- and short-read sequencing methods ( ftp://ftp-trace . ncbi . nlm . nih . gov/giab/ftp/data/AshkenazimTrio/analysis/NIST_SVs_Integration_v0 . 6/README_SV_v0 . 6 . txt ) . It was found that linked-read WGS and PCR-free PE WGS detect the largest number of those variants , while optical maps and MP WGS produce relatively few hits . These results are not surprising given the resolution and size bias that those methodologies present ( Table 5 ) .
It has previously been demonstrated that cytogenetically balanced CCRs often contain cryptic deletions in the breakpoints , explaining the clinical phenotype of the carrier patients in many cases [5] . Here we report two individuals with complex interchromosomal translocations ( Case 1 and Case 2 ) and one individual with a very complex intrachromosomal rearrangement ( Case 3 ) for whom we used multiple combined cytogenetic and molecular methodologies to refine the alterations in their genome content which aided clinical assessment . In Case 1 , a 5 Mb deletion on chromosome 2 and a 14 . 5 Mb deletion on chromosome 8 was identified using FISH mapping . Further analysis using aCGH and WGS , showed that the 5 Mb deletion actually consisted of two deletions , 2 . 1 Mb and 2 . 3 Mb , respectively . WGS could also demonstrate that a 970 kb genomic segment , originally located in-between the two deletions , had been translocated onto chromosome 15 . The deletion on chromosome 8 in Case 1 covers the TRPSII ( Trichorhinophalangeal syndrome type II , TRPSII , Langer Gideon Syndrome , LGS , MIM# 150230 ) locus and most of the characteristic symptoms of TRPSII were present in Case 1 . In Case 2 , the translocated piece of chromosome 10 contained a 14 . 5 Mb deletion , encompassing the typical region for hypoparathyroidism , sensorineural deafness , and renal disease syndrome ( HDRS , Barakat syndrome , MIM# 146255 ) , characterized by the triad hypoparathyroidism , renal dysplasia and hearing loss . The common cause of HDR syndrome is mutations in GATA3 [23] . Deletions of 10p are recurrent , and GATA3 has been pinpointed as the causative gene for HDR syndrome seen in 10p deletions . The size and location of 10p deletions vary , as well as the clinical picture [15] . Case 3 presented with mild dysmorphic features , psychomotor delay , ectopic left kidney , minor hearing disability , dysphasia , feeding difficulties , mild short stature , and developmental delay . Thorough analysis of the 26 breakpoints on chromosome 1 revealed six known disease-related OMIM disease genes to be disrupted or affected by a deletion ( RYR2 , MFN2 , CAMTA1 , SLC9A1 , PRDM16 and PLOD1 ) . Two of the OMIM genes ( CAMTA1 and PRDM16 ) were identified using FISH in a previous study of the same case , while remaining genes were novel findings using WGS [16] . CAMTA1 is known to cause autosomal dominant cerebellar ataxia ( non-progressive ) with mild intellectual disability ( MIM# 614756 ) , with phenotypes including delayed psychomotor development , cerebellar ataxia , intellectual disability , neonatal hypotonia , and variable dysmorphic features , some of them consistent with the phenotype in Case 3 . Remaining genes have not been associated with any phenotypes present in Case 3 and the full phenotype ( ectopic kidney , hearing disability ) in this individual could not fully be explained by the WGS analysis , but needs further investigation of the genes affected by the rearrangement . There are a number of events that could lead to complex rearrangements , including replication-based mechanisms ( fork-stalling and template-switching ( FoSTeS ) model ) [24] and chromothripsis [25] . WGS allowed for detailed analysis of all breakpoint junctions on the nucleotide level in Case 1 and Case 2 , and all junctions but one in the two complex translocations were blunt , with maximum of two nucleotides microhomology . In one breakpoint junction , there were two small insertions of three and four nucleotides , respectively . They did not seem to be duplicated or templated from nearby sequences , which would have indicated a replicative error mechanism , instead the mutational signatures in both Case 1 and Case 2 indicate non-homologous end-joining ( NHEJ ) , characteristic of chromothripsis rearrangement junctions . The characteristics of the breakpoint junctions in Case 3 makes it likely that the complex rearrangement of chromosome 1 formed through a single catastrophic event . These characteristics include randomness of the DNA fragment joins , the DNA fragments appear to be randomly joined in inverted/non-inverted orientation , and the ability to walk along the derivative chromosome [26] . However , the rearrangement does not include a regularity of oscillating copy-number states [26] , but only involves a small number of randomly spread deletions . In addition , the breakpoints of the q and p arms are separated by a 171 Mb DNA fragment , hence the breakpoints are not clustered as would be expected [26] . Further , the p and q arms of chromosome 1 seem to have been brought close together in a ring-like formation . Possibly , the p and q arm were brought together before the scattering of the chromosome , otherwise , the fragments of one arm would either be lost , or the fragments would be less prone to translocate between the arms . Hence , the rearrangement of chromosome 1 in Case 3 could have arisen either from the halted formation of a ring chromosome , or even through a chromothripsis event of a ring chromosome . The four WGS technologies performed were utilized in three different settings: i ) for solving the derivative chromosome structure of the three CCRs , ii ) for a comparison of detection rate of polymorphic CNVs first detected by aCGH in the three cases and iii ) for a general assessment of genome wide SV calls from the three cases as compared to calls present in the public datasets [19 , 21 , 22] . First , we found that no technology provides a significantly higher detection rate than the others regarding the ability to detect and solve the structure of the de novo CCRs presented herein . This is partly a result of the relatively small number of CCR junctions in this study ( 35 ) , but also due to the high detection rate of all four WGS technologies . Moreover , although those CCRs are complex in nature , the majority of the breakpoint junctions could be uniquely mapped to regions that do not present complex repetitive genomic patterns such as LCRs , satellites , centromeric or telomeric repeats . This is exemplified for LCR-containing junctions 1 and 2 of Case 3 that could only be resolved by optical mapping and linked-read WGS . Those technologies require longer DNA molecules and therefore are more appropriated to resolve repeats than short reads . These regions are also more prone to rearrangements [27] , and hence the ability to resolve junctions mapping within those structures is of great importance . Although the detection rates of these methods are similar , the resolutions differ . Both PCR-free PE WGS and linked-read sequencing can resolve most breakpoints to base-pair resolution . In contrast , optical mapping provides the lowest resolution ( ~7 kb ) , which likely explains why it failed to detect breakpoints involving the smaller fragments of Case 1 and Case 3 . To assess whether the CCRs would have been detected in a “WGS-first” scenario the MP and PE WGS data was filtered based on allele frequency , but filtered variant lists of the linked-read and optical mapping calls could not be obtained as there are no frequency databases available for those technologies . Given the low similarity between PCR-free PE WGS and these two methods , the databases such as the SweGen cohort [17] or 1000 Genomes [28] would be of very limited use . Hence , in order to make linked-read WGS and optical mapping usable in a clinical setting , large populations would need to be sequenced using these methods , and the data made available through frequency databases . Finally , comparing the four technologies to polymorphic CNVs , it was found that PCR-free PE WGS provides the highest sensitivity , closely followed by linked-read sequencing . MP WGS and optical mapping performed similarly , with almost half the detection rate of PCR-free PE WGS . Notably , both these two methods failed in detecting smaller CNVs ( <10 kb ) . Furthermore , PCR-free PE WGS did find a significant number of CNVs that linked-read WGS failed to detect: these CNVs did either belong to Case 1 , having partially degraded DNA and too short molecules to be sequenced by linked-read WGS , or the CNVs were subsequently detected using CNVnator instead , which is a read-depth caller and able to detect variants as small as 2 kb [29] . These variants exemplify that the large amounts of high performing Illumina WGS callers provide an edge over these more recent methods , whose pipelines are less mature , and still undergoing rapid development . Lastly , the technologies were evaluated through a general assesment of the calls , as well as through comparison to the DGV [19] , HG002 integrated call-set [22] and Conrad et al . ( 2010 ) datasets [21] . This comparison provided two valuable insights not found through the previous analyses . First , the technologies produce significantly different amounts of variants: both linked-read WGS and optical mapping produce more than 10 , 000 calls on a single individual . In contrast , MP WGS generates less than 1000 calls on a single individual . Given the relatively similar detection rate on the CCRs and the fact that nearly all MP WGS calls are found in DGV , the precision of MP WGS is likely to be high compared to optical mapping and linked-read WGS . Second , it is also clear that each method report variants of different types and sizes and that the reported variant not always reflect the nature of the rearrangement in a given sample . This is particularly observed in the optical mapping results , which do not report any duplications at all , even though optical mapping clearly do detect duplications . Similarly , the Long Ranger SV caller for the linked-read WGS data is the only caller to report variants of “unknown” type . In aggregate , through this comparison , we found that the four WGS methods produce variants of different sizes and these findings are in accordance to the resolution estimates . Notably , there is only a small overlap between the technologies and we only present two orthogonal methods ( Bionano optical mapping and Illumina sequencing ) . Furthermore , many of the calls presented in DGV [19] or Conrad et al . [21] as structural variation are not validated . The results are nevertheless consistent with the previously shown results: MP WGS provides lower detection rate , but higher precision . PCR-free PE WGS , linked-read WGS , and optical mapping detect similar numbers of variants overall , however there are some biases toward certain variant types ( for example , optical mapping reports a large number of insertions ) . Given the similar numbers of detected variants but different numbers of reported variants , the precision of the technologies are likely to be different: we observed that MP WGS provides the highest precision , PCR-free PE WGS the second most precision , and linked-read WGS or optical mapping the worst precision , depending on how the pipelines are combined . The present study is limited by the fact that we have not compared any third-generation sequencing technologies , which have shown great promise for detection of structural variation in complex repeat regions [30 , 31] . However , we found that short-read WGS combined with optical mapping is a powerful combination for analyzing CCRs . Combined , these two technologies would enable detection and validation of most breakpoints in two experiments , at maximum resolution . Given the current high cost of single molecule sequencing , a combined approach could be the most cost efficient . In this study we were able to detect all breakpoint junctions except one using linked-read WGS . The only missing breakpoint junction was in Case 1 , where the DNA was partly degraded and no replacement DNA was available due to the individual being deceased . Taken together , and looking at the linked-read WGS result for Case 2 and Case 3 , we are confident that the missing junction would have been found with better quality input DNA . Hence , in the cases reported here the linked-read sequencing identified all rearrangement breakpoints including those located in repetitive regions and is a valid WGS method of choice to detail complex rearrangements that often have breakpoints in repetitive regions . However , before this can be used in a clinical setting more user-friendly analysis software , as well as better reference data for filtering is desirable . In conclusion , these findings demonstrate how different high throughput genomic methods can add clinically relevant information to conventional molecular analysis methods and enable characterization of the true nature of de novo CCRs . Finally , Case 3 demonstrates the need for long-molecule sequencing or complementary optical mapping to short-read sequencing to be able to map the structure of a highly complex rearrangement with breakpoints in repetitive regions .
Written informed consent was obtained from the legal guardians of all study participants . The local ethical boards in Stockholm , Sweden approved the study ( approval numbers 2012/2106-31/4 and KS 02–145 , 20020506 ) . Three cases were studied following referral to the Clinical Genetics at Karolinska University Hospital , Stockholm , Sweden , due to clinical symptoms indicating genetic testing . Parental samples from Case 2 and Case 3 were analyzed using karyotyping and FISH , showing that the rearrangement had occurred de novo on the paternal allele [15 , 16] . Parental samples from Case 1 and Case 3 were sequenced using linked-read sequencing , and the rearrangement in Case 1 was also originating from the paternal allele . Clinical parameters and phenotypes of the included cases are presented in Table 10 . All three cases were analyzed using a custom designed 400K aCGH [35] and a 1M medical research exome array provided by Oxford Gene Technologies ( OGT ) ( Begbroke , Oxfordshire , UK ) ( Catalog no . 020100 ) with exon-resolution in all known medically relevant genes ( https://www . ogt . com/products/971_cytosure_medical_research_exome_array ) . The resulting CNVs were converted into VCF files using the array2vcf script ( https://github . com/J35P312/convert2vcf ) . The variant calls produced by the WGS technologies or optical mapping were compared to these CNVs using SVDB merge V1 . 1 . 2 . A CNV found by both aCGH and any other technology was considered to be a true positive , all other CNVs were assumed to be false positives . The variant calls of Case 2 were compared to the DGV , an integrated SV call-set produced by GIAB ( ftp://ftp-trace . ncbi . nlm . nih . gov/giab/ftp/data/AshkenazimTrio/analysis/NIST_SVs_Integration_v0 . 6/README_SV_v0 . 6 . txt ) , and the list of CNVs presented in Conrad et al . from 2010 [21] . The DGV and the Conrad et al . datasets were converted into VCF files using the DGV2vcf and Conrad2vcf scripts ( https://github . com/J35P312/convert2vcf ) . The resulting VCF files were then split into one VCF per variant type , and compared to the WGS technologies using SVDB merge V1 . 2 . 2 , which was run using the compare_conrad and compare_dgv scripts ( https://github . com/J35P312/convert2vcf ) . The integrated GIAB call-set was downloaded from the GIAB FTP ( ftp://ftp-trace . ncbi . nlm . nih . gov/giab/ftp/data/AshkenazimTrio/analysis/NIST_SVs_Integration_v0 . 6/HG002_SVs_Tier1_v0 . 6 . vcf . gz ) , and filtered for variants detected using PacBio or Complete Genomics data . The filtering was performed by reading the CGcalls and PBcalls entries in the info column of the VCF file , and any variant having non-zero CGcalls or PBcalls value were kept while all other variants were filtered out . Finally , the four technologies were compared by computing intersect of each pairwise technology-combination , and by calculating the sizes and number of variant calls . These scripts are also made available through ( https://github . com/J35P312/convert2vcf ) . Across all comparisons , two variants were considered similar if their overlap exceed a Jaccard index of 0 . 4 , and if the distance between the breakpoints is less than 100 kb . Any variants not meeting these criteria were considered dis-similar . Throughout these comparisons , only high quality calls were considered: a call was considered to be of high quality if the VCF filter flag was set to PASS . Primers flanking all junctions except junction 1 and junction 2 in Case 3 were designed approximately 500 base pairs away from the estimated breakpoints . Same primers were subsequently used for sequencing using the Sanger method . Primer sequences are available on request . Breakpoint PCR was performed by standard methods using Phusion High-Fidelity DNA Polymerase ( ThermoFisher Scientific , Waltham , MA , USA ) . Sequences obtained were aligned using BLAT ( UCSC Genome Browser ) [34] and visualized in CodonCode Aligner ( CodonCode Corp . , Dedham , MA , USA ) . | Unexpected complexities are common findings in the breakpoints of karyotypically balanced complex chromosomal rearrangements ( CCRs ) . Such findings are of clinical importance , as they may be the cause of mendelian phenotypes in the rearrangement carrier . Whole genome sequencing ( WGS ) allows for high resolution characterization of CCRs , but problems remain for mapping breakpoints located in repetitive regions of the genome , which are known to be prone to rearrangements . In our study , we use multiple complementary WGS experiments to solve the structures of three CCRs originally identified by karyotyping . In all cases , the genomic structure of the derivative chromosomes was resolved and a molecular genetic explanation of the clinical symptoms of the patients was obtained . Furthermore , we compare the performance , sensitivity and resolution of four different WGS techniques for solving these CCRs in a clinical diagnostic laboratory set . | [
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] | 2019 | Comprehensive structural variation genome map of individuals carrying complex chromosomal rearrangements |
A wide variety of biochemical , physiological , and molecular processes are known to have daily rhythms driven by an endogenous circadian clock . While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock , the links between this clock and dependent processes have remained elusive . To address this gap in our knowledge , we have used RNA sequencing ( RNA–seq ) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland . In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue , this approach has revealed novel potential elements of the circadian timing system . We have implicated one rhythmically expressed gene , camk1gb , in connecting the clock with downstream physiology of the pineal gland . Remarkably , knockdown of camk1gb disrupts locomotor activity in the whole larva , even though it is predominantly expressed within the pineal gland . Therefore , it appears that camk1gb plays a role in linking the pineal master clock with the periphery .
All organisms demonstrate a wide variety of physiological , biochemical and behavioral daily rhythms that are driven by intrinsic oscillators , known as circadian clocks . These oscillators work in harmony with the 24 hours periodic changes in environmental conditions . The maintenance and synchronization of the circadian oscillator constitute an adaptive advantage that is evident from the high evolutionary conservation of the circadian system [1] . The current dogma regarding the mechanism of a circadian oscillator is based on positive and negative transcriptional-translational feedback loops with a time period of ∼24 hours . According to this model , in vertebrates , a positive transcription complex , the CLOCK:BMAL heterodimer , activates transcription of the negative clock components , period ( per ) and cryptochrome ( cry ) genes , by binding to E-box elements in their promoters . Negative feedback is achieved by PER:CRY heterodimers that enter the nucleus and suppress their own transcription by physically associating with the CLOCK:BMAL heterodimers , thus closing the feedback loop . Transduction of circadian information from this core oscillator is accomplished by the rhythmic activation of clock-controlled output genes , which in turn regulate downstream processes [2] . Several output genes contribute to the accuracy and stability of the oscillator . These encode transcriptional regulators , which constitute accessory loops that feedback to the core loops , or post-translational modifiers of core clock proteins . This system regulates diverse biochemical pathways which are thought to ultimately lead to the wide variety of physiological and behavioral daily rhythms [2] . In recent years , several factors which receive information from the core clock and schedule various output pathways have been revealed [3]–[5] . It is likely that these factors do not account for all core clock regulated processes . Accordingly , the quest for additional mediators is ongoing . As is the case for many other non-mammalian vertebrates , the zebrafish pineal gland is considered to function as a master circadian clock organ; it is photoreceptive and houses a self-sustained autonomous clock that drives the daily rhythm in the synthesis of melatonin , an important endocrine element of the vertebrate circadian system [6] , [7] . In addition to this hormonal output , neurons of the pineal gland project to brain targets [8] . Through these neuronal and hormonal signals , the pineal gland is thought to convey information regarding the circadian cycle to physiological and behavioral processes [9] . Therefore , this tissue has been extensively studied with the intention of elucidating the molecular components of the core clock [10]–[13] . However , the exact pathways which link the core molecular oscillator within the pineal gland to rhythmic physiological and behavioral processes of the entire organism remain largely unknown . DNA microarray technology is a powerful tool , extensively used to identify circadian changes in the abundance of transcripts ( i . e . , circadian genes ) throughout the animal kingdom . Using this approach in various tissues including the pineal gland , it has been demonstrated that the circadian clock controls groups of genes linked to a large number of molecular and cellular functions [14]–[17] . Surprisingly , different studies show only a moderate level of overlap among the genes identified as circadian in the same tissue from different species and sometimes even in the same species [18] , [19] . These discrepancies could be explained by true biological differences or by the use of different experimental procedures and data analysis methods [18] . However , these discrepancies can also be partially attributed to the inherent limitations of DNA microarray technology , for example cross-hybridization of probe sets [20] . Improvement of circadian profiling is now feasible using next-generation sequencing technology to perform RNA-seq . This method is superior because it provides an unbiased measurement of the entire transcriptome without being restricted to only a subset of genes interrogated by the probe sets on a microarray chip [21] . However , methods to minimize errors and biases generated by RNA-seq are still being developed [22] . Here , we have systematically identified circadian genes in the zebrafish pineal gland , employing both DNA microarrays and RNA-seq; these findings were subsequently confirmed using independent quantitative assays . As described below , this strategy has resulted in the identification of a new element in the circadian timing system that possibly links the core clock with rhythmic locomotor activity in the zebrafish .
Aiming at identifying circadian genes , we extracted RNA through two daily cycles from pineal glands of zebrafish previously adapted to 24 hours light dark cycles and then transferred to constant darkness during sampling ( Figure 1 and Methods ) . This procedure was repeated twice with different sets of fish . The mRNA from the first experiment was quantified using Affymetrix DNA microarrays whereas the mRNA from the second experiment was quantified using RNA-seq ( Methods ) . The data obtained from the DNA microarrays and RNA-seq analysis was subjected to Fourier analysis ( Methods and Levy et al . [23] ) . Demanding 90% true-positives rate , the DNA microarrays and RNA-seq analysis resulted in 112 circadian probe-sets and 309 circadian genes , respectively ( Tables S1 and S2 ) . Altogether , 82 out of the 112 probe-sets identified by the DNA microarray method reliably represent zebrafish mRNAs from GenBank , 66 of which are well-annotated NCBI mRNA reference sequences collection ( RefSeq ) genes ( Table S1 ) . In the analysis of the RNA-seq , only sequencing reads that were aligned to genomic locations of RefSeq genes were used ( Methods ) . The larger number of circadian genes identified using RNA-seq is due in part to the greater number of genes measured; only about half of the RefSeq genes are represented on the DNA microarray ( 7634 out of 14263 RefSeq genes ) . In addition , RNA-seq has a higher detection power due to better accuracy in expression measurement [24]: out of the 309 RefSeq genes identified using RNA-seq , 180 were represented on the DNA microarray but only 30 of them ( 17% ) were identified as circadian . In contrast , about half ( 30 out of 66 ) of the RefSeq genes detected as circadian using DNA microarrays were also identified as being circadian using RNA-seq , demonstrating better accuracy of the RNA-seq and overall reasonable agreement between the two methods . Notably , the 309 circadian genes are enriched with pineal-enhanced genes , i . e . genes with higher expression in the pineal gland compared to other tissues ( 3 out of the 29 pineal-enhanced genes identified in [25] , P-value<0 . 05 , binomial cumulative distribution ) and with genes which were previously reported to have notable expression in the pineal gland ( 28 out of the 485 genes mentioned in the ZFIN database [26] , P-value<10−3 , binomial cumulative distribution ) . Nearly all ( 15 out of 16 ) of the known zebrafish core clock genes were identified as circadian in the RNA-seq analysis ( Figure 2 and Table S3 ) . The only exception was per2 which is known to be light-induced in the pineal gland and not circadian under constant darkness [27] . Notably , the RNA-seq analysis is in agreement with the reported phases of 14 core clock genes ( Table S3 ) . Similarly , most of the genes ( 12 out of 14 ) that are considered to form accessory loops of the molecular circadian oscillator were identified as circadian and their phases are in agreement with previous experimental data ( Table S4 ) . In accordance , functional annotation analysis using DAVID [28] reveals the pathway ‘Circadian rhythms’ as significantly enriched ( Benjamini-Hochberg adjusted P-value<1e-17 ) within the identified circadian genes ( Table S5 and Methods ) . As only a portion of the zebrafish genes are represented on the Affymetrix DNA microarray it is reasonable that the list of circadian genes revealed by RNA-seq is larger . Nevertheless , 8 known clock genes ( 3 core clock and 5 accessory loops-related ) are included within the 82 circadian genes identified in the DNA microarray experiment ( Tables S3 and S4 ) , thereby providing evidence that other results generated by this analysis are reliable . Importantly , the extensively studied pineal gland clock-controlled gene , aanat2 ( arylalkylamine N-acetyltransferase ) [29] , was identified using both the DNA microarray and RNA-seq analyses . Accordingly , it is clear that these methods provide an informative view of circadian changes in the abundance of transcripts in the zebrafish pineal gland . We aimed to characterize new regulators of the pineal master clock or new mediators relaying circadian information to downstream processes . The genes detected using RNA-seq and DNA microarray analysis can serve as a basis for this quest . Thirty genes that were identified using both these two independent methods were considered for further functional analysis ( Table 1 ) . Notably , about one third of these genes ( 9 out of 30 ) were previously reported as core clock or clock-controlled genes ( Table 1 ) . In addition , qRT-PCR and quantitative whole mount in situ hybridization ( ISH ) were performed on selected genes as a validation procedure ( Methods ) ; as expected , nearly all ( 8 out of 9 ) of the tested genes were indeed validated as circadian , showing similar phases to those identified by the DNA microarray and RNA-seq data ( Figure S1 , Table 1 and Methods ) . The use of the two independent genome-wide methods , the re-discovery of previously reported core clock genes and the validation procedure , confirmed that the concise list ( Table 1 ) represent bona fide circadian genes . For further functional analysis we focused on genes that were not previously connected to the core clock or the core clock accessory loops ( Tables S3 and S4 ) . Studying pineal-enhanced genes can aid in elucidating the role of the master clock in coordinating downstream circadian rhythms . We thus selected genes from the concise list based on their expression pattern , focusing only on those showing enhanced expression in the pineal gland . This was determined using whole mount ISH in larvae ( Table 1 and Figure S1 ) . Of the previously unreported genes from the concise list , 5 fulfilled the above requirements: camk1gb , guk1b , arg2 , bmper and ndrg1b ( Table 1 ) . Of these , we chose to focus on camk1gb ( calcium/calmodulin-dependent protein kinase IGb ) . The mammalian Camk1g is a member of a larger family of calcium/calmodulin-dependent protein kinases , the CaMKI family . Like other members of this family , Camk1g requires both calcium/calmodulin and phosphorylation by CaMKK for its full activation [30] . camk1gb is one of two zebrafish homologs of the mammalian Camk1g . Our pineal gland RNA-seq data shows that the expression levels of the other paralog , camk1ga , are similar to those of camk1gb . Yet , it was not found to be expressed in a circadian manner ( Table S2 ) . Interestingly , like camk1gb , the mammalian Camk1g was reported to exhibit enhanced and circadian expression in the rat pineal gland [16] , [31] , suggesting a conserved role in the pineal clock or in other aspects of pineal function . Whole mount ISH of larvae clearly reveals enhanced pineal expression of camk1gb ( Figure 3a and Figure S2 ) . Importantly , the circadian expression pattern in the pineal gland of larvae , characterized by an expression peak at CT6 , is similar to the profile in the pineal gland of adults: Pearson's correlations of 0 . 89 and 0 . 77 to the DNA microarrays and RNA-seq profiles , respectively ( Figure 3b ) . This similarity may suggest that the temporal profile of camk1gb has a functional significance starting from early life stages until adulthood . We examined the effect of camk1gb on clock-regulated zebrafish behavior . Zebrafish larvae exhibit robust circadian locomotor activity with highest activity during the subjective day [32] , [33] . To determine whether camk1gb is required for normal circadian locomotor activity , embryos were injected with either control morpholino or camk1gb morpholino ( Methods ) . camk1gb morpholino treatment results in the inclusion of an intron within the mRNA coding sequence and the consequent introduction of a premature stop codon ( Figure S3 and Text S1 ) . Strikingly , camk1gb knockdown significantly disrupted the circadian activity pattern ( Figure 4a and Methods ) . This experiment was repeated 4 times using a total of 75 larvae injected with camk1gb morpholino and 75 larvae injected with control morpholino ( Methods ) . The disrupted circadian activity pattern is also evident when analyzing individual larvae using Fourier analysis ( Figure 4b , Figure 4c , Figure 4d and Methods ) . Only 8/75 of the camk1gb knockdown larvae have shown a 24 h-period signal that surpasses the median signal for the 75 control larvae ( Figure 4d and Methods ) . Furthermore , we tracked locomotor activity levels at abrupt light to dark transitions [34]; both control and camk1gb knockdown groups showed similar levels of locomotor activity , indicating that camk1gb knockdown does not impair larval movement abilities ( Figure S4 and Methods ) . Lastly , a rescue experiment , in which camk1gb mRNA was co-injected along with the camk1gb morpholino , restored normal circadian activity thereby demonstrating the specificity of the injected morpholino ( Figure 4a and Methods ) . The success of the rescue experiment is remarkable given that the injected mRNA is likely to restore the levels of camk1gb but less likely to restore its rhythmic expression . Therefore , it seems that sufficient expression levels of camk1gb are necessary for proper circadian rhythms of locomotor activity . Alternatively , it is possible that posttranscriptional regulation may restore the rhythmic expression pattern of the protein , thereby contributing to the success of the rescue experiment . The AANAT enzyme drives the rhythmic production of melatonin [35] . Zebrafish pineal aanat2 transcription exhibits a robust circadian rhythm that begins at 2 days post-fertilization [29] , [36] . The transcription of aanat2 is tightly regulated by the core molecular oscillator [29] as well as other transcription factors [37] . Importantly , camk1gb knockdown significantly reduced ( Student's t-test , Bonferroni corrected P-value<0 . 05 ) the amplitude of the aanat2 expression rhythm by half as detected by whole mount ISH ( Figure 5a , Figure 5b and Methods ) . camk1gb knockdown did not affect normal pineal gland development as indicated by whole mount ISH for otx5 [13] ( Figure 5c ) . These results demonstrate that camk1gb is not necessary for aanat2 to be transcribed but is involved in the physiological regulation of the rhythmic transcription of pineal aanat2 . The disruption of the circadian locomotor activity and the reduction in aanat2 rhythmic expression may suggest that camk1gb is a previously unrecognized regulator of the core clock . This notion is in line with findings showing that distant CaMK family members can modulate core clock genes [38]–[41] . We reasoned that if indeed camk1gb is important for normal core clock function , the effect of its knockdown will be manifested in the expression patterns of additional circadian genes . Hence , we tested the effect of camk1gb knockdown on the expression levels of 3 additional pineal-enhanced clock-controlled genes ( sh3gl2 , opn1lw1 and ndrg1b ) and 2 pineal-enhanced core clock accessory loops genes ( dec1 and dec2 ) using whole mount ISH at the peak and the nadir of their rhythm ( Methods ) . However , the expression pattern of the tested genes was not significantly affected by camk1gb knockdown ( Figure S5 ) . Accordingly , over-expression of camk1gb in the zebrafish cell line , Pac-2 , did not disrupt the core clock as indicated by examining the promoter activity of the core clock marker gene , per1b ( Figure S6 and Methods ) . Thus , camk1gb knockdown affects circadian locomotor activity and aanat2 expression levels without affecting the core clock .
In this study we set out to identify new molecular elements that affect the circadian timing system , either directly through the core clock or indirectly by relaying timing information from the core clock to downstream processes . It was previously demonstrated that identification of circadian genes using DNA microarrays can lead to the discovery of previously unrecognized clock components [42] . We reasoned that by using RNA-seq a significant improvement in the number of the detected circadian genes can be achieved , with better detection reliability . However , this new technique introduces non-trivial problems into data analysis and can also bring about biases in genes' quantification [43]–[46] . These hurdles can be overcome by integrating several experimental procedures and employing rigorous and stringent data analysis . Therefore , circadian genes were systematically identified using two independent high-throughput methods , RNA-seq and DNA microarray analyses , followed by computational analysis and extensive in vivo validations . In this study we focused on the zebrafish pineal gland for two main reasons: 1 ) most of the molecular clock components are likely to be functional in this autonomous clock tissue . Indeed , the expression of nearly all the core clock genes was found to be circadian in this tissue ( Figure 2 ) . 2 ) By focusing on pineal genes the link between a master clock and peripheral tissues may be elucidated . Indeed , we demonstrated that camk1gb , which is pineal-enhanced , controls circadian downstream processes within the pineal gland and in the entire animal ( i . e . locomotor activity ) . The link between camk1gb and circadian locomotor activity is intriguing , especially since camk1gb is a pineal-enhanced gene . Although the possibility that camk1gb affects circadian locomotor activity through its low expression in structures outside the pineal gland cannot be ruled out , the enhanced and circadian expression of this gene in the pineal gland ( Figure 3 ) suggests otherwise . At least two possible mechanisms might explain how camk1gb relays circadian timing information from the pineal gland . One is by regulating melatonin secretion: camk1gb knockdown caused a 50% reduction in the night-time expression of aanat2 , the key enzyme in melatonin production [35] . Melatonin administration has a profound effect on locomotor activity rhythms in many organisms including zebrafish [47] , [48] . Therefore , it is tempting to speculate that disruption of melatonin levels leads to the observed alteration in circadian locomotor activity . However , the role of endogenous melatonin rhythms in the maintenance of normal locomotor activity rhythms in zebrafish is still not fully understood and therefore warrants further investigation . A second possible mechanism is by modulating neuronal innervations between the pineal gland and deeper brain regions [8] . Mammalian Camk1g is known to coordinate neuronal morphogenesis . This CaMKI isoform is a membrane-anchored protein , abundant in neurons , which mediates dendritic and axonal outgrowth of neurons in culture [49] , [50] . In teleost fish , the majority of pineal photoreceptor cells form contacts with postsynaptic neurons which send processes to the brain . A fraction of the pineal photoreceptor cells possess long axons that project directly to the brain [9] . Interestingly , we find that camk1gb is indeed expressed within photoreceptors ( Figure S7 and Methods ) . Taken together , these observations point to the possibility that camk1gb is required for the transmission of circadian timing information from the central clock in the pineal gland to the brain . We provide evidence that camk1gb regulates the transcription of aanat2 . Naturally , understanding this regulatory mechanism is of interest . As is the case for most members of the CaMKI family , it was previously demonstrated in vitro that the transcription factor cAMP-response element-binding protein ( CREB ) is one of the phosphorylation targets of the mammalian homolog , Camk1g [30] . It was also reported that Camk1g regulates CREB-mediated transcription [51] . Interestingly , phosphorylated CREB regulates the transcription of Aanat in mammals [52] , [53] , and possibly in other vertebrates [37] . Therefore , it is possible that camk1gb modulates the levels of phosphorylated CREB which in turn affects the transcription levels of aanat2 . We note however that the camk1gb expression peak is in mid-day whereas aanat2 peaks in mid-night ( Figure 3 and Figure 5 ) . A reasonable explanation may be an expected time lag between camk1gb transcription and the appearance of its translated product . An alternative mechanism involves an indirect effect in which camk1gb regulates the pineal core clock and thereby controls aanat2 transcription . However , we found no evidence that camk1gb knockdown affects the core clock mechanism ( Figures S5 and S6 ) . Nevertheless , based on findings showing that distant members of the CaMK family can modulate core clock genes [38]–[41] , further examination of this possibility in tissues other than the pineal gland is justified . A comprehensive view of the pineal gland circadian transcriptome allows a dissection of functions that are clock-related inside the pineal gland . As expected , genes that belong to the ‘Circadian rhythms’ pathway are significantly enriched within the pineal gland circadian transcriptome ( Table S5 and Methods ) . The ‘Glycolysis’ and ‘Pyruvate metabolism’ processes are also significantly enriched ( Table S5 ) , including 4 circadian enzymes out of the 10 required for glycolysis ( Table S6a ) . Recent studies have revealed a close link between the core clock and metabolism that is mediated by REV-ERB transcription factors [3] , [4] . In particular , rev-erb couples glycolysis/gluconeogenesis with the core clock in the mouse liver [54] . Our findings suggest that links between glycolysis and the core clock are not restricted to the liver but may be present in other tissues . Another interesting function which was found to be enriched is ‘Oxidation reduction’ ( Table S5 ) . Twenty-five circadian genes belong to this pathway including catalase and 7 different cytochromes P-450 ( Table S6b ) . Several genes in the ‘Oxidation reduction’ pathway were shown to be clock-regulated [55] . In zebrafish cells , catalase has been implicated in the light-dependent transcription of clock genes [56] . Our data suggest that the link between this pathway and the clock system may be more general and includes both central and peripheral clock tissues . We have constructed a database that contains many interesting candidates for future investigation in the context of either regulating the core clock or in linking of the core clock to downstream pathways . We have focused on camk1gb and showed that this gene is rhythmically expressed in the pineal gland and affects daily rhythms of behavior . In mammals , several genes which connect the master clock to downstream circadian locomotor activity have been discovered [57] . They all share in common the following characteristics: 1 . Rhythmic expression in the master clock ( which is the suprachiasmatic nucleus in mammals ) . 2 . Alterations in their levels disrupt circadian locomotor activity . Since camk1gb shares these properties and regulates aanat2 , and therefore possibly melatonin , we suggest that this gene serves to connect the master clock with circadian locomotor activity in zebrafish . For over a decade , zebrafish seemed to represent an ideal vertebrate model for the quest to identify and characterize novel clock components [10] . However , with the exception of one study [58] , no novel clock components have been identified to date using this model . Instead , the zebrafish has been used to further characterize clock components that were previously identified in mammals . Here , we have demonstrated that the design and analysis of systematic high-throughput experiments based on zebrafish can lead to the discovery of new clock elements .
All procedures were approved by the Tel Aviv University Animal Care Committee and conducted in accordance with the council for experiments on animal subjects , Ministry of Health , Israel . The experimental procedure for the DNA microarrays experiment was performed as follows . Adult ( 0 . 5–1 . 5 years old ) transgenic zebrafish , Tg ( aanat2:EGFP ) Y8 , which express enhanced green fluorescent protein ( EGFP ) in the pineal gland under the control of the aanat2 regulatory regions , were used [29] . Fish were raised under 12-hr light∶12-hr dark ( LD ) cycles , in a temperature controlled room , and transferred to constant darkness ( DD ) for tissue collection . Fish were anesthetized in 1 . 5 mM Tricane ( Sigma ) , sacrificed by decapitation , and pineal glands were removed under a fluorescent dissecting microscope . Starting from circadian time ( CT ) 14 , pineal glands were collected at 4-hr intervals for 48 hours ( 12 time points identified as CT 14 , 18 , 22 , 2 , 6 , 10 , 14b , 18b , 22b , 2b , 6b and 10b ) . Pools of 12 ( DNA microarray ) or 20 ( RNA-Seq ) pineal glands were prepared at each time-point and total RNA was extracted using the RNeasy Lipid Tissue Mini Kit ( QIAGEN ) , according to the manufacturer's instructions . Labeled RNA preparation and hybridization to DNA microarrays were performed according to the Affymetrix manual with the two-cycle target labeling protocol ( http://www . affymetrix . com/support/downloads/manuals/expression_analysis_technical_manual_ . pdf ) . A total of 12 Affymetrix DNA microarrays were hybridized with RNA-pools of pineal glands from 12 time points throughout two daily cycles . Each DNA microarray was normalized using Affymetrix GeneChip Operating Software ( GCOS ) . The entire DNA microarray dataset , logarithmically transformed , was normalized using quantile normalization to guarantee that the distribution of probe intensities was the same in all the chips [59] . The microarray data was deposited to the Gene Expression Omnibus ( GEO ) , under accession GSE41696 . Illumina TruSeq protocol was used to prepare libraries from RNA samples . Overall , 12 libraries ( 12 time points ) were run on 2 lanes of Illumina HiSeq2000 machine using the multiplexing strategy of the TruSeq protocol ( Institute of Applied Genomics ) . On average , ∼30 million paired-end reads were obtained for each library . The reads were 2×100 base pairs for 8 time points ( CTs 22 , 2 , 6 , 10 , 18b , 2b , 6b , 10b ) and 2×50 base pairs for the remaining time points ( CTs 14 , 18 , 14b and 22b ) . TopHat [60] was used for aligning the reads against the zebrafish genome allowing only uniquely aligned reads and up to two mismatches per read . On average , 56% of the reads had unique alignment to the zebrafish genome . Reads aligned to the protein coding regions of known RefSeq genes were used . A custom script written in Perl was used to parse the output of TopHat , which is given in Sequence Alignment/Map ( SAM ) format ( http://samtools . sourceforge . net/ ) , and to convert it into raw number of reads aligned to each position in each RefSeq gene . The RefSeq genes information was obtained from the Table Browser of the UCSC genome browser ( genome . ucsc . edu/ ) using the zebrafish Jul . 2010 ( Zv9/danRer7 ) assembly . To avoid PCR duplicates , only paired-end reads that have unique start position in the genome in both pairs were used [61] . The quality of the sequencing libraries was assessed as described in Levin et al . [61] and the data was normalized using Quantile normalization ( Text S1 ) . We made sure that the normalization scheme properly corrects for different RNA levels and other technical differences between samples ( Text S1 ) . The sequencing data was deposited to the Sequence Read Archive ( SRA ) , under accession SRA054264 . The time-dependent signal was converted into a frequency-dependent signal using the Fast Fourier Transform ( FFT ) . The extent to which the original signal is circadian was quantified by the ratio ( ‘g-factor’ ) of the power ( squared amplitude ) of the frequency which corresponds to 24 hr period to the sum of powers of all frequencies [23] . The higher the g-factor , the higher is the confidence that the transcript is circadian . We note that changing the definition of the g-factor by adding the powers of higher harmonics of the 24 hr period to the numerator , gave similar results compared to the use of the definition above . To determine the true-positive rate for a list of transcripts constructed using a given g-factor cut-off , permutation analysis was conducted as follows: Finally , using the number of transcripts detected for a given g-factor ( step 5 ) and the number of true-positives for a given g-factor ( step 6 ) , the true-positive rate as a function of the number of transcripts detected was calculated and further used to identify circadian genes with high accuracy ( Figure S8 ) . The procedure described here was implemented using in-house MATLAB ( The Mathworks , Inc . ) script . The 308 circadian RefSeq genes identified using the RNA-seq were analyzed to find over-represented molecular functions ( Table S2 ) , using the DAVID bioinformatics tools [28] and focusing on over-represented gene ontology ( GO ) categories and KEGG pathways [62] , [63] . The DAVID's default zebrafish genes background was used . All the significantly enriched ( Benjamini-Hochberg adjusted P-value<0 . 05 ) GO categories and KEGG pathways are presented in Table S5 . The temporal expression pattern of candidate genes was determined by whole mount ISH in zebrafish larvae or by quantitative RT-PCR in the adult pineal gland as previously described [12] , [27] ( Figure S1 and Text S1 ) . Morpholino experiments were conducted as previously described [11] ( Text S1 ) . Rescue experiments were conducted by co-injection of approximately 2 nl volume of camk1gb morpholino ( 1 mM ) and in vitro transcribed camk1gb mRNA ( 100 ng/µl ) . The camk1gb protein-coding sequence was PCR-amplified with a KAPA HIFI PCR kit ( KAPA Biosystems ) using the same set of primers that was used for ISH experiments . The PCR products were cloned into a pCS2+ vector , linearized with NotI restriction enzyme and transcribed using the SP6 mMessage mMachine kit ( Ambion ) , according to the manufacturer's instructions , to generate capped camk1gb mRNA . Embryos were microinjected with either control morpholino , camk1gb morpholino or co-injected with camk1gb morpholino and in vitro transcribed camk1gb mRNA and kept under LD conditions for 3 days . On the fourth day post-fertilization , embryos were placed in 48-well plates in the observation chamber of the DanioVision Tracking System ( Noldus Information Technology ) and exposed , for acclimation , to two days under 12-hr light ( 3400 lux ) ∶12-hr dim light ( 40 lux ) regime followed by 3 days of constant dim light . Live video tracking and analysis was conducted using the Ethovision 8 . 0 software ( Noldus Information Technology ) . Activity was measured at days 6–8 post fertilization , as the distance moved by a larva in 10 min time bins ( Figure 4 ) . The activity record of each individual was subjected to Fourier analysis , and scored with a g-factor ( see Methods section ‘Fourier analysis’ ) . Significant differences in the g-factor distributions between the control and camk1gb morpholino treated groups ( n = 75; combining 4 different experiments ) as well as between the rescue and camk1gb morpholino treated groups ( n = 16 ) were determined by the Kolmogorov-Smirnov test ( Figure 4b and Figure 4d ) . The percent of larvae which are considered circadian depends on the g-factor used as the detection criteria , but for all values of g-factor cutoff tested ( ranging between 0 . 05 and 0 . 3 , Figure 4d ) , significantly more larvae are considered circadian in the control group . To determine whether camk1gb knockdown impairs larval movement abilities , locomotor activity levels were tracked under abrupt light to dark transitions [34] . On day 6 post fertilization , control morpholino and camk1gb morpholino injected larvae ( n = 24 ) were subjected to 3 dark flashes of 5 sec each during the light phase [34] . Activity was measured as the distance moved by each larva during the dark flash . No statistical difference was observed between the activity of the control morpholino and camk1gb morpholino injected groups ( Student's t-test , P-value>0 . 2; Figure S4 ) , indicating that the camk1gb morpholino does not impair larval movement abilities . Transient co-transfection of the Pac-2 cell line with camk1gb and per1b:luciferase constructs was performed as previously described [64] ( Figure S6 and Text S1 ) . For double fluorescence ISH we followed the protocol of Machluf and Levkowitz [65] ( Text S1 ) . | The circadian clock is a molecular pacemaker that drives rhythmic expression of genes with a ∼24-hour period . As a result , many physiological processes have daily rhythms . Many of the conserved elements that constitute the circadian clock are known , but the links between the clock and dependent processes have remained elusive . With its amenability to genetic manipulations and a variety of genetic tools , the zebrafish has become an attractive vertebrate model for the quest to identify and characterize novel clock components . Here , we take advantage of another attraction of the zebrafish , the fact that its pineal gland is the site of a central clock which directly receives light input and autonomously generates circadian rhythms that affect the physiology of the whole organism . We show that the systematic design and analysis of genome-wide experiments based on the zebrafish pineal gland can lead to the discovery of new clock elements . We have characterized one novel element , camk1gb , and show that this gene , predominantly expressed within the pineal gland and driven by the circadian clock , links circadian clock timing with locomotor activity in zebrafish larvae . | [
"Abstract",
"Introduction",
"Results",
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] | [
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] | 2012 | Systematic Identification of Rhythmic Genes Reveals camk1gb as a New Element in the Circadian Clockwork |
The distribution of trachoma in Nigeria is spatially heterogeneous , with large-scale trends observed across the country and more local variation within areas . Relative contributions of individual and cluster-level risk factors to the geographic distribution of disease remain largely unknown . The primary aim of this analysis is to assess the relationship between climatic factors and trachomatous trichiasis ( TT ) and/or corneal opacity ( CO ) due to trachoma in Nigeria , while accounting for the effects of individual risk factors and spatial correlation . In addition , we explore the relative importance of variation in the risk of trichiasis and/or corneal opacity ( TT/CO ) at different levels . Data from the 2007 National Blindness and Visual Impairment Survey were used for this analysis , which included a nationally representative sample of adults aged 40 years and above . Complete data were available from 304 clusters selected using a multi-stage stratified cluster-random sampling strategy . All participants ( 13 , 543 individuals ) were interviewed and examined by an ophthalmologist for the presence or absence of TT and CO . In addition to field-collected data , remotely sensed climatic data were extracted for each cluster and used to fit Bayesian hierarchical logistic models to disease outcome . The risk of TT/CO was associated with factors at both the individual and cluster levels , with approximately 14% of the total variation attributed to the cluster level . Beyond established individual risk factors ( age , gender and occupation ) , there was strong evidence that environmental/climatic factors at the cluster-level ( lower precipitation , higher land surface temperature , higher mean annual temperature and rural classification ) were also associated with a greater risk of TT/CO . This study establishes the importance of large-scale risk factors in the geographical distribution of TT/CO in Nigeria , supporting anecdotal evidence that environmental conditions are associated with increased risk in this context and highlighting their potential use in improving estimates of disease burden at large scales .
Trachoma is the leading infectious cause of blindness worldwide , most recently estimated to be responsible for the loss of 333 , 000 disability-adjusted life years ( DALYs ) in 2010 [1] . Recurrent episodes of infection with the bacterium Chlamydia trachomatis and associated inflammation cause cumulative scarring of the under surface of the upper eyelid which , in some individuals , eventually leads to trichiasis–a clinical stage of trachoma where the eyelashes turn inwards and touch the eye . Without surgical intervention , this condition can progressively damage the cornea and lead to visual impairment and irreversible blindness later in life [2 , 3] . While a number of studies have identified risk factors that are associated with clustering of trachoma within villages , households [4–7] and individuals [8] , only a few studies have quantified associations at larger scales [9 , 10] . Anecdotally , trachoma is believed to be a greater public health risk in dry , dusty and hot environments , although disease may be present anywhere that overcrowding and poor hygiene allow transmission [11 , 12] . Climatic variables are postulated to indirectly influence the transmission of trachoma through the following mechanisms: low rainfall which leads to reduced access or use of water for washing faces; higher temperatures which may influence the distribution and activity of the vector Musca sorbens; and climatic conditions that favour drying of faeces , the fly’s preferred breeding site [13–17] . In addition , there may be a potential role for ocular dryness or environmental irritants to contribute to progression of chronic disease , by aggravating scarring processes [2 , 18–20] . However , robust studies investigating relationships between detailed epidemiological observations and environmental determinants are scarce . Existing studies provide some support for a role of temperature and rainfall in the distribution of trachoma [9 , 21–23] , as well as altitude ( which might be a proxy for temperature ) [24–26] . However , most studies are limited by lack of control for individual level factors [21 , 25 , 27] , and in particular variation in socioeconomic factors [28] . In practice , it is difficult to disentangle the effects of risk factors of trachoma at different spatial levels , due to a complex interplay between large-scale factors such as climate , and mediating factors at smaller scales , like water availability , sanitation access at the household level and individual behaviours , including household water use and personal hygiene [26 , 29 , 30] . Bayesian hierarchical models ( BHM ) are a robust and well established methodology for modelling data that are naturally grouped , and for identifying risk factors at different scales [31] . This approach can be expanded to incorporate information on residual underlying spatial patterns , thus explicitly addressing any remaining spatial correlation between observations that may affect estimates of standard errors [32] . Previous studies have used this approach to identify risk factors at multiple hierarchical levels for a variety of tropical diseases , using data from school-based and community surveys , including malaria [33] , soil-transmitted helminths[34 , 35] , schistosomiasis [36 , 37] and trachoma [9] . A common application in multilevel models is to then apportion the variance in the response according to the different levels of the data , referred to as the variance partition coefficient ( VPC ) [38 , 39] . These methods offer a robust and flexible approach to modelling prevalence data routinely collected as part of disease control programmes in developing countries . Nigeria is a populous country with over 160 million people , comprising approximately 20% of the total population in Africa [40] . There are diverse climatic conditions across the country , and three broad ecological zones: the southern rainforest zone , the central Guinea Savannah zone and the semi-arid northern Sudan Savannah [41] . Trachoma is a significant public health problem in the north of the country and currently only 43% of districts suspected to be endemic have been surveyed by population based prevalence surveys [42] . The 2007 National Blindness and Visual Impairment Survey was conducted in Nigeria to provide estimates of the prevalence and causes of blindness at the national level in order to inform policy and planning for the elimination of avoidable blindness [43] . During this survey , participants were assessed for the presence of trachomatous trichiasis ( TT ) and corneal opacity attributed to trachoma ( CO ) , providing a unique opportunity to describe the distribution of later stages of trachoma in relation to underlying risk factors in Nigerian adults . This analysis aims to use geostatistical BHMs to quantify the relationship between climatic factors and trachomatous trichiasis or corneal opacity ( TT/CO ) amongst adults in Nigeria , while accounting for the effects of risk factors at other levels and any residual spatial correlation .
Available data included field collected data at the individual level and remotely sensed or interpolated environmental variables at the cluster level . An exploratory principal components analysis was conducted on all climatic variables with a correlation coefficient ≥0 . 70 , in order to explore covariance and variance between factors and ultimately inform dataset reduction and model building . All field collected data were used with a reduced set of environmental covariates to build hierarchical multivariate regression models for the presence or absence of TT or CO ( Table 1 ) . Model-building took a spatially explicit approach and incorporated geostatistical random effects to account for spatially-structured residual clustering . There is no simple way to measure variance partition coefficient ( VPC ) for discrete response multilevel models , as the variance at the two levels are measured on different scales and dependent on individual level predictor variables . We used a simulation approach implemented in R 2 , 10 , 1 to estimate the VPC introduced by Goldstein et al . ( 2002 ) , which approximates the variance at each level from a large number of simulations based on the variance in the second-level random effect , beta values from the non-spatial model and average values for each coefficient [38] . Ethical approval for the Nigerian National Blindness Survey was provided by the London School of Hygiene and Tropical Medicine and the Federal Government of Nigeria . The study adhered to the tenets of the Declaration of Helsinki . Written informed consent was obtained from all participants before they were examined . Eye examination and service facilities ( including aphakic spectacles if required ) were provided to all individuals , regardless of their consent to participate in the study .
Complete geolocated survey data were available for 304 clusters , from which 13 , 543 individuals aged 40 years and above , resident in 8 , 621 households , were examined for TT and CO . Overall , 198 ( adjusted prevalence: 1 . 45% ) individuals were diagnosed with either TT or CO in at least one eye , and only two individuals had clinical signs of CO without concurrent TT . Fig 1 presents the distribution of TT/CO among adults aged 40 years and above within clusters ( prevalence ranging from 0 to 28 . 9% ) and highlights the greater burden of trachoma in the northern areas of Nigeria . Summary characteristics of the study population are described in Table 2 and reflect socioeconomic trends across the country . Overall , only 10% of participants used a flush toilet although 64% had access to a pit latrine , and over half ( 56% ) of the participants could not read . Areas within northern geopolitical zones had higher illiteracy ( 62 . 5% ) and unemployment ( 19 . 6% ) compared to southern zones ( 49 . 3% and 12 . 6% ) ( adjusted p-values <0 . 0001 ) . Although fewer people had access to a flush toilet in the northern zones ( 4 . 4% ) than southern zones ( 17 . 5% ) ( adjusted p-value <0 . 0001 ) , open defecation was also reported less in northern zones ( 20 . 9% ) compared to the south ( 31 . 3% ) ( adjusted p-value 0 . 008 ) . All field collected variables and environmental covariates were strongly associated with the presence of TT/CO in univariate logistic regression models , with the exception of mean annual temperature , as summarised in Table 3 . Correlation was observed between a number of variables related to socioeconomic status , including occupation , water availability , literacy and latrine type . Literacy was associated both with occupation ( p<0 . 0001 ) and gender ( p<0 . 0001 ) . Women with a lower literacy status had a higher risk of TT/CO , partly accounting for the increased risk observed in illiterate individuals . A geographic north-south trend in risk of trichiasis was apparent across the country , and the unbounded semi-variogram for the raw TT/CO prevalence supported the presence of spatial autocorrelation in the distribution of disease ( Fig 2A ) . The results from the PCA identified five key groupings of variability in climatic covariates , from each of which a single variable was retained . Mean annual precipitation and land surface temperature were retained from the two contrasting groups from the first component . Mean annual temperature and altitude ( identified in the PCA as a second collinear pair contributing to climatic variation ) and EVI were also retained for further analyses with all other uncorrelated environmental indices . Summary statistics for these variables are presented in Table 1 . During model building , the residual variation in EVI was initially significant after accounting for collinear effects of precipitation and LST , but dropped out after accounting for urban classification . Bayesian hierarchical models retained both individual and cluster-level covariates ( Table 4 ) . The final model reported is Model 2 , which is non-spatial and includes age , gender , and occupation as well as mean annual precipitation , residual variation in LST , mean annual temperature and urban classification . Risk of TT/CO increased with age and was higher in women than men ( OR 2 . 46 , 95% BCI 1 . 82–3 . 39 ) . Lower socioeconomic status , as measured by occupation , was also associated with an increased risk of trichiasis . Despite wide confidence intervals , there was evidence that individuals employed in a professional capacity had the lowest risk of trichiasis while the unemployed were at highest risk ( OR 16 . 71 , 95% BCI 3 . 23–556 . 1 ) . Increased precipitation was associated with a lower risk of TT/CO in Nigeria ( OR 0 . 17 , 95% BCI 0 . 06–0 . 33 ) , and higher residual LST was uniquely associated with an increased risk of TT/CO ( OR 2 . 95 95% BCI 1 . 36–6 . 85 ) additional to the contribution it made through its relationship with precipitation . Although not identified as a risk factor in the univariate analyses , increased mean annual air temperature was associated with lower risk of TT/CO after controlling for the effects of other environmental factors . This variable was kept in the model based on the lower DIC . Finally , the odds of TT/CO were lower in urban areas ( OR 0 . 27 95% BCI 0 . 13–0 . 52 ) , after controlling for individual-level risk factors . Approximation of the VPC using a simulation approach suggested that 14% of the total variation ( based on a null model ) was attributed to the cluster level . After inclusion of both individual and cluster-level risk , only 0 . 7% of the overall residual variation was at the higher level . Although the results from the non-spatial model are reported here , there was evidence of large scale spatial trends as well as local clustering of TT/CO risk in Nigeria . The semi-variogram of the Pearson’s residuals from Model 1 indicated that , compared to the null model , the addition of covariates decreased the proportion of variation that was spatially structured and controlled for large-scale trends ( Fig 2 ) . This residual spatial structure varied within Nigeria ( non-stationarity ) , with a higher proportion of residual variation in North-East and North-West zones showing spatial structure ( Fig 3 ) . Graphs and maps of the residuals from the non-spatial Model 2 suggested that residual variation was localised in a large cluster of higher risk in the north of Nigeria ( Fig 4A and 4B ) . Inclusion of a separate random effect for these northern zones had the effect of reducing overall residual error in the model , as indicated by the reduction in the variance of the non-spatial random effect and narrower confidence intervals ( Model 3 , Table 4 and Fig 4C ) . However , addition of these terms also reduced observed associations with LST and mean annual air temperature , and widened their confidence intervals . This finding suggests that while these environmental factors may be associated with the distribution of risk in the north , they do not explain all observed clustering and are made redundant by inclusion of a spatial random effect . The range of spatial autocorrelation can be calculated by 3/ϕ and is thus 3 . 26 decimal degrees ( approximately 365 km ) in the north . Residual variation in the south was more likely to be aspatial and due to individual level factors .
The present study provides evidence that both individual-level risk factors and broader climatic conditions are associated with later stages of trachoma in adults over the age of 40 years in Nigeria , using uniquely detailed national survey data . The hierarchical approach used in this analysis has the advantage of incorporating risk factors at multiple levels and explicitly modelling residual spatial correlation in TT/CO that could affect the standard errors of estimates of association . A number of well-established individual-level risk factors for trichiasis were identified that included age , gender and occupation , as well as large-scale climatic and environmental factors ( precipitation , LST , temperature and urban classification ) that explained further variation in risk across the country . After adjusting for these factors , there remained a large cluster of higher risk localised in northern Nigeria ( North-East and North-West zones ) . This finding suggests the presence of unknown risk factors which are locally clustered in these areas or spatially-varying relationships between included covariates and disease . Individual-level factors found to be associated with trichiasis are consistent with our general understanding of trachoma epidemiology . These associations replicate those previously found in a number of studies in Nigeria [55] , other countries in sub-Saharan Africa [23 , 28 , 56] and trachoma endemic areas worldwide [57–59] . The risk of TT increases with age , presumably due to cumulative scarring caused by repeated infection over an individual’s life , while the higher risk in females is commonly attributed to close contact with children and greater exposure to infection with the causative agent [3 , 60] . Occupation is a characteristic that captures various dimensions of an individual’s socioeconomic status ( SES ) and may be linked to underlying risk factors for infection , including hygienic behaviours , use of water , human waste disposal , overcrowding or other conditions that encourage the proliferation of flies or increased transmission through contact and fomites . Previous studies have also shown a greater risk of trachoma to associate with lower scores on various socioeconomic measures , including occupation [61] , literacy or formal schooling [59 , 62]; or measures of living standards; but not uniformly across all settings [63] . Variation in relevant socioeconomic measures between settings may reflect differences in underlying transmission dynamics , equity in access to treatment and surgical interventions , as well as unreliability of the measures themselves . After accounting for these risk factors , living in urban areas remained associated with a lower risk of trichiasis . This finding supports anecdotal evidence underlying current trachoma survey strategies that exclude urban areas , and may reflect reduced access to health services or increased contact with flies associated with rural lifestyles . After controlling for individual-level risk factors , lower precipitation , higher land surface temperatures and lower mean annual temperatures were associated with a higher risk of TT in Nigeria . On this scale , climatic factors may influence transmission dynamics through hygienic behaviours related to perceived water availability , actual water availability or as determinants of fly abundance , biological fitness and behaviour [13 , 26 , 29 , 30 , 64 , 65] . Shared variation in precipitation and LST accounted for the most climatic variation , and might be interpreted as variation common to different measures of climatic water availability . Higher LST , after accounting for collinear variation with precipitation , was associated with a further increased risk of TT/CO . These findings are consistent with previous analyses associating a higher risk of active trachoma with higher aridity and lower rainfall [20 , 21 , 23 , 66 , 67] . The higher risk of TT/CO associated with lower air temperatures ( or higher altitudes ) seems counter-intuitive , however this association has been reported in previous studies with limited control for potentially confounding variables [22 , 28] . Lower temperatures are hypothesized to have a biological effect on the fly vector , M sorbens , the life span of which ranges from 12 days at 32°C to 35 days at 24°C [64] . Toyama et al . ( 1981 ) also suggested a mechanism by which increased land surface temperature might be associated with a greater fly abundance [17] . This study recorded temperatures beneath dung pats , and associated higher temperatures with formation of crusts that are hypothesised to protect larvae from predation . It is expected that TT/CO in adults who were ≥40 years old at the time of the survey mainly reflects exposure to risk factors influencing transmission decades previously ( assuming little population movement across clusters/climatic gradients ) . However , it is possible that certain climatic factors may also influence the development or subsequent evolution of trachomatous scarring and hence TT/CO . Ongoing active disease and eye irritants like ocular dryness may be associated with drier climatic conditions and contribute to chronic conjunctival inflammation . This in turn has been associated with a higher risk of TT and faster progression to later disease stages [2 , 18–20] . Despite strong links between water availability and transmission of trachoma , there are a number of potential reasons why water source was not identified as a risk factor in this analysis . First , domestic water consumption and , importantly , its allocation for hygienic purposes will mediate any relationship between water availability and trachoma [30] . Water allocation is difficult to measure , and while distance to water [26 , 68 , 69] and type of source [70] have been associated with trachoma in some studies , they are at best proxy measures of household and individual water use . It is likely that our classifications of water source were not able to capture relevant measures . In addition , a study on water use patterns in Tanzania highlighted the importance of perceived water availability and its impact on water usage , rather than availability itself [29] . It is possible that perceptions around water availability are partly driven by climatic experiences and thus may influence subsequent behaviours , including allocation within the household . Second , this survey was done over 30 months and limited evidence suggests that the water source reported as “main” may vary seasonally in Nigeria [71] . Consequently , any observed relationship between distance and usage may be stronger in the dry season . Third , individual occupation as a socioeconomic measure may have captured any effect of water source , as those with higher incomes had improved water access . And finally , while water is likely to be associated with transmission , trichiasis prevalence is likely to more strongly reflect historical transmission levels , prior to any recent interventions or secular trends . In support of this hypothesis was our finding that an improved water source was associated with an increase in the unadjusted odds of disease in the driest areas , potentially reflecting targeting of water interventions to the driest areas in the last 20–30 years . One of the strengths of this analysis lies in its explicit recognition of the hierarchical structure of the data and ability to incorporate residual spatial variation . After accounting for risk factors at the individual and cluster level , there was evidence that TT/CO was spatially structured over a large ( 365 km ) range in the north . This is likely to be due to a large cluster of residual risk , focused around southern Jigawa , eastern Kano and northern Bauchi states . Approximation of the VPC using a simulation approach suggested that 14% of the total variation was attributed to the cluster level . After accounting for risk factors at both levels , this was reduced to less than 1% . This suggests that risk of TT/CO is more variable within clusters than between clusters , and is consistent with the natural history of trachoma which requires repeated infections of C trachomatis , observed to cluster within households [6 , 28] and individuals [72] . In contrast , a recent study by Hagi et al . attributed nearly 40% of observed variation in active trachoma to the village level [9] . It is not clear what approach was used for this estimate , thus it may not be directly comparable to estimates from this study , but a higher proportion of variation between villages may reflect the importance of environmental factors on transmission dynamics via flies , and water availability . The influence of these factors may give a relatively homogenous “spread” of active disease risk across a community , whereas clustering of TT may reflect the importance of individual-level risk factors which influence the predisposition to infection , duration of infection , or immunological response to infection over longer periods of time . Despite the robust approach used to model these data , there are a number of limitations inherent in the data and methods . First , as anticipated , strong collinearity in environmental , climatic and socioeconomic variables across the country placed limitations on our ability to disentangle their independent effects . Observed associations with climatic factors may reflect socioeconomic factors that we have not taken into account , as rural populations are likely to be dependent on agro-ecological conditions for crop and livestock productivity . Second , this survey was cross-sectional and TT is a condition that represents the cumulative effect of many infections over time . Thus potential decadal climate variability , migration during an individual’s adult life or variation in other risk factors for TF between the period of exposure and time of survey limits any inferences of causality . Expected associations may be masked , or even reversed in some cases , where access to the SAFE strategy ( including surgery and environmental improvement ) has been implemented in high transmission areas . Third , the sample used for this analysis was developed for a national cross-sectional blindness and low vision prevalence survey , and thus may not be an ideal sample to extensively study risk factors for a low prevalence condition like trichaisis . And finally , not all points were able to be geolocated to a specific point location ( 7% ) and the distribution of these points varied across the country . For example , a high proportion ( 24% ) of points were geolocated to the LGA centroid in the North East zone and a low proportion ( 0% ) in the North Central zone . Errors in geolocation could introduce misclassification of cluster-level environmental data and biases in the analysis and interpretation of results . However , exclusion of points geolocated to the centre of LGAs had a minimal effect on associations in the model . For the first time , we have quantified associations between climatic factors and risk of TT/CO in Nigeria while accounting for the effects of individual-level risk factors and residual spatial dependency . While the results indicate that individual-level factors are an important source of variation , individuals living in drier and rural areas of Nigeria were at greater risk of chronic disease stages . This supports anecdotal evidence associating limited water availability with trachoma although other mechanisms may also be important , such as the effect of temperature on the abundance , breeding potential and activity of M sorbens [73] . The current focus on WASH interventions to combat other neglected tropical diseases ( NTDs ) , such as soil-transmitted helminths and schistosomiasis , provides opportunities to strengthen water availability and its use for hygienic purposes , which will have an added impact on the burden of trachoma . Findings from this study may help to more reliably extrapolate trichiasis data within countries and regions and refine estimates of the burden of disease , although further work is required to investigate associations at larger scales and in different endemic contexts . A better understanding of the distribution of the burden of trichiasis and underlying risk factors in Nigeria may aid scale-up of outreach and targeting of surgical interventions . | Trichiasis ( TT ) and corneal opacity ( CO ) are chronic stages of trachoma , which remains an important cause of blindness . This study used multilevel spatial models to investigate risk factors for TT/CO in Nigeria , including data for more than 13 , 500 adults aged 40 years and above collected in the 2007 National Blindness and Visual Impairment survey . Individual-level risk factors were consistent with those identified in other studies , including a higher risk in females , older individuals and those with lower socioeconomic status . After controlling for these factors , there was evidence that a number of environmental and climatic factors are associated with the distribution of TT/CO in Nigeria . These findings establish for the Nigerian context the importance of risk factors at different scales for the later stages of trachoma , supporting anecdotal evidence that hotter , drier environmental conditions are associated with increased risk . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [] | 2015 | Multilevel Analysis of Trachomatous Trichiasis and Corneal Opacity in Nigeria: The Role of Environmental and Climatic Risk Factors on the Distribution of Disease |
The World Health Organization ( WHO ) has targeted the elimination of Human African trypanosomiasis ( HAT ) ‘as a public health problem’ by 2020 . The selected indicators of elimination should be monitored every two years , and we provide here a comprehensive update to 2014 . The monitoring system is underpinned by the Atlas of HAT . With 3 , 797 reported cases in 2014 , the corresponding milestone ( 5 , 000 cases ) was surpassed , and the 2020 global target of ‘fewer than 2 , 000 reported cases per year’ seems within reach . The areas where HAT is still a public health problem ( i . e . > 1 HAT reported case per 10 , 000 people per year ) have halved in less than a decade , and in 2014 they corresponded to 350 thousand km2 . The number and potential coverage of fixed health facilities offering diagnosis and treatment for HAT has expanded , and approximately 1 , 000 are now operating in 23 endemic countries . The observed trends are supported by sustained surveillance and improved reporting . HAT elimination appears to be on track . For gambiense HAT , still accounting for the vast majority of reported cases , progress continues unabated in a context of sustained intensity of screening activities . For rhodesiense HAT , a slow-down was observed in the last few years . Looking beyond the 2020 target , innovative tools and approaches will be increasingly needed . Coordination , through the WHO network for HAT elimination , will remain crucial to overcome the foreseeable and unforeseeable challenges that an elimination process will inevitably pose .
In the last decade of the 20th century , the number of cases of human African trypanosomiasis ( HAT ) , also known as sleeping sickness , reached alarming levels [1 , 2 , 3 , 4] . In reaction to this epidemiological situation of a lethal disease , a number of stakeholders came together to support the affected countries . In the early years of the 21st century , the World Health Organization ( WHO ) launched a public-private partnership that , together with important efforts from bilateral cooperation and non-governmental organizations ( NGOs ) , enabled to reverse the epidemiological trend [5] . In this process , the key role was played by the National Sleeping Sickness Control Programmes ( NSSCPs ) of endemic countries and their committed health workers . The steady reduction in the number of HAT cases reported during the first decade of the current century prompted first the HAT focal points of endemic countries [6] , then the WHO Strategic and Technical Advisory Group on Neglected Tropical Diseases ( NTDs ) [7] and finally the WHO Expert Committee on control and surveillance of HAT [8] to set the elimination of HAT as a goal . The technical viability of HAT elimination rests on the existence of vulnerable points in the transmission cycle , the present as well as prospective availability of control tools , and the evidence of having reached elimination in several HAT transmission areas [9 , 10 , 11] . As a consequence , HAT was included in the WHO NTD roadmap as one of the diseases targeted for elimination as a public health problem by 2020 [7] . The indicators to measure the progress towards elimination were defined , and a reporting calendar was established [8] . The selected indicators for HAT elimination should be monitored every two years , and be presented in HAT stakeholders meetings [12] . A first progress report on HAT elimination looked at the gambiense form only [13] , and it provided an update to 2012 for the main indicators of elimination . HAT elimination was shown to be on track , even though the exclusion of rhodesiense HAT data rendered that progress report incomplete . The present paper provides the first comprehensive biennial update , including both the gambiense and the rhodesiense form , and it covers the progress made in HAT elimination from 2000 to 2014 . Reported data on HAT occurrence are compared to the milestones set by the WHO roadmap on NTDs [7] , in particular as it concerns the target of fewer than 2 , 000 reported cases by 2020 , which is the first global indicator of HAT elimination as a public health problem . Regarding the second global indicator ( i . e . ‘number of foci reporting less than 1 case per 10 , 000 inhabitants’ ) , we present here a revised metric based on the concept of ‘areas at risk of HAT’ [14] , which enables a more robust and objective quantification . This revised metric was recently endorsed by the WHO HAT elimination Technical Advisory Group .
The research does not directly involve human participants . No individual data is used in the paper . All the data used are provided routinely by National Control Programmes as epidemiological information and are fully anonymized . Detection of HAT cases is currently undertaken by NSSCPs , NGOs and Research Institutions . HAT morbidity data in disease-endemic countries are collected by NSSCPs or dedicated departments in the Ministries of Health , and subsequently reported to WHO on an annual basis . Field activities including active and passive case finding are regularly reported . Transboundary cases ( i . e . individuals who contracted the infection in one country but who were detected by the health facilities in a neighbouring country ) are also reported and allocated to the country of infection; national authorities are informed accordingly for appropriate action . Sporadic cases are also detected in non-endemic countries , amongst travellers and migrants . They are reported to WHO thanks to the centralized distribution of anti-trypanosome drugs . Information on the likely area of infection is used to allocate these ‘exported’ cases to the country of infection [15] . All data are entered in the database of the Atlas of HAT [16] . In the present paper , the number of HAT cases reported from 2000 to 2014 is provided for all endemic countries . These figures include a few minor revisions as compared to previously published counts for the period 2000–2013 [8 , 13 , 16 , 17] . The revisions stem from in-depth verifications carried out for the continuous improvement of the Atlas of HAT ( e . g . a more accurate allocation of transboundary cases ) . The geographic distribution of HAT reported cases is mapped at the village level following already described methodologies [16 , 18] . The database includes , from the year 2000 onwards , not only the cases detected actively and passively but also the people examined per village during active screening activities carried out by mobile teams . All records in the database are linked to the source files from which the information was derived , and all source files are safely stored in a digital data repository . In this paper , emphasis is given to the distribution of HAT cases for the five-year period 2010–2014 . Because of the inherent epidemiological features of HAT , and in the context of the elimination strategy , a five-year window is considered as the most useful to analyse and present the updated picture of the extent of the disease [8 , 13] . In particular , the 5 year window is believed to strike a good balance between temporal resolution ( which would call for a shorter window ) and robustness ( which would call for a longer window , so as to smooth the year-to-year variations in screening intensity ) . The risk of HAT infection is estimated from the number of reported cases ( numerator—Atlas of HAT ) and the exposed population ( denominator—Landscan [19] ) . Previously published methods enable point level data from the Atlas of HAT ( village-level mapping ) to be converted into continuous , smoothed surfaces of disease intensity and risk [14 , 20] . Smoothing is based on a 30-km radius kernel density , and although HAT risk was initially estimated over ten-year periods [14 , 20] , more recently five-year periods have been considered more informative to monitor elimination [13] . In the present paper , the progress over time was investigated through a five-year sliding window ( i . e . from 2000–2004 to 2010–2014 ) . The 30-km smoothing for the estimation of HAT risk is meant to account for a variety of complex and not easily quantifiable epidemiological features such as the mobility of people and of the vector , whilst at the same time reducing the effect of mapping inaccuracies in the input data . This methodology and its rationale are described in detail elsewhere [14] . On the basis of the number of HAT cases per annum ( p . a . ) as compared to the exposed population , HAT risk is ranked into five categories: very high ( ≥ 1 HAT case per 102 people ) , high ( ≥ 1 HAT case per 103 people and < 1 per 102 people ) , moderate ( ≥ 1 per 104 people and < 1 per 103 people ) , low ( ≥ 1 per 105 people and < 1 per 104 people ) , and very low ( ≥ 1 per 106 people and < 1 per 105 people ) [13] . Risk is considered ‘marginal’ below the threshold of 1 HAT case p . a . per 106 people . It is noteworthy that below the category of ‘moderate’ , the risk level fits the WHO general definition of elimination of HAT as a public health problem . For the present risk estimates , the Atlas of HAT provided village-level mapping for 92 . 9% of HAT reported cases ( period 2000–2014 ) . For the 7 . 1% of the cases which were not mapped at the village-level , information on the area of occurrence was used ( i . e . unmapped cases were proportionally allocated to the endemic villages of the same area [20] ) . Fixed health facilities play a crucial role in the control and surveillance of HAT . With a view to estimating their physical accessibility and potential coverage of at-risk populations , time-distance analysis was used [21] . In the present paper the coverage of the population at risk of gambiense HAT is updated , and that of rhodesiense HAT is presented for the first time . Data on the fixed health facilities that are active in HAT control and surveillance were provided by NSSCPs through standardized forms . For each health structure , information was collected on the name , location and capacities for HAT diagnosis and treatment . Data were harmonized , mapped and assembled in a geo-spatial database [21] . The survey was conducted between September 2015 and April 2016 . Diagnostic capacities for gambiense HAT were categorized as ‘clinical’ ( DxC ) , ‘serological’ ( DxS ) , ‘parasitological’ ( DxP ) , and ‘stage determination’ ( DxPh ) [21] . For rhodesiense HAT , a serological screening test is not available , but the other three categories do apply . For treatment capacities , gambiense HAT includes treatment of infections in the first-stage , i . e . pentamidine ( Tx1P ) , and in the second-stage , i . e . nifurtimox-eflornithine combination therapy—NECT ( Tx2N ) , eflornithine ( Tx2E ) and melarsoprol ( Tx2M ) [21] . For rhodesiense HAT , treatment of first-stage infections with suramin ( Tx1S ) and of second-stage infections with melarsoprol ( Tx2M ) are available . A time-distance function was used to map the physical accessibility to HAT diagnosis and treatment [21] . A ‘landscape friction’ geospatial layer for Africa provided the travel time through each 1-km/30 arcseconds pixel [22] . Landscape friction takes into account terrain and transportation network . The cumulative travel time was calculated from any location to the nearest health facility ( ‘shortest weighted distance’ or ‘least cumulated time’ ) . The economic cost of travel ( i . e . affordability ) is not considered in our analysis; only travel time is computed . For presentation purposes , results were summarized for three thresholds of travel time ( i . e . ‘one hour’ , ‘three hours’ and ‘five hours’ ) and stratified by risk categories . With a view to exploring trends , results for gambiense HAT are compared to those of a previous survey ( completed in August 2013 [21] ) . For the previous study [21] , stratification relied on a 10-year risk layer ( 2000–2009 ) . To ensure consistency with the present estimates , which are stratified on a 5-year risk layer ( 2010–2014 ) , previous estimates were recalculated on the basis of the corresponding 5-year risk layer ( i . e . 2007–2011 ) .
A total of 3 , 797 new HAT cases ( including both gambiense and rhodesiense HAT ) were reported in 2014 ( Fig 1 ) . For this indicator , the continental target set for ‘HAT elimination as a public health problem’ is fewer than 2 , 000 cases , a level that was planned to be reached by the year 2020 [7] . The intermediate milestone of 5 , 000 cases in 2014 was not only reached but surpassed by 1 , 203 cases . The number of gambiense HAT cases reported by year and by country is shown in Table 1 . In 2014 a total of 3 , 679 cases were reported , corresponding to an 86% reduction compared to 2000 . It is worth noting that the Democratic Republic of the Congo ( DRC ) continues to account for the vast majority of gambiense HAT cases . In 2014 , the DRC accounted for 87% of the total number of cases ( 3 , 205 out of 3 , 679 ) . Fig 2 shows the number of people screened by active case-finding surveys in countries endemic for T . b . gambiense in the period 2000–2014 . The chart shows that , despite year-to-year variations , the overall intensity of active surveillance has been fairly stable over the described time period , and it has stabilized at approximately 1 . 8 million people screened per year over the last four years ( 2011–2014 ) . Concerning rhodesiense HAT , results are shown in Table 2 . With 118 cases reported in 2014 , the rhodesiense form of the disease continues to represent a small part of the total HAT reported cases ( 3% ) . With 709 cases reported in the year 2000 , a reduction of 83% in 14 years was observed . Over the last 4 years , the number of rhodesiense HAT cases has stabilized at around 100 per year . Fig 3 shows the geographic distribution of sleeping sickness cases for the 5-year period 2010–2014 . The locations of active screening activities where no cases were detected are also included ( green circles ) . For the period 2010–2014 , 31 , 188 new HAT cases were reported , 88 . 3% of which could be mapped at the village level . For the whole period 2000–2014 ( 2000 being the start year of the Atlas of HAT ) , a total number of 206 , 570 cases has been included in the database . Of these , 92 . 9% cases have been mapped at the village level , for a total of 30 , 278 mapped villages . The average accuracy for mapped HAT cases is presently estimated at 1 . 3 km , and it is being continuously improved .
The data presented in this paper , covering the 15 years between 2000 and 2014 , indicate clear progress towards HAT elimination as a public health problem , which is on track to be achieved by 2020 . For the first time , in 2014 fewer cases than the set milestone were reported ( i . e . 3 , 797 against the 5 , 000 milestone ) . The level reported in 2014 had been planned to be reached by 2016–2017 . This decrease in reported cases was observed in a context of fairly constant intensity of active screening activities and reinforced passive surveillance in several countries , so the trend is very likely to reflect a real abatement in disease transmission . Preliminary data for 2015 ( not presented here ) show a further reduction in reported cases , thus corroborating the observed trend . Areas at risk of HAT , which are estimated from reported cases and exposed population , are also shrinking . In particular , the areas where HAT is still a public health problem ( i . e . where ≥ 1 HAT case per 104 people p . a . is reported ) , have been decreasing steadily . Between 2004 and 2014 we estimate a reduction of approximately 360 , 000 km2 , i . e . -51% as compared to the 2004 level . The number of fixed facilities providing gambiense HAT diagnosis and treatment increased , and thus their potential coverage of the at-risk population . Improvements were observed in the basic levels of diagnosis ( clinical , serological ) and treatment ( first-stage ) , as well as in the more advanced levels ( i . e . parasitological diagnosis , disease staging , and first-line treatment for second-stage infection with NECT ) . Looking at rhodesiense HAT , we note that this form of the disease continues to represent a relatively small proportion of the total number of HAT reported cases ( i . e . 2 . 7% average for 2000–2014 ) . Against this backdrop , a sizeable decrease was observed between 2000 and 2011 , with a reduction of 594 cases p . a . ( i . e . 84% ) . However , since 2011 progress has stagnated and the number of reported cases has stabilized at around 100 p . a . Several factors may have contributed to the stagnation . One is the expanded use of rapid diagnostic tests ( RDT ) for diagnosing malaria instead of microscopic examination . In fact , microscopic examination enabled the accidental diagnosis of HAT while looking for malaria parasites . Another is the fact that , in the first decade of 2000 , the maximum reduction of cases was observed in areas where livestock is the reservoir of T . b . rhodesiense , and where strengthened veterinary public health brought about the decrease [26] . The wildlife reservoir is much more difficult to manage and it represents the main source of the scattered but constant rhodesiense HAT cases that are reported from protected areas [16] . It is also important to note that for rhodesiense HAT the rate of under-detection is likely to be higher than for gambiense HAT . The causes of this are manifold , and they include a faster disease progression , the poor effectiveness of active screening that makes rhodesiense HAT only detectable by passive screening , a lower incidence and therefore a lower awareness and preparedness of health staff , and the occurrence of the disease in sparsely populated areas . As a result of this likely higher rate of under-detection , the reliability of all indicators based on reported cases is bound to be lower for rhodesiense than for gambiense HAT . Applying to rhodesiense HAT the same risk threshold as for gambiense HAT , we observe that the areas where the disease is still a public health problem are very few and very small . In the period 2010–2014 they were mostly limited to a restricted area in central Uganda ( Kaberamaido and Dokolo Districts [28] ) and the area surrounding the Vwaza Marsh Wildlife Reserve in Malawi [29] . A few additional scattered areas at moderate risk are also found in sparsely populated zones of Zambia ( mostly corresponding to the Luangwa National Parks [30] ) . Looking at the potential coverage of the at-risk populations by passive surveillance , we provide here the first continental survey of fixed facilities having capacity for diagnosis and treatment of rhodesiense HAT . At this stage , we only note that the coverage of population at risk of rhodesiense and gambiense HAT is comparable . One hundred and eleven facilities for rhodesiense HAT were identified , compared with 882 for gambiense HAT , which at first glance seems proportionate to the respective reported burdens . However , when looking at these figures , the substantial differences between the two diseases have to be kept in mind , and especially the generally lower transmission intensity and the likely higher under-detection for rhodesiense HAT . In this context , we argue that there is a need to expand and improve the network of fixed health facilities for rhodesiense HAT ( e . g . in Zimbabwe [27] ) . In this analysis , financial barriers are not considered for both gambiense and rhodesiense HAT , but it is important to underline that in many countries , it can be an important barrier that prevent a wider use of the existing facilities . In this paper , efforts were made to present a comprehensive update of the indicators of elimination , as set by WHO [8] . We show that , in the framework of the Atlas of HAT , data were assembled and methodologies were developed that enable us to follow the progress of HAT elimination with a high level of geographic detail ( village-level mapping ) , and completeness of data in time and space ( comprehensive data from all reporting countries are systematically collated , harmonized and analyzed from the year 2000 onwards ) . However , challenges in monitoring the process of elimination still remain . Regarding the primary global indicators of elimination , while the cumulative number of HAT reported cases has been effectively followed by WHO for a long time , monitoring the second primary indicator , i . e . ‘the number of foci where HAT is no longer a public health problem’ [8] , is proving more challenging . The main reason for this is that the available definition of focus as ‘a zone of transmission to which a geographical name is given ( locality , region , or river ) ’ [31] is useful for operational purposes , but it is vague and not particularly helpful for measuring . In particular , it is difficult to define the geographical boundaries of HAT foci in an objective and standardized way . The challenge is compounded by our incomplete understanding of the focal nature of HAT [16] , and by the fact that different countries use different criteria to define foci . On the other hand , the data collected in the HAT Atlas allow to quantify the area at risk [13 , 14] , which is , in essence , a ‘zone of transmission’ that can be measured in a robust and objective way . As such , it represents a much more suitable metric for the second primary indicator of HAT elimination , originally proposed as ‘number of foci reporting less than 1 case per 10 , 000 inhabitants’ . This metric also easily lends itself to monitoring over time , and at various spatial scales ( from global to subnational and local levels ) . A recently created WHO HAT elimination Technical Advisory Group ( HAT-e-TAG ) recognized the impossibility of enumerating and delineating HAT foci objectively , and endorsed the revised global metric to assess elimination as a public health problem ( i . e . the ‘total area at risk reporting ≥ 1 case /10 , 000 people/year’ , which corresponds to the risk categories of ‘moderate’ , ‘high’ and ‘very high’ ) . HAT-e-TAG also proposed the 2020 target for this indicator , i . e . a reduction of 90% by the year 2020 as compared to the baseline calculated for the period 2000–2004 . Regarding the secondary indicators of elimination , they currently include population at risk , coverage of active and passive screening activities , and the geographic distribution of the disease [8] . For population at risk , the available data and methodologies enable an effective monitoring . Provided that attention is paid to the various levels of risk , the population at risk provides useful complementary information to the primary indicators . Regarding the coverage of passive surveillance , at this stage we are in a position to estimate only a potential coverage ( i . e . physical accessibility ) , and there is still a two-year lag between the survey of health facilities and the risk map used for stratification . In the future , efforts will be made to shorten the lag , and , much more importantly , to estimate the actual coverage of passive surveillance from the number of individuals passively screened by the health facilities . As to active screening activities , data are already systematically included in the Atlas of HAT that will enable the actual coverage to be estimated and mapped . To this end , a methodology is presently being developed . It is worth pointing out that the present methodologies to estimate coverage fail to capture issues of quality of coverage , such as what age , sex , or occupational groups are covered , quality and performance of the services provided and the varying efficacy of detection methods used . As regards geographic distribution , the last secondary indicator , it is still considered as a very useful aspect of the epidemiology of HAT to be monitored . However , measuring this indicator quantitatively is not deemed particularly relevant at this stage , especially because the indicator ‘area at risk’ already captures the main quantitative aspects of the geographic distribution of HAT . One cross-cutting aspect that affects virtually all indicators is their reliance on reported HAT cases , with all the uncertainties that an unknown level of underdiagnosis and underreporting brings . Efforts are being made to estimate and map these uncertainties through geospatial and environmental modelling [32 , 33] . The advances in the process of HAT elimination [13] are confirmed in this new comprehensive report for gambiense and rhodesiense HAT . In particular , the milestone for the number of HAT cases reported in 2014 was achieved and even surpassed . Case-finding efforts were sustained in most of the affected countries , which gives confidence in a real progress in disease elimination . These results were accomplished mainly through sustained efforts in disease surveillance and control by NSSCPs . The strength of the epidemiological knowledge continues to improve . The database of the Atlas of HAT is regularly improved in terms of completeness and accuracy , thus resulting in a robust estimation of the indicators . At the same time , models trying to predict the level of underdetection and the presence or absence of the disease in grey areas are being developed . In a few affected areas , access to diagnosis and treatment is still constrained by insecurity ( e . g . in Central Africa Republic and South Sudan ) and remoteness ( e . g . in some area in the DRC ) . Also , the progressive loss of expertise and motivation of health staff dealing with HAT is one of the inevitable effects of the reduction of cases . New innovative approaches are required to sustain the quality of interventions . Looking to the future , another inevitable consequence of reduced number of cases will be a progressive shift from active screening to a combination of passive surveillance and reactive screening . This shift , and the related integration of gambiense HAT surveillance into the health system , will be one of the main challenges to elimination . Rhodesiense HAT represents a relatively small part of the global HAT problem . Because of its zoonotic dimension , the approach to tackle rhodesiense HAT must consider the epidemiological role of the domestic and wild animal reservoir in a One Health framework . As a result , disease elimination will require a multisectoral approach that should involve the veterinary services and include a vector control component [34 , 35] . As to the interruption of transmission , it is likely to remain elusive for some time to come , unless a breakthrough in control tools enables to tackle the animal reservoir ( especially its wildlife compartment ) . Despite the recent advances , it is crucially important to sustain the commitment of all stakeholders . Appropriate funding must be ensured if the 2020 and the 2030 goals are to be achieved . It is likely that both targets can be met , although the latter ( i . e . interruption of transmission ) is expected to pose a more severe challenge , and it is only applicable to gambiense HAT [8] . In the process of elimination , increased ownership of the fight against HAT by endemic countries must be ensured . The challenges to integration of HAT activities in weak national health systems raise concerns . All efforts and policies aiming to strengthen health systems , especially in rural areas , will contribute to the sustainability of HAT elimination . Looking at gambiense HAT , in this new context of strongly reduced prevalence , human asymptomatic carriers [36] and the possible animal reservoir [37 , 38] need to be studied in more detail , as they could play a role in disease maintenance , resurgence and reintroduction . Development of new control tools , including diagnosis , treatment and vector control , could change the current control and surveillance scenario by enabling innovative , adapted and more cost-effective strategies to be implemented . While the process of HAT elimination is progressing as planned , many challenges still lie ahead . At this juncture , the WHO network for HAT elimination set up in 2014 [12] ensures crucial coordination of stakeholders and maximum effectiveness in the support to endemic countries . Only by maintaining the synergy and coordination of interventions will sustainable elimination of HAT be achieved . | Human African trypanosomiasis ( HAT ) , also known as sleeping sickness , is a neglected tropical disease transmitted by tsetse flies , which has been responsible for devastating epidemics in the 20th century . Since the last alarming spike in disease incidence during the late 1990s , disease surveillance and control have been greatly strengthened , tremendous improvements have been achieved , and the disease is now targeted for elimination by the World Health Organization ( WHO ) . In this paper we provide a comprehensive update of the indicators of HAT elimination to 2014 , including number of reported cases and the areas and populations at risk . Fixed health facilities offering diagnosis and treatment for HAT were also surveyed , mapped , and their potential coverage of populations at risk was estimated . With 3 , 797 reported cases in 2014 , the 2020 global target of ‘elimination as a public health problem’ ( i . e . ‘fewer than 2 , 000 reported cases per year’ ) seems within reach . The 2030 target ( i . e . elimination of transmission ) is expected to pose more severe challenges . The sustained commitment of all stakeholders and close coordination of activities will have to be ensured , if the goal of HAT elimination is to be achieved . | [
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] | 2017 | Monitoring the elimination of human African trypanosomiasis: Update to 2014 |
The host innate immune response mediated by type I interferon ( IFN ) and the resulting up-regulation of hundreds of interferon-stimulated genes ( ISGs ) provide an immediate barrier to virus infection . Studies of the type I ‘interferome’ have mainly been carried out at a single species level , often lacking the power necessary to understand key evolutionary features of this pathway . Here , using a single experimental platform , we determined the properties of the interferomes of multiple vertebrate species and developed a webserver to mine the dataset . This approach revealed a conserved ‘core’ of 62 ISGs , including genes not previously associated with IFN , underscoring the ancestral functions associated with this antiviral host response . We show that gene expansion contributes to the evolution of the IFN system and that interferomes are shaped by lineage-specific pressures . Consequently , each mammal possesses a unique repertoire of ISGs , including genes common to all mammals and others unique to their specific species or phylogenetic lineages . An analysis of genes commonly down-regulated by IFN suggests that epigenetic regulation of transcription is a fundamental aspect of the IFN response . Our study provides a resource for the scientific community highlighting key paradigms of the type I IFN response .
Most emerging human viruses have an animal origin [1] . The increase in the global human population , international travel , and ecological changes , in addition to changes in agricultural practices , has led to complicated interactions between wildlife , domestic species , and humans that has enhanced the opportunities for cross-species transmission of known , as well as newly discovered , viruses [1 , 2] . Physical and molecular components of the innate immune system represent early barriers to incoming viruses that must be overcome in order for an infection to prevail . In vertebrates , one of the key innate immune defences against virus infection is the interferon ( IFN ) system . Type I interferons ( including IFN-β and IFN-α among others; here referred to simply as IFN ) , type II interferons ( IFN-γ ) , and type III interferons ( IFN-λ ) are cytokines with antipathogen , immunomodulatory , and proinflammatory properties . The IFN system is usually stimulated by the detection of pathogen signatures , known as pathogen-associated molecular patterns ( PAMPs ) , resulting in the secretion of IFN . In turn , IFN signalling results in the up-regulation of hundreds of interferon-stimulated genes ( ISGs ) , collectively referred to as the type I ‘interferome’ ( here simply the ‘interferome’ ) [3 , 4] . Unsurprisingly , given the importance of IFN in combatting pathogen invasion , there are numerous examples of coevolutionary arms races between ISGs and invading pathogens [5 , 6] . However , previous studies investigating ISG transcription have focused on the interferomes of single species [7 , 8] . Despite resources such as the Interferome database [9] , variations in experimental and bioinformatic approaches make comparing interferomes derived from divergent species and collected from different studies a difficult prospect if significant technical caveats and confounding factors are to be avoided . Here , we used the same RNA sequencing ( RNAseq ) approach on 10 animal species to deliver a snapshot of the genes that are differentially expressed in cells ( fibroblasts ) in a type I IFN-induced antiviral state . This snapshot of the interferome from a single cell type at one point in time cannot capture the entire temporal and tissue-specific complexity of the interferome . Nonetheless , using this comparative approach , we have uncovered fundamental paradigms of the IFN system .
We first determined the individual interferomes of Homo sapiens ( human ) , Rattus norvegicus ( rat ) , Bos taurus ( cow ) , Ovis aries ( sheep ) , Sus scrofa ( pig ) , Equus caballas ( horse ) , Canis lupus familiaris ( dog ) , Myotis lucifugus ( little brown bat , microbat ) , Pteropus vampyrus ( large flying fox , fruit bat ) , and Gallus gallus ( chicken ) cells . Interferomes were obtained from cells stimulated with type I IFN and experimentally confirmed as being in an antiviral state as described in the Materials and methods ( S1 Fig ) . In our study , we defined an ISG as a gene up-regulated by IFN with a false discovery rate ( FDR ) of <0 . 05 , regardless of the extent to which it was up-regulated . To facilitate mining of the data , we also developed an open access webserver ( http://isg . data . cvr . ac . uk ) capable of filtering the dataset based upon user-defined criteria . The absolute number of ISGs differentially expressed in each species varied ( S1 Table ) , but their pattern of differential expression in response to type I IFN was remarkably similar ( Fig 1A ) . The presence of shared ISGs at specific nodes on a schematic phylogeny provided evidence that interferomes have been sculpted over time by lineage-specific pressures possibly exerted by different pathogens ( Fig 1B ) . As expected , we observed that the most closely related species in our dataset , cows and sheep , showed the greatest similarity in the genes they up-regulate ( Fig 1C ) . However , we also observed substantial levels of similarity in the interferomes of some species that are more distantly related phylogenetically , most notably pigs and humans ( Fig 1C ) . Interestingly , this finding was reflected in a principal component analysis of the 35 one-to-one ( i . e . , single copy ) ISG orthologs up-regulated by every mammalian species in our study ( see below ) , whereby the patterns of differential expression were again similar between humans and pigs ( Fig 1D ) . Every species possessed unique ISGs that were not up-regulated by IFN in any of the other nine species . Furthermore , certain ISGs present in our dataset ( despite being up-regulated by IFN >2 log2 fold change [log2FC] ) had few ( if any ) orthologs in the other genomes in the Ensembl database . Examples include a gene ( RGD1561157 ) that is annotated on chromosome 10 of the rat genome and two chicken genes ( Ensembl IDs ENSGALG00000019325 and ENSGALG00000020899 ) . We identified a core set of 62 genes ( “core vertebrate ISGs” , hereinafter corevert ISGs ) that were up-regulated by IFN by all 10 species analysed in this study , with an additional 28 genes up-regulated specifically in the nine mammalian species ( “core mammalian ISGs” , hereinafter coremamm ISGs ) ( Table 1 ) . The corevert ISGs represent the ancestral functions of the IFN system and include genes encoding proteins broadly involved in ( i ) orchestrating antigen presentation , ( ii ) IFN induction and response , ( iii ) IFN suppression , ( iv ) ubiquitination and protein degradation , ( v ) cell signalling and apoptosis , and ( vi ) antiviral responses ( Table 1 , Fig 1E ) . Nine of the 62 corevert ISGs ( e . g . , various HLA genes , TAP1 , ERAP1 , etc . ) are involved in the generation , trimming , loading , and presentation of MHC-I–restricted antigens , thus providing a direct link between the IFN response and the adaptive immune response via the CD8 T-cell response ( Table 1 ) . Additionally , components of the immunoproteasome ( e . g . , PSMB8 and PSMB9 ) were specifically up-regulated in the mammalian core ( Table 1 ) , reflecting previous studies reporting the absence of the immunoproteasome in birds [10] . We found that various corevert ISGs are involved in IFN induction and response , including pattern recognition receptors , the key adaptor molecule MyD88 , and transcription factors . The classical sensors for RNA PAMPs , including IFIH1/MDA5 , DHX58/LGP2 , and TLR3 ( and RIG-I/DDX58 in the mammals ) were among the corevert ISGs ( Table 1 , Fig 2 ) . RIG-I is known to be absent in chickens ( and other galliformes ) but is present and active in other birds , including ducks and geese [11–13] . Furthermore , TRIM25 , a ubiquitin E3 ligase responsible for ubiquitinating RIG-I , was also a corevert ISG [14] . DAI/ZBP1 , which was originally classified as a DNA sensor but is now thought to be an RNA sensor [15] , was also highly up-regulated by IFN in every mammalian species except humans ( Fig 2 ) . No DNA sensors were found among the corevert ISGs . However , cGAS was found to be an ISG in every mammalian species in this study ( Fig 2 ) . By contrast , the DNA sensor AIM2 was only up-regulated in human cells . Interestingly , whilst the basal expression ( defined in terms of fragments per kilobase mapped values [FPKM] in the absence of IFN treatment ) of RNA sensors was very low , many of the genes in the literature associated with DNA sensing are constitutively transcribed ( Fig 2 ) . With the exception of MyD88 , a key adaptor involved in both the RNA- and DNA-sensing pathways [16] , we observed limited up-regulation among genes involved downstream of nucleic acid detection ( Fig 2 ) . On the other hand , core ISGs included key transcription factors involved in IFN induction and response ( IRF1 , IRF7 , STAT1 and STAT2 are all corevert ISGs in addition to IRF9 among the coremamm ISGs ) ( Fig 2 ) . Importantly , several ISGs that play a role in the suppression of the IFN system , including USP18 , USP25 , IFI35 , and SOCS1 were up-regulated in all species under examination . The encoded proteins of these genes target different points in the IFN response . Thus , negative regulation of the IFN response is multifaceted and a fundamental , ancestral failsafe necessary to avoid excessive/perpetual up-regulation of IFN-induced pathways . Among the corevert ISGs , we found several genes relating to ubiquitination , such as the ring finger proteins RNF213 and RNF19B ( Table 1 , Fig 1E ) , highlighting protein modification as part of the IFN response . Interestingly , N4BP1 , originally identified as a target of Nedd4-mediated ubiquitination , has not previously been directly linked to the IFN response . The corevert ISGs contained 14 IFN-induced antiviral factors such as MX1 , IFIT2 , and viperin ( Table 1 ) . Interestingly , when we assembled a list of 40 genes that either create a cellular environment hostile to or act directly upon the virus lifecycle ( based upon the scientific literature; S2 Table ) , we noticed that 75% of these antiviral genes were ISGs in at least eight of the 10 species analysed in this study ( Fig 3A ) . In addition , antiviral ISGs were up-regulated to a significantly higher extent than randomly sampled ISGs ( P < 0 . 01 for each species , Fig 3B ) . Furthermore , some well-studied antiviral ISGs were not up-regulated by IFN in certain species . For example , OAS2 was not up-regulated in the rat , SAMHD1 was not up-regulated in the horse , OASL was not up-regulated in either the cow or the sheep , and the IFITM genes were not up-regulated in the dog ( Fig 3C ) . In general , genes encoding antiviral factors were transcribed to minimal levels in the absence of IFN . Indeed , the median FPKM level for antiviral genes was lower than that of the overall interferome for every antiviral factor except SAT1 , SHISA5 , and the IFITM genes . Of interest , we noticed particularly high FPKM values for IFITM1 and 3 in the rat and IFITM2 in the microbat ( Fig 3C ) . Two corevert genes , CD47 and IL15RA , encode proteins involved in signalling to components of the adaptive immune system . CD47 is involved in a variety of biological roles , including leukocyte and dendritic cell migration , the development of antigen presenting cells , and immune apoptosis , and it also provides a ‘don’t eat me’ signal [17] . The IL15–IL15Rα axis is well characterised as being important in the promotion of both natural killer cells and a variety of T cell populations , including activated CD8 T cells [18] . Among the corevert ISGs , we identified a number of genes with few or no reported associations with the type I IFN response ( Table 1 ) . Several of these genes have been studied extensively , but not always in the context of the IFN response . DNAJC13 , for example , is reported to be involved in endosome trafficking , and it has been closely linked to Parkinson’s disease [19] . Zinc Finger CCHC-Type Containing 2 ( ZCCHC2 ) has nucleic acid–binding properties and , interestingly , contains a single nucleotide polymorphism ( SNP ) associated with insect bite hypersensitivity in Exmoor ponies [20] . The fragile X mental retardation ( FMR1 ) gene encodes an RNA-binding protein that plays a role in intracellular RNA transport and in the regulation of translation of target mRNAs . FMR1 was not previously linked to the IFN response , although it has recently been shown to be a proviral factor for influenza virus and previously was shown to induce mild restriction of HIV-1 [21 , 22] . Cap methyltransferase 1 ( CMTR1 ) , also known as ISG95 , binds to RNA pol II and is a 2′-O-ribose methyltransferase that participates in the conversion of cap0 to cap1 type transcripts [23] . Interestingly CMTR1 is also known as an important component of IFIT-mediated antiviral activity [24] . We hypothesise that these genes , as corevert ISGs , likely play fundamental roles in host immunity that are underappreciated or have yet to be fully determined . Our data also indicate a relatively underappreciated link between local synthesis of early components of the complement system and the type I IFN response [25 , 26] . C2 was among our coremamm ISGs . In addition , we found that C1r and C1s , essential components of the C1 complex , were up-regulated by IFN in cells from all species analysed in our study with the exception of the cow . Interestingly , this was also the case for a negative regulator of C1r and C1s ( SERPING1/C1-INH ) . Our data enabled an unprecedented opportunity to investigate the interferon-repressed genes ( IRGs ) , which , to date , have received comparatively little attention with regards to their involvement in the innate immune system . Unlike ISGs , the extent of down-regulation of IRGs across all the 10 species used in this study was relatively modest ( overall average −0 . 56 log2FC for IRGs as compared to 1 . 64 log2FC for ISGs ) . We found no IRGs shared by all species , although this may reflect the low fold change in expression and/or greater variability in the response of individual genes . This result could also imply that there has been less conservation of the down-regulated genes over time . The most consistently down-regulated genes were FAM117B and KDM5B , which were both significantly down-regulated in eight of the 10 species analysed in this study . Relatively little is known about the function of FAM117B with the exception that it is a risk factor for sarcoidosis [27] . On the other hand , it has been shown that suppression of the KDM5B gene product , a H3K4 demethylase causing transcriptional repression , results in increased expression of IFN-β and other inflammatory cytokines following infection with respiratory syncytial virus ( RSV ) [28] . We also noticed that , with the exception of the rat , every species analysed down-regulated at least one KDM gene in response to IFN . It is already established that another form of epigenetic control , acetylation , is also required for robust ISG transcription [29] . ANP32A , a protein involved in acetyltransferase inhibition [30] , was down-regulated in five species . Interestingly , ANP32A has been shown to be a host component necessary for influenza virus replication and influences the ability of the virus to replicate in a given animal species [31] . Our data have thus revealed that key epigenetic factors regulating ISG transcription are themselves frequently responsive to IFN treatment . Along these lines , we also investigated the presence of noncoding RNAs ( ncRNAs ) , a class of RNA molecule increasingly recognised as being important in the antiviral response [32 , 33] . We found that in human cells 75 long intergenic noncoding RNAs ( lincRNAs ) were differentially expressed ( 38 up-regulated , 37 down-regulated ) in response to IFN ( S2 Fig ) , including NEAT1 , a lincRNA that has previously been associated with viral infections [34–36] . As other genomes become increasingly well annotated , it will be possible to resolve a more in-depth understanding of the impact that ncRNAs play in the control of the innate immune system . Virus–host coevolution has shaped the innate immune system , most frequently by placing antiviral genes under positive selection . We assessed the dN/dS ratios ( as compared to the human ) for one-to-one ISG orthologs . We observed that the overall distributions of dN/dS values of ISGs were significantly higher than those of randomly selected non-ISGs ( Fig 4A ) . In addition , we assessed the copy number of each ISG across the different species studied here . Strikingly , for each species—with the exception of sheep and , to a lesser extent , cow—the proportion of ISGs with gene expansions was significantly above that of the genome as a whole ( Fig 4B ) . Interestingly , sheep and cows are the only species with multiple copies of the IFN-β gene ( generally a single copy gene in mammals ) . The data described above suggest that , in general , expanded ISGs ( compared to the rest of the genome ) have an increased likelihood of conferring a selective advantage to the host species . Indeed , we observed that ISGs that are shared between multiple species have a higher likelihood of being expanded in the genome compared to other genes ( P < 0 . 001 , Fig 4C ) . Furthermore , among the corevert ISGs , one-to-one orthologs are induced by IFN to a significantly higher level than genes present as paralogs ( two-way ANOVA , F = 2 . 284 , P < 0 . 05 ) . We further analysed gene expansions and deletions among the coremamm ISGs in the genomes of 111 mammalian species using an in silico sequence-similarity screening approach [37] . Although the uneven quality of the genomes used in the analysis make this approach prone to artefacts , we were able to detect coremamm ISG deletions . For example , we observed that XAF1 , which is a negative regulator of inhibitors of apoptosis , is deleted in cats ( Felidae ) ( Fig 5A ) . In addition , we confirmed previously published deletions of IFIT3 among the Scandentia ( tree shrews ) , Cetacea ( whales and dolphins ) , and marsupials ( Fig 5A ) [38 , 39] . Furthermore , we observed that IFIT2 exists as a pseudogene in the Cetacea ( Fig 5B ) .
In this study , we devised a systematic approach to unveil fundamental properties of the type I IFN system in vertebrates . We investigated the IFN response in several mammalian species and the chicken using a consistent experimental framework . By grouping ISGs and IRGs according to the number of species in which they were differentially expressed , we were able to reveal key facets related to the evolution and function of the IFN response . We identified 62 corevert ISGs that were up-regulated both in the chicken and nine mammalian species . Similarities between the chicken and mammalian IFN systems likely reflect fundamental functions that were present in the common ancestor of birds and mammals that have remained conserved over the ensuing circa 300 million years . Orchestration of the adaptive immune response by IFN appears to be a fully conserved and prioritised function among vertebrates . Specifically , within the corevert ISGs , we found MHC class I components , along with genes involved at all levels of the antigen presentation process and genes involved in cell signalling to diverse cells of the adaptive immune system . Interestingly , we found that the type I IFN response may also facilitate local expression of factors of the complement system . Only a limited set of genes relating to PAMP detection ( MDA5 , LGP2 , TLR3 ) and downstream signalling ( IRF1 , IRF7 , STAT1 and STAT2 ) were within the corevert ISGs . In general , we noticed a greater number of IFN-up–regulated RNA sensors ( and a greater level of their up-regulation ) compared to DNA sensors . These data imply that the type I IFN response biases the sensitivity of surveillance for RNA viruses and may also reflect the difficulty , and danger , of differentiating self from nonself RNA in the cytoplasm . Known antiviral ISGs are , in general , shared by many species . It therefore appears that a large proportion of the known antiviral capability of the IFN system evolved at an early point , a finding consistent with the presence of antiviral activity among fish ISGs [40 , 41] . Characterisation of the corevert ISGs has also revealed several genes that hitherto have had little , if any , association in previous studies with the IFN response . Our data suggest that these genes play fundamental roles in the innate immune response of vertebrates that remain yet to be discovered . It is possible that genes such as these have been overlooked in previous studies simply as a result of their relatively modest fold up-regulation in response to IFN treatment in cells derived from individual animal species . This dataset therefore provides additional power with which to uncover novel genes central to the IFN system and an alternative approach by which to prioritise their relative biological significance and evolutionary conservation . We observed that genes have arisen as ISGs throughout evolution , to the extent that certain genes are responsive to IFN only in particular phylogenetic groups . In addition , ISGs shared by multiple species have a higher propensity to be retained in genomes , yet another example of the pressure exerted by invading pathogens shaping vertebrate evolution . Hence , the result of these evolutionary processes is that every species possesses a unique repertoire of ISGs . These findings may help explain the differing sensitivities of certain animal species to specific viruses . For example , it has been widely hypothesised that the bat innate immune system has unique features that allow this species to withstand persistent , asymptomatic infection with viruses that are pathogenic in humans [42–44] . Our data reveal that the overall pattern of the bat interferome is relatively unremarkable: they up-regulate the core ISGs , have similar distributions of up-regulated and down-regulated genes , and up-regulate lineage ( order Chiroptera ) , as well as species-specific , ISGs . However , we found that the basal transcription level of the type I interferome ( including the known antiviral ISGs ) to be higher in both the megachiropteran and microchiropteran cells compared to cells from other species ( S5 Fig ) , a finding consistent with the previous observation that the interferon alpha ( IFN-α ) locus is constitutively active in bats [44] . Hence , bat cells might exist in a relatively constitutively active antiviral state . The IRGs were differentially expressed to a conspicuously lower extent than ISGs , although the overall pattern was largely uniform across the species . The ability to assemble lists of shared IRGs allowed us to suggest that epigenetic control via down-regulation of genes involved in acetylation and methylation may be a relatively underappreciated function of the IFN response . For example , we found that ANP32A , a protein involved in acetyltransferase inhibition [30] , was down-regulated by IFN in human , rat , sheep , cow , and pig cells . ANP32A was recently identified as a key cellular cofactor for avian influenza virus ( AIV ) . Indeed , the avian influenza virus polymerase functions relatively poorly in mammalian cells , and this is due , at least in part , to the inability of AIV polymerase to bind efficiently to mammalian ANP32A [31] . It is intriguing that , in our data , chicken ANP32A is not significantly down-regulated by IFN . Our study , like many of a similar nature , relies on the quality of the annotations of the genomes used . Indeed , many ISGs have been shown to have complex orthologies , and it is possible that some genes are misannotated or not yet annotated in the Ensembl Compara database . In order to decrease the impact of this factor in our data , we manually curated all ISGs that were initially identified in at least eight animal species . In addition , the use of primary cells for most species ( in most cases derived from different individuals ) reduced the possibility of artefacts deriving from cells that were passaged extensively in vitro . We also ensured that all RNAseq experiments were carried out in cells in which IFN stimulation resulted in an antiviral state ( see Materials and methods ) . The system-level nature of RNAseq experiments and downstream bioinformatic analyses complicates direct comparisons between distinct studies . MORC3 , for example , was previously suggested to be specifically up-regulated by the megabats , as it was not up-regulated in human A549 cells [45] . By contrast , in our study , we observed MORC3 among the corevert genes , albeit robustly up-regulated in the fruit bat cells ( > 12-fold ) and minimally in human cells ( < 2-fold ) . Similarly , the scope of this study is limited to type I IFN and a single cell type per species . It will be interesting in future studies to observe the differences in interferomes generated by IFN-γ and IFN-λ and , additionally , the variation that exists between cell types . It is notable that the list of species for which we generated interferomes includes wild ( rat , microbat , and fruit bat ) , as well as domestic companion ( dog and horse ) and livestock ( pigs , chicken , cow , and sheep ) , species . We observed clear species- and lineage-specific ISGs for every species examined , which , as more genomes become sequenced , can be explored for evidence of how , for example , the domestication process has influenced the evolution of ISGs . Overall , the dataset described here represents the most comprehensive , cross-species ‘snapshot’ of the IFN response published to date . Our data provide a framework with which it will be possible to test hypotheses pertaining to the role of host innate immunity on virus emergence , cross-species transmission and pathogenesis .
Ex vivo tissue samples were collected postmortem either at commercial slaughterhouses or at the University of Glasgow School of Veterinary Medicine . In all cases , animals had been euthanized according to protocols approved by the local ethical committee and in accordance with the Council of the European Communities Directive of 24 November 1986 ( 86/609/EEC ) . Ex vivo skin samples were collected from chickens ( n = 3 ) , cows ( n = 4 ) , sheep ( n = 3 ) , horses ( n = 3 ) , a dog ( n = 1 ) , and pigs ( n = 4 ) and primary fibroblasts isolated following an explant procedure . Briefly , the hair or feathers were removed from the skin prior to disinfection by soaking in 70% ethanol . After rinsing in PBS , the skin was removed from the underlying tissues , cut into circa 3-mm square explants , and added to cell culture dishes ( without media , squamous surface uppermost ) for one hour at 37°C before adding Dulbecco’s modified Eagle medium ( DMEM ) ( Gibco ) supplemented with 10% fetal bovine serum ( FBS ) ( Gibco ) , 1% penicillin/streptomycin ( p/s ) ( Sigma ) and 100 U/ml nystatin ( Sigma ) . Human primary dermal fibroblasts were purchased from PromoCell ( catalogue number C-12302 ) . Rat primary dermal fibroblasts were purchased from the European Collection of Authenticated Cell Cultures ( ECACC ) ( catalogue number 06090769 ) . M . lucifugus ( little brown bat , representative of the microbats ) primary dermal fibroblast cells , isolated from individuals caught in Oregon , United States of America , were kindly provided by William Kohler [46] . P . vampyrus ( the large flying fox fruit bat , representative of the megabats ) cells ( PVK4 ) are an immortalised kidney cell line kindly provided by Megan Shaw [45] . The origin of each cell used in this study is summarized in S5 Table . Human , rat , and dog cells were cultured in fibroblast growth medium 2 ( PromoCell ) supplemented with 10% FBS and p/s . 293T and PVK4 were all cultured in DMEM supplemented with 10% FBS and p/s . All cell cultures were incubated at 37°C with 5% CO2 in a humidified atmosphere . IFN- or mock-treated cells were challenged with infectious VLPs of envelope minus vesicular stomatitis virus ( VSV-ΔG-GFP ) decorated with a VSV-G envelope ( provided in trans during VLP production ) in order to confirm the antiviral state of the cells at the time of RNA harvest essentially as already described [47 , 48] . Briefly , cells were harvested by trypsinization and fixed in 5% formaldehyde . The number of infected cells in IFN- and mock-treated cells was assessed by flow cytometry . Parallel sets of cells were plated in multiwell plates 24 or 48 hours prior to IFN treatment and incubated at 37°C . Cells were treated with either 1 , 000 U/ml Universal interferon ( UIFN , PBL InterferonSource ) , 200 ng/ml canine IFNα ( Kingfisher ) , 1 , 000 U/ml porcine IFNα1 ( Stratech ) , or 200 ng/ml chicken IFNα ( AbD Serotec ) . Mock treatment was performed in parallel using DMEM lacking IFN . Cells were incubated for the indicated time period at 37°C , washed with PBS , and either lysed in Trizol ( Thermo Fisher ) for RNA extraction or challenged with VSV-ΔG-GFP to assess the antiviral state . Cells were only further processed for RNAseq analyses when they were in an antiviral state . In this study , cells were considered in an antiviral state when IFN stimulation induced at least 75% inhibition of VSV-ΔG-GFP infectivity ( value chosen as average of three independent experiments ) ( S1 Fig ) . Pilot experiments were performed for each cell type in order to optimise conditions necessary for cells to reach an antiviral state . With the exception of dog cells , all cells reached an antiviral state after four hours of IFN treatment . The antiviral state in the primary canine cells isolated in these experiments required 24 hours of treatment with canine IFNα ( S1 Fig ) . For comparative purposes , a complete set of experiments ranging from IFN stimulation to interferome analysis was performed in parallel in pig cells stimulated with either UIFN or porcine IFN-α ( S3 Fig ) . RNA was extracted using Trizol ( Thermo Fisher ) and RNeasy ( Qiagen ) protocols . Briefly , chloroform was added to the RNA-containing phase of the Trizol sample and centrifuged . The aqueous phase was then mixed with ethanol and purified using RNeasy columns , incorporating an on-column DNase step ( Qiagen ) to ensure the complete removal of genomic DNA . Total RNA samples were quantified using the Qubit ( Thermo Fisher ) and were assessed for integrity using the Bioanalyser pico eukaryotic II RNA chip ( Agilent ) . Only samples with an RNA integrity number ( RIN ) value > 9 were taken forward for library preparation . Libraries of mock- and IFN-treated cell RNA were assembled using equal masses of total RNA . The External RNA Controls Consortium ( ERCC ) spike control was added to the total RNA sample in order to assess library quality following sequencing . RNA samples were first enriched for mRNA by selecting poly ( A ) RNA using the Dynabeads mRNA DIRECT Micro purification kit ( Thermo Fisher ) . The eluted RNA was used to generate RNAseq libraries using the Ion Total RNA-seq Kit v2 ( Thermo Fisher ) following the manufacturer’s instructions , with the exception that RNA samples were sheared for just 1 . 5 minutes . Amplified and purified libraries were checked for quality and quantity using the Agilent Tapestation ( D1000 tape ) and Qubit ( hsDNA assay ) . Libraries were run on the Ion Proton ( Thermo Fisher ) according to the manufacturer’s instructions . Raw data were trimmed and assessed for quality using FASTQC ( https://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) . We performed multidimensional scaling ( MDS ) of normalised counts per million data using EdgeR ( Bioconductor ) in order to assess the impact of IFN and assessed biological covariance as a further control for data quality . In order to assess the presence of cell culture contaminants on the transcriptomic data , we used Kraken to assign taxonomic labels to the reads using the MiniKraken database , which contains all complete bacterial , archaeal , and viral genomes in RefSeq [49] . To assess mycoplasma levels in the cell cultures , we used Bowtie2 to map the data to six different strains of mycoplasma known as frequent contaminants of cell cultures ( S4 Fig ) . Reads were aligned to host genomes ( S6 Table ) using a two-step procedure . A first round of mapping used TopHat2 , followed by a second round of mapping using Bowtie2 in an attempt to map the remaining unmapped reads [50] . HTSeq-Count [51] was used to count reads mapping to genes annotated in . gtf files . Genes with <1 read mapping in at least half of the samples were removed prior to differential gene expression analysis using the EdgeR package ( Bioconductor ) [52 , 53] . FDR values were calculated using the Benjamini–Hochberg method . MDS and statistical analyses of the data were performed in R . We utilised the Ensembl Compara database [54] , combined with our expression data , to generate a table of orthologs with the following associated data for each species: Ensembl ID , Gene name , log2FC , and FDR following IFN treatment . The Compara database provides a thorough account of gene orthology based upon whole genomes available in Ensembl and thus provided us with a standardised approach by which to define phylogenetically the clusters of orthologous genes relative to the chicken taken as an outgroup in the orthology assignment . However , certain gene families relating to innate immunity ( for example , the IFITM genes ) have undergone lineage-specific expansion , potentially resulting in genes not being annotated and clustered within the database [55] . To account for the misannotation and absence of genes in Compara , we improved the table by manually checking ( and annotating , if necessary ) genes initially found to be ‘missing’ in either one , two , or three of the 10 species analysed in this study . For this subset of genes , we searched for the presence of an as-yet-unannotated ortholog in the Ensembl genomes using blastn . In cases where a clear ortholog was detected , we included this gene within the appropriate orthogroup . In total , we identified an additional 18 genes that were added to the final table . In cases where a gene was not annotated in the Ensembl genome but a sequence ( or predicted sequence ) for the homologous species was available in NCBI , we mapped the RNAseq reads to the gene of the homologous species using Bowtie2 . The number of reads mapping from each sample was then counted . In cases where an ortholog was present in NCBI from a closely related species , a relaxed Bowtie2 algorithm was first used to map reads to the sequence . The consensus sequence of the resulting contig was then used to count the reads in individual samples using Bowtie2 as above . Differential expression values were then determined using EdgeR having appended the results to the HTseq file . In total , 64 orthologs were added to the table as a result of the orthologous sequence mapping approach . Finally , we modified the . gtf file of sheep to reannotate STAT4 ( to become STAT1 and STAT4 ) and SOCS1 , and manually annotated the ZCCHC2 gene in the cow . gtf file . Using the human genome as a gold standard for annotation , we next assessed the authenticity of each “species-specific” ISG ( i . e . , an ISG present only in one of the 10 species analysed in this study ) based upon its current annotation within the genomes . We first generated a subset of species-specific genes by applying an arbitrary cutoff of their differential expression of ≥2 log2FC . This cutoff resulted in a total of 102 genes across eight species . A total of 33 genes ( 32% ) were in this category as a result of misannotation . In particular , the current little brown bat and pig genomes appear to be currently less well annotated compared to other genomes , as a large proportion ( 73% and 79% , respectively ) of seemingly species-unique genes were in reality misannotations . We used the database-integrated genome screening ( DIGS ) tool [37] to systematically screen the genomes of 111 mammalian species for sequences disclosing homology to 79 of the 90 coremamm ISGs . The peptide sequences of the human copies of these 79 genes ( obtained from BioMart [56] ) were used as probes for tBLASTn-based searches of each species genome . Sequences disclosing above-threshold similarity to peptide probes were extracted and classified by BLASTx-based comparison to a reference library . This library contained , for each ISG , peptide sequences of human paralogs and orthologs from selected additional mammal species . The DIGS tool captures screening results in a relational database , wherein they could be interrogated using structured query language . The number of significant matches for each gene was determined using a gene-specific bitscore cutoff . These counts were normalised to the median value across the mammalian genomes screened to account for variation in exon numbers . Because DIGS is based upon sequence-similarity screening , high counts for a particular gene do not necessarily reflect bona fide gene expansion . Where no matches to a given gene were identified and no ortholog had been annotated in Ensembl , we attempted to confirm that deletion had occurred by viewing the corresponding genomic region in the UCSC and Genomicus genome browsers [57 , 58] and by comparing alignments of the orthologous genomic regions derived from species with and without the deleted gene . Deletions could not always be confirmed mainly due to low coverage or relatively poor quality assembly of the available genomes . Clusters comprising one-to-one orthologs present in the interferome of each species were extracted and filtered to check for the presence of a gene in species X matching a human Ensembl ID . The human Ensembl ID was then used to query BioMart to extract the corresponding pairwise dN and dS values for species X against the human . Values for differential expression ( log2FC ) and FDR values for species X were merged with the dN/dS values and histograms of both significant ( ISG ) and nonsignificant ( non-ISG ) clusters plotted . dN and dS values were not available in Ensembl for the Large flying fox or the chicken . The overall distributions of dN/dS values for ISGs and non-ISGs were compared using the Kruskal–Wallis rank sum test and Wilcoxon rank sum test . In order to allow mining of our data by the wider research community , we created a web-based public interactive data server , accessible at http://isg . data . cvr . ac . uk . The server hosts a database containing the orthologous ISG clusters studied in this paper . This web tool allows users to search for and download orthologous clusters and the associated experimental results . The search may be based on various search criteria: It should be noted that genes can have different aliases to those in Ensembl , and these must be checked if a gene that is not initially present , e . g . , MDA5 , is present as IFIH1 . These aliases are stated in Ensembl . The webserver for querying the dataset is available at http://isg . data . cvr . ac . uk/ . The DIGS blast framework is available at http://giffordlabcvr . github . io/DIGS-tool/ . The raw fastq files generated during this project have been submitted to the European Bioinformatics Institute ( EBI ) under project accession number PRJEB21332 . | The type I interferon ( IFN ) response is triggered upon sensing of an incoming pathogen in an infected cell and results in the expression of hundreds of IFN-stimulated genes ( ISGs , collectively referred to as ‘the interferome’ ) . Studies on the interferome have been carried out mainly in human cells and therefore often lack the power to understand comparative evolutionary aspects of this critical pathway . In this study , we characterized the interferome in several animal species ( including humans ) using a single experimental framework . This approach allowed us to identify fundamental properties of the innate immune system . In particular , we revealed 62 ‘core’ ISGs , up-regulated in response to IFN in all vertebrates , highlighting the ancestral functions of the IFN system . In addition , we show that many genes repressed by the IFN response normally function as regulators of cell transcription . ISGs shared by multiple species have a higher propensity than other genes to exist as multiple copies in the genome . Importantly , we observed that genes have arisen as ISGs throughout evolution . Hence , every animal species possesses a unique repertoire of ISGs that includes core and lineage-specific genes . Collectively , our data provide a framework on which it will be possible to test the role of the IFN response in pathogen emergence and cross-species transmission . | [
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] | 2017 | Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses |
Because firing properties and metabolic rates vary widely , neurons require different transport rates from their Na+/K+ pumps in order to maintain ion homeostasis . In this study we show that Na+/K+ pump activity is tightly regulated by a novel process , RNA editing . Three codons within the squid Na+/K+ ATPase gene can be recoded at the RNA level , and the efficiency of conversion for each varies dramatically , and independently , between tissues . At one site , a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine , a change that shifts the pump's intrinsic voltage dependence . Mechanistically , the removal of a single methyl group specifically targets the process of Na+ release to the extracellular solution , causing a higher turnover rate at the resting membrane potential .
Within the animal kingdom , the Na+/K+ ATPase is a nearly ubiquitous membrane protein that uses the free energy of ATP hydrolysis to establish and maintain the Na+ and K+ gradients across cell membranes . Na+/K+ pump activity is essential . Without it cells would lack the driving force required for excitability and Na+-coupled transport of solutes in and out of the cell . Because the Na+/K+ pump is costly to operate , using ∼30% of the ATP generated by an organism [1]–[4] , proper regulation of its turnover rate is critical . The Na+/K+ pump is an electrogenic machine , its activity being directly influenced by the transmembrane potential . Turnover rates are maximal at potentials greater than ∼0 mV and decline steadily at negative potentials . This inhibition results from a combination of effects . High extracellular Na+ concentration and negative potentials both tend to drive Na+ back to its binding sites deep within the protein's core [5]–[9] . Interestingly , nature has tuned pump activity so that it is inhibited to a similar extent , irrespective of an organism's ionic environment . For example , at the resting potential Na+/K+ pumps from squid , frogs , or guinea pigs operate at ∼50% activity in the face of drastically different extracellular Na+ concentrations [10]–[13] . It is reasonable to hypothesize that by limiting pumping at negative potentials , activity could be upregulated during periods of heightened activity in order to meet the demands of ion homeostasis . In the nervous system of higher metazoans , RNA editing by adenosine deamination has evolved as an important mechanism for the diversification of the proteome . By removing a single amine that participates in Watson-Crick base-pairing , specific adenosines are converted to inosines within mRNAs and other RNAs . For editing sites within the coding sequence of mRNAs , inosine is read as guanine during translation , causing codons , and protein structure , to change . In both vertebrates and invertebrates , editing targets mRNAs that encode proteins directly involved in action potential conduction and synaptic transmission , and therefore it is assumed that the process is important for regulating rapid electrical signaling [14]–[22] . For some editing sites the specific changes to protein function have been described , however very little is known about their mechanisms of action . In fact , there are just a couple of cases in which we know how edits alter protein function [18] , [21] . In this study we show that RNA editing may regulate ion homeostasis by making specific changes within Na+/K+ pump mRNAs . These changes affect the Na+/K+ pump's intrinsic voltage dependence . Mechanistically , this is achieved by shifting the occupancy of the states of the transport cycle associated with the release of Na+ .
Historically , the Na+/K+ ATPase of the squid giant axon has been one of the most actively studied native pumps . In a previous report we identified the mRNA sequences for the underlying α ( EF467998 ) and β ( EF467996 ) subunits [10] . Because other squid transcripts are regulated by RNA editing [17] , [20] , we examined whether the Na+/K+ ATPase mRNAs were as well . Sequences of 50 individual cDNA clones for the squid NaKα1 subunit , isolated from the giant axon system , showed adenosine-or-guanine variation at specific sites , a hallmark of RNA editing . To explore whether this variation was indeed due to RNA editing , we cloned the gene that encodes squid NaKα1 mRNAs ( Figure 1A ) . The squid NaKα1 gene , which spans over 20 KB , is highly fragmented , containing 19 exons . At four positions , the gene sequence contains an A whereas some or all of the cDNA sequences contain a G ( e . g . , Figure 1B ) . Three of the sites lie at the junction with a nearby intron , as is commonly the case with other RNA editing sites ( Figure 1C ) [23] . Two sites lie within the same codon . Because both were guanosine in all cDNAs sequenced , the lysine at this position was always converted to glycine . To further support the idea that the A→G conversions are caused by RNA editing , we tested whether a squid editing enzyme ( SqADAR2 . 1A ( FJ478450 . 1 ) ; [24] ) could edit these codons in vitro ( Figure S1A ) . Using the genomic form of the full-length , mature squid NaKα1 mRNA as a substrate , recombinant SqADAR2 . 1A could edit all four codons . It is notable that all the information required for editing resides within the exons and that intron sequence was not required , as is commonly the case for other editing sites . Similarly , the structure that drives editing of human Kv1 . 1 channel mRNAs is entirely exonic [18] , [25] . Interestingly , human ADAR2 ( BC065545 . 1 ) can also edit codons K666G and I877V , but not R663G . Predicted folds for NaKα1 mRNA using MFOLD software show an obvious hairpin surrounding the I877V codon ( Figure S1B ) . Using this approach , similar structures are not apparent around codons R663G and K666G . In any case , the combination of our cloning data and the in vitro editing assays verify that the A/G variation observed in Na+/K+ ATPase mRNAs is due to RNA editing . The editing sites R663G and K666G are located within the phosphorylation domain which accepts ATP's γ phosphate during the transport cycle , while the I877V edit lies at the extracellular end of the seventh transmembrane segment . All editing sites recode a highly conserved amino acid ( R663G , K666G , and I877V; Figure 1D ) . In fact , a survey of over 200 NaKα1 cDNA sequences from both vertebrates and invertebrates shows the unedited codon at these positions to be almost invariant . There are a few exceptions , however . The ovine and bovine NaKα1 sequences ( emb CAA26582 . 1 and gb AAI23865 . 1 ) , and a NaK sequence from planaria ( dbj BAA32798 . 1 ) , have an arginine at codon 666 , conceivably being produced by editing at the codon's second position ( AAR→AGR ) . At position 877 , an electric eel NaK cDNA is the only sequence with a valine . As with squid , this could have been caused by RNA editing . Overall , however , our bioinformatics search uncovered little evidence of editing in distantly related organisms . Because of this we tested whether the squid sites are edited in another cephalopod . Using squid specific primers , NaKα1 cDNA and genomic DNA was amplified from Octopus bimaculata collected from Catalina Island , CA . Based on 50 individual cDNA clones , the R663G edit was edited , but at a much lower rate than in Loligo ( 12% versus 96% ) . No editing was apparent in codons K666 or I877 . These data suggest that editing sites are evolving rapidly within cephalopods . In the giant axon , R663G and K666G are edited almost to completion while I887V is scarcely edited . Why undergo a complex process such as editing when a simple mutation to the gene would produce much the same result ? One possibility is that these sites are used for regulating pump function . If this is the case , we would expect the extent of editing at these sites to differ between neuronal tissues . To test this idea , we collected tissue from 10 different regions of the nervous system , both central and peripheral . Using a poison-primer extension assay [26] , we estimated the editing efficiency in each sample ( Figure 2A ) . The extent of variation differed dramatically between sites ( Figure 2B ) . R663G varied only from ∼65%–85% . Editing at codon 666 was more complicated . Because it can be incompletely edited at the first two positions ( AAG ) , a mixed population of pumps with either arginine , glycine , or lysine ( unedited ) can result . In some tissues , as in the giant axon , K666G predominates , while in others K666R or K666 is the dominant species . Although K666E is theoretically possible ( GAG ) , this edit was never observed . The I877V edit is also highly tissue- specific . Barely present in the giant axon system and other peripheral regions , it occurs close to 50% of the time in parts of the central nervous system such as the Inferior Frontal Lobe neurons . These results strongly suggest that RNA editing could be used to regulate Na+/K+ pump function . For any cell maintaining ion homeostasis , the most important aspect of Na+/K+ pump function is the velocity of ion transport . Accordingly , we were interested in determining whether any of the RNA editing events regulates the Na+/K+ pump's turnover rate . Because the Na+/K+ pump is electrogenic and its stoichiometry does not change with voltage [12] , the pump current ( Ip ) is an accurate reflection of the turnover rate at any voltage . Under physiological conditions , the voltage dependence of Ip is approximately sigmoid , reaching a maximum at positive potentials and approaching zero at very negative potentials . We first measured the maximum turnover rate for the unedited pump and all single edited versions ( Figure S2 ) . To estimate this parameter we expressed these constructs in Xenopus oocytes and measured Ip at positive voltages , where it reaches its maximum , while estimating the pump density in the same oocytes . The maximum turnover rate for the unedited pump is 27 . 0±4 . 7 cycles·s−1 ( at 22°C ) . None of the rates determined for the edited versions differed significantly from the unedited construct when compared at the same temperature . Thus , at positive voltages , editing has little effect . The Na+/K+ pump's turnover rate at negative voltages , where it is partially inhibited , is a more relevant measurement because the pump predominantly operates over these potentials . Next we investigated whether editing affects the voltage dependence of the pump's transport velocity . To illustrate our approach , Figure 3A shows a current record of the entire experiment recorded on a slow time scale . The oocyte is held under voltage clamp at 0 mV , where the Ip is maximal . The rapid vertical deflections are the current changes in response to 40 ms voltage pulses from the holding potential to various potentials between −198 mV and +42 mV ( in 10 mV increments ) . After each step the voltage was returned to the holding potential . The voltage protocol was repeated in each experimental condition to verify the stability of the preparation . After the application of 100 µM ouabain , the current trace visibly becomes smaller due to inhibition of Ip . To isolate Ip , current traces after ouabain application ( 3 ) were subtracted from those before ( 2 ) . Figure 3B shows an example of these traces at the extreme voltages ( −198 mV in gray , +42 mV in black ) . Steady-state Ip ( arrow ) was determined at all voltages by averaging the final 5 ms of each trace , after the transients had settled . Similar measurements were performed for the unedited pump , and all single edited versions . I877V differed substantially from the unedited version . Figure 3C shows the normalized voltage dependence of the pump velocity for I877V and the unedited pump . The principal effect of I877V is to shift the Ip-V curve ∼25 mV to more negative potentials , thereby relieving voltage dependent inhibition . Because there is ∼2-fold less extracellular Na+ in oocyte strength solutions than in those used for squid , both curves would be shifted approximately 60 mV to the right , as we have previously shown [10] . From this we estimate that I877V would significantly increase Ip at the resting potential , which is ∼−60 mV in the squid axon [27] . Under physiological conditions the pump's voltage dependence comes mostly from the transitions underlying extracellular Na+ release [5] , [6] , [8] , [10] , [28]–[30] . Therefore , these results suggest that the I877V edit targets this process . To better understand the mechanism by which I877V shifts the Na+/K+ pump's Ip-V relationship , we studied the process of external Na+ binding/release and occlusion/deocclussion in isolation by removing all K+ and maintaining the intracellular ATP concentration at high levels ( Figure 4A ) . As before , the membrane was stepped to a wide range of potentials and stability was assessed by repeating voltage protocols in each condition ( Figure 4B ) . Under these ionic conditions ouabain sensitive currents contain only transient components , reflecting the redistribution of external Na+ between occluded and deocluded states ( see Figure 5A ) . Examples of these currents for the unedited pump at three potentials are given in Figure 4C . Analysis of these traces shows that there are three kinetic components , as in the squid axon where each is thought to reflect the sequential release of one of the three Na+ [6] , [8] . We first focused on the slowest component ( τ∼12 ms at 0 mV ) because it tracks the rate-limiting transition for Na+ release and is therefore responsible for determining the Ip-V relationship's shape . Its voltage-dependence was estimated by integrating the slow component of the off transients and the results are plotted in Figure 4D . As with the steady-state pump currents ( Figure 3C ) , the I877V edit shifts the charge distribution 32 mV towards more negative potentials , indicating that the voltage dependence of the distribution between ( Na3 ) E1-P and P-E2 ( Na2 ) Na states has been targeted . Is this due to a change in rates associated with this transition ? The rate constants between these two states can be estimated by fitting the kinetics of the slow component to a simple model , derived from a Hill equation , that has been used to describe this transition in pumps from a variety of preparations [5] , [8] , [10] , [31] , including the squid clone expressed in Xenopus oocytes ( Figure 4E ) . Conceptually , the model reduces Na+ release to two basic steps: a slow voltage independent conformational change between the occluded and deoccluded states , and a rapid redistribution of ions across a narrow pore that spans part of the membrane's electrical field , which is the step that renders the process voltage dependent [5] , [8] , [31] . In this model , the relaxation rates reach asymptotes at extreme voltages . At positive potentials , the relaxations approach the forward rate , while at negative potentials they approach the sum of the forward and backward rates ( see legend for Figure 4 ) . The steepness of the curve is largely determined by the electrical depth of the access channel , a value that is unchanged by the I877V edit . Data in Figure 4E show that the forward rates for both constructs reach a similar asymptote at positive voltages and fits to the model indicate that the backwards rates do so as well . The small changes that I877V does cause to these rate constants are not sufficient to account for the shift in the voltage dependence of Ip . Of greater significance , the model predicts that I877V considerably reduces the apparent affinity for extracellular Na+ , a change that could be caused by very different physical factors . Because the pump's cation binding sites are thought to be far from position 877 , it is unlikely that this edit directly reduces the pump's affinity for Na+ . In addition , the amino acid change caused by the edit is conservative , making it unlikely that it changes the electrostatics along the ion permeation pathway [32] , [33] , another mechanism that could plausibly affect the apparent affinity [10] , [34]–[37] . An alternative that is more consistent with the amino acid change is that I877V shifts the state occupancy from deeply occluded states towards those that favor release . Which transitions does I877V affect ? Just as the transition from ( Na3 ) E1-P↔P-E2 ( Na2 ) ·Na can be tracked by the slow component of the relaxations , the transitions between P-E2 ( Na2 ) ·Na↔P-E2 ( Na ) Na and P-E2 ( Na ) ·Na↔P-E2·Na can be tracked by the medium ( τ∼1 . 5 ms at 0 mV ) and fast components ( τ<200 µs at 0 mV ) , respectively ( Figure 5A , C ) . In order to estimate the transition rates between these states we would have to accurately measure the kinetics of each component . In our experimental set-up this is not possible because the time constant of the fast component is comparable to that of the clamp . However , by focusing on the proportion of charge carried by each component in the off transients , we can get a snapshot of the state occupancy during the conditioning pulse ( Figure 5C ) . A visual inspection of off transients following a prepulse to −58 mV shows a clear difference caused by I877V ( Figure 5B ) : the fast component is more pronounced and the slow component is reduced . A more rigorous quantification over a broad range of voltages shows that the reduction of the slow component in I877V comes at the expense of the fast component , a trend that is particularly apparent at positive voltages ( Figure 4D ) . The medium component , on the other hand , carries about the same proportion of charge in both pumps at all voltages . From these data we conclude that I877V selectively targets the release of the last Na+ ( fast component ) , thereby shifting the entire equilibrium towards release .
This study demonstrates that RNA editing could have broader physiological impacts than previously supposed , affecting processes outside of fast electrical signaling [14] . Although a large number of editing sites have now been identified in both vertebrates and invertebrates [14] , [38] , in few cases have their mechanistic consequences been worked out . This is probably because they often create but subtle changes . For example , in a human K+ channel editing specifically targets the process of fast inactivation by the removal of a single methyl group in the pore cavity [18] . Here , by making the same change ( I→V ) , editing selectively alters the process of external Na+ release , primarily by increasing the occupancy of the states associated with the extracellular release of the final Na+ . By consequence , the voltage dependence of the transport process is shifted to negative potentials , increasing the Na+/K+ pump's turnover rate over the physiological range . Taking into account the extent of Na+-dependent inhibition for marine osmoconformers ( i . e . those that are isotonic with sea water; [10] ) , from these data we estimate that the I877V edit would cause the Na+/K+ pump's turnover rate to increase by ∼40% at the resting potential . For an organism , it is particularly important to carefully regulate Na+/K+ pump function because of the vast quantity of energy that it consumes . Previous reports have hypothesized that RNA editing fine tunes the nervous system by making small , specific changes to protein function [39]–[41] and our results support this idea . The process of external Na+ release is voltage dependent because of the pump's architecture . After unbinding from the protein , Na+ must exit through a narrow access channel , not unlike the pore of an ion channel , which spans part of the transmembrane electric field . No matter what the ionic environment , evolution has tuned this structure so that Na+/K+ pumps are inhibited by extracellular Na+ and negative voltages to a similar extent . Why depress activity ? An obvious possibility is that the Na+/K+ pump's voltage dependence allows for the turnover rate to be adjusted . Here , using a heterologous expression system , we show that it is indeed a target . By editing Na+/K+ pump mRNAs , the turnover rate could be precisely adjusted in different neurons , presumably to meet the specific demands of ion homeostasis . An I877V mutation in the Na+/K+ pump gene , on the other hand , would uniformly change the physiology of all the pumps it encodes . The molecular data presented in this study clearly demonstrate that pump structure is being regulated in a tissue specific manner . The idea that these changes alter function in response to metabolic requirements is supported by the I877V edit electrophysiological data from oocytes . This site is robustly edited in multiple regions of the central nervous system , areas composed of small neurons with presumably high rates of firing [42] , [43] . I877V is scarcely edited in the giant axon , a structure known to fire at very low rates in vivo [44] . It is worth noting that out of over 250 Na+/K+ pump sequences from different organisms that we surveyed , all but one have an Isoleucine at this position . Only the sequence from the electric organ of the electric eel , an organ exceptionally rich in Na+ channels , has a valine at position 877 [45] . Editing at codons R663 and K666 is also regulated between tissues . Although our electrophysiology approach did not uncover a physiological role for these sites , they could certainly be important for regulating an aspect of Na+/K+ pump physiology not related to ion transport . In both vertebrates and invertebrates , RNA editing plays an important role in diversifying the protein structure and function . Along with other recent reports , these data show that a surprisingly wide variety of cellular functions can be tuned by editing [46] , [47] . Further studies using squid neurons will allow us to directly assess the role that editing plays in regulating ion homeostasis .
The initial cloning of the SqNaKα1 cDNA , which was edited at R663G and K666G but not at I877V , has been described in detail in a previous report [10] . In brief , degenerate PCR primers based on conserved regions of Na/K pump α subunits were used to amplify a partial cDNA fragment from Loligo . The full-length sequence was determined by 5′ and 3′ RACE and a full-length clone was isolated by PCR using a high fidelity polymerase . For this study , an adult Loligo opalescens specimen was collected from Monterey , CA . RNA was extracted from the two giant fiber lobes , which were manually separated from the rest of the stellate ganglion , and used to synthesize cDNA . Full-length SqNaKα1 cDNAs ( genbank EF467998 ) were then amplified using Phusion DNA polymerase ( New England Biolabs ) and 50 individual clones were sequenced . To isolate the SqNaKα1 gene , genomic DNA was extracted from the gill of the same animal that was used for the cDNA . A genomic library was then made using a Fosmid vector system ( EpiFos Copy Control library , Epicentre technologies , UK ) . To construct the library , ∼40 kb pieces of genomic DNA were size selected by pulsed-field gel electrophoresis , packaged , and then transformed into E . coli . Using 32P end-labeled oligos complementary to SqNaKα1 cDNA as hybridization probes , a single positive colony was isolated and sequenced to completion . This clone contained all but the first 138 bp of the cDNA sequence and portions of the first intron . The rest of the genomic sequence was isolated by PCR . Mutagenesis was performed by a standard PCR-based strategy using mutant oligonucleotides . All mutants were generated using Pfu DNA polymerase and verified by DNA sequencing . The poison primer extension assay used to measure editing efficiencies has been described in detail [26] . The following oligonucleotides , all labeled with 5′ Hexachlorofluorescein , were used for the assay: CATTCCAGTCGATCAAGTTAATTC with AcycloG for R663G , GTCGATCAAGTTAATTCAAGGGA with AcycloA for the first edited adenosine of K666G , TCAAGTCGGTTCCATGGATTACAGCT with AcycloT for the second edited adenosine of K666G , and CGCTGGATTTTTCACCTATTTTG with AcycloG for I877V . Functional expression of squid Na+/K+ pumps in Xenopus oocytes has been described before . Na+/K+ pump charge translocation and current-voltage relationships were studied using the cut-open oocyte technique sampling exclusively from the animal pole . Oocytes were clamped with a Dagan CA-1B high performance oocyte clamp . An Innovative Integrations SBC6711 board with the A4/D4 module and GPATCH software ( kindly provided by Dr . Francisco Bezanilla ) were used to control voltage and to digitize analog signals . Data were acquired at 100 kHz and filtered at 20 kHz . Intracellular voltage was measured with a 0 . 2–0 . 3 MΩ pipette filled with 3M NaCl and bridges were filled with 3M Na-MES in 3% agarose . Oocytes were permeabilized with 0 . 2% Saponin in internal solution . The internal solution used for all experiments contained ( in mM ) : 80 Na-Glutamate , 20 TEA-Glutamate , 10 MgS04 , 10 Hepes , 5 EGTA , 5 Na-ATP , pH 7 . 5 . The 5K external solution contained: 100 Na-Glutamate , 5 K-Glutamate , 5 BaCl2 , 2 NiCl2 , 5 HEPES , 2 MgCl2 , 0 . 3 Niflumic acid , pH 7 . 5 . The 0 K external solutions contained: 100 Na-Glutamate , 5 NMG-Glutamate , 5 BaCl2 , 2 NiCl2 , 5 HEPES , 2 MgCl2 , 0 . 3 Niflumic acid , pH 7 . 5 . Oocytes were stepped from a holding potential of 0 mV to voltages between −198 mV and +42 mV for 40 ms before returning to 0 mV . This protocol was repeated before and after the application of ouabain to yield the ouabain sensitive component . The off transient resulting from the return to 0 mV was used for further analysis . Traces were fit to three exponentials . The charge moved by the slow and medium components was determined by multiplying the time constant and the amplitude obtained from the fits . The fast component was isolated by subtracting the fits of the medium and slow components from the current trace . The charge moved by the fast component was estimated by numerical integration of the subtracted trace . For prepulse voltages between −198 mV and −88 mV , the values of the time constants for the slow and medium components were left as free parameters during the fits . For all other voltages , where the amplitudes of these components are much smaller , the time constants were fixed to their average values between −198 mV and −88 mV . | In order for excitable cells like neurons and muscles to generate electrical signals , they require ion gradients across their plasma membranes . For example , sodium concentrations are much lower inside a cell than outside , and for potassium it is the opposite case . The job of maintaining these ion gradients falls squarely on a single protein: the Na+/K+ pump . During each transport cycle , this enzyme moves three sodium ions out of the cell and imports two of potassium . Because this process is the foundation for so many physiological processes , the Na+/K+ pump is costly to operate , using ∼30% of the ATP generated by an organism . Proper regulation of its turnover rate is vital . In this work , we use the giant nerve cell of squid as a model to show that the Na+/K+ pump can be regulated by an unsuspected mechanism . Although the gene that codes for this enzyme can make a perfectly functional pump , sometimes its information changes as it passes through the messenger RNA . This is achieved by editing RNA and as a result subtly different versions of the pump can be made , differing at only three amino acids out of more than a thousand . We demonstrate that RNA editing modulates the Na+/K+ pump's turnover rate and sodium release . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [
"physiology/integrative",
"physiology",
"molecular",
"biology",
"neuroscience/neuronal",
"and",
"glial",
"cell",
"biology",
"physiology/neural",
"homeostasis"
] | 2010 | Regulation of Na+/K+ ATPase Transport Velocity by RNA Editing |
TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 ( TFL2/LHP1 ) is the only Arabidopsis protein with overall sequence similarity to the HETEROCHROMATIN PROTEIN 1 ( HP1 ) family of metazoans and S . pombe . TFL2/LHP1 represses transcription of numerous genes , including the flowering-time genes FLOWERING LOCUS T ( FT ) and FLOWERING LOCUS C ( FLC ) , as well as the floral organ identity genes AGAMOUS ( AG ) and APETALA 3 ( AP3 ) . These genes are also regulated by proteins of the Polycomb repressive complex 2 ( PRC2 ) , and it has been proposed that TFL2/LHP1 represents a potential stabilizing factor of PRC2 activity . Here we show by chromatin immunoprecipitation and hybridization to an Arabidopsis Chromosome 4 tiling array ( ChIP-chip ) that TFL2/LHP1 associates with hundreds of small domains , almost all of which correspond to genes located within euchromatin . We investigated the chromatin marks to which TFL2/LHP1 binds and show that , in vitro , TFL2/LHP1 binds to histone H3 di- or tri-methylated at lysine 9 ( H3K9me2 or H3K9me3 ) , the marks recognized by HP1 , and to histone H3 trimethylated at lysine 27 ( H3K27me3 ) , the mark deposited by PRC2 . However , in vivo TFL2/LHP1 association with chromatin occurs almost exclusively and co-extensively with domains marked by H3K27me3 , but not H3K9me2 or -3 . Moreover , the distribution of H3K27me3 is unaffected in lhp1 mutant plants , indicating that unlike PRC2 components , TFL2/LHP1 is not involved in the deposition of this mark . Rather , our data suggest that TFL2/LHP1 recognizes specifically H3K27me3 in vivo as part of a mechanism that represses the expression of many genes targeted by PRC2 .
Spatial and temporal patterns of gene transcription are central to the developmental programs of plants and animals . Transcriptional repression plays a major role in creating and stabilizing these patterns . In plants , roles for transcriptional repression in reproductive development have been extensively studied . Proteins that repress transcription of genes that promote flowering or confer floral organ identity were identified by analysis of early-flowering mutants , and several of these proteins were predicted to play roles in chromatin regulation [1–7] . For example , CURLY LEAF ( CLF ) and EMBRYONIC FLOWER 2 ( EMF2 ) are both required for the repression of floral organ identity genes and are homologues of Drosophila Enhancer of zeste ( E[z] ) and Suppressor of zeste 12 ( Su[z]12 ) , respectively , two core components of Polycomb repressive complex 2 ( PRC2 ) [8–10] . In Drosophila and mammals , PRC2 catalyzes the tri-methylation of lysine 27 of histone H3 ( H3K27me3 ) of nucleosomes widely located across target developmental genes [11 , 12] . This tri-methylation is then proposed to be recognized by the chromodomain of Polycomb , a central component of PRC1 [13] , which maintains the stable transcriptional repression of target genes , although precisely how PRC1 operates is unclear . Despite the importance of PRC2 in the regulation of gene expression in plants , plant genomes do not appear to encode homologues of the metazoan PRC1 complex [8] . Here we focus on the Arabidopsis protein TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 ( TFL2/LHP1 ) , which was implicated in the repression of flowering and chromatin regulation but is unrelated to known animal PRC1 or PRC2 components [5 , 6 , 14] . Mutations in TFL2/LHP1 cause a range of developmental defects , including early flowering , reduced stability of the vernalized state , conversion of the shoot apical meristem to a terminal flower , curled leaves , and reduced root growth [5 , 6 , 14] . In addition , mutant Arabidopsis display constitutively altered glucosinolate levels and are unable to respond appropriately to heat-shock [15] . The early-flowering phenotype of tfl2/lhp1 mutants results from increased expression of the floral promoter FLOWERING LOCUS T ( FT ) [6] . The instability of vernalization observed in tfl2/lhp1 mutants is due to unstable repression of the floral repressor FLOWERING LOCUS C ( FLC ) [16 , 17] , and it has been proposed that TFL2/LHP1 may have a PRC1-like role in the maintenance of PRC2-mediated repression of FLC [17] . Finally , the curled-leaf phenotype of tfl2/lhp1 mutants is correlated with ectopic expression of the floral organ identity genes AGAMOUS ( AG ) and APETALA3 ( AP3 ) [6 , 18] . These results suggest that TFL2/LHP1 represses transcription of genes that act during different stages of reproductive development . A more general role of TFL2/LHP1 in gene regulation is also suggested by the many misregulated genes detected in a partial transcriptome analysis of tfl2/lhp1 seedlings [19] . TFL2/LHP1 is the only Arabidopsis protein that shows homology to HETEROCHROMATIN PROTEIN 1 ( HP1 ) of metazoans and S . pombe [5 , 6] , and based on the analysis of EST collections it appears to be widely conserved as a single-copy gene in plants . HP1 proteins are characterized by the presence of two conserved domains , the chromodomain and the chromo-shadow domain , and exist in multiple isoforms in metazoans [20] . As their names imply , the first members to be isolated ( HP1a and b in Drosophila , HP1α and β in mammals ) are enriched in heterochromatic regions . These HP1 isoforms , which are involved in the formation and maintenance of heterochromatin , but also participate in the regulation of heterochromatic and euchromatic genes , are believed to associate with target sites via the interaction of their chromodomain with di- or tri-methylated lysine 9 residues of Histone 3 ( H3K9me2 or 3 ) [21–24] , although alternative mechanisms of HP1 association likely exist [25–27] . In contrast to HP1a , b/ α , β isoforms , Drosophila and mammalian HP1c/γ isoforms appear to localize predominantly to euchromatic sites , where they either repress or activate genes through unknown mechanisms [28] . Similar to HP1c/γ , cytological localizations performed with transgenic plants indicate that TFL2/LHP1 is localized primarily in euchromatin , with little or no association with cytologically visible heterochromatin [19 , 29] . Taken together , these observations suggest that TFL2/LHP1 associates with genes present in euchromatin , and indeed such an association has recently been documented for AG , AP3 , FT , PISTILLATA ( PI ) , and FLC [16 , 17 , 30] . In order to gain deeper insight into the nature and chromosome-wide distribution of TFL2/LHP1 target sites and their associated histone marks , we have performed chromatin immunoprecipitation with antibodies that recognize epitope-tagged TFL2/LHP1 and hybridized the precipitated DNA to a DNA tiling array of the entire Arabidopsis Chromosome 4 . We demonstrate that TFL2/LHP1 associates with hundreds of targets across this chromosome , the vast majority of which correspond to genes located within euchromatin . Furthermore , we show that although TFL2/LHP1 binds to H3K9me2 , H3K9me3 and H3K27me27 peptides in vitro , in vivo TFL2/LHP1 associates almost exclusively and nearly co-extensively with H3K27me3 . Moreover , the absence of noticeable changes in the distribution of H3K27me3 along Chromosome 4 in lhp1 mutant plants indicates that TFL2/LHP1 is not involved in the deposition of this mark . Rather , TFL2/LHP1 specifically associates with H3K27me3 in an in vivo context , indicating that it is involved in a general mechanism of gene regulation mediated by PRC2 .
To define the genomic regions that TFL2/LHP1 directly associates with , chromatin immunoprecipitation ( ChIP ) experiments were conducted using transgenic plants expressing a functional HA-tagged version of the protein ( see Materials and Methods ) . PCR analysis of ChIP DNA recovered from seedlings indicated a clear association of TFL2/LHP1 with the putative target gene FT , but not with the WRKY33 gene taken as a negative control ( Figure 1A ) . This association was not restricted to a single site at the FT locus , but spanned the ~700-bp region probed around the transcriptional start site ( Figure 1B , black bars ) . This broad localization was not due to low resolution of the ChIP , because association of the MADS-box transcription factor FLC to the same region of the FT locus resulted in a well-defined and specific enrichment over the first intron , as reported previously [31 , 32] ( Figure 1B , white bars ) . On the basis of these results , we performed a chromosome-wide analysis of TFL2/LHP1-associated regions by hybridizing ChIP DNA to a Chromosome 4 tiling DNA microarray . The array covers the 19-Mb Arabidopsis Chromosome 4 sequence as well as several other genomic regions in the form of 21 , 761 sequential 0 . 3–1 . 2-kb fragments ( [33] , see Materials and Methods ) . Chromosome 4 represents a good model for the 125-Mb Arabidopsis genome , which consists of five chromosomes that vary less than 2-fold in length and have similar sequence features . Importantly , unlike most array designs , which exclude repeats , these were included in order to interrogate repetitive as well as unique genomic sequences . Out of the ~21 , 000 tiles that could be analyzed reliably on the array , 1 , 713 showed robust association with TFL2/LHP1 ( see Materials and Methods and Figure S1 for details of the analysis ) . In the majority of cases , TFL2/LHP1 association was observed over at least two contiguous tiles , as expected from the resolution provided by the array ( 0 . 3–1 . 2 kb ) and the mean size of ChIP fragments ( ~800 bp ) . TFL2/LHP1 domains are 3 . 6 kb long on average , with the vast majority being less than 6 . 5 kb long ( Figure 2A , left panel ) . Most domains are located evenly within the euchromatic parts of Chromosome 4 ( Figures 2B and S2 ) , consistent with cytological observations [19 , 29] . Of note , the 15 largest domains ( 10–27 . 5 kb ) are all located within euchromatin , and may correspond to the granules observed over the diffuse , euchromatic-specific localization of TFL2/LHP1:GFP fusion proteins [19 , 29] . Strikingly , almost all TFL2/LHP1 domains coincide with genes and their flanking sequences ( Figures 2 and S2 ) . On average , preferential targeting of TFL2/LHP1 to proximal promoter regions and 5′ ends of target genes was observed ( Figure 2D ) . Furthermore , the 603 TFL2/LHP1-target genes , which represent 15 % of the genes on the tiling array , showed no skewing towards any particular size class ( Figure 2A , right panel ) . This observation contrasts with Drosophila HP1a , for which a clear preferential association was found with long genes , both in pericentric and nonpericentric regions [34] , and towards the body and the 3′ end of transcription units [27] . Taken together , our results demonstrate that TFL2/LHP1 interacts with the chromatin of numerous individual transcriptional units that are located evenly along the repeat-poor regions of the Arabidopsis genome . There are 249 sets of tandemly repeated genes on Chromosome 4 [35] , corresponding to 679 genes . Thirty percent of these genes are present in the list of TFL2/LHP1 targets , suggesting that they are overrepresented . This was still observed when we excluded from the analysis all tiles that had more than one high-score BLAST hit against the Arabidopsis genome , thus ruling out potential cross-hybridization between highly related sequences as the sole cause of overrepresentation ( Figure 3A ) . Furthemore , segmentally duplicated genes were not similarly enriched ( Figure 3A ) . To explore further the association between TFL2/LHP1 and genes in large tandemly repeated gene clusters , the tandem array of nine glycosylase-18 genes present on Chromosome 4 was analyzed by ChIP-PCR ( Figure 3B ) . Using gene-specific primer pairs , the association of TFL2/LHP1 with each gene of the tandem array was confirmed ( Figure 3B ) . Moreover , IP/INPUT ratios measured by PCR were in agreement with the ChIP-chip data , demonstrating that cross-hybridization was not responsible for the observed broad distribution of TFL2/LHP1 over the glycosylase-18 gene cluster . The Arabidopsis Gene Ontology resource ( http://arabidopsis . org ) was used to categorize genes targeted by TFL2/LHP1 according to their functions . While most categories of “biological processes” were represented among TFL2/LHP1 targets , several categories were significantly enriched or depleted in comparison to their representation on Chromosome 4 ( Figure 3C ) . Thus , protein metabolism ( 3 . 1% versus 6 . 7% ) , cell organization and biogenesis ( 0 . 7% versus 1 . 9% ) , response to stress ( 0 . 9% versus 2 . 5% ) , and environmental stimuli ( 0 . 8% versus 4 . 0% ) are underrepresented , while transcription ( 4 . 9% versus 3 . 0% ) , other biological processes ( 9 . 2% versus 5 . 7% ) , and electron transport or energy pathways ( 3 . 0% versus 1 . 8% ) are all overrepresented . These data demonstrate that TFL2/LHP1 is involved in the regulation of a wide range of biological processes and establish or confirm its association with key developmental regulators such as AG and KNAT1 , which are located on Chromosome 4 , as well as AP3 , FLC , LEAFY , and MEDEA ( MEA ) , which are located on other chromosomes but are represented on the tiling array ( Tables S1 and S2 ) . To analyze the characteristics of TFL2/LHP1 targets further , expression levels were estimated using a comprehensive list of public microarray data compiled at AtGenExpress [36] . Over 80% of them have low or undetectable expression levels in most conditions tested , unlike the majority of other genes present on Chromosome 4 ( Figure 3D and 3E ) . However , some targets are expressed to high levels in specific organs and developmental stages , such as seeds , flowers , apices , or roots ( Figure 3E ) . Similarly , expression of most genes within the tandem array of glycosylase-18 genes is below detection level , unlike that of unrelated flanking genes , which are not associated with TFL2/LHP1 ( Figure 3F ) . Finally , the set of genes with which TFL2/LHP1 is associated was compared with those previously shown to exhibit altered expression patterns in the tfl2 mutant [19] . Of the 41 genes present on the tiling array that are differentially expressed in tfl2–2 , nine are associated with TFL2/LHP1 , and of these , seven show increased expression , including AG and AP3 . The 32 remaining genes exhibit non-significant IP/INPUT ratios , suggesting that they are indirect targets of TFL2/LHP1 ( data not shown ) . By analogy with animal HP1a , b/α , β and the S . pombe homologue Clr4 , it was originally proposed that TFL2/LHP1 recognizes chromatin marked by dimethylation of histone H3 lysine 9 [37] . However , both genetic and cytological evidence have since argued against an association of TFL2/LHP1 with H3K9me2 in plant cells [19 , 29 , 38] . The uncertainty concerning which chromatin mark ( s ) is recognized by TFL2/LHP1 prompted us to assess in vitro the binding of TFL2/LHP1 to histone H3 peptides carrying K9me2 , K9me3 , or K27me3 modifications . The latter two marks were tested because of cytological observations in Arabidopsis indicating that they are preferentially localized within euchromatin , as is TFL2/LHP1 [39–41] . The in vitro assay showed that TFL2/LHP1 binds significantly more to the three modified H3 peptides than to the unmodified one ( Figure 4 ) . To explore further the interaction between TFL2/LHP1 and chromatin , ChIP-chip analyses were carried out using antibodies specific to these three histone H3 modifications . ChIP-chip performed with antibodies against H3K9me2 demonstrated that this chromatin mark is present almost exclusively over transposable elements and related repeats ( Figures 5A and S2; Tables S3 and S4 ) . Consistent with immunolocalization data , H3K9me2 is therefore particularly abundant in the repeat-rich pericentric regions of Chromosome 4 that are covered by the tiling array ( Figure S2 ) , as well as in the heterochromatic knob ( Figure S2 and [42] ) . ChIP-chip analysis of H3K9me3 and H3K27me3 demonstrated that these two marks are detected mostly within the euchromatic parts of Chromosome 4 ( Figures 5A and S2; Tables S5–S8 ) , thus reinforcing cytological observations [43] . However , the higher resolution provided by the ChIP-chip analysis revealed that these two marks do not overlap significantly ( Table S9 ) and are distributed over numerous small domains that typically cover one or two genes and their flanking sequences . Little overlap was observed between TFL2/LHP1 and H3K9me3 localization , and in most cases this limited overlap results from closely juxtaposed domains , not from tight colocalization ( Table S9 ) . In contrast , 87 . 1 % ( 525/603 ) of TFL2/LHP1 gene targets are broadly marked by H3K27me3 ( Figure 5B and Table S9 ) . PCR scanning analysis of AG ( At4g18960 ) and FT ( At1g65480 ) confirmed the broad and coincidental localization of H3K27me3 over TFL2/LHP1 target genes ( Figure 5C and 5D ) . However , the percentage of overlap between H3K27me3-marked genes and TFL2/LHP1 target genes is lower than the reciprocal ( 52 . 8 % versus 87 . 1 %; Table S9 ) . Close examination of the epigenomic maps indicates that this results from a higher signal-to-noise ratio in the case of H3K27me3 ( Figures 5A , 5C , and S2 ) , which leads to H3K27me3 domains appearing somewhat larger in size than the corresponding TFL2/LHP1 domains . When this is taken into account , overlap between H3K27me3-marked genes and TFL2/LHP1 target genes increases from 52 . 8% to over 85% . In addition , some of the remaining differences between TFL2/LHP1 and H3K27me3 localization reflect insertion/deletion polymorphisms between the Arabidopsis accessions Landsberg erecta and Columbia ( data not shown ) , in which the TFL2/LHP1 and the various histone modifications were mapped , respectively . We conclude therefore that TFL2/LHP1 and H3K27me3 are generally colocalized along the genome , and overlap at more than 85%–90% of sites at which they are present . Finally , ChIP-chip analysis of H3K27me3 was also performed in lhp1 mutant seedlings . The H3K27me3 profiles were found to be very similar between wild type and lhp1 ( Figures 5A and S2 ) . This result demonstrates that TFL2/LHP1 is not required for depositing H3K27me3 , and suggests instead that TFL2/LHP1 is involved in interpreting this chromatin mark , which would account for the co-extensive distribution of TFL2/LHP1 and H3K27me3 .
In Drosophila and mammals , silencing by Polycomb group proteins occurs through the deposition of H3K27me3 . This modification is carried out by the SET domain histone H3 methlytransferase E ( z ) , which is present in the evolutionarily conserved PRC2 . Once deposited , this chromatin mark is thought to be recognized by PRC1 , which maintains transcriptional repression by still-unknown mechanisms . The recognition of H3K27me3 by PRC1 is believed to occur via the chromodomain of the Polycomb protein , based on in vitro studies [13] . However , in plants , no homologues of Polycomb or other components of PRC1 have so far been identified , thus raising the question of the nature of the protein that might recognize the H3K27me3 mark . Recent reports have shown that mutations in one of the three genes encoding E ( z ) homologues in Arabidopsis , CLF , SWINGER ( SWN ) , and MEA , reduces H3K27me3 levels at PRC2 target genes , demonstrating that PRC2 generates the same chromatin mark in plants and animals [9 , 44–47] . These results provide strong evidence that the function of PRC2 is conserved between plants and animals . Several genes whose transcriptional repression requires PRC2 are also regulated by TFL2/LHP1 . Thus , clf mutants show a curled-leaf phenotype similar to tfl2/lhp1 as well as ectopic expression of AG and AP3 [48] . Mutations in other PRC2 components also cause developmental defects related to those of tfl2/lhp1 , although these defects are considerably enhanced in some PRC2 mutants . For example , VERNALIZATION 2 ( VRN2 ) and EMF2 , which are homologues of the PRC2 component Su ( z ) 12 in animals , also control processes regulated by TFL2/LHP1 . VRN2 is critical for the maintenance of transcriptional repression of FLC after vernalization [49] and leads to an enrichment of H3K27me3 over FLC [17 , 44] . Stable repression of FLC expression also requires TFL2/LHP1 [16 , 17] . By associating with chromatin that is marked with H3K27me3 , TFL2/LHP1 resembles the Polycomb component of animal PRC1 , which participates in the transcriptional repression of genes targeted by PRC2 . Such a role has in fact already been proposed , notably based on the observation that TFL2/LHP1 is required to maintain stable repression of FLC following vernalization [17 , 41] . In other respects , however , TFL2/LHP1 differs from Polycomb since it is not required for the repression of all genes regulated by PRC2 , as suggested by the milder phenotypes of tfl2/lhp1 mutants compared to mutants in which PRC2 function is affected . Furthermore , tfl2/lhp1 mutants lack fertilization or seed development defects [5 , 6 , 14] , which contrasts with the severe reproductive defects observed with the PRC2 mutants fertilization independent endosperm ( fie ) , fertilization independent seed 2 ( fis2 ) , and mea [50] . These differences may be explained by the small fraction of genes marked by H3K27me3 that may not be associated with TFL2/LHP1 . Alternatively , the repressive function of TFL2/LHP1 may be partially redundant with other protein complexes involved in PRC2-mediated regulation . Such a hypothesis would be consistent with the observation that loss of PRC1 but not PRC2 genes has occurred repeatedly during the evolution of metazoans [51] . While the chromodomain of Drosophila Polycomb recognizes H3K27me3 , the chromodomains of Drosophila and mouse HP1 proteins have low affinity for H3K27me3 peptides and bind to H3K9me2 and H3K9me3 [52–54] . However , none of the mouse Polycomb homologs accumulate at pericentric heterochromatin , which is enriched in H3K9me3 , despite displaying in vitro affinity towards both H3K9me3 and H3K27me3 [55] . Similarly , whereas TFL2/LHP1 is specifically associated with H3K27me3 in vivo , it binds to H3K9me2 , H3K9me3 and H3K27me3 in vitro . These observations demonstrate that predictions of protein binding based on in vitro assays do not always hold up as other factors clearly come into play in vivo . If TFL2/LHP1 takes the place of Polycomb in a plant-specific complex functionally related to PRC1 of animals , other proteins present in this complex may provide additional affinity to H3K27me3 . Alternatively , a second , yet unidentified chromatin mark could codistribute with H3K27me3 and be specifically recognized by other proteins that are part of the complex . Likewise , the ubiquitination mark of H2A lysine 119 is recognized by the Drosophila PRC1 , although here , deposition of the mark appears to be a downstream event of H3K27 trimethylation [56–58] . Both scenarios provide an explanation for the fact that a proportion of the TFL2/LHP1 target genes does not appear to be upregulated in tfl2 and that some mutants of PRC2 have more severe phenotypes than tfl2/lhp1 mutants , since loss of TFL2/LHP1 function could be buffered by other members of the complex [8 , 19 , 59] . As HP1 , TFL2/LHP1 probably recruits accessory repressive proteins via an interaction with its chromo-shadow domain . One of the best characterized interaction partners of HP1 is the SUV ( 39 ) 1/2 histone methyltransferase [20 , 28] . Interaction between the two proteins is thought to be required for the spreading of heterochromatin in animals . So far , there is no genetic evidence in Arabidopsis connecting TFL2/LHP1 to any of the SUV ( 39 ) 1/2 histone methyltransferases [16] . In contrast to lhp1 , single mutants of the ten Arabidopsis homologs of SUV ( 39 ) 1/2 do not interfere with the stable repression of FLC after vernalization [16] . However , genetic redundancy between the Arabidopsis SUV ( 39 ) 1/2 homologs has not been excluded . Nevertheless , suvh4 and suvh2 single mutants affect gene silencing [38 , 39] and PAI1 gene silencing is SUVH4 dependent but does not require a functional TFL2/LHP1 [38] . A connection between transcriptional repression , TFL2/LHP1 recruitment , and di- as well as trimethylation of H3K9 and H3K27 , was previously suggested for the FLC gene , which is silenced during vernalization [16 , 17 , 60] . The finding that TFL2/LHP1 binds H3K9me2 or 3 and H3K27me3 in vitro may corroborate this hypothesis . However , our in vivo data do not support this as a general model for the TFL2/LHP1 mode of function since we found no correlation between TFL2/LHP1 targets and H3K9me2 or H3K9me3 . Nevertheless , FLC is stably repressed only in plants exposed to vernalization , and TFL2/LHP1 might interact with H3K9me2 and H3K9me3 only under specialized conditions such as these . Finally , although 20%–30% of Arabidopsis genes are associated with some degree of DNA methylation [61–63] , we found no correlation between TFL2/LHP1 or H3K27me3 localization and DNA methylation patterns ( data not shown ) . This observation reinforces the notion that DNA methylation does not seem to have a widespread role in regulating the expression of genes in plants [50 , 63] . In Drosophila , repression by Polycomb group proteins occurs through their association with specific DNA sequences . These Polycomb response elements ( PREs ) include many conserved short motifs , but exhibit no overall sequence similarity [13] . In Drosophila , H3K27me3 methylation extends well beyond these PREs , whereas binding of most PRC2 and PRC1 components , with the notable exception of Polycomb , is restricted to the PREs themselves . In mammals however , no PRE has been identified and PRC1 and PRC2 binding extends over larger regions , leaving open the possibility that Polycomb group proteins are recruited by a different mechanism [13] . Whether plants resemble mammals or Drosophila in this respect remains to be determined . Interestingly , TFL2/LHP1 binding and H3K27me3 marking were often more pronounced around the proximal promoter and 5′ coding region of target genes ( Figures 2D and S2 ) . In AG , key regulatory motifs are located within the largest intron , for which maximal association with TFL2/LHP1 and H3K27me3 was detected ( Figure 5C ) . Taken together , these results suggest that transcriptional regulatory elements may provide entry points for PRC2-dependent trimethylation of H3K27 in Arabidopsis . Tandemly repeated genes are frequent targets of TFL2/LHP1 , suggesting that they induce higher order structural changes of their chromatin that provide a mark for TFL2/LHP1 recruitment . Conversely , the fact that these genes are duplicated and highly related may require particular control of their expression and TFL2/LHP1 could therefore actively participate in a dosage compensation mechanism . The observation that genes that are part of segmental duplications are not similarly overrepresented favors the first scenario . TFL2/LHP1 is the only Arabidopsis homologue of HP1 , and therefore our demonstration that TFL2/LHP1 is located almost exclusively in euchromatin , which supports previous cytological data [19 , 29 , 64] , indicates that Arabidopsis does not contain a functional homologue of the heterochromatic isoforms of HP1 . One of the functions of metazoan HP1 a/α and b/β isoforms is the stabilization of condensed pericentromeric heterochromatin . Since Arabidopsis clearly has such heterochromatin , the machinery involved in its stabilization must be different from its metazoan counterpart . There is a relative flexibility as to which histone modifications are associated with heterochromatin in different organisms . The predominant histone H3 modification in mammalian heterochromatin is H3K9me3 , a mark that is not enriched in the analogous heterochromatic regions in Arabidopsis , but shows a diffuse distribution , both at the cytological [39 , 43] and molecular ( this work ) levels . In contrast , the H3K9me2 mark is strongly associated with heterochromatic regions in plants , while in mammals it is not enriched in centromeres but is rather correlated with transcriptional silencing of euchromatic genes [65] . An exception is the inactive X chromosome , which carries both H3K9me2 and H3K27me3 [66] . DNA methylation and the occurrence of the H3K9me2 mark are correlated over repeated sequences in plants and animals , and both are therefore highly enriched in heterochromatin [67] . However , loss of these two marks does not always lead to a loss of cytologically visible heterochromatin [68 , 69] . Recently , VARIANT IN METHYLATION 1 ( VIM1 ) was identified because of its involvement in the maintenance of centromeric heterochromatin . VIM1 is concentrated at chromocenters and possesses both histone and methyl–cytosine interaction domains [70] . If VIM1 provides the glue that stabilizes heterochromatin in Arabidopsis , then TFL2/LHP1 would not have been required for this function in the ancestor of land plants . This , together with the fact that TFL2/LHP1 is euchromatic and that many of the animal HP1 proteins also localize to euchromatin , suggests an evolutionary scenario in which the ancestral role of HP1 was to repress euchromatic genes , with its heterochromatic role being a derived character .
TFL2/LHP1:HA transgenic plants were produced in the Landsberg erecta ( Ler ) accession . The 35S::TFL2/LHP1:HA construct used in this study complements the tfl2/lhp1 mutation , indicating that the addition of the HA epitope does not impair functionality of the protein . Of 22 scored T1 plants , six showed a wild-type growth habit , 15 showed intermediate phenotypes between wild type and tfl2 and only one showed the terminal flower phenotype . Wild-type and lhp1–1 mutant plants used for the ChIP-chip analysis of histone H3 methylation marks were of the Columbia ( Col ) accession . The lhp1–1 mutation was originally isolated in the Wassilewskija accession [5] and was introgressed into Columbia through six crosses . The TFL2/LHP1:HA C-terminal fusion gene was generated by amplifying a full-length TFL2/LHP1 cDNA with primers TFL2_HA_F , 5′-CCATGAAAGGGGCAAGTGGTGCT-3′ and TFL2_HA_R , 5′-CATTAAGTAGTGGGAGAGTCACCGG-3′ that were flanked by Gateway recombination sites , and by recombining the PCR product into a Gateway entry clone ( Invitrogen , http://www . invitrogen . com ) . The TFL2/LHP1 cDNA was then recombined into a modified pJawohl binary destination vector to produce 35S::TFL2/LHP1:HA . Transformation was carried out as described [31] . ChIP assays were carried out as described [31 , 71] using 10-d-old seedlings grown in liquid and the following antibodies: anti-HA ( H6908 , Sigma , http://www . sigmaaldrich . com ) , anti-H3K9me2 ( 07–441; Upstate , http://www . upstate . com ) , anti-H3K9me3 ( 07–442 , Upstate ) , and anti-H3K27me3 ( 07–449 , Upstate ) . Primers used for ChIP-PCR are described in Table S10 . The FT promoter sequence of the accession Ler , which carries a 1 . 5-kb deletion compared with the publicly available Col sequence , has been deposited at EMBL . No signal was detected in mock-antibody precipitations , whereas a weak signal was found in nontarget regions . Since the background bands are at the limit of detection for quantitative PCR and the more sensitive end-point PCR analysis , we expressed our ChIP results as percentage of input fraction and not as fold-enrichment over negative controls . Quantitative PCR values were obtained by averaging results of at least two independent experiments . Note that no significant difference in H3K27me3 distribution was observed between untransformed and 35S::TFL2/LHP1:HA transgenic seedlings , indicating that overexpression of TFL2/LHP1:HA does not affect H3K27me3 distribution ( Figure S3 ) . Arabidopsis Chromosome 4 tiling microarray was designed from the entire sequence of Chromosome 4 and comprised 21 , 405 printed features , each consisting of 0 . 3–1 . 2-kb PCR product amplified with sequential primer pairs along Chromosome 4 . An additional 356 amplicons of similar size were printed that cover 36 genes of interest and neighboring sequences located on the other four chromosomes [63] . Over 50% of tiles represent single-copy regions as identified by BLAST analysis of sequential 100-bp windows against the entire Arabidopsis genome sequence [33] . ChIP-chip analyses of TFL2/LHP1:HA and the three histone marks H3K9me2 , H3K9me3 , and H3K27me3 were performed on two biological replicates , except for that of H3K27me3 in the lhp1 mutant , which was carried out once . DNA recovered after ChIP ( IP fraction ) and directly from input chromatin ( INPUT ) was differentially labeled and hybridized in classical dye-swap experiments to correct dye biases , as previously described [42] . Arrays were scanned ( GenePix 4000A scanner , Axon Instruments ) and fluorescence was quantified using the software GenePix Pro . Data obtained for each array were stored as GenePix reader ( gpr ) files . No background was subtracted , and raw data ( base 2 logarithm of median feature pixel intensity ) corresponding to the 635-nm and 532-nm wavelength channels were extracted for each hybridized array from the corresponding gpr file . Manually flagged spots ( −100 ) were excluded from the analysis . Since the INPUT and IP samples differ substantially , array-by-array normalization such as loess cannot be applied . Instead , normalization between arrays was performed based on the properties of dye-swaps to remove technical biases . Let Yij be the signal of the sample labeled with the dye j on the array i . Given that the second array is a technical replicate of the first one , the distribution of Y21 ( respectively Y22 ) should be close to that of Y12 ( respectively Y11 ) . In practice , the relationship between Y21 and Y12 is linear but it is not the identity function . The parameters of the two linear models are estimated by Y21 = a + bY12 + N ( 0 , σ2 ) and Y22 = c + dY11 + N ( 0 , σ2 ) , and these estimates are used to define the normalized IP and INPUT values of the second array relative to the first one: Y21 = ( Y21 – a ) /b and Y22 = ( Y22 – c ) /d . For each sample and for each tile , the values of the two arrays of the dye-swap are then averaged . For each dye-swap , IP/INPUT ratios were analyzed using a two-step procedure , as follows ( see also Figure S1 ) . First , a hybridization threshold was defined by modeling the distribution of average IP values for the ~12 , 000 tiles of the array that are devoid of repeated sequences , and that cannot therefore lead to cross-hybridization [33] . To this end , truncated and non-truncated Gaussian mixture models were applied [72] , with a component number varying from one to five . Using the Bayesian information criterion , models with two or three components were systematically selected . We then interpret the components to characterize significant IP signals . The selected model is often a mixture with two well-separated components . In this case , tiles classified in the component with greatest mean according to the maximum a posteriori ( MAP ) rule are declared to have significant IP values . When the number of components is three , the component with the lowest mean is well separated from the others and characterizes background hybridization . Consequently , tiles classified in the other two components according to the MAP rule are declared to have significant IP values . In a second step , all IP values above the hybridization threshold were retrieved from the list of ~21000 IP values in order to analyze the corresponding IP/INPUT ratios . To define so-called “enriched” tiles , a similar procedure based on mixture models was used . Given that the Chromosome 4 tiling array contains both unique and repeated sequences , the unavoidable carry over of total genomic DNA leads to possible significant IP values for these sequences . As a matter of fact , most selected models contained two or three components , and the component with the lowest mean was found to correspond mainly to tiles with highly repeated sequences . When the selected model is a mixture with two well-separated components , tiles classified in the component of greatest mean according to the MAP rule are declared enriched . When the number of components is three , we note that the two components with the lowest means are well separated from the last . As above , tiles classified in the component of greatest mean according to the MAP rule are declared enriched . In the case of the model with four components ( H3K9me2 , replicate 2 ) , the component with the second-lowest mean is fully included within the component with the lowest mean . Tiles classified in the two components of greatest means according to the MAP rule are declared enriched ( Figure S1 ) . Lists of tiles with IP/INPUT ratios reporting significant enrichment were compared between biological replicates ( Table S11; Figure S2 ) . Overlap ranged from 94 . 8% for TFL2/LHP1 to nearly 100% for H3K9me3 , reflecting a higher signal-to-noise ratio in the case of most methylation marks compared to TFL2/LHP1 . Selected tiles common to the two replicates were manually curated to remove “singletons , ” as these were not expected from the average size of chromatin fragments ( 800 bp ) and the resolution provided by the array ( 934 bp on average ) . Indeed , the majority of singletons were found to result from cross-hybridization . These curated lists were used for the final analysis ( Tables S1 , S3 , S5 , and S7 ) . Estimation of specificity ( false positive ) and sensitivity ( false negative ) was achieved experimentally by performing ChIP PCR on randomly chosen loci ( Figure S4 ) . Tiles that cover annotated genes by at least 50 bp were called “genic” and were used to obtain the genes lists of Tables S2 , S4 , S6 , and S8 . The TFL2/LHP1 cDNA was recombined into a pTNT vector ( L5610; Promega , http://www . promega . com ) with the Gateway cassette cloned in the XbaI restriction site ( generous gift of S . Jang , Cologne ) . This plasmid allows in vitro transcription of TFL2/LHP1 by the T7 polymerase . Plasmid DNA ( 1 μg ) was transcribed and the resulting RNA translated using the TNT Quick Coupled kit ( L1170 , Promega ) following the manufacturer's instructions . For peptide pull-down assays , 50 ng of biotinylated histone H3 peptides , that were either methylated on K27 ( aa 21–44; 12–565 , Upstate ) , methylated on K9 , or unmethylated ( aa 1–21; 12–430 and 12–403 , Upstate ) were incubated with 10 μl of Dynabeads M-280 Streptavidin ( Dynal/Invitrogen ) in PBS for 2 h at 4 °C and blocked with 0 . 25% BSA in PBS for 30 min . The beads were washed three times with binding buffer ( 20 mM Tris-HCl pH 7 . 9 , 0 . 2 mM EDTA , 1mM DTT , 0 . 2 mM PMSF , and 20% glycerol ) containing 200 mM KCl and mixed with 5 μl of in vitro translated protein and 100 μl of binding buffer for 60 min at 4 °C . After washing five times with binding buffer containing 500 mM KCl , the bound proteins were eluted in SDS loading buffer , resolved by SDS-PAGE , and visualized using a phosphorimager .
Accession numbers for the ArrayExpress ( http://www . ebi . ac . uk/arrayexpress ) data discussed in this paper are A-MEXP-602 , array design and E-MEXP-951 , experimental data . The European Molecular Biology Laboratory database ( EMBL ) ( http://www . ebi . ac . uk/embl ) accession number for the FT promoter sequence in Ler is AM492685 . A browser-based interface to the data is available at the following website: http://dynagen . ijm . jussieu . fr/research/tools/repet/gbrowse/arabidopsis . | Stable repression of gene expression is an important aspect of the developmental programs of higher organisms . In plants and animals , DNA is organized within chromatin , which contains at its core a set of evolutionarily conserved proteins called histones . These proteins can be modified for example by methylation or acetylation of lysines or phosphorylation of serines . Specific combinations of these histone modifications are interpreted by other chromatin proteins and thereby play essential roles in gene regulation . One such potential effector of the histone code in the flowering plant Arabidopsis is TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 ( TFL2/LHP1 ) . Here we present highly detailed “epigenomic” maps that establish that TFL2/LHP1 associates with a subset of Arabidopsis genes that are marked by tri-methylation of Lysine 27 of histone H3 . In plants and animals , an evolutionarily conserved complex called PRC2 deposits this mark . In Drosophila and mammals this modified histone is then read by another complex , called PRC1 , to maintain the stable repression of genes . In Arabidopsis however , no PRC1 complex exists , and our results provide evidence that TFL2/LHP1 may fulfill a related function . | [
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] | 2007 | Arabidopsis TFL2/LHP1 Specifically Associates with Genes Marked by Trimethylation of Histone H3 Lysine 27 |
Ribosome biogenesis is a major energy-consuming process in the cell that has to be rapidly down-regulated in response to stress or nutrient depletion . The target of rapamycin 1 ( Tor1 ) pathway regulates synthesis of ribosomal RNA ( rRNA ) at the level of transcription initiation . It remains unclear whether ribosome biogenesis is also controlled directly at the posttranscriptional level . We show that Tor1 and casein kinase 2 ( CK2 ) kinases regulate a rapid switch between a productive and a non-productive pre-rRNA processing pathways in yeast . Under stress , the pre-rRNA continues to be synthesized; however , it is processed differently , and no new ribosomes are produced . Strikingly , the control of the switch does not require the Sch9 kinase , indicating that an unrecognized Tor Complex 1 ( TORC1 ) signaling branch involving CK2 kinase directly regulates ribosome biogenesis at the posttranscriptional level .
All cells must adapt to a constantly changing environment in order to maintain their intracellular equilibrium and to balance growth with survival . Several signaling pathways monitor concentrations of available nutrients or presence of harmful conditions and switch on or off specific transcriptional programs to ensure the best usage of resources . When essential nutrients become unavailable , cell division is arrested at the G1 phase of the cell cycle , cells change metabolism , and prepare for entry into the reversible quiescent/G0 state , in which they can survive for decades ( reviewed in [1] ) . Most cells from multicellular organisms are also considered to be quiescent , in the G0 phase of the cell cycle , as the end stage of their terminal differentiation . In yeast , like in other microorganisms , several distinct stages of culture growth can be observed in a rich medium , such as yeast extract-peptone-dextrose ( YPD ) : exponential phase , diauxic shift , postdiauxic phase , and stationary/quiescent phase . During exponential phase , with abundant fermentable carbon source ( e . g . , glucose ) , yeast cells use fermentation and divide rapidly . The diauxic shift occurs when glucose becomes depleted from the media and yeast switches to respiratory metabolism concomitantly with a sharp decrease in growth rate . In the subsequent postdiauxic phase , yeast grows at a much slower rate , using respiration to provide energy , and starts to acquire characteristic features of stationary cells until the growth ceases completely and cells become quiescent [2] . Yeast starved of other essential nutrients , such as nitrogen , phosphate , or sulphur , can also enter a quiescent state [3] . An interconnected signaling network of key kinases , the target of rapamycin complex 1 ( TORC1 ) , protein kinase A ( PKA ) , sucrose non-fermenting 1 ( Snf1p ) , and phosphate metabolism 85 ( Pho85p ) , monitors the nutrient availability in yeast . While the initial transcriptional response to limitation of individual nutrients differs [4 , 5] , there is a common core program for preparation of entry into quiescence , regardless of the limiting nutrient [1] . TORC1 is a highly conserved multimeric protein kinase that controls cell growth and aging from yeast to human [6] . TORC1 takes its name from the fact that it is rapidly and specifically inhibited by the macrolide rapamycin [7] . Inhibition of TORC1 by rapamycin leads to G1 cell cycle arrest , decrease in protein synthesis and ribosome biogenesis , initiation of a specific transcription program , induction of autophagy , and eventual entry to quiescence ( reviewed in [8] ) . The activity of TORC1 correlates primarily with the availability of carbon and nitrogen sources; however , TORC1 activity is also sensitive to other nutrients and exposure to heat or oxidative and osmotic stress [9] . Its activity is also down-regulated during the diauxic shift during growth in a rich media [10] . In yeast , TORC1 signals primarily through two key branches: ( 1 ) by phosphorylation of Sch9p ( a yeast ortholog of mammalian S6 kinase ) and ( 2 ) by phosphorylation of Tap42p or Tip41p that are thought to regulate activity of the PP2A phosphatase [9 , 11 , 12] . The Sch9 kinase was described as a master regulator of protein synthesis that controls ribosome biogenesis , translation initiation , and entry to quiescence [9 , 13] . Sch9p affects activity of all three classes of RNA polymerases required for ribosome biogenesis . It controls recruitment of the core initiation factor Rrn3p to the RNA polymerase I ( RNA pol I ) machinery and thus synthesis of ribosomal RNA ( rRNA ) [14 , 15] . Sch9p also regulates RNA polymerase II ( RNA pol II ) transcription of ribosomal proteins ( RPs ) and ribosome biogenesis RiBi genes by controlling activity of several transcription factors and repressors [9 , 13 , 16 , 17] . Active Sch9p promotes activity of the RNA polymerase III ( RNA pol III ) , required for transcription of 5S rRNA and tRNAs , by inhibition of its transcription repressor Maf1p [13 , 18 , 19] . Ribosome biogenesis is a major energy-consuming process in the cell , and it is down-regulated in response to nutrient limitation and stress [20] . The complex pathway of ribosome synthesis is best understood in yeast . It starts by the transcription of the 35S pre-rRNA , a large precursor that is cleaved at multiple sites and processed into mature 18S , 25S , and 5 . 8S rRNAs ( Fig 1A ) , reviewed in [21 , 22] . The processing of 35S pre-rRNA starts with cleavages at sites A0 and A1 , which release the 5′ETS , and is followed by a cleavage at the site A2 that separates the precursors of 40S and 60S ribosomal subunits , 20S and 27SA2 pre-rRNAs , respectively , which are further processed into mature rRNAs . A direct processing at site A3 , producing 23S pre-rRNA , has also been observed [23] . Low levels of 23S pre-rRNA are present in wild-type cells but markedly accumulate in ribosome biogenesis mutants , in which they don’t seem to be further processed and are degraded by exosome [22 , 24] . However , the fate of 23S pre-rRNA remains controversial , and it is usually regarded as a product of aberrant processing [21 , 22 , 25] . Interestingly , in higher eukaryotes , the order of early cleavages is often variable , suggesting that multiple processing pathways exist [25] . Here , we show that TORC1 and casein kinase 2 ( CK2 ) kinases control ribosome biogenesis at the posttranscriptional level of pre-rRNA processing . Upon nutrient limitation or exposure to harmful stress , pre-rRNA processing rapidly switches from the standard A2 site cleavage pathway to an alternative pathway using exclusively the A3 cleavage site and thus producing 23S pre-rRNA . Stable isotope labeling with amino acids in cell culture ( SILAC ) mass spectrometry and in vivo pulse-chase analysis showed that pre-rRNA synthesis continues after nutrients depletion , but the pre-rRNA is processed only into 23S and 27S-A3 pre-rRNAs , and no new ribosomes are made . Inhibition of TORC1 or CK2 kinases induces an identical change in pre-rRNA processing , indicating that TORC1 and CK2 control the choice between the A2 and A3 sites . Analyses of protein composition and phosphorylation changes in preribosomes identified several ribosome biogenesis factors as targets of CK2 kinase and TORC1 pathway . These proteins likely represent the control nodes in ribosome biogenesis .
In exponentially growing yeast , the pre-rRNA processing is initiated by cleavages at sites A0 , A1 , and A2 . We noticed that in yeast cultures undergoing the diauxic shift , rRNA processing at sites A0 , A1 , and A2 was abruptly abolished , and the pre-rRNA was instead processed at site A3 ( Fig 1A and 1B and S1A Fig ) . The switch in processing was completed within 20 min ( S1B Fig ) . Importantly , the steady-state level of 35S rRNA clearly increased , indicating that its processing was delayed , and the ribosomal DNA ( rDNA ) transcription was not inhibited . These changes resulted in production and accumulation of the 23S and 27S-B pre-rRNAs; however , the downstream intermediates 20S and 7S pre-rRNA were hardly detectable , suggesting that further ribosome biogenesis was arrested . The 27SA2 and 23S pre-rRNA are produced from the same primary transcript by a cleavage at sites A2 or A3 , respectively , which are separated by only 76 nucleotides . As both 27SA2 and 23S pre-rRNAs are detected by the same hybridization probe , we can directly compare their steady-state levels . Interestingly , the amounts of 27SA2 before shift and 23S pre-rRNAs after shift were comparable , indicating that at least at the initial time points , the change in the processing was reciprocal and indeed represented a “switch” to an alternative processing . The rapid reduction in the 27SA2 levels likely corresponds to its further processing into mature 25S rRNA during the switch in pre-rRNA processing . The 10–20 min required to switch from the A2 to A3 pathway is a long time compared to a short lifetime of the 27SA2 pre-rRNA , which is 15 s or 90 s for cotranscriptionally or posttranscriptionally produced 27SA2 , respectively [26] . Similarly , the apparent lack of signal for 27SA3 is due to a short lifetime of this intermediate and its immediate processing to 27SB . The addition of fresh glucose to a postdiauxic shift culture led to a fast reversal to the pre-rRNA processing using A2 cleavage site within approximately 10 min ( Fig 1C ) . The yeast continued to grow exponentially for about an hour and , upon depletion of the added glucose , switched again to the A3 processing . This behavior was also consistent in synthetic media . To determine whether the observed switch in pre-rRNA processing is limited only to a carbon source or represents a more general response to a lack of nutrient , we cultured cells under limited nitrogen or amino acid conditions ( Fig 1D and 1E ) . In all cases , the cells reacted in exactly the same manner and changed their pre-rRNA processing abruptly upon depletion of all nutrients tested . We observed identical behavior in three commonly used and well-characterized wild-type strains W303 , BY4741 , and CEN-PK2 ( Fig 1B , S1C Fig ) . We conclude that yeast cells switch to an alternative ribosome biogenesis pathway in response to limited nutrient availability . For simplicity , in the following text , we will refer to the two alternative processing routes as A2 pathway ( occurring in rapidly growing yeast ) and A3 pathway ( slow or arrested growth ) . Ribosome biogenesis is also down-regulated during various cellular stresses . We therefore tested if the switch from A2 to A3 pathway represents a more general mechanism used during the stress response . A wild-type yeast was exposed to a mild heat shock ( shift from 25°C to 37°C ) , osmotic stress ( exposure to 1 M Sorbitol ) , or oxidative stress ( 0 . 2 mM Diamide ) , conditions that were shown to induce a rapid down-regulation of the expression of genes encoding RPs and biogenesis factors [5 , 27] . As can be seen in Fig 1F , the mild heat shock led to an immediate temporal switch to A3 pathway , with a strong accumulation of 35S pre-rRNA . The processing then returned to normal A2 pathway after about 30 min , after adjustment of the cellular metabolism to the growth at higher temperature . An oxidative stress , caused by exposure of the cell to 0 . 2 mM diamide , showed a slightly milder temporal switch to A3 pathway ( Fig 1F ) . We used very mild conditions for both stresses , milder than used standardly , in order not to kill or stop cells growing and thus allow the evaluation of their effect on ribosome biogenesis . Harsher oxidative stress might elicit stronger phenotype . The osmotic shock elicited a less clear change to A3 pathway , with noticeable reduction of 27SA2 levels but no obvious increase of 23S pre-rRNA levels ( S1D Fig ) . These results indicate that the switch from A2 to A3 pathway of pre-rRNA processing is a general response to certain environmental stresses when ribosome biogenesis needs to be repressed . In the previous experiments , we observed that the 35S pre-rRNA continued to accumulate in cells after nutrient depletion , even when the cells had practically stopped proliferating . In addition , the level of 23S pre-rRNA was comparable to levels of 27SA2 in exponentially growing cells . Furthermore , no 20S pre-rRNA was detectable after the switch , raising the question of whether the 23S rRNA intermediate is further processed and new ribosomes are produced . There are two possible explanations: ( a ) the pre-rRNA synthesis ( transcription and processing ) is arrested and preribosomes with either 35S or partially processed pre-rRNA are stalled and waiting for a change in growth conditions; or ( b ) the rDNA transcription is ongoing , the 35S pre-rRNA continues to be synthesized but is processed at the site A3 , and subsequent processing is abolished , and thus no new ribosomes are made . To distinguish between these possibilities , we performed in vivo 3H-uracil pulse-chase . The 3H-uracil can be incorporated into pre-rRNA only during ongoing transcription . Therefore , any radioactive labeling of the 35S and 23S pre-rRNAs clearly indicates ongoing de novo synthesis . If rDNA transcription is repressed ( a ) , no pre-rRNA should be labeled and detected; whereas , in the case ( b ) , both 35S and other pre-rRNA precursors should become labeled and detectable . Exponential ( OD600 = 2 ) or postdiauxic ( OD600 = 10 ) cultures were pulse-labeled for 6 min with 3H-uracil and then chased with cold uracil for 10 min . As expected , in the exponentially growing cells , all the main pre-rRNA processing intermediates and mature 18S and 25S rRNAs were clearly detectable within the first minutes of labeling ( Fig 2A ) . The pre-rRNAs could be clearly chased by the addition of nonradioactive uracil . In contrast , in the postdiauxic culture , only three RNAs were clearly detectable: 35S , 27S-B , and 23S pre-RNAs . No mature 18S or 25S rRNAs or 27S-A and 20S intermediates were detected ( Fig 2B ) . Interestingly , while the 35S pre-rRNA could be slowly chased with cold uracil , the 23S and 27S-B pre-rRNAs seemed to accumulate in the postdiauxic cells , indicating that they are not processed further . The slow and less-efficient chase is likely partially due to a reuse of the 3H-uracil that remains in the intracellular pool of uracil . The 3H-uracil from degraded RNAs ( e . g . , processed spacers of the pre-rRNA or introns ) remains in the cell , as it is not efficiently diluted out by the provided cold uracil , especially in very slowly or non-growing cells , as is the case here . This contributes to the observed accumulation of the longer-lived 23S and 27S pre-rRNA that are not processed further . It is also worth noting that the 35S pre-rRNA was detected prior to the appearance of 27S pre-rRNA , revealing that the A3 cleavage in postdiauxic cells occurred posttranscriptionally . To analyze the effect of fresh media on the postdiauxic cells , one-half of the pulse-labeled culture was diluted with fresh , prewarmed media supplemented with cold uracil to simultaneously initiate a chase ( Fig 2B right ) . The 20S and 27SA2 pre-rRNAs appeared rapidly , and mature 18S and 25S rRNA were detectable after about 6 min . The 35S pre-rRNA was slightly reduced . The 23S pre-rRNA was not detectable after 8 min . It is important to note that in the postdiauxic culture , the total amount of transcription ( radioactive signal ) was strongly reduced , in agreement with previous reports [28] . We estimate from the pulse-chase experiment in Fig 2 that the overall transcription in the postdiauxic cells is about 2% of the transcriptional level in the fast exponentially growing cells . While 2% might seem low , rDNA is very highly transcribed , and it represents a very significant number of transcriptional events , comparable to highly expressed protein-coding genes . It is technically difficult and maybe physiologically irrelevant to distinguish if either the repression of rDNA transcription or change in processing occurs first . It is likely and economically logical that cells would down-regulate both the transcription and processing simultaneously . In the pulse-chase experiment , the signal intensity of 23S pre-rRNA was lower than the signal corresponding to 27S pre-rRNA species . This is seemingly contrary to the results from northern blotting ( Fig 1B ) , in which the 23S pre-rRNA level is clearly equal to 27SA2 pre-rRNA . However , the strong signal in the pulse-chase experiment corresponds to the 27SB pre-rRNA , an intermediate with a longer lifetime accumulating to much higher levels than the 27SA2 pre-rRNA . Nevertheless , the 23S pre-rRNA might also be subject to degradation . We therefore analyzed levels of the 23S pre-rRNA in exosome , TRAMP complex , and Xrn1 exonuclease mutants ( Fig 2E ) . The 23S pre-rRNA levels were clearly increased in the mutants of exosome and TRAMP complex ( rrp6Δ , rrp47Δ , and trf4Δ ) but not in the xrn1Δ strain lacking major 5′-3′ exonuclease . Therefore , the 23S pre-rRNA seems to be turned over by exosome . These results indicate that the A3-type pre-rRNA processing pathway following the diauxic shift is nonproductive and does not lead to detectable synthesis of new ribosomes . This is also in agreement with the observed reduction of the mature 18S and 25S rRNAs content per cell at later time points after the diauxic shift in Fig 1B ( total RNA from the same number of cells were loaded per lane ) . We cannot formally exclude that a small number of ribosomes ( technically undetectable in our experiments ) are being synthesized or that ribosomes with potentially distinct properties are made using the A3 pathway at a certain time point ( see discussion ) . To further understand changes in the ribosome biogenesis after the switch to alternative rRNA processing , we analyzed the protein composition of the early preribosomes ( 90S preribosomes ) by SILAC mass spectrometry . The ribosome biogenesis factor Pwp2p ( Utp1p ) was used as a bait . Pwp2p is commonly used as a bait in tandem affinity purifications , as it is a stable component of early preribosomes , in which the first pre-rRNA processing steps including the cleavages at the A2 or A3 sites occur [29–31] . It also remains associated with the 23S pre-rRNA produced by cleavage at the A3 site . To confirm that Pwp2p associates with newly formed preribosomes after the diauxic shift , we analyzed the RNA copurifying with Pwp2p ( S2 Fig ) . The purified Pwp2-complexes from postdiauxic shift cells contained increased amounts of 35S/32S and 23S pre-rRNA , with majority ( about two-thirds ) of Pwp2p associated with 35S and 32S pre-rRNA . This indicates that Pwp2p is recruited to newly formed preribosomes after the diauxic shift and remains bound following the A3 cleavage . For the SILAC mass spectrometry , an identical number of cells before the diauxic shift ( light media ) and 1 hr after diauxic shift ( heavy media ) were harvested , mixed , and lysed . The 90S preribosomes were purified via Pwp2-FLAG-TEV-ProteinA ( performing both steps of the tandem affinity purification ) . The proteins present in the purified Pwp2-associated preribosomes and the relative ratios between the heavy and light labeled cultures were determined by a mass spectrometry ( S1 Data ) . On average 25%–30% decrease in the purification of the heavy labeled Pwp2p bait ( from postdiauxic shift culture ) was observed . This is in agreement with a slow-down of growth after the diauxic shift . The data was normalized to the H/L ( Heavy/Light ) ratio of the Pwp2p bait to correct for the reduced purification ( the H/L ratio of Pwp2p was set to 1 ) . Fig 2C shows that the overall composition of 90S preribosomes is unaffected , as the relative abundance of the majority of 324 quantified proteins does not change significantly . However , there are the following notable exceptions ( Fig 2D ) . In agreement with the shift from cotranscriptional to posttranscriptional rRNA processing , a number of pre-60S biogenesis factors ( dark blue bars ) and RPs of the large subunit ( RPLs ) ( light blue ) were enriched in preribosomes from the A3 pathway . Several RPs of the small subunit ( RPSs ) were also enriched . Notably , only the Rps27p ( eS27 ) was reduced . A depletion of the Rps27p was reported to lead to 23S pre-rRNA accumulation [32] . Thus , the loss of Rps27p could represent the mechanism underlying the switch to A3 pathway . However , further data analysis excluded this possibility ( see discussion and S8 Fig for more detail ) . Also , several early preribosome factors ( green bars ) remained trapped in the Pwp2p particles compared to prediauxic culture . Importantly , two endonucleases Fcf1 and Rcl1 that were reported to mediate cleavage at the site A2 [33 , 34] remain associated or even enriched in A3 preribosomes ( red dots in Fig 2C ) . Therefore , the loss of the A2 cleavage is not due to the absence of the responsible endonuclease . On the other hand , subunits of the exosome complex , required for the pre-rRNA processing of downstream intermediates , were strongly reduced in the pre-ribosomes from A3 pathway . This is in agreement with the fact that pre-rRNA processing seems to be arrested , and thus substrates for the exosome , which are produced in A2 pathway , are not being made . The methyltransferase Dim1p , which is recruited at later stages of nucleolar processing ( [30] , was also strongly reduced , indicating that the ribosome biogenesis might be arrested before its recruitment . Interestingly , several early factors ( Nop6p , Kri1p , Esf1p , Hca4p , and Mrd1p ) , all required for A2 cleavage , were also released from 90S preribosomes . These proteins might represent potential regulatory nodes of the switch between the two alternative pathways . We conclude that although the early steps of ribosome biogenesis continue after the switch to the A3 pathway , and the overall composition of the preribosomes is largely unaffected , the subsequent maturation of preribosomes is arrested after the A3 cleavage . It is well established that TORC1 regulates rRNA transcription by RNA pol I in response to nutrient availability . Therefore , we investigated if the pre-rRNA processing and , in particular , the switch between A2 and A3 pre-rRNA processing pathways is also under the control of TORC1 . We inhibited TORC1 in exponentially growing cells by rapamycin and analyzed the pre-rRNA processing . This analysis ( Fig 3A ) clearly shows that inhibition of TORC1 caused a rapid switch from the A2 to A3 pathway . The production of the 27SA2 pre-rRNA was abolished , whereas the 23S pre-rRNA accumulated instead in rapamycin-treated cells . The levels of 35S pre-rRNA increased , showing that transcription of rDNA continued and that the switch in processing occurs before or concomitantly with down-regulation of rDNA transcription by TORC1 . The change in pre-rRNA processing induced by rapamycin was specific to TORC1 , as the strain expressing a mutant Tor1-1p , which cannot bind rapamycin , failed to switch to the A3 pathway ( Fig 3B ) . In yeast , in addition to TORC1 , three other core signaling pathways ( PKA , PHO85 , and SNF1 kinases ) are known to contribute to the regulation of cellular metabolism in response to nutrient availability . Interestingly , deletion of either PHO85 or SNF1 or inhibition of PKA in an analog-sensitive PKA strain did not affect the cells’ ability to rapidly switch to A3 pathway ( S3 and S4 Figs ) . Taken together , these findings demonstrate that the regulation of A2 to A3 switch in pre-rRNA processing is specific to TORC1 pathway . It is well established that nutrient depletion or rapamycin treatment also causes the down-regulation of RP genes transcription [28 , 35] . We analyzed the mRNA levels of several RP genes by northern blotting in various conditions used in this study ( S5 Fig ) . Our data shows that the transcription of RP genes is down-regulated simultaneously with the switch of pre-RNA processing to A3 pathway ( see discussion ) . The protein composition of preribosomes after inhibition of TORC1 by rapamycin was analyzed by SILAC in an identical way as in Fig 2 . Exponentially growing cultures at OD600 = 2 were treated for 20 min with rapamycin ( heavy media ) or DMSO only ( light media ) . The preribosomes were isolated via the affinity-tagged Pwp2p . A 25%–30% reduction of bait purification from the rapamycin-treated culture was observed , in agreement with a growth arrest by rapamycin . As can be seen from Fig 3C , the overall composition of preribosomes is unaffected , analogous to the diauxic shift . However , in contrast to the diauxic shift , rapamycin treatment led to a general reduction of copurified ribosome biogenesis factors ( Fig 3D and 3E ) . This corresponds to distinct effects of the rapamycin treatment versus diauxic shift on cell growth . While postdiauxic cells continue to slowly grow , rapamycin rapidly arrests cell proliferation , leading to the observed reduction of ribosome biogenesis . Nevertheless , there are several important parallels between the two conditions , as the same proteins were clearly reduced in preribosomes after either rapamycin treatment or diauxic-shift . Namely , the exosome complex , Dim1p , and prerequisite factors for A2 cleavage Hca4p , Mrd1p , Kri1p , and Nop6p . This finding strengthens the idea that these factors are implicated in the regulation of the switch between the two pre-rRNA processing pathways . We conclude that the TORC1 pathway controls the choice between the two alternative pre-rRNA processing programs . In the current understanding , TORC1 regulates rRNA synthesis at the transcriptional level by promoting the RNA pol I initiation . Our results indicate that the switch between the two alternative pre-rRNA processing pathways occurs at the posttranscriptional level . We were therefore interested to find out if RNA pol I is required for this regulation . We used the strain NOY892 , in which all rDNA repeats are deleted , and the 35S pre-rRNA is transcribed solely from a GAL7 promoter by RNA pol II [36] . The cells were grown in either rich or synthetic media with galactose as a carbon source . Fig 4A reveals that upon depletion of the carbon source , the pre-rRNA processing switched from A2 to A3 pathway in a fashion identical to wild-type yeast . These results indicate that the change in processing is not dependent on transcription by the RNA pol I . The initiation of rDNA transcription by the RNA pol I and thus the synthesis of pre-rRNA is strongly reduced during the diauxic shift or rapamycin treatment . Is it possible that the switch between the A2 and A3 pathways is a simple consequence of changes in the kinetics of pre-rRNA synthesis ( such as the number of transcribing RNA polymerases or the elongation rate ) and/or imbalance in the levels of pre-rRNA and processing factors ? The following experiments were designed to provide an insight into this issue . The previous experiment with the GAL7 promoter demonstrated that the switch in the pre-rRNA processing is not specific to the RNA pol I . To further exclude that the observed change in processing is not due to a general down-regulation of transcription from the GAL7 promoter after depletion of galactose , the exponentially growing strain NOY892 was treated with rapamycin or exposed to a mild heat shock ( Fig 4B ) . In both conditions , 23S pre-rRNA immediately appeared within the first 10 min , concomitant with an increase in 35S pre-rRNA levels . Available transcriptomics data show that the GAL7 promoter activity is not greatly affected by either nutrient depletion ( apart from galactose ) , heat shock , or rapamycin [5 , 10] . Thus , the number of RNA polymerases or the elongation rate of RNA pol II does not change significantly in these conditions . These results argue that the switch from A2 to A3 pathway is unlikely to be caused by a reduced number of RNA polymerases passing through the rDNA or a changed elongation rate . Next , we addressed the question if a cessation of RNA pol I transcription would induce a change in the pre-rRNA processing . We used an anchor-away strain BEN135 [37 , 38] , in which Rpa135 , the second largest subunit of RNA pol I , was fused to FKBP−rapamycin-binding ( FRB ) domain , and large subunit RP Rpl13Ap was fused to FKBP12 . The strain is resistant to rapamycin , and the addition of rapamycin induces dimerization of FKBP12 and FRB domains and , thus , sequesters the Rpa135p in the cytoplasm , effectively abolishing transcription by RNA pol I within minutes . Importantly , the reduced availability of Rpa135p affects the number of RNA pols I available for transcription but not the elongation rate of the transcribing polymerases . Fig 4C shows that the level of all pre-rRNA species reduced equally without a change in the cleavage pattern , confirming that the switch from A2 to A3 pathway is independent of the rate of transcription . Finally , we also asked whether continued robust transcription of pre-rRNA would interfere with the A2 to A3 switch in processing . We took advantage of the CARA ( Constitutive Association of Rrn3 and A43 ) strain , in which RNA pol I subunit Rpa43p is fused with the transcription factor Rrn3p , rendering it resistant to rapamycin [39] . In the CARA strain treated with rapamycin , the RNA pol I is not inhibited , but other cellular processes remain affected by the rapamycin treatment . Therefore , using the CARA strain , we can separate the effects of rapamycin on pre-rRNA processing from transcription . We treated exponentially growing culture of the CARA strain with rapamycin and analyzed the pre-rRNA processing ( Fig 4D ) . The cells accumulated large levels of both 35S and 23S pre-rRNA , corresponding to the usage of the A3 pathway . The ongoing strong pre-rRNA synthesis did not affect the switch in pre-rRNA processing . Our findings provide a strong evidence that the A2 to A3 switch in pre-rRNA processing is neither dependent on the RNA pol I nor likely caused by a change in the elongation rate or the number of transcribing polymerases . Sch9 , the yeast ortholog of the mammalian S6 kinase , was described as a master regulator of ribosome biogenesis in yeast , regulating transcription by all three classes of RNA polymerases [9 , 13] . We tested if Sch9 also controls the switch between A2 and A3 pathways . A yeast strain deleted for Sch9 ( sch9Δ ) was grown until the growth slowed down after the diauxic shift . Fig 5A demonstrates that in the absence of Sch9p , the pre-rRNA processing also changed rapidly from A2 to A3 pathway . Treatment of the exponentially growing sch9Δ strain with rapamycin also elicited a switch from A2 to A3 pathway ( Fig 5C ) . Furthermore , expression of a Sch9-2D3E mutant , which mimics phosphorylated Sch9p and thus remains hyperactive [9] , neither prevented nor induced a premature switch in processing ( Fig 5B ) . Identical results were also obtained in a PtetO7-Sch9 strain , in which the wild-type Sch9p protein ( expressed under the control of the tetracycline tetO7 promoter ) was depleted for 12 hr ( S6 Fig ) , thus excluding the possibility of suppressor mutations in the sch9ΔΔstrain , masking its role in the A2 to A3 switch . We also tested a number kinases or factors acting downstream of Sch9p that were implicated in the regulation of ribosome biogenesis ( Yak1p , Atg1p , Whi2p , Sfp1p , Sic1p , Dot6p , Tod6p , Mpk1c , Rim15p ) . Deletion of none of these factors affected the switch in the pre-rRNA processing ( S3 Fig ) . These findings imply that Sch9p function is dispensable for the control of the choice between A2 and A3 pathways . The second major branch of TORC1 signaling pathway is represented by Tap42p and its negative regulator Tip41p . We first attempted to assess the role of Tap42p using a temperature-sensitive tap42-11 mutant . Unfortunately , the heat-shock due to transfer of the tap42-11 strain from permissive to nonpermissive temperature affects ribosome biogenesis ( see Fig 1F ) ; therefore , this experiment cannot be interpreted . Interestingly , the tap42-11 strain is partially resistant to rapamycin at permissive temperature . Furthermore , a yeast strain lacking Tip41p was also shown to be partially resistant to rapamycin [40–42] . We therefore assessed if the pre-rRNA processing switches to A3 pathway either during diauxic shift or upon rapamycin treatment . Fig 5A ( right panel ) shows that tap42-11 grown at permissive temperature to high density , switches to A3 pathway during the diauxic shift in an identical manner as the control wild-type strain . Similarly , the absence of Tip41p does not affect the switch to A3 pathway in high-density culture ( S3 Fig ) . A treatment of exponentially growing low-density culture by rapamycin induced a switch to A3 pathway in both tap42-11 and tip41Δ strains ( Fig 5D ) . In our preribosome purifications , we detected readily all subunits of CK2 kinase and found that their abundance does not change during diauxic shift ( Fig 2C , green circles ) . The protein kinase CK2 is an essential kinase , the activity of which is required for growth and proliferation . The CK2 kinase is a component of early preribosomes , as a member of the UTP-C subcomplex [43] . It is also a component of a separate CURI complex ( CK2 , Utp22p , Rrp7p , and Ifh1p ) , which was implicated in transcriptional regulation of RP genes [35 , 44] . While the regulation of CK2 activity is largely unclear , it has been recently reported that the CK2 regulatory subunit Ckb1 can be phosphorylated by TORC1 pathway [45] . We therefore assessed whether the CK2 activity is required for the control of the A2 to A3 switch . Wild-type , low-density , exponentially growing cells were treated with 4 , 5 , 6 , 7-Tetrabromobenzotriazole ( TBB ) , a highly selective CK2 inhibitor [46] . The TBB treatment led to an immediate appearance of 23S pre-rRNA and a strong decrease of 27SA2 levels concomitant with the accumulation of 35S pre-rRNA , showing kinetics similar to that of rapamycin treatment ( Fig 6A ) . Interestingly , although the 27SA2 pre-rRNA was strongly reduced , it was not eliminated completely . Sequential treatment of cells by TBB first , followed by rapamycin , showed that the lack of CK2 activity delays the disappearance of 27SA2 pre-rRNA induced by rapamycin treatment ( Fig 6B ) . The activity of CK2 is most likely also required for further 27SA2 processing , and , thus , the inhibition of CK2 delays the disappearance of this species upon rapamycin treatment . Recently , the nonessential kinase Kns1p was identified as an intermediary between TORC1 and CK2 [45] . We assessed whether Kns1p is required for the A2 to A3 switch . The yeast strain kns1Δ lacking Kns1p was grown into high density , and pre-rRNA processing was analyzed by northern blotting ( S3 Fig , top right ) . The switch in pre-rRNA processing from A2 to A3 pathway occurred in an identical fashion to wild-type strains , revealing that the Kns1 kinase is not involved in the regulation of the switch in pre-rRNA processing . We have also analyzed the effect of inhibition of CK2 activity by TBB on the protein composition of preribosomes by SILAC mass spectrometry . Exponentially grown yeast cultures were treated with DMSO ( light ) or TBB ( heavy ) for 15 min , and Pwp2-containing preribosomes were purified . Two independent experiments were performed with two different lots of TBB . Perhaps due to the different lot of TBB used , the second experiment showed similar but smaller changes in the protein composition than the first experiment ( S1 Data ) . In both cases , about 2-fold change in the purification of the bait protein Pwp2p was observed , presumably due to a strong arrest of cell growth by TBB . The H/L ratios of individual proteins were normalized to bait . Fig 6C shows that TBB treatment leads to a robust enrichment of early 90S factors in Pwp2-complexes . Interestingly , Utp22p and Rrp7p , components of the CURI complex , were also strongly enriched . In addition , all four subunits of the CK2 kinase were identified and enriched ( although Cka1p , Cka2p , and Ckb1p were quantified only in one of the two replica experiments; S1 Data ) . Importantly , the 90S factors Hca4p , Mrd1p , Esf1p , Nop6p , and Kri1p , which were strongly reduced in preribosomes from postdiauxic shift or rapamycin-treated cultures , were enriched following CK2 inhibition by TBB . The contrasting behavior of these factors in the three conditions that all cause the switch to A3 pathway makes these factors unlikely candidates to directly regulate the choice between the A2 and A3 cleavage sites . In summary , the CK2 kinase inhibition by TBB produces overlapping but not identical phenotype to diauxic shift or rapamycin treatment . Added together , these results show that CK2 kinase activity is required for A2 pathway pre-rRNA processing and at least partially for control of the choice between A2 to A3 cleavage sites . It most likely acts downstream of TORC1 . Our results suggested that ribosome biogenesis is directly regulated by TORC1 pathway and CK2 kinase at the posttranscriptional level . Several proteomic studies analyzed rapamycin-sensitive phospho-proteome in yeast [13 , 47 , 48] . We mined the data for ribosome biogenesis factors . Surprisingly , only a relatively small number of ribosome biogenesis factors showed significant phosphorylation changes , and these changes in the levels of phosphorylation varies widely between the reports ( S1 Table ) . These studies analyzed whole proteomes and , therefore , might not have detected changes present in different complexes , such as preribosomes . We therefore compared protein phosphorylation pattern of the Pwp2-preribosomes before and after the diauxic shift , rapamycin , or TBB treatment . We identified several hundred phosphorylation sites ( S1 Data ) . Most identified sites were detected in previous phosphoproteome studies ( PhosphoGRID , [49] , and S1 Table ) . Interestingly , we observed that larger changes in phoshorylation were induced by the diauxic shift and TBB compared to rapamycin treatment , which elicited less than 2-fold changes , similar to the results from the above-mentioned previous studies ( S1 Data and S1 Table ) . Importantly , the phosphorylated proteins identified are overlapping between the three conditions . However , the identified phosphorylation sites often differ between experiments , showing that our analyses are not saturating , and not all existing phosphorylation sites were identified . Proteins and their modification sites that changed at least 2-fold during diauxic shift are plotted in Fig 7A . The phosphorylation of some of these proteins ( Cic1p , Esf1p , Sas10p , and Utp14p ) were also found to be affected by rapamycin in previous studies ( S1 Table ) but not in our experiments ( S1 Data ) . Significantly , the majority of the affected proteins are known to be prerequisites for the cleavage of pre-rRNA at the site A2 . However , as all are components of the early preribosomes , it is not clear if they play mechanistically a direct role in the process . Their phosphorylation might be required for proper and directional preribosome assembly but not for cleavage per se . Nevertheless , these proteins are good candidates for the pre-rRNA processing regulator . Importantly , we identified a clear loss of phosphorylation of multiple proteins at predicted CK2 kinase consensus sites ( x-S/T-xx-E/D/pS/pY ) [50–52] ( black arrows in the Fig 7A ) . This is in a good agreement with the observed rapid loss of A2 cleavage and switch to the A3 pathway after inhibition of the CK2 kinase by TBB . To assess if any of these proteins might be direct substrates of CK2 , we analyzed Pwp2-containing preribosomes from cells treated with TBB ( Fig 7B and S1 Data ) . A majority of the proteins that showed at least 1 . 8-fold change in phosphorylation after TBB treatment are among the proteins changed during the diauxic shift , specifically , Mak11p , Mpp10p , Rrp5p , Sas10p , Utp14p , Utp18p , Utp21p , and Utp22p . Intriguingly , phosphorylation of Utp22p , which is a component of the CURI complex containing CK2 kinase itself , was strongly increased at Ser10 ( not a CK2 consensus site ) . It is unclear if CK2 activity controls phosphorylation at this site . In the other factors , we identified increase or decrease of phosphorylation mostly at sites different from the phosphorylation sites identified in the diauxic shift experiment . There are two notable exceptions . One of them is Mak11p , phosphorylation of which was reduced to a similar extent in both the diauxic shift and TBB treatment at sites Ser434/430 , which are part of a CK2 consensus site . Mak11p is involved in early steps of 60S biogenesis . Therefore , it might regulate 60S maturation after diauxic shift . The other exception is Rrp5p , which showed reduced phosphorylation at Ser187 , which lies directly adjacent to CK2 consensus . In addition , phosphorylation of Rrp5p was also changed at Ser1724 , identified in both diauxic shift and TBB experiments . Rrp5p is a huge protein proposed to coordinate pre-rRNA processing and assembly of early preribosomes [53] . It is thus an ideal candidate for regulation of the A2 and A3 cleavage choice . Our results indicate that a subset of the phosphoproteins identified in the diauxic shift experiment are directly or indirectly under the CK2 control; however , with the exception of Mak11p and perhaps Rrp5p , they do not represent direct bona fide CK2 substrates . Future research will also reveal if Mak11p and Rrp5p represent regulatory nodes of the TORC1 pathway controlling the pre-rRNA processing on a posttranscriptional level .
In this study , we show that TORC1 and CK2 kinases regulate pre-rRNA processing at the posttranscriptional level in response to nutrient availability or stress . Ribosome biogenesis clearly switches between two alternative processing routes , the A2 and A3 pathways . Such changes in the pre-rRNA processing occurs , for example , during the postdiauxic growth phase before entry to quiescence or during a transient arrest of ribosome biogenesis upon exposure to stress such as a heat shock . Based on the results presented here , we propose an updated model of the pre-rRNA processing and ribosome biogenesis in yeast ( Fig 7C ) . During a rapid exponential growth , TORC1 is active and yeast uses the A2 pathway to produce mature ribosomes . However , under unfavorable conditions , such as depletion of nutrients or exposure to environmental stress , the growth slows down sharply , or cells are transiently arrested in G1 . TORC1 becomes inactive , leading to a switch in pre-rRNA processing to the non-productive A3 pathway . The 35S pre-rRNA cleavage pattern changes from the A2 to the A3 cleavage site , concomitant with the abolition of 5′ETS and further downstream processing . This leads to accumulation of the 23S and 27S-B pre-rRNAs without further detectable production of new ribosomes . The A3 pathway continues to be used for several days after depletion of carbon source during the preparation for entry to quiescence ( G0 ) . When conditions improve , e . g . , after addition of fresh glucose to media or adjustment of the cell metabolism for higher growing temperature , the pre-rRNA processing reverts rapidly to A2 pathway . The presence of low levels of 23S pre-rRNA in wild-type yeast was reported almost 20 y ago [23] , but its physiological function has not been understood and has remained controversial in the field , with most considering it to result from aberrant processing [21 , 25] . Recently , during preparation of this manuscript , an independent study also observed the appearance of 23S pre-rRNA in high-density cultures or following a treatment with rapamycin [54] . The detection of 23S in exponentially growing yeast cultures seems to be dependent on their handling . Yeast cultures typically are not synchronized and thus contain cells in different stages of cell cycle or age . It is likely that the observed low amount of 23S pre-rRNA is produced in a small percentage of cells that are not growing fully exponentially , perhaps after dilution of a stationary culture or in cultures close to diauxic shift . Our results demonstrate that the 23S pre-rRNA is a physiological intermediate produced when ribosome production is repressed . There is an important difference between the two situations in which 23S pre-rRNA is observed . The appearance of 23S pre-rRNA was most often reported as a consequence of a depletion of early ribosome biogenesis factors or certain early RPSs [32] . However , the depletions were performed in exponentially growing cells in which all other factors remain available , a condition clearly different from the physiological diauxic shift analyzed here . In cases in which the 23S pre-rRNA was observed as a consequence of a mutation or depletion , only the 18S rRNA processing pathway is usually affected , and 25S rRNA continues to be synthesized , leading to an imbalance of the subunits levels . In contrast , we show here that the production of 23S pre-rRNA after diauxic shift or other nutrient depletion is associated with simultaneous arrest of both 18S and 25S rRNA . This strongly suggests an existence of a mechanism that coordinates the synthesis of both subunits under these undesirable but physiological conditions . We would also like to point out that depletions often were ( and still are ) performed for 12 hr or more , by which time the cells had very likely undergone the postdiauxic shift and started to accumulate 23S pre-rRNA . Therefore , results of long depletions need to be evaluated with caution . The switch from A2 to A3 pathway was rapid and completed within 20 min ( Fig 1 and S1 Fig ) . It is worth noting that our yeast cultures are asynchronous , and , therefore , the observed changes represent an average behavior from a large number of individual cells . The actual switch in the processing is likely to be significantly faster in the individual cells . This is corroborated by a more rapid reversal from A3 to A2 pathway upon addition of glucose ( 10 min ) . Cells starved for glucose are presumably more synchronized and , therefore , react more uniformly to the addition of fresh nutrient . The fast kinetics of the switch in processing strongly suggest a direct regulation of the A2/A3 cleavages , e . g . , by the phosphorylation or another posttranslational modification of a responsible biogenesis factor . However , two possible alternative mechanisms are discussed below . The initiation of transcription by the RNA pol I is down-regulated when nutrients are depleted or upon a treatment with rapamycin . We estimate from results of the pulse-chase experiments that rDNA transcription is reduced approximately 50-fold . This means that either only a small number of RNA polymerases initiate pre-rRNA synthesis or that the elongation rate of transcription is strongly reduced . The maturation of pre-rRNA and its assembly into ribosomes is immensely complex and dynamic . It is conceivable that the observed switch in pre-rRNA processing could be an unspecific “side effect” of the down-regulation of RNA pol I transcription . Changes in the initiation and/or elongation rates could , together with an imbalance of ribosome biogenesis factors , affect the folding and assembly and thus the cleavage pattern of the pre-rRNA . The experiments presented in Fig 4 ABCD provide evidence that this is unlikely to be the case . Firstly , the switch from the A2 to A3 pathway was not dependent on the RNA pol I ( Fig 4A and 4B ) . The processing of the pre-rRNA transcribed by the RNA pol II from the GAL7 promoter switched to the A3 pathway after heat shock or rapamycin treatment , conditions that do not significantly affect the transcription from the GAL7 promoter . The RNA pol II is not generally down-regulated during nutrient depletion . The GAL7 promoter also remains active during heat shock and rapamycin treatment [5 , 10] . Therefore , the switch also occurs in conditions in which the initiation and elongation rates are not significantly changed . Secondly , a reduction of the number of available RNA pol I ( but not its elongation rate ) by a rapid sequestering of the core subunit Rpa135p in the cytoplasm led to a sharp overall drop in pre-rRNA levels but did not induce a change in the processing pattern ( Fig 4C ) . Thus , a reduction of pre-rRNA levels leading to an imbalance between pre-rRNA and ribosome biogenesis factors did not affect the processing . Thirdly , in the reverse case , the continued synthesis of pre-rRNA in the CARA strain treated with rapamycin ( Fig 4D ) did not prevent switching to the A3 pathway . Although we cannot formally exclude that a specific change in the elongation rate ( or pausing ) of the RNA pol I in response to nutrient depletion is responsible for the switch to A3 pathway , such an effect would clearly also represent a case of a specific regulation . Taken together , our observations suggest that the switch between the A2 and A3 pathways is a specific regulatory event , most likely controlled at the posttranscriptional level . We also need to address another alternative mechanism: the change in pre-rRNA processing being caused by a general unavailability of RPs following nutrient depletion or rapamycin treatment . It is well known that transcription of genes encoding RPs is down-regulated in response to nutrient depletion [28 , 35] . It has been observed that depletion of several early RPSs leads to pre-rRNA processing defects and accumulation of 23S pre-rRNA [32] . Could reduction of the overall levels of RPs or a specific RP be responsible for the switch to A3 pathway ? Ju and Warner reported that the repression of rDNA transcription appeared to precede the reduction of transcription of RP genes [28] . It seems to suggest that cells ensure that the relative levels of RPs and synthesized pre-rRNAs remain balanced , presumably to avoid ribosome assembly defects observed in the depletion experiments mentioned above . Interestingly , in the case of TBB treatment , the levels of RPs’ mRNAs seem to be only partially reduced 15 min after the TBB exposure , while the change in pre-rRNA processing had already completed ( S5 Fig and Fig 6A ) . Crucially , the SILAC analyses of changes in the composition of preribosomes after the diauxic shift and rapamycin and TBB treatments convincingly exclude this alternative mechanism ( Figs 2D , 3E and 6C and S8 Fig and S1 Data ) . The data clearly show that there is no relevant loss of RPSs in the preribosomes after the switch to A3 pathway . On the contrary , most of the copurified RPSs are , in fact , enriched in the preribosomes . The only exception was the Rps27p , which was lost from postdiauxic preribosomes . However , the same protein was enriched in preribosomes after TBB treatment , which also underwent the switch to A3 pathway . Therefore , the Rps27p is not the cause of the switch , and its loss in the diauxic shift is a consequence of other structural changes in the preribosomes . Critically , we identified all the RPSs , depletion of which causes the appearance of 23S and a block in 20S production [32] ( S8 Fig ) . Depletion of 60S processing factors generally does not cause 23S phenotype . Furthermore , no 23S phenotype upon depletion of RPLs was reported in the extensive study by [55] . For completeness , we show also the general but insignificant ( less than 2-fold ) reduction of RPSs following the rapamycin treatment , in agreement with growth arrest ( S8 Fig ) . However , rapamycin does not fully reflect the physiological conditions of diauxic shift or nutrient depletion . Nevertheless , even in this case , none of the RPSs could be singled out as being more strongly reduced than others . Taking all this data together , the hypothesis that the observed switch in pre-rRNA processing is due to unavailability of RPs can be rejected . A regulation of late nucleoplasmic steps of ribosome maturation by the Tor pathway has been previously reported for 60S subunit synthesis [56] . The authors found that rapamycin treatment of yeast cells causes sequestration of the Nog1p ribosome biogenesis factor in the nucleolus and cessation of 60S ribosomal subunits maturation . Interestingly , our proteomic analyses show that Nog1p is strongly ( ~8-fold ) enriched in the Pwp2p containing preribosomes , following the postdiauxic shift . This observation agrees nicely with the reported nucleolar sequestration of Nog1p . We did not detect a phosphorylation of Nog1p in our proteomic analyses . It remains unclear whether Nog1p plays a more direct role in the Tor pathway-controlled arrest of ribosome biogenesis at the early stages during diauxic shift . Neither deletion of Sch9 nor expression of the hyperactive Sch9-3D2E mutant affected cells ability to switch from the A2 to A3 pathway . This is an important observation , as Sch9 is currently considered to be the master regulator of ribosome biogenesis downstream of TORC1 , controlling transcription by all three RNA polymerases [9] . It indicates that another , Sch9-independent branch or a direct target of TORC1 is responsible for the regulation of the A2 to A3 switch . A similar case of a partial regulation by Sch9 has been reported [19] . While Sch9 is sufficient to control transcription initiation of RNA Pol III by the phosphorylation of Maf1 repression factor , it has been shown that TORC1 can also directly phosphorylate Maf1 and , thus , partially sidestep the requirement for Sch9 [19] . In the presented work we tested all kinases and factors implicated in ribosome biogenesis acting downstream of Sch9 for their role in the A2 to A3 switch ( S3 Fig ) . The fact that they were all dispensable further supports the existence of a Sch9-independent branch of TORC1 required for the regulation of pre-rRNA processing and ribosome biogenesis . Our data also reveals that the A2 to A3 switch functions normally in tap42-11 and tip41Δ strains . Therefore , Tap42p/Tip41p functions disrupted by the mutations in tap42-11p or by a deletion of Tip41p are not involved in the control of A2/A3 cleavages . This is intriguing , as both major branches of TORC1 , Sch9p and Tap42p , do not seem to be involved in the switch regulation; however , we cannot exclude a possible redundancy . We found that CK2 activity is a prerequisite for the A2 pathway . Inhibition of CK2 led to an immediate switch to the A3 pathway . Prior inhibition of CK2 kinase delayed the effect of rapamycin on pre-rRNA processing , indicating that CK2 most likely lies downstream of TORC1 . The role of CK2 in the regulation of pre-rRNA processing is corroborated by our observation of reduced phosphorylation of Serine sites within a CK2 consensus motif in Mak11p and Rrp5p ( Fig 7A and 7B ) . The CK2 kinase is not only a component of preribosome but also forms independent CURI complex with Utp22p , Rrp7p , and Ifh1p that is proposed to be involved in transcription regulation of RP genes and could potentially coordinate it with ribosome biogenesis [35 , 44] . Interestingly , we did not observe loss of Utp22p or Rrp7p from preribosomes; in fact , both factors were enriched in preribosomes from postdiauxic shift and TBB-treated cells ( Figs 2D and 6C and S1 Data ) . Furthermore , Utp22p was hyperphosphorylated at Ser10 , which is not a consensus site for CK2 kinase . Does CK2 activity control phosphorylation of Utp22p at this site by other kinase ( s ) ? Future studies will reveal whether the role of CK2 in CURI complex is independent from its regulation of pre-rRNA processing presented here . The intermediates 23S or 27SB pre-rRNAs generated after the switch to the A3 pathway appear not to be further processed to mature rRNAs . This is strongly supported by the reduced levels of mature rRNAs in cells after the diauxic shift ( Fig 1B , bottom; note that total RNA from the same number of cells was loaded per lane ) . After the diauxic shift and change to A3 pathway , which occurred at OD600 ~ 9 . 5–10 in YPD , the cells grew very slowly and doubled after 2 d . At this time point , the 25S and 18S rRNA levels were reduced to about half , perfectly fitting with the idea that while the cells continue to grow , they do not produce new ribosomes and , thus , the existing ribosomes are divided between the mother and daughter cells . Whether the 23S and 27SB pre-rRNA produced by the A3 pathway can reenter the productive line after improvement of conditions ( fresh nutrients ) remains unclear . On the one hand , we saw a reciprocal disappearance of 23S and appearance of 20S pre-rRNAs in the pulse-chase experiment that could indicated that 23S is being processed into 20S pre-RNA ( Fig 2A ) . On the other hand , the 20S could also be directly produced from prelabeled 35S pre-rRNA that is also accumulating while the 23S is subjected to turnover . Unfortunately , the fast kinetics of the pre-rRNA processing prevented us to answer this intriguing question conclusively . Why do cells continue to synthesize and partially process pre-rRNA if they do not seem to make new ribosomes ? Yeast is a unicellular microorganism , and , as such , it must react fast to environmental changes , in both directions—negative ( e . g . , lack of nutrients when rain washes it away ) or positive ( e . g . , it falls on a new ripe fruit ) . In nature , it needs to compete with other microorganism for the available resources . Therefore , the faster it can start using resources and grow , the more likely it is to outcompete the others and survive ( including the strategy to use fermentation even in the presence of oxygen in order to produce ethanol that inhibits growth of many other organisms ) . It might seem more economical to stop rRNA synthesis completely by repression of RNA pol I activity . However , complete repression of rDNA transcription is likely disadvantageous . Inhibition of RNA pol I transcription , e . g . , by actinomycin D , leads to disruption of nucleolar morphology and diffusion of ribosome biogenesis factors [57 , 58] . In contrast , nutrients starvation or rapamycin treatment leaves nucleoli unchanged [57] . The maintenance of the nucleolus and its structure is intrinsically dependent on the ongoing ribosome biogenesis . We suggest that continued low transcription of rDNA followed by the A3 cleavage of 35S pre-rRNA , both of which occur in the nucleolus , allow cells to maintain nucleolar integrity and to keep high-effective , local concentration of ribosome biogenesis factors . Cells therefore remain poised to switch rapidly to exponential growth should the conditions improve . The capability to rapidly resume growth and compete for resources against other organisms provides an evolutionary advantage that greatly outweighs the energy loss required for sustained pre-rRNA synthesis . In summary , we demonstrate that TORC1 and the CK2 kinase regulate ribosome biogenesis at the posttranscriptional level , independently of the Sch9p kinase branch . We identified Mak11p and Rrp5p as potential targets of CK2 or TORC1 in preribosomes . These findings provide a deep insight into the mechanisms by which cells regulate ribosome biogenesis in response to changing environmental conditions and contribute to our understanding of regulation of cellular processes in preparation for survival , such as entry to quiescence .
The S . cerevisiae strains were grown in YPD , SDC-ura , SDC-his , or YPG ( all from Formedium , United Kingdom ) and harvested as described in the figures . Standard yeast techniques were used as in [59] . The tetracycline depletion strains were constructed and depleted as described in [60 , 65] . Strains used in this study are listed in S2 Table . For treatment with inhibitors , rapamycin was added from a stock in DMSO to final concentration 200 ng/ml . To inhibit CK2 , 4 , 5 , 6 , 7-Tetrabromobenzotriazole ( TBB ) ( Santa Cruz Biotech or Merck ) was added from 10 mM stock in DMSO to final concentration 100 μM . In both cases , the control cultures were treated with DMSO only in the equivalent final concentration of DMSO . Total RNA was extracted with guanidine , phenol-chloroform extractions , and precipitation as described [61] . Northern blotting and hybridization were performed as described [62] , using the total RNA isolated from equivalent number of cells per lane . RNA was denatured with glyoxal and run overnight on 1% agarose-BTPE gel . The RNA was then electro-transferred on positively charged membrane and hybridized in Church buffer with end-labeled oligonucleotide probes ( S3 Table ) . After , washing blots were exposed to phosphor imaging plates ( Fuji ) , scanned on the Fuji FLA7000 imager ( Fuji ) , and quantified with AIDA Image Analyzer software ( Raytest , Germany ) . All experiments were done in two or more biological replicas . Pulse-chase experiments and quantification were performed as previously described [26] . Briefly , to exponential or postdiauxic shift cultures growing at 30°C in SD-Ura media , [5 , 6-3H]-Uracil was added for the time indicated . The chase was initiated by the addition of 100 mM Uracil . Samples of 1 ml volume were collected at the different time points , centrifuged , media removed , and pellets frozen . Total RNA was extracted , separated in 1 . 2% agarose-glyoxal gels , and transferred to nylon membrane . The membranes were exposed to Fuji BAS imaging plates and scanned on the FLA-7000 imager ( Fuji ) . All experiments were done in two or more biological replicas . Cells were grown exponentially in SDC ( light media ) or after diauxic shift/rapamycin treatment in SDC containing 13C , 15N-L-Arginine and 13C , 15N-L-Lysine ( heavy media ) . For diauxic shift , the cells were harvested at OD600 2 and 4 . 5 . For rapamycin or TBB treatment , the OD600 was 2 . 0 . An equal number ( ODs ) of cells were harvested separately ( to avoid exposure of the control cells to rapamycin/TBB , or vice versa of depleted cells to glucose ) , pellets were mixed , lysed , and 90S preribosomes were affinity purified through both affinity steps using Pwp2-FLAG-TEV-ProteinA as bait . For detection of phosphorylation sites , phosphopeptides were enriched on TiMAC . The proteins present in the purified Pwp2-associated preribosomes and their phosphorylation were determined by mass spectrometry at the FingerPrints Proteomics , Dundee ) , and raw data was analyzed by MaxQuant software [63 , 64] with default settings . The H/L ratios of individual proteins were normalized to the H/L ratio of the bait ( which was thus set to 1 ) . For phosphosites , the H/L ratios were normalized to H/L ratio of the protein abundance ( all peptides quantified ) . All experiments were done in two or more biological replicas . | The yeast Saccharomyces cerevisiae must , as a single-celled microorganism , rapidly respond to changes in its environment and carefully balance its energy needs with the available resources . One of the major energy-consuming processes in the cell is the production of ribosomes . Ribosome biogenesis is highly dynamic and complex and requires the coordinated action of myriads of factors and RNAs . It begins with the synthesis of the precursor ribosomal RNA ( rRNA ) , which is concurrently cleaved , modified , folded , and assembled together with ribosomal proteins into mature ribosomal subunits . It is known that in response to depletion of nutrients , yeast quickly represses the transcription of genes encoding rRNAs and ribosomal proteins . In this study , we reveal that yeast also rapidly switches to an alternative rRNA processing pathway , in which the precursor rRNA is cleaved differently and the ribosome biogenesis is arrested at a distinct stage . We demonstrate that the choice between the two alternative pathways is controlled by the target of rapamycin complex 1 ( TORC1 ) and casein kinase 2 ( CK2 ) kinase , but does not require Tap42p and Sch9p , which are currently thought to be the major effectors of TORC1 . Our results indicate the existence of a so far unidentified branch of TORC1 signaling regulating ribosomes biogenesis at the posttranscriptional level . | [
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] | 2017 | Tor1 and CK2 kinases control a switch between alternative ribosome biogenesis pathways in a growth-dependent manner |
Populations change in size over time due to factors such as population growth , migration , bottleneck events , natural disasters , and disease . The historical effective size of a population affects the power and resolution of genetic association studies . For admixed populations , it is not only the overall effective population size that is of interest , but also the effective sizes of the component ancestral populations . We use identity by descent and local ancestry inferred from genome-wide genetic data to estimate overall and ancestry-specific effective population size during the past hundred generations for nine admixed American populations from the Hispanic Community Health Study/Study of Latinos , and for African-American and European-American populations from two US cities . In these populations , the estimated pre-admixture effective sizes of the ancestral populations vary by sampled population , suggesting that the ancestors of different sampled populations were drawn from different sub-populations . In addition , we estimate that overall effective population sizes dropped substantially in the generations immediately after the commencement of European and African immigration , reaching a minimum around 12 generations ago , but rebounded within a small number of generations afterwards . Of the populations that we considered , the population of individuals originating from Puerto Rico has the smallest bottleneck size of one thousand , while the Pittsburgh African-American population has the largest bottleneck size of two hundred thousand .
Effective population size is a key factor in evolutionary genetic processes , such as drift and selection , which have important implications for medical genetics [1 , 2] . With the development of agriculture , human populations have grown super-exponentially during the past few thousand years [3 , 4] . More recently improved sanitation , modern medicine , and industrialized food production have further accelerated population growth . During the last few generations , growth rates have slowed or become negative in many human populations due to the availability of effective birth control methods , higher levels of education for women , urbanization , and other factors [5] . In addition to these global trends , populations in the Americas have experienced bottlenecks due to migrations , introduced diseases , and other effects of colonization . Effective population size is a genetics-based measure of population size [6] . Here we use inbreeding effective population size defined in terms of coalescence probability ( the probability that a given pair of haplotypes are descended from a single haplotype in the previous generation ) . Consider a population of diploid individuals . Randomly select a neutrally evolving locus and a pair of haplotypes from the population . Let qg be the probability that the two haplotypes have a common ancestor g generations ago at that locus , conditional on not having a common ancestor in the last g − 1 generations . In a randomly mating population with Ng breeding individuals g generations ago ( 2Ng haplotypes ) , that probability would be 1/ ( 2Ng ) because there are 2Ng possible ancestors for the second haplotype , each equally likely and only one of which is the ancestor of the first haplotype . Thus , solving qg = 1/ ( 2Ng ) for Ng we obtain the effective population size g generations ago as Ng = 1/ ( 2qg ) . Consequently , to estimate the effective size of a population , we first estimate the conditional coalescence probability qg , and then use the relationship Ng = 1/ ( 2qg ) . The effective size of a population is generally smaller than the census size of a population due to demographic factors such as overlapping generations [7] . In general , populations are not closed , but experience migration . Thus the effective size g generations ago reflects the conceptual population of ancestors that contributed to the current generation , rather than an actual population residing in a certain location g generations ago . Furthermore , the definition of effective size assumes random mating , but actual populations are structured by geography and by cultural factors , with preference for mating within sub-population . Thus the effective size depends on the sampling scheme , which may over-represent some of the sub-populations . Admixed individuals have ancestry that is recently derived from more than one continental population . In the Americas , many individuals have admixed ancestry derived from indigenous peoples of the Americas , European settlers , and enslaved Africans forcibly brought to the Americas , as well as more recent immigration . Because the migration events that brought these continental groups together are recent ( less than 20 generations before present in the Americas ) , the chromosomal segments of single-continent ancestry tend to be long , and it is possible to infer the ancestry at most points in the genome from genotype data [8–11] . Analyses can then be performed using only the parts of the genome that are inferred as derived from a particular ancestry . This enables inference about the ancestral populations . An example of ancestry-specific analysis of admixed data is ancestry-specific principal components analysis , which can be used to investigate the degree of genetic similarity between the ancestors of individuals in an admixed sample and present-day individuals in a geographical region [12 , 13] . When considering the effective size of an admixed population , the overall effective size of the population is generally the most relevant quantity subsequent to admixture . In contrast , preceding admixture the overall effective population size of the combined contributing ancestral populations may not be the quantity of interest . Instead , one may wish to know the historical effective sizes of those individual ancestral populations . Consider two haplotypes of a particular ancestry that are sampled from the admixed population at a fixed locus . Looking back in time , if those two haplotypes have not coalesced at this locus by the time of the onset of admixture , the probability that they coalesce in the previous generation depends ( as noted above ) on the effective size of the population in which the haplotypes were located at that time , which is the particular source ancestral population . For example , in a sample of admixed individuals from a Latin American location , the ancestral populations may be from Europe ( primarily Spain ) , Africa ( primarily West Africa ) , and America . The ancestral American populations are primarily those that were resident pre-admixture in the region around the sampling location , but can include a broader region if the sampling location is home to significant numbers of migrants from elsewhere in Latin America . When we calculate ancestry-specific effective population size for pre-admixture times , we are estimating the effective sizes of the source populations that participated in the admixture , which could be subsets of larger populations ( e . g . Spanish colonizers within the larger Spanish population ) . Although we can also calculate ancestry-specific effective sizes post-admixture , these may not be particularly meaningful because they do not correspond to any actual population of individuals . Identity-by-descent ( IBD ) sharing in population samples can be used to estimate recent effective population size [14 , 15] . Segments of IBD can be detected in genotype data . We consider segments with genetic length greater than 2 centiMorgans ( cM ) . These segments are due to inheritance from recent common ancestors within the past several hundred generations . The numbers and lengths of IBD segments contain information about coalescence probabilities . Shorter segments of IBD represent coalescent events that occurred further back in time ( up to several hundred generations ago ) , while longer segments represent coalescent events that occurred in the past few generations . If the number of IBD segments is high , a larger number of coalescence events have occurred , indicating that the coalescence probability is high and thus the effective size is low . Similarly , if the number of IBD segments is low , the effective size is high . These relationships can be quantified mathematically for estimation of effective size , including estimation of changes in effective population size over time . A previous study estimated ancestry-specific historical effective population size using the site frequency spectrum of alleles in ancestry segments from admixed individuals [16] . The site frequency spectrum interrogates a much more distant time period than the IBD-based method that we use here . Site frequency spectrum methods also require sequence data , whereas our IBD-based method can use single nucleotide polymorphism ( SNP ) array data , increasing the range of existing data to which it can be applied . We demonstrate the effectiveness of our ancestry-specific effective population size estimation methodology with simulated data , and then use our methodology to estimate ancestry-specific recent effective population size in populations from the Hispanic Community Health Study/Study of Latinos ( HCHS/SOL ) , and in African-American and European-American populations in two US cities from the Healthy Aging and Body Composition ( Health ABC ) study .
We first summarize the key points of the IBDNe method for estimating the effective population size of homogeneous populations ( further details may be found in [14] ) , and then describe how this method can be applied to ancestry-specific effective size in admixed populations . Consider a population with an effective size of Ng diploid individuals g generations before the present ( generation 0 is the current generation , generation 1 is the most recent previous generation , and so on ) . If qg is the probability that a pair of haplotypes randomly sampled from the current generation coalesce ( have most recent common ancestor ) at generation g given that they have not coalesced by generation g − 1 , then Ng = 1/ ( 2qg ) by definition ( see Introduction ) . Suppose we have a sample of individuals from the current generation , and we identify long segments of identity by descent between pairs of haplotypes drawn from different individuals , finding all those segments of identity by descent that exceed some length threshold C . In most settings , we use C = 2 cM , since we have high power to detect such segments using existing methods such as Refined IBD [17] . Then , if the past trajectory of effective size N = {Ng: g ≥ 1} was known , we could calculate Eg , the expected total length of detected IBD genome-wide that is attributable to most recent common ancestry at generation g . Also , if the effective size trajectory N was known , we could calculate the probability that an IBD segment of length l is due to most recent common ancestry at generation g , and hence obtain Og , the expected total length of detected IBD genome-wide that is attributable to most recent ancestry at generation g , conditional on the observed IBD segments . Both Eg and Og are functions of N . Finding values of Ng that give equal values of Eg and Og provides a methods of moments estimate of N . We iterate estimating N and re-calculating Eg and Og until convergence of our estimate of N . During the iterative estimation process we also impose a smoothness requirement on Ng as a function of g to aid in the estimation . Now consider an admixed population , in which local ancestry has been determined . The ancestry-specific effective size Ng ( a ) for ancestry a considers only those haplotypes that are descended from ancestry a . If qg ( a ) is the probability that a pair of such haplotypes randomly sampled from the current generation coalesce at generation g given that they have not coalesced in generations 1 to g − 1 , then Ng ( a ) =1/ ( 2qg ( a ) ) . For generations prior to the admixture event , this ancestry-specific effective size Ng ( a ) represents the total effective size of the ancestral population that contributed to ancestry a in the admixed population . For example , if considering European ancestry , and the European ancestors came from some population in Spain , the pre-admixture European-specific effective population size will be the effective size of that population in Spain . Our interest in the ancestry-specific effective population size is mainly for the pre-admixture effective population sizes , but we also obtain estimates of post-admixture ancestry-specific effective population sizes . If the post-admixture population is randomly mating , and has proportion p ( a ) of its ancestry being of ancestry a , then it is straightforward to show that Ng ( a ) =p ( a ) Ng where Ng is the overall effective size of the admixed population . If there is assortative mating or ongoing migration , this relationship will not hold . We now discuss how to estimate N ( a ) ={Ng ( a ) :g≥1} using the IBDNe framework . The IBDNe framework needs the following information: the IBD lengths , in order to obtain Og; and the number of pairs of sampled haplotypes , which are needed to obtain Eg . With a homogenous population , we obtain the IBD lengths directly from the detected IBD segments , and we calculate the number of pairs of sampled haplotypes from the number of sampled individuals . With ancestry-specific analysis , there are differences because we are only interested in IBD between haplotypes of the given ancestry , but ancestry is not constant along the genome . This affects both the way in which the IBD lengths are handled , and the way in which the number of pairs of sampled haplotypes is calculated . Some IBD segments in an admixed population will have a very recent common ancestor ( from the generations post-admixture ) , and since this ancestor is admixed , the IBD segment may include more than one ancestry . Only those parts of the segment that are derived from the ancestry of interest will contribute to the total length of detected IBD for this ancestry , but we still need to know the length of the whole IBD segment in order to calculate the probability that the most recent common ancestor lived in generation g ( see Methods ) . Further , the number of pairs of sampled haplotypes of the given ancestry now varies from one genomic position to another , because the ancestry of each individual’s DNA varies along the genome , but the expected number of pairs of sampled haplotypes of the given ancestry can be calculated from the genome-wide ancestry proportions ( see Methods ) . Apart from these differences , estimation of ancestry-specific effective population size is the same as for estimation of overall effective population size , and the existing IBDNe software may be used . An example analysis pipeline is provided at http://faculty . washington . edu/sguy/asibdne/ . We simulated an admixed population with ancestry from three continental groups in order to test the accuracy of our methods . The simulation includes a low rate of genotype error ( 0 . 1% ) and the simulated data have a marker density that is similar to a 1M-feature SNP array . A full description of the data simulation can be found in Methods ( in the “Simulated data” section ) . Briefly , we used a coalescent-based simulator to simulate the Africa-Europe-Asia demographic history estimated by the 1000 Genomes Project [18] , and added population bottlenecks and admixture occurring 12 generations ago . Fig 1 shows true and estimated ancestry-specific effective population size . We see that important aspects of the effective population size trajectory are represented in the estimated trajectories , including the approximate timing of the population bottleneck , the approximate size of the ancestral population , and the effective size of the ancestry-specific fraction of the admixed population after the bottleneck . The bootstrap confidence intervals do not always cover the true value , but they provide an approximate measure of the precision of the estimates . In order to keep the simulation from becoming overly complex , the simulated population size decrease associated with migration occurs instantaneously , resulting in a sharp bottleneck in population size . The effective population size estimation procedure cannot fully capture this sharp change because it applies smoothing by fitting exponential growth curves to groups of 8 generations . In real life admixture events , such as that associated with the colonization of the Americas , we would not expect population changes to occur instantaneously . Rather , migration and population decline would have taken place over the course of several generations , resulting in smoother effective size trajectories . We simulated two admixture scenarios that don’t include bottlenecks . One has the three populations merging without population size reductions , while the other has continuous migration from two of the populations into the third . We estimated ancestry specific effective population sizes and show the results for the merging scenario in S1 Fig and for the continuous migration scenario in S2 Fig . Because these scenarios don’t include large population size changes over short time intervals , the estimates closely match the underlying true values , and the bootstrap intervals mostly cover the true values . We also simulated an admixture scenario with recent population structure , with or without biased sampling across the sub-populations , and show the results in S3 Fig . With unbiased sampling the results are similar to those for the main simulation without structure ( Fig 1 ) , while with biased sampling the ancestry-specific effective sizes are underestimated in the first few generations . We group individuals in HCHS/SOL into populations based on the reported country-of-origin of their grandparents . Individuals with missing grandparental origins or grandparents from different countries are omitted from the analysis . Fig 2 shows estimated ancestry-specific effective population sizes and overall effective population sizes for the HCHS/SOL populations for the past 100 generations . S1 Table shows average total length of detected IBD segments shared by unrelated pairs of samples , which is a summary of the data used to estimate the overall effective population size . Table 1 gives sample sizes for each population . When the overall sample size is low , the amount of data for estimating the overall effective population size is low and the estimates will have a high level of uncertainty . Similarly , when the average genome-wide ancestry proportion multiplied by the sample size is low , the amount of data for estimating the ancestry-specific effective population size is low . The bootstrap intervals give approximate measures of precision of the estimates . Estimates with wide intervals should be disregarded because the bootstrap intervals may not capture the full extent of the uncertainty in these estimates . The widest intervals occur when the sample size and/or average ancestry proportion is lowest . Wide pre-bottleneck confidence intervals are also seen for Puerto Rico , despite its high sample size , due to the extremely small bottleneck that occurred in this population . The small bottleneck means that many of the possible coalescences between haplotypes occurred in the post-bottleneck period , leaving few independent haplotypes to provide information about the pre-bottleneck period . In consequence , we recommend against drawing conclusions about the pre-bottleneck sizes of the populations ancestral to Puerto Ricans from this analysis . The results for Puerto Rico show an apparent severe drop in overall effective population size in the most recent couple of generations . This is an artefact resulting from an excess of relatives in the Puerto Rican sample , particularly at the level of 2nd to 3rd cousins . Although we exclude IBD from relatives that are half-siblings or closer ( including parent-offspring pairs and full siblings ) , it is not straightforward to exclude IBD from more distant relatives without the use of pedigree information , and the IBDNe program is not designed to allow for the removal of these more distant relatives . Inclusion of these relatives in the analysis results in an excess of long IBD segments which depresses the estimate of effective population size in the last few generations . This effect may also somewhat depress estimates of effective population size in the last few generations in the other HCHS/SOL populations , though clearly not to such an extreme as for the Puerto Rican sample . Most of the populations and ancestries show a clear population bottleneck around 12 generations ago . Colonization began earlier , around 17 generations ago ( approximately 500 years ago , assuming 30 years per generation ) , but occurred over the course of multiple generations . Overall bottleneck sizes vary considerably across the populations , ranging from 1 , 000 for Puerto Rico to 60 , 000 for Mexico . Growth in overall effective population size subsequent to the bottleneck is estimated to have been very high in all the populations , with estimated current effective sizes in the hundreds of thousands or millions ( Table 2 ) . When interpreting the drop in ancestry-specific effective population size at colonization , one must consider population structure . Populations are not closed units , since there is always migration between regions . When one considers the effective size of a “population” , one is considering the effective number of ancestors of the individuals in that population . For example , if the population is a village in a region with low migration , most of the parents and grandparents ( corresponding to effective size at generations 1 and 2 , respectively ) will be derived from that village and the effective size will reflect the effective size of the village . However , when one looks back 20 generations , many of the ancestors may have come from nearby villages , and the effective size will reflect the effective size of the region containing those villages . Thus the effective size for the village may be lower in recent generations than in more distant generations , even if the census population size in the village and region has been stable . This effect was noted in a previous IBD-based analysis [15] . In the context of HCHS/SOL , the effective size 20 or so generations before admixture may reflect larger regional effective sizes while the immediate pre-bottleneck sizes reflect smaller sub-populations . Thus the changes in size between the maximum pre-admixture effective population size and the bottleneck effective population size ( Fig 2 and Table 2 ) reflect these population structure effects as well as the effects of colonization . Fig 3 shows selected population-ancestry combinations for which the precision is relatively high . African-American and European-American populations from Memphis are included for comparison . Considering each ancestry in turn , we see similarities and differences between populations in the estimated pre-admixture effective population sizes . In the African component , we see smaller estimated pre-admixture effective sizes for Cuba ( 150 , 000 ) and Mexico ( 100 , 000 ) than for the Dominican Republic ( 700 , 000 ) , suggesting that the African ancestors of the former two populations came from smaller sub-populations of Africa than the African ancestors of the latter two populations . In the European component we see smaller estimated pre-admixture effective sizes for Cuba ( 200 , 000 ) , Mexico ( 150 , 000 ) , and Nicaragua ( 120 , 000 ) than for the Dominican Republic ( 400 , 000 ) . In the American ancestral component , the estimated pre-admixture effective sizes are similar between Nicaragua ( 400 , 000 ) , Ecuador ( 700 , 000 ) , and Mexico ( 600 , 000 ) . We can also look at the relative magnitude of the bottleneck population sizes to the pre-admixture sizes , bearing in mind the potential effects of population structure discussed above . For the African and European ancestral components , the drops in size are presumably mostly related to founder effects induced by migration . In contrast , for the American ancestral components , the drops in effective population size are likely due to the negative impacts of colonization including war and disease . Mexico had a relatively smaller estimated reduction in American-specific effective population size compared to the other populations ( Fig 3 and Table 2 ) . In interpreting these results , it is important to recognize that the sampled individuals were residents of four major cities in the United States . Thus the results apply to those particular urban populations , and not necessarily to the entire countries-of-origin represented . If the populations in the US are derived from regional subsets of the countries-of-origin , the estimated effective sizes will be smaller than would be found for the countries as a whole if one had samples of individuals drawn randomly from those countries . This would be expected to have a significant influence on the estimated effective size of the most recent generations , and less influence on more distant generations due to mixing within the population over time . In order to further investigate the demographic history of US populations we analyzed data from the Health ABC study , which is comprised of samples from Memphis and Pittsburg ( Table 3 ) . As in the HCHS/SOL populations , the Health ABC populations show significant growth in the most recent generations , as expected . The overall bottleneck effective sizes for the Memphis populations and for the Pittsburgh African-American population are 130 , 000–190 , 000 which is more than twice as large as those for any of the HCHS/SOL populations . The pre-admixture African-specific effective population sizes for the Memphis and Pittsburg populations are 1–2 million , and thus are higher than those for HCHS/SOL populations ( Fig 3 ) . The pre-admixture European-specific effective sizes for the African-American populations and for the Memphis European-American population are around 1 million , and thus are also higher than those for most of the HCHS/SOL populations . The estimated ancestry-specific effective population sizes for the African-American populations in these two cities are similar to each other ( Fig 4 ) . The similarity of the estimated demographic histories of the Memphis and Pittsburgh African-American populations suggests significant historical mixing within the larger African ancestry population that encompasses these cities , so that the two populations have a shared demographic history . In particular , the similarity of the estimated demographic histories of the European ancestry component is consistent with previous analysis of genetic data from African Americans [19] , which indicates that most of the European admixture in US African American populations occurred in the southern US prior to the Great Migration movement of African Americans from the South to elsewhere in the US . In contrast , the demographic histories of the European-American populations in Memphis and Pittsburgh differ , both before and after the founding bottleneck . Prior to the founding bottleneck , the effective population size of the Memphis European-American population was similar to that of the European component of the two African-American populations , suggesting that the European ancestors of these populations were drawn from the same European source , which again is consistent with the European admixture in the African-American populations having occurred in the South . Memphis European-Americans have a higher estimated current effective size than Pittsburgh European-Americans , which contrasts with a previous report of more long segments of IBD between European Americans in the South than in the Northeast [19] . Urban areas often differ from the general population in having more immigrants , both domestic and foreign . During the period 1910–1950 , Memphis grew rapidly , tripling in size from 130 thousand to 400 thousand , while Pittsburgh’s population only increased slowly , from 530 thousand to 680 thousand [20] . Since 1950 , Memphis’s population has continued to grow , while Pittsburgh’s population has declined . Thus , it is likely that Memphis’s European American Health ABC population has more diverse geographical origins on average than Pittsburgh’s European American Health ABC population , leading to the larger current effective population size . A striking difference between the two demographic histories is that the estimated bottleneck size in Pittsburg European-Americans is significantly smaller than that in Memphis , and the timing of the bottleneck appears to be earlier ( > 20 generations ago versus around 10 generations ago ) , which is earlier than the timing of European migration to North America . Also the decrease in population size approaching the bottleneck is very gradual , which suggests that the ancestors came from sub-populations that had quite slow rates of mixing with the broader European ancestral population . One possible explanation that could fit both of these characteristics is that many of the ancestors of the European-American population in Pittsburgh may have been members of groups that formed relatively small separated populations within Europe prior to their migration to the US . An example of such an ancestral group that fits the historical record would be the Anabaptists ( including Mennonites and Amish ) , who separated from other European populations around 500 years ago and migrated to the US in large numbers to escape religious persecution . Many of these Anabaptists settled in Pennsylvania [21] . Although this is one possible explanation , it is not the only possibility , and our data do not address the question of origins .
In this paper we presented a method for calculating ancestry-specific recent effective population size by integrating local ancestry calls with inferred IBD segments . With this approach , one can estimate the demographic history over the past several thousand years of populations ancestral to current-day admixed populations . We applied our method to data from admixed populations sampled in the United States . Our method is based on iterated method of moments . As such , it is not guaranteed to make full use of all information contained in the data , although we show that it is able to make accurate inferences from moderately large samples . One source of information that we do not incorporate in our method is the observation that any IBD segment that contains a switch in ancestry must necessarily be due to a post-admixture ancestor . This information could improve the estimation of the number of generations to the most recent common ancestor of the IBD segment if the time of the onset of admixture is known . However only a small proportion of IBD segments would provide this additional information because most IBD segments will not have a switch in ancestry and could be inherited from either pre- or post-admixture ancestors . Previous methods for ancestry-specific effective population size estimation have not been able to estimate changes in effective size in the recent past , so the approach presented here opens new avenues for inference of demographic history . Whereas Gravel et al . [16] estimate constant American-specific effective population sizes over the past ~12 , 000 years , our method allows for estimation of population growth and bottlenecks during the past 500 years . Our estimates ( Table 2 and Fig 2 ) of the American-specific effective size of Mexico are fairly flat over the past 500 years , and are in agreement with Gravel et al . ’s estimate of 62 , 000 . We estimate an order of magnitude bottleneck around 13 generations ago for Colombia’s American ancestry population , and a three orders of magnitude bottleneck around 13 generations ago for Puerto Rico’s American ancestry population . The estimates of American-specific effective size given by Gravel et al . ( 7 , 000 for Colombia and 2 , 000 for Puerto Rico ) are intermediate between our bottleneck and maximal pre-admixture estimated sizes . A caveat of our approach , and that of other methods based on local ancestry calls , including that of Gravel et al . [16] , is that it depends on the accuracy of the local ancestry calls and inferred IBD segments . In simulated data that was designed to have similar characteristics to real human data , the results produced with our analysis pipeline had good accuracy , giving confidence that the results presented here are sound . Our approach requires at least a few hundred samples . Additional samples are required when considering an ancestral component that forms a relatively small proportion , p , of the overall ancestry of the population . For most human populations , we recommend the use of sample sizes , n , that are sufficiently large so that np > 100 . The precision of the estimates depends on the total number of IBD segments detected , which depends not only on np but also on the effective population size that is being estimated , so that a larger sample size will be needed in populations of larger effective size . In addition , in populations with extremely small bottleneck sizes the pre-bottleneck estimates will have low precision due to a high proportion of haplotypes coalescing around the time of the bottleneck , leaving few haplotypes to provide information about the pre-bottleneck sizes . When the sample size is small , or when considering effective sizes prior to a strong bottleneck event , the bootstrap intervals are larger , indicating lack of precision . In our experience , when the bootstrap intervals are particularly large they have significantly lower coverage than the nominal 95% and hence the results may be unreliable . For this reason , we suggest avoiding making inferences about the apparently large pre-bottleneck sizes of the populations ancestral to Puerto Ricans . Our method estimates effective population size , which provides much information about population history including the approximate timing of population bottlenecks associated with migration and colonization . Admixture likely began at approximately the same time as the bottlenecks , however our method does not assume this or prove it to be the case . Other approaches can be used to directly estimate admixture times and other aspects of population history such as specific source populations [12 , 16 , 22] . We can compare our results to historical records , when those are available . The Trans-Atlantic Slave Trade Database ( www . slavevoyages . org ) provides numbers of disembarked slaves in different regions of the Americas . The total number of slaves disembarked in Mainland North America was 303 , 209 which is two to three times as high as the estimated bottleneck African-specific sizes in Memphis and Pittsburgh . The number of slaves disembarked in Puerto Rico was 23 , 779 while the estimated bottleneck size was 540; 728 , 809 slaves disembarked in Cuba while the estimated bottleneck size was 8400; 28 , 818 slaves disembarked in Hispaniola while the estimated bottleneck size in the Dominican Republic was 3700 . Thus the estimated African-specific bottleneck effective population sizes are all much smaller than the numbers of arriving slaves . Several factors may play a role in this: first , many slaves died soon after arrival in the New World; second the sex-ratio in the slave trade was imbalanced with around 60% of imported slaves being male [23]; third , there are factors of geographically-based population structure involved , in that not all slaves disembarking at a regional port would have stayed in that region to contribute to the ancestry of the current-day population of that region . In modern human populations with low levels of immigration , the overall effective population size tends to be around one-third of the census size , largely because the generation time is approximately one-third of the expected lifespan [24] . In a previous analyses of the European-ancestry populations of the UK and Finland , the recent estimated effective sizes were in line with this expectation [14] . In contrast , in this study , we did not find that estimates of overall recent effective size were around one-third of the census values . There are several reasons for this . First , in the case of Pittsburgh and Memphis , the populations are cities and experience large amounts of migration , including immigration from other regions of the US . Thus it is not surprising that the estimated current effective sizes ( Table 2 ) are significantly larger than the census sizes of these cities ( less than 1 million in each case , although the populations of the broader metropolitan areas are significantly larger ) . Second , in the HCHS/SOL analysis , the individuals sampled are far from being a random sample of individuals from their respective countries of origin: they are immigrants to the US , and they were sampled by household in four US communities . The household design results in an excess of relatives in some of the populations , which reduces most recent effective size estimates . The restriction to immigrants and then further to four US communities also reduces post-immigration effective population sizes . Finally , immigrants are a self-selected group that is unlikely to be representative of the country-of-origin as a whole , and will likely have over-representation from certain sub-populations . Our simulations showed that biased sampling of a structured population results in underestimation of most recent effective population size . When we compare the estimated current effective sizes of HCHS/SOL country-of-origin populations to World Bank population sizes ( accessed via Google Public Data Explorer ) from 1995 ( when the average age of the sampled individuals was around 25 ) , we find that the ratio of current estimated effective size to 1995 population size ranges from approximately 1/60 ( Ecuador ) to approximately 1/4 ( Cuba ) , with typical values around 1/10 . Although estimates of effective size in the most recent generations are affected by these issues , our simulations also showed that less recent generations are not affected . Thus our estimates are useful for learning about the effective population sizes at and before admixture . We found that the overall bottleneck ( founder ) effective population size for the population of individuals of Puerto Rican origin was significantly lower than for the other populations considered . Populations with small founder sizes can have medically important genetic variants at moderate frequencies that would be extremely rare elsewhere in the world [2 , 25] . Such populations are valued for genetic studies , because relatively small samples from such populations are often sufficient to determine the locations of disease-associated variants . In analysis of data from two US cities ( Memphis and Pittsburgh ) , we found that the estimated historical effective sizes of the African-American populations from the two cities were very similar , consistent with a shared demographic history . In contrast , the estimated demographic histories of the European-American population in each city were different . In conclusion , estimates of ancestry-specific recent effective population size can shed light on past demographic events and suggest directions for future medical genetics research . A caveat for the results presented in this paper is that they apply specifically to the sampled populations . The HCHS/SOL individuals were sampled in four major US cities , and may not be representative of their countries of origin or of Hispanics throughout the US . The individuals sampled in the Health ABC study were at least 70 years old when they were recruited in 1997–1998 , and thus they are not necessarily representative of current-day individuals living in Memphis and Pittsburgh .
We used msprime [26] to simulate three continental populations ( Africa , Europe and Asia ) with recent admixture . Our pre-admixture model is based on a published model inferred from the 1000 Genomes project data [18] . The initial population size ( in Africa ) was 7310 , which increased 5920 generations ago to 14474 . The out of Africa event occurred 2040 generations ago , with the out-of-Africa population size being 1861 , and the migration rate between Africa and out-of-Africa being 1 . 5 × 10−4 ( migrant proportion of population , per generation ) . The Asian and European populations split from the out-of-Africa population 920 generations ago , with sizes of 1032 for Europe and 554 for Asia , and growth rates of 3 . 8 × 10−3 for Europe and 4 . 8 × 10−3 for Asia ( the African population size remained constant at 14474 ) . Migration rates post-split were 2 . 5 × 10−5 between Africa and Europe , 7 . 8 × 10−6 between Africa and Asia , and 3 . 11 × 10−5 between Europe and Asia [18] . We create an admixed population with admixture occurring 12 generations ago . The admixed population had an initial size of 30 , 000 and grew at a rate of 5% per generation , with 1/6 of the population of African ancestry , 1/3 European , and 1/2 Asian . We simulated sequence data with a mutation rate of 1 . 25 × 10−8 per basepair per meiosis , and a constant recombination rate of 1 × 10−8 per basepair per meiosis ( i . e . , 1 cM = 1 Mb ) . We simulated 500 individuals from the simulated admixed population , and 100 reference individuals from the three ancestral populations ( Africa , Europe , and Asia ) . We simulated 30 chromosomes each of length 100 Mb . Our simulation code can be found in S1 Source Code . After simulating the data , we removed all variants with minor allele frequency less than 5% , and 70% of the remaining variants . After removing these variants , 926 , 159 variants remained for analysis across the 30 simulated chromosomes , so that the data are similar to those on a 1M SNP array . We then added genotype error: 0 . 1% of genotypes were randomly chosen to have a random allele changed . We followed the same analysis pipeline as for the real data , using RFMix [8] to infer local ancestry , Refined IBD [17] with a gap filling procedure to infer segments of identity by descent of length 2 cM and longer , and IBDNe [14] to estimate ancestry-specific effective population sizes . Further details are given below . The analysis pipeline can be found at http://faculty . washington . edu/sguy/asibdne/ . In addition , for the simulated data we needed to obtain the ground-truth effective population sizes for comparison with the estimated values . It is not straightforward to determine the populations’ effective sizes directly from the simulation parameters . So we simulated coalescence trees from the same simulation model as in the main simulation , and we utilized the known coalescence times and ancestral origins to determine the ground-truth ancestry-specific effective sizes . Specifically , we simulated 5000 replicate trees , each with 10 , 000 haplotypes sampled from the current-day admixed population . For each sampled haplotype , we determined its ancestral origin by tracing its path back through the tree to the first pre-admixture coalescence node . Considering a given ancestral population and a given generation ( g < 100 ) before present , in simulated tree i we determined the number of branches , bi , that are ancestral to haplotypes derived from the given ancestral population . Any pair of branches could coalesce , so the number of opportunities for coalescence is bi choose 2 , or bi ( bi − 1 ) /2 . We also determined the actual number of coalescences occurring in this generation for this ancestry population , ci . Combining results across the simulated trees , we obtained the conditional coalescence rate qg^= ( ∑i=15000ci ) / ( ∑i=15000bi ( bi−1 ) /2 ) . The large number of simulated trees ( 5000 ) and large number of sampled haplotypes per tree ( 10 , 000 ) result in an estimated qg^ that is close to the true value qg . We then obtained the ancestry-specific effective size at generation g from the relationship Ng = 1/ ( 2qg ) . In our previous development of IBDNe [14] we used IBDseq [27] to detect IBD segments because we were analyzing homogenous data and because assignment of an IBD segment to a particular haplotype was not necessary . However , IBDseq overestimates IBD segment lengths in heterogeneous data such as the admixed data analyzed in this study , because it does not account for linkage disequilibrium induced by population structure . The haplotype-based IBD detection method Refined IBD [17] is robust to genetic heterogeneity because the requirement that two haplotypes are identical over an extended region is very strong , and because haplotype frequencies estimated using appropriate approaches such as the Beagle model account for linkage disequilibrium . Further , Refined IBD assigns IBD segments to individual haplotypes which is necessary for determining the local ancestry within the IBD segment , whereas IBDseq does not assign phase to the IBD segments . We used the Beagle 4 . 1 version of Refined IBD with default settings unless otherwise noted , and we applied a 2 cM IBD segment length threshold . In order to successfully apply IBDNe , we need an IBD detection method that has high power to detect IBD segments with length greater than a threshold ( 2 cM in this study ) , and that estimates the lengths of these segments accurately . Genotype errors and haplotype phase errors can result in gaps in the estimated IBD segments when using a haplotype-based approach such as Refined IBD . A single long IBD segment may be reported as two shorter segments with a gap between them . This leads to underestimation of IBD segment lengths . We developed a software tool merge-ibd-segments . jar ( available from the Beagle Refined IBD website http://faculty . washington . edu/browning/refined-ibd . html ) to fill these gaps between reported segments . In this study we filled a gap between two detected IBD segments if the length of the gap was less than 0 . 6 cM and there was at most one pair of genotypes inconsistent with IBD ( e . g . opposite homozygotes ) in the gap . This strategy produces estimates of IBD segment length that are reasonably accurate for segments > 2 cM length , even in admixed populations ( S4 Fig ) . We used RFMix [8] version 1 . 5 . 4 to estimate local ancestry in each data set , simulated and real . For the Health ABC data , we used 112 CEU ( CEPH from Utah ) and 147 YRI ( Yoruba from Ibadan Nigeria ) samples from Hapmap3 [28] as reference samples . For the HCHS/SOL data , we used reference data from 195 West Africans , 63 Amerindians , and 527 Europeans obtained from the 1000 Genomes project and the Human Genome Diversity Project as previously described [13] . We used the “PopPhased” option and “-n 5” argument as recommended in the RFMix documentation . RFMix requires phased input data , and we used the phased genotypes generated by Beagle in the Refined IBD analysis . RFMix performs some rephasing of the data while inferring local ancestry . In order to match the local ancestry haplotypes to the IBD segment haplotypes , we adjusted the phasing of the RFMix Viterbi local ancestry calls to match the genotype phasing used in the IBD segment detection . Moving along the chromosome for an individual , we checked whether the phase of heterozygous genotypes matched between the RFMix rephasing and the Beagle phasing for the individual . We inferred a switch in the RFMix phasing relative to the Beagle phasing whenever the relative phasing of consecutive heterozygous genotypes changed . These switches were then applied to the RFMix Viterbi local ancestry calls for the individual in order to make the phasing of the ancestry calls consistent with the phasing of the IBD segments . The local ancestry proportion for an IBD segment was then determined from the phase-adjusted local ancestry calls for the corresponding haplotypes , using the mean called local ancestry from the two haplotypes . For example , if 41% of the length of the IBD segment had called local ancestry 1 for the first haplotype , and 43% of the length of the IBD segment had called local ancestry 1 for the second haplotype , then we considered the IBD segment to have 42% ancestry 1 . We investigated the concordance between the called local ancestry proportions for pairs of IBD haplotypes in the HCHS data . Considering one ancestry at a time , we record the proportion of that ancestry for each IBD haplotype , and calculate the correlation of these proportions between the pairs of IBD haplotypes . For African ancestry , we obtain a correlation of 0 . 980; for European ancestry , 0 . 982; for Native American ancestry , 0 . 987 . An IBD segment may span a change in ancestry if the most recent common ancestor lived more recently than the commencement of admixture . The total length of an IBD segment provides information about the time to the most recent common ancestor , so one cannot simply cut the IBD segment into smaller ancestry-homogeneous segments . In order to estimate effective size , it is necessary to consider both the length of detected ancestry-specific IBD ( which gives information about coalescence probabilities ) and the length of the overall IBD segment ( which gives information about the coalescence time ) . Specifically , the IBDNe program assigns each IBD segment fractionally to various coalescence times ( measured in discrete numbers of generations ) depending on the IBD segment length and the current estimates of effective population size history , and then uses the total ( sum of fractional counts across IBD segments ) for each generation to re-estimate the effective population size of that generation . For ancestry-specific effective size , we only want to consider pairs of haplotypes of the particular ancestry , that is those parts of IBD segments that are of that ancestry , but we need to use the full IBD segment lengths to estimate the coalescence times . One way to achieve this would be to weight the IBD segments by their proportion of the given ancestry . Implementing this approach would add complexity to the IBDNe program so instead we randomly assign the IBD segment to an ancestry based on the ancestry proportions of the segment . For example , if the segment has 80% of its length called as ancestry 1 and 20% as ancestry 2 , we assign it to ancestry 1 with probability 80% and to ancestry 2 with probability 20% . This approach has the same average input as the fractional assignment approach , and results in an unbiased estimate of the observed total length of IBD corresponding to a particular ancestry . The expected total length of IBD attributable to generation g , Eg , is proportional to the number of pairs of haplotypes that can generate that IBD . Two haplotypes can only be IBD with respect to a given ancestry at genomic positions where both haplotypes have that ancestry . We assume the local ancestry of individuals i and j is independent , which is approximately true for all but close relatives . If individuals i and j have proportion pi and pj of the given ancestry , respectively , then the expected proportion of genome over which a random haplotype drawn from individual i and a random haplotype drawn from individual j both have that ancestry is pipj . If there are n individuals , then the ancestry-adjusted number of pairs of haplotypes is ∑i=1n−1∑j=i+1n4pipj . Estimation of effective population size assumes a random population sample . Non-random ascertainment can affect the estimated effective size for the first few generations before present . In particular , an excess of close relatives in a data set will result in an excess of very large IBD segments , and hence a downward-biased estimate of the most recent effective size . By default , the IBDNe program searches for close relatives ( half-sibs and closer ) by looking for high genome-wide levels of IBD sharing and removes such pairs from consideration [14] . In the ancestry-specific case , the total proportion of the genome that could be IBD for the specific ancestry will tend to be low , and hence IBDNe won’t identify the pairs . We therefore identify pairs of close relatives using all IBD segments , and manually remove pairs with over 1200 cM of IBD genome-wide from the ancestry-specific analysis by removing the corresponding IBD segments and removing the pairs from the calculation of the ancestry-adjusted numbers of pairs of sampled haplotypes . We obtain confidence intervals for the estimated effective population size trajectories by bootstrap resampling of chromosomes . The bootstrapping is performed by the IBDNe program . Each bootstrap replicate resamples from the chromosomes with replacement to obtain the same number of chromosomes ( 22 for autosomal human data ) as in the original data . The program estimates the effective population size trajectory for each bootstrap replicate in the same manner as for the original data . We use the default number of bootstrap replicates ( 80 ) and show the 2 . 5th and 97 . 5th percentiles . These intervals provide an approximate measure of the precision of the effective population size estimates . In the course of this project , we made some changes to the IBDNe software to simplify the algorithm and reduce computation time . The original version of the IBDNe program performs 50 runs of an iterated method of moments algorithm and reports the harmonic mean of the population size at each generation from the 50 runs . Each run starts with a random initial effective population size at each generation before the present ( a trajectory ) and updates this estimate at each iteration for 50 iterations . In each iteration , the algorithm fits a piecewise exponential growth curve , with a new growth rate every 8 generations . For each run , the first change in growth rate is randomly chosen to occur between 5 and 12 ( inclusive ) generations before the present [14] . The revised IBDNe program performs 8 runs of the iterated method of moments algorithm and uses a fixed initial effective size trajectory for each run ( a constant effective population size of 100 ) . As before the algorithm fits a piecewise exponential growth curve , with a new growth rate every 8 generations . The first change in growth rate still occurs between generations 5 and 12 ( inclusive ) , and each of the 8 runs uses a different first time of change in growth rate . As before the program averages results from runs of the algorithm using a harmonic mean . We increased the default number of iterations per run from 50 to 1000 , as we found continuing improvements in fit up to this point . These changes are implemented in the revised release of the IBDNe program ( version 1 . 1 ) . The program can be freely downloaded from http://faculty . washington . edu/browning/ibdne . html . To allow ancestry-specific estimation of effective population size , we further modified IBDNe to include a parameter that specifies the ancestry-adjusted number of pairs of sampled haplotypes ( see Ancestry-adjusted number of pairs of sampled haplotypes ) . If this parameter is missing , IBDNe will assume that overall ( rather than ancestry-specific ) effective size is being estimated , and determine the number of sampled haplotype pairs directly from the data . Thus one can give IBDNe ancestry-specific IBD segments and an ancestry-adjusted number of pairs of sampled haplotypes ( both of which are obtained using the procedures described above ) in order to obtain ancestry-specific estimates of effective population size . HCHS/SOL is a study of 16 , 415 self-identified Hispanic/Latino individuals aged 18–74 ( mean 41 ) , with baseline examination in 2008–2011 . The individuals were sampled by household in four US field centers ( Chicago , IL; Miami , FL , Bronx , NY; San Diego , CA ) . The individuals were genotyped on an Illumina Omni 2 . 5M array with additional custom content , and the genotype data and phenotype data for 12 , 437 individuals are posted on dbGaP ( accession numbers phs000880 . v1 . p1 and phs000810 . v1 . p1 ) . In our IBDNe analysis , we excluded data from individuals who were not included in the dbGaP posting . Segments of IBD were called with Beagle version 4 ( r1203 ) [17] using genotyped SNPs with minor allele frequency > 0 . 02 . Local ancestry calls were made previously with RFMix [13] . In analysis of effective population size , we considered only individuals whose four grandparents all had the same country of origin . Countries with fewer than 120 sampled individuals were excluded . Sample sizes by country can be found in Table 1 . Due to the household-based sampling design , the data include many close relatives . We excluded IBD from close relative pairs as described above . We used the HapMap recombination map [29] for analyses with RFMix , for analyses with Beagle , and to determine IBD segment lengths . Individuals from the Healthy Aging and Body Composition ( Health ABC ) study were genotyped for the CIDR Visceral Adiposity Study . Genotype data were obtained for around 600 self-identified black and 800 self-identified white individuals ( here referred to as African American and European American , respectively ) from each of Memphis and Pittsburgh ( Table 2 ) . All individuals were 70–79 years old at recruitment in 1997–1998 . Participants were identified from a random sample of white Medicare beneficiaries and all age-eligible community-dwelling black residents in designated zip code areas surrounding the field centers in Pittsburgh , Pennsylvania , and Memphis , Tennessee [30] . Genotype data from an Illumina Human1M-Duo BeadChip array were downloaded from dbGaP ( study accession phs000169 . v1 . p1 ) . We removed SNPs with call rate <99% in either the African American or the European American samples , Hardy-Weinberg equilibrium p-value < 10−6 in either the African American or the European American samples , or minor allele frequency < 1% in either the African American or the European American samples . We called segments of IBD using Beagle 4 . 1 . We used HapMap phase 3 CEU and YRI samples ( Utah residents with ancestry from northern and western Europe and Yoruba in Ibadan , Nigeria respectively ) [28] as reference panels for the local ancestry calling , utilizing SNPs genotyped in both the HapMap phase 3 and Health ABC data . We phased the reference HapMap data using the phased Health ABC data from our Beagle Refined IBD analysis as a phasing reference panel . We used the HapMap recombination map [29] for analyses with RFMix , for analyses with Beagle , and to determine IBD segment lengths . The Health ABC data did not contain any close relatives . | Using genome-wide genetic data on several hundred individuals sampled from a population , we can estimate the current effective size of the population and the changes in effective size that have occurred over the past hundred generations . Many populations in the Americas are admixed , having ancestry from Europe , Africa , and the Americas . In such cases , one can learn not only about the effective population size history of the admixed population since admixture , but also about the effective population size histories of the contributing ancestral populations . In this paper we develop methodology for estimating past effective population size and analyze data from Hispanic , African-American , and European-American populations resident in the United States . We observe differences between populations in their historical effective sizes . These differences are useful for understanding differences in disease incidence between populations and for identifying populations that will maximize power in genetic association studies . | [
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] | 2018 | Ancestry-specific recent effective population size in the Americas |
WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum , called SGE1 , is required for pathogenicity and expression of key plant effector proteins . F . graminearum , an important pathogen of cereals , is not known to employ switching and no effector proteins from F . graminearum have been found to date that are required for infection . In this study , the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus . Deletion of the WOR1 ortholog ( called FGP1 ) in F . graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro . The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat . Under toxin-inducing conditions , expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains . FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro . The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini . Interchanging the N- and C- terminal portions of proteins from F . oxysporum and F . graminearum resulted in partial to complete loss of function . Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi , likely by adaptations to the C-terminal portion of the protein .
Pathogenic fungi have evolved sophisticated ways to infect their hosts , mainly by adapting to the host environment and by producing pathogenicity-related products such as toxic secondary metabolites , effector proteins and/or extracellular enzymes . The expression of genes involved in the adaptation to a host and synthesis of pathogenicity factors are under tight regulation to assure successful infection and survival . The pathogenic fungus Fusarium graminearum is a devastating pathogen of wheat and barley [1] , [2] and modulates its pathogenicity largely by regulating a cluster of genes encoding enzymes for the biosynthesis of trichothecene toxins [3] , [4] , [5] . These mycotoxins are required during wheat infection to breach the rachis node of spikelets which acts as a barrier to systemic infection and maximal head blight symptoms . Toxin production during infection depends on multiple cellular and environmental factors ( see recent review [6] ) and yet , exactly how the genes for trichothecene biosynthesis are regulated is still largely unknown . Another member of the Fusarium genus , F . oxysporum [7] , contains both pathogenic and non-pathogenic strains . Pathogenic F . oxysporum strains modulate their pathogenicity in part by secreting small secreted proteins which may act both as virulence and as avirulence factors [8] . In the tomato wilt pathogen F . oxysporum f . sp . lycopersici , the nuclear protein Sge1 was demonstrated to be required for parasitic growth and expression of the small-secreted proteins genes SIX1 , SIX2 , SIX3 and SIX5 , during conditions mimicking in planta growth [9] . A Δsge1 mutant was still able to colonize roots , but was unable to reach the normally infected xylem vessels . Recently an orthologous gene in the necrotrophic plant pathogen Botrytis cinerea , REG1 , was shown to be required for infection of bean leaves [10] , indicating that these genes may have a conserved role in fungal plant pathogenicity . SGE1 and REG1 are orthologs of WOR1 from Candida albicans and RYP1 in Histoplasma capsulatum [11] , [12] . In these human pathogenic fungi , both proteins are involved in the dimorphic switch , a transition correlated with the ability to cause disease [13] , [14] . This suggests that the orthologous proteins found in plant pathogens also may be involved in switching from a saprophytic to a parasitic lifestyle . In this study we characterize the ortholog of WOR1 in F . graminearum , and show that it is absolutely required for pathogenicity and regulates the expression of the trichothecene biosynthetic ( TRI ) genes in planta as well as in vitro . It also apparently mediates a morphological change during toxin production , and , as determined by transcriptome analysis , regulates the expression of other genes , many of which are related to pathogenicity . In order to study functional conservation of the orthologs from the two Fusarium species , we interchanged the genes and as well as swapped the C-terminal portions of the genes between the species . We found that the gene from the opposite species was not fully functional in the other species , nor were the chimeric genes . Additionally , very little overlap was found among genes regulated by each protein during vegetative growth . The results from these experiments show that the family of Wor1-like proteins regulates pathogenicity in many fungi through transcriptional reprogramming . Additionally , two members in two closely related Fusarium species have evolved different function , presumably in order to adapt to successfully infect their different hosts .
Most fungi have two homologous proteins encoded by the WOR1 gene family [9] , which are recognized by a common GTI1/PAC2 domain , named after the WOR1-like genes GTI1 and PAC2 of Schizosaccharomyces pombe [15] , [16] . Phylogenetically , the two paralogous genes from each species sort into two different clades [9] with e . g . GTI1 , WOR1 , RYP1 and SGE1 residing in one clade [9] and e . g . PAC2 from S . pombe and PAC2 from F . oxysporum in the other clade [9] . In F . oxysporum SGE1 is required for pathogenicity whereas PAC2 is not [9] . F . graminearum also has these two WOR1-like homologs . The gene that shows the highest similarity to SGE1 from F . oxysporum and aligns with the WOR1 clade is FGSG_12164 that we have named FGP1 ( F . graminearum GTI1/PAC2 1 ) . The predicted FGP1 gene is 1029 bp , intronless and encodes a 342 amino acid protein . The paralogous gene from F . graminearum that shows the highest similarity to PAC2 from F . oxysporum and aligns with the PAC2 clade is FGSG_10796 that we have renamed FGP2 ( F . graminearum GTI1/PAC2 2 ) . The predicted FGP2 gene is 1266 bp , intronless and encodes a 421 amino acid protein . In order to assess the conservation of the Fgp1/Sge1 and Fgp2/Pac2 proteins in more Fusarium species , we obtained the sequences of the respective proteins from two other sequenced Fusarium strains , F . verticillioides and Fusarium solani f . sp . pisi ( also known as Nectria haematococca ) , using the BLAST function [17] on the websites of the Broad Institute and the DOE Joint Genome Institute , respectively . As for F . oxysporum and F . graminearum , two genes were found for F . verticillioides as well as for F . solani . The genes that show the highest similarity to SGE1 and FGP1 and align with the WOR1 clade are FVEG_09150 and Fs_81912 of F . verticillioides and F . solani , respectively . The genes that show the highest similarity to PAC2 and FGP2 and align with the PAC2 clade are FVEG_11476 and Fs_60837 of F . verticillioides and F . solani , respectively . When we aligned the four protein sequences of the Wor1 clade ( Fo Sge1 , FVEG_09150 , Fg Fgp1 and Fs_81912 ) , we found great divergence between the sequences of the four Fusarium proteins . Conservation is mainly restricted to the N-terminal portion ( first ±220 amino acids in Figure 1A ) of which the total similarity percentage ranges from 64–90 . 5% ( Figure 1B ) . The C-terminal portions ( last first ±140 amino acids , grey shaded in Figure 1A ) of the Fusarium orthologs from the Wor1-clade are highly diverged despite of the high numbers of glutamine residues in all four sequences ( 5 . 6–13 . 6% , Table S1B ) . Overall sequence similarity is as low as 37% between F . graminearum and F . verticillioides . The sequences of the F . verticillioides ortholog and F . oxysporum Sge1 share the highest similarity; 65% ( Figure 1B ) . On the contrary , when we aligned the four protein sequences of the Pac2 clade ( Fo Pac2 , FVEG_11476 , Fg Fgp2 and Fs_60837 ) , high similarities and conservation among the four Fusarium proteins ( 80–97% , Table S1A ) are observed throughout the N-terminal and C-terminal regions ( Figure S1 ) . The N-terminal portion of all the proteins from the four Fusarium strains in both clades contains the conserved WOPRa box ( black line above the sequence , Figure 1 and S1 ) and WOPRb box ( dashed black line above the sequence , Figure 1 and S1 ) [18] . The WOPRa and WOPRb boxes , previously recognized as the GTI1/PAC2 domain , have been shown to be involved in DNA binding of Wor1 in C . albicans . In the WOPRa box , a conserved threonine residue is present ( boxed , Figure 1 ) that functions as a putative phosphorylation site . Mutation of this site in Gti1 , Wor1 and Sge1 impairs the function of the respective proteins [9] , [15] , [19] . It has been shown previously that the N-terminal portion containing the WOPRa and WOPRb boxes from Fo Sge1 and Fo Pac2 align with the same domains from other proteins of the Wor1 family [18] and the same likely holds for the other Fusarium species because of the high conservation of these domains among the Fusarium proteins . The C-terminal portions of the Wor1-like proteins from Fusarium however have not only greatly diverged from each other but also from other fungal species . For example , the C-termini of the Fusarium proteins do not align at all with the C-terminus of Wor1 or Reg1 due to differences both in sequence and in length ( data not shown ) . Strains deleted for FGP1 or FGP2 were generated in F . graminearum using constructs produced in plasmid pPK2HPH-GFP [20] containing a cassette for a hygromycin B resistance gene fused at the C-terminus to GFP and regulated by a GPD1 promoter and TRPC terminator . In the respective plasmids , the cassette is flanked by the up- and downstream sequences ( +/−2000 bp ) of FGP1 or FGP2 . Using Agrobacterium tumefaciens mediated transformation , multiple independent transformants were obtained of which five were selected for both FGP1 or FGP2 . For each gene , all five proved by PCR and Southern blot to be homologous gene deletions , indicating that no ectopic transformants were obtained ( Figure S2 ) . Wheat heads of the variety “Norm” , point-inoculated with the wild type ( WT ) strain PH-1 and four Δfgp1 strains were assessed after two weeks for the number of diseased spikelets . Wild type strain PH-1 was able to spread from the inoculated spikelet ( arrow ) to other spikelets in the head causing blight symptoms; bleached spikelets and deformed anthers ( Figure 2B , left panel , lower head ) . In contrast , the Δfgp1 strains did not spread from the inoculated spikelet ( arrows ) to other spikelets ( Figure 2A ) . In the inoculated spikelet , the Δfgp1 strains only cause minor disease symptoms; some browning of the palea ( Figure 2B , left panel , upper four heads ) . When the inoculated spikelets were analyzed for trichothecene content , no toxin was detected in the spikelets inoculated with the Δfgp1 strains , in contrast to spikelets inoculated with the wild type , which accumulated significant levels of the toxins deoxynivalenol ( DON ) and 15-deoxynivalenol ( 15-ADON ) ( Figure 2C ) . Reintroduction of the wild type gene FGP1 into a Δfgp1 strain resulted in complemented strains that were tested for spread through the wheat head and for toxin production in the inoculated spikelet . In total , four independent complemented strains used regained the ability to spread throughout the wheat head ( Figure 2A ) and cause disease symptoms similar to wild type; spread was present from the inoculated spikelets ( arrows ) to other spikelets in the head ( Figure 2B , right panel ) . The four complemented strains also regained the ability to produce toxin similar to wild type ( Figure 2C ) . In contrast , Δfgp2 strains were not reduced in the ability to cause disease . The Δfgp2 strains were able to cause head blight symptoms almost to the same extent as wild type PH-1 ( Figure 2D ) and accumulated mycotoxin levels comparable to wild type ( Figure 2E ) . These results affirm previous observations that genes from the WOR1 clade are involved in pathogenicity whereas genes from the PAC2 clade are not and suggest that WOR1 orthologs may regulate secondary metabolite synthesis . As previously mentioned , no spread of disease symptoms was observed with the Δfgp1 mutant compared to wild type . To determine the reason for this , we investigated whether the symptoms in the Δfgp1 and Δfgp2 mutants are correlated to the growth of the mutant strains within the wheat head , using bright light and fluorescent microscopy . Since no differences in symptoms were observed between wild type PH-1 and Δfgp2 , the latter was used as a fluorescent positive control strain . After inoculation , spread of the Δfgp1 and Δfgp2 strains were monitored through the spikelet for two weeks . At three days after inoculation , “fluffy” mycelium growing from of the inoculated spikelet was observed for the Δfgp1 strain to the same extent as wild type ( data not shown ) and the Δfgp1 strain had infected the palea and a portion of the lemma but had not spread towards the glume on the outer part of the spikelet ( Figure 3A ) . Even after two weeks , the glume was still green and apparently healthy . Presence of the fungus in the palea and lemma is manifested by browning of the tissue and was verified by microscopy ( data not shown ) . In contrast to Δfgp1 , the Δfgp2 strain had infected the palea , the lemma and had spread to the glume after three days ( Figure 3B ) . For both Δfgp1 and Δfgp2 strains , GFP is coupled to the constitutively expressed hygromycin B resistance gene HPH , making it possible to follow the growth inside the plant using hyphal fluorescence . Doing so , we identified the rachis node as the flower tissue where growth of the Δfgp1 mutant was halted . No fluorescence from the Δfgp1 mutant was found in the rachis node ( Figure 3C , red circle ) nor was fluorescence detected beyond the rachis over the complete time period of two weeks . Growth of Δfgp1 was observed in other spikelet parts after three days ( Figure 3C , white arrow ) . In contrast , fluorescence of the Δfgp2 mutant was found in the flower ( arrow ) as well as in the rachis node and beyond after three days ( Figure 3D , red circle ) . The inability of the Δfgp1 mutant to penetrate the rachis node and the consequential pathogenicity loss might be caused solely by the lack of trichothecene production by the fgp1 mutant . This inference is made because Δtri5 mutants of F . graminearum , lacking the gene for the first enzymatic step in toxin synthesis , are unable to produce trichothecene toxins in planta and also are stalled during infection at the rachis node [21] . To determine whether the inability to cause disease could be correlated to a major growth or developmental deficiency , the growth characteristics of Δfgp1 strains were assessed on different media . Germination rate was assessed by placing freshly produced spores on PDA agar . Spores from WT PH-1 show almost complete ( ≥95% ) germination after 8 hours , while Δfgp1 spores show a slight delay with complete ( ≥95% ) germination observed after 12 hours ( data not shown ) . On PDA and minimal medium , the deletion of FGP1 in wild type results in a slightly reduced radial growth phenotype , which can be attributed to the delayed germination . Complementation of the Δfgp1 strain with the wild type FGP1 gene restored the wild type growth phenotype . No growth differences were observed on complex solid media including V8 , carrot or mung bean agar plates ( data not shown ) . Fgp1 is required for full asexual spore formation as was observed for Sge1 in F . oxysporum [9] . During growth on mung bean agar ( MBA ) , fewer ( p≤0 . 05 , Student's t-Test ) macroconidia are formed in four independent Δfgp1 deletion strains compared to wild type ( Figure 4A ) . Additionally , spores of the Δfgp1 strain are smaller during growth on mung bean agar ( MBA ) ( p≤0 . 05 , Student's t-Test ) or in carboxymethylcellulose ( CMC ) ( p≤0 . 01 , Student's t-Test ) ( Figure 4B ) or may exhibit precocious germination or appear not fully developed ( three middle pictures , Figure 5C representative spores are shown ) . In the complementation strain , the quantity of spores is restored , albeit incompletely , still , the spores produced resemble wild type ( Figure 4A , B and C , representative spores of wild type and complemented strain are shown ) . Using Calcofluor white and Hoechst staining , no defects in cell wall composition or nucleus quality were observed in the Δfgp1 strain ( data not shown ) . When strains were grown on carrot agar to induce perithecium and ascospore formation , it was observed that perithecia formed by the Δfgp1 strain are comparable to wild type ( data not shown ) . Ascospore formation on the other hand was delayed by one week in the Δfgp1 strain and altogether only a few ascospores emerged from the perithecia . Cirrhi of wild type PH-1 and a FGP1 complementation strain appear one week after the formation of the perithecia and consist of strings of ascospores emerging from perithecia ( data not shown ) . Cirrhi of the FGP1 deletion strains appear two weeks after the formation of the perithecia and contain only a few ascospores at the perithecial apex ( data not shown ) . When the ascospores were harvested and counted , fewer ascospores ( p≤0 . 01 , Student's t-Test ) were produced by the Δfgp1 strains compared to wild type ( Figure 4D ) . This defect was restored in the complementation strains ( Figure 4D ) . These observations indicate that FGP1 is involved the developmental processes of conidiogenesis and ascospore formation although these processes are not fully abolished in the deletion mutant . As a consequence of this impaired conidiogenesis , the Δfgp1 strain probably displayed delayed germination on certain media . On the other hand , Fgp1 has no impact on normal vegetative growth . Since the Fgp2 homolog Pac2 is involved in the sexual cycle of Schizosaccharomyces pombe , we also tested whether perithecium and ascospore formation was altered in the Δfgp2 strain . No major differences with respect to perithecium and cirrhi formation were noted when compared to wild type ( data not shown ) . Previous studies have shown that F . graminearum produces trichothecene toxins when grown on medium containing polyamine compounds [22] and that this medium induces the expression of genes involved in trichothecene production [5] . Since trichothecene toxins did not accumulate in wheat spikelets inoculated with Δfgp1 , we investigated whether deletion strains also are impaired in production of toxin in vitro . In order to do this , the wild type , four independent Δfgp1 strains and two independent complemented strains were inoculated into putrescine containing medium and after one week assessed for the presence of trichothecene toxins . Toxin was found in wild type cultures but no toxin was detected in the cultures of the Δfgp1 strains ( Figure 5A ) . Toxin was also found in the cultures of the two complemented strains albeit to slightly lower levels than wild type ( Figure 5A ) . The lack of toxin production in the Δfgp1 strains could be due to the inability to express the genes from the TRI cluster . In order to test for this , a northern blot experiment was performed . Both wild type PH-1 and a Δfgp1 strain were grown in minimal medium containing the polyamine putrescine or in control minimal medium with NaNO3 as the sole nitrogen source instead of putrescine . Samples for RNA isolation were taken 8 , 16 , 24 , 32 , 40 or 48 hrs after inoculation and the RNA was used for gel electrophoresis and capillary blotting . Northern blots were hybridized with TRI14 , a gene from the trichothecene biosynthetic cluster [23] or with a constitutively expressed actin gene used as the loading control . TRI14 is expressed to high levels during growth in polyamine medium [24] and is therefore a good marker gene for the expression of the TRI cluster . No TRI14 transcript was observed in the Δfgp1 mutant strain grown in putrescine medium at any time point in contrast to wild type PH-1 in which expression of TRI14 was detected at 32 , 40 and 48 h ( Figure 5B ) . RNAs corresponding to TRI14 were not detected in cultures grown in minimal medium for either wild type or Δfgp1 ( data not shown ) . Actin transcripts were detected in all samples of wild type and Δfgp1 , indicating equal loading . Analysis of the cultures filtrates of the different samples revealed that trichothecene toxins were present in the putrescine medium containing wild type PH-1 at 32 h and accumulated to higher levels at 40 and 48 h ( Figure 5B ) . No toxin was detected for the Δfgp1 strain grown in putrescine medium at any time point nor in control medium , which is consistent with the previous toxin analysis experiment and the lack of TRI14 gene expression . The Δfgp1 strain exhibits growth equal to wild type in putrescine medium ( data not shown ) , suggesting that it likely can take up putrescine and use it as a nitrogen source but this does not lead to TRI gene expression . In minimal medium lacking any nitrogen source , growth of both wild type and the Δfgp1 strain was noticeably reduced and no toxin was detected ( data not shown ) . This suggests that TRI gene expression is not triggered by the absence of a nitrogen source but rather that putrescine is a specific inducer . Clearly , Fgp1 is required for putrescine induced trichothecene production . The full HPLC spectra of the wild type and Δfgp1 samples grown for 40 hours in putrescine medium were , besides used for trichothecene toxin identification , also inspected for other differences in metabolite profiles . Doing so , no significant peaks other than the peaks corresponding to DON and 15-ADON are missing in the spectrum from the Δfgp1 strain compared to the wild type ( Figure S3 ) . This suggests that Δfgp1 can still produce a majority of the other metabolites present in the spectrum and that it does not appear to be impaired in production of metabolites in general . To examine in more detail why TRI gene expression was not observed in the Δfgp1 strain , wild type PH-1 , Δfgp1 and complemented strain were grown in putrescine medium and examined microscopically . In all strains , a distinct morphological change in hyphae was observed when cultures were grown in putrescine medium for 40 h compared to control medium . In control medium , hyphae display a uniform thickness over their entire length and grow in long branches in wild type , Δfgp1 , and complemented strain ( Figure 6 , upper panel ) . In putrescine medium , the wild type and complemented strains display hyphae that produce bulbous sub-apical structures , which are to some extent also observed in the Δfgp1 strain ( Figure 6 , center panel ) . In wild type and complemented strain , bulbous structures up to 21 µm in diameter are observed among numerous other , but fewer and smaller bulbous structures are observed in the Δfgp1 strain ( Figure 6 , lower panel ) . Another independent Δfgp1 and complemented strain displayed similar growth morphology as the Δfgp1 and complemented strain given in Figure 6 . To investigate whether this morphological change in the putrescine medium parallels the expression of TRI genes and trichothecene accumulation we also examined the cultures at the different time points . After 16 and 24 h , some hyphae begin to swell and form bulbous structures in both wild type and Δfgp1 strains ( see arrows , Figure S4 ) . These structures become more abundant at 32 h in the wild type but not in the Δfgp1 strain ( see arrows , Figure S4 ) . At 40 and 48 h , large bulbous structures appear in the wild type which are less apparent in the Δfgp1 strain ( see arrows , Figure S4 ) . This suggests that the formation of these bulbous structures might be involved in the production of trichothecene toxins and that somehow the Δfgp1 strain is less capable to form the same structures as in wild type . To examine whether the bulbous structures observed in wild type are formed as a consequence of toxin production or whether the structures may accommodate toxin production , a Δtri6 mutant was grown in putrescine and control minimal medium . TRI6 encodes a transcription factor that is absolutely required for the expression of the genes in the TRI cluster and for trichothecene synthesis [25] . The Δtri6 strain lacks detectable trichothecene accumulation in the putrescine medium ( data not shown ) , but the hyphae show the same degree of hyphal swelling and bulbous structure formation as the wild type ( data not shown ) . We conclude from this that the bulbous cells do not form in response to trichothecene synthesis because they appear to develop before transcription of the TRI genes , and the toxins themselves , can be detected . It remains unclear what the exact role this hyphal swelling plays in toxin synthesis and how Fgp1 might be involved in the facilitation of this process . To investigate the underlying genetic basis for the differences in toxin accumulation and hyphal morphology seen in putrescine medium , RNA was extracted from wild type PH-1 ( three independent samples ) and Δfgp1 strains ( three independent samples ) after growing 40 h in putrescine and labeled for microarray experiments . Additionally , in order to find genes regulated by Fgp1 during in planta growth , RNA was extracted from wheat heads inoculated for 72 hours with wild type ( three independent samples , three heads per sample with ten inoculated spikelets per head ) or Δfgp1 ( three independent samples , three heads per sample with ten inoculated spikelets per head ) and labeled for microarray experiments . The number of genes showing a >2-fold difference in average expression between wild type PH-1 and Δfgp1 strains during growth on putrescine medium and during wheat infection were determined ( Figure S5 ) . Fgp1 appears to regulate gene expression positively as well as negatively during growth in putrescine medium and infection of wheat with hundreds of genes differentially expressed . In total , 654 genes show a >2 . 0-fold ( P<0 . 05 ) lower expression in Δfgp1 compared to wild type when grown in putrescine and 536 genes show higher expression in Δfgp1 ( Table S2 ) . Additionally , 634 genes were ≥2-fold lower expressed ( P<0 . 05 ) in the Δfgp1 strain compared to wild type during wheat infection and 39 genes were found >2 fold higher expressed ( Table S3 ) . Some overlap is found between genes positively or negatively regulated by Fgp1 in putrescine medium and those regulated during wheat infection ( 91 and 3 genes , respectively ( Figure S5 ) ) . Recently , a gene set considered to be expressed “in planta only” was described containing genes from F . graminearum exclusively expressed during plant infection but not in axenic cultures [26] . Out of the 369 genes from this gene set , 243 were found expressed in the experiments described here , either in wild type or the Δfgp1 strain grown in putrescine medium ( 45 genes ) or in infected wheat ( 199 genes ) ( Table S4 ) . Overlap was found between these 243 “in planta only” genes and genes lower or higher expressed in the Δfgp1 strain in putrescine medium or during wheat head infection ( Figure S5 ) . Altogether , Fgp1 regulates a remarkably large proportion of genes considered “in planta only”; 99 out of 244 or 41% were positively regulated and another nine ( 3 . 7% ) were negatively regulated . Fgp1 positively regulates thirteen “in planta only” genes in putrescine as well as in wheat heads . Among these thirteen are six genes from the trichothecene biosynthetic cluster ( FGSG_03534 , FGSG_03536 , FGSG_03537 , FGSG_03538 , FGSG_03540 and FGSG_03543 ) as well as four other genes involved in trichothecene toxin synthesis or co-expressed with TRI5 [24] ( FGSG_00007 , FGSG_01819 , FGSG_07562 and FGSG_10397 ) . Among the remaining three are FGSG_08079 a gene involved in butenolide synthesis , FGSG_08309 encoding an ABC transporter with orthologs in other fungi and FGSG_02120 for which there is no annotated function or ortholog found in any other fungi with a sequenced genome . Fgp1 negatively regulates one “in planta only” gene expressed in putrescine medium and during infection: FGSG_11033 . TRI6 ( FGSG_03536 ) encodes a transcription factor that regulates the expression of other TRI genes and genes involved in the isoprenoid biosynthesis pathway . Since TRI6 and an ancillary transcription factor , TRI10 , are both extremely down-regulated in the Δfgp1 strain , genes positively regulated by Tri6 and Tri10 might be expected to be expressed at lower levels in the Δfgp1 strain . Indeed , 56 and 62 genes expressed at lower levels in the Δfgp1 strain in putrescine and wheat head , respectively ( Table S5 ) , were also found to be expressed at lower levels in the Δtri6/tri10 strains 96 h after wheat inoculation . Among the common regulated genes are the ones involved in the isoprenoid biosynthesis pathway . This suggests that Fgp1 may assume a portion of its regulatory control by acting upstream of the Tri6 and Tri10 transcription factors . As co-regulated clusters of genes often are associated with secondary metabolite biosynthesis , expression of putative gene clusters from F . graminearum identified previously [27] was examined using the expression values obtained from the microarray experiment . Unlike in wild type , expression of all genes involved in trichothecene biosynthesis was absent or was greatly reduced in the Δfgp1 strain during growth in putrescine medium as well as during wheat infection ( Figure 7A ) . This loss of expression was confirmed by the absence of TRI14 transcript in the northern blot experiment ( Figure 5 ) and lack of the trichothecene toxins produced by the Δfgp1 strain . In addition to the twelve genes from the TRI cluster , 19 of the 38 genes that showed co-expression with TRI5 in agmatine medium [24] are also regulated by Fgp1 , either during growth in putrescine medium or during wheat infection ( Table S6 ) . Fgp1 negatively regulates two of the 19 genes: FGSG_01832 and FGSG_03132 . Five of the 17 genes that are positively regulated by Fgp1 are also regulated by Tri6 ( Table S6 ) . Genes involved in butenolide synthesis , also reside in a cluster [28] . Other than FGSG_08077 , FGSG_08078 and FGSG_08079 , expression of genes from the butenolide cluster was not detected in putrescine medium for both wild type and Δfgp1 . However , during wheat infection expression of all genes from the cluster were detected on the microarray chip in wild type and in the Δfgp1 strain , albeit to a significantly lesser extent for the mutant ( Figure 7B ) . A cluster of genes involved in the production of the yellow or purple pigment aurofusarin [29] as well as genes comprising the non-ribosomal protein synthase 8 ( NPS8 ) [24] gene cluster also appear to be regulated by Fgp1 . In the Δfgp1 strain , the aurofusarin cluster genes are up-regulated when compared to wild type suggesting a role for Fgp1 in the negative regulatory control of this cluster ( Figure 7C ) . The genes of the NPS8 cluster are highly down-regulated or not detected in putrescine medium in the Δfgp1 strain when compared to wild type ( Figure 7D ) . For both gene clusters no gene expression was detected during wheat infection . Fgp1 also regulates other genes that encode polyketide synthases ( PKS ) [30] and non-ribosomal protein synthases ( NPS ) [31] that may be involved in secondary metabolite synthesis . During wheat infection NPS9 ( FGSG_10990 ) , NPS12 ( FGSG_11294 ) , NPS14 ( FGSG_11395 ) , PKS7 ( FGSG_13295 ) and PKS15 ( FGSG_04590 ) are expressed at lower levels in the Δfgp1 mutant compared to wild type . During growth in putrescine medium NPS11 ( FGSG_03245 ) is expressed at lower levels in the Δfgp1 mutant . Higher expression was observed for NPS18 ( FGSG_13783 ) in the Δfgp1 mutant during growth in putrescine medium . Transcriptome experiments therefore show that Fgp1 regulates gene clusters and genes predicted to encode secondary metabolite synthesis proteins and genes previously found solely expressed during plant infection . To determine whether Fgp1 and Sge1 share common regulatory targets , a comparative transcriptional study was conducted during growth in culture . Both wild type strains Fol4287 and PH-1 as well as deletion strains Δsge1 and Δfgp1 from F . oxysporum and F . graminearum , respectively , were grown in complete medium ( CM ) for 48 h . RNA was extracted , reverse-transcribed into cDNA , labeled and hybridized to microarray chips . In F . graminearum , 119 genes show a ≥2 fold lower expression in the Δfgp1 strain on CM and 83 genes a higher expression compared to WT ( Table S7 ) . For F . oxysporum , 394 genes show a ≥2 fold lower expression in the Δsge1 strain on CM and 819 a higher expression compared to WT ( Table S7 ) . When the gene sets regulated by either Fgp1 or Sge1 were compared , only a few orthologous genes were found to be regulated by both Sge1 and Fgp1 in the two Fusarium species ( Figure S6 ) . A set of 16 down-regulated and eight up-regulated orthologous genes were found in both deletion strains ( Table S8 ) . Remarkably , eleven genes up-regulated in Δsge1 were found down-regulated in Δfgp1 and one gene up-regulated in Δfgp1 was found down-regulated in Δsge1 ( data not shown ) . Altogether , the results from this comparative transcriptomic experiment suggest that Fgp1 and Sge1 regulate largely non-overlapping sets of genes . Because of the effect of the Δsge1 mutation on sporulation in F . oxysporum it was noteworthy that there was differential regulation between wild type and the Δsge1 mutant for several genes known to effect sporulation in Fusarium and other fungi . REN1 [32] , an ortholog of MEDUSA [33] from Aspergillus nidulans , ABA1 , an ortholog of ABACUS [34] from A . nidulans and FOXG_01756 , an orthologs of FlbC [35] from A . nidulans , were among the genes expressed at lower levels in the Δsge1 mutant . The lower expression of these genes could account for the fewer number of spores produced by the Δsge1 mutant [9] . F . graminearum does not produce conidia in CM , so , in order to study these genes , we grew wild type and Δfgp1 in liquid carboxymethylcellulose ( CMC ) medium or mung bean agar ( MBA ) , two media that induce sporulation . Quantitative PCR experiments were conducted using cDNA obtained from F . graminearum wild type and Δfgp1 and from F . oxysporum wild type and Δsge1 strains during growth in conidia inducing medium . We used the constitutive expressed gene FRP1 [36] as a reference in both F . oxysporum and F . graminearum . The expression levels of FRP1 proved not to be influenced by deletion of SGE1 or FGP1 ( results not shown ) . The results showed that in F . oxysporum all three genes ( REN1 , ABA1 and FOXG_01756 ) were expressed at lower levels in the Δsge1 strain , confirming the microarray results , but in the Δfgp1 strain of F . graminearum , only the expression of ABA1 was significantly reduced during growth on MBA and CMC compared to wild type ( Figure S7 ) ; the expression of REN1 and FGSG_ 07052 ( FLBC ortholog ) were not significantly lower; both genes even show a slightly higher expression in the Δfgp1 strain compared to wild type when grown on CMC . However , the expression of FGSG_ 07052 was lower in the Δfgp1 strain compared to wild type when grown on CM ( Figure S7 ) . These results suggest that Sge1 regulates a number of sporulation genes and that Fgp1 only regulates Aba1 during conidia formation . This expression difference may be attributable to the difference in spores that are formed under these conditions: microconidia in F . oxysporum versus macroconidia in F . graminearum . To study the functional conservation of FGP1 and SGE1 , the two genes were introduced in the opposite species in order to test whether they can take over each other's function . Doing so , SGE1 was introduced into the Δfgp1 F . graminearum strain and FGP1 into the Δsge1 F . oxysporum strain . Two independent transformants that carry the SGE1 complement in the Δfgp1 strain in F . graminearum were obtained and were used to inoculate wheat spikelets . In contrast to the transformants harboring the FGP1 complement , both SGE1 complements were unable to restore disease causing ability on wheat heads ( Figure 8A , bars below the FGP1 box and SGE1 box , respectively ) . This suggests that SGE1 has diverged from FGP1 in such way that it is unable to restore its function . This observation led to the hypothesis that the functional specificity of the protein may be present in the highly diverged C-terminal portion of the protein . To test this , combinations of the two genes from F . oxysporum and F . graminearum were made and transformed into a Δfgp1 strain of F . graminearum and into a Δsge1 strain of F . oxysporum . A combination consisting of the N-terminal portion of Fgp1 ( amino acids ( aa ) 1–219 ) and the C-terminal of Sge1 ( aa 220–330 ) , expressed from the native FGP1 promoter was used to complement the Δfgp1 strain . The strains obtained showed less spread ( p≤0 . 01 , Student's t-Test ) and disease in inoculated wheat spikelets after two weeks compared to the full length FGP1 complements but more disease than complements with full length SGE1 ( Figure 8A , bars below the FGP1/SGE1 box ) . This suggests that the highly diverged C-terminus of Fgp1 is required for full function in F . graminearum . Reintroduction of the complete wild type gene FGP1 of F . graminearum ( Figure 8B , bars below the FGP1 box ) used to complement the Δfgp1 strain into a Δsge1 strain of F . oxysporum failed to restore pathogenicity towards tomato in contrast to complementation with full length SGE1 ( Figure 8B , bars below the SGE1/FGP1 box ) . The combination of the N-terminal domain of SGE1 ( encoding aa 1–218 ) and the C-terminal domain of FGP1 ( encoding aa 219–342 ) expressed from the native SGE1 promoter also failed to complement the mutant phenotypes in the Δsge1 strain . This suggests that the diverged C-terminal portions of the genes are critical for their function in the species of origin .
This study is the first to demonstrate that a Wor-1 like protein has the ability to control the expression of genes for mycotoxin biosynthesis and well as other gene clusters for synthesis of fungal secondary metabolites such as aurofusarin and butenolide . Fgp1 positively regulates the TRI cluster but negatively the AUR cluster responsible for aurofusarin production . This phenotype is also seen in a heterochromatin protein encoding gene deletion mutant of F . graminearum , Δhep1 [37] . The similarity in phenotype between Δfgp1 and Δhep1 could suggest that Fgp1 regulates chromatin modification too . Trichothecene toxins produced by F . graminearum are central to its pathogenicity to wheat [21] . These toxins normally are synthesized during pathogenic growth and are induced within specific plant parts [3] . As trichothecene biosynthesis is strongly under the control of Fgp1 , especially through the transcription factor genes TRI6 and TRI10 , the protein must have evolved to regulate the genes for mycotoxin synthesis that are peculiar to this disease . A morphological change that accompanies trichothecene accumulation and gene expression is noticeably altered in the Δfgp1 mutant , suggesting that Fgp1 may be involved in a morphological change required for pathogenicity . A similar morphological change occurs during infection of wheat; in planta , F . graminearum forms thickened hyphae and coralloid structures that resemble the bulbous hyphae that are observed in putrescine medium [38] . This morphological phenomenon has been associated with toxin biosynthesis gene expression in planta in various studies [38] , [39] , [40] , however , the necessity of the morphological change for toxin accumulation remains to be determined . Using microarray analysis many putative downstream targets of Fgp1 were identified . Among these are several pathogenicity related genes [26] , including ones previously described as infection specific , such as genes from the TRI cluster and others involved in toxin production [24] . We also found many putative downstream targets of Fgp1 and of its F . oxysporum counterpart Sge1 when grown on CM . Both proteins show a high degree of specificity towards their putative targets as very little overlap was found between orthologous genes in the two species regulated by either Fgp1 or Sge1 . Additionally , of the hundreds of genes that are regulated by Δfgp1 in putrescine medium and during wheat infection , only eight are also regulated by Fgp1 during growth in CM ( Table S9 ) , suggesting that Fgp1 regulates specific sets of genes during toxin induction conditions ( putrescine or wheat head ) or during growth in rich , toxin non-inducing medium ( CM ) . A few target genes of Sge1 and Fgp1 identified in this study and confirmed by quantative PCR analysis , are involved in sporulation and some may also play a role during pathogenicity . For example , Sge1 regulates the expression level of REN1 , a conserved gene required for adherence , biofilm formation and virulence in A . fumigatus [41] and absolutely required for microconidia formation but not for pathogenicity in F . oxysporum [32] . The lower expression level of REN1 in Δsge1 might explain , at least partly , the lower number of microconidia produced in CM . F . graminearum does not produce microconidia under any condition tested and perhaps as a consequence , no difference in REN1 expression is observed between wild type and Δfgp1 strains during macroconidia formation . Sge1 also regulates the expression level of ABA1 , another gene involved in conidium formation . In F . oxysporum f . sp . melonis , a pathogen of melon , Aba1 regulates production of both micro- and macroconidia and is required for full virulence . The Δaba1 mutant of this strain shows delayed pathogenicity towards melon probably due to fewer spores produced inside the xylem vessels ( personal communication Dr . Tsutomu Arie , Tokyo University of Agriculture and Technology , Japan ) . In addition to sporulation , the Aba1 ortholog in Penicillium marneffei is involved in the dimorphic switch [42] and in A . fumigatus in autolysis and cell death [43] . The expression levels of ABA1 of F . graminearum are also regulated by Fgp1 during conidia formation but not during growth in putrescine medium or infection of wheat heads . The role of ABA1 during infection is therefore still elusive . Another gene known to control conidia formation in fungi that is regulated by Sge1 is FlbC . In A . nidulans , FlbC regulates the developmental processes of conidia formation and sexual fruiting and is required for normal vegetative growth [35] . To the contrary , we found that Fgp1 does not regulate the expression of FLBC of F . graminearum during conidia formation . Instead we found that expression of FLBC is regulated by Fgp1 during growth on CM and putrescine medium . Interestingly , FlbC in F . graminearum is absolutely required for wheat infection but not for toxin production [44] . How FlbC regulates virulence independent of toxin production and whether and how FLBC is regulated by Fgp1 during wheat infection is not yet known . FLBC is an interesting candidate gene to investigate further regarding its role in pathogenicity and its putative function downstream of both Fgp1 and Sge1 . A transcription factor not involved in sporulation but nevertheless regulated both by Fgp1 and Sge1 during growth on CM is DAL81 . Dal81 is a general activator of nitrogen metabolic genes in yeast , including those for γ-aminobutyrate ( GABA ) [45] . Its exact role in Fusarium species is not yet known but this transcription factor ( FGSG_02068 ) is required for toxin production in F . graminearum but , paradoxically , apparently not for virulence [44] . Whether Fgp1 and Sge1 are transcription factors and bind DNA directly is not known . Their conserved N-terminal regions contain putative DNA binding domains , previously called the GTI1/PAC2 domain [15] , [16] but recently renamed the WOPR box ( Wor1 , Pac2 and Ryp1 ) [18] . This motif consists of two globular peptide domains: WOPRa and WOPRb . Via these two domains Wor1 is able to bind a 14-bp DNA sequence and thereby activates its target genes and itself via a positive feedback loop [18] . Of the fourteen base pairs , the ones located in positions 6 through 14 ( TTAAAGTTT ) are absolutely required for binding . By scanning the upstream regions of both FGP1 and SGE1 , variations to the WOR1 motif were found 557 upstream of the ATG of FGP1; TTAAAGTTC and 644 bp upstream of the ATG of SGE1: TTAACGCTT . Whether these domains in the promoters of FGP1 and SGE1 are DNA binding sites required for FGP1 and SGE1 expression is unknown . However , a search conducted for these patterns in upstream regions in both the genomes did not unveil any enrichment for this pattern in the genes found up- or down-regulated by either Sge1 or Fgp1 ( data not shown ) . Likewise , a specifically conserved DNA pattern was not found upstream of genes regulated by Fgp1 or Sge1 under different conditions using search engines like RSAT [46] . The evolution of the Wor1-like proteins involved a duplication of the ancestral Wor1 like gene sequence prior to the divergence of the yeast -like and filamentous ascomycetous fungi . The duplication resulted in paralogous genes ( FGP1/WOR1 and FGP2/PAC2 ) that apparently have evolved quite different regulatory functions . While FGP2/PAC2 orthologs have been shown to be dispensable for pathogenicity , several studies now have established a role for FGP1/WOR1 in pathogenicity for a surprisingly diverse array of fungal pathogens of both plants and animals [9] , [10] , [11] , [12] . The function of FGP1/WOR1 however , clearly extends beyond its role in pathogenicity since transcriptome studies demonstrate its regulatory control over many other functions such as reproduction and secondary metabolism . FGP1/WOR1 and FGP2/PAC2 orthologs also are found in strictly non-pathogenic fungi like Podospora anserina ( data not shown ) ; however , interestingly , in the non-pathogenic fungus Neurospora crassa only the FGP2/PAC2 ortholog is present . Unlike the N-termini , the divergent glutamine-rich C-termini of FGP1/WOR1 genes in Fusarium likely allow specificity of regulatory control that has evolved independently in each species . Indeed the sets of genes regulated by orthologs FGP1 and SGE1 in F . graminearum and F . oxysporum , respectively , show little overlap and swapping experiments indicate the function of the C-terminal domains is largely specific to the species of origin . How WOR1/FGP1 genes are regulated themselves is still elusive although the mitogen activated protein kinase ( MAPK ) , as well as the protein kinase A ( PKA ) pathway could be involved . In B . cinerea , REG1 expression levels are regulated by two mitogen activated kinases: BcSAK1 and Bmp3 [10] . For SGE1 , higher expression levels ( ±5-fold ) are observed during in planta growth compared to growth in axenic culture [9] , which might indicate that SGE1 is regulated through expression levels too , but for FGP1 no significant differences in expression levels were observed during the conditions tested ( data not shown ) . In F . oxysporum and F . graminearum , both mitogen activated kinases are required for pathogenicity [47] , [48] of which the Δgpmk1 mutant in F . graminearum lacks the ability to form bulbous infection hyphae in planta [38] which might indicate that this strain is also defective in production of the bulbous hyphea in putrescine medium . In C . albicans , Wor1 phosphorylation and subsequent activation is believed to be performed by Tpk2 , a subunit of the PKA [19] . The conserved phosphorylation site of the Wor1-like proteins resembles a PKA site , making it likely to be phosphorylated by PKA . But whether Sge1 or Fgp1 are also phosphorylated and which kinase may be responsible for that is still unknown . However , a PKA mutant in F . oxysporum ( ΔfocpkA ) is also impaired in root penetration and virulence [49] . In C . albicans , Wor1 negatively regulates Efg1 transcription levels in opaque cells directly and indirectly via Czf1 and is itself regulated by Wor2 [18] , [50] . The conserved EFG1 ortholog in Fusarium species is called STUA and has been studied in F . graminearum ( FgSTUA ) [51] and F . oxysporum ( FoSTUA ) [52] . Czf1 and Wor2 have no conserved ortholog in Fusarium species as sequences homologous to these genes cannot be located using low stringency BLAST searches of the Fusarium genomes . The expression level of FGP1 seems to be negatively regulated in F . graminearum by StuA . During growth of the ΔfgstuA mutant in CMC and in a two-stage toxin induction medium levels of FGP1 transcripts are ±10 and ±100-fold higher compared to wild type . During growth of the ΔfgstuA mutant on wheat head , on the other hand , no significant difference in FGP1 levels were observed [51] . In contrast , FgSTUA or FoSTUA expression levels are not significantly different from wild type in Δfgp1 and Δsge1 mutants , respectively ( [9] and data not shown ) . The ΔfostuA mutant is still able to infect its host but the ΔfgstuA mutant is impaired in pathogenicity and toxin production [51] . In both F . graminearum and F . oxysporum , StuA is involved in conidia formation [51] , [52] . Overall , the ΔfgstuA demonstrates a more severe phenotype than the Δfgp1 mutant with greatly reduced vegetative growth and spores almost entirely absent [51] . These observations suggest that regulation of STUA and FGP1 in Fusarium species occurs differently than their orthologs WOR1 and EFG1 in C . albicans . In-depth phosphorylation experiments and expression studies of FGP1 and SGE1 with different mutant strains will be needed to identify other putative upstream activation factors . Additional work also will be required to fully understand the divergent roles of the N- and C- terminal domains on protein function and target specificity . Lastly , further investigations into the genome-wide impact of Fgp1 and Sge1 regulation on cell homeostasis , spore development and host infection will be needed to grasp a better understanding of this very interesting protein family .
The fungal isolates used in this study are the sequenced strains of F . graminearum PH-1 and F . oxysporum f . sp . lycopersici ( Fol ) strain 4287 . Also used were the Fol Δsge1 strain SGE1KO4 and complementation strain SGE1com79 reported earlier [9] . All fungal strains were kept at −80° and revitalized on potato dextrose broth plus agar ( PDB and Bacto agar , Difco ) . F . graminearum strains were grown for five days in carboxymethylcellulose ( CMC ) medium and F . oxysporum in rich complete medium ( CM ) for macroconidia and microconidia production , respectively . Agrobacterium tumefaciens EHA105 [53] used for Agrobacterium mediated transformations was grown in LB containing 20 µg/ml rifampicin and at 28°C . The wheat varieties “Norm” and “Bobwhite” and the wilt susceptible tomato variety “Bonny Best” ( Reimer Seeds , North Carolina USA ) were used for plant infection studies . Wheat pathogenicity assays were performed with cultivar “Norm” using point inoculation [54] . Pathogenicity was scored two weeks after inoculation by counting the number of infected spikelets . Wheat infection used for microarray studies were performed with cultivar “Bobwhite” using point inoculations of 10 spikelets in the head . 72 hour after inoculation , anthers were cut off and the infected spikelets were detached from rachis , collected and frozen until RNA processing [25] . Tomato infections were performed with two-week-old seedlings sown in vermiculite and given 20-20-20 NPK fertilizers after one week . Seedlings were inoculated using the root dip method and disease was scored as described previously [9] . In order to generate deletion constructs of FGP1 and FGP2 , PCR was used to amplify the up- and down- stream sequences of each gene using primers 2 & 3 and 6 & 7 ( FGP1 ) and primers 9 & 10 and 12 & 13 ( FGP2 ) ( Table S10 ) . PCR products were ligated into plasmid pPK2hphgfp [20] after both product and plasmid were digested using the appropriate restriction enzymes . The upstream flanks of FGP1 and FGP1 were cut with KpnI and PacI and the downstream flanks with HindIII and XbaI . For FGP1 , first the upstream flank was ligated into the plasmid and then the downstream flank . For FGP2 , first the downstream flank was ligated into the plasmid and then the upstream flank . A FGP1 complementation construct was generated using PCR and primers 14 & 15 ( Table S10 ) , which contain KpnI and EcoRI restriction sites , and ligated in pGEMT-easy ( Promega ) and sequenced . A correct product was ligated into plasmid pRW1p [55] , which was cut using the same enzymes: KpnI and EcoRI . The complementation constructs for chimeric FGP1 and SGE1 genes were prepared using the complementation constructs for both SGE1 and FGP1 in plasmid pRW1p . The combination of the N-terminal SGE1 with the C-terminal FGP1 was amplified using primers 16 & 17 and 18 & 19 ( Table S10 ) . The combination of the N-terminal FGP1 part and the C-terminal SGE1 part was amplified using primers 20 & 21 and 22 & 23 ( Table S10 ) . PCR products were ligated in pGEMT-easy and sequenced . Correct N-terminal SGE1 and C-terminal FGP1 products were cut from pGEMT-easy using enzymes BglII and XbaI and XbaI and PvuII , respectively . Correct N-terminal FGP1 and C-terminal SGE1 products were cut from pGEMT-easy using enzymes AdhI and XbaI and XbaI and BglII , respectively . Plasmid pRW1pSGE1 [9] was cut using enzymes BglII and PmeI . Using a three-point ligation strategy , the N-terminal SGE1 and C-terminal FGP1 products were ligated into plasmid pRW1pSGE1 cut using enzymes BglII and PmeI and the N-terminal FGP1 and C-terminal SGE1 products were ligated into plasmid pRW1pFGP1 cut using enzymes BglII and AdhI . For transformations of F . graminearum , a neomycin resistance cassette was ligated into the different pRW1p plasmids . This neomycin cassette was previously cut from pSM334 [56] using the flanking XbaI site and ligated into plasmid pAG1 [57] cut with XbaI . Subsequently , the neomycin cassette was cut from pAG1-Neo with BamHI and ligated into pRW1p cut with the same enzyme . Agrobacterium mediated transformation used for F . oxysporum was performed as described previously [58] . Agrobacterium mediated transformation used for F . graminearum was performed as described previously [29] with the following alterations: A different A . tumefaciens strain was used ( EHA105 ) , filters containing resistant colonies were not transferred to fresh selection plates but instead an agar plug containing a drug resistant colony was placed in liquid CMC medium and after two days of growth , spores were filtered through one layer of sterile miracloth and plated onto PDA plates containing cefoxitin ( 300 µg/ml ) and hygromycin or geneticin ( 150 µg/ml ) . Single spore colonies were subsequently transferred to a fresh PDA plate and mycelial plugs were stored at −80°C . Deletion mutants were tested by PCR and Southern analysis . Transformants made to complement the different deletion strains were checked by PCR for presence of the inserted construct ( data not shown ) and strains containing an insertion of the gene of interest were used . DNA was extracted using the CTAB protocol [59] and 5–10 µg was used for restriction and loaded for gel electrophoresis . Transfer of DNA to HyBond N+ ( GE Health Care ) was performed using standard alkaline procedures according to the manufacturer's protocol . Probes for FGP1 and FGP2 were amplified by PCR using primers 24 & 25 and 26 & 27 , respectively ( Table S10 ) . Probe hybridization and detection was performed using an AlkPhos kit and CDP-Star chemiluminescent solution ( GE Health Care ) according to the manufacturer's protocol . For quantification of microconidia , three independent experiments were performed , each with two replicates Microconidia were harvested after five days of growth in 100 ml CMC medium and 1 ml of 2*104 spores/ml were inoculated into 25 ml of fresh CMC medium . Alternatively 50 µl of a 1*106/ml spore suspension were inoculated into 5 ml of CMC in a 24-deep well plate ( 1*104 spores per well ) and incubated for 5 days . the third method used was to count spores produced on mung bean agar ( MBA ) , using 2 µl of a 1*106/ml spore suspension ( 2*103 spores ) to inoculate a mung bean agar plate . After one week of incubation , two ml of water was added to the plate and spread over the mycelium to collect spores . One ml of the spore suspension was pipetted into a tube and spores within a volume of 10 µl were counted using a haemocytometer . To assess the spore length , 40–75 spores produced in either CMC or on MBA were placed under a Nikon Eclipse 90i microscope and their length was measured . Perithecium formation was analyzed using the modified carrot agar method as described previously [60] in three replicas . To count the amount of ascospores , two ml of water was spread over the perithecia to collect ascospores . One ml of the ascospore suspension was pipetted into a tube and spores within a volume of 10 µl were counted using a haemacytometer . For in vitro toxin analysis , conidia ( 1*104 sp/ml ) of each strain were inoculated in six wells containing 2 ml of putrescine medium ( 30 g/l sucrose , 1 g/l KH2PO4 , 0 . 5 g/l MgSO4 , 0 . 5 g/l KCl , 0 . 8 g/l putrescine , 2 ml/l FeSO4*H2O solution ( 5 mg/ml ) and 200 µl trace elements ( 50 g/l citrate , 50 g/l ZnSO4*7H2O , 2 . 5 g/l CuSO4*5H2O , 0 . 5 g/l H3BO3 , 0 . 5 g/l NaMoO4*2H2O , 0 . 5 g/l MnSO4*H2O ) and grown for 1 week in the dark at 25°C . For microscopy and the time series experiment , flasks containing 25 ml of putrescine medium or minimal medium ( 30 g/l sucrose , 1 g/l KH2PO4 , 0 . 5 g/l MgSO4 , 0 . 5 g/l KCl , 2 g/l NaNO3 , 2 ml/l FeSO4*H2O solution ( 5 mg/ml ) and 200 µl trace elements ( 50 g/l citrate , 50 g/l ZnSO4*7H2O , 2 . 5 g/l CuSO4*5H2O , 0 . 5 g/l H3BO3 , 0 . 5 g/l NaMoO4*2H2O , 0 . 5 g/l MnSO4*H2O ) were inoculated with 2000 spores/ml and grown in the dark at 25°C with shaking 150 rpm . For each time point , the filtrate was collected by passing it through one layer of miracloth and 250 µl of culture filtrate was placed in a glass vial and lyophilized . In planta trichothecene analysis was performed by placing the inoculated spikelet in a glass vial and measuring its weight . Determination of DON , 3ADON and 15ADON concentration per unit mass in the vials was performed as described earlier [54] . RNA was extracted using Trizol ( Invitrogen ) according to manufacturer's protocol with an alternative precipitation step using ½ volume of isopropanol and a ½ volume of salt solution ( 0 . 8 M Sodium Citrate , 1 . 2 M NaCl ) . RNA was extracted from mycelium growing in complete medium for 48 hours in the dark at 25°C with shaking 150 rpm , from CMC grown cultures in a 24 well plate in 12 h light cycle at 25°C with shaking 150 rpm , from MBA grown cultures in 12 h light cycle at 25°C and from putrescine and minimal medium grown cultures for each time point as described above . Mycelium from cultures was harvested by filtration over one or two layers of miracloth , washed with water and frozen in liquid nitrogen . Mycelium was then lyophilized and ground in a mortar and pestle prior to Trizol extraction . RNA was also isolated from inoculated wheat spikelets , which were harvested , frozen in liquid nitrogen and ground in a mortar and pestle prior to Trizol extraction . For northern blotting 15 µg RNA in loading buffer ( 0 . 5× 3- ( N-morpholino ) propanesulfonic acid buffer ( MOPS ) , 1 M deionized glyoxal , 50% DMSO ) was loaded onto a 1× MOPS 1% agarose gel . The RNA was subsequently blotted onto Hybond-N+ ( GE Health Care ) using a capillary blotting protocol provided by the manufacturer with 20× SSC as transfer buffer . The TRI14 probe was amplified using primers 28 & 29 ( Table S10 ) followed by BamHI digestion and gel purification of the 897 bp fragment containing the portion of TRI14 downstream of the preditcted intron . The ACTIN probe was amplified using primers 30 & 31 ( Table S10 ) . Probe hybridization and detection was performed using the AlkPhos kit and CDP-star chemiluminescent solution ( GE Health Care ) according to the manufacturer's protocol . RNA cleanup was done using the RNeasy Mini Kit ( Qiagen ) prior to reverse transcriptase or microarray labeling . RNA labeling reactions were performed according to the standard Affymetrix protocols . The putrescine and minimal medium samples were hybridized to the Affymetrix F . graminearum GeneChips [61] and the complete medium and wheat infection samples were hybridized to an updated Affymetrix nine fungal plant pathogen GeneChip ( www . plexdb . org ) . Hybridizations were performed at the BioMedical Genomics Center of the University of Minnesota . RNA ( 2 µg ) was treated with DNase ( Invitrogen ) and used for RT-PCR with SuperScript III Reverse Transcriptase ( Invitrogen ) according to manufacturer's protocol . The cDNA obtained by different methods was used as template for Quantitative PCR ( qPCR ) , which was performed in two replicates with DyNamo™ SYBR® Green qPCR ( Finnzymes ) using a DNA-Engine Peltier thermal cycler ( BioRad ) equipped with a Chromo4™ real-time PCR detector and MJ Opticon Monitor™ analysis software . To quantify mRNA levels of genes of interest the ΔCt method was used . Primers used for constitutively expressed FRP1 genes ( primers 32–35 ) and the respective sporulation genes ( primers 36–47 ) are listed in Table S10 . Fungal infection in spikelets was monitored using an Olympus SZX16 Research Stereo Microscope and perithecia and cirrhi formation was observed using an Olympus SZX12 Research Stereo Microscope . Hyphal morphology of fungal strains growing in putrescine and control minimal medium was observed using a Nikon Eclipse 90i microscope . CEL files were imported in Refiner 5 . 3 software ( Expressionist ) and RMA preprocessing was applied . Signal values ( p-value 0 . 04 ) obtained in the Analyst software ( Expressionist ) were normalized to the median . Fold-expression filters were applied as described in the results . The probe sets and the corresponding probe descriptions from the F . graminearum GeneChips were converted from the annotation of the FG1 assembly to the FG3 assembly using the Fusarium graminearum database of MIPS ( http://mips . helmholtz-muenchen . de/genre/proj/FGDB/ ) in order to compare the experiments [62] . Probe sets on the nine fungal plant pathogen genome array GeneChips was designed based on the FG3 assembly for F . graminearum and the FO2 assembly for F . oxysporum ( Fusarium Comparative Sequencing Project , Broad Institute of Harvard and MIT ( http://www . broadinstitute . org/ ) ) . For the experiments with both F . graminearum and F . oxysporum grown in CM , a F . oxysporum ortholog was queried by BLAST [17] for every F . graminearum gene showing altered expression . In order to establish whether a gene was conserved in both F . oxysporum and F . graminearum , we searched for the respective orthologs using BLAST and a hit was considered a full ortholog at a bit score of >200 . Data and CEL files for microarray experiments are available at www . plexdb . org [63] under accession numbers NF2 ( wheat infection ) , NF3 ( growth on complete medium ) and FG18 ( growth on putrescine medium ) . | Plant pathogenic fungi can have devastating effects on crop yield and quality . In addition , these fungi may generate mycotoxins that pose health risks when the contaminated crops are consumed . The pathogen Fusarium graminearum infects wheat heads and grows through the rachis by synthesizing trichothecene toxins . The mechanisms and environmental cues triggering the production of trichothecene toxins have been studied for many years . Here , we describe a fungal gene , Fgp1 , that is absolutely required for pathogenicity and mycotoxin synthesis during infection and in culture . Fgp1 is not required for vegetative growth of F . graminearum but is important for reproductive development and potentially for a putative switch from a vegetative to a pathogenic phase . Deletion of Fgp1 results in reduced expression of the trichothecene biosynthetic gene cluster and genes specifically expressed during toxin synthesis in planta and in vitro . Fgp1 contains a conserved N-terminal domain and a divergent in the C-terminal region . The corresponding C-terminus from a sister species , F . oxysporum , is unable to function fully in F . graminearum when fused to the conserved F . graminearum N-terminal domain , suggesting the gene function is highly species specific . | [
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] | 2012 | The Wor1-like Protein Fgp1 Regulates Pathogenicity, Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum |
Epithelial morphogenesis generates the shape of tissues , organs and embryos and is fundamental for their proper function . It is a dynamic process that occurs at multiple spatial scales from macromolecular dynamics , to cell deformations , mitosis and apoptosis , to coordinated cell rearrangements that lead to global changes of tissue shape . Using time lapse imaging , it is possible to observe these events at a system level . However , to investigate morphogenetic events it is necessary to develop computational tools to extract quantitative information from the time lapse data . Toward this goal , we developed an image-based computational pipeline to preprocess , segment and track epithelial cells in 4D confocal microscopy data . The computational pipeline we developed , for the first time , detects the adherens junctions of epithelial cells in 3D , without the need to first detect cell nuclei . We accentuate and detect cell outlines in a series of steps , symbolically describe the cells and their connectivity , and employ this information to track the cells . We validated the performance of the pipeline for its ability to detect vertices and cell-cell contacts , track cells , and identify mitosis and apoptosis in surface epithelia of Drosophila imaginal discs . We demonstrate the utility of the pipeline to extract key quantitative features of cell behavior with which to elucidate the dynamics and biomechanical control of epithelial tissue morphogenesis . We have made our methods and data available as an open-source multiplatform software tool called TTT ( http://github . com/morganrcu/TTT )
Epithelial cells form cohesive sheets of cells that play diverse structural and functional roles in multicellular organisms such as the covering of internal and external surfaces , compartmentalization of the body into discrete organs , and the regulation of surface and trans-epithelial transport . The formation of structurally and functionally distinct embryonic structures requires that epithelial tissues change shape during development in a process called epithelial morphogenesis . A range of cellular behaviors drives these epithelial tissue shape changes , including cell shape change , rearrangements of cell-cell contacts , migration , proliferation , and programmed cell death . These behaviors , in turn , depend on intracellular molecular dynamics that allow cells to generate and transmit mechanical forces to one another , while maintaining epithelial cohesion [1 , 2] . This dual requirement is fulfilled by the adherens junction ( AJ ) , a specialized protein complex that links epithelial cells together . The AJs form a planar belt-like structure below the apical surface of the epithelium composed primarily of the single pass adhesion protein E-cadherin ( E-cad ) and associated proteins . The extracellular domain of E-cad forms trans homo-dimers to promote cell adhesion . The intracellular domain of E-cad associates with the force-generating actomyosin cytoskeleton and functions as a site for the transmission of mechanical forces that can remodel cell-cell contacts and cell shape by influencing the dynamics of the AJs themselves [3] . Despite advances in understanding the roles of the AJs and their regulators in controlling epithelial morphogenesis , we still do not understand how intracellular forces and cell behaviors coordinate at the tissue level to drive epithelial morphogenesis . Specifically , cell shape may be controlled by either autonomous or non-autonomous behaviors or forces , while the interaction between such local dynamics can lead to emergent effects on cell or tissue morphology . Live imaging of cell and molecular dynamics using fluorescently-tagged proteins is a key method to investigate these processes [4] . However , to fully leverage these experimental methods , quantitative approaches to automatically identify , track and interrelate molecular , cell and tissue level dynamics are required . The analysis of this quantitative information could then suggest molecular , cellular , and tissue level mechanisms that drive morphogenetic processes , and guide experimental approaches to test these possible mechanisms [5–9] . Several methods for segmentation and tracking of epithelial cells have been developed . These methods are based on the detection of the AJs in projections of 3D information into 2D planes . These methods provide approximations of epithelial shape but often lead to inaccurate representations of cell shape , especially in curved regions of epithelial sheets . The projection of an image volume into a 2D plane also increases the image noise , which may interfere with image preprocessing and quality of segmentation . Therefore , to circumvent these problems and provide a more accurate representation of epithelial cells , methods to segment and track the AJs in 3D need to be developed . Most methods for the segmentation and tracking of biological entities in 4D microscopy data are designed to establish the identity of particle-like objects such as nuclei [10–12] , organelles or molecular assemblies [13 , 14] . These methods consider objects as particles moving independently without restrictions imposed by cell packing in an epithelial sheet , and thereby do not model cell shape and cell-cell contact dynamics . Only a few methods have been developed for the segmentation and tracking of cells in tissues . A common approach is to perform a dual analysis of multichannel time lapse images labeled for both nuclei and membrane . Locations of cell nuclei are first identified and later employed to find the location of the cell membrane . Using this approach Bourgine et al . [15 , 16] have built a Partial Differential Equation framework to filter image noise , segment cell nuclei , locate the cell membrane and model the evolution of cell shapes to track the cells . A similar scheme has been developed by Luengo-Oroz et al . who employed a 4D morphological structuring element framework to denoise image volumes and locate cell nuclei , and a viscous watershed algorithm to infer cell nuclei locations as seeds for detecting cell extent properties [17] . This process is performed in 4D , implicitly tracking the cells . The same group has developed an alternative 3D based scheme to first identify nuclei , employ the viscous watershed method to determine cell extents and then track the cells and identify mitotic events [18] . Recently , several methods have been developed to detect the plasma membrane in tissues labeled with a membrane marker only . Mosaliganti et al . have built the ACME system to detect the planar structure of the cell membrane , and employed it to segment the spatial extent of cells in zebrafish embryos [19] . The EDGE system has been developed to segment and track epithelial cells in Drosophila embryos using a spatial alignment of 2D slices of a membrane marker to reconstruct the structure of the plasma membrane . The 2D slices are first denoised and thresholded , and then polygonal approximations are fitted to the segmented regions and stacked in 3D . Finally , the cells are tracked based on a custom polygon matching method [8] . A new version of the software called EDGE4D was recently published [20] , employing fluorescently labeled nuclei in the cell segmentation . All the previous methods focused on recovering the full spatial extent of the cells delimited by membranes . However , our final goal is to study the role of molecular forces remodeling AJs so we sought to develop methods to segment and track cells in epithelial tissues using only AJ markers . Although the AJs arrange in a belt-like structure , methods to detect this structure in 3D have not been reported . To the best of our knowledge , segmentation and tracking of AJs have only been done using 2D max-intensity projections of confocal time lapse data . Watershed algorithms are employed by Packing Analyzer [6] and by SeedWater Segmenter [21] to segment the AJs in 2D . A 2D simplification of the 3D Partial Differential Equation framework introduced in [15] has been reported in [22] . AJs assume the role of membranes when computing cell extent properties from cell nuclei . All the above methods are not easily generalized to 3D because the AJs do not surround the cells in 3D as they do in 2D . Successful segmentation of AJs and tracking of cells in confocal image volumes require solutions to several challenges that arise from the dynamic properties of the cells and the fluorescent E-cad∷GFP reporter used to highlight the AJs . In morphogenetic epithelia , E-cad molecules are constitutively endocytosed and recycled back to the cell surface providing epithelial cells with the plasticity to dynamically rearrange cell shape and cell-cell contacts [23–25] . As a result , E-cad is highly enriched in endocytic vesicles that can produce false positive membrane detections . At the cell surface , the distribution of E-cad is not always uniform increasing the complexity of detecting the AJs as a continuous structure . Certain cell types such as sensory cells accumulate high levels of E-cad thus increasing the complexity of segmenting these cells . Membrane detection methods have to distinguish between membrane and cytoplasmic signal , bridge discontinuities in signal distribution , and tolerate high signal intensity in certain locations . During epithelial morphogenesis , the cells grow or shrink , narrow or elongate , move and exchange neighbors , divide and die . Cell tracking methods in epithelial tissues need to identify cells as they change shape and contacts with their neighbors . To ensure cell track consistency it is also important to detect dividing cells and properly label the two daughter cells of mitotic events instead of creating a new track for one of them . Similarly , when a cell dies , the event should be properly identified and distinguished from a cell that leaves the scene . Tissue drift causes new cells to enter the scene and others to leave it . The cells that enter and leave the scene should be distinguished from cells that are generated by mitosis and lost by apoptosis , respectively . Tissue drift also introduces a global motion that should be compensated for . Here we present a novel approach for the automated segmentation and tracking of epithelial cells labeled with genetically encoded E-cad∷GFP using 4D confocal microscopy data of two developing epithelia: the Drosophila leg and notum . We chose these tissues for their unique morphological and developmental characteristics . The leg is a tube-like epithelium , which narrows and so elongates dramatically during development , while the epithelium of presumptive joints invaginates by apical constriction [26] . By contrast , the notum is a more planar epithelium that undergoes more subtle morphological changes [27] . The proposed approach to detect the AJs is independent of a prior detection of nuclei . Instead , cells are detected and tracked based on segmentation of the AJs and the determination of the connectivity between cells in the tissue . The methods we developed: 1 ) directly detect the vertices of the AJs where three or more cells meet in image volumes; 2 ) compute a planar graph approximation to the AJs network structure; 3 ) compute cell locations in the tissue and spatial associations between cells; 4 ) track cells and discover mitotic and apoptotic events . Additionally , we show how the computed motion of cell centroids can be used to describe the global dynamic behavior of a tissue . Finally , we provide a framework to assess the performance of the methods over a range of parameter values .
We have developed a system to segment the AJs of epithelial cells highlighted by AJ markers such as E-cad∷GFP and find the correspondence among them in adjacent temporal frames . The system is able to segment and track from just a few to hundreds of cells in 3D under different imaging conditions , including planar epithelial tissues such the Drosophila notum or tube-like epithelial tissues such the Drosophila leg . Fig 1A outlines the computational pipeline we developed to preprocess , segment and track epithelial cells . First , confocal time lapses of epithelial tissue morphogenesis ( Fig 1B ) are acquired . To enhance the representation of the AJs , timelapses are deconvoluted to eliminate both out-of-focus signal , and the Poisson noise introduced by the photodetector during imaging ( Fig 1C ) . If required , the images are locally equalized to obtain an even intensity of the E-cad∷GFP signal employing Contrast Limited Adaptive Histogram Equalization ( CLAHE ) [28] . Second , the AJs are located in the input volumes and encoded into a spatial graph to mimic their approximate polygon structure . A pair of filters based on the second order spatial derivatives of the image intensity are employed to measure the likelihood of each voxel being part either of a vertex or an edge [29] ( Fig 1D ) . The vertices of the graph are initialized with the local maxima of the output of the vertex detection filter . A level-set based region growing algorithm is employed to establish the connectivity among vertices in the input volume , adding an edge to the AJ graph for each pair of adjacent vertices ( Fig 1E ) . See materials and methods for details . Third , the cells in the tissue are identified and encoded in the Cell graph . The AJ graph is planar and has a face for each cell . The Cell graph ( Fig 1F ) is obtained as the dual of the AJ graph , with a vertex for each cell and an edge for each pair of adjacent cells . Each cell is associated with a list of vertices in the AJ graph defining its spatial extent . The spatial moments of this polygonal representation provide a set of spatial features for each cell such as perimeter , area , centroid , size or rotation . Finally , the correspondence among cells in adjacent frames is resolved ( Fig 1G ) . To this end we employ a min-cost max-flow cell tracking framework capable of detecting cellular mitosis and apoptosis events [10] ( Fig 2 ) . We consider that the detection of these events is as important as the correct association of cells among frames , although their relatively low frequency makes their detection even harder . However , understanding where and when cells proliferate and die is as important as studying how cells change their shape . Our system is able to successfully detect cellular apoptosis as the area of the dying cells tends to vanish and to detect cellular mitosis as the spatial moments of the parent cell are similar to the moment of the union of the daughter cells . The segmentation and tracking algorithms employed provide reliable outputs with some errors as we show later . We developed tools to fix errors in the output of the vertex location , edge segmentation and cell tracking algorithms ( Screenshot shown in S11 Fig ) . Error correction is done after each step as errors at one step are highly magnified in the next . Error correction ensures that the data to be analyzed accurately reflects the observed data . We illustrate the usage of the system to study the morphogenesis of a region of a Drosophila notum 24 hours after pupariation ( apf ) exploring the collective cell behaviors that contribute to tissue deformation . The timelapse captured the dynamics of cells in an area around the midline of the mid-scutum . We have employed the system to recover AJ graphs and cell graphs ( Fig 3B and 3C ) , identifying the cells in the tissue and establishing the temporal correspondence among them ( Fig 3D ) . The output of the system included some error that we corrected manually with validation tools that we developed to obtain accurate data . A visual inspection of the recovered cell trajectories shown in Fig 3D reveals a velocity gradient , increasing from posterior ( left ) to anterior ( right ) . To understand the reason for this , we have built a model to quantify the process that contributes to the global deformation of the tissue at this developmental stage . The strain rate tensor of a vector field describes the instantaneous rate of change of the deformation of a continuous material [31] . Because the epithelium of the notum is relatively planar , we dropped the third dimension of the cell trajectories and we built a strain rate tensor for the tissue , taking the velocity vector field of the cell centroids . The strain rate tensor components ( ∂ x . ∂ x , ∂ x . ∂ y , ∂ y . ∂ x , ∂ y . ∂ y ) and the mean velocities ( x . 0 , y . 0 ) of the vector field are shown in Fig 3E and 3F . Their values confirm that the velocity vectors depend on the position of each cell in the tissue and their signs show they are higher the farther they are from the posterior scutellum . We decomposed the strain rate tensor at each frame into their symmetric and antisymmetric components to respectively compute the expansion coefficient ℰ ( Fig 3G ) and the coefficient of rotation of the tissue θ ( Fig 3H ) . The expansion coefficient is always positive as the surface area of the tissue increases during the process . The coefficient of rotation remains almost constant , reporting a global rotation of the tissue of about one degree between frames , very likely resulting from a drift of the tissue in the medium . Prior to computing the strain rate tensors we have preprocessed the cell centroid trajectories to remove the noise in them employing a Kalman filter [32] ( see Materials and Methods ) . The Kalman filter assumes that cells obey a linear motion model among frames , providing a smoothed estimation of the position and velocity of each cell at each instant . We assessed the performance of the algorithms employed to segment the AJs and track the cells in 4 different time lapse data sets . Performance metrics were computed by comparing the outputs of the system to a ground truth , which was established through manual correction of the system output . For the notum dataset we corrected ( added , moved or deleted ) about 100 vertices for a total of 824 vertices and 60 edges ( added or deleted ) for a total 1207 it contains . For the leg dataset we corrected 120 vertices for a total of 403 and 93 edges for a total of 586 . The aim of this performance evaluation framework is not only to assess the quality of the proposed algorithms but also to provide a comparison framework to assess the quality of future alternatives . First , we analyzed the performance of the vertex location method . We limited the assessment to the first frame of the notum and leg time lapses . A vertex in the ground truth is considered as detected if the algorithm marks a vertex location closer than 2 voxels from the manually annotated position , i . e . , a vertex is detected if there is a detection in any of the voxels around it . The vertex location method depends on the interval of spatial scales employed to control the size of the AJ vertices detected and a detection threshold controlling the strength of the detected vertices . The spatial scale interval is easy to set up visually to an acceptable value , so we only assess the performance regarding variations in the vertex detection threshold . See supplementary S4 Fig for guidelines on how to select the proper spatial scales . Fig 4A shows Precision-Recall curves generated for the different datasets at different threshold detection levels . Briefly , Precision measures the number of truly detected vertices found with a given threshold level , while Recall measures the number of vertices undetected for that threshold level . The curve for the Notum dataset has an anomalous behavior for low recall values that arises from the very high values of the Vertexness function at sensory bristle cell locations that are mistaken for AJ locations . S6A Fig presents an example of this effect . Another common vertex location error that is produced at AJs are indentations between adjacent vertices as shown in S6B Fig . These are likely to arise by displacement of cell contacts by contractile forces generated by the actomyosin cytoskeleton . Vertex locations errors are more common in areas where cells are more parallel to the Z axis , where they are more difficult to locate due to voxel anisotropy and in areas with a low signal to noise ratio ( Fig 4C and 4D ) . Second , we conducted a performance assessment of the AJ edge detector . The AJ graph was initialized with the ground truth vertex locations curated for each frame in the time lapses . Fig 4E presents the Precision-Recall curves generated for the different data sets for different propagation thresholds Tℰ of the algorithm employed to detect edges . Fig 4F shows the variations of the performance according to the value of the threshold . The method achieves high detection scores detecting vertices in the different datasets as soon as the threshold is given an appropriate value . When the threshold is too low many cells are not be detected , while when the threshold is too high extra cells that don’t exist are detected . The optimal detection results are shown in Fig 4G and 4H . Note that areas surrounding bristles and areas with low signal-to-noise ratio produce more detection errors ( Fig 4G and 4H ) . In addition , as the orientation of edges become more parallel to the Z-axis they become more difficult to detect . Last , we evaluated the performance of establishing cell correspondences across frames . To this end we employed all the timelapses . The method depends on 11 parameters to compute the association costs ( see materials and methods for detail ) . As it is not practical to adjust all values for each new dataset , we searched for a combination of parameter values that works wells for the different tissues . We have exhaustively explored the parameter space and have found a combination that provides accurate tracking results across the different scenarios , with a mean AF1 = 0 . 93 . This represents a very high value as the AF1 metric highly penalizes failures in rare events such as mitosis and apoptosis . Fig 4I , 4J , 4K and 4L present cell trajectories recovered from the different timelapses . The position over time of each cell is projected in 2D . The variations of the performance according to the perturbation of the different parameters are provided as supplementary material ( S7 and S8 Figs ) . To check how the sampling rate of the timelapse influences tracking performance , we repeated the search for optimal parameter values including a decimated copy of the four datasets discarding every odd frame . The performance drops to a mean AF1 − score = 0 . 822569 . Computing the AF1 for the complete and decimated data set with the new parameters reveal that the AF1complete = 0 . 92 and the AF1decimated = 0 . 736818 . This shows that the sampling rate of the data set highly influences tracking performance . Interestingly , if we compare the values found for the weights in previous and current experiments ( S9 Fig ) , the weight given to the distance among centroids is reduced and the weight given to other features such as cell perimeter and rotation is increased . Additionally , we compared a 2D simplification of our system to the SeedWaterSegmenter , which utilizes a watershed algorithm to detect cell extents [21] , in the detection of cells , and found that our method performs better in obtaining cell locations . See supplementary material for further experimental details .
We have developed a computational pipeline that successfully transforms input 3D time lapse data into a rich description of cells features , connectivity and dynamics . We have shown that the pipeline detects and tracks cells with a high accuracy when properly parameterized . Clearly the most difficult part of the segmentation process is to locate the AJ vertices , as indicated by a lower F1-score , compared to the detection of edges and the computation of correspondences across frames . However , we described the errors produced by the vertex detection algorithm , so it should be straightforward to manually correct these errors to improve the accuracy of segmentation . We have also provided an example of how to use the generated cell centroid trajectories to study global cellular behaviors during tissue morphogenesis . Our methods were developed to segment and track epithelial cells in both simple and pseudostratified epithelial monolayer such as those present in Drosophila embryo and imaginal discs and early embryos of other species including mouse and zebra fish . Thus , our methods should be generally applicable to the analysis of epithelial morphogenesis in developing embryos in a range of species . We are currently using the system to explore the morphogenesis of the epithelium of the notum and leg imaginal discs to detect patterns of cellular behavior that correlate with the regional subdivision of these structures . We are particularly interested in exploring the behaviors that contribute to the narrowing and elongation of the epithelium of the leg imaginal disc and the relative contribution of presumptive joints and segments to this process [33 , 34] . In the notum , we are particularly interested in the relationship between the anterior-posterior subdivision of the notum and the pattern of cell rearrangements in each region [35 , 36] . Addressing these questions will help us understand how patterning of epithelial sheets at early stages of development affect epithelial dynamics and mechanics that generate the final form of adult structures at later stages . The proposed system can be used to investigate the topology of epithelial cells at each developmental time point , the deformation of cells and domains over time and the contributions of mitosis , apoptosis and cell movement to tissue morphogenesis . The detection of cell outlines and motion can be used to examine the evolution of cell topologies and the relative contribution of subcellular and cellular deformations to tissue morphogenesis . As the system stores the locations of vertices , edges and centroids in the data structure , it could be used to parameterize force models of epithelial remodeling such as cell vertex models [37 , 38] . The detection of mitosis and apoptosis can be used to investigate the contribution of lineage relationships and patterns of apoptosis to tissue development . A major challenge in the study of epithelial dynamics and tissue mechanics is to characterize the forces that drive planar cell rearrangements in epithelial sheets , how the forces are generated , how they affect cellular behavior [2 , 22] and tissue remodeling . The activity of contractile actomyosin networks and their coupling to the AJs can alter cell shape and cell contacts , as well as spatial patterns of cell proliferation and apoptosis . In contrast , adhesive forces mediated by cell adhesion proteins promote formation and expansion of cell contacts . Differential regulation of these contractile and adhesive networks in space and time can be used to deform epithelial sheets in predictable ways . Understanding how the activities of these networks are related to the changes in cell behavior can suggest mechanisms that drive tissue morphogenesis . The proposed system might be extended to relate changes in abundance , dynamics and polarity of contractile and adhesive molecular assemblies to the changes in cell and tissue shape . The segmentation and tracking of AJs in 3D and the curation of vertices , edges , cells and trajectories would enable the extension of the pipeline to relate molecular dynamics with cell and tissue remodeling for multiscale analysis of epithelial tissue morphogenesis .
Pupa were collected at the white prepupal stage ( 0 APF ) and aged in a humidified chamber at 25°C . For notum imaging , the pupal case was removed to expose the head and dorsal thorax at the desired developmental stages . For mounting , a slab of 1 . 5% agarose gel was placed atop a 30 mm coverslip . 2 intact pupae were mounted in tandem on the coverslip through a slit made in the agarose slab . A silicone gasket ( Sylgard 184 , Dow Corning ) was fitted to surround the agarose slab and a chamber constructed from acrylic was fitted atop the gasket to seal the chamber [39] . For leg imaging , legs were dissected in Shields and Shang M3 insect medium and then placed in a 35-mm glass bottom microwell petri dish ( P35G-1 . 5-14-C; MatTek ) in eversion medium composed of M3 medium ( S3652; Sigma ) , 2% fetal calf serum ( 10438; Gibco ) , 0 . 5% penicillin-streptomycin ( 15140–122; Invitrogen ) , 0 . 1 μg/mL Ecdysone ( 20-hydroxyecdysone H5142; Sigma ) , 2 . 5% /vol methyl-cellulose ( M0387-100G; Sigma ) as previously described [40] . The precise settings for acquisition of time lapse movies varied for each experiment but were dictated by the need to minimize photobleaching of the E-cad∷GFP reporter , tissue damage and image corruption by noise , and to maximize image resolution ( supplementary S1 Table ) . Approximately 10 sections at 1 to 1 . 5 micrometer intervals with 50% overlap were collected every 10 minutes to provide sufficient temporal resolution to track the cells and capture mitosis and apoptosis events . The 3D volumes were deconvolved employing a custom implementation of the Richardson-Lucy algorithm [41] to remove the effects of the lens blur and the Poisson noise process introduced in the photodetector . We employ an Elastic-Net Prior [42] to perform the deconvolution , as many of the voxels are expected to be zero . CLAHE was employed to locally normalize the intensity of the 3D volumes , enhancing the AJ structures . An AJ graph is built to represent the structure of the AJs in the input 3D volumes . It is an undirected graph defined by the tuple GA = ( VA , EA ) , where VA is the set of AJs vertices—the points where three or more cells touch—and the set of edges EA is such that there is an edge e ∈ EA for each pair of contiguous vertices in the tissue . We build the AJ graph from the output of the plateness function 𝒫 ( x ) proposed by Mosaliganti et al . [29] measuring how likely each voxel is part of the AJs and a vertexness function 𝒱 ( x ) measuring how likely each voxel is an AJ vertex . Both functions are built from spatial second order derivatives of the image intensity . See supplementary material for an accurate description of the functions . The vertex locations VA = ( {v1 , … , vn} are obtained as the local maxima of the vertexness function 𝒱 ( x ) . A threshold value TV is set up to reject spurious detections so ∀x ∈ V T𝒱 ≤ 𝒱 ( x ) . To identify edges based on segmented vertices , we devised a method based on dividing the input space into supervertices ( Fig 5B ) and inferring their connectivity relationships . A supervertex is the 3D region around each vertex v ∈ VA such that v is the vertex minimizing a time of travel cost function to be introduced later . We build the formal definition of a supervertex from the notion of Voronoi Region around a vertex . The Voronoi Region [43] around a vertex v ∈ VA is the subset of all the points in the 3D space that are closer to v than to any other vertex in VA: Voronoi ( v ) = [ x ∈ ℝ 3 ∣ arg min v ′ ∈ V A ∥ v ′ - x ∥ 2 = v ] ( 1 ) The partition of the space given by the different Voronoi Regions is called a Voronoi Diagram ( Fig 5A ) . For each point on the Voronoi Region of a vertex v we compute the time needed to travel to v , where the travel speed at each point x ∈ ℝ3 is given by F ( x ) = 𝒫 ( x ) + 𝒱 ( x ) > 0 . The time needed to travel from each point x ∈ Voronoi ( v ) to the vertex v is found as the solution to the partial differential equation: F ( x ) | ∇ T v ( x ) | = 1 ( 2 ) with boundary condition T ( v ) = 0 . This equation is a well known partial differential equation known as the Eikonal equation . It is employed to obtain the travel time for a wave to reach a point when traveling through a scalar field . Based on the notion of a Voronoi region around a vertex and the Eikonal equation employed to measure the travel-time cost from a point to its nearest vertex we provide a formal definition for a supervertex . The supervertex of a vertex v ∈ VA is defined to be the subset of the points in the Voronoi Region of v such that the solution T v * of Eq 2 with initial vertex v is lower than a threshold Tℰ: Supervertex ( v ) = [ x ∈ Voronoi ( v ) ∣ T v ( x ) ≤ T ℰ ] ( 3 ) Given the set of AJ vertices VA we obtain their corresponding supervertices employing a custom implementation of the Fast Marching algorithm [44] to solve Eq 2 that keeps track of the origin of the propagating wave and does not allow propagation across different Voronoi regions . An edge ( v , w ) is added to EA if and only if supervertex ( v ) and supervertex ( w ) touch . The computed AJ graph is shown in Fig 5C . The AJ graph computed in the previous section provides a symbolic representation of the AJ structure . However , the AJ graph does not explicitly model cells and their spatial extent and connectivity . To build a tissue model at the level of cells and edges and perform inferences , it is possible to exploit the properties of the AJ graph , and transform it into another graph to represent cells and their neighborhood relationships . The AJ graph belongs to a class of graphs known as planar graphs [45] . Intuitively , a graph is planar when it might be drawn in a plane with none of its edges crossing one another . Formally , a graph G = ( V , E ) is planar if there exist an embedding of the vertices in ℝ2 , f : V → ℝ2 such that for all pairs of edges ( a , b ) , ( c , d ) ∈ E , with a ≠ b ≠ c ≠ d ∈ V , the line segment from f ( a ) to f ( b ) does not cross the line segment from f ( c ) to f ( d ) . The edges of any planar graph G divide the space into regions called faces . The main implication for a graph being planar is the existence of an equivalent dual graph . For every planar graph G drawn in a plane there exists a graph G* whose vertices correspond to the faces of G and there is an edge between every pair of adjacent faces , i . e . the faces have at least one edge in common . The planarity property of the AJ graph allows the identification of the cells that form the tissue from the stained AJs without the need to first identify cell nuclei . The Cell graph GC = ( VC , EC ) is defined as the dual graph of GA , removing the vertex corresponding to the outer face . Each one of the vertices v ∈ VC represents a cell . Each one of the edges ( v , w ) ∈ EC represents cell adjacency among v and w . Thus , for each cell in the tissue there is a vertex in the Cell graph . A cell is defined by the pair ( c , 𝒜 ) where c ∈ VC is the vertex representing the cell in the cell graph and the set A = a1 , … , aK , ak ∈ VA is the clockwise ordered list of the vertices of the AJ graph delimiting the cell . The polygonal representation of Aj allows the derivation of moments such as cell perimeter p ( c ) ∈ ℝ+ , cell area a ( c ) ∈ ℝ+ , cell centroid b ( c ) ∈ ℝ3 , cell width w ( c ) ∈ ℝ+ , length h ( c ) ∈ ℝ+ or rotation r ( c ) ∈ ( −π , π ) . All these features are later employed to match cells among adjacent frames for cell tracking . This representation also provides an explicit model of cell neighborhood necessary to compute tissue measurements such as those proposed in [46] or [47] . We next developed methods to establish the correspondence among cells over time . The developed cell tracking methods rely on the methods we have developed to identify cell locations and their spatial properties . The method we employed to solve the cell correspondence problem among frames is a variation of the coupled min cost-max flow framework reported in [10] . The solution to the cell correspondence problem is obtained as the solution to a flow transportation problem in a directed graph . A total of N + M units of flow need to be sent from a source vertex T+ to a sink vertex T− traversing a network formed by set of vertices and a set of arcs connecting the vertices that encodes the cell association problem . Arcs have a maximum capacity and an associated cost for sending units of flow through them . The set of arcs minimizing the cost for sending the N + M units of flow through the network gives the solution to the cell correspondence problem . Flow has to be preserved among the arcs of the network , i . e . , the same amount of flow that gets into a vertex needs to be sent to others , except at source and sink vertices . The cell association cost attached to the arcs is computed from the cell moments obtained in the previous section . The cost of associating a cell to a given cell in the next frame is computed as a weighted sum of the difference among their moments . In case of a mitosis event , the cost is computed in a similar way but from the union of the polygons corresponding to a pair of adjacent hypothesized sibling cells . The weight for cells entering and leaving the scene is proportional to the distance from the centroid to the tissue perimeter . The weight for the apoptosis hypothesis is proportional to the area of the dying cell . The exact formulation of the coupled min-cost max-flow framework employed is provided in supplementary material . The proposed framework differs from the original in the way correspondence hypotheses are formulated . As we track cells embedded in an epithelial sheet rather than freely moving particles we exploit the neighborhood relationships among cells to drop association hypothesis not corresponding to adjacent cells . To assess the performance of the proposed algorithms to respectively detect AJ vertices , AJ edges and compute cell correspondences among frames we neeed to define a measure of the quality of a given configuration . There are two type of errors that detection algorithms might produce , known as False Positives ( FP ) and False Negatives ( FN ) . A FP is produced when the algorithm marks a detection that is not real , while a FN is produced when the algorithm misses a real detection . Thus , to assess the quality of a given set of detections , it is possible to compute three metrics known as Precision , Recall and F1 measure defined as follows: Precision = | Marked detections ∩ Real detections| | Marked Detections | ( 7 ) Recall = | Marked detections ∩ Real detections| | Real Detections | ( 8 ) F1 Measure = 2 × Precision × Recall Precision + Recall ( 9 ) Precision measures the amount of FP errors produced , Recall measures the number of FN the detector produces , while the F1 measure is the harmonic mean of both measures and might be understood as a summary of them . Detectors commonly have a detection threshold ( the proposed here do ) that has to be adjusted a priori to some value and conditions Precision and Recall values of the algorithm . Note that Precision and Recall are conflicting measures: a high detection threshold produces high precision and low recall ( very few but true detections , but many missed detections ) , while a low detection threshold produces low precision but a high recall ( not a lot of missed detections , but a lot of false detections ) . Thus , algorithms assessment should include tests for many threshold levels to produce Precision-Recall curves . The sensitivity of parameters was explored based on the changes of the F1 measures . Similar metrics are employed to evaluate cell tracking algorithm . To this end we consider it as a label prediction problem . For each cell in a given frame the task is to predict the correspondence of a cell with the same cell in next frame , if it should disappear following apoptosis , or if it should be associated with two new cells following mitosis . Thus , an average F1 measure might be obtained for each tracking configuration as the harmonic mean of the individual F1 measures: A F 1 = 4 F 1 a s s o c i a t i o n - 1 + F 1 c r e a t i o n - 1 + F 1 t e r m i n a t i o n - 1 + F 1 m i t o s i s - 1 ( 10 ) | Epithelia are the most common tissue type in multicellular organisms . Understanding processes that make them acquire their final shape has implications to pathologies such as cancer progression and birth defects such as spina bifida . During development , epithelial tissues are remodeled by mechanical forces applied at the Adherens Junctions ( AJs ) . The AJs form a belt-like structure below the apical surface that functions to both mechanically link epithelial cells and enable cells to remodel their shape and contacts with their neighbors . In order to study epithelial morphogenesis in a quantitative and systematic way , it is necessary to measure the changes in the shape of the AJs over time . To this end we have built a complete computational pipeline to process image volumes generated by laser scanning confocal microscopy of epithelial tissues where the AJs have been marked with AJ proteins tagged with GFP . The system transforms input voxel intensity values into a symbolic description of the cells in the tissue , their connectivity and their temporal evolution , including the discovery of mitosis and apoptosis . As a proof of concept , we employed the data generated by our system to study aspects of morphogenesis of the Drosophila notum . | [
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] | [] | 2015 | Segmentation and Tracking of Adherens Junctions in 3D for the Analysis of Epithelial Tissue Morphogenesis |
The question whether fatty acids can be converted into glucose in humans has a long standing tradition in biochemistry , and the expected answer is “No” . Using recent advances in Systems Biology in the form of large-scale metabolic reconstructions , we reassessed this question by performing a global investigation of a genome-scale human metabolic network , which had been reconstructed on the basis of experimental results . By elementary flux pattern analysis , we found numerous pathways on which gluconeogenesis from fatty acids is feasible in humans . On these pathways , four moles of acetyl-CoA are converted into one mole of glucose and two moles of CO2 . Analyzing the detected pathways in detail we found that their energetic requirements potentially limit their capacity . This study has many other biochemical implications: effect of starvation , sports physiology , practically carbohydrate-free diets of inuit , as well as survival of hibernating animals and embryos of egg-laying animals . Moreover , the energetic loss associated to the usage of gluconeogenesis from fatty acids can help explain the efficiency of carbohydrate reduced and ketogenic diets such as the Atkins diet .
It is well known that excess sugar in the human diet can be converted both into glycerol and fatty acids and , thus , into lipids such as triglycerides . A related question biochemistry students are often asked in their exams is whether the reverse route is also feasible , that is , whether the human body can convert fatty acids back into glucose . As for even-chain fatty acids , the expected answer is “No” ( odd-chain fatty acids practically do not occur in mammals ) . This summarizes the result of a debate that dates back to the late 19th century . However , it was not until the 1950s that such a conversion could be monitored using 14C labeled fatty acids [1] . It was found that part of the label arrives at glucose , proving that there is a connected route from acetyl-CoA to glucose . However , as shown mathematically [1] , [2] , there cannot be any sustained conversion at steady state along the tricarboxylic acid ( TCA ) cycle due to stoichiometric constraints . In particular , oxaloacetate would not be balanced ( Fig . 1A ) . A possible route that does allow this conversion in some prokaryotes [3] , [4] , plants [5] , fungi [6] and nematodes [7] is the glyoxylate shunt . It produces an additional oxaloacetate , thus balancing this compound ( Fig . 1B ) . However , the corresponding enzymes have not been found in mammals in spite of controversial speculations [8] . Reports that the glyoxylate shunt would be present in some hibernating animals [9] were not confirmed . In a recent experimental work they have been genetically introduced into mice [10] . In summary , there is a general consent that carbohydrates cannot be produced from even-chain fatty acids in humans although the opposite conversion is feasible . In consequence , this statement can be found throughout prominent biochemistry textbooks [11] , [12] , [13] . It has even been used as a benchmark criterion for the reconstruction of whole-cell metabolic networks in hepatocytes [14] . An alternative reconstruction of hepatocyte metabolism [15] however , does not use that criterion . This problem is of particular importance with respect to the provision of energy to the brain in situations of drastically reduced carbohydrate uptake . Although the brain can use ketone bodies in these situations , it still needs a certain amount of glucose [16] , which has critical implications upon starvation and similar conditions . Recently , we re-investigated the problem in question in a small model of human central metabolism [2] . We used the concept of elementary flux modes [17] , which allows one to detect all feasible metabolic pathways in small to medium-scale reaction networks . We were able to corroborate the results of Weinman et al [1] . However , only a small part of metabolism rather than the entire human metabolic network was considered . Hence , alternative potential pathways for gluconeogenesis from fatty acids via acetone , such as those proposed in the 1980s [18] , [19] could not be detected . In order to check for the existence of such pathways in a holistic approach , we used the most detailed reconstruction of the human metabolic network [20] , which had been reconstructed on the basis of experimental results . Moreover , we employ the concept of elementary flux patterns , which allows an analysis in whole-cell metabolic networks similar to elementary mode analysis [21] . An elementary flux pattern corresponds to a set of reactions within a subsystem of a reaction network that is compatible with feasible pathways through the entire system ( Fig . 2 ) . Since the entire system is explicitly taken into account , this method allows one to detect all feasible routes on which one compound can be converted into another ( for details see Materials and Methods ) . Equipped with this new modeling technique and a genome-scale model of human metabolism , we want to answer the question whether carbohydrates can be produced from fatty acids in humans . The question whether fatty acids can be converted into carbohydrates has many biochemical implications: effect of starvation ( cf . Owen et al . [16] ) , sports physiology [22] , the traditional diet of inuit , which is practically carbohydrate-free [23] and high in fish and seal oil . In earlier times , low-carbohydrate diets were also typical for native Americans hunting in the plains . Also the survival of hibernating animals [24] and of embryos of egg-laying animals is crucially dependent on gluconeogenesis [25] . Moreover , the efficiency of carbohydrate reduced and ketogenic diets such as Atkins diet is worth being studied from this viewpoint . In particular , we will introduce two quantities and compute them for fatty acids and various other storage compounds: gluconeogenic energy efficiency and glucose storage efficiency . The former measure quantifies how much energy in form of ATP is regained if a compound is used for gluconeogenesis and is subsequently catabolized . The latter measure quantifies how much glucose is regained if a compound is produced from glucose and subsequently converted back into glucose .
Overall we detected nine possible pathways for the conversion of acetyl-CoA into mitochondrial acetoacetate ( see Text S1 ) . In acetone degradation we identified 58 possible pathways . In addition to the work of [26] in which acetone metabolism was reviewed , we identified a new intermediate of acetone metabolism , D-lactaldehyde , which is used in 14 out of the 58 pathways . This metabolite can be produced from either methylglyoxal or lactoyl-glutathione , an intermediate in the conversion of methylglyoxal to D-lactate . The conversion of methylglyoxal to D-lactaldehyde is catalyzed by an aldo-keto reductase or by a glyoxylate reductase . Aldo-keto reductase is only weakly expressed in the liver [27] . Thus , this pathway might be of greater importance in other tissues . Another important point is the import of pyruvate into mitochondria . Pyruvate can be either directly imported into the mitochondrion or indirectly via a lactate shuttle [28] ( Fig . 4 ) . We will not consider the indirect import since its importance is disputed [29] . Therefore , only 22 pathways for the conversion of acetol into pyruvate remain ( Text S1 ) . One important feature of the described pathways is that many of them involve the reduction of NAD+ or oxidation of NADPH . Overall , 1–3 moles of NADPH are oxidized and 0–4 moles of NAD+ are reduced during the conversion of one mole of acetone into pyruvate ( see Text S1 ) . Special emphasis can be put on the oxidation of methylglyoxal to pyruvate . In this case , one mole of NADP+ can be reduced leading to pathways in which the net balance in oxidized NADPH is only one ( pathways 7 and 14 , depicted in Fig . 3 ) . Pathways not using this reaction have a net balance of at least two moles of NADPH oxidized . As discussed below , these requirements on the reduction potential impose constraints on the utilization of gluconeogenic routes from fatty acids . Besides the concept of elementary flux patterns , other techniques to determine pathways in genome-scale metabolic networks exist . Among the most widely used techniques is flux balance analysis [30] that allows one to identify physiologically feasible fluxes within a metabolic network that optimize a certain objective function . While a single pathway for the conversion of fatty acids in gluconeogenesis can be obtained with this method , a systematic identification of all possible pathways is not possible . Another frequently used tool is elementary flux mode analysis [17] that allows one to enumerate all physiological feasible flux distributions in a metabolic network . However , the number of extreme pathways [31] , a subset of the elementary flux modes , is estimated to be 1029 for the human genome-scale network [32] , which makes the full enumeration of all the elementary flux modes impossible . Nevertheless , recently two approaches to enumerate subsets of elementary flux modes with increasing number of reactions have been developed [33] , [34] . Here , we used the K-shortest EFM method to compute the 100 shortest elementary flux modes producing cytosolic glucose 6-phosphate from mitochondrial acetyl-CoA . Within these elementary flux modes only nine of the 22 pathways for the conversion of acetone to pyruvate were present ( Text S1 ) . Hence , the complete enumeration of all possible pathways for the conversion of acetone to pyruvate on the pathway of gluconeogenesis from fatty acids could not be achieved by this approach .
We performed a global survey of gluconeogenic routes from fatty acids in human metabolism using a genome-scale metabolic model and elementary flux pattern analysis . Even though prominent biochemistry textbook negate the existence of such a route in mammals and humans in particular [11] , [12] , [13] , we were able to confirm several routes that have been proposed earlier [18] , [19] . Additionally , we were able to identify 14 new pathways that proceed via a new intermediate in acetone metabolism , D-lactaldehyde . These results further underline the utility of elementary flux pattern analysis in assessing the metabolic capabilities of organisms . Gluconeogenesis becomes important when the glucose level in the body cannot be sustained by the glycogen store in the liver , which is sufficient for up to one day in humans [16] . Such a situation arises , for instance , during starvation , fasting , prolonged physical exercise , upon carbohydrate-reduced and ketogenic diets like Atkins diet as well as in hibernating animals . In addition to glucogenic amino acids from proteins , the glycerol component of lipids can be converted into glucose while fatty acids serve as principal metabolites to fuel oxidative phosphorylation . In prolonged starvation , not only muscular protein but also proteins essential for the maintenance of principal body functions are broken down to serve for gluconeogenesis [16] . However , there is obviously a limit to such protein degradation . While the principal fuel of the brain under normal conditions is glucose , in starvation , the brain starts using ketone bodies in addition to reduced consumption of glucose . The increase of the production of ketone bodies leads to rising levels of acetoacetate , which is constantly decarboxylated into acetone . For example , in 21 days fasted humans , 37% of the acetoacetate is converted into acetone [35] . The putative enzyme catalyzing this reaction , acetoacetate decarboxylase , has been characterized in terms of catalytic activity [36] and inhibitors [37] , but has not yet been identified [26] . 2–30% of acetone is excreted via the urine and breath [35] , while the remainder is metabolized further and could account for up to 11% of gluconeogenesis during starvation [35] . Glucose formation from acetone was indeed suggested as an explanation for the finding that common gluconeogenic precursors alone could not fully account for renal gluconeogenesis [16] . Additional supporting evidences come from the increased activity of cytochrome P450 ( Cyp2e1 ) , an enzyme essential in the presented pathways , during starvation [38] and the observation that increased survival time of obese rats during starvation correlates with the activity of acetone metabolism [39] . Moreover , increased levels of methylglyoxal , an intermediate of some of the gluconeogenic routes from fatty acids , have been observed in subjects on the carbohydrate reduced Atkins diet during which ketogenesis is particularly active [40] . Thus , these pathways play an important role in gluconeogenesis also in other situations like the ones mentioned in the Introduction in which ketogenesis is particular active . In hibernating animals , for example , protein may be saved by not using it as the only source for gluconeogenesis . This hypothesis is confirmed by recent results in which it was found that Cyp2e1 and phosphoenolpyruvate carboxykinase ( Pck1 ) , both enzymes within the described pathways , are significantly ( p = 0 . 005 ) upregulated in the liver of hibernating black bears ( FoldChange 5 . 990 and 13 . 17 , respectively ) [41] . In a more recent work , also Pck1 ( FC = 10 . 7 ) and Pc ( FC = 2 . 62 ) were found to be upregulated in the liver of the animals during hibernation [42] . Important aspects related to the presented pathways are energetic requirements that constrain their capacity . Experimental investigation of the gluconeogenic role of acetone showed differences in its utilization between species: the net synthesis of glucose from acetone as observed in murine hepatocytes [43] does not seem to occur in perfused rat liver [44] . Instead , net synthesis from acetone could be demonstrated in either case when also other gluconeogenic substrates were given [43] . This can be explained by the increased requirement for NADPH in the conversion of acetone to pyruvate . Each possible pathway requires the oxidation of at least two moles of NADPH ( see Fig . 4 ) . However , there are only two pathways which reduce the net balance in NADPH to-1 moles by the direct conversion of methylglyoxal to pyruvate ( pathways 7 and 14 , Fig . 3 ) . During gluconeogenesis , cytosolic pathways for the replenishment of NADPH are impaired since they would further deplete gluconeogenic metabolites [45] . In fact , NADPH has to be supplied from mitochondrial sources and its availability represents the rate-limiting factor in acetone metabolism [39] , [46] . There are two known pathways of NADPH replenishment from mitochondria [45] ( Fig . 5 ) . One of them involves the transport of mitochondrial NADPH into the cytosol and the other the transfer of electrons from mitochondrial NADH to cytosolic NADP+ . The direct transport from the mitochondrion involves a citrate∶oxoglutarate shuttle . Since both metabolites are intermediates of the TCA cycle , they would potentially reduce mitochondrial fatty acid oxidation when used for this shuttle system . The other pathway of NADPH replenishment uses a route via cytosolic malic enzyme , which decarboxylates malate to pyruvate . Both metabolites are intermediates of gluconeogenesis from acetone . Hence , each oxidized NADPH would require an additional cycle in the conversion of pyruvate to malate . Each of these cycles involves the hydrolysis of one ATP to ADP and the oxidation of one NADH . Especially pathways 7 and 14 in the conversion of acetol to pyruvate are of interest since they only require a net consumption of one mole of NADPH per mole of pyruvate produced . Thus , only a single pyruvate-malate cycle would be necessary to balance NADPH consumption . Since there are several possible pathways in acetone metabolism , there might be species differences in the principal pathways which are used for acetone degradation . Another reason for the limited capacity of the pathways in question is that several of their intermediates , such as methylglyoxal and acetone , are toxic in higher or even moderate concentrations . Thus , the organisms can use these pathways only to a limited extent . A further limiting factor may arise if the decarboxylation of acetoacetate indeed proceeds spontaneously . As it had been observed that Caucasian people can adapt to the diet of inuit within about three weeks [23] , the limitation in capacity of these pathways is likely to get less severe over time , probably due to induction of specific enzymes along those pathways . We investigated the energetic requirements of the presented pathways in terms of ATP consumption under the assumption that all other metabolites , including NADPH and NADH need to be balanced ( Fig . 6A and Text S1 ) . We found that the presented pathways consume 6–22 moles of ATP for the production of one mole of glucose and two moles of CO2 from four moles of acetyl-CoA ( requiring a flux of two through the described pathways ) . The most efficient pathway in terms of ATP consumption proceeds via the oxidation of methylglyoxal to pyruvate , oxidizes two moles of NADPH and reduces two moles of NADH ( pathway 7 , displayed in Fig . 3 ) . However , since the mitochondrial NADH concentration is already high during ketogenesis [47] , pathway 14 might be more favorable because even two moles of cytosolic NADH are reduced on this pathway . Using this pathway the cost for the synthesis of one mole of glucose is increased to 16 moles of ATP . Moreover , we computed the Gibbs free energy change of the overall pathways to check whether they are thermodynamically feasible ( Fig . 6A ) . We performed these computations for gluconeogenesis starting from acetyl-CoA and palmitate , respectively . Thus , we found that the overall Gibbs free energy change is in the range of −1162 to −1449 for gluconeogenesis from acetyl-CoA and in the range of −1345 to −1610 for gluconeogenesis from palmitate . Thus , the presented pathways are all thermodynamically feasible . In order to analyze the role of the described pathways during prolonged starvation and fasting , we computed for amino acids , fatty acids , lactate and glycerol how much energy in form of ATP is regained in the net balance if they are used for gluconeogenesis and subsequently catabolized . This quantity , termed gluconeogenic energy efficiency , is defined as the ratio of the above mentioned net amount of energy and the energy that would be obtained by catabolizing the substrate directly ( Fig . 7A ) . That quantity equals unity if both pathways provide the same amount of ATP . Furthermore , we determined how well amino acids , fatty acids , lactate and glycerol are suited for the storage of glucose . This measure , termed glucose storage efficiency , is defined as the relative amount of glucose regained if these compounds are produced from glucose and subsequently converted back into glucose ( Fig . 7B ) . Details on the calculation are given in Text S1 . Gluconeogenic energy efficiency and glucose storage efficiencies of a selected list of compounds are displayed in Fig . 6B . We found that 53–74% of the energy remains if fatty acids are used for gluconeogenesis using the most efficient and most inefficient pathways , respectively ( pathways 7 and 20 ) . Thus , 26–47% of the energy contained in fatty acids is lost if they are used for gluconeogenesis . For glucogenic amino acids , in contrast , the energy loss is much smaller; the gluconeogenic energy efficiency is in the range from 73% ( leucine ) –96% ( valine ) with a value of 87% for the amino acid composition of a typical dietary protein . For glycerol , the gluconeogenic energy efficiency is 95% . These values can explain the particular efficiency of carbohydrate reduced and ketogenic diets like Atkins diet for weight reduction . The reason is likely to be the increased energy loss of gluconeogenesis from fatty acids and ketogenic amino acids in comparison to gluconeogenesis from glucogenic amino acids . Indeed , intermediates of the pathway for gluconeogenesis from fatty acids have been observed in subjects on the Atkins diet [40] . This is also supported by the observation that the traditional diet of inuit does not lead to obesity in spite of the high content in fat . In contrast , new dietary habits of inuit implying a higher consumption in carbohydrates often lead to obesity [48] . Comparing the glucose storage efficiency of the described compounds ( Fig . 6B ) , we find fatty acids , using pathway 7 , at the lower end with a storage efficiency of 50% in comparison to 39% for glycine , 78% for alanine and 86% for glycerol . Hence , the conversion of glucose to fatty acids and gluconeogenesis from fatty acids results in a loss of half of the glucose . These values show that fatty acids are not very well suited as glucose storage since their use as such is associated to a higher loss of glucose equivalents of carbohydrates in comparison to glycerol and amino acids that are the major carbohydrate storage compounds besides glycogen . Nevertheless , as discussed above , the utilization of fatty acids as glucose storage gives the body additional flexibility in the utilization of its storage compounds and appears to be used as such in situations during which gluconeogenesis is active . Moreover , both quantities can be useful for examining the effect of caloric restriction on ageing which is known to extend life span in a large number of organisms [49] . Summarizing our findings , it can be concluded that a thorough , systematic and detailed in-silico investigation of the stoichiometrically feasible routes from fatty acids to glucose based on an experimentally corroborated genome-scale metabolic network provides new insight into human metabolism under glucose limitation . It confirms earlier , anecdotal evidence and hypotheses about gluconeogenesis from fatty acids via acetone and provides hitherto unrecognized pathways for that conversion . This provides a plausible explanation for the surprising independence from nutritional carbohydrates over certain periods ( e . g . upon the low-carbohydrate diet of inuit , in hibernating animals and embryos of egg-laying animals ) . Moreover , we provided a detailed analysis of the energetic balance of these pathways , which explains their limited capacity and their contribution to the particular efficiency of carbohydrate reduced and ketogenic diets .
Elementary flux patterns have been introduced as a new theoretical tool for the analysis of metabolic pathways in genome-scale metabolic networks [21] . Within a subsystem of metabolism , that is , a set of reactions of interest , elementary flux patterns correspond to basic route through that subsystem that are compatible with steady-state fluxes through the entire network . Thus , every elementary flux pattern is associated to at least one steady-state flux of the entire system and corresponds to the set of reactions used by this steady-state flux in the subsystem . The property of elementarity in the definition of elementary flux pattern requires that no elementary flux pattern can be written as set union of other elementary flux patterns . A formal definition will be provided next . Given the stoichiometric matrix of a reaction network we assume for simplicity that the first reactions make up the subsystem of interest . A flux pattern is defined as a set of reactions within the subsystem that is compatible with at least one steady-state flux of the entire system . Hence , a set of reaction indices is called a flux pattern if there exists at least one flux vector that fulfills the following conditions: Furthermore , we call a flux pattern elementary if this set of reactions cannot be written as set union , of other flux patterns . For more details see Kaleta et al ( 2009 ) . Elementary flux patterns can be used to elucidate all possible pathways consuming a certain compound and producing another . This process builds upon a successive expansion of the subsystem under study to reactions that belong to alternative pathways . It will be outlined by way of a small example network comprising glycolysis and the pentose phosphate pathway ( Fig . 8 ) . Within this system we want to find all pathways producing ribose-5-phosphate ( R5P ) , a precursor of histidine and nucleotide syntheses from glucose ( Glc ) . We start with a subsystem encompassing the inflow reaction of Glc and the outflow of R5P ( Fig . 8A ) . Two elementary flux patterns are found . One of them only contains the inflow reaction of Glc and the other the inflow of Glc as well as the outflow of R5P . The first elementary flux pattern indicates the existence of a pathway consuming Glc at steady state without using the outflow of R5P . This corresponds to the glycolytic pathway producing glycerol-3-phosphate ( G3P ) which is subsequently drained from the system ( Fig . 8C ) . The other flux pattern corresponds to a pathway producing R5P ( Fig . 8B ) . Since we found no elementary flux pattern containing the outflow of R5P without the inflow of Glc we can conclude that the inflow reaction is required for the production of R5P . Otherwise we would have obtained a second elementary flux pattern containing only the outflow of R5P . Next , we need to determine an elementary mode through the entire system using exactly the reactions of the elementary flux pattern in the subsystem . Such an elementary mode can be obtained using linear programming [21] . This elementary mode corresponds to a first pathway for the production of R5P from Glc . From this initial pathway it is possible to deduce reactions that are essential for the conversion of R5P to Glc ( see Text S1 for more details ) . Thus , we find that in addition to the inflow of Glc , the conversion of Glc to glucose-6-phosphate ( G6P ) and the conversion of ribulose-5-phosphate ( Ru5P ) to R5P are required for production of R5P at steady state . The knowledge of essential reactions can simplify the analysis in two ways . First , we do not need to include essential reactions into the subsequent subsystems since every pathway producing R5P will use them anyway . Second , if we find several sequences of essential reactions the task of searching for pathways can be split into sub-tasks . Each sub-task then consists in the search for a pathway connecting a product of a sequence of essential reactions and the educt of the next sequence of essential reactions . In order to determine reactions that belong to alternative pathways , we include the reactions of the first detected pathway into the subsystem of the next step . As noted above , we need not to add essential reactions and consequently , the subsystem of the second step comprises three reactions ( Fig . 8D ) : the inflow of glucose , the conversion of G6P to Ru5P and the outflow of R5P . This subsystem gives rise to three elementary flux patterns . One of them contains the inflow of G6P and the outflow of R5P ( Fig . 8E ) . This elementary flux pattern corresponds to a pathway for the production of R5P . This flux pattern is associated to an elementary mode that also uses reactions that do not belong to the subsystem ( and are not essential reactions ) . Hence , we have identified reactions that belong to an alternative route . These reactions are subsequently added to the subsystem of the third step ( Fig . 8F ) . In this subsystem we find eight elementary flux patterns , two of which contain the outflow of R5P ( Figs . 8G and 8H ) . Determining the elementary modes associated to these elementary flux patterns , we find that both use only reactions that are either essential for the pathway or belong to the subsystem . Thus , we have identified all pathways producing R5P from Glc . These pathways correspond to the elementary modes associated to each of the two flux patterns containing the outflow of R5P . We computed the 100 shortest elementary flux modes producing glucose 6-phosphate from acetyl-CoA using a previously described method [33] . This method allows one to enumerate elementary flux modes with increasing number of reactions . The entire set of elementary flux modes cannot be computed for the genome-scale metabolic network of humans since their number is too large [32] . For the computation we set the metabolites H2O , CO2 , Pi , Ppi , protons , O2 , ATP , ADP , AMP , GTP , GDP , NADH , NAD+ , NADPH , NADP+ , coenzyme A and HCO3+ to external status to obtain elementary flux modes differing in the underlying carbon conversion pathway rather than in co-factor balancing reactions . We calculated the Gibbs free-energy changes of the described pathways . We performed these calculations for two scenarios: gluconeogenesis from acetyl-CoA and from palmitate . For the first scenario , we assumed that additionally to the energetic balance as depicted in Fig . 6 four moles of acetyl-CoA are converted into one mole of glucose , four moles of coenzyme A and two moles of carbon dioxide . For the second scenario , we assumed that biosynthesis starts from 0 . 5 moles of palmitate yielding an additional 3 . 5 moles of NADH as well as FADH and hydrolyzing one mole of ATP through β-oxidation of fatty acids . We calculated the Gibbs free energy change bywhere corresponds to the Gibbs free energy of formation , to the universal gas constant , to the temperature , to the stoichiometric coefficient of the th product and to the stoichiometric coefficient of the th substrate of the pathway . We assumed a pH-value of 7 . 2 and a temperature of 37°C . We used measured Gibbs free-energy of formation when available [50] , [51] and estimated values otherwise [52] . For concentrations we used measured values if they were available . The corresponding values and references are given in Text S1 . | That sugar can be converted into fatty acids in humans is a well-known fact . The question whether the reverse direction , i . e . , gluconeogenesis from fatty acids , is also feasible has been a topic of intense debate since the end of the 19th century . With the discovery of the glyoxylate shunt that allows this conversion in some bacteria , plants , fungi and nematodes it has been considered infeasible in humans since the corresponding enzymes could not be detected . However , by this finding only a single route for gluconeogenesis from fatty acids has been ruled out . To address the question whether there might exist alternative routes in humans we searched for gluconeogenic routes from fatty acids in a metabolic network comprising all reactions known to take place in humans . Thus , we were able to identify several pathways showing that this conversion is indeed feasible . Analyzing evidence concerning the detected pathways lends support to their importance during times of starvation , fasting , carbohydrate reduced and ketogenic diets and other situations in which the nutrition is low on carbohydrates . Moreover , the energetic investment required for this pathway can help to explain the particular efficiency of carbohydrate reduced and ketogenic diets such as the Atkins diet . | [
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] | 2011 | In Silico Evidence for Gluconeogenesis from Fatty Acids in Humans |
The paramyxovirus matrix ( M ) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane . Transient nuclear residence of some M proteins hints at non-structural roles . However , little is known regarding the mechanisms that regulate the nuclear sojourn . Previously , we found that the nuclear-cytoplasmic trafficking of Nipah virus M ( NiV-M ) is a prerequisite for budding , and is regulated by a bipartite nuclear localization signal ( NLSbp ) , a leucine-rich nuclear export signal ( NES ) , and monoubiquitination of the K258 residue within the NLSbp itself ( NLSbp-lysine ) . To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins , we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera . Using quantitative 3D confocal microscopy , we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus , Hendra virus , Sendai virus , and Mumps virus . Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine , which inhibits M ubiquitination , also results in nuclear and nucleolar retention of these M proteins . Recombinant Sendai virus ( rSeV-eGFP ) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type . However , foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type , and infection did not spread . Recombinant Mumps virus ( rMuV-eGFP ) bearing the homologous mutations showed similar defects in viral morphogenesis . Finally , shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex , nuclear transport receptors , and nucleolar proteins . We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype .
Paramyxoviruses include pathogens of global medical and agricultural concern . These viruses occupy broad ecological niches infecting a wide range of hosts including mammals , reptiles , birds and fish , and they cause diverse outcomes ranging from asymptomatic infection to lethal disease . Measles virus ( MeV ) , mumps virus ( MuV ) , the human parainfluenza viruses ( hPIVs ) , respiratory syncytial virus ( RSV ) , and human metapneumoviruses remain significant causes of human morbidity and mortality [1] . Animal pathogens , such as Newcastle disease virus ( NDV ) and the recently eradicated Rinderpest virus [2] , have caused significant rates of lethal disease in birds and cattle , respectively . The newly emergent zoonotic paramyxoviruses Nipah virus ( NiV ) and Hendra virus ( HeV ) are among the most deadly known pathogens , showing case-fatality rates in excess of 70% in humans , and are classified as biosafety level 4 pathogens due to the absence of vaccines or therapeutics approved for human use [3–6] . Paramyxoviruses are released as enveloped virions from the host cell plasma membrane . Virions are ~150–300 nm in diameter and are spherical , pleomorphic or filamentous in shape , depending on the virus and the producer cell-type . The non-segmented , single-strand , negative-sense RNA genomes of paramyxoviruses consist of six principal genes: nucleocapsid ( N ) , phosphoprotein ( P ) , matrix ( M ) , fusion ( F ) and attachment ( HN , H or G ) glycoproteins , and polymerase ( L ) [1 , 5 , 7] . The attachment and fusion glycoproteins mediate binding to sialic acid moieties or to specific protein receptors on the cell surface and the fusion of the viral envelope with the host cell plasma membrane [8–10] . Within the virion , the ribonucleoprotein ( RNP ) consists of the RNA-dependent RNA polymerase complex formed by P and L associated with the N-encapsidated RNA genome . L is required for viral RNA synthesis during viral replication [1 , 5] . M is the primary viral structural protein [1 , 5 , 7] . A number of studies have found that M proteins oligomerize , bind lipids , and form a grid-like array on the inner surface of the viral membrane ( _ ( ( ( ( xxx ) ) ) ) _ ) [7 , 11–25] . M proteins can serve as a molecular scaffold by interacting with the cytoplasmic tails of the transmembrane glycoproteins and the RNP via N [7 , 17 , 25–35] . Many paramyxoviral M proteins ( NiV-M , MeV-M , NDV-M , SeV-M , and hPIV1-M ) can drive viral budding and form virus-like particles ( VLPs ) in the absence of other viral components [13 , 31 , 36–42] , albeit with varying efficiencies . However , the budding of some others ( PIV5-M and MuV-M ) requires coexpression of N and/or the envelope glycoproteins [43 , 44] . MeV and SeV engineered with budding-defective or deleted M proteins have been found to have severe defects in viral replication [45–47] . Although paramyxoviruses are classic cytoplasmic replicating viruses , some paramyxoviral M proteins have been observed to traffic through the nucleus . For example , SeV-M , NDV-M and RSV-M can be detected in the nucleus at early stages of infection [48–53] . These findings suggest that paramyxoviral M proteins may perform roles beyond viral assembly at the plasma membrane . However , with the exception of RSV , which belongs to the Pneumovirinae subfamily , the cell biology of M protein nuclear trafficking has not been examined in a systematic fashion for most Paramyxovirinae subfamily members . We previously found that NiV-M translocates to the nucleus at early stages of infection . The high homology between NiV-M and HeV-M ( ~90% amino acid identity ) suggests that HeV-M also localizes to the nucleus , and it was recently found that overexpression of ANP32B , a nuclear protein , results in nuclear accumulation of HeV-M and NiV-M [54] . We have shown that nuclear-cytoplasmic trafficking of NiV-M is mediated by a classical bipartite nuclear localization signal ( NLSbp ) , homologous to NDV-M’s NLSbp , and a leucine-rich nuclear export signal ( NES ) [39 , 48] . We further demonstrated that nuclear trafficking is regulated by ubiquitination , presumably on a conserved lysine residue ( K258 ) located within the NLSbp of NiV-M ( 244RR-X10-RRK258 ) . The K258A mutant is defective in nuclear import , while the K258R mutant retains a functional NLS but is defective in nuclear export; both mutants have decreased levels of ubiquitination and have budding defects [39] . The canonical NES and NLSbp that we functionally characterized in NiV-M are highly conserved across most , if not all members of the Paramyxovirinae . Therefore , it is important to resolve whether ubiquitin-dependent nuclear-cytoplasmic trafficking of M is unique to NiV , or to what extent other members of the subfamily also exhibit a nuclear-trafficking phenotype . Uncovering the mechanisms that govern paramyxovirus M protein trafficking has direct bearing on the fundamental biology of paramyxoviral replication , and may reveal host-dependent pathways and factors that can be exploited for antiviral strategies . Here , we specifically analyze ubiquitin-dependent nuclear-cytoplasmic trafficking of M proteins across representative viruses from all five major genera of Paramyxovirinae ( Respirovirus , Rubulavirus , Morbillivirus , Henipavirus , and Avulavirus ) . We use a panoply of methods including quantitative 3D confocal microscopy analysis of M nuclear localization , bimolecular fluorescence complementation ( BiFC ) assays of M ubiquitination , and introduction of M mutations into live recombinant viruses with the use of reverse genetics . Our findings demonstrate that ubiquitination of M , regulated by a lysine within the second basic patch of the NLSbp , critically modulates the subnuclear and nuclear-cytoplasmic trafficking of M proteins from prototypic viruses of the Henipavirus , Rubulavirus and Respirovirus genera . Proteomic identification of nuclear transport receptors and nuclear pore complex components that copurify with paramyxoviral M proteins further supports a model for active transport of M in and out of the nucleus , and also hints at possible non-structural functions of M proteins .
Since the nuclear-cytoplasmic trafficking of the Nipah virus matrix protein ( NiV-M ) is regulated by its monoubiquitination [39] , we wondered whether the ubiquitin-proteasome system similarly regulates the nuclear sojourn of other Paramyxovirinae M proteins . We cloned 3X-Flag- and GFP-tagged-M from prototypical members of the five Paramyxovirinae genera: NiV-M ( genus Henipavirus ) , Hendra virus M ( HeV-M , genus Henipavirus ) , Sendai virus M ( SeV-M , genus Respirovirus ) , Mumps virus M ( MuV-M , genus Rubulavirus ) , Newcastle disease virus M ( NDV-M , genus Avulavirus ) , and Measles virus M ( MeV-M , genus Morbillivirus ) . To biochemically detect ubiquitination of M proteins , we cotransfected HEK 293T cells with HA-UbK0 and each of 3X-Flag-tagged NiV-M , HeV-M , SeV-M , MuV-M , NDV-M or MeV-M [55] . HA-UbK0 functions as a ubiquitin ( Ub ) chain terminator or as monoubiquitin because all lysines have been mutated to arginines . We used this construct to visualize discrete ubiquitin bands and to determine if matrix proteins can be monoubiquitinated since this posttranslational modification can regulate the function of proteins without promoting proteasome-dependent protein degradation [56] . Cell lysates were subjected to anti-Flag immunoprecipitation ( IP ) and immunoblots were simultaneously probed with anti-HA and anti-Flag antibodies . As shown in Fig . 1A , for all the M proteins the majority of M is unmodified ( M0 ) at steady state . However , a detectable minority of M ( M1 ) is size-shifted by the molecular weight of at least one ubiquitin monomer ( Ub , ~8 . 5 kDa ) ( Fig . 1A , merge ) . These results indicate that all 3X-Flag-tagged M proteins investigated are ubiquitin substrates . We have found that proteasome inhibition results in nuclear retention of NiV-M in transfected and in NiV-infected cells ( S1 Fig . ) [39] . Proteasome inhibition stabilizes polyubiquitinated proteins and depletes the cellular levels of free ubiquitin available for conjugation [57–62] . To determine whether ubiquitination is involved in the nuclear export of the other Paramyxovirinae M proteins , we treated GFP-M-expressing HeLa cells with the proteasome inhibitor MG132 ( Fig . 1B-G ) . We used quantitative 3D confocal microscopy to characterize the subcellular localization of M . The cells were counterstained with DAPI to visualize nuclei , and with fluorescent phalloidin to visualize the entire cell , and the proportion of nuclear M was determined computationally as described in Materials and Methods . As with GFP-NiV-M , ubiquitin depletion via proteasome inhibition resulted in significant nuclear retention of GFP-tagged HeV-M , SeV-M and MuV-M ( Fig . 1B-E ) [39] . We further confirmed biochemically that MG132 reduces the direct conjugation of ubiquitin to 3X-Flag-tagged NiV-M , HeV-M , SeV-M , MuV-M , MeV-M and NDV-M by co-IP of each and HA-UbK0 , as described above , with quantification of immunoblot band integrated intensities as described in Materials and Methods ( S2 Fig . ) . The ubiquitination of the various M proteins were differentially sensitive to proteasome inhibition with NDV-M and MeV-M being the least and most sensitive to proteasome inhibition , respectively ( S2E–F Fig . ) . For NDV-M ( Avulavirus ) and MeV-M ( Morbillivirus ) , reduction of ubiquitin conjugation did not result in a nuclear retention phenotype under the conditions and cell type examined ( Fig . 1F-G ) , suggesting there is no strict correlation between the degree of matrix ubiquitination per se and M nuclear localization . In contrast , the ubiquitin-proteasome system appears to regulate the nuclear-cytoplasmic trafficking of the Henipavirus ( NiV-M , HeV-M ) , Respirovirus ( SeV-M ) , and Rubulavirus ( MuV-M ) matrix proteins . We have shown that the nuclear-export of NiV-M is regulated by a leucine-rich nuclear export signal ( NES ) as well as by the K258 lysine residue located within the second basic patch of the bipartite nuclear localization signal ( NLSbp; Fig . 2A , blue residues ) [39] . A K258A mutation partially disrupts the NLSbp and decreases nuclear localization of NiV-M , while a K258R mutation is unexpectedly retained in the nucleus despite having intact NES sequences and preservation of the positive charge necessary for NLSbp function . However , both mutants are impaired for ubiquitination [39] . Sequence alignment of M proteins indicates that a lysine is present in the homologously aligned position across the Paramyxovirinae genera . Thus , we hypothesized that this residue might be conserved for regulation of M ubiquitin-dependent nuclear export ( Fig . 2A , bold and underlined blue residues ) . To interrogate our hypothesis , we mutated the NLSbp-lysine to an arginine in all M proteins studied and analyzed their subcellular localization by quantitative 3D confocal microcopy as described above ( Fig . 2B-G , quantified in Fig . 2H ) . Since NDV-M contains another lysine adjacent to this position we mutated both ( Fig . 2A , bold and underlined blue residues ) . A lysine to arginine mutation is expected to preserve the nuclear import function of the putative NLSbp , but prevents posttranslational modification at that position . As a comparison for nuclear retention , we also mutated the leucines that correspond to the NES of NiV-M within all M proteins ( Fig . 2A , bold and underlined blue residues ) [39] . Mutation of the NES of GFP-NiV-M ( ML106A L107A ) resulted in a significant increase in nuclear localization of the protein , which confirms our previous findings ( Fig . 2B , 2H ) [39] . Similarly , but to varying degrees , GFP-tagged HeV-ML106A 107A , SeV-ML102A L103A , and MuV-ML106A also exhibited significantly increased nuclear retention compared to their respective wild type ( WT ) proteins ( Fig . 2C-2E , 2H ) . In contrast , GFP-NDV-ML103A L106A had an apparent nuclear exclusion phenotype ( Fig . 2F , 2H ) contrary to expectations , while the nuclear localization of GFP-MeV-ML90A 191A was not significantly different than WT ( Fig . 2G , 2H ) , indicating that these motifs are either redundant or non-functional in NDV-M and MeV-M . Mutation of the NLSbp-lysine resulted in significantly enhanced nuclear localization of GFP-tagged NiV-MK258R , HeV-MK258R , SeV-MK254R , MuV-MK261R , and NDV-MK259R K260R , but not MeV-MK240R ( Fig . 2B-2G ) . These phenotypic differences are quantified in Fig . 2H . Note that the spread in the degree of nuclear localization for any given M mutant also emphasizes the need to score a sufficient number of cells by computationally defined volumetric criteria ( see Materials and Methods ) in order to obtain robust statistics from inherently variable cell biological data . Thus , each data point in Fig . 2H ( as in Fig . 1B-G ) represents reconstructed volumetric data from a single cell , acquired from ~20–30 confocal optical Z-stacks ( at 0 . 3–0 . 5 μm/step ) per cell . In contrast to the NLSbp-lysine-to-arginine mutations , our attempt to disrupt the NLSbp consensus sequence through alanine substitutions in the second patch of basic residues ( bp2 ) resulted in diffuse cytoplasmic localization of GFP-tagged NiV-M , HeV-M , SeV-M , and MuV-M ( S3 Fig . ) . However , similar mutations did not appear to disrupt the localization of MeV-M or NDV-M ( S3 Fig . ) indicating that this motif does not function as the NLSbp in MeV-M or that additional mutations are necessary to fully disrupt the function of the NLSbp as has been previously shown for NDV-M [48] . Since the proteasome inhibitor MG132 inhibits the nuclear export of GFP-tagged NiV-M , HeV-M , SeV-M , and MuV-M ( Fig . 1 ) , we wanted to test whether the NLSbp-lysine that regulates their nuclear export ( Fig . 2B-E ) also regulates their ubiquitination . In Fig . 3 , we first assessed the ability of 3X-Flag-tagged NiV-MK258R , HeV-MK258R , SeV-MK254R , MuV-MK261R , MeV-MK240R and NDV-MK259R K260R to be ubiquitinated biochemically , via co-IP of 3X-Flag-tagged-M with HA-UbK0 , as described above ( Fig . 3A-D ) . Although NDV-M and MeV-M did not exhibit a ubiquitin-dependent nuclear trafficking phenotype ( Fig . 1F-G ) , we included NDV-MK259R K260R and MeV-MK240R in this experiment since the ubiquitin conjugation of WT NDV-M and MeV-M was sensitive to MG132 inhibition , albeit to varying degrees ( S2E–F Fig . ) . We controlled for protein abundance by normalizing the integrated intensity of the Ub band by the integrated intensity of the total M ( Ub/M0+M1 ) . Using this measure , 3X-Flag-tagged NiV-MK258R ( Fig . 3A ) , HeV-MK258R ( Fig . 3B ) and MeV-MK240R ( Fig . 3E ) exhibited the greatest reduction in relative monoubiquitination compared to the WT proteins ( >70% ) , while 3X-Flag-tagged SeV-MK254R ( Fig . 3C ) and MuV-MK261R ( Fig . 3D ) showed only a modest to mild impairment in monoubiquitination ( 36% and ~15% reduction , respectively ) . 3X-Flag-tagged NDV-MK259R K260R did not display reduced ubiquitination ( Fig . 3F ) . Residual ubiquitination of the matrix mutants indicates that other lysines within the 3X-Flag-tagged M proteins are also targets of ubiquitin conjugation . Table 1 summarizes the results obtained thus far: although ubiquitinated species can be detected for all six matrix proteins examined ( Fig . 1A , S2 Fig . ) , only NiV-M , HeV-M , SeV-M , and , MuV-M displayed a ubiquitin-dependent nuclear-cytoplasmic trafficking phenotype ( Fig . 1B-1E , S2B–S2E Fig . ) that was also dependent on a lysine in the NLSbp ( Fig . 2B-2E ) . Ubiquitination is a dynamic process determined in part by the rates of conjugation versus de-conjugation , but our co-IP and immunoblot analysis of HA-UbK0-modified 3X-Flag-tagged M is a steady-state assay . It is possible that this assay for ubiquitinated M might not efficiently detect subtle differences that arise from such dynamic processes since i ) ubiquitinated 3X-Flag-tagged M proteins are of low stoichiometry relative to unmodified native protein , ii ) 3X-Flag-tagged M proteins might be mono- and/or polyubiquitinated on multiple lysines , further obscuring a contribution of any single lysine to the sum total ubiquitination , iii ) ubiquitination is reversible , and iv ) HA-UbK0 must compete with endogenous ubiquitin . In an attempt to overcome these issues , and to further assess whether NiV-MK258R , HeV-MK258R , SeV-MK254R and MuV-MK261R are impaired for ubiquitination , we developed a bimolecular fluorescence conjugation ( BiFC ) ubiquitination assay in which ubiquitin-conjugation of M produces an irreversible fluorescence signal ( S4 Fig . ) [63 , 64] . We fused Ub and M to split N- and C-terminal fragments of the fluorescent protein Venus , VN173 and VC155 , respectively . Covalent conjugation of Ub to M brings the spilt Venus fragments into close proximity and allows the two otherwise non-fluorescent Venus fragments to reconstitute a functional fluorophore [65 , 66] . This complemented Venus will remain associated with M ( via VC155-M ) even if the VN173-Ub moiety is subsequently cleaved from M by a deubiquitinating enzyme ( DUB ) , preserving an atemporal record of ubiquitin conjugation ( S4A Fig . ) . Analysis of total cellular fluorescence showed that the Ub-M BiFC signal was decreased by almost 80% for the Henipavirus-M K258R mutants , confirming the significant role of K258 in ubiquitination of NiV-M ( Fig . 3A , Fig . 4A ) and HeV-M ( Fig . 3B , Fig . 4B ) . In addition , we determined that the Ub-M BiFC signals for SeV-MK254R ( Fig . 4C ) and MuV-MK261R ( Fig . 4D ) were also significantly decreased in BiFC signal by nearly 70% compared to the WT proteins . We observed a punctate localization of GFP-tagged NiV-M , HeV-M , SeV-M , and MuV-M within DNA-void regions of the nucleus when cells were treated with MG132 ( Fig . 1B-E ) . Native untagged NiV-M also exhibited similar subnuclear localization in NiV infected cells treated with bortezomib , an FDA-approved proteasome inhibitor ( S1 Fig . ) . We determined that MG132 redistributes GFP-tagged NiV-M , HeV-M , SeV-M and MuV-M to nucleoli by counterstaining cells with anti-nucleolin antibodies ( Fig . 5A-D , second vertical panels ) . Similarly , treatment of cells with MG132 caused a significant increase in the nucleolar localization of SeV-M during infection with live eGFP-expressing recombinant Sendai virus ( rSeV-eGFP ) . This rSeV-eGFP is derived from a Fushimi strain engineered with mutations that permit replication in mammalian cells without the addition of trypsin as described in Materials and Methods ( Fig . 6 ) [67] . The nucleolar localization of M proteins during ubiquitin depletion predicts that mutations in M that prevent efficient ubiquitination would also cause nucleolar retention . Indeed , GFP-tagged NiV-MK258R , HeV-MK258R , SeV-MK254R and MuV-MK261R phenocopied the MG132-induced nucleolar localization of the WT proteins ( Fig . 5A-D , compare the second and third vertical panels ) . In contrast , the nuclear localized NES mutants , GFP-tagged NiV-ML106A 107A , HeV-ML106A 107A , SeV-ML102A L103A , and MuV-ML106A , were primarily enriched within the nucleoplasm and not the nucleolus . Thus , NES mutants are stalled at a different stage of subnuclear trafficking compared to the NLSbp-lysine mutants ( Fig . 5A-D , fourth vertical panels ) . In sum , for the cognate paramyxovirus matrix proteins that exhibit a consistent nuclear trafficking phenotype that is both ubiquitin- and motif-dependent , our data supports a model where proper matrix ubiquitination is required for efficient nucleolar exit and/or preventing retention in the nucleolus . We previously determined that NiV-ML106A 107A and NiV-MK258R are defective at budding virus like particles ( VLPs ) [39] . We wanted to compare the effects of the corresponding mutations in 3X-Flag-tagged SeV-M or MuV-M , however these proteins have a poor budding efficiency that is less than 10% of 3X-Flag-tagged Henipavirus-M proteins ( Fig . 7A ) . This may be due to the presence of the 3X-Flag-tag or to the fact that SeV-M and MuV-M do not efficiently bud VLPs without the support of other viral proteins [41–43] . To overcome these technical difficulties and to study these mutations in a biologically relevant context , we engineered SeV-ML102A L103A and SeV-MK254R into a recombinant T7-driven , GFP-expressing Sendai virus genome ( rSeV-eGFP ) that can be rescued as live virus via the cotransfection of support plasmids expressing N , P , L ( comprising the necessary replication complex ) and a codon-optimized T7 polymerase . This highly efficient reverse genetics system allows us to quantify the number of rescue events directly in transfected producer cells at early time-points ( see Materials and Methods ) . At two days post-transfection , GFP-positive cells ( rescue events ) could be observed by epifluorescence and quantified by FACS analysis . As a control for background GFP expression in the absence of virus production , we found that cotransfection of WT rSeV-eGFP and T7 polymerase without the N , P and L support plasmids resulted in no GFP-positive cells . We determined that rSeV-eGFP-ML102A L103A , and rSeV-eGFP-MK254R rescued at similar if not higher efficiencies than rSeV-eGFP-MWT ( Fig . 7B ) . However , only rSeV-eGFP-MWT produced infectious viral titers ( ~107 I . U . /ml ) at day 6 post-rescue , while the mutants did not produce detectible infectious virus ( <10 I . U . /ml ) ( Fig . 7C ) . To determine the nature of the defect in viral replication , we counterstained the viral rescue cells with anti-SeV-M or anti-SeV-F antibodies and analyzed them by 3D confocal microscopy . By day 6 post-rescue of rSeV-eGFP-MWT , infection has spread to all cells without evidence of cell-cell fusion ( Fig . 7D ) . It is known that SeV replication in cell culture does not result in cell-cell fusion [45 , 47 , 68] , an unusual phenotype as most paramyxovirus infections result in extensive cell-cell fusion ( e . g . see S1 Fig . for NiV ) . Interestingly , although the rSeV-eGFP-ML102A L103A and rSeV-eGFP-MK254R rescue cells did not produce infectious virus , the GFP-positive rescue cells did initiate the formation of large foci of fused cells ( Fig . 7D , second and third horizontal panels ) . Virus-cell and cell-cell fusion require the presence of F and HN [9 , 10] , and we confirmed that SeV-F is expressed on rSeV-eGFP-ML102A L103A and rSeV-eGFP-MK254R foci ( Fig . 7E ) . Recombinant MuV-eGFP genomes engineered with M nuclear export mutants were also unable to efficiently spread beyond the fused cells formed at sites of rescue ( S5 Fig . ) . These data indicate that proper M nuclear-cytoplasmic trafficking is necessary for viral morphogenesis . To determine the nuclear localization of SeV-M , we counterstained rSeV-eGFP rescue cells with DAPI to visualize nuclei and anti-fibrillarin antibodies to visualize nucleoli . SeV-MWT was primarily extranuclear at the cell periphery ( Fig . 7D , 7F ) . SeV-ML102A L103A did not have an obvious nuclear localization in the viral context , although intracellular inclusions were apparent , suggesting that this mutant nonetheless had an altered localization ( Fig . 7F ) . SeV-MK254R , on the other hand , was strongly nuclear and enriched within the nucleoli ( Fig . 7D , 7F ) . These results are consistent with the previous transient transfection experiments in which SeV-MK254R was also more strongly localized to the nucleus than SeV-ML102A L103A ( Fig . 7H ) . Thus , these live virus results support our model that ubiquitination regulates the nuclear and subnuclear trafficking of SeV-M . Having characterized determinants of Paramyxovirinae M nuclear-cytoplasmic trafficking encoded within some M proteins , we turned to identifying potential cellular regulators of this process . We generated inducible 3X-Flag-M-expressing stable HEK 293 cell lines to efficiently copurify M-interacting proteins and analyzed their composition using multidimensional protein identification technology ( MudPIT ) as described in Materials and Methods ( S1–S4 Tables ) . We opted to determine the protein interactomes of NiV-M , HeV-M , SeV-M and NDV-M since these are the Paramyxovirinae M proteins with confirmed nuclear trafficking during live virus infection ( Fig . 6 , Fig . 7 , S10 Fig . ) [39 , 48 , 51–54 , 69] , and because these proteins cover the widest range of sequence homology to NiV-M: ~90% amino acid identity for HeV-M , ~37% amino acid identity for SeV-M , and ~20% amino acid identity for NDV-M . S6 Fig . shows our experimental schema and stringent filtering that resulted in our list of putative M protein interactors detailed below and listed in S1–S4 Tables . Nonspecific interactions in MudPIT analyses tend to be independent of the bait of interest . Rather , they are background contaminants related to the cell type and the affinity purification scheme [70] . To remove background contaminants , our putative M interactomes represent only those proteins identified in the sample purifications that are absent in 3 independent negative-control purifications using lysates from the parental/isogenic Flp-In T-REx-293 cells , irrespective of relative abundances ( S6 Fig . , Worksheet 1 in S1–S4 Tables ) . As an independent confirmation of stringency , comparison of the putative NiV-M , HeV-M , SeV-M , and NDV-M interactomes to 21 relevant control experiments in the mass spectrometry contaminant repository , CRAPome , revealed relatively few additional proteins that are common sources of contamination ( S6 Fig . , Worksheet 2 in S1–S4 Tables ) [70] . These were primarily actins , tubulins , histones and ribosomal proteins , which are also the most common contaminants across the entire CRAPome ( Worksheet 2 in S1–S4 Tables ) [70] . During manuscript revisions , another group published the identification of ~130 HeV-M-interacting proteins using affinity-purification , in-gel digestion and mass spectrometric identification , and further characterized AP3B1 as a Henipavirus M interactor that regulates VLP production [71] . A majority of their proteins either went undetected by our global analyses or were excluded as background contaminants because they were present in the control purifications . For the purpose of comparison , the proteins in their paper that are also present in our HeV-M and/or NiV-M interactomes , excluding some ribosomal proteins , are KRI1 , RFC1 , FAM120A , SMC1A , SART3 , UPF1 , Nat10 , Smarca5 , UTP14A , POP1 , ZC3HAV1 , RAD18 , AP3D1 , USP7 , Tat-SF1 , SKIV2L2 , PARP-1 , and Importin-7 ( Worksheet 1 in S1 and S2 Tables ) . Other than PARP-1 , these proteins were unlikely to be present as contaminants in the 21 relevant CRAPome control experiments . We noted that the E3 ubiquitin ligase RAD18 was not present as a contaminant in any of the 21 CRAPome control experiments and was the least likely to be encountered in the entire CRAPome database; it was found in only 4 of 411 experiments with an average of only 1 . 3 spectra per experiment , whereas we measured 11 unique spectra ( 22 . 6% coverage ) in the NiV-M affinity purification [70] . We confirmed the interaction of NiV-M with RAD18 by co-IP and immunoblot analysis ( S7A Fig . ) , indicating that a combination of experimental and computational approaches to background contaminant subtraction can facilitate the identification and characterization of bona fide protein-protein interactions ( S6 Fig . ) . Other ubiquitin ligases identified in our proteomics experiments ( UBE2O and Cullin ring ligases ) were also confirmed for copurification with M proteins by co-IP and immunoblot analysis ( S7B–C Fig . ) . Confident that our putative M interactomes were largely reflective of true protein-protein interactions , we further analyzed the interactomes bioinformatically and biochemically ( Fig . 8 , S7–S9 Fig . ) . Comparisons of our putative NiV-M , HeV-M , SeV-M , and NDV-M interactomes to one another revealed significant overlap; over 60% of the proteins found in any single interactome were also found in the interactomes of one or more of the other three ( Fig . 8A ) . Furthermore , we identified 178 proteins common to all M interactomes , the majority of which are not present in the 21 historical control experiments ( Worksheet 3 in S4 Table ) . This common set of proteins represents 24–48% of all the proteins in any single viral M interactome ( Fig . 8A , S1–S4 Tables ) . Interestingly , proteins associated with the nuclear pore complex were significantly enriched within individual M interactomes as well as the subset of common interacting proteins ( Fig . 8B , -log10 ( p-value ) >10 ) . These include nuclear pore complex components ( RanBP2 , Nup37 , Nup93 , Nup107 , Nup155 , Nup205 , Sec13 , Seh1 ) , nuclear transport receptors ( NTRs ) required for nuclear import of proteins ( α/β-importins ) , nuclear export of proteins ( Exp1/CRM1 , Exp2 ) , nuclear export of dsRNA/dsRNA-binding proteins ( Exp5 ) , nuclear export of tRNA ( Exportin-T ) , nuclear export of mRNA ( Rae1 ) , and a regulator of the RanGTP/GDP cycle that modulates the association/dissociation of cargo with NTRs ( RanGAP1 ) ( Fig . 8C , Worksheet 1 in S1–S4 Tables ) . All of these proteins were unlikely background contaminants ( Worksheet 2 in S1–S4 Tables ) and we confirmed that NiV-M interacts with α-importins and Exp1/CRM1 ( S8 Fig . ) . Thus Paramyxovirinae M proteins interact with a highly interconnected network of proteins necessary for transport of NLS and NES containing cargo proteins across the nuclear pore . Our cell biological and proteomic findings are synthesized into a working model for ubiquitin-regulated nuclear-cytoplasmic trafficking of M proteins shown in Fig . 8D . Consistent with the nucleolar transit phase exhibited by the Paramyxovirinae M proteins under study , the M interactomes also revealed a significant enrichment of resident or transient nucleolar proteins ( Fig . 8E and 8F ) . One of these , the RNA polymerase I transcription factor UBF-1 ( UBTF ) had abundant spectral counts in the SeV-M and NiV-M interactomes but was essentially absent in the matched CRAPome control experiments ( S1 Table and S3 Table ) . We determined that UBF-1 sequesters NiV-M in the nucleus and inhibits NiV-M budding when overexpressed ( S9 Fig . ) . Thus , our proteomic and functional data indicate that M proteins interact with an array of nuclear and nucleolar proteins , at least some of which can modulate M nuclear-cytoplasmic trafficking .
Whether or not they replicate in the nucleus , many viruses are known to target , modify , and hijack nuclear components and nuclear functions to promote the infectious life cycle . It is generally thought that paramyxoviruses replicate in the cytosol without a nuclear stage . However , it is becoming increasingly clear that nuclear trafficking of M is shared by a number of paramyxoviruses . It was previously observed that SeV-M , NDV-M and RSV-M traffic through the nucleus [49 , 50 , 69] and a functional bipartite nuclear localization signal ( NLSbp ) has been defined within NDV-M [48] . Here , we show that the NLSbp of NDV-M is functionally conserved for nuclear import along with NiV-M , HeV-M , SeV-M and MuV-M ( Fig . 8D , S3 Fig . ) [39 , 48] . NLSs specify translocation through the nuclear pore through high-affinity interactions with importins , which in turn interact with cognate nuclear pore components on the cytoplasmic side [72] . In our proteomic analyses we identified numerous importins , exportins and nuclear pore complex components as common candidate interactors of NiV-M , HeV-M , SeV-M and NDV-M ( Fig . 8 , S8 Fig . , S1–S4 Tables ) . Thus , the size of Paramyxovirinae M proteins ( >40 kDa ) , the presence of a functional NLSbp within M proteins , and the interaction with nuclear transport receptors are strong evidence that the nuclear localization of M proteins is an active and regulated transport process . That the putative M interactomes show such a strong enrichment of proteins involved in nuclear-cytoplasmic transport also suggests that M proteins may antagonize the nuclear-cytoplasmic trafficking of host proteins and RNA to facilitate viral replication [73] . In addition to the NLSbp , we show that a leucine-rich NES sequence is functionally conserved within NiV-M , HeV-M , SeV-M and MuV-M ( Fig . 2 , Fig . 5 , Fig . 8D ) . We note that mutation of the corresponding region in NDV-M resulted in decreased nuclear localization . However , this sequence is not as well conserved as in the other M proteins ( Fig . 2A ) and a recent study identified other functional NES motifs within different regions of NDV-M [52] . Thus , Paramyxovirinae M proteins appear to have both shared and unique determinants of nuclear-cytoplasmic trafficking depending on their evolutionary heritage . We also acknowledge that our experimental system utilizing human cells may not fully recapitulate the regulation of NDV-M trafficking since NDV is an avian virus , while NiV , HeV , SeV , MuV and MeV are mammalian viruses . Nuclear export of NiV-M , HeV-M , SeV-M and MuV-M is also regulated by a lysine within the second basic patch of the NLSbp . Mutating this lysine to an arginine results in decreased ubiquitination and a nuclear retention phenotype that is phenocopied by pharmacological depletion of free ubiquitin with a proteasome inhibitor ( Fig . 1B-E , Fig . 2B-E , Fig . 5 , Fig . 6 ) . We hypothesize that mutation of this lysine prevents its ubiquitination . Alternately , mutation of this lysine may prevent the ubiquitination of a nearby lysine within the NLSbp by preventing the interaction of M with a ubiquitin ligase . Whether ubiquitination regulates MeV-M or NDV-M function ( s ) remains indeterminate . The subcellular localization phenotypes of the NES and the various NLSbp mutants for NDV-M and MeV-M are not congruent ( compare S3 Fig . and Fig . 2F-G ) . Furthermore , NDV-M and MeV-M also exhibit divergent sensitivity with regards to ubiquitin conjugation ( S2E–F Fig . ) , and neither were found to be sensitive to MG132 induced nuclear retention ( Fig . 1F-G ) . Altogether , our results suggest that the degree of the nuclear trafficking phenotypes of transfected M proteins mutated at the putative NES , the putative NLSbp , or the homologously aligned lysine within the NLSbp , are strongest for the Henipavirus M proteins , moderate for SeV-M and MuV-M , variable for NDV-M , and inconclusive/absent for MeV-M under the cell type and conditions examined ( Table 1 ) . Although we did not find conclusive evidence for ubiquitination of NDV-M on the lysine that corresponds to the Henipavirus M K258 , it is probable that NDV-M is a biological target of posttranslational modification on other lysines within the NLSbp . Our proteomic analysis detected a peptide from the NLSbp of NDV-M containing 114 . 0492 Da mass signatures indicative of the vestigial diglycine of ubiquitin that remains attached to the modified lysine ( K[114 . 04292] ) after trypsin cleavage ( 249GKK[114 . 04292]VTFDK[114 . 04292]LEKKIRSLDLSVGLSDVLGPSVLVK281 ) [74] . How could NLS ubiquitination regulate nuclear trafficking ? Protein import into the nucleus is regulated by the affinity of importins for cargo NLSs , which can be modulated by intermolecular or intramolecular masking of the NLS itself [75 , 76] . For example , ubiquitination of the NLS of p53 by MDM2 has been shown to block p53 nuclear import by preventing the binding of importin-α3 [77] . It was recently shown that the ubiquitin-conjugating enzyme UBE2O multi-monoubiquitinates tumor suppressor BAP1 on its NLSbp to promote cytoplasmic localization . It was further determined that UBE2O specifically binds and ubiquitinates a number of similar bipartite NLSs within nuclear trafficking proteins known to regulate RNA processing , transcription , DNA replication , and chromatin remodeling [78] . We hypothesize that ubiquitination of M on a lysine within the NLSbp itself prevents importin binding as a means to prevent nuclear re-entry once the protein has completed its nuclear sojourn ( Fig . 8D , S8A Fig . ) . Given this model for ubiquitin-dependent nuclear-cytoplasmic trafficking , it is possible that M utilizes a nuclear-resident E3 ubiquitin ligase [79] . Our proteomic analyses identified a number of candidate ubiquitin ligases that interact with M proteins including UBE2O , which was found within the NiV-M and NDV-M interactomes ( S7B Fig . , S1 Table , S4 Table ) . Further study of these ubiquitin ligases will help resolve the spatiotemporal dynamics of M ubiquitination vis-à-vis nuclear trafficking ( S7 Fig . ) . Nuclear-cytoplasmic trafficking is a prerequisite for M budding and viral morphogenesis . We previously showed that NiV-M mutants defective in either nuclear import or nuclear export were also defective at budding VLPs [39] . Alternately , overexpression of a nuclear NiV-M-interacting protein UBF-1 can sequester NiV-M in the nucleus and inhibits efficient budding of VLPs ( S9 Fig . ) . Other viruses have been reported to interact with UBF-1 and utilize or modify UBF-1 function . For example , UBF-1 is inactivated during Poliovirus infection as part of a viral strategy to inhibit host cell transcription globally [80] . The DNA viruses Adenovirus and HSV-1 co-opt UBF-1 into viral DNA replication centers , and it has been hypothesized that UBF-1 is used as a cofactor in viral DNA replication [81–83] . Finally , the SV40 large T antigen and the HCV NS5A protein stimulate RNA Pol I transcriptional activity and enhance rRNA synthesis by hyperphosphorylation of UBF-1 [84 , 85] . Such rRNA transcriptional activation is thought to contribute to the cell transformation caused by these tumorigenic viruses . For NiV , even though NiV-M clearly interacts with overexpressed UBF-1 , and is colocalized with UBF-1 in the nucleus , it is unclear whether NiV utilizes UBF-1 to benefit viral replication under physiological expression levels and conditions ( S9 Fig . ) . Nonetheless , the inhibitory effects of nuclear UBF-1 on VLP budding supports the model that functional trafficking to the plasma membrane requires properly regulated nuclear import and export ( Fig . 8D ) . Here , we also engineered M nuclear export mutants into recombinant SeV . rSeV-eGFP-ML102A L103A or rSeV-eGFP-MK254R were completely attenuated for production of infectious virus , but formed large foci of fused cells at sites of viral rescue . Moreover , nuclear localization of SeV-MK254R , the ubiquitination mutant , was observed in both virus rescue and transient transfection experiments ( Fig . 2D , Fig . 5C , Fig . 7F ) . It is known that mutations that abrogate the interaction of M with the glycoproteins , including M deletion , can increase cell-cell fusion in SeV and MeV , while mutations that enhance their interaction can decrease cell-cell fusion [33 , 34 , 45–47 , 68] . Since M and F proteins were expressed in the foci of fused cells , our results indicate that rSeV-eGFP-ML102A L103A and rSeV-eGFP-MK254R are defective in proper assembly of viral components at the plasma membrane rather than in expression of viral components necessary for budding per se . The link between viral replication and M ubiquitin-dependent nuclear-cytoplasmic trafficking may explain why proteasome inhibitors that deplete free cellular pools of ubiquitin have been found to inhibit SeV and NiV replication [39 , 86] . Beyond regulating nuclear import/export itself , we previously found that ubiquitination of NiV-M is necessary for membrane targeting and budding [39] . It is possible that the ubiquitination of M proteins promotes recognition by cellular factors such as ESCRT complexes known to mediate transport and budding of many enveloped viruses [56 , 87 , 88] , especially in light of known sequence motifs in PIV5-M , SeV-M and MuV-M that can bind ESCRT complex components [27 , 43 , 89] . The status of M ubiquitination may also regulate the interactions of M with cellular factors inside the nucleus and within subnuclear compartments such as the nucleolus . A number of cellular proteins become enriched in the nucleolus upon proteasome inhibition , including p53 [90–99] . Similarly , pharmacological or genetic inhibition of NiV-M , HeV-M , SeV-M , and MuV-M ubiquitination sequesters these proteins in the nucleolus ( Fig . 5 , Fig . 6 ) , and nucleolar localization of SeV-MK254R was also confirmed in the context of rSeV-eGFP rescue ( Fig . 7F ) . Nucleolar localization of M proteins is a natural feature of the nuclear sojourn of some M proteins , with M enriched at nucleoli during the early stage of live NiV and NDV infections ( S10 Fig . ) [69 , 100] . Although M proteins do not have an evident nucleolar localization signal ( NoLS ) that would have predicted this observation [101] , our proteomics experiments suggest that Paramyxovirinae M proteins can interact with a number of nucleolar hub proteins ( Fig . 8E and F , S1–S4 Tables ) [102] . Most , if not all , viral families interact with the nucleolus , often to usurp cellular functions and promote viral replication [103–105] , as the nucleolus is a dynamic structure involved in a vast array of biological functions beyond ribosome biogenesis , including tRNA and mRNA processing and export from the nucleus , cell cycle regulation , and response to cellular stress . Additionally , there is growing recognition that NLS containing viral proteins target the nuclear pore complex to alter the export of macromolecules and mRNA , thereby counteracting antiviral responses and promoting viral gene expression at the expense of host gene expression [73] . For example , influenza NS1 is a multifunctional protein known to translocate to the nucleolus and to the nuclear pore where it inhibits host mRNA export factors resulting in impaired immune responses and enhanced viral virulence [106] . Vesicular stomatitis virus M also inhibits mRNA nuclear export through interaction with nuclear pore components [73 , 107] . Further , RSV-M is shuttled to the host cell nucleus where it inhibits host gene expression and induces cell cycle arrest , indicating that paramyxovirus M proteins also antagonize nuclear functions [108 , 109] . We hypothesize that ubiquitin-dependent nuclear and subnuclear trafficking of some Paramyxovirinae M proteins is part of a viral strategy to promote viral replication . Therefore , the study of M interactions with the nucleolus and the nuclear pore complex represents an opportunity to gain new insights into the cell biology of the nucleus and to identify novel antiviral targets .
HeLa , Vero , and HEK 293T cells were maintained at 37°C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) and 1% 100X penicillin/streptomycin solution ( Gibco/Life Technologies , Gaithersburg , MD ) . For confocal microscopy imaging , cells were seeded on 22 mm #1 . 5 coverglass coated with Collagen Type I ( BD Biosciences , San Jose , California ) . Cells were transfected using Lipofectamine LTX per the manufacturer’s instructions ( Invitrogen/Life Technologies ) . 3X-Flag-M Flp-In T-REx-293 cell lines , generated as described below , were maintained at 37°C in a 5% CO2 atmosphere in DMEM supplemented with 10% dialyzed FBS and 1% 100X penicillin/streptomycin solution . 3X-Flag-M protein expression was induced and immunoprecipitated as described below . 3X-Myc-tagged Cullin constructs ( Addgene plasmids 19896 , 19892 , 19893 , 19951 , 19922 , 19895 and 20695 ) are described in [110–113] . GFP-UBF-1 ( Addgene plasmid 17656 ) is described in [114] . Flag-tagged Karyopherin constructs were the kind gift of Dr . Christopher F . Basler ( Icahn School of Medicine at Mount Sinai , New York , NY ) . 3XFlag-CRM1 ( Addgene plasmid 17647 ) is described in [115] . The Myc-UBE2O construct was the kind gift of Dr . El Bachir Affar ( Maisonneuve-Rosemont Hospital Research Center , Department of Medicine , University of Montreal , Montreal ) and is described in [78] . HA-UbK0 ( Addgene; plasmid 17603; all lysines mutated to arginines ) is described in [55] . Codon optimization and cloning of untagged , 3X-Flag-tagged and 3X-Flag-GFP-tagged Nipah virus matrix ( NiV-M ) and generation of corresponding NiV-M mutants is described in [39] . We similarly codon optimized and cloned the open reading frames encoding M from Hendra virus ( HeV-M , genus Henipavirus ) , Sendai virus ( SeV-M , genus Respirovirus ) , Mumps virus ( MuV-M , genus Rubulavirus ) , and Measles virus ( MeV-M , genus Morbillivirus ) , and also a non-codon optimized Newcastle disease virus M ( NDV-M , genus Avulavirus ) : briefly , eGFP was fused to the N-terminus of M by overlap extension PCR ( OE-PCR ) . WT or GFP-fused HeV-M , SeV-M , RSV-M , MuV-M , and MeV-M were inserted within the HindIII and XhoI sites of pCMV-3Tag-1 , while NDV-M was inserted within HindIII and ApaI sites of pCMV-3Tag-1 ( Agilent Technologies , Santa Clara CA ) to generate 3X-Flag- and 3X-Flag-GFP-tagged-M constructs . Alignment of M sequences using Clustal Omega identified sequences motifs corresponding to NiV-M’s nuclear export sequence ( NES ) and bipartite nuclear localization sequence ( NLSbp ) [39 , 116] . Mutations were generated using the QuikChange II site-directed mutagenesis kit using PAGE-purified mutagenesis primers designed using the online QuikChange primer design tool ( Agilent Technologies ) . A RAD18 cDNA clone was purchased from Origene ( SC323786 ) . 3X-Flag-GFP-RAD18 was generated by replacement of the NiV-M insert in HindIII/XhoI digested 3X-Flag-NiV-M . The Flp-In T-REx system ( Invitrogen ) was used to generate doxycycline-inducible 3X-Flag-M cell lines . Codon-optimized 3X-Flag-tagged NiV-M , HeV-M , SeV-M , and NDV-M were inserted within the KpnI and XhoI sites of pcDNA5/FRT/TO . The constructs and pOG44 were cotransfected into Flp-In T-REx-293 cells , and stable cell lines were selected with hygromycin and blasticidin according to the manufacturer’s instructions . Constructs for bimolecular fluorescence complementation ( BiFC ) analyses were generated with split Venus residues 1–172 ( VN173 ) and 155–238 , A206K ( VC155 ) [65 , 117] . VN173 and VC155 were PCR amplified from pBiFC-VN173 ( Addgene plasmid 22010 ) and pBiFC-VC155 ( Addgene plasmid 22011 ) , and were fused to the N-termini of Ub and M proteins via a flexible linker encoding GGGGSGGGGGR by OE-PCR . VN173-Ub and VC155-M were inserted within the NotI and XhoI sites of pcDNA3 . 1 ( + ) ( Life Technologies ) . Mutations within VC155-M constructs were generated using the QuikChange II site-directed mutagenesis kit using PAGE-purified mutagenesis primers designed using the online QuikChange primer design tool ( Agilent Technologies ) . The recombinant Sendai virus ( rSeV ) anti-genome RGV0 , a Fushimi strain construct with F1-R strain mutations in F and M , and helper plasmids encoding SeV N , P and L were the kind gift of Dr . Nancy McQueen and are described in [67] . The encoded virus has the ability to replicate in mammalian cells without the addition of trypsin . We further modified the rSeV anti-genome construct by inserting an eGFP reporter flanked at the 3’ end by a unique NotI site between the N and P genes . A hammerhead ribozyme sequence was inserted between the optimal T7 promoter and the start of the anti-genome . Mutations were introduced into the SeV-M ORF by OE-PCR using primers containing the desired mutations , followed by insertion into NotI and AfeI sites in the parental rSeV-eGFP construct . The full-length construct encoding recombinant Mumps virus ( rMuV ) anti-genome of the Jeryl Lynn 5 ( JL5 ) vaccine strain and helper plasmids encoding MuV-JL5 N , P and L proteins were a kind gift from Dr . W . Paul Duprex and are described in [118] . We modified the rMuV anti-genome construct by inserting an eGFP reporter between the NP and P genes . A hammerhead ribozyme sequence was inserted between the optimal T7 promoter and the start of the anti-genome . Mutations were introduced into the MuV-M ORF by OE-PCR using primers containing the desired mutations , followed by insertion into SalI and SbfI sites in the parental rMuV construct . 3X-Flag-M Flp-In T-REx-293 cell lines were grown to ~80% confluency and induced for protein expression with 100 ng/mL doxycycline for 24h . Cells were washed three times in dPBS and lysed in 100 mM Tris-HCL pH 8 , 150 mM NaCL , 5 mM EDTA , 5% glycerol , 0 . 1% NP40 , complete protease cocktail ( Roche ) , PhosSTOP ( Roche ) and 25 mM N-ethylmaleimide . Cell lysate was clarified by centrifugation at >15 , 000×g for 15 min at 4°C and incubated with lysis buffer-equilibrated anti-Flag M2 affinity gel ( Sigma-Aldrich , St . Louis , MO ) for 2 hours at 4°C . The affinity gel was extensively washed with lysis buffer and then with elution buffer consisting of 100 mM Tris-HCL pH 8 , 150 mM NaCL , 5 mM EDTA , and 5% glycerol . Bound proteins were eluted from the affinity gel with elution buffer containing 3X-Flag peptide ( Sigma-Aldrich ) , were precipitated with trichloroacetic acid , washed with acetone twice , dried , and stored at -20°C until further processing . Protein samples were resuspended in 8M urea in 100 mM Tris pH 8 . 5 , reduced , alkylated and digested by the sequential addition of lys-C and trypsin proteases as previously described [119] . The digested peptide solution was fractionated online using strong-cation exchange and reverse phase chromatography and eluted directly into an LTQ-Orbitrap mass spectrometer ( Thermofisher ) [119 , 120] . MS/MS spectra were collected and subsequently analyzed using the ProLuCID and DTASelect algorithms [121 , 122] . Database searches were performed against a human database containing the relevant paramyxovirus M protein sequence . Protein and peptide identifications were further filtered with a false positive rate of less than 5% as estimated by a decoy database strategy [123] . Normalized spectral abundance factor ( NSAF ) values were calculated as described [124] . Proteins were considered candidate M-interacting proteins if they were identified in the relevant affinity purification but not present in 3 independent control purifications using lysates from the parental Flp-In T-REx-293 cells . Analysis of other potential background contaminants was performed using CRAPome [70] . Venn diagrams were generated using jvenn [125] . Gene-annotation enrichment analysis was performed using DAVID Bioinformatics Resources 6 . 7 [126 , 127] . Physical and predicted protein interaction networks were visualized using the GeneMANIA plugin for Cytoscape 3 . 1 [128 , 129] . Transfected HEK 293T cells were washed once in dPBS and lysed in 100 mM Tris-HCL pH 8 , 150 mM NaCL , 5 mM EDTA , 5% glycerol , 0 . 1% NP40 , complete protease cocktail ( Roche ) and 25 mM N-ethylmaleimide . The cell extract was clarified by centrifugation at >15 , 000×g for 15 min at 4°C before incubation overnight at 4°C with lysis buffer-equilibrated anti-Flag M2 affinity gel ( Sigma-Aldrich , St . Louis , MO ) . The affinity gel was extensively washed with lysis buffer and then with elution buffer consisting of 100 mM Tris-HCL pH 8 , 150 mM NaCL , 5 mM EDTA , and 5% glycerol . Bound proteins were eluted from the affinity gel with elution buffer containing 3X-Flag peptide ( Sigma-Aldrich ) , were subjected to SDS-PAGE and transferred to immobilon-FL PVDF membrane ( EMD Millipore , Billerica , MA ) . To analyze M ubiquitination by immunoblot , HEK 293T cells were cotransfected with 3X-Flag-M and HA-UbK0 for 24h and subjected to immunoprecipitation as described above . Membranes were simultaneously probed with mouse anti-Flag M2 primary antibodies ( Sigma-Aldrich ) and rabbit anti-HA primary antibodies ( Novus Biologicals , Littleton , CO ) followed by anti-mouse-680 and anti-rabbit-800 secondary antibodies ( LI-COR , Lincoln , Nebraska ) and imaged on an Odyssey infrared scanner ( LI-COR ) according to the manufacturer’s instructions . To quantify relative ubiquitination , the background subtracted integrated fluorescence intensities of the monoubiquitin bands ( Ub ) normalized to total M ( M0+M1 ) was determined using LI-COR Odyssey software . For 3D confocal microscopy analysis , transfected HeLa cells were treated with 50 μM MG132 or 0 . 5% DMSO at 16 h post-transfection for 8 hours , then fixed and processed for quantitative image analysis as described below . For immunoblot analysis of M ubiquitination during ubiquitin depletion , transfected HEK 293T cells were treated with 10 μM MG132 or 0 . 1% DMSO at 18 h post-transfection for 6 hours , and 3X-Flag-M was immunoprecipitated as described above . VLP budding assays were performed as described in [39] . Briefly , precleared supernatants from 3X-Flag-M transfected HEK 293T were ultracentrifuged through a 20% ( w/v ) sucrose at 36 , 000 rpm for 2 h at 4°C ( AH-650 rotor , Thermo Scientific ) . VLP pellets and cells were resuspended in lysis buffer and subjected to SDS-PAGE and anti-Flag immunoblotting . Relative integrated intensity of VLP/cell lysate bands were quantified and normalized relative to the budding of 3X-Flag-NiV-M . HeLa cells were infected with Nipah virus under biosafety level 4 ( BSL-4 ) conditions as described in [39] . For rescue of WT or mutant rSeV-eGFP , 2X106 HEK 293T cells were transfected with recombinant plasmid encoding the anti-genome ( 4 μg ) along with the cognate accessory plasmids encoding SeV NP ( 1 . 44 μg ) , P ( 0 . 77 μg ) , and L ( 0 . 07 μg ) , and a codon optimized T7 RNA polymerase ( 4 μg ) using Lipofectamine LTX ( 8 . 9 μL ) and Plus Reagent ( 5 . 5 μL ) , according to manufacturer’s instructions . Cells were harvested for FACS analysis at 48 hours post-transfection ( the earliest time point when GFP-positive cells can be observed by epifluorescence microscopy , yet when supernatant titer is still not detectable ) to quantify rescue efficiency . The number of GFP-positive cells ( rescue events ) was determined from 500 , 000 cells analyzed with a FACSCalibur Flow Cytometer ( BD Biosciences ) and FlowJo software ( TreeStar Inc . , Ashland , OR ) . Cells plated on coverslips were fixed at day 6 post-transfection for analysis of rescued virus infection by 3D confocal microscopy . Supernatant was collected from rescue cells at day 6 post-transfection for quantification of viral titers . Briefly , supernatant stored at -80°C was thawed on ice and serial diluted 2-fold in serum-free DMEM . 100 μL of each dilution was used to infect ~60 , 000 Vero cells in a 24-well plate for 1 hour . After 1 h , 500 μL of DMEM 10% FBS was added to each well and the cells were incubated at 37°C . Cells were harvested for FACS analysis at 24 h post infection and titers were calculated based on percent infection in the linear range of supernatant dilutions . For rescue of WT or mutant rMuV , 4X105 BSR-T7 cells were transfected with recombinant plasmid encoding the anti-genome ( 5 μg ) along with the cognate accessory plasmids encoding MuV NP ( 0 . 3 μg ) , P ( 0 . 1 μg ) , and L ( 0 . 2 μg ) , and a codon optimized T7 RNA polymerase ( 2 μg ) using Lipofectamine LTX ( 18 . 75 μL ) and Plus Reagent ( 7 . 5 μL ) , according to manufacturer’s instructions . Nipah virus-infected cells were fixed in 10% formalin solution for a minimum of 24 h prior to removal from the BSL-4 laboratory . For all other immunofluorescence microscopy , samples were fixed with 2% paraformaldehyde in 100 mM phosphate buffer ( pH 7 . 4 ) for 15 min . Fixed cells were permeabilized in blocking buffer containing PBS , 1% saponin , 3% bovine serum albumin , and 0 . 02% sodium azide . After incubation with antibodies/probes in blocking buffer , samples were extensively washed in blocking buffer and mounted on glass slides with Vectashield mounting medium with DAPI ( Vector Laboratories , Burlingame , California , United States ) . The samples were imaged with a Leica SP5 confocal microscope ( Leica Microsystems , Buffalo Grove , IL ) , acquiring optical Z-stacks of 0 . 3–0 . 5 μm steps . Z-stacks were reconstructed and analyzed in three dimensions using Volocity 5 . 5 software ( Perkin Elmer , Waltham , Massachusetts ) . Widefield microscopy was performed using a Cytation 3 Cell Imaging Multi-Mode Reader ( BioTek , Winooski , VT ) or a Nikon TE300 microscope . NiV-M was detected with rabbit anti-NiV-M antibodies ( 1:1000 ) [39] . SeV-M was detected with mouse anti-SeV-M ascites ( 1:200 ) , and SeV-F was detected with mouse anti-SeV-F ascites ( 1:200 ) kindly provided by Dr . Toru Takimoto [130] . Nucleoli were detected with mouse anti-nucleolin antibodies ( 1:500 ) ( Invitrogen/Life Technologies ) or rabbit anti-fibrillarin antibodies ( 1:500 ) ( Abcam , Cambridge , MA ) . Alexa-fluor conjugated Anti-IgG antibodies of appropriate species reactivity and fluorescence spectra were used for secondary detection ( 1:300–1:1000 ) ( Invitrogen/Life Technologies ) . F-actin was visualized by incubating samples with Alexa-fluor conjugated phalloidins ( 1:300 ) ( Invitrogen/Life Technologies ) . Immunoblots were imaged on an Odyssey infrared scanner ( LI-COR ) using secondary antibodies of appropriate species reactivity and fluorescence spectra ( LI-COR ) . An antibody to the C-terminus of GFP ( LS-C51736 , LifeSpan BioSciences , Inc . , Seattle , WA ) was used for immunoblot detection of VC155-fusion proteins . Anti-Flag M2 ( F3165 , Sigma-Aldrich ) was used to for immunoblot detection of Flag-tagged proteins . Anti-HA ( NB600–363 , Novus Biologicals , Littleton , CO ) was used for immunoblot detection HA-tagged proteins . Anti-Myc Tag , clone 4A6 ( 05–724 , Millipore , Temecula , CA ) was used for immunoblot detection of Myc-tagged proteins . Anti-UBE2O ( NBP1–03336 , Novus Biologicals ) was used for immunoblot detection of UBE2O . Anti-β-Tubulin ( T7816 , Sigma-Aldrich ) and Anti-COX IV ( 926–42214 , Licor ) were used for loading controls . Random 40X fields were imaged using acquisition settings ensuring no under-saturated or over-saturated pixel intensities . Volocity 5 . 5 software was used for quantitative analysis of 3D confocal images . To determine the quantity of nuclear M , the nuclear compartment was defined with the find objects function within the DAPI-fluorescence channel . Holes in objects ( DNA-absent regions such as nucleoli ) were filled , and fluorescent objects smaller than nuclei were excluded . The entire cell body was defined by drawing a region of interest ( ROI ) encompassing all F-actin staining . The sum of voxel intensities in the GFP channel was measured within these defined sets . The average voxel fluorescence of untransfected cells was used for background subtraction . To determine the quantity of nucleolar M , the nucleolus was defined with the find objects function within the Fibrillarin-stained-fluorescence channel . Nucleolar objects were grouped within their respective nuclei , defined as above based on the DAPI-fluorescence channel . To quantify fluorescence from bimolecular fluorescence complementation images , a ROI was drawn around cells fluorescent in the YFP channel . The average voxel fluorescence of untransfected cells was used for background subtraction . For analysis of M nuclear localization , p-values were generated with a Student’s t test when analyzing two sample groups . To analyze three or more sample groups , p-values were generated by ANOVA with Bonferroni correction for multiple comparisons . To analyze BiFC experiments , p-values were generated using a Mann-Whitney test . All graphs and statistical analyses were generated with Prism 6 ( GraphPad Software , La Jolla , CA ) . | Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection . Paramyxoviruses include human and animal pathogens of medical and agricultural significance . Their matrix ( M ) structural protein organizes virion assembly at the plasma membrane and mediates viral budding . While nuclear localization of M proteins has been described for some paramyxoviruses , the underlying mechanisms of nuclear trafficking and the biological relevance of this observation have remained largely unexamined . Through comparative analyses of M proteins across five Paramyxovirinae genera , we identify M proteins from at least three genera that exhibit similar nuclear trafficking phenotypes regulated by an NLSbp as well as an NES sequence within M that may mediate the interaction of M with host nuclear transport receptors . Additionally , a conserved lysine within the NLSbp of some M proteins is required for nuclear export by regulating M ubiquitination . Sendai virus engineered to express a ubiquitination-defective M does not produce infectious virus but instead displays extensive cell-cell fusion while M is retained in the nucleolus . Thus , some Paramyxovirinae M proteins undergo regulated and active nuclear and subnuclear transport , a prerequisite for viral morphogenesis , which also suggests yet to be discovered roles for M in the nucleus . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2015 | Evidence for Ubiquitin-Regulated Nuclear and Subnuclear Trafficking among Paramyxovirinae Matrix Proteins |
Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole . Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus ( GA ) into ministacks , which facilitates the acquisition of host lipids into the growing inclusion . GA fragmentation results from infection-associated cleavage of the integral GA protein , golgin-84 . Golgin-84-cleavage , GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk . Here we identify the bacterial protease chlamydial protease-like activity factor ( CPAF ) as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition . WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C . trachomatis and C . pneumoniae . The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system . Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation . GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity . These results define CPAF as the first protein that is essential for replication of Chlamydia . We suggest that this role makes CPAF a potential anti-infective therapeutic target .
Chlamydiae are a group of obligate intracellular bacteria that infect humans and animals . Chlamydia trachomatis is the most common bacterial agent of sexually transmitted disease with a prevalence of up to about 5% in young women as well as a common cause of eye infections . Clinically , the most relevant aspect of infection with C . trachomatis is its propensity for chronic infection , which may lead to female infertility and to blinding trachoma [1] , [2] , [3] . Chlamydia pneumoniae is a very common cause of ( typically mild ) airway infection but it has also been proposed to cause chronic infection of artery walls , contributing to atherosclerosis [4] . During its developmental cycle Chlamydia switches between two morphologically distinguishable forms . The infectious but metabolically inactive elementary body ( EB ) is taken up by the host cell , where it resides in the cytosol , within a membranous vacuole , termed the inclusion . Within the inclusion EBs differentiate into reticulate bodies ( RBs ) , which divide by binary fission . During this intracellular growth phase the inclusion substantially increases in size , often filling almost the entire cell at later time points . Towards the end of the cycle RBs re-differentiate into EBs , which are subsequently released from the cell . In vitro this cycle takes approximately 2 days for C . trachomatis and 3-4 days for C . pneumoniae [5] . To be able to grow , Chlamydia must escape cellular defenses and acquire nutrients and macromolecules from the host cell . In particular , trafficking of host lipids to the chlamydial inclusion has been noted where they are probably required for the expanding lipid membrane of the growing inclusion as well as for bacterial membranes [6] , [7] , [8] . Numerous alterations of other host cell systems have also been described during infection , including massive changes in gene transcription , cytoskeletal rearrangement and the inhibition of apoptosis [9] . Although molecular details often remain unclear these changes are probably mainly the result of the translocation of bacterial effectors into the host cytosol . Chlamydia possesses a type III secretion system and a number of bacterial proteins have been described to be secreted into the inclusion membrane or beyond [10] , [11] . One chlamydial protease , chlamydial protease-like activity factor ( CPAF ) , is known to translocate from the inclusion to the cytosol approximately mid-cycle [12] . Several host cell proteins have been identified as proteolytic targets of CPAF although the immediate relevance of such cleavage events for the development of the infection has not been established [13] . We recently identified the fragmentation of the Golgi apparatus ( GA ) as a consequence of infection with C . trachomatis [14] , [15] . This fragmentation coincides with the cleavage of an integral GA matrix protein , golgin-84 , and can be phenocopied by silencing of golgin-84 by RNAi . Intriguingly , golgin-84-cleavage can be inhibited by a modified tetrapeptide , z-WEHD-fmk ( WEHD-fmk ) [14] , and WEHD-fmk blocks the replication of C . trachomatis [14] , [16] . Conversely , silencing of golgin-84 enhances bacterial replication [14] . This strongly suggests that the cleavage of golgin-84 during infection causes fragmentation of the GA and that this fragmentation is required for chlamydial growth , most likely because it facilitates transport of essential lipids to the inclusion . Here we identify CPAF as the chlamydial protease responsible for golgin-84-cleavage and as the target of WEHD-fmk during infection . WEHD-fmk , which has been developed as an inhibitor of caspase-1 is shown to inhibit CPAF-activity in a number of experimental situations . When expressed ectopically in human cells , CPAF is found to cause the fragmentation of the GA . Chlamydia thus depends on the secretion of CPAF into the host cell to ensure its intracellular growth . Targeting this individual bacterial protease can prevent replication of Chlamydia in human cells .
We have previously shown that WEHD-fmk-treatment dramatically ( several hundredfold ) inhibits the generation of infectious EB during cell culture infection with C . trachomatis [14] . Confocal analysis confirmed that development of C . trachomatis was strongly reduced and inclusions were reduced in size when cultures were treated with WEHD-fmk ( Fig . 1A ) . We also determined that WEHD-fmk inhibits the growth of a chlamydial species distinct from C . trachomatis , C . pneumoniae . As shown in Fig . 1B , WEHD-fmk substantially inhibited growth and development of C . pneumoniae in infected epithelial cells . In C . pneumoniae infection WEHD-fmk impaired the production of infectious EBs more than 10-fold at 3 days post-infection ( d p . i . ) ( Fig . 1B , left panel ) although this effect was not quite as striking as in the case of C . trachomatis ( see [14] ) . Genome copy number was measured at 48 h ( following WEHD-fmk addition at 24 h ) and was found to be about 4-fold reduced ( Fig . 1B , bottom panel ) . Inhibitor treatment was also demonstrated to impede the fusion of individual , smaller bacterial inclusions to larger inclusions during C . pneumoniae infection ( Fig . 1C ) . During acute C . pneumoniae infection the chlamydial protease CPAF is secreted from the inclusion into the cytosol approximately mid-cycle [17] . We analysed CPAF localisation in C . pneumoniae infected cells 3 d p . i . by confocal microscopy and found that secretion of CPAF was blocked when cells were cultured in the presence of WEHD-fmk ( Fig . 1C ) . An unusual , patchy staining pattern for CPAF was observed inside the inclusions in the presence of WEHD-fmk , whereas inhibitor treatment did not affect lipopolysaccharide ( LPS ) staining pattern . This suggests that less CPAF is secreted from the chlamydial inclusion into the host cell during C . pneumonia infection ( Fig . 1C ) ( we used C . pneumoniae here because we were able to obtain better staining with the antibodies available to us in this species ) . As will become clear below , all of this probably reflects the general growth inhibition imposed by WEHD-fmk . As previously reported for infection with various C . trachomatis strains [14] , the infection with C . pneumoniae also caused the cleavage of the GA matrix protein , golgin-84 ( Fig . 2A ) . However , the protease responsible for this degradation had not been identified . We considered the participation of the chlamydial protease CPAF mainly for two reasons . First , the time course of golgin-84-cleavage during infection ( beginning around mid-cycle ) correlates very well with the appearance of CPAF-activity [12] , [17] . Indeed , CPAF-mediated degradation of vimentin correlated well with the degradation of golgin-84 during C . pneumoniae infection ( Fig . 2A and see below ) . Secondly , we observed that WEHD-fmk not only inhibited the degradation of golgin-84 during C . pneumoniae infection but also prevented the cleavage of the known CPAF substrate vimentin ( Fig . 2A ) . To gain an overview of WEHD-fmk-specificity we tested a number of related tetrapeptide inhibitors . Screening of a library of inhibitors , which has been designed to inhibit various caspases , showed that among these modified peptides WEHD-fmk had the strongest CPAF-inhibitory effect in the cell-free assay ( using recombinant CPAF ) while two inhibitors , VEID-fmk and LEVD-fmk , came close in terms of inhibitory potential ( Fig . S1A; we here used as a source of active CPAF 293T cells where CPAF had been activated by dimerisation; Text S1; [18] and see below ) . Others ( for instance DEVD-fmk and LEHD-fmk; also YVAD-fmk , which has like WEHD-fmk been devised as a caspase-1-inhibitor ) had no detectable activity . Closer investigation of some of the inhibitors showed that VEID-fmk was comparable to WEHD-fmk in the cell-free system ( Fig . S1B; Text S1 ) and showed some , although less marked , activity in inhibiting CPAF-dependent cleavage inside human cells ( Fig . S1C ) or growth of Chlamydia ( Fig . S1E , Text S1 ) , most likely due to different cell-permeability . We had previously found that a calpain inhibitor also blocked Chlamydia-mediated degradation of golgin-84 [14] . This inhibitor was also active in inhibiting CPAF-mediated vimentin cleavage in T-Rex-293-gyrB-CPAF cells ( Fig . S1C ) . When tested during infection , LEHD-fmk and DEVD-fmk were inactive in inhibiting CPAF-mediated cleavage of vimentin or bacterial growth ( Fig . S1D , E ) . This suggests that during chlamydial infection WEHD-fmk acts by targeting CPAF rather than a caspase . For a closer analysis of the link between WEHD-fmk and CPAF we returned to the C . trachomatis cell culture infection model . Again , WEHD-fmk not only inhibited the degradation of golgin-84 but also the cleavage of the CPAF-substrates vimentin , cytokeratin-8 ( CK8 ) , cyclin B1 and RFX5 [13] , [18] ( Fig . 2B ) . Remarkably , WEHD-fmk also reduced the detectable levels of active CPAF ( Fig . 2B ) . This is probably the consequence of a feedback-loop: since WEHD-fmk blocks the growth of Chlamydia , fewer bacteria develop in the cells and this reduces the total production of CPAF . Addition of CPAF also blocked the appearance of the strong anti-apoptotic effect that Chlamydia establishes in infected cells ( i . e . in the presence of WEHD-fmk infected cells remained sensitive to staurosporine while they became resistant in its absence as reported before [19]; data not shown ) . In the presence of WEHD-fmk , there was a modest reduction in the amounts of chlamydial proteins , as assessed by probing Western blots with Chlamydia-reactive antisera from human patients ( Fig . 2C ) , suggesting inhibition of growth or development . This inhibition very likely also leads to the defects in CPAF secretion and chlamydial development ( see above ) . These data were suggestive of a link between growth inhibition of Chlamydia by WEHD-fmk and CPAF . Possible mechanisms of the observed effects included the reduced secretion of CPAF from the vacuole to the cytosol and the direct inhibition of CPAF by WEHD-fmk . It could also not be excluded at this stage that the loss of CPAF activity was secondary to a different inhibitory mechanism of WEHD-fmk . To distinguish between these possibilities we used a system for the ectopic expression of active CPAF in human cells . As reported earlier , CPAF can be activated in the absence of infection by the experimental dimerisation of CPAF ( through the dimerisation of its fusion partner gyrase B ( gyrB ) , which is achieved by the dimeric gyrB ligand coumermycin ( CM ) [18] ) . We utilised a system based on the human cell line 293T , which stably carries a tetracycline-inducible gyrB-C . trachomatis-CPAF construct ( the cells are designated T-REx-293-gyrB-CPAF ) . Addition of anhydrotetracycline ( AHT , a tetracycline-derivative lacking antibiotic activity ) and CM to these cells induces CPAF-activation as measured as autocatalytic cleavage and cleavage of all known CPAF-substrates [18] . Expression of active CPAF in T-REx-293-gyrB-CPAF cells caused the cleavage of vimentin as reported earlier ( Fig . 3A ) [18] . With a similar time course golgin-84 was degraded to fragments that were indistinguishable from those generated during chlamydial infection ( Fig . 3A ) suggesting that CPAF was responsible for the cleavage of golgin-84 in both situations . Addition of WEHD-fmk at the time of CPAF-induction prevented the cleavage of golgin-84 as well as the degradation of the known CPAF-substrates ( Fig . 3B , C ) . Lactacystin , which is known to inhibit both the human proteasome and CPAF [12] , [20] , also reduced cleavage of the CPAF-substrates tested while a specific inhibitor of the proteasome ( epoxomicin ) had no effect ( Fig . 3C ) . WEHD-fmk has been developed as an inhibitor of caspase-1 . Since caspase-1-activation during infection of HeLa cells has recently been demonstrated in Chlamydia infected cells [16] , it was also a possibility that part of the effect of WEHD-fmk was due to caspase-1 inhibition . We have above shown evidence that WEHD-fmk is not acting by inhibiting a caspase , and the evidence is overwhelming that the CPAF-dependent cleavage of the host cell proteins tested ( vimentin , CK8 , RFX5 , cyclin B1 ) does not depend on caspase-1 [12] , [13] , [18] , [20] , [21] , [22] . The pan-caspase-inhibitor z-VAD-fmk further failed to inhibit the tested cleavage events ( Fig . 3C ) , excluding the possibility that WEHD-fmk acted by inhibition of caspases in this system . These results suggested that WEHD-fmk acted as an inhibitor of CPAF . This notion is further supported by the finding that WEHD-fmk also blocked the morphological effects that CPAF produces when expressed in T-REx-293-gyrB-CPAF cells ( Fig . S2A; Text S1 ) at concentrations that blocked cleavage of vimentin ( Fig . S2B ) . In a cell-free assay , lysate from AHT/CM-induced T-REx-293-gyrB-CPAF cells ( i . e . lysate containing active CPAF ) degraded myc-tagged CK8 ( separately expressed by transfection of 293T cells ) ; this cleavage was inhibited by WEHD-fmk and lactacystin ( Fig . 3D ) . Further , bacterial lysate containing active recombinant CPAF degraded the substrates vimentin and CK8 in lysates from human cells and this cleavage was again inhibited by WEHD-fmk ( Fig . S3A , B; Text S1 ) . Thus , cleavage of all tested proteins that are known to be cleaved in a CPAF-dependent way during infection was inhibited by WEHD-fmk . Further , the cleavage of the same proteins was also blocked by WEHD-fmk when active CPAF was expressed in human cells , as was the cleavage of vimentin and CK8 by bacterial recombinant CPAF . This makes it very unlikely that another protease was involved and strongly suggests that WEHD-fmk acted as an inhibitor of chlamydial CPAF . We have shown previously that most of the inhibitory effect of WEHD-fmk is reversed when golgin-84 is knocked down , suggesting that the effect of the inhibitor is limited to preventing the cleavage of golgin-84 ( and therefore to inhibiting CPAF [14] ) . Inhibition of chlamydial replication by WEHD-fmk is therefore very likely due to CPAF inhibition and not effects on additional targets . As reported previously , Chlamydia causes the fragmentation of the GA to ensure its replication , very likely because this facilitates lipid transport to the bacterial inclusion [14] , [15] . Since GA fragmentation is linked to the cleavage of golgin-84 and golgin-84-cleavage is the result of CPAF-activity , CPAF-activity may be expected to be responsible for the fragmentation of the GA during infection . Indeed , the isolated expression of active CPAF induced GA fragmentation in T-REx-293-gyrB-CPAF cells ( Fig . S4; Text S1 ) as well as in HeLa cells transiently expressing the gyrB-CPAF expression construct ( Fig . 4 ) . The presence of WEHD-fmk during expression of active CPAF prevented GA fragmentation as expected ( Fig . 4 , Fig . S4 ) . We have recently shown that Chlamydia-induced GA fragmentation requires the small GTPases Rab6A and Rab11A [15] . If CPAF is responsible for GA fragmentation , this effect of CPAF is expected also to depend on Rab6 and Rab11 . We tested this prediction and found that indeed the reduction of either Rab6 or Rab11 by RNAi had a clear inhibitory effect on GA fragmentation induced by the expression of active CPAF in HeLa cells ( Fig . 5 ) while knock-down per se did not affect GA morphology ( Fig . S5; Text S1 ) [15] . Taken together , these results provide strong evidence that CPAF is the enzyme that cleaves golgin-84 and thereby contributes to GA fragmentation , which in turn is linked to lipid transport to the chlamydial inclusion . WEHD-fmk blocks CPAF activity , interrupting these events and inhibiting normal chlamydial growth .
These results establish a link between the production of CPAF by Chlamydia , the cleavage of golgin-84 and subsequent GA fragmentation , which again is required for efficient chlamydial replication . CPAF activity is thus indispensable for the intracellular replication and development of Chlamydia , and the targeting of CPAF may be a strategy to reduce the burden of chlamydial infection . WEHD-fmk blocked the degradation of all known and investigated CPAF substrates when recombinant CPAF was tested in cell extracts and when active CPAF was expressed in human cells . This suggests a direct binding of the inhibitor to the protease . Although the protease has been crystallised together with its known inhibitor lactacystin , the great width of the active site makes it difficult to predict how this inhibitory mechanism might work . The differences in inhibitory potential of other peptide inhibitors tested suggest a binding of individual amino acids from the inhibitor sequence . There is no clearly defined substrate recognition and cleavage sequence of CPAF . The possibility should also be considered that substrate recognition of CPAF occurs at a site distinct from the actual cleavage site . If that was the case , WEHD-fmk might bind at a domain outside the active site of the protease . During infection , WEHD-fmk not only inhibits CPAF-dependent substrate cleavage but also growth of Chlamydia , as already reported earlier [14] . This is linked to the cleavage of golgin-84 and the subsequent GA fragmentation . CPAF has a high number of known substrates [13] and very likely many more that have not been identified . All of these substrates may have an effect on chlamydial growth . However , the cleavage of golgin-84 may be of particular importance . As we have found earlier , knock-down of golgin-84 ( inducing GA fragmentation ) reverses the inhibitory effect of WEHD-fmk to a considerable extent [14] . As cleavage of all other CPAF substrates is presumably still blocked by the inhibitor in this situation , these cleavage events may not be critical , at least in cell culture . The group of chlamydiae and Chlamydia-related bacteria are very widespread and successful as infectious agents of many diverse species throughout the animal kingdom [23] . The forms of related bacteria range from organisms that have been isolated as symbionts of free-living amoebae ( for instance Protochlamydia amoebophila [24] ) over agents that parasitize amoebae but may also infect humans ( Parachlamydia acanthamoebae [25] ) to a number of species that frequently infect animal ( such as C . caviae , C . abortus ) and human hosts ( C . trachomatis , C . pneumoniae , C . psittaci ) . All of these bacterial species share the lifestyle of intracellular development in a membranous cytosolic inclusion . This developmental peculiarity carries the advantage of a degree of protection against host cell defense systems afforded by the lipid membrane , and probably makes it easier to generate a specialised environment for optimal replication . It is however also associated with the requirement of arranging for vacuole growth by continuous substitution of lipids as well as the acquisition of lipids and nutrients from the host cell for bacterial growth . Sphinogmyelin acquisition by the chlamydial inclusion occurs both early ( within hours of infection ) and late in the developmental cycle , and has been linked to multivesicular bodies and to interception of post-Golgi traffic [7] , [8] , [26] . We have recently found that fragmentation of the GA facilitates this transport at later stages of infection [14] . The results shown here suggest that this late sphingolipid acquisition is the result of CPAF activity , which may be an important aspect of the function of this protease in chlamydial intracellular development . Identifiable CPAF-orthologs have been found in Chlamydia-related organisms as distant from human pathogens as P . amoebophila [24] . This suggests that CPAF indeed serves a function that is indispensable to the lifestyle of these organisms . It is at least conceivable ( although highly speculative at this stage ) that the function in vesicle acquisition that we identify here during infection of human cells with C . trachomatis was an original function of CPAF already in Chlamydia-related organisms parasitizing amoebae . Acute chlamydial infections can be easily and successfully treated with antibiotics and although antibiotic resistance may develop this is not a major problem at this stage . The biggest problem clinically concerns chronic infections . At least in vitro , chlamydiae can enter a persistent state that likely reflects chronic infection and during which Chlamydia is resilient to antibiotic treatment [27] . We cannot say whether Chlamydia also depends on CPAF for the maintenance of a chronic infection but it is intriguing that serum antibodies to CPAF are characteristic of long-term infections in humans [28] , suggesting that CPAF is indeed expressed during chronic infection . The inhibition of CPAF , the principal feasibility of which is shown here , may therefore turn out to be a valuable approach to the therapy of human chlamydial infection .
The human embryonic kidney cell line T-REx-293 , which stably expresses the tetracycline repressor ( Invitrogen , Darmstadt , Germany ) was maintained in humidified air at 5% CO2 and 37°C in Dulbecco modified Eaglès minimal essential medium ( DMEM ) supplemented with 10% fetal calf serum ( FCS , tetracycline negative; PAA Laboratories , Pasching , Austria ) and 5 µg/ml blasticidin ( PAA Laboratories , Pasching , Austria ) . Stable , gyrB-CPAF expressing T-REx-293 clones were generated by electroporation with the pcDNA4/TO/myc-His-gyrB-CPAF ( C . trachomatis CPAF ) construct and subjected to antibiotic selection as previously reported [18] . The cells were cultured as above but in the presence of zeocin ( 350 µg/ml; InvivoGen , San Diego , CA , USA ) . To induce and activate CPAF 5 ng/µl anhydrotetracycline ( AHT; IBA , Göttingen , Germany ) and 1 µM coumermycin ( CM; Sigma-Aldrich , Steinbach , Germany ) were added . HeLa and HEp-2 cells were grown in DMEM supplemented with 10% fetal calf serum but lacking blasticidin and zeocin . WEHD-fmk ( R&D Systems , Wiesbaden-Nordenstedt , Germany ) , z-VAD-fmk ( Bachem , Bubendorf , Switzerland ) , epoxomicin ( Calbiochem , Darmstadt , Germany ) or Clasto-lactacystin β-lactone ( Sigma-Aldrich , Steinbach , Germany ) were added as indicated . The C . trachomatis strain ( serovar L2 ) was obtained from the American Type Culture Collection ( ATCC ) . For infection of T-REx-293 or HeLa cells , the culture medium was replaced with serum free medium without antibiotics and bacteria were added using the specified multiplicity of infection ( MOI ) . Two hours later the medium was supplemented with 10% FCS . C . pneumoniae strain VR1310 was a kind gift of Dr . Gunnar Christiansen ( Institute of Medical Microbiology and Immunology , University of Aarhus , Denmark ) . Prior to infection , culture medium was removed and C . pneumoniae was added in DMEM containing 1 µg/ml cycloheximide . The cells were then centrifuged at 2 , 000 g for 35 min at 35°C and further cultivated for the times indicated . Cell extracts were prepared using RIPA-buffer ( 1% Triton X-100 , 0 . 5% SDS , 0 . 5% deoxycholate , 1 mM EDTA , 150 mM NaCl , and 50 mM Tris , pH 8 . 0 ) supplemented with a protease inhibitor cocktail ( Roche , Mannheim , Germany ) . Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes . Antibodies used were directed against β-actin ( Sigma-Aldrich , Steinbach , Germany ) , chlamydial Hsp60 ( Alexis Biochemicals , San Diego , CA , USA ) , Bim , cyclin B1 , myc ( all from Cell Signaling Technology , Beverly , MA , USA ) , CK8 , RFX5 , vimentin ( all from Acris , Herford , Germany ) , golgin-84 ( BD Bioscience , Heidelberg , Germany ) and CPAF ( a polyclonal serum generated by immunization of rabbits with a peptide from the C-terminal fragment of C . trachomatis CPAF; Pineda Antibody-Service , Berlin , Germany ) . Antibodies specific for C . pneumoniae CPAF were a generous gift of Dr . Guangming Zhong ( San Antonio , Texas , USA ) . In one series of experiments , Western blots were probed with human sera from patients that had tested positive for C . trachomatis-specific antibodies by line blot ( Mikrogen , Munich , Germany ) . A mixture of five sera was used . Proteins were visualized using peroxidase-conjugated secondary antibodies and a chemoluminescence detection system ( GE Healthcare , Uppsala , Sweden ) . As a source of active CPAF , T-REx-293-gyrB-CPAF cells were stimulated with AHT/CM for 18 h and lysed in NP-40 buffer ( 1% NP-40 , 150 mM NaCl , 1 mM EDTA , and 20 mM MOPS , pH 7 . 4 ) . Cell extracts were mixed with equal volumes of lysate from T-REx-293 cells transiently transfected with a myc-tagged CK8-construct . Reactions were incubated at 37°C for 1 h and subjected to Western blot analysis . Clasto-lactacystin β-lactone or WEHD-fmk was added 30 min prior to substrate addition . siRNAs were purchased from QIAGEN ( QIAGEN , Hilden , Germany ) . HeLa cells were seeded into 12-well plates one day before transfection . Transfection was performed using QIAGEN RNAiFect according to the manufacturer's guidelines . 1 µg siRNA was added to 96 µl Opti-MEM medium ( Invitrogen , Darmstadt , Germany ) , vortexed , mixed with 6 µl RNAiFect and incubated for 15 min at room temperature . The liposome/RNA mix was added to cells with 600 µl growth medium [15] . After 24 h , cells were trypsinised and seeded into new cell culture plates and incubated for another 24 h . Cells were seeded onto coverslips and treated as indicated . Cells were then fixed with 2% PFA for 30 min at room temperature . The Golgi marker GPP130 ( Covance , Princeton , NJ , USA ) as well as CPAF were detected using specific antibodies . Bacteria were stained using either a chlamydial LPS specific antibody ( Milan Analytica AG ) or a chlamydial Hsp60 specific antibody ( Alexis Biochemicals , San Diego , CA , USA ) . Binding was visualized using fluorescence labeled secondary antibodies . Cells were mounted in Mowiol . For the localisation of CPAF , HeLa cells were infected with C . pneumoniae VR1310 ( MOI = 3 ) and WEHD-fmk ( 80 µM ) was added 24 h p . i . ( DMSO was added in the controls ) . Fixation was followed by treatment with 0 . 2% Triton X-100 in PBS supplemented with 0 . 2% BSA . Cells were analysed with an LSCM ( Leica TCS SP-1 , 63x/1 . 32 HCX PL APO CS oil lens , Leica Microsystems , Wetzlar , Germany ) and images were processed using Adobe Photoshop . Genome copy numbers were determined by real-time LightCycler PCR as described previously [29] . Briefly , DNA extraction was performed using NucleoSpin RNA/DNA according to the manufacturer's recommendations ( Macherey-Nagel , Düren , Germany ) : The 16S rRNA was amplified ( forward primer , CAT CGT TTA CGG CAA GGA CTA; reverse primer , AGG CCT TAG GGT TGT AAA GCA ) . Absolute numbers of genome copies were obtained by calculating the cp-values of the respective samples against a standard curve ( ten-fold dilutions of C . pneumoniae infection inoculums ) with known IFUs/ml . | Chlamydiae are bacteria that replicate only inside host ( for instance human ) cells and that are frequent agents of human disease , in particular sexually transmitted disease . Chlamydia lives in a vacuole inside the cell , surrounded by a lipid membrane , and must acquire nutrients and other factors from the host cell for its replication and for the growth of the vacuole . Recent results show that for this , Chlamydia relies on its ability to induce the loss of an individual protein of the Golgi apparatus ( a cellular structure that sorts materials for transport in the cell ) called golgin-84 . In this work we find that Chlamydia does this using its protein-cleaving enzyme CPAF ( which is made by Chlamydia and transported from the vacuole into the cell ) . CPAF cleaves golgin-84 and thereby induces changes in the Golgi apparatus that are linked to the acquisition of some cellular material by Chlamydia . We further show that a synthetic inhibitor , which was recently found to block chlamydial growth , does that by inhibiting CPAF . CPAF therefore seems necessary for chlamydial growth and blocking CPAF may be a therapeutic strategy against infections with Chlamydia . | [
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] | 2011 | Targeting of a Chlamydial Protease Impedes Intracellular Bacterial Growth |
Cooperative breeding is an extreme form of cooperation that evolved in a range of lineages , including arthropods , fish , birds , and mammals . Although cooperative breeding in birds is widespread and well-studied , the conditions that favored its evolution are still unclear . Based on phylogenetic comparative analyses on 3 , 005 bird species , we demonstrate here that family living acted as an essential stepping stone in the evolution of cooperative breeding in the vast majority of species . First , families formed by prolonging parent–offspring associations beyond nutritional independency , and second , retained offspring began helping at the nest . These findings suggest that assessment of the conditions that favor the evolution of cooperative breeding can be confounded if this process is not considered to include 2 steps . Specifically , phylogenetic linear mixed models show that the formation of families was associated with more productive and seasonal environments , where prolonged parent–offspring associations are likely to be less costly . However , our data show that the subsequent evolution of cooperative breeding was instead linked to environments with variable productivity , where helpers at the nest can buffer reproductive failure in harsh years . The proposed 2-step framework helps resolve current disagreements about the role of environmental forces in the evolution of cooperative breeding and better explains the geographic distribution of this trait . Many geographic hotspots of cooperative breeding have experienced a historical decline in productivity , suggesting that a higher proportion of family-living species could have been able to avoid extinction under harshening conditions through the evolution of cooperative breeding . These findings underscore the importance of considering the potentially different factors that drive different steps in the evolution of complex adaptations .
Cooperative breeding is an extreme form of cooperation that occurs when individuals help raise conspecific offspring that are not their own [1] , often while temporarily foregoing their own reproduction [2 , 3] . This common form of cooperation has intrigued evolutionary biologists since Darwin [4] and is thought to have evolved multiple times in a range of lineages , including insects , fish , birds , and mammals , usually as a product of kin selection [5] . Even though life history and ecological correlates of cooperative breeding have been particularly well studied in birds [6–9] , large-scale comparative analyses in this group have yielded contradictory findings [6 , 7 , 9 , 10] . Thus , the conditions that favor the evolution of cooperative breeding are currently unclear [2] . Earlier theoretical work suggested that delayed dispersal ( i . e . , family formation ) is a critical step in the evolution of cooperative breeding [3 , 11 , 12] , reflecting the fact that helping at the nest in birds is overwhelmingly kin-based [13–15] . These studies proposed that family living arises when parents can afford to invest in offspring beyond independence , which is more likely in long-lived species [12 , 16] and in stable and productive environments that allow for a prolonged association of offspring with their parents [17 , 18] . However , subsequent work has generally overlooked that many bird species live in families that do not breed cooperatively [14] . Consequently , prior comparative analyses have investigated the evolution of cooperative breeding by contrasting cooperative and noncooperative species [7 , 9 , 10 , 19–23] and have provided equivocal predictions about the occurrence of cooperative breeding . For example , these studies suggest that cooperative breeding may be favored either when living in saturated habitats with a slow turnover in breeding opportunities ( i . e . , stable environments with a long mean growing season [MGS] [3 , 7 , 10 , 11 , 24] ) or when living in unpredictable environments , where helpers at the nest can buffer reproductive failure in harsh years ( i . e . , high degree of unpredictability [3 , 6 , 9 , 23 , 25] ) . Under both of these hypotheses , cooperative breeding is predicted to evolve preferentially in species with a high survival probability [10] , because high survival increases the time offspring have to queue for breeding opportunities , increases habitat saturation , and enhances opportunities to act as helper at the nest [26] . Here , we test the hypothesis that the evolution of cooperative breeding from a noncooperative ancestor may have involved 2 distinct transitions: one to a continued parent–offspring association beyond the period when offspring are actively provisioned by their parents ( i . e . , the formation of families [14] ) and a subsequent one to the evolution of helping at the nest . We posit that , by considering only 1 transition from noncooperative breeding to cooperative breeding , prior studies may have obscured the role of potential ecological and life history drivers because the factors that promote family living may have been inadvertently confused with those that promote helping at the nest [14 , 18] . Thus , a more nuanced understanding of the evolutionary steps through which cooperative breeding arose may help clarify the current debate on the conditions favoring its evolution [6 , 7 , 9 , 10] . We took advantage of the extensive natural history data on the social life of birds ( N = 3 , 005 terrestrial species , including species from all major orders and bioregions , see S1 Table for details ) to categorize species into 1 of 4 social systems . The 3 most common systems are: ( i ) “non-family-living species , ” in which parent–offspring associations do not extend beyond nutritional independence and individuals do not engage in cooperative breeding ( 55% in our data set; Fig 1A ) ; ( ii ) "family-living species , ” in which offspring remain with their parents beyond nutritional independence but the retained offspring do not assist their parents in rearing activities [14] ( this includes species with both biparental and uniparental brood care; 31% in our data set; Fig 1B ) ; and ( iii ) “cooperatively breeding species , ” in which offspring remain with their parents beyond nutritional independence and help them in subsequent breeding attempts or engage in redirected helping at nests of relatives ( 13% in our data set; Fig 1C ) . Family-living and cooperatively breeding species differ not only in terms of helping at the nest but also in that offspring in 91% of family-living species disperse before the onset of the next breeding season . Finally , the fourth social system involves a very limited number of bird species that exhibit helping at the nest among unrelated individuals [8 , 13] ( “non-kin cooperatively breeding species”; 1% in our data set; Fig 1D ) . To test the suitability of the proposed 2-step model for the evolution of cooperative breeding , we first estimated the relative rates of evolutionary transitions among different avian social systems and investigated whether family living was a necessary precursor for the evolution of cooperative breeding . We then evaluated the ecoclimatic correlates of each social system to gain insight into the potential pressures of selection that drove each of these evolutionary transitions , with a particular focus on distinguishing the conditions that promoted the formation of family groups from those that favored the evolution of cooperative breeding .
Given the rarity of non-kin helping ( see above ) , we began our analyses by focusing on the 3 major avian social systems ( i . e . , non-family-living , family-living , and cooperatively breeding families ) . Based on a recent class-wide phylogeny [27] and a model of discrete trait evolution [28] , we estimated evolutionary transitions between these social systems and confirmed that the ancestral social system in birds was very likely to be non-family living ( Fig 2 ) . Transitions between non-family living and family living , as well as those between family living and cooperative breeding , were common ( i . e . , transition rates range from 0 . 01 to 0 . 04; Fig 3 ) . Importantly , however , direct transitions from non-family living to cooperative breeding were exceedingly rare ( transition rate = 0 . 002; Fig 3 ) . Including non-kin cooperatively breeding species in the analysis showed that this system mostly arose from non-family-living species but does not have an evolutionary link to family-based cooperative breeding ( S2 Fig ) . These results strongly suggest that the evolution of family living was a pivotal precondition for the evolution of cooperative breeding in the majority of birds . Thus , to examine the possible conditions favoring cooperative breeding in birds , we now ask how the predictors of cooperative breeding differ from those of family living . To investigate the conditions favoring the evolution of family living and cooperative breeding in birds , we used a phylogenetically controlled multinomial generalized linear mixed model [29 , 30] . Our model explored the effects of putative ecoclimatic , social , and life-history predictors of cooperative breeding explored in previous analyses ( i . e . , sedentariness [10] , stable climatic conditions [3 , 7] , environmental unpredictability [3 , 6 , 9 , 25] , nesting modus [31] , low annual mortality [10] , and altricial offspring that require active food provisioning [32] ) . We also controlled for the potentially confounding effects of having classified social systems using 3 different sources of information ( see Materials and methods ) . We calculated mean values , predictability indices , and within-year variances for precipitation , temperature , and net primary productivity ( NPP ) by computing values locally ( cell size: 0 . 5° x 0 . 5° ) and subsequently averaging them across the entire breeding distribution of each species . Because climatic unpredictability during the breeding season is thought to be particularly important for the evolution of cooperative breeding [6] , we calculated ecoclimatic correlates both across the entire year and exclusively during the likely breeding season at each location . The duration of avian breeding seasons at a given locality was estimated from the length of the growing season of local plants [33] ( see S1 Text ) . We used principal component analysis ( PCA ) to reduce the dimensionality of our original set of 23 continuous predictors because most of them exhibited moderate to strong collinearity . The first 8 principal components ( PCs ) in this analysis captured 92% of the variance in continuous predictors ( S2 Table ) . Fifteen out of the 19 original environmental variables loaded primarily on the first 2 PCs ( PC1 and PC2 ) . PC1 , dubbed “variable rainfall among years , ” reflects a gradient toward environments where rainfall is higher on average ( along with an associated increase in NPP ) but more variable among years . PC2 , dubbed “mean growing season duration , ” reflects a gradient toward longer breeding seasons and more stable temperatures throughout the year . The remaining components capture the residual variance in 1 to 3 variables each , after accounting for correlations with other components ( S2 Table ) . We could not include non-kin cooperatively breeding species as an independent social system in the multinomial analysis because the number of species in this category is too small to derive meaningful estimates of statistical parameters . Our multinomial analysis ( using family living as the reference level ) reveals that non-family living and family living are associated with very different ecoclimatic and life-history variables , while the predictors associated with family living and cooperative breeding are nearly identical ( Table 1 ) . Compared to non-family-living species , family-living species have a higher probability of occurrence at localities where rainfall is more abundant and variable ( PC1 ) , MGSs are longer ( PC2 ) , and the among-year variance in productivity during the growing season is higher ( PC5 ) ( Table 1 , Figs 4 and 5 ) . Moreover , family-living species are typically larger ( PC8 , Fig 4 ) , more sedentary , live in denser habitats ( PC7 , Fig 4 ) and exhibit a higher degree of food specialization than non-family-living species ( Table 1 ) . Thus , many of the ecological conditions currently believed to promote the transition to cooperative breeding are likely to have driven the initial transition to family living instead . In contrast , our analyses revealed very few differences in the predictors of cooperative breeding and family living . An important difference is that cooperatively breeding species are more likely to occupy environments with a high within year variability in environmental productivity , whereas family-living species are more common in localities where the within year variance in productivity is intermediate ( PC3 , Figs 4 and 5 ) . This result suggests that helping at the nest evolved where family-living species faced environments with more variable productivity , supporting the hard life hypothesis [25] and the environmental unpredictability hypothesis [6 , 9] . Earlier studies suggested that prolonged parental investment [14 , 16] and cooperative breeding [35] are associated with a high survival probability . Given that survival is poorly studied in most species , we included longevity instead as its proxy in our models ( available for N = 1 , 023 species ) . However , this model did not reveal any additional effects on the distribution of social systems ( S3 Table ) , although we note that longevity can be a poor surrogate for annual survival . We also note that the subsample of birds for which longevity is known is biased toward temperate , non-family-living species and that estimates of longevity are highly influenced by sampling effort [36] .
Overall , our results help unravel the potential sequence of evolutionary steps in the evolution of cooperative breeding and provide a clearer picture of the role of ecoclimatic factors in this process . Our comparative analyses show that almost all cooperatively breeding species evolved from family-living ancestors and that many of the ecoclimatic correlates that were previously thought to promote helping at the nest [3 , 6 , 37] may have favored instead the prerequisite transition toward family living . This finding highlights that helping at the nest is not the only social adaptation that can help birds deal with variable environmental conditions . For example , family living can reduce the mortality of independent juveniles [38] through parental protection against predators [39–41] , easier offspring access to resources [42 , 43] , increased offspring foraging efficiency [44] , and a potential reduction of per capita investment in territoriality [45] . Furthermore , family living is associated with ample opportunities to socially acquire critical life skills [46] and potentially increase cognitive abilities [47] . These benefits of family life may improve offspring survival in productive but variable environments [38] and lead to higher grand-offspring fitness even in the absence of helping at the nest [48] . Notably , these direct fitness benefits accrue both during and outside of the breeding season and , most importantly , suggest that cooperation outside of the reproductive context facilitates the evolution of family living [18] . These insights allow us to reconsider the role of limited dispersal options ( i . e . , ecological constraints [3] ) for the evolution of cooperative breeding . Both family living and cooperative breeding are associated with productive but variable habitats that may limit dispersal options; however , it is more likely that these conditions in fact facilitate family living by reducing the cost to parents [16] and offspring [18 , 26] . Thus , delayed dispersal is an adaptive life-history decision rather than a “best of a bad job” strategy reflecting dispersal constraints [49] . Earlier studies have reported a rather weak and variable influence of ecoclimatic factors on the distribution of cooperative breeding [9 , 23 , 50] . However , the effects of these predictors are likely to have been inadvertently misinterpreted by considering a single transition from non-family living to cooperative breeding . As shown above , the initial formation of family groups was likely to be associated with the occupancy of productive environments that facilitate family living [14 , 18] . In contrast , the subsequent evolution of cooperative breeding was likely to have been associated instead with a secondary occupancy of environments with more variable productivity . In years of low productivity , helping at the nest benefits both parents [23 , 25] and offspring [2 , 5 , 8] , as these conditions increase the chance for parents to breed successfully and limit the chances of offspring to successfully breed independently , particularly in long-lived species [26] . In some short-lived cooperative breeders , mature offspring disperse nearby to breed independently , and proximity allows relatives to provide help at each other’s nests [8] . Low environmental productivity has also been suggested to favor cooperative breeding in mammals [51] and humans [52] . Moreover , a high within-group relatedness ( i . e . , family living ) has been proposed to facilitate the evolution of eusociality in insects as well as cooperative breeding in mammals [21 , 53] . Therefore , a high enough but variable level of resources throughout the year favors the evolution of persistent kin groups and cooperation outside the reproductive context , while an additional increase in the variation in productivity may act as the condition favoring the subsequent evolution of cooperative breeding . A recent comparative study suggested that high annual survival facilitates the evolution of cooperative breeding in birds [35] . Using these data but separating noncooperative breeders into non-family-living and family-living species shows that both cooperatively breeding and family-living species have a higher annual survival than non-family-living species ( Phylogenetic Generalized Least Squares [PGLS] model: non-family living versus cooperative breeding: p = 0 . 00001 , non-family living versus family living: p = 0 . 03; controlling for body size; N = 189 species ) . High annual survival allows prolonged parental investment into offspring [16 , 47] by providing offspring an incentive to remain with the parents beyond independence [38] . Moreover , it favors a delayed onset of independent reproduction [26] , making cooperative breeding adaptive , particularly in variable environments where helpers at the nest can buffer reproductive failure in harsh years [6] . Our findings provide novel insights into the geographic distribution of different social systems , which has currently defied full explanation [9] . We speculate that the answer lies with an increase in the within year variance in environmental productivity . For example , several of the previously identified geographic hotspots of cooperative breeding ( Southern Africa , Australia , Northern South America; [9] ) underwent drastic climatic changes throughout the Eocene , from subtropical and tropical climates to seasonal savanna habitats or arid environments [54] . Accordingly , these environmental changes suggest these hotspot locations likely changed over time from favoring family living to favoring cooperative breeding . In conclusion , our analyses reveal 2 key findings that provide a novel way of understanding the evolution of cooperation in birds and suggest a resolution for earlier equivocal findings [6 , 7 , 9 , 10 , 23] . First , family living enables coping with variable environmental conditions and increases offspring survival both within and outside the breeding season [38] . Subsequently , it sets the scene for the secondary evolution of cooperative breeding [5] when environments have become more variable throughout the year and during the breeding season . Second , we found that cooperative breeding among unrelated individuals is exceptional and likely has different evolutionary origins than family-based cooperative breeding ( S2 Fig ) . Previous work suggested that this form of cooperative breeding arose through an alternative pathway , namely direct fitness benefits from reproductive sharing [13] . Overall , our analysis shows that considering path dependence is essential for understanding the evolution of complex adaptations , such as cooperative breeding , that may involve multiple independent evolutionary steps to be achieved [55] .
We collected data on the social system , life history , and ecological parameters of bird species from the literature ( see S1 Text ) . We used 3 different criteria to differentiate between the different social systems , using the known duration of family associations ( i . e . , the time offspring remain with parents beyond nutritional independence [14] ) , the occurrence of family groups during the non-breeding season ( when the exact time offspring remain with parents beyond independence was unknown ) , or the occurrence of cooperative breeding [15] and the kin relationship of helpers [13] ( see S1 Text ) . We did not categorize occasional cooperative breeding species as cooperative breeders [1] ( based on the first 2 criteria above ) . Occasional cooperative breeding resembles interspecific feeding , in which individuals feed offspring of another species , and thus , different factors are likely to be associated with occasional cooperative breeding and regular cooperative breeding [1] . Species were categorized as sedentary ( maximally engage in local movements ) or migratory ( short- , long-distance , and altitudinal migrants ) . Species that only use 1 food category were categorized as food specialists , whereas species that used at least 2 different food types were categorized as food generalists ( see S1 Text for details on the food categorization ) . Habitat openness was calculated based on aerial images , following the International Union for Conservation of Nature ( IUCN ) Habitat Classification Scheme [56] . Nest type was categorized as a binary variable ( cavity breeders: nests in cavities , cliffs , and caves; other nests: all other nest constructions ) . We used the mean body weight ( combining male and female weight ) and distinguished precocial from altricial species ( categorizing semiprecocial species as precocial and semialtricial species as altricial ) . Climatic variables were computed from data provided by the Climatic Research Unit Time Series 3 . 21 database at the University of East Anglia ( http://catalogue . ceda . ac . uk/uuid/ac4ecbd554d0dd52a9b575d9666dc42d; downloaded 7 April 2014 ) and NASA ( http://neo . sci . gsfc . nasa . gov/view . php ? datasetId=MOD17A2_M_PSN; downloaded 5 December 2013 ) . We calculated for each species: Since the variance often increases with the mean ( i . e . , Taylor’s law [58] ) , it has been suggested that the coefficient of variance may be a more appropriate measurement to assess climatic variability . Thus , we re-ran our analyses using the coefficient of variance where appropriate ( for variables measured on absolute scales , i . e . , precipitation , temperature ) . Both the PCA ( S4 Table ) and the multinomial model ( S5 Table ) resulted in qualitatively similar results as our main analyses , indicating that our choice of variability metric did not bias our results . All statistical analyses were performed in R with the packages Diversitree [28] , phytools [59] , and MCMCglmm [29] . Ancestral state estimation was performed using the MuSSE function of the Diversitree package [28] on a consensus phylogeny estimated from a sample of 1 , 000 phylogenetic trees [27] with the maximum parsimony matrix method using the Hackett tree backbone . We note that using a consensus phylogeny with the Ericsson backbone returned qualitatively identical results . Also , using a model in which speciation and extinction rates were allowed to vary resulted qualitatively in the same results as the main models with diversification rates fixed to be equal across breeding modes ( S6 Table ) . We fitted phylogenetically controlled multinomial models to our data using MCMCglmm [29] . The response variable in all models was a categorical representation of social system ( 3 nominal levels: non-family living , family living , and cooperative breeding ) . The phylogenetic random effect was modelled based on a recent phyla-wide phylogeny [27] . To account for the uncertainty of phylogeny estimation , we refitted the main model with 50 randomly selected trees from the posterior distribution of trees published in Jetz et al . [27] . Given that ancestral character reconstruction may be biased when characters influence diversification [60] , we also used a phylogenetic controlled PCA ( phyloPCA function in phytools ) [59] , resulting is a somewhat different PC structure ( S7 Table ) . However , running our main model with this PC resulted qualitatively in the same results ( S8 Table ) . | Cooperative breeding is a common form of cooperation in which individuals help raise conspecific offspring that are not their own . It has evolved in a range of lineages , including arthropods , fish , birds , and mammals . In birds , cooperative breeding is widespread and well-studied; however , the conditions that favored its evolution are still unclear . Based on an analysis of 3 , 005 bird species , we show that the evolution of this social system required 2 transitions . First , families formed by prolonging parent–offspring associations , and second , retained offspring began helping at the nest . We then show that the formation of families is associated with more productive and seasonal environments and that the subsequent evolution of cooperative breeding is linked to an increase in the variability of environmental productivity . These findings are consistent with patterns in insects and mammals ( including humans ) and clarify current disagreements on the role of environmental forces in the evolution of cooperation . | [
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] | 2017 | Family living sets the stage for cooperative breeding and ecological resilience in birds |
Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli . RNA editing is one of such control mechanisms . We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast , and contributes to control over the extent of ndhB transcript editing . ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex ( NDH ) involved in cyclic electron flow ( CEF ) around photosystem I . In ocp3 mutant strains , ndhB editing efficiency decays , CEF is impaired and disease resistance to fungal pathogens substantially enhanced , a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins ( i . e . CRR21 , CRR2 ) . Furthermore , we observed that following a pathogenic challenge , wild type plants respond with editing inhibition of ndhB transcript . In parallel , rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue . Therefore , NDH complex activity and plant immunity appear as interlinked processes .
Plastid function relies on nuclear gene expression , and the import of nuclear gene products into plastids [1] . In fact , the plastid genome of current land plants encodes 75–80 proteins [2] , whereas nuclear-encoded chloroplast proteins are estimated between 3500 and 4000 [3] . Current data approximates several hundred nuclear-encoded proteins are involved in post-transcriptional regulation of plastid gene expression [4] , [5] . One such regulation is mediated through RNA editing , a post-transcriptional process that alters specific cytidine residues to uridine ( C-to-U ) in different plastid RNAs [6] . Thirty-four sites are edited in 18 transcripts of Arabidopsis plastids [7] . Among the nuclear-encoded proteins regulating RNA editing , the pentatricopeptide repeat ( PPR ) protein family has attracted notable interest [8] . This family comprises 450 members defined by a tandem array of PPR motifs . PPRs are also involved in almost all stages of plastid gene expression , including splicing , RNA cleavage , translation , and RNA stabilization [9] . The pioneer work of Kotera et al . [10] , [11] revealed the Arabidopsis PPR protein CHLORORESPIRATORY REDUCTION4 ( CRR4 ) acts as a site-specific recognition factor for RNA editing of the site 1 ( ndhD-1 ) in the plastid ndhD transcript . ndhD encodes the D subunit of the chloroplast NADH dehydrogenase-like complex ( NDH ) , involved in cyclic electron flow ( CEF ) around photosystem I ( PSI ) [11] , [12] . Consequently , crr4 mutants are defective in ndhD transcript editing at the ndhD-1 site , and CEF is compromised [10] , [11] . Subsequently , the number of PPR-encoding genes participating in editing control in the chloroplast has enlarged [9] . Although empirical evidence has been demonstrated for only a few PPR proteins , it is currently accepted that PPR proteins act as sequence-specific RNA binding adaptors , and hypothetical inferences suggest PPRs recruit effector enzymes or proteins to the target RNAs [13] , [14] . While the mechanism by which specific PPR proteins recognize specific editing sites is becoming understood , questions still remain to be completely solved including the characterization of the molecular components that conform the RNA editing apparatus ( editosome ) or the still unsolved identification of editing enzyme itself . Therefore , identification of additional components modulating editing activities in plastids , and ascertaining how control of the post-transcriptional mechanism of chloroplast function influences other biological processes , in particular immune responses , is of great importance . Despite the critical role of chloroplasts as a site for production of integral mediators of plant immunity such as salicylic acid , jasmonic acid , and ABA [15] , the molecular link between chloroplasts and the nuclear-encoded immune system remains largely unexplored . MEcPP , a plastidial metabolite previously shown to be involved in activating plant immunity in Arabidopsis [16] has been shown to mediate a retrograde signaling regulating expression of nuclear stress-response genes [17] . Nomura et al . [18] recently reported the chloroplast calcium-sensing receptor ( CAS ) , involved in transducing changes in cytosol Ca2+ concentrations into chloroplast responses , regulates plant immunity in Arabidopsis , possibly through chloroplast-derived ROS signals ( i . e . 1O2 and H2O2; [19] ) . These ROS signals may function through a retrograde signaling pathway to activate the expression of nuclear genes . Terashima et al . [20] demonstrated CAS is a crucial component of the machinery driving CEF around photosystem I in Chlamydomonas reinhardtii , and suggested CAS mediates changes in CEF activity , however the mechanism remains unresolved . In contrast to linear photosynthetic electron flow , where light drives ATP and NADPH synthesis; during CEF , light only drives ATP production by cycling electrons around PSI and Cyt b6f complexes , providing the molecular basis for this major energetic switch . Concurrently , CEF leads to the reduction of the plastoquinone pool , thereby increasing the frequency of charge recombination events in PSII; and as a result , altering the chloroplast redox status [21] . Consequently , CAS and Ca2+ via CEF alter ROS homeostasis , and may activate ROS-mediated retrograde signaling , which in a plant-pathogen interaction may have an impact on the outcome of plant disease resistance . CEF is also interrelated with nonphotochemical quenching ( NPQ ) which protects plants against damage resulting from ROS formation [22] . Göhre et al . [23] recently reported defense activation during PAMP-triggered immunity ( PTI ) in Arabidopsis resulted in rapid NPQ decrease , and NPQ also influenced immune responses , suggesting that NPQ and CEF are integral components regulating plant defense response . Similarly , chloroplast-generated ROS following the recognition of pathogen-derived effectors by plant R proteins , resulted in HR-type programmed cell death ( PCD ) , demonstrating chloroplast contribution to effector-triggered immunity ( ETI ) [24] . These evidences emphasize the importance of chloroplasts in plant immunity , and indicate the potential for future discoveries in this area of research . We show the disease resistance regulator OVEREXPRESSOR OF CATIONIC PEROXIDASE3 ( OCP3 ) is targeted to the chloroplast , and controls editing efficiency of plastid ndhB transcripts . We also show that NDH activity , and therefore CEF around PSI , is an important control point in plant immunity . Furthermore , a previously undescribed signaling pathway linking editing control with plant immunity via CEF activity modulation in the chloroplast was elucidated in this study .
OCP3 was classified as a transcription factor as it contained a 60-amino acid domain resembling a homeodomain and carried also two canonical bipartite nuclear localization signals [25] ( Figure 1A ) . These features were interpreted as indicative of targeting OCP3 to nuclei , where it would be functioning as a negative regulator of plant immunity and was congruent with ocp3 plants exhibiting a remarkable enhanced resistant to fungal pathogens due to a primed immune state [25]–[27] . However , when Arabidopsis were transformed with a gene construct carrying the fluorescent YFP protein fused to the OCP3 N-terminus or , alternatively , to the C-terminus ( i . e . 35S::YFP-OCP3 and 35S::OCP3-YPF constructs , respectively ) , confocal microscopy revealed different subcellular localizations for each protein ( Figure 1B ) . YFP-OCP3 expression led to YFP-specific fluorescence at dispersed positions within the cell , while that derived from OCP3-YFP was unequivocally localized to the chloroplast ( Figure 1B ) . These different protein localizations were reproduced upon transfecting tobacco protoplasts using the same constructs ( Supplemental Figure S1 ) . Western blot using an anti-GFP antibody , revealed YFP-OCP3 accumulated as a YFP immunoreactive band similar to that observed for free YFP ( Figure 1C ) . This was interpreted as partial trimming or processing of YFP-OCP3 . Conversely , the OCP3-YFP protein was stable , and accumulated as two low migrating immunoreactive bands; the molecular weight of these polypeptides congruent with that expected for a fusion OCP3 with YFP ( Figure 1C ) . Furthermore , the 35S::OCP3-YFP gene construct , but not 35S::YFP-OCP3 , complemented the ocp3 phenotype following stable transformation ( Figure 1D ) . The ocp3 mutant line carried a copy of the pathogen- and H2O2-responsive Ep5C::GUS transgene that became constitutively active in the mutant ( revealed after staining with X-gluc ) [25] , [28] , therefore , complementation was recorded as Ep5C::GUS expression repression ( Figure 1D ) . Eight independent ocp3/35S::OCP3-YFP lines were assayed , and all showed repressed GUS expression ( Figure 1D ) , indicative of effective complementation; in all cases the OCP3-YFP protein was targeted to the chloroplast ( Figure 1E ) . However , all 12 independent ocp3/35S::YFP-OCP3 transformed lines we generated retained GUS expression driven by the Ep5C gene promoter , demonstrating the inability of YFP-OCP3 to complement ocp3 . Inspection of OCP3 with TargetP ( http://www . cbs . dtu . dk/services/ChloroP/ ) revealed a predicted 69 amino acid chloroplast signal peptide ( SP ) ( Figure 1A ) . Correspondingly , N-terminal amino acid sequencing using Edman degradation of OCP3-YFP protein revealed processing at the predicted site ( Polyp2; Supplemental Figure S2A ) . Furthermore , fusion of the first 81 amino acids of OCP3 to YFP ( i . e . OCP31–81-YFP ) was sufficient to target and internalize YFP to the chloroplast ( Supplemental Figure S2B–C ) . Interestingly , chloroplast targeting , but not internalization , occurred when a short deletion ( from aa 68-to-74 ) was introduced in the constructs ( OCP3Δ68–74-YFP; Supplemental Figure S2B–C ) , indicating amino acids at position 68-to-74 were critical for proteolytic maturation of OCP3 in the chloroplast . These results were congruent with the absence of ocp3 complementation with deleted OCP3Δ68–74-YFP , and lack of mature OCP3-YFP protein accumulation in transformed plants ( Supplemental Figure S2D-E ) . Immunoblot analysis of chloroplast suborganellar fractionations derived from plants expressing OCP3-YFP revealed incorporation of the protein into the chloroplast , and enrichment in the stroma and thylakoid fractions ( Supplemental Figure S2F ) . Collectively , these results indicated a functional OCP3 protein resides in the chloroplast . OCP3-YFP distribution within the chloroplast showed protein accumulated in the form of speckles or punctate patterns . We noted similarities among proteins targeting different plastid structures or molecules , including plastoglobules ( i . e . PGL34 , [29] ) , plastid nucleoids ( PEND , [30] ) , targeting associated with introns containing RNAs ( i . e . WHIRLY , [31] ) and with RNAs undergoing editing ( i . e . pTAC2 ) ( Figure 2A ) . We co-expressed the OCP3-mCherry protein with PGL34-YFP , PEND-RFP , WHIRLY-GFP , or pTAC2-YFP in protoplasts to demonstrate possible co-localizations . OCP3-mCherry fluorescence overlapped consistently with the fluorescence derived from the pentatricopeptide repeat ( PPR ) protein pTAC2-YFP ( Figure 2B ) . Transcriptionally coordinated genes tend to be functionally related [32] . We hypothesized that identification of genes that are co-expressed with OCP3 ( at5g11270 ) in Arabidopsis would provide clues into the biological processes of OCP3 in the chloroplast . Therefore , we initially identified a co-expressed gene vicinity network for OCP3 using the AraGenNet platform ( http://aranet . mpimp-golm . mpg . de/ ) [33] where OCP3 matched cluster 49 ( Supplemental Figure S3 ) . Based on functional annotation using MapMan ontology terms ( http://aranet . mpimp-golm . mpg . de/ ) , the co-expression network contained 207 genes significantly enriched for biochemical and regulatory aspects related to chloroplast development and function ( see Table S1 ) . A closer vicinity network including only genes two steps away from OCP3 , identified 31 genes that were all related to plastid processes ( Figure 3A and Table S2 ) . Nine of these genes encoded PPR proteins , and the biological role in only one of these PPR genes , CRR21 ( at5g55740 ) , has been elucidated . CRR21 acts as a site-specific factor recognizing RNA editing site 2 ( ndhD-2 site ) in plastid ndhD transcript , suggesting that the Ser128Leu change has important consequences for the function of the NDH complex [13] . The remaining eight PPR genes , were tentatively named as follows: PPRa ( at4g21190 ) , PPRb ( at4g30825 ) , PPRc ( at3g29230 ) , PPRd ( at3g46610 ) , PPRe ( at5g14350 ) , PPRf ( at1g15510 ) , PPRg ( at3g14330 ) , and PPRh ( at3g49140 ) ( Figure 3A ) . As for pTAC2 , plastidial overlapping localization pattern was observed for OCP3-mCHERRY and PPRa-YFP ( Figure 3B–C ) . Similarly , OCP3-mCHERRY overlaps with CRR21-YFP both following the same punctate distribution pattern ( Figure 3B–C ) . The common co-localization pattern of OCP3 with different PPR proteins was not followed by all other proteins whose genes where co-expressed with OCP3 , as deduced from the non-overlapping localization pattern in OCP3-mCHERRY and AT1G63680-YFP ( Figure 3B ) . The observed common localization of OCP3 and different PPR proteins suggested involvement of OCP3 in some aspects of plastidial RNA editing processes To test the involvement of OCP3 in RNA editing , we systematically examined the editing status of chloroplast transcripts derived from wild type and ocp3 plants using high-resolution melting ( HRM ) screen of the 34 sites undergoing editing in Arabidopsis [7] . We identified major defects in the RNA editing of ndhB-6 , nhdB-4 , ndhB-3 , and ndhB-2 sites in ocp3 plants ( Figure S4 ) . The comparison of the sequencing electrophoregrams of the RT-PCR products surrounding the editing sites confirmed that editing was compromised at the four indicated sites , if not totally at least partially , in ocp3 plants ( Figure 4A and Figure S6 ) . All other known sites appeared similarly edited in ocp3 plants as in Col-0 plants . Editing defects were further confirmed by poisoned primer extension ( PPE ) assays ( Figure 4B–E ) . ndhB-6 , ndhB-4 , and ndhB-3 sites were edited in Col-0 at estimated efficiencies of approximately 72% , 95% , and 88% , respectively , while in ocp3 plants efficiencies were reduced approximately to 55% , 89% , and 72% , respectively ( Figure 4B and 4D ) . The ndhB-2 editing site possessed a contiguous cytosine residue adjacent to the cytosine to be edited , which impeded a reliable PPE assay . Therefore , ndhB-2 was not further studied by this method . The ndhB-5 site exhibited no editing variation between Col-0 and ocp3 , with efficiencies in the range of 82 . 5% and 81% , respectively; therefore , it served as an internal editing control site for the ndhB transcript not affected in ocp3 plants . Sequencing of individual cDNA clones , in sufficient quantities , is considered the most accurate method to measure editing extent , but is not cost effective for large-scale studies . We sequenced individual cDNA clones derived from RNAs obtained from equivalent leaves from Col-0 and ocp3 plants . cDNA cloning strategy was designed to include the ndhB-4 , ndhB-3 , and ndhB-2 sites in one amplicon ( amplicon I ) and the ndhB-6 site , along with non-altered ndhB-5 , and ndhB-7 sites , in another amplicon ( amplicon II ) . One hundred cDNA clones for each amplicon , and for each genotype were analyzed by direct sequencing . Editing efficiency comparison is shown in Figure 4F . For the ndhB-4 site , Col-0 showed a 92% ( 92 of 100 sequenced clones ) editing extent , which was reduced to 71% in ocp3 plants . Col-0 showed a 94% editing extent for ndhB-3 , reduced to 77% in ocp3 . The ndhB-2 site exhibited an editing extent of 86% in Col-0 , which was reduced to 64% in ocp3 . These values were comparable to those observed for the PPE assays . Interestingly , ocp3 plants exhibited concurrent editing inhibition at two sites within the same cDNA clone ( of the three potential ones in amplicon I ) in 21% of the sequenced clones while in Col-0 it was only 3% . Furthermore , lack of concurrent editing at the three sites remained notable in ocp3 , and was observed in 8% of the sequence clones while in Col-0 it was 0% . These results suggested the concomitant editing inhibition at more than one site on the same ndhB transcript was a common feature in editing defects in ocp3 plants . The ndhB-6 site showed an editing extent of 81% in Col-0 plants , which was reduced to a 61% in ocp3 plants ( Figure 4F ) . ndhB-5 and ndhB-7 served as controls for non-variation sites within the same transcript , and the editing extent was similar between Col-0 and ocp3 plants ( 83% reduced to 81% for ndhB-5; and 76% increased to 78% for ndhB-7 ) ( Figure 4F ) . Collectively these data indicated that OCP3 is required for efficient ndhB transcript edition . To directly assess the association of OCP3 with ndhB transcripts , leaves from Col-0 and from a transgenic line expressing a 35S::OCP3:GFP:HA gene construct were treated with folmaldehyde to generate protein-RNA cross-links and subsequently subjected to RNA immunoprecipitation ( RIP ) , an analysis that serves to detect the presence of the corresponding RNA in the protein immunoprecipitate by reverse transcription PCR ( RT-PCR ) . Immunoprecipitation of crude protein extracts with an anti-HA antibody selectively enriched the chimeric OCP3 protein in samples derived from the 35S::OCP3:YFP:HA overexpressing line ( Figure 4G , upper panel ) . Interestingly , the immunoprecipitated OCP3 complexes were shown to specifically co-precipitate ndhB transcripts as revealed by comparative RT-PCR analysis of the corresponding samples derived from the transgenic line and Col-0 plants ( Figure 4G , lower panel ) . ndhD transcripts , which served as a negative control , did not show association with the OCP3 complex ( Figure 4G , lower panel ) . The results thus indicate that OCP3 associates in vivo with ndhB transcript . Whether this association is the result of a direct interaction of OCP3 with the RNA molecule , or rather a consequence of the interaction of OCP3 with an RNA binding protein recognizing specifically RNA sequences of the ndhB transcript remains unknown . Future characterization of such protein complex and the elucidation of its associated biochemical function will shed light on how editing of the ndhB RNA at their multiple editing sites is regulated . Normal RNA editing at ndhB-6 , ndhB-4 , ndhB-3 and ndhB-2 sites converts a Ser codon to a Leu codon at aa279 , a Ser to Phe at aa249 , a His to Tyr at aa196 , and a Pro to a Leu at aa156 in the NdhB protein . NdhB is one of the eleven chloroplast-encoded subunits of the chloroplast NDH complex . We hypothesized ndhB editing defects observed in ocp3 plants would affect encoded protein function , which in turn would alter photosynthetic parameters in the mutant . NDH complex activity can be monitored as a transient increase in chlorophyll fluorescence reflecting plastoquinone pool reduction after turning off actinic light , as originally demonstrated by Shikanai et al . [11] . Figure 5A shows a typical chlorophyll fluorescence trace from Arabidopsis Col-0 and its comparison with crr21 , a mutant lacking NDH activity . In ocp3 , the post-illumination increase in chlorophyll fluorescence was modified in a manner similar to what occurred in crr21 plants , indicating that NDH activity was compromised . This result strongly indicated OCP3 is a chloroplast factor pivotal in normal NDH complex function . This important phenotype was confirmed by employing additional mutant alleles . Due to the absence of T-DNA insertions mutants for the OCP3 locus , and being the ocp3 mutant currently used a loss-of-function EMS mutant , we generated additional mutant alleles of this gene by artificial microRNA ( amiRNA ) interference . Two independent homozygous amiRNA lines ( i . e . amiRNA-2 and amiRNA-3 ) phenocopying the original ocp3 mutant ( Figure S5A–D ) , were selected . These lines were designated ocp3-2 and ocp3-3 , respectively , and the original ocp3 now designated ocp3-1 . Defective NDH activity was recorded in these mutants ( Figure 5A ) . Complementation of ocp3-1 plants with an OCP3 wild-type sequence fully restored the post-illumination increase of chlorophyll fluorescence ( Figure 5A ) . These results confirmed the importance of OCP3 for appropriate NDH complex function . RNA editing results in amino acid changes that directly alter protein translation , function , or even may act to destabilize multiprotein complexes . The NDH complex is unstable when NdhD subunit is absent due to editing-mediated translation defects [34] , [35] . The NdhB subunit defects observed in ocp3 plants were evaluated to determine the effects on NDH stability in vivo . Protein blots were analyzed using antibodies against the NdhI and NdhJ subunits , which served to monitor NDH complex stability ( Figure 5B ) . NdhI and NdhJ accumulation levels did not experience noticeable changes in ocp3 mutants compared to Col-0 plants . Similarly , NDH complex stability remained intact in crr21 plants ( Figure 5B ) . Although the exact function and organization of the whole set of subunits of the NDH complex in plants remains to be fully elucidated [36] , our results indicate that the four amino acid residues in the NdhB subunit which were derived from editing-mediated codon conversion appear important for activity , but not for assembly of the NDH complex . We hypothesized that via NDH complex inhibition , plants could develop an alerted immune status . This might explain why ocp3 plants exhibited enhanced disease resistance to fungal pathogens resulting from earlier and more intense callose synthesis and deposition following pathogen exposure [25] , [26] . If so , then mutants showing similar chloroplast NDH complex defects would activate the same immune status , and become resistant to fungal attack . Consequently , we challenged crr21 and crr2 mutants with P . cucumerina , and studied disease susceptibility in comparison to the resistant ocp3 plants , and the susceptible Col-0 plants . CRR2 is a distinct PPR protein that functions in the intergenic RNA cleavage between rps7 and ndhB , which is essential for subunit B translation , and crr2 mutants are compromised in NDH activity [35] . ppra , a previously uncharacterized T-DNA mutant , defective in the expression of PPRa ( Figure S8 ) encoding a PPR protein of unknown function that is highly co-expressed with CRR21 and OCP3 ( Figure 3A ) , was also evaluated . Similarly , pprb , a T-DNA mutant defective in another co-expressed PPR of unknown function ( Figure S7 ) was included in these experiments for comparison . Following inoculation with P . cucumerina , disease was scored 12 d after inoculation by following necrosis and chlorosis extent present in inoculated leaves . As expected , Col-0 plants were highly susceptible to P . cucumerina , and all inoculated plants showed extended necrosis accompanied by extensive proliferation of fungal mycelia ( Figure 6A–B ) . The same disease susceptibility was observed in the pprb mutant , indicating this PPR gene is not essential in plant's defense activation ( Figure 6A–B ) . In marked contrast , the inoculated crr21 , crr2 , and ppra plants responded with a substantial increase in disease resistance to P . cucumerina infection that was of a magnitude similar to that attained in ocp3 plants ( Figure 6A–B ) . Comparative cytological observations were performed at the sites of attempted fungal infection and the degree of induced callose deposition induction in inoculated leaves was monitored after staining with aniline blue , and examination by fluorescence microscopy . Results indicated none of the mutants exhibited aniline blue staining in control leaves ( Figure 6C ) . Col-0 and pprb plants deposited callose locally at sites demarcating the zones of extended fungal growth . In marked contrast , crr21 , crr2 , ppra , and ocp3 plants all exhibited intensified and highly localized callose deposition in response to fungal infection , which occurred at zones where fungal growth and colonization was impeded ( Figure 6C–D ) . Consequently , heightened disease resistance , and increased callose deposition were concurring traits in mutants defective in the correct editing of RNAs encoding subunits of the chloroplast NDH complex . The above results indicated that editing efficiency , chloroplast NDH activity , and disease resistance to fungal pathogens are linked traits mediated by OCP3 . Fungal infection provokes local down regulation of OCP3 in wild type plants [25] , therefore we hypothesized that following Col-0 inoculation with a fungal pathogen , editing inhibition of ndhB would very likely arise and , the NDH complex would consequently be affected . Therefore , we inoculated Col-0 plants with the fungal pathogen P . cucumerina and examined the editing status of RNAs corresponding to chloroplast-encoded subunits of the NDH complex ( i . e , NdhB , NdhF , NdhG and NdhD ) by bulk sequencing of RT-PCR products . We identified major alterations in the RNA edition of ndhB . Eight sites normally edited in the ndhB transcripts ( i . e . ndhB-1 to ndhB-8 ) showed inhibition at 48 h . p . i . with P . cucumerina ( Figure S6 ) . Results for the other NDH subunit RNAs indicated only the editing status of ndhD transcript was notably affected , and only at position 117166 ( ndhD-1 site ) which controls NdhD translation ( Supplemental Figure S6 ) . These results surpass the four distinct defective editing sites identified in the ocp3 mutant ( Figure S6 and Figure 4 ) . Therefore , in addition to OCP3 , other factors appeared to be targeted for the realization of the fungal-promoted editing inhibition . Temporal recording in a time course experiment following P . cucumerina inoculation revealed that editing inhibition is an early plant response to fungal attack . Most inhibition changes at the identified pathogen-sensitive ndhB editing sites were induced early following P . cucumerina inoculation ( at 12 h . p . i . ) , and were sustained up to 48 h . p . i ( Figure 7A ) , indicating the special vulnerability of ndhB editing to pathogenic cues . Results showed the specific editing defects at the ndhD-1 site lagged behind ndhB editing inhibition , reaching maximal inhibition at 48 h . p . i ( Figure 7A ) . Some of these early effects were further corroborated by specific PPE assays , which provided estimates that editing at ndhB-6 , ndhB-5 , and ndhB-3 sites were inhibited following pathogen inoculation at different efficiencies and declining rates . ndhB-6 editing inhibition was the most prominent , with an efficiency that abruptly dropped at 12 h . p . i . and progressively decayed thereafter ( Figure 7B ) . Following these previous observations we asked if stability of the NDH complex could become also altered following fungal infection . To assess this , NDH complex stability was monitored by Western blots using antibodies against one of the NDH subunits ( i . e . NdhI ) . We observed NdhI accumulation level decayed very early following pathogen inoculation , with apparent reduction occurring at 6 h . p . i . ( Figure 7C ) . The decay was progressive and showed an approximate 50% reduction in NdhI protein at 24 h . p . i . ( Figure 7C ) . The results suggested decay specificity for NDH complex protein , since chloroplast integrity , measured using other marker proteins ( i . e . , Pet-D , PSAD-2 and APX ) , did not change or even increase in response to the fungus ( Figure 7C ) . The decay process was set in motion at early stages of infection , and was inversely correlated with fungal growth ( Figure 7D ) . Furthermore , the observed pathogen-triggered dismantling of the NDH complex subunit preceded the activation of other plant responses , which are diagnostic of an activated plant immune response . Deposition of the cell wall β-1 , 3-glucan polymer callose , identified and quantified following aniline blue staining of inoculated leaves , clearly lagged behind observed editing defects and dismantling of NDH complex ( Figure 7E ) . Similarly , transcriptional activation of the defense related gene PDF1 . 2 followed editing defect accumulation ( Figure 7F ) . Furthermore , the inhibitory effect on NdhI accumulation was mirrored by Botrytis cinerea inoculation , another fungal pathogen ( Figure 7G ) . The decay in NDH subunit content promoted by B . cinerea ( Figure 7G , right graph ) was notable , but not as progressive as observed in P . cucumerina , presumably reflecting different infection styles for the two distinct fungal pathogens . The rapid editing inhibition and the parallel dismantling of the NDH complex constitutes two early chloroplast responses to pathogens , evoking integration of these processes as part of the mechanism governing immune response activation . Therefore , we verified if editing inhibition and NDH complex destabilization could be similarly triggered by application of chitosan ( 2-amino-2-deoxy- ( 1-4 ) -β-D glucopiranan ) , a naturally-occurring pathogen-associated molecular pattern ( PAMP ) compound able to elicit plant innate immune responses similar to those activated by complex fungal pathogens [37] . Results showed strong and rapid ( within 2 h ) down-regulation of NDH subunit accumulation was promoted by the sole application of chitosan to wild type Arabidopsis seedlings ( Figure 7H ) . Interestingly , we observed also an early and abrupt down-regulation of OCP3 , CRR21 and PPRa gene expression following chitosan treatment , which contrasted with the concurring high transcriptional activation of MYB51 ( Figure 7I ) , the latter a transcription factor required for PAMP-triggered callose deposition in Arabidopsis [38] . Since CRR21 , OCP3 and PPRa are nuclear-encoded chloroplast factors required for an effective plant immune response ( Figure 6 ) , the results indicate the existence of a nuclear PAMP-mediated transcriptional regulation of NDH complex-related editing regulatory genes as an integral component of innate immunity . This thus represents an additional layer of control of chloroplast NDH complex activity interconnecting nuclei and chloroplasts . Moreover , electrophoregrams comparison of bulk sequencing of RT-PCR products , generated at different times after chitosan treatment , revealed that chitosan-induced NDH complex dismantling was also accompanied by an early editing inhibition at seven ndhB editing sites , and at three ndhD editing sites ( Figure S7 ) . Therefore , these results provided additional support for the engagement of plastid RNA editing inhibition in plant immunity . Moreover , the observed rapid and transient dismantling of the NDH complex that follows perception of pathogenic cues suggests the engagement of a highly regulated proteolytic system in the chloroplast . The identification and characterization of such proteolytic system remains a challenge for the future .
This study provides new insights into the control of disease resistance in plants , and reinforces the importance of the chloroplasts in plant immunity . Our data identified OCP3 targeted to chloroplast , a finding that conceptually changed the previous assumption that OCP3 could function as a nuclear transcription factor . Furthermore , confocal microscopy revealed that OCP3 accumulated in plastids matching several PPR proteins . Moreover , OCP3 was found to be closely co-expressed with a cluster of 9 genes encoding PPR proteins including CRR21 . CRR21 is responsible for site 2 editing at ndhD transcript , and ndhD encodes the D subunit of the chloroplast NDH complex [13] , a crucial component of the CEF machinery around PSI [11] . In crr21 plants , NDH complex activity is impaired and CEF activity compromised [13] , supporting a predominant post-transcriptional level of control . All these observations prompted us to hypothesize that OCP3 was involved in RNA editing in plastids . Therefore , we performed a comparative systematic study of the editing status of chloroplast transcripts between Col-0 and different ocp3 mutants . This study revealed that OCP3-defective plants carry specific editing defects at ndhB-6 , ndhB-4 , ndhB-3 , and ndhB-2 sites . The observation that OCP3 associates in vivo with the ndhB transcript , as revealed by RIP assays , reinforce the consideration that OCP3 contributes to control over the extent of ndhB transcript editing . However , OCP3 appears not to carry any structural motif resembling the conserved RNA recognition motif ( RRM ) , not even the motifs characteristic of other proteins functioning as trans-factors essential for editing , such as those present in the large subclasses of the pentatricopeptide repeat ( PPR ) -containing family proteins [13] , [14] . This may suggest that the association of OCP3 with the ndhB RNA molecule may be likely indirect , presumably though the interaction with canonical RNA binding proteins recognizing appropriate cis-elements present in the ndhB RNA molecule such as those RNA-binding proteins mentioned above . Therefore , OCP3 may serve a regulatory role on the editing apparatus by regulating and/or adjusting the editing extent of the ndhB transcript according to external environmental cues . This appears to be the case also for other described editing accessory proteins such as the recently identified multiple organellar RNA editing factor ( MORF ) and members of the RNA-editing interacting protein ( RIP ) family [39] , [40] . ndhB encodes the NDH complex B subunit , therefore we next hypothesized that the absence of a functional OCP3 protein should result in a defective NDH complex . Results indicated that the observed alterations in ndhB editing in ocp3 plants affected NDH activity but not NDH complex stability . This is a feature also found in other editing-related mutants ( i . e . crr21 ) . However , in other cases , the defective gene results in lack of NDH complex accumulation , as seen when editing defects impedes translation initiation of NdhD subunit ( i . e . crr4 , [10] ) or when appropriate maturation of ndhB transcript is blocked ( i . e . crr2 , [35] ) . The editing defects in ocp3 plants resulted in an inactive NDH complex that compromised normal CEF around PSI . We therefore concluded that OCP3 is an integral plastidial factor required for fine-tuning CEF around PSI , and this control is exerted post-transcriptionally through the regulation of ndhB transcript editing . This finding has important consequences as it represents the first evidence interfacing plant immunity , RNA editing and CEF . Therefore , one can propose that when OCP3 fails , as occurs in ocp3 plants , then accurate ndhB transcript editing is impeded , and in turn NDH complex is altered and eventually CEF inhibited . Since in chloroplast the NDH complex is considered to alleviate various oxidative stresses [41] , [42] , it can be speculated that a defective CEF pathway could generate ROS locally that eventually may resulted in disease resistance activation . Since ocp3 plants carry constitutive enhanced production of ROS species , particularly H2O2 , and also show constitutive expression of ROS-inducible genes [25] , [28] , and OCP3 gene expression was rapidly down regulated following fungal attack [25] , we reasoned that editing will fail , and consequently NDH impaired , when a plant encounters a pathogen . This led us to find that Arabidopsis respond to attempted P . cucumerina infections by activating a rapid mechanism of editing inhibition which affected the 8 major editing sites in ndhB , therefore including those requiring OCP3 , plus an additional editing site at ndhD . This suggests involvement of other factors functioning as pathogen-sensitive regulators of the editing process . In fact , a similar cause-effect relationship was observed in crr2 , crr21 and ppra plants , which exhibited the same response as ocp3 plants when inoculated with P . cucumerina . Their characteristic heightened disease resistance was accompanied by increased callose deposition in response to fungal infection , evoking activation of a mechanism for priming of callose deposition in these mutants similar to that previously discovered in ocp3 plants [26] . In wild type plants , in addition to the pathogen-induced editing inhibition of ndhB transcripts , we observed the NDH complex becoming rapidly destabilized and therefore dismantled , presumably by the action of chloroplast proteases . Therefore , either NDH complex activity and/or stability constitute distinct hallmarks of the plant's defense response to fungal pathogens . Whether or not editing inhibition and NDH complex stability are linked processes or rather represent independent processes remains unknown and is a challenging issue for future research . Furthermore , strong repression of OCP3 and CRR21 gene expression , severe editing inhibition of ndhB and ndhD transcripts , and NDH destabilization were also rapidly triggered by the sole application of chitosan , which functions as a PAMP mimicking fungal structures . Consequently , editing inhibition and dismantling of the NDH complex appeared definitively engaged during activation of innate immunity . Therefore , when appropriately and timely activated following pathogen perception , the mechanisms leading to alteration of the NDH complex in the chloroplast should serve to set in motion a signaling process leading to an effective defense response to halt the advance of the pathogen . Cumulatively , these observations reinforced the idea that maintaining NDH complex integrity is pivotal to normal CEF functioning during photosynthesis , however its timely inhibition following pathogen attack is fundamental for plant immunity . Therefore , modulation of the NDH complex activity must be under a delicate balance , requiring precise but flexible control . The breadth of our data indicate control occurs at the RNA editing level , a process where the described proteins ultimately serve as sensors regulating the rate of NDH complex activity . Therefore , OCP3 , and presumably those PPRs and accessory proteins mediating editing extent of NDH complex subunits , exhibit a Janus-faced function , serving reciprocally as negative regulator of plant immunity and as positive regulator of CEF during oxygenic photosynthesis .
Arabidopsis thaliana plants were grown in a growth chamber ( 19–23°C , 85% relative humidity , 100 mEm−2 sec−1 fluorescent illumination ) on a 10-hr-light and 14-hr-dark cycle . All mutants are in Col-0 background . For the OCP3-GFP , -YFP and -mCHERRY constructs , the OCP3 full length cDNA was amplified by PCR using Pfu DNA polymerase ( Stratagene , San Diego , CA ) and specific primers including Gateway adapters , and recombined into pDONR221/207 using BP ClonaseMixII kit ( Invitrogen ) . After sequencing , all constructs were recombined into pEarleyGate101 destination vector using LR ClonaseMixII kit ( Invitrogen ) and introduced into ocp3 plants for complementation analysis or when indicated in Col-0 via Agrobacterium transformation . Cloning of the different ORFs employed in the present work and their fusion with the indicated fluorescent tag was done is a similar way . List of primers used for cloning purposes is provided in Supplementary information . Chlorophyll fluorescence was measured using a MINI-pulse-amplitude modulation portable chlorophyll fluorometer ( Dual-PAM-100 , Walz , Effeltrich , Germany ) . The transient increase in chlorophyll fluorescence after turning off actinic light ( AL ) was monitored as described [12] . Plant tissue was observed with a Leica TCS LS spectral confocal microscope using and HCX PL APO ×40/1 . 25-0 . 75 oil CS objective . GFP- or YFP-derived fluorescence was monitored by excitation with 488- and 514-nm argon laser lines , respectively , and emission was visualized with a 30-nm-width band-pass window centered at 515 nm . When RFP and CHERRY were used , excitation was performed by means of a 543-nm green-neon laser line , and fluorescence emission was collected at 695 to 630 nm . The artificial microRNA designer web WMD3 ( http://wmd3 . weigelworld . org/cgi-bin/webapp . cgi ) was employed for designing the 21 mer amiRNA sequence specific for OCP3 ( At5g11270 ) and for subsequent cloning and amplifications protocols . The target region selected in OCP3 was 5-′GCGTCGTAAAACTAGTATTAA-3 ( positions 625 to 645 ) and the 4 oligonucleotide sequences used to engineer the artificial miRNA into the endogenous miR319a precursor by site-directed mutagenesis were: As a template for the PCRs the pRS300 was used . The amiRNA sequence was cloned behind a 35S gene promoter and the binary vector used to transform Arabidopsis Col-0 plants . Eight independent transformants were initially selected and two homozygous lines showing remarkable reduced expression ( amiRNA-2 and amiRNA-3 ) of OCP3 were selected for further studies . Total RNA was extracted using TRIzol reagent ( Invitrogen ) following the manufacturer's recommendations and further purified by lithium chloride precipitation . For reverse transcription , the RevertAid H Minus First Strand cDNA Synthesis Kit ( Fermentas Life Sciences ) was used . Quantitative PCR ( qPCR ) amplifications and measurements were performed using an ABI PRISM 7000 sequence detection system , and SYBR-Green ( Perkin-Elmer Applied Biosystems ) . ACTIN2/8 was chosen as the reference gene . All 34 known Arabidopsis chloroplast RNA editing C targets were assayed by high resolution melt ( HRM ) as described [7] . Chloroplast RNA editing sites were assayed by RT-PCR bulk sequencing using similar set of primers . Primers for amplicons covering each of the 34 editing sites present in plastids are listed below in Supplemental information . Poison primer extension ( PPE ) analysis on chloroplast sites were conducted as described [43] and sequence of the corresponding fluorescent primers used are listed below in Supplemental information . Protoplasts isolation and transfection protocol with the different gene constructs was as described [44] . Protein crude extracts were prepared by homogenizing ground frozen leaf material with Tris-buffered saline ( TBS ) supplemented with 5 mM DTT , protease inhibitor cocktail ( Sigma-Aldrich ) . Protein concentration was measured using Bradford reagent; unless otherwise indicated 20 µg of total protein was separated by SDS-PAGE ( 12% acrylamide w/v ) and transferred to nitrocellulose filters . The filter was stained with Ponceau-S after transfer , and used as a loading control . Unless otherwise indicated , immunoblots were incubated with the indicated primary antibodies at the appropriate dilution and developed by chemiluminescence using an anti-IgG peroxidase antibody ( Roche ) at a 1∶1000 dilution and Western Lighting plus-ECL substrate ( Perkin-Elmer ) . Protein samples resolved by SDS-PAGE were blotted to PVDF membranes and the band of interest identified by Ponceau staining . The band sector corresponding to OCP3 was recovered and subjected to five/six cycles of automated microsequencing by sequential Edman degradation in an Applied Biosystems , Procise 494 . In both B . cinerea and P . cucumerina infections , five-week-old plants were inoculated as described [25] , [26] , with a suspension of fungal spores of 2 . 5×104 and 5×106 spores/mL respectively . The challenged plants were maintained at 100% relative humidity . Disease symptoms were evaluated by determining the lesion diameter of at least 50 lesions 3 or 12 days after inoculation . For pathogen-induced callose deposition analyses , infected leaves were stained with aniline blue and callose deposition quantifications were performed as described by Garcia-Andrade et al . [26] . Approximately 15 sterilized Col-0 seeds were sown per well in sterile 12-wells plates , containing filter-sterilized MS mediums without Gamborg's vitamins and with 0 , 5% of sucrose . Seedlings were cultivated under standard growth conditions ( 15 h day cycle; 20°C/17°C ) with a light intensity of 150 µM/m2/s . After 7 days , the growth medium was replaced by fresh MS medium . One day later , plants were mocked or challenged with chitosan at the final concentration of 10 µg/mL in the growth medium , and at the indicated times the samples were collected and immediately frozen in liquid nitrogen . Fractionation of total chloroplasts preparations from full expanded Arabidopsis leaves into stromal , thylakoids and membrane envelope was performed as described [45] . Each suborganellar fraction was identified and validated by developing Western blots with anti-BCCP ( stroma ) , anti-NIP ( thylakoids ) and anti-OEP21 ( membrane envelope ) antibodies . Antibodies were obtained from Uniplastomic ( Gieres , France ) . RIP assays were performed as described [46] with minor modifications . Essentially , 2 g of leaf tissue from Arabidopsis plants ( 4 weeks old plants ) were ground to a fine powder with a mortar and pestle in liquid nitrogen and homogenized in 12 . 5 mL/g lysis buffer ( 50 mM Tris-HCl , pH 7 . 4 , 2 . 5 mM MgCl2 , 100 mM KCl , 0 . 1% Nonidet P-40 , 1 µg/mL leupeptin , 1 µg/mL aprotonin , 0 . 5 mM phenylmethylsulfonyl fluoride , one tablet of Complete proteinase inhibitor tablet ( Roche ) , and 50 units/mL RNase OUT ( Invitrogen ) . Cell debris was pelleted by centrifugation for 5 min at 12 , 000 rcf at 4°C . Clarified lysates were incubated with 4 µg/mL of anti-HA antibody ( Roche ) for 15 min at 4°C and then with 100 µL of Protein-A agarose ( Roche ) per milliliter for 30 min at 4°C . Beads were washed six times for 10 min with lysis buffer at 4°C and then divided for protein and RNA analysis . RNAs were recovered by incubating the beads in 0 . 5 volumes of proteinase K buffer ( 0 . 1 M Tris-HCl , pH 7 . 4 , 10 mM EDTA , 300 mM NaCl , 2% SDS , and 1 µg/µL proteinase K ( Roche ) ) for 15 min at 65°C , extraction with saturated phenol , phenol∶chloroform∶isoamyl alcohol and chloroform , and ethanol precipitation . For RT-PCR assays , 1 µg of total RNA was used for the input fraction , and 20% of the RNA immunoprecipitate was used for the immunoprecipitation . PCR to amplify fragments corresponding to ndhB and ndhD cDNAs was done using specific oligos , CHLORO 187 FW/RV and CHLORO 212 FW/RV , respectively , as listed in Supplemental material . For protein blot assays , 10 µL of clarified eluate was loaded for the input fraction , and 3% of the immunoprecipitated beads was used for the immunoprecipitation . OCP3:GFP:HA was detected by immunoblotting and chemiluminescence using and anti-GFP peroxidase antibody ( Roche ) at a 1∶1000 dilution and Western Lighting plus-ECL substrate ( Perkin-Elmer ) . Homozygous lines of ppra and pprb T-DNA insertion mutants were identified by PCR using primers listed in Supplemental information . | Plastids originated from cyanobacteria that were incorporated into the eukaryotic cell through an endosymbiotic relationship . During the gradual evolution from endosymbiont to organelle , most genes of the cyanobacterial genome were transferred to the nuclear genome . Therefore , plastid biogenesis and function relies on nuclear gene expression and the import of these gene products into plastids , with the molecular dialogue between these two plant cell compartments therefore needing a precise coordination . Nuclei-to-chloroplast communication , and vice versa , are thus regulated through anterograde and retrograde signaling pathways , respectively . Post-transcriptional RNA editing of plastid RNAs by nuclear encoded regulatory proteins , such as pentatricopetide repeat ( PPRs ) proteins , represents one of such mechanisms of control . Through the characterization of the nuclear-encoded OCP3 protein , previously found to function as a disease resistance regulator in Arabidopsis , we have discovered a pathogen-sensitive editing-mediated control of the plastidial NDH complex involved in cyclic electron flow ( CEF ) around photosystem I . This led us to find that different PPRs controlling editing extent of transcripts for plastidial NDH complex are modulated by pathogenic cues . Our results thus represent the first series of evidence indicating engagement of chloroplast RNA editing and chloroplast NDH activity in plant immunity . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2013 | Mediated Plastid RNA Editing in Plant Immunity |
The large conductance , voltage- and calcium-dependent potassium ( BK ) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders . In addition to membrane depolarization , activation of the BK channel requires a rise in cytosolic calcium . Localization of the BK channel near calcium channels is therefore critical for its function . In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel , SLO-1 , we identified ctn-1 , which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin . ctn-1 mutants resemble slo-1 loss-of-function mutants , as well as mutants with a compromised dystrophin complex . We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons . In muscles , CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels . In neurons , CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex . Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains , thereby playing a crucial role in muscles and neurons .
Precise control of membrane excitability , largely determined by ion channels , is of utmost importance for neuronal and muscle function . The regulation of ion channel localization , density and gating properties thus provides an effective way to control the excitability within these cells [1] . Indeed , the localization and gating properties of ion channels are often regulated or modified by cytoskeletal and signaling proteins , or auxiliary ion channel subunits expressed in a cell-type specific manner [2] . Potassium channels are critical in determining the excitability of cells , because potassium ions are dominant charge carriers at the cell resting potential . Among potassium channels , the large conductance , voltage- and calcium-dependent potassium BK channels ( also called SLO-1 or Maxi-K ) are uniquely gated by coincident calcium signaling and membrane depolarization [3] , [4] . This feature of BK channels provides a crucial negative feedback mechanism for calcium-induced functions , and plays an important role in determining the duration of action potentials [3] . BK channels are widely expressed in a variety of cell types and are implicated in many physiological processes , including the regulation of blood pressure [5] , neuroendocrine signaling [6] , smooth muscle tone [7] , and neural network excitability [8] , [9] . Mounting evidence indicates that BK channels can interact with a variety of proteins that modulate channel function , or control membrane trafficking . For example , the Drosophila BK channel , dSLO , interacts with SLO binding protein ( slob ) , which in turn modulates the channel gating properties [10] . Similarly , mammalian BK channels associate with auxiliary beta subunits that influence channel activation time course and voltage-dependence [11] . In yeast two hybrid screens , the cytoplasmic C-terminal tail of mammalian BK channels has been shown to interact with several proteins , including cytoskeletal elements , such as actin-binding proteins [12] , [13] and a microtubule-associated protein [14] . These cytoskeletal proteins are partially co-localized with BK channels , and appear to increase cell surface expression of BK channels in cultured cells [12] , [13] . However , it remains to be determined whether these proteins have any role in controlling the localization of BK channels to specific areas of the plasma membrane in vivo . Robust activation of BK channels requires higher intracellular calcium concentrations ( >10 µM ) , which only occur in the immediate vicinity of calcium-permeable channels [4] . Hence , the localization of BK channels to specific areas ( i . e . calcium nanodomains ) where calcium-permeable ion channels are located is physiologically important for BK channel activation . In C . elegans , loss-of-function mutations in slo-1 partially compensate for the synaptic release defects of C . elegans syntaxin ( unc-64 ) mutants [15] and lead to altered alcohol sensitivity [16] . Recent studies in C . elegans have also implicated SLO-1 in muscle function [17] . slo-1 mutants display an exaggerated anterior body angle , referred to as the head-bending phenotype that is shared by mutants that are defective in the C . elegans dystrophin complex [18]–[20] . Recent evidence that the C . elegans dystrophin complex interacts with SLO-1 channels via SLO-1 interacting protein , ISLO-1 , explains this phenotypic overlap [21] . However , C . elegans dystrophin complex mutants do not appear to alter the biophysical properties of BK channels per se [17] . Similarly , ISLO-1 does not modify SLO-1 channel properties [21] . Rather , ISLO-1 tethers SLO-1 near the dense bodies of muscle membranes , where L-type calcium channels ( EGL-19 ) are localized [21] . Consequently , defects in the dystrophin complex or ISLO-1 cause a large reduction in SLO-1 protein levels in muscle membrane , which in turn causes muscle hyper-excitability leading to enhanced intracellular calcium levels . This perturbation of calcium homeostasis has been postulated to be one of the first steps in the degenerative muscle pathogenesis associated with disruption of the dystrophin complex [22] . In this study , we performed a forward genetic screen to identify additional genes responsible for SLO-1 localization and function in C . elegans . We identified ctn-1 , an orthologue of α-catulin , as a novel gene that controls SLO-1 localization and function in muscles and neurons . Our analysis showed that ctn-1 uses different strategies to localize SLO-1 in these two cell types . In muscles , CTN-1 utilizes the dystrophin complex to localize SLO-1 near L-type calcium channels via ISLO-1 . In neurons , CTN-1 localizes SLO-1 independent of the dystrophin complex .
Loss-of-function slo-1 mutants exhibit a jerky locomotion and head bending phenotype [15] . By contrast , gain-of-function slo-1 mutants exhibit sluggish movement combined with low muscle tone [16] . When slo-1 ( gf ) mutant animals are mechanically stimulated , they fail to make a normal forward movement , and tend to curl ventrally ( Video S1 ) . To identify genes that regulate slo-1 function , we performed a forward genetic screen to isolate mutants that suppress the phenotypes of the slo-1 ( ky399 ) gain-of-function mutant . Based on a previous genetic study [21] , suppressor genes were expected to encode slo-1 , components of the dystrophin complex , as well as novel proteins that control neuronal or muscular function of SLO-1 . As expected , several loss-of-function alleles of slo-1 were isolated . In addition to these intragenic suppressors , several mutants could be segregated away from slo-1 ( gf ) ( Figure 1A and Video S1 ) and exhibited the head bending phenotype . Genetic mapping and complementation testing determined that these extragenic suppressors include dyb-1 and stn-1 which encode two homologous components of the dystrophin complex , dystrobrevin and syntrophin respectively . Additionally we isolated cim6 and eg1167 suppressors that represent novel genes . Compared to slo-1 ( ky399 ) and cim6;slo-1 ( ky399 ) mutants , eg1167;slo-1 ( ky399 ) mutants exhibited a profound improvement in the locomotion speed ( Figure 1A ) . It was previously observed that slo-1 ( gf ) mutants retain significantly more eggs than wild-type animals due to low activity of the egg-laying muscles [16] . We found that suppressor mutants abolish an egg laying defect of slo-1 ( gf ) mutants and retain eggs in uteri at levels similar to wild-type animals ( Figure 1B ) . To understand the role of novel genes in slo-1 function , we pursued the identification of genes that mapped to chromosomal locations neither previously implicated in BK channel function , nor encoding known components of the dystrophin complex . Two mutations , cim6 and eg1167 , both mapped to the left side of chromosome I and failed to complement each other for head bending , suggesting that these two mutations represent alleles of the same gene . Our quantitative analysis for locomotion and egg laying phenotypes showed that the locomotion speed of eg1167;slo-1 ( gf ) was higher than that of cim6;slo-1 ( gf ) whereas egg laying was comparable in both strains ( Figure 1A and 1B ) . We further mapped eg1167 to a 250 kb interval and rescued the phenotype of eg1167 by generating transgenic animals with the fosmid WRM0621cC01 ( Figure S1 ) . Next , we rescued the head bending phenotype of eg1167 with a transgene consisting of the ctn-1 gene ( Y23H5A . 5 ) and approximately 4 kb upstream of the translation initiation codon ( Figure 2A and 2B ) . The same transgene caused eg1167;slo-1 ( gf ) double mutants to revert to the slo-1 ( gf ) phenotype , displaying sluggish movement and retention of late-staged eggs in uteri ( Figure 2C and 2D ) . These results indicate that a genetic defect in ctn-1 is responsible for suppression of the slo-1 ( gf ) phenotypes . The ctn-1 gene is orthologous to mammalian α-catulin ( 39 . 4% identity to human α-catulin ) , and is named on the basis of sequence similarity to both α-catenin and vinculin ( Figure 2A ) [23] . Vinculin and α-catenin are membrane-associated cytoskeletal proteins found in focal adhesion plaques and cadherens junctions . In C . elegans , vinculin ( DEB-1 ) is localized to the dense bodies of body wall muscle and is essential for attachment of actin thin filaments to the sarcolemma [24] , whereas α-catenin ( HMP-1 ) is localized to hypodermal adherens junctions and is essential for proper enclosure and elongation of the embryo [25] . Based on its homology to vinculin/α-catenin and the localization of mammalian α-catulin [26] , CTN-1 is likely to interact with other cytoskeletal proteins , which may in turn affect SLO-1 function . Additionally , the ctn-1 gene encodes a predicted coiled-coil domain . Such a coiled-coil domain mediates the interaction between dystrophin and dystrobrevin [27] , two components of the dystrophin complex , although we do not know if the coiled-coil domain of CTN-1 is important for the interaction with these proteins ( Figure 2A ) . We determined nucleotide sequence of the predicted exons and exon-intron boundaries of the ctn-1 gene in eg1167 and cim6 . The mutation sites found in both alleles create translation-termination codons ( R144>STOP in eg1167 , Q521>STOP in cim6 ) ( Figure 2A ) . eg1167 exhibits complete suppression of slo-1 ( gf ) phenotypes ( see below ) and is hence considered as a severe loss-of-function or null allele . All subsequent experiments were carried out with eg1167 , unless mentioned otherwise . Although both eg1167 and cim6 mutants alone exhibit the head-bending phenotype , they differ with respect to suppression of slo-1 ( gf ) phenotypes . Whereas ctn-1 ( eg1167 ) suppresses all aspects of the slo-1 ( gf ) phenotype , ctn-1 ( cim6 ) completely suppresses the egg-laying defect of slo-1 ( gf ) ( Figure 1B ) , but not the locomotory defect ( Figure 1A ) . These results suggest that the C-terminal third of CTN-1 is required for normal egg laying and head bending , but is not necessary to mediate the locomotion speed defect of slo-1 ( gf ) mutants . To elucidate the function of CTN-1 , we examined the expression pattern of the ctn-1 gene using a ctn-1 promoter-tagged GFP reporter ( Figure 2E–2H ) . We observed GFP fluorescence in body wall muscles , pharyngeal muscle , egg-laying muscle and enteric muscle of transgenic animals as well as in most , if not all , neurons of the nerve ring and ventral nerve cord . Based on the ctn-1 expression pattern and the phenotypic differences between eg1167 and cim6 , we investigated whether the head-bending phenotype and the suppression of sluggish movement of slo-1 ( gf ) mutants are separable by expressing ctn-1 minigenes under the control of either muscle- or neuron-specific promoters in ctn-1 and ctn-1;slo-1 ( gf ) mutant animals . Muscle , but not neuronal , expression of ctn-1 rescued the head-bending phenotype of the ctn-1 mutant ( Figure 2B and Figure S1C ) . These results are consistent with previous reports that the head-bending phenotype is due to perturbations in muscle function [17]–[19] . Furthermore , muscle expression of ctn-1 in ctn-1;slo-1 ( gf ) mutants resulted in egg retention to the level observed in slo-1 ( gf ) mutants , whereas neuronal expression of ctn-1 did not alter the number of eggs retained in the uteri of ctn-1;slo- ( gf ) mutants ( Figure 2C ) . Conversely , neuronal expression of ctn-1 in ctn-1;slo-1 ( gf ) mutants reverted the seemingly normal locomotion of ctn-1;slo-1 ( gf ) to the sluggish , uncoordinated locomotion of the slo-1 ( gf ) mutant , whereas muscle expression of ctn-1 did not ( Figure 2D ) . These results indicate that the sluggish , uncoordinated locomotory phenotype of slo-1 ( gf ) mutants comes from presynaptic depression , but not from direct suppression of muscle excitability . Together with the allele specific phenotypic differences indicating different regions of CTN-1 are required for normal locomotory speed and head bending , these results suggest that CTN-1 uses two distinct mechanisms for mediating SLO-1 function in muscle and neurons by interacting with different sets of genes . Most , if not all , of the mutants that exhibit the head bending phenotype have a defect in either a component of the dystrophin complex or proteins that interact with the dystrophin complex [17]–[19] . The dystrophin complex is localized near muscle dense bodies [21] . Because ctn-1 mutants exhibit the head bending phenotype , we determined the subcellular localization of CTN-1 using a GFP-tagged CTN-1 transgene , which rescues the head bending phenotype ( data not shown ) . GFP::CTN-1 exhibited a punctate expression pattern that resembled that of the dense bodies ( Figure 3A ) . To further define the localization of CTN-1 , we stained GFP-tagged CTN-1 transgenic animals with GFP antibodies and vinculin/DEB-1 antibodies that recognize the attachment plaque and dense bodies . CTN-1::GFP is localized in close proximity to , or partially colocalized with , vinculin/DEB-1 in dense bodies , but not in the attachment plaques , indicating that CTN-1 is localized near dense bodies ( Figure 3A ) . This expression pattern of CTN-1 , along with the head bending phenotype of ctn-1 mutants , prompted us to examine whether the ctn-1 mutation disrupts the integrity of the dystrophin complex . We compared the expression pattern of a component of the dystrophin complex , SGCA-1 ( an α-sarcoglycan homolog ) in wild-type , dys-1 , slo-1 and ctn-1 animals using a GFP-tagged SGCA-1 that rescues the head bending phenotype of sgca-1 mutants [21] ( Figure 3B ) . GFP::SGCA-1 exhibited a punctate expression pattern in the muscle membrane of wild-type and slo-1 mutant animals . By contrast , GFP puncta were greatly diminished in dys-1 and ctn-1 mutants . These results indicate that ctn-1 is critical for maintaining the dystrophin complex near the dense bodies . We previously demonstrated that ISLO-1 interacts with STN-1 through a PDZ domain-mediated interaction , thereby linking SLO-1 to the dystrophin complex [21] . Because we failed to observe a component of the dystrophin complex in the muscle membrane of ctn-1 mutants , we examined mCherry-tagged ISLO-1 in the muscle membrane of wild-type and ctn-1 mutant animals . The punctate mCherry::ISLO-1 fluorescence was observed in wild-type muscle membranes , but was greatly reduced in ctn-1 mutant ( Figure 3C ) . These results further strengthen the notion that CTN-1 is required for maintaining the integrity of the dystrophin complex . Based on the genetic interaction between ctn-1 and slo-1 , and the observation that the integrity of the dystrophin complex and ISLO-1 localization are disrupted in ctn-1 mutants , we hypothesized that CTN-1 regulates the localization of SLO-1 in muscle . To test this hypothesis , we examined the localization of GFP-tagged SLO-1 in muscles of wild-type , dys-1 , and ctn-1 animals ( Figure 4A and 4B ) . The punctate SLO-1::GFP expression pattern in the muscle membrane of wild-type animals was greatly diminished in the muscles of either dys-1 or ctn-1 mutant . Interestingly , the protein levels of SLO-1::GFP were not significantly different in wild-type , dys-1 and ctn-1 animals ( Figure S2B ) , indicating that mislocalized SLO-1 does not necessarily undergo degradation . The mislocalization of SLO-1 in dys-1 mutants is consistent with the requirement of the dystrophin complex for ISLO-1 localization [21] . These results further indicate that CTN-1 stabilizes or maintains the punctate muscle expression of SLO-1::GFP in a dystrophin complex-dependent manner . In mammals , BK channels are found in neuronal somata , dendrites and presynaptic terminals [28] , [29] . An immunoelectron microscopy study indicates that BK channels are not homogeneously distributed in neurons , but are clustered , presumably near calcium channels [30] . We addressed whether SLO-1 is evenly distributed or clustered in C . elegans neurons by examining SLO-1::GFP . Wild-type animals displayed patches of fluorescence along the ventral nerve cord or near cell bodies under high magnification ( Figure 4C and 4D , Figure S2 ) . Tissue-specific rescue experiments demonstrated that ctn-1 mediates SLO-1 function in neurons independent of the dystrophin complex ( Figure 2D ) . Therefore , we compared neuronal SLO-1::GFP expression in dys-1 and ctn-1 mutant animals . The clustered GFP expression observed along the ventral cord of both wild-type and dys-1 mutant animals contrasted with the uniform GFP localization in ctn-1 mutants ( Figure 4C and 4D ) . These results indicate that ctn-1 mutation disrupts the neuron-specific clustering of SLO-1::GFP independent of the dystrophin complex . SLO-1 contributes to the repolarization of the synaptic terminal following neuronal stimulation , thereby terminating neurotransmitter release . Consequently loss-of-function slo-1 mutants are hypersensitive to the paralyzing effects of aldicarb , an acetylcholinesterase inhibitor , a phenotype indicative of enhanced acetylcholine release . Consistent with this interpretation , electrophysiological recordings from neuromuscular junctions of slo-1 loss-of-function mutants exhibit prolonged evoked synaptic responses [15] , [16] . If CTN-1 regulates SLO-1 localization in motor neurons and thus slo-1 function , we would expect ctn-1 mutants to exhibit similar pharmacological and synaptic changes . Indeed , we found that ctn-1 mutants were hypersensitive to aldicarb compared to wild-type animals ( Figure S3 ) . To confirm this observation directly , we measured synaptic responses from the neuromuscular junctions of dissected wild-type and ctn-1 mutant animals engineered to express channelrhodopsin-2 in motor neurons [31] ( Figure 5 ) . Evoked synaptic responses were elicited by blue light activation of channelrhodopsin-2 and recorded from voltage-clamped post-synaptic body wall muscle cells . Consistent with our pharmacological data and localization results , recordings from ctn-1 showed prolonged evoked synaptic responses similar to those of slo-1 ( lf ) mutants ( Figure 5B and 5C ) . Furthermore , muscular expression of ctn-1 in ctn-1 mutant animals rescued the head-bending phenotype ( Figure 2B ) , but did not rescue prolonged evoked synaptic responses ( Figure S3B ) . These data strongly suggest that altered synaptic responses of ctn-1 mutants result from a neuronal defect . In contrast to the slo-1 ( lf ) mutants , evoked responses of slo-1 ( gf ) mutants were short-lived ( Figure 5C ) , and the charge integral , a measure of total ion flux during the evoked response , was significantly reduced ( Figure 5E ) . Our genetic analyses demonstrated that the ctn-1 mutation suppresses the sluggish locomotory phenotype of slo-1 ( gf ) mutants and disrupts SLO-1 localization ( Figure 1A , Figure 4C and 4D ) . If this is due to loss of neuronal SLO-1 ( gf ) channels , the ctn-1 mutation should suppress the evoked response defects of slo-1 ( gf ) . Consistent with this prediction , the decay time of the ctn-1;slo-1 ( gf ) double mutants ( t1/2 = 6 . 61±0 . 53 ms ) was significantly longer than slo-1 ( gf ) ( t1/2 = 3 . 23±0 . 21 ms ) ( Figure 5D ) , and the charge integral was restored to wild-type levels ( Figure 5E ) . Interestingly , ctn-1 mutants did not convert the decay time of slo-1 ( gf ) evoke responses to that of slo-1 ( lf ) , indicating that residual SLO-1 function may be mediated by dispersed SLO-1 channels .
In a genetic screen to identify novel regulators of SLO-1 , we found two alleles of ctn-1 , a gene which encodes an α-catulin orthologue . CTN-1 mediates normal bending of the anterior body through SLO-1 localization near the dense bodies of body wall muscles . CTN-1 also maintains normal locomotory speed through SLO-1 localization within neurons . Based on our data , we propose a model for ctn-1 function in localizing SLO-1 ( Figure 6 ) . In muscles , CTN-1 interacts with the dystrophin complex . It is also possible that CTN-1 may influence the stability of another protein that directly interacts with the dystrophin complex . Loss of CTN-1 function disrupts the integrity of the dystrophin complex , thus compromising ISLO-1 and SLO-1 localization near muscle dense bodies , where L-type calcium channels are present . Disruption of SLO-1 localization is expected to uncouple local calcium increases from SLO-1-dependent outward-rectifying currents , resulting in muscle hyper-excitation . Previous studies have shown that the head bending phenotype , shared among mutants that have a defect in the dystrophin complex or its associated proteins , results from muscle hyperexcitability [17]–[19] , [32] . Our data further show that this head-bending phenotype does not result from a synaptic transmission defect , but from a muscle excitation and contraction defect . In neurons , SLO-1 localization is not mediated through the dystrophin complex , suggesting that CTN-1 interacts with other proteins to localize SLO-1 to specific neuronal domains . Why does CTN-1 use two distinct mechanisms to localize SLO-1 to subcellular regions of muscles and neurons ? BK channels are functionally coupled with several different calcium channels ( including voltage-gated L-type and P/Q-type calcium channels and IP3 receptors ) that are localized in different subcellular regions [30] , [33] , [34] . Although it has not been determined whether all of these calcium channels are functionally coupled with SLO-1 in C . elegans , these calcium channels are distributed in different regions of neurons . For example , the L-type calcium channel ( EGL-19 ) is mainly expressed in the cell body and the P/Q type calcium channel ( UNC-2 ) is concentrated at the presynaptic terminals [35] , [36] . A distinct set of proteins is perhaps required for SLO-1 channel localization near different calcium channels . How CTN-1 interacts with the dystrophin complex in muscle remains to be determined . It has been suggested that mammalian α-catulin interacts with the hydrophobic C-terminus of dystrophin resulting from alternative splicing [37] . However , the C . elegans dys-1 gene does not encode a hydrophobic C-terminus . Thus , CTN-1 may interact with a different domain of dystrophin , or with another component of the dystrophin complex . In this regard , it is noteworthy that both mammalian dystrophin and C . elegans DYS-1 have multiple spectrin repeat domains , and that the N-terminal region of vinculin which exhibits homology to that of α-catulin ( Figure S1 ) is known to bind the spectrin repeat domain of α-actinin [38] . By extension , we speculate that the N-terminal region of CTN-1 may bind the spectrin repeat domain of DYS-1 directly . Alternatively , the coiled-coil domain of dystrophin , which is known to interact with the coiled-coil domain of dystrobrevin [27] , may potentially bind the coiled-coil domain of CTN-1 . Interestingly , CTN-1 exhibits high homology to vinculin in both the N-terminal and C-terminal regions ( Figure S1B ) . The C-terminal region of vinculin interacts with cytoskeletal molecules or regulators ( F-actin , inositol phospholipids and paxillin ) in focal adhesion and adherens junctions [39] . Because the C-terminal region of CTN-1 is also necessary for normal head bending , we speculate that this C-terminal region may be important for tethering the dystrophin complex to other cytoskeletal proteins . In mammalian striated muscle , dystrophin is enriched in costameres [40] which are analogous to C . elegans dense bodies . A costamere is a subsarcolemmal protein assembly that connects Z-disks to the sarcolemma , and is considered to be a muscle-specific elaboration of the focal adhesion in which integrin and vinculin are abundant . Compromised costameres have been postulated to be an underlying cause of several different myopathies [40] . It was recently shown that ankyrin-B and -G recruit the dystrophin complex to costameres [41] . Based on overall high homology of ctn-1 to vinculin and α-catenin , we speculate that CTN-1 similarly interacts with cytoskeletal proteins in the dense bodies , and links the dystrophin complex to the dense bodies . Another intriguing conclusion from our data is that loss of CTN-1 does not completely abolish SLO-1 function . Complete abolishment of SLO-1 function in ctn-1 mutant should alter the decay time for evoked synaptic responses of ctn-1;slo-1 ( gf ) to the same degree as slo-1 ( lf ) mutants , rather than to that of wild-type animals ( Figure 5D ) . Mutants including slo-1 ( gf ) , that have defects in neural activation or membrane depolarization , are reported to cause str-2 , a candidate odorant receptor gene , to be expressed in both AWC olfactory neurons whereas wild-type animals express str-2 in only one of the AWC pair [42] . We find that ctn-1 mutation does not suppress the misexpression of str-2 in both AWC neurons in slo-1 ( gf ) mutants , suggesting that ctn-1 mutations do not completely abolish SLO-1 function ( unpublished observations , HK ) . It is thus likely that the defect in SLO-1 localization in ctn-1 mutants makes it less responsive to local calcium nanodomains found at presynaptic terminals and dense bodies , but still able to respond to depolarization-induced global calcium increases , albeit at a lower level . In conclusion , we have identified ctn-1 , a gene encoding the C . elegans homolog of α-catulin , and demonstrated that CTN-1 mediates SLO-1 localization in muscles and neurons by dystrophin complex-dependent and -independent mechanisms , respectively . How SLO-1 is localized to certain neuronal domains will require further screening of slo-1 ( gf ) suppressor mutants . Given that proteins affecting components of the dystrophin complex are likely to contribute to the pathogenesis of muscular dystrophy , α-catulin is a candidate causal gene for a form of muscular dystrophy in humans .
The genotypes of animals used in this study are: N2 ( wild-type ) , CB4856 , dys-1 ( eg33 ) I , stn-1 ( tm795 ) I , ctn-1 ( eg1167 ) I , ctn-1 ( cim6 ) I , slo-1 ( eg142 ) V , slo-1 ( ky399gf ) V and sgca-1 ( tm1232 ) X . The following transgenes were used in this study: cimIs1[slo-1a::GFP , rol-6 ( d ) ] [21] , cimIs5[mCherry::islo-1 , ofm-1::GFP] [21] , zxIs6[unc-17::chop-2 ( H134R ) -yfp; lin-15 ( + ) ] [31] , cimIs6[GFP::sgca-1 , rol-6 ( d ) ] , cimEx5[ctn-1 , ofm-1::GFP] , cimIs7[GFP::ctn-1 , rol-6 ( d ) ] , cimEx6[Pmyo-3ctn-1 , Pmyo-3GFP , ofm-1::GFP] and cimEx7[PH20ctn-1 , PH20GFP , ofm-1::GFP] . Gain-of-function slo-1 ( ky399 ) mutants were mutagenized by exposure to 50 mM EMS ( ethane methyl sulfonate ) for 4 h [43] . Suppressors that suppress or ameliorate the sluggish locomotory phenotype of slo-1 ( ky399gf ) mutants were selected from F2 progeny of the mutagenized animals . We screened approximately 5 , 000 haploid genome size for suppressor mutants and identified a total of 17 suppressor mutants . Genetic analysis of these suppressor mutants indicates that three of these have a second mutation in the slo-1 gene . In addition , we found that eight have mutations in genes causing head-bending phenotype ( 2 alleles of dyb-1 , 3 alleles of stn-1 and 2 alleles of ctn-1 ) . The remaining six mutants do not exhibit distinct locomotory phenotypes when segregated from slo-1 ( gf ) . For genetic mapping , slo-1 ( ky399 ) mutants were outcrossed 12 times to the CB4856 strain . The resulting strain was used for SNP ( single nucleotide polymorphism ) mapping [44] . Alternatively , we used CB4856 as a mapping strain when mapping is based on the head-bending phenotype . For transgenic rescue , fosmid clones purchased from Gene services Inc . ( Cambridge , UK ) were injected into the gonad of ctn-1 mutant at 2 ng/µl along with ofm-1::GFP marker ( 30 ng/µl ) . Once we rescued the head bending phenotype of ctn-1 with a single fosmid , we rescued ctn-1 mutant with a genomic DNA fragment encompassing the entire coding sequence of ctn-1 and approximately 4 kb upstream of the putative translation site . To verify the predicted coding sequence of ctn-1 , we first performed BLAST search analysis using the genomic sequences of C . briggsae and C . remanei . This analysis suggested that the first and 12th exons are longer than predicted in WormBase ( WS208 ) , and that an additional exon ( 10th exon ) is present . Second , we sequenced C . elegans ORF ctn-1 clone ( 9349620 ) and confirmed the 10th and the 12th exon sequences . Third , we performed sequence analysis of the DNA fragment obtained from RT-PCR with a primer set ( SL1 and an internal primer ) and identified the trans-splicing site which is 29 bp upstream of the newly-defined translation initiation site . Our analysis indicates that ctn-1 encodes a predicted protein with 784 amino acids ( Figure S1B ) . The ctn-1 genomic DNA ( approximately 4 kb upstream of the promoter and the entire coding sequence ) was amplified by the expand long template PCR system ( Roche Applied Science ) and used directly for rescue . For PH20ctn-1 and Pmyo-3ctn-1 constructs , the neuron-specific H20 [45] or muscle-specific myo-3 promoter sequences were fused to the translation initiation site of the ctn-1 genomic DNA in frame by the overlapping extension PCR ( Roche ) . For localization of CTN-1 , we inserted the GFP sequence to the translation initiation site of ctn-1 cDNA , and then the ctn-1 promoter sequence was inserted before the GFP sequence . The resulting construct rescued the head-bending phenotype of ctn-1 mutants and was used for generating integrated transgenic animals . For GFP::sgca-1 construct , the GFP sequence was inserted in-frame right after the signal sequence of sgca-1 open-reading frame as described previously [21] . Transgenic strains were made as described [46] by injecting DNA constructs ( 2–10 ng/µl ) along with a co-injection marker DNA ( pRF4 ( rol-6 ( d ) ) or ofm-1::GFP ) into the gonad of hermaphrodite animals at 100 ng/µl . We obtained at least 3 independent transgenic lines for rescue , and found that all lines show similar results . To remove bacteria attached to animals , approximately fifteen age-matched ( 30 hr after L4 stage ) hermaphrodite animals for each genotype were placed on a NGM ( nematode growth medium ) agar plate without bacteria for 15 min . The animals were then placed inside one of two copper rings embedded in a NGM plate . We found that age of agar plate influences the speed of animals , probably because the surface tension resulting from the liquid surrounding animals slows down movement . We used approximately one week-old plates for our assay , and compared with the speed of wild-type control animals . Video frames from two different genotypes were simultaneously acquired with a dissecting microscope equipped with Go-3 digital camera ( QImaging ) for 2 min with a 500 ms interval and 20 ms exposure . We measured the average speed of animals by using Track Objects from ImagePro Plus ( Media Cybernetics ) . The activity of egg laying muscle was measured indirectly by counting eggs retained in uteri . Single age-matched ( 30 hrs post-L4 ) animals ( total 15 for each genotype ) were placed in each well of a 96 well plate that contains 1% alkaline hypochlorite solution . The eggshells protect embryos from dissolution by alkaline hypochlorite . After 15 min incubation , the remaining eggs were counted in each well . Body curvature analysis was previously described [47] . A single animal was transferred to an agar plate and its movement was recorded at 20 frames per second . We limited image acquisition within 15 to 60 seconds after transfer , because the head bending phenotype is prominent when animals are stimulated to move forward rapidly . A custom-written software automatically recognizes the animal and assigns thirteen points spaced equally from the tip of nose to the tail along the midline of the body , and produces the pixel coordinates of thirteen points . First supplementary angles were calculated from the coordinates of the first three points with MATLAB software . First angle data were obtained when the head swing of an animal reached the maximal extension to the dorsoventral side . Mixed stage worms were washed and collected in M9 buffer . Equal volume of 2× Laemmli sample buffer was added to the worm pellets . The resulting worm suspension was heated at 90 °C for 10 min , centrifuged at 20 , 000 g for 10 min , and then immediately loaded on 7 . 5% SDS-PAGE gel . The Western blot analysis was performed using anti-GFP antibody ( Clontech , JL-8 ) and anti-α-tubulin antibody ( Developmental hybridoma bank , AA4 . 3 ) . Fixation and immunostaining procedures are previously described [21] . Fluorescence images were observed under a Zeiss Axio Observer microscope with 40× objective ( water-immersion , NA: 1 . 2 ) or an Olympus Fluoview 300 confocal microscope with a 60× objective ( oil-immersion , NA: 1 . 4 ) or 100× objective ( oil-immersion , NA: 1 . 4 ) . We typically observed more than 50 animals for each genotype . Images for quantification were acquired under an identical exposure time , gains and pinhole diameter . The intensity of puncta from acquired images was analyzed using linescan ( Metamorph , Molecular Devices ) and presented as values obtained by subtracting background levels from the peak grey levels of puncta . Electrophysiological methods were as previously described [48] . Briefly , animals raised on 80 µM retinal plates , were immobilized with cyanoacrylic glue and a lateral cuticle incision was made to expose the ventral medial body wall muscles . Muscle recordings were made in the whole-cell voltage-clamp configuration ( holding potential −60 mV ) using an EPC-10 patch-clamp amplifier and digitized at 2 . 9 kHz . The extracellular solution consisted of ( in mM ) : NaCl 150; KCl 5; CaCl2 5; MgCl2 4 , glucose 10; sucrose 5; HEPES 15 ( pH 7 . 3 , ∼340mOsm ) . The patch pipette was filled with ( in mM ) : KCl 120; KOH 20; MgCl2 4; ( N-tris[Hydroxymethyl] methyl-2-aminoethane-sulfonic acid ) 5; CaCl2 0 . 25; Na2ATP 4; sucrose 36; EGTA 5 ( pH 7 . 2 , ∼315mOsm ) . All of the animals carry a transgene ( zxIs6 ) that expresses channelrhodopsin-2 under the control of the cholinergic motor neuron ( unc-17 ) -specific promoter . Evoked currents were recorded in a body-wall muscle after eliciting neurotransmitter release by a 10 ms illumination using a 470 nm LED ( Thor labs ) triggered with a TTL pulse from the EPC10 pulse generator [31] . Evoked post-synaptic responses were acquired using Pulse software ( HEKA ) run on a Dell computer . Subsequent analysis and graphing was performed using Pulsefit ( HEKA ) , Mini analysis ( Synaptosoft Inc ) and Igor Pro ( Wavemetrics ) . The data were analyzed with one-way ANOVA followed by Dunnett's multiple comparison . | Calcium ions are essential for many physiological processes , including neurosecretion and neuronal and muscle excitation . Paradoxically , abnormal accumulation of calcium ions is associated with cell death and has been documented as an early event in muscle and neural degenerative diseases . One mechanism to avoid detrimental calcium accumulation is to link the calcium increase with activation of calcium-dependent potassium ion channels , thereby reducing cell excitability and preventing further calcium influx . This negative feedback requires these potassium channels to be localized in close proximity to sites of calcium entry . In a Caenorhabditis elegans genetic screen , we identified α-catulin , known as a cytoskeletal regulatory protein in mammals , important for the localization of calcium-dependent potassium channels in both muscles and neurons . In muscle , α-catulin controls the localization of the dystrophin complex , a multimeric protein complex implicated in muscular dystrophy . The dystrophin complex in turn tethers the calcium-dependent potassium channels near calcium channels . In neurons , the α-catulin-mediated localization of the potassium channels is independent of the dystrophin complex . Lack of α-catulin results in mislocalization of the potassium channels , and in turn causes defects in neuromuscular function . Our results support the idea that cytoskeletal proteins function as anchor molecules that localize ion channels to specific cellular domains . | [
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] | 2010 | An Alpha-Catulin Homologue Controls Neuromuscular Function through Localization of the Dystrophin Complex and BK Channels in Caenorhabditis elegans |
Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches . Amongst these , S-palmitoylation is unique in that it confers hydrophobicity . Due to technical difficulties , the understanding of this modification has lagged behind . To investigate principles underlying dynamics and regulation of palmitoylation , we have here studied a key cellular protein , the ER chaperone calnexin , which requires dual palmitoylation for function . Apprehending the complex inter-conversion between single- , double- and non- palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling . We found that calnexin , due to the presence of two cooperative sites , becomes stably acylated , which not only confers function but also a remarkable increase in stability . Unexpectedly , stochastic simulations revealed that palmitoylation does not occur soon after synthesis , but many hours later . This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells . Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition , most of which is degraded . This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6 .
Reversible post-translational modifications of proteins allow cells to regulate processes in time and in space [1–5] . Amongst these , S-palmitoylation is unique in that in confers hydrophobicity to proteins by covalent attachment of a fatty acid chain to cysteine residues [6 , 7] [8] . In the cytoplasm , this enzymatic reaction is mediated by palmitoyltransferases of the DHHC family and reversed by acyl protein thioesterases ( APTs ) [6 , 7 , 9] . Recent large-scale palmitoyl-proteome profiling studies have jointly revealed that hundreds of proteins , with major cellular functions , undergo this lipid modification in mammalian cells [10–14] . Although S-palmitoylation was identified more than 30 years ago , our understanding of this modification , its dynamics , its regulation and its consequences on protein properties , is still rudimentary . The aim of this paper is to study the palmitoylation events , and their dynamics , occurring on a key component of the endoplasmic reticulum ( ER ) , the type I transmembrane protein calnexin . Here we report the step-by-step design and output analysis of the first model of a palmitoylation network . Besides studying palmitoylation , another valuable objective of this work has been the estimation of system parameters which cannot be estimated by simple experiments , such as the time required for calnexin to get double palmitoylated or the half life of the palmitoylated species , but instead , they require the consideration of the system as a whole . Calnexin is best known for its function as a lectin-like chaperone involved in the folding of glycosylated proteins in the lumen of the ER [15] . It is also involved in regulating calcium homeostasis at ER-mitochondria contact sites [16] . More recently , we have found that calnexin can act as an ER sensor , modulating the transcriptional response of cells to EGF in an ER-stress dependent manner [17] . Importantly , the ability of calnexin to assist folding of newly synthesized proteins , to control calcium signalling and to modulate the EGF signalling response , all require its palmitoylation [16–19] . Calnexin is composed of a large well-folded N-terminal luminal domain that carries the chaperone activity [20] . It is followed by a single transmembrane domain that terminates with two cysteine residues at positions 502 and 503 , which are the sites of palmitoylation [18 , 19] . Even though the ER contains numerous DHHC enzymes [21] , calnexin palmitoylation is mediated exclusively by DHHC6 [19] . The palmitoylation sites are followed by a 90 residue cytosolic tail that is predicted to be disordered and contains multiple phosphorylation sites [20] . This cytosolic domain has multiple functions . It allows association of calnexin with the ribosome translocon complex [19 , 22] but can also be proteolytically released following specific stimuli such as apoptotic drugs [23] or EGF [17] . In the latter case , the released cytosolic tail binds to PIAS3 ( Protein Inhibitor of Activated STAT ) and thereby promotes EGF-induced STAT3-mediated transcriptional response to EGF [17] . Since calnexin has two sites of palmitoylation , it can exist in cells under different forms: non-modified , palmitoylated on one site or the other , or on both . To grasp the full complexity of the system , we combined mathematical modelling of the system with experimental determination of the kinetics of palmitate acquisition and turnover , and of protein degradation for wild type ( WT ) and palmitoylation-deficient mutants . We set up a general model consisting of ordinary differential equations ( ODE ) , describing the dynamic transitions between states/species of the network . We defined the topology of our network as a combination of well-known inter-convertible cycles described by Goldbeter and Koshland [24] . Combinations of such subunits have been used to successfully describe biological systems in which proteins undergo multiple covalent modifications , especially in the context of protein kinase signalling networks [25–27] . Since palmitoylation has , as phosphorylation , the potential to control protein function in a switch-like manner , a similar approach appeared suitable to model calnexin palmitoylation . Mathematical modelling allowed us to access an unprecedented level of understanding of the dynamics and the complexity of inter-convertible species of the same protein undergoing various post-translational modifications on multiple sites and access key parameters that are not directly measurable through experimentation . We could in particular estimate the half-life of single or dual palmitoylated calnexin , the off-rate of palmitate from a specific site upon occupancy of the other site or the time that separates calnexin synthesis from palmitoylation events , all together revealing the existence of a regulatory system that allows cells to post-translationally control the cellular levels and thus activity of this key ER chaperone .
Calnexin was recently shown to rely on palmitoylation to perform its major functions [17–19 , 28] . This raises the question as to which percentage of the total calnexin population is at a given time palmitoylated . At present , no reliable method enables to determine the percentage of a protein that is palmitoylated and to differentiate single from double palmitoylation . To estimate the species distribution and understand the dynamics of the inter-conversion between them , we therefore developed a mathematical model of the calnexin palmitoylation cycle . Modelling was performed as an open system , including protein synthesis , and degradation of all species ( S1 Table , Fig 1A ) . Calnexin is synthesized by ER-associated ribosomes and inserted into the ER membrane ( represented by rCAL ) . Presumably already co-translationally , the calnexin luminal domain undergoes folding , a process that is very efficient as discussed below . The cytosolic tail of calnexin is predicted to be highly disordered ( IUPRED [29] , http://iupred . enzim . hu/ ) . fCAL represents folded but non-palmitoylated calnexin . This species can be modified on the first or second palmitoylation site leading to c1CAL and c2CAL , respectively . Single palmitoylated species can acquire a second palmitate both leading to c12CAL ( Fig 1A ) . The palmitoyltransferase DHHC6 catalyses the palmitoylation reaction for each site [19] . Depalmitoylation , which requires acyl protein thioesterases [8] , the identity of which remain to be established for calnexin , can occur from both sites . The individual palmitoylation and depalmitoylation steps were assumed to follow irreversible Michaelis-Menten kinetics , similar to the kinetics used in signalling network models [30] . These kinetics were determined using the total quasi steady state approximation ( tQSSA ) to a mass action model [31–33] reported by Pedersen et al . for the study of phosphorylation and dephosphorylation cycles [34 , 35] . This approach included a step-by-step application of tQSSA to a variety of biochemical reactions and contained a formulation of tQSSA for systems with competitive inhibition . As Gunawardena and colleagues pointed out [36] this avoids any ad hoc assumption inherent in the common Michaelis-Menten equations and takes into account sequestration effects when enzymes have multiple substrates . In these systems , a single enzyme can catalyse the same reaction on multiple substrates , which is conceptually similar to multiple sites on the same substrate . The equations could therefore be adapted to palmitoylation and depalmitoylation of the two cytoplasmic calnexin cysteines . Although this approximation is valid for a very wide range of enzyme-substrate concentrations [34] , in the case of calnexin palmitoylation , the validity of the Michaelis-Menten equations was ensured by the relative concentration of DHHC6 with respect to calnexin . Based on quantitative proteomic studies , the ratio of DHHC6-to-calnexin is indeed in the order of 1:500 [37–39] . Since the two sites might not be equivalent and since rates might depend on the occupancy of the neighbouring site , we introduced different Km values for each palmitoylation and depalmitoylation step . Also since the same enzyme catalyses the palmitoylation of both sites , a competition term between the two sites was implemented in the enzymatic kinetics as described in [35] . A similar competition term was introduced for depalmitoylation . The model also includes degradation rates for each species , with different first-order rate constants . The description of the rate expressions , the definition of the parameters , and the assumptions used in the development of the model are described in detail in the Expanded View ( S1 Fig and S1–S3 Tables ) . Sets of experimental data were generated to calibrate and test the predictability of the model . Since degradation of all species was included in the model , we first monitored protein degradation kinetics using 35S Cys/Met metabolic pulse-chase experiments . This requires immuno-precipitation of calnexin which we performed either using a polyclonal antibody against the C-terminus to follow the endogenous protein , or using an anti-HA antibody to follow transiently transfected WT and calnexin-HA mutants . WT calnexin , endogenous or HA-tagged , showed identical biphasic decays , with a t1/2 of ca . 8h ( shown only for calnexin-HA Fig 1B–1E ) . The faster initial decay made us wonder what the contribution is of the luminal domain , the largest domain of calnexin , in shaping this decay curve . We generated a truncated version of calnexin consisting of the luminal domain fused to the KDEL sequence to ensure ER localization . HA-calnexin-KDEL was far more stable than the full-length protein ( Fig 1B and 1C ) . In particular there was no decay at early time points indicating that the luminal domain undergoes efficient folding . Thus the initial decay phase observed for the full-length protein must be dictated by transmembrane domain and/or the cytosolic tail of calnexin . Decay of full-length calnexin was accelerated by silencing the DHHC6 enzyme ( Fig 1D ) , or by mutating one or both of the palmitoylation sites ( Fig 1E ) . In reverse , calnexin decay was slowed down by overexpression of DHHC6 ( Fig 1D ) . Based on these metabolic 20 min pulse-chase experiments , the apparent half-life of WT calnexin is ca . 8h , while that of non-palmitoylated calnexin , obtained either through enzyme silencing or site mutation , is ca . 5h . To further fuel the mathematical model of the palmitoylation cycle with experimental data , we determined the turnover of the palmitate moiety once attached to calnexin . To do so , we performed pulse-chase experiments using 3H-palmitate , followed by immunoprecipitation of calnexin . Following a 2 h 3H-palmitate pulse , loss of 3H-palmitate from WT calnexin occurred with an apparent half-life of ca . 8 hrs and was somewhat accelerated in single cysteine mutants ( Fig 2A and 2B ) . We also monitored the kinetics of incorporation of 3H-palmitate into calnexin ( Fig 2C and 2D ) . This was performed in the presence of cycloheximide , to prevent synthesis of new proteins during the labelling time . Degradation was not prevented with any drug . Palmitate incorporation increased as a function of time and did not reach a plateau within the 7 h time frame of the experiment . Experiments were kept within this time frame to avoid toxic/indirect effects of prolonged inhibition of protein synthesis . Altogether these experiments show that WT calnexin can undergo palmitoylation hours after it has been synthesized and that turnover of palmitate is very slow . A subset of the above 35S Cys/meth and 3H-palmitate pulse-chase experiments were used to calibrate the system , i . e . for parameter estimation , namely: decays of WT , the AC and the double AA mutants , incorporation of 3H-palmitate into WT calnexin , 3H-palmitate turnover for WT and the CA mutant . Since there is not a unique set of parameters that fits such a dataset , we employed a stochastic optimization method , which allowed us to generate a population of models , each having different combinations of parameter values while all being consistent with the calibration experiments . From a population of 10’000 models , we selected 382 models that best fitted the experimental data used as objective function ( see Expanded View , results of the fitting on the calibration dataset are shown in S2A–S2F Fig ) . The pool of selected models was subsequently used for the simulations and analyses . Note that all generated predictions were obtained by simulating each model independently . The outputs of all the models were then averaged and the standard deviation with respect to the mean was used as a measure of the variability among the different models ( S2A–S2L Fig ) . The remaining set of experiments were used to validate the output of the model , i . e . test its predictability ( see Expanded View ) , namely the WT calnexin decay upon DHHC6 silencing or overexpression , the decay of the single CA cysteine mutant as well as palmitate turnover for this mutant . As illustrated in Figs 2E and S2G–S2L , the predictions were in close agreement with the experimental data , indicating that the model reliably describes the events of the calnexin palmitoylation/depalmitoylation cycle . The model was first used to determine the distribution , and the evolution thereof , of the 5 palmitoylation species during the 35S-Cys/Meth pulse-chase experiments . We set up an in-silico 20 minutes labelling experiment ( see Expanded View , S3 Fig ) and calculated the relative concentrations of calnexin in the different palmitoylation states at different time points of the chase ( Fig 3A ) . At the end of the metabolic pulse ( t = 0 ) , the population is predicted to be exclusively of non-palmitoylated , ca . 25% of which are already folded . Ten hours after the pulse , the entire calnexin population was predicted to be folded , as can be expected , but , more unexpectedly , ca . 50% was still non-palmitoylated ( Fig 3A ) . Almost the palmitoylated species , the dually-palmitoylated was predicted to be the most populated , with barely and single palmitoylated species . As chase time proceeded , the non-palmitoylated species decreased again to the benefit of the dual palmitoylated form , which approached 90% at the end of the chase period ( Fig 3A ) . Note that distributions are expressed as percentages of the remaining population , not of the initial population , which decreased by 80% between the beginning and the end of the chase period . This analysis indicates that the initial faster phase of degradation occurs when most of calnexin is non-palmitoylated . Once a significant percentage of the population becomes acylated , the rate of decay drastically decreases , confirming a stabilizing effect of palmitoylation . We next determined the distribution of WT calnexin at steady state , since it may differ from the distribution at the end of our chase period . At steady state in our Hela cells and under our experimental conditions , the model predicts that ca . 70% of calnexin is dual-palmitoylated and the remaining population is free of palmitate . The prediction that the great majority of cellular calnexin is palmitoylated is consistent with our previous experimental evidence [19] . We noted that palmitoylated calnexin poorly migrates in 2D gels . We subsequently compared the calnexin signal by western blotting on 2D gels of control cells vs . cells silence for the DHHC6 palmitoyltransferase , and found that the signal was increased by ≈9 fold . This value is rather qualitative given the non-linearity of western blotting , but clearly indicated that the majority of calnexin is palmitoylated at steady state . We next utilised the model to estimate the steady state distribution of calnexin species in cells over-expressing DHHC6 and found that the dually-modified population increased to almost 100% while it fell to zero upon silencing of the enzyme ( Fig 3B ) , as expected . By regulating DHHC6 amounts and/or activity , cells thus have the potential to control the percentage of calnexin that is dually-palmitoylated , and thus functional . Consistent with the low population of single palmitoylated species for WT calnexin , determination of the steady state species distribution of the CA and the AC calnexin mutants revealed that >80% of the molecules are non-palmitoylated . This indicates that to obtain a significant population of palmitoylated calnexin , two sites are necessary . That the single palmitoylated species do not get significantly populated was initially unexpected given the relatively small difference in the apparent palmitate turnover rates of WT and single cysteine mutants ( Fig 2B ) . To understand this apparent inconsistency , we used the model to predict the distribution of the species labelled during the 2 hrs 3H-palmitate pulse ( corresponding to t = 0 in Fig 2B ) ( S4 and S5 Figs , Supporting information ) . In silico , 3H-palmitate labelling shows that in 2 hrs , only a minute percentage of the total WT population–less than 3%–undergoes labelling ( S5 Fig ) . This is consistent with the fact that 70% of the steady state population is dual palmitoylated at steady state and can thus not be further modified upon addition of 3H-palmitate . Moreover , Fig 2D indicates that palmitoylation can be very slow , since some proteins undergo palmitoylation 7 h or more after having been synthesized ( Fig 2D ) . Thus Fig 2B represents the loss of 3H-palmitate from a population of 3H-palmitate labelled species composed roughly of 50% c1CAL and 50% c12CAL ( Fig 4A , top panel ) , jointly representing just 3% of the total population . To estimate the rate of palmitate release from dually-palmitoylated calnexin ( c12CAL ) , which is not readily accessible experimentally , we determined what the 3H-palmitate decay would be if the starting point was that of steady state palmitoylation , i . e . 70% calnexin dually labelled ( Fig 4A , bottom panel ) . Under these conditions , the loss of palmitate was significantly slower ( Fig 4B ) . In fact , we measured an average turnover rate for palmitate of ~32h , which is almost 3 times slower than the apparent turnover rate estimated by our 2 hrs labelling experiment . This analysis indicates that the rate constant of palmitate removal from a given site depends on the occupancy of the second site . Our predictions indeed indicate that , in situations of single site occupancy , the rate constants of loss of palmitate from site 1 are drastically higher than the rate constants of loss from either site in the situation of double occupancy ( Fig 4C ) . Thus , single palmitoylated species do not get significantly populated as compared to the double palmitoylated one , because they lose their palmitate at a far higher rate . Altogether this analysis indicates that two sites are required to stably palmitoylated calnexin , because double occupancy drastically slows down depalmitoylation . 35S Cys/Met pulse-chase experiments of WT and cysteine mutants indicate that palmitoylation has a stabilizing effect and that the half-life of palmitoylation deficient calnexin is ca . 5 hrs . They however do not allow direct identification of the half-life of single or dually palmitoylated calnexin . We therefore made use of the model to predict the decay kinetics of each of the calnexin species ( Table 1 and Fig 5A ) . Palmitoylation of site 1 has a mildly stabilizing effect , the half-life of c1CAL being ca . 6 . 5 hrs ( Fig 5A ) . Palmitoylation of site 2 , whether site 1 is or not modified , leads to a spectacular stabilization , with dual-palmitoylated calnexin having a predicted half-life of 45 hrs ( Table 1 and Fig 5A ) . We sought for an experimental confirmation for this almost 10-fold increase in protein stability . At steady state , 70% of calnexin is predicted to dually-modified ( Fig 3B ) . To assess the half-life of this population , we chose to monitor the stability of calnexin tagged with SNAP , a widely used protein that self-labels when incubated with O6-benzylguanine derivatives [40] . We labeled cells for 30 min with SNAP-cell-TMR-star , a red fluorescent substrate of SNAP , in order to label the entire , steady state , population of calnexin-SNAP . Following different periods of chase , cells were harvested and the level of fluorescent calnexin was monitored by SDS-PAGE and fluorescence scanning . As shown in Fig 5B , the decay of calnexin-SNAP-TMR-star was slower than that observed by 35S pulse-chase , yet still biphasic . At t = 0 of SNAP-cell-TMR-star labeling , the population of calnexin-SNAP molecules have different”ages” , the “youngest” having just been synthesized . We therefore tested whether pretreatment of cells with cycloheximide for different times , to block protein synthesis , would affect the apparent stability of the SNAP-cell-TMR-star labeled population . A 2 hrs cycloheximide pretreatment already led to an increase in apparent half-life of the population ( Fig 5B ) . We extended the pretreatment to 6 h , so that all calnexin molecules would be at least 6 h “old” . With this treatment , no decay was observed for the first 24 hrs , and this was followed by a slow decline , leading to an apparent half-life of ca . 47 hrs ( Fig 5B ) . Altogether , the mathematical modeling and the calnexin-SNAP tagged decay analysis indicate that palmitoylation of the two juxtamembranous sites leads to a dramatic increase in the stability of calnexin . The analysis of the steady state distribution of WT and cysteine mutants ( Fig 3B ) indicates that two sites are required for stable palmitoylation and suggest that there might be cooperativity between sites . We therefore estimated the rates of palmitate incorporation at each site , depending on whether the other site was occupied or not . As shown in Fig 5C , palmitoylation on site 1 is predicted to be drastically more efficient than on site 2 . However , if site 1 is already occupied , site 2 is readily modified . In marked contrast , if site 2 is occupied , site 1 by being positioned between the transmembrane and the palmitoylated site 2 might be less accessible to the enzyme . This reaction flux analysis indicates that site 1 is preferentially modified and when this has occurred , site 2 is rapidly acylated , indicating positive cooperatively between sites 1 and 2 . We next simulated the degradation fluxes of the different calnexin species ( Fig 5D ) . Even though nearly 70% of the protein is dual-palmitoylated at steady state ( Fig 3B ) , the degradation flux was highest for the non-palmitoylated state . Doubly-palmitoylated calnexin did undergo degradation , but at a 3 to 4 times slower rate , which is due to the 3 to 4 times lower values of the degradation rate constants ( by parameter estimation using the corresponding experimental information , S2 Table ) . Therefore , efficient degradation of calnexin appears to require prior depalmitoylation by thioesterases . An unexpected and intriguing observation in this study is the slow appearance of the palmitoylated population following synthesis ( Fig 3A ) and the absence of a plateau in the palmitate incorporation experiment ( Fig 1H ) . This suggests that there is a lag time between synthesis and palmitoylation of calnexin . To evaluate this lag time , we derived a stochastic formulation of the original model of the palmitoylation process , and performed stochastic simulations that allowed us to track single proteins in the system from synthesis to dual-palmitoylation ( Supporting information , S5 Fig ) . Simulations were performed for 5000 molecules . Of these about 3000 were degraded before any palmitoylation event occurred . For the remaining population , we determined the frequency distribution of the time required to reach dual-acylation ( Fig 6 ) . Some molecules underwent palmitoylation within a few hours of synthesis ( Fig 6 ) . Most however remained in the non-palmitoylated form for extended periods of time , leading to an average time of synthesis-to-palmitoylation of 8 hrs ( Fig 6 ) . Calnexin and its palmitoylating enzyme DHHC6 are two membrane proteins that reside within the same two-dimensional space of the ER membrane . The lag time between synthesis and palmitoylation of calnexin could potentially be due to slow diffusion of one of the two molecules . We therefore determined their mobility using Fluorescence Recovery after Photobleaching ( FRAP ) of C-terminally GFP-tagged variants . Both calnexin and DHHC6 showed rapid diffusion with rates of 0 . 63±0 . 09 and 0 . 67±0 . 13 μm2/s respectively , in close agreement with previously published rates for calnexin ( Fig 7A ) [41] . As a control of a slow diffusing membrane protein [42] , we confirmed that Climp63/CKAP4 has a diffusion rate of 0 . 06±0 . 01 μm2/s ( Fig 7A ) [42] . Given the high rates of DHHC6 and calnexin diffusion , slow palmitoylation of calnexin cannot be explained by a low probability of encounter between the enzyme and the substrate . This raised the possibility that calnexin palmitoylation is actively prevented and led us to investigate the effect of calnexin phosphorylation on its acylation . We generated a triple mutant in which all three serine phosphorylation sites in the calnexin tail ( Ser- 554 , 564 , 583 ) were mutated to alanine ( S3A mutant ) . Remarkably , mutation of the serine phosphorylation sites led to a ~200% increase in palmitoylation during the 2h labelling period ( Fig 7B and 7C ) . Also , kinetics of 3H-palmitate incorporation , in the absence of protein synthesis , were faster for the S3A mutant than for WT calnexin ( Fig 7D ) . Consistently , 35S Cys/Meth pulse-chase experiments revealed that the S3A mutant was far more stable than the WT protein ( Fig 7E ) , in particular due to the disappearance of the initial rapid decay phase , which in WT is due to degradation of the non-palmitoylated species . Altogether these observations indicate that palmitoylation of calnexin is under the negative control of serine phosphorylation .
S-palmitoylation is a post-translational modification that is receiving increasing attention as more and more key cellular events and pathways appear to rely on the reversible acylation of specific proteins [7 , 14] . The dynamics of this modification and the regulatory mechanisms are however poorly understood . We have here investigated palmitoylation of the key ER chaperone calnexin [15] . Calnexin shares its ability to promote folding of N-glycosylated proteins with calreticulin , a soluble ER protein . Calnexin in contrast spans the ER membrane and harbors a 90 residue cytosolic tail . While the transmembrane nature of calnexin was initially thought to favor the chaperoning of transmembrane proteins , it is increasingly clear that the transmembrane domain and cytosolic tail confer additional properties and functions to calnexin which involve its palmitoylation [17 , 20 , 28 , 43] . We developed a mathematical model of the palmitoylation cycle that accurately captures the properties of the system as shown by its predictive power . This combination of modeling and experimentation led to a variety of interesting , often unexpected , analyses , predictions and conclusions . First , it highlights the under-appreciated complexity of classical metabolic pulse-chase experiments . What is referred to as WT–of calnexin or any other protein–is in fact a complex population of species , the distribution of which evolves with time . In our model , we have distributed calnexin into 5 species , defined by the folding and palmitoylation status . In future studies we will increase the complexity of the model , including additional species with different types of post-translational modifications , such as phosphorylation on three serines of its cytosolic tail [20] , a modification which we find affects its palmitoylation status . Our analysis also indicates that , while 35S Cys/Met labels a well-defined sub-population–the one that has been synthesized by the cell during the pulse– , this is by no means the case when labeling cells with 3H-palmitate . Radiolabeled palmitate can indeed be incorporated into any molecule that has an unoccupied palmitoylation site but importantly cannot be incorporated into fully palmitoylated proteins , which in the case of calnexin compose ca . 70% of the total cellular population at steady state . These fully palmitoylated proteins are thus silent in such a 3H-palmitate pulse-chase analysis . Most importantly our model provides unprecedented understanding of the palmitoylation process . In the context of calnexin , we found that the molecule undergoes palmitoylation first on site 1 . Once this has occurred , site 2 can be readily modified . There is thus cooperativity between site 1 and 2 . Depalmitoylation can rapidly occur if a single site is occupied but removal is drastically slowed down if the two sites are acylated . As a consequence , calnexin is either not modified or acylated on both sites , the single palmitoylated state being barely populated . Importantly , the percentage of calnexin that is modified at steady state can be tuned between 0 to 100% by the activity of the DHHC6 palmitoyltransferase . It has recently been shown that the activity of DHHC6 , at least for certain substrates , depends on its association with Selenoprotein K ( selK ) [44] . Selk was found to be upregulated by ER stress [45] , i . e . when increased chaperone activity is necessary , which would lead to an increase in DHHC6 activity and thereby in calnexin protein . Interestingly , we found that palmitoylation not only affects calnexin function—through association with the ribosome-translocon complex or ER-mitochondrial interaction sites [17 , 19 , 20 , 28]–but has a drastic effect on its stability , increasing the average half-life of the molecules from 5 to 45hrs . Interestingly however , palmitoylation only occurs , on average , 8 hrs after the calnexin molecule has been synthesized . This means that , at the population levels , some 80% of the calnexin molecules that a resting cell synthesizes are degraded before they acquire palmitate and thereby activity . Considering that a resting cell has 500’000 to 1 million copies of calnexin at steady state [37–39] , this implies that 2 . 5 to 5 million copies were actually synthesized . While 80% of these are degraded in resting cells , they can potentially be mobilized if palmitoylation occurred earlier after synthesis . This unexpected apparent inefficiency for a protein that has no folding problems ( Fig 1C ) , combined with the fact that both calnexin–the substrate–and DHHC6 –the enzyme–rapidly diffuse within the same membrane and thus must have a high probability of encounter , led us to search for a mechanisms that would actively prevent palmitoylation . Since calnexin is known to undergo serine phosphorylation , we investigated the potential impact of this modification on palmitoylation and found that a serine phosphorylation deficient calnexin mutant undergoes greatly accelerated palmitoylation . Our study thus indicates that cells can post-translationally tune the expression level of calnexin by controlling the kinetics of its palmitoylation via phosphorylation of its cytosolic domain . In addition to providing important novel insight into the mechanisms by which cells control the level of chaperone activity in the ER , the mathematical model that we have elaborated provides a framework to study palmitoylation of other proteins . In particular , a great variety of type I membrane proteins harbor one or two palmitoylation site in the vicinity of their transmembrane domains . It will be of interest to determine whether the rules of cooperativity and 2 site-requirement revealed by calnexin apply to other proteins and how palmitoylation kinetics of these are controlled . Considering that palmitoylation and depalmitoylation are mediated by enzymes that maybe themselves undergo cycles of palmitoylation and depalmitoylation [46 , 47] , this post-translational modification is particularly in need of mathematical modeling to understand the complexity of its regulation .
Hela cells were grown in complete MEM ( Sigma ) supplemented with 10% foetal bovine serum ( FBS ) , 2 mM L-glutamine , penicillin and streptomycin . Rabbit antibodies against calnexin were produced in our laboratory against the C-terminal peptide: CDAEEDGGTVSQEEEDRKPK; anti phospho S583-calnexin were from Abcam ( ab58503 ) , anti-HA and anti-HA-agarose conjugated beads were from Roche ( Applied Science , IN ) , protein G-agarose conjugated beads from GE Healthcare , HRP secondary antibodies from Pierce . Human calnexin-HA and human calnexin-C501A-C502A-HA , calnexin-C501A-HA ( AC ) , calnexin-C502A-HA ( CA ) , calnexin S554A-S564A-S583A-HA ( S3A ) , calnexin-C501A-C502A-S554A-S564A-S583A-HA ( AAS3A ) and human-calnexin-SNAP-HA , dog-HA-calnexin-KDEL and human DHHC6-myc were cloned in pcDNA3 [19] . For control transfections , we used an empty pcDNA3 plasmid . Plasmids were transfected into Hela cells for 24 hrs ( 2 μg cDNA/9 . 6 cm2 plate ) using Fugene ( Roche Diagnostics Corporation ) . To generate GFP fusions , calnexin and DHHC6 were cloned into the peGFP vector and CKAP4 was cloned into the peCE vector . siRNA against human DHHC6 were purchased from Qiagen ( target sequence: gaggtttacgatactggttat ) . As control siRNA we used the following target sequence of the viral glycoprotein VSV-G: attgaacaaacgaaacaagga . For gene silencing , Hela cells were transfected for 72 h with 100pmol/9 . 2cm2 dish of siRNA using interferin ( Polyplus ) transfection reagent . To follow palmitoylation , calnexin expressing cells were incubated 2h or several hours at 37°C in incubation medium ( Glasgow minimal essential medium buffered with 10 mM Hepes , pH 7 . 4 ) with 200 μCI /ml 3H palmitic acid ( American Radiolabeled Chemicals , Inc ) , washed and incubated different times at 37°C with complete medium prior to immunoprecipitation using anti-calnexin or anti-HA antibodies . Beads were incubated 5 min at 90°C in reducing sample buffer prior to SDS-PAGE . Immunoprecipitates were split into two , run on 4–20% gels and analyzed either by autoradiography ( 3H-palmitate ) after fixation ( 25% isopropanol , 65% H2O , 10% acetic acid ) , gels were incubated 30 min in enhancer Amplify NAMP100 ( Amersham ) and dried or submitted to Western blotting ( anti-calnexin ) . Autoradiograms and western blotting were quantified using the Typhoon Imager ( Image QuantTool , GE healthcare ) . For metabolic labeling , Hela cells were transiently transfected ( 24h ) or not with calnexin-HA cDNAs , washed with methionine /cysteine free medium , incubated 20 min pulse at 37°C with 50 μCi/ml 35S-methionine/cysteine ( Hartman Analytics ) , washed and further incubated for different times at 37°C in complete medium with a 10-fold excess of non-radioactive methionine and cysteine . Calnexin were immunoprecipitated and analyzed by SDS-PAGE . Protein synthesis was blocked by 30 min treatment with 10 μg/ml cycloheximide ( Sigma ) at 37°C . For immunoprecipitations , cells were lysed 30 min at 4°C in IP buffer ( 0 . 5%NP40 , 500 mM Tris-HCl pH 7 . 4 , 20 mM EDTA , 10 mM NaF , 2 mM benzamidine , and a cocktail of protease inhibitors , Roche ) , centrifuged 3 min at 2000 g and supernatants were pre-cleared with protein G-agarose conjugated beads and supernatants were incubated 16 h at 4°C with antibodies and beads . To follow SNAP labeling , calnexin-SNAP expressing cells were incubated 6 hours with 10 μg/ml cycloheximide at 37°C in complete medium , then 30min at 37°C in complete medium with 1 μM SNAP-cell-TMR-star ( Biolabs ) and cycloheximide , then washed three times and incubated different times at 37°C with complete medium prior lysis of the cells . Total cell lysates were analyzed by SDS-PAGE and the fluorescence was measured using the Typhoon Imager ( Image QuantTool , GE healthcare ) . HeLa cells were seeded on FluoroDish ( glass bottom: 0 . 17mm thickness , from World Precision Instrument Inc . USA ) and GFP-tagged proteins ( calnexin , DHHC6 and Climp63 ) were transiently expressed for 24 hours . Fluorescence recovery after photobleaching ( FRAP ) experiments were performed on a Leica SP5 microscope using a 63x oil-immersion objective ( 1 . 4 NA ) . The microscope was operated using the software supplied with the instrument ( LAS AF 2009 ) . The 488nm line of the argon laser was set at 60% output and 100% transmission . During the experiment , the cells were kept in a chamber at 37°C and 5% CO2 . The pinhole was wide-open . The scanner speed was set at 1400Hz . The digital zoom was set at 6 . The detector gain was set at 740V . The frame size was set at 512x32 . The resulting scanning time was 32ms per frame . Point bleach measurements were performed and the effective radius of the bleached area was calculated as 0 . 9μm according to [48] . 50 iterations using 4% transmission of the laser were acquired as pre-bleach reference scans . Then the GFP-tagged proteins localized in the ER were bleached a single iteration for 5ms . Afterwards 500 post-bleach iterations were acquired with same settings as for the pre-bleach acquisitions . FRAP experiments were conducted for each condition on 12 different cells expressing the same level of GFP-tagged proteins . Longer post-bleach acquisitions were conducted for Climp63 to enable a better exponential fitting of the post-bleach curves . Diffusion coefficients were extracted ( according to [49] ) from the fitting of an exponential equation to the post-bleach recovery curves using a hand-made code in matlab ( MATLAB 8 . 0 and Statistics Toolbox 8 . 1 , The MathWorks , Inc . USA ) . | The endoplasmic reticulum ( ER ) is the largest intracellular organelle of mammalian cells . It is responsible for many fundamental cellular functions , such as folding , quality control of membrane and secreted protein , lipid biosynthesis , control of apoptosis and calcium storage . Recent studies have shown that many ER membrane proteins are lipid modified . We therefore hypothesized that palmitoyltransferases , the enzymes responsible for this modifications , act as a regulator of the mammalian ER , controlling the function of a network of key proteins through reversible acylation . In this work we combine computational methods with experimental determination of parameters to study the mechanisms and properties of ER palmitoylation , using as a model the palmitoylation of the ER protein calnexin . The systematic analysis of the mathematical model , built and calibrated with the help of experimental data , shows that Calnexin palmitoylation leads to a 9-fold increase in half-life and that a long delay separates synthesis from palmitoylation in unstimulated cells . Surprisingly during this delay , 75% of synthesized calnexin is degraded before being palmitoylated . We hypothesize that this unexpected apparent inefficiency is a design principle that provides the cell with a means to post-translationally tune the calnexin content . | [
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"sciences"
] | 2016 | Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin |
The complex correlation structure of a collection of orthologous DNA sequences is uniquely captured by the “ancestral recombination graph” ( ARG ) , a complete record of coalescence and recombination events in the history of the sample . However , existing methods for ARG inference are computationally intensive , highly approximate , or limited to small numbers of sequences , and , as a consequence , explicit ARG inference is rarely used in applied population genomics . Here , we introduce a new algorithm for ARG inference that is efficient enough to apply to dozens of complete mammalian genomes . The key idea of our approach is to sample an ARG of chromosomes conditional on an ARG of chromosomes , an operation we call “threading . ” Using techniques based on hidden Markov models , we can perform this threading operation exactly , up to the assumptions of the sequentially Markov coalescent and a discretization of time . An extension allows for threading of subtrees instead of individual sequences . Repeated application of these threading operations results in highly efficient Markov chain Monte Carlo samplers for ARGs . We have implemented these methods in a computer program called ARGweaver . Experiments with simulated data indicate that ARGweaver converges rapidly to the posterior distribution over ARGs and is effective in recovering various features of the ARG for dozens of sequences generated under realistic parameters for human populations . In applications of ARGweaver to 54 human genome sequences from Complete Genomics , we find clear signatures of natural selection , including regions of unusually ancient ancestry associated with balancing selection and reductions in allele age in sites under directional selection . The patterns we observe near protein-coding genes are consistent with a primary influence from background selection rather than hitchhiking , although we cannot rule out a contribution from recurrent selective sweeps .
At each genomic position , orthologous DNA sequences drawn from one or more populations are related by a branching structure known as a genealogy [1] , [2] . Historical recombination events lead to changes in these genealogies from one genomic position to the next , resulting in a correlation structure that is complex , analytically intractable , and poorly approximated by standard representations of high-dimensional data . Over a period of many decades , these unique features of genetic data have inspired numerous innovative techniques for probabilistic modeling and statistical inference [3]–[9] , and , more recently , they have led to a variety of creative approaches that achieve computational tractability by operating on various summaries of the data [10]–[17] . Nevertheless , none of these approaches fully captures the correlation structure of collections of DNA sequences , which inevitably leads to limitations in power , accuracy , and generality in genetic analysis . In principle , the correlation structure of a collection of colinear orthologous sequences can be fully described by a network known as an ancestral recombination graph ( ARG ) [18]–[20] . An ARG provides a record of all coalescence and recombination events since the divergence of the sequences under study and specifies a complete genealogy at each genomic position ( Figure 1A ) . In many senses , the ARG is the ideal data structure for population genomic analysis . Indeed , if an accurate ARG could be obtained , many problems of interest today—such as the estimation of recombination rates or ancestral effective population sizes—would become trivial , while many other problems—such as the estimation of population divergence times , rates of gene flow between populations , or the detection of selective sweeps—would be greatly simplified . Various data representations in wide use today , including the site frequency spectrum , principle components , haplotype maps , and identity by descent spectra , can be thought of as low-dimensional summaries of the ARG and are strictly less informative . An extension of the widely used coalescent framework [1] , [2] , [9] that includes recombination [21] is regarded as an adequately rich generative process for ARGs in most settings of interest . While simulating an ARG under this model is fairly straightforward , however , using it to reconstruct an ARG from sequence data is notoriously difficult . Furthermore , the data are generally only weakly informative about the ARG , so it is often desirable to regard it as a “nuisance” variable to be integrated out during statistical inference ( e . g . , [22] ) . During the past two decades , various attempts have been made to perform explicit inference of ARGs using techniques such as importance sampling [19] , [22] ( see also [23] ) and Markov chain Monte Carlo sampling [24]–[27] . There is also a considerable literature on heuristic or approximate methods for ARG reconstruction in a parsimony framework [28]–[35] . Several of these approaches have shown promise , but they are generally highly computationally intensive and/or limited in accuracy , and they are not suitable for application to large-scale data sets . As a result , explicit ARG inference is rarely used in applied population genomics . The coalescent-with-recombination is conventionally described as a stochastic process in time [21] , but Wiuf and Hein [36] showed that it could be reformulated as a mathematically equivalent process along the genome sequence . Unlike the process in time , this “sequential” process is not Markovian because long-range dependencies are induced by so-called “trapped” sequences ( genetic material nonancestral to the sample flanked by ancestral segments ) . As a result , the full sequential process is complex and computationally expensive to manipulate . Interestingly , however , simulation processes that simply disregard the non-Markovian features of the sequential process produce collections of sequences that are remarkably consistent in most respects with those generated by the full coalescent-with-recombination [37] , [38] . In other words , the coalescent-with-recombination is almost Markovian , in the sense that the long-range correlations induced by trapped material are fairly weak and have a minimal impact on the data . The original Markovian approximation to the full process [37] is known as the sequentially Markov coalescent ( SMC ) , and an extension that allows for an additional class of recombinations [38] is known as the SMC' . In recent years , the SMC has become favorite starting point for approximate methods for ARG inference [39]–[42] . The key insight behind these methods is that , if the continuous state space for the Markov chain ( consisting of all possible genealogies ) is approximated by a moderately sized finite set—typically by enumerating tree topologies and/or discretizing time—then inference can be performed efficiently using well-known algorithms for hidden Markov models ( HMMs ) . Perhaps the simplest and most elegant example of this approach is the pairwise sequentially Markov coalescent ( PSMC ) [42] , which applies to pairs of homologous chromosomes ( typically the two chromosomes in a diploid individual ) and is used to reconstruct a profile of effective population sizes over time . In this case , there is only one possible tree topology and one coalescence event to consider at each genomic position , so it is sufficient to discretize time and allow for coalescence within any of possible time slices . Using the resulting -state HMM , it is possible to perform inference integrating over all possible ARGs . A similar HMM-based approach has been used to estimate ancestral effective population sizes and divergence times from individual representatives of a few closely related species [39]–[41] . Because of their dependency on a complete characterization of the SMC state space , however , these methods can only be applied to small numbers of samples . This limits their utility with newly emerging population genomic datasets and leads to reduced power for certain features of interest , such as recent effective population sizes , recombination rates , or local signatures of natural selection . An alternative modeling approach , with better scaling properties , is the product of approximate conditionals ( PAC ) or “copying” model of Li and Stephens [43] . The PAC model is motivated primarily by computational tractability and is not based on an explicit evolutionary model . The model generates the th sequence in a collection by concatenating ( noisy ) copies of fragments of the previous sequences . The source of each copied fragment represents the “closest” ( most recently diverged ) genome for that segment , and the noise process allows for mutations since the source and destination copies diverged . The PAC framework has been widely used in many applications in statistical genetics , including recombination rate estimation , local ancestry inference , haplotype phasing , and genotype imputation ( e . g . , [44]–[48] ) , and it generally offers good performance at minimal computational cost . Recently , Song and colleagues have generalized this framework to make use of conditional sampling distributions ( CSDs ) based on models closely related to , and in some cases equivalent to , the SMC [49]–[52] . They have demonstrated improved accuracy in conditional likelihood calculations [49] , [50] and have shown that their methods can be effective in demographic inference [51] , [52] . However , their approach avoids explicit ARG inference and therefore can only be used to characterize properties of the ARG that are directly determined by model parameters ( see Discussion ) . In this paper , we introduce a new algorithm for ARG inference that combines many of the benefits of the small-sample SMC-based approaches and the large-sample CSD-based methods . Like the PSMC , our algorithm requires no approximations beyond those of the SMC and a discretization of time , but it improves on the PSMC by allowing multiple genome sequences to be considered simultaneously . The key idea of our approach is to sample an ARG of sequences conditional on an ARG of sequences , an operation we call “threading . ” Using HMM-based methods , we can efficiently sample new threadings from the exact conditional distribution of interest . By repeatedly removing and re-threading individual sequences , we obtain an efficient Gibbs sampler for ARGs . This basic Gibbs sampler can be improved by including operations that rethread entire subtrees rather than individual sequences . Our implementation of these methods , called ARGweaver , is efficient enough to sample full ARGs on a genome-wide scale for dozens of diploid individuals . Simulation experiments indicate that ARGweaver converges rapidly and is able to recover many properties of the true ARG with good accuracy . In addition , our explicit characterization of the ARG enables us to examine many features not directly described by model parameters , such as local times to most recent common ancestry , allele ages , and gene tree topologies . These quantities , in turn , shed light on both demographic processes and the influence of natural selection across the genome . For example , we demonstrate , by applying ARGweaver to 54 individual human sequences from Complete Genomics , that it provides insight into the sources of reduced nucleotide diversity near functional elements , the contribution of balancing selection to regions containing very old polymorphisms , and the relative influences of direct and indirect selection on allele age . Our ARGweaver software ( https://github . com/mdrasmus/argweaver ) , our sampled ARGs ( http://compgen . bscb . cornell . edu/ARGweaver/CG_results ) , and genome-browser tracks summarizing these ARGs ( http://genome-mirror . bscb . cornell . edu; assembly hg19 ) are all freely available .
The starting point for our model is the Sequentially Markov Coalescent ( SMC ) introduced by McVean and Cardin [37] . We begin by briefly reviewing the SMC and introducing notation that will be useful below in describing a general discretized version of this model . The SMC is a stochastic process for generating a sequence of local trees , and corresponding genomic breakpoints , such that each describes the ancestry of a collection of sequences in a nonrecombining genomic interval , and each breakpoint between intervals and corresponds to a recombination event ( Figure 1B ) . The model is continuous in both space and time , with each node in each having a real-valued age in generations ago , and each breakpoint falling in the continuous interval , where is the total length of the genomic segment of interest in nucleotide sites . The intervals are exhaustive and nonoverlapping , with , , and for all . Each is a binary tree with for all leaf nodes . We will use the convention of indexing branches in the trees by their descendant nodes; that is , branch is the branch between node and its parent . As shown by Wiuf and Hein [36] , the correlation structure of the local trees and recombinations under the full coalescent-with-recombination is complex . The SMC approximates this distribution by assuming that is conditionally independent of given , and , similarly , that depends only on and , so that , ( 1 ) where is the effective population size , is the recombination rate , and it is understood that . Thus , the SMC can be viewed as generating a sequence of local trees and corresponding breakpoints by a first-order Markov process . The key to the model is to define the conditional distributions and such that this Markov process closely approximates the coalescent-with-recombination . Briefly , this is accomplished by first sampling the initial tree from the standard coalescent and setting , and then iteratively ( i ) determining the next breakpoint , , by incrementing by an exponential random variate with rate , where denotes the total branch length of ; ( ii ) sampling a recombination point uniformly along the branches beneath the root of , where is a branch and is a time along that branch; ( iii ) dissolving the branch above point ; and ( iv ) allowing to rejoin the remainder of tree above time by the standard coalescent process , creating a new tree ( Figure 1B ) . As a generative process for an arbitrary number of genomic segments , the SMC can be implemented by simply repeating the iterative process until then setting equal to and equal to . Notice that , if the sampled recombination points are retained , this process generates not only a sequence of local trees but a complete ARG . In addition , a sampled sequence of local trees , , is sufficient for generation of aligned DNA sequences corresponding to the leaves of the trees ( Figure 1C ) . Augmented in this way , the SMC can be considered a full generative model for ARGs and sequence data . We now define an approximation of the SMC that is discrete in both space and time , which we call the Discretized Sequentially Markov Coalescent ( DSMC ) . The DSMC can be viewed as a generalization to multiple genomes of the discretized pairwise sequentially Markov coalescent ( PSMC ) used by Li and Durbin [42] . It is also closely related to several other recently described discretized Markovian coalescent models [39] , [40] , [50] . The DSMC assumes that time is partitioned into intervals , whose boundaries are given by a sequence of time points , with , for all ( ) , and equal to a user-specified maximum value . ( See Table 1 for a key to the notation used in this paper . ) Every coalescence or recombination event is assumed to occur precisely at one of these time points . Various strategies can be used to determine these time points ( see , e . g . , [50] ) . In this paper , we simply distribute them uniformly on a logarithmic scale , so that the resolution of the discretization scheme is finest near the leaves of the ARG , where the density of events is expected to be greatest ( see Methods ) . Each local block is assumed to have an integral length measured in base pairs , with all recombinations occurring between adjacent nucleotides . The DSMC approaches the SMC as the number of intervals and the sequence length grow large , for fixed and . Like the SMC , the DSMC generates an ARG for ( haploid ) sequences , each containing nucleotides ( Figure 1B ) . In the discrete setting , it is convenient to define local trees and recombination events at the level of individual nucleotide positions . Assuming that denotes a recombination between and , we write , with for positions and . Notice that it is possible in this setting that and . Where a recombination occurs ( ) , we write where is the branch in and is the time point of the recombination . For simplicity and computational efficiency , we assume that at most one recombination occurs between each pair of adjacent sites . Given the sparsity of variant sites in most data sets , this simplification is likely to have , at most , a minor effect during inference ( see Discussion ) . Like the SMC , the DSMC can additionally be used to generate an alignment of DNA sequences ( Figure 1C ) . We denote such an alignment by , where each represents an alignment column of height . Each can be generated , in the ordinary way , by sampling an ancestral allele from an appropriate background distribution , and then allowing this allele to mutate stochastically along the branches of the corresponding local tree , in a branch-length-dependent manner . We denote the induced conditional probability distribution over alignment columns by , where is the mutation rate . In this work , we assume a Jukes-Cantor model [53] for nucleotide mutations along the branches of the tree , but another mutation model can easily be used instead . Notice that , while the recombinations are required to define the ARG completely , the probability of the sequence data given the ARG depends only on the local trees . In the case of an observed alignment , , and an unobserved ARG , , the DSMC can be viewed as a hidden Markov model ( HMM ) with a state space given by all possible local trees , transition probabilities given by expressions of the form , and emission probabilities given by the conditional distributions for alignment columns , . The complete data likelihood function of this model—that is , the joint probability of an ARG and a sequence alignment given model parameters —can be expressed as a product of these terms over alignment positions ( see Methods for further details ) : ( 2 ) This HMM formulation is impractical as a framework for direct inference , however , because the set of possible local trees—and hence the state space—grows super-exponentially with . Even with additional assumptions , similar approaches have only been able to accommodate small numbers of sequences [32] , [35] , [54] . Instead , we use an alternative strategy with better scaling properties . The key idea of our approach is to sample the ancestry of only one sequence at a time , while conditioning on the ancestry of the other sequences . Repeated applications of this “threading” operation form the basis of a Markov chain Monte Carlo sampler that explores the posterior distribution of ARGs . In essence , the threading operation adds one branch to each local tree in a manner that is consistent with the assumed recombination process and the observed data ( Figure 2 ) . While conditioning on a given set of local trees introduces a number of technical challenges , the Markovian properties of the DSMC are retained in the threading problem , and it can be solved using standard dynamic programming algorithms for HMMs . The threading problem can be precisely described as follows . Assume we are given an ARG for sequences , , a corresponding data set , and a set of model parameters Assume further that is consistent with the assumptions of the DSMC ( for example , all of its recombination and coalescent events occur at time points in and it contains at most one recombination per position ) . Finally , assume that we are given an th sequence , of the same length of the others , and let The threading problem is to sample a new ARG from the conditional distribution under the DSMC . The problem is simplified by recognizing that can be defined by augmenting with the additional recombination and coalescence events required for the th sequence . First , let be represented in terms of its local trees and recombination points: . Now , observe that specifying the new coalescence events in is equivalent to adding one branch to each local tree , for , to obtain a new tree ( Figure 2 ) . Let us denote the point at which each of these new branches attaches to the smaller subtree at each genomic position by , where indicates a branch in and indicates the coalescence time along that branch . Thus , the coalescence threading of the th sequence is given by the sequence . To complete the definition of , we must also specify the precise locations of the additional recombinations associated with the threading—that is , the specific time point at which each branch in a local tree was broken before the branch was allowed to re-coalesce in a new location in tree . Here it is useful to partition the recombinations into those that are given by , denoted , and those new to , which we denote ( Figure 3A&B ) . Each is either null ( ) , meaning that there is no new recombination between and , or defined by , where is a branch in and is the time along that branch at which the recombination occurred . We call the recombination threading of the th sequence . For reasons of efficiency , we take a two-step approach to threading: first , we sample the coalescence threading , and second , we sample the recombination threading conditional on . This separation into two steps allows for a substantially reduced state space during the coalescence threading operation , leading to significant savings in computation . When sampling the coalescence threading ( step one ) , we integrate over the locations of the new recombinations , as in previous work [42] , [50] . Sampling the recombination threading ( step two ) can be accomplished in a straightforward manner independently for each recombination event , by taking advantage of the conditional independence structure of the DSMC model ( see Methods for details ) . The core problem , then , is to accomplish step one by sampling the coalescence threading from the distribution , ( 3 ) where the notation indicates that random variable is held fixed ( “clamped” ) at a particular value throughout the procedure . This equation defines a hidden Markov model with a state space given by the possible values of each , transition probabilities given by and emission probabilities given by ( Figure 3C ) . Notice that the location of each new recombination , , is implicitly integrated out in the definition of . Despite some unusual features of this model—for example , it has a heterogeneous state space and normalization structure along the sequence—its Markovian dependency structure is retained , and the problem of drawing a coalescent threading from the desired conditional distribution can be solved exactly by dynamic programming using the stochastic traceback algorithm for HMMs . Additional optimizations allow this step to be completed in time linear in both the number of sequences and the alignment length and quadratic only in the number of time intervals ( see Methods for details ) . The main value of the threading operation is in its usefulness as a building block for Markov chain Monte Carlo methods for sampling from an approximate posterior distribution over ARGs given the data . We employ three main types of sampling algorithms based on threading , as described below . We implemented these sampling strategies in a computer program called ARGweaver , that “weaves” together an ARG by repeated applications of the threading operation . The program has subroutines for threading of both individual sequences and subtrees . Options allow it to be run as a Gibbs sampler with single-sequence threading or a general Metropolis-Hastings sampler with subtree threading . In either case , sequential sampling is used to obtain an initial ARG . Options to the program specify the number of sampling iterations and the frequency with which samples are recorded . The program is written in a combination of C++ and Python and is reasonably well optimized . For example , it requires about 1 second to sample a threading of a single 1 Mb sequence in an ARG of 20 sequences with 20 time steps . Our source code is freely available via GitHub ( https://github . com/mdrasmus/argweaver ) . To summarize and visualize samples from the posterior distribution over ARGs , we use two main strategies . First , we summarize the sampled ARGs in terms of the time to most recent common ancestor ( TMRCA ) and total branch length at each position along the genome . We also consider the estimated age of the derived alleles at polymorphic sites , which we obtain by mapping the mutation to a branch in the local tree and calculating the average time for that branch ( see Methods ) . We compute posterior mean and 95% credible intervals for each of these statistics per genomic position , and create genome browser tracks that allow these values to be visualized together with other genomic annotations . Second , we developed a novel visualization device for ARGs called a “leaf trace . ” A leaf trace contains a line for each haploid sequence in an analyzed data set . These lines are ordered according to the local genealogy at each position in the genome , and the spacing between adjacent lines is proportional to their TMRCAs ( Figure S2 ) . The lines are parallel in nonrecombining segments of the genome , and change in order or spacing where recombinations occur . As a result , several features of interest are immediately evident from a leaf trace . For example , recombination hot spots show up as regions with dense clusters of vertical lines , whereas recombination cold spots are indicated by long blocks of parallel lines . Having demonstrated that ARGweaver was able to recover many features of simulated ARGs with reasonable accuracy , we turned to an analysis of real human genome sequences . For this analysis we chose to focus on sequences for 54 unrelated individuals from the “69 genomes” data set from Complete Genomics ( http://www . completegenomics . com/public-data/69-Genomes ) [58] . The 54 genome sequences were computationally phased using SHAPEIT v2 [59] and were filtered in various ways to minimize the influence from alignment and genotype-calling errors . They were partitioned into ∼2-Mb blocks and ARGweaver was applied to these blocks in parallel using the Extreme Science and Engineering Discovery Environment ( XSEDE ) . For this analysis , we assumed generations , , and , implying . We allowed for variation across loci in mutation and recombination rates . For each ∼2-Mb block , we collected samples for 2 , 000 iterations of the sampler and retained every tenth sample , after an appropriate burn-in ( see Methods for complete details ) . The entire procedure took ∼36 hours for each of the 1 , 376 2-Mb blocks , or 5 . 7 CPU-years of total compute time . The sampled ARGs were summarized by UCSC Genome Browser tracks describing site-specific times to most recent common ancestry ( TMRCA ) , total branch length , allele ages , leaf traces , and other features across the human genome . These tracks are publicly available from our local mirror of the UCSC Genome Browser ( http://genome-mirror . bscb . cornell . edu , assembly hg19 ) .
Several decades have passed since investigators first worked out the general statistical characteristics of population samples of genetic markers in the presence of recombination [21] , [80]–[83] . Nevertheless , solutions to the problem of explicitly characterizing this structure in the general case of multiple markers and multiple sequences—that is , of making direct inferences about the ancestral recombination graph ( ARG ) [19] , [20]—have been elusive . Recent investigations have led to important progress on this problem based on the Sequentially Markov Coalescent ( SMC ) [17] , [37]–[42] , but existing methods are still either restricted to small numbers of sequences or require severe approximations . In this paper , we introduce a method that is faithful to the SMC yet has much better scaling properties than previous methods . These properties depend on a novel “threading” operation that can be performed in a highly efficient manner using hidden Markov modeling techniques . Inference does require the use of Markov chain Monte Carlo ( MCMC ) sampling , which has certain costs , but we have shown that the sampler mixes fairly well and converges rapidly , particularly if the threading operation is generalized from single sequences to subtrees . Our methods allow explicit statistical inference of ARGs on the scale of complete mammalian genomes for the first time . Furthermore , the sampling of ARGs from their posterior distribution has the important advantage of allowing estimation of any ARG-derived quantity , such as times to most recent common ancestry , allele ages , or regions of identity by descent . Despite our different starting point , our methods are similar in several respects to the conditional sampling distribution ( CSD ) -based methods of Song and colleagues [49]–[52] . Both approaches consider a conditional distribution for the th sequence given the previous sequences , and in both cases a discretized SMC is exploited for efficiency of inference . However , the CSD-based methods consider the marginal distribution of the th sequence only given the other sequences and never explicitly reconstruct an ARG , while ours considers the joint distribution of an ARG of size and the th sequence , given an ARG of size and the previous sequences . In a sense , we have employed a “data augmentation” strategy by explicitly representing full ARGs in our inference procedure . The main cost of this strategy is that it requires Markov chain Monte Carlo methods for inference , rather than allowing direct likelihood calculations and maximum-likelihood parameter estimation . The main benefit is that it provides an approximate posterior distribution over complete ARGs and many derived quantities , including times to most recent common ancestry , allele ages , and distributions of coalescence times . By contrast , the CSD-based methods provide information about only those properties of the ARG that are directly described by the model parameters . We view these two approaches as complementary and expect that they will have somewhat different strengths and weaknesses , depending on the application in question . Our explicit characterization of genealogies can be exploited to characterize the influence of natural selection across the genome , as shown in our analysis of the Complete Genomics data set . In particular , we see clear evidence of an enrichment for ancient TMRCAs in regions of known and predicted balancing selection , reduced TMRCAs near protein-coding genes and selective sweeps , and reduced allele ages in sites experiencing both direct selection and selection at closely linked sites . Interestingly , the genealogical view appears to have the potential to shed light on the difficult problem of distinguishing between background selection and hitchhiking . Our initial attempt at addressing this problem relies on a genealogy-based summary statistics , the relative TMRCA halflife ( RTH ) , that does appear to distinguish effectively between protein-coding genes and partial selective sweeps identified by iHS . However , more work will be needed to determine how well this approach generalizes to other types of hitchhiking ( e . g . , complete sweeps , soft sweeps , recurrent sweeps ) and whether additional genealogical information can be used to characterize the mode of selection more precisely . Additional work is also needed to determine whether our ARG-based allele-age estimator—which is highly informative in bulk statistical comparisons but has high variance at individual sites—can be used to improve functional and evolutionary characterizations of particular genomic loci . A related challenge is to see whether our genome-wide ARG samples can be used to improve methods for association/LD mapping ( see [34] , [84]–[88] ) . In addition to natural selection , our methods for ARG inference have the potential to shed light on historical demographic processes , an area of particular interest in the recent literature [16] , [17] , [51] , [52] , [89] . To explore the usefulness of ARGweaver in demography inference , we attempted to infer a population phylogeny with admixture edges for the 11 human populations represented in the Complete Genomics data set , based on the genealogies sampled under our naive ( panmictic ) prior distribution . We extracted 2 , 304 widely spaced loci from our inferred ARGs , obtained a consensus tree at each locus , and reduced this tree to a subtree with one randomly selected chromosome for each of the 11 populations ( see Text S1 for details ) . We then analyzed these 11-leaf trees with the PhyloNet program [90] , [91] , which finds a population tree that minimizes the number of “deep coalescences” required for reconciliation with a given set of local trees , allowing for both incomplete lineage sorting and hybridization ( admixture ) events between groups . PhyloNet recovered the expected phylogeny for these populations in the absence of hybridization and generally detected complex patterns of gene flow where they are believed to have occurred , but it had difficulty reconstructing the precise relatinonships among source and admixed populations ( Supplementary Figure S20 ) . These experiments suggested that the posterior distribution of ARGs does appear to contain useful information about population structure even when a noninformative prior distribution is used , but that additional work will be needed to fully exploit the use of ARG inference in demographic analysis . An alternative strategy would be to extend our methods to incorporate a full phylogenetic demographic model , such as the one used by G-PhoCS [92] , thereby generalizing this fully Bayesian method to a setting in which recombination is allowed and complete genome sequences are considered . Importantly , the use of the complete ARG would allow information about demographic history from both patterns of mutation and patterns of linkage disequilibrium to be naturally integrated ( see [92] ) . However , as with CSD-based methods [51] , [52] , an extension to a full , parametric multi-population model for application on a genome-wide scale would be technically challenging . In our case , it would require the ability to sample “threadings” consistent with the constraints of a population model ( e . g . , with no coalescent events between genetically isolated populations ) and exploration of a full collection of population parameters , which would likely lead to slow convergence and long running times . Nevertheless , a version of this joint inference strategy may be feasible with appropriate heuristics and approximations . Our methods may also be useful for a wide variety of related applications , including local ancestry inference [47] , [93] , [94] , haplotype phasing/genotype imputation [46] , [48] , [95] , [96] , and recombination rate estimation [22] , [97] . Our initial implementation of ARGweaver relies on several simplying assumptions that appear to have minimal impact on performance with ( real or simulated ) human sequence data , but may produce limitations in other settings . Following Li and Durbin [42] , we compute probabilities of recombination between discrete genomic positions under the assumptions of the continuous-space SMC [37] . When recombination rates are low , the discrete and continuous models are nearly identical , but the differences between them can become significant when recombination rates are higher [98] . Similarly , our assumption of at most one recombination event per site and our use of the SMC rather than the improved [38] may lead to biases in cases of higher recombination rates , larger numbers of sequences , or more divergent sequences . In addition , our heuristic approach of accommodating zero-length branches by randomly sampling among “active” branches for coalescence and recombination events ( see Methods ) may lead to biases when the discretization scheme is coarse relative to evolutionary events of interest . Finally , we currently assume haploid genome sequences as input , which , in most cases of current interest , requires computational phasing as a pre-processing step . Phasing errors may lead to over-estimation of recombination and mutation rates and associated biases , because the sampler will tend to compensate for them with additional recombination and/or mutation events . In principle , most of these limitations can be addressed within our framework . For example , it should be fairly straightforward to extend ARGweaver to use the SMC' and Hobolth and Jensen's finite-loci transition density . In addition , we believe it is possible to enable the program to work directly with unphased data and integrate over all possible phasings ( see , e . g . , [92] , [99] ) . The ability to perform explicit ARG inference on the scale of complete genomes opens up a wide range of possible applications , but the long running times required for these analyses and the unwieldy data structures they produce ( numerous samples of ARGs ) are potential barriers to practical usefulness . In our initial work , we have attempted to address this problem by precomputing ARGs for a highly informative public data set and releasing both our complete ARGs and various summary statistics as browser tracks for use by other groups . We have also developed a simple web interface that allows users to retrieve local trees and several useful summary statistics for specified genomic intervals , populations , and individuals ( http://compgen . bscb . cornell . edu/ARGweaver/CG_results ) . In future work , it may be possible to improve data access by providing more sophisticated tools for data retrieval and visualization . For example , sampled ARGs could be stored in a database in a manner that allowed researchers to efficiently extract features such as regions of IBD or recombination maps for designated subsets of samples . A related possibility would be to support on-the-fly threading of user-specified query sequences into precomputed ARGs . This operation would be analogous to local ancestry inference [47] , [93] , [94] , but would reveal not only the population sources of query sequence segments , but also additional information about recombination events , coalescence times , approximate mutation ages , and other features . The same operation could be used to allow our sampling methods to scale to thousands of genomes: one could infer ARGs for , say , 100 genomes , then simply thread in hundreds more , without full MCMC sampling . In general , we believe that posterior samples of ARGs will be a rich resource for genetic analysis , but additional work is needed on data storage and query interfaces for these samples to become practically useful to large numbers of genomic researchers .
To sample ARGs genome-wide , we split each sequence alignment into non-overlapping segments of 2 Mb , flanked on each side by 100 kb of overlapping sequence . We chose a core set of 12 individuals ( 24 haplotypes ) randomly such that each major population group was represented . We then used ARGweaver to sample ARGs for these genomes , assuming a population size of time steps , and a maximum time of generations . Our prior estimate of was based on an empirical estimate of from the CG sequence data , and an assumption of mutations per site per generation for non-CpG sites ( see previous section ) . This initial step involved 500 sampling iterations , consisting of 100 initial iterations under an infinite sites assumption , and 400 iterations with the full finite sites model . The final sample from this initial step was used as a starting point for threading in the remaining genomes . Once these were threaded , we applied ARGweaver with infinite sites for 100 iterations , followed by 2400 iterations with the finite sites model . Samples were recorded every 10 iterations for the final 2000 iterations , for a total of 200 samples . For our genome-wide analyses , we integrated the separate 2 . 2 Mb analyses by setting a switchpoint at the middle of each overlapping 100 kb segment , in order to minimize boundary effects at the analyzed sites . To compute the neutral CDFs in Figure S17 , we used a set of putatively neutral regions obtained by removing all GENCODE ( v15 ) genes plus 1000 bp flank on either side of each exon , as well as all mammalian phastCons elements plus 100 bp of flanking sequence . From the remaining portion of the genome , we sampled 1000 sets of 69 regions with the same distribution of lengths as the non-CpG regions identified by [77] . To estimate the allele age at each polymorphic site , we considered all local genealogies sampled at that position , discarding any sampled genealogies that required more than one mutation to explain the observed data . In addition , we required that all of the retained genealogies implied the same derived allele , excluding positions that violated this condition from our analysis . For the remaining cases , we estimated the allele age for each sample as the average age of the branch on which the mutation leading to the derived allele was assumed to occur by parsimony , and averaged this value across samples . | The unusual and complex correlation structure of population samples of genetic sequences presents a fundamental statistical challenge that pervades nearly all areas of population genetics . Historical recombination events produce an intricate network of intertwined genealogies , which impedes demography inference , the detection of natural selection , association mapping , and other applications . It is possible to capture these complex relationships using a representation called the ancestral recombination graph ( ARG ) , which provides a complete description of coalescence and recombination events in the history of the sample . However , previous methods for ARG inference have not been adequately fast and accurate for practical use with large-scale genomic sequence data . In this article , we introduce a new algorithm for ARG inference that has vastly improved scaling properties . Our algorithm is implemented in a computer program called ARGweaver , which is fast enough to be applied to sequences megabases in length . With the aid of a large computer cluster , ARGweaver can be used to sample full ARGs for entire mammalian genome sequences . We show that ARGweaver performs well in simulation experiments and demonstrate that it can be used to provide new insights about both demographic processes and natural selection when applied to real human genome sequence data . | [
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] | 2014 | Genome-Wide Inference of Ancestral Recombination Graphs |
Long-term antibiotic use generates pan-resistant super pathogens . Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens . A candidate target pathway is quorum sensing ( QS ) , which many bacterial pathogens use to coordinately regulate virulence determinants . The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline ( HHQ ) and 3 , 4-dihydroxy-2-heptylquinoline ( PQS ) , whose syntheses depend on anthranilic acid ( AA ) , the primary precursor of 4-hydroxy-2-alkylquinolines ( HAQs ) . Here , we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression . These compounds restricted P . aeruginosa systemic dissemination and mortality in mice , without perturbing bacterial viability , and inhibited osmoprotection , a widespread bacterial function . These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P . aeruginosa pathogenesis , and possibly other clinically significant pathogens .
Current treatment of human bacterial infections depends on bactericidal and bacteriostatic antibiotics whose long-term effectiveness is limited by the development of drug resistance and can devastate the host commensal microbial community . An alternative approach to combat bacterial pathogens is the use of anti-infective drugs that selectively disrupt pathways that mediate virulence , such as regulation of pathogenesis genes [1] . Compounds that do not disrupt survival or growth should be less likely to generate resistance than traditional antibiotics . Ideally , these reagents should not disrupt bacterial and host metabolism , and should not cause harmful side effects . To date , the development of such drugs has been limited [2–4] . Here , we validated the utility of selective anti-infective compounds to combat infections caused by the opportunistic human pathogen Pseudomonas aeruginosa . This ubiquitous Gram-negative bacterium readily develops antibiotic resistance , and it is a principal agent of deleterious and fatal infections in immunocompromised patients and in pan-antibiotic-resistant outbreaks [5–7] . As such , identification of compounds that selectively disrupt P . aeruginosa pathogenesis should lead to improved clinical treatments of human P . aeruginosa infections . Bacterial pathogens express certain virulence genes at high cell density . Since this population-dependent regulation controls virulence , but not viability , it is a potential Achilles' heel through which to attack pathogenicity [3 , 4 , 8 , 9] . Many differentiated bacterial behaviors are similarly triggered in response to cell density , and such coordinated intercellular regulation is achieved via quorum sensing ( QS ) , a chemical communication system mediated by small extracellular signal molecules [10] . Signal synthesis is autoinducible , and as such , signal molecule concentration rises as the population density increases until a critical threshold concentration is reached , which then triggers expression of certain sets of genes . However , further studies suggest that the activation of most QS-controlled genes is not solely triggered by the accumulation of signal but also requires additional factors [11 , 12] . The archetypal QS system uses an acyl homoserine lactone ( AHL ) intercellular signal that , when the minimum threshold is attained , binds to and activates its cognate LuxR-type transcriptional regulator [13] . This coligand–protein complex then binds as a homodimer to a “lux-box” sequence within the promoters of target loci , including the AHL synthase gene , to activate or repress their transcription [14 , 15] . Significantly , QS signal molecules occur in infection sites , suggesting that QS inhibition might disrupt virulence [16 , 17] . Such inhibition could be achieved by interfering with one or more QS components , including signal synthesis , signal regulator binding , or synthase or regulator stability . Although compounds have indeed been identified that inhibit QS in infection sites , to date none have been shown to combat infection effectively . For example , certain AHL derivatives enhance bacterial clearance in lung , and delay death in infected mice , yet fail to reduce overall mortality [18–20] . Similarly , furanones that stimulate LuxR turnover [21] give similar results , yet again do not reduce overall mortality . Three distinct regulatory pathways have been identified that control QS-dependent expression in P . aeruginosa . Two of these systems utilize the LuxR regulatory proteins LasR and RhlR and their cognate AHL autoinducers and synthases [22] . In contrast , the third system utilizes a LysR-type transcriptional regulator ( LTTR ) , MvfR , which is activated by its coligands , 4-hydroxy-2-heptylquinoline ( HHQ ) and 3 , 4-dihydroxy-2-heptylquinoline ( PQS ) . Although both HHQ and PQS bind to and activate MvfR , PQS is 100-fold more potent than HHQ [23] . Interestingly , in contrast to the in vitro findings , HHQ is highly produced in vivo , where it is not fully converted into PQS [23] . These coligands are part of a large family of 4-hydroxy-2-alkylquinolines ( HAQs ) that comprise five distinct congener series , including N-oxides , such as 4-hydroxy-2-heptylquinoline N-oxide ( HQNO ) and dihydroxylated derivatives [24–26] . Once MvfR is activated , it binds to a “lys-box” in its target promoters [23 , 27 , 28] . MvfR coligand synthesis is also autoinducible [23 , 29 , 30] . Production of the coligands is controlled by MvfR , which regulates the production of multiple QS-regulated virulence factors , including pyocyanin , hydrogen cyanide , elastase , and lectins [25 , 29] . mvfR− mutant cells have greatly reduced pathogenicity in several infection models [31] , yet sustain wild-type viability . The attenuated virulence of lasR− mutant cells is mediated , at least in part , via MvfR [32] . Consequently , selective inhibition of MvfR/HAQ regulation should restrict P . aeruginosa pathogenesis , but not cell viability . Such selective inhibition would also interfere with the ability of P . aeruginosa cells to intercept host stress molecules required to activate P . aeruginosa virulence factors via MvfR and the HAQ signaling molecules PQS and HHQ [33] . Other important bacterial pathogens , such as Burkholderia species , also synthesize HAQs [34] , which suggests that compounds that selectively inhibit HAQ production could be used as anti-infectives against other pathogens beyond P . aeruginosa . MvfR coligand synthesis is essential for MvfR activation . This synthesis requires two MvfR-regulated operons: phnAB and pqsA-D . The operon phnAB directs production of anthranilic acid ( AA ) , which , in conjunction with ß-keto fatty acids , is the primary HAQ precursor; while pqsA-D directs production of the HAQ congener family [26 , 35 , 36] . Also , pqsH directs the final HHQ to PQS conversion . An early step in HAQ biosynthesis is hypothesized to be formation of the quinoline 3–4 carbon bond ( see Figure 1B ) via activation of the AA carbonyl that then reacts with the ß-keto fatty acid methylene . PqsA encodes a predicted coenzyme A ligase , and such ligases can activate aromatic carboxylic acid compounds [37] . According to this scheme , AA analogs that compete with AA for the PqsA protein should inhibit HHQ and PQS synthesis , but not viability , and consequently restrict MvfR activation and bacterial pathogenesis . Here , we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis in both P . aeruginosa and Burkholderia thailandensis cells , disrupted MvfR-dependent gene expression , and reprogrammed the expression of both QS-dependent and QS-independent genes . Significantly , these reagents restricted P . aeruginosa virulence in mice and increased host survival , without perturbing bacterial viability . In addition , they reduced osmoprotection , an environment-related cell response common to many human bacterial pathogens . These compounds provide a starting point for the design and development of selective anti-infective drugs that restrict human–P . aeruginosa pathogenesis , and also possibly other significant pathogens .
The AA analog methylanthranilate ( MA; Figure 1A ) has been previously shown to reduce P . aeruginosa PQS levels [38] . Nevertheless , MA is a poor candidate anti-infective , as 1 . 5 mM MA only partially reduced the levels of HHQ and PQS , while those of HAQ N-oxides were unaffected ( Figure 2 ) . Figure 1A presents the five AA analogs tested that carry a fluorine or chlorine atom ortho or para to the AA carbonyl group: 2-amino-5-fluorobenzoic acid ( 5FABA ) , 2-amino-4-fluorobenzoic acid ( 4FABA ) , 2-amino-6-fluorobenzoic acid ( 6FABA ) , 2-amino-6-chlorobenzoic acid ( 6CABA ) , and 2-amino-4-chlorobenzoic acid ( 4CABA ) . These analogs were chosen because the electron-withdrawing effects of the halogen atoms should restrict formation of an activated carbonyl such as a CoA ester , and thus synthesis of the second aromatic ring of the HAQ quinoline backbone would be prevented ( Figure 1B ) . However , we cannot exclude the possibility of a steric hindrance effect or the result of an interaction of the halogen with some residues in or near the active site of PqsA ( see below ) . Our results showed that 6FABA , 6CABA , and 4CABA , but not 5FABA and 4FABA , greatly reduced the levels of HHQ , PQS , and HQNO , which are representative congeners of the three principal HAQ series , in PA14 cultures ( Figure 2; unpublished data ) . Furthermore , no chloro-HAQs were detected with 4CABA or 6CABA , and only traces of halogenated N-oxides were detected in the presence of 6FABA ( unpublished data ) , indicating that these halogenated AA analogs were not incorporated into HAQs . Also , up to 6 mM 6FABA or 6CABA , and up to 1 . 5 mM 4CABA did not significantly perturb PA14 growth kinetics ( Figure S1 ) . Thus , these concentrations were used in all subsequent experiments , unless noted otherwise . The AA analogs 6FABA , 6CABA , and 4CABA markedly reprogram global gene expression in PA14 cells at late exponential growth , when many virulence-related genes are most highly expressed . Comparing the whole-genome transcriptome profile of control cells to the profiles of cells grown in the presence of 6FABA , 6CABA , or 4CABA showed that 354 , 618 , and 683 genes , respectively , were differentially expressed in response to these compounds ( Tables 1 and S1 ) , or 6 . 2% , 10 . 8% , and 12% of the 5 , 684 predicted ORFs assayed by the P . aeruginosa Genechip array . As a high number of genes were identified as differentially expressed , to limit the false positives , we focused on the genes that were affected by all three compounds . Together , these compounds altered the expression of a common set of 205 genes , of which 173 were repressed , and 32 activated ( Tables 1 and S1 ) . Inhibition of HHQ and PQS coligand synthesis should prevent MvfR activation , and consequently MvfR-dependent gene regulation . To this end , the AA analogs were found to significantly reprogram MvfR-dependent gene expression , including the suppression of several positively regulated loci that direct HAQ synthesis , or the production of key virulence factors . One hundred forty-four total PA14 genes are MvfR dependent , with 122 positively and 22 negatively regulated [25] . One hundred eleven ( 78% ) of these loci were differentially expressed in response to at least one of the AA analogs ( Table S2 ) . Individually , 6FABA , 6CABA , and 4CABA altered the expression of 54 , 89 , and 102 MvfR-dependent loci , respectively ( Table S2 ) . Of the 122 positively regulated MvfR-dependent genes , 56 have known functions , many of which promote P . aeruginosa pathogenicity , including HAQ production [25] . Indeed , all three AA analogs strongly repressed the HAQ biosynthetic operons pqsA-E and phnAB and the pyocyanin production–mediating operons phzABCDEFG , phzH , phzM , and phzS ( Table S2 ) . In addition , the analogs inhibited several loci that direct the synthesis of additional virulence-related factors , including hydrogen cyanide ( hcnABC ) , chitinase ( chiC ) , lectins ( lecA and lecB ) , and elastase ( lasB ) [19 , 25 , 31 , 32 , 39 , 40] . Functional assays confirmed that the AA analogs effectively eliminated HAQ biosynthesis ( Figure 2 ) and pqsA-lacZ reporter gene expression ( Figure S2A ) , and significantly reduced production of the MvfR-dependent virulence factors , pyocyanin and elastase ( Figure S2B and S2C ) . QS plays a critical role in the activation of virulence gene expression in many pathogens , and Table 1 shows that the AA analogs strikingly reprogrammed QS-dependent gene expression ( also Table S1 ) . Of the 173 common genes that were downregulated by all three analogs , 104 ( 60% ) were under QS control [19 , 25 , 41 , 42] , with 37 MvfR-dependent , and 67 solely LasR- and/or RhlR-dependent . Similarly , of the 32 common upregulated loci , 10 were QS dependent ( Table 1 ) . This broad perturbation of QS-regulated gene expression further suggested that the AA analogs should potentially limit P . aeruginosa virulence . Functional classification of the 205 common genes that were differentially expressed in response to all three of the AA analogs revealed different sets of cell activities ( unpublished data ) . Specifically , the repressed genes were overrepresented for activities that include secreted factors , for which the repressed genes strikingly account for almost 20% of all such genes , adaptation and protection functions , such as osmoprotection , and chemotaxis . In contrast , the activated genes were overrepresented for activities that mediate the general cellular machinery and contribute to the cell metabolome . High resolution nuclear magnetic resonance ( NMR ) analysis of PA14 cells grown minus and plus 4CABA provided functional evidence that the AA analogs stimulate the metabolome , as the assigned resonances for ATP , ADP , and NAD peak resonances were all significantly higher ( p < 0 . 05 ) in the 4CABA-treated cells ( Figure S3 and Table S3 ) . Note that 6FABA , 6CABA , and 4CABA upregulated metabolome genes , which further demonstrated they did not inhibit bacterial growth at the concentrations used . Through what molecular target do the AA analogs restrict HAQ synthesis ? Figure 3A shows that AA accumulated in the cell supernatants of PA14 cells grown in 6FABA , 6CABA , or 4CABA; accordingly , the antABC genes , which mediate AA degradation , were strongly upregulated ( Table S1 ) . As such , 6FABA , 6CABA , or 4CABA did not restrict HAQ production by inhibiting AA production via PhnAB or another AA synthase . Instead , they most likely shut down HAQ synthesis by competing with AA for the PqsA active site at the start of the HAQ biosynthetic pathway ( Figure 1B ) . To this end , pqsA− mutant cells also accumulated AA ( Figure 3A , and [26] ) , indicating that the analogs inhibited HAQ biosynthesis activity that utilized AA . Also , exogenous AA partially restored HHQ and PQS production in PA14 cells grown in 6FABA or 4CABA , but not in 6CABA ( Figure 3B; unpublished data ) . If the analogs do directly compete with AA , they should also perturb tryptophan production , since TrpD , the initial enzyme in the tryptophan biosynthesis pathway , utilizes AA as a substrate . Indeed , these compounds inhibited cell proliferation in minimal media , and exogenous tryptophan rescued this defect for the cells grown in 6FABA , or 6CABA , but not 4CABA ( Figure S4A ) . Together , these results suggest that the AA analogs inhibit HAQ production , and at least 6FABA and 6CABA inhibit tryptophan synthesis by respectively competing with AA for the PqsA and TrpD active sites . The AA analogs should restrict P . aeruginosa virulence , as they prevent the expression of MvfR-dependent virulence genes . Indeed , these compounds limited P . aeruginosa virulence in a thermal injury mice model , where mice were burned and then infected ( B+I ) with PA14 , and subsequently administered with a single intravenous injection of each AA analog at 6 h post-B+I . Figure 4A shows that while only 10% of the uninjected B+I controls survived P . aeruginosa infection , 35% , 37% , and 50% of the mice treated with 6FABA , 6CABA , and 4CABA , respectively , survived infection . In addition , the kinetics of mortality was significantly delayed in the treated mice . As expected , reduced protection was seen for later single injection post-B+I , and essentially no protection was afforded by 24 h ( unpublished data ) . PA14 cells produce HHQ and PQS in the infection wound site [23] . Figure 4B shows that the AA analogs strongly inhibited this production . The injection of 6FABA or 6CABA at 6 h post-B+I greatly limited in vivo HHQ levels at 12 h post-B+I in rectus abdominus muscle that directly underlies the infection site , versus comparable muscle from untreated B+I mice . This reduction was not due to reduced PA14 proliferation , as the AA analogs did not alter bacterial CFU/mg counts at 12 h post-B+I in the muscle ( unpublished data ) . Although 4CABA was less effective in reducing in vivo HHQ synthesis by 12 h than 6FABA or 6CABA , which could be because the mice received 2 . 5-fold less 4CABA , it was a better inhibitor of in vitro HAQ synthesis and of MvfR-dependent gene expression than 6FABA or 6CABA . Nevertheless , 4CABA , even with a lower HAQ inhibitory efficacy in vivo , was capable of limiting infection over time similar to 6FABA or 6CABA ( Figure 4A ) . PA14 cells inoculated intradermally into the midline crease of the mouse burn eschar proliferate in the wound , invade the intact underlying rectus abdominus muscle , and then spread via the blood to infect adjacent muscle tissue [43 , 44] . This systemic dissemination , which is a significant problem in human P . aeruginosa infections , was greatly reduced in mice injected with 6FABA , 6CABA , or 4CABA at 6 h post-B+I , versus uninjected control mice . Figure 5A shows that the bacterial counts in muscle at the infection site were statistically the same for treated and control mice , which further confirmed that the AA analogs did not restrict in vivo bacterial proliferation . Conversely , Figure 5B shows that the PA14 CFU/mg counts in adjacent muscle were 2–3 log units lower in the experimental versus control animals , and Figure 5C shows that the blood bacterial counts were similarly reduced . This inhibition of the systemic dissemination of PA14 cells is likely a major component of the protective in vivo anti-infective efficacy of the AA analogs to limit PA14 pathogenesis . Note that this efficacy is most likely due to inhibition of virulence factor gene expression , versus metabolic perturbation , as isogenic trpE and trpC mutant cells proliferated and were as pathogenic as PA14 cells in the B+I mice ( unpublished data; Figure S4B ) , indicating that tryptophan inhibition was non-limiting for in vivo growth and virulence . Other bacterial pathogens also likely utilize HAQ-based QS signaling and HAQ congeners as virulence mediators . As such , the AA analogs could have broad efficacy as anti-infective reagents against human pathogens beyond P . aeruginosa via their inhibition of HAQ synthesis pathways . To this end , both the highly virulent Gram-negative human pathogen Burkholderia pseudomallei and its close relative B . thailandensis encode functional homologs of the P . aeruginosa pqsA-E operon [34] and produce unsaturated HAQs , including HEHQ , NEHQ , and UDEHQ , plus lower levels of saturated HAQs , including HHQ , but not PQS . [34] . Figure 6 shows that 3 mM 6FABA , 6CABA , and 4CABA effectively abolished B . thailandensis HAQ synthesis . Virulence genes encode a diversity of activities , several of which permit pathogens to withstand hostile conditions in the host environment , such as high osmolarity . Betaine , a key bacterial osmoprotectant , is synthesized from betaine aldehyde via the betB gene product . PA14 cells grown in the presence of 4CABA exhibited significantly higher betaine aldehyde levels versus control cells , as assessed by high resolution NMR ( Figure S3 and Table S3 ) , correlating with the lower betaine levels as determined by liquid chromatography/mass spectrometry ( LC/MS ) and with the reduced betB expression in the presence of AA analogs ( unpublished data ) . Figure 7 shows that 6 mM 6FABA , 6 mM 6CABA , and 1 . 5 mM 4CABA exposure caused PA14 cells to be far less resistant to high salt , versus control cells . This heightened osmosensitivity was not mediated via AA analog inhibition of AHL- or HAQ-mediated QS , as wild-type , pqsA− , and lasRrhlR− mutant cells were equally salt resistant , and betB expression is QS independent . Instead , this effect was likely due to reduced betaine levels , plus additional QS-independent factors , as betB− mutant cells were more sensitive to high salt than control cells , but less so than cells grown in the presence of AA analogs . Figure 7 also shows that the AA analogs , especially 6CABA , similarly increased the osmotic sensitivity of the Gram-negative bacteria B . thailandensis and Yersinia pseudotuberculosis , and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis . Note that 4CABA was highly effective in reducing the salt resistance of B . thailandensis , but failed to affect the other tested pathogens . These osmoprotection results further suggest that the AA analogs could have broad-spectrum anti-bacterial activity against pathogens beyond P . aeruginosa .
Combating super pathogens with broad-spectrum resistance may require new anti-infective compounds that selectively interfere with pathways that mediate virulence without affecting cell viability . Here we showed that three halogenated AA analogs , 6FABA , 6CABA , and 4CABA , restricted P . aeruginosa pathogenesis in mice by inhibiting the MvfR-dependent QS system that regulates the expression of key virulence genes . Significantly , these compounds did not perturb cell viability . Four principal results demonstrated the anti-MvfR and anti-pathogenesis efficacy of these AA analogs: 1 ) they blocked in vivo and in vitro production of HHQ and PQS , and consequently MvfR function; 2 ) they disrupted the expression of MvfR-dependent virulence genes , including pqsA-D , which mediate coligand synthesis; 3 ) they increased host survival to P . aeruginosa infection; and 4 ) they restricted systemic bacterial dissemination . These compounds also inhibited HAQ synthesis in B . thailandensis and restricted the production of several non-MvfR-dependent metabolites , including the osmoprotectant betaine , which may promote bacterial survival under harsh host conditions . As such , these compounds could have a multifactorial effect and possibly a broad-spectrum anti-infective activity against several clinically significant human pathogens . How do the AA analogs disrupt MvfR coligand synthesis ? The pqsA gene product , which is closely related to coenzyme A ligases that activate aromatic carbonyl compounds [37] , is thought to generate the HAQ quinoline backbone from AA and a ß-keto fatty acid . Our hypothesis is that an electron-withdrawing group could inhibit the formation of an activated carbonyl such as a CoA ester , and our results showed that indeed these halogenated AA analogs inhibited HAQ synthesis . We propose that the halogenated analogs compete with AA for the PqsA active site since AA accumulates in PA14 cells grown in the presence of these compounds to levels equivalent to those in pqsA− mutant cells . Furthermore , exogenous AA reversed 6FABA and 4CABA inhibition of HAQ synthesis , suggesting that they reversibly bind to PqsA . Conversely , AA did not reverse 6CABA inhibition , which suggests that this compound is a non-competitive inhibitor , has much higher PqsA affinity , or has a higher intracellular concentration versus 6FABA or 4CABA . However , unless the PqsA enzyme is purified , it cannot be determined whether AA analogs act on PqsA only through such mechanisms , as it is also possible that the halogen atom could interact with other PqsA residues either inside or close to the active site . The three compounds also had additional differences to each other , including quantitative and qualitative effects on gene expression , inhibition of osmoprotection , and restoration of tryptophan auxotrophy to cells grown in 6FABA or 6CABA , but not in 4CABA . That 6CABA and 4CABA behave differently—non-competitively in one assay and competitively in another one—in rescuing HHQ synthesis and tryptophan auxotrophy may be due to the fact that PqsA and TrpD enzymes do not have any similarity in protein sequence or domain structure . Therefore , it is possible that these enzymes possess a different AA binding site . The comparison of TrpD and PqsA crystal structures , when they become available , might confirm this . Nevertheless , all three compounds strongly inhibited HHQ and PQS coligand synthesis , and consequently MvfR-dependent gene regulation , and reduced P . aeruginosa pathogenicity . Such effects require that the concentrations of the AA analogs are in the mM range , possibly due to at least either an intrinsic low affinity for the PqsA active site , a reduced ability to enter into the cell , or because they are actively pumped out . If either of the latter two cases occurs , a low concentration of the compounds inside the cells is achieved . The AA analogs reprogrammed MvfR-dependent and MvfR-independent gene expression . This differential expression response demonstrated the efficacy of the analogs to disrupt MvfR regulon activation and their broader effect on a large number of MvfR-independent loci . Of the 205 genes affected by all three compounds , 173 were repressed , and 32 were activated . That the compounds altered the expression of MvfR-independent loci , indicates they affect additional regulatory pathways , and suggests a possible multifactorial effect . The mechanism by which AA analogs restrict pathogenicity has not yet been demonstrated . We propose that their anti-infective efficacy is principally due to their inhibition of the MvfR-dependent QS regulatory pathway , though we cannot exclude a multifactorial effect . Interestingly , a single injection of any of the compounds impacted the course of the disease over days . Bacterial numbers and systemic dissemination are major contributing factors to the potential lethality of P . aeruginosa infection , especially in immunocompromised individuals . Indeed , while the AA analogs did not alter bacterial proliferation at the infection site at 12 and 24 h , they significantly lowered bacterial counts in adjacent muscle tissue and prevented systemic spread . These effects , which were not due to reduced tryptophan synthesis and/or viability , likely resulted from inhibition of HAQs , at least up to 12 h , which consequently impacted the expression of the virulence factors dependent on their production , and thus greatly aided the host's ability to clear the infection . As such , MvfR/HAQ inhibition is likely an important component of the anti-infective efficacy of the AA analogs in improving mouse survival to P . aeruginosa infection . Several plant and human pathogens , including Pseudomonas , Bordetella , Burkholderia , Ralstonia , Streptomyces , Mycobacteria , and the Archeae Sulfolobus , encode putative PqsA and MvfR homologs , and in some cases , pqsBCD-like loci . It would be of interest to determine whether these homologs mediate HAQ production and promote virulence , and if so , whether AA analogs also inhibit them . B . thailandansis and B . pseudomallei , a potential biowarfare agent , carry functional pqsA-D genes that direct HAQ synthesis [34] , and we showed that the AA analogs blocked B . thailandensis HAQ production . These results suggest that the AA analogs may have anti-infective efficacy against bacterial pathogens beyond P . aeruginosa . Furthermore , as virulence functions are often encoded in bacterial pathogens by QS-dependent genes , the effects of AA analogs on MvfR-independent QS-regulated genes also suggests that they may have broad-spectrum activity . Although QS inhibitors can disrupt QS in vivo , to date , no such compounds have been demonstrated to increase host survival to P . aeruginosa infection [3] . We present here a new class of QS inhibitors that blocked the expression of MvfR-dependent virulence genes . These reagents are the first example of QS pathway inhibitors that restrict P . aeruginosa pathogenesis and improve host survival . Their efficacy confirmed the importance of the MvfR-dependent QS pathway for P . aeruginosa virulence and demonstrated its utility as a therapeutic target for selective anti-infective reagents . Their potential as therapeutic molecules was further supported by the fact that the alternative pathways for AA production , shikimate and tryptophan , are absent in humans . In addition , the ability of AA analogs to restrict production of MvfR-independent compounds , including betaine , could further contribute to their anti-infective activity . Notably , these compounds did not impede bacterial cell viability at the site of infection . Furthermore , the presence of mvfR and HAQ synthetic gene homologs in other bacterial pathogens , together with the ability of AA analogs to increase the osmosensitivity of several clinically significant bacteria , suggests that these compounds could be used to treat infections caused by other bacterial pathogens besides P . aeruginosa . It remains to be seen if these compounds cause any significant side effects . Nevertheless , they provide the basis for the design and development of compounds that block the MvfR regulatory pathway . Such reagents should have significant clinical utility in treating acute and chronic P . aeruginosa infections , and possibly other bacterial pathogens .
The RifR P . aeruginosa human clinical isolate UCBPP-PA14 [44] , and its pqsA− [26] , lasRrhlR− , ( this study ) , trpC− , trpE− , and betB− [45] isogenic mutant derivatives , were grown at 37 °C on Luria Bertani ( LB ) agar plates , in LB broth , or in minimal M9 medium plus 2 mM MgSO4 , 0 . 4% glucose , and 0 . 1 mM CaCl2 . The lasRrhlR− double mutant was generated by allelic exchange using the previously constructed single rhlR::Tc PA14 [25] and lasR::Gm PA14 mutants [26] . B . thailandensis E264 , S . aureus 8325 , and B . subtilis QPB467 were grown at 37 °C on LB agar or in LB broth , and Y . pseudotuberculosis IP32953 was grown at 30 °C on LB agar or in LB broth . One hundred μg ml−1 rifampicin , 300 μg ml−1 carbenicillin , 30 μg ml−1 gentamycin , tetracycline ( 100 μg/ml ) , 1 mM tryptophan , and different concentrations of AA were used as required . For bacterial assays , a fresh solution of each compound ( Sigma-Aldrich , http://www . sigmaaldrich . com/ ) was prepared in culture medium . For mice injections , fresh 20-mM solutions of 6FABA or 6CABA were prepared in 0 . 9% NaCl , dissolved at 50 °C for 30 min , and filtered through 0 . 22-μm filters . For 4CABA , a 20-mM solution in 50% ethanol was first prepared , as 4CABA has a maximal solubility of 3 mM in 0 . 9% NaCl . Overnight PA14 cultures were grown in LB minus or plus AA analog and diluted the following day in fresh media minus or plus compound . Bacterial growth kinetics were determined by measuring OD600 . The maximal concentrations that do not restrict PA14 growth in LB were found to be 6 mM 6FABA , 6 mM 6CABA , and 1 . 5 mM 4CABA . These concentrations were used in all subsequent experiments , unless otherwise noted . Overnight cultures of PA14 and pqsA− cells harboring pGX5 , which carries the PpqsA-lacZ reporter gene [27] , were diluted to OD600 0 . 05; and β-galactosidase activity , expressed as Miller Units [46] , and OD600 were measured at selected time points . Assays were performed in triplicate . Pyocyanin concentration ( μg/ml ) was determined by measuring OD520 [47 , 48] . Elastase activity was determined by measuring the OD495 of 1 ml of OD600 = 4 culture supernatant mixed with 10 mg of elastin congo red ( Sigma-Aldrich ) , following incubation at 37 °C for 3 h with agitation and centrifugation . PA14 cells were grown in 5 ml of LB at 37 °C with agitation minus and plus 6 mM 6FABA , 6 mM 6CABA , or 1 . 5 mM 4CABA . Triplicate samples of two independent cultures for each AA analog were harvested at OD600 = 2 . 5 , and total RNA was purified using the RNAeasy spin column ( Qiagen , http://www . qiagen . com/ ) , and assayed using the GeneChip P . aeruginosa Genome Array ( Affymetrix , http://www . affymetrix . com/ ) . Affymetrix DAT files were processed using the Affymetrix Gene Chip Operating System ( GCOS ) to create . cel files . The raw intensity . cel files from the 12 chips , three replicates each for four different conditions , were normalized by robust multi-chip analysis ( RMA ) ( Bioconductor release 1 . 7 ) with PM-only models . Array quality control metrics generated by Affymetrix Microarray Suite 5 . 0 were used to assess hybridization quality . Normalized expression values were analyzed with SAM ( Significance Analysis of Microarray ) [49] using the permuted unpaired two-class test . The control group consisted of sample replicates in the absence of AA analogs . Each of the three experimental groups consisted of the AA analog sample replicates . Genes whose transcript levels exhibited an up or down absolute fold change >2 , and q value <6% in response to all three analogs versus control were further analyzed . Functional annotation for the differentially regulated genes is from http://v2 . pseudomonas . com/ . The likelihood of overrepresentation of functional categories in the upregulated or downregulated genes relative to the background of all array genes was calculated using Fisher's exact test . The statistical significance differences of PA14 CFU/mg between experimental and control groups were calculated using the Wilcoxon rank sum test . The statistical significance differences in the metabolome of PA14 cells grown in absence or presence of 4CABA were calculated using the independent samples t-test ( equal variances , two-tailed; α = 0 . 05 ) . Significance of survival kinetics was calculated using Kaplan–Meier analysis with assessment of statistical significance using the Mantel–Cox log-rank test and Cox model . The statistical significance difference in HHQ levels in vivo between control and treated mice was calculated using the t-test and Wilcoxon rank sum test . The quantification of HAQs in bacterial culture supernatants and in infected mouse tissue was performed as described [23 , 50] . The HAQs were separated on a C18 reverse-phase column connected to a triple quadrupole mass spectrometer , using a water/acetonitrile gradient [50] . Positive electrospray in MRM mode with 2 × 10−3 mTorr argon and 30 V as the collision gas and energy was employed to quantify HAQs , using the ion transitions HHQ 244>159 , HHQ-D4 248>163 , HQNO 260>159 , PQS 260>175 , and PQS-D4 264>179 . B . thailandensis HAQs were assessed as above . The pseudomolecular ions of each compound were monitored in full scan mode , using the unsaturated PA14 HAQ response factors . PA14 cells were grown in triplicate minus or plus 1 . 5 mM 4CABA to OD600 = 3 . The culture samples were centrifuged and the pellets were washed with ice cold PBS , resuspended in 100 μl of 75% prewarmed ethanol , and sonicated . Four hundred μl of 75% prewarmed ethanol was added and the samples were incubated at 100 °C for 4 min . Bacterial debris were removed by centrifugation , and the supernatants were dried in a SpeedVac ( Thermo Scientific , http://www . thermo . com/ ) and dissolved in 100 mM phosphate prepared with D2O . An aliquot of 10–20 μl of each sample was pipetted into a 4-mM rotor with a spherical insert , and 10–20 μl D2O containing 50 mM TSP ( trimethylsilyl propionic-2 , 2 , 3 , 3-d4 acid , Mw = 172 , d = 0 ppm ) was added to provide the deuterium lock and external chemical shift references , respectively . High-resolution magic angle spinning proton nuclear magnetic resonance ( HRMAS 1H NMR ) was performed on a Bruker BioSpin ( http://www . bruker-biospin . com/ ) Avance NMR spectrometer ( proton frequency at 600 . 13 MHz , 89-mm vertical bore ) using a 4-mM triple resonance ( 1H , 13C , 2H ) HRMAS probe ( Bruker ) . Temperature was maintained at 4 °C by a BTO-2000 unit in combination with a MAS pneumatic unit ( Bruker ) . The MAS speed was stabilized at 4 . 0 ± 0 . 001 kHz by a MAS speed controller . One dimensional 1H NMR spectra were acquired for all the samples using a rotor-synchronized Carr-Purcell-Meiboom-Gill ( CPMG ) spin echo pulse sequence , ( 90°- ( t-180°-t ) n-acquisition ) , which works as a T2 filter to remove the spectral broadening . The inter-pulse delay ( t ) was synchronized to the MAS speed of 250 μs . The number of transients was 256 with 32 , 768 ( 32 k ) data points . A line-broadening apodization function of 1 . 0 Hz was applied to all HRMAS 1H FIDs prior to Fourier transformation . Metabolite chemical shifts were according to Bundy et al . [51] and Chauton et al . [52] . The animal protocol was approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee . A thermal injury mouse model [43] was used as described previously [44] to assess bacterial pathogenicity in 6-wk-old CD1 mice ( Charles River Laboratories , http://www . criver . com/ ) . Following mouse anesthetization , a full-thickness thermal burn injury involving 5%–8% of the body surface area was produced on the dermis of the shaved abdomen , and an inoculum of 5 × 105 PA14 cells was injected intradermally into the burn eschar . To assess the anti-infective efficacy of the AA analogs to limit P . aeruginosa virulence , B+I mice received a single IV injection of 100 μl of 20 mM 6FABA ( 12 μg/g body weight ) , 100 μl of 20 mM 6CABA ( 13 . 6 μg/g ) , or 40 μl of 20 mM 4CABA ( 5 . 4 μg/g ) . Note that these compounds were injected at 6 h post-B+I because HHQ and PQS were not detected in vivo at that time point . Mice received a lower dose of 4CABA , as they appeared disoriented when a higher amount was used . Such an effect may be due to the fact that 4CABA was dissolved in EtOH instead of saline , which was used for both 6FABA and 6CABA . Mice survival was subsequently assessed over 7 d . Experiments were repeated at least in duplicate . Mice infected with pqsA− mutant cells , whose virulence was attenuated in B+I mice versus PA14 cells [25] , served as additional controls . Note that injection of each of the compounds tested is nontoxic . Four sets of B+I mice were burned and infected with PA14 . Three of these sets were injected 6 h post-infection with 6FABA , 6CABA , or 4CABA , with the uninjected mice serving as controls . Five to 12 mice from each set were sacrificed at 12 or 22 h post-B+I , and blood samples and tissue biopsies of rectus abdominus muscle directly underlying the infection site , or adjacent to this site , were collected for quantification of bacterial CFU/mg tissue . Blood samples of 50 μl or 5 μl were immediately plated on agar , muscle tissue was homogenized in 2 ml of PBS , and serial dilutions were plated on LB Rif plates . Following 24 h incubation at 37 °C , PA14 CFU were determined . Note that although bacteria can directly invade muscle that closely underlies the infection site , they require systemic delivery via the blood to infect adjacent muscle . Starting from OD600 = 3 bacterial cultures , a series of six 10-fold dilutions were spotted onto high-salt LB agar tester plates minus AA analog , or plus 6 mM 6FABA , 6 mM 6CABA , or 1 . 5 mM 4CABA . Concentrations of 0 . 9 M , 0 . 6 M , and 0 . 7 M NaCl were respectively used to osmotically stress P . aeruginosa; B . thailandensis; and S . aureus , B . subtilis , and Y . pseudotuberculosis cells . Plates were read after overnight incubation . Note that the compounds do not perturb bacterial growth under the low osmotic conditions of regular LB plates . | Current treatments of human bacterial infections depend on antibiotics , whose long-term effectiveness is limited as they select for multidrug-resistant pathogens . An alternative approach that is likely to limit the development of bacterial super pathogens is to selectively disrupt bacterial virulence mechanisms without affecting bacterial viability . Quorum sensing ( QS ) , a highly regulated bacterial communication system , is a promising candidate target because it is used by numerous pathogens to stimulate and coordinate the expression of many virulence determinants , and its disruption does not affect bacterial cell viability . We have identified three compounds that efficiently inhibited the synthesis of molecules required for the activation of the human opportunistic pathogen Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway that controls the expression of key virulence genes . We showed that prevention of MvfR pathway activation disrupted MvfR-dependent gene expression , and limited P . aeruginosa infection in mice , without perturbing bacterial viability . In addition , the compounds identified limited the ability of a number of bacterial pathogens to tolerate salt , a widespread bacterial function , and possibly other functions relevant to pathogenesis . These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P . aeruginosa pathogenesis , and possibly other clinically significant pathogens . | [
"Abstract",
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] | 2007 | Inhibitors of Pathogen Intercellular Signals as Selective Anti-Infective Compounds |
Leishmania ( L . ) infantum is the causative agent in an endemic focus of canine leishmaniasis in the Mont-Rolland district ( Thiès , Senegal ) . In this area , the transmission cycle is well established and more than 30% of dogs and 20% of humans are seropositive for L . infantum . However , the sand fly species involved in L . infantum transmission cycle are still unknown . Between 2007 and 2010 , 3654 sand flies were collected from different environments ( indoor , peridomestic , farming and sylvatic areas ) to identify the main L . infantum vector ( s ) . Nine sand fly species were identified . The Phlebotomus genus ( n = 54 specimens; Phlebotomus ( Ph ) duboscqi and Phlebotomus ( Ph ) . rodhaini ) was markedly under-represented in comparison to the Sergentomyia genus ( n = 3600 specimens; Sergentomyia ( Se ) adleri , Se . clydei , Se . antennata , Se . buxtoni , Se . dubia , Se . schwetzi and Se . magna ) . Se . dubia and Se . schwetzi were the dominant species indoor and in peridomestic environments , near humans and dogs . Blood-meal analysis indicated their anthropophilic behavior . Some Se . schwetzi specimens fed also on dogs . The dissection of females in the field allowed isolating L . infantum from sand flies of the Sergentomyia genus ( 0 . 4% of Se . dubia and 0 . 79% of Se . schwetzi females ) . It is worth noting that one Se . dubia female not engorged and not gravid revealed highly motile metacyclic of L . infantum in the anterior part of the midgut . PCR-based diagnosis and sequencing targeting Leishmania kinetoplast DNA ( kDNA ) highlighted a high rate of L . infantum-positive females ( 5 . 38% of Se . dubia , 4 . 19% of Se . schwetzi and 3 . 64% of Se . magna ) . More than 2% of these positive females were unfed , suggesting the parasite survival after blood-meal digestion or egg laying . L . infantum prevalence in Se . schwetzi was associated with its seroprevalence in dogs and humans and L . infantum prevalence in Se . dubia was associated with its seroprevalence in humans . These evidences altogether strongly suggest that species of the Sergentomyia genus are probably the vectors of canine leishmaniasis in the Mont-Rolland area and challenge one more time the dogma that in the Old World , leishmaniasis is exclusively transmitted by species of the Phlebotomus genus .
Leishmaniases are vector-borne diseases with complex ecology and epidemiology . In humans , they are caused by more than 20 species of Leishmania parasites that live in a wide range of ecosystems and may have different clinical manifestations ( mainly cutaneous , mucocutaneous or visceral ) [1] . It is classically acknowledged that the sand flies involved in Leishmania transmission belong to the genus Phlebotomus in the Old World and to the genus Lutzomyia ( sensu Young & Duncan ) in the New World [2 , 3] . In the rural community of Mont-Rolland ( Senegal , West Africa ) , L . infantum is the causative agent in an endemic focus of canine leishmaniasis described since 1970 [4 , 5] . More recent studies have clearly shown that L . infantum circulation is well established in this focus and more than 30% of dogs and 20% of humans have a positive serologic test result [6] . However , to our knowledge , only one human clinical case has been recorded: one child with several cutaneous lesions [6] . This L . infantum canine leishmaniasis focus is particularly interesting because of its unusual location ( i . e . , outside the Mediterranean basin , Central and Southwest Asia , China , Middle East , Central and South America etc . ) [7 , 8] and because of the absence of the usual vectors ( Phlebotomus sand flies ) for this parasite . Therefore , the main objectives of this study were to identify the vector ( s ) of L . infantum and to describe the transmission cycle in Mont-Rolland . Sand flies were caught in various environments ( indoor , peridomestic , farming and sylvatic areas ) to determine their degree of endophily and exophily . The presence of Leishmania promastigotes was investigated using a classical parasitological method ( dissection under the microscope and culture ) and/or by PCR detection of Leishmania kinetoplast DNA ( kDNA ) and sequencing . The physiological status of females ( engorged , gravid or unfed ) and the blood-meal origin in blood-fed females were also determined . Finally , the association of PCR-positive specimens with the rate of infected dogs and seropositive humans was investigated .
The rural community of Mont-Rolland ( population: 18 , 000 inhabitants ) is located about 15 km north of Thiès city ( Western Senegal ) , at latitudes 14°55’–14°56’N and longitudes 16°50’–16°55’W ( Fig 1 ) . The climate is tropical , typical of the Soudan-Sahel region . The rainy season lasts generally from July to October . The annual rainfall in this area is between 500 and 650 mm with an average annual temperature of 26 . 7°C . The lowest temperatures are recorded during the dry season , with a minimum of 24 . 4°C , and the highest ones during the rainy season , with a maximum of 29 . 2°C . Hygrometry presents seasonal and daily variations . The maximum is about 90% relative humidity ( RH ) during the second half of the night in the rainy season and the minimum is about 25% RH during the day at the end of the dry season [9] . Sand flies were collected during seven days in April and then in June or July ( before the rainy season ) , each year , from 2007 to 2010 . Two types of interception and attraction methods ( CDC miniature light trap ( John W . Hock Co . FL , U . S . A . ) , sticky paper ) and pyrethroid spraying were used according to the procedures described in Niang et al . [10] . and Abonnenc [11] Sticky traps and CDC light traps were set before sunset and retrieved the following day , early in the morning . Indoor spraying with pyrethroid insecticides was carried out between 7 and 10 am . Collections were carried out in seven villages ( Fouloum , Guidieur , Khaye Diagal , Ndiaye Bopp , Pallo Youga , Pallo Dial , and Thiaye ) of the Mont-Rolland community ( Fig 1 ) , where previous studies reported the presence of a large number of sick dogs [6] and various sand fly species [12] . In each village , sand flies were caught in various environments with different levels of anthropization ( indoor , peridomestic , farming and sylvatic areas ) , according to the following plan: In 2008 and 2009 , female sand flies caught by indoor spraying were dissected in a drop of sterile saline ( 0 . 9% ) and examined microscopically for the presence of Leishmania promastigotes in the digestive tract . Females collected with CDC light traps were kept alive and immobilized with cigarette smoke before dissection . They were then transferred to a sterilized microscope slide in a drop of sterile saline solution ( 0 . 9% ) . The head and genitalia were used for species identification , as detailed above , while the gut was carefully dissected for promastigote detection by microscopic examination . When flagellates were observed in the gut , the cover glass was gently removed and more physiologic solution added . The liquid containing the digestive tract was then aspirated and inoculated in a culture tube containing Novy-MacNeal-Nicolle ( NNN ) medium with 0 . 75 ml of penicillin diluted in physiological serum ( final concentration: 100 , 000 UI/ml ) . Tubes were placed at 26°C and monitored under a microscope after three days and then twice a week for four to six weeks . In positive cultures , the Leishmania species was identified by using a nested PCR-based method [13] followed by sequencing . As isolation of promastigotes from female sand flies caught on sticky paper is difficult because they are generally dead for too long ( i . e . several hours ) and that we could not keep them refrigerated until dissection , these specimens were stored in 70% ethanol and were used for species identification and kDNA detection . After species identification by microscopic analysis , whole DNA ( including kDNA ) was extracted from the rest of the body using the Qiagen DNeasy Blood & Tissue Kit . To validate the microscopic identification , Leishmania kDNA was amplified by nested PCR , according to Noyes et al . [13] , to detect the presence of parasites and to differentiate the main Old World Leishmania species , particularly L . major , L . tropica , L . infantum and L . tarentolae . The amplification products were analyzed on 1 . 6% agarose gels . We used the following reference strains for species identification: L . infantum , MHOM/MA/67/ITMAP263; L . major , MHOM/IL/80/Friedlin or MHOM/SU/73/5ASKH; L . tropica , MHOM/SU/74/K27; L . tarentolae , RTAR/SN/67/G10 . The amplicons obtained from the positive cultures ( see above ) were then sent to Eurofins MWG Operon for purification and sequencing to confirm the species . Blood-fed females collected with sticky paper , light traps and indoor spraying were stored individually in Eppendorf tubes containing 70% ethanol . Females were identified and DNA extraction was carried out as detailed above . To identify the blood-meal source , first the presence of human DNA was investigated by PCR amplification of the human-specific AluYb8 repeat , according to the procedure described by Deininger and Batzer [14] . In the negative samples , the presence of the mammalian prepronociceptin ( PNOC ) gene was then assessed as described by Haouas et al . [15] . Amplicons were then sequenced by Eurofins Genomics . Sequences were identified using BLAST ( Basic Local Alignment Search Tool , National Center for Biotechnology Information ) . All data collected in this study and the results of the serological studies performed in dogs and humans by Faye et al . [9 , 16] were used for statistical analyses . The association between each species and the environment was studied by logistic regression , where the response was the proportion of sand flies of the given species among captured sand flies . Global significance was assessed by likelihood ratio tests , and partial Wald tests were used to test the nullity of each estimated parameter . Post-hoc analysis was performed using single-step adjustment of P-values . We also measured the relationship between the sand fly infection rate and environment on one hand and Leishmania infantum seroprevalence in dogs and humans on the other hand . All computations were carried out with the R software ( R-core team 2015 ) [17] and specifically the multcomp package [18] .
A total of 3654 sand fly specimens ( 1070 males and 2584 females ) was captured . Microscopic identification showed that sand flies belonging to the Phlebotomus genus ( 54 specimens caught ) were much less abundant than those belonging to the Sergentomyia genus ( 3600 specimens caught ) . Nine species were identified . Two belonged to the Phlebotomus genus ( Ph . duboscqi Neuveu-Lemaire , 1906 and Ph . Rodhaini Parrot , 1930 ) and the other seven to the Sergentomyia genus ( Se . schwetzi Adler , Theodor et Parrot 1929 , Se . dubia Parrot , Mornet et Cadenat , 1945 , Se . buxtoni Theodor , 1933 , Se . magna Sinton , 1932 , Se . clydei Sinton , 1928 , Se . adleri Theodor , 1933 and Se . antennata Newstead , 1912 ) ( Table 1 ) . Their distribution in the seven villages of the rural community of Mont-Rolland is shown in Fig 1 . The complete logistic regression results are shown in supplementary data ( S1 Table ) . Sergentomyia schwetzi was most commonly found in the peridomiciliary environment , and its abundance in this environment is significantly different from the three other environments ( P-values less than 1 . 0E-07 , ( see S1 Table for details ) . Sergentomyia dubia is most commonly found in the intradomiciliary environment and in this case too , this environment is significantly different from all three other environments ( P-values always <2 . 0E-16 ) . Sergentomyia . magna was similarly distributed in the different environments . The species Se . clydei , Ph . duboscqi , Se . antennata , Se . adleri were rarely found indoors and in peridomestic areas , but and were caught mainly in farming areas ( 59% , 68% , 91% and 72% , respectively ) . Sergentomyia buxtoni was negatively associated with intradomiciliary- , peridomiciliary- and farming habitats , supporting its association with the sylvatic environments ( ( P-values always <2 . 0E-16 ) ; logistic regression analysis , supplementary data ( S1 table and Fig 2 ) . Among the captured females , 612 specimens belonging to the nine phlebotomine species were dissected and their digestive tract was examined under a microscope to detect flagellated parasites . Only four females ( two Se . dubia and two Se . schwetzi ) were infected . The parasite strains isolated from these four females were inoculated in culture tubes with NNN medium and flagellated forms were clearly observed after three days of culture . Molecular identification of the cultured parasites using the nested PCR-based method indicated that the four Sergentomyia specimens were infected by Leishmania species . A band of about 750pb similar to the amplicon size of the L . infantum reference ( Table 2 , S1 Fig ) was amplified from the cultures of one Se . dubia and two Se . schwetzi specimens ( SEN27 , SEN19 and FR011 , respectively ) . A band of about 800bp , matching the amplicon size of the L . tarentolae reference , was detected in the culture ( SEN15 ) from the other Se . dubia specimen ( Table 2 , S1 Fig ) . To confirm the identifications , we sequenced the amplicons of the four isolates . All the sequences showed a high average quality that allowed a nucleotide reading between 70% and 97% of the amplicons . These sequences were submitted to GenBank and were assigned the accession numbers KU587707 to KU587710 corresponding to the following isolates , SEN15 , SEN19 , SEN27 , FR011 respectively . BLAST analysis showed 94% homology between SEN15 sequence ( KU587707 ) and the minicircle sequence of L . tarentolae ( GeneBank accession number: AF380693 . 1 ) . The SEN19 sequence ( KU587708 ) was 93% similar to a L . chagasi ( synonymous L . infantum ) kinetoplast minicircle sequence ( GeneBank accession number: JX156608 . 1 ) , SEN27 sequence ( KU587709 ) was 98% similar to another L . chagasi ( syn . L . infantum ) kinetoplast minicircle sequence ( GeneBank accession number: AF308682 ) and FR011 99% similar to a kinetoplast minicircle sequence obtained from a sand fly isolate ( GeneBank accession number: AJ270104 . 1 ) and 79% similar to a L . infantum kinetoplast minicircle sequence ( GeneBank accession number: AF027577 . 1 ) . These data support the conclusion that the SEN15 isolate from one Se . dubia female belonged to L . tarentolae and that the three other isolates , SEN19 isolate from Se . schwezi female and SEN27 and FR011 from Se . dubia females belonged to L . infantum . The Se . dubia female infected with L . infantum ( SEN27 ) was caught in a light trap inside a house in the village of Thiaye . It was not engorged and not gravid . We could distinguish a mixture of forms in which highly motile metacyclic promastigotes were clearly observed ( with relatively short body length and flagellum length two times body length ) in the anterior part of the midgut as described in Bates et al . [19] . Of the two Se . schwetzi females infected by L . infantum ( SEN19 and FR011 ) , one was collected in a peridomestic environment by CDC light trap in Pallo Diale . Many motile promastigotes were found in the midgut , which contained brown-colored blood corresponding to an old ( 36–48 hours ) partially digested blood-meal . The second one was caught by indoor spraying in Khaye Diagal . This female was not blood-fed or gravid and its anterior midgut contained many metacyclic promastigotes . The Se . dubia specimen positive for L . tarentolae ( SEN15 ) was caught indoor , in Khaye Diagal . The gut contained many promastigotes and digested blood . Then , 2113 females , of which 156 were blood-fed and 199 gravid , from the seven villages were screened for Leishmania parasite infection by using the nested kDNA PCR assay [13] . L . infantum kDNA could be amplified in 69 specimens ( 3 . 26% ) ( Table 3 ) . The positive females belonged to three species: Se . dubia ( 29 specimens , 5 . 38% of all captured Se . dubia ) , Se . schwetzi ( 32 specimens , 4 . 19% ) and Se . magna ( 8 specimens , 3 . 64% ) . The females from the other Sergentomyia species and the two Phlebotomus species were all negative . L . tarentolae kDNA was found in 24 specimens , mainly in Se . dubia females , the proven L . tarentolae vector in Senegal [20] , but also in Se . schwetzi , Se . clydei and Se . buxtoni specimens ( Table 3 ) . Among the 69 females infected with L . infantum , 15 had a blood-meal and 13 were gravid . Thus , the proportion of L . infantum-positive specimens was higher among blood-fed ( 9 . 62% ) and gravid ( 6 . 53% ) than among unfed females ( 2 . 32% ) ( Table 4 ) . It is worth noting that the positive individuals were distributed over the years of collection , supporting the fact that Leishmania is circulating constantly in sand flies . The specimens identified as L . infantum-positive by PCR assay were mostly captured in areas where both humans and dogs live: indoor ( 33 of the 562 indoor specimens; 5 . 87% ) and in peridomestic environments ( 28 of the 573 peridomestic specimens; 4 . 88% ) ( Table 5 ) . In environments less frequented by dogs and humans during the period of sand fly activity ( night and dusk ) , such as farming areas , only 8 females out of the 642 tested ( 1 . 25% ) were infected by L . infantum . In the sylvatic area , none of the collected specimens was positive for L . infantum . Logistic regression analysis showed that the probability of infection for the species Se . dubia ( p-value = 0 . 011 ) and Se . schwetzi ( p-value = 0 . 0013 ) was significantly associated with the environment . Specifically , the probability of infection was higher indoor and in peridomestic environments for Se . dubia sand flies and in peridomestic areas for Se . schwetzi . PCR analysis of 141 blood-meals in females from seven species using primers to amplify the AluYb8 repeat and the PNOC gene gave positive results in 43 samples ( 30 . 5% ) . Some PNOC amplicons could not be identified because the obtained sequence was too short for sequence comparison with BLAST and thus the blood-meal source was reported as “non-human mammals” . The blood-meal analysis revealed a large variety of blood sources ( Table 6 ) . Sergentomyia . dubia blood-meals were mainly from humans . Sergentomyia . schwetzi appeared to feed on a wide range of hosts , such as humans , dogs , horses , cows and mice . For the other species , the sequencing results indicated either human blood or blood from “non-human mammals” . Logistic regression analysis was also employed to explore the effect of L . infantum prevalence in sand flies on the probability for dogs and humans to be seropositive for L . infantum . For each village , and for each sand fly species , the prevalence of infection by L . infantum was computed and then log-transformed . The serological status ( L . infantum positive/negative ) of 315 people ( 73 positive ) and 160 dogs ( 74 positive ) in these villages was retrieved from Faye et al . [6 , 16] . The probability for a dog to be seropositive for L . infantum was strongly correlated with L . infantum prevalence in Se . schwetzi in the dog's village . The odds ratio ( OR ) associated with a 10% increase of prevalence was 8 . 5 , 95% CI ( 1 . 88–35 . 3 ) , p-value = 0 . 0041 ( Fig 3A ) . There was no association with L . infantum prevalence in the other sand fly species . The probability of a human being seropositive for L . infantum was correlated with L . infantum prevalence in Se . schwetzi in the seven villages . The OR associated with a 10% increase of prevalence was 1 . 96 , 95% CI ( 1 . 16–3 . 31 ) , p-value = 0 . 012 ( Fig 3B ) . The probability of a human being infected was also correlated with L . infantum prevalence in Se . dubia in the village [OR = 7 . 65 , 95% CI = ( 2 . 15–27 . 16 ) , p-value = 0 . 0013] ( Fig 3C ) . There was no association with L . infantum prevalence in the other sand fly species .
In this study , almost all the sand fly specimens captured around the habitats of dogs and humans belonged to the Sergentomyia genus . Indeed , the two Phlebotomus species were poorly represented ( Ph . rodhaini: 8 specimens , 0 . 22%; Ph . duboscqi: 46 specimens , 1 . 26% of all specimens ) . Phebotomus duboscqi sand flies were caugh mainly in farming areas in the sandy ecosystem , where canine leishmaniasis is less frequent [6 , 10] , Fig 1 ) . Concerning Ph . rhodlaini , a previous study showed that this species is probably underestimated because of unsuitable traps and could play a role in L . donovani transmission between animal reservoir hosts [24] . Rodent or dog-baited traps were not used in this study but light traps were placed above and in rodent burrows , around dogs in farming and peridomestic areas and we collected all residual fauna ( all the insects staying inside after night ) indoor . It is thus likely that the low number of Ph . rhodaini collected reflect the population of this species in the environments where L . infantum is transmitted . Sergentomyia dubia , Se . schwetzi , and to a lesser extent Se . magna , were the most abundant species captured indoors and in peridomestic environments , around infected dogs and serologically positive humans . As previously reported [11] , Se . dubia , the vector of the gecko leishmaniasis in Senegal , showed the most endophilic behavior . This species was significantly associated with the indoor environment where the majority of blood-fed and gravid females were also caught . Blood-meal analysis confirmed that Se . dubia feeds frequently on humans ( Table 6 ) . These results were unexpected because previous studies associated its presence indoors with its preference for reptiles , and particularly for geckos [25] . Nevertheless , this is consistent with the strong adaptability of this species [11 , 25] . Sergentomyia schwetzi , the most abundant species captured , was found predominantly outdoors ( peridomestic and farming environments ) , although it was also well represented indoors . Blood-fed and gravid females were more numerous in peridomestic and farming areas , suggesting a more exophilic behavior . In accordance with previous studies [25] , the results of the blood-meal source analysis ( often humans and dogs ) confirmed this opportunistic behavior [11 , 25] . Sergentomyia magna was present in the different sites of capture . Although slightly more abundant in farming areas , its presence in peridomestic areas and indoors was not negligible , suggesting a regular contact with dogs and humans . One of the Se . magna females had fed on humans . The other Sergentomyia species were very rare or almost absent indoors and in peridomestic areas , although the blood-meals of one Se . clydei and two Se . buxtoni , caught indoor , were of human origin . Sergentomyia clydei , Se . antennata and Se . adleri were most abundant in farming areas . In agreement with previous studies [9 , 12 , 25] , Se . buxtoni was mainly collected in the sylvatic area , suggesting a very exophilic behavior and feeding preferences focused on wild animals . The finding of naturally infected sand flies is essential to incriminate a vector and also to study the infection rates in endemic areas [2 , 19 , 21] . In the present study , microscopic examination indicated that 0 . 4% of Se . dubia and 0 . 79% of Se . schwetzi were infected with living L . infantum promastigotes of which two nongravid and unfed individuals ( one S . schwetzi and one S . dubia ) had mature forms in the anterior midgut . Natural infections in unfed specimens strongly suggest that the parasites have overcome the main barriers to metacyclogenesis ( i . e . , the digestive enzymes and the peritrophic membrane ) in the sand fly midgut [21] . Consequently , the mature and metacyclic promastigotes observed in unfed and non-gravid females correspond more likely to infective forms . To our knowledge , these results are the first report of natural infection of Sergentomyia species by L . infantum . It is consistent with the prevalence of L . infantum infection in many sand fly vectors [26–29] . As the microscopic detection of promastigotes in dissected flies is difficult to carry out , the prevalence rates reported using this method are generally low ( 0 . 01–1% ) even in competent vectors and in endemic areas [2 , 27] . Therefore , the isolation of L . infantum by dissection in Se . dubia and Se . schwetzi individuals is an important finding . The results of the nested PCR diagnostic assay confirmed the dissection data . They revealed the presence of L . infantum kDNA in Se . dubia ( 5 . 38% of all tested specimens for this species ) , in Se . schwetzi ( 4 . 19% ) and also in Se . magna ( 3 . 64% ) . These percentages are in agreement with the infection rates reported in proven vectors by Aransay et al . [21] and Kishor et al . [30] . Nevertheless , it is essential to keep in mind that the detection of Leishmania DNA in a sand fly does not prove the vector competence [31 , 32] . Indeed , a positive PCR result does not allow establishing whether the Leishmania kDNA was due to Leismania ingested while feeding , or to the presence of well-established or developing parasites [21 , 29 , 30 , 32] . In the current study , the percentage of PCR-positive sand flies was significantly different in blood-fed ( 9 . 62% ) , gravid ( 6 . 53% ) and unfed and non-gravid females ( 2 . 32% ) . The PCR-positive unfed/non-gravid females ( 2 . 74% of Se schwetzi , 4 . 44% of Se . dubia and 2 . 76% of Se . magna ) may reflect the survival of parasites ( or persistence of DNA ) in these sand fly species . Indeed , the presence of Leishmania kDNA in these females strongly suggests that the parasites were ingested several days before the capture and that they had started their developmental cycle . However , we cannot exclude the persistence of DNA without any infective role , especially because the sand flies were caught in the vicinity of infected dogs . Nevertheless , the microscopic observation of mature L . infantum promastigotes in unfed females strongly suggests that they are not only carrying parasite DNA , but that they could be competent vectors . None of the other species was found to be infected with L . infantum both by dissection and PCR testing . Currently , phlebotomine sand flies remain the exclusive proven vectors of leishmaniasis , although other insects , such as midges , are suspected to act as leishmaniasis vectors [33] and few cases of vertical transmission ( mother to child ) have been reported [34 , 35] The Sergentomyia genus is still not considered to be involved in leishmaniasis transmission [35]; however , several studies have detected Leishmania DNA and in one of them live parasites of L . major in various Sergentomyia and Spelaeomyia species , suggesting that this genus could play a role in Leishmania transmission [31 , 36–40] Our working hypothesis was that the vector species should belong to the Phlebotomus genus , which contains the classical vectors of L . infantum in the Old World . However , in the Mont-Rolland area , the two species belonging to the Phlebotomus genera ( Ph . duboscqi and Ph . rodhaini ) were not abundant and not infected by L . infantum parasites . These results are in agreement with previous works showing that Ph . duboscqi , the main L . major vector , is refractory to infection by L . infantum [41] . Conversely , some Sergentomyia species satisfied several criteria for vector incrimination . First , Se . dubia , Se . schwetzi and , to a lesser extent , Se . magna were the most abundant species captured indoors and in peridomestic environments surrounding infected dogs and serologically positive humans . Sergentomyia dubia showed an endophilic and anthropophilic behavior , feeding frequently on humans and Se . schwetzi a more exophilic and opportunistic behavior . Sergentomyia magna was moderately collected in the different sites , but displayed also a regular contact with dogs and humans . Sergentomyia dubia and Se . schwetzi females were found to be infected with Leishmania parasites both by dissection and by PCR-based diagnostic assay . Se . magna was found to be infected with Leishmania only by PCR . The isolates obtained from the dissections were successfully cultured and characterized as L . infantum , similarl to the parasites isolated from the dogs in this region [16] . Furthermore , unfed and non-gravid females were also infected , strongly suggesting that L . infantum survived after digestion of the blood-meal or egg laying . These data suggest that L . infantum can develop in Se . dubia and in Se . schwetzi . Nevertheless , a recent experimental study on the susceptibility of Se . schwetzi to L . donovani , L . infantum and L . major reported that this sand fly species is refractory to infection by L . infantum because the parasites were defecated with the blood remnants [42] . It is essential to underline that the conditions in natural populations and in laboratory setups can be quite different . Moreover , for a rigorous analysis of the Sergentomyia capacity to transmit Leishmania , it is important to work with parasites and sand flies collected from the area in which the transmission is suspected . Indeed , the sand fly-Leishmania interactions could be the results of a specific and recent co-evolution in that area and the ability of Sergentomyia species to transmit L . infantum could be region-specific . On the other hand , in the study by Sadlova et al . [42] , the Se . schwetzi colony was established from specimens collected in north-western Ethiopia and the L . infantum strain was isolated in Turkey from a sand fly specimen belonging to the Ph . tobbi species [42] . No experimental data are available either on Se . dubia or Se . magna . Our hypothesis is also supported by the results of the statistical analyses that demonstrate a strong ecological association between flies , humans and hosts ( criteria 4 of Ready , [23] . Indeed , Se . dubia and Se . schwetzi were strongly associated with humans and dogs ( indoor and peridomestic environments ) ; the probability of infection was higher indoors and in peridomestic environments for Se . dubia sand flies and in peridomestic areas for Se . schwetzi; the PCR-positive Se . schwetzi and Se . dubia specimens were significantly correlated with the seroprevalence data in dogs and/or humans . Taken together , these data strongly suggest , for the first time , that Se . dubia and Se . schwetzi are possible vectors of canine leishmaniasis and responsible for the contact between the Leishmania parasites and humans in the Mont-Rolland area . This study challenges one more time the dogma claiming that Phlebotomus is the only genus responsible for Leishmania transmission in the Old World . Thus , these findings should be confirmed by experimental infection using Se . schwetzi and Se . dubia colonies and L . infantum parasites from the Mont-Rolland community . | Leishmaniases , neglected tropical vector-borne diseases , remain today a problem of public health . Classically , the sand flies involved in Leishmania transmission belong either to the Phlebotomus genus ( Old World ) or to the Lutzomyia genus ( New World ) . In the rural community of Mont-Rolland ( Senegal , West Africa ) , Leishmania infantum is the causative agent in an endemic focus of canine leishmaniasis . Recent surveys revealed more than 30% of dogs and 20% of humans with a positive serological test for Leishmania in this community . However , the sand fly species involved in L . infantum transmission were still unknown . Between 2007 and 2010 , we carried out a study in this community to identify the sand fly species responsible for L . infantum transmission . We collected nine species belonging mainly to Sergentomyia genus and in low proportion to Phlebotomus genus . The abundance around dogs and humans , the detection of live and mature parasites in anterior midgut , the high rate of L . infantum-positive females using molecular analyses and the identification of dog and human blood in the fed females incriminates Se . schwetzi and Se . dubia as possible vectors of L . infantum . This hypothesis is strongly supported by statistical analyses performed to compare the prevalence of infected sand flies with the seroprevalence data in humans and dogs . | [
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] | 2016 | Transmission of Leishmania infantum in the Canine Leishmaniasis Focus of Mont-Rolland, Senegal: Ecological, Parasitological and Molecular Evidence for a Possible Role of Sergentomyia Sand Flies |
Rebound of HIV viremia after interruption of anti-retroviral therapy is due to the small population of CD4+ T cells that remain latently infected . HIV-1 transcription is the main process controlling post-integration latency . Regulation of HIV-1 transcription takes place at both initiation and elongation levels . Pausing of RNA polymerase II at the 5′ end of HIV-1 transcribed region ( 5′HIV-TR ) , which is immediately downstream of the transcription start site , plays an important role in the regulation of viral expression . The activation of HIV-1 transcription correlates with the rearrangement of a positioned nucleosome located at this region . These two facts suggest that the 5′HIV-TR contributes to inhibit basal transcription of those HIV-1 proviruses that remain latently inactive . However , little is known about the cell elements mediating the repressive role of the 5′HIV-TR . We performed a genetic analysis of this phenomenon in Saccharomyces cerevisiae after reconstructing a minimal HIV-1 transcriptional system in this yeast . Unexpectedly , we found that the critical role played by the 5′HIV-TR in maintaining low levels of basal transcription in yeast is mediated by FACT , Spt6 , and Chd1 , proteins so far associated with chromatin assembly and disassembly during ongoing transcription . We confirmed that this group of factors plays a role in HIV-1 postintegration latency in human cells by depleting the corresponding human orthologs with shRNAs , both in HIV latently infected cell populations and in particular single-integration clones , including a latent clone with a provirus integrated in a highly transcribed gene . Our results indicate that chromatin reassembly factors participate in the establishment of the equilibrium between activation and repression of HIV-1 when it integrates into the human genome , and they open the possibility of considering these factors as therapeutic targets of HIV-1 latency .
Following integration into the host cell genome , HIV-1 transcription is the most important step regulating viral replication . The main factor involved in this regulation is the viral Tat protein , which binds TAR , a structured RNA element present at the 5′ end of the viral mRNAs . The structure of the mRNA 5′ end also contributes to the pausing of RNA polymerase II ( RNApolII ) at the LTR [1] . This pausing is characteristic of HIV-1 transcription and appears to play a role in maintaining low levels of basal transcription when the promoter is not activated [2] , [3] . Tat activates transcription by both inducing chromatin remodeling and recruiting P-TEFb , a cell factor required for productive transcription elongation , onto the viral LTR [4] , [5] . Tat also stimulates transcription by direct , TAR-independent activation of the viral promoter [6] . Induction of the host transcription factors NF-α B cooperates with Tat in completing HIV activation [7] . Chromatin plays an essential role in the transcriptional regulation of HIV ( reviewed by [8] , [9] ) . The transition from basal to activated transcription correlates with drastic changes in the acetylation levels of the nucleosomes covering the HIV promoter [10] and with the rearrangement of nucleosome positioning on the 5′ LTR [11] . These chromatin alterations are catalyzed by histone modifying enzymes and ATP-dependent chromatin remodeling complexes , which are recruited by Tat to the LTR [12]–[14] . Tat action on HIV chromatin is also mediated by the nucleosome assembly protein hNAP-1 [15] . Several host factors , including the receptor tyrosine kinase RON [16] and a subunit of the CPSF complex [17] , contribute to maintaining the repressive state characteristic of HIV latency , but most elements directly responsible for HIV postintegration latency are also related to chromatin . Histone deacetylases ( HDAC ) are involved in the transcriptional repression of the LTR [18] and their recruitment by CBF-1 promotes HIV-1 entry into latency [19] . HP1 , binding trimethylated histone H3-K9 , also plays a role in HIV-1 silencing [20] . Consistent with this role of chromatin in HIV latency , the chromatin environment of the integration site influences the transcriptional behavior of the provirus , whose level of basal transcription is undetectable in some integrants [21] . One of the most interesting phenomena that takes place during the transcriptional activation of a latent HIV-1 provirus is the precise , transcription-independent remodelling of nucleosome-1 , positioned at the 5′ end of HIV-1 transcribed region ( 5′HIV-TR ) , immediately downstream of the transcription start site [11] . In this work , we performed a genetic analysis of the role of the 5′HIV-TR in basal transcription , making use of the yeast Saccharomyces cerevisiae , which has already been successfully used to investigate other aspects of HIV-1 biology [22]–[24] . We show that the 5′HIV-TR is critical in repressing basal transcription in yeast and that this phenomenon is mediated by FACT , Spt6 and Chd1 , proteins involved in co-transcriptional chromatin reassembly . Finally , we confirm that this group of factors plays a role in maintaining low levels of basal transcription in human cells .
It has been previously described how the entire HIV-1 LTR is transcriptionally inactive in yeast [25] . Therefore , in order to investigate transcription elongation through the 5′HIV-TR in Saccharomyces cerevisiae , we constructed a chimeric yeast-HIV transcription unit . We located a fragment of the HIV-1 transcribed region ( +1/+671 ) , under the transcriptional control of a Ty1 promoter , which drives a retroelement with a low , but detectable , level of basal transcription [26] . The fragment included all the sequences that have been shown to be relevant in regulating HIV-1 transcriptional elongation . We did not choose a longer piece of HIV-1 to avoid the complex pattern of spliced forms that characterize this virus . In order to ensure its detection by northern blot , we increased the length of the mRNA by adding the coding region of the yeast PHO5 gene ( Figure 1A ) . A transcript of the expected length ( 2 . 1 kb ) was detected when we transformed three different wild-type strains of Saccharomyces cerevisiae with a centromeric plasmid containing the Ty1-HIV construct ( Figures 1A and Figure S1 ) . In order to explore whether the 5′HIV-TR can influence transcription in yeast , we deleted a 203 bp fragment of this region , including most of the TAR-encoding R domain . The new transcription unit , Ty1-HIVTARless , also expressed a transcript of the expected length ( 1 . 9 kb ) . Quantification of the transcripts revealed that the deletion produced a clear increase in the mRNA amounts ( Figures 1A and Figure S1 ) . To confirm that this difference was due to RNApolII pausing , we performed ChIP experiments with a Myc-tagged form of Rpb1 , the biggest subunit of RNApolII . The results obtained with Ty1-HIV showed that the amounts of RNApolII bound to the 5′HIV-TR were higher than the levels detected downstream ( Figure 1B ) . This enrichment was not due to the proximity to the initiation site , since we did not detect a significant accumulation of RNApolII at the equivalent region of Ty1-HIVTARless , immediately downstream of its initiation site ( Figure 1B ) . One of the main factors regulating HIV-1 transcription is DSIF , which exerts a negative influence on RNApolII transcription during early elongation [27] . We analyzed the mRNA levels of Ty1-HIV and Ty1-HIVTARless in an spt4Δ mutant lacking one of the subunits of yeast DSIF . As expected , the absence of Spt4 partially abolished the repressive role of the 5′HIV-TR ( Figure 1A ) . In order to ascertain whether the TAR structure contributes to the repressive role of the 5′HIV-TR in yeast , we constructed a mutant version of Ty1-HIV ( Ty1-HIVTARmut ) , in which we replaced the promoter-distal part of the TAR-encoding sequence ( 5′-GCTCTCTGGCTAACTAGGGAACCC-3′ ) by a complementary string ( 5′-CGAGAGACCGATTGATCCCTTGGG-3′ ) . The new transcription unit , encoding an mRNA without the ability to form a TAR structure , showed levels of expression similar to Ty1-HIV and clearly below Ty1-HIVTARless ( Figure 1C ) , indicating that the phenomenon that we are describing does not depend on the TAR element . Non-active HIV-1 LTR is occupied by a set of positioned nucleosomes , one of which covers the 5′HIV-TR [11] . To complete the characterization of Ty1-HIV , we digested chromatin and naked DNA samples with micrococcal nuclease ( MNase ) and analyzed nuclesome positioning by quantitative PCR . As shown in Figure 1D , the transcribed region of Ty1-HIV showed a clearly defined nucleosomal pattern , similar to HIV-1 in human cells . In contrast , the pattern on the Ty1-HIVTARless transcribed region is incompatible with a unique translational phase of nucleosomes , suggesting a more dynamic chromatin structure with several alternative distributions ( Figure 1D ) . The higher accessibility of Ty1-HIVTARless chromatin was also confirmed by hybridization of MNase–treated samples with probes covering the 5′end of its transcribed region ( Figure S2 ) . Hybridization of Ty1-HIV samples with probe 1 , covering the 5′HIV-TR produced a ladder of signals , compatible with a regular nucleosomal structure , whose shortest fragment was approximately 150 bp long . Similar results were obtained with probe 3 . In contrast , hybridization of Ty1-HIVTARless samples with probe 2 , covering the 5′ end of its transcribed region , produced a nucleosomal ladder that included a smeared signal of DNA fragments shorter than 100 bp . Rehybridization with probe 3 , also showing a less regular nucleosomal ladder , excluded the possibility that this smear was due to DNA degradation ( Figure S2 ) . Taken together , the results shown so far indicate that Ty1-HIV is a good tool for investigating transcription elongation through the 5′HIV-TR , and suggest that the repressive role of this DNA element in yeast is related to its chromatin structure . It has been described how nucleotide deprivation stimulates Ty1 transcription [28] . We found that the addition of 6-azauracil , an NTP-depleting drug , to the medium produced a rapid increase in the overall level of Ty1 mRNA ( Figure S3 ) . We made use of this simple method of activating the Ty1 promoter in order to study the effect of promoter activation on 5′HIV-TR transcription . We observed that promoter activation eliminated the functional differences between Ty1-HIV and Ty1-HIVTARless , which became equally expressed in the presence of 6-azauracil . Similar results were obtained in the absence of the RNApolII cleavage-factor TFIIS ( dst1Δ ) , suggesting that RNApolII does not become arrested when transcribing the 5′HIV-TR ( Figure 2A ) . To further confirm that promoter activation abolishes the repressive effect of the 5′HIV-TR , we replaced the Ty1 promoter by the one of GAL1 , a tightly regulated gene that becomes strongly activated when galactose is the carbon source . Under conditions of weak activation ( 2% raffinose plus 0 . 02% galactose ) a clear difference was observed between GAL1-HIV and GAL1-HIVTARless ( Figure 2B ) , indicating that the 5′HIV-TR does not only repress Ty1-driven transcription . As expected , when we added high levels of galactose ( 2% ) to the medium , we observed a further increase in mRNA accumulation . However , in this case , the levels of mRNA accumulation were the same in cells containing the GAL1-HIV construct as in those transformed with GAL1-HIVTARless ( Figure 2C ) . This result shows again that the repressive role of the 5′HIV-TR is not effective under activating conditions . When HIV-1 promoter becomes active in human cells , the TAR domain is required for full activation by Tat and P-TEFb . We wondered whether the activation of the promoter in yeast still allows a supplemental induction by Tat and P-TEFb . We expressed hCDK9 , hCycT1 and Tat by cloning their cDNAs in yeast expression vectors ( Figure S4A and B ) . We did not detect any influence of Tat and P-TEFb on Ty1-HIV basal transcription ( Figure 2D ) . We were also interested in testing the effect of Tat and P-TEFb on Ty1-HIV expression under activating conditions . The addition of 6AU to these cells produced very irregular results , likely due the presence of three different plasmids in this strain and to the plasmid instability produced by 6-azauracil [29] . As an alternative approach , we tested the effect of Tat and P-TEFb on a more active Ty1-HIV by repeating the assay in an spt4Δ strain . In this case we obtained consistent results and a weak but significant effect was detected ( Figure 2D ) . We also detected a positive effect of P-TEFb on activated GAL1-HIV expression , which was strictly dependent on CycT1 and partially dependent on CDK9 ( Figure S4C ) . We analyzed whether this second level of activation was mediated by the 5′HIV-TR , encoding the TAR RNA domain . As expected , Tat and P-TEFb were unable to enhance the expression of GAL1-HIVTARless ( Figure 2C ) . This set of results suggests that the repressive role of the 5′HIV-TR and the TAR-dependent regulation of HIV-1 transcription can be functionally separated . We then analyzed the expression of Ty1-HIV and Ty1-HIVTARless in a selected group of mutants related to the elongation step of transcription . The results of these analyses are shown in Figure S5 and 3A . Some of the mutants tested , such as swi2Δ , lacking a subunit of the SWI-SNF chromatin remodeling complex , set1Δ , affecting the COMPASS histone-methylation complex , or rpd3Δ , lacking one of the main histone deacetylases , did not produce a significant effect on the expression of any of the two chimeric transcription units . We concluded that these factors do not contribute significantly to either elongation through the 5′HIV-TR or Ty1 promoter activity . Some other mutants , such as paf1Δ , lacking the main subunit of the PAF1 complex; spt2-150 , affected in a HMG component of yeast chromatin; those affecting the SAGA complex ( gcn5Δ , spt3Δ , spt8Δ ) , or those lacking elements of cyclin-dependent kinases involved in transcription ( bur2Δ , ctk1Δ ) produced a negative effect on the expression of both Ty1-HIV and Ty1-HIVTARless ( Figure S5 ) . The simplest interpretation of these results is that this set of factors is required for the basal activity of the Ty1 promoter and/or for transcription elongation through both Ty1-HIV and Ty1-HIVTARless . In any case , it seems that these factors are not related to the repressive effect of the 5′HIV-TR . The absence of Isw1 , the catalytic subunit of the ISW1 chromatin remodeling complexes , stimulated the expression of both Ty1-HIV and Ty1-HIVTARless ( Figure S5 ) . This result is fully consistent with the described role of Isw1 in inhibiting Ty1 expression [30] . A similar result was obtained with the ioc2Δ mutant , lacking one of the subunits of the ISW1b complex . However , the absence of Ioc3 or Ioc4 , belonging to the ISW1a and ISW1b complexes respectively , enhanced the expression of Ty1-HIV but decreased that of Ty1-HIVTARless ( Figure S5 ) . These results confirm the intricacy of the roles played by the ISWI complexes [31] and might suggest their involvement in the repressive effects of the 5′HIV-TR , but are difficult to interpret properly without further research . A last group of mutants included chd1Δ , lacking a chromodomain protein involved in transcription elongation; spt16-197 , affecting one of the subunits of the FACT elongation factor; and spt6-140 , encoding a defective form of a factor related to chromatin and mRNA transactions during elongation [32]–[34] . Both SPT16 and SPT6 are essential genes and , like CHD1 , are involved in the reassembly of chromatin during transcription elongation [35] . Strong functional interactions have been detected amongst these three genes [36]–[38] . This group of mutants did not significantly affect the expression of Ty1-HIVTARless but did increase the levels of Ty1-HIV mRNA , a phenotype very similar to that of spt4Δ ( Figure 3A , 3B and S5 ) . Spt16 , FACT and Chd1 are general chromatin factors that are associated with actively transcribed regions in all eukaryotes investigated so far [39]–[41] . Since they seemed to play a role in the weakly transcribed Ty1-HIV , we tested the presence of these factors on this transcription unit by ChIP . We found significant enrichments for the three proteins on Ty1-HIV ( Figure 3C ) . In all three cases , the enrichment was higher on the transcribed region than on the promoter , confirming the well-known connection of these factors with transcription elongation . These results suggest that the repressive function of the 5′HIV-TR depends on chromatin reassembly . To further corroborate this hypothesis , we analyzed the expression of Ty1-HIV and Ty1-HIVTARless in hta1Δ-htb1Δ , a mutant suffering from a deficit of H2A and H2B histones . The expression of Ty1-HIVTARless was not significantly affected in this mutant , whereas the mRNA levels of Ty1-HIV showed a clear increase ( Figure 5S ) . Thus , hta1Δ-htb1Δ grouped with spt4Δ and the three chromatin-reassembly mutants ( Figure 3A ) . In order to verify that this group of mutants was indeed affecting the chromatin structure of Ty1-HIV , we analyzed nucleosome positioning on Ty1-HIV in some of the mutants ( Figure 3D ) . spt4Δ caused a general alteration of nucleosome positioning on the transcribed region of Ty1-HIV , clearly affecting nucleosomes 1 , 2 and 3 . In the overlapping region , this pattern was very similar to the one exhibited by Ty1-HIVTARless in the wild type ( Figure 1D ) . spt6-140 also showed a less positioned pattern than the wild type , although in this case nucleosome 2 was almost unaffected . In turn , chd1Δ almost eliminated the signal corresponding to nucleosome 2 without significantly affecting the other two nucleosomes ( Figure 3D ) . In order to investigate whether chromatin reassembly factors really do play a role in the repression of HIV-1 basal transcription in human cells , we knocked down the expression of human Spt6 and Chd1 in HIV latently infected cells . A model of HIV-1 latency in Jurkat cells infected with an HIV minigenome encoding Tat and GFP has been previously reported [42] . Upon infection , latent cells are defined as those that do not express constitutively GFP , but need further stimuli of HIV promoter by mitogens ( PMA ) or cytokines ( TNF-α ) . After removal of stimuli , the HIV promoter becomes repressed again and GFP-negative cells can be purified and maintained as a population; alternatively , individual cells representing unique latent viral integrations can be cloned . These cells are useful for investigating maintenance of HIV promoter repression and viral latency . We hypothesized that , if chromatin reassembly factors were involved in HIV promoter repression , its depletion would cause gene reactivation . We used lentiviral shRNA expression vectors to deplete Spt6 and Chd1 in HIV latently infected cell populations ( Figure 4A ) . 10–15 days after shRNA infection and puromycin selection , the percentage of Spt6 and Chd1 knocked-down cells expressing GFP reached ca . 5% , a significantly higher proportion that obtained with a control shRNA vector ( Figure 4B ) . Because the latently-infected cell population is heterogeneous , representing a myriad of HIV integrations at different genome locations and chromatin environments , only a small proportion of latent HIV provirus are activated by solely depleting these factors . A similar degree of reactivation was obtained with an shRNA against YY1 , a host transcription factor previously reported to be involved in histone deacetylase ( HDAC ) recruitment and HIV promoter repression [43] , [44] ( Figure 4B ) . The HDAC inhibitor trichostatin A ( TSA ) activates the HIV promoter in ca . 9 . 5% of the latently-infected cell population , indicating again that not all HIV integrations are equally sensitive to the inhibition or depletion of a repressive chromatin factor ( data not shown ) . Next , we investigated the effect of Spt6 and Chd1 depletion on HIV expression in particular clones containing single latent integrations either in centromeric alphoid repeats ( clones H2 and C1 ) , an intergenic region ( clone A1 ) or in an intron of the highly transcribed gene UBXD8 ( clone 27 ) [42] ( and unpublished results ) . The percentage of reactivated cells varied depending upon the clones , reaching ca . 30% reactivation in clone 27 after knocking-down Spt6 ( Figure 4C and 4D ) . Reactivation by TSA is also dissimilar between clones , ranging from 27% in clone C1 to 47% in clone 27 ( Figure 4C and data not shown ) . Similarly , Spt6 and Chd1 depletion promoted reactivation of the silent HIV promoter in a newly-generated model of HIV latency in HeLa cells ( Figure S6 ) . Moreover , these effects were observed with several different shRNA sequences and correlated well with the degree of target protein depletion achieved , discarding unspecific off-target effects ( Figure S6 ) . Altogether , our data indicates that these two chromatin reassembly factors identified in the yeast screening contribute to maintain HIV repression in infected human cells .
In this work we have made use of yeast genetic analysis to investigate the influence of the 5′HIV-TR on basal transcription . Several attempts to study HIV-1 transcription in yeast have been described , all of them focused in the transactivation capacity of Tat . Although a fusion of Tat with the DNA binding domain of Gal4 can activate the GAL1 promoter [45] , no Tat-dependent transactivation of the HIV-1 LTR had yet been achieved [25] . Therefore , the present work is the first successful reconstruction in yeast of a transcriptional system based on HIV-1 elements . The artificial character of the Ty1-HIV transcription unit raises the possibility of the conclusions extracted from this work being of no relevance for HIV-1 biology . Several results presented in this piece of research argue against that point of view . We show that the 5′HIV-TR not only represses transcription driven by the Ty1 promoter but can also act on a completely different promoter ( GAL1 ) when this is weakly active . We also show that the 5′HIV-TR induces an accumulation of RNApol II immediately downstream of the promoter , a very common situation throughout the human genome but extremely infrequent in yeast [46] . It has been recently proposed that the reason for this difference is the chromatin organization of the transcription start site , which is usually covered by a positioned nucleosome in yeast and immediately upstream of a positioned nucleosome in most metazoan genes [47] . We show in this work that the chromatin organization of the HIV fragment present in Ty1-HIV closely resembles the distribution of positioned nucleosomes of HIV-1 proviruses in the human genome . We also present evidence that transcription through the 5′HIV-TR in yeast is influenced by factors that have been previously shown to govern HIV-1 transcription elongation: yDSIF contributes to the repressive role of the 5′HIV-TR in basal transcription , whereas Tat and P-TEFb enhance active transcription in a 5′HIV-TR-dependent manner . Finally , the main conclusion of the genetic analysis , which is the involvement of chromatin reassembly factors in repressing HIV-1 basal transcription , has been confirmed in a human model of HIV-1 latency . Based on these considerations , we believe that the chimeric yeast-HIV system is a valid complementary tool for HIV research . The role of the 5′HIV-TR in basal transcription has scarcely been studied , and the data available is sometimes contradictory [48] , [49] , likely due to the use of transiently transfected DNA , which does not ensure a proper organization of DNA in chromatin [50] . In fact , mutation in this region produced different effects on HIV-1 transcription when an integrated version was compared to a transiently transfected one [51] , [52] . The data shown in the present work indicates that the 5′HIV-TR contributes to maintaining low levels of basal transcription without interfering with promoter activation . The genetic analysis that we have performed shows a contribution of chromatin reassembly factors to this repressive function of the 5′HIV-TR . We have also confirmed that Spt6 and Chd1 favor a close chromatin configuration on the transcribed region of Ty1-HIV . The role of chromatin reassembly factors at this level seems to be as significant as the one played by DSIF , since the combination of the chromatin alterations produced by spt6-140 and chd1Δ fully matches those caused by spt4Δ . Deletion of the 5′HIV-TR causes a disruption of nucleosome positioning on the rest of the HIV fragment present in Ty1-HIVTARless , mimicking the patterns of spt4Δ and , to a lesser extent , of spt6-140 . It is possible that this chromatin difference between the two transcription units is a consequence of the higher transcription level of Ty1-HIVTARless . Alternatively , the absence of the +1 nucleosome may destabilize the overall chromatin configuration and this would , in turn , give rise to increased transcriptional activity . The differences in the chromatin patterns of spt4Δ , spt6-140 and chd1Δ , all showing similar levels of expression , indicate that the chromatin differences are likely to be responsible for the transcription increase and not vice versa . This explanation also fits better with the accumulation of RNApolII on the 5′HIV-TR and with the results of our genetic analysis . In this scenario , productive elongation would be infrequent under non-activating conditions due to both the low number of initiation events and the positioned nucleosomes sitting on the 5′HIV-TR but , in those rare occasions when RNApol II gets through the chromatin boundary , the immediate action of chromatin reassembly factors ( recruited by elongating RNApolII itself ) would contribute to rebuilding the repressive chromatin configuration , avoiding a transition into an activation-prone chromatin environment ( Figure 5A ) . Several chromatin-mediated mechanisms contribute to regulating HIV-1 transcription [8]: the activation of the LTR promoter is mediated by the acetylation state of its chromatin , especially by the nucleosome located upstream in the LTR ( nucleosome 0 ) [10] . An additional role of chromatin in regulating HIV-1 transcription takes place at the level of early elongation , since the positioned nucleosome covering the 5′HIV-TR ( nucleosome 1 ) becomes remodeled in response to promoter activation , in a transcription-independent manner [11] . Although transcription of Ty1-HIV is more intensively repressed by chromatin reassembly factors than Ty1-HIVTARless , we do not believe that the 5′HIV-TR is specifically required for their recruitment . Spt6 , FACT and Chd1 are general elongation factors , whose association to actively transcribed regions , in an RNA polymerase II-dependent manner , is well documented from yeast to metazoa [33] , [38]–[41] , [53]–[55] . We favour the idea of the 5′HIV-TR being an optimal DNA sequence for nucleosome positioning . Recent genome-wide studies show the importance of 5′ sequences in specifying the location of +1 nucleosomes . In turn , these act as barriers against which other nucleosomes are packed [56] . In such a DNA context the repressive action of chromatin reassembly factors would be maximal . Basal transcription of HIV-1 is highly dependent on the chromatin environment of integration sites [21] . Nevertheless , mutations affecting the sequences located at the 3′ border of nucleosome 1 , which increase its stability , produce a general reduction in basal transcription , irrespective of the integration site [57] . In contrast , the deletion of 60 nucleotides within the sequence covered by this nucleosome , which presumably destabilize it , makes basal transcription even more dependent on the integration site than the wild type [57] . This data is fully compatible with our yeast results and provides support for chromatin configuration playing a role in repressing HIV-1 transcription at the level of early elongation . A similar mechanism has been reported for the human hsp70 gene [58] , where the repressive chromatin configuration covering its transcribed region becomes remodeled during activation , allowing poised RNApolII to complete elongation [59] . The most relevant conclusion of our genetic analysis is the involvement of chromatin-reassembly factors in repressing HIV-1 basal transcription . We have confirmed that their function is not restricted to our chimeric yeast-HIV system but they are also playing a role in HIV-1 basal transcription in human cells . It has been reported that integration into regions of compacted chromatin , i . e . centromeric heterochromatin , causes HIV promoter inactivity , probably due to the inaccessibility of the basal transcriptional machinery or the inability of transcription factors to overcome the repressive chromatin state imposed [42] . In the latent integrations , this repressive state can be overcome by exogenous stimulation with mitogens or cytokines . We have found that , by knocking-down Spt6 and Chd1 , the HIV promoter integrated in the context of latency increases its level of expression . When we deplete Spt6 or Chd1 from the heterogeneous population of HIV latently infected cells , only a small proportion of integrant promoters are activated . This indicates that only a subset of integrations are either negatively regulated by these factors , or able to be reactivated by solely depleting them . In some chromatin environments , transcription may be completely inhibited , such that chromatin disruption by basal transcription would not be an issue and chromatin reassembly factors would play no role . Similarly , TSA treatment and YY1 depletion are not able to reactivate all latent integrations . We had predicted that individual integrations would have a more consistent response to the depletion of reassembly factors . In fact , we observed that individual clones responded differently , both amongst themselves and to the two distinct shRNAs . Still , not all cells of a clonal population respond equally . This behavior is also observed in response to TSA and resembles position effect variegation [42] . Depletion of Spt6 and Chd1 may cause a deficit of chromatin reassembly in those rare events , during non-activated conditions , in which RNApolII would be able to overcome the chromatin barrier ( Figure 5A ) . This chromatin change would facilitate additional rounds of transcription under non-activating conditions and , eventually , an increase in transcription factor loading and PIC assembly . If transcription were enough to produce some Tat protein , then promoter activity would be reinforced . Inhibition of HDAC activity with TSA has a similar effect , supporting the idea that discrete modifications of chromatin compactness may be sufficient to switch a repressed provirus to active . It has been shown absence of chromatin reassembly factors in yeast provokes activation of cryptic promoters located in the body of transcribed genes . In the absence of transcription , no PIC assembles on these cryptic promoters , due to the inhibitory action of chromatin; under active transcription , chromatin reassembly factors ensure rapid nucleosome deposition after RNApol II passage , avoiding the activation of cryptic promoters [54] , [60] . HIV proviruses integrated in highly transcribed genes are usually latent [61] . Several mechanisms of transcriptional interference have been proposed to explain this phenomenon [62] . It has been shown that read-through transcription from an upstream promoter can interfere with HIV transcription by disturbing PIC assembly [63] . Recent published evidence indicates that transcriptional interference is caused by the elongating form of RNA pol II , transcribing through the latent HIV copy [64] , [65] . This situation parallels that of yeast cryptic promoter and , accordingly , it is likely that chromatin reassembly factors also play an important role in maintaining HIV latency by transcriptional interference ( Figure 5B ) . The integration site of one of the individual clones tested in our depletion experiments ( clon 27 ) is a highly transcribed gene . We found a clear reactivation of the latent HIV copy of this clone when chromatin reassembly factors were depleted . In fact , when Spt6 was depleted , this clone showed the maximal reactivation level reached in the whole set of experiments . When taken together , our results indicate that Spt6 and Chd1 participate in the mechanism that controls the equilibrium between activation and repression of HIV-1 expression when the provirus is integrated in the human genome , which depends greatly on the chromatin environment of the integration site . Disturbance of this equilibrium , by depleting chromatin reassembly factors for example , makes some of the latent integrations become activated . These factors may also play a general role in repressing basal transcription throughout the human genome . Therefore , the specificity of chromatin reassembly factors in repressing viral basal transcription should be carefully evaluated before considering them as therapeutic targets against HIV-1 latency .
Yeast strains used are listed in Table S1 and are isogenic to the S288C derivative BY4741 [66] . Plasmids are described in Table S2 . Yeast cells were grown following standard procedures [67] . mRNA levels were measured by Northern analysis as described [29] . Laemmli boiled crude extracts were run on a 10% SDS-polyacrylamide gel and transferred to nylon membranes ( Hybond-ECL ) . After blocking with Tris-buffered saline containing 0 . 1% Tween 20 and 5% milk , proteins were detected using anti-FLAG antibodies ( monoclonal , Santa Cruz ) and human cyclin T1 ( polyclonal , Santa Cruz ) and peroxidase-conjugated goat anti-mouse and rabbit anti-goat IgG respectively . Blots were washed with tris-buffered saline and 0 . 1% Tween 20 , and developed by enhanced chemiluminescence reactions ( PIERCE ) . Signals were detected with Hyperfilms ECL ( Amersham ) , exposing from 15 sec to 5 min . ChIP analyses of Rpb1-Myc were performed using a monoclonal anti-cMyc antibody ( 9E10 ) as described previously [55] . For crosslinking , cells were treated with 1% formaldehyde for 15 min at room temperature . As a non-transcribed control , we amplified a region adjacent to FUS1 . Primer mixes were empirically adjusted for balanced signals . Immunoprecipitation was defined as the ratio of each gene-specific product in relation to that of the non-transcribed region , always after normalization with the signal of its corresponding whole-cell extract . Several dilutions of the whole cell extract were tested to ensure that the assays were in the linear range . Spt16-Myc , Spt6-HA and Chd1-HA abundances on Ty1-HIV were assayed as indicated , by using ChIP with monoclonal anti-cMyc ( 9E10 ) or anti-HA antibodies and protein A-Sepharose . We used 20- to 30-bp oligonucleotides for PCR amplification of fragments of the Ty1 promoter ( positions −157 to −107 , relative to the transcription start site ) and the 5′HIV-TR ( positions +102 to +162 ) . Real-time quantitative PCR was performed with SYBR green dye in the 7500 Real Time PCR system of Applied Biosystems by following the manufacturer's instructions . A non-transcribed telomeric fragment was used to normalize the signals . No-antibody controls were performed to exclude unspecific amplification . Yeast spheroplasts and micrococcal nuclease digestions were performed according to [68] with the modifications of [69] . Spheroplasts were prepared from mid-log phase cultures grown in SC-Ura with 2% glucose . Cells were lysed and immediately digested with 7 . 5 to 125 mU of micrococcal nuclease . Digested DNA was resolved in 1 . 5% agarose gels and analyzed by Southern blot with the probes indicated in Figure S2 . Alternatively , DNA was quantified by real time PCR performed with SYBR green dye in a Applied Biosystems 7500 Real Time PCR system , following the manufacturer's instructions . In order to correct for the sequence-specificity of MNase , naked-DNA samples digested with MNase were also quantified by real-time PCR . The chromatin/naked-DNA ratio was considered to be a valid estimation of chromatin-dependent resistance to MNase . Pools or particular clones of Jurkat cells latently infected with an HIV-derived minigenome [42] were infected with Control , YY1 , Spt6 or Chd1 shRNA-expressing lentiviruses ( vector pLKO . 1-Puro ) obtained from Sigma ( MISSION™ shRNAs ) . The protocol for viral particles production and cell infections has been described elsewhere [42] . Upon puromycin ( 2 mg/ml ) selection of infected cells , HIV reactivation was followed by FACS analysis of GFP-positive cells . shRNA-mediated inhibition was tested by Western blot with specific anti-human YY1 antibodies ( Santa Cruz sc-281 ) , Spt6 ( Abcam ab32820 ) and Chd1 ( Abnova H00001105-A01 ) . Anti-tubulin antibody was from Sigma . A pool of latently infected HeLa cells was constructed as previously reported [42] and infected with several Spt6 and Chd1 shRNA-expressing vectors . Further details are described in the legend of the Figure S6 . | Acquired immunodeficiency syndrome ( AIDS ) is caused by the human immunodeficiency virus ( HIV ) . Drugs used for anti-viral therapy are very efficient in controlling the presence of viral particles in infected patients . However , if this therapy is interrupted , rebound of viremia occurs due to the small population of cells that remain latently infected . A specific region of the HIV genome ( the beginning of the transcribed region ) plays an important role in viral expression and may contribute to its silent state in latently infected cells . We reconstructed a minimal HIV system in yeast to perform a genetic analysis of the role played by that specific region of the viral genome . We found that the repressive role played by this region is mediated by a group of proteins ( chromatin reassembly factors ) so far associated with other functions during gene expression . We confirmed that this group of factors plays a role in controlling HIV-1 basal transcription in human cells . | [
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] | 2009 | Yeast Genetic Analysis Reveals the Involvement of Chromatin Reassembly Factors in Repressing HIV-1 Basal Transcription |
Lymphatic filariasis ( LF ) is among the 10 neglected tropical diseases targeted for control or elimination by 2020 . For LF elimination , the World Health Organization ( WHO ) has proposed a comprehensive strategy including ( i ) interruption of LF transmission through large-scale annual treatment ( or mass drug administration ( MDA ) ) of all eligible individuals in endemic areas , and ( ii ) alleviation of LF-associated suffering through morbidity management and disability prevention . In Cameroon , once-yearly mass administration of ivermectin and albendazole has been implemented since 2008 . The aim of this study was to assess progress towards the elimination goal , looking specifically at the impact of six rounds of MDA on LF transmission in northern Cameroon . The study was conducted in the North and Far North Regions of Cameroon . Five health districts that successfully completed six rounds of MDA ( defined as achieving a treatment coverage ≥ 65% each year ) and reported no positive results for Wuchereria bancrofti microfilariaemia during routine surveys following the fifth MDA were grouped into three evaluation units ( EU ) according to WHO criteria . LF transmission was assessed through a community-based transmission assessment survey ( TAS ) using an immunochromatographic test ( ICT ) for the detection of circulating filarial antigen ( CFA ) in children aged 5–8 years old . A total of 5292 children ( male/female ratio 1 . 04 ) aged 5–8 years old were examined in 97 communities . Positive CFA results were observed in 2 , 8 and 11 cases , with a CFA prevalence of 0 . 13% ( 95% CI: 0 . 04–0 . 46 ) in EU#1 , 0 . 57% ( 95% CI: 0 . 32–1 . 02 ) in EU#2 , and 0 . 45% ( 95% CI: 0 . 23–0 . 89 ) in EU#3 . The positive CFA cases were below WHO defined critical cut-off thresholds for stopping treatment and suggest that transmission can no longer be sustained . Post-MDA surveillance activities should be organized to evaluate whether recrudescence can occur .
Lymphatic filariasis ( LF ) is among the most widespread neglected tropical diseases . In the mid-1990s , it was reported that about 1 . 4 billion people were exposed to the disease worldwide , of whom 120 million were infected and more than 40 million disfigured by the disease [1] . One of the core resolutions of the 50th World Health Assembly held in 1997 was to eliminate LF as a public health problem ( resolution WHA50 . 29 ) . To address this global concern , the World Health Organization ( WHO ) proposed a comprehensive elimination strategy including ( i ) transmission interruption in endemic communities ( so-called mass drug administration or MDA strategy ) , and ( ii ) implementation of interventions to prevent and manage LF-associated disabilities ( so-called morbidity management and disability prevention or MMDP strategy ) [2] . The Global Programme to Eliminate Lymphatic Filariasis ( GPELF ) was launched in 2000 , by the WHO , to elaborate specific plans and coordinate control efforts to reach this ambitious goal . In the MDA strategy , LF must be mapped and preventive chemotherapy ( PC ) implemented to treat the entire eligible population ( areas where prevalence of antigenaemia is ≥ 1% ) . In areas where onchocerciasis is endemic and where Wuchereria bancrofti prevails , the recommended PC is a single dose of a bi-therapy ( 150 μg/kg of body weight ivermectin in combination with 400 mg albendazole ) , administered once yearly [3 , 4] . Since this treatment is not macrofilaricidal , adult worms can remain viable for about six years and the delivery of several rounds of MDA appeared crucial . It is now accepted that annual MDA should be repeated for at least 5 years at adequate levels of coverage , estimated to be at least 65% of the total population in endemic areas ( “effective” MDA ) , to ascertain that the level of infection in the community will be reduced to levels below which transmission cannot be sustained , even after MDA has been stopped [5] . Recent estimates of the impact of MDA during the past 13 years revealed that more than 96 million LF cases were prevented or cured , although as many as 36 million cases of hydrocele and lymphedema remain [1] . However , data reporting interruption of LF transmission are scanty , especially in Sub Saharan Africa where the disease represents one-third of the global burden [6] . Cameroon is known to be endemic to onchocerciasis [7 , 8] and bancroftian filariasis [9 , 10] , and MDA against LF have been implemented since 2008 . Indeed , ivermectin and albendazole have been distributed by community drug distributors ( CDDs ) following the community directed treatment with ivermectin ( CDTI ) approach . This strategy has already been implemented 15–20 years earlier to fight against onchocerciasis . As such , the strategy was already well mastered by CDDs and was ongoing smoothly at the time MDAs against LF were implemented , following a door-to-door approach . This study aimed at assessing whether the transmission of LF has been successfully halted in areas where six MDA rounds have already been delivered .
This study was carried out in 2014 in the North and Far North Regions of Cameroon , situated between latitudes 7° and 12°N , and longitudes 12° and 16°E . Five health districts or implementation units ( Ngong , Poli , Tcholliré , Rey-Bouba in the North Region and Mokolo in the Far-North Region ) , with rural to semi-urban settings , were included in this study . These implementation units ( IU ) were organized into three evaluation units ( EU ) ( Fig 1 ) according to the criteria described in the WHO monitoring and evaluation manual [2] . In 2014 , the population of each of these two Regions was estimated to two millions , children aged 6–7 years old representing about 10% of the general population [11] . A cross sectional study was carried out following the recommendations described in the WHO manual for national elimination programs [2] . The flow chart below ( Fig 2 ) describes the different steps taken in the LF elimination process , thus conferring the eligibility of the targeted implementation units to the transmission assessment survey step . All relevant data for this study were recorded into a purpose-built Microsoft Access database and subsequently exported into PASW Statistics version 18 ( SPSS Inc . , Chicago , IL , USA ) for statistical analyses . The prevalences of infection were expressed as the percentage of infected children ( harboring CFA ) among the total number of children examined; the 95% confidence interval ( CI ) was calculated using the Wilson method not corrected for continuity [18] . Chi-square tests were used to compare LF prevalence between sexes and age groups , as well as the computed threshold of infection prevalence below which transmission is likely no longer sustainable , so-called critical cut-off threshold , against the observed proportion of ICT positive cases . This study was conducted as part of the action plan of the national program to eliminate lymphatic filariasis in Cameroon . Ethical clearance was granted by the Cameroon National Ethics Committee for Human Health Research ( N°2014/09/491/CE/CNERSH/SP ) . Before enrolment , the objectives and schedule of the study were explained to the eligible population and individuals willing to participate signed two inform consent forms , and kept a copy . The second copy was stored at the Centre for Research on Filariasis and other Tropical Diseases . Even after minors assenting , the approval of their parents or legal guardians was necessary before any procedure . Each enrollee was assigned a unique identifier and his data analyzed anonymously . Positive cases were referred to CDDs and health officers for a close follow-up during next treatments , and their parents or legal guardians warned about the situation to further insure a better compliance . Although no guidelines are given in the TAS manual [2] , the number of positive cases- that can be up to 18 as was the case in the present study - , appears as a real concern in a context where MDA has to be halted if the EU passes TAS . In this context , we have recommended to treat these rare positive cases with ivermectin during the MDA campaign plan just after the survey , then by a long course of doxycycline ( 4–6 weeks ) when they get above 8 years and MDA no longer available .
A total of 97 communities ( EAs ) were surveyed in the three EUs , and 5292 children ( 48 . 9% females ) examined . These children were aged 5–8 ( median age: 6 ) years old . Among the 5292 enrollees , 4171 ( 78 . 8% ) were aged 6–7 years old ( initial target ) , a small proportion being aged 5 ( 11 . 8% ) or 8 ( 9 . 4% ) years old . A total of 1595 children were examined in EU#1 , 1919 in EU#2 and 1778 in EU#3 , the expected sample size being reached in all the three EUs ( Table 1 ) . Prevalence of W . bancrofti circulating antigens , assessed using ICT card test , was equal to 0 . 13% ( 95% CI: 0 . 04–0 . 46 ) in EU#1 , 0 . 57% ( 95% CI: 0 . 32–1 . 02 ) in EU#2 , and 0 . 45% ( 95% CI: 0 . 23–0 . 89 ) in EU#3 ( Table 1 ) . The overall prevalence was 0 . 40% ( 95% CI: 0 . 26–0 . 61 ) , with 80 . 95% positive cases aged 6–7 years old . The prevalence of LF was similar , both between age groups and sexes ( p > 0 . 7408 ) . The spatial distribution of positive cases was in general over-dispersed ( both among health districts and EAs ) , except in the EU#2 where 8 children ( 1 . 07%; 95% CI: 0 . 54–2 . 10 ) with W . bancrofti circulating antigens were found in the Ngong health district , 6 of them belonging to two EAs . The total number of LF positive cases was 2 in EU#1 , 11 in EU#2 and 8 in EU#3 , all below the critical cut-off threshold ( 18 in each EU ) generated by the Survey Sample Builder . As compared to the threshold of infection prevalence below which transmission is likely no longer sustainable , the proportion of positive cases was significantly lower in the EU#1 ( Chi-square = 12 . 68; p = 0 . 0004 ) and EU#3 ( Chi-square = 5 . 27; p = 0 . 02 ) , but not significantly lower in EU#2 ( Chi-square = 3 . 48; p = 0 . 06 ) .
In Cameroon , MDA against LF , using the combination of ivermectin and albendazole , started in 2008 in the North and Far North Regions . In 2014 , five health districts ( Mokolo , Ngong , Poli , Rey-Bouba and Tcholliré ) completed six MDA rounds , and successfully passed the assessment of impact of MDA on LF infection after the fifth round of MDA ( post 5th MDA survey ) . The objective of the present study was thus to check whether the transmission of the disease has been successfully halted . Based on historical data [10] , sentinel sites’ survey data and/or kriging data [9 , 12] , the North and Far North Regions were previously highly endemic for LF , and were reported among the most prevalent over the country . In 2014 , LF prevalence observed in each of the three EUs investigated—in average equal to 0 . 40% ( 95% CI: 0 . 26–0 . 61 ) —was significantly lower than the threshold below which the transmission of the disease can no longer be sustained . Indeed , it was accepted that in areas where W . bancrofti is endemic and Anopheles or Culex is the principal vector , this target threshold must be < 2% antigenaemia prevalence [2] . In Cameroon , LF entomological data are very scanty but malaria data can be informative . Although Anopheles gambiae and Anopheles funestus have been found naturally infected with W . bancrofti [10] , malaria entomological data have shown that the most abundant vectors in the Northern Cameroon are from genera Anopheles and Culex ( Nwane , personal communication ) . The number of positive antigenaemia cases observed in each of the three EUs surveyed was below the critical cut-off generated by the SSB ( 18 CFA positive cases ) , indicating that the area successfully “passed” TAS , and a cessation of MDA in the constituting communities should be envisioned . Indeed , the sample sizes and critical cut-off values were chosen so that an EU has ( i ) at least a 75% chance of passing TAS if the true prevalence of antigenaemia is 1 . 0% ( half the target level if the vector is Anopheles or Culex ) , and ( ii ) no more than about a 5% chance of passing ( incorrectly ) TAS if the true prevalence of antigenaemia is ≥2 . 0% [19 , 20] . The importance of transmission assessment surveys as an evaluation tool for stopping MDA have been previously demonstrated in a multicenter evaluation using different approaches or study designs [21] . Moreover , the validity of TAS was also proven in long term post-MDA surveillance , although complementary test ( antibody and xenomonitoring ) appear of interest to ascertain the interruption of transmission during post-MDA surveillance [22–24] . It is important to notice that the interruption of transmission was achieved despite the fact that in some health districts , the effective treatment coverage was not reached for one or two rounds , although globally higher than 65% ( S1 Table ) . TAS was considered for these health districts for three main reasons: ( i ) long lasting insecticidal nets ( LLINs ) have been distributed in the study area in the framework of malaria control program activities . Indeed , between 2003 and 2010 , more than two millions LLINs have been distributed to pregnant women and children under 5 years old . In the framework of LLINs universal coverage for the control of malaria , a total of 21 , 028 , 770 LLINs have been distributed in the entire country in 2011 and 2016 ( with 73% coverage in 2011 and 88% coverage in 2016 ) , on the basis of one LLIN for every 2 . 2 households [25] . The impact of LLINs on prevalence and intensity of LF infection is now widely accepted [26 , 27] , and it was shown that a sustained reduction in LF prevalence can be reached in spite of missed rounds of MDA [28] . In addition to these efforts related to the known usefulness of LLINs , the relatively poor compliance observed at the beginning of this large scale control strategy against malaria , and to some extent against LF , was improved over time thanks to communication and sensitization of populations [29] . It seems worth to mention that although insecticide resistance has been reported in several foci in Cameroon , it was demonstrated that LLINs might still offer some protection against the resistant Anopheles gambiae s . l . populations in northern Cameroon [30] . ( ii ) Ivermectin has been widely distributed in the study area since 1987 ( Fig 2 ) . Indeed , in 1987–1989 , limited MDA campaigns were organized in the framework of a phase IV trial of ivermectin conducted in the Vina Valley located in the North Region of Cameroon [31] . In 1992 , the Ministry of Health ( MoH ) and the River Blindness Foundation ( RBF ) began to broaden distribution of ivermectin , with the assistance of non-governmental developmental organizations ( NGDOs ) , through mobile teams/outreach approach [32] . Since 1997–1998 , the African Program for Onchocerciasis Control ( APOC ) joined the coalition to support annual delivery of ivermectin through community-directed treatment with ivermectin ( CDTI ) [33] . Although ivermectin is not macrofilaricidal , it is highly microfilaricidal and repeated treatments might have significantly contributed to the interruption of LF transmission [34 , 35] . Moreover , it was demonstrated that the transmission can be interrupted earlier than expected in areas previously treated for onchocerciasis [36] . ( iii ) Last but not the least , the prevalences in the study areas were relatively low when MDAs against LF began , suggesting that in such context , LF endemicity can be quickly lowered to level under which transmission cannot be sustained . After six years of MDA ( ivermectin in combination with albendazole ) , the transmission of LF was interrupted in five IUs ( Mokolo , Ngong , Poli , Tchollire and Rey-Bouba heath districts ) of the North and Far North Regions . These results support the cessation of MDA in these IUs , but this decision needs further thinking . It was demonstrated that MDA can be safely stopped in some but not all local government areas of Plateau and Nasarawa States in Nigeria [37] , suggesting that the cessation of MDA can be feasible in the IUs investigated in northern Cameroon , even if the transmission of LF might be ongoing in the neighboring IUs . This is likely in accordance with the focal LF transmission that might be occurring in Cameroon . Also , the LF prevalences were relatively low at the beginning of MDAs , and the neighboring EUs has already completed at least four effective rounds of MDA when mass treatments can be halted as a consequence of transmission interruption . However , epidemiological surveys conducted in northern Cameroon in 2008–2010 showed that mass ivermectin distributions had significantly lowered prevalence and intensity of onchocerciasis , but the transmission of the disease was yet to be interrupted [33] . In such circumstances where onchocerciasis transmission is still ongoing in these ( and the neighboring ) IUs , the interruption of treatments ( IVM + ALB ) might need further thinking . It is accepted that in areas where onchocerciasis is endemic , ivermectin can be used solely after interruption of LF transmission but this might be challenging while conducting surveillance activities to investigate potential recrudescence of LF . Another important challenge to take into account is the endemicity of soil transmitted helminthiasis ( STH ) since both IVM and ALB are effective against the parasites responsible of these diseases , especially in areas where STH control is not performing well . In such circumstances , it appears useful to investigate the situation of onchocerciasis and STH , especially now rapid diagnostic tests are being releasing for these diseases . This will help taking the decision about stopping MDA not only according to the evidence of LF transmission interruption , but also to the situation of STH and onchocerciasis in the selected EU . These additional data , collected in an integrated manner during TAS surveys , will be really cost effective and provide more insights in decision making . | Lymphatic filariasis ( LF ) affects more than 120 million people worldwide , and is considered the second leading cause of permanent and long-term disability . In response to the important burden of this disease , the World Health Organization ( WHO ) elaborated a strategic plan to eliminate LF as a public health problem through annual preventive chemotherapy ( PC ) , repeated for at least six years , and reaching at least 65% of the population at risk . To date , about 5 . 63 billion cumulative treatments have been delivered since 2000 , and more than 300 million people no longer require PC thanks to successful implementation of the WHO strategy . In Cameroon , PC for LF has been implemented since 2008 . The aim of this study was to assess whether the transmission of LF has been interrupted . Cross-sectional surveys were conducted in three evaluation units ( EU ) in northern Cameroon . The LF prevalence observed in each of these EU was lower than the threshold of infection below which transmission is likely no longer sustainable , suggesting that the transmission of LF has been interrupted in the study area . | [
"Abstract",
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] | 2017 | First evidence of lymphatic filariasis transmission interruption in Cameroon: Progress towards elimination |
Leishmania ( Leishmania ) infantum is the zoonotic agent of visceral leishmaniasis ( VL ) , a disease with a global distribution . The transmission scenario of VL has been undergoing changes worldwide , with the biologic cycle invading urbanized areas and dispersing the parasites into other previously free areas . The epidemiological cycle in Brazil has dispersed from the Northeast to other regions of the country . In this study , an integrative approach , including genotyping Brazilian strains of L . ( L . ) infantum for 14 microsatellite markers and reviewing historical records of the disease , was used to assess dispersion routes throughout central-southern Brazil . Our results support three L . ( L . ) infantum dispersion routes: A ) dispersion from Bolivia to the states of Mato Grosso , Mato Grosso do Sul and São Paulo via the Bolivia-Brazil gas pipeline from 1998 to 2005; B ) VL dispersion from Paraguay to the Brazilian side of the triple border ( Foz do Iguaçu and Santa Terezinha de Itaipu ) during after 2012; and C ) emergence of a new L . ( L . ) infantum cluster in western Santa Catarina State and its dispersion to southern Paraná State ( municipality of Pato Branco ) , after 2013 . Hypotheses regarding possible entries of Leishmania ( L . ) infantum into the area of the triple border are presented and discussed . Understanding how VL has dispersed is vital to the development of control measures for this disease and to avoid future dispersion events .
Human Visceral Leishmaniasis ( hVL ) is a widely distributed neglected disease caused by the protozoans Leishmania ( Leishmania ) infantum in Asia , Africa , Europe and Americas , and L . ( L . ) donovani in Asia and Africa [1] . These parasites use the domestic dog as a reservoir , in which it causes canine Visceral Leishmaniasis ( cVL ) , and Phlebotominae sand fly species of Phlebotomus and Lutzomyia longipalpis as vectors in the Old and New World , respectively [2] , although other phlebotominae species have been hypothesized as secondary vectors in the latter region ( see Thomaz-Soccol et al . [3] for further discussion ) . VL has recently experienced changes in its transmission profile in both the Old and New World [1 , 4–6] . The disease has dispersed to places where it had not been previously described ( e . g . United States , Uruguay , Madrid Spain ) , and has expanded its geographical distribution into previously free areas in endemic countries [7–11] . Thus , the number of cases of VL has increased in recent last years in both the Old ( e . g . [6 , 12] ) and New World [e . g . [13 , 14] . Currently , 1 . 69 billion people are estimated to be living in VL transmission areas worldwide , the disease presented 2 . 27 cases per 100 , 000 habitants in 2015 , and 90% of global VL cases occurred in six countries , including Brazil [14–16] . Although known since 1913 [17] L . ( L . ) infantum is likely an invasive species in Brazil , arriving first in the Northeast Region carried by dogs transported with colonizers from Portugal and Spain [18–21] . Between 1920 and 1980 , VL was restricted to rural areas in Northeast Brazil , where it has remained endemic [4 , 22–24] . However , the disease subsequently began to invade urban and peri-urban areas in other regions of the country [25–29] , with epidemics in the north region , especially in Teresina , state of Piaui , in 1981 and in São Luis , state of Maranhão , in 1982 [30 , 31] . In the subsequent decade , several epidemic outbreaks were reported , especially in the Southeast and Central-West regions , with high rates of cVL cases followed by clinical human cases in Belo Horizonte , state of Minas Gerais , Campo Grande , state of Mato Grosso do Sul , and Araçatuba , state of São Paulo . Now , L . ( L . ) infantum has spread throughout the states of Minas Gerais , Goiás , São Paulo , Mato Grosso , Mato Grosso do Sul , Rio de Janeiro and Espírito Santo [1 , 4 , 26 , 32–37] Dispersion of VL in the Southern Region of Brazil has been more recent . The first records of cVL and hVL in this region were in the state of Rio Grande do Sul in 2006 and 2008 , respectively [38] , followed by the state of Santa Catarina in 2011 [39 , 40] . In the state of Paraná the first detection of vectors and dogs diagnosed with cVL was in 2012 , while the first human case was recorded in 2016 [41–45] . In the South Region of Brazil , VL occurs primarily in cities bordering Paraguay , Argentina and Uruguay [3 , 9–11 , 46] . Currently , the disease is classified as ‘controlled’ in Brazil according with to its epidemiological scenario [47] . Several hypotheses have been proposed for the spread of L . ( L . ) infantum throughout central-southern Brazil . For instance , the construction of the east-west route of the Bolivia-Brazil gas pipeline is thought to have allowed the dispersion of L . ( L . ) infantum into the central-southern Brazil through the migration of workers and infected dogs and deforestation in the 1990s [1 , 48–53] . Moreover , the construction of railways and the immigration of infected dogs from other endemic areas seemed to have also facilitated the spread of the disease throughout central-southern Brazil [36 , 50 , 52 , 54] . Deforestation and climate and environmental changes have also been proposed as assisting the expansion of VL in different parts of Brazil [22 , 49] . Most of these studies used different data to test these hypotheses , including molecular markers ( i . e . microsatellite markers , see Ferreira et al . [51] ) and historical spatial data ( e . g . [49 , 50 , 55] ) . However , no study has tested these hypotheses using an integrative approach that combines both methods . As part of the IDRC #107577–002 research project ( idrc . ca/en/project/ addressing-emergence-and-spread-leishmaniasis-bordersargentina-brazil-and-paraguay ) , the objective of the present study was to evaluate the dispersion of L . ( L . ) infantum in central-southern Brazil by integrating molecular markers and historical records for hVL and cVL in the region . Assessing which cluster of L . ( L . ) infantum is present in each city allows reconstructing potential dispersion routes , while integrating spatial-temporal analysis of the first descriptions of VL cases helps to determine the direction of dispersion . Knowing dispersion routes is essential for developing strategies to control this emergent disease and restrain its future dispersion .
One hundred and thirty-two isolates from dogs , humans and sand flies were genotyped for 14 loci of microsatellite markers to assess the dispersion of L . ( L . ) infantum in central-southern Brazil ( Table 1 ) . For this study , seventy samples were collected in four areas in the municipality of Foz do Iguaçu , Paraná ( 62 from dogs , four from sand flies and four from humans ) , and four samples from dogs collected in Santa Terezinha de Itaipu , Paraná , between 2013 and 2016 ( see Thomaz Soccol et al . [45] for collection and parasite isolation details ) . Briefly , Leishmania strains from Foz do Iguaçu and Santa Terezinha de Itaipu were isolated from bone marrow , aspiration of lymph nodes and leukocyte layer of dogs , intestines of sand flies , and leukocyte layer of humans with clinical symptoms . These samples were inoculated in Neal , Novy and Nicole ( NNN ) culture medium with 0 . 9% saline solution for four weeks at 24ºC [56] . The promastigote cultures from the other samples were cultivated in Brain Heart Infusion ( BHI ) with 0 . 9% saline solution at 24ºC . After culture , parasites were centrifuged at 3 , 500 g at 4°C and washed three times ( 0 . 9% saline solution , 0 . 3% saline solution and again 0 . 9% saline solution ) . The DNA of cultured promastigotes and biological samples was extracted using the phenol/chloroform/isoamyl alcohol method [57] . The isolates from other regions of Brazil were acquired from the Molecular Biology Laboratory of the Graduate Program in Bioprocess Engineering and Biotechnology of Universidade Federal do Paraná ( UFPR ) . Additionally , 10 samples from Asunción ( Paraguay , PY ) were provided by the Laboratorio de Medicina Tropical of Instituto de Investigaciones en Ciencias de la Salud of Universidad Nacional de Asuncion , and seven samples from the Old World ( MON-1 from France , Spain and Portugal , MON-24 from Algeria , MON-98 from Egypt , MON-108 from France and MON-198 from Spain ) were kindly provided by the Molecular Ecologie Laboratory of the Medecine Faculty of the University of Montpelier , France . The collection of human sampling was conducted in accordance with the International Ethical Guidelines for Biomedical Research in Humans . The samples were taken by the doctors . In addition , ethical approval was obtained from the Universidade Federal do Paraná Ethical Committee ( number 684 . 244 ) and we complied with the minimum requirements of the Southern Common Market Treaty ( Mercosur ) , Resolution No . 129/96 . All individuals have signed the free consent clause indicating that they agree to use this sample . For dogs , all procedures were carried out in strict compliance with the rules defined by the National Council for the Control of Animal Experiments ( CONCEA ) . Every effort was made to minimize suffering of the dogs . The work was approved by the Ethics Committee of the Federal University of Paraná ( protocol number 044/2014 ) . The owners have signed a consent form for the use of the samples . Fourteen microsatellite markers ( Li46-67 , Li41-56 , Li71-7 , Li71-33 , Li23-41 , Li22-35 , Lm2TG , Lm4TA , Li45-24 , CS20 , Li71-5/2 , TubCA , List7031 , List7039 ) described by Jamjoom et al . [58] , Ochsenreither et al . [59] and Kuhls et al . [60] were selected to assess the genetic profile of the populations in central-southern Brazil . The 10 μL PCR reactions were performed with 10x buffer , 1 . 5 mM MgCl2 , 0 . 2 mM dNTP , 0 . 3 units of Platinum Taq DNA Polymerase ( Invitrogen ) , 0 . 3 pmol of fluorescence conjugated forward primer ( 0 . 5 pmol for Li23-41 , Li22-35 , Lm4TA , Li45-24 and List7039 ) and the same quantity of the reverse primer , 10 ng of DNA template ( 5 ng for the loci Li71-7 and Lm2TG ) and ultrapure water to complete the final volume . The PCR cycles were set to run for 3 min at 95°C for initial denaturation; 35 cycles of 30 s at 95°C for denaturation; 60 s at 50°C ( Li46-67 , Li41-56 , Li71-7 and Li71-33 ) , 52°C ( Li23-41 and Li22-35 ) , 54°C ( Lm4TA and Li45-24 ) , 55°C ( Lm2TG ) , 56°C ( CS20 , Li71-5/2 and List7039 ) and 58°C ( TubCA and List7031 ) for primer annealing , and 60 s at 72°C for DNA extension; and a final extension at 72°C for 60 min . The amplified products were genotyped in an automated capillary sequencer ABi 3130 ( Applied Biosystems ) . The amplification of the 14 microsatellite markers and assessment of their fragment size were performed using Gene Marker V2 . 4 . 2 ( SoftGenetics ) . The presence of null alleles , allele dropout and scoring errors was analyzed with Micro-Checker 2 . 2 . 3 [61] . The presence of loci under selection was assessed in the BayeScan v2 . 1 [62] only for populations with more than five individuals . Hardy-Weinberg disequilibrium , diversity ( gene diversity , Ho and He ) and genetic differentiation ( FST and AMOVA , only for populations with more than five strains ) analyses were performed using the software Arlequin [63] . Allelic richness was calculated in FSTAT 2 . 9 . 3 . 2 [64] . The critical p value was corrected using the B-Y method [65] in analyses with multiple comparisons . The probable number of genetic populations was assessed using the assign method implemented in STRUCTURE 2 . 3 . 3 [66] with three runs for each K ( K between 1 and 8 ) , composed of a burn-in period of 500 , 000 itinerations and 5 , 000 , 000 Markov Chain Monte Carlo ( MCMC ) iterations , and no-admixture model . The ad hoc method of Evanno et al . [67] , implemented on the online tool Structure Harvester [68] , was used to assess the most likely value of K . However , the main assumptions of Structure Analysis are that the population present Hardy-Weinberg and linkage equilibrium , while species of Leishmania species frequently deviate from these assumptions ( see the Results section and [21 , 60 , 69] ) regarding caution in interpreting Structure Analysis results for Leishmania spp . ) . Thus , we also assigned strains of L . ( L . ) infantum using Discriminant Analysis of Principal Components ( DAPC ) , which is free from the assumptions of H-W and linkage equilibrium [70] . This analysis was performed using the package 'adegenet' [71] in R 3 . 5 . 0 software ( R development core team [72] ) . The optimum number of retained PCs was assessed using both α-score and cross-validation , while the numbers of clusters was chosen based on the results of structure analysis . Due its more flexible assumptions , the results of the DAPC were preferably used to assess the dispersion of L . ( L . ) infantum in central-southern Brazil . Strains with posterior probability of belong to a cluster higher than 0 . 80 in the DACP analysis were assigned to that cluster , while strains with posterior probability of belong to a cluster lower than 0 . 80 remained undetermined . Subestructuration within the clusters was assessed in 3 runs of 5 , 000 , 000 MCMC ( burn-in of 500 , 000 iterations ) with K between 1 and 4 in STRUCTURE for each cluster . The dispersion of L . ( L . ) infantum in central-southern Brazil was assessed using historical data of the first records of VL cases in dogs and humans in each city of the states of the region ( Mato Grosso ( MT ) , Mato Grosso do Sul ( MS ) , São Paulo ( SP ) , Goiás ( GO ) , Minas Gerais ( MG ) , Rio de Janeiro ( RJ ) , Espírito Santo ( ES ) , Paraná ( PR ) , Santa Catarina ( SC ) and Rio Grande do Sul ( RS ) . Additionally , cases reported in neighboring countries ( Bolivia ( BO ) , Argentina ( AR ) , Paraguay ( PY ) , Uruguay ( UR ) ) were also added to the database to assess possible dispersion of the parasite from these countries . For this , a search was performed for publications available in the Scopus , PubMed , Google Scholar , and Scielo portals between 1913 and 2017 using the following keywords: “first case visceral leishmaniasis” , “visceral leishmaniasis in dogs” , “visceral leishmaniasis in human” or “Leishmania infantum" . Descriptions of errant dogs were not considered due to uncertainty regarding origin ( autochthonous or allochthonous ) . Records of autochtonous human VL cases in the SINAN database ( Sistema de Informação de Agravos de Notificação , available in http://portalsinan . saude . gov . br/ ) were also considered . The SINAN is the database that presents the records of diseases with mandatory notification in Brazil , includinhg hVL . The hVL cases recorded in the SINAN ranges between 2001 and 2017 . All cases were categorized into five ranges of years according the following events: 1 . 1913 to 1980: population migration from the Northeast Region to central-southern Brazil ( see [73–75] ) ; 2 . 1981 to 1997: beginning of rural exodus , with migration of people and their animals from rural to urban areas ( see [74–76] ) ; 3 . 1998 to 2005: construction of the Bolivia-Brazil gas pipeline and migration of employees and their pets to the states of Mato Grosso do Sul ( MS ) and São Paulo ( SP ) ; epidemics of VL to large cities in the states of São Paulo ( SP ) , Minas Gerais ( MG ) and Mato Grosso do Sul ( MS ) ( e . g . [4 , 35 , 36 , 49 , 77–79] ) ; 4 . 2006 to 2010: first VL cases registered Argentina and in the state of Rio Grande do Sul , and dispersion from cities in Paraguay ( e . g . [9 , 38 , 80–82] ) ; 5 . 2011 to 2018: Dispersion of VL cases in South Brazil ( e . g . [39 , 40 , 42–45 , 83] ) .
Among the 14 microsatellite markers assessed , the loci List 7031 , Li 41–56 , Li 45–24 and TubCA exhibited recurrent evidence of null alleles for some populations and were thus removed from further analyses . No loci presented evidence of balancing or positive selection . Greater allelic diversity was observed in Brazilian populations from Campo Grande ( MS2 ) and Foz do Iguaçu ( PR5 ) , while the populations from Foz do Iguaçu and Paraguay ( PY ) had greater intra-population allelic richness ( see Table 1 ) . The AMOVA ( performed only with populations with more than five strains genotyped ) , revealed that 62% of the genetic variation of populations is at the inter-population level ( FST = 0 . 617 , p value = 0 . 000 ) . Pairwise genetic differentiation found significant genetic differentiation ( p < 0 . 017 after B-Y correction between Asunción ( PY ) and São Miguel do Oeste ( SC1 ) , Asunción ( PY ) and Descanso ( SC2 ) , São Miguel do Oeste ( SC1 ) and Belo Horizonte ( MG ) , Descanso ( SC2 ) and Belo Horizonte ( MG ) , São Miguel do Oeste ( SC1 ) and Foz do Iguaçu ( PR5 ) , and Descanso ( SC2 ) and Foz do Iguaçu ( PR5 ) ( Table 2 ) . The ad hoc method of Evanno et al . ( 2005 ) [67] supported two ( ΔK: 43 . 4 ) as the most probable number of clusters of the assignment analysis implemented in Structure , followed by three ( ΔK: 27 . 0 ) . Since both analyses were informative , the results of K = 2 and K = 3 are presented . The Structure and DACP analyses with 2 clusters indicated that the populations of São Miguel do Oeste ( SC1 , except one strain ) , Descanso ( SC2 ) , Aracaju ( SE , undetermined in Structure analysis ) , Rondonópolis ( MT ) , Curitiba ( PR1 , only in Structure analysis ) , Pato Branco ( PR4 ) , and one strain from Campo Grande ( MS2 ) compose a cluster ( named Cluster 1 ) , while the other populations from South America compose the other cluster ( named Cluster 2 ) ( Fig 1 ) . The Structure and DACP analysis with K = 3 divided Cluster 2 into two other clusters ( named Clusters 2 . 1 and 2 . 2 ) ( Fig 2 ) . The Cluster 1 contained only populations São Miguel do Oeste ( SC1 , except two strains ) , Descanso ( SC2 ) , Curitiba ( PR1 , undetermined in the DACP analysis; and Pato Branco ( PR4 ) , as well as MON-24 from Algeria , MON-108 from France and MON-198 from Spain . Cluster 2 . 1 encompassed the strains from Aracaju ( SE ) , Rondonópolis ( MT ) , Campo Grande ( MS1 ) , Três Lagoas ( MS2 ) , Bauru ( SP1 ) , Andradina ( SP2 ) , a strain from Descanso ( SC1 ) , MON-1 from France , Spain and Portugal , and MON-98 from Egypt . The Structure analysis also assigned five strains from Asunción ( PY ) , one from Fortaleza ( CE ) , one from Santa Terezinha de Itaipu ( PR3 ) , and ten from Foz do Iguaçu ( PR5 ) to Cluster 2 . 1 . The strains from populations Asunción ( PY ) , Fortaleza ( CE ) , Palmas ( TO ) , Belo Horizonte ( MG ) , Maringá ( PR2 ) , Santa Terezinha de Itaipu ( PR3 ) , Foz do Iguaçu ( PR5 ) and a strain from São Miguel do Oeste ( SC1 ) were assigned to Cluster 2 . 2 . In the subestructuration analysis , each cluster presented a cohesive genetic group , with no signal of genetic subestructuration . The research for first records of VL cases in dogs and humans resulted in 52 , 029 articles published between 1913 and 2018 , of which 350 were pre-selected due their epidemiological information or the report of the first case of VL in cities of central-southern South America . Among these articles , 55 were selected due to human or canine VL reports in Bolivia , Paraguay , Argentina , Uruguay or Brazil . Cases of VL were described for 672 cities ( Table 3 and Fig 3 ) , of which 611 were the first record in humans ( hVL ) and 61 the first record in dogs ( cVL ) . The SINAN database contained 456 human VL records between 2001 and 2017 , while the literature revealed 216 cVL and hVL cases between 1913 and 2017 . Between 1913 and 1980 ( Fig 3A ) , VL was described for the first time in 16 cities ( 15 hVL , 1 cVL ) in Argentina ( Northwest and Chaco regions ) and Brazil ( Central-West and Southeast regions ) . The number of cases remained low between 1981 and 1997 ( Fig 3A ) , with first records of VL in 11 cities ( 8 hVL , 3 cVL ) , including 10 new records in Brazil and one in Bolivia . Between 1998 and 2005 , the first years with records in SINAN database , the number of cities with their first records of VL increased between 1998 and 2005 ( Fig 3B ) , with 283 ( 270 hVL , 13cVL ) from Paraguay ( 1 ) , Bolivia ( 1 ) and Brazil ( 281 ) , with the Brazilian states of Minas Gerais ( 106 ) and São Paulo ( 100 ) containing most these records . The new records of VL in the state of São Paulo were registered in cities close to the Bolivia-Brazil gas pipeline , most of them described in the literature , while for Minas Gerais they were registered mainly in the SINAN database in cities close to the states in the Northeast Region of the country ( i . e . Bahia ) and near the state capital Belo Horizonte . Between 2006 and 2010 ( Fig 3C ) , 173 cities in Argentina ( 3 ) , Paraguay ( 5 ) , Bolivia ( 1 ) and Brazil ( 164 ) had their first VL cases ( 141 hVL , 32 cVL ) . In Brazil , VL spread to cities northward and southward from the cities in the São Paulo state that is close to the Bolivia-Brazil gas pipeline and had their first records in previous period . Moreover , the first records of VL for the South Region of Brazil were during this period in three cities in the state of Rio Grande do Sul . New records of VL in Argentina occurred in cities close to gas pipelines and the frontiers with Paraguay and Brazil ( i . e . Rio Grande do Sul state ) . Between 2011 and 2017 ( Fig 3D ) , 189 new cities recorded VL ( 177 hVL , 12 cVL ) , three in Paraguay , one in Argentina , one in Uruguay and 184 in Brazil . The number of new cities with cases increased in the South Region of Brazil , with five in Paraná , five in Santa Catarina and two in Rio Grande do Sul ) . In the state of São Paulo , VL continued its spread to cities northward and southward of the Bolivia-Brazil gas pipeline .
In summary , our results highlight the need of the development of plans that efficiently avoid the dispersion of the visceral leishmaniasis in the central-southern Brazil that includes monitoring of this diseases and joint policies with countries bordering this Brazilian region . | The dispersion of visceral leishmaniasis is an enigma . The State of Paraná , in southern Brazil , borders the states of São Paulo and Mato Grosso , which have experienced LV epidemics over the past 20 years . Therefore , we expected that the disease would enter this state through the contiguity of epidemics from other regions following by "ghost shadows" . However , in 2012 , the vectors of the parasite were reported in the western region ( Foz do Iguaçu ) of Paraná state , far from the epidemic regions . In the cross-sectional study , 23 . 8% of the dogs were infected , which is more than the eyes can see , showing an unexpected scenario where the disease was already widespread in the city . Now the question was: where does the life cycle element came from ? In this study , we used genetic markers to understand the dispersion of Leishmania infantum throughout central-southern Brazil . Our results showed two possible agent inputs in the Paraná state , one coming from Paraguay and , another from Santa Catarina state . When we verify our results we perceived the monitoring importance of the distribution of these agents by diverse hypotheses , not only those that the scientific literature presents . Another relevant factor is always to be attentive to the environmental and socioeconomic events that can provide this dispersion . | [
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] | 2019 | Dispersion of Leishmania (Leishmania) infantum in central-southern Brazil: Evidence from an integrative approach |
The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite Plasmodium falciparum . While recent studies assigned important roles for reversible histone modifications , silent information regulator 2 and heterochromatin protein 1 ( PfHP1 ) in epigenetic control of variegated expression , factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown . Here , we describe the purification and characterization of PfSIP2 , a member of the ApiAP2 family of putative transcription factors , as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains . Interestingly , SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain , PfSIP2-N , which is released during schizogony . Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding . Genome-wide in silico mapping identified 777 putative binding sites , 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements . Immunofluorescence and chromatin immunoprecipitation ( ChIP ) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends . Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites . Consistent with this specialized distribution pattern , PfSIP2-N over-expression has no effect on global gene transcription . Hence , contrary to the previously proposed role for this factor in gene activation , our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity . These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P . falciparum and other eukaryotes , and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology .
Throughout the eukaryotic kingdom , the overall structure of chromosome ends is conserved and characterized by the telomeric tract , composed of short G-rich repeats , and an extensive subtelomeric region consisting of various types and lengths of repeats , also known as telomere-associated sequences ( TAS ) [1] . This conservation underscores the functional importance of these domains in genome function and maintenance . Due to the heterochromatic nature of subtelomeric regions , genes located nearby are subject to epigenetic control and variegated expression [2]–[5] . Furthermore , subtelomeric domains promote frequent recombination events driving the evolution and diversity of gene families located close to chromosome ends [1] , [3] . Pathogenic microorganisms exploit this system for antigenic variation of surface antigens to evade adaptive immune responses or to respond to other changes in environmental conditions [6] . The apicomplexan parasite Plasmodium falciparum causes the most severe form of malaria in humans with up to two million deaths annually [7] . Malaria symptoms are entirely associated with the erythrocytic phase of infection where repeated rounds of intra-erythrocytic parasite multiplication take place . Sequestration of infected red blood cell aggregates in the microvasculatory system , which is mediated by the binding of P . falciparum erythrocyte membrane protein 1 ( PfEMP1 ) to a variety of endothelial receptors [8]–[11] , represents one of the main contributors to severe disease , including cerebral and placental malaria [12]–[14] . PfEMP1 is encoded by the var gene family comprising approx . 60 mostly subtelomeric members [15]–[18] . Importantly , due to mutually exclusive transcription of var genes , only one PfEMP1 variant is exposed per parasite at any time and switches in var gene expression result in antigenic variation of PfEMP1 [17] , [19] facilitating immune evasion and chronic infection . Recent studies highlighted the important contribution of the specific biology and dynamics of heterochromatic chromosome ends in the regulation of var genes and additional subtelomeric gene families coding for proteins involved in host-parasite interactions [20]–[27] . P . falciparum chromosome ends consist of a stretch of telomeric GGGTT ( T/C ) A repeats with an average size of 1 . 2 kb , followed by an extensive 20 to 40 kb TAS domain [28] . This region is composed of a conserved arrangement of so-called telomere-associated repeat elements ( TAREs 1 to 6 ) , each of which consists of distinct non-coding repeat arrays of varying length and sequence [29] . On all chromosome ends , the coding part of the genome directly downstream of TARE 6 is characterized by members of multiple antigen gene families including var , rif , stevor and pfmc-2tm [18] . Similar to other unicellular eukaryotes , P . falciparum chromosome ends associate into clusters that are anchored to the nuclear periphery [30]–[32] . This structurally conserved context facilitates meiotic recombination between var genes on heterologous chromosomes [30] . Interestingly , spontaneous chromosome breakage and telomere healing events create chromosome ends lacking the entire TARE region; while such chromosomes are still tethered to the nuclear periphery , they display a reduced association with other chromosome ends implicating a role for TARE in cluster formation [33] . Expression of P . falciparum subtelomeric gene families is clonally variant and restricted to only one member ( or a few ) in each family [19] , [34]–[36] . Transgenes inserted into TARE 6 as well as endogenous var genes are reversibly silenced in a manner reminiscent to telomere-postion effect in other eukaryotes [25] . Recent genome-wide studies highlighted the striking and exclusive association of the repressive histone 3 lysine 9 tri-methylation mark ( H3K9me3 ) and heterochromatin protein 1 ( PfHP1 ) throughout the TAS region and adjacent gene families on all chromosomes [23] , [27] , [37] . These heterochromatic marks are also important in telomere-proximal gene silencing in S . pombe and higher eukaryotes [38] , [39] , indicating the existence of conserved epigenetic control strategies in highly divergent eukaryotes . The epigenetic changes underlying mutually exclusive var gene transcription and switching have been studied in some detail . Active var loci are enriched in acetylated H3K9 and H3K4me2/me3 [21] , and the process of activation is linked to locus repositioning into an ill-defined transcriptionally active zone at the nuclear periphery [24] , [31] , [40] . Silenced var loci lack these activation marks and are enriched in H3K9me3 and PfHP1 instead [21] , [22] , [41] . Furthermore , silencing of var and a subset of rif genes is dependent on the two P . falciparum orthologs of silent information regulator 2 ( PfSIR2 ) [20] , [25] , [26] . Overall , these results show that conserved epigenetic mechanisms that are also in place in other eukaryotes dictate heterochromatic silencing in P . falciparum . However , it remains completely unknown which proteins and cis-acting sequences are involved in the recruitment and organization of P . falciparum heterochromatin , and how they contribute to the important role of chromosome end biology in this pathogen . Our understanding of sequence-specific DNA-protein interactions in P . falciparum is negligible and mostly limited to the description of upstream sequence elements and their interaction with unknown nuclear proteins , and to in silico mapping of over-represented motifs in candidate promoter sequences [42] . This lack of knowledge is related to the extreme diversity of specific DNA-binding proteins in eukaryotes and the poor representation of such factors in the apicomplexan lineage [43]–[45] . Until recently , PfMYB1 was the only sequence-specific DNA-binding protein that had been investigated to some extent in vivo [46] . New impulses were given by the discovery of the lineage-specific expansion of the ApiAP2 family of putative transcription factors in apicomplexan parasites , characterized by the presence of plant-like AP2 DNA-binding domains [47] . The binding of three parasite AP2 domains to specific cis-acting elements upstream of P . falciparum genes in vitro has recently been demonstrated [48] , and another study described an essential role for PbAP2-O in stage-specific transcription in P . berghei ookinetes [49] . Here , we identified a member of the ApiAP2 family as the unknown protein binding to SPE2 arrays upstream of subtelomeric var genes at the border of the non-coding and coding parts of P . falciparum chromosomes [50] . The SPE2-interacting protein , termed PfSIP2 , contains two adjacent AP2 domains and is proteolytically processed in vivo to release a 60kDa functional N-terminal domain , PfSIP2-N . We show that both AP2 domains are strictly required for binding to the bipartite SPE2 motif . In vivo , PfSIP2-N is associated with over 700 SPE2 consensus sites that cluster upstream of subtelomeric var genes and in TARE2/3 , but not to single chromosome internal sites . Consistent with this striking and exclusive binding of PfSIP2-N to heterochromatic regions , we found no effect of PfSIP2-N over-expression on global gene transcription . Instead , our results imply major roles for PfSIP2-N in var gene silencing and chromosome end biology .
The bipartite SPE2 motif consists of two imperfect 6bp repeats separated by 4bp and its sequence-specific interaction with the unknown nuclear protein is only detectable after the onset of S-phase [50] . We used a high salt schizont stage nuclear extract , pre-cleared by incubation with single-stranded DNA , to purify the SPE2-binding activity based on its affinity to immobilized concatenated SPE2 elements . As control , mutated SPE2M motifs that are unable to interact with the protein were used [50] . EMSA monitoring showed that the SPE2-binding activity was efficiently depleted only after incubation with SPE2 but not SPE2M . Likewise , the activity was eluted only from beads carrying SPE2 but not SPE2M motifs ( Figure 1A ) . Total protein eluted from both beads were precipitated separately , trypsinized and analyzed by LC-MS/MS . Peptide spectra were searched against a combined human and P . falciparum annotated protein database using TurboSequest software [51] . 89 and 82 P . falciparum proteins represented by two or more unique peptides were identified in the SPE2- and the SPE2M-bound fractions , respectively ( Tables S1 and S2 ) . Interestingly , of the 18 proteins exclusively detected in the SPE2 sample ( Table 1 ) , two proteins belong to the ApiAP2 family of transcription factors carrying putative sequence-specific AP2 DNA-binding domains , including the second-ranked protein encoded by PFF0200c . Furthermore , six co-purifying proteins have predicted roles in DNA and chromatin metabolism . In contrast , the 15 proteins detected exclusively in the control sample showed no such enrichment ( Table S2 ) . Due to the high peptide coverage and a temporal expression profile matching the presence of the SPE2-binding activity in stage-specific nuclear extracts [50] , [52] , [53] , we considered PFF0200c the most likely candidate to encode the SPE2-binding activity . PFF0200c encodes a large 230kDa protein containing two N-terminal AP2 domains as the only annotated features ( Figure 1B ) . However , UV-crosslinking experiments revealed a 70kDa SPE2-protein complex , which is consistent with an estimated size of 50–60kDa of the SPE2-binding protein ( Figure 1C ) . This discrepancy , and the fact that all seven PFF0200c-derived tryptic peptides mapped to the region containing both AP2 domains ( Table S1 ) , prompted us to consider possible proteolytic processing and release of a DNA-binding N-terminal fragment . We therefore expressed epitope-tagged N-terminal fragments of PFF0200c containing both AP2 domains as recombinant proteins in both P . falciparum and E . coli ( Figure 1B ) . Nuclear extracts from 3D7/SIP2-N-HA parasites produced a SPE2-specific complex similar to the one observed in wild-type parasites ( Figure 1D ) , and anti-HA antibodies specifically supershifted the complex obtained with the 3D7/SIP2-N-HA-derived extract only . Similarly , E . coli lysates containing the same fragment as 6×HIS tagged version ( SIP2-N-HIS_A ) produced a shift of similar size and specificity that was supershifted in presence of anti-6×HIS antibodies . Consistent with the smaller size of the SIP2-N-HIS_B protein , we observed a faster migrating complex of identical specificity . For completeness , we also expressed a 150kDa protein in P . falciparum containing all three AP2 domains of the second ApiAP2 protein PF10_0075 but were unable to detect binding to SPE2 ( data not shown ) . Together , these results unambiguously identified PFF0200c as the P . falciparum SPE2-binding protein , which we termed PfSIP2 ( SPE2-interacting protein ) . As indicated by gel shift and UV-crosslinking experiments , the sizes of the endogenous PfSIP2 activity and the N-terminal PfSIP2-N-HA protein were of similar size . To test if PfSIP2 is at all expressed as a full-length protein we generated a transgenic line expressing C-terminally tagged full-length PfSIP2 from the endogenous locus ( 3D7/SIP2-Ty ) ( Figure S1 ) . Western analysis of nuclear extracts identified a 250kDa band , consistent with the predicted size of full-length PfSIP2-Ty , specifically in early and late schizonts ( Figure 1E , top panel ) . An additional 150kDa C-terminal fragment ( SIP2-C-Ty ) appeared in late schizonts indicating that indeed a specific proteolytic event releases an N-terminal DNA-binding isoform . In line with this result , the same early and late schizont extracts produced a typical SPE2/PfSIP2-N complex in EMSA ( Figure 1E , bottom panel ) . These results further suggested that full-length PfSIP2 is unable to interact with SPE2 in vitro . To test this , we performed SPE2 pull-down experiments and confirmed that only N-terminal PfSIP2-N-Ty bound to SPE2 beads , but not full-length PfSIP2-Ty nor the C-terminal fragment PfSIP2-C-Ty ( Figure 1F ) . Together , these results substantiate the existence of a specific proteolytic event to activate the release of the functional DNA-binding protein PfSIP2-N during schizogony . Since SPE2 arrays occur in conserved positions upstream of subtelomeric upsB var genes we expected PfSIP2-N to mark chromosome end clusters . Consistent with this assumption , indirect immunofluorescence ( IFA ) microscopy identified discrete PfSIP2-N-HA foci at the nuclear periphery with increasing numbers of foci in replicating stages ( Figure 2A ) . To confirm that these signals indeed represented chromosome end clusters we compared the PfSIP2-N-HA signals with those of the heterochromatic marker PfHP1 [27] in a double transgenic line co-expressing PfSIP2-N-HA and PfHP1-Ty simultaneously . As expected , double-labeling IFAs revealed that both proteins co-localized at the nuclear periphery ( Figure 2B ) . Next , we tested if PfSIP2-N binds to SPE2 elements in vivo by targeted chromatin immunoprecipitation ( ChIP-qPCR ) . We observed specific enrichment of PfSIP2-N-HA at the SPE2 array upstream of the upsB var gene PFL0005w while three regions further downstream showed no association ( Figure 2C ) . Together , these findings demonstrate that PfSIP2-N binds specifically to SPE2 arrays upstream of upsB var genes in vivo . We recently demonstrated that var gene promoters driving expression of the drug-selectable marker hdhfr are silenced by default . Challenge with the antifolate WR99210 allowed selection for activated promoters , which displayed a ring stage-specific temporal activity profile similar to the endogenous promoters [24] , [54] . To explore if PfSIP2 participates in the regulation of upsB var gene promoters , we used quantitative reverse transcriptase-PCR ( qRT-PCR ) to compare the activities of an episomal upsB promoter and a truncated version lacking a 500bp region containing the entire SPE2 array in transfected parasites lines 3D7/upsBR [54] and 3D7/upsBRΔSPE2 , respectively , in a time course experiment ( Figure S2 ) . As expected , the default state of the wild-type upsB promoter in 3D7/upsBR was silenced . Interestingly , deletion of the region including the SPE2 array resulted in a ten-fold increase in default activity in all three ring stage samples , which is in line with previous results obtained by transient transfection [50] . In their activated states , however , both promoters displayed strong and almost identical activities and temporal profiles ( Figure S2 ) . These findings indicate that PfSIP2 may contribute to , but is not the only determinant of , upsB promoter-mediated silencing , and has no role in stage-specific var promoter activity . As an important step towards understanding the role of PfSIP2-N in parasite biology , we were interested in scrutinizing the specificity of the PfSIP2-N/SPE2 interaction . Our earlier work demonstrated that two point mutations in either half of the bipartite SPE2 sequence abrogated binding [50] . Intriguingly , a recent study identified GTGCA ( which is identical to the first half of the bipartite SPE2 sequence ) as consensus motif for PfSIP2-N [48] . These authors also argued that the first AP2 domain alone was sufficient for binding . To clarify these conflicting results , we compared the ability of PfSIP2-N to bind to a bona fide SPE2 motif and to a DNA sequence of identical length carrying the GTGCA motif only . Figure 3A shows that under identical conditions PfSIP2-N binds only to SPE2 but not GTGCA . We next speculated that the bipartite nature of both the SPE2 element and PfSIP2-N with two adjacent AP2 domains reflects the strict requirement of both intact modules for successful interaction . We expressed both AP2 domains separately or in combination as GST-fusions in E . coli and used gel shift assays to confirm that indeed both adjacent AP2 domains are required for binding ( Figure 3B , right panel ) . Next , we used gel shift competition assays to pinpoint as accurately as possible the minimal sequence requirements for a functional SPE2 consensus motif ( Figure 3C and S3 ) . A first set of 16 competitors incorporated single or multiple base deviations within the first and/or second half , and tested the importance of spacing between the half sites . The only changes tolerated were G to C at position one or two in the first or second repeat , respectively; all other changes completely averted binding . The spacing between the half sites was also critical with only four base pairs tolerated . A second set of competitors included eleven SPE2-like motifs naturally occurring upstream of genes coding for invasion-related proteins [55] , and two untested SPE2 versions naturally present in SPE2 arrays upstream of var genes ( M1 . C1 , M1 . A1C2 ) . Only three motifs competed efficiently ( rap3 , rhoph3 , M1 . A1C2 ) and two competed moderately ( MAL6P1 . 292 , M1 . C1 ) . These motifs are most closely related to the original SPE2 sequence . Furthermore , in two instances a 5bp spacer was tolerated . Interestingly , the rap2 ( non-competing ) and rap3 ( competing ) sequences are identical except for the fifth base in the spacer , indicating that these positions can also contribute to specificity . The competition EMSAs were repeated several times with independent batches of competitors and input protein ( both nuclear extracts and E . coli lysates ) and yielded identical results ( Figure S3 and data not shown ) . This high degree of sequence-specificity of the PfSIP2-N/SPE2 interaction allowed us to deduce a functional SPE2 consensus motif ( Figure 3C ) . Genome-wide in silico prediction using the consensus motif as query revealed a striking distribution of 777 putative PfSIP2-N binding sites throughout the genome ( Tables S3 and S4 ) . 330 sites ( 42 . 5% ) are associated with the full set of 24 upsB var genes encoded in the genome . The majority of these ( 262 ) occur in sense-oriented tandem arrays approx . 2 . 2kb upstream of 23 subtelomeric upsB loci with an average of eleven motifs spaced by 12bp per locus , and the only chromosome-internal upsB var gene contains two upstream SPE2 sites . 66 sites define a second highly conserved cluster of upsB-associated SPE2 sites approx . 2 . 7 kb upstream of every subtelomeric locus . Interestingly , 393 sites ( 51 . 7% ) are located in TAS concentrated in the TARE2/3 region on every chromosome end , with conserved position and orientation and an average of 18 motifs per chromosome end , most of which are spaced by 120bp . Figure 3D shows the right end of chromosome three as a representative example for the conserved arrangement of subtelomeric SPE2 sites on all chromosome ends . Of the remaining 45 sites ( 5 . 8% ) , 30 are located as single motifs in sense orientation upstream of mainly centrally located single copy genes coding for hypothetical proteins , and 15 sites map to coding regions or introns . In summary , this analysis identified a surprising pattern of putative PfSIP2-N-binding sites throughout the genome , with 94% of all predicted motifs confined to two major landmark regions in the subtelomeric domains of P . falciparum chromosomes . To test our in silico prediction and to identify potential additional PfSIP2-N target sites we performed genome-wide ChIP ( ChIP-on-chip ) on a high-density whole genome tiling array ( NimbleGen Systems Inc . ) [27] , [37] . Comparison of the genome-wide PfSIP2-N-HA occupancy pattern with the in silico prediction revealed a high degree of overlap ( Figure 4 and Table S3 ) . Strikingly , the in vivo association of PfSIP2-N-HA was restricted to SPE2 sites in the predicted landmarks in TARE2/3 and upstream of subtelomeric var genes , both of which are located within H3K9me3/PfHP1-enriched heterochromatin [23] , [27] , [37] . In contrast , none of the chromosome-internal sites was bound by PfSIP2-N-HA and we observed no enrichment of PfSIP2-N-HA at non-SPE2 loci . To validate the ChIP-on-chip data we performed ChIP-qPCR on selected loci in 3D7/SIP2-N-HA schizont stage parasites . Figure 5A shows that in addition to SPE2 upstream of upsB var genes ( see Figure 2C ) , PfSIP2-N-HA was also bound to TARE2/3 , and interestingly also to the only internal upsB var locus PFL0935c containing two juxtaposed SPE2 motifs . However , we found no specific enrichment at five promoters of internal genes carrying a single SPE2 site . As negative control , we tested the promoters of five genes that are not associated with SPE2 . To confirm these results , and to test the anticipated co-occupancy of PfSIP2-N with PfHP1 , which was previously shown to be enriched at upsB loci [27] , we performed parallel ChIP using chromatin isolated from 3D7/SIP2-N-HA/HP1-Ty schizonts . Indeed , PfHP1 and PfSIP2-N were both enriched at a subtelomeric and the internal upsB locus ( Figure 5B ) , corroborating the findings presented in Figures 2C and 5A . In independent experiments , we used ChIP-re-ChIP to directly confirm the co-occupancy of PfSIP2-N and PfHP1 on the same chromatin fragments in 3D7/SIP2-N-HA/HP1-Ty parasites ( Figure S4 ) . Interestingly , in both instances PfHP1 showed a marked reduction directly over the SPE2 array at the PFL0005w locus indicating that in vivo occupancy by PfSIP2-N might be incompatible with local nucleosome formation . In summary , our combination of in vitro , in silico and in vivo characterisation uncovers a highly specific association of PfSIP2-N with SPE2 consensus motifs on a genome-wide level . The exclusive local restriction of this interaction to telomere-proximal non-coding regions implies important structural and functional roles for PfSIP2-N in subtelomeric heterochromatin formation and chromosome end biology . To test a possible role for PfSIP2-N in transcriptional regulation by alternative means we compared the global transcript levels in 3D7/SIP2-N-HA parasites to a mock-transfected line at four stages during the intra-erythrocytic developmental cycle ( IDC ) . Over-expression of PfSIP2-N-HA was evident by up to eight-fold higher levels of pfsip2 transcripts in 3D7/SIP2-N-HA compared to the control ( Figure 6 ) . The overall effect of PfSIP2-N-HA over-expression was surprisingly minor with only 21 genes up- or down-regulated by more than three-fold in at least one time point . None of the de-regulated genes is associated with an upstream SPE2 motif and none of the putative PfSIP2-N target genes identified here or by others [48] , [55] was affected , arguing strongly against a role of PfSIP2-N in transcriptional activation . Most of the affected genes , including seven non-upsB var genes , are located in heterochromatic regions , which can be explained by stochastic variation in the expression of heterochromatic genes between different isogenic cell lines .
Sequence-specific DNA-protein interactions serve to target specific activities to specific sites in the genome and are instrumental in genome organization and gene regulation . Here , we identified PfSIP2 , a member of the ApiAP2 family of putative transcription factors , as the unknown nuclear protein binding to SPE2 tandem arrays upstream of subtelomeric var genes . Our comprehensive analysis reveals important novel insights into the nature and specificity of the PfSIP2/SPE2 interaction and provides compelling evidence for a major role of PfSIP2 in chromosome end biology . Surprisingly , we found that the SPE2-binding activity is not exerted by the full-length protein but rather by a processed N-terminal fragment , PfSIP2-N . The inability of full-length PfSIP2 to interact with SPE2 in vitro indicates that the release of PfSIP2-N does not occur accidentally during extraction but reflects a true proteolytic cleavage event required to activate the DNA-binding activity . Our thorough re-definition of the specificity of the PfSIP2-N/SPE2 interaction in gel shift competition assays allowed us to determine a highly specific SPE2 consensus motif . Both 6bp half sites of the bipartite SPE2 element are strictly required for binding , which is in agreement with our earlier findings [50] . We also show that both adjacent AP2 domains are necessary for binding , probably through reinforcing interactions of each domain with each half of the bipartite motif . Such a scenario is consistent with the binding of single AP2 domains to single 5–6bp motifs in Plasmodium [48] , [49] and plants [56] , [57] , and the specific interaction of the Arabidopsis tandem AP2-domain protein AINTEGUMENTA to a 16bp sequence , which also requires both AP2 domains for binding [58] . These findings challenge the previously proposed role of PfSIP2 in transcriptional activation of genes carrying an upstream GTGCA motif [48] . We clearly show that PfSIP2-N does not bind to GTGCA neither in vitro nor in vivo and , for that matter , consider PfSIP2-N unlikely to act as transcriptional activator of GTGCA-associated genes . We explain these conflicting results by fact that the protein binding microarray technology used in the former study was limited to the screening of random 10mers only , which precluded identification of the 16bp SPE2 element as high affinity binding motif . Our results are highly relevant for our understanding of the role of PfSIP2 in parasite biology , and provide important information for the future investigation of specific ApiAP2-DNA interactions . Our genome-wide in silico prediction identified 777 PfSIP2-N target sites , 94% of which are located in two distinct landmark regions within subtelomeric heterochromatin on all chromosomes . One cluster corresponds to the previously described tandem arrays upstream of subtelomeric var genes [50] , and a second newly identified cluster lies within TARE2/3 . In addition , we detected 30 internal SPE2 sites located upstream of single copy genes . Importantly , genome-wide and targeted ChIP analysis of PfSIP2-N occupancy correlated strongly with the in silico prediction of subtelomeric target sites , showing that both subtelomeric landmark regions were bound by PfSIP2-N in vivo . In contrast , PfSIP2-N was absent at internal sites , except for the only internal upsB locus with two upstream SPE2 motifs . Therefore , binding of PfSIP2-N is restricted to heterochromatic regions including the entire subset of upsB var genes . The co-localization of PfSIP2-N with PfHP1 at perinuclear chromosome end clusters and upstream of upsB var genes corroborates this exclusive association . The observed increase in default upsB promoter activity upon deletion of the SPE2 array suggests a role for PfSIP2-N in var gene silencing , possibly through direct or indirect recruitment of effector proteins . However , the overall distribution pattern of PfSIP2-N implies important roles in structural and functional organization and maintenance of P . falciparum chromosome ends that go beyond regulating var gene expression . In contrast to the well-established and conserved roles of telomere repeat-binding proteins ScRAP1 , SpTAZ1 , HsTRF1/2 in telomere position effect , telomere length regulation and heterochromatin formation in yeasts and humans , respectively [2] , [59]–[64] , specific DNA-protein interactions in TAS are hardly known and have only been investigated in detail in S . cerevisiae . One such factor is ABF1 , which is involved in silencing , initiation of DNA replication , alteration of chromatin structure and nucleotide excision repair [65]–[70] . Our results are in agreement with similar roles of PfSIP2-N in P . falciparum . First , multiple attempts to generate a PfSIP2 knockout line failed due to refractoriness of the pfsip2 locus to disruption ( data not shown ) . Since pfsip2 was readily accessible for 3′ replacement , we believe PfSIP2 is essential for parasite survival . Second , given the close connection between DNA replication and heterochromatin formation , the specific co-purification of several DNA replication/repair and chromatin remodeling factors ( RFC , DNA pol ε , SNF2L , PRS ) with PfSIP2-N ( Table 1 ) supports a role in chromosome end maintenance . DNA pol ε and RFC , as well as members of the ATP-dependent chromatin remodeling complexes SWI/SNF , are central players in chromosome end replication and repair [71]–[75] and have important roles in silencing [76] , [77] . Interestingly , these factors are also associated with DNA repair at stalled replication forks [78] , [79] , which can be induced by tight DNA-protein interactions and are crucially involved in recombination at rDNA repeats and mating type switching in S . cerevisiae and S . pombe , respectively [80] . Third , PfSIP2 expression and subsequent release of PfSIP2-N correlate with the DNA replication and nuclear division cycles during schizogony , and it is tempting to speculate that proteolytic activation of PfSIP2-N may occur in a cell cycle-dependent manner . A similar process has been described in activation of the CDP/Cut transcription factor by S phase-specific cleavage [81] , and proteolytic processing of various targets , including cyclins , DNA replication factors and cohesin is an important regulatory strategy in cell cycle progression [82] . In summary , these results and observations are consistent with a potential multifunctional role of PfSIP2-N in chromosomal replication and/or segregation and in the nucleation of subtelomeric heterochromatin on newly replicated chromosomes . ApiAP2 factors have been proposed to act as regulators of stage-specific expression [47] , [48] , and this was experimentally demonstrated for AP2-O in P . berghei ookinetes [49] . We mapped 45 SPE2 consensus sites in internal regions , mostly located upstream of single copy genes that are transcribed late during the IDC ( Table S5 ) . Another study identified SPE2-like motifs upstream of eleven genes coding for invasion-related proteins with similar expression profiles [55] . Interestingly , our ChIP experiments failed to reveal binding of PfSIP2-N to any of these sites . Although we cannot exclude that this lack of association is due to insufficient ChIP sensitivity or physical masking of the HA epitope at internal loci , we believe this finding reflects the true absence of this protein since PfSIP2-N was undoubtedly bound to the only chromosome-central upsB var locus carrying two SPE2 motifs . In addition , over-expression of PfSIP2-N had no effect on transcription of any of these genes . These observations are clearly inconsistent with a role for PfSIP2-N in transcriptional activation . However , we do not rule out a possible function of full-length PfSIP2 in regulation of target loci . First , given the overlap in expression of PfSIP2 with that of SPE2-asscociated genes , a role for PfSIP2 in their activation is conceivable . Although full-length PfSIP2 did not bind to SPE2 in vitro , it is still possible that PfSIP2 binds to and regulates target sites in vivo , possibly in association with other factors . Second , deletion of the SPE2 motif from the rap3 promoter resulted in reduced activity [55] and , conversely , introduction of SPE2 into a heterologous promoter activated transcription in late-stage parasites [54] . Third , PfSIP2 orthologs exist in a subset of other apicomplexan parasites including all sequenced Plasmodium species , yet subtelomeric SPE2 arrays are unique to P . falciparum . In the P . vivax and P . knowlesi genomes , for instance , we predicted 120 and 80 SPE2 consensus sites , respectively , all of which occur as single sites mostly in chromosome-internal regions ( data not shown ) . Hence , if SIP2 has the same binding specificity in other species ( which is likely due to the remarkable sequence identity in their DNA-binding domains ) then SIP2 must have a function other than being involved in subtelomere biology . It will be interesting to test if processing of PfSIP2 reflects a specific gain-of-function process during evolution of the P . falciparum lineage in order to cope with the massive expansion and control of subtelomeric virulence gene families . In conclusion , we have identified the first sequence-specific component of subtelomeric regions in P . falciparum . To the best of our knowledge , PfSIP2-N/SPE2 represents a novel type of sequence-specific interaction at chromosome ends that has not been reported in any other eukaryote . Our results are highly relevant in the dissection of the specific biology of P . falciparum chromosome ends , which is key to the evolution and variable expression of subtelomeric virulence gene families , and will help to understand similar processes in other systems . Efforts to analyze a loss-of-function phenotype and to identify PfSIP2 interaction partners will be important future steps into this direction .
P . falciparum 3D7 parasites were cultured as described previously [83] . Growth synchronisation was achieved by repeated sorbitol lysis [84] . Transfection constructs are described in Protocol S2 . Transfections were performed as described [24] and selected on either 5µg/ml blasticidin-S-HCl or 4nM WR99210 , or both . To obtain C-terminally tagged endogenous PfSIP2 parasites transfected with pSIP2-2×Ty_3′RP were subject to 3 cycles of growth in presence and absence of WR99210 . Plasmid integration was verified by Southern analysis . High salt nuclear extracts and EMSAs were prepared and carried out as described [50] ( see also Protocol S1 ) . E . coli lysates were diluted to avoid excess input of recombinant protein . Competition EMSAs were performed in presence of non-specific competitor DNA ( 1µg sheared salmon sperm DNA and 200fmol random 30 base ss oligonucleotide per reaction ) and a 50- to 100-fold molar excess of specific ds competitors . Protein samples were incubated for 20min in EMSA buffer with 20fmol 32P-labeled SPE2 probe in presence or absence of a 25-fold molar excess of SPE2 or SPE2M competitors . DNA-protein interactions were UV-crosslinked for 60min ( 107 Joule ) in a Stratalinker 1800 ( Stratagene ) and separated by SDS-PAGE . Gels were directly exposed to X-ray film . The complete protocol is explained in detail in Protocol S1 . Briefly , the SPE2-binding activity was purified by incubation of schizont stage nuclear extracts with streptavidin magnetic beads carrying immobilized biotinylated SPE2 or SPE2M elements ( see Protocol S4 for oligonucleotide sequences ) . Bound proteins were eluted with 2M KCl , precipitated with 10% TCA and dissolved in 50µl 100 mM Tris-HCl , pH 8 . 0 . Proteins were digested with trypsin and analysed by capillary liquid chromatography tandem mass spectrometry ( LC-MS/MS ) using an Orbitrap FT hybrid instrument ( Thermo Finnigan , San Jose , CA , USA ) . MS/MS spectra were searched against a combined P . falciparum/human annotated protein database . To verify successful 3′ replacement at the PFF0200c locus , gDNA from 3D7 wild-type parasites and drug-cycled 3D7/SIP2-Ty parasites was digested with BamHI and HindIII and analysed by Southern blot . The blot was probed with a 32P-dATP-labeled 702bp fragment derived from the 3′ end of PFF0200c . Plasmids are described in Protocol S2 . Recombinant proteins were expressed in E . coli Tuner ( DE3 ) cells ( Novagen ) replicating pMICO [85] . After 4h induction using 1mM IPTG at 30°C , bacteria were pelleted and resuspended in 50mM Tris-HCl ( pH7 . 5 ) containing protease inhibitors ( Roche Diagnostics ) . The suspension was frozen , thawed and sonicated . NaCl , Triton X-100 , β-ME and glycerol were added to final concentrations of 0 . 3M , 0 . 5% , 10mM and 5% , respectively , followed by centrifugation for 15min at 15 , 000g and 4°C . Supernatants were used in gel shift assays without further purification . Primary antibody dilutions were: anti-HA 3F10 ( Roche Diagnostics ) 1∶2 , 000; anti-Ty BB2 ( kind gift of K . Gull ) 1∶10 , 000; anti-6×HIS ( R&D Systems ) 1∶5 , 000 . High salt nuclear extracts from 3D7/SIP2-Ty and 3D7/SIP2-N-Ty schizonts were incubated for 1hr with streptavidin agarose beads carrying immobilized SPE2 or mutated SPE2M motifs in EMSA buffer supplemented with non-specific competitor DNA . After centrifugation at 2000rpm the supernatant was saved and beads washed three times in binding buffer . Bound proteins were eluted with 2M KCl . Methanol-fixed cells were analysed using rat anti-HA 3F10 ( 1∶100 ) or mouse anti-Ty BB2 ( 1∶1 , 000 ) antibodies . Alexa-Fluor® 568-conjugated anti-rat IgG ( Molecular Probes ) 1∶500; FITC-conjugated anti-mouse IgG ( Kirkegaard Perry Laboratories ) 1∶300; TexasRed-conjugated anti-mouse IgG ( Molecular Probes ) 1∶500 . Images were taken on a Leica DM 5000B microscope with a Leica DFC 300 FX camera and acquired via the Leica IM 1000 software and processed and overlayed using Adobe Photoshop CS2 . To identify the full complement of SPE2 consensus elements in P . falciparum , P . vivax and P . knowlesi , genome sequences available at PlasmoDB ( version5 . 5 ) were searched using the PfSIP2-N-binding consensus sequence determined in competition gel shift assays ( Figure 3C and Figure S3 ) . A regular expression search engine ( DREG ) from the EMBOSS software package was used . ChIP and ChIP-on-chip using formaldehyde-crosslinked chromatin was performed as described in detail elsewhere [27] . PfSIP2-N-HA enrichment at selected loci was tested by ChIP-qPCR using three independent chromatin preparations isolated from 3D7/SIP2-N-HA parasites , and by ChIP- and ChIP-re-ChIP-qPCR using two independent chromatin preparations isolated from the double-transfectant 3D7/SIP2-N-HA/HP1-Ty ( qPCR primers are listed in Protocol S4 ) . Enrichment values were calculated by dividing the recovery values obtained by ChIP with anti-HA 3F10 ( Roche Diagnostics ) /anti-Ty BB2 antibodies with that of the non-specific control IgG antibody . For ChIP-re-ChIP , chromatin fragments immuno-precipitated with anti-Ty BB antibodies were eluted with 50µl elution buffer ( 1% SDS and 0 . 1M NaHCO3 ) . After incubation at 65°C for 10min to inactivate the BB2 antibody , the eluate was diluted 6× in incubation buffer lacking SDS ( resulting in SDS concentration comparable to that of the first ChIP ) . Re-ChIP reactions were carried out in the presence of 0 . 2mg/ml Ty peptide to avoid immuno-precipitation by the BB2 antibody carried over from the first ChIP . Recovery values were defined in the percentage of the input material of the first ChIP . For genome-wide analysis , immunoprecipitated DNA was amplified by a modified T7 linear amplification method and analyzed on a tiling array ( based on the May 2005 NCBI sequence of the P . falciparum genome; 385 , 000 probes with a median spacing of 48bp , Roche NimbleGen ) [37] . qPCR was performed on reverse transcribed total RNA and gDNA isolated from synchronous parasite cultures at six timepoints across the IDC . A detailed protocol , relative transcript calculation and primer sequences are provided in Protocols S3 and S4 . Growth of 3D7/SIP2-N-HA parasites was tightly synchronized in parallel three times by sorbitol treatment to achieve a ten-hour growth window . Total RNA was isolated at four timepoints across the IDC at early ring stages ( 4–14 hours post-invasion ( hpi ) ) , late ring stages ( 14–24 hpi ) , trophozoites ( 24–34 hpi ) and schizonts ( 32–42 hpi ) by lysis of pelleted RBCs in TriReagent ( Sigma ) . Transcript levels in 3D7/SIP2-N-HA were compared to those of the control line 3D7/camHG [27] . RNA samples were analyzed using a P . falciparum microarray as previously described [86] . RNA from each time point was hybridized against a RNA pool assembled from equal amounts of total RNA collected from the 3D7 strain at every eight hours across the IDC . PlasmoDB ( www . plasmoDB . org ) accession numbers for genes and proteins discussed in this publication are: PfSIP2 ( PFF0200c ) ; PfHP1 ( PFL1005c ) ; upsB var genes ( PFL0005w , PFL0935c ) ; rhoph1/clag2 ( PFB0935w ) ; rhoph1/clag3 . 1 ( PFC0120w ) ; rhoph1/clag9 ( PFI1730w ) ; rhoph2 ( PFI1445w ) ; rhoph3 ( PFI0265c ) ; rap1 ( PF14_0102 ) ; rap2 ( PFE0080c ) ; rap3 ( PFE0075c ) ; rama ( MAL7P1 . 208 ) ; rnp3 ( PFL2505c ) ; imp , putative ( PFF0645c/MAL6P1 . 292 ) ; gap45 ( PFL1090w ) ; asp ( PFD0295c ) ; msp1 ( PFI1475w ) ; sera4 ( PFB0345c ) ; conserved Plasmodium proteins ( PFL1025c , MAL7P1 . 119 ) ; RFC , subunit 2 ( PFB0840w ) ; PfSNF2L ( PF11_0053 ) ; DNA pol ε , subunit A ( PFF1470c ) ; transcription factor with AP2 domains ( PF10_0075 ) . | Plasmodium falciparum , like many other unicellular pathogens , uses antigenic variation of surface proteins as a prime mechanism for immune evasion . Subtelomeric gene families , including the major virulence family var , encode these antigens , and recent studies highlighted the important contribution of the specific biology of chromosome ends in regulation of their expression . P . falciparum chromosome ends are enriched in epigenetic marks of eukaryotic heterochromatin; however , it remains unknown which proteins and regulatory DNA motifs are involved in the recruitment and organization of heterochromatin , and how they contribute to the role of subtelomere biology in this important pathogen . Here , we present the experimental identification and genome-wide characterization of PfSIP2 , a member of the ApiAP2 family of transcription factors that are specific to Plasmodium and related apicomplexan parasites . PfSIP2 binds to a conserved recognition sequence exclusively in heterochromatic domains upstream of subtelomeric var genes and within telomere-associated repeat elements . Our results suggest important roles for this protein in several aspects of chromosome end biology , including var gene silencing . They are furthermore highly relevant for future efforts to dissect the specific biology of P . falciparum chromosome ends , and for our understanding of the role of ApiAP2 factors in parasite biology . | [
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] | 2010 | A Major Role for the Plasmodium falciparum ApiAP2 Protein PfSIP2 in Chromosome End Biology |
HIV superinfection ( reinfection ) has been reported in several settings , but no study has been designed and powered to rigorously compare its incidence to that of initial infection . Determining whether HIV infection reduces the risk of superinfection is critical to understanding whether an immune response to natural HIV infection is protective . This study compares the incidence of initial infection and superinfection in a prospective seroincident cohort of high-risk women in Mombasa , Kenya . A next-generation sequencing-based pipeline was developed to screen 129 women for superinfection . Longitudinal plasma samples at <6 months , >2 years and one intervening time after initial HIV infection were analyzed . Amplicons in three genome regions were sequenced and a median of 901 sequences obtained per gene per timepoint . Phylogenetic evidence of polyphyly , confirmed by pairwise distance analysis , defined superinfection . Superinfection timing was determined by sequencing virus from intervening timepoints . These data were combined with published data from 17 additional women in the same cohort , totaling 146 women screened . Twenty-one cases of superinfection were identified for an estimated incidence rate of 2 . 61 per 100 person-years ( pys ) . The incidence rate of initial infection among 1910 women in the same cohort was 5 . 75 per 100pys . Andersen-Gill proportional hazards models were used to compare incidences , adjusting for covariates known to influence HIV susceptibility in this cohort . Superinfection incidence was significantly lower than initial infection incidence , with a hazard ratio of 0 . 47 ( CI 0 . 29–0 . 75 , p = 0 . 0019 ) . This lower incidence of superinfection was only observed >6 months after initial infection . This is the first adequately powered study to report that HIV infection reduces the risk of reinfection , raising the possibility that immune responses to natural infection are partially protective . The observation that superinfection risk changes with time implies a window of protection that coincides with the maturation of HIV-specific immunity .
Development of a safe and effective prophylactic HIV vaccine remains enormously challenging , due to the virus's high diversity and our limited understanding of immune correlates of protection . While most effective vaccines are designed to mimic natural infection and protective immune responses to it , such a template for HIV vaccine design remains elusive , since sterilizing immune responses to natural infection have not been observed . A priority of HIV vaccine development is , therefore , to identify settings where natural infection elicits some immune functions desired in a vaccine . For example , HIV-infected individuals who spontaneously control viral replication have provided insights into immune mechanisms of HIV control [1] . However , models where the response , rather than delaying disease , prevents infection – the ultimate goal of a prophylactic vaccine – remain less well characterized . Studies of superinfection ( reinfection from a different partner ) provide a unique model in which to investigate the impact of pre-existing responses on susceptibility to infection by diverse circulating viral variants , which include multiple subtypes with up to 30% sequence variation . HIV superinfection has been reported in a number of settings [2]–[13] , implying that HIV acquisition can occur despite the immune response to initial infection . However , it remains an open question whether pre-existing infection affords some protection from superinfection , and individuals who do become superinfected are a select subset deficient in a particular aspect of immunity . Published estimates of superinfection incidence vary from no identified cases [1] , [14]–[16] to rates roughly similar to initial infection [2]–[13] , [17] , [18] . These discrepancies are largely explained by differences in participant inclusion criteria and study design . The studies that have directly compared initial and superinfection incidence have had limited statistical power due to cohort size [5] , [12] , [17] , [18] or number of cases of superinfection identified [3] , [8] . Additionally , methods used to identify superinfection have evolved . Superinfection is most reliably detected in longitudinal samples by the presence of a single viral clade initially followed by introduction of a second phylogenetically distinct clade [19] . Detection sensitivity is dependent on the number of genomic regions analyzed [12] , as well as sequencing depth [20] . Until recently , sequences were obtained by limiting dilution amplification and Sanger sequencing [5] , [6] , [12] , [17] , which limits detection to cases where the second virus is relatively abundant . The development of next generation sequencing ( NGS ) has enabled higher-throughput , deeper sequencing of large cohorts [20] , [21] . To date , the largest study to examine the rate of superinfection in a prospective seroincident cohort was a NGS screen by Redd et al . of 149 individuals in which 7 cases were identified [8] . No statistically significant difference was found between the incidences of initial infection and superinfection , though the relatively small number of cases may have resulted in limited statistical power . A greater number of cases was found in a high-risk cohort in Mombasa , Kenya , with 12 cases of 56 women screened [5] , [12] , [17] . However , this study used Sanger sequencing to sample ∼7 clones per sample , which could miss lower frequency variants , and was not powered to compare incidences . In the present study , we developed a NGS method for identification of superinfection , and used it to screen 129 women in the same Mombasa cohort , including those classified as singly infected in the prior study . We identified 9 additional cases of superinfection , for a total of 21 cases in this cohort . These combined data enabled comparison of the incidence rates of initial infection and superinfection .
In order to conduct a sensitive , high-throughput screen for superinfection in the Mombasa cohort , we developed a pipeline for amplification , next-generation sequencing ( NGS ) , data cleaning , and phylogenetic and sequence diversity analysis of longitudinal plasma RNA ( Fig . 1 ) . One-hundred thirty-two women met our selection criteria for the NGS superinfection screen , with a median follow-up time of 2046 days ( IQR 1265–2848 ) . We successfully amplified gag , pol and env at three timepoints in 115 women and at least two genomic regions in at least the first and last timepoints in 129 . The remaining 3 women were dropped from analysis . In total , ∼1 . 7 million raw sequencing reads were obtained , with ∼1 . 25 million passing quality filtering: a median of 901 per amplicon per sample . Women were considered putative superinfection cases if the posterior probability of monophyly supported single infection at the earliest studied timepoint followed by introduction of a distinct viral clade and increased viral diversity consistent with that seen in simulated dual infection ( Fig . 1e&f ) . Putative cases of superinfection were confirmed and their timing specified by analyzing intervening timepoints . Nine cases of superinfection were detected and their timing specified . One case of suspected dual infection was detected , in which two clades were detected at the earliest sample analyzed ( 60 days post-infection ( dpi ) ) and throughout infection ( data not shown ) . Example data from two cases of superinfection are summarized in Figures 2 and 3 . Initial screening of subject QD151 ( Fig . 2 ) showed monophyletic subtype A infection at 39 dpi and two subtype A clades in all three genes at 938 and 1701 dpi . In subsequent analysis of intervening timepoints the second clade was first detectable at 801 dpi ( Fig . 2a ) . At this time , pairwise distance increased sharply , for example in gag from 0 . 27% at 241 dpi , to 12 . 75% at 801 dpi ( Fig . 2b ) , into the range observed in simulated dual infections . These observations supported introduction of a second subtype A variant between 241 and 801 dpi . The initial clade was no longer detectable in pol at 1701 dpi , suggestive of a genomic recombination event ( Fig . 2c ) . Similarly , subject QB210 ( Fig . 3 ) showed initially monophyletic infection with a subtype A/D virus , followed by introduction of a subtype C/D virus at 163 dpi , evidenced by polyphyly and a shift in pairwise distance ( >10% ) in all 3 genes ( Fig . 3a and 3b ) . In intervening timepoints , the second variant could be detected in all genes at 163–170 dpi , but was undetectable in gag and pol after 170 dpi , indicating recombination ( Fig . 3c ) . Characteristics of the 9 new cases of superinfection are summarized in Table 1 and Figure S2 . In all but two cases the superinfecting variant was detected in all 3 amplicons in at least one timepoint . In all cases , the superinfecting variant was detected at multiple timepoints in at least one amplicon . In one case ( QC369 ) , the initial variant became undetectable in any amplicon following superinfection , suggesting it was replaced , to our detection limit , by the superinfecting variant . Both variants were detected at two timepoints each , the initial variant at 17 dpi and 28 dpi , and the superinfecting variant at 143 dpi and 451 dpi ( Fig . S2 ) , indicating this result was not due to contamination . Further , the possibility of sample mix-up was excluded by HLA-typing ( data not shown ) . As illustrated in Figures 2 , 3 and S2 , in the other 8 cases , variants were intermittently detected in different amplicons at different times , suggestive of genomic recombination and dynamic turnover of the circulating viral population . Combining the data here with those from previous studies in the Mombasa cohort [5] , [12] , [17] , a total of 146 women were examined for superinfection: 90 were tested using NGS , 39 using both NGS and Sanger sequencing , and 17 using only Sanger sequencing . Among the 39 women previously identified as singly infected by Sanger sequencing and tested by NGS here , no new cases of superinfection were identified , suggesting older methods were sensitive enough to detect superinfection . Twenty-one cases of superinfection were confirmed based on detection of the superinfecting virus in two or more samples . The timing windows of all 21 superinfection events are summarized in Figure 4 and Table S2 . The midpoint of the timing window of the 9 new cases ranged from 81 to 1041 dpi , with 6 occurring within the first year of infection . The window of superinfection events was defined to a median of within 127 days , with window sizes of 90 to 1253 days . Timing of all 21 cases ranged from 63 to 1895 dpi , defined to a median of within 146 days . We detected both inter-subtype and intra-subtype superinfections . In 6 of 9 cases identified by NGS , the superinfecting variant was the same subtype as the initial variant in every gene where both were detected . In all 9 cases , the variants were the same subtype in the env amplicon ( Table 1 ) . Among all 21 cases of superinfection ( Table S2 ) , the majority of superinfection events we detected were intrasubtype , regardless of genomic region: 53 . 8% were intrasubtype based on gag sequence , 62 . 5% based on pol , and 70 . 6% based on env . We further investigated the possibility of a bias in sequence similarity of superinfecting variants to initial variants by analyzing amino acid diversity . We compared the pairwise amino acid distance between initial and superinfecting variants within each superinfection case to the distance that would be expected by chance . The latter was modeled by simulated mixtures of sequences from all possible pairs of singly infected individuals in the Mombasa cohort ( Fig . 5 ) . Using NGS data from the 9 superinfection cases and 120 singly infected women screened here , we found no significant differences between the sequence similarity within superinfected individuals and that expected by chance ( Fig . 5a ) . Including Sanger sequencing data from the additional 12 superinfected women previously screened yielded a similar result ( Fig . 5b ) The incidence of superinfection among women who were screened was compared to the incidence of initial infection in the entire cohort at risk . Only incident HIV infections ( occurring after enrollment in the cohort ) were included . Fourteen women who were seronegative but HIV RNA positive at enrollment were excluded for this reason . Seven of these had been screened for superinfection , and one was found to be superinfected , which mirrors the frequency of superinfection observed in the entire group . The individual with evidence of dual infection at the earliest timepoint was also excluded , since we were unable to distinguish coinfection from superinfection . After exclusions , 1910 women were at risk of initial infection , contributing 5124 person-years , and 138 women were screened for superinfection , contributing 764py following first infection . There were 295 initial infections , giving a crude incidence rate of 5 . 7 per 100pys , and 20 superinfections , giving a crude incidence rate of 2 . 61 per 100 pys . The incidence of superinfection and initial infection over time is summarized in Figure 6 . We used Andersen-Gill proportional hazards analysis to generate a hazard ratio ( HR ) relating the incidence of superinfection to that of initial infection . The unadjusted HR for this comparison was 0 . 49 ( CI 0 . 31–0 . 76 , p = 0 . 0018 ) . Variables previously shown to influence HIV exposure risk in this cohort [22] , [23] were included as adjustments in the model ( summarized in Table 2 ) . These included self-reported sexual risk behavior , place of work , hormonal contraceptive use , genital tract infections , years in sexwork , age at first sex , total follow-up time in the cohort and calendar year . The HR for superinfection compared to initial infection , adjusted for these variables , was 0 . 47 ( CI 0 . 29–0 . 75 , p = 0 . 0019 ) . Since proportional hazards analysis is based on time to infection and the precision with which superinfection timing was determined varied between cases , we performed sensitivity analyses setting infection timing for all cases to the start or midpoint of the timing windows rather than the end , as done for the above analysis . In both of these analyses , significant differences in incidence were also observed: setting infection timing to the start of the windows , the adjusted HR was 0 . 33 ( CI 0 . 18–0 . 58 , p = 0 . 00012 ) ; using the window midpoints , the adjusted HR was 0 . 39 ( CI 0 . 23–0 . 63 , p = 0 . 00016 ) . We assessed whether the risk of superinfection varied with time since initial infection by dividing our data into infection events occurring early or late in follow-up and estimating the HR , as above , in each subset . We found that within the first 6 months at risk , the incidence rates of initial and superinfection did not differ significantly ( adjusted HR 0 . 73 , p = 0 . 51 ) , whereas after 6 months the rate of superinfection was lower than that of initial infection ( adjusted HR 0 . 40 , p = 0 . 0017 ) . A similar result was observed when considering events within or beyond one year at risk: within the first year , the incidence rates of initial and superinfection did not differ significantly ( adjusted HR 0 . 54 , p = 0 . 14 ) , but after one year the rate of superinfection was significantly lower ( adjusted HR 0 . 43 , p = 0 . 0059 ) . Sensitivity analyses setting infection time to the start and midpoint of the timing windows as above reproduced the same results ( data not shown ) . We noted that the previous screens in the cohort appeared to detect a higher frequency of superinfection than the NGS screen ( 12 cases of 56 women screened , compared with 9 cases of 90 ) , with a greater fraction of the events occurring later after initial infection ( Fig . 4 ) . Since the NGS screen spanned later years in the cohort than the previous studies , such a difference could be due to the known decline in infection risk in the cohort over calendar time [22] , [23] . However , the numbers of events are small when the datasets are considered separately and the difference both in superinfection incidence rate and post-infection timing between the two studies was not statistically significant ( data not shown ) .
In this study we used NGS to screen for superinfection in 129 high-risk women and identified 9 cases of superinfection . Combined with previous studies[5] , [12] , [17] , a total of 21 cases of superinfection were detected among 146 women screened in this cohort . There was a statistically significant difference between the incidence of superinfection ( 2 . 61 per 100pys ) and initial infection ( 5 . 75 per 100 pys ) , with a hazard ratio of 0 . 47 after adjusting for potential confounding factors . This suggests that HIV infection provides partial protection from subsequent infection . The relatively large size of this cohort and high number of superinfection cases enabled us to detect for the first time a statistically significant difference between the incidence of initial infection and superinfection . This possibility has been proposed previously , though the studies were not designed and/or powered to detect a difference [17] , [18] . In the largest incidence study prior to the present study , Redd et al . screened a comparable number of individuals ( 149 ) in a lower-risk cohort and identified 7 cases of superinfection . The incidence of superinfection was not found to differ significantly from initial infection , but there was a trend for lower incidence of superinfection when controlling for baseline sociodemographic differences between the groups at risk of initial and superinfection . Analysis of our data using the same methods as Redd et al . – Poisson regression with propensity score matching [8] – was consistent with the results of our Andersen-Gill analysis , showing a significant difference in incidence , with an estimated incidence ratio of 0 . 48 ( p = 0 . 011 ) comparing superinfection to initial infection . In addition to sample size , two strengths of our incidence analysis were our specification of infection timing to within a few months on average and our comparison of initial and superinfection risk within the same cohort . These enabled us to adjust for the same potential confounding factors in both the initial infection and the superinfection risk sets , using frequently collected time-varying covariate data . Particularly important , given the sequential nature of superinfection , was adjustment for calendar year to control for decline in infection risk in the cohort over time . The distributions of initial and superinfection events over calendar time were similar ( Fig . S3 ) , suggesting community-level changes over time did not severely bias our analysis . The ∼two-fold reduction we found in the incidence of superinfection has a number of possible interpretations . First , it may indicate that the adaptive immune response elicited by initial infection provides partial protection from second infection . If this were the case , superinfection might preferentially occur early in infection , before the response has matured [2] , [13] , [24] . In support of this idea , we found that , although superinfection occurred throughout the course of first infection , the incidence of superinfection was significantly lower than initial infection after the first 6 months of infection , but not earlier . This suggests that susceptibility to superinfection decreased over time , coincident with broadening and strengthening of HIV-specific immunity . Indeed , this has been suggested by two earlier studies , each documenting three cases of superinfection that occurred within the first year after initial infection [3] , [18] . If the difference in incidence we observed is due to a partially protective adaptive immune response , we would anticipate superinfection would preferentially occur with more distantly related viruses , more likely to escape the response . Using viral subtype and pairwise amino acid distance as surrogate measures of antigenic distance , our data provided no evidence of this effect . The majority of the 21 superinfection events we detected were intrasubtype , and the proportion of subtype A , C and D viral sequences was similar for the initial and superinfecting viruses , consistent with the subtype distribution in this cohort [25] . The pairwise distance between initial and superinfecting variants was no higher than the distribution of distances between random pairs of singly-infected individuals from the Mombasa cohort . This may potentially be explained by limited sample size or insufficient simultaneously circulating subtypes . It also may be that sequence relatedness is a poor indicator of susceptibility to the immune response or the genome regions we analyzed are not critical antigenic determinants of protection . Alternatively , it is possible that protective immune responses are not driving the protective effect we observed . Another potential explanation for the lower risk of superinfection is that HIV infection itself may reduce infection risk by depleting permissive target cells . On the other hand , chronic immune activation and immunodeficiency following HIV infection could increase susceptibility , potentially blunting protective effects [26] . Thus , there may be a complex interplay of biological factors impacting HIV risk in an HIV-positive individual . So far , studies of immune correlates of superinfection have yielded variable results – some suggesting neutralizing antibody deficits in superinfection [27] , [28] , while others , including studies in the Mombasa cohort , detected no differences in antibody [29] , [30] or cellular [31] responses . A major challenge in these studies has been the identification and analysis of large enough numbers of superinfection cases: the small sample sizes in studies to date ( three to twelve superinfected individuals ) would restrict detection to only very large effects . Small sample size is just one factor that has made detecting immune deficits associated with superinfection challenging and contributed to variable results among studies . There has also been variation among published studies in the control groups used for comparison , including the time at which the response was analyzed relative to the time of superinfection and initial infection . Given the dynamic nature of the immune response , sample timing could impact measures in both controls and cases . Furthermore , precision in the estimated timing of superinfection varies between studies , and between cases , providing an additional variable . Divergent findings between studies may also reflect differences in the assays used and subtleties in the immune parameters they capture . Our finding of lower risk of superinfection than initial infection provides greater impetus for larger-scale comprehensive analysis of multiple immune mechanisms , including both those analyzed in the smaller studies to date and , perhaps of more interest , those not characterized in prior studies . If the discrepancies in earlier studies reflect the fact that multiple immune parameters are at play , then examining a variety of immune responses in the same individuals in a larger cohort may be needed to define responses that contribute to HIV susceptibility following initial infection . Like all studies , the study presented here has a number of limitations . Firstly , while our screening methods are among the most sensitive developed , it remains possible that some cases of superinfection were missed . In particular , reinfection by the same source partner is not captured by any existing methods . Additionally , our specification of the timing of superinfection was limited by the samples available to us . While follow-up was generally frequent in this study population , there were six superinfection cases where sample availability limited our ability to define the time of superinfection to within a one-year period . This uncertainty in superinfection timing did not affect our findings , as we found that whether we assumed in the incidence analysis that the true timing of superinfection was at the start , midpoint or end of the timing window , the results indicated that the incidence of superinfection was significantly lower than that of initial infection . Finally , as in all observational studies , residual confounding of our incidence estimate by behavioral changes and sexual network-level factors not measured or accounted for in our analyses remains a possibility . However , the fact that we compared initial and superinfection risk within the same cohort and collected covariate data at frequent intervals enabled us to minimize this issue to an extent not possible in previous studies . This study provides the first robust evidence that HIV infection reduces the risk of subsequent infection . The underlying mechanism remains unclear , but this finding prompts exploration of correlates of protection from HIV in high-risk individuals who continue to be exposed after first infection . Furthermore , this study reinforces that superinfection occurs at a considerable rate , calling for studies of its impact on the clinical progression , transmission , and epidemiology of HIV .
The study was approved by the ethical review committees of the University of Nairobi , the University of Washington and the Fred Hutchinson Cancer Research Center . Written informed consent was obtained from all participants . Seronegative women in Mombasa , Kenya , attended monthly visits , at which clinical examinations , interviews and sample collection took place , as previously described [22] . Following seroconversion , sample collection took place quarterly . Individuals were selected for superinfection screening based on sample availability <6 months and >2 years post-initial HIV infection , and an approximately equally spaced intervening sample . Within these limitations , samples with maximal plasma viral load , >1000 copies/ml , and prior to initiation of antiretroviral therapy were selected . Thirty-nine of 44 women previously screened for superinfection by Sanger sequencing and identified as singly infected [5] , [12] , [17] were rescreened; the remaining 5 women did not have adequate samples available . HIV virions were isolated from heparinized plasma using the μMACS VitalVirus HIV Isolation kit ( Miltenyi Biotec ) and viral RNA extracted from 140–420 µl , depending on viral load , using the Qiamp viral RNA Mini kit ( Qiagen ) . Nested RT-PCR of ∼500 bp in gag , pol and env was conducted in duplicate ( see Table S1 ) . RNA input into each reaction was normalized to 3000 viral genomes according to plasma viral load , or the maximum possible where viral load was too low . RT-PCRs for the three genes were multiplexed . Nested PCR reactions were carried out separately for each region with primers containing adaptors for Roche 454 sequencing and a unique 8 bp barcode sequence to identify each sample . PCR products were purified using AMPure XP PCR purification beads ( Agencourt ) and quantified using the Qubit dsDNA HS assay ( Invitrogen ) . PCR products were sequenced on the Roche 454 GS-Junior or GS-FLX titanium platform . Where initial sequencing suggested superinfection ( see below ) , timing was inferred by sequencing intervening timepoints . Sequences are available upon request from the authors . 454 sequences were error-corrected using AmpliconNoise [32] . Chimeric sequences were identified and removed using UCHIME [33] . Cross-contamination between samples sequenced together and contamination by other lab samples was identified by all-against-all BLAST against a local database of published HIV sequences and sequences from the same sequencing run . Sequences with high identity hits to known laboratory stains or other samples from the same sequencing run were removed . Sequences with abundance <5 reads or 0 . 5% of the sample , whichever was higher , were excluded from further analyses as lower abundance variants were not reproducibly detected in repeated deeper sequencing of two selected samples where rare variants formed a distinct phylogenetic clade . An amplicon-specific profile HMM was created from an alignment of representative sequences from multiple subtypes . For each subject and amplicon , 20 reference sequences were selected by placing 454 reads on a tree of candidate reference sequences [34] and minimizing the average distance to the closest leaf [35] . These reference sequences , representatives from subtypes common to the region , and 454 reads were aligned to the HMM using hmmalign [36] and non-consensus columns removed . Any sequences <200 bp long after alignment and trimming were removed . We used BEAST [37] to calculate a posterior probability of monophyly for the sequences . A posterior sample of trees was obtained using a strict molecular clock , Bayesian Skyline Plot population model and the HKY substitution model . Each MCMC chain ran 20 million iterations , sampling every 2000 , discarding the initial 25% of samples as burn-in . Chains were assessed for convergence by examining effective sample size ( ESS ) and by visual inspection of traces of key parameters . A strict clock was used as poor mixing was frequently observed under relaxed clock models . BEAST runs with intermediate posterior probabilities ( 0 . 2–0 . 8 ) were manually examined for recombinant sequences and run again with putative recombinants removed . Pairwise distances were calculated for all sequence pairs under the TN93 model using APE [38] , reporting the maximum within-subject distance . For comparison , 95% confidence limits of pairwise distances were calculated for sequences from known single infections ( previously screened in [5] , [12] , [17] ) and simulated dual infections . Dual infections were simulated by combining all pairs of sequences from previously screened singly infected samples . Pairwise distances calculated from 454 sequences obtained in this study were compared to the upper bound of the 95% quantile of single infection distances , and the lower bound of the 95% quantile of simulated dual infection distances . This pipeline was validated and refined by processing monophyletic viral isolates , known mixtures of isolates , and known cases of superinfection detected by Sanger sequencing [17] . These methods were found to be sensitive enough to distinguish two subtype A isolates mixed at abundances of 5%∶95% genome copies in all three genomic regions , and at 1%∶99% in two of three genomic regions ( Fig . S1 ) . Sequences were aligned as for the phylogenetic analysis . Insertions relative to the reference alignment were removed , and sequences with <60% coverage or identified as recombinants between initial and superinfecting variants upon visual inspection were excluded . For each case of superinfection , viral sequences were annotated as the initial strain or the superinfecting strain . We calculated the mean Hamming distance between amino acid sequences of the superinfecting strain from the time of superinfection detection and sequences of the initial strain up to and including this time . In calculating the mean distance , each pairwise comparison was weighted using the product of the multiplicities of the two reads . To investigate whether these distances deviated from what would be expected by chance , an artificial set of mock superinfections was generated by combining sequences from singly infected individuals . All pairs of singly infected individuals screened by 454 sequencing were enumerated . In each pair , one individual was randomly chosen to be the source of the ‘initial’ virus in the simulated superinfection . A time of ‘superinfection’ was chosen randomly from the available sampled timepoints and sequences from all timepoints up to and including this time were used for analysis . The other individual in the pair acted as the source of the ‘superinfecting’ virus . A time of ‘transmission’ was chosen randomly from the available sampled timepoints and sequences from this timepoint were used . Mean distances within pairs were calculated as above . The analysis was repeated including gag and env Sanger sequences from previously published cases [5] , [12] , [17] , trimmed to the genome region amplified for NGS , and given unit weight . A two-sample Wilcoxon test was used to test for a difference between the distances observed in true superinfections and those simulated in mock superinfections . Statistical analysis was performed using R ( www . r-project . org ) . The incidences of initial and superinfection were compared by Andersen-Gill proportional hazards analysis . The predictor was inclusion in the screen for superinfection , modeled as a time-dependent variable , and the outcome was time to HIV infection ( initial and super ) . Timing of infection events for the incidence analysis was set to the study visit of their detection ( for initial infection events the visit after inferred infection timing; for superinfection events , the time at which the superinfecting virus was first detected ) . Individuals who were HIV infected but not screened for superinfection were censored after acquisition of initial infection . Individuals who became superinfected were censored after acquisition of superinfection . Individuals who were screened and not found to be superinfected were censored at the last timepoint screened . Since samples after initiation of antiretroviral treatment were excluded from superinfection screening , no follow-up after treatment initiation was included . The model was adjusted for time-varying variables at each visit: calendar year , age , years in sexwork , number of weekly sexual partners , number of weekly unprotected sex acts , hormonal contraceptive use in the prior 70 days and any genital tract infection in the prior 70 days ( bacterial vaginosis , cervicitis , genital ulcer disease , gonorrhea , trichomoniasis ) ; place of work and age at first sex recorded at enrollment; and total follow-up time in the study . Incidences of initial and superinfection were also estimated as described in [8] , using Poisson regression and propensity score matching to select a subset of women at risk of initial infection whose baseline risk profiles most closely matched those of women screened for superinfection . | HIV-infected individuals with continued exposure are at risk of acquiring a second infection , a process known as superinfection . Superinfection has been reported in various at-risk populations , but how frequently it occurs remains unclear . Determining the frequency of superinfection compared with initial infection can help clarify whether the immune response developed against HIV can protect from reinfection – critical information for understanding whether such responses should guide HIV vaccine design . In this study , we developed a sensitive high-throughput method to identify superinfection and used this to conduct a screen for superinfection in 146 women in a high-risk cohort . This enabled us to determine if first HIV infection affects the risk of second infection by comparing the incidence of superinfection in this group to the incidence of initial infection in 1910 women in the larger cohort . We found that the incidence of superinfection was approximately half that of initial infection after controlling for behavioral and clinical differences that might affect infection risk . These results suggest that the immune response elicited in natural HIV infection may provide partial protection against subsequent infection and indicate the setting of superinfection may shed light on the features of a protective immune response and inform vaccine design . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"immunodeficiency",
"viruses",
"medicine",
"virology",
"epidemiology",
"biology",
"microbiology"
] | 2013 | HIV-1 Superinfection Occurs Less Frequently Than Initial Infection in a Cohort of High-Risk Kenyan Women |
Nuclear pore complexes ( NPCs ) are highly selective filters that control the exchange of material between nucleus and cytoplasm . The principles that govern selective filtering by NPCs are not fully understood . Previous studies find that cellular proteins capable of fast translocation through NPCs ( transport receptors ) are characterized by a high proportion of hydrophobic surface regions . Our analysis finds that transport receptors and their complexes are also highly negatively charged . Moreover , NPC components that constitute the permeability barrier are positively charged . We estimate that electrostatic interactions between a transport receptor and the NPC result in an energy gain of several kBT , which would enable significantly increased translocation rates of transport receptors relative to other cellular proteins . We suggest that negative charge is an essential criterion for selective passage through the NPC .
The defining feature of eukaryotic cells is the separation of nuclear and cytoplasmic compartments by the nuclear envelope . All nuclear proteins , for example polymerases and transcription factors , are made in the cytoplasm and imported into the nucleus . Conversely , RNAs that function in translation are made inside the nucleus and exported to the cytoplasm . The transport of material between the nucleus and cytoplasm occurs through nuclear pore complexes ( NPCs ) , aqueous channels that are embedded in the nuclear envelope . Translocation through NPCs is fully reversible and uncoupled from NTP hydrolysis [1] , [2] , [3] , [4] , [5] . Kinetic measurements demonstrate that a single NPC can selectively translocate nearly one thousand molecules per second [6] , [7] . Proteins above 30–40 kDa typically only traverse the NPC at appreciable rates with the aid of dedicated nuclear transport receptors ( for reviews see [8] , [9] , [10] , [11] ) . Nuclear transport receptors are soluble proteins that bind to their substrates and translocate together with them through the NPC channel . Importinβ-like transport receptors mediate the vast majority of nuclear transport , and are typically classified as importins or exportins , depending on whether they mediate nuclear import or export . The importinβ superfamily includes at least 21 members in the human proteome Homo sapiens , and 14 in the yeast Saccharomyces cerevisiae [8] . The NPC consists of approximately 30 proteins ( termed nucleoporins ) in S . cerevisiae , and roughly the same number in vertebrates [12] , [13] , [14] , [15] , [16] . Many nucleoporins contain a series of phenylalanine-rich repeats ( FG-repeats ) , which typically occur within the amino acid motifs FxFG or GLFG ( here “x” stands for a variable amino acid; [17] , and references therein ) . The FG-repeats are separated by largely unfolded and hydrophilic spacer sequences [17] , [18] , [19] . Immuno-electron microscopy data reveal that FG-repeat domains occupy the NPC channel as well as the cytoplasmic and nuclear rim of the NPC [19] , [20] , [21] , [22] . Several research groups have established that relatively specific interactions between transport receptors and FG-repeats within nucleoporins are necessary to facilitate selective translocation through the NPC barrier [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] , [37] . Transport receptor-FG-repeat interactions are mediated by specific hydrophobic regions on the surface of the transport receptors [6] , [24] , [25] , [26] , [28] , [29] , [32] , [33] , [38] , [39] , [40] . Indeed , the crystal structures of importinβ [41] , transportin [42] , NTF2 [43] , [44] , and TAP [45] reveal that their surfaces are characterized by a high proportion of hydrophobic regions . Here , we observe that nuclear transport receptors carry more negative charge than the majority of cellular proteins . The influence of surface charge on the translocation reaction has not been addressed so far . We note that most components of the selectivity barrier within the NPC are characterized by net positive charge [17] , [19] , [34] . We calculate that electrostatic interactions between a negatively charged transport receptor and positively charged nucleoporins could result in an energy gain of multiple kBT . This could help compensate for the energy barrier that transport receptors encounter on translocating through the spatially confined NPC channel . We propose that positively charged nucleoporin domains are an important component of the selective filter and promote the specific translocation of negatively charged transport receptors , while imposing a large energy barrier against translocation of positively charged cellular proteins .
To understand the biophysical properties that facilitate NPC translocation we first compared the amino acid composition of translocation-competent particles ( nuclear transport receptors and cognate transport receptor-cargo complexes ) with that of cargo proteins . We assembled a collection of nuclear transport receptors and their cognate cargos from both S . cerevisiae and H . sapiens ( Table S1 ) . In addition , we compiled a list of biophysical properties including measures of charge , polarity , and 27 different empirical metrics for hydrophobicity ( Table S2 ) . For every protein , the value of each property ( with the exception of the isoelectric point ) is obtained by summing the contribution from each amino acid in its sequence , and normalizing by sequence length . Note that these metrics do not take into account the solvent accessibility of each residue; this is not feasible due to the lack of structural information for many cargo proteins . The results of this analysis are displayed as a heat map in Figure 1A . Every property is normalized over the entire set of proteins to have mean zero . Bright red and green correspond to 3 standard deviations above and below this mean , respectively . The proteins are clustered according to the similarity of their properties . Each column corresponds to a different property and each row to a different protein or protein complex . The individual rows in the top panel of Fig . 1A reveal that the profiles of individual nuclear transport receptors resemble each other , but are visibly different to the profiles of individual cargo proteins ( middle panel , Fig . 1A ) . This suggests that particles with translocation-promoting properties are distinct from the average cargo protein with regard to the properties measured here . To eliminate redundancy in the set of physical properties , we applied principal component analysis ( PCA ) to the 27 hydrophobicity scales . PCA transforms a number of correlated variables into a smaller number of uncorrelated variables called principal components , ordered according to the amount of variability that they explain . For the 27 hydrophobicity scales , the first principal component captures 75% of the total variance , and thus serves as an “aggregate” hydrophobicity metric that correlates with each of the individual hydrophobicity scales . Moreover , we find that polarity correlates strongly with the aggregate hydrophobicity scale ( correlation coefficient r2 = −0 . 93 , Fig . S1 ) , and so can be eliminated as an independent property . In summary , the original 29 dimensional property space could be reduced to just two properties , the aggregate hydrophobicity scale and isoelectric point . Since the NPC is an aqueous channel that is freely permeable to ions , we assume that the pH within the NPC channel is comparable to that in the cytoplasm and so report the net charge at pH 7 . 2 of each protein or protein complex , instead of its isoelectric point hence forth . The charge of a protein at pH 7 . 2 can be predicted from its amino acid sequence according to , where the sum is over all ionized amino acids in the protein , and the pKX values were obtained from the emboss iep application [46] . We note that representations of protein properties in terms of hydrophobicity and charge have been employed elsewhere [19] . Fig . 1B depicts the hydrophobicity and charge at pH 7 . 2 of proteins from the yeast proteome . This plot reveals that transport receptors have extreme properties compared to the majority of yeast proteins: Transport receptors and receptor-cargo complexes ( light and dark blue squares , and purple triangles , respectively ) are collectively more negatively charged at pH 7 . 2 . In contrast , cargo proteins ( red circles ) tend to be positively charged . The amount of positive charge on a typical cargo protein is far greater than the five to ten charges conferred by a monopartite or bipartite nuclear localization signal [47] . In addition , transport receptors are more hydrophobic than most cellular proteins; as mentioned in the introduction several experiments suggest that specific hydrophobic regions on the surface of nuclear transport receptors are critical for translocation . Note that the segregation of transport receptors from their cargo proteins on the basis of charge and hydrophobicity is also apparent in H . sapiens ( Fig . 1C and S2 ) . Together , these data suggest that net negative charge at pH 7 . 2 is an evolutionarily conserved property that distinguishes translocation competent particles from cargo proteins . To address the role of charge in the translocation reaction we next analyzed the amino acid sequences of nucleoporins from S . cerevisiae ( Fig . 2A; Table S3 ) . Upon clustering according to the similarity of their properties , the nucleoporins segregate into two visually distinct categories , which correspond to their location within the NPC [16] , [22] . The top half of Fig . 2A contains structural nucleoporins that form the core scaffold of the NPC . These nucleoporins coat the surface of the nuclear membrane in which the NPC is embedded [16] . The bottom half of Fig . 2A ( labeled N1–N13 ) comprises all nucleoporins that contain FG-repeats . These FG-nucleoporins are anchored to the NPC scaffold and protrude into the inner of the NPC channel , presumably forming the selective barrier [16] . Fig . 2B plots the hydrophobicity and charge at pH 7 . 2 of the nucleoporins from Fig . 2A . This plot reveals that the majority of FG-nucleoporins ( red circles ) are characterized by net positive charge and low hydrophobicity , while the scaffold nucleoporins ( blue circles ) are both more negatively charged and more hydrophobic . Note that mammalian nucleoporins also segregate into distinct groups ( Table S3 and Fig . S3 ) . FG-nucleoporins contain both structured and unstructured domains . The unstructured domains are comprised of numerous hydrophobic FG-repeats separated by hydrophilic spacers [17] , [19] , [34] . Part of the FG-domain from the yeast nucleoporin Nup100 is shown in Fig . 2C , note that positive charge is present within the hydrophilic spacers that separate the hydrophobic FG-repeats . In this case the positive charge is predominantly derived from the amino acid lysine . It is presumably the unfolded FG-domains that interact with transport receptors and thus determine selectivity ( [25] , [28] , [29] , [36] , [37] , [39] ) . In Fig . 2D we analyze the unfolded FG-domains of all 13 FG-nucleoporins ( Fig . 2A ) as described by [19] , [36] . The data suggest that these FG-nucleoporin domains ( red circles ) are positively charged and hence complementary to the transport receptors ( blue squares ) . This indicates a potential role for electrostatic interactions between the transport receptors and the unfolded domains of the FG-nucleoporins in NPC selectivity .
The rate at which a protein ( or protein complex ) translocates through the NPC depends upon the energy barrier that it must overcome to enter the NPC [48] , [49] . The size of this energy barrier is given by ΔG = ΔH−TΔS , where the enthalpy change ( ΔH ) describes the binding energy of a protein to NPC components , T is the temperature , and ΔS is the change in entropy as a protein enters the NPC . When the charged protein enters the NPC and becomes spatially confined , the entropy of the system decreases , increasing ΔG and disfavoring translocation . However , ΔG can be lowered if specific interactions between a translocating protein and the NPC decrease enthalpy and thereby compensate for the decrease in entropy . We next estimate whether electrostatic interactions between a transport receptor and the NPC interior could in principle be large enough to compensate for the loss of entropy . Within the cell , proteins are surrounded by counter ions that screen their charge; the screening length within the cytoplasm is estimated to be ∼1nm [50] . We calculate the size of the interaction between each charge on the surface of the receptor , ( where many charged amino acids reside; Fig . S4 ) and those nucleoporin charges within a hemisphere of radius 1nm ( QNPC , Fig . 3 ) . We estimate the total charge of the transport receptor ( QNTR ) as the sum of its charged residues . With this approach the electrostatic interaction energy between a transport receptor and the NPC can be approximated by the Coulombic interaction between QNTR and QNPC , separated by a screening length : ( 1 ) where QNTR is the charge derived from the transport receptor , QNPC the charge derived from the NPC and is the dielectric constant of water . A direct derivation of equation ( 1 ) follows from assuming the electrostatic interactions are governed by Debye-Huckel theory , and then computing the interaction energy between the transport receptor and a hemisphere of size of uniform background charge density filling the nuclear pore . Sophisticated treatments of electrostatic interaction energy are possible [51] , [52] , and such analyses will likely modify the ΔHElectrostatic predicted by ( 1 ) , however they should not change the order of magnitude . For human transport receptors at pH 7 . 2 , we find that , with median , where is the electron charge . To calculate QNPC , we approximate the yeast NPC as a cylinder with a radius of ∼19nm and a height of ∼37nm [16] , and thus a pore volume of . The yeast NPC is thought to contain 13 different types of FG-nucleoporins , with either eight or 16 copies of each [16] , [19] . A conservative estimate of the average number of nucleoporins within a hemisphere of radius 1nm is therefore . The charge of an individual interior ( yeast ) nucleoporin ranges from to , with median , resulting in QNPC∼ . Equation ( 1 ) implies that , where QNPC and QNTR are expressed in units of the elementary charge . Using we have that ( 2 ) Thus , for a transport receptor with negative charge Q = , the energy gain due to direct interaction with the NPC interior is ΔHElectrostatic∼−2 . 5 kBT . We emphasize that the model underlying Equation ( 2 ) is simplistic , for example: ( i ) Our treatment of the electrostatic interactions assumes Debye Huckel interactions , which are quantitatively modified when surface charge densities are sufficiently high; ( ii ) Detailed structural information for the charge distribution in the NPC channel is not available; ( iii ) We ignore potential entropic contributions to the electrostatic energy . Equation ( 2 ) thus represents an order of magnitude estimate . Calculation of the entropic cost ΔS for a particle to enter the pore is complicated [12] , requiring detailed knowledge of the environment both inside and outside the pore [53] . However , it is significant that the ΔHElectrostatic predicted by Eqn . 2 is the same order of magnitude as ΔS [12] . Note that the entropic gain upon leaving the pore will compensate for the ensuing reduction in ( electrostatic or other ) binding energy [48] . The ΔHElectrostatic magnitude calculated here would decrease the energy barrier for a transport receptor , increasing translocation efficiency . We therefore propose that electrostatic interactions between negatively charged particles and the positively charged selective barrier components provide a substantial part of the binding energy needed to mediate entry of a particle into the pore .
Our study finds that transport receptors are more negatively charged than the majority of cellular proteins . Existing crystal structures of transport receptors such as importinβ reveal that negative charge is distributed over the surface of the protein ( Fig . S4; [40] , [45] ) . High sequence homology of importinβ-like transport receptors , both within species and between species , suggests that net negative surface charge is a conserved property of this protein family . How could negative surface charge promote the translocation of transport receptors through the NPC channel ? Current models of translocation through the NPC postulate that hydrophobic FG-repeats within the selective barrier principally determine NPC selectivity [6] , [12] , [32] , [36] , [39] , [48] , [54] , [55] . We suggest that charge within the NPC channel may be a second feature relevant for translocation . The unfolded domains that separate FG-repeats are characterized by net positive charge ( Fig . 2C , D ) , and we suggest that they represent a critical element of the selective barrier . Indeed , the positive charge of the spacers is conserved across multiple species [56] , suggesting a functional constraint on the design of the spacer elements . We propose that their negative surface charge allows transport receptors to adsorb to the positively charged nucleoporin domains via electrostatic interactions , facilitating selective partitioning of transport receptors and transport receptor-cargo complexes into the NPC ( Fig . 4 ) . Those soluble cellular proteins that are positively charged ( Fig . 1B , C ) should fail to enter the NPC efficiently because the corresponding energy barrier is too high . Note that according to this model , a translocating particle could become trapped within the NPC if its charge is too negative . The translocation rate is maximal only if the charge of a particle compensates for the decrease in entropy , rendering the total free energy barrier flat . We also note that repulsive electrostatic interactions between the patches of positive charge on the FG-domains may compete with the meshwork forming inter-FG linkages . Electrostatic interactions as proposed here would help the NPC control entry of particles according to their surface charge , independently of their size , and thereby efficiently hinder passive diffusion of positively charged proteins . This is illustrated by histones , relatively small proteins that do not diffuse efficiently through the pore channel by themselves . Using the simple model in ( 2 ) we estimate the contribution of electrostatic interactions to the free energy of translocation for histone1 ( H1 ) . H1 has a molecular weight of 21kD and a charge Q = at pH 7 . 2 . Thus it would encounter an energy barrier of ∼2 . 6 kBT at the NPC , making spontaneous diffusion highly inefficient . By binding to its two transport receptors , importinβ and importin7 [57] , H1 acquires a net charge of , resulting in an energy barrier of ∼−3 . 2 kBT . Combining this electrostatic energy gain with the entropic cost of entering the pore , and possible interactions with FG-repeats , could flatten the energy barrier , thus maximizing the H1 translocation rate . Numerous signal transduction molecules are capable of rapidly shuttling between the nucleus and cytoplasm . How their entry into or exit from the nucleus is regulated is a matter of intense investigation . Fig . 5 shows the charge of a small number of well-characterized signaling proteins ( listed in Table S4 ) plotted against their hydrophobicity . Two conclusions can be drawn from this plot . First , a number of shuttling proteins fall in the translocation competent regime , so are predicted to efficiently self-translocate through the NPC without association to transport receptors . One example is beta-catenin ( marked with asterisk ) , which has a molecular weight of 84kDa and is above the passive diffusion limit of the NPC . Indeed , beta-catenin was discovered to translocate through the NPC without associating to a transport receptor [58] . Secondly , a number of signaling proteins , for example STAT3 and SMAD2 , fall on the edge of the translocation competence regime . Our analysis predicts that addition of negative charge , as occurs through phosphorylation , could contribute to regulating the translocation rate of such particles . Using Eqn . ( 2 ) we estimate that the addition of a single negative charge ( corresponding to phosphorylation of a single site ) would lead to a decrease in the energy barrier by ∼0 . 05 kBT . Thus , phosphorylation of a small number of residues may tune the rate of translocation , even in the absence of a transport receptor . Specific hydrophobic regions on the surface of nuclear transport receptors are critical for translocation . One important future challenge is to dissect the contribution of both a particle's negative charge and its hydrophobicity to the translocation reaction . This question could be addressed with direct experimental tests that measure a particle's translocation rate as a function of its charge , size and hydrophobicity . The charge and size of particles can be independently varied , and doing so would disentangle their relative contributions to translocation through the pore . The results of such experiments will enable the correlation of a protein's translocation rate through the NPC with its charge and hydrophobicity properties . Moreover , this information could facilitate a novel prediction tool for transport receptor-cargo matching . | All proteins that move between the cytoplasm and the nucleus must pass through nuclear pore complexes , large aqueous channels around 40nm in diameter . In some cases the nuclear envelope is perforated with several thousand nuclear pore complexes , while in other cases they are few and far between . Macromolecular transport through nuclear pores is highly regulated; an elaborate system , involving the binding and unbinding of accessory proteins ( transport receptors ) , allows regulation of which proteins can pass through the pores . The basic principles that govern this selective filtering are not fully understood . Some proteins pass through the pore without binding to transport receptors , while others require the binding of multiple transport receptors for efficient translocation . How does the pore select which proteins can pass through , and which cannot ? This paper carries out a biophysical analysis of the properties of proteins that can translocate through the nuclear pore . We find that proteins capable of fast translocation are highly negatively charged , whereas proteins that cannot pass through the pore are positively charged . Moreover , proteins that constitute the interior of the pore channel itself are net positively charged . This suggests that electrostatic interactions between translocating proteins and the pore are an essential part of the selective filtering mechanism . | [
"Abstract",
"Introduction",
"Results",
"Model",
"Discussion"
] | [
"cell",
"biology",
"biophysics/macromolecular",
"assemblies",
"and",
"machines",
"computational",
"biology/systems",
"biology",
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"matter"
] | 2010 | Charge as a Selection Criterion for Translocation through the Nuclear Pore Complex |
A total of 1 , 596 laboratory-confirmed imported dengue cases were identified in Taiwan during 2011–2016 . Most of the imported cases arrived from Southeast Asia as well as the Indian subcontinent , the Pacific region , Latin America , Australia and Africa . Phylogenetic analyses of the complete envelope protein gene sequences from 784 imported dengue virus ( DENV ) isolates were conducted , and the results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant serotype/genotype of DENV strains circulating in Southeast Asia . The DENV-1 genotype III , DENV-3 genotype III and DENV-4 genotype I and II strains were found to be newly emerging in several Southeast Asian countries . Our results also showed that geographical restrictions of DENV-1 genotype I , DENV-1 genotype III and DENV-2 Cosmopolitan genotype are becoming blurred , indicating the extensive introductions and continuous expansions of DENV strains between nations in Southeast Asia . In this study , we present the geographic distribution and dynamic transmission of DENV strains circulating in Southeast Asian countries . In addition , we demonstrated local dengue epidemics caused by several imported DENV strains in Taiwan during 2011–2016 .
Dengue is the most prevalent mosquito-borne viral infection of humans in tropical and subtropical regions of the world [1] . In recent decades , the incidence of dengue has grown dramatically; approximately half of the world’s population is now at risk [2] . An estimated 390 million dengue infections occur annually , of which 96 million dengue infections manifest clinically [3 , 4] . Dengue virus ( DENV ) belongs to the genus Flavivirus in the family Flaviviridae . The DENV genome consists of a single-stranded , positive-sense RNA , which is of approximately 10 , 700 nucleotides and contains a long open reading frame that encodes three structural proteins ( capsid [C] , premembrane/membrane [prM] and envelope [E] proteins ) and seven nonstructural ( NS ) proteins ( NS1 , NS2A , NS2B , NS3 , NS4A , NS4B , and NS5 ) [5 , 6] . There are 4 genetically and antigenically distinct DENV serotypes ( DENV-1 to DENV–4 ) that cause dengue . DENVs are transmitted to humans through the bite of an infected female Aedes mosquito [7] . Dengue disease can manifest as mild dengue fever or the more severe and potentially fatal dengue hemorrhagic fever or dengue shock syndrome [8 , 9] . Dengue is endemic to most countries in Southeast Asia , the Western Pacific region and the Americas , with a very high morbidity rate and disease burden [4 , 10] . A large number of cases are reported each year , and all four DENV serotypes currently circulate in hyperendemic countries . The rapid expansion of DENV strains to different parts of the world has been accelerated by the increase in worldwide travel and trade . Studies on DENV infection in travelers can thus provide useful information on the geographic distribution and global movement of DENV [11–14] Taiwan is an island off the southeastern coast of mainland China in the western Pacific Ocean . The island straddles the Tropic of Cancer , giving it a warm tropical-subtropical climate . Aedes albopictus is found throughout Taiwan , whereas Ae . aegypti is distributed in the south [15] . Dengue is not considered endemic in Taiwan; thus , the close commercial links and air travel between Taiwan and other countries are responsible for the constant importation of multiple DENVs and the outbreaks that occur each year [16 , 17] . To reduce the introduction of DENV strains into Taiwan and prevent local epidemics , both passive and active surveillance of DENV infections have been implemented in Taiwan . We previously reported the molecular characterization of DENV strains imported into Taiwan during 2003–2010 [18 , 19] . The results provided information on the geographic distribution and dynamic transmission of DENV strains in Southeast Asian countries . In this study , we continued to perform laboratory-based surveillance and provide essential information on the molecular epidemiology of DENV strains circulating in Southeast Asian countries during 2011–2016 .
Dengue is a reportable infectious disease in Taiwan , and suspected cases must be reported within 24 hours of clinical diagnosis . To provide effective surveillance , both passive ( the hospital-based reporting system ) and active ( such as fever screening at airports , self-reporting , and expanded screening for contacts of confirmed cases ) surveillance systems were implemented by the central and local health departments in Taiwan . Human serum samples of suspected dengue cases were submitted to the Centers for Disease Control , Taiwan ( Taiwan CDC ) , for confirmation of DENV infection . The human serum samples used in this study were derived from confirmed dengue cases submitted to the Taiwan CDC during 2011–2016 . All samples analyzed were anonymized . The study protocol was reviewed and approved by the Taiwan CDC Institutional Review Board ( IRB 104121 ) . The informed consent requirement was waived by the board . An imported dengue case was defined as a laboratory-confirmed dengue case with travel history to endemic countries within 14 days before the date of onset of dengue . An indigenous case was recorded when no overseas travel was indicated . DENV infection was defined as a febrile illness associated with the detection of DENV RNA by reverse transcription-polymerase chain reaction ( RT-PCR ) , isolation of DENV by cell culture , detection of DENV nonstructural protein 1 ( NS1 ) antigen , or a seroconversion or at least a four-fold increase in the titer of IgM or IgG antibodies against DENV in paired acute and convalescent serum samples tested by capture IgM and IgG enzyme-linked immunosorbent assays ( ELISA ) . Isolation of DENV was performed using a mosquito cell line ( clone C6/36 of Ae . albopictus cells ) as previously described [18] . Briefly , for each acute-phase serum sample , 50 μL of the sample diluted at ratios of 1:20 , 1:40 , 1:80 , and 1:160 with RPMI 1640 medium ( Gibco/BRL , Life Technologies , Auckland , New Zealand ) containing 1% fetal calf serum was added to a 96-well microtiter plate . Then , 1x105 cells/100 μL/well of C6/36 were added to the microtiter plate and incubated for 7 days at 28°C . Cells were harvested , and infection was confirmed by immunofluorescence assay using dengue serotype-specific monoclonal antibodies , including 5F3-1 ( DENV-1 specific , ATCC HB-47 ) , 3H5-1 ( DENV-2 specific , ATCC HB-46 ) , 5D4-11 ( DENV-3-specific , ATCC HB-49 ) and 1H10-6 ( DENV-4-specific , ATCC HB-48 ) . The viruses were subcultured in C6/36 cells and harvested for nucleotide sequencing after the first or second passage . Isolated viruses were identified using the nomenclature of serotype/country of origin/strain/year of isolation . To detect and differentiate DENV serotypes in acute-phase samples , we performed one-step , SYBR Green I-based , real-time RT-PCR ( QuantiTect SYBR Green RT-PCR kit , Qiagen , Hilden , Germany ) using the LightCycler 96 Real-Time PCR System ( Roche Diagnostics , Mannheim Germany ) . Real-time RT-PCR was performed using two sets of consensus primers , one primer set targeting a region of the nonstructural protein 5 ( NS5 ) genes to detect all of the flaviviruses and the other primer set targeting a region of the C gene to detect all of the DENV serotypes . The DENV serotypes of the positive samples were then confirmed by DENV serotyping using four sets of serotype-specific primers targeting the C gene [20] . A commercial DENV NS1 Ag strip rapid test kit ( Bio-Rad Laboratories , Marnes La Coquette , France ) and SD Dengue NS1 Ag test ( Standard Diagnostics , Inc . Kyonggi-do , Korea ) were used to detect the DENV NS1 antigen in serum samples . Envelope ( E ) /Membrane ( M ) -specific capture IgM and IgG ELISA were used to detect DENV-specific IgM and IgG antibodies as previously described [21] . Viral RNA was extracted from acute-phase serum samples or the culture supernatant of C6/36 cells infected with each of the isolated DENV strains using a QIAamp Viral RNA Mini Kit ( QIAGEN , Hilden , Germany ) . Primers used for amplification and sequencing of C , prM and E gene sequences of DENVs were described previously [17] [18] . The RT-PCR reaction was carried out with the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity ( Invitrogen ) . The cDNA synthesis step was performed at 55°C for 30 min; PCR at 94°C for 2 min; 40 cycles of 94°C for 15 sec , 50°C for 30 sec , and 68°C for 1 min; and prolonged elongation at 68°C for 5 min . PCR products were purified using a Qiagen QIA quick Gel Extraction Kit ( QIAGEN ) . Nucleotide sequences were determined by an automated DNA sequencing kit and an ABI Prism 3730XL DNA sequencer ( Applied Biosystems , Foster City , CA ) according to the manufacturer’s protocols . Overlapping nucleotide sequences were combined for analysis and edited with the Lasergene software package ( DNASTAR Inc , Madison , WI ) . Nucleotide sequences of the complete E gene of the DENV strains described in this study were submitted to GenBank with the following accession numbers: 334 DENV-1 strains ( KT175076-KT175078 , KT175082-KT175101 , KT175103-KT175110 , KU365900 , KY496854 , KY496855 , and MG894671-MG894970 ) , 234 DENV-2 strains ( KT175111-KT175140 , KU365901 , and MG894971-MG895173 ) , 133 DENV-3 strains ( KP176703-KP176710 , KP175715 , MG895174-MG895297 ) , and 99 DENV-4 strains ( MG895298-MG895396 ) . All the strain identifiers and their accession numbers are shown in the S1 Table . The nucleotide sequences of the complete E gene of 784 imported and 16 epidemic strains in Taiwan in combination with sequences of epidemic strains from Southeast Asian countries and various global reference strains of different genotypes available from GenBank were analyzed . In addition , sequences representing the most closely related to the epidemic strains in Taiwan obtained using BLAST were selected for phylogenetic analyses . Sequences of DENV strains were aligned , edited and analyzed using Clustal W software [22] . The phylogenetic analysis was performed using MEGA version 7 ( http://www . megasoftware . net/ ) [23] . To construct the phylogenetic trees , the maximum likelihood method using the general time reversible as a substitution model and the neighbor-joining method using the maximum composite likelihood as a substitution model were utilized . The reliability of the analysis was evaluated by a bootstrap test with 1 , 000 replications . Sequences of D2/New Guinea/NGC/1944 strain/M29095 , D2/Senegal/DAKHD10674/1970/AF231720 , D1/USA/Hawaii/1945 strain/AF425619 and D2/New Guinea/NGC/1944 strain/M29095 , were used as outgroups to root the tree of the DENV-1 , DENV-2 , DENV-3 and DENV-4 strains , respectively .
A total of 1 , 596 laboratory-confirmed imported dengue cases ( both visitors to Taiwan and local returning residents ) were identified in Taiwan during 2011–2016 . Among them , 703 cases ( 44 . 0% ) were identified by fever screening at airports ( Table 1 ) and most ( >90% ) of these cases were in their viremic stages with positive real-time RT-PCR and negative IgM and IgG results . Most cases arrived from Southeast Asian countries , with Indonesia ( 24 . 8% , 396 cases ) , the Philippines ( 19 . 2% , 306 cases ) , Malaysia ( 14 . 2% , 226 cases ) , Thailand ( 12 . 0% , 192 cases ) , and Vietnam ( 12 . 0% , 191 cases ) being the most frequent country sources of importation . Cases were also imported from other Asian countries ( 16 . 6% , 265 cases , including Myanmar , Singapore , Cambodia , India , China , Bangladesh , Maldives , Sri Lanka , Laos , Saudi Arabia , and Japan ) , the Pacific region ( 0 . 7% , 11 cases , including Palau , Papua New Guinea , Nauru , Fiji , Solomon Islands , Tuvalu , and French Polynesia ) , Australia ( 0 . 1% , 2 cases ) , Latin America ( 0 . 4% , 7 cases , including Brazil , Costa Rica , and Saint Lucia ) , and Africa ( 0 . 1% , 2 cases , one from South Africa and the other from Kenya ) . Comparing the numbers of imported dengue cases between 2003–2010 and 2011–2016 , we found there is an increasing trend of imported cases from the Philippines , Malaysia and Singapore during the study period . Fig 1 shows the country sources of importation of DENVs in Taiwan during 2011–2016 . Fig 2 shows the number of imported dengue cases in Taiwan during 2003–2016 . From the 1 , 596 imported dengue cases , 380 , 303 , 171 and 114 cases were determined to be infected with DENV-1 , DENV-2 , DENV-3 , and DENV-4 , respectively . Table 1 summarizes serotype and genotype distributions of imported DENV strains from 29 countries . The serotype distributions of DENV strains imported from the most common Southeast Asian countries each year during 2003–2016 are shown in Fig 3 . Yearly changes in serotype distribution were observed , and all four serotypes of DENV were found to circulate in each of these countries during 2011–2016 . The number of imported dengue cases from Malaysia increased sharply during 2014–2016 , and the main serotypes were DENV-1 and DENV-2 . The main serotype of imported DENV strains from Vietnam shifted from DENV-1 during 2007–2010 to DENV-2 during 2012 and 2015 and then back to DENV-1 during 2016 . The number of imported dengue cases from Singapore increased significantly during 2013–2016 , and the main serotypes in recent years have been DENV-1 and DENV-2 . A relatively high number of imported cases was observed from Myanmar in 2015 , and DENV-1 , DENV-2 and DENV-4 were the main serotypes . All 4 serotypes of DENV were found to cocirculate in Cambodia during 2015–2016 . Among the 1 , 596 imported dengue cases , 784 DENV strains were isolated from acute-phase serum samples of patients infected in 23 countries ( Table 1 ) . Phylogenetic analyses of the E gene sequences of imported DENV strains were conducted to determine the genotype and genetic relationship of these viral strains . The designations of DENV genotypes are based on the classification of A-Nuegoonpipat et al . [24] , Twiddy et al . [25] , Lanciotti et al . [26] , and Klunthong et al . [27] for the DENV-1 , DENV-2 , DENV-3 and DENV-4 strains , respectively . The genotype distributions of the DENV-1 to DENV-4 strains imported from the 8 most common Southeast Asian countries during 2003–2016 are shown in Figs 4–7 , respectively . Fig 4 shows genotype distributions of imported DENV-1 strains . The DENV-1 strains obtained from Asia and the Pacific can be classified into three genotypes ( I , II and III ) . Genotype I contains the majority of strains from Asia , while genotype II comprises a smaller set of Asian and Pacific strains . Genotype III contains viruses from a wide geographic area [24] . Genotype I of DENV-1 was the predominant genotype imported from Southeast Asian countries , including Indonesia , Malaysia , Vietnam , Thailand , Myanmar and Cambodia . Before 2006 , genotype II of imported DENV-1 was the main genotype in Indonesia; however , since 2007 , the main genotype has shifted to genotype I . Genotype II was the predominant genotype of imported DENV-1 from the Philippines . The numbers of genotype III of imported DENV-1 strains from Malaysia and Singapore increased during 2013–2014 . Fig 5 shows genotype distributions of imported DENV-2 strains . The DENV-2 strains can be classified into six genotypes . The Cosmopolitan genotype has a wide geographic distribution . The Asian genotype 1 and 2 contain viruses from Asia , and the Asian/American genotype comprises viruses from Southeast Asia and Latin America . The American genotype consists of viruses from Latin America and older isolates collected from Indian subcontinent and the Pacific , and the Sylvatic genotype contains sylvatic strains from Asia and Africa [25] . The Cosmopolitan genotype was the predominant genotype of imported DENV-2 strains from Indonesia , the Philippines , Malaysia , and Singapore , and Asian genotype 1 was the main genotype of imported DENV-2 strains from Vietnam , Thailand , Myanmar and Cambodia . Fig 6 shows genotype distributions of imported DENV-3 strains . The DENV-3 strains can be classified into four genotypes . Genotype I consists of viruses from Indonesia , Malaysia , the Philippines , the Pacific islands and Australia . Genotype II contains viruses from Southeast Asia . Genotype III has a wide geographical distribution which includes Asia , Africa and Latin America . Genotype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus isolates [26] . Genotype I was the predominant genotype of imported DENV-3 strains from Indonesia and the Philippines . In Malaysia , the number of imported genotype III strains increased between 2015 and 2016 . Genotype II was the main genotype of imported DENV from Vietnam , Myanmar and Cambodia . In Thailand , the main detected genotype has shifted from genotype II to genotype III in recent years . Fig 7 shows genotype distributions of imported DENV-4 strains . The DENV-4 strains were separated into four genotypes . Genotype I contains viruses from Asia . Genotype II consists of viruses from Asia , the Pacific and Latin America . Genotype III contains viruses from Thailand and genotype IV contains sylvatic strains from Malaysia [27] . Genotype I was the predominant genotype of imported DENV-4 from Vietnam , Thailand , Myanmar and Cambodia , whereas Genotype II was the main genotype from Indonesia and Malaysia . In the Philippines , the main detected genotype shifted from genotype I during 2003–2009 to genotype II during 2010–2016 . We first made trees for all E gene sequences of the imported and epidemic strains in Taiwan in combination with sequences of epidemic strains from Southeast Asian countries and various global reference strains of different genotypes available from GenBank . In addition , sequences representing the most closely related to the epidemic strains in Taiwan obtained using BLAST were selected for phylogenetic analyses . The results are shown in S1 Fig–S4 Fig for DENV-1 to DENV-4 , respectively . The representative E gene sequences based on country source of importation and date of sample collection , were selected to build the trees in Figs 8–11 . Except for the Philippines , most of the DENV-1 strains isolated from imported cases from Southeast Asian countries belonged to genotype I ( Table 1 and Fig 8 ) . Imported DENV-1 genotype I strains from Indonesia and Malaysia showed a high degree of genetic diversity and strains in different lineages that were co-circulating in these countries . Some of the strains from Thailand , Singapore , Myanmar , Laos and China were clustered with strains from Indonesia and Malaysia . Viral strains from Cambodia were closely related to viruses from Vietnam and Thailand , whereas viral strains from Myanmar and Sri Lanka were clustered with viruses from Thailand . Genotype II contained imported viral strains from the Philippines , Malaysia and Indonesia . Genotype III contained imported viral strains from diverse geographical regions , including Asia ( Singapore , Bangladesh , Malaysia , Maldives , Thailand , China and India ) and the Americas ( USA and Costa Rica ) . It is interesting to note that the strains for DENV-1 tend to be less geographically clustered than in the other serotypes . The DENV-2 strains isolated from imported cases during 2011–2016 fell into two genotypes , the Cosmopolitan genotype and Asian genotype 1 ( Table 1 and Fig 9 ) . The Cosmopolitan genotype strains from imported cases can be divided into three clusters . Cluster 1 contains viral strains from Malaysia , Singapore and Indonesia . Some of the imported viral strains from Thailand , Maldives , Vietnam and China also fell into this cluster . Cluster 2 contains imported viral strains from the Philippines , Tuvalu and Palau . Cluster 3 contains imported strains from India , Saudi Arabia and Kenya . A strain from Thailand and two strains from Vietnam were also found to cluster with strains from India . Asian genotype 1 contains viral strains from Thailand , Vietnam , Cambodia , Lao , and Myanmar . Imported strains from Malaysia also fell into this genotype . No Asian/American genotype and Asian genotype 2 strains were found among imported cases during 2011–2016 . The DENV-3 strains isolated from imported cases fell into three genotypes , genotype I , II and III ( Table 1 and Fig 10 ) . Genotype I can be divided into two clusters: one contains viral strains from Indonesia , Malaysia , Singapore and Solomon Islands , and the other contains viral strains from the Philippines . Genotype II contains imported strains from Vietnam , Thailand , Cambodia , and Laos . Genotype III contains viral strains from diverse geographical localities , including India , Singapore , Malaysia , Thailand , Vietnam and Cambodia . The DENV-4 strains isolated from imported cases fell into two genotypes , genotype I and II ( Table 1 and Fig 11 ) . Genotype I contains two major clusters: one cluster contains viral strains from the Philippines , and the other contains viral strains from Vietnam , Thailand , Myanmar and Cambodia . In 2016 , an imported strain from the Maldives also fell into this cluster and was closely related to virus strains from Sri Lanka . Genotype II contains imported viral strains from the Philippines , Indonesia , Malaysia and Singapore . Imported viral strains from Papua New Guinea and Brazil also belonged to this genotype . Table 2 lists the major dengue outbreaks and epidemic DENV strains circulating in Taiwan during 2011–2016 . Our results showed that a DENV-1 strain ( D1/Taiwan/700TN1109a/2011 ) caused outbreaks in southern Taiwan during 2011–2013 . This strain belongs to genotype III of DENV-1 and is closely related to viral strains from the Americas . This is the first time that an American DENV strain caused an epidemic in Taiwan . In 2011 , the other 3 epidemic strains , DENV-1 ( D1/Taiwan/111TP1110a/2011 ) , DENV-2 ( D2/Taiwan/802KH1108c/2011 ) and DENV-3 ( D3/Taiwan/811KH1109a/2011 ) , caused outbreaks in Taipei City , Kaohsiung City and Penghu County . These strains were likely introduced from Myanmar and Vietnam . In 2012 , in addition to the epidemic strain D1/Taiwan/700TN1109a/2011 , the other four DENV strains ( D1/Taiwan/234NP1209a/2012 , D2/Taiwan/802KH1208a/2012 , D3/Taiwan/832KH1210a/2012 and D4/Taiwan/811KH1207a/2012 ) caused outbreaks in New Taipei City and Kaohsiung City . These strains were likely introduced from Cambodia , Indonesia , Thailand and the Philippines . In 2013 , in addition to the epidemic strain D1/Taiwan/700TN1109a/2011 , which was transmitted to Pingtung County , the other three strains ( D2/Taiwan/920PT1306a/2013 , D2/Taiwan/900PT1308a/2013 and D3/Taiwan/932PT1305b/2013 ) caused outbreaks in southern Taiwan . These strains were likely introduced from Indonesia . During 2014–2015 , there was a large outbreak caused by a DENV-1 strain ( D1/Taiwan/806KH1405a/2014 ) in southern Taiwan; this epidemic strain belonged to genotype I and is closely related to virus strains from Indonesia . In 2014 , a DENV-2 strain ( D2/Taiwan/807KH1411a/2014 ) also caused a small outbreak in Kaohsiung City . In 2015 , a DENV-2 strain ( D2/Taiwan/704TN1505a/2015 ) caused a large outbreak in Tainan City and later in Kaohsiung City , this strain belonged to the Cosmopolitan genotype and is closely related to strains from Indonesia . From January to April 2016 , a total of 372 indigenous cases were identified . These cases represented the last wave of the 2015 outbreak in southern Taiwan . In 2016 , there were only 8 indigenous cases identified between May and December in Taiwan . A DENV-1 strain ( D1/Taiwan/114TP1611a/2016 ) caused a small outbreak in Taipei City in November 2016; this strain belonged to Genotype II and is closely related to strains from the Philippines . ( Figs 8–11 )
A total of 1 , 596 laboratory-confirmed imported dengue cases were identified in Taiwan during 2011–2016 , most of which arrived from Asian countries and other regions , including the Pacific Islands , Australia , Africa and the Americas . Among them , 92 . 7% of cases ( 1 , 480 cases ) arrived from the eight most common country sources of importation: Indonesia , the Philippines , Malaysia , Thailand , Vietnam , Myanmar , Singapore and Cambodia . An analysis of imported DENV strains from these countries showed changes in serotype distributions during the study period . As expected , all 4 serotypes of DENV were found to cocirculate in each of the most common country sources of importation during 2011–2016 . A total of 784 DENV strains , namely , 329 DENV-1 , 227 DENV-2 , 130 DENV-3 , and 98 DENV-4 strains , were isolated during 2011–2016 . Phylogenetic analyses of E gene sequences of imported DENV strains suggested that genotype I of DENV-1 and the Cosmopolitan genotype of DENV-2 were the predominant DENV strains imported from Southeast Asian countries during 2011–2016 . Notably , genotype III of the DENV-1 strain was found to newly emerge in Malaysia , Vietnam , Thailand and Singapore . In addition , genotype III of the DENV-3 strain also emerged in Malaysia , Thailand and Singapore . However , Asian genotype 2 and the Asian/American genotype of the DENV-2 strain were not found in imported cases from Southeast Asian countries in the last decade , suggesting a low prevalence of these two genotypes in this region . Previous studies have shown that DENV-1 genotype I was the predominant DENV genotype circulating in Southeast Asian countries [18 , 19 , 28–32] . In our study , the numbers of imported DENV-1 genotype I strains increased sharply in Indonesia and Malaysia and became the predominant genotype in the last decade . Phylogenetic analysis of E gene sequences of imported DENV-1 genotype I strains from Indonesia , Malaysia , Thailand and Vietnam showed a high degree of genetic diversity . Interestingly , we found that most of the DENV-1 genotype I strains did not segregate into a distinct clade in each country but that viral strains from Indonesia , Malaysia and a few strains from Singapore , Thailand , and Laos were clustered together . In addition , most of the imported DENV-1 genotype I strains from Thailand , Myanmar , Cambodia , China and Sri Lanka formed another cluster [33–35] . A DENV-1 genotype I strain ( D1/Laos/1508aTw/2015 ) isolated from a case imported from Laos in 2015 is closely related to virus strains from Malaysia . Although only a few DENV strains have been isolated from imported cases from Laos in Taiwan during 2003–2016 , genotype distribution of these imported DENV strains within each serotype is consistent with the results of a study by Castonguay-Vanier et al . [36] . Genotype III of DENV-1 strains imported from China , Vietnam , Malaysia , Singapore and Bangladesh were clustered together . The results suggest a close genetic relationship and frequent transmission of DENV-1 among Southeast Asian countries and may reflect frequent trade and travel between these countries [37 , 38] . Genotype distribution of DENV-2 in Southeast Asian countries remains largely unchanged in the last decade . However , it is interesting to note that the number of DENV-2 Cosmopolitan genotype strains imported from Vietnam and Thailand increased and that Asian genotype I strains were found in Malaysia , indicating that these two genotypes of DENV-2 have expanded into new territories . Imported DENV-2 strains from the Maldives during 2015–2016 belonged to the Cosmopolitan genotype and are closely related to virus strains from Malaysia and Singapore . A DENV-2 Cosmopolitan genotype strain ( D2/Palau/1612aTw/2016 ) isolated from a case imported from Palau is closely related to virus strains from the Philippines , Papua New Guinea and Fiji , suggesting cocirculation of these virus strains among these countries . Except for Malaysia and Thailand , the genotype distribution of DENV-3 strains imported from Southeast Asian countries remains largely unchanged . It is interesting to note that the genotype of DENV-3 strains from Malaysia shifted from genotype I to genotype III . In addition , the genotype of DENV-3 strains from Thailand shifted from genotype II to genotype III in recent years . Recent studies have shown that the DENV-3 genotype III strains are emerging in Asian countries [39–41] . In this study , the number of DENV-3 genotype III strains has been increasing in Malaysia and Thailand , and these strains were clustered together with strains from Cambodia , Vietnam and Singapore . Although a relatively low prevalence of DENV-4 strains was found in Southeast Asia [42] , there was an increase in the numbers of DENV-4 strains imported from the Philippines , Myanmar and Cambodia , during 2011–2016 . A DENV-4 genotype II strain ( D4/Papua New Guinea/1608aTw/2016 ) isolated from a case imported from Papua New Guinea in 2016 is closely related to virus strains from Indonesia . Interestingly , we found that two DENV-1 genotype I strains ( D1/Maldives/1604aTw/2016 and D1/Maldives/1608aTw/2016 ) , a DENV-2 Cosmopolitan genotype strain ( D2/Maldives/1612aTw/2016 ) and a DENV-4 genotype I strain ( D4/Maldives/1605aTw/2016 ) were cocirculating in the Maldives in 2016 , indicating multiple introductions of DENV strains into the Maldives in the recent dengue epidemic . We previously reported that the genetic relationship and genotype distribution of DENV depend largely on the geographical location [18 , 19]; however , our study demonstrates that geographical restrictions of DENV genotypes are becoming blurred . For example , DENV-1 genotype I , DENV-1 genotype III and DENV-2 Cosmopolitan genotype strains from different geographical locations or countries were clustered together , indicating the extensive introductions and continuous expansions of DENV strains between nations in Southeast Asia . Due to the small sample size of imported cases from country sources of importation in Taiwan , the potential limitations of our study include: ( 1 ) Tourists/travelers may primarily travel to specific locations within a country , and thus , viruses circulating within other regions may not be represented often in our dataset; ( 2 ) Potentially changing patterns in travel ( i . e . the number of travelers between Taiwan and country sources of importation and the purpose of travel ) may affect the numbers of imported dengue cases and the representativeness of our analyses; ( 3 ) Because of the limited epidemiological data from country sources of importation , it is unclear whether the serotype/genotype dynamics described in this study are representative of the epidemiological situation within country . Because air travel has become increasingly popular and convenient , DENV strains will be transmitted by travelers to other countries , sometimes leading to extensive outbreaks . During 2011–2016 , several dengue outbreaks occurred in Taiwan , and most epidemic strains were introduced from neighboring Southeast Asian countries , including Indonesia , the Philippines , Vietnam and Myanmar ( Table 2 ) . In accordance with our previous studies , the patterns of imported DENV strains observed among the travelers are connected to the overall patterns of the dengue dynamics in Taiwan [19 , 43] . It is worth noting that an epidemic DENV-1 strain ( D1/Taiwan/700TN1109a/2011 ) , which caused the dengue outbreak in southern Taiwan for three consecutive years ( 2011–2013 ) , is closely related to virus strains from Central America . This is the first time that a dengue outbreak in Taiwan was caused by an American strain and suggests not only that imported DENV strains from neighboring Southeast Asian countries cause local outbreaks but also that viral strains introduced from the Americas or other continents may establish transmission chains within Taiwan . In this study , we conducted molecular epidemiological analyses to monitor DENV serotype and genotype distributions and dynamic movements in Southeast Asian countries . The DENV strains isolated from imported dengue cases and the availability of a DENV genome sequence database can provide essential information on the global expansion and genetic evolution of DENV , which is useful for disease surveillance , laboratory diagnoses , pathogenesis investigation and vaccine development . Our results also indicate that it is important to reinforce active surveillance and travel and border health measures for dengue prevention and control in Taiwan . | Dengue is the most prevalent mosquito-borne viral disease in the world . The expansion of dengue viruses to different parts of the world has been accelerated by the increase in worldwide travel and trade . In this study , we present the results of a laboratory-based dengue surveillance in Taiwan during 2011–2016 . A total of 1 , 596 laboratory-confirmed imported dengue cases were identified . The travelers were infected in 29 countries in Southeast Asia , the Indian subcontinent , the Pacific region , Latin America , Australia and Africa . Phylogenetic analyses of the envelope gene sequences of 784 imported dengue virus isolates suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant serotype/genotype DENV strains circulating in Southeast Asia . Our results also showed that geographical restrictions of some of the DENV genotypes are becoming blurred , indicating the extensive introductions and continuous expansions of DENV strains between countries in Southeast Asia . In addition , we demonstrated dengue outbreaks in Taiwan caused by viruses imported from Asia and the Americas . The DENV envelope gene sequences from this study will contribute to a better understanding of the genetic evolution , dynamic transmission and global expansion of dengue viruses . | [
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] | 2018 | Molecular characterization and phylogenetic analysis of dengue viruses imported into Taiwan during 2011-2016 |
The size of a cell sets the scale for all biochemical processes within it , thereby affecting cellular fitness and survival . Hence , cell size needs to be kept within certain limits and relatively constant over multiple generations . However , how cells measure their size and use this information to regulate growth and division remains controversial . Here , we present two mechanistic mathematical models of the budding yeast ( S . cerevisiae ) cell cycle to investigate competing hypotheses on size control: inhibitor dilution and titration of nuclear sites . Our results suggest that an inhibitor-dilution mechanism , in which cell growth dilutes the transcriptional inhibitor Whi5 against the constant activator Cln3 , can facilitate size homeostasis . This is achieved by utilising a positive feedback loop to establish a fixed size threshold for the Start transition , which efficiently couples cell growth to cell cycle progression . Yet , we show that inhibitor dilution cannot reproduce the size of mutants that alter the cell’s overall ploidy and WHI5 gene copy number . By contrast , size control through titration of Cln3 against a constant number of genomic binding sites for the transcription factor SBF recapitulates both size homeostasis and the size of these mutant strains . Moreover , this model produces an imperfect ‘sizer’ behaviour in G1 and a ‘timer’ in S/G2/M , which combine to yield an ‘adder’ over the whole cell cycle; an observation recently made in experiments . Hence , our model connects these phenomenological data with the molecular details of the cell cycle , providing a systems-level perspective of budding yeast size control .
Balanced growth of proliferating cells requires some coordination between the increasing size of a growing cell and its probability of undergoing DNA synthesis and division . In particular , the average time between two successive cell divisions must allow for a doubling in cell mass ( or volume , which we will use interchangeably in the following ) . Any systematic deviation from this balance would lead to progressive changes in size over consecutive generations , eventually leading to the breakdown of biochemical processes . However , despite mounting evidence for active size control in various cell types and across different organisms [1] , if and how cells measure their size and relay this information to the cell cycle remains controversial [2] . An elegant way to coordinate cell division and growth is to restrict passage through a certain cell cycle stage to cells that are larger than a particular target size [1] . Such ‘size checkpoints’ have been proposed to underlie size control at the Start transition in budding yeast [3–5] , and at the G2/M transition in fission yeast [6–8] and slime mould plasmodia [9–11] . The critical size required to pass these transitions depends , among other things , on the ploidy of the cell and its nutritional status [2] . To establish a size checkpoint , cells need to generate a size-dependent biochemical signal . Yet , most cellular macromolecules increase in abundance proportionally to cell volume , so that their concentration remains constant and the reactions they are involved in are independent of size [12] . Several proteins that defy this general rule have been indicated in size control . The mitotic activator Cdc25 , for instance , increases in concentration with size in fission yeast [8] , while Whi5 , an inhibitor of Start in budding yeast , is diluted by cell growth [13] . This suggest a general mechanism , in which size control emerges from the interplay between size-dependent and size-independent cell cycle regulators . Here , we study this intriguing possibility , focusing on the budding yeast cell cycle . The budding yeast Saccharomyces cerevisiae divides asymmetrically , with size control mainly operating in the new-born daughter cell when it commits to enter the cell cycle anew at the Start transition [3–5] . Passage through Start is driven by activation of the transcription factor SBF [14] . In early G1-phase , before Start , SBF is kept inactive by its stoichiometric inhibitor Whi5 [15 , 16] . To enter the cell cycle , the cyclin-dependent kinase Cdk1 ( encoded by the CDC28 gene ) in conjunction with its regulatory binding partner Cln3 phosphorylates Whi5 , which partially liberates SBF from inhibition and induces the synthesis of other G1 cyclins ( Cln1 and Cln2 ) . Cln1/2:Cdk1 complexes then accelerate the phosphorylation of Whi5 and activation of SBF , thereby promoting the Start transition [15–17] . Recent experiments show that during G1 the concentration of Cln3 , the activator of Start , is constant , while the concentration of Whi5 decreases , suggesting that an inhibitor-dilution mechanism facilitates size control [13] . However , previous theoretical considerations and experimental data suggested a different mechanism based on the titration of an activator that increases in molecule number during growth–as would be the case if its concentration is kept constant–against a fixed number of nuclear sites [18–20] . To test these hypotheses , we developed a mechanistic mathematical model of the budding yeast cell cycle . At its core , the model comprises a simple description of gene expression in which both size-dependent and size-independent synthesis of proteins emerge seamlessly from the assumption of differential affinity of genes for ‘transcription machinery’ . This allows size-dependent proteins to maintain a fixed concentration during growth without the need for complex , gene-specific regulation and for size-independent proteins to maintain a fixed number of molecules per cell . Together , such size-dependent and -independent proteins can generate size-dependent biochemical signals for progression through the cell cycle . Using this model , we show that an inhibitor-dilution mechanism can facilitate size homeostasis and correctly account for changes in protein synthesis observed in experiments that perturb the number of gene copies of cell cycle regulators as well as the overall ploidy of the cell . However , the model fails to reproduce changes in cell size seen in some of these mutants . Intriguingly , a combination of inhibitor dilution and the titration of an activator against genomic sites correctly recapitulates these changes in cell size . Such a model also produces cell size patterns consistent with a ‘sizer’ mechanism in G1 and a ‘timer’ period comprising S , G2 and M-phase , which combine to yield an ‘adder-type’ behaviour over the entire cell cycle; an observation recently made experimentally [21] . Hence , our model unites various experimental findings that were previously thought incompatible .
Experimental evidence suggests that size control emerges from the interplay of regulatory proteins whose synthesis rates depend on cell size and their size-independent counterparts [8 , 13] . To simulate the expression of such proteins we propose a simple mathematical model based on the differential binding of transcription machinery ( TM ) to genes ( Fig 1A ) . We model cell growth by assuming that components of the TM are themselves synthesised from size-dependent genes , which makes the production of TM autocatalytic , and that products of size-dependent genes control the increase in cell volume . These simple assumptions result in an exponential rise in both the amount of TM and cell size over time ( Fig 1B ) , as is characteristic for budding yeast both in single cells and at the population level [2 , 21] . We note that the accumulation of TM in our simulations is compatible with experimental data on RNA polymerase II , which has been implicated in global transcriptional control [22] . Moreover , cell growth in the model depends on proteins that are themselves made by TM , which naturally leads to a direct proportionality between cell volume and transcriptional capacity . More precisely , as cells produce more and more TM their volume growth rate increases by the same extent , such that the number of TM molecules per unit cell volume remains constant . The fact that larger cells contain more TM translates into an increased occupation of size-dependent genes by TM , while size-independent genes are already fully occupied in small cells due to their high affinity for TM ( Fig 1C ) . Consequently , the transcriptional output from size-dependent genes increases with cell size , allowing their proteins to maintain a constant concentration during exponential cell growth ( Fig 1D ) . By contrast , expression from size-independent genes remains almost constant , such that their proteins are diluted by cell growth . Note that protein transcription is a highly complex , non-equilibrium process involving the binding of transcription factors , chromatin remodelling and multiple layers of regulation [23 , 24] , e . g . cell cycle and nutrient-dependent control . We propose that the basic size-related regulation shown here operates alongside these other mechanisms to compensate for changes in cell size . Furthermore , the two protein classes in Fig 1A represent extremes on either end of the binding-affinity spectrum . Intermediate expression patterns , including proteins that switch from being size-dependent to size-independent during cell growth , can arise in between these extremes ( S1 Fig ) . Our gene expression model predicts that the two principal gene types react differently to a ploidy increase , i . e . , a doubling of their copy number and of the rest of the genome . In particular , size-dependent genes compensate for ploidy by splitting TM between the two gene copies and the genome , whereas their size-independent counterparts compete efficiently for TM with other genes and increase in expression ( Fig 1E ) . However , an additional gene copy in the absence of a ploidy increase leads to a higher expression of either gene type ( Fig 1F ) . Hence , protein synthesis depends on the copy-number-to-ploidy ratio for size-dependent genes and strictly on the gene copy number for size-independent genes . In summary , our model uses a simple mechanism to explain why size-independent proteins are diluted by cell growth , whereas size-dependent proteins keep a constant concentration , without the need for complex , gene-specific regulation . Next , we asked whether the differential expression of cell cycle regulators according to the above model would allow budding yeast cells to control their size . In budding yeast , size control acts at Start [3–5] , where cells commit to cell cycle entry . Hence , we developed a cell cycle model centred on this transition ( Fig 2A ) . In this model , passage through Start is facilitated by the activation of SBF , which is opposed by the stoichiometric inhibitor Whi5 . Through the phosphorylation of Whi5 , Cln3 liberates SBF from inhibition , thus driving cell cycle entry ( S2A Fig ) . Based on experimental observations [13] , we assume that Whi5 is a size-independent gene , while all other proteins in our model are size-dependent . Consequently , cell growth in G1 dilutes the inhibitor of Start , Whi5 , while the activator Cln3 is maintained at constant concentration ( Fig 2B ) , as has been observed experimentally [13] . Our model shows that this inhibitor-dilution mechanism can establish a size threshold for Start , where SBF is relieved from Whi5 inhibition only after sufficient growth has occurred ( Fig 2C ) . This transition is rapid and switch-like because of positive feedback via Cln1 and Cln2 , which are expressed in response to SBF activation and further phosphorylate Whi5 [25 , 26] . The positive feedback loop creates a bistable switch , which implements the threshold response to graded changes in Whi5 concentration caused by cell volume growth , providing a sensitive size-sensing mechanism . After Start has been passed , growth is restricted to the bud [4] , and it continues until the end of the cycle , when the degradation of Clb1 and Clb2 initiates the separation of mother and daughter cell ( Fig 2D ) . Intriguingly , our model readily shows size homeostasis over multiple generations ( Fig 2D , lower panel ) . In particular , daughter cells , which we follow in our simulations because they show strong size control , reach the same size as their mothers , suggesting that Whi5 dilution can indeed couple cell division to cell growth . In order to actively regulate cell size , i . e . , to reduce size differences between cells , the inhibitor-dilution model requires that larger than average cells are born with lower than average Whi5 concentration so that they progress faster through G1 , while smaller than average cells have higher Whi5 concentration , which gives them more time to grow . It has been proposed that this negative correlation between cell size at birth and Whi5 concentration results from the synthesis of a fixed amount of Whi5 during a period of fixed duration , which encompasses S- , G2- and M-phases in budding yeast [13] . By design our model accounts for this synthesis pattern , restricting Whi5 synthesis to the post-Start period ( Fig 2E ) . We find that new-born cells do indeed exhibit a size-dependent Whi5 concentration ( Fig 2F ) . This allows for the adjustment of G1 duration to a cell’s birth size ( Fig 2G ) . In summary , our model demonstrates that size-independent synthesis of Whi5 and its dilution during G1 can allow cells to maintain their size over multiple generations by creating a cell-size threshold for Start . Furthermore , the synthesis of a fixed amount of Whi5 per cell cycle can adjust for size differences by tuning G1 duration . To further explore the model’s ability to reproduce size control , we compared it to experiments that vary the copy number of CLN3 and WHI5 , as well as the cell’s overall ploidy [13] . These data were originally used to prove that Whi5’s synthesis rate is independent of cell size , while Cln3’s synthesis rate increases in larger cells ( [13] and Fig 3A ) . These experiments also highlight that Whi5 synthesis is largely independent of ploidy , with only a slight decrease seen between haploid and diploid cells that harbour the same number of WHI5 copies ( Fig 3A , left panel ) . Yet , when the copy number of its gene is doubled , the Whi5 synthesis rate changes proportionally . Cln3 expression , in contrast , does change with ploidy , i . e . , the slope of the synthesis rate decreases in diploid cells with one copy of CLN3 compared to their haploid counterparts ( Fig 3A , right panel ) . However , an increase in CLN3 copy number does not affect the Cln3 synthesis rate as long as the ratio between copy number and ploidy is kept constant . Crucially , diploid cells ( with two copies each of WHI5 and CLN3 ) were shown to be roughly twice the size of haploid cells ( with one copy each of WHI5 and CLN3 ) . We simulated these copy-number mutants using the inhibitor-dilution model , which includes the features of gene expression shown in Fig 1 . The resulting simulations correctly predict the changes in protein synthesis rates for both Whi5 and Cln3 ( Fig 3B ) . In particular , they recapitulate the copy-number dependence of Whi5 synthesis rate and the copy-number-to-ploidy dependence of Cln3 synthesis rate . The model also correctly predicts the two-fold size increase between haploid and diploid cells . However , our simulations fail to reproduce the size increase observed between haploid and diploid cells with the same number of WHI5 copies ( Fig 3C ) . More precisely , since protein synthesis rates for both Whi5 and Cln3 are similar in haploid and diploid cells with one WHI5 copy ( S3A Fig ) , the model predicts a similar size threshold for Start ( Fig 3D ) . In fact , considering the slight decrease in Whi5 synthesis rate observed in experiments [13] , diploid cells with one WHI5 should show a slight decrease in size compared to haploid cells according to our model . Reference [13] attributes the observed increase in cell size between haploid and diploid cells ( with one or two copies of WHI5 ) to a delay in S/G2/M progression for diploid cells . Testing this hypothesis , we find that it only partially accounts for the observed size changes ( S3B Fig ) . In particular , diploid cells with one WHI5 are predicted to be smaller than haploid cells with two WHI5 , suggesting a larger influence of Whi5 synthesis rate than ploidy ( S3C Fig ) . However , in experiments the opposite is observed ( [13] and Fig 3A ) . Moreover , a delay in S/G2/M progression together with the observed increase in Whi5 synthesis rate would lead to a more than two-fold difference between haploid and diploid cells ( S3C Fig ) , in contradiction to experimental data [13] . Taken together , the inhibitor-dilution model thus correctly captures protein synthesis rates in copy-number and ploidy mutants but fails to reproduce the observed size increase for some diploid cells . Previous theoretical and experimental studies attributed the effects of ploidy on cell size to an alternative control mechanism relying on the titration of a protein with constant concentration against a fixed number of nuclear sites [9–11 , 18–20] . In particular , it has been suggested that Cln3 is titrated against SBF bindings sites on the genome [20] . Based on these suggestions , we augmented the inhibitor-dilution model with a titration mechanism to test whether these two concepts can be brought into unison ( Fig 4A ) . In the pure inhibitor-dilution model , SBF , Whi5 and Cln3 interact in a strictly concentration-based manner ( S2A Fig ) . By contrast , the titration model assumes that SBF occupies a fixed number of sites on the genome . In early G1 ( i . e . , in small daughter cells ) , these sites are filled with Whi5-inhibited SBF complexes to which Cln3 can bind tightly in a stoichiometric fashion . Once bound , Cln3 slowly hypo-phosphorylates Whi5 and dissociates in the process . However , it can rapidly rebind to unphosphorylated SBF:Whi5 ( S2B Fig ) . As the cell grows larger , the number of Cln3 molecules per cell increases ( Cln3 is a size-dependent protein , whose concentration is maintained constant in G1 ) ( Fig 4B ) . This leads to a gradual accumulation of Cln3:Cdk1 heterodimers on Promoter:SBF:Whi5 complexes until all sites are filled , at which point free Cln3:Cdk1 kinase complexes emerge in the nucleus . Free Cln3:Cdk1 then promotes rapid hyper-phosphorylation of SBF-bound and free Whi5 , facilitating the Start transition ( Fig 4B ) . Similar to the inhibitor-dilution model , the titration model readily yields size homeostasis in consecutive generations ( Fig 4C ) by coupling the passage through Start to cell size ( Fig 4D ) . When simulating changes in gene copy number , we observe that , similar to inhibitor dilution , the titration model correctly predicts protein synthesis rates ( Fig 4E ) . However , the titration mechanism also captures the increase in size between haploid and diploid cells with the same number of WHI5 copies ( Fig 4E ) . In particular , diploid cells harbour twice the number of SBF binding sites , which require a higher amount of Cln3 , and therefore a larger cell size , to be filled ( S4A Fig ) . Note that our model overestimates the size of diploid cells with one copy of CLN3 ( Fig 4F ) . The cause for this discrepancy is that the absence of a second CLN3 copy in diploid cells only reduces Cln3 synthesis rate by ~15% ( compare diploid cells with 1xCLN3 and 2xCLN3 in Fig 3A , right panel ) , whereas the model predicts a reduction by ~50% ( Fig 4E , right panel ) . After accounting for this , cell size predictions are much more accurate ( S4B and S4C Fig ) . It is not yet clear why a single CLN3 can partially compensate for the second copy’s expression rate in diploid cells . Further experimental evidence for a titration mechanism comes from an observed increase in cell size upon transformation of otherwise wild-type cells with a high copy number plasmid containing perfect SBF binding sites [20] . These decoy sites were proposed to change the size threshold for Start by binding Cln3 such that an increased number of Cln3 molecules , and hence a larger cell size , is required to initiate the transition . Simulating this setup , our model does indeed show such an increase in size ( S4D Fig ) , providing further support for the existence a titration mechanism . In summary , a combination of Whi5 dilution and Cln3 titration against SBF binding sites is not only able to capture protein synthesis rates but also the size of WHI5- and CLN3-mutant haploid and diploid cells and of cells harbouring an increased number of SBF binding sites . Historically , three different strategies have been proposed to maintain cell size homeostasis: the sizer , where a cell cycle transition is triggered once the cells reaches a critical target size; the timer , whereby the cell cycle takes a constant amount of time; and the adder , postulating that cells add a constant volume each generation [27 , 28] . Each of these concepts may apply to the complete cell cycle or only to a certain cell cycle phase , and all of them generate characteristic size patterns that can be probed experimentally ( Fig 5A ) . An ideal sizer mechanism suggests that the final volume at the end of the sizer period is independent of the initial volume , such that the added volume shows a linear slope of minus one , i . e . , small cells need to grow more to reach the critical size . By contrast , exponentially growing cells that employ a perfect timer show a slope of plus one in the added volume as small cells grow less during the same time increment . Note that a slope of exactly one is only observed if cells double their mass within the phase that uses a timer , e . g . if the timer is employed over the whole cell cycle of a symmetrically dividing cell . Finally , an adder leads to a slope of zero since the added volume is assumed to be constant . We wanted to understand how these concepts connect to the mechanistic model of cell cycle control presented above . Simulations of our titration model reveal that G1-phase behaves like an imperfect sizer with smaller cells adding more volume during G1 ( slope of -0 . 64; Fig 5B , right panel ) and cell size at S-phase entry showing a slight positive correlation with birth size ( Fig 5B , left panel ) . S/G2/M-phases , by contrast , exhibit a timer ( see also S5A Fig ) . The combination of a mechanistic sizer and a mechanistic timer yields a phenomenological adder with the added volume being virtually independent of cell size at birth ( R = -0 . 02; Fig 5B , right panel ) . However , the added volume is not directly sensed by the system in any way . Instead the negative slope of the sizer compensates for the positive slope of the timer . The results above raise the question as to why cells employ two seemingly different strategies in G1 and S/G2/M-phases , a sizer and a timer , respectively . Presumably , S , G2 and M-phase are completed fast , with a size-independent timing , to allow the mother cell to start the next budding event , while size control is relegated to the daughter cell’s G1 phase . In addition , a timer period of constant length in combination with a size-independent Whi5 synthesis allows the cell to produce a constant , size-independent amount of Whi5 per cell cycle ( Figs 5C and 2E ) . This constant Whi5 amount is part of the mechanism that tunes G1 length with respect to birth size . Hence , the S/G2/M timer helps to set up the G1 sizer . We also note that our simulations show an imperfect sizer with a slight positive correlation between the cell volume at Start and the birth volume ( Fig 5B , left panel ) , as has been found experimentally [21 , 27] . Whereas an ideal sizer requires the size threshold for Start to be independent of birth size , we find that cells which are larger at birth progress through Start at a slightly larger size ( Fig 5B and 5D ) . According to our model , the main reason for this threshold change is the distribution of Whi5 molecules at cell division . In particular , larger cells are born with slightly higher amounts of Whi5 ( Fig 5E ) , since some of the Whi5-containing complexes are distributed according to the volume ratio of mother and daughter cell ( Fig 5F ) . It is primarily by this mechanism that birth size affects the size threshold for Start in our model , as shown in S5B Fig , where we manually set the Whi5 amount at birth to a constant value ( birth size-independent ) and find that the model behaves as an almost ideal G1 sizer . In summary , our model shows that size control in budding yeast uses an S/G2/M timer that helps to produce a constant amount of Whi5 per cell cycle and to facilitate a sizer in the G1 phase of daughter cells . Both mechanism combine to yield a phenomenological adder over the whole cell cycle . However , the size-dependent distribution of Whi5 at cell division can cause an imperfect adjustment to size differences at birth .
Balanced growth , achieved by coupling cell division to the increase in cell mass , is crucial to cell survival as progressive changes in size over generations would eventually lead to a breakdown of biochemical processes . In this study , we developed a mechanistic mathematical model for size control in budding yeast based on the differential expression of cell cycle regulators in growing cells . We show that the interplay of size-dependent and size-independent synthesis of these regulators can establish a size threshold at Start and facilitate size homeostasis . It has long been recognised that the amounts of most proteins in a cell increase with cell size [29 , 30] , such that protein concentrations remain constant and reaction rates are unaffected by growth [12] . This has also been observed for the majority of cellular mRNAs , suggesting that adaptation to volume growth occurs at the transcriptional level [22 , 29 , 31–33] . Based on these observations , we propose a general mathematical model for gene expression in growing cells which assumes that a limiting component of the transcription machinery , which we named TM and that may correspond to an RNA polymerase or factors influencing chromatin accessibility [18] , is produced in an autocatalytic manner by transcribing its own mRNA . Under conditions where nutrients and precursors are not limiting , this leads to an exponential increase in TM . If we assume that the increase in cell volume depends on proteins that are themselves transcribed by TM , the exponential rise in TM directly translates into an exponential increase in cell volume and it naturally leads to a direct proportionality between both , i . e . , protein synthesis rates per unit volume remains constant . This scaling is an emergent property of the system and does not require complex regulation or a dedicated mechanism that measures size and tunes transcriptional capacity accordingly . In very large cells , genes become saturated , at which point transcription rates remain constant and cell growth transitions into a phase of linear increase . These features of the model are consistent with a large body of experimental literature showing exponential growth of cell volume and transcription for small cells which plateaus when cells exceed a certain size [12 , 21 , 22 , 27 , 34] . Given this model of gene expression , two different types of genes emerge in our simulations based on their affinity to TM . Genes that bind TM with high affinity are saturated early , in small cells , and thus show size-independent protein synthesis . Consequently , they give rise to size-independent proteins , whose amount is constant , leading to a decreasing concentration in growing cells . Whi5 is an example of such a protein [13] . Due to their high affinity , size-independent genes compete efficiently for TM and an increase in their copy number , due to gene or genome duplication , directly translates into an increased synthesis and concentration . We hence propose that , in the context of size control , size-independent genes can act as gene-copy-number sensors . Beyond size regulation , the genes might encode proteins that need to be present in a fixed proportion to the genome content , e . g . , transcription factors or histones . By contrast , size-dependent genes bind TM with lower affinity , such that their occupation by TM increases proportional to cell volume . Through this mechanism , their proteins can maintain a constant concentration until the gene is saturated . We propose that the majority of proteins uses this type of control , Cln3 being a concrete example [13] . Due to their characteristics , size-dependent genes share TM among themselves , such that an overall ploidy increase does not result in an increase in protein concentration . Size-dependent genes can hence act as sensors for the copy-number-to-ploidy ratio , the gene dosage . Variations of the affinity constants between the two extremes may lead to intermediate expression patterns , including genes that can switch from size-dependent to size-independent expression within a given range of cell sizes . We propose that this simple mechanism of gene expression operates alongside other forms of transcription control , which involves non-equilibrium processes and stochastic phenomena [23 , 24] , to compensate for cell growth . By incorporating the gene expression model into a model of the yeast cell cycle , we show that size-independent synthesis of the inhibitor Whi5 and size-dependent synthesis of the activator Cln3 , a mechanism termed inhibitor dilution [13] , can indeed establish size control at Start . It is important to note that , because Whi5 is a stoichiometric inhibitor of SBF without catalytic activity [15 , 16] , we have assumed in our inhibitor-dilution mechanism that Whi5 and SBF form a tightly bound complex . In addition , we assumed that phosphorylation of Whi5 by Cln3 breaks up the complex and liberates SBF . Considering that SBF maintains a constant concentration , as has been shown experimentally for one of its subunits [13] , Whi5 is therefore in fact countered by two size-dependent activators , Cln3 and SBF . Given these molecular interactions , our results suggest that , in the inhibitor-dilution paradigm , the rising number of SBF molecules in a growing cell eventually overcomes inhibition by exceeding the constant number of Whi5 molecules ( see S1 Text for details ) . Cln3 merely sets the threshold at which SBF activation occurs by keeping a fraction of Whi5 molecules in a phosphorylated ( inactive ) state . Because this fraction does not change appreciably with cell size , Cln3 is not directly involved in creating the size-dependent signal that facilitates Start in our version of the inhibitor-dilution model . Hence , between the inhibitor-dilution model and the titration-of-nuclear-sites mechanism there exists an intriguing symmetry , in which Whi5 and nuclear sites are very much alike . Both are constant in number and proportional to DNA content and both titrate away an activator . We also show that the gradual increase in SBF activity in response to cell volume growth that is caused by Whi5 dilution is converted into an all-or-nothing decision by a bistable switch located at Start . This switch is created by a positive feedback loop on SBF activity and it establishes a strict size threshold of Start . Hence , positive feedback and bistability are used to implement a size checkpoint in G1 . While inhibitor dilution is able to maintain size homeostasis and reproduce the size increase seen in diploid cells , it fails to explain why an increase in ploidy at a constant number of WHI5 copies leads to larger cells . Such a change does not alter the expression of Whi5 and Cln3 and hence should not affect cell size at Start . Even the delay in S/G2/M progression observed experimentally [13] is unable to reproduce these size changes in our model , suggesting that ploidy influences cell size beyond an effect through Whi5 and Cln3 expression and S/G2/M duration . Such an effect could be mediated by an as-yet-unknown inhibitor of Start which is produced in a size-independent manner similar to Whi5 . In this case , an increased expression of this inhibitor in diploid cells , due to a higher copy number of its gene , would cause the observed size increase . However , a much more appealing hypothesis is that the genome itself acts as an inhibitor of Start . In particular , the binding of SBF to a limited number of genomic sites , which was proposed based on experiments [20] , essentially converts SBF into a variable that does not change in number with cell size , as only the SBF that is bound to the genome would affect the Start transition . Since the number of Whi5 molecules is constant as well , the amount of Whi5:SBF complexes , assuming tight binding between both , is not changing with cell size . However , the number of Cln3 molecules increases , such that Cln3 titrates against Whi5:SBF complexes on the genome . At a particular threshold size , Cln3 exceeds the number of Whi5:SBF complexes , leading to a sharp increase in free Cln3 that can trigger Start . Positive feedback is again used to convert this increase into an all-or-none decision . In this context , a diploid cell is larger because it contains twice the number of SBF binding sites , requiring more Cln3 molecules to trigger Start . Hence , the genome itself , through providing SBF binding sites that titrate Cln3 , acts as a Start inhibitor , using a form of distributed control ( binding sites distributed throughout the genome ) instead of a single gene product such as Whi5 . We show that this Cln3 titration model is consistent with WHI5 and CLN3 mutant phenotypes and with experiments in which additional SBF binding sites are expressed and consequently cell size at Start is increased [20] . Also note that size-independent synthesis of Whi5 in the titration model is beneficial because increasing Whi5 production in large cells would impair their progress through Start , thereby compromising size control . Moreover , the proportional increase in Whi5 synthesis with gene copy number allows for a constant ratio between Whi5 and SBF molecules on binding sites in cells with increasing ploidy , providing an intriguing hypothesis for why Whi5 is synthesised in a size-independent manner . A recent study of cell cycle commitment in buddy yeast called into question the dilution of Whi5 , arguing instead that a size-dependent increase in the concentrations of G1/S transcription factors helps to set the size threshold for Start [35] . In S6 Fig , we show that such a model is indeed able to achieve size homeostasis but is incompatible with data on Whi5 synthesis rates and the size of some mutant strains ( see S2 Text for details ) . Hence , our model in combination with careful measurements of not only protein concentrations but also protein synthesis rates and cell sizes in mutants could help to resolve such discrepancies . In recent years , studies of bacterial size control have argued for an ‘adder-type’ mechanism , whereby cells add a constant increment of cell mass per cycle [36 , 37] . A similar type of behaviour was found between two budding events in S . cerevisiae [34] . Yet , it remained unclear whether cells actively sense the added mass and use this information to regulate cell cycle events , a scenario later referred to as a mechanistic adder [21] . From our simulations , we indeed observe the presence of an adder over the whole cell cycle , with no correlation between the added cell mass and the volume at birth . However , this behaviour does not result from a direct mechanism , but rather from a combination of a mechanistic sizer in G1 and a mechanistic timer in S/G2/M , which is in excellent agreement with a recent study arguing that the adder phenomenon emerges from independent pre- and post-Start controls [21] . Similar to these and other experiments [5 , 21] , our titration model shows an inverse proportionality between G1 length and birth size , and an imperfect sizer mechanism . We propose that adaptation is imperfect because of a volume-dependent distribution of Whi5 . An ideal sizer , where Start size is independent of birth size , requires that each daughter cell receives a constant amount of Whi5 . However , Whi5 complexes that diffuse freely in the nucleus or cytoplasm would be distributed based on the size of the daughter cell , with large cells receiving a larger increment of Whi5 that keeps them in G1 for longer . In our model , this results in a weak birth-size dependence of the Start threshold and imperfect size control . This might be one reason why cells do not rely on a pure inhibitor-dilution mechanism , which would exacerbate the influence of Whi5 distribution , but instead use a combination of Whi5 dilution and Cln3 titration . In addition , Cln3 is a highly unstable protein [38] , and thus provides a snapshot of the current transcriptional capacity and volume of a cell , while Whi5 was produced in the previous cycle , inevitably introducing some form of memory of past growth conditions . In summary , our study provides a mechanistic model of gene expression and cell cycle regulation in budding yeast that readily shows size homeostasis . Since the control network of Start in budding yeast is structurally similar to restriction point control in mammalian cells , similar mechanisms could be at work during mammalian size control .
Our models for budding yeast size control comprise sets of ordinary differential equations ( ODEs ) . These ODEs describe the dynamics of genes and proteins in terms of their molecule number rather than concentration , which is used by most biochemical models that do not account for cell volume growth . In the following , we explain each of the two models ( inhibitor dilution and titration of nuclear sites ) in detail , starting with a generic description of gene expression that underlies both models . Both size-control models were prepared in the Systems Biology Toolbox 2 [44] for MatLab ( version 9 . 1 . 0 R2016b ) and simulated with the CVODE routine [45] . Bifurcation diagrams were calculated using the freely available software XPP-Aut [46] . Models are provided as S1–S3 Files in the Supplement and different versions are available at www . cellcycle . org . uk/publication . Model files were also deposited in BioModels [47] and assigned the identifiers MODEL1803220001 and MODEL1803220002 . Parameter values and initial conditions are listed in S1–S4 Tables and S5 Table shows the changes required to simulate ploidy mutants in Figs 3 , 4 , S3 and S4 . In order to simulate cells of different sizes ( e . g . in Fig 2F and 2G ) , we varied the specific growth rate , with higher growth rates producing larger cells . In particular , the specific growth rate ( μ ) in our model follows from Eqs 9 and 10 as μ=1Vtd ( Vt ) dt=kVoSy∙GDTMVt∙GCNGDt . Since GDt ≫ GIt and almost all of the TM is bound to genes for the cell sizes we study here , the amount of transcriptionally active size-dependent genes can be approximated by the total number of TM ( GDTM ≈ TMt ) . Moreover , we can calculate the transcriptional capacity per unit cell volume as TMtVt=kTmSykVoSy . Taken together this gives μ≈kVoSy∙kTmSykVoSy∙GCNGDt , demonstrating that by changing both kVoSy and kTmSy by the same factor , we can change the specific growth rate , while still maintaining the same transcriptional capacity per unit cell volume and thus similar protein expression . Accordingly , for simulations in Fig 2F and 2G , kVoSy and kTmSy were multiplied by a factor f ∈ [0 . 75 , 1 . 25] . For simulations in Figs 5 and S5 , we followed a single cell lineage over a large number of divisions to correlate cell sizes at different cell cycle stages . To obtain different cell sizes , we again varied the growth rate as described above , assuming that it changes at cell division . In particular , we assumed that the specific growth rate in the next cycle ( μn+1 ) is partly inherited from the mother cell’s growth rate ( μn ) and partly influenced by stochasticity , e . g . , by the random distribution of molecules at cell division , using μn+1=0 . 5∙μn+0 . 5∙μ¯∙ ( 1+N ( 0 , 0 . 04 ) ) , where μ¯ is the average growth rate and N ( 0 , σ ) a normally distributed random variable with mean 0 and variance σ . | Proliferating cells need to coordinate the initiation of genome replication and cell division with cell growth . In particular , the average time between two division events must precisely allow for a doubling in cell volume . Any systematic deviation from this balance would lead to progressive changes in cell size over consecutive generations and to a breakdown of biochemical processes . Here , we study two molecular mechanisms by which budding yeast cells might achieve this coordination . Through mathematical modelling , we show that the dilution of an inhibitor of cell cycle progression by cell growth can facilitate size homeostasis . But this mechanism fails to reproduce the size of mutant cells in which parts of the control machinery have been altered . By contrast , the titration of an activator against a constant number of genomic sites recapitulates these data and achieves size homeostasis . Since the control network of cell cycle progression in budding yeast is structurally similar to mammalian cells , our model could indicate a common mechanism for size control . | [
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] | 2018 | Dilution and titration of cell-cycle regulators may control cell size in budding yeast |
Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts . These antibodies can be measured by enzyme-linked immuno sorbent assays ( ELISAs ) using salivary gland homogenates ( SGHs ) as the source of antigens and serve as a markers for exposure to biting sand flies . Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins . In East Africa , Phlebotomus orientalis is the main vector of Leishmania donovani , a trypanosomatid parasite causing visceral leishmaniasis . We tested recombinant salivary proteins derived from Ph . orientalis saliva to study exposure of domestic animals to this sand fly species . Antigenic salivary proteins from Ph . orientalis were identified by immunoblot and mass spectrometry . Recombinant apyrase rPorSP15 , yellow-related protein rPorSP24 , ParSP25-like protein rPorSP65 , D7-related protein rPorSP67 , and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L . donovani foci in Ethiopia where Ph . orientalis is present . Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen , displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH . This recombinant protein was the most suitable one for testing sera of dogs , sheep , and goats . In addition , a different antigen , rPorSP65 was found efficacious for testing canine sera . Recombinant salivary proteins of Ph . orientalis , specifically rPorSP24 , were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph . orientalis , the vector of L . donovani . The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph . orientalis antibody-mediated reactions also in other host species .
Phlebotomine sand flies are the vectors of Leishmania parasites causing leishmaniasis , the disease responsible for an estimated 1 . 3 million new human cases and 20 000 to 30 000 deaths annually [1] . During blood-feeding , sand fly females inoculate saliva into the host skin . Over the last three decades , various research groups have investigated the composition and biological activities of saliva , as well the potential use of salivary antigens in an anti-Leishmania vaccine ( reviewed in [2] ) . Sand fly salivary molecules are also highly antigenic and elicit strong antibody-mediated response in repeatedly exposed hosts . This response can be utilized as a marker for exposure to biting sand flies . In animals experimentally-exposed to sand fly bites the production of specific anti-saliva IgG antibodies is positively correlated with the number of blood-fed sand flies [3 , 4] . The elevated antibody levels persisted in bitten hosts for weeks or even months [3–6] but decreased after the last exposure to sand flies , suggesting that screening for anti-saliva antibodies can be used also for estimating the timing of exposure [7 , 8] . As a reliable epidemiological tool , anti-sand fly saliva antibodies have already been successfully employed to evaluate the effectiveness of vector control interventions [4 , 9] , to estimate the risk of Leishmania transmission [4 , 10–12] , and to indicate the feeding preferences of sand flies [13–15] . Screening for anti-sand fly saliva antibodies in large populations is impractical due to the amount of work required to obtain sufficient quantities of salivary gland homogenate ( SGH ) . However , the use of recombinant salivary proteins enables to circumvent the necessity for sand fly colony maintenance , laborious dissections of salivary glands and potential cross-reactivity with non-vector species [8] . The main salivary antigens in several sand fly species have already been characterized [4 , 12 , 16–18] , however , recombinant salivary proteins from only three species—Lutzomyia longipalpis , Ph . perniciosus , and Ph . papatasi—have been tested so far in seroepidemiological studies [13 , 19–25] . Here , we focus on Ph . orientalis , the most important vector of human visceral leishmaniasis ( VL ) in East Africa [reviewed in [26]] . In Ethiopia , the main endemic areas of VL are located in the lowlands of southwestern Ethiopia and in the Metema-Humera plains in the northwest [27] , where Ph . orientalis was found to be an abundant sand fly species [28] . This opportunistic sand fly feeds on different mammals , depending on the host availability [29–31] . Indeed , anti-Ph . orientalis antibodies have recently been detected in several domestic animal species in Ethiopia—dogs , donkeys , sheep , goats , and cows using SGH as antigen [15] . In the present study , five proteins from saliva of Ph . orientalis were expressed in Esherichia coli and evaluated as markers for exposure using sera of domestic animals , namely dogs , sheep , and goats , from L . donovani endemic foci in northern Ethiopia .
BALB/c mice were maintained and handled in the animal facility of Charles University in Prague in accordance with institutional guidelines and the Czech legislation ( Act No . 246/ 1992 coll . on Protection of Animals against Cruelty in present statutes at large ) , which complies with all relevant European Union and international guidelines for experimental animals . The experiments were approved by the Committee on the Ethics of Animal Experiments of the Charles University in Prague ( Permit Number: 24773/2008-10001 ) and were performed under the Certificate of Competency ( Registration Number: CZ 02439 , CZ 02457 ) in accordance with the Examination Order approved by Central Commission for Animal Welfare of the Czech Republic . Sera of domestic animals were collected within the study by [15] . Their collection was approved by the Ethiopian National Research Ethics Review Committee ( NRERC ) under approval no . 3 . 10/3398/04 . For more details see [15] . Murine sera were obtained from animals exposed at least ten-times to about 150 insectary-bred sand fly females of a single species in two-week interval . Ten mice were exposed to Ph . orientalis , four to Ph . papatasi , and four to Sergentomyia schwetzi . Four mice served as non-exposed controls . Serum samples of Ethiopian domestic animals , obtained during the previous study by [15] included 179 sheep , 36 dog , and 233 goat sera . Sera from 30 sheep , 14 dogs , and 15 goats non-exposed to sand flies were used as negative controls . More details about samples from domestic animals ( both of Ethiopian origin and controls ) are provided in [15] . The colony of Ph . orientalis ( originating from Ethiopia , Melka Werer ) was established in 2008 [32] and reared under standard conditions as described in [33] . Salivary glands were dissected from 4–6 day old female sand flies in 20 mM Tris buffer with 150 mM NaCl and stored at -20°C . Before use , salivary glands were disrupted by freeze-thawing three times in liquid nitrogen [34] . Antigenic proteins were selected based on the reactivity of two pools of canine sera ( five sera each ) from endemic area in Ethiopia with SGH using one pool ( five sera ) of non-exposed dogs as control . Salivary proteins ( equivalent to 20 glands per well ) were separated by SDS-PAGE on 12% polyacrylamide gel under non-reducing conditions . Proteins were transferred from the gel to nitrocellulose membranes using an iBLOT dry system ( Invitrogen ) . Membranes were cut into strips and blocked overnight with 5% nonfat dry milk in Tris buffer with 0 . 05% Tween ( Tris-Tw ) and incubated for 1 hour with dog sera diluted 1:50 in Tris-Tw . Then , the strips were incubated with peroxidase conjugated anti-dog IgG ( Bethyl Laboratories ) diluted 1:3000 in Tris-Tw . Antigenic protein bands were visualized using the substrate solution with diaminobenzidine . The protein profile was compared with Ph . orientalis SGH studied by [35] and the identity of antigenic bands was confirmed by proteome analysis and mass spectrometry; according to the protocol described in [35] . Five proteins from Ph . orientalis salivary glands were chosen for expression in E . coli: PorSP15 , PorSP24 , PorSP65 , PorSP67 , and PorSP76 ( in [35] marked as PorASP15 , PorMSP24 , PorMSP65 , and PorMSP67 , and PorASP76 , respectively ) ( Table 1 ) . The PCR products from a previously constructed cDNA library [35] were used as the starting material . Products were amplified by PCR under the following conditions: initial incubation ( 3 minutes in 94°C ) , then 30 cycles of denaturation ( 30 seconds in 94°C ) , annealing ( 1 minute in 57°C ) , and elongation ( 1 minute in 72°C ) . The whole reaction was terminated by heating to 72°C for 10 minutes . Specific primers were synthesized according to the sequences of the mature protein ( without the signal peptide ) ( Table 1 ) . Thereafter , we followed the procedure described in [5] . Briefly , PCR products were ligated into E . coli pGEM-T Easy Vector ( Promega ) using TA cloning and the ligation products were transfected into E . coli competent cells TOP10 ( Invitrogen ) . Vectors were replicated in bacteria and after that , the gene for yellow-related protein was restricted using Spe I and Xho I and the genes for the remaining four proteins were restricted using Nde I and Xho I enzymes . Restricted E . coli pET-42 Expression Vectors ( Novagen ) were ligated and ligation products were transformed into E . coli competent cells TOP10 ( Invitrogen ) again . Plasmids were isolated from the bacteria , and transfected into E . coli BL21 ( DE3 ) gold ( Agilent ) for expression . E . coli lysates were prepared under denaturing conditions and His-tagged proteins ( with six histidins ) were purified under denaturing conditions with 8M urea in Ni-NTA column ( 731–1550: Bio-Rad , USA ) . Purity of the recombinant proteins was verified on immunoblot using the monoclonal anti-polyHistidine-peroxidase ( A7058-1VL: Sigma Aldrich , UK ) and protein concentration was measured by the Lowry method ( Bio-Rad ) following the manufacturer’s protocol . The ELISA protocol described in [15] was used with the following modification: The ELISA plates Immulon 4HBX ( 96w flat bottomed plate , 735–0465: VWR , USA ) were coated in concentrations of 5 μg/ml ( 0 . 5 μg/well ) for recombinant proteins and 28 ng/well for SGH ( corresponding to 0 . 2 of salivary gland/well ) . In all ELISA tests , serum samples were tested individually . In the first series of experiments ( evaluation step ) , sera from ten experimentally-bitten mice and sera of 10 dogs , 10 goats , and 35 sheep with the highest anti-Ph . orientalis SGH titer values found by [15] were used to evaluate the antigenicity of the five recombinant proteins with anti-Ph . orientalis saliva IgG . Sera from non-exposed animals ( 3 mouse , 3 dogs , 3 goats , and 8 sheep ) were used as negative controls . In the second series of experiments , 16 murine sera ( 4 exposed to Ph . orientalis , 4 to Ph . papatasi , 4 to Se . schwetzi , and 4 non-exposed controls ) were used to verify specificity of selected recombinant proteins . Based on the results of evaluation experiments with murine sera , three recombinant proteins with significant correlation with SGH ( rPorSP24 , rPorSP67 , and rPorSP76 ) were selected . In the third series of experiments ( validation step ) , selected recombinant proteins with correlation coefficient higher than 0 . 7 from evaluation experiments ( rPorSP15 , rPorSP24 , rPorSP65 , and rPorSP67 ) were tested with the whole set of serum samples from Ethiopia ( 179 sheep , 36 dogs , and 233 goats ) and an appropriate number of non-exposed controls ( 30 sheep , 14 dogs , and 15 goats ) . The non-parametric Spearman test was used to assess correlations between total anti-SGH and anti-recombinant protein IgG levels using GraphPad Prism version 6 ( GraphPad Software , Inc . , San Diego , CA ) . For evaluating the possible cross-reactivity with other sand fly species non-parametric Wilcoxon Rank-Sum test in GraphPad Prism version 6 ( GraphPad Software , Inc . , San Diego , CA ) was used . Statistical significance was considered when the p-value was<0 . 05 . Cut-off values were calculated from the mean optical density of control sera plus 3 standard deviations . The optical density values of anti-SGH antibodies were used as the gold standard to validate recombinant proteins in ELISA tests using positive and negative predictive values , sensitivity , and specificity . Accession numbers of proteins used in this study: AGT96431 , AGT96428 , AGT96466 , AGT96467 , AGT96441 . Accession numbers of proteins discussed in this study: AAL16051 , AHA49643 , AAL11049 , AAL11048 , AFY13224 , ABI20147 , AHF48995 , AHF48996 , AAD32198 , AAS05318 , AHF49000
To identify antigenic proteins in Ph . orientalis salivary glands , two pools of canine sera ( five sera each ) from naturally-exposed dogs were tested with SGH of Ph . orientalis . Individual bands were identified based on the proteomic analysis , immunoblot , and mass spectrometry of Ph . orientalis SGH . Canine sera reacted with at least 10 protein bands ( Fig 1 ) ; five of them were identified as ParSP25-like protein PorSP65 , yellow-related protein PorSP24 , apyrase PorSP15 , antigen 5-related protein PorSP76 , and D7-related protein PorSP67 ( for GenBank ACCN refer to Table 1 ) . These five antigenic proteins were chosen for expression in E . coli . Very weak reaction was observed between SGH and negative control sera around 30 , 40 , and 90 kDa ( Fig 1 ) . To evaluate the reactivity of anti-Ph . orientalis saliva IgG with five recombinant proteins , we screened them first with selected sera of naturally-exposed domestic animals from Ethiopia ( dogs , sheep , and goats ) , mice experimentally-bitten by Ph . orientalis , and non-exposed controls . The antigenicity of recombinant proteins was evaluated based on the correlation of antibody reactions with SGH for each of the twenty combinations between recombinant proteins and animal species ( Table 2 ) . In canine sera , a significant correlation was achieved for all tested proteins; the highest correlation coefficient was found for rPorSP24 ( ρ = 0 . 868 ) , followed by rPorSP65 , and rPorSP15 . Similarly , in sheep and goat sera the best correlation ( above 0 . 8 ) was found for rPorSP24 , other recombinant proteins with correlation coefficient above 0 . 75 were rPorSP67 and rPorSP15 for goats and rPorSP65 for sheep ( Table 2 ) . Sera from experimentally-bitten mice showed significant correlation with three out of five proteins tested; the highest correlation coefficient was achieved for rPorSP24 ( ρ = 0 . 857 ) . Specificity of recombinant proteins was tested only with murine sera due to the absence of positive control samples from other host species . Three recombinant proteins with significant correlation to SGH in evaluation experiments with murine sera ( Table 2 ) were selected to verify their specific reaction with anti-Ph . orientalis IgG . Fig 2 shows the strong reactions of sera from mice exposed to Ph . orientalis bites with SGH and with all tested recombinant proteins ( rPorSP67 , rPorSP76 , and rPorSP24 ) , while the recognition of these antigens by sera from mice exposed to solely to Ph . papatasi or Se . schwetzi were similar to the negative controls ( sera of unexposed mice ) . Four recombinant proteins with the highest correlation from evaluation experiments ( rPorSP15 , rPorSP24 rPorSP65 , and rPorSP67 ) were chosen for further validation using the whole set of Ethiopian serum samples ( 179 sheep , 36 dogs , and 233 goats ) and non-exposed controls . For canine sera , the highest correlation coefficient was achieved with rPorSP65 ( ρ = 0 . 906 ) followed by rPorSP24 and PorSP15 . For sheep as well as for goats , the highest correlation coefficient was detected with rPorSP24 ( ρ = 0 . 818 and ρ = 0 . 522 , respectively ) followed with rPorSP65 for sheep and with rPorSP15 and rPorSP67 for goats ( Fig 3 ) . All results from correlation analyses between SGH and four recombinant proteins were highly significant and cut-off values for individual recombinant proteins were the lowest for the proteins with the highest correlation coefficient ( Fig 3 ) . Additionally , rPorSP24 reached the highest values of positive and negative predictive values ( PPV and NPV ) in all host species as well as the sensitivity in dogs and the specificity in goats and sheep . The specificity in dogs was the best with rPorSP15 and the sensitivity in goats with rPorSP67 . The best combinations ( the lowest cut-off value , the highest correlation coefficient , PPV , NPV , specificity , and sensitivity values ) between SGH and recombinant protein for each animal species tested are shown in Fig 4 .
We have studied antigenic salivary proteins of Ph . orientalis , the most important vector of VL in Ethiopia and Sudan , using sera of naturally-exposed hosts . Antigenic proteins were identified on immunoblot based on their recognition by canine sera . These were: D7-related protein ( PorSP67 ) , antigen 5-related protein ( PorSP76 ) , apyrase ( PorSP15 ) , yellow-related protein ( PorSP24 ) , and ParSP25-like protein ( PorSP65 ) . The antigenicity of their recombinant counterparts expressed in E . coli was validated in large-scale tests using sera from naturally-exposed dogs , sheep , and goats . The utilization of recombinant proteins as markers for exposure may help to highlight the specificity of the reaction and to evade nonspecific binding as observed when SGH was recognized by negative control . sera . Sand fly salivary proteins from the D7-related family are well known antigens; they were recognized by sera of mice bitten by Ph . papatasi [10] , dogs bitten by Lu . longipalpis and Ph . perniciosus [4 , 36] , and humans bitten by Ph . papatasi [12] . As far as we are aware , five recombinant D7-related ( rD7 ) proteins from sand fly saliva have already been tested as exposure markers; one from Lu . longipalpis ( AAL16051 , also known as LJL13 ) [13] , another from Ph . perniciosus ( AHA49643 ) [21] , and three from Ph . papatasi ( AAL11049 , AAL11048 , and AFY13224 ) [5 , 20] . However , only some of them bound anti-saliva IgG and their antigenicity was host-specific . The rD7 protein ( AAL11049 ) was specifically recognized by sera from mice bitten by Ph . papatasi [5] , but the same protein was not recognized by human sera from Tunisia [20] . Another rD7 protein ( AAL11048 ) from Ph . papatasi did not react with sera from mice immunized by sand flies [5] . The rD7 protein LJL13 was recognized by dogs naturally-exposed to Lu . longipalpis but not by sera from foxes and humans from endemic focus of L . infantum [13] . In the present study , rPorSP67 showed promising results with limited number of goat sera during the evaluation test but was not validated in a broader test , when medium or low correlation coefficient , NPV , PPV , sensitivity , and specificity with values ranging between 0 . 28 and 0 . 6 were observed . This suggests that this recombinant protein would not be useful as an exposure marker to Ph . orientalis bites . Proteins of the antigen 5-related family from various sand fly species were repeatedly shown to be potent salivary antigens , being recognized by sera of mice bitten by Ph . papatasi and Ph . arabicus [5 , 16] , dogs bitten by Ph . perniciosus [4] , rabbits exposed to Ph . tobbi [18] , and hamsters bitten by Ph . argentipes [37] . In our study , rPorSP76 showed high correlation with SGH only with sera from experimentally-bitten mice ( ρ = 0 . 8 ) , thus its use in field studies with domestic animals is not justified . Apyrases are well-known salivary enzymes with anti-haemostatic properties [38] . Antigenic properties of apyrases were described in SGHs of various vector-host models such as Ph . perniciosus and dogs [4 , 17] or Ph . argentipes and hamsters [37] . Recombinant apyrase ( ABI20147 ) was recognised by sera of mice immunized with Ph . duboscqi saliva [39] . Two apyrases in a recombinant form ( AHF48995 , AHF48996 ) were used as exposure markers for dogs , hares , and rabbits bitten by Ph . perniciosus [21 , 22] , however , the most recent work by Kostalova et al . [23] revealed that neither of these recombinant proteins gave optimal ELISA results in large-scale tests with naturally-exposed dogs . Our results showed a significant correlation between antibody response against SGH of Ph . orientalis and recombinant apyrase rPorSP15 in sera of all domestic animals tested but not in mice sera . The best correlation ( ρ = 0 . 7 ) was observed with canine sera with medium values of NPV , PPV , sensitivity , and specificity ( ranging between 0 . 59 and 0 . 68 ) suggesting necessity of further validation before its utilization as antigen for detecting dog exposure to Ph . orientalis . The most promising and universal candidate for an exposure marker to sand fly bites belongs to the family of yellow-related proteins . Strong antibody responses to these proteins were previously demonstrated for various sand fly and host species , including dogs and humans [3–7 , 10 , 12–14 , 17 , 36 , 40] . Ph . perniciosus recombinant yellow-related protein AHF49000 was successfully used as an antigen both in ELISA and immunoblot reacting well with sera of mice and dogs experimentally-bitten by Ph . perniciosus [21] . Yellow-related recombinant proteins were also validated as exposure markers to sand fly bites in endemic areas; Lu . longipalpis AAD32198 and AAS05318 for humans [19 , 13] and Ph . perniciosus AHF49000 for dogs , rabbits , and hares [22 , 23] . In the present study , rPorSP24 from Ph . orientalis confirmed the high reactivity of yellow-related protein family with antibodies from sera of bitten hosts and this advocates for its use as an exposure marker in large-scale field studies . It reached high correlation with SGH in mice ( ρ = 0 . 9 ) , sheep ( ρ = 0 . 8 ) , and dogs ( ρ = 0 . 8 ) and the best in goats ( ρ = 0 . 5 ) . Similarly , high correlation between SGH and recombinant yellow-related protein was previously attained with dogs ( ρ > 0 . 7 [21–23] ) , hares ( ρ = 0 . 9 [22] ) , and rabbits ( ρ = 0 . 7 [22] ) . Moreover , rPorSP24 achieved the highest values of specificity , sensitivity , PPV and NPV with majority of the host species tested . In dogs , this recombinant protein showed 100% NPV and sensitivity but lower values of PPV and specificity ( 0 . 66 and 0 . 41 , respectively ) , indicating higher probability of false positivity among non-exposed dogs . On the other hand , in sheep , very high values of PPV and specificity were achieved ( both 0 . 9 ) but medium values of NPV and sensitivity ( 0 . 58 and 0 . 68 , respectively ) could indicate possible false negative results . However , this statistical analysis is based on data from naturally-exposed hosts and negative controls; further validation is needed using sera of experimentally-bitten animals as a positive control . The fifth recombinant protein tested belongs to the ParSP25-like family . Antigenicity of this protein family was previously demonstrated in Ph . perniciosus [4 , 17] . So far as we are aware , no recombinant protein from this group was prepared and used for measuring the antibody reaction with sera of bitten hosts . Our results suggest significant correlation between rPorSP65 and antibody response against SGH of Ph . orientalis . The highest correlation coefficient was observed with canine sera ( ρ = 0 . 9 ) , accompanied by high degree of sensitivity ( 0 . 95 ) . Nevertheless , the specificity of the test with rPorSP65 was very low ( 0 . 1 ) suggesting high probability of false positivity among non-exposed dogs . Antigenic specificity of recombinant proteins was confirmed by using murine sera experimentally-exposed to Sergentomyia schwetzi or Phlebotomus papatasi . These two sand fly species are present in Ethiopia , in some places sympatrically with Ph . orientalis [27] . Antibodies from sera of mice bitten by Ph . papatasi or Se . schwetzi did not react with the recombinant proteins with significant correlation from evaluation experiments ( rPorSP24 , rPorSP67 , and rPorSP76 ) and confirmed that they are species-specific . The reactivity of recombinant proteins might be affected by the expression system or conditions of protein purification . Antibodies from bitten hosts can be targeted also to the glycosylated parts of the antigen that are lacking in proteins from the E . coli expression system , therefore some authors prefer to express recombinant proteins in mammalian cells [20 , 24] . Nevertheless , some of the recombinant proteins without posttranslational modifications proved to be as efficient markers of exposure as native antigens [5 , 21–23] . Similarly , several proteins prepared in this study were found as suitable antigens , despite their being expressed in E . coli . In conclusion , our study suggests rPorSP24 , the recombinant protein from the yellow-related family , as the most reliable and universally efficacious antigen for measuring exposure of dogs , sheep and goats to Ph . orientalis bites . In addition , the recombinant protein rPorSP65 from ParSP25-like group was found as a good antigen to screen for canine exposure but its low specificity suggests possible false positivity in some specimens . Serological tests using these proteins could be a highly practical and economical tool for screening of domestic animals for exposure to the main vector of L . donovani in East Africa . Moreover , the promising characteristics of rPorSP24 suggest a potential use of this antigen for screening sera of other hosts , including humans . The availability of recombinant salivary proteins should enable to measure anti-Ph . orientalis antibodies in large-scale experiments to evaluate vector control programs in areas affected by VL in East Africa . However , further studies are needed to validate such recombinant protein-based test for routine use . | The sand fly Phlebotomus orientalis is the main vector of Leishmania donovani , the causative agent of visceral leishmaniasis in East Africa . During bloodfeeding , sand flies inject saliva into the host skin and repeated bites result in a specific antibody response in the bitten hosts . Antibody responses are directed against sand fly salivary proteins and the levels of these antibodies reflect the intensity of exposure to biting sand flies . The antibody reactions can be measured using salivary gland homogenates ( SGHs ) , but for large-scale testing its use is impractical because of the amount of work required to obtain sufficient quantities of SGH . Recombinant proteins prepared based on the antigens in the sand fly saliva can substitute whole SGH in large-scale studies . We tested five recombinant proteins from Ph . orientalis saliva expressed in Escherichia coli and demonstrated that the yellow-related protein rPorSP24 can replace the SGH in estimating exposure to sand flies of dogs , goats , and sheep in Ethiopia . Immune reactions to vector saliva in endemic areas , provides useful information on levels of exposure and , thereby , on the effectiveness of vector control programs . | [
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] | 2016 | Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites |
Severe acute respiratory syndrome virus ( SARS-CoV ) that lacks the envelope ( E ) gene ( rSARS-CoV-ΔE ) is attenuated in vivo . To identify factors that contribute to rSARS-CoV-ΔE attenuation , gene expression in cells infected by SARS-CoV with or without E gene was compared . Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene . In addition , genes involved in signal transduction , transcription , cell metabolism , immunoregulation , inflammation , apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene . Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus , or treated with drugs , such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms . In addition , SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 ( IRE-1 ) of the unfolded protein response , but not the PKR-like ER kinase ( PERK ) or activating transcription factor 6 ( ATF-6 ) pathways , and reduced cell apoptosis . Overall , the activation of the IRE-1 pathway was not able to restore cell homeostasis , and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination . The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells , suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE .
Severe acute respiratory syndrome coronavirus ( SARS-CoV ) was identified as the etiological agent of a respiratory disease that emerged in Guandong Province , China at the end of 2002 , and spread to 32 countries in a few months [1] , [2] , [3] , [4] , [5] , [6] , [7] . SARS-CoV infected 8000 people in 2002–2003 , with an average mortality of 10% . After July 2003 , only a few community and laboratory-acquired cases have been reported ( http://www . who . int/csr/sars/en/ ) . Nevertheless , coronaviruses similar to the one that caused the epidemic are widely disseminated in bats circulating all over the world , making a future outbreak possible [8] , [9] , [10] . SARS-CoV is an enveloped , single-stranded positive sense RNA virus , with a genome of 29 . 7 kb . The coronavirus replicase gene is encoded within the 5′ two thirds of the genome , and includes two overlapping open reading frames ( ORFs ) named ORF1a and ORF1b . Translation of both ORFs in the cytoplasm of infected cells results in the synthesis of two large polyproteins , pp1b and pp1ab , processed by two viral proteases to yield 16 non structural proteins ( nsps ) [11] , [12] . The nsps are involved in genome replication and transcription of subgenomic mRNAs ( sg mRNAs ) that encode structural proteins such as the nucleocapsid ( N ) , envelope ( E ) , membrane ( M ) , and spike ( S ) , and a set of group-specific proteins whose sequence and number differ among the different coronavirus species [13] . In the case of SARS-CoV , the group-specific proteins 3a , 6 , 7a and 7b , are also structural proteins [14] , [15] , [16] , [17] , [18] . SARS-CoV E protein , a small integral membrane protein of 76 amino acids , contains a short hydrophilic amino-terminus followed by a hydrophobic region and a hydrophilic carboxy-terminus [19] . The hydrophobic region forms at least one amphipathic α-helix that oligomerizes to form an ion-conductive pore in membranes [19] . Furthermore , HCoV-229E , murine hepatitis virus ( MHV ) , SARS-CoV , and infectious bronchitis virus ( IBV ) E proteins form ion channels permeable to monovalent cations [20] , [21] , [22] . The E protein from genus α transmissible gastroenteritis coronavirus ( TGEV ) is essential for the generation of propagation competent viruses [23] , [24] , [25] . In contrast , genus β MHV and SARS-CoV E proteins are not completely essential for the generation of infectious viruses [26] , [27] , [28] . SARS-CoV lacking the E protein is attenuated in different animal models for SARS , such as hamsters and transgenic mice that express the SARS-CoV receptor , human angiotensin converting enzyme 2 ( hACE-2 ) [26] , [27] . Virus infection may result in the expression of stress proteins , like heat shock proteins ( hsps ) , glucose-regulated proteins ( GRPs ) and ubiquitin [29] . Some of these proteins are constitutively expressed , while others are induced by proteotoxic stresses such as protein overload , heat shock , hypoxia , ischemia , heavy metals , radiation , calcium increase , reactive oxygen species , and drugs , in addition to virus infection [30] . Stress proteins may act as molecular chaperones , participating in protein synthesis , folding , transport , cell viability [31] , and modulating the immune response [32] . Increasing evidence suggests that certain hsps play a role in both innate and adaptive immunity [32] , [33] . Hsps can act independently of chaperoned peptides to directly stimulate innate immune responses , such as the maturation and activation of dendritic cells , and the activation of natural killer cells ( reviewed in [33] ) . Coronavirus infection generates double membrane vesicles [34] , [35] derived from the endoplasmic reticulum ( ER ) , in which the RNA virus genome is replicated and transcribed [36] . In addition , enveloped viruses modify and perturb membranes to generate new virus particles . This extensive use of intracellular membranes for virus replication and morphogenesis likely overloads the ER during infection , causing ER stress responses and triggering the unfolded protein response ( UPR ) . The UPR increases the production of chaperones that facilitate protein folding , promotes the synthesis of lipids that constitute cellular membranes and inhibits translation in order to reduce ER stress [37] . The UPR is mediated by three ER-resident transmembrane proteins that are activated through binding to unfolded proteins: PKR-like ER kinase ( PERK ) , activating transcription factor 6 ( ATF6 ) , and inositol-requiring enzyme 1 ( IRE-1 ) [38] , [39] , [40] . Upon activation , PERK dimerizes and autophosphorylates . This protein phosphorylates eIF2α , leading to the inhibition of translation . ATF6 activation involves the translocation of this protein to the Golgi compartment , where site 1 and site 2 proteases process the 90 KDa form to create a 50 KDa form , the ATF6α ( C ) , a soluble transcription factor that translocates to the nucleus and upregulates the expression of genes involved in protein folding . IRE-1 mediates the splicing of the mRNA encoding the transcription factor X box-binding protein 1 ( XBP-1 ) , leading to a frame shift and translation of a functional XBP-1 protein . The active transcription factor ( sXBP-1 ) can then stimulate the transcription of genes encoding proteins that promote the folding , transport , and degradation of ER proteins , and lipid biosynthesis . The ER stress response acts to restore ER homeostasis . However , when homeostasis cannot be restored , persistent or intense ER stress can also trigger programmed cell death or apoptosis [41] , a physiological mechanism to control the number of cells during development and to respond to infections . Autopsy studies have revealed signs of apoptosis in SARS-CoV-infected tissues from patients , such as lung , spleen and thyroid [42] , [43] . Accordingly , it has been shown that the infection by SARS-CoV triggers apoptosis in cell cultures via protein kinase R ( PKR ) [44] and that at least eight SARS-CoV-encoded proteins induce apoptosis [45] . The expression of genes leading to hyperinflammation has been associated with SARS-CoV-induced pathology . In fact , highly elevated expression of inflammatory mediators such as interleukin ( IL ) -1 , -6 , and -8 , CXCL10/interferon-inducible protein ( IP ) -10 , CCL2/monocyte chemoattractant protein ( MCP ) -1 , CCL5/regulated on activation , normal T expressed and secreted ( RANTES ) , and CXCL9/monokine induced by interferon gamma ( MIG ) , has been described within the circulation and lungs of SARS patients [46] , [47] , [48] , [49] , [50] , [51] . In this study , the effect of SARS-CoV E protein on host cell responses during virus infection was analyzed for the first time by comparing the transcriptomes of rSARS-CoV-ΔE and rSARS-CoV-infected cells using microarrays and quantitative reverse transcription polymerase chain reaction ( qRT-PCR ) . We showed that SARS-CoV E protein influenced the expression of genes associated to stress response , immunoregulation , inflammation , apoptosis , and cell cycle and differentiation . Among these changes , the effect on stress response was most robust , based on both the number of differentially expressed genes regulating this activity and on the extent of the changes observed . This downregulation of the stress response in the presence of gene E was specific as this process was reversed by providing E protein in trans . In addition , we showed that E protein reduced the cellular stress caused by another respiratory virus , respiratory syncytial virus ( RSV ) , and two drugs ( tunicamycin and thapsigargin ) that induce stress by different mechanisms . Furthermore , the presence of E protein reduced the activation of the IRE-1 mediated pathway during the UPR . However , the activation of these signaling pathways in the absence of E protein was not sufficient to reverse the cellular stress induced by rSARS-CoV-ΔE since infected cells underwent apoptosis . In addition , the absence of E protein increased the expression of the double specificity phosphatases ( DUSP ) -1 and DUSP-10 , and down regulated proinflammatory cytokines such as CCL2 and CXCL2 . Therefore , the effect of E protein on the stress response , including the UPR , and on proinflammatory cytokine expression may explain the attenuation of rSARS-CoV-ΔE in vivo .
To study the host response elicited by SARS-CoV , it is essential to use cell lines , such as Vero E6 , MA-104 , CaCo-2 , Huh7 , FRhK-4 , PK15 , HepG2 , 293 and 293T cells , that are highly susceptible to infection with SARS-CoV [52] , [53] , . To determine whether these cell lines were also susceptible to rSARS-CoV-ΔE , virus growth kinetics studies were performed . rSARS-CoV-ΔE passaged 16 times in Vero E6 cells ( P16 ) was analyzed , as this virus grew with titers similar to those of rSARS-CoV , around 10-fold higher than virus passaged only once ( P1 ) . rSARS-CoV-ΔE-P16 contained only a single nucleotide substitution at amino acid 607 of the S gene ( S607F ) compared to the P1 virus [56] . Both rSARS-CoV-ΔE P1 and the P16 were attenuated in the highly susceptible transgenic mice model [56] , showing that the deletion of the E gene , and not the amino acid substitution in S protein , was responsible for virus attenuation . All the cell lines indicated above were infected with SARS-CoV with and without E gene at a multiplicity of infection ( moi ) of 1 , 3 and 5 and the percentage of infected cells at 24 hours post infection ( hpi ) was determined using an immunofluorescence assay . Similar results were obtained both in SARS-CoV and rSARS-CoV-ΔE-infected cells , so only the results obtained with SARS-CoV-infected cells are provided in Supplementary Table SI . An increase in the moi led to a higher proportion of infected cells in all cell lines . The percentage of infected cells was below 40% in all cases , except for African green monkey kidney Vero E6 and MA-104 cells ( Supplementary Table S1 ) , which have or do not have , respectively , a defect in interferon ( IFN ) production [57] , [58] . More than 90% of Vero E6 cells were infected with rSARS-CoV-ΔE or rSARS-CoV at 24 hpi , whereas in the case of MA-104 cells , more than 80% of the cells were infected with both viruses at 24 hpi ( Supplementary Table S1 ) . The growth kinetics of rSARS-CoV-ΔE and rSARS-CoV in Vero E6 cells at an moi of 2 showed similar profiles and titers for both viruses , reaching maximum titers and cytopathicity at 15 hpi ( Fig . 1A ) . In contrast , in the case of MA-104 cells , although growth kinetics for rSARS-CoV-ΔE and the parental virus were similar , a 10-fold reduction in virus titers was observed in cells infected with rSARS-CoV-ΔE virus ( Fig . 1A ) . This difference is not unexpected , as the ΔE virus that was used in these experiments was passaged and adapted to growth in Vero E6 , but not in MA-104 cells . The cytopathic effect in MA-104 cells was evident at 48 hpi and maximum virus titers were reached at 65 hpi ( Fig . 1A ) . The kinetics of genomic RNA and N gene sg mRNA accumulation were similar in rSARS-CoV-ΔE and rSARS-CoV-infected Vero E6 and MA-104 cells , as determined by qRT-PCR ( Fig . 1B and 1C ) , indicating that SARS-CoV E protein had no influence on the accumulation of viral RNAs . Maximum levels of both types of viral RNA were observed at 15–22 hpi , in the case of infected Vero E6 cells and at 48 hpi in the case of MA-104 cells . These data showed that although Vero E6 and MA-104 cells were susceptible to SARS-CoV , the kinetics of the infection was slower in MA-104 than in Vero E6 cells , which needs to be considered when cellular mRNAs are collected for differential gene expression studies . To analyze the impact of E protein on host gene expression during SARS-CoV infection , the transcriptomes of rSARS-CoV-ΔE and rSARS-CoV-infected Vero E6 and MA-104 cells were compared . Taking into account the data obtained in Figure 1 , early ( 7 hpi in the case of Vero E6 , and 24 hpi in the case of MA-104 cells ) , and late ( 15 and 65 hpi , in Vero E6 and MA-104 cells , respectively ) times post-infection ( pi ) , were analyzed . Microarray-based studies of global gene responses were performed in triplicate in each case . As there are no commercially available microarrays specific for African green monkey species , and the sequence homology between humans and monkeys is very high [59] , human U133 plus 2 . 0 microarrays were used . The results of the microarray analysis have been deposited in the Gene Expression Omnibus ( GEO , NCBI , accession code GSE30589 ) . Only those genes showing significant expression changes ( i . e . , 2 . 0-fold and false discovery rate ( FDR ) <0 . 01 ) at each time point were selected for further investigation . Comparison of gene expression in cells infected with rSARS-CoV with or without E gene versus mock-infected cells showed that more that 800 cellular genes were differentially expressed at late time post-infection ( Fig . 2 ) and that the number of genes differentially expressed increased over time ( i . e . in the case of Vero E6 cells , 4940 annotated genes for rSARS-CoV versus mock-infected cells at 15 hpi , compared to 1324 annotated genes at 7 hpi; for MA-104 cells , 971 annotated genes for rSARS-CoV versus mock-infected cells at 65 hpi , compared to 11 annotated genes at 24 hpi ) . Interestingly , the number of annotated genes differentially expressed in cells infected with rSARS-CoV-ΔE compared to rSARS-CoV , in which the only difference is the expression of E gene , was reduced to 57 ( Vero E6 cells ) or to 72 ( MA-104 cells ) at 15 or 65 hpi , respectively ( Fig . 2 ) . These genes were classified according to their most commonly accepted functions ( Fig . 3 ) . A high number of genes related to stress responses ( 19 out of 57 in Vero E6 cells , and 19 out of 72 in MA-104 cells ) were differentially expressed , with 2- to 35-fold increases . The pattern of genes upregulated in rSARS-CoV-ΔE compared to rSARS-CoV-infected cells was very similar in Vero E6 and MA-104 cells , and included different isoforms of heat shock protein ( hsp ) ( hsps-10 , -27 , -40 , -60 , -70 , -90 and -105/110 ) , and different genes encoding ubiquitins and chaperonins ( Fig . 3 ) . These data clearly indicated that the cellular stress induced by the infection was significantly reduced in the presence of E protein . Nevertheless , it is worthy to mention that not all cellular stress genes were differentially expressed in cells infected with SARS-CoV lacking E protein versus those infected with rSARS-CoV . In fact , a set of genes coding for different isoforms of hsp40 , hsp70 , and hsp 90 , also modified their expression between −11 . 0 and and +4 . 0-fold but to a similar extent in rSARS-CoV-ΔE and rSARS-CoV-infected cells when compared with mock infected ones ( Fig . S1 ) . Differentially expressed genes were also involved in signal transduction , transcription , cell metabolism , immunoregulation , inflammation , apoptosis and cell cycle and differentiation , although to a lower extent ( Fig . 3 ) . Among the genes involved in signal transduction , the upregulation of DUSP1 and DUSP10 may be relevant in rSARS-CoV-ΔE attenuation , as these genes are involved in down regulating cellular responses associated with different types of stress . Furthermore , these genes reduce the inflammatory response induced by viral infections by negatively regulating mitogen-activated protein kinase ( MAPK ) signaling [60] . Accordingly , the expression of the proinflammatory cytokines CCL2 and CXCL2 was reduced in rSARS-CoV-ΔE-infected , compared to rSARS-CoV-infected MA-104 cells . Consistent with the mRNA results , we detected increases in the levels of representative stress proteins , such as hsp60 and hsp90 although differences were not as great as observed when mRNA levels were assessed ( Fig . 4 ) . Lesser effects on protein levels may reflect inhibitory effects of SARS-CoV on non-viral protein synthesis [61] or , alternatively to the presence of pre-existing stress proteins in cells prior to infection . To better understand the biological relevance of the SARS-CoV E protein on host gene expression , all of the genes that were significantly upregulated or downregulated in rSARS-CoV-ΔE-infected compared to rSARS-CoV-infected Vero E6 and MA-104 cells were clustered in functional groups based on gene ontology ( GO ) classification . A summary is shown in Fig . 5 . In contrast , no enriched GO terms were found for genes that were downregulated in MA-104-infected cells . All of the functionally enriched GO terms were related to cellular stress ( chaperone binding , response to biotic stimulus , unfolded protein binding , protein folding ) , cellular death ( anti-apoptosis ) , cellular transport ( protein import , nucleocytoplasmic transport ) , transcription ( transcription repressor activity ) and metabolism ( protein catabolic process , cellular protein catabolic process ) . Remarkably , similar , highly significant ( FDR<0 . 01 ) changes in levels of genes related to cellular stress response to biotic stimulus , unfolded protein binding and protein folding were identified in both Vero E6 and MA-104-infected cells ( Fig . 5 ) . To validate the results obtained with the cDNA microarrays , the differential expression of a wide set of cellular genes observed in cells infected with rSARS-CoV with or without the E gene was evaluated by qRT-PCR . 18S ribosomal RNA ( rRNA ) was used in all cases to normalize the data because differences in levels of this RNA were always lower than 1 . 5-fold and because the 18S rRNA has also been used successfully in similar reports [62] , [63] . The patterns of differential gene expression obtained by qRT-PCR analysis were similar to those observed with the microarray data ( Figs . 3 and 6 ) , validating the results obtained with both techniques . Nevertheless , in the case of genes with large differences in expression between rSARS-CoV and rSARS-CoV-ΔE-infected cells determined using microarrays , changes were even larger when evaluated by qRT-PCR . To confirm the effect of the E protein on the stress response , total RNA from infected cell cultures were analyzed at different times pi ( 15 , 22 and 28 hpi in the case of Vero E6 cells , and 24 , 48 , 65 and 75 hpi , in the case of MA-104 cells ) for the expression of genes related to cytosolic ( hsp70 A1B and hsp90 AB1 ) , ER ( hspA5/GRP78 ) , and mitochondrial ( hsp60 D1 ) stress by qRT-PCR . Maximal differences in the upregulation of the three types of stress responses in rSARS-CoV-ΔE compared to rSARS-CoV were observed at 22 and 65 hpi in Vero E6 and MA-104 cells , respectively ( Fig . S2 ) . Consequently , these time points were selected to further analyze the stress responses elicited by these viruses ( Fig . 6 ) . Using microarrays , we observed that nineteen genes involved in cytosolic stress were upregulated at least 2 . 5-fold ( FDR<0 . 01 ) in rSARS-CoV-ΔE-infected compared to rSARS-CoV-infected Vero E6 cells ( 15 hpi ) and MA-104 cells ( 65 hpi ) . Changes in expression of these cytosolic stress genes were confirmed by qRT-PCR ( Fig . 6 ) and shown to be highly significant ( from 2 . 4 to 42 . 5-fold in Vero E6 cells , and from 3 . 1 to 372 . 3-fold in MA-104 cells ) . In addition , we confirmed the effect of E protein on ER ( GRP78 , GRP94 , DNAJC3 and SERPINH1 ) and mitochondrial ( hspA9 , hsp10 E1 , and hsp60 D1 ) stress , using infected Vero E6 and MA-104 cells ( Fig . 6 ) , with differences in gene expression that were up to 23 . 4 or 13 . 0-fold greater for ER and mitochondrial stress , respectively . These data reinforced the conclusion that SARS-CoV E protein reduced cellular stress induced by SARS-CoV , and that this reduction affected the cytosol , ER , and mitochondria . In the experiments described above , virus without E protein was passaged 16 times , resulting in a virus with a 10-fold increase in titer , and a single point mutation in the S gene . To rule out the possibility that the mutation in the S gene was responsible for the observed increase in cellular stress , and not the absence of E protein , the induction of stress genes in cells infected with rSARS-CoV-ΔE-p1 , which has an RNA genome sequence identical to that of the parental virus except for the deletion of gene E , was analyzed . Total RNA from Vero E6 cultures infected with the original viruses ( P1 ) with or without E protein , and with the virus lacking E protein passaged 16 times , was extracted at 22 hpi . The expression of cellular genes involved in cytosolic , ER , and mitochondrial stress was evaluated by qRT-PCR . Cellular stress genes were upregulated to similar extents in cells infected with the viruses lacking the E gene ( either from P1 or P16 ) compared to cells infected with virus expressing the E gene ( Fig . S3 ) . These data indicated that the mutation in gene S was not responsible for the observed differences in stress response , and confirmed that E protein itself down regulated the cellular stress in virus-infected cells . To reinforce the conclusion that SARS-CoV E protein was responsible for the reduction of cellular stress , we transfected the E gene into rSARS-CoV-ΔE-infected cells together with controls . Vero E6 cells were infected with viruses lacking the E gene ( P1 and P16 ) or with virus expressing the E gene , and 90 min later , cells were transfected with the plasmid pcDNA3 . 1-E , encoding the E protein , or with empty plasmid as a control . E protein was expressed in cells transfected with plasmids expressing this protein , although levels were 10-fold lower than in SARS-CoV infected cells , as shown by Western-blot analysis ( Fig . 7A ) . As an additional control , the effect of E protein expression on the replication of SARS-CoV with or without E gene was studied ( Fig . 7B ) . E protein added in trans had no significant effect on rSARS-CoV-P16 or rSARS-CoV-ΔE titers , indicating that the absence of E protein in rSARS-CoV-ΔE , and not the amount of virus produced , was responsible for the increase in cell stress response . The expression of stress genes hsp70 A1A , hsp90 AA1 , hspH1 , SERPINH1 , and hsp10 E1 in cells infected with rSARS-CoV-ΔE viruses ( P1 and P16 ) or rSARS-CoV , in the presence or absence of the transfected E gene , was analyzed by qRT-PCR ( Figs . 7C and S4 ) . The expression of all analyzed stress-induced genes was clearly upregulated in cells infected with virus lacking E protein , compared to those infected with rSARS-CoV . When E protein was provided in trans , the expression of these genes in rSARS-CoV-ΔE-infected cells was clearly reduced . To analyze whether the decreased expression of stress-related genes in the presence of E protein was specific , the expression of the gene encoding DNA polymerase theta ( polQ ) was evaluated . No significant differences were observed in the expression of polQ , irrespective of the presence or absence of E protein ( Figs . 7C and S4 ) , suggesting that the reduction of stress-related genes was specific . In addition , the expression of 18S rRNA was analyzed as an endogenous control for the amount of RNA in all samples ( Figs . 7C and S4 ) . These data indicated that E protein reduced the stress caused by SARS-CoV infection . To analyze whether E protein alone could reduce the cellular stress caused by another virus , the effect of SARS-CoV E protein on the stress induced by RSV was analyzed . Vero E6 cells were transfected with a plasmid encoding E protein or with empty plasmid as control . At 24 hours post-transfection ( hpt ) , Vero E6 cells were infected with RSV or left uninfected , and RNA was extracted at the indicated hpi . The expression of E protein in cells infected with RSV was confirmed by Western-blot analysis and the levels were similar to those of rSARS-CoV infected cells ( Fig . 8A ) . In addition , no significant effect of E protein expression on RSV titers was detected . The expression of the stress response genes hsp90 AA1 , UBB , hspH1 , SERPINH1 and hsp10 E1 was analyzed in the presence or absence of SARS-CoV E protein by qRT-PCR ( Fig . 8B ) . The expression of these stress response genes was significantly induced by RSV infection at almost all times ( Fig . 8B ) . In the presence of E protein , the induction of these stress genes was significantly reduced ( Fig . 8B ) in a specific manner as no significant differences were observed in the expression of polQ gene , irrespective of the presence or absence of E protein ( Fig . 8B ) . These data indicated that SARS-CoV E protein alone reduced different types of stress , such as cytosolic ( genes hsp90 AA1 , UBB , hspH1 ) , ER ( gene SERPINH1 ) and mitochondrial stress ( gene hsp10 E1 ) , produced by infection with at least two different respiratory viruses ( SARS-CoV and RSV ) . Coronavirus infection induces ER stress [64] due to the extensive use of intracellular membranes for the generation of replication complexes and for the assembly of virus particles [36] , [65] . In addition , viral glycoproteins can induce ER stress during infection as a result of incomplete glycosylation and incorrect folding or accumulation in the ER lumen [66] , [67] . Accordingly , we decided to focus our attention on ER stress . To determine whether E protein alone was responsible for the downregulation of the ER stress response , Vero E6 and MA-104 cells were transfected with a plasmid encoding SARS-CoV E protein or with empty plasmid as a control . At 24 hpt cell cultures were treated with thapsigargin and tunicamycin , which induce ER stress by altering intracellular Ca++ levels or by preventing protein glycosylation , respectively [68] , for 8 or 20 h , or left untreated . The levels of E protein were monitored by Western-blot analysis and were similar to E protein levels after SARS-CoV infection of Vero E6 ( Fig . 9A ) or MA-104 cell ( data not shown ) . Total cellular RNAs were collected and the expression of the ER-stress inducible genes GRP78 and GRP94 was evaluated by qRT-PCR . The effect of E protein expression at the times post-induction when upregulation of these genes was highest is shown ( Fig . 9B and C ) . Treatment with thapsigargin and tunicamycin clearly induced the expression of ER stress genes in Vero E6 and MA-104 cells transfected with the empty plasmid ( Fig . 9B and C ) . The expression of GRP78 and GRP94 was significantly reduced in the presence of E protein ( Fig . 9B and C ) . No decrease in the expression of polQ gene was observed in the presence of E gene , suggesting that the reduction in the expression of stress related genes was specific ( Fig . 9B and C ) . These data indicated that E protein alone was sufficient to reduce cellular stress caused by different mechanisms . Cells induce the UPR to reduce the burden imposed by unfolded or misfolded proteins in the ER . To analyze the mechanisms by which the E protein can reduce ER stress , the effect of E protein on the three branches of the UPR ( PERK , ATF6 , and IRE-1 ) was analyzed . The PERK pathway involves the phosphorylation and subsequent activation of this kinase . Accordingly , the levels of phosphorylated PERK in Vero E6 cells infected with rSARS-CoV-ΔE or rSARS-CoV were compared at different times pi . As a control , levels of the house-keeping gene GAPDH were measured and used for normalization . Phosphorylated PERK was detectable in rSARS-CoV-ΔE and wt-infected cells at 6 hpi , in contrast to mock-infected cells , in which no phosphorylated PERK was detected . No significant differences in the phosphorylation levels of PERK were detected between cells infected with rSARS-CoV with or without E protein ( Fig . S5 ) , suggesting that E protein had no significant influence on the phosphorylation of PERK . To analyze whether E protein inhibited the ATF6 pathway , the extent of ATF6α processing in cells infected with rSARS-CoV with or without the E gene , or mock-infected cells was measured by Western blot using an ATF6-specific antibody that recognizes the full-length and the cleaved N-terminal domain of the protein . No significant activation of ATF6 was observed in infected cells , compared to mock-infected cells ( data not shown ) , suggesting that SARS-CoV infection did not efficiently activate this pathway . Activation of IRE-1 mediates cytoplasmic splicing of the mRNA encoding the transcription factor XBP-1 , leading to a frame shift and subsequent translation of a functional XBP-1 transcription factor . To evaluate whether SARS-CoV E protein has an impact on the activation of this pathway , Vero E6 cells were infected with rSARS-CoV with or without the E gene and RNA was collected at different times pi . RT-PCR was used to amplify fragments representing both the unspliced ( u ) and spliced ( s ) forms of XBP-1 mRNA , differing by 26 nt [69] ( Fig . 10 ) . The relative abundance of these XBP-1 mRNAs was independent of PCR efficiency as the corresponding mRNAs were amplified using the same primer pair . A third slowly migrating species ( h ) , corresponding to a heterohybrid formed by the amplified unspliced and spliced forms was also detected ( Fig . 10 ) . The activation of IRE-1 was estimated as a ratio between the spliced and unspliced forms of XBP-1 . Levels of spliced XBP-1 were higher in rSARS-CoV-ΔE-infected compared to rSARS-CoV infected cells from 15 to 28 hpi ( Fig . 10 ) . This result indicated that in the presence of E protein , activation of the XBP-1 pathway was reduced . Persistent or intense ER stress can trigger apoptosis [41] . To analyze whether SARS-CoV E protein modulated apoptosis induced by SARS-CoV infection , the induction of apoptosis was analyzed in cells infected with rSARS-CoV lacking or expressing E gene . Cells infected either with rSARS-CoV or rSARS-CoV-ΔE were simultaneously stained with propidium iodide ( PI ) and Annexin V , and monitored by flow cytometry . Mock infected cells remained viable ( Annexin V− , PI− ) throughout the experiment , indicating that the treatment did not induce apoptosis by itself ( Fig . 11 ) . rSARS-CoV induced low levels of apoptosis ( Annexin V+ ) from 15 hpi , and a minor cell population in late apoptosis ( Annexin V+ , PI+ ) was evident from 24 hpi ( Fig . 11 ) . rSARS-CoV-ΔE triggered apoptosis more rapidly and to a greater extent than rSARS-CoV , with a 3 to 4-fold increase in early apoptotic cells at 4 and 15 hpi , and a 4 and 5- fold increase in late apoptotic cells between 15 and 24 hpi ( Fig . 11 ) .
We previously showed that rSARS-CoV-ΔE is attenuated in vivo [26] , [27] . In this work , to identify possible mechanisms for this attenuation , the effect of E protein on host cell responses during virus infection was analyzed by comparing the transcriptome of rSARS-CoV-ΔE and rSARS-CoV-infected cells . Among the genes differentially expressed , a large number of genes corresponding to cellular stress were upregulated in rSARS-CoV-ΔE compared to wt virus infected cells , clearly indicating that the presence SARS-CoV E protein reduced the stress response during infection . Upregulation of the stress response was also confirmed at the protein level , as the expression of representative stress response proteins , such as hsp60 and hsp90 was also increased . The addition of E protein in trans reversed the increase in stress response gene expression observed in rSARS-CoV-ΔE-infected cells , confirming the specific suppression of the stress response by E protein . Interestingly , levels of E protein were 10-fold lower than those expressed in SARS-CoV-infected cells , but were sufficient to reduce the increase in stress response genes , indicating the robust effect of E protein . In addition , rSARS-CoV-ΔE titers were not significantly increased by providing E protein in trans , probably due to the low levels of E protein expressed in rSARS-CoV-ΔE infected cells , indicating that the presence or absence of E protein , and not the amount of virus , was responsible for the increase in stress response and apoptosis . In addition , stress induced by another virus , RSV , was also downregulated by SARS-CoV E protein . Furthermore , expression of E protein in the absence of virus infection reduced stress induced by tunicamycin or thapsigargin . SARS-CoV E protein also inhibited a subset of the stress response . Specifically , E protein inhibited the activation of the XBP-1-mediated pathway of the UPR , and apoptosis induced by SARS-CoV . We have shown that in MA-104 cells infected with rSARS-CoV-ΔE , two important pro-inflammatory cytokines ( CCL2/MCP-1 and CXCL2/macrophage inflammatory protein 2 [MIP-2] ) were downregulated , indicating that the E protein reduces virus-induced inflammation . SARS-CoV is the most pathogenic human coronavirus known [70] . Besides pneumonia , SARS-CoV causes diarrhea [71] , lymphopenia [72] , haematological disorders [47] , pulmonary vasculitis , and thrombosis [73] , [74] . In previous reports , we showed that rSARS-CoV-ΔE was attenuated in hamsters and hACE2 transgenic mice [26] , [27] . The relevance of virus-host interaction in virus attenuation is high as differences in virulence are frequently due to differences in host responses , rather than to virus growth kinetics [75] , [76] . Coronavirus infection induces an ER stress response due to the extensive use of ER membranes for RNA synthesis [35] , [36] and virion assembly at the ER-Golgi intermediate compartment [64] , [77] . Further , it has been shown that SARS-CoV structural proteins S , 6 , and 3a [66] , [78] , [79] , [80] , and the accessory protein 8ab [81] induce ER stress responses . Using genomic approaches , the upregulation of stress genes in SARS-CoV-infected Huh-7 [82] , Vero [59] , and blood mononuclear cells [83] , [84] has been reported in cell cultures and also in vivo [75] , [85] . We show , for the first time , that SARS-CoV E protein limits the stress response elicited by SARS-CoV infection , which probably represents a selective advantage for the virus . In fact , we have shown that rSARS-CoV-ΔE is cleared faster than rSARS-CoV with E protein [26] , [27] . We observed that genes related to hsps were upregulated in rSARS-CoV-ΔE infected compared to wt virus-infected cells . The presence of hsps on the cell surface facilitates the elimination of infected cells by natural killer ( NK ) and T cell subsets [32] . Hsps facilitate the presentation of antigenic peptides by the major histocompatibility complex I ( MHC I ) , helping clearance of infected cells by CD8+ T cells [86] . SARS-CoV E protein expressed in trans reduced the stress response induced by rSARS-CoV-ΔE , by a heterologous virus such as RSV ( without affecting the amount of virus in both cases ) , and by non-viral agents , such as thapsigargin and tunicamycin . Therefore , E protein limited the ER stress caused by the unbalance of ER Ca++ ion concentrations , and by the inhibition of N-glycosylation leading to the accumulation of misfolded or unfolded proteins . Overall , these results showed that the downregulation of the stress response by SARS-CoV E protein was a general phenomenon . In order to analyze the specific pathways modulated by SARS-CoV E protein , the three branches of the UPR were analyzed . Only the XBP-1 pathway was significantly activated in cells infected with rSARS-CoV-ΔE compared to infection with the wt virus . Possibly , the partial activation of the UPR was not sufficient to alleviate cellular stress , and cell apoptosis was induced to help virus clearance [31] , [41] . The ectopic expression of coronavirus E protein induces apoptosis in the absence of infection [87] , [88] , whereas in this manuscript we describe that the expression of E protein in the context of SARS-CoV infection , limited the levels of apoptosis in infected cells , which may represent an advantage for virus production and dissemination [89] . This is not surprising , as previous experiments were performed in transfected cells and not in the context of viral infection , and as many other viral proteins such as 3C-like protease , spike , membrane , nucleocapsid , 3a , 3b , and 7a ( reviewed by Tan et al . in [45] ) , and proteins 6 , 7b , and 8a [78] , [90] , [91] also elicit apoptosis . Removal of the E gene from SARS-CoV led to an increase in stress responses and UPR . Nevertheless , the stress and UPR responses were not able to balance the homeostasis of the system and apoptosis was increased as a defense mechanism that may have contributed to the attenuation observed in rSARS-CoV-ΔE-infected hamsters and mice [26] , [27] . Overall , these data indicate that the regulatory influence of E protein on signaling pathways leading to apoptosis still needs further clarification . The control of the stress response and apoptosis by a viral protein has also been observed in infections by human cytomegalovirus , in which the UL38 protein suppresses ER stress-induced death , preventing premature cell death and facilitating efficient virus replication [92] , [93] . The expression of genes leading to exuberant inflammation has been associated with SARS-CoV-induced pathology [75] , [76] . The upregulation of stress genes observed in SARS-CoV-infected cells when the E gene was deleted probably diminished proinflammatory processes , leading to a decrease in pathology [94] , [95] . In fact , we have observed that MAPK phosphatases DUSP1 and DUSP10 were upregulated in rSARS-CoV-ΔE-infected cells when compared to wt virus-infected cells . DUSP proteins are critical regulators of innate immune responses [96] . Using DUSP1 and DUSP10 knock out cell cultures and mice , it has been shown that these genes limit the expression of inflammatory genes such as TNF , IL-6 , CCL2/MCP-1 , CCL3 , CCL4 and CXCL2/MIP-2 [60] , [97] , [98] , [99] . Interestingly , we observed a decrease in the expression of CXCL2/MIP-2 and CCL2/MCP-1 in rSARS-CoV-ΔE infected MA-104 cells compared to wt virus-infected cells , probably contributing to the reduction of lung inflammation that we observed in vivo [26] , [27] . In human SARS , increases in IL-6 , CCL2/MCP-1 and CXCL10/IP-10 expression were detected in the lungs of human patients with fatal SARS [48] , [49] , [100] . Furthermore , persistent expression of CCL2/MCP-1 , CXCL9/MIG and CXCL10/IP-10 was observed in the blood of SARS patients with fatal disease [48] , [49] , [100] , reinforcing the idea that elevated expression of proinflammatory cytokines significantly contributes to the pathogenicity of the virus . In summary , we found that deletion of the E gene from SARS-CoV increased the expression of host genes involved in stress response and immunoregulation , among others , and decreased those involved in inflammation . Further , SARS-CoV E protein reduced the stress caused by two viruses , SARS-CoV and RSV , and by drugs . E protein may represent a novel strategy used by SARS-CoV to increase its virulence and may also serve as a potential therapeutic target in outbreaks of SARS-CoV or other coronaviruses .
rSARS-CoV and rSARS-CoV-ΔE were rescued from infectious cDNA clones as previously described [26] , [101] . rSARS-CoV-ΔE was passaged 16 times in Vero E6 cells and characterized in vitro and in vivo ( rSARS-CoV-ΔE-P16 ) [56] . Remarkably , only a single mutation , at position 23312 , which resulted in a serine to phenylalanine mutation in the gene S , was detected in the rSARS-CoV-ΔE passaged 16 times [56] . All work with infectious viruses was performed in biosafety level ( BSL ) 3 facilities by personnel wearing positive-pressure air purifying respirators ( 3M HEPA AirMate , St . Paul , MN ) . African Green monkey kidney-derived Vero E6 cells were kindly provided by Eric Snijder ( Medical Center , University of Leiden , The Netherlands ) . African monkey kidney-derived MA-104 cells were kindly provided by J . Buesa ( Universidad de Valencia , Valencia , Spain ) . Human colon carcinoma-derived CaCo-2 cells were obtained from the European Collection of Cell Cultures ( EACC 86010202 ) . Human hepatocarcinoma-derived Huh7 cells were provided by R . Bartenschlager ( Department for Molecular Biology , University of Heidelberg , Germany ) . Rhesus monkey kidney-derived FRhK-4 cells were obtained from the American Type Culture Collection ( ATCC CRL-1688 ) . Porcine kidney-derived PK15 cells were provided by A . Carrascosa ( Centro de Biología Molecular , Madrid , Spain ) . Human hepatocarcinoma-derived HepG2 cells were provided by M . Esteban ( Centro Nacional de Biotecnología , Madrid , Spain ) . Human kidney-derived 293 cells were obtained from the American Type Culture Collection ( ATCC CRL-1573 ) . The 293-derived clone 293T , which expresses the SV40 T antigen , was obtained from the American Type Culture Collection ( ATCC CRL-11268 ) . In all cases , cells were grown in Dulbecco's modified Eagle's medium ( GIBCO ) supplemented with 25 mM HEPES and 10% fetal bovine serum ( Biowhittaker ) . Virus titrations were performed in Vero E6 cells following standard procedures using closed flasks or plates sealed in plastic bags , as previously described [26] . Subconfluent monolayers ( 90% confluency ) of Vero E6 and MA-104 cells were infected at an moi of 2 with rSARS-CoV-ΔE , or rSARS-CoV . Culture supernatants were collected at different hpi and virus titer was determined as previously described [26] . Subconfluent Vero E6 , MA-104 , CaCo-2 , Huh7 , FRhK-4 , PK15 , HepG2 , 293 and 293T cells grown in 9 cm2 flasks were infected at an moi of 1 , 3 or 5 . At different times pi , cells were washed in ice-cold phosphate-buffered saline ( PBS ) and fixed with 4% paraformaldehyde for 30 min at room temperature . The cells were then permeabilized with 0 . 2% saponin in blocking solution ( PBS , pH 7 . 4 , containing 10% FBS ) for 1 h at room temperature and incubated with a SARS-CoV N protein-specific monoclonal antibody ( SA46-4 ) , kindly provided by Ying Fang ( Center for Infectious Disease Research and Vaccinology , Brookings , South Dakota , USA ) for 90 min at room temperature . Cells were then washed three times with PBS , incubated with Alexa 488-conjugated mouse antibodies ( Molecular Probes ) at 1∶500 dilution in blocking solution for 30 min at room temperature and washed five times with PBS . The slides were removed , mounted with glass coverslips and analyzed with a Zeiss Axiophot fluorescence microscope . Vero E6 or MA-104 cells were mock-infected or infected at an moi of 2 with rSARS-CoV or rSARS-CoV-ΔE . Total RNA was extracted using a RNeasy mini kit ( Qiagen ) according to the manufacturer's instructions and RNA integrity was measured in a bioanalyzer ( Agilent Technologies , Inc . ) . RNAs were biotin-labeled using the One cycle target-labeling kit ( Affymetrix , Santa Clara , CA ) . Briefly , cDNA was synthesized from 5 µg total RNA using an oligo-dT primer with a T7 RNA polymerase promoter site added to the 3′ end . After second-strand synthesis , in vitro transcription was performed using T7 RNA polymerase to produce biotin-labeled cRNA . cRNA preparations ( 15 µg ) were fragmented at 94°C for 35 min into 35–200 bases in length and added to a hybridization solution ( 100 mM 4-morpholinopropanosulfonate acid , 1 M Na+ , 20 mM EDTA and 0 , 01% Tween-20 ) . The cRNAs ( 10 µg ) were hybridized to Human Genome U133 plus 2 . 0 Arrays ( Affymetrix , Santa Clara , CA ) at 45°C for 16 hours . The arrays were stained with streptavidin-phycoerythrin and read at 1 . 56 µm in a GeneChip Scanner 3000 7G System ( Affymetrix , Santa Clara , CA ) . Three independent microarrays were hybridized for each experiment . Data analysis was performed with the system affylma GUI R [102] . Robust Multi-array Analysis ( RMA ) algorithm was used for background correction , normalization and presentation of the expression levels [103] . Next , analysis of differential expression was performed with the Bayes t-statistics using microarray data ( limma ) linear models , included in the affylmGUI package . P-values were corrected for multiple-testing using the Benjamini-Hochberg's method ( False Discovery Rate ) [104] , [105] . Genes were considered differentially expressed if the FDR were <0 . 01 . In addition , only genes with a signal log ratio of more than one or less than minus one were considered for further analysis . To understand the biological significance underlying the gene expression data , gene set enrichment analysis ( GSEA ) was used [106] . This method analyzes all of the gene expression data to identify genes coordinately regulated in predefined gene sets . GSEA was applied independently to gene expression results obtained at 15 hpi and to those obtained at 65 hpi . Gene expression results were sorted by their logRatios . Gene Sets based on Gene Ontology keywords as defined in the subset C5 of Molecular Signatures Database ( MSigDB v2 . 5 ) [106] were used . 1402 Gene Sets containing more than 4 and less than 501 members were considered . 1000 permutations were performed . In each case , the top 20 Gene Sets showing positive correlation with upregulated genes in our data were further analyzed . Total RNA from Vero E6 , or MA-104-infected cells was extracted using the Qiagen RNeasy kit according to the manufacturer's instructions and used to determine N gene subgenomic ( sg ) mRNA and genomic RNA levels by qRT-PCR . Reactions were performed at 37°C for 2 h with a High Capacity cDNA transcription kit ( Applied Biosystems ) using 100 ng of total RNA and the antisense primers Q-NsgSARS-RS ( 5′-TGGGTCCACCAAATGTAATGC-3′ ) , complementary to nt 44 to 64 of N gene; and Q-SARS-2015-RS ( 5′- ATGGCGTCGACAAGACGTAAT-3′ ) , complementary to nt 1995 to 2015 of genomic RNA . cDNAs were amplified by PCR using the Power SYBR Green PCR Master Mix ( Applied Biosystems ) and oligonucleotides Q-NsgSARS-VS ( 5′-AAGCAACCAACCTCGATCTC-3′ ) , complementary to the virus leader sequence , and Q-SARS-1931-VS ( 5′-ACCACTCAATTCCTGATTTGCA-3′ ) , complementary to nucleotides 1931 to 1952 of genomic RNA , and the oligonucleotides RS previously described [1] . All the primers were designed using Primer Express software ( Applied Biosystems ) . Data were acquired with an ABI PRISM 7000 sequence detection system ( Applied Biosystems ) and analyzed with ABI PRISM 7000 SDS version 1 . 0 software . Levels of viral RNAs are represented in comparison to reference levels from cells infected with rSARS-CoV at 0 hpi . For qRT-PCR of cellular genes , total RNA from Vero E6 , and MA-104-infected cells was extracted as described above . Reactions were performed at 37°C for 2 h using a High Capacity cDNA transcription kit ( Applied Biosystems ) using 100 ng of total RNA and random hexamer oligonucleotides . Cellular gene expression was analyzed using TaqMan gene expression assays ( Applied Biosystems ) specific for human or monkey genes ( Table 1 ) . Data were acquired with an ABI PRISM 7000 sequence detection system ( Applied Biosystems ) and analyzed with ABI PRISM 7000 SDS version 1 . 0 software . Gene expression in rSARS-CoV-ΔE and rSARS-CoV-infected cells were compared . Alternatively , gene expression in rSARS-CoV-ΔE or SARS-CoV-infected cells was compared to mock-infected cells . Quantification was achieved using the 2−ΔΔCt method , which is a convenient way to analyze relative changes in gene expression in qPCR experiments [107] . The data represent the average of three independent experiments . Vero E6 cells grown to 90% confluence in M24 wells , were infected at an moi of 0 . 5 with rSARS-CoV-ΔE-P1 and -P16 and rSARS-CoV . Ninety min after infection , cells were transfected with 1 µg of the plasmid pcDNA3 . 1-E expressing the SARS-CoV E protein [77] , or empty plasmid as control , using 1 µg of Lipofectamine 2000 ( Invitrogen ) according to manufacturer's instructions . Total RNA from mock- infected or rSARS-CoV-infected cultures was extracted at different times pi as described above and used to quantify the expression of the stress-response genes hspA1A , hsp90AA1 , hspH1 , SERPINH1 and hspE1 by qRT-PCR as described . Vero E6 cells grown to 90% confluence in M24 multiwell plates were transfected with 1 µg of the plasmid pcDNA3 . 1-E , or empty plasmid as control , using 1 µg of Lipofectamine 2000 ( Invitrogen ) according to the manufacturer's instructions . After an incubation period of 5 h at 37°C , the transfection media were replaced and cells were incubated at 37°C for 24 h . Then , the cells were infected at an moi of 2 with RSV , Long strain [108] . RSV was provided by Dr . Blanca Garcia-Barreno ( National Institute of Microbiology , Madrid ) , and titrated on Hep-2 cells as previously described [109] . Total RNA from mock-infected or RSV-infected cultures was extracted at different times pi as described above and used to quantify the expression of the stress-response genes hspAA1 , UBB , hspH1 , SERPINH1 and hspE1 by qRT-PCR as described . Vero E6 and MA-104 cells were transfected with plasmid pcDNA3 . 1-E or empty plasmid as above . Twenty-four hpt , cells were cultured in media containing 1000 nM thapsigargin , or 2 µg/ml of tunicamycin and incubated for another 8 or 20 hours , before analysis of expression of the UPR-induced genes , GRP78 and GRP94 . Cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE ) . Proteins were transferred to a nitrocellulose membrane with a Bio-Rad mini protean II electroblotting apparatus at 150 mA for 2 h in 25 mM Tris-192 mM glycine buffer , pH 8 . 3 , containing 20% methanol . Membranes were blocked for 1 h with 5% dried skim milk in TBS ( 20 mM Tris-HCl , pH 7 . 5 , 150 mM NaCl ) and incubated with antibodies specific for hsp60 ( Cell Signaling , Ref . 4870 ) , hsp90 ( Cell Signaling , Ref . 4877 ) , SARS-CoV E protein ( kindly provided by Shen Shuo , Institute of Molecular and Cellular Biology , Singapore ) , phospho-PERK ( Santa Cruz Biotechnology , Ref . sc-32577 ) , GAPDH ( Abcam , Ref . ab9485 ) , and ATF-6 ( Abcam , Refs . ab11909 and ab37149 ) . Bound antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies ( Cappel ) and the ECL detection system ( Amersham Pharmacia Biotech ) . Total RNA from mock-infected or rSARS-CoV or rSARS-CoV-ΔE-infected cells was used for RT-PCR analysis of XBP-1 mRNA . cDNA was prepared using the specific oligonucleotide XBP1-RS ( 5′-CTGGGTCCTTCTGGGTAGAC-3′ ) . cDNAs were amplified by PCR using the sense primer XBP1-VS ( 5′-CTGGAACAGCAAGTGGTAGA-3′ ) , and XBP1-RS , flanking the splicing region of XBP-1 mRNA [69] . The RT-PCR products were resolved by electrophoresis in 2% agarose gels . Vero E6 cells were grown to confluence in 12 . 5 cm2 flasks and infected at an moi of 4 with rSARS-CoV or rSARS-CoV-ΔE . At 4 , 15 and 24 hpi , cells were treated with fluorescein isothiocyanate ( FITC ) -conjugated annexin V ( Southern Biotech ) to identify apoptotic cells measured by flow cytometry , as previously described [110] . Cells were then treated with 1 volume of 4% paraformaldehyde in PBS to inactivate virus . At the end of the process , propidium iodide ( PI ) staining was performed to differentiate cells in early apoptosis ( Annexin V+ , PI− ) from those in late apoptosis ( Annexin V+ , PI+ ) stage . | To identify potential mechanisms mediating the in vivo attenuation of SARS-CoV lacking the E gene ( rSARS-CoV-ΔE ) , the effect of the presence of the E gene on host gene expression was studied . In rSARS-CoV-ΔE-infected cells , the expression of at least 25 stress response genes was preferentially upregulated , compared to cells infected with rSARS-CoV . E protein supplied in trans reversed the increase in stress response genes observed in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus , and by treatment with drugs causing stress by different mechanisms . Furthermore , in the presence of the E protein a subset ( IRE-1 pathway ) , but not two others ( PERK and ATF-6 ) , of the unfolded protein response was also reduced . Nevertheless , the activation of the unfolded protein response to control cell homeostasis was not sufficient to alleviate cell stress , and an increase in cell apoptosis in cells infected with the virus lacking E protein was observed . This apoptotic response was probably induced to protect the host by limiting virus production and dissemination . In cells infected with rSARS-CoV-ΔE , genes associated with the proinflammatory pathway were down-regulated compared to cells infected with virus expressing E protein , supporting the idea that a reduction in inflammation was also relevant in the attenuation of the virus deletion mutant . | [
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] | 2011 | Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Regulates Cell Stress Response and Apoptosis |
Approximately 680 million people are at risk of infection with Opisthorchis viverrini ( OV ) and Clonorchis sinensis , with an estimated 10 million infected with OV in Southeast Asia alone . While opisthorchiasis is associated with hepatobiliary pathologies , such as advanced periductal fibrosis ( APF ) and cholangiocarcinoma ( CCA ) , animal models of OV infection show that immune-complex glomerulonephritis is an important renal pathology that develops simultaneously with hepatobiliary pathologies . A cardinal sign of immune-complex glomerulonephritis is the urinary excretion of immunoglobulin G ( IgG ) ( microproteinuria ) . In community-based studies in OV endemic areas along the Chi River in northeastern Thailand , we observed that over half of the participants had urine IgG against a crude OV antigen extract ( OV antigen ) . We also observed that elevated levels of urine IgG to OV antigen were not associated with the intensity of OV infection , but were likely the result of immune-complex glomerulonephritis as seen in animal models of OV infection . Moreover , we observed that urine IgG to OV antigen was excreted at concentrations 21 times higher in individuals with APF and 158 times higher in individuals with CCA than controls . We also observed that elevated urine IgG to OV antigen could identify APF+ and CCA+ individuals from non-cases . Finally , individuals with urine IgG to OV antigen had a greater risk of APF as determined by Odds Ratios ( OR = 6 . 69; 95%CI: 2 . 87 , 15 . 58 ) and a greater risk of CCA ( OR = 71 . 13; 95%CI: 15 . 13 , 334 . 0 ) than individuals with no detectable level of urine IgG to OV antigen . Herein , we show for the first time the extensive burden of renal pathology in OV endemic areas and that a urine biomarker could serve to estimate risk for both renal and hepatobiliary pathologies during OV infection , i . e . , serve as a “syndromic biomarker” of the advanced pathologies from opisthorchiasis .
Foodborne trematodiases represent an important group of communicable diseases , and some of the most clinically significant neglected tropical diseases ( NTDs ) affecting East Asia . Approximately 680 million people are at risk of infection with the human liver flukes Opisthorchis viverrini and Clonorchis sinensis [1] . In Southeast Asia alone , up to 67 million people are at risk of infection with O . viverrini ( OV ) , with 10 million people estimated to be infected with this pathogen in the Mekong Basin Subregion of Thailand and Lao PDR [2] , [3] . Humans become infected with OV by consuming raw or undercooked fish that contain the infective metacercarial stage ( for review see [4] ) . Although the infection can be eliminated by the anthelminthic praziquantel , environmental and cultural factors of the Mekong Basin region strongly favor re-infection [4] . Despite mass drug administration ( MDA ) efforts in the northeast region of Thailand ( Isaan ) , the prevalence of OV remains intransigently high [5] , [6] . Our community-based ultrasound studies in O . viverrini endemic areas along the Chi River Basin in Khon Kaen , Thailand have revealed that significant morbidity occurs early during the course of chronic OV infection , including advanced hepatobiliary pathologies such as advanced bile duct ( periductal ) fibrosis ( APF ) and bile duct cancer ( cholangiocarcinoma or CCA ) [7] , [8] . As individuals do not become symptomatic until the late stages of these diseases , early detection remains an important public health objective [4] , [6] . Although renal disease is not usually considered among the more critical pathologies of chronic opisthorchiasis , as with many other parasitic infections ( e . g . Plasmodium spp , Schistosoma spp , Filarioidea ) [9] , glomerulopathy has been reported in laboratory animal models of OV infection [10] , [11] . More specifically , early during experimental OV infection ( 8 weeks ) , hamsters develop a “mesangioproliferative glomerulonephritis” , characterized by the deposition of immune complexes ( ICs ) consisting of immunoglobulin ( Ig ) G , complement component 3 ( C3 ) , and OV tegumental antigen [10] . After 12 weeks , the infected hamsters show a complete obsolesce of the glomeruli characterized by deposition of amyloid ( AA protein ) , tubular atrophy , interstitial inflammation , and tubular fibrosis , all of which are co-incident with APF and CCA [10] . It is interesting to note that a deterioration in renal function has been reported in humans with obstructive jaundice due to OV-associated CCA in endemic areas of Thailand [12] , although this is likely a manifestation of ‘hepatorenal syndrome’ ( HRS ) , a common end stage complication of chronic hepatic diseases , such as liver cirrhosis and liver cancer [13] . Previous studies have attempted to show a correlation between the intensity of OV infection and levels of urine IgG to various crude OV antigen extracts [14]–[16] . Although urine can contain small quantities of ‘intact’ immunoglobulin as well as light and heavy chain fragments of immunoglobulin , the restrictive pore radius of the renal glomerular filter in a healthy human kidney would not filter macromolecules the size of intact IgG ( for review see [17] ) . As such , the frequent observation of elevated levels of urine IgG to OV antigen in areas of high OV transmission [14]–[16] most likely reflects structural damage from immune complex deposition in the glomeruli as observed in the hamster model of OV infection [10] , [11] . In the current manuscript , we investigated the presence of urine IgG to a crude adult OV antigen extract ( OV antigen ) in residents from OV endemic areas along the Chi River Basin , in Khon Kaen Thailand . Our hypothesis is that if levels of urine IgG to OV antigen are elevated in individuals with renal and hepatobiliary pathologies , then this non-invasive and easily assayed biomarker could serve as a single marker for both pathologies , i . e . , as a “syndromic biomarker” of advanced pathologies from chronic opisthorchiasis .
This study uses baseline data from the Khon Kaen Cancer Cohort ( KKCC ) , which was conducted in seven ( 7 ) villages with high OV transmission along the Chi River Basin in Khon Kaen Thailand . A detailed description of the KKCC and the methods used to assemble this cohort can be found in several manuscripts [7] , [8] , [18] . The dataset from the KKCC included 296 individuals divided into three clinical groups described below and shown in Table 1 . In brief , 148 males and 148 females were enrolled in the KKCC . Of the males and females in this dataset , 256 ( 86 . 4% ) were infected with OV as determined by microscopic fecal examination . Participants in the KKCC were classified into groups based on abdominal ultrasound ( US ) examination and microscopic fecal examination for OV infection . Group 1 consisted of 40 individuals considered “Endemic Normals” ( EN ) , who were age , sex and ‘nearest-eligible-neighbor’ matched with cases ( Group 3 ) and were OV negative ( OV− ) and APF negative ( APF− ) as determined by abdominal US . Group 2 consisted of 139 individuals considered “controls” , who were age , sex and ‘nearest-eligible-neighbor’ matched to cases ( Group 3 ) and were OV positive ( OV+ ) and APF negative ( APF− ) . Group 3 consisted of 117 individuals considered “cases“ who were APF positive ( APF+ ) . Group 4 was not part of the KKCC and consisted of 98 individuals with histologically proven opisthorchiasis-associated CCA whose serum and urine samples were obtained from the biological specimen repository of the Liver Fluke and Cholangiocarcinoma Research Center , Khon Kaen University , Thailand . Individuals positive for infection with OV were referred to the local public health outpost for treatment with praziquantel . All subjects in Groups 1–3 provided written informed consent using forms approved by the Ethics Committee of Khon Kaen University School of Medicine , Khon Kaen , Thailand ( reference number HE480528 ) and the Institutional Review Board of the George Washington University School of Medicine , Washington , D . C ( GWUMC IRB# 020864 ) . The serum and urine from Group 4 was obtained from the biological specimen repository of the Liver Fluke and Cholangiocarcinoma Research Center , Khon Kaen University , Thailand using a protocol approved by the Ethical Committee on Human Research , Faculty of Medicine , Khon Kaen University , Thailand ( reference Nos . HE450525 and HE531061 ) . Assessment of hepatobiliary status was done by abdominal US with positive findings scored as APF+ or APF− as previously described [7] , [8] . Two fecal samples were collected on consecutive days from each participant in Groups 1–3; fecal samples were not available for Group 4 patients ( CCA cases ) . OV infection was determined and quantified ( eggs per gram of feces or epg ) by microscopic fecal examination using the formalin-ethyl acetate concentration technique ( FECT ) as described by Elkins et al [19] on two consecutive days of fecal samples . In addition , the following samples were also collected from Groups 1–3: thirty ( 30 ) milliliters ( ml ) of venous blood collected into siliconized tubes after overnight fasting and first morning mid-stream urine samples collected into sterile containers . Venous blood samples were allowed to clot at room temperature for 30 minutes after collection , centrifuged , and the serum removed and aliquoted for storage stored at −20°C in a temperature-monitored freezer . Urine samples were centrifuged and the supernatant aliquoted and stored at −20°C in a temperature-monitored freezer . In the case of CCA patients ( Group 4 ) , serum or urine specimens were obtained by simple random sampling from the collection of biological specimens in the repository of the Liver fluke and Cholangiocarcinoma Research Center , Khon Kaen University , Thailand . Individual subjects were asked to provide morning urine into clean polypropylene containers that were kept on ice during transportation to the laboratory . Unprocessed urine samples were screened for protein by urine strip ( ARKRAY's AUTION Sticks , Japan ) and then analyzed using an Automated Urine Chemistry Analyzer ( AUTION MAX AX-4280 , Arkray , USA ) . Adult O . viverrini worms from experimentally infected hamsters were washed three times with sterile phosphate buffered saline ( PBS pH 7 . 2 ) containing 0 . 149 M sodium chloride ( Fisher Scientific , NJ ) , 8 . 29 mM disodium hydrogen phosphate ( Acros Organics , NJ ) and 18 mM sodium dihydrogen phosphate monohydrate ( Fisher Scientific , NJ ) in deionized ( DI ) water . A 100× Protease Inhibitor Cocktail ( Calbiochem , CA ) was added to the worms in PBS , which were then homogenized using a tissue grinder on ice . The worm pellet was homogenized by ultrasonic disintegrator ( MISONIC sonicator 3000 , US ) and then centrifuged at 4°C , 14000 rpm for 30 min . The BCA™ Protein Assay kit ( PIERCE , IL ) was used to determine the protein yield of the crude somatic O . viverrini adult antigen extract . The supernatant was collected and stored at −80°C until used . Flat-bottom 96-well microtiter plates ( Maxisorb , NUNC , DN ) were coated with 1 µg/ml of crude somatic O . viverrini adult antigen in PBS buffer ( pH 7 . 2 ) , which was then covered with sealing film and incubated overnight at 4°C in the dark . On the next day , the plates were washed 5 times with a buffer containing 0 . 05% Tween20 in PBS ( pH 7 . 2 ) using an automated plate washer ( Thermoelectron , MA ) . After washing , 250 µl of a blocking buffer containing 5% BSA ( Fitzgerald , MA ) in PBS and 0 . 5% Tween-20 ( Fisher , NJ ) was added to all wells , and the plates incubated at room temperature ( RT ) for 1 hour . Serum samples were diluted in a buffer which contained 5% BSA ( Fitzgerald , MA ) in PBS and 0 . 5% Tween-20 ( Fisher , NJ ) , and added to wells ( 100 µL/well ) in duplicates and incubated overnight at 4°C . Undiluted urine supernatants were added to wells ( 100 µL/well ) in duplicate and incubated overnight at 4°C . The plates were then washed 5 times with a buffer of PBS and 0 . 5% Tween-20 , and a horseradish peroxidase ( HRP ) -conjugated secondary antibody was added to all wells and incubated for 2 hours at RT . HRP-goat anti human IgG ( Zymed , CA ) was used to detect IgG in serum and urine . HRP-mouse anti-human IgG1 ( Southern Biotech , AL ) , and an HRP-mouse anti-human IgG4 ( Zymed , CA ) were used to detect IgG1 and IgG4 in serum , respectively . After incubation and washing , a substrate solution , which consisted of Ortho phenylenediamine ( Sigma , MO ) , 53 mM citric acid anhydrous ( Fisher , NJ ) , 102 mM dibasic sodium phosphate dodecahydrate ( Acros Organics , NJ ) and 30% w/w hydrogen peroxide ( Fisher , NJ ) in DI water was added to the wells and incubated at RT in the dark for 30 min . The reaction was stopped by the addition of 2N sulfuric acid ( BDH , PA ) and the plates were read using a plate reader ( SpectraMax 340PC384 system ) at 492 nm . Following the method of Quinn and colleagues [20]–[22] , we developed a diagnostic assay using an indirect ELISA that incorporates “homologous interpolation” to determine the concentration of an analyte ( e . g . , anti-OV IgG ) in either diluted serum or undiluted urine supernatant samples by interpolation of test serum or urine supernatant OD at 492 nm onto a Standard Calibration Curve ( SCC ) run on each microtitre plate . Briefly , a Standard Reference Sera ( SRS ) and urine Standard Reference Solution ( SRS ) were made by pooling of sera or urine supernatants with known high levels of IgG and its subclasses against Ov antigen from individuals who were O . viverrini egg positive ( see references for details of this method [20]–[22] ) . Each serum SRS and the urine SRS are serially diluted on each microtiter plate in two-fold steps using a dilution buffer ( 5% Bovine Serum Albumin in PBS and 0 . 5% Tween-20 at a pH of 7 . 2 ) . The ODs of each dilution point are then used to generate the SCC by 4-PL regression modeling ( SOFTmax PRO version 5 . 4 software ) [23] , [24] . To generate the SCC , Arbitrary Units ( AU ) of antibody are assigned to the Standard Calibration Curves as shown in Table S1 . The 4-PL function is used to model the characteristic curve for the SRS . As shown by Quinn et al [22] , the SRS in serial dilution should exhibit a sigmoidal shape when plotted on an OD-log10 dilution scale . The 4-PL function fits these data with a high degree of accuracy and extends the range of the assay , thus providing a more precise measurement of antibody concentration for patient sera [20]–[22] . Furthermore , 8 wells per ELISA plate were assigned as internal controls , consisting of two blanks ( no sample with/without secondary antibody ) , a positive serum and urine control , and negative serum and urine control . The ELISA was qualified for accuracy and precision as previously described [22] . Figure S1 shows a graphic representation of the methods used to derive the RDL , which was used as the threshold above which a serum or urine sample was considered positive for antibodies against OV antigen . Figure S1 Panels A , C , E and G show the mean of the combined SCCs for each antibody-antigen pair ( e . g . , serum IgG1 to OV antigen ) and their 95% CI intervals . Figure S1 Panels B , D , F and H show the derivation of the RDL . In a manner similar to Quinn et al . [22] , the RDL was derived from the level of AUs for each anti-OV-antigen antibody corresponding to the interpolated intersection of the upper 95% CI asymptote with the lower-95% CI of the standards data as shown Figure S1 Panels B , D , F , and H . A human serum or urine supernatant sample with Arbitrary Antibody units above the RDL was defined as “reactive” ( positive ) and those with Arbitrary Antibody units below the RDL as “nonreactive” ( negative ) as shown in Table 2 . Again following Quinn et al . , [22] , we defined ELISA assay “accuracy” as the exactness of the assay to measure a known , true value of urine anti-OV IgG and to measure it repeatedly and expressed assay accuracy as the percent error between the assay-determined values and the assigned value for that serum . A percent error of ≤20% was considered the acceptable level of accuracy for the ELISAs presented herein [22] , [25] . We also defined assay “precision” according to Quinn et al [22] , [25] as the measure of the degree of repeatability of an assay under normal operating conditions , and expressed assay precision as the coefficient of variation ( CV ) of the concentrations calculated for the SCC dilutions within a single assay plate ( intra-assay precision ) and between different assay plates ( inter-assay precision ) determined over time and controlling for different operators . Acceptable levels of intra-assay and inter-assay precision are 10% and 20% , respectively [22] , [25] . The “goodness of fit” of each SCC was used to determine how closely each SRS fit the 4-PL model . Goodness of fit was expressed as the regression coefficient ( R2 ) of the SCC . An R2 value that approached unity ( 1 . 00 ) was indicative of a good fit for the data to the curve [22] , [25] and these are shown for each SCC in Figure S1 Panels A , C , E and G . Data distributions were assessed for normality . For normally-distributed data , differences between AUs of antibody to OV antigen were compared between different matrices ( urine or sera ) by clinical groups or by different matrices using the intensity of OV infection by Student's t-test . Non-parametric data were compared using the Mann-Whitney U-test . One-way ANOVA ( normally distributed data ) or a Kruskal-Wallis tests ( non-normally distributed data ) were used to determine statistically significant associations among the aforementioned groups followed by a Bonferonni corrected pairwise comparison when comparing pairs in each group . All statistical analyses were performed using SAS 9 . 2 . Results were considered significant when the p-value was less than <0 . 05 . Sensitivity was calculated as the number of individuals with serum or urine IgG to OV antigen above the cut-off set for each assay as determine by Receiver Operator Characteristic ( ROC ) curves obtained with ROCKIT1 . 1 software . Specificity was calculated as the number of individuals with serum or urine IgG to OV antigen below the “cut-off” set for each assay divided by the total number of control individuals ( EN or APF− ) . As we propose these assays as screening tests , we selected the cut-offs to achieve the highest sensitivity without losing specificity: i . e . , the best trade off between high sensitivity and modest specificity . The area under the curve ( AUC ) is a measure of the ROC's validity . The 45-degree line in each ROC curve analysis subsumed an area equal to 0 . 50 and is equivalent to using a coin toss procedure to classify participants . To calculate the positive predictive values ( PPV ) for each assay , we used a 50% prevalence of infection OV as determined by microscopic fecal exam in the age range of 20 to 60 years from our previous studies of the KKCC [7] , [8] , [18] . The following formula was used to estimate the Positive Predictive Value with prevalence set at 0 . 50 for each:
Table 1 shows the age of the study participants by clinical group . Of the two hundred and ninety-six ( 296 ) individuals who provided fecal specimens for examination by FECT ( Groups 1 , 2 and 3 ) , 256 ( 86 . 4% ) were confirmed to be infected with OV by microscopic fecal exam . One hundred and seventeen individuals ( n = 117 ) were assigned the status of APF+ and assigned the status of case . Additionally , 139 OV+ and APF− individuals were assigned the status of age , sex and “nearest-eligible-neighbor” matched controls ( Group 2 ) . Note that the larger sample size of the controls is due to the fact that 22 of the cases were matched with two controls for greater sample size . Forty ( n = 40 ) OV− and APF− individuals were assigned the status of Endemic Normals . Finally , 98 serum samples were obtained by simple random sampling from the biological specimen bank of the Liver Fluke and Cholangiocarcinoma Research Center , Khon Kaen University , Thailand; only 8 of these individuals has urine samples . Fecal specimens were not available for the CCA patients . Figure S2 shows the parallelism of each SCC by plotting linearized versions of each SCC for serum IgG ( P = 0 . 225 ) ( Figure S2 Panel A ) , serum IgG1 ( P = 00 . 240 ) ( Figure S2 Panel B ) , serum IgG4 ( P = 0 . 136 ) ( Figure S2 Panel C ) , and urine IgG ( P = 0 . 402 ) ( Panel D ) . The regression slopes of the fitted SCCs were not significantly different as determined by ANOVA ( p>0 . 05 ) , indicating parallelism among each group of SCCs [24] . Using the RDL shown in Figure S1 Panel B as the detection threshold , Table 2 shows that serum IgG against OV antigen was detected in all individuals ( 100%; n = 394 ) in the study . Similarly , Table 2 also shows that using the RDL shown in Figure S1 Panel D as the detection threshold for anti-IgG1 OV antigen , nearly all ( 98%; n = 387 ) of individuals in the study had detectable levels of this serum antibody to OV antigen , again , regardless of OV or clinical status . Finally , using the RDL shown in Figure S1 Panel F as the detection threshold , Table 2 shows that a third to half of the individuals in each clinical group had detectable levels of IgG4 to OV antigen . Using the RDL as the detection threshold ( Figure S1 Panel H ) , Table 2 shows that over 60% of APF+ individuals had detectable levels of IgG to OV antigen in their urine . A lower proportion ( 38% ) of individuals who were OV+ and APF− ( matched controls ) had detectable levels of urine IgG to OV antigen . All eight ( n = 8 ) of the CCA patients who had urine samples had detectable levels of IgG to OV in their urine . Levels of serum IgG ( Figure 1 Panel A ) and serum IgG1 ( Figure 1 Panel B ) to OV antigen were significantly higher ( P<0 . 001 , for both ) in individuals with both lighter ( 1–499 epg ) or heavier ( ≥500 epg ) OV infections compared to EN individuals ( no eggs in feces ) . In addition , individuals with heavier OV infection had higher levels of serum IgG1 to OV antigen than individuals with lighter OV infection ( Figure 1 Panel B ) . Levels of urine IgG were not significantly elevated in any of the infection groups ( Figure 1 Panel D ) . Figure 2 Panel D shows that urine levels of IgG to OV antigen were significantly higher ( P<0 . 001 ) in APF+ individuals than individuals in the EN or APF− groups: i . e . , on average , 21 times higher in APF+ individuals than EN individuals and 7 times higher in APF+ individuals than APF− individuals . Similarly , Figure 2 Panel D shows that urine levels of IgG to OV antigen were significantly higher ( P<0 . 001 ) in CCA+ individuals than individuals in the EN , APF− , and APF+ groups . On average , urine levels of IgG to OV antigen were 158 times higher in CCA+ individuals than EN individuals; 21 times higher in CCA+ individuals than APF− individuals; and 7 times higher in CCA+ individuals than APF+ individuals . Figure S3 Panel A shows that there is no association between levels of serum IgG to OV antigen and urine levels of IgG to OV antigen in the same individuals . This was found for all infection and clinical categories . Figure S3 Panel B shows that few of OV+ individuals in either group ( APF− or APF+ ) had proteinuria as determined by point-of-care testing using a strip-based urine reagent device , with a positive urine dipstick test for protein defined by a color change of “+” or greater that equates to at least 30 mg/L of protein . Table 3 shows the area under the curve ( AUC ) for ROC curve analyses with PPVs determined using 50% prevalence: e . g . , serum IgG against OV antigen had an AUC of 0 . 68 and a PPV of 0 . 60 using the cutoff of 35 . 66 AUs resulting from ROC curve analysis of the highest possible sensitivity without a decrease in the specificity of the assay . Using these cutoffs , we estimated crude and adjusted Odds Ratios ( 95%CIs ) for the risk of OV-positivity . The ROC curves for serum IgG1 against OV-antigen had the best AUC with 0 . 68 using an antibody cutoff of 9 . 21 AUs . Using this cutoff resulted in a PPV of 0 . 61 , as well as an adjusted OR of 2 . 51 ( 95%CI 1 . 26 , 5 . 00 ) for the risk of OV positivity . Despite a poor AUC and a weak PPV , urine IgG indicated significant risk for OV infection with a crude OR of 7 . 60 ( 95%CI 3 . 56 , 16 . 20 ) and an adjusted OR of 7 . 68 ( 95%CI 3 . 58 , 16 . 50 ) . Table 4 shows that elevated levels of serum IgG1 to OV antigen had the best combination of sensitivity and specificity for the prediction of heavy OV infection ( >500 epg ) compared to individuals negative for OV . In addition , elevated levels of serum IgG and IgG1 to OV antigen showed excellent capacity to indicate risk of high OV infection as determined by significant crude and adjusted ORs ( Table 4 ) . Though serum IgG4 to OV antigen and urine IgG to OV antigen showed moderate discriminatory capacity for OV infection , Table 4 shows that elevated levels of urine IgG to OV antigen could still predict risk of OV infection as shown in an adjusted OR of 7 . 88 ( 95%CI: 2 . 64 , 23 . 51 ) . Table 5 shows that elevated levels of urine IgG could discriminate between individuals who were APF positive from EN individuals , with an AUC of 0 . 72 and a PPV of 0 . 67 using a cutoff of 4 . 00 AUs of urine IgG to OV antigen . In addition , urine IgG to OV antigen showed significant crude and adjusted ORs for predicting risk of APF: crude OR = 6 . 34 ( 95%CI: 2 . 75 , 14 . 66 ) and adjusted OR = 6 . 69 ( 95%CI: 2 . 87 , 15 . 58 ) . Elevated levels of urine IgG to OV antigen could also differentiate individuals with APF from matched APF− controls as well as an OR which indicated risk of APF in adjusted and unadjusted models . Table S2 shows that serum IgG to OV antigen could also modestly discriminate APF positive individuals from individuals from the EN group , with an AUC of 0 . 52 and a PPV of 0 . 52 and an adjusted OR of 2 . 71 ( 95%CI: 1 . 26 , 5 . 84 ) . Table 5 also shows that elevated levels of urine IgG to OV antigen can discriminate better than serum antibodies individuals with confirmed CCA versus endemic normals , with an AUC of 0 . 97 for the ROC curve analysis and a PPV of 0 . 79 when the antibody cutoff of 2 . 84 AU is used . In addition , Table 5 shows that elevated levels of urine IgG to OV antigen are strongly associated with CCA: crude OR = 85 . 00 ( 95%CI: 19 . 89 , 362 . 0 ) and adjusted for sex and age OR = 71 . 13 ( 95%CI: 15 . 13 , 334 . 0 ) . Table S3 shows that serum antibodies to OV antigen do not have the sensitivity nor specificity to be predictive of CCA nor the crude and adjusted Odds Ratios to indicate risk for CCA compared to urine IgG to OV antigen .
In the current manuscript , we show that more than half of the individuals resident in endemic areas along the Chi River Basin in Khon Kaen , northeastern ( Isaan ) Thailand have detectable levels of urine IgG to adult OV antigen extract . Moreover , elevated levels of urine IgG to OV antigen were not associated with either the intensity of OV infection ( as measured by fecal egg counts ) or the levels of serum antibodies to OV antigen . These findings support our hypothesis that urine IgG to OV antigen most likely represents renal pathology in the form of immune complex-mediated structural damage to the glomeruli ( injury of podocytes ) and to the tubular interstitium as observed in animal models of OV infection [10] , [11] . Interestingly , we found that IgG to OV antigen was detectable in the urine at concentrations 21-times higher in individuals with OV-induced APF and 158-times higher in individuals with OV induced CCA compared to controls . As shown in Table 5 , individuals with elevated urine IgG to OV antigen had an increased risk for APF ( adjusted OR of 6 . 69; 95%CI: 2 . 87 , 15 . 58 ) and an increased risk for CCA ( adjusted OR of 71 . 13; 95%CI: 15 . 13 , 334 . 0 ) than individuals who had no detectable levels of urine IgG to OV antigen . We also found that a single measurement of urine IgG to OV antigen had good predictive value for the detection of both APF and CCA compared to age , sex , and nearest-neighbor matched non-cases ( either EN or APF negative controls ) . Hence , as shown in Figure 3 , our study adds to the literature on the pathophysiology of opisthorchiasis the possibility that renal pathology occurs simultaneously with the advanced hepatobiliary pathologies more commonly associated with this infection and that renal pathology can be detected by elevated levels of urine IgG to OV antigen [10] , [11] . A urine-based assay that could simultaneously evaluate the clinical status of individuals for both renal and hepatobiliary pathologies from chronic opisthorchiasis would be of profound benefit in Southeast Asia , especially in the resource-limited settings of the Mekong Basin region countries of Thailand , Laos and Cambodia . Antibodies have long been known to play a central role in the immune response to Opisthorchis viverrini infection [14] , [26]–[36] . Individuals and animals infected with this food-borne trematode show high serum/plasma levels of the classic antibodies associated with helminth infections such as IgG , IgG1 , IgG4 and IgE to crude OV antigen extracts . As such , it has been hypothesized that circulating antibodies to OV antigens may “leak” from the plasma into the urine at levels proportionate to the intensity of OV infection [14] , [16] . However , as seen in these other studies [14] , [16] , we found that urine IgG to OV antigen is a poor method for diagnosing OV infection and an even poorer method for predicting the intensity of OV infection ( Tables 3 and 4 ) . In addition , we observed only weak correlations between circulating levels of serum IgG to OV antigen and levels of urine IgG to OV antigen . These findings are consistent with our current understanding of the pathophysiology of urine proteinuria ( e . g . IgG in urine ) from various clinical settings [9] , [11] , [17] , [37] , [38] . A healthy glomerular capillary wall should efficiently restrict the passage of IgG from the blood ( plasma ) into Bowman's space on the basis of this intact immunoglobulin's molecular size , electrical charge , and steric configuration; for example , the restrictive pore radius of the renal glomerular filter is 45 Ångstroms ( Å ) , whereas intact IgG has a molecular radius of 55 Å ( see [17] for excellent review ) . Additionally , IgG is a cationic protein , which means it binds strongly to the negatively charged proximal tubule cells [17] . Hence , even if small amounts of IgG are filtered into Bowman's space and , thereby into the tubular lumen , they would be readily reabsorbed in the proximal tubule . In keeping with similar findings in experimental animal models of OV infection [10] , we hypothesize that the frequent observation of IgG in the urine of OV infected individuals reflects glomerulopathy . More specifically , we suspect that it reflects structural damage to the glomerular capillary wall characterized by injured podocytes , resulting in increased glomerular permeability or increased glomerular pore size that allows for passage of macromolecules such as IgG . In addition , the reabsorption capacity of the epithelial cells of the proximal tubules may also be impaired . Both glomerular and tubular damage are cardinal signs of kidney disease [17] , [37] , [38] . The fact that the IgG detected in the urine is specific for OV antigen leads us to postulate that the observed renal pathology is the result of immune complex deposition similar to that observed in animal models of OV [10] . Renal pathology caused by immune complex deposition has been described in association with other parasitic helminth infections such as schistosomiasis [9] . Immune complexes are putatively deposited in the glomerular subendothelium , resulting in activation of complement , chemoattraction of leukocytes , and an inflammatory reaction that leads to disruption of the glomerular basement membrane with enlargement of glomerular barrier pores , thus permitting passage of high molecular weight ( HMW ) macromolecules such as IgG to which the basement membrane is normally impermeable [17] , [38] . The increased load of HMW macromolecules in the tubular lumen leads to saturation of the re-absorptive mechanism by tubular cells . As mentioned above , the renal pathology observed in OV-infected hamsters is a mesangial proliferative glomerulonephritis with the immune complexes consisting of IgG , complement component 3 ( C3 ) , and OV antigen [10] , [11] , [39] . Immune complexes accumulate in the glomeruli of the hamster kidney either by ( a ) passive trapping of circulating immune complexes or ( b ) in situ formation by the binding of antibody to OV-antigen that was previously deposited in the glomeruli . During the course of a single experimental OV infection , hamsters develop progressive sclerosis of glomeruli , tubular atrophy , as well as interstitial inflammation and fibrosis that appear to be coincident with the development of bile duct fibrosis and bile duct cancer [10] . However , not all individuals , who are chronically infected with O . viverrini , develop renal or hepatobiliary abnormalities . From our community-based studies in OV endemic areas in northeastern Thailand , we have observed that a subset of individuals infected with OV respond to the chronic inflammation from OV infection response with pathologies [7] , [8] , [18] . We have termed these individuals as having a ‘pro-inflammatory’ phenotype [40] . The current study adds to our hypothesis evidence that the ‘pro-inflammatory’ phenotype extends to renal pathology associated with chronic opisthorchiasis ( i . e . , APF and CCA ) . We suspect that individuals with the ‘pro-inflammatory phenotype’ have a dysregulation of inflammatory cytokine production in response to chronic fluke infection , which manifests as inflammation in the renal filters ( glomeruli ) , enlarging them to allow passage of macroproteins ( e . g . , intact IgG to OV antigen ) from the plasma . As seen in the hamster model , we also suspect that over time there is a progressive obsolescence of the glomeruli , tubular atrophy , interstitial inflammation , and renal fibrosis associated with this proteinuria [10] , [11] , [39] . As urine IgG to OV antigen is an easily accessible biomarker , we suggest that it could serve as a biomarker for the multiple inflammation-related pathologies from opisthorchiasis . An important question arising from our study is the absence of detectable proteinuria by means of point-of-care testing using a strip-based urine reagent device . Theoretically , the increased permeability of the glomerular barrier that allows the filtering of IgG should also result in proportional losses of albumin into the urine , which is the principal protein component detected by the urine dipstick test [37] . There are two possibilities that explain the false negative urine dipstick results . First , total protein concentration in urine depends on degree of hydration ( i . e . , the specific gravity of the urine ) . False negative results may relate to the manner in which the urine samples were collected [37] , including collection of the entire urine sample and not a mid-stream urine sample ( which represents urine from the kidneys ) or the collection of first morning urine ( 24 hour urine sample ) , which represents an accumulation of urine that could result in a dilution of the sample that would decrease the sensitivity of the dipstick test to detect proteinuria [37] . On the other hand , the indirect ELISA would be much more sensitive to the presence of IgG in urine than a dipstick test because ( a ) the urine is concentrated in a preparation step prior to immunoassaying and ( b ) the monoclonal antibodies to human IgG used in the ELISA are much more sensitive and specific than the dipstick's colorimetric method which is used to primarily detect albumin in the urine . The second hypothesis is the possibility of a restrictive mechanism that prohibits the filtering of albumin into urine but that allows leakage of IgG . Numerous investigators ( see [37] for review ) have observed that the movement of albumin into Bowman's space is not restricted by pore size , but by its negative charge and the consequent repulsive electrostatic interactions with the negatively charged glomerular endothelium . Hence , it is quite possible that the presence of IgG in the urine due to OV infection is not accompanied by appreciable albuminuria—a hypothesis that clearly deserves further study . A final issue is the relationship between the concurrent renal and hepatobiliary pathologies observed in this study . The strongest associations in the current study were between elevated urine IgG to OV antigen and APF or CCA: individuals with elevated levels of urine IgG to OV antigen had a 6 times greater risk of having APF and a 71-times greater risk of having CCA ( Table 5 adjusted OR ) than individuals with no detectable IgG to OV antigen in their urine . Additionally , a single measurement of urine IgG to OV antigen had a good positive predictive value for the detection of both APF and CCA . However , as shown in the hamster model for OV-induced bile duct fibrosis and CCA [10] , no plausible physiologic relationship exists between the hepatobiliary and renal pathologies induced by chronic OV infection . It appears that they are the result of two distinct pathological mechanisms that develop simultaneously during OV infection . As we have written extensively , hepatobiliary pathology from chronic opisthorchiasis is likely the result of repeated injury sustained by the biliary epithelium from a combination of the mechanical , toxic , and immune insults associated with the presence of the fluke in the bile duct [for review see [8] , [18] ) . As individuals are infected with O . viverrini for many years ( often a lifetime ) , a persistent cycle of tissue damage and repair takes place in the intrahepatic biliary ducts , creating a chronic inflammatory milieu that stimulates periductal fibrogenesis and tumorigenesis [8] , [18] . The renal injury observed herein is the likely the consequence of chronic OV infection , resulting from the sustained systemic effects of the parasitic infection on the host immune response ( i . e . , immune complex–mediated glomerulopathy ) . Despite the lack of a common pathogenic mechanism , the renal and hepatobiliary pathologies associated with OV infection develop simultaneously in the animal model and probably in humans as well . As such , a biomarker for renal pathology could be equally indicative of risk for APF and CCA . It should also be noted that as early as 1990 , Mairiang et al . [12] , reported acute renal failure in nearly all patients with obstructive jaundice due to CCA caused by opisthorchiasis . This is in keeping with our own findings of elevated urine IgG to OV antigen in CCA cases . However , in the case of end-stage CCA , the finding of proteinuria may reflect “hepatorenal syndrome” ( HRS ) , which is a common complication of patients with advanced forms of liver disease such as CCA and cirrhosis [13] and is caused by intense vasoconstriction of the renal circulation , leading to a pronounced reduction in glomerular perfusion and filtration [13] . HRS generally occurs in late stages of severe liver disease , when patients have already manifested significant complications of cirrhosis . HRS is an acute condition with a very poor prognosis . As such , there remains some question as to whether the renal pathology , presumably chronic in nature , that is seen among individuals with opisthorchiasis-induced APF is the same as that seen among CCA cases , as the latter may reflect HRS rather than immune complex associated glomerulonephritis . As shown by our community-based studies [7] , [8] , [18] , chronic O . viverrini infection results in a persistent immunological and inflammatory challenge to the human host . For the first time , we have shown that chronic OV infection may also result in a significant burden of renal disease in the form of immune complex-mediated glomerulopathy . The importance of this study is the observation that this renal pathology can be readily detected in the urine by an immunoassay for IgG against OV-antigen and that elevated levels of urinary IgG to OV-antigen are also strongly associated with hepatobiliary pathologies . In future studies , we plan to improve on the sensitivity and specificity of this biomarker by screening urine for the specific antigens recognized by IgG in the crude adult OV-antigen extract used here . Recent advances in immunomics , in which the O . viverrini proteome can be assembled on a microarray chip , allows for high-throughput screening of urine samples to determine the most abundantly recognized proteins . These could subsequently be developed as recombinant proteins as reagents for urine diagnostic tests . As such , screening for urinary IgG to specific recombinant OV antigens might be used to indicate risk of several pathologies that can arise from chronic opisthorchiasis , and thereby be used as a “syndromic biomarker” of chronic opisthorchiasis . | Approximately 680 million people risk infection with food-borne trematodes , including Opisthorchis viverrini ( OV ) . Animal models show that significant kidney pathology results from OV infection as detected by antibodies in urine ( microproteinuria ) . However , kidney pathology in humans infected with OV is often overlooked because it develops alongside more severe pathologies such as bile duct fibrosis and bile duct cancer . In Northeastern Thailand , the researchers observed that OV infected individuals had elevated levels of urine IgG against OV antigen that was not associated with the level of OV infection . The researchers observed that urine IgG to OV antigen was associated with bile duct fibrosis and bile duct cancer . Moreover , individuals with urine IgG to OV antigen also had elevated risk of bile duct fibrosis and bile duct cancer than individuals with no urine IgG to OV antigen . For the first time , OV infection has been shown to result in significant kidney disease in humans , which is also strongly associated with bile duct pathology . A urine-based assay that could indicate both renal and bile duct pathology from OV infection would be of profound benefit in Southeast Asia , especially in the resource-limited settings of the Mekong Basin region countries of Thailand , Laos and Cambodia . | [
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] | 2013 | Microproteinuria during Opisthorchis viverrini Infection: A Biomarker for Advanced Renal and Hepatobiliary Pathologies from Chronic Opisthorchiasis |
Azoospermia is one of the major reproductive disorders which cause male infertility in humans; however , the etiology of this disease is largely unknown . In the present study , six missense mutations of WT1 gene were detected in 529 human patients with non-obstructive azoospermia ( NOA ) , indicating a strong association between WT1 mutation and NOA . The Wilms tumor gene , Wt1 , is specifically expressed in Sertoli cells ( SCs ) which support spermatogenesis . To examine the functions of this gene in spermatogenesis , Wt1 was deleted in adult testis using Wt1flox and Cre-ERTM mice strains . We found that inactivation of Wt1 resulted in massive germ cell death and only SCs were present in most of the seminiferous tubules which was very similar to NOA in humans . In investigating the potential mechanism for this , histological studies revealed that the blood–testis barrier ( BTB ) was disrupted in Wt1 deficient testes . In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs . Further studies found that the expression of cell polarity associated genes ( Par6b and E-cadherin ) and Wnt signaling genes ( Wnt4 , Wnt11 ) were downregulated in Wt1 deficient SCs , and that the expression of Par6b and E-cadherin was regulated by Wnt4 . Our findings suggest that Wt1 is important in spermatogenesis by regulating the polarity of SCs via Wnt signaling pathway and that WT1 mutation is one of the genetic causes of NOA in humans .
Infertility is a common health problem which affects about 15–20% of couples . Among infertile couples , about 50% are related to male infertility [1] , a major cause of which is azoospermia . Genetic causes of azoospermia include autosomal chromosome abnormalities , Y chromosome microdeletions , and single gene mutations . Several genes have been reported to play a role in azoospermia , including PRM1 , SPATA16 , AURKC , and KLHL10 [2]–[5] . As support cells , Sertoli cell ( SCs ) play central roles in testis development and spermatogenesis . During mouse embryogenesis , SCs emerge at E10 . 5 and are involved in seminiferous cord formation and prevent germ-cell entry into meiosis [6] . Mullerian duct regression in males is induced by anti-Müllerian hormone ( AMH ) which is secreted by immature SCs [6] , [7] . At puberty , SCs undergo terminal differentiation and develop complex morphological interactions with each other and with adjacent germ cells . Mammalian spermatogenesis is dependent on the proper functioning of SCs which provide structural support and nutrition to developing germ cells , phagocytose degenerating germ cells and residual bodies , release spermatids at spermiation , and produce a host of proteins that regulate or respond to pituitary hormone [8]–[10] . Our previous studies have demonstrated that Wt1 , which encodes a nuclear transcription factor , is important for testes development with inactivation of Wt1 in SCs between E12 . 5–E14 . 5 , resulting in testicular cord disruption and testes dysgenesis [11] . However , the functional significance of Wt1 in adult testis has been unclear , in part due to the gonadal agenesis of Wt1−/− [12] and Wt1R394W/R394W [13] mice . Rao et al reported that knock-down of Wt1 using siRNA in postnatal SCs caused reduced sperm count [14] , suggesting that Wt1 plays a role in spermatogenesis . However , the exact function of Wt1 in spermatogenesis and underlying mechanism by which it plays a role are still largely unknown . In this study , we demonstrated that inactivation of Wt1 in adult SCs resulted in massive germ cell death with only SCs surviving in the seminiferous tubules . Six WT1 missense mutations were detected in 529 NOA patients by mutational analysis , indicating a strong association between WT1 mutation and spermatogenic defects in human . We further demonstrated that Wt1 is critical for maintaining the polarity of SCs , likely via Wnt signaling pathways . Inactivation of Wt1 resulted in loss of polarity in SCs and abnormal tight junction assembly which in turn caused germ cell death .
Wt1−/flox; Cre-ERTM and control mice ( Wt1−/flox , Wt1+/flox; Cre-ERTM ) were obtained by intercrossing of Wt1flox/flox and Wt1+/−; Cre-ERTM mice . The growth of Wt1−/flox; Cre-ERTM mice were indistinguishable from that of control mice and the morphology and histology of Wt1−/flox; Cre-ERTM testes were completely normal ( data not shown ) . To induce Cre activity , Wt1−/flox; Cre-ERTM and littermate control mice were injected with 9 mg/40 g ( body weight ) Tamoxifen for two consecutive days at 8 weeks of age . The testes were collected at 1 , 2 , and 3 weeks after Tamoxifen injection . The efficiency of Tamoxifen induced Cre recombination was examined by Wt1 Real-time PCR ( Figure S2G ) and western blot ( Figure S3 ) . Compared to control testis Wt1 mRNA was reduced about 50% in Wt1−/flox; Cre-ERTM testis , indicating that Wt1 was deleted in about 50% of SCs . Because the Cre activation results in the in-frame deletion of exons 8 and 9 , the Wt1Δ allele results in a truncated protein . Our previous work indicated that this truncation has the same phenotypic effect as Wt1 deletion [11] . As shown in Figure S3 , approximately the same amount of wild type and truncated Wt1 protein was observed in Wt1−/flox; Cre-ERTM testes 1 week after Tamoxifen induction , indicating that Wt1 function was lost in approximately 50% of Sertoli cells . This was consistent with real time PCR results . The size of testes from Wt1−/flox; Cre-ERTM mice was dramatically reduced 3 weeks after Tamoxifen treatment ( Figure 1C ) . Histological analysis results revealed that the seminiferous tubules were grossly normal in Wt1−/flox; Cre-ERTM testis 1 week after Tamoxifen induction ( Figure 1G ) , although vacuolization was first noted in a small number of tubules . Severe epithelial vacuolization ( Figure 1H , arrows ) was noted in mutant testis 2 weeks after Tamoxifen induction . At 3 weeks post-Tamoxifen , atrophic seminiferous tubules with massive cell loss were evident in mutant testes ( Figure 1I ) , whereas the control testis was completely normal ( Figure 1F ) . In addition to germ cell loss , inactivation of Wt1 in adult mice also resulted in multiple phenotypes , including accumulation of ascitic fluid ( Figure S1A ) , atropic spleen ( Figure S1B ) , abnormal pancreas ( Fig . S1C ) , and renal failure ( Figure S1D ) , consistent with a previous report [15] . In this study , however , the defect in spermatogenesis was not observed , likely because mice were treated with Tamoxifen for 5 consecutive days and , died earlier in our model system , Apoptotic cells ( Figure S4C , white arrows ) were noted by TUNEL assay at the peripheral region of seminiferous tubules in Wt1−/flox; Cre-ERTM testis at 1 week after Tamoxifen induction , and the number of TUNEL positive cells ( Figure S4D , white arrows ) was dramatically increased at 2 weeks . In contrast , very few apoptotic cells were detected in control testis at both 1 ( Figure S4A ) and 2 weeks ( Figure S4B ) . The results of statistical analysis showed that the number of apoptotic cells was significantly increased in Wt1−/flox; Cre-ERTM mice at both 1 and 2 weeks after Tamoxifen treatment compared to control testes ( Figure S4E ) . To identify the cells which survived in Wt1-deficient testes , immunohistochemistry analyses were carried out . Because the truncated WT1 protein encoded by the recombined Wt1Δ allele is recognized by the Wt1 antibody , Wt1 antibody could be employed to identify Sertoli cells . Wt1 positive Sertoli cells were localized at the periphery of the seminiferous tubules in both control ( Figure 2A , arrows ) and Wt1−/flox; Cre-ERTM ( Figure 2B , arrows ) testes . Multiple layers of GCNA1-positive germ cells were noted in seminiferous tubules of control testes ( Figure 2C ) . However , very few GCNA1 positive germ cells were present in Wt1−/flox; Cre-ERTM testis ( Figure 2D , arrows ) , and some tubules were complete devoid of germ cells ( Figure 2D , asterisks ) . The cauda epididymes of control mice were filled with normal mature sperm ( Figure 2E , arrows ) . In contrast , only cellular debris and round , prematurely released spermatocytes were noted in Wt1−/flox; Cre-ERTM mice ( Figure 2F , arrow heads ) . These results indicate that Wt1 is essential for SCs function such that Wt1-deficient SCs cannot support the development of germ cells , eventually resulting in germ cell death . Azoospermia is one of the major causes of male infertility in human . The testes histology of human non-obstructive azoospermia ( NOA ) was very similar to Wt1-deficient testes . As shown in Figure S6 , only Sertoli cells ( black arrows ) were presented in the seminiferous tubules ( asterisks ) of human NOA patients . To determine whether WT1 mutation is associated with spermatogenic defects in humans , following exonic capture we sequenced ( mean depth of 43× ) the exons of WT1 in 529 non-obstructive azoospermia ( NOA ) patients and 709 men with proven fertility . On average per sample , 96% of target bases were covered at least once , and 78% were covered sufficiently for variant calling ( Table S1 ) . The first coding exon was poorly captured due to its extremely high GC content . As shown in Figure 3 and Table S2 , 6 WT1 missense mutations were detected in 6 patients while no mutations were found in the control group . By Chi-square analysis , there was a statistically significant ( p = 0 . 004 ) difference in WT1 status between the NOA population and the control group . The WT1 variants present in the NOA patients have not been detected as SNPs in the 1000 Genomes Project , further indicating the significance of the observation . Two mutations were in the two zinc finger domains ( encoded by exons 8 and 9 ) which are most important for DNA-protein interaction [16] . The other four mutations were localized to the transcription regulatory domain ( Exons 3 , 4 , 6 ) . The functional significance of the NOA-associated mutations was predicted using a combination of several approaches as previously described [17] . All 6 mutations were predicted to be deleterious by several complementary nsSNV scoring algorithms ( Table S2 ) , including SIFT [18] , PolyPhen2 [19] , PhastCons [20] and GERP scores [21] ( Table S2 ) . The observation of these predicted deleterious mutations specifically in the NOA population strongly indicates that WT1 is also important for spermatogenesis in human and that WT1 mutation plays an etiologic role in azoospermia . One of the major functions of SCs is to maintain the integrity of the BTB , which , when disrupted , results in germ cell death and spermatogenic defects [22] , [23] . To test whether the integrity of the BTB was damaged in Wt1-deficient testes , surface biotinylated reagent was injected into the testicular interstitium of both control and Wt1−/flox; Cre-ERTM testes at 1 week after Tamoxifen treatment and before obvious histological changes were observed . In control mice , biotin tracer was restricted to the testicular interstitium and the basal compartment of the seminiferous tubules with no tracer being observed in the tubular lumen ( Figure 4A , Figure S8A and C ) . However in Tamoxifen treated Wt1−/flox; Cre-ERTM testes , biotin tracer was also present along the SCs plasma membrane from the basement membrane to the lumen in about 30% of seminiferous tubules ( Figure 4B , Figure S8B and D , asterisks ) . This difference was significant ( Figure S8E ) . These results indicated that the integrity of BTB in Wt1−/flox; Cre-ERTM testes was disrupted after Wt1 inactivation such that it was permeable to the biotin tracer . The BTB structure in Wt1-deficient testes was further examined by transmission electron microscope ( TEM ) . As shown in Figure 4E , a normal BTB ( bracket ) structure with F-actin bundles ( black arrows ) was observed in control testes . In contrast , in Wt1-deficient testes , the BTB was aberrant with intermittent loss of F-actin bundles ( Figure 4F , black arrows ) and the occurrence of vacuoles , likely formed as a result of loss of interaction between SCs ( Figure 4F , asterisks and black arrowheads ) . We also found that the structure of apical ES ( Ectoplasmic Specialization ) was damaged in Wt1−/flox; Cre-ERTM testes at 1 week after Tamoxifen treatment . In control testes , the apical ES between Sertoli cell and elongated sperm was well organized with F-actin bundles ( Figure 4C , arrows ) , whereas the apical ES structure was disrupted and there was a loss of F-actin bundles in Wt1-deficient testes ( Figure 4D , arrows ) . The expression of BTB components was examined by immunofluorescence at 1 week after Tamoxifen treatment . As shown in Figure S7 , tight junction protein Claudin11 ( A , B ) , adhesion junction proteins N-cadherin ( C , D ) and β-catenin ( E , F ) , and gap junction protein CX43 ( G , H ) were detected at the peripheral region of seminiferous tubules where tight junctions are formed in control testes ( A , C , E , G ) . In contrast , the expression of these genes was significantly reduced in Wt1−/flox; Cre-ERTM testes ( B , D , F , H ) , indicating the BTB structure was disrupted in Wt1-deficient testes at 1 week after Tamoxifen induction . To explore the physiological function of Wt1 in spermatogenesis , control and Wt1−/flox; Cre-ERTM SCs were isolated from 2 months old mice and cultured in vitro . Wt1 was deleted by addition of 4-OH-tamoxifen to the culture medium . As shown in Figure 5A , the control SCs had a cuboidal epithelial morphology as visualized by F-actin staining ( Figure 5A ) . In contrast , SCs assumed a mesenchymal-like morphology when Wt1 was inactivated by Tamoxifen induction ( Figure 5C ) . The quantitative results showed that the number of mesenchymal-like cells was dramatically increased after Wt1 inactivation , and this difference was significant ( Figure 5E ) . Tight junctions were well established in control SCs after a few days culture indicating by ZO-1 staining ( Figure 5B , white arrows ) . However , in Wt1-deficient SCs , ZO-1 protein was diffused in the cytosol and no obvious staining was noted at the cell junctions ( Figure 5D ) . The tight junction formation by SCs was further examined by Paracellular FITC-dextran Flux assay . As shown in Figure 5F , the permeability of Wt1-deficient SCs was significantly increased compared to control cells . This result further confirmed that the tight junction formation of SCs is disrupted when Wt1 is inactivated , consistent with the Zo-1 staining data . Our previous study found that deletion of Wt1 in SCs during early stage of embryonic development resulted in downregulation of Sox9 and AMH [11] . To test whether deletion of Wt1 in adult testes also affects the expression of SC specific genes , immunohistochemistry and real time PCR were conducted . The results of immunohistochemistry showed that the expression of Sox9 ( Figure S2A , B ) , AR ( Figure S2C , D ) , and Gata4 ( Figure S2E , F ) was not changed in Wt1−/flox; Cre-ERTM testes ( Figure S2B , D , F ) 3 weeks after Tamoxifen treatment . Real time PCR further confirmed these results ( Figure S2G ) . The expression of SC-specific genes was also examined in Wt1-deficient SCs by immunofluorescence and real time PCR . As shown in Figure S9 , inactivation of Wt1 in cultured primary SCs did not affect the expression of SC specific genes , such as Sox9 , Dmrt1 , Gata4 , AR , Gata1 , and Nr5a1 . To explore the molecular mechanism of the spermatogenic defect in Wt1-deficient testes , RNA-Seq analysis were performed using mRNA from control and Wt1-deficient SCs . A total of 710 differentially ( p-value<0 . 05 ) expressed genes were identified ( 456 upregulated and 254 downregulated genes ) , the raw data has been uploaded to http://www . ncbi . nlm . nih . gov/geo/ , the accession number is GSE46664 . As listed in Table S3 , the genes were differentially expressed in multiple pathways based on the pathway term analysis . However , no specific pathway was significantly altered in Wt1-deficient SCs . In the present study , we found that the morphology of SCs was transformed from epithelium into mesenchyme with a concurrent loss tight junction formation after Wt1 inactivation . It has been reported previously that Wt1 is involved in EMT process during kidney and heart development [24] , [25] . Therefore , EMT and cell polarity related genes were selected for further analysis . RNA-Seq analysis revealed that cell polarity-associated genes , such as E-cadherin , Par6b , were significantly decreased in Wt1-deficient SCs . In contrast , the expression of the EMT related genes was not significantly changed . We also found that Wnt signaling gene , Wnt11 was downregulated in Wt1-deficient SCs . The expression of Wnt4 was also decreased in Wt1-deficient SCs , but this was not statistically significant . To further verify the RNA-Seq results , the expression of genes related to EMT and cell polarity was analyzed by real time PCR . As shown in Figure 6A , the expression of Par6b , E-cadherin , Wnt11 , and Wnt4 was significantly decreased in Wt1-deficient SCs . Western blot analysis also showed that the expression of E-cadherin and Par6b was dramatically reduced in Wt1-deficient SCs , however , ZO-1 and N-cadherin expression was not changed ( Figure 6B ) . In contrast , Snail1 , Snail2 , Twist , Zeb , Sip , Mmp2 were not differentially expressed between control and Wt1-deficient SCs ( Figure S10 ) . The expression of cell polarity related genes in Wt1-deficient testes was also examined by immunofluorescence and real time PCR . The results of immunofluorescence ( Figure S5A–D ) showed that the localization of E-cadherin and Par6b was disorganized in Wt1−/flox; Cre-ERTM testes at 1 week after Tamoxifen induction . Real time PCR results showed that the mRNA level of Par6b and Wnt4 was significantly reduced in Wt1−/flox; Cre-ERTM testes at 1 week after Tamoxifen treatment . mRNA level of E-cadherin and Wnt11 was also reduced , but this was not statistically significant ( Figure S5E ) . These results were consistent with the results from the in vitro study . Notably , when Tamoxifen treated Wt1−/flox; Cre-ERTM SCs were transfected with Wt1 expressing adenovirus , the mRNA level of Par6b , E-cadherin , Wnt4 , and Wnt11 was completely rescued , indicating that the expression of these genes was regulated by Wt1 directly or indirectly ( Figure 6C ) . To examine whether Wt1's role in regulating SCs polarity is mediated by Wnt signaling , Wnt4 was knocked down in cultured SCs using siRNA . As shown in Figure 6F , compared to treatment with scrambled siRNA , the mRNA level of Wnt4 was reduced about 70% after transfection with Wnt4-specific siRNA , and the expression of E-cadherin and Par6b was also reduced about 30–50% respectively ( Figure 6F ) , suggesting that the expression of E-cadherin and Par6b is regulated by Wnt4 in SCs . To further confirm these results , Tamoxifen treated Wt1−/flox; Cre-ERTM SCs were transfected with Wnt4- and Wnt11- expressing adenovirus . We found that the expression of Par6b was dramatically increased upon over-expression of both Wnt4 and Wnt11 in Wt1-deficient SCs ( Figure 6D ) . However , the expression of E-cadherin was not rescued ( Figure 6E ) , suggesting that the expression of E-cadherin was also directly regulated by Wt1 . Although the WT1 mutations detected in NOA patients were predicted to be deleterious by several complementary nsSNV scoring algorithms ( Table S2 ) , the effect of mutations on Wt1 function was further assessed by in vitro functional analyses . Two Wt1 expressing adenovirus carrying the zinc finger domain mutations detected in patient W643 and W606 were generated by site-directed mutagenesis and designated as Wt1R362Q and Wt1K386R . We found that both of these mutations did not affect the nuclear localization of Wt1 protein ( Figure S11 ) . However , functional analysis showed that both these mutated Wt1 could not induced the expression of E-cadherin , Par6b , Wnt4 , and Wnt11 in Wt1-deficient SCs ( Figure 7A ) , indicating that these mutations caused Wt1 protein loss of function . To examine whether these mutation affect the ability of Wt1 protein binding to the promoter of a Wt1 targeting gene , a ChIP assay was performed using HepG2 cells . As shown in Figure 7B , C , a 211 bp DNA fragment in the Wnt4 promoter region was pulled down by Wt1 , but not Wt1R362Q and Wt1K386R . These results indicated that these mutations affected the interaction between Wt1 protein and DNA .
Wt1 is specifically expressed in SCs of the testes and our previous study has demonstrated that it is critical for testicular development [11] , [12] . However , the functional significance of Wt1 in adult testes is largely unknown . In the present study , we have demonstrated for the first time in vivo , using a conditional Wt1 knockout mice strain , that Wt1 plays an important role in maintaining SC polarity and that inactivation of Wt1 leads to the loss of epithelial characteristics of SCs which in turn results in germ cell death . In adult mammalian testes , SCs are polarized epithelial cells which , as nurse cells , are essential for germ cell division and differentiation [26] , providing structural support and creating an immunological barrier from the systemic circulation [26] , [27] . The latter function is conferred by the blood-testes barrier ( BTB ) , which is formed by tight junctions ( TJs ) , basal ectoplasmic specialization ( ES ) , and desmosome-like junctions between SCs [23] , [28] . The integrity of the BTB structure is very important for spermatogenesis , and abnormal assembly of BTB is known to cause germ cell loss and male infertility [29] . In this study , we have demonstrated , both by biotin tracer injection experiments ( Figure 4B ) and TEM ( Figure 4F ) , that loss of Wt1 disrupted the normal BTB structure . While Wt1 is a transcriptional regulator , the expression of genes known to be important in spermatogenesis , AR [30] , Dmrt1 [31] , Gata4 [32] , Connexin43 [33] , Claudin11 [34] , was not changed in Wt1-deficient testes , suggesting that the spermatogenic defect of Wt1-deficient mice was mechanistically different . Analysis of cultured SCs revealed that upon Wt1 deletion , cells underwent a morphologic transformation from cuboidal epithelium into mesenchyme-like cells with a concomitant loss of tight junctions and a significant reduction in E-cadherin expression , both of which are normal features of epithelial cells . These results indicate that Wt1 is critical for the maintenance of epithelial characteristics of SCs and that the loss of these characteristics results in germ cell death . A recent study has shown that Wt1 is essential for epithelial to mesenchymal transition ( EMT ) in epicardial cells by controlling the expression of Snai1 [25] . However , we found that the expression of the EMT-related genes ( such as Snail , Twist , Zeb , Sip , et al ) was not changed in Wt1-deficient SCs . Instead , genes important for cell polarity maintenance ( such as Par6b , Cdc42ep5 ) and Wnt pathway signaling genes ( Wnt4 and Wnt11 ) were significantly decreased in SCs when Wt1 gene was deleted . These results suggest that the function of Wt1 is different in different cell types which is probably dependent on the interaction with different cofactors . PAR6b directly binds to PAR3 , aPKC , and CDC42 [35] . This complex is involved in tight junction formation in epithelial cells [35] , and inhibiting the expression of Par3 , Par6 , or aPKC blocks tight junction assembly in MDCK cells [35] , [36] . The PAR3/PAR6b/aPKC complex is also involved in regulating apical ectoplasmic specialization ( ES ) and blood testes barrier ( BTB ) restructuring in the testis . Knockdown of Par6b or Par3 results in defective tight junction formation in cultured SCs [37] . These data suggest that the spermatogenesis defect we observed following Wt1 ablation is due to the loss of SC polarity . Non-canonical Wnt signaling is known to play an important role in regulating cell polarity and motility [38] . In vertebrates , Wnt4 , Wnt5a and Wnt11 encode ligands that activate the Wnt signaling pathway [39]–[41] that is critical for regulating epithelial versus mesenchymal cell characteristics . Wnt4 is necessary and sufficient for MET during kidney development [42] , [43] , and Wt1 directly activates Wnt4 expression in developing kidney . Interestingly , by recruiting different cofactors , Wt1 represses Wnt4 expression and induces an epithelial to mesenchymal transition in epicardium [24] . In this study , we found that the expression of Wnt4 and Wnt11 was activated by Wt1 in SCs and that deletion of Wt1 resulted in downregulation of these genes and loss of epithelial cell polarity . Wnt4 RNAi knockdown experiments showed that the expression of Par6b and E-cadherin was regulated by Wnt4 in SCs . On the other hand , overexpression of Wnt4 and Wnt11 in Wt1-deficient SCs induced Par6b expression . These data suggest that Wt1 controls SCs polarity indirectly through Wnt4 . However , studies in 3T3 cells suggest that Wt1 can directly regulates E-cadherin expression [44] , and our data cannot exclude this possibility in SCs . WT1 germline mutation is known to result in a predisposition to Wilms tumors , male sex differentiation disorder , and early-onset renal failure [45]–[51] . These WT1 mutations are truncating mutations or missense mutations that are predicted , based on structural studies , to alter the ability of zinc finger domains to bind to DNA . We have now identified 6 novel missense mutations in NOA patients . These data strongly suggest that WT1 is also important for spermatogenesis in humans and that WT1 gene mutations play an etiologic role in azoospermia . Four of these mutations occur in the regulatory domain of the protein . Two occur in exons encoding zinc finger domains known to be critical for DNA binding , and functional studies revealed that they affected the ability of Wt1 to bind to Wnt4 promoter and result in a failure to induce targeting gene expression . Of not , these Wt1 mutants did not act in a dominant negative manner . Interestingly , neither aberrant sex determination nor renal failure was noted in the NOA patients carrying WT1 mutations . We speculate that the NOA-associated mutations represent a functionally new type of WT1 alteration which probably also affects the ability to bind other transcription factors and/or chromatin-remodeling factors critical for its regulatory role . In summary , our data strongly support a model by which loss of Wt1 in SCs results in downregulation of non-canonical Wnt signaling genes ( Wnt4 and Wnt11 ) and cell polarity genes ( E-cadherin and Par6b ) . This altered expression subsequently leads to loss of the epithelial characteristic and BTB integrity in SCs and concomitant germ cell loss . Such disruption of the BTB is also known to result in germ cell loss and male infertility in humans , and our observation of WT1 mutations in NOA patients strongly suggests that altered WT1 function constitutes one genetic cause of azoospermia in humans . Wt1 mutational analysis will be potentially useful clinically for characterizing NOA patients .
Peripheral blood samples from 529 patients with non-obstructive azoospermia ( NOA ) and 709 men with proven fertility were collected from Peking University Shenzhen Hospital and the Center of Reproductive Medicine , Tongji Medical College , Huazhong University of Science and Technology . The inclusion criteria for NOA patients included: 1 ) no sperm detected in the pellets of semen samples on three different occasions , 2 ) no inflammation and injury of the reproductive system or pelvic cavity , 3 ) no endocrinological defect , and 4 ) no karyotypic abnormality nor Y chromosome microdeletion . Testicular biopsy and histological analysis were conducted for azoospermic men wherever possible . All the control men had fathered at least one child . This study was approved by the ethical committees of Peking University Shenzhen Hospital and Tongji Medical College , and all participants signed the consent form permitting the collection and use of their blood samples in the study . Genomic DNA was isolated from the blood samples using QIAamp DNA Mini Kits ( QIAGEN ) . The exons of WT1 and other 600 genes were selectively captured by NimbleGen custom arrays ( Roche NimbleGen , Inc , USA ) and sequenced following the standard Illumina-based resequencing procedures as described [52] . Reads were mapped to the UCSC human reference genome build hg19 by SOAPaligner [53] . Mutation analysis for all the exons of WT1 were performed with SOAPsnp [54] . Novel coding mutations in WT1 were defined as those variants that had not been annotated in dbSNP nor in the publicly available dataset from the 1000 Genome Project . To further refine those novel mutations that may be associated with NOA , all the genetic variants detected in the fertile men were also eliminated for subsequent analysis . To validate the novel mutations identified in the WT1 gene by the massively parallel sequencing , primers flanking the point mutations were designed with Primer3 software ( primer sequences are given in Table S5 ) . PCR was performed using the following conditions: 94°C for 7 minutes; 30 cycles of denaturation at 94°C for 30 seconds , annealing at 57–60°C for 45 seconds , extension at 72°C for 1 minute , and a final extension at 72°C for 7 minutes . PCR products were checked on 1 . 5% agarose gel . The amplification product was directly sequenced using the 3730 DNA analyzer ( Applied Biosystems ) . All animal work was carried out in accordance with institutional animal care and use committee ( IACUC ) regulations . All the mice were maintained in a C57BL/6;129/SvEv mixed background . Wt1+/flox [11] mice were mated with mice carrying the Wt1-null allele ( Wt1+/− ) [12] and Cre-ERTM [55] transgenic mice to produce Wt1−/flox; Cre-ERTM offspring . DNA isolated from tail biopsies was used for genotyping . Genotyping was performed by PCR as described previously [11] , [56] . Tamoxifen ( Sigma ) was dissolved in corn oil at a final concentration of 20 mg/ml . Two month old control ( Wt1+/flox; Cre-ERTM , Wt1−/flox , and Wt1+/flox ) and Wt1−/flox; Cre- ERTM males were injected intraperitoneally with 9 mg/40 g body weight for 2 consecutive days . Testes were dissected from mutant and control mice immediately after euthanasia and fixed in 4% paraformaldehyde for up to 24 hr , stored in 70% ethanol , and embedded in paraffin . Five-micrometer-thick sections were cut and mounted on glass slides . After deparaffinization , slides were stained with H&E for histological analyses . IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC ( avidin–biotin–peroxidase ) kit ( Vector Laboratories , Burlingame , CA ) as recommended and using antibodies to WT1 ( Santa Cruz , sc-192 ) and GCNA1 ( gift of Dr . George Enders ) . The IHC procedure was performed as described previously [13] . Stained slides were examined with a Leica DMR Epifluorescence Microscope , and images were captured by a Hamamatsu CCD camera . Total RNA was prepared from cultured SCs isolated from 2 month old control ( Wt1+/flox; Cre-ERTM , Wt1−/flox , and Wt1+/flox ) and Wt1−/flox; Cre-ERTM mice after Tamoxifen treatment using an RNeasy Minikit ( Ambion , Austin , TX ) . The main reagents and instruments used for RNA library construction and deep sequencing were the Illumina Gene Expression Sample Prep Kit , Solexa Sequencing Chip ( flowcell ) , Illumina Cluster Station and Illumina HiSeq 2000 System . Sequence tags were prepared using the Illumina Digital Gene Expression Tag Profiling Kit , according to the manufacturer's protocol . The RNA-Seq was performed as described by Zhang et al [57] . In brief , raw data was filtered to remove adaptor tags , low quality tags and tags with a single copy number . Clean tags were classified according to their copy number and the saturation of the library was analyzed . All clean tags were mapped to the reference sequences , filtered and the remainder of the clean tags was designated as unambiguous clean tags . The number of unambiguous clean tags for each gene was calculated and normalized to the number of transcripts per million clean tags ( TPM ) . To identify DEGs between control and Wt1-deficient Sertoli cells , the number of raw clean tags in each library was normalized to the TPM to obtain the normalized gene expression level . DEGs was identified as previously described [58] using a false discovery rate ( FDR ) ≤0 . 001 and a threshold absolute log2-fold change ≥1 for the sequence counts across the libraries . A modified method was used to isolate primary Sertoli cells from the testes of 6-week-old mice [59] . Testes were decapsulated under the dissection microscope . The seminiferous tubules were pooled and washed with phosphate-buffered saline ( PBS ) three times . The tubules were incubated with 2 mg/ml collagenase I ( Sigma ) and 0 . 5 mg/ml DNase I ( sigma ) in DMEM for 30 minutes at 37°C on a shaker , then washed twice with DMEM and further digested with 2 mg/ml collagenase I , 0 . 5 mg/ml DNase I and 1 mg/ml hyaluronidase type III ( Sigma ) for 20–30 minutes at 37°C . The tubules were allowed to settle and were then washed twice with DMEM before being digested with 2 mg/ml collagenase I , 0 . 5 mg/ml DNase I , 2 mg/ml hyaluronidase , and 1 mg/ml trypsin for 40–60 minutes at 37°C . This final digestion step resulted in a cell suspension containing primarily Sertoli cells and type A spermatogonia . The dispersed cells were then washed twice with DMEM and placed into culture dishes in DMEM containing 10% fetal calf serum and incubated at 37°C and 5% CO2 . Spermatogonia were unable to attach to the dish and were removed after the medium change on the next day . 4-OH-Tamoxifen ( Sigma , H7904 ) was dissolved in ethanol to generate a 1 mM stock solution and further diluted to appropriate concentrations prior to use . Recombination was initiated by adding 4-OH-TM to cultured Sertoli cells at a final concentration of 1 µM . After 3 days culture , total RNA and protein were extracted as described below . Total RNA was extracted from cultured Sertoli cells or testes using a Qiagen RNeasy kit in accordance with the manufacturer's instructions . To quantify gene expression , real-time SybrGreen assay was performed with the isolated RNA . Gene expression was quantified relative to the expression of the gene for Gapdh ( glyceraldehyde-3-phosphate dehydrogenase ) . Primers used for the RT-PCR are listed in sTable 4 . Cells were lysed in radioimmune precipitation assay lysis buffer ( RIPA ) containing complete Mini protease-inhibitor cocktail tablets ( Roche , Mannheim , Germany ) . The protein concentration in the supernatants was estimated using a Bradford assay ( Bio-Rad Laboratories , Hercules , CA ) . The proteins were electrophoresed under reducing conditions in 12% SDS-PAGE gels and transferred to nitrocellulose membranes . Blots were incubated overnight at 4°C with primary antibody and followed by 1 h of incubation at room temperature with HRP-labeled secondary antibody . Specific signals were detected using the ECL Western blotting detection system . The permeability of the BTB was assessed by using a biotin tracer ENREF-37 [22] . Two-month-old control and Wt1−/flox; Cre- ERTM animals were injected with 9 mg/40 g body weight of tamoxifen . A week later they were anesthetized with avertin , and 50 µl of 10 mg/ml EZ-Link Sulfo-NHS-LC-Biotin ( Pierce Chemical Co . ) freshly diluted in PBS containing 1 mM CaCl2 was injected into the interstitium of one testis and the other testis was injected with 50 µl of 1 mM CaCl2 in PBS as an internal control . The animals were euthanized 30 min later , and the testes were removed immediately and embedded with OCT . Cryosections were prepared for further staining . Testes were collected from control and Wt1−/flox; Cre-ERTM males 1 week after Tamoxifen treatment and fixed overnight in 2 . 5% glutaraldehyde in 0 . 1 M phosphate buffer ( pH 7 . 4 ) . They were then washed in phosphate buffer ( two changes ) , postfixed with 1 . 0% osmium tetroxide , dehydrated in a graded series of ethanol , and embedded in EPON/Araldite resin . Thin sections were cut , mounted on 200-mesh grids , and stained with uranyl acetate and lead citrate . Mutant Wt1 cDNA was generated using the QuikChange Site-Directed Mutagenesis Kit ( Stratagene , La Jolla , CA ) . Mouse Wt1 cDNA ( -KTS ) was cloned by our lab previously . The WT1 mutations detected in patients W643 and W606 caused amino acid changed from Arg to Gln and Lys to Arg respectively ( Table S2 ) . We generated the same mutations with mouse Wt1 cDNA using the following primers ( Wt1R362Q: forward , 5′CTTCAAGGACTGCGAGCAAAGGTTTTCTCGCTCAG3′ , reverse , 5′CTGAGCGAGAAAACCTGTTCTCGCAGTCCTTGAAG3′; Wt1K386R: forward , 5′ CATTCCAGTGTAGAACTTGTCAGCG3′ , reverse , 5′ CGCTGACAAGTTCTACACTGGAATG3′ ) . The adenovirus containing wild type Wt1 , Wt1R362Q , and Wt1K386R cDNA were generated using the Gateway Expression System ( Invitrogen ) . The candidate genes were amplified by PCR and inserted into the pEntr 3C vector ( Invitrogen ) . The resulting plasmids were then generated by homologous L/R recombination . Viral constructs were transduced into a 293A cell line , and high titer ( 108 IU/ml ) viral particles were obtained by 4 rounds of amplification . The titer of virus was determinated as previous described [60] . Chromatin immunoprecipitation ( ChIP ) assays were performed according to the protocol provided by Upstate Biotechnology ( Charlottesville , VA ) . In brief , HepG2 cells were transiently transfected with Wt1 , Wt1 R362Q , and Wt1K386R expressing adenovirus . 72 hr after transfection , cells were crosslinked with 1% formaldehyde in medium at 37°C for 15 min . Cells were then washed in ice-cold phosphate-buffered saline ( PBS ) and resuspended in 200 µl of SDS lysis buffer containing protease inhibitor mixture . The suspension was sonicated on ice and pre-cleared with protein A-agarose beads blocked with sonicated salmon sperm DNA ( Upstate Biotechnology ) for 30 min at 4°C . The beads were removed , and the chromatin solution was immunoprecipitated with anti-Wt1 antibody at 4°C , followed by incubation with protein A-agarose beads for an additional 1 h at 4°C . Normal rabbit IgG was used as a negative control , anti-Histone antibody was used as a positive control . The immune complexes were eluted with 100 µl of elution buffer ( 1% SDS and 0 . 1 M NaHCO3 ) , and formaldehyde cross-links were reversed by heating at 65°C for 6 h . Proteinase K was added to the reaction mixtures and incubated at 45°C for 1 h . DNA of the immunoprecipitates and control input DNA were purified and then analyzed by standard PCR using mouse Wnt4 promoter specific primers ( Forward , 5′ ATAGCAAGCTCATGTGGTGTGCAG3′ , reverse , 5′ ATATAGGCCGCCGCACTTATCAGA3′ ) . Experiments were repeated at least three times . The data were evaluated for statistical differences using student T-test . A p-value<0 . 05 was considered significant . | Infertility is one of the most common health problems , affecting about 15% of the couples in the world . In about half of these couples , infertility is related to male reproductive defect . Azoospermia is one of the major causes of male infertility in humans . Previous studies have found that the mutation or deletion of some genes is associated with azoospermia; however , the genetic cause of this remains largely unknown . In the present study , we detected Wt1 missense mutations in men with non-obstructive azoospermia ( NOA ) . An essential function for WT1 in male spermatogenesis was confirmed by the use of a Wt1 conditional knockout mouse strain . Inactivation of Wt1 resulted in germ cell loss in mice , which was similar to NOA in human patients . Our data indicate that WT1 mutation is one genetic cause of male infertility and suggest that WT1 mutational analysis will be useful for diagnosis in a clinical setting . | [
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] | 2013 | The Wilms Tumor Gene, Wt1, Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans |
HIV infection and the associated chronic immune activation alter T cell homeostasis leading to CD4 T cell depletion and CD8 T cell expansion . The mechanisms behind these outcomes are not totally defined and only partially explained by the direct cytopathic effect of the virus . In this manuscript , we addressed the impact of lymphopenia and chronic exposure to IFN-α on T cell homeostasis . In a lymphopenic murine model , this interaction led to decreased CD4 counts and CD8 T cell expansion in association with an increase in the Signal Transducer and Activator of Transcription 1 ( STAT1 ) levels resulting in enhanced CD4 T cell responsiveness to IFN-α . Thus , in the setting of HIV infection , chronic stimulation of this pathway could be detrimental for CD4 T cell homeostasis .
Homeostatic forces regulate the number and survival of T cell clones throughout life , allowing only a limited degree of non-antigen driven expansion for each individual cell in order to preserve the diversity of the T cell repertoire [1] . This is achieved by a balance between signals that mediate survival and proliferation , which are regulated by homeostatic cytokines such as IL-7 and IL-15 . Through homeostatic mechanisms , the size of the T cell pool remains relatively constant despite the expansion of T cell clones during antigen-specific responses . In an immune competent host , homeostatic proliferation is controlled by the limited availability of homeostatic cytokines . However , under lymphopenic conditions , a robust homeostatic proliferation occurs leading to polyclonal T cell expansion and accumulation of cells with a highly activated memory phenotype [1] . This is observed in certain human clinical conditions such as bone marrow transplants and HIV infection , where an increased availability of IL-7 is detected in the serum of the patients [2]–[4] . During HIV infection , in addition to HIV-specific immune responses , there is a generalized immune activation that alters the homeostasis of the CD4 and CD8 T cell pools leading to CD4 T cell depletion and CD8 T cell expansion . The mechanisms behind these extreme outcomes are not totally understood . The direct cytopathic effects of HIV do not appear adequate to explain this dichotomy . HIV-induced CD4 T cell depletion triggers a homeostatic response that occurs in an inflammatory environment rich in Type-I IFNs and driven by HIV replication . Both lymphopenia and viral load contribute to the immune activation observed in the CD4 T cell pool . In contrast , the expansion and activation of the CD8 T cell pool is tightly correlated with levels of HIV replication and does not appear influenced by homeostatic forces [5]–[9] . The Type-I IFN activity associated with HIV infection is reflected by increased mRNA transcripts of genes such as OAS1 , ISG15 , IFNAR1 and STAT1 in both CD4 and CD8 T cells [10]–[12] . Type-I IFN signals through a receptor consisting of two subunits ( IFNAR1 and IFNAR2 ) complexed with JAK1 and TYK2 . Activation of these tyrosine kinases leads to the phosphorylation of Signal Transducers and Activators of Transcription 1 , 2 , 3 and 5 ( STAT1 , -2 , -3 and -5 ) [13] . While Type-I IFNs are critical for antiviral immunity , in the setting of chronic HIV/SIV infection , chronic exposure has been suggested to play a role in the pathogenesis of the infection , a distinguishing feature of pathogenic from non-pathogenic SIV infection [10] , [14] , [15] . To understand the mechanisms by which HIV infection alters the homeostasis of CD4 and CD8 T cell pools , we hypothesized that lymphopenia and the chronic exposure to IFN-α may both play a role in this dysregulation . In the present manuscript , we show that IL-7 in vitro or lymphopenia in vivo can upregulate the total levels of STAT1 , -2 and -3 , rendering CD4 T cells more sensitive to IFN-α . Levels of total STAT1 ( t-STAT1 ) were associated with the degree of lymphopenia and IL-7 serum levels in HIV-infected patients . In a murine model , lymphopenia and chronic treatment with IFN-α led to diminished survival of CD4 T cells and an expansion of CD8 T cells , thus recapitulating the alterations of the homeostasis of these pools observed in patients with HIV infection . In addition , these data provide new evidence that IL-7 in vitro can enhance Type-I IFN responses by modulating the levels of the STATs . This effect could enhance T cell effector differentiation and be advantageous in host defense against pathogens . However , chronic stimulation of this pathway in the setting of lymphopenia and uncontrolled HIV viral replication could be detrimental for CD4 T cell homeostasis and may contribute to the aberrant immune activation and eventual CD4 T cell depletion observed in these patients . The analysis of these pathways can contribute to the development of new strategies to reverse the dysregulation in the T cell pools seen in patients with HIV infection .
Previous studies have demonstrated that in vivo CD4 T cell proliferation in patients with HIV infection is correlated with both CD4 T cell counts and HIV-RNA levels [11] , [12] . In addition , CD4 T cells from viremic HIV-infected patients showed an enhanced response to in vitro stimulation with IFN-α measured by phosphorylation of STAT1 [12] . Because of the potential implication of chronic stimulation of this pathway in CD4 T cell homeostasis , we investigated the mechanisms involved in this process . We hypothesized that this enhanced response could be due to increases in the levels of proteins involved in Type-I IFN signaling pathway , such as IFNAR1 and STATs . To test this hypothesis , we first studied a longitudinal cohort of patients to determine the levels of t-STAT1 before and after combination anti-retroviral therapy ( cART ) and suppressed viremia to <50 copies/ml . Following therapy , CD4 T cell counts increased from 198 to 264 cells/µl ( Table 1 ) . The analysis of the intracellular expression of t-STAT1 in naïve ( CD45RA+ CD27+ ) and memory ( CD45RA− CD27+ ) CD4 and CD8 T cell subsets by flow cytometry showed that both naïve and memory CD4 T cells expressed high levels of t-STAT1 prior to therapy that were significantly reduced with treatment ( p<0 . 01 ) ( Fig . 1 ) . Similar results were observed in naïve CD8 T cells ( p<0 . 01 ) . Memory CD8 T cells expressed lower levels of t-STAT1 than naïve CD8 T cells and their expression was also further reduced by treatment ( p = 0 . 02 ) ( Fig . 1 ) . These results indicate that levels of t-STAT1 are upregulated during HIV infection . Additionally , the differential expression of t-STAT1 in CD4 and CD8 memory T cells suggests potential differences in their responses to Type-I IFNs . To define the mechanisms involved in modulating t-STAT1 levels during HIV infection , we examined the role of two cytokines associated with CD4 T cell lymphopenia ( IL-7 ) [16] , [17] and viral replication ( IFN-α ) [10] , [14] , [15] . PBMCs from healthy donors cultured with IL-7 for 3 days showed significant increases in t-STAT1 levels in naïve and memory CD4 T cells and naïve CD8 T cells when compared to cells cultured with media alone ( p<0 . 01 ) ( Fig . 2a and Fig . S1a , b ) . Consistent with the observations from the longitudinal cohort of patients ( Fig . 1 ) , the smallest increases were seen in memory CD8 T cells ( p<0 . 01 ) ( Fig . 2a ) . We next tested the ability of cells cultured with IL-7 or IFN-α to respond to further in vitro stimulation with IFN-α . T cells cultured with IL-7 showed enhanced phosphorylation of STAT1 ( p-STAT1 ) in response to in vitro IFN-α stimulation ( Fig . 2b and Fig . S1c ) . In contrast , cells cultured for 3 days with IFN-α were refractory to further in vitro stimulation with IFN-α and did not show phosphorylation of STAT1 ( Fig . 2b and Fig . S1c ) . These results suggested interplay between IL-7 and Type-I IFN in T cells that is mediated by the levels of STAT1 induction and subsequent phosphorylation . We next examined the effect of these cytokines on other STATs known to be involved in Type-I IFN signaling pathway ( STAT2 , -3 and -5 ) [13] . Freshly isolated CD4 T cells from healthy volunteers cultured for 3 days with IL-7 showed 8 . 3- , 2 . 1- and 2 . 7-fold increases in t-STAT1 , t-STAT2 and t-STAT3 respectively ( Fig . 3a ) . No changes were noted in total levels of STAT5 . Cells cultured with IL-7 and then stimulated with IFN-α exhibited 3 . 7- , 2- , 2 . 5- and 2 . 5-fold increases in p-STAT1 , -2 , -3 and -5 respectively . No such effects of IL-7 were observed on total CD8 T cells likely due to the predominance of CD8 memory cells ( Fig . 3b ) . These changes in CD4 T cells were mainly attributed to the modulation of t-STAT expression levels since IL-7 had no significant effect on the expression of IFNAR1 on T cells ( Fig . S2 ) . In contrast , cells initially cultured in the presence of Type-I IFN were generally refractory to further in vitro stimulation with IFN-α and phosphorylated only small amounts of STAT proteins ( Fig . 3 ) . These results demonstrate that IL-7 can induce upregulation of multiple components of the Type-I IFN signaling pathway leading to an enhanced response to IFN-α . Thus , in the context of HIV infection , the increased availability of IL-7 associated with HIV-induced CD4 T cell lymphopenia , could render CD4 T cells more susceptible to the chronic effects of Type-I IFNs . We next evaluated the contributions of in vivo lymphopenia ( CD4 T cell counts ) and serum levels of IL-7 to t-STAT1 expression and activation ( p-STAT1 ) in patients with chronic HIV infection . To limit the contributions of viremia , we studied a cohort of patients receiving cART who had HIV-RNA levels of <50 copies/ml for more than 9 months ( IQR 9–35 months , Table 2 ) . Levels of t-STAT1 and detection of p-STAT1 after in vitro stimulation with IFN-α were analyzed by flow cytometry . CD4 T cells from patients with CD4 counts <200 and between 200–500 cells/µl showed significantly higher levels of t-STAT1 expression when compared with those patients with CD4 counts >500 cells/µl ( p<0 . 01 ) . The latter showed t-STAT1 expression levels similar to those observed in healthy controls ( Fig . 4a ) . Although significant , less marked differences were observed in CD8 T cells as a function of CD4 counts ( Fig . 4a ) . In addition , an inverse correlation was noted between t-STAT1 levels in both CD4 and CD8 T cells and CD4 T cell counts ( r = −0 . 52 , p<0 . 01 and r = −0 . 34 , p<0 . 01 respectively ) ( Fig . 4b ) . Serum levels of IL-7 were negatively associated with CD4 T cell counts ( r = −0 . 56 , p<0 . 01 ) ( Fig . 4c ) and positively associated with t-STAT1 levels in CD4 ( r = 0 . 38 , p = 0 . 01 ) but not CD8 T cells ( Fig . 4d ) . Because of the association between sustained expansion of CD8 T cells and immune activation in patients with suppressed viremia [18] , we next examined the relationship between t-STAT1 expression and IL-7 serum levels with CD4/CD8 T cell ratio . A similar negative association was observed between serum levels of IL-7 and t-STAT1 in CD4 and CD8 T cells with CD4/CD8 T cell ratio ( r = −0 . 56 , p<0 . 01 , r = −0 . 36 , p<0 . 01 and r = −0 . 56 , p<0 . 01 , respectively ) ( Fig . S3a , b ) . The expression levels of t-STAT1 in T cells from HIV infected patients were associated with p-STAT1 levels after in vitro stimulation with IFN-α in both CD4 and CD8 T cells ( r = 0 . 47 , p<0 . 01 and r = 0 . 48 , p<0 . 01 , respectively ) ( Fig . 4e ) . Patients with CD4 counts of >500 cells/µl and healthy controls showed similar activation of STAT1 after in vitro stimulation with IFN-α ( Fig . S3c ) . Taken together , these results indicate that , in the setting of HIV infection , CD4 T cell lymphopenia can contribute to upregulating t-STAT1 expression in CD4 T cells and this subsequently leads to increased levels of p-STAT1 following exposure to IFN-α . To experimentally examine the interaction between lymphopenia and Type-I IFN in vivo and its potential impact in CD4 and CD8 T cell homeostasis , we performed a series of experiments in which lymphocytes were adoptively transferred into lymphopenic mice that were receiving chronically-administered IFN-α . Initially , we analyzed t-STAT1 and p-STAT1 expression in donor T cells transferred into lymphopenic or lymphoreplete mice . Lymph node ( LN ) cells from wild type C57BL/6 ( B6 CD45 . 2 donor ) mice were adoptively transferred into either congenic lymphoreplete B6 . SJL ( B6 CD45 . 1 host ) or lymphopenic B6 . SJL-[KO]RAG1 ( RAG−/− host ) mice . At day 7 after transfer , both CD4 and CD8 donor T cells proliferated under lymphopenic conditions , whereas they did not divide when transferred into lymphoreplete hosts ( Fig . 5a ) . Donor T cells transferred into lymphopenic RAG−/− mice showed significantly increased levels of t-STAT1 , in LNs as well as in the spleen , when compared to cells transferred into lymphoreplete B6 hosts ( Fig . 5a , b ) . The lower upregulation of t-STAT1 in splenic cells was associated with an increased proportion of highly proliferating T cells with an effector memory and effector phenotype when compared to cells derived from the LNs ( Fig . S4 ) . In the LNs , increased levels of t-STAT1 were associated with enhanced phosphorylation of STAT1 in response to in vitro IFN-α stimulation in CD4 donor T cells ( Fig . 5a , c ) . In contrast , lower levels of STAT1 phosphorylation were noted in splenic cells after in vitro stimulation with IFN-α when comparing cells that had been transferred into lymphopenic mice to those transferred into lymphoreplete hosts ( Fig . 5c ) . Identical levels of t-STAT1 expression and activation after in vitro stimulation with IFN-α were observed in donor and recipient T cells from the lymphoreplete B6 host ( Fig . S5 ) . These data suggest that a lymphopenic environment can induce upregulation of t-STAT1 expression , leading to enhanced p-STAT1 following IFN-α stimulation . Since IL-7 in vitro was able to upregulate t-STAT1 , we next analyzed its contribution in the setting of lymphopenia . We performed adoptive cell transfer experiments into RAG−/− and IL-7−/− x RAG−/− mice . Because IL-7 is critical for T cell survival , these experiments were analyzed 5 days after cell transfer . IL-7 deficiency in the RAG−/− background reduced IL-7 dependent lymphopenia-induced slow proliferation and survival of the transferred cells ( Fig . 6a , Fig . S6a ) [19] , [20] . In contrast , the rapid proliferation , which is known to be TCR dependent and IL-7 independent was not affected [21] . T cells derived from the LNs of IL-7 deficient RAG−/− mice expressed reduced levels of t-STAT1 when compared with cells transferred into RAG−/− hosts , although remained slightly higher than cells transferred into a lymphoreplete B6 host ( Fig . 6a , b ) . To eliminate the rapid TCR driven proliferation in the RAG−/− background , we next assessed the role of IL-7 in sublethally irradiated hosts . In this model , the lymphopenia is not as severe as in RAG−/− mice and transferred cells compete with the host cells during reconstitution by undergoing slow homeostatic proliferation which is IL-7 dependent [21] , [22] . 24 hours after sublethal irradiation , mice were treated with either anti-IL-7 and anti-IL7R or isotype control mAbs before cell transfer [23] , [24] . LNs and spleens were analyzed 5 days after transfer ( Fig . 6c ) . In this model , donor cells undergoing homeostatic proliferation upregulated the levels of t-STAT1 when compared to cells transferred into a lymphoreplete B6 host ( Fig . 6c , d ) . Treatment with anti-IL-7 and anti-IL7R mAbs blocked homeostatic proliferation . Because blockade of IL-7 signaling reduced survival of donor CD4 and especially CD8 T cells , in LNs and spleen , only CD4 donor T cells were analyzed ( Fig . S6b ) [19] , [20] . Expression levels of t-STAT1 in donor T cells were reduced with α-IL-7+α-IL7R mAbs , however remained slightly higher than those observed in lymphoreplete B6 hosts ( Fig . 6d ) . These results suggest that lymphopenia in vivo drives increased expression of t-STAT1 in CD4 T cells that is partially dependent on IL-7 . We next analyzed the impact of chronic exposure to IFN-α on T cell homeostasis . Lymphopenic RAG−/− mice received LN cells from congenic B6 CD45 . 2 mice . Administration of recombinant murine ( rm ) IFN-α was started on day 5 after transfer . Mice were treated with therapeutic doses of 10 , 000 U of rmIFN-α , daily ( ∼4×105 U/kg/day ) , for one month [25]–[27] . Control mice , which were otherwise identical to the experimental mice , were treated for the same period of time with an equivalent volume of PBS rather than rmIFN-α . Analysis of the CellTrace Violet profiles of CD4 and CD8 donor T cells showed extensive proliferation in LNs and spleens for both groups ( Fig . 7a ) . We found no differences between the proliferation profiles of CD4 donor T cells from treated and non-treated animals . However , an increased proliferation of CD8 donor T cells was observed in both LNs and spleen of IFN-α treated mice , suggesting differences in the response to Type-I IFN by these two pools . Similar proportions of the naïve , central memory , effector memory and effector phenotype of CD4 donor T cells were observed in both LNs and spleen of the control and rmIFN-α treated animals ( Fig . S7a , b ) . In contrast , in CD8 donor T cells , the proportion of naïve phenotype was reduced and increased proportions of central memory phenotype were observed in both organs of IFN-α treated mice ( Fig . S7a , b ) . While the absolute numbers of CD4 and CD8 donor T cells recovered from LNs did not differ substantially between IFN-α treated and control mice ( Fig . 7b ) , significant decreases in CD4 donor T cell numbers were noted in the spleens of the rmIFN-α treated mice . In contrast , there was a significant increase in CD8 donor T cell numbers in the spleens of the treated mice ( Fig . 7b ) . In addition , decreased CD4 T cell numbers in the spleens of IFN-α treated animals were primarily effector memory phenotype , suggesting that CD4 T cells may undergo activation induced cell death in this condition ( Fig . S7c ) . Thus , chronic exposure to Type-I IFN in the setting of lymphopenia led to enhanced CD8 T cell expansion and impaired CD4 T cell homeostasis .
Patients with HIV infection show a unique form of immune activation leading to CD4 T cell depletion and CD8 T cell expansion . The CD4 T cell depletion observed in these patients is only partially explained by a direct cytopathic effect of the virus . The small number of actively infected cells at any given point suggests that other mechanisms may play a role [28] , [29] . The mechanisms behind these extreme outcomes for both CD4 and CD8 T cells pools remain unresolved . Because CD4 T cells are under continuous homeostatic pressure , we hypothesized that these outcomes could be associated with differences in homeostatic regulation of these two pools and in their response to the combination of cytokines associated with lymphopenia and viral replication . In the present manuscript , we have shown that chronic exposure to Type-I IFN under lymphopenic conditions can lead to impaired CD4 T cell homeostasis resulting in diminished CD4 T cell counts in association with CD8 T cell expansion . This phenotype recapitulates the alterations of the CD4 and CD8 T cell pools seen in patients with HIV infection . Our data support a model in which CD4 T cells under lymphopenic conditions become more responsive to IFN-α by modulating the levels of STATs . This continuous stimulation of CD4 T cells may lead to activation induced cell death ( AICD ) and decreased survival . In contrast , the same set of stimuli lead to CD8 T cell expansion . The different outcomes observed in the CD4 and CD8 T cell pools in the setting of untreated HIV infection are associated with distinct mechanisms that regulate the homeostasis of these pools . For instance , in healthy controls and HIV infected patients , CD4 T cell homeostatic proliferation is tightly associated with CD4 T cell counts . This association is weak in CD8 T cells [12] . The overall size of the CD4 T cell pool is highly controlled and does not undergo large expansions following antigenic exposure in vivo . In contrast , such expansions are common in the CD8 T cell pool . Lymphopenia-induced proliferation is a mechanism triggered to maintain the CD4 T cell pool at a relatively constant size . IL-7 is a central homeostatic cytokine in this process that promotes T cell survival . Cells undergoing slow ( IL-7 dependent ) or fast ( IL-7 independent ) proliferation acquire new properties such as , memory phenotype and secretion of cytokines like IL-2 and IFN-γ [21] , [30] . These effects are also observed in humans T cells in vitro [31] and in patients after bone marrow transplant [32] . Therefore , lymphopenia and increased availability of IL-7 generates a complex environment . In patients with HIV infection , the relatively weak association observed between IL-7 and levels of t-STAT1 suggests that lymphopenia in combination with other factors potentially driven by ongoing viral replication may play a role . Several reports have shown that IL-7 upregulates expression of CD95 in T cells and IL-7 serum levels correlate with CD95 expression and increased susceptibility to CD95-induced apoptosis [33]–[35] . Our data extend these observations by demonstrating that IL-7 in vitro can also modulate the levels of STATs in T cells and thus modulate their responses to Type-I IFNs . This is an unexpected characteristic of IL-7 and suggests that this pathway may be advantageous in host defense against pathogens . Accordingly , recent reports in an animal model of acute influenza A virus suggested a role for IL-7 in adaptive immunity against viruses [36] . Similarly , administration of IL-7 in chronic LCMV infection model overcame infection and limited organ damage [37] . In contrast , in a scenario in which the host is chronically lymphopenic , such as HIV infection [16] , [17] , in association with an environment rich in Type-I IFNs maintained by viral replication , this interaction may lead to more immune activation and dysregulation of the homeostasis of the CD4 T cell pool . By modifying this relationship , such as by the administration of super physiological doses of IL-2 or IL-7 to patients with HIV infection receiving cART , one can see increased T cell counts [38]–[40] . Thus increasing the levels of homeostatic cytokine and reducing the inflammatory environment by cART may lead to improvement in T cell counts . Type-I IFNs are important cytokines with anti-viral and regulatory functions [41] . They can inhibit thymopoiesis and B cell development [42] and can induce proliferation and exhaustion of hematopoietic stem cells [43] . The potential detrimental effects of chronic exposure to Type-I IFN in HIV/SIV pathogenesis have been largely reported . For example , Type-I IFN can induce CD4 T cell-TRAIL/DR5-mediated apoptosis [44] . Chronic exposure to Type-I IFN may have detrimental consequences on T cell homeostasis and survival [27] , [44]–[48] . In vivo , HIV and SIV infection trigger a strong Type-I IFN response [10] , [14] , [15] . Paradoxically , the administration of super physiological doses of Type-I IFN have been shown to have both anti-tumor effects in the treatment of Kaposi sarcoma and anti-viral effects leading to reduction in HIV-RNA levels in patients with high CD4 T cell counts [49] , [50] . These effects were associated with decreases in both CD4 and CD8 T cells . Because of the potential detrimental effects of chronic Type-I IFN signaling in patients with HIV infection , blockade of this pathway has been explored as a potential therapeutic target . A recent report using an antagonist of TLR7/9 that blocks IFN-α production by plasmacytoid dendritic cells ( pDCs ) in SIV infected rhesus macaques had shown that pDCs are not the only source of IFN-α production in this infection model [51] . In addition , blockade of IFN-α production by pDCs did not prevent activation of T cells nor was it effective at reducing viral loads and had minimal effect on IFN-stimulated genes [51] . The anti-viral properties of IFN-α are best established and successfully used in the treatment of infectious diseases and cancer in humans [49] , [50] . In this regard , it is not surprising that therapies blocking IFN-α signaling may compromise immunity against HIV and other pathogens . In contrast to the anti-viral effects of Type-I IFNs , the immune modulatory properties of these cytokines are broad and less understood . For example , while IFN-β is used to treat the inflammatory autoimmune disease , multiple sclerosis , its mechanism of action in this setting is poorly understood [52] . The present model will help to dissect some of these properties of IFN-α signaling pathway in the setting of lymphopenia and may allow for identifying more direct targets of these immune modulatory functions . IL-7 in vitro and lymphopenia in vivo differentially regulated expression of t-STATs in CD4 and CD8 T cells as well as their responses to Type-I IFN . Memory CD8 T cells from healthy controls and HIV-infected patients expressed lower levels of t-STAT1 than naïve CD8 T cells , suggesting differences in their responsiveness to Type-I IFN in vivo as a function of T cell phenotype and state of activation . Previous reports have suggested that differences in the expression levels and activation of STATs in human PBMCs may explain differences in the induction of apoptosis after exposure to Type-I IFNs [53] . In animal models of viral infection , it has been reported that T cell responsiveness to cytokines is controlled by their relative expression of the STAT transcription factors [54] . For instance , regulation of expression levels of t-STAT1 in CD8 T cells has been described as a mechanism by which CD8 T cells can escape the anti-proliferative effects of Type-I IFNs during LCMV infection [55] and basal and temporal changes in the expression levels of several STATs have been demonstrated as significant in shaping the immune responses in the LCMV model [56] . Altogether these findings suggest that during HIV infection , the interplay of lymphopenia and inflammation ( Type-I IFN ) may lead to alterations in the ways that CD4 and CD8 T cells respond to stimulation with Type-I IFNs . Further dissection of the relative roles of these forces during HIV infection may provide us with better approaches to correct the immunologic abnormalities seen in HIV infected patients .
This human study was conducted according to the principles expressed in the Declaration of Helsinki . Patients were studied under a NIAID Institutional Review Board approved HIV clinical research study protocol in the NIAID/CCMD intramural program . Patients and healthy controls provided written informed consent for the collection of samples and subsequent analysis . Healthy volunteers were obtained through the NIH Blood Bank . All animal work was conducted according to relevant national and international guidelines . The animal experiments were performed under a study protocol approved by the NIAID Animal Care and Use Committee . Patients and healthy controls were consented and studied in NIAID/CCMD intramural program IRB approved HIV clinical research studies . Healthy volunteers were obtained through the NIH Blood Bank . The majority of the patients studied had chronic HIV infection . The longitudinal cohort of HIV infected patients used to analyze the expression levels of t-STAT1 consisted of patients ( n = 10 ) with viral loads of >30 , 000 copies/ml before starting cART and viremia suppressed to <50 copies/ml for more than 6 months ( Table 1 ) . The cohort of HIV infected patients used to assess the contributions of lymphopenia ( CD4 counts and IL-7 serum levels ) to t-STAT1 expression consisted of patients ( n = 53 ) who had successfully suppressed viremia ( viral load <50 copies/ml ) with cART for more than 6 months . The patients' characteristics are described in Table 2 and viral infection history in Table S1 . PBMCs from healthy controls were obtained by Ficoll gradient centrifugation . CD4 and CD8 T cells were isolated by negative selection ( Miltenyi Biotec ) and cultured in X-VIVO 15 medium ( Lonza ) in absence or presence of the previously tested optimal concentration of recombinant human ( rh ) IL-7 ( Peprotech ) and rhIFN-α ( PBL Biomedical laboratories ) . After 3 days of culture , the cells were harvested , washed with cytokine-free medium and adjusted at a concentration of 2×106 cells/ml prior to resting overnight for subsequent in vitro stimulation with IFN-α ( see below ) . Cells that had been rested overnight were washed and labeled with live/dead ( Invitrogen ) . Adjusted at a concentration of 2×106 cells/ml , cells were then incubated for 30 minutes at 37°C and 5% CO2 with rhIFN-α ( 100 U/ml; PBL Biomedical laboratories ) and p-STAT1 levels were analyzed by flow cytometry . IFN-α stimulations were stopped by fixation with 4% paraformaldehyde followed by a permeabilization step with 1∶1 methanol/acetone mix for 30 minutes on ice . For human PBMCs , after three washes , cells were incubated for 10 minutes with 10 µg/ml human IgG ( Sigma-Aldritch ) to block potential Fc receptor binding and stained for 1 hour at room temperature with anti-CD3 Qdot 605 ( Invitrogen , clone UCHT1 ) , anti-CD4 Pacific blue ( BD Biosciences , clone RPA-T4 ) , anti-CD45RA PerCP-Cy5 . 5 ( eBioscience , clone HI100 ) , anti-CD27 PE ( BD Biosciences , clone L128 ) , anti-STAT1 N-terminus Alexa Fluor 647 ( BD Biosciences , clone 1/Stat1 ) , and anti-tyrosine 701-phosphorylated STAT1 Alexa Fluor 488 ( Cell Signaling , clone 58D6 ) . For murine experiments ( see below ) , donor T cells were detected by virtue of their CD45 . 2 expression . After three washes , mouse lymphocytes were stained for 30 minutes at room temperature with a mix of anti-STAT1 N-terminus Alexa Fluor 647 ( BD Biosciences , clone 1/Stat1 ) and anti-tyrosine 701-phosphorylated STAT1 Alexa Fluor 488 ( Cell Signaling , clone 58D6 ) , followed by an additional 20 minutes incubation at room temperature with a mix of anti-CD3 V500 ( BD Biosciences , clone 500A2 ) , anti-CD4 PerCP-Cy5 . 5 ( eBioscience , clone RM4-5 ) , anti-CD8α eFluor 650NC ( eBioscience , clone 53–6 . 7 ) , anti-CD45 . 2 PE ( eBioscience , clone 104 ) and anti-CD16/CD32 ( BD Biosciences , clone 2 . 4G2 ) . Full-minus-one controls were performed in the Alexa Fluor 647 and Alexa Fluor 488 channel for control of compensation spread [57] . The intensity of CellTrace Violet fluorescence was analyzed in fresh , unstimulated mouse lymphocytes incubated with anti-CD45 . 2 PE ( eBioscience , clone 104 ) , anti-CD3 APC ( eBioscience , clone 17A2 ) , anti-CD4 PerCP-Cy5 . 5 ( eBioscience , clone RM4-5 ) and anti-CD8α eFluor 650NC ( eBioscience , clone 53–6 . 7 ) . This was performed for CD4 and CD8 donor T cells by gating respectively on CD45 . 2+ CD3+ CD4+ T cells and CD45 . 2+ CD3+ CD8+ T cells . Human samples were collected on a BD LSR II and mouse samples were collected on a BD LSRFortessa using FACSDiva software . Data were subsequently analyzed using FlowJo ( Tree Star ) . CD4 and CD8 T cells from healthy controls were isolated by negative selection ( Miltenyi Biotec ) , cultured for 3 days in absence or presence of rhIL-7 and rhIFN-α and further stimulated in vitro with rhIFN-α as described above . The stimulation was stopped by adding an equal volume of cold-temperature PBS containing phosphatase inhibitor to prevent de-phosphorylation of the activated STATs ( Thermo Scientific ) . After three washes with cold-temperature PBS containing phosphatase inhibitor , the samples were stored at −20°C as dry pellets . Whole cell lysates were prepared from these samples with RIPA lysis buffer containing 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 1% Nonidet P-40 , 0 . 1% SDS , 0 . 5% Sodium Deoxycholate supplemented with protease inhibitor and phosphatase inhibitor cocktails ( Pierce , Thermo Scientific ) before use . Antibodies to STAT1 ( clone E-23 ) , STAT2 ( clone C-20 ) , STAT5 ( clone C-17 ) were from Santa Cruz Biotechnology; and antibody to STAT3 was from Cell Signaling Technology . Phosphorylation-specific antibodies to STAT1 ( Tyr701 ) , STAT2 ( Tyr690 ) , STAT3 ( Tyr705 ) and STAT5 ( Tyr694 ) were from Cell Signaling Technology . After detection of the target protein , the membrane was stripped and reprobed with anti-beta-Actin antibody ( clone mAbcam 8226; Abcam ) to assess loading equivalency . Densitometric values of band densities on western blots were measured with ImageQuant TL 7 . 0 ( GE Healthcare Life Sciences ) . Human IL-7 levels were quantified by ELISA , in cryopreserved plasma from HIV-infected individuals , using a commercial kit ( R&D System ) in accordance with the manufacturer's instructions . Samples were tested in duplicate . All experiments were approved by the NIAID Animal Care and Use Committee ( ASP #: LIR5 ) . B6 . SJL-[KO]RAG1 ( RAG−/− ) , B6 . SJL ( B6 CD45 . 1 ) and C57BL/6 ( B6 CD45 . 2 ) mice were purchased from Taconic . IL-7−/− x RAG−/− mice were kindly provided by Dr . Scott K . Durum ( NCI , Frederick , MD ) . All mice used in these studies were between 5 and 13 weeks of age . B6 CD45 . 1 mice were sublethally irradiated ( 600 rads ) utilizing a Cesium 137 source ( J . L . Shepherd Mark 1 ) . Under these conditions , depletion of host T cells was approximately 90% at 24 h after irradiation . Mice were used for adoptive transfer experiments 24 hours after irradiation . For adoptive transfer studies , lymph node ( LN ) cells ( 2×107 cells/ml ) from B6 CD45 . 2 donor mice were labeled with 5 µM CellTrace Violet ( Molecular Probes ) in PBS for 10 minutes at 37°C . Lymphoreplete B6 CD45 . 1 and lymphopenic RAG−/− , IL-7−/− x RAG−/− and irradiated B6 CD45 . 1 recipient mice were injected i . v . with 9×106 of B6 CD45 . 2 congenic cells . B6 CD45 . 1 mice received every day alternately 0 . 5 mg/mouse of anti-IL-7 ( clone M25; BioXCell ) and 0 . 5 mg/mouse of anti-IL-7R ( clone A7R34; BioXCell ) by i . p . injection starting at 18 hours after irradiation and up to day 5 . Control mice received the same amount of the matched isotype control mAbs ( clone MPC-11 , mouse IgG2b and clone 2A3 , rat IgG2a , respectively; BioXCell ) . For in vivo treatment with IFN-α , rmIFN-α ( eBioscience: 10 , 000 U/mouse of rmIFN-α4 diluted in 0 . 2 ml PBS ) was administered by subcutaneous ( s . c . ) injection every day starting on day 5 after adoptive transfer . Mice were treated for a month . Control mice were injected with 0 . 2 ml PBS . Five to thirty-five days after transfer , spleen and a mixture of inguinal , axillary , cervical , mandibular , popliteal , mesenteric and pancreatic LN were excised . Spleen and LNs were processed separately to obtain single cell suspensions by mechanic disruption on Nitex filters in RPMI 1640 medium ( Cellgro ) containing 10% FCS , 55 µM β-mercaptoethanol , 2 mM L-glutamine and 50 µg/ml gentamicin ( Complete media ) at 4°C . After counting , cells from LNs and spleen were stained with the indicated Abs and/or stimulated in vitro with 500 U/ml of recombinant murine IFN-α4 ( rmIFN-α; eBioscience ) for 30 minutes at 37°C and 5% CO2 , after being rested for at least 1 hour in a cytokine free complete medium . For the human studies , Wilcoxon signed-rank tests were performed for within-group comparisons . Spearman's rank correlation coefficients were used to assess the association of t-STAT1 expression and plasma level of IL-7 with CD4 T cell counts , as well as the association among t-STAT1 expression , STAT1 phosphorylation after in vitro IFN-α stimulation , and IL-7 level in Figure 4 . Nonparametric unpaired Mann-Whitney tests were used to analyze the data from the mouse experiments . Data were considered to be statistically different if the p value was ≤0 . 05 . | While the acute CD4 depletion observed in the initial phase of HIV infection is likely due to direct cytopathic effects of the virus , the mechanism/s underlying the steady decline of the CD4 T cell pool during the chronic phase of infection are unclear and are felt to be associated with “immune activation . ” We hypothesized that the combination of two distinct forces: homeostatic ( CD4 T cell depletion ) and inflammatory ( HIV-driven IFN-α ) , lead to a form of T cell activation that results in a decline in the CD4 T cell pool and an increase in the CD8 T cells . IL-7 and lymphopenia enhanced CD4 T cell responsiveness to IFN-α by modulating expression of the Signal Transducers and Activators of Transcription 1 , 2 and 3 . In a murine model , CD4 T cell depletion and CD8 T cell expansion were observed in a lymphopenic host chronically treated with IFN-α . These findings suggest that a synergistic interaction between lymphopenia and IFN-α may play a role in the pathogenesis of HIV infection . The analysis of this pathway may contribute to the development of new strategies to reverse the dysregulation of the T cell pools seen in patients with HIV infection . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
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] | 2014 | Chronic Exposure to Type-I IFN under Lymphopenic Conditions Alters CD4 T Cell Homeostasis |
Loss of mitochondrial function is a fundamental determinant of cell injury and death . In heart cells under metabolic stress , we have previously described how the abrupt collapse or oscillation of the mitochondrial energy state is synchronized across the mitochondrial network by local interactions dependent upon reactive oxygen species ( ROS ) . Here , we develop a mathematical model of ROS-induced ROS release ( RIRR ) based on reaction-diffusion ( RD-RIRR ) in one- and two-dimensional mitochondrial networks . The nodes of the RD-RIRR network are comprised of models of individual mitochondria that include a mechanism of ROS-dependent oscillation based on the interplay between ROS production , transport , and scavenging; and incorporating the tricarboxylic acid ( TCA ) cycle , oxidative phosphorylation , and Ca2+ handling . Local mitochondrial interaction is mediated by superoxide ( O2 . − ) diffusion and the O2 . −-dependent activation of an inner membrane anion channel ( IMAC ) . In a 2D network composed of 500 mitochondria , model simulations reveal ΔΨm depolarization waves similar to those observed when isolated guinea pig cardiomyocytes are subjected to a localized laser-flash or antioxidant depletion . The sensitivity of the propagation rate of the depolarization wave to O2 . − diffusion , production , and scavenging in the reaction-diffusion model is similar to that observed experimentally . In addition , we present novel experimental evidence , obtained in permeabilized cardiomyocytes , confirming that ΔΨm depolarization is mediated specifically by O2 . − . The present work demonstrates that the observed emergent macroscopic properties of the mitochondrial network can be reproduced in a reaction-diffusion model of RIRR . Moreover , the findings have uncovered a novel aspect of the synchronization mechanism , which is that clusters of mitochondria that are oscillating can entrain mitochondria that would otherwise display stable dynamics . The work identifies the fundamental mechanisms leading from the failure of individual organelles to the whole cell , thus it has important implications for understanding cell death during the progression of heart disease .
The spatial and temporal organization of the mitochondrial network is crucial for understanding its function in cells [1] , [2] . Complex spatiotemporal factors not only contribute to physiological signaling [3]–[6] , but determine the fate of the cell under stress . In heart cells , we have previously studied how the lattice-like packing arrangement of the mitochondrial network lends itself to propagation of bioenergetic signals [7] and to synchronization of self-organized oscillations in ROS , redox potential , and mitochondrial inner membrane potential ( ΔΨm ) in response to pathological stimuli [8]–[10] . Moreover , the scaling of instability of the mitochondrial network to the whole-cell and whole-organ levels has been shown to underlie the electrophysiological and contractile dysfunction associated with cardiac disease [11]–[13] . Only recently have we begun to understand the mechanisms responsible for stable , unstable , and oscillatory modes of mitochondrial bioenergetics , by combining experiments with mathematical model development . In studies of isolated cardiomyocytes subjected to localized oxidative stress , we elucidated the role of ROS in triggering autonomous synchronized oscillations of mitochondrial energetics [8] , [9] . An emergent low frequency ( 0 . 01Hz ) , high amplitude , limit cycle oscillation of ΔΨm was observed that spanned the length and breadth of the cardiomyocyte , but only after a considerable delay following the initial perturbation ( local laser flash ) . We found that the abrupt transition in ΔΨm was preceded by the gradual increase of oxidative stress in the network , more specifically , when ∼60% of the mitochondria accumulated ROS to a threshold level . We referred to the state of the mitochondrial network just before depolarization as the point of “mitochondrial criticality” [9] . In the critical state , a small perturbation anywhere in the network can lead to the propagation of a ΔΨm depolarization wave . The mechanism of this phenomenon involves a mitochondrial ROS-induced ROS release mechanism ( a term originally coined by Zorov et al [14] , [15] ) , triggering an energy dissipating inner membrane anion channel ( IMAC ) that is inhibited by mitochondrial benzodiazepine receptor ( mBZR ) ligands , but not by inhibitors of the permeability transition pore [8]–[10] . A computational model of a ROS-dependent single mitochondrion oscillator was constructed to study the dynamics of the system [16] and it could reproduce the main features observed experimentally , including bursts of ROS release to the extramitochondrial space during rapid ΔΨm depolarization . Interestingly , in addition to the slow , large amplitude oscillation mode most closely associated with the pathological state , the model displayed a wide range of frequencies and amplitudes , suggesting the possibility that mitochondrial ROS release may be operating under physiological conditions as well . A subsequent study provided experimental evidence to support the idea that the mitochondrial network is organized as a collection of weakly coupled oscillators under physiological conditions that couple more strongly under stress to produce slow , synchronized waves and oscillations [3] . Because the initial collapse of the mitochondrial network initiates a cascade of events including activation of sarcolemmal KATP channels , alteration of the electrical excitability of the cardiomyocyte , and ultimately cardiac arrhythmias [11] , [17] , it is important to gain a precise understanding of the mechanisms responsible for signal propagation between mitochondria in the network , in order to find ways to interrupt potentially catastrophic events . Thus , in the present work , we test the proposal that O2 . − diffusion from one mitochondrion to its nearest neighbors is responsible for the propagation of ΔΨm depolarization waves and synchronization of oscillation in the mitochondrial network by building one dimensional ( 1D ) and two dimensional ( 2D ) reaction-diffusion models of the mitochondrial network . We show that model simulations closely resemble ΔΨm depolarization wave propagation observed in experiments in response to oxidative stress , and remarkably , we find that a few oscillating mitochondria can entrain the entire network into an oscillatory mode even if the majority of the mitochondrial lattice is not in the oscillatory parametric domain . We propose that perturbing a minimal number of mitochondria is sufficient to trigger cell-wide responses through ROS-dependent coupling of mitochondria in the network .
The present results demonstrate that the observed emergent macroscopic properties of the mitochondrial network can be reproduced in a reaction-diffusion model of RIRR . Moreover , the simulations have uncovered a novel aspect of the synchronization mechanism , which is that clusters of mitochondria that are in the oscillatory domain of the parametric space can entrain mitochondria that would otherwise display stable dynamics . Propagation of electrical and Ca2+ signals within and between cells in the heart is essential for cardiac function , but less is understood about propagation of signals between organelles [18] . Several hypothetical mechanisms have been previously suggested to explain how ΔΨm depolarization may spread throughout the mitochondrial network of the cardiac myocyte . Amchenkova et al [19] proposed that there may be direct electrical continuity between mitochondria; however , more recent data showing that individual mitochondria or groups of mitochondria can be depolarized with little effect on their immediate neighbors argues against direct coupling . Ichas et al [20] provided evidence for propagated mitochondrial Ca2+-induced Ca2+ release mediated ΔΨm depolarization that involved the activation of the PTP . A mitochondrial ROS-induced ROS release mechanism was described by Zorov et al [14] to explain how laser-induced depolarization generates a large burst of O2 . − generation from the electron transport chain upon activation of the PTP , which can occur independently of Ca2+ . In previous work we investigated whether or not the PTP and/or Ca2+ was involved in the mechanism of whole-cell ΔΨm oscillation ( [8]; see [21] for a review ) . Briefly , several lines of evidence ruled out both the PTP and Ca2+ as playing a role in the mitochondrial oscillations observed in heart cells . With regard to Ca2+: ( i ) the myocytes were studied under quiescent , minimally Ca2+-loaded conditions , and no sarcomere shortening was evident; ( ii ) inhibition of the sarcoplasmic reticulum Ca2+ pump or mitochondrial Ca2+ handling did not influence flash-induced mitochondrial oscillations; ( iii ) extensive buffering of intracellular Ca2+ with 1 mM EGTA did not affect flash-induced oscillations . The possible contribution of the PTP was ruled out by the following evidence: ( i ) cyclosporin A did not block the transitions , and ( ii ) small ( 600 MW ) fluorophores were not lost from the mitochondrial matrix upon depolarization . More recently , we have provided extensive evidence showing that IMAC and PTP open sequentially as a function of the glutathione redox status in permeabilized cardiomyocytes [22] . Studies from our laboratory demonstrated that in cardiomyocytes , mitochondrial ΔΨm depolarization and redox potential during metabolic stress can be highly synchronized throughout the mitochondrial network , displaying complex behaviors including wave propagation within and between cells [7] and limit cycle oscillations [7] , [8] , [16] . Using highly localized laser excitation of less than 1% of the cellular volume to induce O2 . − release and ΔΨm depolarization , we showed that after several minutes of delay , spatiotemporally synchronized oscillations in ΔΨm , O2 . − , NADH , and GSH [8] , [9] , [16] , spanning the entire mitochondrial network of the cardiomyocyte , can occur . This phenomenon could be prevented or acutely reversed by interrupting mitochondrial O2 . − generation , increasing antioxidant capacity , or blocking IMAC , and neither Ca2+ nor PTP opening were involved in this response [8] . These responses could be described by a computational model involving RIRR and the activation of IMAC [16] . Employing the RD-RIRR mitochondrial network model , the present work successfully reproduces several experimental observations , including: i ) ΔΨm redox wave propagation and its spatial-dependence on O2 . − diffusion , production , and scavenging , and ii ) synchronization of independent mitochondrial oscillators . Interestingly , a new feature was revealed by the 2D model simulation - entrainment of mitochondrial ΔΨm oscillation in mitochondria that would otherwise show stable behavior and low O2 . − production . Finally , another important achievement was the direct confirmation of a key component of the oscillatory mechanism and model , which was the experimental demonstration that O2 . − itself can induce ΔΨm depolarization and mitochondrial O2 . − accumulation in permeabilized cardiomyocytes . The role of O2 . − was previously inferred from the effects of superoxide dismutase mimetic compounds [8] . Reaction-diffusion theory ( pioneered by Turing [23] ) , as a basis for pattern formation in biological or chemical systems , emphasizes the importance of two components; an autocatalytic reaction producing a local product ( mediator ) , and the transport of this product by diffusion away from the source . This process can give rise to spontaneous symmetry-breaking and the appearance of self-organized spatial patterns including waves and oscillations [24]–[30] . With respect to the present model , the reaction consists of the reduction of O2 to produce ROS ( specifically O2 . − ) driven by mitochondrial electron donors ( e . g . , NADH ) . The local concentration of O2 . − around the mitochondrion is shaped by several others factors , including buffering by the antioxidant reactions and transport of O2 . − across the mitochondrial membrane . As in many reaction-diffusion systems , the mitochondrial oscillator also displays an inhibitory , or a self-limiting , mechanism; the concentration of O2 . − at the activator site on IMAC decreases during ΔΨm depolarization because i ) the rate of scavenging by SOD increases as O2 . − accumulates and ii ) the driving force for O2 . − production and transport diminishes . Diffusion of the O2 . − to neighboring mitochondria is shaped by the O2 . − diffusion coefficient ( DO2 . −i ) and the amount of the O2 . − scavenger enzyme , etSOD , which consequently determines the rate of propagation of ΔΨm depolarization through the network . As expected , increasing etSOD slowed down the depolarization wave . The rate of propagation of the depolarization wave in the model corresponded to 26 µm s−1 with low DO2 . −i ( on the order of 10−14 ) , which compares well with the experimentally determined 22 µm s−1 [9] at 37°C . A restricted diffusion range of O2 . − in cells is consistent with experimental data; however , the actual diffusion coefficient of O2 . − in cells ( with antioxidant systems disabled ) has not been determined and is likely to be influenced by local reactions with other molecules and molecular crowding around mitochondria , which would decrease the effective volume and increase the viscosity of the medium . This assumption of restricted diffusion is represented by the low DO2 . −i in the model . Scaling of local interactions in complex dynamic systems to produce global emergent behavior is common in physical , social , financial and biological networks [31] . The RD-RIRR model illustrates how local neighbor-neighbor interactions ( 1–2 µm distance ) can lead to long distance spatiotemporal patterns . Mechanistically , in cell-wide mitochondrial oscillations , propagation is mediated by regenerative RIRR between neighboring mitochondria . Our previous work treated the network as a percolation lattice [9] and we postulated that near the percolation threshold of the system , any mitochondrion within the spanning cluster ( i . e . , one which is close to the critical level of ROS accumulation in the matrix ) can depolarize , producing a burst of O2 . − , which diffuses to its neighbor with a particular spatial concentration profile that is a function of the rate of O2 . − scavenging , to elicit a cell-wide response . A suprathreshold level of cytoplasmic O2 . − must reach the neighboring mitochondrion for the response to be regenerative , eliciting the O2 . −-mediated opening of IMAC . In the model , the open probability of IMAC increases as a function of O2 . − , which alters the IMAC conductance versus ΔΨm relationship ( see Eq . 50 in the Supplementary Materials ) . The initial IMAC opening leads to a regenerative increase in ROS because the increased energy dissipation accelerates respiration , in turn increasing the number of electrons shunting to O2 . − , which crosses the inner membrane to further activate IMAC . It is important to note that the global response is very much dependent on both the arrangement of mitochondria in the network and also the number of mitochondria close to the threshold . Before the first ΔΨm depolarization , the balance of O2 . − production to O2 . − scavenging must approach the threshold of oxidative stress in approximately 60% of the mitochondria , at which point a small perturbation can cause the synchronous collapse ( and/or oscillation ) of ΔΨm in the mitochondrial network . We referred to this vulnerable state as mitochondrial criticality [2] , [9] . A limitation of this first RD-RIRR model is that it does not entirely reproduce the collective properties of network close to the critical state . For example , when we trigger ΔΨm oscillations by laser flash , the local increase in ROS load on the network is enough to induce a gradual accumulation of oxidative stress throughout the cell , and the bulk of the network gets closer and closer to criticality . The first global wave of depolarization does not usually happen instantaneously after the trigger , but often occurs after several minutes of delay: it presumably originates from a region having a suprathreshold level of ROS to trigger IMAC opening , and neighbors that are also close to the threshold . The phase of initial spreading of the “oxidative load” after laser stimulation is a collective network property that is difficult to represent with this simple reaction-diffusion model ( indeed , modeling the behavior of critical systems is a nascent science ) . At the edge of criticality , depolarization of even a single mitochondrion can evoke collapse of almost the entire network ( Fig . 10 , see also Supplemental Materials Video S4 ) . Alternatively , we can induce a critical state of the mitochondrial network by depleting the antioxidant defenses of the cell ( globally ) with diamide ( see [10] ) . In this condition , we hypothesize that a local increase of ROS above threshold in any part of the 3D network could evoke RIRR . Specifically , the fast synchronized ΔΨm depolarization-repolarization cycles and waves are the aspects of the phenomenon that are well-reproduced by the 2D RD-RIRR model . Another limitation of current 1D and 2D models is that in the actual experiments , the O2 . − can , of course , diffuse both laterally and vertically to other mitochondria , that is , in all 3 dimensions . Presumably , the ROS will have a similar diffusion rate in all directions , although this might depend on the structural organization of the organelles , membranes , and myofilaments . A 3D network would introduce additional triggerable ROS sources above and below the mitochondrion , which could , theoretically , alter the rate of wave propagation . This could be explored in the future by extending the model to three dimensions; however , we do not think that the major conclusions of the study regarding the properties of the excitable system will be substantially different . The RD-RIRR simulations revealed a novel aspect of RIRR-mediated coupling . A small number of oscillating mitochondria could elicit oscillations in the entire network in both the 1D and 2D models , even though the parameters were set for stable behavior in most of the network ( i . e . , the percentage of respiration leaking to ROS was below threshold for independent oscillation ) . This indicates that entrainment by forced oscillation might also occur in the mitochondrial network . In this case , the combined effects of subthreshold ROS release and ROS diffusion from nearby mitochondria exceed the threshold for a regenerative response . This behavior is evident as recruitment of more and more mitochondria into the oscillatory cluster after several cycles of oscillation ( as shown in Fig . 8D and the corresponding movie in the Supplemental Materials , Video S3 ) . While focusing on a specific mechanism of RIRR ( i . e . , IMAC-mediated ) , the present findings also provide general theoretical support for mitochondrial communication via RIRR . The RD-RIRR model simulations confirm that O2 . − diffusion occurring locally between neighboring mitochondria over a distance of a few microns is sufficient for propagation and synchronization of ΔΨm depolarization over a larger distance . RIRR involving PTP-dependent depolarization [15] , [32] can be readily incorporated into the model in the future; however , at present it is unclear what additional factors besides ROS may be required to evoke this more pronounced , and typically irreversible , cell death event . In our own experiments , we have demonstrated that the IMAC mechanism occurs with mild-to-moderate oxidative stress ( by GSH depletion or lower O2 . − levels ) while PTP activation occurs during more severe stress , e . g . with further GSH depletion [10] , or , in the present study , at higher O2 . − concentrations . Mitochondrial Ca2+ overload is also thought to be a requirement for PTP activation as well [33] , [34] . Hence , it is necessary to gain a better understanding of the interplay between the ROS species and the mitochondrial Ca2+ load in the activation of the PTP in the intact myocyte before a comprehensive PTP activation model can be constructed . In summary , we show that the autocatalytic release of O2 . − and diffusion between mitochondria are the essential components of propagated RIRR between mitochondria in a closely packed array like that found in the cardiomyocyte . The kinetics and emergent spatiotemporal patterns of ΔΨm depolarization in the RD-RIRR mitochondrial network are modulated by O2 . − production , O2 . − scavenging , and diffusion . The highly interdependent nature of the mitochondria as a network of oscillators also suggests that synchronization by forced oscillation may occur when only a part of the network is perturbed . | Cardiac cell injury and death is a key component of cardiac diseases such as heart failure or myocardial infarction , thus it is important to understand the earliest steps leading up to irreversible cell damage . Mitochondria are the organelles responsible for generating the energy required to keep the cell running , yet they are particularly vulnerable to damage by toxic byproducts of metabolism , which include reactive oxygen species ( ROS ) . ROS wreak havoc on cellular functions by attacking proteins , lipids and DNA , so the cardiac cell has evolved sophisticated defenses to remove them . The work we present in this paper using a computer model of the mitochondrial network describes how ROS generated inside the cell can spread from one mitochondrion to the next in a positive feedback process known as ROS-induced ROS release . Understanding this process will help us to find ways to intervene in this catastrophic mechanism to prevent loss of cell function and the associated cardiac arrhythmias and contractile problems leading to sudden cardiac death . | [
"Abstract",
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] | 2010 | A Reaction-Diffusion Model of ROS-Induced ROS Release in a Mitochondrial Network |
Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy , and its efficacy against Leishmania donovani has been well documented . Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil . Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient , we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient , who had not been previously treated with miltefosine . In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L . amazonensis type strains . Due to this difference in susceptibility , we evaluated whether genes previously associated with miltefosine resistance were involved . No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes . These analyses were conducted in parallel with the characterization of L . amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro , i . e . , exposure of promastigotes to increasing drug concentrations . In these mutant lines , a single nucleotide mutation G852E was found in the miltefosine transporter gene . In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy . Miltefosine was effective in the treatment of BALB/c mice infected with the L . amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate . On the other hand , animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy . These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo . This study contributed to establish that the miltefosine transporter is essential for drug activity in L . amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients .
Leishmania spp . are the etiological agents of a spectrum of diseases collectively known as leishmaniasis , endemic in tropical and subtropical areas of the world [1] . Leishmania braziliensis and Leishmania amazonensis are the main causative agents of cutaneous leishmaniasis ( CL ) in Brazil , with more than 20 , 000 cases in 2010 [2] . L . amazonensis is also the etiological agent of diffuse cutaneous leishmaniasis ( DCL ) , a severe pathology associated with defective cell mediated immune responses to the parasite [3] . Since there is no effective vaccine for prevention , control is essentially based on chemotherapy . Meglumine antimoniate ( Glucantime , Aventis ) is the drug of choice for the treatment of CL in Brazil [2] . Unresponsive cases are treated with pentamidine or amphotericin B in liposomal or conventional forms . There is , however , no effective treatment for DCL . Clinical improvement is noted during and shortly after the courses of treatment , but in general relapses follow the discontinuation of therapy [4] . Miltefosine ( MF ) , an antitumoral oral compound , has recently emerged as an effective drug against visceral leishmaniasis ( VL ) , with cure rates as high as 95% in India [5]–[7] . For CL , reported cure rates vary between 53% and 91% depending on the Leishmania species [8] . MF's leishmanicidal mode of action is not completely understood and most information on its properties was obtained from MF-resistant parasites generated in vitro . MF resistance in Leishmania is mainly due to a drastic reduction in drug accumulation [9] , associated with the inactivation of a P-type ATPase , also known as Miltefosine Transporter ( MT ) , responsible for the translocation of phospholipids across the plasma membrane of the parasite [10] . Mutated MT were observed in resistant L . donovani [10] , L . major and L . infantum [11] . The uptake of MF is also dependent on another protein , LdRos3 , a non-catalytic subunit of the MT . LdRos3 inactivation also led to a significant reduction of MF accumulation in L . donovani [12] . Pyridoxal kinase ( PK ) , an enzyme involved in the formation of pyridoxal 5′-phosphate ( the active form of vitamin B6 ) also has a role in MF susceptibility [11] . MF susceptibility data for L . amazonensis strains is limited , especially in Brazil , where the drug has yet to be approved for leishmaniasis treatment . Miltefosine's EC50 has been determined for three L . amazonensis type strains [13]–[15] , but whether differences in MF susceptibility occur in L . amazonensis field isolates is entirely unknown . In this study , we evaluated the susceptibility to MF of L . amazonensis 2506 , a strain recently isolated from a chronic DCL patient in Teresina city , Piauí state , Brazil . The patient had previously received multiple courses of antimony and amphotericin B , with only partial responses to chemotherapy but had not been exposed to miltefosine . Known molecular signatures for MF resistance and phospholipid uptake were evaluated in this isolate and in an in vitro selected MF resistant L . amazonensis mutant .
Animal experiments were approved by the Ethics Committee for Animal Experimentation ( Protocol: 178/138/02 ) in agreement with the guidelines of the Sociedade Brasileira de Ciência de Animais de Laboratório ( SBCAL ) and of the Conselho Nacional de Controle da Experimentação Animal ( CONCEA ) . MF was purchased from Sigma-Aldrich ( St . Louis , MO , USA ) ; 2- ( 6- ( 7-nitrobenz-2-oxa-1 , 3-diazol-4-yl ) amino ) hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine and N- ( 7-nitrobenz-2-oxa-1 , 3-diazol-4-yl ) -1 , 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine , triethylammonium salt ( NBD-PC and NBD-PE respectively ) were obtained from Molecular Probes . The clinical isolate 2506 ( MHOM/BR/2008/2506 ) was obtained in 2008 from a diffuse cutaneous leishmaniasis patient . Parasites were cultured from aspiration of skin lesions performed as part of the follow up procedure and with the patient's consent . Material collected from the border of the skin lesion was suspended in biphasic Schneider's and Novy , McNeal , and Nicolle media and incubated at 26°C . Promastigotes identified in the primary culture were subsequently subpassaged in Schneider's medium and aliquots were frozen . This isolate , named MHOM/BR/2008/2506 , was typed by PCR of the internal transcribed ribosomal DNA as described [16] and identified as L . amazonensis ( data not shown ) . L . amazonensis reference strain ( MHOM/BR/1973/M2269 ) [17] and the clinical isolate ( MHOM/BR/2008/2506 ) were grown at 25°C in M199 medium ( Sigma-Aldrich , St . Louis , MO , USA ) supplemented with 10% heat inactivated fetal calf serum ( FCS ) and 0 , 25% of hemin . L . amazonensis independent mutants were selected from the reference strain M2269 by exposing promastigotes to increasing MF concentrations . Four independent mutants were recovered in the presence of 100 µM MF and cultured in 150 µM of MF , allowing the selection of the MF150 . 3 line . To obtain clonal lines of mutants , the MF150 . 3 resistant population was plated onto M199 medium containing 1% of agar ( Gibco , Invitrogen Corporation ) . After 15 days , colonies were picked and expanded in liquid M199 medium containing 150 µM of MF . The stability of mutants was tested by trying to get revertants . This was done by culturing the MF150 . 3 resistant cloned lines in the absence of MF for 20 passages . Total genomic DNA was isolated using DNAzol reagent ( Invitrogen ) as recommended by the manufacturer . Genes involved in MF susceptibility/resistance in Leishmania spp . were amplified by PCR using Phusion High-Fidelity DNA Polymerase ( New England Biolabs Inc . ) or Accuprime Taq DNA polymerase system ( Life Technologies ) . Primers were designed based on the L . mexicana ( MHOM/GT/2001/U1103 ) genome data [18] available at TriTrypDB ( http://www . tritrypdb . org/ ) . Internal primers were also designed for nucleotide sequencing of studied genes . All primers were designed using Primer3 software [19] and are listed in Table S1 . PCR amplified products were excised from 0 . 8% agarose gels after electrophoresis and purified with QIAquick PCR purification kit ( Qiagen ) . Nucleotide sequences were determined automatically with the Big Dye Terminator v3 . 1 Cycle Sequencing kit ( Applied Biosystems ) . Nucleotide sequence analysis was performed using Lasergene Software ( DNASTAR ) and Clone Manager 9 . 0 Software . Nucleotide sequences are available at GenBank under accession number: KF993340 , KF993341 and KF993342 . While writing this paper , the genome sequence of the L . amazonensis M2269 strain was published [20] and the sequence is now available at http://bioinfo08 . ibi . unicamp . br/leishmania/ . Log-phase promastigotes parasites were incubated in HPMI buffer ( 5×106/mL ) ( 20 mM HEPES , 132 mM NaCl , 3 . 5 mM KCl , 0 . 5 mM MgCl2 , 5 mM glucose , 1 mM CaCl2 , pH 7 . 4 ) containing 0 . 3% ( w/v ) BSA and 10 µM of NBD-PC or NBD-PE for 30 minutes at 25°C [21] . Before the addition of NBD-phospholipids , parasites were incubated for 15 minutes in buffer containing 500 µM PMSF to inhibit the catabolism of phospholipids . After labelling with NBD , parasites were washed twice with ice-cold HPMI containing 0 . 3% BSA to remove short-chain NBD-phospholipids from the external plasma membrane [22] , [23] and then finally resuspended in PBS for flow cytometry analysis . Labeled parasites were analysed at room temperature using Guava EasyCyte Mini Flow Cytometer System ( Millipore ) . Data from 5 , 000 cells , defined by gating at data acquisition , was collected and analysed using CytoSoft version 4 . 2 . 1 software ( Guava Technologies ) and FlowJo version 9 . 4 . 9 software ( Tree Star , Ashland , Oregon ) . Alternatively , 1×106 labeled parasites ( 5×106/mL in PBS ) at room temperature were analysed in a microplate reader ( POLARstar Omega , BMG Labtech ) with excitation at 460 nm and emission at 530 nm . At least three independent experiments were done in duplicate . Drug activity was determined by incubating promastigotes in the presence of increasing MF concentrations ( 3–400 µM ) . After 24 h of incubation , the number of viable cells was determined as described [24] . Briefly , cells were incubated with MTT ( 3-[4 , 5-dimethyl-2-thiazolyl]-2 , 5- diphenyl-2H-tetrazolium bromide; Sigma-Aldrich ) and the optical density was determined in a plate reader ( POLARstar Omega , BMG Labtech ) with a reference wavelength of 690 nm and a test wavelength of 595 nm . Results were expressed as the mean percentage reduction of parasite numbers compared with untreated control wells calculated for at least three independent experiments performed in triplicate . The EC50 was determined by sigmoidal regression curves using Graph Pad Prism 6 . 0 software . The activity of MF against intracellular amastigotes was analysed using infected bone marrow-derived macrophages ( BMDM ) from BALB/c mice as previously described [25] . BMDM were plated on round glass coverslips in 24-well culture dishes , at a density of 4×105 cells in 500 µL of RPMI 1640 medium ( Gibco , Invitrogen Corporation ) supplemented with 10% FCS ( Gibco , Invitrogen Corporation ) in a 5% CO2 atmosphere for 24 h at 37°C allowing macrophages to adhere . Macrophages were then infected with stationary-phase promastigotes ( 20 parasites per macrophage ) for 3 h at 33°C . Non-internalized parasites were removed by washing with warmed PBS , followed by the addition of fresh medium containing increasing MF concentrations ( 2 . 5–40 µM ) . After 72 h , the cells were fixed in methanol and stained with the Instant Prov kit ( Newprov , Pinhais , PR , Brazil ) . The percentage of infected macrophages was determined by counting 100 cells in three independent experiments . Female BALB/c mice were obtained from Instituto de Ciências Biomédicas , Universidade de São Paulo . Considering that the reference , isolate and MF150 . 3-1 mutant parasites were in different passages in culture in vitro as promastigotes and that the number of passages can affect the infectivity of the parasites [26] , we normalized the inoculum for BALB/c infections using amastigotes . These were obtained from in vitro infections using 1×107 BMDM plated in 148 cm2 dishes and infected with stationary phase promastigotes . After 72 h , infected BMDM were detached using a cell scraper and amastigotes were counted with a Neubauer hemocytometer . Amastigotes ( 1×106 ) of L . amazonensis M2269 , 2506 and mutant MF150 . 3 were injected subcutaneously in the right hind footpad in a volume of 30 µL . MF treatment was initiated 5 weeks after infection in experimental groups of 5 animals . An untreated group was used as a control for each line studied . MF was prepared daily with sterile water and the treatment was administered in doses of 13 mg/kg/day by oral gavage for 15 consecutive days . Lesion size was evaluated once a week using a caliper ( Mitutoyo Corporation , Kawasaki , Kanagawa , Japan ) by measuring the difference in the thickness between the infected and contralateral uninfected footpad . Parasite burden in three infected animals from each group was determined by limiting dilution as described [27] , one week after the end of treatment . Animals with absence of detectable parasites by limiting dilution and histopathological examination of infected tissues at the end of the treatment were considered cured . MF150 . 3-1 parasites were recovered from mice after the end of treatment , differentiated into promastigotes in vitro in M199 medium and used in MF susceptibility assays during the first passages in vitro using MTT as described above . Statistical analysis was performed using GraphPad Prism 6 . 0 software . For data on in vitro intracellular amastigotes , lesion progression and limiting dilution , we used One Way ANOVA , followed by the Tukey post-test . A result was considered significant at P<0 . 05 .
The susceptibility tests on isolate 2506 were performed in parallel with a reference strain of L . amazonensis ( M2269 ) , isolated from a patient in the Amazon region ( Pará state , Brazil ) [17] and that has been maintained in culture for 40 years . Promastigotes of both strains had a similar pattern of growth in vitro ( data not shown ) . The EC50 of MF against the 2506 isolate was found to be 2 . 4-fold higher than the type strain ( Figure 1 and Table 1 ) . To verify whether the decreased sensitivity to MF observed for promastigotes of the isolate 2506 was also present in amastigotes , intracellular killing assays were performed comparing the reference strain M2269 with the isolate 2506 . Initially , the percentage of infection and the average of amastigotes per infected BMDM were evaluated and compared between the two strains . In vitro infectivity to macrophages evaluated as the percentage of infected cells and number of parasites per infected cell were similar for the two lines ( Figure S1 ) . Treatment of infected BMDM with MF showed that the drug inhibited the in vitro intracellular growth of L . amazonensis M2269 and 2506 amastigotes in a dose dependent manner ( Figure 2 , A and B ) . The decreased susceptibility of the 2506 isolate , previously observed in promastigotes , was confirmed against intracellular amastigotes; L . amazonensis 2506 amastigotes were less sensitive to MF than the type strain , with a 4 . 5-fold increase in the EC50 ( Table 1 and Figure 2A ) . The natural decreased susceptibility of the 2506 isolate to MF led us to ask whether previously described mechanisms of resistance to this drug would be used by L . amazonensis . To investigate that , we decided to characterize the natural 2506 isolate and to try and obtain in vitro selected drug resistant parasites , using a classical strategy for drug resistance selection . The mutant line MF150 . 3 , selected using this strategy , exhibited an EC50 of MF 8 . 4-fold higher than the EC50 calculated for the reference wild-type strain ( Figure 1 and data not shown ) . Three independent clones from this resistant population were obtained and tested for MF susceptibility . Similar levels of susceptibility were observed for the three clones ( Figure 1 ) , suggesting a homogenous resistance phenotype . We selected the MF150 . 3 clone 1 ( MF150 . 3-1 ) to be further characterized . This mutant was highly resistant to MF compared to M2269 and 2506 strains ( Table 1 and Figure 1 ) and this phenotype was stable , since parasites cultured in the absence of MF for 20 passages displayed the same level of resistance ( Figure 1 ) . As expected , amastigotes of the MF150 . 3-1 line were also highly resistant to MF ( EC50 higher than 40 µM ) , with similar infection levels in untreated macrophages and in cells treated with the highest concentration of MF used in these experiments ( Figure 2 , A and B ) . It was not possible to determine the EC50 of this resistant line , because MF concentrations higher than 40 µM were toxic to macrophages ( Figure 2A and data not shown ) . To understand the basis for the altered susceptibility of the isolate and lines studied here , we chose to determine the nucleotide sequence of genes previously linked to MF resistance . The sequences of MT , Ros3 and PK encoding genes of M2269 strain , 2506 isolate and MF150 . 3 mutant were determined . A summary of these results is presented in Table 2 . When the translated sequence of the 2506 MT was subjected to comparison against the M2269 sequence , 100% or 99 . 8% identity were detected depending on the allele examined . When compared to the ortholog in L . mexicana , sequences were 98 . 6% identical , while approximately 90% identity was obtained when comparing the L . amazonensis MT polypeptide sequence with the corresponding sequences from L . major , L . infantum or L . donovani . Polymorphisms were found between MT and PK genes of the L . amazonensis 2506 isolate compared to the M2269 strain , with one non-synonymous SNP in each gene ( Table 2 ) . However , these amino acid changes were conservative and located outside the conserved motifs found in these proteins ( Table 2 ) . For MT and PK sequences , synonymous SNPs were also found while no SNP was found in the Ros3 gene between these two parasites ( Table 2 ) . Therefore , no molecular signature was found in the genes previously associated with MF resistance that could justify the natural decreased susceptibility found in the 2506 isolate . On the other hand , a single MT gene mutation at nucleotide position 2555 that causes an amino acid substitution , Gly-852→Glu ( G852E ) was present in three independent clones of the mutant line MF150 . 3 ( Table 2 and data not shown ) . This amino acid substitution is located in the large cytosolic loop between the consensus sequences characteristics of P-type ATPases and the 5th trans-membrane domain . Moreover , MF selection led to a loss of heterozigosity of the MT gene in MF150 . 3 clones , evidenced by the presence of just one allele ( SNPs 129 and 294 in L . amazonensis M2269 reference strain represent independent alleles that became homozygous in the MF150 . 3 resistant strain ) ( Table 2 ) . No SNPs were detected when nucleotide sequences of Ros3 and PK genes were compared between the MF150 . 3 highly resistant mutant clones and the parental strain M2269 ( Table 2 ) . The MT is responsible for the translocation of phospholipids across the plasma membrane [10] . To verify whether the amino acid substitution identified in the MT gene of the mutant line could be correlated with an altered phenotype , we quantified the transport of fluorescent-labelled NBD-phospholipids in the reference strain , in the in vitro selected line and in the natural 2506 isolate . The accumulation of NBD-PC and NBD-PE was investigated by flow cytometry and fluorescence intensity . No significant differences in NBD-PC accumulation were found between M2269 and 2506 parasites ( Figure 3 , A and B ) . On the other hand , NBD-PC accumulation was approximately 5-fold lower in the MF150 . 3-1 line as compared with the parental strain M2269 ( Figure 3 , A and B ) . Therefore , a phenotypic change was apparent supporting a correlation between the reduced accumulation of PC and the G852E substitution in the MT gene in this MF-resistant line . Strains M2269 and 2506 and MF 150 . 3-1 line displayed limited accumulation of NBD-PE ( Figure 3 , C and D ) . No significant differences were observed in the accumulation of PE among the three lines studied . To evaluate whether the decreased susceptibility observed in vitro for the isolate and for the mutant line would have an impact on the response to chemotherapy in vivo , we evaluated the efficacy of oral MF treatment in mice infected with the different parasites . When the control untreated groups were compared , no significant differences in the progression of disease were observed , as judged by the size of the lesion ( Figure 4 and data not shown ) and by the quantification of parasite burden in the three lines studied ( Figure 5 ) . Infections with M2269 or 2506 parasites responded similarly to MF , with animals completely cured at the end of treatment ( 8 weeks post-infection ) ( Figure 4 , A and B ) . Histopathological examination of tissues indicated that treatment of M2269 and 2506 infections with MF was translated into complete tissue healing with absence of detectable parasites ( Figure S3 ) . Limiting dilution assays confirmed the absence of parasites in the infected footpads of mice inoculated with M2269 or 2506 and treated with MF ( Figure 5 ) . No relapse was found in treated animals until 15 weeks after the interruption of treatment ( data not shown ) . On the other hand , infections with the MF150 . 3-1 line were completely refractory to MF treatment . MF150 . 3-1 infected mice treated with MF showed an indistinguishable progression of disease and parasite burden as compared with the control untreated group ( Figures 4C , 5 and S3 ) . These findings indicate that the resistant phenotype of MF150 . 3-1 line observed in promastigotes and intracellular amastigotes in vitro persisted in vivo . To investigate whether the drug susceptibility parameters were stable after in vivo passage and maintenance in the absence of drug , parasites were recovered from mice infected with the MF150 . 3-1 line ( treated with MF and untreated ) 8 weeks post-infection and allowed to transform to promastigotes . The EC50 of MF for promastigotes recovered after infection from treated and untreated mice was 154 . 5±11 . 7 µM and 149 . 8±8 . 1 µM respectively ( Figure S2A ) , therefore not significantly different from values determined prior to in vivo passage ( Table 1 ) . These parasites also maintained a reduced accumulation of NBD-PC as compared to the wild-type strain M2269 ( Figure S2B ) . Finally , sequencing of the MT gene from the parasites isolated from treated and untreated mice confirmed the MT gene mutation at nucleotide position 2 , 555 ( G852E substitution ) ( Figure S2C ) , previously identified in the selected resistant MF150 . 3-1 line used for infections in mice , confirming the stability of the mutation . Based on renal failure grading , susceptibility assays and drug availability , a course of treatment was administered to the DCL patient associating MF and pentamidine . Miltefosine 100 mg bid was given for 40 days while 10 doses of 200 mg pentamidine ( 3 mg/kg/day ) were administered over 60 days . The patient responded with a sharp decrease in ulcerated areas .
This work was driven by the necessity of finding a new therapeutic scheme for a DCL patient that , over 25 years , had received multiple courses of antimony and amphotericin B , with only partial responses to chemotherapy . The therapeutic options for the treatment of this patient were very limited . Antimonials were ineffective and renal insufficiency precluded further use of amphotericin B . MF was then contemplated as a possible alternative . The initial evaluation of the clinical isolate indicated , however , that L . amazonensis 2506 was less suscetible to MF when compared with the reference strain in vitro . The EC50 were 2 . 4 and 4 . 5-fold greater in 2506 promastigotes and amastigotes , respectively , as compared with the M2269 type strain . These findings confirm previous observations reporting variable MF sensitivity in Leishmania species and strains [28]–[31] . It should be emphasized that the 2506 strain was never exposed to MF , indicating that these parasites are intrinsically more tolerant to MF . We then decided to compare the phenotype of this less susceptible isolate with parasites selected for MF resistance in vitro . Stepwise increase in MF concentrations led to the selection of promastigotes displaying 8 . 4-fold increased EC50s in about 30 passages . Previous studies have demonstrated that the inactivation of MT through point mutations is a trait found in L . donovani , L . major and L . infantum resistant mutants selected in vitro [10] , [11] . Similarly , in the L . amazonensis resistant mutant MF150 . 3-1 a single point mutation in the MT gene was identified , leading to an amino acid substitution ( G852E ) . Both alleles were mutated owing to a loss of heterozigosity observed in all clones isolated from the initial MF150 . 3 population , suggesting the initial selection of a homogenous population ( data not shown ) . This amino acid change is located in the large cytosolic loop between the consensus sequences characteristic for P-type ATPases and the 5th transmembrane domain . This particular region of the protein seems important for MF transport , since other Leishmania MF resistant mutants contained mutations in the same region . In L . donovani and L . major selected resistant mutants , respectively L856P and G852D substitutions in MT sequence were found [10] , [11] . Interestingly , a single point mutation in the same region ( L832F ) was also identified in L . infantum isolated from an HIV co-infected patient after treatment with MF [32] . On the other hand , we did not find evidence of a MT inactivation on the 2506 isolate . Other genes potentially involved in MF resistance were also sequenced in this study . These genes were selected based on recent findings using whole genome sequencing technology in L . major MF resistant mutants selected in vitro [11] . We looked for mutations/polymorphisms in Ros3 and PK genes in L . amazonensis 2506 isolate and MF150 . 3 line . No SNP was found in the Ros3 gene in the parasites studied . PK is responsible for the phosphorylation of pyridoxal 5′phosphate , the active form of vitamin B6 [33] . The PK gene was found mutated in L . major , but not in L . infantum lines resistant to MF [11] . In this study , no mutation in the PK gene was found in three independent clones of the MF150 . 3 line . This gene was in heterozygosity in the type strain M2269 and contained two SNPs , while in 2506 strain just one allele was present with these SNPs in homozygosity . One of these SNPs codes for two different amino acids , however this change is conservative and not located in the conserved motifs of PK . In agreement with the MT sequence data , the accumulation of NBD-PC was reduced in the MF150 . 3 line but not in the 2506 isolate , as compared with the reference strain M2269 . A reduced accumulation of PC was previously described in L . donovani MF resistant mutants displaying MT gene inactivation [10] . On the other hand , these same L . donovani resistant mutants had a reduced accumulation of PE [10] . In L . amazonensis the accumulation of NBD-PE was extremely low and did not change when the reference strain was compared with the parasites less susceptible to MF . Differences in phospholipid internalization patterns have been noted for different Leishmania species [13] and may be due to the MT activity and substrate specificity . These analyses indicated , therefore , that while in L . amazonensis mutations in the MT are related to MF resistance as shown previously for other species , this was not the cause of the reduced MF tolerance of the 2506 isolate . Recently , an alternative method was described to obtain resistant parasites using intracellular amastigotes in vitro [34] , [35] . This method mimics the disease more closely and was employed to select paromomycin resistant parasites [35] . Interestingly , when amastigotes were selected by exposure to MF , no shift in the EC50 was found . However , promastigotes differentiated from these selected amastigotes were harvested at increased MF concentrations as compared to the control parasites [34] . In our study , the decreased susceptibility to MF was a stable phenotype observed equally for promastigotes and amastigotes . The different resistant patterns observed in parasites selected using different methods support the existence of more than one mechanism leading to MF resistance . Notwithstanding the success in selecting L . amazonensis resistant mutants in vitro , we were still left with a problem . Our data showed that L . amazonensis 2506 was less susceptible to MF than the M2269 type strain and that this phenotype was not due to mutations in the MT gene or in other genes previously linked to MF resistance . What was the significance of the decreased susceptibility ? Was this resistance or , in other words , was this level of reduction in susceptibility sufficient to impair the clinical outcome if MF was used ? The outcome of treatment was then compared in BALB/c mice infected with the L . amazonensis type strain , with the 2506 isolate or with the MF150 . 1 line . The World Health Organization recommends 2 . 5 mg/kg/day MF for 28 days for the treatment of CL or VL in humans . This scheme is not effective for obtaining complete cure in L . amazonensis infected BALB/c mice [36] . Previous studies in our laboratory indicated that MF was effective in the treatment of L . amazonensis infections in BALB/c mice if used at 13 mg/kg/day for 15 days by the oral route ( data not shown ) . After MF treatment , we observed clinical and parasitological cure in mice infected with L . amazonensis M2269 and 2506 . On the other hand , infections with MF150 . 3-1 were completely refractory to the same MF dose , with similar number of parasites recovered at the end of treatment in treated and untreated animals . Moreover , promastigotes recovered from lesions of treated and untreated MF150 . 3-1 infected animals were equally resistant to MF . Similar findings were described in a L . donovani resistant line that contained the MT gene mutated , with no response to MF therapy in mice [37] . Based on these findings , we can state that MT has an essential role in MF susceptibility in vivo across the genus Leishmania and its activity may affect MF response in vivo . Previous reports studying the correlation between in vitro and in vivo suscetibility/outcome of therapy in leishmaniasis showed no correlation between in vitro susceptibility to antimonials and the corresponding in vivo treatment outcome [38] , [39] . Similar findings were described here . Promastigotes and amastigotes of the 2506 isolate were less susceptible to MF when compared with the reference strain M2269 in vitro but not in vivo , prompting the reevaluation of MF's prospects in the treatment of the DCL patient . The administration of an association of MF and pentamidine was employed successfully , leading to healing of most of the cutaneous lesions ( data not shown ) . Obviously , long-term efficacy is still uncertain and will have to be evaluated during the clinical follow-up in months to come . The characterization of clinical or field isolates of Leishmania always raises concerns related to the potential changes in phenotype determined by the adaptation to laboratory conditions . Even when restricting the analysis to parasites with a low passage in vitro , we cannot completely rule out differences between parasites inhabiting the natural hosts and the ones obtained from culture . As far as the parameters evaluated in this study were concerned , the isolate 2506 maintained the growth pattern , infectivity and virulence observed previously for other clinical isolates [24] . In conclusion , our findings showed that MF was effective in vivo against a L . amazonensis isolate obtained from a Brazilian DCL patient , despite the decreased pattern of MF susceptibility in vitro . The mechanisms that justify the 2506 isolate decreased susceptibility to MF in vitro are still undetermined . On the other hand , MT activity proved to be essential for drug effectiveness , with parasites becoming completely refractory when this transporter was inactivated . These findings highlight the need to consider in vitro resistance determinations with caution as well as the relevance of MT as a potential molecular marker of MF unresponsiveness in leishmaniasis patients . | Leishmania amazonensis is the etiological agent of diffuse cutaneous leishmaniasis . The disease is extremely difficult to treat and frequently relapses once the treatment is interrupted . Although not yet approved in Brazil , miltefosine is an attractive alternative for leishmaniasis treatment due to its oral administration and low incidence of side effects . Here , we evaluated the efficacy of miltefosine against a L . amazonensis line that was isolated from a chronic diffuse cutaneous leishmaniasis patient to ascertain whether miltefosine could be considered as a therapeutic option in this case . Parasites isolated from this patient were less susceptible to miltefosine than a reference strain in vitro . The mechanisms underlying this decreased susceptibility were studied in this natural parasite isolate in parallel with mutant strains selected in vitro for miltefosine resistance . A mutation in the gene encoding the miltefosine transporter was identified in the mutants selected in vitro but not in the line isolated from the patient . Notwithstanding the decreased susceptibility in vitro , when used to treat infected mice , miltefosine was equally effective against the isolate from the patient and the type strain , but completely ineffective against the resistant line . | [
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] | 2014 | In Vitro and In Vivo Miltefosine Susceptibility of a Leishmania amazonensis Isolate from a Patient with Diffuse Cutaneous Leishmaniasis |
The microrchidia ( MORC ) family proteins are chromatin-remodelling factors and function in diverse biological processes such as DNA damage response and transposon silencing . Here , we report that mouse Morc2b encodes a functional germ cell-specific member of the MORC protein family . Morc2b arose specifically in the rodent lineage through retrotransposition of Morc2a during evolution . Inactivation of Morc2b leads to meiotic arrest and sterility in both sexes . Morc2b-deficient spermatocytes and oocytes exhibit failures in chromosomal synapsis , blockades in meiotic recombination , and increased apoptosis . Loss of MORC2B causes mis-regulated expression of meiosis-specific genes . Furthermore , we find that MORC2B interacts with MORC2A , its sequence paralogue . Our results demonstrate that Morc2b , a relatively recent gene , has evolved an essential role in meiosis and fertility .
The microrchidia ( MORC ) protein family forms a conserved class of chromatin remodeling factors found in diverse species from Arabidopsis to human [1] . MORC proteins contain GHKL-type ( Gyrase , Hsp90 , histidine kinase , MutL ) ATPase domain and PHD zinc finger domain , implying functions related to DNA metabolism and epigenetic regulation . Arabidopsis AtMORC1 and AtMORC6 repress transposable elements in a methylation-independent manner and are essential for heterochromatin formation and gene silencing [2] . In mammals , four different MORC proteins ( MORC1-4 ) have been identified [1] . Human MORC2 recruits histone deacetylases to promoter regions , causing local histone H3 deacetylation and transcriptional repression [3 , 4] . MORC2 also modulates chromatin relaxation in response to DNA damage [5 , 6] . MORC3 binds to H3K4me3 ( trimethylated histone H3 lysine 4 ) in vitro and localizes to H3K4me3-marked genomic sites [7] . Collectively , these studies reveal a conserved role for MORC proteins in the regulation of high-order chromatin organization . Mutations in MORC2 cause axonal Charcot-Marie-Tooth disease ( CMT ) in humans [8] . CMT is a neural disorder characterized by muscle weakness and atrophy , and changes in the sensation in the body periphery . In neuronal cells , MORC2 is recruited to heterochromatin by the HUSH ( human silencing hub ) complex to compact chromatin and thus is required for epigenetic silencing [9] . The HUSH complex mediates H3K9me3 deposition in heterochromatin by SETDB1 ( H3K9 trimethyltransferase ) to maintain transcriptional silencing [10] . In addition , MORC2 promotes breast cancer invasion/metastasis and gastric tumorigenesis [6] . These studies demonstrate the critical role of MORC2 in human diseases . Genetic requirements of Morc1 and Morc3 in mouse have been reported . Mouse Morc1 , the founding member of the MORC family , is specifically expressed in the male germline and its ablation results in male sterility with meiotic arrest [11 , 12] . Morc1 deficiency is associated with de-silencing of transposable elements in the male germline [13] . Morc3-/- mice die at birth or within one day after birth [14] . Study of mice heterozygous for a Morc3 mutation reveals a role in bone homeostasis [15] . However , the physiological functions of Morc2 and Morc4 are not known . In mouse , two paralogues of Morc2 are present: Morc2a and Morc2b . Mouse Morc2b was reported to be a transcriptional target of PRDM9 , a histone H3 trimethyltransferase required for meiotic progression and involved in speciation [16] . PRDM9 is the only known mammalian speciation gene [17] . PRDM9 specifies sites of preferred meiotic recombination ( i . e . hotspots ) and drives recombination away from functional genomic elements such as gene promoter regions [18] . Following sequence-specific DNA binding through its array of zinc fingers , PRDM9 catalyzes trimethylation of H3K4 ( H3K4me3 ) and H3K36 ( H3K36me3 ) [19–23] . In Prdm9-deficient testes , Morc2b is not expressed and H3K4me3 at the Morc2b promoter is low , suggesting that PRDM9 normally induces Morc2b expression via H3K4me3 [16] . However , the function of Morc2b remains unknown . Here , we report that Morc2b is required for chromosomal synapsis and meiotic recombination in both sexes . Inactivation of Morc2b causes mis-expression of a number of genes including meiosis-specific genes . We find that MORC2B interacts with MORC2A . The Morc2b-null mouse mutant exhibits meiotic defects similar to the Prdm9-null mutant , suggesting that MORC2B may be a key downstream effector of PRDM9 in meiosis .
Sequence comparison of the five murine MORC members revealed that MORC2A and MORC2B exhibited the highest sequence homology within the family , with 73% amino acid sequence identity ( S1A Fig ) . MORC2A and MORC2B were also the closest homologues according to phylogenetic analysis ( S1B Fig ) . Both MORC2A and MORC2B contain the conserved GHKL-type ATPase and PHD zinc finger domains shared by MORC proteins ( S2 Fig ) . The gene structure of Morc2a and Morc2b differs fundamentally: Morc2a contains 26 introns , whereas Morc2b lacks introns in the coding region ( Fig 1A ) . This gene structure implies that Morc2b is a retrotransposed homologue of Morc2a that arose from reverse transcription of a processed transcript followed by integration into the genome . Although most retrotransposition events produce truncated or otherwise non-functional pseudogenes , a small number of retrotransposed genes have retained functionality [24 , 25] . An annotated Morc2a gene ( referred to as Morc2 in non-rodent species ) is found in more than 100 mammalian species in the NCBI database . In contrast , Morc2b is only present in mouse and rat but not in other non-rodent eutherians , suggesting that the Morc2b retrotransposition event occurred 12–24 million years ago prior to the radiation of mouse and rat ( Fig 1B ) [26–28] . Western blot analysis with a MORC2B-specific polyclonal antibody showed that MORC2B migrates at the predicted size of ~120 kDa ( Fig 1C ) . MORC2B protein was abundant in testes but not detected in other adult mouse tissues examined , whereas MORC2A , migrating at the predicted size of ~120 kDa , was highly expressed in both testis and skeletal muscle ( Fig 1C ) . Thus , the Morc2b gene encodes a bona fide testis-expressed protein and represents a functional retrotransposed gene rather than a pseudogene . We next examined the spatiotemporal localization pattern of MORC2B in adult testis . MORC2B was detected in germ cells with a distinct developmental-specific expression pattern but not in somatic cells such as Sertoli cells ( Fig 1D and 1E ) . MORC2B was present in meiotic spermatocytes , abundant in post-meiotic haploid round spermatids , and absent from elongated spermatids ( Fig 1D ) . MORC2B localized to the nucleus in spermatocytes and strongly to the nucleus in round spermatids . Absence of immunofluorescence signals in Morc2b-deficient spermatocytes supported the specificity of the MORC2B antibody ( Fig 1E ) . The observed tissue- and cell type-specific expression of MORC2B and its stage-specific subcellular localization suggest a germ cell-specific nuclear function of Morc2b . We further examined the expression of Morc2b using juvenile testes ( day 8 through day 20 ) ( Fig 1F and 1G ) . The first wave of spermatogenesis is synchronized [29] . At postnatal day 8 , testes contain spermatogonia but no spermatocytes . Pre-leptotene and leptotene spermatocytes first appear at day 10 , zygotene spermatocytes at day 12 , pachytene spermatocytes at day 14 , and round spermatids at day 20 . Morc2b expression was absent prior to day 12 , was detected at a low level at day 12 , and increased significantly at day 14 and beyond ( Fig 1F and 1G ) . This expression pattern was consistent with the immunofluorescence analysis of MORC2B in adult testis ( Fig 1D ) . In conclusion , Morc2b is not expressed in spermatogonia , begins to express in zygotene spermatocytes at a low level , and increases expression from pachytene spermatocytes through round spermatids . To assess the function of Morc2b , we generated a Morc2b-null allele by targeted deletion of exon 2 through homologous recombination in embryonic stem cells ( Fig 2A ) . Exon 2 includes the entire Morc2b coding region . The offspring from intercrosses of heterozygous ( Morc2b+/- ) mice exhibited a normal Mendelian distribution of genotypes ( wt , 72; Morc2b+/- , 119; Morc2b-/- , 65; χ2 test , p = 0 . 44 ) , suggesting that Morc2b is dispensable for embryonic and postnatal development . Morc2b-/- mice were viable and appeared to be grossly normal . However , both Morc2b-/- males and females were sterile . Adult males of all genotypes were of similar body weight ( age 2–3 months; wt and Morc2b+/- , 26 . 0 ± 3 . 9 g; Morc2b-/- , 26 . 6 ± 3 . 6 g ) , but Morc2b-/- males had significantly smaller testes than Morc2b+/- control males ( Fig 2B ) . Morc2b-/- testes weighed approximately 70% less than control testes ( Morc2b-/- , 53 . 4 ± 8 . 3 mg vs wt and Morc2b+/- , 166 . 4 ± 17 . 5 mg , n = 4 , p = 0 . 0001 ) . Western blot analysis confirmed reduced levels and absence of MORC2B protein in Morc2b+/- and Morc2b-/- testes respectively; MORC1 and MORC2A were present at reduced abundance in Morc2b-/- testes ( Fig 2C ) . Histological analysis of testes revealed that spermatogenesis in Morc2b-/- males did not progress beyond meiotic stages . Seminiferous tubules of heterozygous ( Morc2b+/- ) testes contained germ cells at all stages including pachytene spermatocytes , round and elongating spermatids , whereas Morc2b-deficient tubules contained early meiotic germ cells including pachytene-like spermatocytes but were devoid of any post-meiotic spermatids ( Fig 2D ) . TUNEL analysis revealed that apoptosis was strongly increased in Morc2b-/- testes , suggesting that Morc2b-null spermatocytes were eliminated by apoptosis due to the activation of the pachytene checkpoint in response to meiotic defects ( Fig 2E ) [30 , 31] . As expected , sperm were absent in Morc2b-/- epididymides . The ovaries of adult Morc2b-/- female mice were much smaller than those from heterozygous littermates and were devoid of oocytes ( Fig 3A ) . To determine the time point of oocyte loss , we performed immunofluorescence analysis of ovaries with anti-YBX2 antibodies to label oocytes [32] . Oocytes were present in Morc2b-/- ovaries at birth ( Fig 3B ) but disappeared by postnatal day 2 ( Fig 3C ) . TUNEL analysis showed increased apoptosis of oocytes in Morc2b-/- ovaries at birth ( Fig 3D ) . Perinatal loss of oocytes was observed in several recombination-defective mouse mutants ( Dmc1 , Msh5 , Atm , Meiob , or Prdm9 ) [16 , 33 , 34] . The early postnatal loss of oocytes in Morc2b-/- mice therefore suggests severe defects in female meiosis . This data is consistent with the expression of Morc2b during meiosis in embryonic ovaries [16] . The Morc2b ( previously referred to as 4932411A10Rik ) transcript is only present in embryonic ovaries at E13 . 5 and E14 . 5 , but not at E15 . 5 and beyond including adulthood [16] . Collectively , our results show that Morc2b is essential for meiosis and fertility in both sexes . We assessed chromosomal synapsis by immunofluorescence analysis of spread nuclei using antibodies against SYCP2 , a component of synaptonemal complex ( SC ) axial elements , and SYCP1 , a component of SC transverse elements [35 , 36] . SC axial elements are formed at the leptotene stage , initiate synapsis through physical juxtaposition at the zygotene stage , achieve full synapsis on autosomes at the pachytene stage , and subsequently separate at the diplotene stage [37] . Wild-type pachytene spermatocytes contained fully synapsed autosomes , whereas the most advanced spermatocytes from Morc2b-/- males were at a pachytene-like stage , characterized by apparent chromosome pairing and formation of SC axial elements ( SYCP2 ) but absence of full chromosomal synapsis ( Fig 4A ) . We quantified spermatocytes at different stages from juvenile wild type and Morc2b-/- males and found that diplotene spermatocytes were absent in Morc2b-/- males , indicating meiotic arrest at the pachytene-like stage ( Fig 5 ) . We identified similar defects in meiotic progression in Morc2b-/- oocytes ( Fig 4B ) . Female germ cells enter meiosis shortly after sex determination during embryogenesis . At embryonic day 17 . 5 ( E17 . 5 ) , wild type pachytene oocytes had all 20 chromosome pairs fully synapsed , whereas Morc2b-/- ovaries did not contain normal pachytene stage oocytes . The most advanced oocytes were at a pachytene-like stage as characterized by pairing and alignment of chromosomes and absence of extensive synapsis ( Fig 4B ) . The defects in chromosomal synapsis were strikingly similar between Morc2b-deficient spermatocytes and oocytes . These results demonstrate that MORC2B is required for chromosomal synapsis during meiosis in both sexes . HORMAD1 is associated with unsynapsed chromosomes [38–40] . In both wild type and Morc2b-/- spermatocytes , HORMAD1 localized to the SC axial elements ( SYCP3 ) of unsynapsed chromosomes but was excluded from synapsed regions ( Fig 4C ) . This result is consistent with the synapsis defects in Morc2b-/- mice . To monitor meiotic recombination in Morc2b-deficient spermatocytes , we evaluated the formation of DNA double strand breaks ( DSBs ) and localization of recombination nodules in spread nuclei of Morc2b-/- spermatocytes . During meiosis , following PRDM9-mediated chromatin changes at recombination hotspots , the SPO11 protein catalyses the formation of DSBs , which elicits the DNA damage response [19 , 21 , 41–43] , leading to activation of the ATM kinase and subsequent phosphorylation of H2AX ( termed γH2AX ) . At the leptotene and zygotene stages , γH2AX is present on autosomal chromatin and distributed widely throughout the nucleus ( Fig 5A ) . The presence of strong γH2AX signals suggested that DSBs are formed in Morc2b-/- leptotene and zygotene spermatocytes ( Fig 5B ) . In normal spermatocytes , γH2AX disappears from the autosomes following meiotic DSB repair and becomes restricted to the XY chromatin during the pachytene and diplotene stages , concomitant with meiotic sex chromatin inactivation ( Fig 5A ) . However , the pachytene-like Morc2b-/- spermatocytes remained γH2AX-positive throughout the nucleus and failed to form a sex body ( Fig 5B ) , suggesting a failure in the repair of meiotic DSBs . Meiotic recombination is executed through coordinated actions of a large number of DNA repair proteins [37] . We examined four single-stranded DNA-binding proteins: RPA , MEIOB , RAD51 , and DMC1 ( Fig 6 ) . These recombination proteins form distinct foci on meiotic chromosomes . The RPA heterotrimer consists of RPA1 , RPA2 , and RPA3 . RPA binds to ssDNA ends of the meiotic DSBs [44] . MEIOB forms a heterodimer with SPATA22 and interacts with RPA [34 , 45 , 46] . RAD51 and DMC1 are recombinases . RAD51 and DMC1 form filaments on RPA-coated ssDNA and direct strand invasion into the homologous chromosome , which is required for crossover formation , homologue pairing , and chromosomal synapsis [47] . Consistent with the formation of DSBs , unsynapsed chromosomes in Morc2b-/- spermatocytes contained abundant foci of these recombination proteins ( Fig 6 ) . The initial number of RPA2 , MEIOB , and RAD51 was similar between Morc2b+/- and Morc2b-/- spermatocytes at the leptotene stage . With the progression of meiotic recombination , the number of RPA2 and RAD51 foci decreased progressively in control ( Morc2b+/- ) spermatocytes , however , the number of RPA2 foci was sharply higher in Morc2b-/- spermatocytes at both zygotene-like and pachytene-like stages ( Fig 6A ) and the number of RAD51 foci was higher at the pachytene-like stage ( Fig 6C ) . The number of MEIOB foci also increased in Morc2b-/- spermatocytes at the zygotene-like and pachytene-like stages ( Fig 6B ) . These defects further suggested that meiotic DSBs were not repaired in the absence of MORC2B . In contrast , the number of DMC1 foci decreased significantly in Morc2b-/- spermatocytes , suggesting defects in strand invasion and/or stabilization of homologue pairing ( Fig 6D ) . Such defects were consistent with the failure in chromosomal synapsis in Morc2b-deficient germ cells ( Fig 4 ) . Furthermore , we did not detect MLH1 foci , representing sites of future crossovers , in Morc2b-/- spermatocytes . These results demonstrate that MORC2B is required for meiotic recombination . Loss of MORC1 causes upregulation of retrotransposons in male germ cells [13] . We examined the expression of LINE1 and IAP retrotransposons in Morc2b-/- testes . In contrast with the upregulation of LINE1 and IAP in Mov10l1-/- testes ( positive control ) [48] , retrotransposons were not de-silenced in Morc2b-/- testes ( S3 Fig ) , implying functional divergence of these two MORC family members . PRDM9 catalyzes trimethylation of H3K4 and consequently loss of PRMD9 reduces the level of H3K4me3 in male meiotic germ cells [16] . We confirmed the reduced level of H3K4me3 in spermatocytes from Prdm9-/- testes ( S4 Fig ) . Morc2b was reported to be a PRDM9 target gene [16] . We found that the H3K4me3 level was comparable in spermatocytes between wild type and Morc2b-/- males ( S4 Fig ) , suggesting that loss of MORC2B is not responsible for reduced H3K4me3 in Prdm9-/- testes . Since the MORC family proteins are involved in chromatin remodelling , we sought to examine the transcriptome in Morc2b-/- testes by RNA-seq . We chose testes at postnatal day 12 for two reasons . First , Morc2b begins its expression at day 12 ( Fig 1G ) . Second , the histology of testes is comparable between wild type and Morc2b-/- males at day 12 , when the most advanced germ cells are at the zygotene stage . Analysis of RNA-seq data identified 71 differentially expressed genes: 57 downregulated and 14 upregulated in Morc2b-/- testes ( Fig 7A and S1 Table ) . Seven genes ( six downregulated and one upregulated ) were chosen for validation by real-time PCR . The differential expression was confirmed for all seven genes at day 12 ( Fig 7B ) . As expected , their expression was comparable between wild type and Morc2b-/- testes at day 10 ( Fig 7B ) . Gene ontology analysis identified meiotic cell cycle as the most affected biological process ( Fig 7C ) . Interestingly , two meiosis-specific genes Msh5 and Ccnb3 are downregulated and upregulated respectively . MSH5 , a DNA repair protein , is required for chromosomal synapsis [49 , 50] . CCNB3 is a meiosis-specific cyclin [51] . These data strongly suggest that mis-regulated expression of meiosis-specific genes may contribute to the meiotic defects in Morc2b-/- mice . To identify MORC2B-interacting partners , we performed immunoprecipitation ( IP ) with testicular protein extracts using the MORC2B antibody . Two protein bands were present in immunoprecipitated proteins from wild type testis but not detected in Morc2b-/- testis IP ( Fig 8A ) . The upper band had the same apparent molecular weight as MORC2B and contained both MORC2A and MORC2B and three other proteins ( Fig 8A and S2 Table ) . Mass spectrometry of the lower unique band identified three proteins: DDX41 , HSP72 , and ARID3B ( S3 Table ) . We verified the association of MORC2A and MORC2B in testes by co-IP and western blot analyses and confirmed that MORC2B was present in the immunoprecipitated proteins from wild type testes with anti-MORC2A antibody ( Fig 8B ) . Additionally , co-expression and co-IP in HEK 293T cells also validated the association between MORC2A and MORC2B ( Fig 8C ) . Our results suggest that MORC2B may regulate meiosis through interaction with MORC2A .
The MORC family proteins are involved in chromatin remodelling , transcriptional regulation , and transposon silencing . Here we find that Morc2b is required for meiosis and fertility in both males and females . Interestingly , the Morc2b gene evolved in the rodents via retrotransposition from Morc2a . Strikingly , the relatively young Morc2b gene has evolved an essential role in meiosis and fertility , suggesting a strong selection pressure . Among the Morc gene family , Morc1 and Morc2b are germ cell-specific but exhibit distinct functions . This is evident from differences in the phenotype of the corresponding mouse mutants . The fertility of Morc1 mutant is sexually dimorphic: males are sterile but females are fertile [11 , 12] , whereas inactivation of Morc2b causes sterility in both sexes . The Morc1 mutant phenotype is similar to that of piRNA ( Piwi-interacting RNA ) pathway mutants: male-only sterility and de-repression of transposable elements ( LINE1 and IAP ) in male germ cells [13 , 52] . Since the piRNA pathway appears to be intact in Morc1 mutant germ cells , MORC1 most likely protects genome integrity in male germ cells by silencing transposable elements through a different yet unknown mechanism [13] . In contrast , MORC2B deficiency does not cause de-silencing of LINE1 and IAP retrotransposons in testes ( S3 Fig ) but leads to a failure in chromosomal synapsis and meiotic recombination in both sexes . Human MORC2 plays a critical role in chromatin remodelling in DNA damage response and transcriptional gene silencing [3–6] . MORC2 acts as a transcriptional repressor of the CAIX gene ( carbonic anhydrase IX ) by decreasing histone H3 acetylation at the CAIX promoter . MORC2 binds to the CAIX promoter and recruits HDAC4 ( histone deacetylase 4 ) to deacetylate histone H3 , which is associated with a repressed chromatin state [3] . Similarly , MORC2 represses p21 in gastric cancer cells by recruiting HDAC1 to the p21 promoter [4] . MORC2 also modulates chromatin configuration during the DNA damage response [5 , 6] . Upon DNA damage , MORC2 becomes phosphorylated by p21-activated kinase 1 ( PAK1 ) , exhibits DNA-dependent ATPase activity , and facilitates chromatin relaxation [5] . Given the known function of MORC proteins in chromatin remodelling , MORC2B might play a role in the regulation of high-order chromatin structure during meiosis . Loss of MORC2B results in mis-expression of 71 genes in testes . Out of the 71 genes , 30 genes have been disrupted in mice ( S1 Table ) . Twelve knockout mice exhibit sterility or impaired fertility: Adam2 , Adam3 , Clgn , Crisp1 , Fmr1 , Krt8 , Msh5 , Piwil1 , Rsph1 , Tnp1 , Tnp2 , and Ybx2 . The remaining 18 knockout mice exhibit lethality , or no defects , or somatic defects but normal fertility . Several affected genes are known to play critical roles in meiosis: Msh5 , Fmr1 , and Ccnb3 . Msh5 is downregulated in Morc2b-/- testes , whereas Fmr1 and Ccnb3 are upregulated in Morc2b-/- testes . MSH5 forms a heterodimer with MSH4 and functions in meiotic recombination . Inactivation of Msh5 causes a failure in chromosomal synapsis and thus meiotic arrest [49 , 50] . FMR1 localizes to chromatin and regulates DNA damage response [53] . CCNB3 ( cyclin B3 ) is specifically expressed in leptotene and zygotene spermatocytes [51] . Strikingly , mis-expression of the human CCNB3 transgene in mouse spermatocytes after the zygotene stage disrupts spermatogenesis [54] . Therefore , mis-expression of these meiosis genes could contribute to the meiotic defects in Morc2b-/- mice . It is possible that MORC2B regulates the transcription of these genes through chromatin relaxation or epigenetic modifications . Our biochemical studies demonstrate that MORC2B interacts with MORC2A . The interaction among MORC proteins is also present in Arabidopsis . AtMORC6 interacts with AtMORC1 and AtMORC2 in two mutually exclusive protein complexes [55] . Both AtMORC1 and AtMORC2 are needed to repress the set of genomic loci silenced by AtMORC6 . The interaction between MORC proteins and the non-redundant nature of their functions are conserved between Arabidopsis and mouse and possibly so in other species . In addition , MORC2B may function through other interacting proteins such as ARID3B –a member of the ARID ( AT-rich interaction domain ) family of DNA-binding proteins ( S3 Table ) [56 , 57] . Genetic studies of MORC2A and ARID3B in germ cells are not available yet but are necessary to determine their functional requirement for meiosis and the physiological significance of their interaction with MORC2B . As DNA-dependent ATPases , MORC proteins have been found to modulate chromatin superstructure in DNA damage response , heterochromatin formation , and gene silencing . Further studies are necessary to elucidate a possible role of MORC2B in chromatin remodelling in the regulation of meiosis . PRDM9 , a meiosis-specific histone H3 methyltransferase , is a major determinant of meiotic recombination hotspots in mice , primates , and humans [19–21] . PRDM9 binds to recombination hotspots in a sequence-specific manner through its variable number of zinc fingers . Disruption of Prdm9 results in meiotic failure and sterility in both males and females [16] . The cause of meiotic failure in Prdm9-null mice has not been identified , and the relationship between control of meiotic recombination hotspots and meiotic progression remains unclear . While PRDM9 catalyses H3K4me3 at hotspots , it also affects the expression of one target gene–Morc2b . In Prdm9-deficient testes , Morc2b is not expressed [16] . Morc2b expression is also nearly absent in sterile hybrids of mouse subspecies [17] . Furthermore , both Morc2b and Prdm9 mutants exhibit a failure in chromosomal synapsis and meiotic recombination . The similar phenotype of these two mutants raises the intriguing possibility that the absence of Morc2b might be responsible for or at least contribute to the meiotic failure in Prdm9-null mice .
Mice were maintained and used for experimentation according to the protocol approved by the Institutional Care and Use Committee of the University of Pennsylvania . The mouse MORC2B C-terminal fragment ( aa 823–1022 ) and MORC1 C-terminal fragment ( aa 751–950 ) were expressed as GST fusion proteins in E . coli using the pGEX4T-1 vector and affinity purified with glutathione sepharose . Two rabbits were immunized with each fusion protein ( Cocalico Biologicals Inc . ) . The resulting working antisera are: anti-MORC2B , UP2419 and UP2420; anti-MORC1 , UP2424 . Affinity-purified antibodies were used for immunofluorescence analysis and Western blotting . The following additional antibodies were used: MORC2A ( 1:250 , catalogue number PAB15729 , Abnova ) and ACTB ( 1:7 , 500 , catalogue number A5441 , clone AC-15 , Sigma ) . The MORC2A antibody ( Abnova ) was produced against its C-terminal fragment ( aa 791–1030 ) , which displays 69% aa sequence identity with MORC2B . The MORC1 antigen ( aa 751–950 ) shows 30% aa sequence identity with MORC2B . In the targeting vector , the 3 . 2-kb Morc2b coding exon was replaced with the PGKNeo selection cassette ( Fig 2A ) . The two homologous arms were amplified from a Morc2b-containing BAC clone ( RP24-63E7 ) by high-fidelity PCR . The HyTK negative selection cassette was cloned after the right arm . V6 . 5 embryonic stem ( ES ) cells ( on a C57BL/6 x 129S4/SvJae hybrid background ) were electroporated with the ClaI-linearized targeting vector . ES cells were cultured in the presence of 350 μg/ml G418 and 2 μM ganciclovir . Out of 96 double-resistant ES cell clones , nine targeted clones were identified by long-distance PCR . Clone 1F3 was injected into blastocysts . Germline transmission of the knockout allele was obtained through breeding of chimera males with C57BL/6 females . Offspring of intercrosses of Morc2b+/- mice were used for all the analyses . Wild-type allele ( 220 bp ) was assayed by PCR with primers TGCACTGAACCCGACACTAC and GGTAGGAGCGGCAGAGATTC . The Morc2b mutant allele ( 415 bp ) was assayed by PCR with primers ATAGCAGGCATGCTGGGGATGCGGT and TGCACCTACACCAGGCAGCTCAGG . For histological analysis , testes and ovaries were fixed in Bouin’s solution , embedded with paraffin , and sectioned . Sections were stained with haematoxylin and eosin . Color histological images were captured on a Leica DM5500B microscope with a DFC450 digital color camera ( Leica Microsystems ) . For immunofluorescence and TUNEL analysis , testes and ovaries were fixed in 4% paraformaldehyde for 3 h or overnight at 4°C , dehydrated , embedded , and sectioned using a cryostat . TUNEL assays were performed with the ApopTag Fluorescein In Situ Apoptosis Detection Kit ( Catalogue number S7110 , EMD Millipore ) . Nuclear spread analysis of spermatocytes and oocytes was performed as previously described [58 , 59] . The following antibodies were used for immunofluorescence: SYCP1 ( 1:50 , catalogue number ab15090 , Abcam ) , SYCP2 [35] , SYCP3 ( 1:200 , catalogue number ab97672 , Abcam ) , HORMAD1 [39] , γH2AX ( 1:500 , catalogue number 16-202A , Clone JBW301 , EMD Millipore ) , RPA2 ( 1:100 , catalogue number 2208S , clone 4E4 , Cell Signaling Technology ) , MEIOB [34] , RAD51 ( 1:30 , catalogue number sc-8349 H-92 , Santa Cruz Biotechnology ) , DMC1 ( 1:30 , catalogue number sc-22768 H-100 , Santa Cruz Biotechnology ) . FITC- and Texas red-conjugated secondary antibodies were used . Fluorescence images were captured with an ORCA Flash4 . 0 digital monochrome camera ( Hamamatsu Photonics ) on a Leica DM5500B microscope ( Leica Microsystems ) . Sections of postnatal day 14 testes ( wild type , Morc2b-/- , and Prdm9-/- ) were immunostained with H3K4me3 antibody ( 1:200 , catalogue number ab8580 , Abcam ) . Prdm9 targeted mice were obtained from Jackson Laboratory ( Stock No: 010719 ) [16] . Images were acquired under the same condition . The relative intensity of H3K4me3 fluorescence signal was quantified using the ImageJ software . One pachytene or pachytene-like spermatocyte and one Sertoli cell were randomly selected from each tubule cross-section ( 10 tubules/genotype ) . The relative H3K4me3 signal intensity in the spermatocyte was normalized to that in the Sertoli cell . Total RNA was isolated from eight pairs of postnatal day 12 mouse testes ( ~16 mg/pair; 4 pairs of wild type and 4 pairs of Morc2b-/- ) using TRIzol reagents ( Thermo Fisher Scientific ) . The RNA concentration was determined using a NanoDrop 2000 Spectrophotometer ( Thermo Fisher Scientific ) . Equal amounts ( 1 μg ) of total RNA from each sample were used to generate RNA-seq libraries using TruSeq Stranded mRNA Library Preparation Kit Set A ( Cat . No . RS-122-2101 , Illumina ) according to the manufacturer’s instruction . The concentration of DNA library templates was determined using a Qubit 3 . 0 Fluorometer ( Thermo Fisher Scientific ) . The quality of libraries was evaluated using the Agilent 4200 TapeStation ( Agilent Technologies ) . Eight individual libraries ( 4 wild type and 4 Morc2b-/- ) were pooled in equal amounts for sequencing using the Illumina NextSeq 500/550 High Output v2 kit ( Illumina , 75 cycles , FC-404-2005 ) and the NextSeq 500 system ( Illumina ) . The RNA-seq data are available under GEO accession no: GSE103127 . After trimming the adapter sequences and removing the low-quality reads , the clean reads were mapped to the mouse reference genome ( NCBI37/mm9 ) using Tophat with default parameters . Mapped reads were subjected to Cufflinks to estimate gene expression levels [60] . The expression of each gene was normalized by calculating fragments per kilobase of exon per million fragments mapped ( FPKM ) . The FPKM values of each gene for both wild type and Morc2b-/- group were used to assess the differential expression with Cuffdiff . The expression cutoff of ≥ 1FPKM in either wild type or Morc2b-/- testes was applied . Differentially expressed genes were determined by an adjusted P value ( false discovery rate , FDR ) < 0 . 05 based on Benjamini and Hochberg multiple testing correction . A volcano plot was constructed to illustrate the differentially expressed genes by plotting log2 of the fold change on the X axis and the negative log10 of the p value on the Y axis ( Fig 7A ) . The expression of seven differentially expressed genes was analyzed using independent testis samples from postnatal day 10 and 12 mice ( 3 testis samples per genotype per time point ) by real-time PCR . Expression of LINE1 and IAP retrotransposons in wild type and Morc2b-/- testes at postnatal day 14 was assayed by real-time PCR ( S3 Fig ) . Postnatal day 14 Mov10l1-/- testes were used as a positive control for de-silencing of LINE1 and IAP [48 , 61] . Real-time PCR primers are listed in S4 Table . Each sample was assayed in triplicates . Quantification was normalized to Actb using the Ct method ( ABI Prism 7700 Sequence Detection System , Applied Biosystems ) . Co-immunoprecipitation was performed with postnatal day 20 mouse testes using affinity-purified MORC2B antibodies as previously described [34] . Immunoprecipitated proteins were resolved by SDS-PAGE . The protein bands specific to the wild type testis sample were subjected to mass spectrometry for protein identification . The full-length coding sequences of mouse Morc2a and Morc2b were cloned into pcDNA3 . 1/myc-His vector and pcDNA3 . 1/V5-His-TOPO vector respectively . Plasmids were transfected into HEK 293T cells . Forty-eight hours after transfection , cells were collected and lysed in whole cell lysis buffer ( 50 mM HEPES , pH 7 . 5 , 140 mM NaCl , 1 mM DTT , 10% glycerol , 0 . 5% NP-40 , 1 mM PMSF ) . Immunoprecipitation on protein lysate was performed with anti-V5 antibody ( Catalogue number R96025 , Invitrogen ) , followed by Western blotting with anti-Myc antibody ( Catalogue number 631206 , Clontech ) . Statistical analysis was performed with Student’s t-test or χ2 test . | In sexually reproducing organisms , meiosis , a process unique to germ cells , produces haploid gametes . Abnormalities in meiosis can lead to infertility , loss of pregnancy , or genetic diseases such as Down syndrome . The meiotic processes are tightly regulated by a large number of genes including many meiosis-specific ones . The majority of meiosis-specific factors are conserved , however , species-specific factors have evolved . Here we report functional studies of a rodent lineage–specific gene named Morc2b . Morc2b belongs to a family of chromatin-remodelling factors . Morc2b is specifically expressed in germ cells . Disruption of Morc2b causes meiotic arrest and infertility in both sexes . Notably , MORC2B regulates the expression of a number of meiosis-specific genes . Interestingly , MORC2B interacts with its sequence homologue MORC2A . These functional studies have uncovered a new protein complex in the regulation of key meiotic processes and suggested the presence of continued selection pressure for evolution of new meiosis-specific factors . | [
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] | 2018 | MORC2B is essential for meiotic progression and fertility |
The MHC class I Dk molecule supplies vital host resistance during murine cytomegalovirus ( MCMV ) infection . Natural killer ( NK ) cells expressing the Ly49G2 inhibitory receptor , which specifically binds Dk , are required to control viral spread . The extent of Dk-dependent host resistance , however , differs significantly amongst related strains of mice , C57L and MA/My . As a result , we predicted that relatively small-effect modifier genetic loci might together shape immune cell features , NK cell reactivity , and the host immune response to MCMV . A robust Dk-dependent genetic effect , however , has so far hindered attempts to identify additional host resistance factors . Thus , we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV . We discovered and validated many quantitative trait loci ( QTL ) ; these were mapped to at least 19 positions on 16 chromosomes . Intriguingly , one newly discovered non-MHC locus ( Cmv5 ) controlled splenic NK cell accrual , secondary lymphoid organ structure , and lymphoid follicle development during MCMV infection . We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements , to enhance lymphoid remodeling and to increase viral clearance in spleen .
Yellow fever virus ( YFV ) , once a major scourge of humanity , was one of the first viruses studied experimentally in mammalian hosts [1] . In pioneering studies , Nobel Laureate Max Theiler developed an inactivated YFV vaccine [2] , and laid the groundwork for investigations into the genetic basis of host resistance to virus infection . Sawyer and Lloyd later observed that different strains of white mice are differently susceptible to YFV [3] , and Lynch and Hughes solidified the point that YFV susceptibility is a heritable trait [4] . Many years later , the underlying cause of disease and effect of genetic variance on host resistance to viral infection and pathogenesis is still of vital interest [5] , as it promises to reveal yet unknown molecular targets , signaling pathways and cellular networks with relevance to human health and disease . Genetic analysis of host resistance to MCMV has been especially rewarding [6–8] . Recently discovered genes encode virus sensors and ligands , cytokines and receptors , signal transducers , and effector molecules that either increase or decrease host resistance to infection [9–16] . Often , these molecules are related to cellular immunity , including a clutch of polymorphic NK cell receptors that specifically bind ligands on virus-infected cells [17–24] . Yet , our understanding of the genetic influences on NK cells in the response to viral infection remains incomplete . We established several mouse models to explore the effect of MHC class I ( MHC I ) polymorphism on NK cells in viral immunity [25] . MHC I Dk confers dominant MCMV resistance in MA/My and C57L-derived transgenic Dk mice , while Db-expression in C57L and MA/My-derived congenic M . H2b mice does not [26] . The Dk resistance effect requires NK cells that express Ly49G2 ( G2 ) , an inhibitory receptor that binds Dk and protects against viral spread [24] . Thus , decreased Dk expression on infected cells might release the G2-specific brake on NK stimulatory signals , therefore helping to eliminate MCMV targets [25 , 27] . However , G2's precise role in host resistance is still under investigation . While analyzing the MHC effect on NK cells , we found that Dk-dependent MCMV resistance is greater in C57L-derived mice , relative to MA/My mice [26] . Thus , C57L genetic modifiers may increase host resistance to infection . However , modifier genetic loci have so far eluded detection , likely due to the prominent role of Dk . Although forward and reverse genetics approaches have uncovered many pathogen resistance genes , neither strategy is ideally suited to resolve smaller genetic effects . Moreover , reverse genetics relies on introducing novel mutations that result in phenotypic abnormalities , so it is not a practical way to identify or characterize natural allele variants with distinct effects on immune function or pathogen resistance . We thus set out to map and characterize variable genetic effects ( quantitative trait loci , QTL ) that shape immune features in naïve and MCMV-infected mice . We expected that virus-responsive immune cell traits and those that provide critical MCMV resistance would be revealed through common QTL linkages at defined chromosome positions across the genome . To guard against spurious results , we generated an additional cohort of MHC I Dk-disparate offspring that were given lower dose MCMV infection . We found that defined genomic regions controlled either naïve or MCMV-responsive NK cell features , while others affected both sets of traits . While the MHC I D molecule has a central role in virus resistance and NK cell functionality , genetic dissection allowed many additional immune cell and host response traits to be separately mapped and verified . One such newly discovered non-MHC QTL remarkably controlled the integrity of the splenic environment , the prevalence of NK cells , and overall resistance to virus infection .
Previously we assessed the immune response to MCMV with a deep phenomic approach [23] . The original high dose ( HD ) cohort [23] and a second cohort of MA/My x C57L hybrid mice given lower dose ( LD ) infection are included in the current analysis ( S1 Fig ) . All mouse genome-wide genotypes were assessed and verified using automated SNP analysis on an Illumina platform . We performed genome scans to detect and map host resistance and immune response modifier QTL . Briefly , pre- and postinfection quantitative traits from both cohorts were separately analyzed using the quantitative genetic mapping package R/qtl in the statistical computing program R [28] . Table 1 consolidates the data , conservatively reporting the most significant genetic intervals ( QTL ) detected . Results are ordered by chromosome , and then by position with overlapping genetic intervals for the same trait detected in both cohorts , or distinct traits detected in at least one cohort listed . As expected , sex-related naïve mouse body weight ( Mbw-X ) QTL mapped to overlapping genetic intervals on chr-X in both cohorts , but with slightly different peak logarithm of the odds ( LOD ) linkage positions , which relates the probability that an observed linkage was not due to chance ( Fig 1A and 1B , Table 1 ) . MCMV-induced body weight postinfection ( Vbwp-X ) QTL also mapped to this interval on chr-X , with near identical positions detected in LD and HD cohorts . As the only Vbwp QTL detected after LD infection , the results further suggested that MCMV-induced weight change was especially sensitive to chr-X weight control . Several smaller-effect mouse body weight QTL ( Mbw-3 , Mbw-7 , Mbw-19 ) were also found and these coincided with postinfection weight control QTL ( Vbwp-3 , Vbwp-7 , Vbwc-7 , Vbwp-19 ) detected on chromosomes 3 , 7 and 19 , respectively ( Table 1 ) . Thus , several QTL were discovered that affected body weight in naive and MCMV-infected mice . Host immune cell and response traits were likewise analyzed . Of the fifteen newly discovered NK cell trait modifier ( Nktm ) QTL , eight mapped respectively in both cohorts ( Table 1 ) . Twenty-four cohort-specific QTL were also identified ( Table 1 ) , which suggested that subtle differences in infectious dose , cohort-size , or genetic make-up ( see S1 Fig ) could have revealed additional genetic associations . In general , broadly profiling genetically diverse mice with genome scans for immune traits provided a sensitive and reliable measure of the host response to viral infection , and led to the discovery of many novel QTL in control of immune cell homeostasis and reactivity . As expected based on prior studies [20 , 25 , 29 , 30] , a strong MCMV ( log spleen ) resistance QTL mapped to H-2D on chr-17 in both cohorts ( Fig 1A and 1B , Table 1 ) . No other single QTL was mapped with greater precision . Two critical MCMV-induced weight-control QTL ( Vbwp-17 , Vbwc-17 ) also mapped at the H-2D locus , but only in HD-infected mice ( Fig 1 , Table 1 ) . Thus , these data demonstrated that H-2D controlled viral spread and the severity of weight loss ( i . e . morbidity ) solely after HD infection . The prominence of Dk in host resistance led us to next assess its effect on NK cells in the response to MCMV . In fact , several NK cell modifier QTL in control of NKp46+ or G2+ NK cells and their response to MCMV were detected on chr-17 . Interestingly , both NK cell trait ( Nktmg2sp-17 , Nktmg2spf-17 ) and response ( Nkrmg2sp-17 , Nkrmg2spf-17 ) modifiers of G2+ NK cells mapped to the H-2D locus . In contrast , QTL regulating Ly49I/U+ ( I/U+ ) NK cells had no association to chr-17 , and instead mapped to the NKC on chr-6 ( Fig 1A and 1B , Table 1 ) . Importantly , H-2D polymorphism specifically controls baseline G2+ NK cell features in naïve mice in addition to the G2+ NK response to MCMV , virus clearance and morbidity indices . To pursue genetic modifiers of MCMV immunity , we performed two-dimensional genome scans in R/qtl and analyzed each trait in possible two-locus QTL models . A higher LODav1 value is indicative of an additive effect , without evidence of epistasis , while a higher LODint value is suggestive of genetic interaction . Two-locus modeling in R/qtl detected numerous QTL on a range of chromosomes with decidedly significant LODav1 values , including many already discovered in Table 1 . Thus , these QTL added to H-2D’s ( pos 17 . 8 in Table 2 ) effect on MCMV burden or body weight , without evidence of interaction ( Table 2 , S2A Fig ) . Interestingly , seven common QTL positions precisely mapped on chromosomes 1 , 3 , 6 ( pos 21 . 7 , separate from the NKC ) , 11 , 17 ( pos 12 . 8 , separate from the MHC ) , 18 and X added to both MCMV and body weight control , which suggested that a single shared QTL on each chromosome enhanced H-2D's effect on both traits . Three additional QTL that mapped at similar positions on chromosomes 7 , 8 and 12 also added to H-2D's effect on MCMV and body weight control , which suggested these too mediate a shared locus effect . Five additional QTL on chromosomes 2 , 9 , 10 , 13 and 19 had wider-ranging positions that added to H-2D's control of either trait . Thus , we could not resolve whether common or distinct QTL on these chromosomes had a role . Nonetheless , both chr-13 ( pos 37 . 1 ) and chr-19 ( pos 49 . 1 ) QTL intervals that affected MCMV burden also overlapped with postinfection body weight intervals ( Table 2 ) . Several Nktm and Nkrm QTL mapped at the same chr-13 interval ( Tables 1 and 2 ) , further established strong support of at least one QTL on chr-13 . While a chr-2 MCMV control QTL ( pos 16 . 8 ) coincided with Nktmg2n-2 and Nktmg2sp-2 , a weight-control QTL ( pos 31 . 8 ) corresponded better with two MCMV-induced weight control ( Vbwp-2 , Vbwc-2 ) QTL locations and the NK response modifier , Nkrmdn-2 ( Tables 1 and 2 ) . Thus , two QTL were reliably detected on chr-2 . Lastly , a chr-10 QTL for MCMV burden coincided with NK trait modifier QTL ( Nktmg2n-10 , Nktmg2sp-10 ) , but a chr-10 ( pos 13 . 4 ) weight control QTL could not be independently verified . In aggregate , the results implicated at least sixteen new QTL that regulate NK cells , virus clearance and morbidity indices , and most corroborated QTL reported in Table 1 . Evidence of epistasis or interactive QTL ( iQTL ) pairs with significant LODint values that affected MCMV burden or weight control was also uncovered ( Table 2 , S2B Fig ) . Of these , all but the QTL on chr-5 and -14 could be independently verified in the genome scans of other traits . Interestingly , a chr-4 weight control iQTL that mapped similarly to Cmv6 , coincided with an additive H-2D effect on MCMV burden ( Table 1 ) , in addition to its interaction with a chr-12 iQTL that further added to H-2D-dependent control of MCMV clearance and morbidity ( Table 2 ) . Thus , two-dimensional genome profiling uncovered and validated at least 18 novel QTL that affected viral spread or weight control during MCMV infection ( S2 Fig ) . We infer that these genetic modifiers of NK cells and body weight together strongly influence overall host resistance to viral infection in both H-2D-dependent and independent ways . NKC-encoded receptor polymorphism has been shown to affect NK cells in the response to MCMV , and MHC-NK gene complex ( NKC ) epistasis contributes to MCMV control [20 , 30] . As expected , several modifier-QTL in control of NK cells , and I/U+ or G2+ NK subsets , were mapped to the NKC on chr-6 ( Tables 1 and 2; Fig 2A and 2B ) . Chr-6 , however , had no obvious effect on viral clearance or morbidity , so it was unlikely that Ly49 ( Klra ) polymorphism alone affected the variance in MCMV resistance in this genetic comparison . Nonetheless , genome profiling had uncovered a chr-6 QTL that added to H-2D’s effect on MCMV burden and weight control ( Table 2 ) . This result corresponds to a prior study showing that the C57L-derived NKC ( NKCl ) enhanced NK cell-mediated resistance after HD-infection in C57L mice without Dk [31] . Thus , we tested NK cell responsiveness by stratifying the phenotypic results by MHC and NKC genotypes . Interestingly , LD mice without Dk ( i . e . H-2b homozygous mice designated LL ) had a lower viral burden if they also had a C57L-derived NKC ( Fig 2C ) . A similar relationship was observed in HD mice , though not significant . While a single amino acid variation distinguishes G2 receptors in MA/My and C57L [24 , 32] , the results suggested that G2 receptor polymorphism could have affected NK cell sensitivity to MCMV . Further analysis of H-2b homozygous offspring revealed that naïve G2-SP NK cells with C57L-derived G2c57l receptors were fewer with lower G2 receptor MFI values , in comparison to mice solely expressing MA/My-derived G2mamy receptors ( Fig 2C ) . Whereas MHC I ligands of inhibitory NK receptors are known to affect NK cell and receptor expression features [33 , 34] , the data suggested that the variant G2 receptors might differently bind H-2b class I or class I-related molecules , which could have further resulted in MCMV resistance differences . To corroborate our analysis of NK cells in H-2b homozygous offspring , we next examined NKC congenic mice . As with the hybrid NK cells , naïve G2+ NK cell percentages and G2 MFI values were lower in NKCc57l than NKCmamy congenic mice ( Fig 2D ) , which suggested that the disparate G2 receptors might also differently license NK cells . However , comparisons of NK cells in NKC congenic mice ( S3 Fig ) , or C57L and M . H2b mice ( S4A Fig ) revealed similar sensitivity to ex vivo triggering via several different NK stimulatory receptors , and consequently no difference in NK cell licensing . Lower viral loads in NKCc57l than in NKCmamy spleens ( Fig 2C and 2D , [31] ) , nonetheless prompted further analysis of G2+ NK cell reactivity to MCMV . In agreement with results from C57L-derived NKC congenic mice , lower G2+ NK cell percentages and G2 receptor MFI trends were observed in C57L mice , in comparison to MHC-matched , but NKC-disparate M . H2b mice ( S4B Fig ) . More intriguingly , host resistance to MCMV in C57L exceeded M . H2b , which directly corresponded to higher postinfection G2+ NK cell percentages and G2 receptor MFI ( S4B–S4D Fig ) . C57L mice also exhibited significantly increased frequencies and numbers of total NK and G2+ NK cells producing greater amounts of IFN-γ ( S4E–S4G Fig ) . Dk-independent host resistance to MCMV therefore was also magnified in the C57L genetic background . We infer that G2+ NK cell phenotypes and effector functions are distinctly shaped by and sensitive to both NKC and non-NKC genetic effects , which further controls host-specific variations in virus response features . Given the robust Dk effect on NK cells and virus resistance , it was not surprising that the postinfection percentage of NKp46+ NK cells in spleen also mapped to chr-17 ( Table 1 , Fig 3A and 3B ) . Precision mapping in the HD cohort , however , resolved a peak QTL location ( rs13483002 ) distal to H-2D ( Fig 3B ) . The data implicated that a distinct locus outside of the MHC on chr-17 affected NK cell percentages after infection . To address the question , we compared allele effect plots for several pre- and postinfection traits . We found that Dk had a profound effect on G2 receptor expression and NK cell subsets in naïve animals ( Fig 3C ) . It also had a dominant effect on spleen MCMV burden after LD- or HD-infection ( Fig 3D , compare H-2D genotype classes , LL and LM ) . More remarkable , Dk had a major impact on postinfection percentages of G2-SP NK cells and the severity of weight loss after infection ( Fig 3D ) . In contrast , a C57L-derived locus ( rs13483002 ) either dominantly prevented or failed to support NKp46+ NK cell accrual after HD-infection ( Fig 3D , right panel ) . This effect was specific to NK cells since Dk affected the overall balance of total lymphocytes in the spleen ( Table 1 , Fig 3E ) . A CI for the QTL controlling percentages of NKp46+ NK cells was mapped distal to Dk in the HD-infected cohort ( Fig 3E ) . In support of this location , a very narrow R7-MHC congenic block ( ~300-kb Dk-spanning segment [25] ) was included in the genetic crosses used to generate the HD cohort so that many offspring had recombinant chromosomes between MHC I D and rs13483002 . The results demonstrated that the Dk-resistance factor alone was inadequate to support NK accumulation in spleen after HD-infection . Thus , at least two different chr-17 QTL control NK cells responding to MCMV . To corroborate the rs13483002 QTL effect , we compared the NK-cell response to infection in MA/My and two recently generated MA/My-derived strains , M . H2b and M . H2b-TgDk ( hereafter referred to as Tg1 ) , which expresses a genomic Dk transgene [25] . MA/My and Tg1 mice display MCMV resistance , whereas M . H2b mice are highly susceptible to infection . Moreover , the M . H2b congenic interval spans the rs13483002 locus ( for maps of R7 and M . H2b congenic blocks , see Ref [23] ) . Overall NK cell percentages and numbers , in addition to spleen sizes , were equivalent in uninfected mice ( Fig 4A and 4B ) . Within days after infection , however , MA/My spleens had grown much larger with significantly increased frequencies of NK1 . 1+ ( S5 Fig ) and NKp46+ NK cells ( Fig 4A and 4B ) , which corresponded to lower viral loads ( Fig 4C ) . In contrast , M . H2b spleens failed to increase in size or frequency of NK cells after infection , regardless of viral dose , which resulted in greatly increased viral burden . Similar differences distinguished MA/My and Tg1 after HD-infection , regardless of enhanced viral control in Tg1 ( compared to M . H2b mice ) . Thus , Dk expression by itself is not sufficient to support accumulation of spleen NK cells in response to MCMV . Higher virus levels in Tg1 than MA/My mice further suggested that a C57L-derived susceptibility allele in the M . H2b congenic interval is sufficient to decrease overall resistance . To test this , we next examined ( MA/My x M . H2b ) F1 cross mice after MCMV infection . Remarkably , M . H2k/b spleens were smaller with significantly higher MCMV levels , in comparison to MA/My ( Fig 4D ) . Thus , these data demonstrated that a C57L-derived susceptibility allele , provisionally designated Cmv5s , interfered with the NK-mediated resistance effect of Dk . Moreover , the results established that diminished control in M . H2k/b is unlikely related to a Dk gene dose effect , since the same allele also interfered with host resistance in Tg1 mice with multiple integrated Dk gene copies and slightly higher protein expression [32] . Significantly higher percentages of NKp46+ NK cells and larger spleens in infected Cmv5r resistant MA/My mice provide added support of this interpretation . We infer that enhanced host resistance in MA/My due to increased accrual of NK cells is prevented by expression of at least one Cmv5s allele in more susceptible MA/My-derived ( e . g . M . H2b ) mice . Bekiaris et al . have previously shown that Ly49H+ NK cells are needed to protect spleen white pulp after MCMV infection [35] . Thus , we investigated whether NK cells similarly protected splenic architecture in Cmv5-disparate MA/My mice by comparing secondary lymphoid organ ( SLO ) structures after MCMV infection . Remarkably , within four days after infection , we observed decidedly ill-defined white pulp ( WP ) regions that were smaller and had less clearly delineated marginal zones ( MZ ) in M . H2b and Tg1 mice , in comparison to infected MA/My or uninfected control mice ( Fig 5A ) . Infected MA/My mice had more abundant lymphoid-like cells , in addition to pseudo-nodules of loosely packed mononuclear cells that were heterogeneous in size and shape , appeared to be activated with many mitotic figures , and located at the MZ at the edge of the WP ( Fig 5B ) . In parallel , we observed severe necrosis in the expanded red pulp ( RP ) , which was infiltrated with cells with viral inclusions , granulocytes , and large cells that resemble plasma cells in M . H2b and Tg1 spleens , as compared to infected MA/My or uninfected controls ( Fig 5B ) . To validate the histological findings and Cmv5 genetic regulation of SLO structure after MCMV infection , we next examined spleen sections for NK cells and SLO structural features by IF microscopy . As expected , we observed equivalent numbers of intact NKp46+ cells ( ring-like staining patterns ) in splenic RP of each strain before infection ( Fig 5C ) . Afterward , NKp46+ NK cells were more closely associated with MAdCAM+ cells at the MZ ( Fig 5C ) . In infected MA/My spleens , we observed intact NKp46+ cells , in addition to fragmented NKp46+ granular material , possibly representing NK cell debris . In contrast , intact NKp46+ cells were rare in infected M . H2b and Tg1 spleens . Instead , extensive NKp46 staining of fragmented granular material , frequently concentrated in foci several times larger than an intact NK cell , was typical of infected M . H2b and Tg1 spleens . These data suggested that necrotic NK cells and debris were much more prevalent in mice without Cmv5 protection . We also examined MAdCAM+ marginal sinus-lining cells and CD169+ macrophages at the MZ . At four days postinfection , MA/My spleens consistently had more discrete ( rather than more diffuse ) MZ staining patterns , than either M . H2b or Tg1 ( Fig 5D ) . Nonetheless , IF staining for T and B cells verified even more consistent differences and a selective loss of SLO structural features in M . H2b and Tg1 mice after MCMV infection ( Fig 5E ) . Prominently , in M . H2b and Tg1 spleens , WP was marked by a lack of association between B220+ cells and the MZ . In addition , we observed disorganized regions of T and B cells , and a loss of B cell zone integrity . Together , these results establish that the M . H2b congenic segment failed to protect spleen structures and NK cell accrual after infection . Related occurrences in Tg1 mice verified that Dk was inadequate to protect SLO structure after infection , even though it aided viral clearance compared to M . H2b ( Fig 4 ) . As the extent of viral control differs in MA/My and Tg1 spleens ( Fig 4 ) , we considered that apparent differences in splenic SLO integrity could be due to the variance in viral titers . In line with previous studies [23–25] , we reasoned that G2+ NK-mediated resistance in MA/My and Tg1 is equivalent . Thus , we further assessed the effect of G2+ NK cells on SLO structure under both LD- and HD-infection conditions . As expected , G2+ NK cell depletion resulted in significantly diminished spleen weight , splenocyte numbers , and NK cell recovery after infection in both strains ( S6A and S6B Fig ) . Virus levels were correspondingly higher in G2-depleted mice ( S6C Fig ) . Thus , G2+ NK cells are critical . Nonetheless , residual MCMV resistance in G2-depleted MA/My still exceeded that observed in G2-depleted Tg1 , which revealed that Cmv5 exerted G2+ NK-independent effects on spleen and MCMV clearance ( S6C Fig ) . Moreover , although spleen structure again waned with increased viral burden , G2-depleted MA/My spleens retained better defined WP and a higher density of small lymphocytes , in comparison to virus level-matched Tg1 ( S6C and S6D Fig; compare LD G2-depleted Tg1 to HD G2-depleted MA/My , and HD Tg1 spleen sections ) . Despite that infected cell inclusion bodies were observed in both strains , Tg1 spleen was remarkable for increased granulocytosis and fibrinoid necrosis ( S6C and S6D Fig ) . These results therefore demonstrate that Cmv5's effect on SLO structural integrity during infection is independent of MCMV titers , and G2+ NK-mediated resistance . We infer that the combined effect of Dk and Cmv5r homozygosity in MA/My intensifies host resistance by protecting or regenerating spleen SLO structural features , preventing severe necrosis in spleen RP and promoting NK cell expansion needed to mediate specific virus clearance .
Although small-effect QTL were thought to increase the extent of Dk-dependent resistance to MCMV in C57L , compared to MA/My background mice [26] , until now they have eluded detection likely due to the robust effect of Dk . A genome-driven integrated approach with multiparametric flow cytometry revealed 56 novel immune and MCMV responsive QTL . Though improbable that any two separately measured traits ( e . g . splenic MCMV level and percentage G2+ NK cells postinfection ) would map similarly , unless controlled by the same , or another closely linked gene , the combined approach helped to distinguish significant genomic linkages . Importantly , the combined genome scanning approach resulted in the discovery and mapping of multiple QTL , which together shape strain-specific variances associated with MCMV infection . Consistent with its role in licensing G2+ NK cells [25 , 36] , we observed that the H-2D locus regulated both naïve G2+ NK cell percentages and G2 receptor expression without specific effects on features in other NK cell subsets . H-2D also controlled G2+ NK cell percentages , and the severity of weight loss after MCMV exposure . Together the findings confirm that much of the genetic impact on host resistance is through Dk-specific regulation of G2+ NK cells . Intriguingly , an interactive NKC-linked QTL further shaped MHC I D genetic regulation of G2+ NK cells since G2c57l and G2mamy receptors were expressed differently on NK cells in non-Dk mice . Lower G2c57l receptor expression on a smaller percentage of NK cells is suggestive that these NK cells could be licensed , though not confirmed by ex vivo stimulation experiments . As the majority of NK cells express the given G2 receptor allele in C57L-derived NKCm or NKCl congenic mice , further analysis is needed to exclude potential effects due to other common licensing receptors . Moreover , whereas increased host resistance due to NKCl further suggests that G2 receptor polymorphism might affect MCMV clearance differences , further analysis is needed to precisely define the role of G2+ NK cells . In aggregate , these findings suggest that chr-6 holds two or more QTL: one or more in the NKC that influence NK cell traits , and another more proximal locus , reported in Table 2 , that regulates postinfection body weight . Therefore , the best interpretation is that an additive chr-6 effect with H-2D on MCMV resistance requires a more proximal locus , possibly in addition to a NKC-linked QTL . Given H-2D’s profound influence over NK cells , it was not surprising that overall postinfection percentages of NKp46+ NK cells were controlled by a chr-17 QTL . The newly discovered Cmv5 locus , however , segregated away from H-2D in both the LD and HD analyses . Two-dimensional genome scans of MCMV resistance and morbidity indices validated that at least one non-MHC chr-17 iQTL added to the genetic effect of H-2D . Though mapped proximally in the LD cohort such that a Cmv5 CI spans the H-2D locus , we favor a more distal map location as obtained in the HD analysis , for several reasons . First , in addition to its larger size , the HD cohort included two different MHC congenic blocks ( R7 and M . H2b ) , which increased both resolution ( smaller CIs ) and precision ( more informative crossovers ) in the genetic linkage analysis . Second , less morbidity amongst LD mice also might have limited the map analysis as Cmv5’s effect was most clear after HD-infection . Lastly , analysis of MA/My-derived congenic strains verified that Cmv5 is distinct from and resides distal to H-2D on chr-17 . In addition to its effect on NK cells in infected spleen , we found that Cmv5 also regulates spleen size , SLO structure and organization , and red pulp necrosis , which is at least partly via its direct effect in spleen and independent of G2+ NK cells . One possibility is that the Cmv5r resistance allele protects against the loss of splenic organization and other changes by increasing NK-mediated control of viral replication . An intrinsic modifier of cell proliferation or effector function , for example , might facilitate a more rapid NK response and sensing of viral targets . In fact , m157-specific Ly49H+ NK cells were recently shown to protect lymphoid organization in spleen red and white pulp within days after MCMV infection [35] . Extrinsic cues ( i . e . cytokines or growth factors ) , on the other hand , could also aid in accrual of NK cells via increased recruitment , retention or enhanced proliferation in infected spleen tissue . Cmv5 protection of , or enhancement of IL-15 production by , CD8α DC [37–39] or VCAM-1+ CD31- stromal cells in spleen [40] might explain increased NK accumulation . FMS-like tyrosine kinase 3 ligand ( Flt3-L ) , another interesting candidate , is needed to generate pDC and resident DC from Lin-Flt3+ bone marrow precursors [41] , which subsequently stimulates DC to increase NK cell activation after MCMV infection [42] . Alternatively , Cmv5’s action may be directed to support specific spleen cell function ( s ) independent of NK-mediated viral control , especially given that resistance in Tg1 surpasses that in M . H2b mice after LD- or HD-infection , without affecting other Cmv5 features . Thus , Dk-dependent MCMV resistance per se is inadequate to guard against virus-induced loss of splenic architecture . Whether a cell intrinsic effect or not , greater numbers of competent NK cells in the spleen that can limit viral spread would be expected to minimize excessive inflammation and tissue injury [43] . Recently , we performed whole exome sequencing of MA/My , C57L and M . H2b to elucidate potential genetic candidates for Cmv5’s effect ( H . Lee , A . G . and M . G . B . , manuscript in preparation ) . In the critical genetic interval ( ~17-Mb ) flanked by H2D and the M . H2b congenic recombination crossover marked by Sgol1 , 117 expressed sequences were assessed , including many with non-synonymous or frame shift variants that distinguish MA/My and C57L alleles . Notable among immune-related positional candidates are Tnfrsf21 , and Pla2g7 implicated in influenza control [44] , though its effect was limited to male mice . The Trem /Trem-like gene cluster [45] also maps nearby Cmv5's peak position . As these molecules have been shown to regulate innate immune cells [45] , inflammation [46 , 47] , dead cell clearance [48 , 49] and disease [50–53] , they too represent interesting positional candidates . During recovery from viral infection , the spleen and other organs can serve as sites of blood formation or extramedullary hematopoiesis ( EMH ) [54] . Indeed , spleen NK cells were also shown to promote effective EMH by specifically controlling viral spread [55] . It will be important to determine whether Cmv5 regulates EMH , either apart from or in unison with Dk-dependent NK-mediated MCMV resistance . Thus , this genetic model may extend beyond investigating the effect of MHC polymorphism on NK-mediated virus resistance to examine EMH regulation generally , and during viral infection . Genetic regulation of splenic NK cell accumulation might lend additional precision to the host immune response . Whether due to increased splenic recruitment , retention , or enhanced proliferation that result from intrinsic or extrinsic effects , the fewer numbers of NK cells in the spleen of mice that carry a C57L-derived Cmv5s susceptibility allele should likewise decrease NK-mediated virus resistance . Alterations in NK numbers , and possibly activation status , have the potential to influence other immune cells in the response to infection . Specific NK-mediated control of MCMV has been shown to protect CD8α DC , which reciprocally affect the expansion of NK cells after infection [37] , and the balance of NK-DC crosstalk has considerable potential to affect T cell immunity [56 , 57] . During persistent LCMV infection , NK cells were also shown to regulate adaptive immunity through direct lysis of CD8+ and CD4+ T cell effectors [58 , 59] , which is subject to type I IFN regulation of MHC I expression on T cells themselves [60 , 61] . Likewise in MCMV infection , too few NK cells or inefficient NK activity may result in over exaggerated effector T-cell function , while too many NK cells can also harm otherwise protective T cell immunity [62 , 63] . Intriguingly , this effect of NK cells extends to humoral immunity , as follicular helper T cells are also a target of NK-mediated lysis after LCMV and other viral infections [64 , 65] . Thus , Cmv5 regulation of the innate immune response to viral infection is an additional pivotal mechanism governing NK cell numbers and consequently , their impact on other critical immune cells in the host response , inflammation and tissue injury .
All animal experiments conducted in this study were carried out in accordance with the Animal Welfare Act and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . All experiments were approved by the University of Virginia Animal Care and Use Committee ( Protocol Number: #3050 ) . MA/My x C57L crossed offspring given HD MCMV were described previously [23] . Congenic blocks were included in the crosses to aid identification of small-effect , and possibly MHC- or NKC-linked QTL . Crossed mice without congenic blocks were also generated and analyzed following LD MCMV infection . A diagram of the breeding strategy is shown in S1 Fig . MA/My-derived MHC congenic M . H2b ( i . e . MA/My . L-H2b ) and M . H2b-TgDk ( Tg1 ) were described before [25 , 26] . Tg1 mice have been backcrossed to M . H2b mice for 10+ generations , with marker-assisted genetic selection in the first 4–5 generations . Mice used in the study were managed with a Colony Management System ( Jackson Labs , Version 4 . 1 . 2 ) , maintained in a dedicated animal care facility under SPF conditions and treated in accordance with IACUC regulations and guidelines . Abs and isotype controls for flow cytometry were purchased from BD Biosciences , Biolegend , and R&D Systems . They included anti-CD16/32 ( 2 . 4G2 ) , Ly49G2 ( 4D11 and AT8 ) , Ly49D ( 4E5 ) , Ly49A ( YE1/48 . 10 . 6 ) , Ly49I/U ( 14B11 ) , CD3 ( 145-2C11 ) , CD19 ( 6D5 ) and NKp46 ( 29A1 . 4 ) . Abs were conjugated to FITC , PE , PerCP-Cy5 . 5 , APC , APC-Cy7 or biotin followed by streptavidin conjugated to APC-Cy7 ( Biolegend ) . Leukocyte stains , dead cell exclusion , compensation and cytometric analyses were performed as described [23 , 36] . To determine the licensing status and functional cytokine production by NK cells , ex vivo stimulation and intracellular cytokine staining were performed as previously described [66] . For IF staining of leukocytes and MZ , spleens were snap frozen in liquid nitrogen , processed at the UVA Research Histology Core and examined as described [67] . Briefly , frozen sections ( 6 μm ) were fixed with a 1:1 mix of acetone/ethanol ( 100% ) and stained with Abs against B220 ( RA3-632 , BD Bioscience ) , CD3 ( 145-2C11 , Ebiosciences ) , MAdCAM-1 ( MECA-367 , BioXcell ) , CD209b/SIGN-R1 ( eBio22D1 , Ebiosciences ) , NKp46 ( polyclonal , R&D systems ) , and CD169 ( MOMA-1 , AbDSerotec ) . Non-specific staining and endogenous peroxidase activity were blocked using the Avidin/Biotin blocking kit ( Vector Labs ) , and 3%H2O2 + 10mM NaN3 solution , respectively . The reaction was amplified using the tyramide signal amplification technique ( Perkin Elmer , Boston MA ) . Slides were incubated with primary Abs ( 1h ) , followed by secondary biotinylated or FITC-conjugated Abs ( Vector Labs ) . Slides were then incubated with streptavidin-conjugated horseradish peroxidase and with biotinylated or FITC conjugated tyramide . Finally , streptavidin conjugated with Texas Red ( Southern Tech ) was used to visualize the reaction . Sections in which the primary Ab was omitted served as negative control . Sections were counterstained with DAPI . Spleens fixed with 10% phosphate-buffered formaldehyde were processed and stained at the UVA Research Histology Core and then examined as described [67] . Mice given HD ( 2 x 105 PFU ) MCMV were described previously [23] . Additional crossed mice were infected with LD ( 1 x 104 PFU ) MCMV and analyzed as described before [23] . Spleen and liver fragments were processed for genomic ( g ) DNA genotyping and quantitative real-time PCR ( qPCR ) analysis of MCMV genome level as described [23 , 68] . MA/My , M . H2b and M . H2b-TgDk received HD or LD MCMV and tissues were analyzed 3-7d after infection . Spleen and liver gDNA samples were prepared using a Gentraprep kit . A DNA 'footprint' of MHC , NKC and chromosome 19 genotypes was obtained for each mouse using gene-specific PCR strategies as described [23 , 32 , 69] . High-resolution melt PCR for H-2D exons 5 and 8 was used to validate cross and congenic mouse genotypes . Genome-wide genotyping was performed at DartMouse ( Dartmouth , NH ) using an Illumina medium density SNP panel . Mouse genotypes were concatenated and transposed in Excel . Genomic profiles were reviewed for marker alignment and reliability . Those with greater than 20% failure rate ( double recombination or non-informative ) were discarded . Genomic profiles were then merged with quantitative trait data and stored in a Csv file . LD- and HD-infected mice were studied and analyzed in two separate cohorts . QTL mapping was performed using R/qtl in the statistical computing program R [28] . Csv files were imported into R using read . cross . Calc . genoprob ( 5 cM step ) was used to predict genotypes between markers . Each trait was analyzed using a One QTL scan ( Haley-Knott method ) . QTL significance thresholds were calculated based on 10 , 000 permutations . QTL effect plots were generated using effectplot after running sim . geno ( 1 cM step ) and n . draws ( = 64 ) to obtain imputations . In some cases , the phenotype x genotype command was run to view phenotype values for individual animals at a given marker . Traits analyzed with two-dimensional genome scans ( scantwo ) in R/qtl had significance thresholds based on 1000 permutations . Assessment of data integrity for HD mice has been described [23] . Similar methods were used to assess and validate LD results . Statistical analyses included paired and unpaired Student T tests , Pearson correlations and multiple linear regression tests performed in R [70] with select plots drawn using the ggplot2 package [71] . Results obtained for MA/My-derived congenic strains were analyzed using one-way ANOVA in conjunction with Tukey’s test using Prism software ( version 6 . 0d , 2013 ) . | Uncovering the genetic basis of resistance to viral infection and disease is critical to learning about how immune defenses might be adjusted , how to design better vaccines , and how to elicit effectual immune protection in human populations . Prior studies have shown that both MHC and non-MHC genes support host defenses , or endow specialized immune cells with efficient sensing or responsiveness to infection . Many additional resistance genes remain to be identified , including difficult to detect smaller-effect alleles , which might add to or interact with other genetic factors . Our grasp of the complex interaction involving these genetic elements is thus inadequate . We combined genomic and multiparameter phenotypic analyses to map and identify host genes that control immune cells or sensitivity to viral infection . We reasoned that some might also affect viral clearance . Thus we enumerated a range of immune cell traits in mice before and after infection , which permitted genomic analysis of viral immunity , and mapping of genetic modifiers for each trait . Our study demonstrates that distinct loci collectively regulate both NK cells and host resistance , which provides a framework to understand the genetic interactions , and a variety of potential novel targets to adjust NK cell functionality and host resistance to infection . | [
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] | 2016 | Genomic Modifiers of Natural Killer Cells, Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection |
A hallmark of visual rhabdomeric photoreceptors is the expression of a rhabdomeric opsin and uniquely associated phototransduction molecules , which are incorporated into a specialized expanded apical membrane , the rhabdomere . Given the extensive utilization of rhabdomeric photoreceptors in the eyes of protostomes , here we address whether a common transcriptional mechanism exists for the differentiation of rhabdomeric photoreceptors . In Drosophila , the transcription factors Pph13 and Orthodenticle ( Otd ) direct both aspects of differentiation: rhabdomeric opsin transcription and rhabdomere morphogenesis . We demonstrate that the orthologs of both proteins are expressed in the visual systems of the distantly related arthropod species Tribolium castaneum and Daphnia magna and that their functional roles are similar in these species . In particular , we establish that the Pph13 homologs have the ability to bind a subset of Rhodopsin core sequence I sites and that these sites are present in key phototransduction genes of both Tribolium and Daphnia . Furthermore , Pph13 and Otd orthologs are capable of executing deeply conserved functions of photoreceptor differentiation as evidenced by the ability to rescue their respective Drosophila mutant phenotypes . Pph13 homologs are equivalent in their ability to direct both rhabdomere morphogenesis and opsin expression within Drosophila , whereas Otd paralogs demonstrate differential abilities to regulate photoreceptor differentiation . Finally , loss-of-function analyses in Tribolium confirm the conserved requirement of Pph13 and Otd in regulating both rhabdomeric opsin transcription and rhabdomere morphogenesis . Taken together , our data identify components of a regulatory framework for rhabdomeric photoreceptor differentiation in Pancrustaceans , providing a foundation for defining ancestral regulatory modules of rhabdomeric photoreceptor differentiation .
Rhabdomeric ( r ) photoreceptors are one of two fundamental types of photoreceptors that have been described [1] . Typically , r- photoreceptors populate the visual systems of protostomes including insects , crustaceans , and annelids ( reviewed in [2] ) . This wide phylogenetic distribution and the presence of both types of photoreceptors in many species imply that r- photoreceptors , like their deuterostome counterparts , ciliary photoreceptors , were present before the split of bilaterian animals [3]–[6] . Despite this wide utilization of r- photoreceptors , knowledge about r- photoreceptor differentiation has been virtually exclusively defined from studies in the Drosophila ( fruit fly ) visual system ( reviewed in [7] , [8] ) . Generally , two features characterize r- photoreceptor differentiation . The first is the expression of an r- opsin for light detection , which upon the absorption of a photon leads to the activation of a Phospholipase C cascade and the depolarization of the photoreceptor . This phototransduction cascade permits the amplification of responses to single photons of light [9] . The second program concerns the generation of the rhabdomere , an expansion of the photoreceptor apical membrane to house the phototransduction machinery . This adaptation is necessary for increasing the accuracy of measuring light intensity required for vision [10] . Therefore , understanding the development and evolution of rhabdomeric photoreceptor differentiation requires clarification of how these processes are transcriptionally regulated and whether this regulation is conserved within all rhabdomeric photoreceptor types . In Drosophila , two homeodomain proteins have been identified that are critical for regulating r- photoreceptor differentiation . The first , Orthodenticle ( Otd ) , is the Drosophila ortholog of a conserved family of Otd/Otx homeodomain transcription factors , which are essential for head and brain development across species [11] , [12] . In the r- photoreceptors of Drosophila eyes , otd is required for both aspects of differentiation [13] . Otd promotes the proper morphogenesis of rhabdomeres and directs multiple aspects of the differential expression of r- opsin paralogs , which characterizes the complex visual organization of the Drosophila retinas ( for a review see [14] ) . In particular , Otd is required for the expression of rh3 , an ultra-violet ( UV ) sensitive r- opsin , and rh5 , a blue ( B ) sensitive r- opsin in the two inner photoreceptors of Drosophila ommatidia [15] , [16] . In addition , Otd is critical for repressing rh6 , the Drosophila ancestral long-wave ( LW ) opsin [17] in the six outer photoreceptors of the Drosophila ommatidium [16] . The second critical transcription factor is PvuII-PstI homology 13 ( Pph13 ) , a paired-class homeobox protein that is similar to the vertebrate Aristaless-related homeodomain ( Arx ) proteins [18] , [19] . Like Otd , the loss of Pph13 results in defects in rhabdomere morphogenesis in the Drosophila eye [19] . Interestingly , the concurrent removal of Pph13 and Otd results in complete elimination of the rhabdomeres in Drosophila , suggesting that the two proteins cooperate and have overlapping functions with respect to photoreceptor morphology [20] . Pph13 is also essential for the expression of r- opsins rh6 and rh2 [20] . In contrast to otd mutants , phototransduction is abolished in Pph13 mutants due to the loss and reduced transcription of several key components of the phototransduction machinery [19] , [20] . Lastly , Pph13 regulates photoreceptor differentiation by binding to a subset of Rhodopsin core sequence I ( RCSI ) elements [20] , [21] , which are conserved elements present in Drosophila Rhodopsin promoters [22]–[24] . Consistent with this , Drosophila Pph13 is necessary and sufficient for driving photoreceptor specific reporter gene expression from the artificial 3XP3 promoter [20] , which has been assembled from a Pax6 homeodomain binding site [25] . Given the central role of both Pph13 and Otd in r- photoreceptor differentiation in Drosophila , the question we address here is whether Pph13 and Otd functions represent a common regulatory pathway of arthropod r-visual photoreceptor differentiation . To examine whether Pph13 and Otd could represent a common set of transcription factors required for r- visual photoreceptor differentiation , we chose to investigate their orthologs from two key nodal species , Tribolium castaneum ( red flour beetle ) , a second insect , and Daphnia ( water flea ) , a crustacean . Together , insects and crustaceans define the superclade Pancrustacea within the Arthropoda [26] , [27] and any similarities between Daphnia , Tribolium , and Drosophila r- photoreceptor differentiation would indicate a pathway common to the ancestor that generated both lineages , at least 500 million years ago [28] , [29] . First , we demonstrate that Otd and Pph13 orthologs are present and expressed in the visual systems of both species . Consistent with conservation of Pph13 mediated r- opsin regulation , the Pph13 RCSI binding site is conserved in the promoters of the r-opsin genes of both Tribolium and Daphnia and found only within LW r- opsins . Further , the Tribolium and Daphnia Pph13 homologs have retained similar DNA binding capabilities to their respective endogenous RCSI sites and we confirmed their functional equivalency to direct photoreceptor differentiation in Drosophila photoreceptors by transgenic rescue . The Otd paralogs of Tribolium and Daphnia are comparable in their ability to direct rhabdomere morphogenesis but exhibit differential abilities with respect to r- opsin regulation in Drosophila . Lastly , functional analyses in Tribolium reveal that both Pph13 and Otd homologs are essential for both aspects of photoreceptor differentiation , rhabdomere creation and r-opsin expression . In particular , Pph13 is a critical factor for LW r-opsin expression and Otd2 is necessary for the transcription of UV sensitive r-opsin . In summary , our data identify common components for rhabdomeric photoreceptor differentiation among Pancrustaceans , providing a foundation for defining the ancestral transcriptional mechanisms for rhabdomeric photoreceptor differentiation throughout Bilateria .
As a first step towards examining whether the role of Pph13 and Otd in Drosophila r- visual photoreceptor differentiation was conserved , we investigated the conservation of orthologs in the genome sequences of the distantly related arthropod species , Tribolium castaneum and Daphnia pulex [30] , [31] . Tribolium had been previously shown to possess two paralogs of Otd: Otd1 and Otd2 [32] . The same state was described in the Crustacean Parhyale hawaiensis [33] . However , the relationships of the crustacean and coleopteran Otd homologs to the singleton homolog of Drosophila were previously considered unresolved due to the low level of sequence conservation outside the homeodomain; within Diptera there has been a reduction to only one otd paralog ( Figure 1A , S1 and [33] ) . As in Parhyale , our search in Daphnia pulex as well as Daphnia magna identified two Otd homologs . Protein sequence alignment of an expanded set of Otd homologs ( Figure 1A ) revealed a highly conserved leucine ( L ) residue at the C-terminal end of the Otd1 homeodomain , which was unique for Paired-class homeodomain proteins in general [34] , distinguished the insect representatives of the Otd1 subfamily , including all dipteran homologs . This finding established Drosophila Otd as a member of the insect Otd1 subfamily , implying the loss of insect Otd2 in the evolutionary lineage to Diptera . This conclusion was tentatively supported in a molecular phylogenetic analysis of the relationships between Otd homologs ( Figure S1 ) . The latter approach and amino acid residues in the homeodomain that were unique to each of the Parhyale and Daphnia Otd sequences further suggested that the latter duplicates represented the results of independent gene duplications in crustacean and insect lineages . Thus , the use of the previously introduced acronyms Otd1 and Otd2 for Parhyale and Daphnia paralogs do not imply specific orthology to insect Otd1 and Otd2 . In contrast to the well-characterized deep conservation of the Otd/Otx gene family , homologs of Pph13 have thus far been identified only in a limited number of insects . Previous efforts identified Pph13 homologs in the Tribolium [17] and honey bee genomes [35] but not in the Daphnia pulex genome [36] . Closer inspection of candidate Daphnia pulex orthologs revealed one annotated locus encoding a 5′ truncated Pph13-related homeodomain , suggesting that the annotation for this locus ( JGI_V11_8835 –wfleabase . org ) was incorrect . Further examination of upstream genomic regions revealed that the DNA encoding the 5′ portion of the homeodomain was present . This conclusion was confirmed by RT-PCR ( data not shown ) and in the genome draft of a second related species: Daphnia magna . The complete Daphnia pulex Pph13 cDNA sequence thus included sequence from previously annotated loci JGI_V11_8835 and JGI_V11_313449 . Sequence conservation between Daphnia , Tribolium , and Drosophila homologs was confined to the homeodomain ( Figure 1B ) . However , examination of a larger sample of Pph13 homeodomain sequences in combination with that of members of the Aristaless ( Al/Arx ) gene family revealed amino acid residues that defined each subfamily and added further support of their hypothesized common descent ( Figure 1B and Figure S2 ) [35] . A tyrosine ( Y ) residue at the fourth homeodomain position , otherwise not observed in the Drosophila Paired-class homeodomain proteins [34] , characterized all members of the Arx gene family ( Figure 1B ) . Furthermore , a phenylalanine ( F ) residue at homeodomain position 30 specifically marked the Pph13 orthologs , in contrast to the threonine ( T ) residue in the large majority of Al and Arx orthologs . While this variation was in line with the overall variability at homeodomain position 30 , we noted the singularity of the Pph13 characteristic phenylalanine ( F ) among all Drosophila homeodomain proteins [34] . Based on these clues , we also identified putative orthologs of Pph13 and Al in the mollusk Aplysia californica . Taken together , these alignment data indicated that a gene duplication at least predating the origin of the Pancrustacea gave rise to Pph13 . Finally , we noted the presence of a third Al-related Arx gene family member in the Tribolium genome ( Tcas A12 ) that may be of similar ancient origin based on conservation in non-arthropod invertebrates ( Figure 1B ) . The presumed functional conservation of the Pph13 and Otd homologs predicted their expression in photoreceptors of Tribolium and Daphnia . We therefore assayed the spatial expression patterns of Pph13 , otd1 and otd2 during adult eye development in both Tribolium and Daphnia . Like Drosophila , the Tribolium adult compound eye consists of individual ommatidia each of which contains six outer and two inner photoreceptors [37] . Approximately 30–40 hours after pupation we detected a signal from antisense probes to each transcript in all eight photoreceptors of a single ommatidium ( Figure 2 ) , whereas the corresponding sense probes did not generate any specific pattern ( Figure S3 ) . Pph13 and otd1 appeared to have similar expression patterns , with equal levels of transcript in each photoreceptor ( Figure 2A and B ) . An antibody raised against Tribolium Pph13 confirmed its expression in the nucleus of each photoreceptor ( Figure 2D–F ) . Together the Pph13 RNA in situ pattern and immunofluorescence staining suggested Pph13 is expressed in every photoreceptor of the eye as observed in Drosophila . We also detected otd2 in all eight photoreceptors but its expression in the two central photoreceptors , R7 and R8 , was considerably stronger than in the outer photoreceptors ( Figure 2C ) . In Daphnia , the adult eye consists of a single bilaterally symmetrical compound eye , containing a total of twenty-two ommatidia with eight photoreceptors in each ommatidium [38] . Each Daphnia eye is generated by the fusion of two lateral groups of ommatidia along the midline late in embryogenesis . Due to the lack of molecular markers , the exact biogenesis of the photoreceptors has not been described . However , previous transmission electron microscopy ( TEM ) studies of the development of the axonal photoreceptor connections with lamina neurons predict a model in which the photoreceptors begin to differentiate at the midline and move laterally as they mature [39]–[41] . To confirm and differentiate the developing photoreceptors we first examined the expression of a limited set of r-opsins in Daphnia magna . In Daphnia pulex , there are 27 annotated r-opsin paralogs [30] . Like Drosophila , Daphnia pulex contain representatives of UV , LW and B- light sensitive r-opsins . This diversity includes 23 LW opsins that split between the LOPA and LOPB clades [30] ( Figure S4 ) . The r-opsin family has not been defined for Daphnia magna but for our examination we assayed the expression of a pool of three putative representatives from LOPA ( Figure 3A ) and LOPB ( Figure 3B ) and the putative UV opsin ( Figure 3C ) . Besides observing a differential display of expression between all three groups , the RNA in situ hybridization patterns confirmed and delineated the embryonic tissue that gives rise to the photoreceptors of the eye and ocellus ( Figure 3 and Figure S5A–F ) . Our expression analysis of Otd1 , Otd2 , and Pph13 in Daphnia magna also yielded results that were consistent with this model of eye formation ( Figure 4 and Figure S5G–J ) . The expression of Otd1 was limited to the midline region of the embryo and not necessarily associated with visual photoreceptors ( Figure 4A , C ) . In contrast to Otd1 , Otd2 was expressed in an increasing number of cells in two lateral symmetrical regions of the head during embryogenesis ( Figure 4B–F ) . By 48 hours after egg deposition ( AED ) , each lateral cluster contained approximately 75–78 Otd2 positive cells ( Movie S1 ) . Considering that the adult eye consists of two lateral clusters of eleven ommatidia , this suggested that there were seven Otd2 positive photoreceptors per ommatidium . Furthermore , the additional Otd2 staining present in the central portion of the embryo corresponded to the region where the ocellus develops ( Figure 4A–F and Figure 3B ) . Similar and consistent expression patterns were detected for Daphnia magna homolog of Pph13 , which was expressed in two symmetrical lateral clusters of cells , in the cells of the presumptive ocellus ( Figure 4G–I ) potentially colocalizing with Otd2 protein expression ( Figure 4H , I ) . To further explore the possibility of conserved regulatory roles of Pph13 and Otd in visual photoreceptor differentiation , we probed for the conservation of the RCSI site in candidate target genes of Pph13 in Tribolium and Daphnia . Previous work has shown that Pph13 binds a subset of RCSI sites and that this binding site is essential for transcriptional activation [19] , [20] . The same studies defined a Pph13 RCSI site as a palindromic sequence of TAAT spaced by three nucleotides with one half site matching the consensus sequence of 5′-CTAATTG-3′ [20] . In Drosophila , these Pph13-specific RCSI sites are present in the 5′ cis-regulatory DNA of r- opsin genes and in several other key phototransduction proteins , including the heterotrimeric G-protein β subunit ( Gβ76C ) [19] , [20] . Tribolium contains two r- opsins , one of which belongs to the LW opsin subfamily and one of which belongs into the UV sensitive subfamily [42] . Scanning their upstream regions for the RCSI motif , we found a potential RCSI site in both of them . Furthermore , in the upstream region of the Tribolium homolog of Drosophila visual Gβ ( Gβ76C ) , LOC662674 ( beetlebase . org ) , we also detected a RCSI site ( Table S1 and Figure S6 ) . Examining the immediate upstream regions of Daphnia LW , UV , and B opsins revealed putative RCSI motifs only in the LOPB clade of LW opsins ( Figure S4 and Table S1 ) . In addition , the closest homolog to both Drosophila and Tribolium visual Gβ subunit in Daphnia ( JGI_V11_210534 -wfleabase . org ) contained a potential RCSI site ( Figure S6 and Table S1 ) . The presence of potential RCSI sites in photoreceptor-expressed genes of all three species suggested that these sites could serve as Pph13 binding sites . To test this possibility , we investigated whether the Pph13 homologs could bind the putative endogenous RCSI sequences with electrophoretic mobility shift assays ( EMSAs ) . These experiments revealed that Tribolium and Daphnia Pph13 have similar binding abilities to a consensus RCSI site ( P3 ) [24] , [43] as well as specific Drosophila RCSI sites ( Figure 5 and data not shown ) . Furthermore , each has the capability to bind to their endogenous RCSI sites ( Figure 5 and Figure S7 ) . Interestingly , in Tribolium like Drosophila [20] , we observed a differential affinity of Pph13 to the identified RCSI sites of UV and LW r-opsins . Tribolium Pph13 bound efficiently to the LW opsin RCSI site but binding was barely detectable on the UV opsin RCSI element , suggesting that the simple presence of a correctly spaced palindromic sequence of TAAT was not sufficient to bind Pph13 . The in vivo expression patterns and in vitro binding assays provided strong evidence that Pph13 and Otd regulate r- visual photoreceptor cell differentiation in Drosophila , Tribolium and Daphnia . To test for functional equivalency among the orthologs and , more importantly , the ability to direct photoreceptor differentiation , we examined whether Daphnia and Tribolium orthologs were capable of rescuing the photoreceptor defects observed in Drosophila Pph13 and otd mutants . Drosophila Pph13 mutants have two distinct characteristics . First , Pph13 is necessary for expression of opsin rh6 ( Figure 6A , B ) in 70% of the R8 photoreceptor cells [44] . Second , Pph13 mutants have severe defects in rhabdomere morphology ( Figure 7A , B ) . The morphological defects are acute enough to hamper the detection and accumulation of other r-opsins [20] , [21] ( Figure 6B ) . For rescue experiments , each homolog was placed under the control of GAL4 transcription [45] and inserted into the identical locus in the Drosophila genome . To drive expression , we generated a GAL4 driver under the control of the endogenous Drosophila Pph13 cis-regulatory region – Pph13-Gal4 . In testing the Pph13 homologs of Daphnia and Tribolium , we evaluated both the restoration of Rh6 opsin expression and rhabdomere morphogenesis as compared to rescue with Drosophila Pph13 . We found that all Pph13 homologs were capable of restoring Rh6 expression . In addition , we also detected a mosaic expression pattern of Rh6 in the R8 photoreceptors ( Figure 6C–E ) . Of note , this result also demonstrated the specific rescue of rhabdomere morphology in the R8 photoreceptors that express Rh5 opsin in a Pph13 independent manner . To assay rhabdomere morphology directly , TEM analysis of each rescue condition was performed . We observed wild-type rescue of rhabdomere morphology with all three Pph13 homologs ( Figure 7 ) . However , the rescue was not fully penetrant with Tribolium and Daphnia Pph13 . In particular , we observed photoreceptors missing rhabdomeres with Tribolium Pph13 ( Figure 7D′ ) and with Daphnia Pph13 , the rhabdomeres did not maintain their position and morphology along the proximodistal axis of the photoreceptor ( compare Figure 7E and E′ ) . For examination of functional equivalency among Otd orthologs , we used a previously established rescue paradigm [46] , [47] . Otd is required for rh3 opsin expression in the distally located inner photoreceptor , the activation of rh5 in the proximal inner photoreceptor , repression of rh6 opsin in the outer photoreceptors , and correct rhabdomere morphology in every photoreceptor [13] , [15] , [16] . In our rescue experiments , we assayed for all these functions . With respect to rhabdomere morphology ( Figure 8 ) , TEM analysis demonstrated that both Tribolium Otd paralogs ( Figure 8D , E ) and Daphnia Otd1 ( Figure 8F ) could direct rhabdomere morphogenesis in Drosophila . In these three rescue experiments , we observed the return of symmetrical wild-type pattern of rhabdomere shape and size . However , the rescue was not fully penetrant with Tribolium Otd2 and Daphnia Otd1 . For example , some ommatidia have photoreceptors that lack a detectable rhabdomere . The expression of Daphnia Otd2 resulted in an adult eye that contained a mosaic of intact and dead tissue , regardless of the presence of endogenous Otd ( Figure S8 ) ; this phenotype was not observed with any of the other Otd orthologs . TEM analysis confirmed the lack of photoreceptors in the discolored regions ( data not shown ) . In the normal pigmented regions we observed the presence of rhabdomeres that were not characteristic of the otd mutant or normal rhabdomeres in Drosophila suggesting these defects resulted from the misexpression of the non-endogenous Otd ( Figure 8G ) , which prevented further analysis . As for rescue of r- opsin regulation ( Figure 9 and Figures S9 and S10 ) , our results exposed differential effects among the Otd orthologs . For example , Tribolium Otd1 could activate rh3 expression ( Figure 9D ) but failed to repress rh6 expression ( Figure S9D ) . Tribolium Otd2 in contrast was capable of both ( Figure 9E and S9E ) . On the other hand , Daphnia Otd1 could activate rh3 expression and repress rh6 expression ( Figure 9F and S9F ) , even though Daphnia Otd1 does not appear to be expressed in photoreceptors . Despite the associated cell death and irregular rhabdomere morphology with expression of Daphnia magna Otd2 , we observed that the expression of Rh6 appeared to be limited to a single photoreceptor of each ommatidium suggesting Daphnia magna Otd2 can execute the rh6 repression function ( Figure S9G ) . However , we did not detect any expression of Rh3 , suggesting that Daphnia magna Otd2 failed to execute the rh3 activation function ( Figure 9G ) . In Drosophila , the expression of opsin rh5 in a subset of R8 photoreceptors is dependent on direct activation by Otd . Moreover , Otd also regulates the feedback loop responsible for generating the correct ratio of Rh5 and Rh6 expressing R8 photoreceptors [16] , [48] . In agreement with previous results utilizing this rescue paradigm Drosophila Otd is relatively insufficient in activating rh5 expression [47] and only detected Rh5 expression upon the rescue with Tribolium Otd2 ( Figure S10 ) . Taken together , our expression and rescue assays supported a common role of Pph13 and Otd in r- visual photoreceptor differentiation among Pancrustaceans . For further functional verification , we took advantage of the effective RNAi protocol in Tribolium [49] . Thus to examine the in vivo role of the Tribolium Pph13 , otd1 , and otd2 genes , we generated double stranded RNA ( DsRNA ) against each corresponding mRNA for injection into Tribolium larvae . None of the DsRNAs affected developmental timing , viability , or external morphological structures as compared to mock injections; scanning electron microscopy of the adult eye did not reveal any major effects on the external organization of the compound eyes ( Figure 10A–E ) . To investigate the possible role of Pph13 and Otd in rhabdomere morphogenesis , RNAi knockdown adults were prepared for TEM analysis within twelve hours after eclosion to minimize any potential later disruptions of photoreceptor morphology as a result of long-term degeneration . In these specimens , we found that the knockdown of Tribolium Pph13 resulted in the complete absence of rhabdomeres ( Figure 10G ) . The knockdown of either Tribolium otd1 or otd2 caused a completely distinct set of defects . In otd1 knockdowns , the rhabdomeres were present but reduced compared to wild-type controls ( Figure 10H ) . The ordered array of microvillar projections was normal in each of the rhabdomeres but the microvilli were smaller but no evidence of photoreceptor degeneration could be detected . In otd2 knockdown animals , the rhabdomeres were in a state of disarray suggesting degeneration ( Figure 10I and Figure S11 ) . We detected whole rhabdomeres that however appeared to be unraveling; as suggested by the presence of large membrane protrusions into the photoreceptor cell body and enlarged distances between microvillar projections . In addition some photoreceptors completely lacked rhabdomeres . Lastly , the combinatorial removal of both otd paralogs resulted in the absence of all rhabdomere structures ( Figure 10J ) . As a first approximation of whether Pph13 and Otd homologs may be required for the second step defining r- visual photoreceptor differentiation , i . e . the transcription of the phototransduction machinery , we asked if 3XP3-RFP , an in vivo transcriptional reporter for Pph13 activity in Drosophila [20] , was disturbed upon reduction of Pph13 , otd1 or otd2 ( Figure 11 ) . DsRNAs against each transcript were injected into Tribolium m26 larvae , which express RFP from a 3XP3-RFP reporter transgene . In these experiments , only the Pph13 knockdown resulted in the elimination of the photoreceptor specific expression of RFP ( Figure 11B and Table S2 ) . Single as well as combinatorial injection of the Tribolium otd1 and otd2 dsRNAs did not affect the expression of 3XP3 reporter ( Figure 11C–E ) . To further explore whether Tribolium Pph13 and Otd were required for r- opsin expression in Tribolium , we assayed the transcription of the Tribolium LW and UV opsins by RT-PCR ( Figure 11F ) and by RNA-seq ( Figure 11G , H and Table S5 and S6 ) in the RNAi knockdown conditions . Based on our DNA binding assays and the requirement of Pph13 for 3XP3 expression , we predicted that the knockdown of Pph13 should affect only LW r- opsin transcription . Indeed , the knockdown of Tribolium Pph13 was associated with the reduction of LW transcription but not UV opsin transcription ( Figure 11F , G ) . There was a 3 . 27 Log2 fold decrease in LW opsin expression in Pph13 knockdown animals as compared to mock injections . The knockdown of Otd2 , however , resulted in the virtual absence of UV opsin transcription ( Figure 11F , H ) , as indicated by 5 . 73 Log2 fold decrease compared to control animals . Finally , DsRNA directed against both Tribolium otd paralogs had no discernible effect on LW opsin transcription ( Figure 11F , G ) . Although Tribolium otd1 is expressed in all photoreceptors , we did not detect any significant effect on UV expression alone or enhancement in combination with knockdown of otd2 ( Figure 11F , H ) .
Our results demonstrate a key role of both Pph13 and Otd for visual r- photoreceptor differentiation among Pancrustaceans . While the conservation of Otx transcription factors in metazoan eye development has been documented by numerous studies [47] , [50]–[53] , our data provide the first evidence of a deeper evolutionary conservation of Pph13 . In combination with the initial failure to detect orthologs in other species beyond Drosophila , the specificity of Pph13 expression and function to a single developmental context , terminal photoreceptor differentiation and maintenance [19] , [20] , raised the possibility that Pph13 represented a more recently evolved regulator in insect retinal development; our findings here refute this scenario . Moreover , our comparative sequence analyses consolidate that Pph13 arose by duplication of an ancestral singleton member of the Arx gene family of homeodomain transcription factors , as previously hypothesized [18] , [19] . Further identification of orthologs will be required to date the exact time point of this gene duplication . In our preliminary analyses we have also identified putative Pph13 and Al orthologs in the mollusk Aplysia califonica . However , searches in genome and transcriptome data from other invertebrates as well as non-Pancrustacean arthropods recovered only orthologs of Al/Arx at this point . Here , in this study , we have deployed a combination of assays to assess the functional conservation of Pph13 over hundreds of millions of years of Pancrustacean evolution . Our expression analyses revealed that Pph13 is specifically expressed in the visual systems of Daphnia and Tribolium . Furthermore , this study and previous work now identifies the binding of Pph13 to the RCSI site as the driver of default activation of LW-opsins in both Drosophila and Tribolium . Our findings demonstrate the conserved ability of Pph13 homologs to discriminate between RCSI sites , preferably binding the RCSI sites in LW r-opsins [20] . Moreover , the functional analysis in both Drosophila as well as Tribolium reveals that Pph13 is required for transcription of only the LW r- opsins . We therefore believe that activation through Pph13 at the RCSI site was a module in the cis-regulatory control of the ancestral singleton LW-opsin . Consistent with the conservation of this evolutionarily conserved target sequence , all three homologs are capable of rescuing both aspects of r- photoreceptor differentiation in Drosophila Pph13 mutants: rhabdomere biogenesis , and opsin regulation . The observed decrease in Pph13 rescue efficiency with evolutionary distance may be the result of a failure to interact with the required cofactors in Drosophila or to activate transcription through the Drosophila RCSI sites . Given the conserved binding activity of all Pph13 homologs , it is most likely that the lack of sequence conservation outside the homeodomain compromises interactions with cofactors in the across-species rescue experiments . Our data also demonstrate conserved critical roles of Otd in both aspects of rhabdomeric photoreceptor differentiation . However , the presence of two paralogs in Tribolium and Daphnia , and their differential expression patterns and functional abilities complicate defining the ancestral role of Otd in visual r- photoreceptor differentiation . In all three species at least one Otd ortholog is expressed in developing photoreceptors . Further , the downregulation of Otd orthologs leads to a disruption of rhabdomere formation in both Drosophila and Tribolium . Moreover , with the exception of Daphnia magna Otd2 , each Otd homolog that we tested has maintained the ability to direct rhabdomere morphogenesis in Drosophila . The expression of Daphnia magna Otd2 in Drosophila resulted in cell death and as such precluded assessment of its ability to rescue the Drosophila otd mutant . Taken together , these data suggest that the requirement of Otd in rhabdomere morphogenesis is ancestral . In agreement with this , previous studies demonstrated that all three vertebrate OTX paralogs were capable of rescuing rhabdomere morphogenesis when expressed in Drosophila otd mutant photoreceptors [47] . Consistent with the evidence of independent duplication events in the insect and crustacean lineages , we find that there is no simple correlation between the expression profiles of otd paralogs and their ability to direct r- opsin expression in our data set . Most conspicuously , Daphnia magna Otd1 , which is not detected in photoreceptor cells within Daphnia magna , has the ability to execute both the repression of rh6 and activation of rh3 r- opsins in the Drosophila Otd rescue paradigm . Daphnia magna Otd2 , on the other hand , which is endogenously expressed in photoreceptor cells , was only able to rescue the appropriate repression of Drosophila rh6 , which however works in conjunction with defective proventriculus ( Dve ) [48] . It is also noteworthy that while we could not establish that Daphnia Otd1 is orthologous to Drosophila Otd or Tribolium Otd1 , it has the ability to rescue both rh6 repression and rh3 activation whereas Tribolium Otd1 the ortholog of Drosophila Otd rescued only the activation of rh3 . Moreover , even though Tribolium Otd1 is expressed in all photoreceptors and has the ability to activate r- opsin transcription in Drosophila , this ortholog is apparently dispensable for r- opsin expression within Tribolium . Interestingly , the three vertebrate Otx homologs also exhibited differential rescuing activities of rh3 , rh5 and rh6 regulation [47] . The sum of these data indicates that the paralogs , which originated through independent duplications of the otd locus in insect and crustacean lineages contributed to different subfunctionalization trajectories . While the functional diversification that resulted from this appears bewildering , it implies continued evolutionary interchangeability , which may have been key to consolidating all Otd-related functions onto a single homolog during the loss of otd2 in the lineage to dipteran species . The activation of structural gene batteries forms the endpoint in the gene regulatory network control of cell differentiation [54] . The synergistic activation of the structural genes by both Otd and Pph13 in the Drosophila eye is a good example of this paradigm . Interestingly , our functional analysis in Tribolium reveals differences in how Pph13 and Otd are employed in directing rhabdomere morphogenesis compared to Drosophila . First , within Tribolium , the reduction of either Otd1 or Otd2 generates non-overlapping defects in rhabdomere morphogenesis while the simultaneous knockdown of both genes leads to complete failure of rhabdomere formation . This outcome could result from incomplete subfunctionalization , leaving a limited degree of genetic redundancy in place . Alternatively , the two paralogs may have limited capacities to compensate for the downregulation of the sister paralog via expression level increase . Second , in Drosophila , both Pph13 and Otd are providing independent and overlapping functions to generate the rhabdomere [20] . As a result , the removal of both Otd and Pph13 is required to generate photoreceptors that lack rhabdomeres . In Tribolium , the knockdown of either Pph13 alone or the knockdown of both otd paralogs is sufficient to eliminate rhabdomeres . Thus , there appears to be significantly less redundant control of rhabdomere formation in Tribolium in contrast to Drosophila . Assuming the general conservation of rhabdomere structure target genes , this finding implies evolutionary turnover of Otd and Pph13 dedicated target genes in the context of rhabdomere formation . Given the well defined binding properties of Pph13 and Otd and the conservation of the Pph13 binding sites between Drosophila and Tribolium , it should be feasible to elucidate the diverged distribution of Otd versus Pph13 targets in Tribolium versus Drosophila . Such studies will expand our understanding of the evolution of gene regulatory networks by specifically testing the proposal that downstream network components enjoy greater degrees of evolutionary freedom than intermediate upstream components [55] . Ever since Darwin pondered about the evolution of the eye [56] , the process has and continues to be a challenge to investigators [57] . To date , with respect to photoreceptors , it is now accepted that the two fundamental types , ciliary and rhabdomeric , were present before the split of bilaterian animals and share a common ancestor [3]–[6] . However , compounding the study of the evolution of photoreceptors is the fact that the photoreceptors are utilized in various visual and non-visual light detection systems and obtain diverged morphological states in both protostomes and deuterostomes . Therefore , a critical component to provide clarity for defining homologous photoreceptors [3] is to identify the conserved regulatory proteins and switches required for the differentiation of the various classes of animal photoreceptors . Our studies have now identified two common regulators of one type of photoreceptor: rhabdomeric visual photoreceptors . Nonetheless , a key for a complete understanding will be to not only document their presence in but also confirm their functional roles in the visual systems of emerging model systems; to date our attempts with RNAi against Daphnia Pph13 and otd2 have not been successful . Fortunately , with the advent of TALEN and CRISPR technology [58] , [59] functional studies may no longer be the limiting step . Thus future work will seek to define the ancestral mode of rhabdomeric photoreceptor differentiation among protostomes and in addition determine how the regulatory cascade is modified to generate diversity . For example , it will be interesting to explore how the origin of Pph13 relates to the diversification of ciliary and rhabdomeric photoreceptors during early metazoan evolution and whether Pph13 or Otd is required for the differentiation of non-visual rhabdomeric photoreceptors , as exemplified by intrinsic photosensitive retinal ganglion cells ( ipRGC ) [60] , [61]; ipRGCs express an r- opsin but do not develop the characteristic membrane folds of a rhabdomere . With respect to Pph13 , we have not observed a vertebrate ortholog . While , Arx transcription factors are known to carry out patterning functions in the developing forebrain that could affect the visual system [62] , [63] no Arx functions have been reported that relate to the terminal differentiation of photoreceptors . Furthermore , the identification of Otd orthologs as critical components in both rhabdomeric and ciliary photoreceptor cell differentiation [50]–[53] raises the question whether Otd represents the ancestral mechanism for regulating photoreceptor differentiation in both fundamental types of photoreceptors . Overall , these answers will come from comprehending how the differences in phototransduction and morphology between the two fundamental types of photoreceptors are transcriptionally regulated , whether the regulation is conserved within each photoreceptor type , and how transcriptional regulation is modified dependent upon whether the photoreceptor is incorporated into a visual or non-visual system .
cDNAs representing Tribolium Pph13 and otd2 were constructed from RT-PCR reactions from total RNA isolated from beetle heads . The otd1 cDNA was a gift from Dr . G . Bucher . cDNAs representing Daphnia magna Pph13 and otd2 were constructed from RT-PCR reactions from total RNA isolated from whole adults . The Daphnia magna cDNA of otd1 was isolated by screening an embryonic cDNA library [64] . For transgenics , all cDNAs were cloned into pUASTattB vector and integrated into genome position 65B2 ( Rainbow Transgenic Flies , Inc . ) . Pph13-GAL4 was generated by inserting the immediate upstream 1 . 6 kb of genomic DNA extending from the first coding Methionine of the Pph13 locus into pCHS-GAL4 . Drosophila strains utilized: cn bw , cn bw Pph13hzy [19] , otduvi [65] , [66] , y w; Sp/Cyo; UAS-Flag-otd/TM2 [46] , [47] , and otduvi; otd-GAL4 , Pwiz6/Cyo; TM2/TM6B [46] , [47] . For otd rescue experiments females of otduvi; otd-GAL4 , Pwiz6/Cyo; TM2/TM6B were crossed to w; +; UAS – otd X transgenic lines and only non CyO;TM6B male progeny were examined . For Pph13 rescue experiments w; cn bw Pph13/CyO; Pph13-GAL4/TM6B homozygous flies were crossed to w; cn bw Pph13/CyO; UAS- Pph13 X/TM6B and only non CyO; TM6B progeny were examined . All cDNAs were cloned into pCDNA 3 . 1 ( Invitrogen ) and EMSAs were performed as described in [19] , [67] . Proteins were generated in reticulocyte lysates ( Promega ) . The sequences of probes utilized are listed in Table S1 . To generate RNAi knockdown animals , 1 ug/ul of total probe , DsRNA was injected into pu11 , m26 , or vw late stage larvae; pu11 and m26 contain a 3XP3-GFP and a 3XP3-RFP reporter , respectively [68] , [69] . Two independent DsRNAs for each gene were tested and eyes from at least five different subjects were examined to confirm phenotypes . The regions listed for each gene are listed in Table S3 . For all the results reported here a mixture of the two DsRNAs for each gene were utilized . The mixtures of DsRNAs contained equal amounts of each individual DsRNA and were used and a final concentration of 1 ug/ul was injected . Dark-reared , newly eclosed ( <12 hours old ) beetles were collected , scored and utilized for various procedures . Total RNA was isolated using Trizol and DNAase treated . First strand synthesis was accomplished with the Superscript III ( Invitrogen ) kit . Each reaction contained 2 ug of total RNA and oligo-dT as primers . PCR amplification was performed with 1 ul ( 1/20th ) of RT reaction and samples were collected at 25 or 30 cycles . All reactions were repeated three times with two independent sets of total RNA . Equal amounts of PCR reactions were analyzed by gel electrophoresis . The list of PCR primers used can be found in Table S4 . For each condition , duplicate sets of total RNA from entire animals ( <12 hours old ) were isolated . Stranded RNA sequencing libraries were constructed using the TruSeq Stranded mRNA Sample Preparation Kit ( Illumina , San Diego , CA ) according to manufacturer's instructions . Libraries were quantified using the KAPA SYBR FAST Roche LightCycler 480 2X qPCR Master Mix ( Roche , Indianapolis , IN ) , pooled in equal molar amounts , and sequenced on a HiSeq2000 instrument ( Illumina , San Diego , CA ) using a 100 bp paired-end run . HISeq read sequences were cleaned using Trimmomatic version 0 . 30 [70] to remove adapter sequences and perform quality trimming . Trimmomatic was run with the following parameters , “3:30:10 LEADING:3 TRAILING:3SLIDINGWINDOW:4:20 MINLEN:75” . The resulting reads were mapped against the Tribolium release 3 . 0 gene models ( http://www . Beetlebase . org/ ) using TopHat2 version 2 . 0 . 9 [71] with the parameters “–b2-very-sensitive–read-edit-dist 2 –max-multihits 100 –library-type fr-firststrand” . Read counting was done for each gene using htseq-count from the HTSeq package version 0 . 5 . 4p3 [72] with the “–stranded = reverse” parameter . For Tribolium , read counts were normalized across samples using the DESeq package ( version 1 . 12 ) in R/Bioconductor [73] . DESeq [72] was further used to detect statistically significant differences in expression between two conditions using the binomial test with a . 05 adjusted p-value cutoff . The complete data set will be presented and discussed elsewhere but total read data and reads specific to Tribolium LW and UV opsin are listed in Tables S5 and Tables S6 . Drosophila and Tribolium heads were prepared as previously described [20] , [74] . All samples were from newly emerged adults and for each genotype at least three retinas from three different heads were examined . Samples were observed under transmission electron microscope ( TEM ) , operated at 60 KV and digital images were captured and imported into Adobe Photoshop . The Tribolium Pph13 polyclonal antibody , 171 , was created by injecting rats with a GST-fusion protein representing amino acids 104–220 of the protein . The Daphnia magna Otd2 polyclonal antibody was prepared in guinea pigs against a bacterially expressed C-terminal portion of the protein ( amino acids 235–395 ) . The Daphnia magna Otd1 polyclonal antibody was prepared in rats against a bacterially expressed portion of the protein ( amino acids 220–351 ) . Whole mount RNA in situ hybridization in Tribolium and Daphnia was performed as previously described [42] , [75] . The regions of the sense and ant-sense probes are listed in Table S3 . The following primary antibodies were used: rabbit anti-Rh6 ( 1∶2500; Dr . C . Desplan ) , rat anti- Tribolium Pph13 ( 1∶20 ) , rat anti- Daphnia Otd1 ( 1∶500; Dr . Y . Shiga ) , guinea pig anti- Daphnia Otd2 ( 1∶500; Dr . Y . Shiga ) mouse anti-Rh3 ( 1∶50; Dr . Steve Britt ) , mouse anti-Rh5 ( 1∶50; Dr . Steve Britt ) and rabbit anti-Rh6 ( 1∶1000; Dr . Claude Desplan ) . For Otd2 , in Daphnia , signals were amplified with the Tyramide Signal Amplification ( TSA ) system ( PerkinElmer ) . FITC and Rhodamine conjugated secondary antibodies were utilized ( Jackson ImmunoResearch ) . Immunofluoresence studies were performed as previously described [47] , [76] . All Drosophila samples were from newly emerged adults and for each genotype at least three retinas from three different heads were examined . Many of the sequences utilized in this study can be found in Table S7 . | Visual systems are populated by one of two fundamental types of photoreceptors , ciliary and rhabdomeric . Each photoreceptor type is defined by the opsin molecule expressed and the final morphological form adapted to house the phototransduction machinery . Here we address whether a common transcriptional mechanisms exists for the differentiation of rhabdomeric photoreceptors . We demonstrate that orthologs of two Drosophila ( fruit fly ) transcription factors , Pph13 and Orthodenticle , are expressed in photoreceptors of Pancrustaceans , Tribolium ( red flour beetle ) and Daphnia ( water flea ) , and are capable of executing conserved functions of rhabdomeric photoreceptor differentiation . In particular , Tribolium and Daphnia orthologs are capable of substituting and rescuing the photoreceptor differentiation defects observed in their corresponding Drosophila mutants . Furthermore , loss of function analysis in Tribolium of both Pph13 and orthodenticle genes demonstrate they regulate opsin transcription and morphogenesis of the photoreceptor apical membrane . Our data illuminate a framework for rhabdomeric photoreceptor differentiation and provide the foundation for defining the ancestral regulatory modules for rhabdomeric differentiation and potential modifications that underlie the functional diversity observed in rhabdomeric photoreceptors . | [
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] | 2014 | Common Transcriptional Mechanisms for Visual Photoreceptor Cell Differentiation among Pancrustaceans |
Newly emerged wheat blast disease is a serious threat to global wheat production . Wheat blast is caused by a distinct , exceptionally diverse lineage of the fungus causing rice blast disease . Through sequencing a recent field isolate , we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains . No mini-chromosomes were observed in an early field strain , and at least two from another isolate each contain different effector genes and core chromosome end sequences . The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends . Additionally , transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point ( RIP ) mutation genome defenses . Our results , collectively , indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories , and mini-chromosomes and core chromosome ends are coupled as a mobile , fast-evolving effector compartment in the wheat pathogen genome .
Wheat blast is an explosive emerging disease capable of 100% yield losses . Little resistance is available in cultivated wheat varieties , and fungicides are not effective under disease favorable conditions [1 , 2] . The disease emerged in Brazil in 1985 and spread within South America , limiting wheat production ( Fig 1 ) . Wheat blast jumped continents in 2016 , causing major yield losses in Bangladesh with this first report [3 , 4] . Wheat blast has now established in South Asia , enhancing fears about further disease spread , disruption of global grain trade by this seed-borne pathogen , and endangerment of global food security [5] . Wheat blast is caused by a wheat-adapted lineage of Magnaporthe oryzae ( synonymous with Pyricularia oryzae ) [6] , known as the Triticum pathotype ( MoT ) . MoT strains are distinct from rice pathogens in the M . oryzae Oryza pathotype ( MoO ) and millet pathogens in the Eleusine ( MoE ) and Setaria ( MoS ) pathotypes ( S1 Fig ) . A serious turf grass disease emerged in the United States in the late 1980s , caused by the Lolium pathotype ( MoL ) with ryegrass as its major host . Although some MoL strains can infect wheat [7] , MoT strains are distinguished as highly aggressive wheat pathogens that are so far restricted to certain countries in South America and South Asia ( Fig 1A ) . Although little is known about wheat blast , studies on rice blast disease have identified numerous effector genes , generally encoding small proteins that are specifically expressed in planta and play roles in host invasion [8–10] . Some effectors , termed avirulence ( AVR ) effectors , determine either rice cultivar or host species specificity through blocking infection upon recognition by corresponding cultivar- or species-specific resistance ( R ) genes and triggering hypersensitive resistance . For example , strains of several M . oryzae pathotypes are not able to infect weeping lovegrass , Eragrostis curvula , because they carry a host species-specific AVR effector PWL2 [11 , 12] . Planting of wheat varieties lacking the R gene Rwt3 in Brazil likely enabled MoL strains with the corresponding host species-specific AVR effector PWT3 to adapt to wheat , and subsequent loss of PWT3 function played a role in the wider emergence of the MoT subgroup [13] . So far , characterization of 11 MoO AVR effectors together with their corresponding R gene products has identified direct or indirect protein interactions that control rice cultivar specificity [9 , 14] . In contrast , understanding how individual effectors function in host invasion has been difficult due to apparent functional redundancy . That is , deletion of individual effector genes rarely dramatically impacts the pathogen's ability to cause disease . Effector genes in diverse filamentous eukaryotic pathogens generally reside in rapidly evolving , transposon-rich chromosomal regions , which , together with slowly evolving core chromosome regions containing housekeeping genes , results in a 'two-speed' genome [15 , 16] . M . oryzae effectors from the Oryza pathotype are known to reside in transposon-rich regions , often near chromosome ends [9 , 17] . Two AVR effector genes [18 , 19] have been localized to dispensable mini-chromosomes ( also known as supernumerary , accessory or B chromosomes [16 , 20 , 21] ) that show non-mendelian inheritance and are present in some , but not all individuals in a population [22–24] . Effectors are associated with frequent presence/absence polymorphisms between and/or within the different M . oryzae lineages [18 , 25] . Deletion of the corresponding AVR effector gene could be a response to deploying R genes in a crop . In one well-studied case , AVR-Pita1 , which corresponds to the periodically-deployed Pita rice R gene , has been mobile in the M . oryzae genome [18] . Specifically , AVR-Pita1 is found on different chromosomes in different strains , often near telomeres , and sometimes on mini-chromosomes . Understanding AVR effector gene dynamics is key to combating the ability of the blast fungus to rapidly overcome deployed R genes and to developing sustainable disease control . Wheat blast disease is proving even harder to control than the ancient , still-problematic rice blast disease . Potential wheat resistance identified using strains isolated soon after disease emergence in 1985 are no longer effective in controlling recent aggressive field isolates from wheat in South America and South Asia . The global threat now posed by wheat blast disease makes it critical to generate genomic resources to further understand the wheat blast fungus . Here , a reference genome of an aggressive MoT strain was generated and compared to genomes of early and recent wheat pathogens and other host-adapted strains . We report that the genome structures of the 7 wheat blast core chromosomes have not diverged significantly from the rice blast core chromosomes . However , mini-chromosomes present in zero , one or two copies in different strains serve as a highly variable compartment for effector genes .
We sequenced and generated a near-complete genome assembly of the highly aggressive Bolivian field isolate B71 [4 , 26] , which exhibits high sequence similarity with MoT isolates from Bangladesh ( Fig 1A , S1 Table and S1 Fig ) . An assemblage containing 31 contigs ( S2 Table ) was produced from >12 . 4 Gb of whole genome shotgun ( WGS ) PacBio long reads ( S2 Fig ) . Genome polishing utilizing ~10 Gb Illumina sequencing data corrected 37 , 982 small insertions and deletions as well as 350 base-pair substitutions in the PacBio draft assembly ( S1 Data ) . Corrected assembled contigs were in the range of 44 . 2% to 52 . 5% GC content with the exception of a contig of 28 . 4% , which was predicted to be from mitochondria of B71 owing to its high similarity ( 99% identity ) to the mitochondrial sequence of M . oryzae rice pathogen 70–15 [27] . A circularized B71 mitochondrial sequence was obtained after removing redundant sequences at the contig ends . We developed a novel scaffolding technology , LIEP ( Long Insert End-Pair sequencing ) to improve the continuity of the assembly ( Fig 2A ) . Briefly , LIEP involved construction of millions of vectors , each of which contains a unique DNA barcode pair of 22 nt and 21 nt random barcodes . Barcodes for each vector were sequenced to establish a sequence database of barcode pairs . The vectors were then used to construct clones with 20–30 kb long inserts of B71 genomic DNA flanked by the two vector barcodes . Both ends of the insert were sequenced , generating clone-end sequences with paired barcode sequences . Barcode sequences were used to recover clone-end pairs . All steps were performed with pooled clones rather than individual clones . After scaffolding , a small contig ( ~12 kb ) with the poor support from Illumina reads was discarded . Scaffolding and filtering condensed the assembly to 12 contigs , which were then reoriented and renamed based on the MG8 genome assembly of rice pathogen 70–15 [27] . Consequently , the final B71 genome assembly ( B71Ref1 ) is comprised of ~44 . 46 Mb in seven chromosomes and five unanchored scaffolds ( Fig 2B ) . Telomere repeat sequences ( TTAGGG ) n or M . oryzae telomeric retrotransposons ( MoTeRs ) that integrate in telomere repeats [28] were identified on both ends of chromosomes 2 , 4 , 5 , 6 , 7 and on one end of chromosome 1 , indicating that B71Ref1 is a near end-to-end assembly . The B71Ref1 and MG8 assemblies show high end-to-end co-linearity for chromosomes 2 , 4 , 5 , and 7 ( Fig 2C , S2 Data ) . A two-megabase rearrangement was identified between chromosomes 1 and 6 , of which part of chromosome 1 of MG8 was located on chromosome 6 of B71 . The rearrangement was supported by eight pairs of LIEP sequences ( S3 Fig ) and by 50 single PacBio long reads . This rearrangement is not MoT specific because it was also observed in a MoO field isolate , evidenced by a long PacBio assembled sequence spanning both chromosome 1 and chromosome 6 of MG8 [29] . A large sequence in B71Ref1 , from 1 . 3 to 2 . 9 Mb on chromosome 3 , was absent in MG8 . The unanchored 70–15 MG8 contig , supercont8 . 8 , was mapped at the beginning of B71 chromosome 7 , implying supercont8 . 8 is the missing end of chromosome 7 in the MG8 reference genome . None of five unanchored scaffolds of B71Ref1 can be mapped to MG8 , with the requirement of , at minimum , a 10-kb match and 95% identity . Annotation of B71 identified 12 , 141 genes , with 1 , 726 harboring signal peptide domains ( Fig 3 , S3 and S4 Data ) . Of the 248 highly conserved core set of eukaryotic genes , 243 ( 98 . 0% ) orthologs from the B71 annotation were identified by CEGMA , compared to 97 . 6% orthologs in MG8 . Therefore , completeness and annotation of the B71 genome are at least comparable to that of MG8 , which was produced using Sanger sequencing and multiple technologies . Comparison of RNA-Seq data of MoT-infected wheat from the field in Bangladesh [3] and culture-grown MoT identified 335 and 153 genes that were only expressed in planta and in culture , respectively ( SI Materials and Methods ) ( S5 Data ) . Secretion signal domains occurred in 173 in planta-specific genes , and in 18 culture-specific genes . The in planta-specific genes included homologs of five MoO effector genes , including PWL2 and PWL4 ( an inappropriately expressed homolog from a weeping lovegrass pathogen that fails to block infection of Eragrotis spp . ) [11 , 12] , AVR-Pib and AVRPiz-t that determine rice cultivar specificity [30 , 31] , and the cytoplasmic effector BAS1 [32] ( Fig 3G and S5 Data ) . The remaining 168 in planta-specific genes were considered putative effectors ( S6 Data ) . Both known and putative effector genes tended to be located towards the ends of core chromosomes ( Fig 3G ) . We also generated RNA-Seq data from both B71 in planta leaf samples enriched with fungus at 40 hours post inoculation ( HPI ) and from B71 grown in liquid medium , which was referred to as the second RNA-Seq experiment . Differential expression analysis identified 2 , 891 up-regulated genes and 2 , 429 down-regulated genes of in planta B71 samples as compared to in vitro cultured samples . Considering genes with high fold changes in expression ( at least 16x fold-change ) between the two groups , we found many more highly up-regulated genes than highly down-regulated genes in planta ( 863 vs . 44 ) . Of 174 known or putative effector genes , 110 were highly up-regulated at 40 HPI . We sequenced eight additional field isolates , including less-aggressive early strain T25 isolated in Brazil in 1988 [26] , five other MoT strains , a MoL strain , and a MoE strain ( S1 Fig and S1 Table ) [6] . A read depth approach was employed to detect genomic copy number variation ( CNV ) between B71 and each isolate , focusing on the identification of genomic regions with conserved copy number ( CNequal ) , higher copy number ( CNplus ) , or lower copy number ( CNminus ) in non-B71 isolates ( S4 Fig ) . Among ~41 . 7 Mb of low repetitive regions , 36 . 4 Mb ( 87 . 3% ) exhibited CNequal among all nine isolates . In total , 4 . 9 Mb ( 11 . 8% ) displayed CNV between B71 and at least one other isolate , with 2 . 7 Mb ( 6 . 5% ) being CNplus and 3 . 4 Mb ( 8 . 2% ) CNminus ( Fig 3D , 3E and 3F ) . Ten effector homologs [9] ( PWL4 , AVR-Pik-chr3 , AVR-Pi54 , BAS1-chr1 , BAS2 , BAS3 , BAS4 , AVR1-CO39 , AVR-Pi9 , and AVRPiz-t ) resided in CNequal regions ( chromosome identifier added to distinguish paralogs ) . Four ( AVR-Pii-chr3 , AVR-Pib , PWL2 , and BAS1 ) were in CNminus regions and four ( PWT3 , AVR-Pii-scaf1 , AVR-Pib , and AVR-Pik ) in CNplus ( S3 Table ) . CNV analysis of effector genes was supported by Illumina draft assemblies of the eight strains ( S4 Table ) . Sequences from Illumina draft assemblies also showed sequence variation of some effector genes among these strains , such as DNA insertions in PWT3 and AVR-CO39 , two AVR genes governing host specificity [13 , 33 , 34] . Thus , some AVR homologs are equal in copy number and highly conserved across all strains , while many are subject to sequence changes , including copy number changes . Of 1 . 2 Mb genomic sequences exhibiting CNplus in some isolates but CNminus in others , ~819 kb ( 68 . 5% ) were from the five scaffolds ( scaf1-5 ) , which constitute only 4 . 3% of the genome . CNV variation of sequences in the B71 scaffolds indicated they are absent in the less aggressive MoT strain T25 ( Fig 4A ) . The P3 and B71 comparison , however , suggested that most scaffold sequences are duplicated in P3 , an aggressive isolate from Paraguay in 2012 ( Fig 4B ) . In summary , extensive copy number variation was observed among M . oryzae field isolates , especially in five scaffolds that were not anchored to the seven chromosomes . Variability in the five scaffolds led us to hypothesize that some or all scaffolds might correspond to mini-chromosome sequences in B71 . Electrophoretic karyotypes of B71 using contour-clamped homogeneous electric field ( CHEF ) electrophoresis confirmed that B71 , indeed , contained a mini-chromosome or multiple mini-chromosomes of ~2 . 0 Mb in size ( Fig 4C ) . Mini- and core chromosomal DNAs were separately excised from the gel for Illumina sequencing . The five scaffolds were highly over-represented among reads obtained from the mini-chromosome DNA and highly under-represented among the core chromosome reads , confirming that all five scaffolds are from the mini-chromosome ( Fig 4D ) . Roughly equal mean depths of B71 WGS reads mapped on all seven core chromosomes or the mini-chromosome supported that B71 contains a mini-chromosome . The mini-chromosome contains 192 protein-coding genes . Of those , 58 . 9% ( 113/192 ) of the genes were expressed ( S5 Data ) . Approximately half expressed genes ( N = 56 ) were highly regulated in expression with at least 16 fold changes comparing 40 HPI in planta samples with in vitro cultured samples , and , significantly , they were all up-regulated in planta , which indicated that genes in the mini-chromosome are likely to be associated with pathogenicity . Of 113 expressed genes , 23 were functionally annotated . Notably , the mini-chromosome contains four of all six genes in the genome that encode plasma membrane fusion proteins , and all four were highly up-regulated in planta at 40 HPI . Three functionally annotated genes exhibited in planta specific expression in the field samples or the B71 in planta leaf sheath samples , namely BSY92_12116 , BSY92_11977 , and BSY92_12070 , encoding endochitinase B1 , a gentisate 1 , 2-dioxygenase , and a heat-labile enterotoxin ( a putative effector gene ) , respectively . A transcriptional regulatory gene , an Sge1 homologous gene ( BSY92_12088 ) , governing expression of secondary metabolite biosynthetic genes [35] was highly up-regulated in planta . Most other functionally annotated expressed genes are associated with putative enzymatic activities . A gene BSY92_11993 encoding ubiquitin-like-specific protease 2 was expressed in both in planta and in vitro cultured samples , but it was highly up-regulated in planta . Gene ontology ( GO ) enrichment analysis identified that cysteine-type peptidase activity ( GO:0008234 , p-value = 0 . 0001 ) was over-represented in genes on the mini-chromosome ( S7 Data ) . Eight out of all 11 genes associated with cysteine-type peptidase activity are located on the mini-chromosome , and 7 out of these 8 were expressed in either in planta or in vitro cultured samples . Known effector genes PWL2 and BAS1 ( S5 and S6 Figs ) , which are located on different core chromosomes in MG8 , were located immediately adjacent to one another and surrounded by various transposon sequences on the B71 mini-chromosome ( Fig 5A ) . This configuration was supported by 211 PacBio long reads and by Sanger sequencing of a PCR product obtained with a PWL2 and BAS1 primer pair ( S7 Fig ) . No PWL2 or BAS1 homologs , with at least 70% identity , were identified on core chromosomes , supported by an under-represented sequencing coverage on the PWL2 or BAS1 regions from CHEF sequencing of B71 core chromosomes ( Fig 5D ) . Both genes exhibited in planta-specific expression on the mini-chromosome ( Fig 5B and S8 Fig ) . Therefore , mini-chromosomes harbor effector genes that show similar in planta-specific expression patterns to effector genes residing on core chromosomes . Further CHEF analyses showed no evidence of mini-chromosomes in T25 and supported at least two mini-chromosomes in P3 , consistent with predictions from the CNV results . The P3 mini-chromosomes are ~1 . 5 Mb and ~3 Mb in length ( Fig 4C ) . Sequences of both P3 mini-chromosomes exhibited similarities to the B71 mini-chromosome but also marked differences ( Fig 4D and Fig 5E ) . The large P3 mini-chromosome contained both PWL2 and BAS1 genes ( Fig 5C and 5D ) , plus it harbored ~33 kb ( assembly location 6 , 007 to 6 , 039 kb ) of duplicated DNA from a region near the end of chromosome 6 . This duplicated DNA segment included a homolog of the MoO effector AVR-Pib [30] . In contrast , the small P3 mini-chromosome lacked the PWL2 and BAS1 genes , but it contained a duplication of approximately 0 . 39 Mb of the chromosome 7 end ( assembly location ~3 . 65 to 4 . 04 Mb ) ( Fig 5D ) . Retention of this segment in the core chromosome explains the large CNplus segment at this region of P3 chromosome 7 ( Fig 4B ) . The CNV result indicated that both sequences of ends of chromosome 6 and chromosome 7 found in separated mini-chromosomes have only one extra copy , supporting that P3 mostly likely has no more than two mini-chromosomes . Notably , this segment contained five putative effector genes and a homolog of the known MoO effector gene AVR-Pik [36] . Another notable region from the end of chromosome 3 was present in both P3 mini-chromosomes , but not present in the B71 mini-chromosome ( Fig 4D ) . Sequencing P3 core chromosomes identified sequences homologous to the B71 mini-chromosome that were not present in B71 core chromosomes ( Fig 4D ) . Taken together , these three MoT mini-chromosomes contain different sets of known or predicted effector genes and other core-chromosome end sequences , which are either missing or duplicated on the core chromosomes of the same or other strains . The highly variable structure of MoT mini-chromosomes indicates frequent acquisition of sequences from core chromosomal ends . Repeat annotation showed approximately 12 . 9% of the B71 genome consisted of transposons and other repetitive elements , and transposons accounted for 9 . 7% and 52 . 8% of the core and mini-chromosomes , respectively ( Fig 6A and S5 Table ) . Many of the transposons that were over-represented in the mini-chromosome occurred frequently on chromosome arms , particularly at chromosome ends ( S9 Fig ) . Four transposon subclasses made up a greater proportion of the total transposon sequences on the mini-chromosome versus core chromosomes , including three LINEs ( Tad1 , Jockey and I ) and the DNA transposon TcMar-Fot1 ( Fig 6B ) . These four are among the top five elements enriched in the core chromosomal 20% ends relative to the 20% middle core chromosome regions ( Fig 6C ) . Besides similarities in transposon composition between chromosome ends and the mini-chromosome , alignment of the B71 mini-chromosome sequence to core chromosomes identified duplications of >10 kb fragments with at least 95% identity . Duplications were located at ends of chromosomes 3 , 4 , and 7 ( S9 Fig ) , and they were highly enriched for telomere-associated MoTeRs ( LINE/CRE element ) . Therefore , a subset of MoT transposons is implicated in dynamic interactions between MoT mini-chromosomes and core chromosome ends . Nucleotide composition analysis indicated that , overall , repetitive sequences along core chromosomes were highly negatively correlated with GC content ( Fig 3B and 3C , S10 Fig ) . However , the highly negative correlation did not hold in the mini-chromosome , which is highly repetitive while maintaining relatively high GC content ( S10 Fig ) . Repetitive sequences in many fungi , including M . oryzae MoT strains , are subject to repeat-induced point ( RIP ) mutation resulting in C-to-T or G-to-A transitions and , thereby , leading to reduced GC content [37–40] . Given higher GC content of repetitive sequences in the mini-chromosome versus core chromosomes , we explored the possibility of different levels of RIP in these genomic regions by assessing their RIP-type mutation rates . Of six high-abundance transposons examined , all exhibited reduced levels of RIP-type mutations in the mini-chromosome relative to core chromosomes ( S11 Fig ) . We examined transposons MGR583 ( LINE/Tad1 element ) and Pot2 ( DNA/TcMar-Fot1 element ) that are present with multiple copies in both core and mini-chromosomes . RIP analysis indicated that no sequences of MGR583 ( N = 7 ) or Pot2 ( N = 22 ) from the mini-chromosome were subjected to extensive RIP-type mutations , while 14/20 MGR583 and 3/19 Pot2 from core chromosomes contained abundant RIP-type mutations ( Fig 6D and S12 Fig ) . Therefore , unlike transposons in core chromosomes , transposons in MoT mini-chromosomes do not appear to be inactivated by the RIP genome defense mechanism .
The B71 reference genome for the wheat blast fungus has shown a high degree of macrosynteny for the core chromosomes relative to the rice pathogen reference genome 70–15 ( MG8 ) , which supports the recent report maintaining M . oryzae as a single species [6] . In contrast , mini-chromosomes present in B71 and another recent MoT field isolate P3 ( P3-large and P3-small mini-chromosomes ) are highly variable , with each one containing shared and different MoO effector homologs , putative effector genes , and other sequences from core chromosome ends . The B71 and P3-large mini-chromosomes contain the only copies of known MoO effectors PWL2 and BAS1 in these strains and neither gene was present in the early MoT strain T25 , which we show lacks mini-chromosomes . PWL2 and BAS1 are located on different core chromosomes in 70–15 , but they are found side-by-side on the B71 mini-chromosome . Both effectors show similar in planta specific expression on the MoT mini-chromosomes and on the MoO core chromosomes . Only the P3-large mini-chromosome contains a homolog of the MoO AVR-Pib gene , and only the P3-small mini-chromosome contains a homolog of AVR-Pik . Each mini-chromosome contains many other sequences that are either duplicated from core chromosome ends or missing from core chromosomes altogether . In one case , a P3 core chromosome sequence was homologous to the B71 mini-chromosome but not present in B71 core chromosomes . Taken together , our findings provide new insight on the M . oryzae two-speed genome [15] previously known to involve effector localization in transposon-rich regions near chromosome ends . We expand understanding of this effector compartment to include two apparently interchangeable regions , non-dispensable core chromosome ends coupled to dispensable mini-chromosomes . We show that the M . oryzae accessory mini-chromosomes have a unique set of properties relative to accessory chromosomes in other fungi , including the well-studied accessory chromosomes in Fusarium species [41 , 42] and in Zymoseptoria tritici ( syn . Mycosphaerella graminicola ) [43 , 44] . M . oryzae mini-chromosomes , like lineage-specific chromosomes in Fusarium spp . [41 , 42] and the mini-chromosome in Leptosphaeria maculans [45] contain multiple genes associated with virulence and host-specificity . However , extensive recombination with core chromosomes has been so far only observed in M . oryzae mini-chromosomes . The rich set of accessory chromosomes in Z . tritici lack genes with an obvious role in pathogenicity , although some contribute quantitative pathogenicity effects in some strains [46] . The Z . tritici accessory chromosomes appear to be relatively ancient based on apparent survival through at least one speciation event [43 , 46] . M . oryzae mini-chromosomes resemble the accessory chromosomes of F . poae in lacking signs of the fungal specific genome defense mechanism known as RIP [47] , therefore differing from the mini-chromosome and AT-isochore regions of L . maculans for which RIP appears to be a major mechanism for effector gene mutation during response to R gene deployment [48 , 49] . The gene and transposable element crosstalk between the core and supernumerary genomes reported in F . poae does not preferentially involve effectors and core chromosomes ends such as we report for M . oryzae [47] . Although supernumerary chromosomes in many systems appear heterochromatic , with low levels of gene expression [21] , effector genes in M . oryzae mini-chromosomes show in planta specific expression characteristic of these genes on core chromosomes . Therefore , mini-chromosomes in the wheat blast pathogen differ in degree of variability of effector gene content and extent of recombination with core chromosome ends compared to dispensable chromosomes characterized so far in other fungi . The mechanism for sequence exchange between core- and mini-chromosomes is unknown . However , the enrichment in mini-chromosomes of multiple subclasses of LINE retro-transposons and a DNA transposon that are also enriched at core chromosome ends , points to a transposon-mediated recombination mechanism involving non-allelic homology . Such a mechanism has been shown to facilitate genome rearrangements in another phytopathogenic fungus [50] . In contrast to seemingly RIPed core chromosome copies , the multiple copies of both MGR583 ( LINE element ) and Pot2 ( DNA element ) in the mini-chromosomes are nearly devoid of RIP-type mutations . This suggests that transposons on the mini-chromosomes remain active , facilitating multiplication and recombination . Telomere-associated MoTeR elements , found in MoL strains but not in MoO strains , are present on MoT mini-chromosomes . MoTeR elements have been reported to account for the extreme sequence variability of MoL telomeres compared to MoO telomeres [28] , suggesting these elements might enhance mini-chromosome dynamics in MoT and MoL strains through destabilization of telomere regions . Transposon-rich genomic regions have been linked to increased sequence and structural variation in fungal plant pathogens [15 , 25 , 46] . Therefore , transposon-rich mini-chromosomes that also carry a number of genes , including many putative effectors , likely serve as genomic hotspots promoting genomic variation . Exceptional genomic variation produced in mini-chromosomes , and capable of flowing into core chromosomes , could accelerate the evolutionary potential of the pathogen . Dynamic interchange between mini-chromosomes and core chromosome ends would contribute to AVR-Pita1 effector gene mobility , which is especially characteristic of rice pathogens [18] . M . oryzae rice pathogens are notorious for their ability to rapidly overcome deployed R genes . AVR-Pita1 and AVR-Pita2 , which each confer avirulence to rice carrying the corresponding Pita resistance gene , belong to a subtelomeric gene family ( S4 Table ) and show a high rate of spontaneous mutations , including frequent deletions [51 , 52] . AVR-Pita1 and AVR-Pita2 occur in zero , one or more copies in different M . oryzae isolates and show highly variable genomic locations , usually near ends on core chromosomes 1 , 3 , 5 , 6 , 7; in 3 separate locations on chromosome 4; and on supernumerary chromosomes [18] . In contrast , avr-pita3 , which lacks AVR activity , is stably located on chromosome 7 across the host-adapted lineages of M . oryzae . Therefore , extremely high genomic mobility , particularly of AVR-Pita1 , appears to be a response to the periodic deployment of the Pita gene in rice . Mini-chromosomes would provide a population-wide repository for AVR genes that are deleted from individual strains and a means for rapid loss of AVR gene function from individual strains , because mini-chromosomes are frequently lost during meiosis and mitosis [22–24 , 53] . Individual strains lacking AVR-Pita1 could regain it through acquiring AVR-Pita1 containing mini-chromosomes from other individuals through the parasexual cycle and lateral gene transfer [18] . This would explain how the gene became integrated into new locations on the core chromosomes , typically at chromosome ends . The dynamic coupling we report between mini-chromosomes and core chromosome ends supports the multiple translocation hypothesis for AVR genes responding to periodic negative selection pressure of R gene deployment . Collectively , we propose that the mini-chromosome plays a role for gene movements like a shuttle , in which mutation , duplication , loss , and rearrangements of DNA occur at a faster pace than normal genomic changes , hence , accelerating genomic evolution for adaptation . Growing evidence suggests that avirulence-conferring PWL family members ( S4 Table ) may be undergoing multiple translocation similarly to AVR-Pita family members [18] . PWL2 from a rice isolate and PWL1 from an Eleusine isolate each confer avirulence toward Eragrostis spp . [11 , 12] . The well-studied PWL2 gene , like AVR-Pita1 , occurs in zero to four copies in different strains and is subject to frequent spontaneous deletion [12] . Genetic analyses showed that PWL2 and PWL1 map to different chromosomal locations , with PWL1 linked to a telomere . Homology between the PWL2 and PWL1 genes begins 70 bp upstream of the PWL1 initiation codon and ends immediately after the stop codon , and sequences beyond this conserved region are completely unrelated . In contrast , the apparently allelic , non-AVR conferring PWL3 and PWL4 genes mapped to a third genomic region and share conserved flanking sequences [11] . Two copies of PWL2 are present on chromosomes 3 and 6 in the reference rice genome MG8 , the intact PWL2 sequence was found in three assembled contigs of a highly aggressive rice isolate 98–06 [54] , and we report that PWL2 resides on a mini-chromosome in some wheat pathogens . Further research is needed to track chromosomal dynamics of PWL2 , as well as PWL1 , in host-adapted forms of M . oryzae . AVR-Pita1 effector gene mobility is reported to be in response to periodic deployment of the corresponding Pita gene in rice , raising the question of comparable selection pressure that might be acting in the Eragrostis system . Introduction of weeping lovegrass , native to South Africa , and other Eragrostis spp . , around the world for forage and erosion control in the past decades could have provided conditions promoting loss and recovery of PWL family members . Our results will inspire further exploration of function and evolutionary roles of mini-chromosomes in the fungal phytopathosystem , and facilitate answering important questions for blast on wheat and other cereal crops . Our early MoT strain T25 , isolated in 1988 , lacks mini-chromosomes , as was previously reported for 7 other MoT strains isolated in Brazil between 1986 and 1988 [23] . This raises the question of whether mini-chromosomes have contributed in any way to the enhanced aggressiveness characteristic of recent field isolates such as B71 and P3 . It is critical to monitor further evolution , including potential recombination with other M . oryzae pathotypes , of the complex MoT population in South America and the initially clonal MoT population in South Asia [1 , 4] . Localization of the PWL2 host species-specificity gene on mini-chromosomes in wheat pathogens raises the question of a role for mini-chromosomes in host jumps . Effector gene dynamics , so far only associated with a small number of MoO AVR effector genes corresponding to periodically deployed R genes , raises the question of what roles known MoO AVR effector homologs and BAS1 ( lacking known AVR activity in MoO strains ) play in wheat infection by MoT strains . Finally , it is critical to identify and deploy effective wheat blast resistance .
All M . oryzae strains examined were field strains from South America ( S1 Table ) . MoT isolates B71 , T25 , and P3 were isolated in Bolivia ( 2012 ) , Brazil ( 1988 ) , and Paraguay ( 2012 ) , respectively . All work with living wheat blast fungus in the U . S . was performed with proper USDA-APHIS permits and monitoring in BSL-3 laboratories in the Biosecurity Research Institute at Kansas State University . Single spore isolates of each pathogen strain were cultured in complete medium for mycelium propagation . Mycelium was harvested and frozen using liquid nitrogen . To avoid excessive mitochondrial DNA , mycelial nuclei were collected by gradient centrifugation as described [55] . The CTAB ( cetyltrimethylammonium bromide ) DNA extraction method was applied to isolate genomic DNA from the nuclear samples [56] . The 3–20 kb WGS libraries were constructed using B71 nuclear genomic DNAs . The library was sequenced with P6-C4 chemistry on ten SMRTcells of PacBio RS II . Nuclear genomic DNAs were also subjected to 2x250 bp paired-end Illumina sequencing . To increase the assembly continuity , LIEP was devised and used to generate 20–30 kb long-distance paired sequences for scaffolding . PacBio long reads were assembled using the Canu pipeline [57] . Self-correction using PacBio reads did not correct all PacBio sequencing errors . Illumina reads and the Illumina assembly sequences assembled using DISCOVAR de novo [58] were both utilized for further error correction . The resulting assembled contigs were scaffolded using LIEP long-distance paired sequences with the software SSPACE [59] . Two RNA-Seq experiments were performed . In the first RNA-Seq experiment , an in vitro cultured mycelium sample was collected for the total RNA extraction using RNeasy Plant Mini Kit . Total RNA was used for RNA sequencing on a MiSeq to generate 2x150bp paired-end data . Clean data after adaptor and quality trimming were de novo assembled using Trinity [60] , which were then aided in genome annotation . In the second RNA-Seq experiment , we attempted to compare B71 gene expression in planta and in vitro culture with three biological replicates in each group . RNAs of in planta samples were isolated from B71-infected epidermal cells of leaf sheaths from 3–4 weeks old wheat plants at 40 HPI . The B71 in vitro culture RNAs were extracted from mycelium grown in liquid swirling cultures with minor modifications to the method of Mosquera et al . [32] . The total RNAs were subjected to library preparation for mRNA sequencing to produce single-end 75bp reads . Clean data after adaptor and quality trimming were aligned to the B71Ref1 reference genome with STAR [61] . Read counts per genes were used for differential expression analysis with DESeq2 [62] with 1% false discovery rate ( FDR ) as the threshold to declare significantly differentially expressed genes between in planta and in vitro culture groups [63] . A Maker pipeline was used for the B71 genome annotation [64] . Both evidence-driven prediction and ab initio gene prediction were employed [65] . Transcriptional evidence was provided using assembled sequences from RNA sequencing data of the B71 strain that was cultured in media and field wheat leaf samples infected by Bangladesh wheat blast strains , which were genetically almost identical to B71 . CEGMA was used to assess the completeness of the genome assembly or annotation [66] . Publically available RNA-Seq data of MoT infected wheat were used as in planta expression data to compare with in vitro culture RNA-Seq data from the first RNA-Seq experiment . Field RNA-Seq data includes samples 5 and 7 from Bangladesh wheat fields [3] . These MoT isolates have been demonstrated to be almost identical to B71 . All data from samples 5 and 7 were merged to represent field in planta transcriptomes . Genes with read abundance higher than 0 . 1 FPKM ( fragment per kilobase of coding sequence per million reads ) in either in planta or in culture samples were considered to be expressed genes . Genes with read abundance higher than 1 FPKM from the in planta data set but no reads from the cultured sample were considered to be in planta specific expression . In planta specific genes containing classical signal peptide domains [67] were considered putative effectors . Read depth approach was employed to identify CNV between each of some M . oryzae strains and B71 for each of sequence bins ( e . g , 300 bp ) . Segmentation with the R package of DNACopy was performed to identify genomic CNV segments merged from multiple bins [68] . MoT protoplasts were prepared and mixed with 1 . 5% low melting-temperature agarose [23] . Suspensions were loaded into disposable plug molds . Protoplasts in plugs were lysed with proteinase K and washed . A Biorad CHEF electrophoresis system was used for separating chromosomes embedded in the plugs . After the CHEF gel electrophoresis , DNAs from individual mini-chromosomes , and from core chromosomes as one unit , were excised and purified from the agarose gels . Purified DNAs were subjected to Illumina 2x151 bp paired-end sequencing . Repetitive sequences were identified using MGEScan [69] , LTR_Finder [70] , LTRharvest [71 , 72] , and RepeatModeler ( github . com/rmhubley/RepeatModeler ) . Merging discovered repetitive sequences and previously characterized M . oryzae repeats [73] produced a non-redundant database , which served as a repeat library to identify repeats in the B71 genome using RepeatMasker ( www . repeatmasker . org ) . Some transposable elements were subjected to analysis of RIP-type polymorphisms , nucleotide changes of C to T or G to A . | The emerging blast disease on wheat is proving even harder to control than the ancient , still-problematic rice blast disease . Potential wheat resistance identified using strains isolated soon after disease emergence are no longer effective in controlling recent aggressive field isolates from wheat in South America and South Asia . We construct a high-quality assembly of an aggressive , recently-isolated wheat blast fungal strain and the first assembled mini-chromosome genome sequence of wheat and rice blast pathogens . We report that recent wheat pathogens can contain one or two highly-variable dispensable mini-chromosomes , each with an amalgamation of fungal effector genes and other sequences that are duplicated or absent from indispensable core chromosome ends . Well-studied effectors found on different core chromosomes in rice pathogens appear side-by-side in wheat pathogen mini-chromosomes . The rice pathogen often overcomes deployed resistance genes by deleting triggering effector genes . We propose that the fast-evolving effector-rich compartment of the wheat blast fungus is a combination of core chromosome ends and mobile mini-chromosomes that are easily lost from individual strains . Localization of effectors on mini-chromosomes would therefore accelerate pathogen adaptation in the field . | [
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] | 2019 | Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus |
Infection by the autonomous parvovirus minute virus of mice ( MVM ) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication . Although p53 remains activated , p21 protein levels remain low throughout the course of infection . We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers . PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA . PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro . Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication , while a stable p21 mutant which lacked this interaction did not . Thus , while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers , efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA .
Minute Virus of Mice ( MVM ) is an autonomously-replicating parvovirus which induces a DNA damage response resulting in substantial p53 activation which persists throughout the course of viral replication [1] . p53 is a well-established activator of p21WAF1/Cip1 ( hereafter referred to as p21 ) expression . Transient expression of the MVM NS1 protein alone also leads to an increase in p21 levels [2] , [3] . However , surprisingly , while these signals lead to an increase in p21 RNA accumulation , p21 protein levels remain low throughout the course of infection , including during the prolonged G2 phase in which the viral genome is replicated [2] . p21 can be a potent antiviral factor and possesses several potentially inhibitory activities including cyclin-dependent kinase ( CDK ) inhibition and repression of E2F1-mediated expression [4] . In addition , p21 has been shown to be an effective inhibitor of the DNA polymerase δ cofactor PCNA [5]–[7] , and it has been shown to inhibit MVM replication in vitro [8] . p21 depletion during MVM infection was shown to be proteasomally mediated , suggesting that an E3 ubiquitin ligase was involved in targeting p21 for degradation [2] . Viruses often make use of the ubiquitin conjugation machinery to target for degradation cellular proteins that might otherwise negatively affect viral replication [9] . The Cullin-RING Ligase ( CRL ) CRL4Cdt2 consists of the scaffold protein Cullin 4 and the homo-trimeric protein DDB1 which serves as an adaptor for the putative substrate recognition protein Cdt2 . This ligase has been shown to program the ubiquitination and subsequent degradation of p21 in response to DNA damaging agents such as UV treatment in order to ensure low p21 levels during S-phase [10]–[12] . Upon DNA damage or S-phase entry CRL4Cdt2 is recruited to chromatin via PCNA interaction where it targets substrate proteins for degradation [13] . We show here that efficient MVM replication in S/G2 arrested cells required the targeting for proteasomal degradation of p21 by the CRL4Cdt2 E3-ubiquitin ligase which was re-localized to viral chromatin within active MVM replication centers . PCNA provides a molecular platform that aids substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase , and p21 targeting to this ligase during MVM infection required its interaction with PCNA . PCNA is also an important co-factor for DNA polymerase δ-dependent MVM replication which can be antagonized by p21 in vitro . Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication , while a stable p21 mutant which could no longer interact with PCNA did not . Introduction of a p21-derived peptide that bound to PCNA also substantially decreased viral replication . Our results suggest that interaction with PCNA was important for targeting p21 to the re-localized CRL4Cdt2 ubiquitin ligase , yet subsequent depletion of p21 was required to prevent its sustained interaction with PCNA which otherwise inhibited efficient viral replication .
The CRL4Cdt2 E3 ubiquitin ligase has been implicated in targeting p21 for proteasomal degradation upon S-phase entry and after cellular DNA damage [10]–[12] . Was this ubiquitin ligase also enlisted to target p21 at late times during MVM infection when cells were blocked at the G2/M border ? To test this possibility , DDB1 and Cdt2 , components of this ligase which are not present in other E3 ubiquitin ligases known to modify p21 [13] , [14] , were targeted via RNAi in the protocol illustrated in Figure 1A . For these experiments cells were parasynchronized prior to infection to maximize the number of cells progressing uniformly through S-phase . This both synchronized infection and the characterization of p21 depletion . At the time of release from the synchronization procedure ( as the cells progressed from G0 to G1 ) there were high levels of p21 expression ( Mock T0 , Figures 1B and 1C , lanes 1 ) , which were reduced 24 hr post MVM infection ( Figures 1B and 1C , lanes 2 ) , and remained low up to 40 hr pi ( Figures 1B and 1C , lanes 4 ) . Targeting of endogenous DDB1 ( panel 1B ) or Cdt2 ( panel 1C ) by RNAi , which led to significant depletion of these proteins ( Figure 1B and 1C , lanes 3 and 5 ) , substantially prevented the loss of p21 both at 24 hr pi ( Figures 1B and 1C , lanes 3 ) , and also 40 hr pi ( Figure 1B and 1C , lanes 5 ) – the latter being a point well past S-phase when infected cells are known to be arrested in G2 [1] . Expression of cyclin A indicated unperturbed entry into S-phase ( Figures 1B and C , lanes 2–5 ) , and expression of the viral NS1 protein indicated that the initiation of viral infection was unaffected by the RNAi protocol ( Figures 1B and C , lanes 2–5 ) . Similar results were also obtained following knockdown of Cullin 4A , a component of the CRL4Cdt2 ligase also present in several other ubiquitin ligases ( data not shown ) . Taken together , these results indicated that the CRL4Cdt2 ligase was responsible for p21 degradation throughout virus infection . CRL4Cdt2 also targets Set8 and Cdt1 for degradation during S-phase and in response to DNA damaging agents [15]–[17] . We also observed loss of Set8 in response to MVM infection ( Figure S1 ) . Cdt1 levels were not reduced for reasons not yet clear . We next examined the functional consequence of CRL4Cdt2 E3 ligase depletion for viral replication using the protocol illustrated in Figure 2A . DDB1 knockdown resulted in an approximate 2-fold decrease in accumulated viral replicative forms at each time point compared to application of negative control siRNA ( Figure 2B , compare lanes 1 to 2 , 3 to 4 , 5 to 6 ) . Cdt2 knockdown during infection resulted in an approximate 2 . 5-fold decrease in accumulated viral replicative forms at each time point when compared to negative control siRNA ( Figure 2C , compare lanes 1 to 2 , 3 to 4 , 5 to 6 ) . Importantly , as mentioned above , expression of NS1 ( Figure 1 ) , and flow cytometric analysis ( Figure S2 ) , confirmed normal entry into S-phase following this synchronization and RNAi protocol which thus did not affect the S-phase dependent initiation of infection . These results demonstrated that the activity of the CRL4Cdt2 E3 ubiquitin ligase was necessary for efficient viral replication . MVM replicates in nuclear compartments termed autonomous parvovirus associated replication ( APAR ) bodies which have been shown to be enriched for cellular proteins such as DNA polymerase δ , RPA , cyclin A , and PCNA , which are also essential for parvovirus replication [18] . These nuclear bodies have been shown , via BrdU staining , to serve as sites of ongoing viral replication in infected cells and can be visualized by staining for the viral replicator protein NS1 [19] . Importantly , whereas punctate staining distributed throughout the nucleus was observed in mock treated cells , we detected recruitment of both DDB1and Cdt2 to NS1-containing viral replication compartments . Resistance to detergent pre-extraction prior to immunofluorescence ( Figure 3A and 3B , note the merged images for each ) also suggested that it may be bound to viral chromatin . These results indicated that MVM infection redirected the viral replication-enhancing CRL4Cdt2 ligase complex to APAR bodies . The recruitment was specific for the CRL4Cdt2 ligase because the APC/CCdc20 E3 ligase , which targets p21 for degradation after mitotic entry [20] , was not similarly recruited ( Figure S3 ) . This is the first demonstration of the specific recruitment of a cellular E3 ubiquitin ligase to parvoviral replication compartments . The DNA polymerase δ cofactor PCNA is known to target the CRL4Cdt2 E3 ubiquitin ligase to cellular chromatin via its interaction with Cdt2 during normal cell division [13] , [21] . Ubiquitin modification of p21 by the CRL4Cdt2 ligase requires Cdt2 interaction with the p21 degron motif , as well as interaction between p21 and PCNA via its PCNA-interaction ( PIP ) box [22] . p21 is recruited to UV-induced DNA lesions via interaction with PCNA , and a p21 mutant defective in binding to PCNA was resistant to degradation following UV treatment [10] , [12] . To investigate the importance of these interactions for the MVM-dependent targeting of p21 by the CRL4Cdt2 ligase we generated stable murine cell lines via lentivirus transduction that conditionally expressed FLAG-tagged wild-type or mutant p21 ( p21WT , p21ΔDegron , p21ΔPCNA , mutations shown in Figure 4A ) in a doxycycline-responsive manner . As expected , MVM infection of a p21WT expressing cell line resulted in degradation of the tagged p21 ( Figure 4C and 4E , compare lanes 2 to 4 ) , which could be prevented by treating cells with the proteasome inhibitor MG132 ( Figure S4 , panel A ) , or via siRNA knockdown of CRL4Cdt2 components ( Figure S4 , panels B and C ) . These results suggested that the depletion of p21 in these cell lines occurred via a similar mechanism to that of the endogenous p21 . The lysine at the p21 +4 position , 3′ to its PIP box , has been reported to be required for interaction of p21 with the CRL4Cdt2 ligase complex via Cdt2 . Mutation of the +3 to +5 amino acids KRR to AAA ( p21Δdegron ) abolished p21 interaction with the CRL4Cdt2 complex following its transient transfection as reflected by loss of interaction with DDB1 ( Figure 4B , compare lanes 2 to 3 ) . Murine cell lines that expressed the p21Δdegron mutant were generated , and when infected with MVM , in contrast to cell lines expressing wild-type p21 ( p21WT ) , the p21Δdegron protein was resistant to degradation ( Figure 4C , compare lanes 6 and 8 to lanes 2 and 4 ) . This suggested that MVM-induced p21 degradation required interaction of p21 with the CRL4Cdt2 ubiquitin ligase complex . The CRL4Cdt2 ligase also requires PCNA as a cofactor for targeting of its substrates [13] . Mutation of the p21 PIP box ( p21ΔPCNA , Figure 4A ) abrogated interaction with PCNA ( Figure 4D , compare lane 3 to 2 ) , and the p21ΔPCNA protein expressed in murine cells was not degraded as efficiently as the wild-type protein ( p21WT ) following infection ( Figure 4E , compare lanes 8 and 6 to 4 and 2 ) . These results suggested that PCNA-binding was also essential for effective CRL4Cdt2 targeting of p21 during MVM infection . p21 interaction with PCNA has been shown to interfere with DNA polymerase δ−mediated cellular DNA replication [5] , [6] . PCNA is an important cofactor for MVM replication; it contributes to MVM replication in vitro [8] , and is recruited to APAR bodies during infection . Thus , we investigated whether MVM-dependent depletion of p21 during infection , mediated by the CRL4Cdt2 E3 ubiquitin ligase , promoted efficient replication of the MVM genome by abrogating its inhibition of PCNA . Unexpectedly , the p21ΔDegron mutant , although stable , interacted poorly with PCNA for reasons not yet clear ( data not shown ) . As a result , we could not use cell lines expressing this mutant to determine whether stabilized , p21 affected MVM replication via PCNA binding . Thus we generated a murine cell line conditionally expressing a mutant p21 in which all seven lysines in p21 were changed to arginine [p21K7R , a similar mutation has been reported by others [23]] . Similar to the p21ΔDegron mutant , the p21K7R protein was resistant to degradation following MVM infection ( Figure 5A , compare lanes 6 and 8 to 2 and 4 ) , yet retained substantial interaction with PCNA in transient transfection assays ( Figure 5B , compare lane 3 to lanes 2 and 4 ) . Whereas induction of p21WT expression for 8 hrs after infection had little effect on MVM replication ( Figure 5C , compare lanes 1 and 2 ) , p21K7R expression reduced replication by up to 3 fold ( Figure 5C , compare lanes 3 and 4 ) . Importantly , in contrast , the p21ΔPCNA mutant-expressing cell line , which expressed a stable p21 which no longer could interact with PCNA ( Figure 4D and 4E ) , failed to inhibit MVM replication upon induction ( Figure 5D , compare lanes 3 and 4 ) . This was also the case with the p21ΔDegron mutant-expressing cell lines ( data not shown ) . Furthermore , cell lines expressing a mutant of p21K7R in the PIP box-mutated background ( p21K7RΔPIP ) ( see Figure 5B , lane 4 ) also failed to inhibit MVM replication ( Figure S5 ) , demonstrating that absent PCNA binding , the K7R mutation itself had no deleterious effect on MVM replication . All the mutants tested were recruited to APAR bodies during MVM infection ( Figure S6 ) . To confirm that the p21-PCNA interaction mediated the inhibitory role of p21 during infection , we made use of a previously described peptide containing 20 residues derived from sequences comprising the p21 PIP box [24] fused to a 16-mer penetratin motif to facilitate cellular entry [25] . A scrambled version of the p21 peptide linked to the penetratin peptide was used as control . Following application to murine cells , the wild-type ( Figure 5E , lane 4 ) , but not the scrambled version ( Figure 5E , lane 3 ) , disrupted the interaction between over-expressed FLAG-tagged p21 and endogenous PCNA , demonstrating that the peptide could competitively interact with PCNA . Subsequently , while treatment of cells with the scrambled peptide had no effect on MVM replication ( Figure 5F , compare lanes 2 and 4 ) , treatment of cells with the wild-type p21 PCNA-binding peptide significantly inhibited MVM replication ( Figure 5F , compare lanes 2 and 3 ) . These results indicated that a stabilized p21 interaction with PCNA was detrimental to viral replication . Thus , while interaction with PCNA was important for targeting p21 to the re-localized CRL4Cdt2 ligase , efficient viral replication required subsequent depletion of p21 and consequent abrogation of its inhibition of PCNA .
Parvoviruses and other small DNA viruses rely on host polymerases to replicate their genomes . How the replication machinery is exploited to sustain parvovirus replication in G2-arrested cells , which normally contain potentially inhibitory cellular proteins such as p21 , is not fully understood . p21 levels are high in G1 , low in S-phase , restored in G2 phase , and are regulated by proteasomal degradation during cell cycle progression [4] . We and others have previously reported that expression of the parvoviral NS1 protein leads to increases in p21 levels [2] , [3] . Additionally , p53 , a transcriptional activator of p21 , is significantly up-regulated and activated throughout MVM infection . Remarkably however , while these signals lead to increased p21 RNA accumulation , p21 protein levels remain low throughout infection [2] . Here we have identified the mechanism by which p21 was degraded upon parvovirus infection , and identified the consequence of this for virus replication . Degradation of p21 during S-phase and in response to DNA damaging agents such as UV treatment is programmed by the CRL4Cdt2 ubiquitin ligase [13] and in this manuscript we have demonstrated that the same ligase targets p21 for degradation during MVM infection . The circumstances surrounding p21 degradation and the signals leading to it in the context of parvoviral infection , however , differ from how it occurs during S-phase . During MVM infection p21 loss persists for extended periods of time while virus is replicating in G2 arrested cells in the presence of high amounts of activated p53 and NS1 [2] . Additionally , whereas ATR activity is important for p21 degradation in response to various DDR-inducing agents [23] , [26] , the ATR substrate Chk1 is not activated during MVM replication ( Adeyemi and Pintel , in preparation ) , suggesting that p21 degradation during infection may occur independently of ATR activity . During MVM infection the CRL4Cdt2 ligase is recruited to viral replication centers . Recently , the CRL4Cdt2 ligase was shown to be recruited to cellular chromatin via direct PCNA interaction [21] . It is not yet clear whether similar mechanisms mediate CRL4Cdt2 recruitment to MVM APAR bodies; however , it appears that PCNA recruitment to MVM chromatin may represent a critical step leading to viral hijacking of the CRL4Cdt2 ligase . Neither interaction with PCNA nor the ligase was required for recruitment of p21 to replication centers , as all the mutants tested were relocalized to APAR bodies . Stabilization of p21 via CRL4Cdt2 depletion led to reduced MVM replication subsequent to the initiation of genome replication following S-phase entry . This is the first published demonstration of the requirement and re-localization to replication centers of a specific cellular ubiquitin ligase during autonomous parvovirus replication . p21 is a potent inhibitor of CDKs and PCNA . Exactly how stabilized p21 inhibited MVM replication is not fully clear; however , interaction with PCNA mediated its inhibitory role . Using inducible cell lines expressing wild-type and mutant p21 proteins we demonstrated that p21 degradation during infection required motifs that mediate interaction with both Cdt2 and PCNA . PCNA is recruited to MVM chromatin [18] and is essential for parvovirus replication [27] , [28] . Overexpression of a stable mutant p21 that retained interaction with PCNA , but could not be targeted for degradation by the CRL4Cdt2 ligase , led to reduced virus replication . Stable p21 mutants unable to bind PCNA did not affect MVM replication , indicating that other potential inhibitory functions of p21 , such as CDK binding and promoter repression , were not detrimental to viral replication absent PCNA interaction . Additionally , introduction of a p21-derived peptide which maintained PCNA interaction abolished viral replication . Thus , p21 interaction with PCNA ( and Cdt2 ) was necessary for targeting of p21 to the co-localized CRL4Cdt2 ligase , and its subsequent depletion also prevented its sustained interaction with PCNA that would otherwise be inhibitory to viral replication . Although our earlier work had suggested that Cdk2 activity was required for MVM replication and that p21 degradation might be necessary to prevent inhibition of CDKs [2] , we have recently shown that Chk2 activation during MVM infection results in CDC25A degradation leading to partial CDK2 inhibition , independent of p21 [29] . Thus , while some level of CDK activity is required for MVM replication , prevention of CDK inhibition is not likely to be the critical reason for p21 degradation . p21 has been shown to inhibit MVM replication in vitro , and this effect was shown to be overcome by the addition of increasing amounts of PCNA [8] . p21 binds to PCNA via its PIP box , a conserved motif shared by substrates of the CRL4Cdt2 ligase , as well as cellular proteins such as DNA polymerase δ , that are essential for replication but escape ubiquitin targeting due to the absence of a PIP degron [13] . The p68 subunit of DNA polymerase δ binds to PCNA via a similar hydrophobic pocket recognized by the p21 PIP box . Thus , while the mechanism of p21 inhibition of the DNA polymerase δ/PCNA complex has not been clearly resolved , a stabilized high-affinity interaction of p21 with the DNA polymerase δ binding pocket within PCNA could directly inhibit viral replication by competing with DNA polymerase δ for binding to its cofactor [30] . Due to its myriad effects on cell cycle progression and cancer , several viruses make use of different strategies to inactivate p21 during infection . For example , several oncogenic DNA viruses indirectly inhibit p21 by targeting p53 [31] . Additionally , papillomaviruses HPV E7 proteins sequester p21 thereby preventing its interaction with PCNA and CDKs [32] , [33] . KSHV encodes a microRNA that down-regulates p21 in order to prevent cell cycle arrest [34] . p21 restricts HIV in myeloid cells of certain patients [35] , [36] , and a recent report demonstrated that this restriction may occur via ribonucleotide reductase-2 repression resulting in inhibition of dNTP biosynthesis [37] . Thus , cellular components required for viral genomic replication , such as dNTPs and polymerase cofactors , may be critically dependent on p21 depletion . Our findings present first a novel mechanism of p21 antagonism by a virus , namely , the use of a cellular E3 ligase recruited to viral replication factories . Further , we shown that efficient virus replication depends on the depletion of p21 to prevent its inhibition of PCNA . PCNA is an important cofactor for the replication of several DNA viruses . PCNA-mediated degradation of p21 in the context of viral infection may emerge as an important paradigm for allowing sustained viral replication of DNA polymerase-δ dependent viruses in infected cells .
Murine A9 and Human 293T cell lines were propagated as previously described [38] , [39] . Stable doxycycline-inducible A9 cell lines were generated by infection of A9 cells with pseudotyped virus using the pINDUCER20 lentiviral system [40] . Cell lines were selected with 800 µg/ml of geneticin ( Gibco ) and maintained like regular A9s except for addition of geneticin . A9 cells were parasynchronized in G0 by isoleucine deprivation as previously described [1] . pINDUCER20 lentiviral transformed cell lines were induced with 500 ng/mL doxycycline hydrochloride ( MP Biomedical ) . MG132 ( Calbiochem ) was added at a final concentration of 10 µM . Wild-type MVMp was propagated and titered in murine A9 cell lines as described [1] . Pseudotyped viruses were generated by co-transfecting equal concentrations of HIV Gag/Pol , VSV-G and pINDUCER20 plasmids into 293T cells . Supernatants were collected and used to infect A9 cells . Murine wild-type p21 cDNA ( Origene ) was cloned into p3XFLAG-CMV 7 . 1 ( Sigma ) . Additionally , p21 was tagged with a 3× HA tag by PCR mutagenesis . p21K7R was generated by PCR mutagenesis . p21ΔPCNA , p21Δdegron and p21K7RΔPIP ( Q139A , L142A , F145A , Y146A ) mutants were generated by site-directed mutagenesis ( Agilent ) . FLAG and HA-tagged wild-type and mutant p21 were cloned into pDONR221 ( Invitrogen ) and pINDUCER20 using BP and LR clonase kits ( Invitrogen ) respectively . pINDUCER reagents were a gift from Guang Hu at NIH/NIEHS . DNA transfection was performed using LipoD293 ( Signagen ) or Lipofectamine ( Invitrogen ) . A9 cells were transfected twice with 40 nM Control siRNA ( Qiagen #1022076 ) , Cdt2 ( DTL ) siRNA ( Dharmacon #L-045921-01-0005 ) , Cul4A ( Dharmacon #L-052208-00-0005 ) or DDB1 siRNA ( Dharmacon #L-043146-01-0005 ) . siRNA transfections were performed using HiPerfect ( Qiagen ) . Wild-type p21 ( KRRQTSMTDFYHSKRRLIFSRQIKIWFQNRRMKWKK ) and scrambled p21 ( KSTARHTKLSAQRYIRSFARRQIKIWFQNRRMKWKK ) were purchased from Peptide 2 . 0 Inc . The peptides consist of p21-derived or scrambled sequences fused to penetratin – a 16 amino acid nuclear internalization sequence derived from the Antennapedia homeodomain [25] . Peptides were added to cells at 25 µM . Commercially available antibodies used in this study were obtained from Bethyl ( Cdc20 , cat# A301-180A ) , Cell Signaling ( Set8 , cat# 2996S ) , Invitrogen ( DDB1 , cat# 399901; p21 , cat# 556430 ) , Millipore ( Cdt1 , cat# 06-1295; PCNA , cat# CBL407 ) , Novus ( Cullin 4A , cat# NB100-2267 ) , Pierce ( Actin , cat# MA515739 ) , Sigma ( FLAG , cat# F1804; HA , cat# H9658; Tubulin , cat# T4026 ) , and Upstate ( Cyclin A , cat# 06-138 ) . Cdt2 antibody was a generous gift from Anindya Dutta ( University of Virginia ) . All secondary antibodies were from Invitrogen . NS1 ( CE10 ) and NS1/2 ( M55 ) were previously described [1] . Immunoblots were performed as described previously [1] . Protein concentrations were quantified by Bradford assay and equal amounts of lysates were run . FLAG and HA-tagged p21 was immunoprecipitated from 293T cells as previously described [12] . FLAG beads ( Sigma ) and HA antibodies were used for IP . For IF , A9 cells were grown on glass coverslips in 35 mm dishes and infected with MVMp at an MOI of 10 . At 24 hr pi , cells were washed twice with cold phosphate-buffered saline ( PBS ) solution and then with cytoskeleton buffer [10 mM piperazine-N , N′-bis ( 2-ethanesulfonic acid ) ( PIPES ) , pH 6 . 8 , 100 mM NaCl , 300 mM sucrose , 1 mM MgCl2 , 1 mM EGTA] . Afterwards cells were pre-extracted for in cytoskeletal buffer containing 0 . 5% Triton X-100 , protease and phosphatase inhibitors for 3 minutes on ice , washed , fixed with 4% paraformaldehyde and stained for the indicated proteins . Nuclei were visualized by staining with To-Pro-3 ( Invitrogen ) . The coverslips were mounted in Fluoromount-G ( Southern Biotech ) and images were acquired using a Zeiss LSM 510 META confocal microscope . All images were captured using an objective of 63× . Southern blots were carried out as previously described [38] , using whole MVM genome probes . Loading of DNA samples was normalized using a nanodrop spectrophotometer . This procedure was verified using probes on Southern blots against mitochondrial DNA . Unless otherwise indicated , infections were carried out at a low MOI in order to maximize effects of siRNA knockdowns and overexpression analyses on viral replication . Representative Southern Blots are shown; quantifications in the text reflect multiple knockdown experiments . A9 cells were fixed in 70% ethanol overnight at 4°C . Cells were then pelleted , washed in PBS and resuspended in PBS containing 0 . 2 mg/ml RNAse A for 1 hr at 37°C , then propidium iodide was added at 40 µg/ml . Flow cytometry was performed using FACScan ( BD biosciences ) . Data were analyzed using Summit software ( Beckman Coulter ) . | Many DNA viruses induce and then exploit host cellular DNA damage responses to generate a suitable environment for their continued replication . Parvoviruses , important disease agents in both humans and animals , rely on host DNA polymerases to replicate their genomes in cell-cycle arrested cells . We show that efficient parvovirus replication requires the recruitment to viral replication compartments of a host cellular E3-ubiquitin ligase , CRL4Cdt2 , to target the potent cell cycle regulator p21 for subsequent degradation . The DNA polymerase-δ cofactor PCNA provides a molecular platform for initial substrate recognition by this ligase , and subsequent p21 depletion prevents its continued interaction with PCNA which otherwise inhibits efficient viral replication . Virally-induced p21 degradation represents another way of promoting efficient replication of DNA polymerase-δ-dependent viruses . | [
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] | 2014 | Efficient Parvovirus Replication Requires CRL4Cdt2-Targeted Depletion of p21 to Prevent Its Inhibitory Interaction with PCNA |
Segmented negative strand RNA viruses of the arena- , bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism . This involves cleavage of host mRNAs close to their capped 5′ end by an endonuclease ( EN ) domain located in the N-terminal region of the viral polymerase . We present the structure of the cap-snatching EN of Hantaan virus , a bunyavirus belonging to hantavirus genus . Hantaan EN has an active site configuration , including a metal co-ordinating histidine , and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs ( orthobunyavirus and orthomyxovirus respectively ) , but is more active in cleaving a double stranded RNA substrate . In contrast , Lassa arenavirus EN has only acidic metal co-ordinating residues . We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan , La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive . The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase .
Segmented negative strand viruses ( sNSVs ) represent one of the most threatening groups of emerging viruses for global health [1] . They are classified in three main families: Orthomyxoviridae , Bunyaviridae and Arenaviridae with respectively six to eight , three and two genome segments [2] . Seasonal and pandemic influenza A virus ( IAV , orthomyxovirus ) strains rapidly propagate worldwide with human to human transmission being the key factor for spread . In contrast , arenaviruses ( e . g . Lassa virus ) or bunyaviruses ( e . g . Hantaan , La Crosse , Rift Valley , Crimean Congo Haemorrhagic viruses ) , as well as some highly pathogenic avian influenza strains , are zoonotic viruses that result in generally limited outbreaks through contact with animal vectors but with high mortality rates and lack of effective treatments . The future spread of some of these infectious agents from their traditional geographical niches due to vector species redistribution arising through climate change is a potential threat [3 , 4] , emphasising the need to develop new , ideally broad-spectrum , drugs against sNSV zoonotic viral diseases . Despite the diversity in the infectious cycles of sNSVs there are common mechanisms that can be potentially targeted for broad spectrum inhibitors , such as genome and mRNA synthesis by the conserved RNA dependent RNA polymerase ( RdRpol ) or their characteristic cap-snatching transcription mechanism [5–8] . This mechanism , most extensively characterized for IAV virus , involves the recognition of capped cellular mRNAs by a cap-binding domain located in the polymerase and its subsequent cleavage 10–14 nucleotides downstream by the polymerase’s endonuclease ( EN ) to provide a primer for viral mRNA transcription [5 , 9] . The cap-binding and the EN domains were first identified in the IAV hetero-trimeric polymerase and are located in the middle region of the PB2 and the N-terminal region of the PA subunits respectively [10 , 11] . The recent crystal structures of influenza A and B heterotrimeric polymerases show the relative disposition of these two domains within the full RdRpol domains allowing an integrated structural model for the cap-snatching mechanism to be proposed for orthomyxoviruses [9 , 12] . Studies on La Crosse ( LACV ) bunyavirus and Lymphocytic Choriomeningitis ( LCMV ) arenavirus allowed the structural and functional characterization of the cap-snatching EN domains in the amino terminal region of their monomeric polymerases ( L proteins ) [13 , 14] and showed them to be essential for viral transcription . Similar results were subsequently obtained for Lassa arenavirus and the bunyaviruses Rift Valley Fever Virus ( RVFV ) and Crimean Congo Haemorragic Fever Virus ( CCHFV ) [15–18] . However the location of the putative cap-binding domain still remains elusive for bunya- and arenaviruses . The sNSV cap-snatching ENs belong to the PD-D/ExK superfamily of cation dependent nucleases . The available structures of the influenza orthomyxovirus and LACV orthobunyavirus show the canonical conformation of the active site with two divalent metal ions directly coordinated by the acidic conserved residues of the PD and the D/ExK motifs as well as with a conserved histidine ( His+ ENs ) . The two metal ions bind aligned towards the catalytic lysine [14] . The arenavirus EN crystal structures reported to date ( LCMV and Lassa ) are structurally homologous to LACV EN [13 , 16] , but there are important differences in their active sites . The main divergence is that the metal co-ordinating histidine , conserved in most bunya- and orthomyxoviruses , is replaced by an acidic residue in arenavirus ENs ( His- ENs ) . No metal ions were present in the LCMV EN structure [13] and the Lassa EN structure was reported with two magnesium ions in the active site coordinated by some catalytic residues through bridging water molecules , instead of the direct coordination shown by His+ ENs . The reported ion preference for the catalytic activity also changes , Lassa EN preferring magnesium and LCMV EN or His+ ENs preferring manganese [16] . Here we focus on two sNSV , Hantaan bunyavirus and Lassa arenavirus , that are both transmitted to humans by rodents and can cause severe haemorrhagic fevers with up to 50% fatality rates [19 , 20] . To demonstrate the presence of a cap-snatching endonuclease domain in hantavirus L proteins we determine the crystal structure of the isolated Hantaan virus EN in complex with Mn2+ ions and characterize its endonuclease activity . By comparing the activity and ion binding with the IAV ( Orthomyxoviridae ) , LACV ( Orthobunyaviridae ) and Lassa ( Arenaviridae ) ENs we find that the catalytic histidine present in Hantaan and other His+ ENs correlates with high endonuclease activity . Subsequent structural characterization of the Lassa endonuclease with bound ions reveals an active site with a non-canonical coordination of the catalytic metal ions and this correlates with low intrinsic activity . Therefore the histidine of His+ ENs appears to promote the canonical binding of metal ions in the active site and is a determinant for efficient in vitro catalytic activity . These results are relevant for understanding possible differences in the mechanism of regulation of EN activity and have strong implications in the development of new antiviral drugs targeting transcription of sNSV .
By sequence alignment the Hantaan virus EN is predicted to be at the N-terminus of the L protein [14] . We could express and purify protein constructs comprising residues 1–179 and 1–182 that crystallized with 2 mM MnCl2 as thick plates diffracting to 1 . 7 Å resolution . The structure was solved by SAD experiment ( see Materials and Methods and Table 1 ) . Each protein binds two Mn2+ ions in the active site in a similar fashion to that observed for LACV and influenza [10 , 14] . A third Mn2+ stabilises the interface between crystallographic symmetry related neighbour proteins ( S1A Fig ) . The structure of the Hantaan virus cap-snatching EN ( molecule A ) is shown in comparison with Lassa ( form X3 , see below ) , LACV orthobunyavirus ( PDB: 2xi7 ) and IAV H1N1 virus ( PDB: 4avq ) in Fig 1 . Despite sequence identities below 20% , all structures present root mean square deviations of around 3 . 5 Å for at least 98 residues alignment ( Dalilite ) [21] ( S1 Table ) . Overall , Hantaan EN conserves the two lobe shape of LACV but with the active site , which lies between the two lobes , being more accessible ( S2 Fig ) . The central β-sheet made by the four conserved strands βa-d is extended by βe until the C-terminus of the construct ( Figs 2A , 2B and S1B ) . LACV EN does not have an equivalent of this βe strand and instead , the 30 C-terminal residues of LACV fold into three α-helices , two integrated into the helical bundle which also includes the N-terminal α-helices ( S1B Fig ) . The central β-sheet is surrounded by the conserved helices αb-e . A specific insertion between helix αc and the β-sheet , consisting of helices αc’ and αc” partially covers the β-sheet on the catalytic side ( Figs 1 and 2 ) . The flexible loop linking αb and αc ( highlighted in green in Fig 1 ) , harbouring a catalytic acidic residue ( see below ) , plays the same structural and functional role as in IAV and LACV ENs . In Hantaan EN helix αa points outwards compared to the other ENs ( Fig 1 ) . This conformation , identical for the two molecules in the asymmetric unit , is stabilized by crystallographic contacts partially mediated by a shared , non-active site Mn2+ ion ( S1A Fig ) , and might also be a consequence of the lack of the C-terminal α-helices stabilising the helical bundle , thus allowing helix αa to move ( S1B Fig ) . A structure based alignment of sNSV ENs of known crystal structure illustrates not only the overall conservation of secondary structures and catalytic residues but also the specific features of each family ( Fig 2A and 2B ) . The structures show an identical secondary structure organization in the central region , starting from helix αb and ending at strand βc . However the different lengths of helices αc and αd change the overall shape . The longer helix αc and much shorter helix αd of IAV EN confer a globular shape in contrast with the elongated shape of bunya- and arenavirus ENs ( Figs 1 and 2 ) . Hantaan EN has a unique two α-helix insertion after αc , whereas IAV has a longer unique insertion between helices αb and αc . The structural alignment of the N- and C-terminal regions is poor because of the different arrangement of terminal alpha helices building the helical bundle around conserved helix αb . The 182 residue long Hantaan construct has only 16 residues after strand βc instead of 45 in IAV or 50 in LACV . Thus our structure lacks part of the helical lobe ( see [22] , co-submission ) . However we were not able to express longer constructs with the wild-type sequence in E . coli . The active site of Hantaan EN structure is configured very similarly to that of LACV and IAV ENs with two divalent cations bound in a canonical way ( Fig 3A , 3B and 3C ) . The two ions ( denoted Mn1 and Mn2 ) were identified as Mn2+ by the anomalous scattering signal detected at their respective positions ( S1C Fig ) . Mn1 is octahedrally coordinated by the side chains of amino acid residues H36 , D97 ( from the conserved PD motif ) and E110 , and the main chain carboxyl oxygen of V111 . The putative catalytic lysine K124 ( see below ) is deployed from helix αd as is K134 in IAV ( Fig 3A and 3B ) whereas the LACV catalytic lysine ( K94 ) is deployed from strand βb ( Fig 3C ) . Mn2 is coordinated by D97 and E54 coming from the conserved flexible loop preceding helix αc . Together all these residues constitute the conserved bunya/orthomyxovirus motif H . PD . D/E . K . The octahedral coordination of each Mn2+ is completed by two and four water molecules for Mn1 and Mn2 respectively , one central water being shared by both ions ( S1C and S1D Fig ) . To study the ion binding specificity of Hantaan EN we measured the melting temperature ( Tm ) increase by a Thermal Shift Assay ( TSA ) in the presence of 2 mM of several metal ions ( Fig 4A ) . This follows previous work showing that divalent metal ion binding increases EN thermal stability [10 , 14 , 24] . The Hantaan EN has a Tm of 40 . 5°C in the absence of metal ions . The Tm increases in the presence of MnCl2 ( +5 . 6°C ) , CaCl2 ( +4 . 9°C ) and MgCl2 ( +1 . 2°C ) , does not change in the presence of CoCl2 and is slightly lower with NiCl2 ( -2°C ) . To define the specific ion binding preferences of Hantaan EN we compared , in parallel experiments , the stabilisation effect of metal ions on Lassa , LACV and IAV ENs ( Fig 4A ) . The results show that Hantaan EN is generally the least stable , MnCl2 induces the highest stability increase for all ENs , MgCl2 also stabilise all ENs but less so for Hantaan EN and Ca2+ stabilizes Hantaan , IAV and Lassa ENs almost as much as Mn2+ , but not LACV EN . The effects of CoCl2 and NiCl2 on Hantaan EN stabilization are similar to LACV and different to IAV and Lassa ENs ( Fig 4A ) . Increase of the MgCl2 concentration from 2 mM to 5 mM further stabilises the Hantaan EN by 1 . 7°C ( Fig 4B ) . Therefore , each EN has a specific ion stabilization pattern , but with the common feature that the highest Tm shift results from Mn2+ binding . Subsequent TSA experiments were performed in the presence of 2 mM MnCl2 and 200 μM 2 , 4-dioxo-4-phenylbutanoic acid ( DPBA ) a known EN inhibitor [14 , 25] ( Fig 4B ) . The addition of 2 mM Mn2+ and 200 μM DPBA induces a dramatic increase of the Hantaan EN stability ( 13 . 2°C ) , even larger than found for the other ENs tested ( Fig 4B ) . DPBA has been shown to chelate the two metal ions in the active site in the homologous LACV and IAV endonucleases [14 , 26] . To confirm that Hantaan EN also binds two metal ions in solution , Isothermal Titration Calorimetry ( ITC ) experiments were performed to determine the binding affinities to Mn2+ and Mg2+ . The Mn2+ binding profile could be fitted by a model that assumes sequential binding of two ions ( S3A Fig ) . The first Mn2+ ion binds with a Kd1 of 48 . 5±2 . 6 μM and the second showed a much lower affinity with a Kd2 of 1 . 1±0 . 06 mM . The binding to Mg2+ is much weaker making it impossible to calculate reliable affinity values . The calculated affinities could be underestimated because of protein precipitation observed during the ITC experiment . However , these data are consistent with the two metal ion binding in the active site observed in the structure of the Hantaan EN and the stronger binding of Mn2+ ions compared to Mg2+ . We next investigated the ion dependence of the nuclease activity of Hantaan EN using IAV and LACV and Lassa ENs for comparison ( Fig 4C ) . The reactions were carried out at room temperature for one hour with 2 mM of each metal ion , 7 . 5 μM of G-rich RNA and 10 μM protein . Hantaan EN shows clear nuclease activity with Mn2+ and also some with Co2+ , but not with the other ions tested ( Mg2+ , Ca2+ , Ni2+ , and Zn2+ ) . DPBA was able to inhibit the reaction with 2 mM MnCl2 at 200 μM . The control ENs , LACV and IAV , behaved as previously described [10 , 14 , 24] and showed a similar ion dependence to Hantaan EN . Surprisingly , under the same conditions Lassa EN showed no nuclease activity with any ion . In order to test the substrate specificity the same experiment was carried out in the presence of 2 mM MnCl2 on three different RNAs: unstructured U-rich and G-rich and the structured Alu RNAs ( Fig 4D ) . The Hantaan EN is able to degrade all three RNAs efficiently showing no sequence or secondary structure specificity . Indeed Hantaan EN cleaves the structured Alu RNA more efficiently than LACV EN , which itself is more efficient than IAV EN ( as previously reported [14] ) . Again , Lassa EN showed no activity against any RNA . In all cases no RNA degradation was detected in the absence of MnCl2 or in presence of 200 μM DPBA . The cleavage efficiency of the more structured RNA correlates with higher substrate accessibility to the active site ( S2 Fig ) . The inhibitory effect of DPBA on Hantaan EN was further analysed by titrating increasing amounts of DBPA in EN assays with G-rich and Alu RNA in the presence of 2 mM Mn2+ . LACV EN was used as a control . DPBA had a slightly higher inhibitory effect on Hantaan virus , with the IC50 estimated between 15 and 31 μM compared to the 62 μM estimated for LACV , in agreement with previously reported values for LACV ( ~50 μM , [14] ) and with the TSA experiments where DPBA has a higher stability effect on Hantaan than on LACV EN ( S4 Fig ) . In conclusion , Hantaan EN is thermally stabilized most by Mn2+ , consistent with it having the highest binding affinity for this ion . Mn2+ also most efficiently promotes the nuclease activity which is non sequence specific and inhibited by DPBA . The Hantaan EN efficiency for single stranded RNA appears similar to IAV and LACV but it is able to cleave structured RNA more efficiently than either IAV or LACV , probably due to the higher accessibility to the active site . Under the same experimental conditions , Lassa EN showed no nuclease activity at all . Intrigued by the differences in activity found between ENs from different families and genera we analysed nuclease activity rates with a more sensitive and quantitative real-time assay . This was done by measuring the fluorescence increase upon RNA substrate cleavage using a doubly labelled RNA ( see Materials and Methods ) . For different protein concentrations , initial reaction velocities were determined as the initial slope of the reaction progression as monitored by the fluorescence signal . The linear relationship between the initial reaction velocity ( V = ru/min , where “ru” is fluorescence relative units ) and protein concentration is shown for Hantaan EN in Fig 5A . From the slope , a specific reaction rate of 14 . 38 ru min-1 μM-1 was derived . This activity is five-fold lower than for IAV or LACV ENs with rates of 74 . 88 and 69 . 45 ru min-1 μM-1 respectively ( Fig 5A ) . The difference could be explained by the lower stability of the truncated Hantaan EN construct . On the other hand , the Hantaan EN is 180-fold more active than Lassa EN , which has a rate of 0 . 086 ru min-1 μM-1 ( Fig 5B ) . Since Lassa EN was reported to prefer Mg2+ as catalytic ion we also compared the activity with Hantaan , IAV , LACV and Lassa ENs in the presence of 5 mM MgCl2 . Hantaan EN showed fifty-fold lower activity in the presence of 5 mM MgCl2 than with 2 mM MnCl2 at 1 μM of protein concentration and no activity was detected in the absence of metal ions ( Fig 5C ) . IAV and LACV showed at the same protein concentration 20 fold slower activity with 5 mM MgCl2 than with 2 mM MnCl2 ( Fig 5E ) . For 60 μM Lassa EN ( compared to typically 1 μM for the other ENs ) , the activity did not change significantly in the presence of 5 mM MgCl2 and in the absence of metal ions . This suggests that the observed weak Lassa EN activity must be , at least partially , ion-independent , perhaps due to contaminants which become significant at very high protein concentrations ( Fig 5D ) . Concerned by this lack of activity , we measured the ion binding to Lassa EN by ITC . Lassa EN showed two Mn2+ binding sites with Kd values of 21 . 2 ± 0 . 7 μM and 120 . 6 ± 4 . 7 μM . The interaction with Mg2+ gave a lower signal that could be fitted by a one site model with a much higher Kd of 352 . 0 ± 7 . 3 μM ( S3B Fig ) . In Fig 5F we summarise the quantitative nuclease activity results . Hantaan virus EN has between four and five fold less activity than LACV and IAV and Lassa virus 800 fold less activity in presence of 2 mM MnCl2 . The activity drops to 6 . 5 , 5 . 5 and 2 . 2% respectively for LACV , IAV and Hantaan upon substituting 2 mM MnCl2 by 5 mM MgCl2 . We conclude that Hantaan , together with LACV and IAV ENs are active ENs that preferentially use Mn2+ but can also use Mg2+ with much lower activity rates . By comparison Lassa EN is virtually inactive in the presence of either Mg2+ or Mn2+ metal ions , even if it is able to bind them with similar affinities . To elucidate the role for the Hantaan EN active site residues in ion binding and catalytic activity the mutants H36A , E54G , D97A , K124A and K127A were produced . We first analysed the effect of mutation on protein stability by TSA with either no ions , 5 mM MgCl2 or 2 mM MnCl2 with and without 200 μM DPBA ( Fig 6A ) . In the absence of ions , removal of negatively charged sidechains ( D97 and E54 ) resulted in a greater than 5°C protein stability increase , whereas removal of positive charges ( H36 , K124 , K127 ) had a lower effect on stability changes . Mutation of H36 slightly reduced the Mn2+ and Mg2+ stabilization effect but reduced more DPBA stabilisation , to 50% of the wild-type . Mutation of the D97 , which coordinates both ions , resulted in a complete lack of ion or DPBA stabilisation . Mutation of E54 , in the flexible loop and coordinating Mn2 , impairs both the ion stabilization effect and reduces the DPBA super shift . When the putative catalytic lysine K124 and its neighbour K127 were mutated to alanine the stabilization by ions and DPBA was not impaired and even slightly enhanced . These results are consistent with the crystal structure where H36 , D97 and E54 are engaged in ion coordination but K124 and K127 are not . Subsequently , ITC experiments were performed with mutants E54G and D97A . E54G shows , instead of the wild-type two ion binding profile , a binding profile consistent with one Mn2+ binding site with a Kd = 387 . 6 ± 6 . 2 μM , higher than the wild-type for Mn1 binding ( Kd = 48 . 5 ± 2 . 6μM , see above ) whereas for D97A almost no binding was detected ( S3C Fig ) . This confirms the loss of one binding site ( Mn2 ) and both binding sites ( Mn1 and Mn2 ) upon removal of respectively E54 and D97 side chains , in agreement with the ion coordination observed in the Hantaan EN crystal structure . To determine the role of the mutated residues in the catalytic activity we carried out EN assays with 2 mM MnCl2 and the three different RNAs in parallel with the wild-type EN ( Fig 6B ) . All mutations abolished Hantaan EN activity . The mutant’s activity was also tested by the more sensitive FRET based EN assay . Again , the mutants showed a dramatic drop of reaction rate compared to the wild-type . Only K127A showed clear EN activity above the other mutants , but still much lower than the wild-type ( Fig 6C ) . Therefore those active site conserved residues that are observed to coordinate the metal ions in the crystal structure are important in solution for both ion binding and endonuclease activity , as is the putative catalytic lysine K124 . A neighbouring residue , K127 , is also important for EN activity , possibly in substrate binding , but is not essential since its mutation still allows a low activity rate . To investigate the cause of the lack of activity of Lassa EN we structurally characterized the Mn2+ ion bound form by X-ray crystallography to see how it differs from the active ENs . Using a construct encompassing residues 1–174 from Lassa L protein ( see Materials and Methods ) we obtained three different crystal forms: one with no ions bound ( crystal form X1 ) , and two with one or two Mn2+ ions bound in the active site ( crystal forms X2 and X3 respectively ) . The X1 structure was solved by molecular replacement using LCMV EN structure ( PDB: 3JSB ) and refined at 1 . 85 Å resolution ( see Materials and Methods and Table 1 ) . It is similar to the reported Lassa EN structure in complex with Mg2+ [16] . Despite the presence of 2 mM MnCl2 in the crystallization buffer no anomalous scattering was detected in the active site . The X2 data , solved by molecular replacement using the X1 structure , reached an ultra-high resolution of 1 . 09 Å , thus providing a highly accurate electron density map of the Lassa EN . Compared with the X1 form , the X2 and X3 forms show a 17 degree rotation between the helical bundle lobe ( residues 4–49 and 149–167 ) and residues 50–148 , with the hinge being at the base of helix αb ( Fig 7A ) . The closure of the two lobes slightly changes the orientation of the active site residue E51 ( see below and Fig 7B ) . In the X2 form , data measured close to the manganese absorption edge , showed a 50σ anomalous peak corresponding to one Mn2+ ion in the active . X3 was obtained by co-crystallization with the inhibitor DPBA in the presence of 5 mM MnCl2 . The structure was solved from the X2 model and refined to 2 . 36 Å resolution . Whereas structures X2 and X3 both exhibit the closed form ( S5A and S5B Fig ) , X3 has two Mn2+ ions in the active site ( as detected by anomalous scattering , S5C Fig ) , but no extra density for DPBA . Figs 1 and 2 compare the fold of the Lassa X3 structure with the other sNSV ENs . Lassa EN has the basic EN fold made by three beta strands ( βa-c ) and two alpha helices ( αc-d ) without any insertion . The helical bundle lobe , in comparison with the LACV EN , comprises four long α-helices , the N terminal αpre-a being additional . The active site residues are between the two lobes . The loop connecting αb and αc is also conserved , but instead of approaching the active site , as in Hantaan , LACV and IAV , it is turned outwards and the acidic residue D66 , equivalent to the catalytic residues E80 ( IAV ) D52 ( LACV ) and E54 ( Hantaan ) , is distanced from the active site ( Fig 3D ) . Both the X2 and X3 structures share a common Mn2+ site ( Mn1 ) which is coordinated by the D89 and E102 side chains with 2 Å bond distances , but , unlike other ENs , the carbonyl oxygen of the C103 backbone is too distant for a direct interaction . In the X3 structure , a second Mn2+ ion ( Mn2 ) is found in the active site coordinated by D89 and one carboxyl oxygen of E51 at 2 . 4 Å distance ( Fig 3D ) . This two metal ion binding is additionally stabilised by crystal contacts with N-terminal residue E3 from the neighbouring symmetry related molecule also coordinating Mn2 ( Fig 3D ) . The Mn2+ ion pair in Lassa EN is differently orientated with respect to the catalytic residues than in Hantaan , LACV and IAV ENs , and only four of the eight possible octahedral coordination bonds for the two Mn2+are satisfied ( Fig 3 ) . The previously reported Lassa EN structure in complex with Mg2+ ions [16] has an open conformation of the two lobes , similar to the X1 apo-structure ( Fig 7C ) . The active site residues have the same rotamer conformation than in the X3 structure ( Fig 7B and 7D ) but the helix αb is more open , slightly enlarging the active site . The two Mg2+ ions are both directly coordinated by D89 . Furthermore , Mg1 interacts directly with the C103 backbone carbonyl oxygen and indirectly with E51 and E102 by bridging water molecules . However the full canonical ion co-ordination observed in the active His+ ENs is not achieved . The two Mn2+ ions binding mode reported here for Lassa EN is non-canonical when compared to the mode of ion binding in the His+ ENs . This is likely a consequence of several factors , including the replacement of the histidine by a glutamic acid ( E51 ) , the loss of one residue potentially coordinating the metal ions ( D66 , that is far from the active site ) and the observed flexibility of the active site that is able to alter configuration because of the hinge at the base of helix αb . In comparison , Hantaan , LACV and IAV ENs have more constrained active site with higher ion coordination provided by an acidic residue from the flexible loop and the presence of a histidine that helps stabilise the ion binding in an active configuration . To further investigate these findings on Lassa EN , we mutated to alanine the amino acid residues E51 , D89 and E102 , which are engaged in ion coordination , D66 from the flexible loop and the putative catalytic lysine K115 ( whose side-chain amide group superposes exactly with that of catalytic K137 in IAV EN ) . TSA experiments again show a stability increase , in the absence of ions , when active site acidic residues are mutated to alanine , presumably due to the loss of electrostatic repulsion between these residues ( Fig 8A ) . The stability increase induced by metal ions and DPBA was severely impaired for the E51A mutation that removes one Mn2 coordination in the X3 Lassa EN structure , and for E102A , that removes one Mn1 coordination , and completely abolished for D89A , which removes the coordination with both Mn1 and Mn2 ions . Mutations D66A and K115A , which do not contact the catalytic ions in the X3 structure , did not significantly affect the ion stabilisation effect . For the D66A and D89A mutants , Mn2+ binding was tested by ITC . The D66A thermogram fits a two ion binding model with affinities of Kd1 = 16 . 9 ± 0 . 6 μM and Kd2 = 153 . 8 ± 62 . 3 μM , similar to the wild-type protein ( Kd1 = 21 . 2 ± 0 . 7 μM , Kd2 = 120 . 6 ± 4 . 7 μM , see above ) . D89A resulted in a complete loss of ion binding ( S3D Fig ) . Altogether this data is consistent with the non-canonical two ion binding of Lassa EN observed in the X3 crystal structure . We also tested whether an E51H substitution could increase the activity of Lassa EN , by potential conversion of a His- to a His+ EN , but observed the same lack of endonuclease activity shown by the wild-type Lassa EN . LCMV , another His- EN , was included in this experiment showing that , in our experimental conditions and like Lassa EN , it lacks activity in comparison with the LACV and Hantaan ENs included in the experiment as positive controls ( Fig 8B ) . Therefore changing the catalytic E51 to a histidine is not enough to confer efficient EN activity to Lassa EN . Despite the fact that the isolated Lassa EN is almost inactive in vitro , the full length polymerase is clearly able to carry out cap-snatching dependent transcription in the cellular context . Indeed , using a minireplicon system , it has already been confirmed that active site residues D89 , E102 , D129 [17] and E51 and K115 [16] are essential for cap-dependent transcription . Because of the very low activity of the isolated Lassa EN in vitro we decided to test the activity of new mutants in the full length L protein using the same approach , with the E102A mutant as negative control ( Fig 8C and 8D ) . D66 was mutated to alanine truncating the sidechain , to glutamic to extend the side chain by one carbon , and to asparagine for the removal of the negative charge . All mutants showed about a 50% increase of transcriptional activity confirming that this residue does not play an essential metal-binding role , consistent with the TSA and ITC experiments just described , but unlike the equivalent residues in Hantaan ( E54 ) , LACV ( D52 ) and IAV ( E80 ) . Consistent with in vitro results , which showed the E51H mutant EN to be inactive ( Fig 8B ) , we also found that in the full-length context this mutant did not support any transcription . Some conservative mutations were performed on the critical catalytic residues ( E51D , D89E and K115R ) and residues involved in hydrogen bonding close to the active site ( K122R and D119T , which makes hydrogen bonds with both K115 and K122 ) . The mutations of the acidic residues showed a certain drop of transcriptional activity , but not as much as the E102A mutation negative control . Interestingly both lysine to arginine mutations did not change the transcriptional activity . Based on these results we can conclude that the acidic residue on the flexible loop ( D66 ) in Lassa EN is not essential for activity ( as it is in the His+ ENs ) , D51 is not replaceable by histidine , and that even if conservative mutations in the putative catalytic K115 or neighbouring residues ( K122 and D119T ) do not significantly change the transcriptional activity yields , these residues seems to be important in natural infection as shown by their conservation in sequence alignments of arenavirus ENs ( S6 Fig ) .
The cap-snatching mechanism for transcription is exclusively used by sNSVs . The partial picture provided by the isolated endonucleases and cap-binding domain structures has been dramatically extended recently with the structure determination and biochemical characterization of the heterotrimeric influenza polymerase and the major part of the LACV L protein , allowing the understanding of how the cap-snatching EN domains integrate with the RdRpol domain [9 , 12 , 27–30] . Furthermore , in influenza virus polymerase , the EN domain ( as well as several other PB2 domains including the cap-binding domain ) is connected to the polymerase core through a flexible hinge , which allows it to adopt multiple conformations including those competent for cap-snatching and others where access to the cap-snatching domains is hindered [28 , 30] . 5' vRNA end binding is required to induce the correct relative positioning of the cap-binding and cap-snatching endonuclease domains to increase the RNA cleavage efficiency by 100-fold compared to the isolated EN domain [30] . This allows , for instance , an efficient EN activity with MgCl2 in the context of the full length polymerase that is not achievable by the isolated domain [9] . This shows that the activity of the cap-snatching endonuclease can be significantly modified in the context of the full length polymerase and this is likely to be also the case in the arena- and bunyavirus L proteins . Here we demonstrate that hantavirus L proteins also have a cap-snatching endonuclease that shares with LACV bunyavirus and IAV the same configuration of the active site and contributes to define the canonical binding of metal ions in the active sites by which all the catalytic residues of the H . PD . E/D motif are directly coordinating the two metal ions oriented towards the catalytic lysine ( Fig 3 ) . The mutation of any of these residues affects the ion binding and , results in the complete loss of catalytic activity , as does mutation of the catalytic lysine . To be able to express the Hantaan EN we had to delete part of its C-terminus , which may impair the activity to some extent , although the measured activity rate is comparable to those of LACV and IAV . Indeed , the wild-type Hantaan EN activity could be much higher , resulting in toxicity for E . coli cells , as observed in the accompanying article ( [22] , co-submitted ) for the full length wild-type Andes virus EN , where only reducing the activity by mutagenesis near the active site made expression possible . In the case of arenavirus ENs the most significant active site differences correspond to Lassa EN E51 , that substitutes the bunya- and orthomyxovirus conserved histidine , and D66 , which is not any more engaged in the active site , unlike the equivalent acidic residue in the bunya- and orthomyxovirus EN flexible loop . This study shows that in Lassa EN these differences result in the non-canonical binding of metal ions to the isolated enzyme causing a dramatic drop of endonuclease activity in comparison with the His+ ENs . However the Lassa EN PD-D/E-K catalytic residues are essential for transcription in the minireplicon context , showing that in the full length polymerase the nuclease efficiently cleaves cellular mRNA . Since arenavirus EN active site is more open than His+ ENs the catalytic residues cannot directly coordinate the two metal ions in the same way . Even in the Lassa EN X3 crystal form , were the active site is more closed than X1 form , the open helix αb conformation maintains E51 2 Å away from the Mn1 coordination site ( S5D Fig ) . Therefore we speculate that to achieve the canonical binding required for activity , the Lassa active site needs to be activated for instance by changing E51 and E102 rotamers coupled to a slight movement of helix αb that would close the active site allowing the coordination of Mn1 as shown by the active His+ ENs ( S7 Fig ) . Another possibility is that residues from other parts of the polymerase might contribute to the ion coordination . These changes could be induced by other parts of the L-protein in response for example to vRNA binding ( as in IAV ) or induced by substrate binding . The shift between an active or an inactive enzyme would provide arenaviruses with an “on and off” transcription switch . With the addition of the Hantaan EN structure and new results of Lassa EN , this comparative study puts previously reported work on the isolated cap-snatching endonucleases from bunyavirus , orthomyxovirus and arenavirus in a more general context . We find two different kinds of endonucleases , one with the characteristic catalytic histidine ( His+ ) as in orthomyxovirus and bunyavirus , which have efficient endonuclease activity in isolation , and a second , without the histidine ( His- ) and conserved among arenaviruses which shows very poor activity in vitro . This classification should be taken into account in further development of inhibitor screening assays targeting sNSV ENs . Furthermore , the structure of the active Hantaan EN provides another tool towards the comprehensive development of broad spectrum antivirals against sNSV .
E . coli codon optimised coding sequences were synthesised ( Geneart ) for residues 1–250 of Lassa polymerase ( Lassa 250 ) and residues 1–182 of Hantaan polymerase ( Hanta 182 ) ( UniProt accession code Q6GWS6 and P23456 respectively ) . A histidine tag and a Tobacco Etch Virus ( TEV ) cleavage site ( MGHHHHHHDYDIPTTENLYFQG- ) were added to the amino terminus of all protein constructs . For the final Hantaan constructs a SUMO tag was inserted between the histidine tag and the TEV cleavage site . All protein variants were cloned into a modified pET9a ( Novagen ) vector as described [14] . Mutagenesis of the proteins expressed in E . coli was performed on Lassa196 and Hanta182 . Mutant constructs were obtained by site directed mutagenesis using overlapping oligonucleotides and Pfu DNA polymerase . Proteins were expressed in Escherichia coli strain BL21 ( DE3 ) in LB media with 25 mM kanamycin at 18°C overnight after induction with 0 . 2 mM of IPTG . The protein was purified as previously described , removing the histidine or His-SUMO tags by TEV protease resulting in an additional glycine before the first translated methionine of the original sequence . The resulting untagged proteins were concentrated and purified by gel filtration chromatography using a SD75 column ( Pharmacia ) with lysis buffer ( 20 mM Tris-HCl pH 8 . 0 , 150 mM NaCl , 5 mM β-mercaptoethanol ) for in vitro experiments and crystallization trials . Hantaan required 1mM TCEP in the lysis buffer to avoid aggregation . IAV A/H1N1 EN ( PA residues 1–198 ) and La Crosse EN ( LACV-L 1–183 ) were expressed as described elsewhere [26] [14] and purified as Lassa and Hantaan ENs . Several protein constructs including the first 179 to 250 aa of the Hantaan L protein N-terminus were tested for expression in E . coli . Only the shorter constructs ( 179–185 aa ) were expressed , but insolubly into inclusion bodies . This could be circumvented by insertion of an N-terminal SUMO tag linked by a TEV cleavage site . The length of the proteolytically stable amino terminal domain was defined from the purified Lassa 250 protein by limited papain digestion with 1:500 ( w:w ) papain: protein ratio . Products were characterized by N-terminal sequencing ( Edmann degradation ) and mass spectrometry ( Electrospray ) . Two papain resistant fragments were obtained with molecular weights of 22 . 0–22 . 8 kDa and 25 . 4 kDa corresponding to the first 191–198 or 222 residues respectively of the Lassa L-protein . Proteins Lassa 190 , 192 , 196 , 205 , 221 were subsequently produced for crystallization trials . Based on the first crystal structure obtained , the constructs Lassa 174 , 177 , 180 , 186 were cloned . Finally , the protein construct Lassa 196 was used for in vitro biochemical experiments and Lassa 174 for structural studies . The influence of manganese , magnesium and DPBA binding on protein stability was measured by thermal stability assays ( TSA ) [31] at a protein concentration of 7 . 5 μM in lysis buffer implemented with 2 mM or 5 mM metal ion concentration or 2 mM Mn2+ plus 200 μM DPBA concentration . For nuclease activity experiments , 10 μM of Influenza-PA 1–209 , LACV-L 1–183 , Lassa 1–196 and Hantaan 1–182 wild-type and mutant proteins were incubated with 10 μM of Alu RNA ( 110 nucleotides of the Alu domain of Pyrococcus horikoshii SRP RNA ) or 15 μM of 44 nucleotides U-rich ( 5’-GGGCCAUCCU GCUCU4CCCU11CU11-3’ ) and G-rich ( 5’-GGGCCAGGAAAGGGAGGAGA AAG11AAAAGG AGAAA-3’ ) RNAs for 1 or 2 h at room temperature in the same buffer . The metal ion concentration was 2 mM . The reaction was stopped by adding loading buffer , 10 M urea and twofold concentrated Tris-borate-EDTA buffer ( TBE ) . The reaction products were loaded onto 15% acrylamide 8 M urea TBE gels and stained with methylene blue . For the FRET based real-time quantitative endonuclease activity assays 500 nM of synthetic double labelled RNA , 6-FAM-5′-CUCCUCAUUUUUCCCUAGUU-3′-BHQ1 ( IBA ) , were mixed with the endonuclease proteins . The reaction buffer was 20 mM Tris-HCl pH 8 , 150 mM NaCl , 1 mM TCEP and 2 mM MnCl2 or 5 mM MgCl2 . The fluorescence increase upon the RNA cleavage was measured in a TECAN ( infinite M200 pro ) at 26°C using 465 nm excitation and 520 nm emission wavelengths . The initial reactions velocities were determined by the slope of the linear part of the reaction and where the fitting quality for a straight line was above R2 = 0 . 99 . ITC measurements were performed at 25°C , using an ITC200 Micro-calorimeter ( MicroCal , Inc ) . Experiments comprised 26 injections of 1 . 5 μL of 2 . 5–5 . 0 mM manganese or magnesium solutions into the sample cell containing 200 μL of 100–160 μM of Lassa EN ( wild-type , E51A or D89A ) or Hantaan EN ( wild-type , E54G or D97A ) . All binding studies were performed in the lysis buffer . For data analysis the heat produced by the metal ion dilution in the buffer was subtracted from the heat obtained in the presence of the protein . Binding isotherms were fitted to a one-site binding or two-site sequential binding model using Origin Software version 7 . 0 ( MicroCal , Inc ) . The initial data point was routinely deleted . Hantaan EN crystals were obtained with the NH179 and NH182 constructs at 10 mg/ml in lysis buffer with 2 mM MnCl2 mixed at 1:1 ratio with mother buffer Hepes 0 . 1 M pH7 , 20% PEG 6K , 1 M LiCl2 . The crystals grow at 20 0C after 24 h and were frozen with mother buffer plus 30% glycerol and 2 mM MnCl2 . LACV and IAV were expressed and purified as previously described [14] [26] . Protein constructs denoted Lassa 174 , 177 , 180 , 186 , 190 , 192 , 196 , 205 , 221 were expressed and screened for crystallization using a Cartesian robotic system [32] . Only Lassa 174 and Lassa 190 crystallised and Lassa 174 was used for all following work . The first crystals ( X1 ) were obtained by mixing 1:1 ratio protein: reservoir solution of 5 mg/ml Lassa174 protein in lysis buffer with 2 mM MnCl2 , and a reservoir composition of sodium citrate 0 . 191 M , PEG 3350 4% , 0 . 1 M Hepes pH7 and 0 . 1 M strontium chloride . Form X2 crystals were obtained with Lassa 174 in lysis buffer with 10 mM GMP and 2 mM MnCl2 and a reservoir composition of 0 . 1 M MES pH6 and 20% v/v 2-Methyl-2 . 4-pentanediol . Form X3 was crystallized with Lassa 174 in lysis buffer with 5 mM DPBA and 2 mM MnCl2 and a reservoir composition of 0 . 005 M magnesium chloride hexahydrate , 0 . 05 M HEPES-Na pH7 and 25% ( v/v ) PEG MME 550 . All crystals grow at 20°C overnight . The crystals were frozen in liquid nitrogen in the reservoir buffer with 30% glycerol after adding 5 mM MnCl2 and 10 mM of GMP or 5 mM of DPBA for the co-crystallization derived crystals . The Hantaan EN crystals are of monoclinic space group ( P21 ) , with two molecules in the asymmetric unit , and the structure was solved by a SAD experiment performed at 1 . 77 Å wavelength on beamline ID23-1 ( ESRF ) with high redundancy ( Table 1 ) . The data were processed with XDS and solved with CRANK [33] within the CCP4i package . The three Mn2+ ions and Met94 of each molecule gave the eight anomalous sites enabling structure solution . The structure was refined with a native dataset to Rwork/Rfree of 0 . 166/0 . 216 at 1 . 7 Å resolution obtained at 0 . 984 Å wavelen sequence alignment sequence alignment gth on ID23-1 with NH182 crystals ( Table 1 ) . Molecule A in the asymmetric unit shows density for residues 1–179 , whereas molecule B shows residues 1–171 with a gap between residues 165 to 167 . Lassa EN form X1 crystals are of space-group P212121 with two molecules in the asymmetric unit . Data were collected on ID14-4 at the European Synchrotron Radiation Facility ( ESRF ) to 1 . 8 Å resolution using a wavelength of 0 . 939 Å . Data were processed and scaled with the XDS package [34] and subsequent analysis performed with the CCP4i package . Statistics of data collection and refinement are given in Table 1 . The structure was solved by molecular replacement using Phaser and the LCMV EN model ( PDB: 3jsb ) split into two parts ( residues 1–60 plus 147–170 and residues 61–145 ) . The resultant map was excellent and could be largely built automatically by ARP/wARP [35] . The structure was refined to Rwork/Rfree of 0 . 205/0 . 259 at 1 . 85 Å resolution using REFMAC [36] ( Table 1 ) . Lassa EN crystal forms X2 and X3 were obtained by co-crystallization with GMP and DPBA respectively . They are of space-group P41212 with one molecule in the asymmetric unit . Two X2 datasets were collected on ID23-1 to 1 . 09 Å resolution , one at a wavelength of 0 . 976 Å and another close to the manganese edge ( 1 . 892 Å ) . Data were processed , scaled and the structure solved by molecular replacement using the X1 structure , which only succeeded after searching separately for the two lobes of the EN ( residues 1 to 60 and 145 to 170 , including most of the alpha helical bundle , and residues 65 to 140 including the β-sheet core of the protein ) . Data were refined to Rwork/Rfree 0 . 161/0 . 179 at 1 . 09 Å resolution ( Table 1 ) . Extra density for manganese is observed in the X2 active site , with a peak > 50 σ in the anomalous difference map calculated from the data collected at the Mn2+ edge . The X3 data were collected at SOLEIL on beamline PROXIMA1 to 2 . 5 Å resolution at a wavelength of 0 . 976 Å . The structure was solved from the X2 model and refined to Rwork/Rfree of 0 . 204/0 . 288 at 2 . 36 Å resolution . Extra density for two manganese ions is observed in the active site , consistent with the anomalous difference map . A third Mn2+ also is found bound to His75 on the protein surface . There is no extra density consistent with DPBA or GMP in the active sites of the X2 or X3 structures . The T7 RNA polymerase-based Lassa virus replicon system was used as described previously [37 , 38] [17] . Briefly , generation of L genes with single mutations in the endonuclease domain was performed by mutagenic PCR using pCITE-L as a template and Q5 High-Fidelity DNA Polymerase ( NEB ) for amplification . After purification and spectrophotomeric quantification the PCR-products were directly used for transfection . To make sure the specific mutations were present the PCR-products were sent for sequencing . For Luciferase measurements and RNA extractions BSR-T7/5 cells stably expressing T7 RNA polymerase ( kindly provided by Ursula Buchholz and Karl-Klaus Conzelmann ) [39] were transfected in a 24 well plate with the following amounts of DNA per well: ( a ) 250 ng of minigenome expressing Renilla luciferase ( Ren-Luc ) , ( b ) 250 ng of L gene PCR-product , ( c ) 250 ng of pCITE-NP expressing NP , as well as ( d ) 10 ng of pCITE-FF-Luc expressing firefly luciferase ( FF-Luc ) as an internal transfection control . About 24 hours after transfection , either total cellular RNA was purified for Northern blotting using an RNeasy minikit ( Qiagen ) or cells were lysed and assayed for FF-Luc and Ren-Luc activity using the dual-luciferase reporter assay system ( Promega ) . To compensate for differences in transfection efficiency and cell density Ren-Luc levels were corrected with the FF-Luc levels resulting in standardized relative light units [sRLU] . For Northern blot analysis , 500 ng of total cellular RNA was separated in a 1 . 5% agarose-formaldehyde gel and transferred onto a HybondN+ membrane ( GE Healthcare ) . After UV crosslinking and methylene blue staining to visualize 28S rRNA the blots were hybridized with a 32P-labeled riboprobe targeting the Ren-Luc gene . Transcripts of Ren-Luc genes and RNA-replicates of the minigenome were visualized by autoradiography using an FLA-7000 phosphorimager ( Fujifilm ) . To provide proof for expression of L protein mutants in BSR-T7/5 cells the cells were transfected with 500 ng of PCR product expressing C-terminally 3xFLAG-tagged L protein mutants per well in a 24-well plate . To boost the expression levels and thus facilitate detection by immunoblotting cells were additionally infected with modified vaccinia virus Ankara expressing T7 RNA polymerase ( MVA-T7 ) [40] . After cell lysis and electrophoretic separation in a 3 to 8% Tris-acetate polyacrylamide gel proteins were transferred to a nitrocellulose membrane ( GE Healthcare ) , and FLAG-tagged L protein mutants were detected using peroxidase-conjugated anti-FLAG M2 antibody ( 1:10 , 000 ) ( A8592; Sigma-Aldrich ) . Detected bands were visualized by chemiluminescence using Super Signal West Femto substrate ( Thermo Scientific ) and a FUSION SL image acquisition system ( Vilber Lourmat ) . The structure factors and PDB models are deposited in the PDB database as PDB: 5IZE for Hantaan EN , PDB: 5IZH for Lassa X1 , PDB: 5J1N for Lassa X2 and PDB: 5J1P for Lassa X3 . | Segmented negative strand viruses ( sNSV ) such as Influenza , Lassa or Hantaan viruses are responsible for a large number of severe human infectious diseases . Currently , there are vaccines and antiviral treatments available for influenza but none for the infections caused by other sNSV . All carry out transcription by the cap-snatching mechanism , which requires the action of a metal ion dependent endonuclease ( EN ) , a domain within their large viral polymerases . Here we provide the crystal structure of the Hantaan virus ( family Bunyaviridae ) and Lassa virus ( family Arenaviridae ) cap-snatching ENs in complex with manganese and a comparative functional study of their catalytic activity . Despite the high structural homology between the two ENs a few changes in the active site , involving a catalytic histidine , cause a different binding of the metal ions with dramatic consequences for their in vitro activity . Hantaan EN binds the metal ions as Influenza A ( family Orthomyxoviridae ) and LACV ( family Bunyaviridae ) ENs and all three are active in vitro . In contrast Lassa virus EN is inactive in the same experimental conditions . We can now classify sNSV into two functionally distinct groups ( His+ and His- ENs ) , providing a broad view of the sNSV cap-snatching ENs properties that will be determinant for the comprehensive development of broad-spectrum antiviral drugs . These results also have implications for the viral transcription regulation in the light of recent studies on full-length sNSV polymerases . | [
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] | 2016 | Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases |
Emerging influenza viruses are a serious threat to human health because of their pandemic potential . A promising target for the development of novel anti-influenza therapeutics is the PA protein , whose endonuclease activity is essential for viral replication . Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding , and the necessary mRNA caps are cleaved or “snatched” from host pre-mRNAs by the PA endonuclease . The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain . In this study , we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity . Using an in vitro endonuclease activity assay , we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain . Using X-ray crystallography , we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues . Two structures also reveal an induced-fit mode of inhibitor binding . The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation . Taken together , our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors .
Influenza viruses can cause sporadic global pandemics , and they can result in high mortality rates such as the 1918 pandemic that resulted in 30 to 50 million deaths worldwide [1] . The recent 2009 pandemic was caused by a novel H1N1 virus that originated in swine [2] , but of more concern is the impending threat of the highly pathogenic avian influenza H5N1 viruses that cause mortality rates approaching 60% when transmitted to humans [3] . Although H5N1 viruses have yet to naturally acquire the capacity for efficient human-to-human transmission , this has recently been demonstrated in animal models [4] , [5] and they remain an ever-present threat due to their continued circulation in avian species . The development of a new vaccine requires several months , and effective antiviral therapies are therefore important at the beginning of a fast-spreading pandemic . Antivirals that target the M2 ion channel ( amantadine and rimantadine ) or neuraminidase ( zanamivir and oseltamivir ) have proven to be effective at reducing the severity of illness ( reviewed in [6] ) , but the rapid emergence of resistant strains has highlighted the need for new therapeutic options [7] . Influenza virus contains a negative-strand segmented RNA genome comprising eight ribonucleoprotein assemblies . The RNA-dependent RNA polymerase ( RdRp ) catalyzes both the transcription and replication steps that are essential in the virus life cycle . The RdRp is a heterotrimeric complex comprising subunits PA , PB1 , and PB2 that associates with the 3′ and 5′ ends of each RNA genome segment [8] , [9] . Translation of viral mRNAs by the host ribosome requires 5′ capping , and the necessary mRNA caps are cleaved or “snatched” from host pre-mRNAs . This “cap-snatching” mechanism begins with the binding of PB2 to the cap of a host pre-mRNA , followed by the cleavage of the pre-mRNA by the endonuclease functionality [10] , [11] , [12] . The resulting 10- to 14-residue cap-containing oligonucleotide is then used as a primer for viral mRNA transcription by PB1 [13] , [14] . The endonuclease activity is an excellent target for the development of new anti-influenza inhibitors [15] , and recent crystallographic studies have facilitated this approach . Two groups found that the endonuclease activity resides not in PB1 as previously suggested [11] but in an independently folded N-terminal domain of PA ( PAN ) [16] , [17] . This explains previous findings that PA-specific siRNA can down-regulate viral mRNA production and block virus replication in cell culture [18] . The crystal structures revealed that PAN is a member of the PD- ( D/E ) XK nuclease superfamily , although there was disagreement as to whether there is a single magnesium ( Mg2+ ) ion in the active site [17] or two manganese ( Mn2+ ) ions [16] . However , PAN has greater thermal stability and higher endonuclease activity in the presence of Mn2+ ions than other divalent cations [16] , and isothermal titration calorimetry ( ITC ) [19] and earlier studies [20] also support the presence of two Mn2+ ions . During the past 5 years , structural studies have revealed that the influenza RdRp comprises multiple , independently-folded , sub-domains with defined functionalities , and the PAN domain structure is particularly important with implications for structure-based drug discovery [10] , [16] , [17] , [21] , [22] , [23] , [24] , [25] . Mutational analyses support the idea that the PAN domain is a valuable vehicle for drug discovery [12] , [17] , [19] . Previous studies have reported inhibitors of influenza transcription and/or endonuclease activity , but there are no structural data demonstrating their molecular mechanisms [15] , [26] , [27] , [28] , [29] , [30] . Here , we present crystal structures of PAN from strain A/Vietnam/1203/2004 ( H5N1 ) in complex with six known or predicted inhibitors that allow us to precisely describe their interactions with the PAN active site . In an accompanying article by Kowalinski and coworkers , structures of a complementary set of inhibitors in complex with PAN from strain A/California/04/2009 ( H1N1 ) are reported [31] . Together , our structures provide a molecular explanation for the structure-activity relationship ( SAR ) of several related influenza inhibitors , reveal the mechanism of drug-resistance by a PA mutation , and provide a solid basis for future structure-based drug discovery efforts .
The structure of the PAN domain has been reported in two studies [16] , [17] , but neither construct was considered suitable for drug discovery . In one structure , a 22-residue loop of one PAN molecule packs into the active site of a neighboring molecule [16] making it unavailable for inhibitor binding . In the second structure , although these loop residues are disordered and the PAN active site is suitably exposed , we were unable to reproduce these crystals at high resolution [17] . We therefore designed a new truncated construct of PAN , termed PANΔLoop , from strain A/Vietnam/1203/2004 ( H5N1 ) ( Fig . 1A ) , in which the loop is replaced by a Gly-Gly-Ser linker and which ends at residue 196 , the last visible residue in both of the crystal structures . PANΔLoop readily crystallized in a new crystal form that diffracted to 2 . 05 Å ( Table 1 , PANΔLoop–Apo ) with four molecules in the asymmetric unit and all active sites exposed ( Fig . S1A ) . The PANΔLoop structure is essentially identical to the previously reported structures of PAN ( backbone alpha-carbon RMSD of 0 . 45 Å ) . Importantly , the active site residues are virtually superimposable ( Fig . 1B ) , two metal ions are clearly present ( Fig . 1B ) , and the dose-dependent endonuclease activity is unaffected by the truncations ( Fig . 1C , 1D ) . This suggests that the function of the loop is architectural rather than catalytic , presumably to mediate interactions with another subunit of the influenza RdRp or with a host cell factor . Previous structural studies raised the question as to whether there is a single Mg2+ ion [17] or two Mn2+ ions [16] in the PAN active site . Because of this uncertainty , we included both 10 mM MgCl2 and 5 mM MnCl2 in our crystal soaking solutions . We eventually modeled two Mn2+ ions into the active sites of all of our structures for the following reasons . First , PANΔLoop–Apo crystals soaked in a solution containing only 5 mM MnCl2 revealed strong electron density in both metal sites ( Fig . S1B ) . Second , refinements of all our structures consistently favored Mn2+ over Mg2+ ions to account for the observed electron densities . Third , ITC studies have shown that two Mn2+ ions bind tighter than one Mg2+ ion [19] . Finally , in the accompanying article by Kowalinski and coworkers , a strong anomalous signal for Mn2+ was observed in both metal sites when diketo inhibitors or mononucleotides are bound to PAN [31] . We first investigated three known inhibitors of the influenza RdRp , compounds 1–3 ( Fig . 2 ) . Compound 1 is an N-hydroxyimide that has been shown to inhibit transcription in vitro [29] , and it is structurally related to Flutimide that was found to specifically inhibit transcription , endonuclease activity , and influenza virus replication [30] . Compounds 2 ( 2 , 4-dioxo-4-phenylbutanoic acid , or DPBA ) and 3 ( L-742 , 001 ) are members of a series of 4-substituted 2 , 4-dioxobutanoic acids that were found to inhibit both transcription and endonuclease activities by purified RdRp in vitro [15] . Compound 3 is one of the most potent inhibitors of influenza transcription , and it exhibits dose-dependent inhibition of viral replication in cell culture ( IC50 value 0 . 35 µM ) and in mice [15] , [26] . Purified , recombinant PAN was incubated with single-stranded DNA substrate and increasing concentrations of 1 , 2 , and 3 ( Fig . 3 ) , and each inhibited PAN enzymatic activity in a dose-dependent manner . While this activity has been reported for 2 [16] , this is the first evidence that 1 and 3 also inhibit the isolated PAN domain . To investigate the mechanisms of action of 1 , 2 , and 3 , we determined their co-crystal structures with PANΔLoop ( Table 1 ) . Clear difference electron density showed each compound adjacent to the active site Mn2+ ions ( Figs . 4 , S2 ) . In each structure , the three adjacent and planar oxygen atoms on the inhibitor chelate the two Mn2+ ions in a pairwise fashion such that the central oxygen atom is shared by the ions . Thus , Mn2+ ion 1 ( Mn1 ) is octahedrally coordinated to His41 , Asp108 , Glu119 , Ile120 ( carbonyl ) and two oxygen atoms in the inhibitor , and Mn2 is tetrahedrally coordinated by Glu80 , Asp108 , and two oxygen atoms in the inhibitor . The side oxygen atom of the former pair also forms hydrogen bonds to Lys134 , a key catalytic residue [12] , [16] , [17] , [19] , and an ordered water molecule ( H2O122 ) . The orientation of compound 1 in the active site was not entirely clear . Two of the four molecules in the asymmetric unit showed convincing electron density for the orientation shown in Figures 4A and S2A , while the orientations of the other two molecules were ambiguous . This ambiguity may reflect the weak electron density , possibly due to the poor solubility of 1 in the crystal soak solution . Alternatively , the benzene ring forms no obvious interactions with PANΔLoop , and 1 may be free to adopt two alternate docking modes . Compound 2 has also been structurally characterized in complex with the La Crosse virus endonuclease , and it engages the two-metal active site in the same fashion [32] . However , in the PAN complex , two copies of the molecule are bound in the active site . Molecule A engages the Mn2+ ions and molecule B π-stacks onto molecule A in a parallel fashion via the phenyl group and the planar side chain ( Figs . 4 , S2 ) . This arrangement was present in all four active sites in the asymmetric unit . The carboxyl group of molecule A forms a salt bridge to Lys134 and hydrogen bonds to metal-coordinating residues His41 , Glu119 , and Ile120 ( carbonyl ) and to H2O122 . Molecule B engages a pocket comprising Ala20 , Met21 , Glu26 , Lys34 , and Ile38 ( Figs . 4B ) , and its carboxyl side chain also forms hydrogen bonds to His41 and H2O122 in a fashion similar to that of molecule A . The phenyl groups of both molecules form an edge-to-face interaction with the side chain of Tyr24 that is pushed out approximately 2 . 0 Å in comparison with the PANΔLoop-Apo structure . This suggests that the binding of compound 2 involves an induced-fit mechanism ( Figs . 4 , S3 ) , and the relatively high B-factors in helix-α3 that contains Tyr24 reveal that this region is suitably mobile ( Fig . S3 ) . Kowalinski and coworkers also describe the structure of PAN bound to compound 2 and reveal an identical mode of binding [31] . However , they did not observe the second bound molecule , and we suggest that this is due to the higher concentration of 2 used in our structural studies . To confirm the stoichiometry of binding of compound 2 at the higher concentration , we carried out ITC experiments ( Fig . S4 ) . Analysis of the data strongly supports a 1∶2 molar ratio for PAN∶compound 2 ( N = 1 . 86 , Fig . S4A ) , and an alternative analysis using a sequential binding model ( Fig . S4B ) also supports the second bound molecule of 2 , albeit with a nearly 100-fold lower affinity . These ITC analyses are therefore consistent with the structures in both studies where one or two molecules bind PAN depending on the concentration of compound 2 . Compound 3 binds in a similar orientation as 2 , with the carboxylic acid interacting with Lys134 ( Fig . 4C ) . The increased potency of 3 is likely due to the additional interactions formed by the benzylpiperidine and chlorobenzyl groups that splay in opposite directions perpendicular to the dioxobutanoic acid . The chlorobenzyl group engages the pocket occupied by the phenyl groups of molecules A and B in 2 ( Fig . 4 ) . The piperidine moiety directs the benzyl group into a narrow pocket comprising Arg84 , Trp88 , Phe105 , and Leu106 ( Fig . 4C ) . Although the electron density for 3 was relatively poor ( Fig . S2 ) , our model is supported by several lines of evidence . First , molecular docking of 3 into the PAN active site yields a strikingly similar orientation to that found in our crystallographic model ( Fig . S5 ) . Second , the chlorobenzyl group causes a similar movement in Tyr24 that is seen for 2 , which suggests that 3 also binds via an induced-fit mechanism ( Figs . 4 , S3 ) . Finally , mutation of Thr20 to alanine within the pocket occupied by the chlorobenzyl group caused a 3-fold reduction in virus inhibition in cell culture and a 2–3-fold reduction in inhibition of transcription by 3 ( L-742 , 001 ) [18] . In our PANΔLoop construct , residue 20 is naturally an alanine , and a reduced affinity for 3 could explain the weak electron density for the chlorobenzyl group . We hypothesize that the larger threonine side chain mediates tighter interactions with the chlorobenzyl group and thereby increases affinity and inhibition . Kowalinski and coworkers report the structure of PAN bound to compounds related to 3 [31] , but the most closely-related compound ( R05-2 ) adopts a significantly different orientation . The cyclohexane group of R05-2 is rotated 180° to coincide with the chlorobenzyl group of 3 , and the chlorobenzyl group of R05-2 enters a completely different pocket . The orientation of R05-2 is incompatible with the electron density of 3 and the reverse is also true [31] . The difference in conformations is not entirely surprising because Kowalinski and coworkers demonstrate that a similar compound ( R05-3 ) binds in two distinct conformations [31] . We suggest that these compounds may adopt various conformations within the large PAN active site cleft depending on the microenvironment . Two-metal active sites similar to the one observed in PAN are present in many enzymes that process nucleic acids , and they mediate a common catalytic reaction [33] . Raltegravir is an antiretroviral drug developed to treat HIV infections , and it targets the two-metal active site of HIV integrase [34] . The drug is built around a central pyrimidinol ring scaffold that contains in its plane three adjacent oxygen atoms similar to compounds 1–3 , and these oxygen atoms also coordinate the two-metal center in the active site of foamy virus integrase [35] , [36] . In keeping with our hypothesis that the pyrimidinol scaffold can serve as a general inhibitor of two-metal enzymes [37] , we predicted that compounds 4 and 5 , which also contain the pyrimidinol scaffold ( Fig . 2 ) , would inhibit PAN activity , and showed this to be the case ( Fig . 3 ) . Structural characterization of the two compounds bound to PAN ( Table 1 ) confirmed their interaction with the two Mn2+ ions , but we were surprised to find that their carboxyl groups are not in same location as the carboxyl group in compounds 2 and 3 ( Figs . 4 , S2 ) . Compared with 2 and 3 , the pyrimidinol scaffold is flipped by 180° and there is no electrostatic interaction between the carboxyl groups and Lys134 . We suggest that the flipped orientation of compounds 4 and 5 is necessary to maintain the optimal metal coordination for Mn1 ( see discussion ) . The imidazole and phenyl moieties of compounds 4 and 5 , respectively , show no obvious interactions with the PAN active site cleft , but similar to what we observed with compound 2 , a second molecule ( B ) of compound 5 π-stacks onto molecule A ( Figs . 4E , S2E ) . Molecule B is rotated 180° compared to molecule A and they interact via π-stacking interactions between the pyrimidinol and phenyl groups . Molecule B is further stabilized by hydrogen-bonding and ionic interactions with Lys34 and Arg124 ( Fig . 4E ) . Attempts to determine the binding stoichiometry of compound 5 using ITC were not successful due to compound solubility problems , but similar to compound 2 , the electron density is unequivocal . Finally , two recent studies have identified several compounds , including marchatins , green tea catechins , and dihydroxy phenethylphenylphthalimides , that inhibit PAN endonuclease activity and influenza virus growth [27] , [28] , [38] , [39] . The common moiety in these inhibitors is a dihydroxyphenethyl group , and we predicted that dihydroxybenzoic acid ( compound 6 ) , which contains this moiety and has oxygen atoms in positions similar to those in compounds 4 and 5 , would be able to bind and inhibit PAN ( Fig . 2 ) . Although the compound shows little ability to inhibit PAN endonuclease activity ( Fig . 3F ) , we were able to determine the structure of 6 bound to PAN at a resolution of 2 . 50 Å ( Table 1 ) . Compound 6 interacts with the two Mn2+ ions in the same orientation as the pyrimidinol scaffold ( Figs . 4F , S2F ) . These data suggest that the dihydroxyphenethyl group binds to the PAN active site in the same manner as 4 and 5 , but that additional interactions available in the marchatins , green tea catechins , and dihydroxy phenethylphenylphthalimides are required to inhibit PAN activity . Indeed , Kowalinski and coworkers report the structure of PAN bound to the green tea catechin EGCG and this reveals these additional interactions [31] . Figure 5 shows the inhibitory concentration ( IC50 ) values of a series of compounds related to 1 , including the natural product inhibitor Flutimide ( 7 ) [29] , [30] . Using the co-crystal structure with 1 ( Fig . 4 ) , we analyzed the SAR of this series . We suggest that the increased potency of Flutimide compared with 1 is the result of an interaction between one of the two isobutyl groups and Tyr24 , and that this is further enhanced by the larger fluorobenzyl group of 8 , as reflected by the 6-fold increase in potency compared with Flutimide . Docking studies support our hypothesis that compounds 7 and 8 form molecular interactions with Tyr24 ( Fig . S5 ) . Finally , the presence and positioning of all three Mn2+-binding oxygen atoms is confirmed by the lack of potency observed in compounds 9–11 . Our co-crystal structures with 2 and 3 also provide molecular insights into the SAR of several 4-substituted 2 , 4-dioxobutanoic acids ( Fig . 6 ) [15] , [26] . The addition of an extra phenyl group to 2 as seen in 12 results in a 6-fold gain in potency , and this can be rationalized by additional interactions with Tyr24 . Consistent with this , replacement of the phenyl group in 2 with shorter hydrophobic groups in 13 and 14 results in 2 . 6- and 14-fold reductions in potency , respectively . The importance of the electrostatic interaction between the carboxyl group and Lys134 is confirmed by 15 , in which the replacement of the carboxyl with a methyl ester severely compromises potency . Similar to the effect seen in the Flutimide-related compounds , deletion or repositioning of metal-coordinating oxygen atoms eliminates activity ( 16–19 ) . Compounds 20 , 21 and 22 were found to inhibit in vitro transcription and endonuclease activity with high potency similar to 3 ( Fig . 6 ) , and to exhibit dose-dependent inhibition of viral replication in cell culture [15] , [26] . While the additional groups at the 4-position of the dioxobutanoic acid scaffold clearly increase the activity of these compounds , the differences between our structure with compound 3 and the structures in the accompanying article with 20 , 21 and 22 [31] make it difficult to characterize their SAR . However , the observed conformational differences do suggest that the potencies of these compounds can be significantly improved now that structural information is available . Finally , we recently used a fluorescence polarization assay to identify several additional PAN inhibitors that are related to 4 and 5 ( Fig . 7 ) [40] . In compounds 23 , 25 and 26 , the carboxylic acid has been replaced with marginal impact on potency as reflected in the Ki values . This is consistent with the co-crystal structures of 4 and 5 , in which the carboxylic acid does not interact with Lys134 and there is available space for the substituent ( Fig . 4 ) . The significant gain in potency of 26 may reflect an interaction with Tyr24 as observed in 2 and 3 ( Fig . 4 ) . The increase in potency of 26 is also reflected in the increase in antiviral activity of this compound ( Figs . 7 , S6 ) .
Our studies , and those described by Kowalinski and coworkers in the accompanying article [31] , provide the first molecular insights into the mechanism of inhibition of the essential influenza enzyme PA endonuclease , and we have confirmed that it represents an ideal target for drug discovery . Previous mutagenesis studies have shown a direct correlation between PAN endonuclease activities and RdRp transcription activities , suggesting that the isolated PAN domain contains the same structure in the context of the intact RdRp [12] , [17] , [19] . Our biochemical studies show that inhibitors of RdRp transcription also inhibit PAN endonuclease activity , and this validates the use of the isolated PAN endonuclease domain for drug development . Our structural studies provide the framework to develop novel inhibitors of the influenza virus PA endonuclease . However , two-metal active sites are ubiquitous in enzymes that process nucleic acids , and it may be challenging to develop drugs that specifically target PAN endonuclease . We therefore analyzed the PAN active site for conserved and unique features for drug discovery by aligning ∼13 , 000 PA amino acid sequences to identify the consensus sequence for PAN of influenza types A , B , and C ( Fig . 8A ) . Thirty residues are highly conserved and 17 are more than 99 . 9% identical . Unsurprisingly , most are in the active site pocket and include the metal-binding residues His41 , Glu80 , Asp108 , and Glu119 and the catalytic residue Lys134 ( Fig . 8B ) . The central scaffolds of our characterized inhibitors interact with these residues and are likely to be resistant to mutation but are unlikely to be useful for specificity . Our studies have shown that interactions with residues further away from the two-metal center substantially increase potency . The same conclusion has been drawn by Kowalinski and coworkers who specifically identified four pockets that can be exploited for inhibitor optimization [31] . Figure 8C maps out how compounds 1–6 engage these pockets , and it can be seen that none of the compounds bind pockets 1 and 2 , which only appear to become available upon side-chain rotation and inhibitor binding [31] . However , our structures reveal two additional pockets 5 and 6 . Compounds 2 and 3 occupy pocket 3 and interact with Tyr24 , which is a highly conserved aromatic residue . The biological role of Tyr24 is revealed in the studies of Kowalinski and coworkers which show that it forms a crucial stacking interaction with the base of the mononucleotide [31] . The new pocket 5 is revealed by the binding of the benzylpiperidine group of compound 3; it comprises conserved residues Arg84 , Trp88 , Phe105 , and Leu106 , and is an excellent target for further exploration ( Figs . 4C , 8C ) . The same is true for the new pocket 6 that engages the acetamide group of compound 5 and comprises highly conserved residues Thr123 , Tyr130 , Lys134 and Lys137 ( Figs . 4E , 8C ) . Mutation of Arg84 , Tyr130 , or Lys137 to Ala reduces but does not eliminate endonuclease activity , suggesting that inhibitor resistance could develop , although possibly at a cost to virus fitness [12] , [19] . Similarly , the interactions between molecule B of compound 5 and pocket 4 residues Lys34 and Arg124 are unlikely to be useful for drug development because these residues are not well conserved . However , π-stacking interactions have been shown to be very productive in terms of increasing potency [35] , [41] , [42] , and Tyr24 , His41 , F105 , Tyr130 , and F150 offer potential opportunities . These data reveal the potential for the use of growing and linking strategies to design potent inhibitors . The entropic contribution to binding can be substantial when ordered water molecules are displaced [43] , [44] , [45] , and the PAN active site offers opportunities in this regard . PAN contains a large , deep active site ( over 3000 Å3 ) with several ordered water molecules , 17 of which are found in at least three of the four PAN molecules in the asymmetric unit ( Fig . 8E ) . A large network of water molecules near Val122 becomes displaced by molecule B of compound 5 , and a network of four water molecules between Mn2 and Arg84 is displaced by the benzylpiperidine group of compound 3 , and both can be targeted for inhibitor optimization . Ordered water molecules can also be mimicked by oxygen atoms introduced during inhibitor optimization ( see for example [46] ) . Our studies provide an example of this . One water molecule ( H2OMn1 ) that interacts with Mn1 , Glu119 , and Lys134 becomes displaced by an oxygen atom from compounds 1–6 ( Figs . 8D , 8E ) . H2OMn1 also forms a hydrogen bond with water molecule H2O122 , which in turn forms hydrogen bonds with Val122 ( backbone amide ) , Tyr130 , and another water molecule . Modification of inhibitors that displace H2O122 but preserve its hydrogen bonds should significantly improve inhibitor binding via gains in both entropy and enthalpy . Another important consideration in the design of optimal inhibitors is the location and coordination sphere of each Mn2+ ion in the PAN active site . Detailed structural analyses on the Bacillus halodurans RNase H revealed that the distance between the metal ions changes at different stages of phosphodiester hydrolysis [47] , [48] . Consistent with this is the observation that the metals are approximately 2 . 9 Å apart in PANΔLoop–Apo and move to 3 . 8–4 . 0 Å apart when an inhibitor is bound . This mobility seems to occur in Mn2 because Mn1 is in a similar location in both the unbound and inhibitor-bound structures . Our data suggest that the inhibitor-bound form of PAN represents the enzyme-substrate complex stage in which the metals are separated by about 4 . 0 Å [47] , [48] . Thus , computational modeling or docking of inhibitors may best be suited with the inhibitor-bound form of PAN and Mn2+ ions . Furthermore , metal coordination appears to play an important role in compound binding . Specifically , the compound oxygen atoms that coordinate Mn1 in all the complexes described here and in the accompanying article [31] are separated by two atoms ( Fig . 8D ) , and this allows them to ideally contribute to the octahedral geometry completed by the Mn1-coordinating oxygen atoms from H41 , D108 , E119 , and I120 . Finally , our studies support the potential for developing antiviral inhibitors that target the endonuclease activity of other negative strand and cap-snatching segmented RNA viruses , specifically the Orthomyxoviridae , Bunyaviridae , and Arenaviridae families . Recent crystal structures of the endonuclease domains from La Crosse orthobunyavirus L protein and lymphocytic choriomeningitis virus L protein reveal clear structural homology to the influenza A virus PAN endonuclease domain with dependence on manganese ions for activity [32] , [49] ( Fig . S7 ) . However , low sequence homology and structural variation between virus family endonucleases suggest opportunities for developing virus family-specific inhibitors .
The activity , but not synthesis , of compound 1 ( an N-hydroxyimide ) was described previously [29] . We produced compound 1 using synthetic conditions described by Birch et al . [50] . Briefly , hydroxylamine HCl ( 0 . 9 M ) was added to anhydride ( 1 . 0 M ) in pyridine in a microwavable vessel . The reaction was incubated under a nitrogen atmosphere at 120°C for 60 min under high absorption in a Biotage initiator 60 microwave . Methyl tert-butyl ether was used to precipitate the hydroxylsuccinate product that was isolated via filtration . Compound 1 was further re-crystallized with methanol:chloroform . Compounds 2 ( 2 , 4-dioxo-4-phenylbutanoic acid , or DPBA ) and 3 ( L-742 , 001 ) were prepared with a slight modification to published methods [15] . Instead of producing a methyl ester intermediate , a tert-butyl ester intermediate was produced and then converted to the acid form with trifluoroacetic acid . Compound 4 ( 5-hydroxy-2- ( 1-methyl-1H-imidazol-4-yl ) -6-oxo-1 , 6-dihydropyrimidine-4-carboxylic acid ) and compound 5 ( 2- ( 3-acetamidophenyl ) -5-hydroxy-6-oxo-1 , 6-dihydropyrimidine-4-carboxylic acid ) were synthesized in a similar manner as related compounds described previously [37] , [51] . Compound 6 ( dihydroxybenzoic acid ) was purchased from Sigma-Aldrich and used without further purification . Compound purities were determined by ultra-high-pressure liquid chromatography on a BEH C18 column with a gradient elution of solvent A ( 0 . 1% formic acid in water ) to solvent B ( 0 . 1% formic acid in acetonitrile ) using an evaporative light scattering detector ( ELSD ) and an ultraviolet ( UV , 210 to 400 nm ) detector . Purities are: compound 1 ( ELSD: >99% , UV: 97% ) , compound 2 ( ELSD: 92% , UV: 85% ) , compound 3 ( ELSD: >99% , UV: 98% ) , compound 4 ( ELSD: >99% , UV: 81% ) , compound 5 ( ELSD: >99% , UV: 97% ) , and compound 6 ( ELSD: >99% , UV: 92% ) . Nuclear magnetic resonance ( NMR ) spectra measured on a Brooker-400 ( 400 MHz ) spectrometer showed that all compounds are consistent with their assigned structures . NMR experimental results have previously been published [40] . The tautomeric form of compound 2 shown in Figure 2 was confirmed by solving the high resolution ( 0 . 84 Å ) x-ray crystal structure of the compound alone . PAN ( residues 1–209 ) or PANΔLoop ( residues 1–50 and 73–196 with a 3-residue linker Gly-Gly-Ser between residues 50 and 73 ) from H5N1 influenza virus A/Vietnam/1203/2004 ( Accession #AY818132 ) was cloned between the NcoI and NotI sites in the pET52b plasmid in-frame with a C-terminal thrombin cleavage site followed by a 10-histidine purification tag . PAN and PANΔLoop were expressed and purified with modifications to previously published methods [16] . The recombinant proteins were overexpressed in E . coli strain BL21 ( DE3 ) , and the proteins were purified from soluble lysates by HisTrap affinity chromatography . The 10-histidine purification tags were removed by digestion with biotinylated thrombin , which was later removed by incubation with streptavidin-agarose beads . Undigested protein was removed with cobalt-NTA beads . PAN and PANΔLoop were then purified by size-exclusion chromatography on a Superdex 75 column in 10 mM Tris pH 8 . 0 , 100 mM NaCl , and 1 mM DTT . Proteins were concentrated to 5–10 mg/ml . In vitro endonuclease activity assays were done with modifications to previously published methods [16] . Single-stranded DNA plasmid M13mp18 ( 50 ng/µl ) was incubated in digestion buffer ( 10 mM Tris pH 8 . 0 , 100 mM NaCl , 10 mM β-mercaptoethanol , and 2 . 5 mM MnCl2 ) in the presence of 3 , 10 , or 30 µM PAN or PANΔLoop for 2 h at 37°C . The reaction was stopped by adding 50 mM EDTA . For studies with inhibitors , 10 mM inhibitor in DMSO was diluted 3-fold in series with DMSO and then used at a 10% concentration in enzymatic reactions containing 15 µM PAN . Reaction products were resolved on a 1 . 0% agarose gel stained with ethidium bromide . PANΔLoop protein crystals were grown by the hanging-drop vapor diffusion method at 18°C in a well solution of 1 . 50 M ammonium sulfate , 2% PEG1500 , 0 . 1 M Tris pH 8 . 0 , and 1 mM MnCl2 . Crystals grew after 3–4 days . Crystals were transferred into a soak solution ( 1 . 65 M ammonium sulfate , 2% PEG1500 , 0 . 1 M Tris pH 8 . 0 , 5 mM MnCl2 , and 10 mM MnCl2 ) containing ∼20 mM inhibitor and incubated overnight at 18°C . Crystals were quickly transferred into a cryo-protection solution ( 0 . 4 M ammonium sulfate , 2% PEG1500 , 0 . 1 M Tris pH 8 . 0 , 5 mM MnCl2 , 10 mM MnCl2 , and 25% PEG400 ) containing 10 mM inhibitor before flash freezing in liquid nitrogen . In the case of PANΔLoop-Apo , crystals were mock-soaked in soak solution without inhibitor and cryo-protected without inhibitor . Diffraction data were collected at cryogenic temperature at X-ray wavelength 1 . 00 Å from the Southeastern Regional Collaborative Access Team's 22-ID and 22-BM beamlines at the Advanced Photon Source ( Argonne National Laboratory , Chicago , IL ) . Data processing and reduction were completed with HKL-2000 software [52] . The PANΔLoop-Apo structure was determined by molecular replacement using the program Phaser [53] . A solution was obtained by using a model of the avian PAN crystal structure ( PDB code 3EBJ , residues 1–50 and 73–196 ) [17] . The model was corrected to encode PA residues from A/Vietnam/1203/2004 , and residues 80 , 108 , and 119 were mutated to alanine to remove model bias from these metal-coordinating active-site residues . Simulated annealing was then done using Phenix [54] . Residues 80 , 108 , and 199 were corrected and model building was performed using Coot [55] followed by restrained refinement using the CCP4 software suite's REFMAC5 [56] . Refinement was monitored by following the Rfree value calculated for a random subset ( 5% ) of reflections omitted from refinement . For the PANΔLoop-inhibitor structures , simulated annealing was done with PANΔLoop-Apo without Mn+2 ions and with residues 80 , 108 , and 119 mutated to alanine to remove model bias . Purified PAN protein was dialyzed against 25 mM HEPES pH 8 . 0 , 100 mM NaCl , and 1 mM MnCl2 . ITC titrations were performed with an Auto-iTC200 Isothermal Titration Calorimeter ( MicroCal ) at 25°C . Nineteen injections of 2 µl each of 2 mM compound 2 were titrated into 100 µM protein solution . 5% DMSO was added to the ITC buffer for the titration experiment . Data were analyzed using MicroCal Origin 7 . 0 software using a One-Site binding model and Sequential Binding Sites model with two sites . The experiments were performed independently twice and showed very similar results . Docking of compounds 3 , 7 ( Flutimide ) , and 8 into PANΔLoop active site was performed by Glide module in Schrodinger software . For compound 3 , the docking model was generated from the crystal structure of the PANΔLoop–compound 2 complex , with the 2 , 4-dioxobutanoic acid group defined as the reference core structure for guiding the corresponding functional group in compound 3 into the correct orientation ( tolerance set to 0 . 8 Å RMSD ) . For compounds 7 and 8 , the docking model was generated from the crystal structure of the PANΔLoop–compound 1 complex , with the N-hydroxyimide group defined as the reference core structure for guiding the corresponding functional group in compounds 7 and 8 into the correct orientation ( tolerance set to 0 . 8 Å RMSD ) . Two Mn2+ ions in the active site were kept as part of the protein . The binding pocket is defined as residues within 20 Å radius of the reference core structure . All water molecules were deleted from the protein structure before docking . The compound geometries were built and optimized by SYBYL program . The standard precision of Glidescore scoring functions was used to rank binding poses . Antiviral activity assays were carried out exactly as done previously [40] . Briefly , avian H1N1 influenza A virus ( A/PuertoRico/8/34 ) grown in embryonated eggs was used for infection [50–100 PFU of PR8 virus per well ( MOI = 0 . 0001 ) ] in Madin-Darby canine kidney ( MDCK ) cells ( 3×105 cells/well ) . After 1 h , each well was overlaid with medium containing agarose and compound ( at least 10 concentrations of each compound ) . After 72 h , plaques were visualized with crystal violet and counted . The concentration of compound required for 50% inhibition of plaque formation ( IC50 ) was determined for triplicate measurements by nonlinear least-squares analysis using GraphPad Prism 4 . 03 . Compound cytotoxicity assays were carried out exactly as done previously [40] . Briefly MDCK cells ( 3×105 cells/mL , 20 µL per well ) were incubated with compound at 2-fold serial dilutions from 60 µM . The negative control was 0 . 6% DMSO and the positive control was 60 µM staurosporine . After 72 h , 20 µL CellTiter-Glo reagent was added and luminescence was measured . The concentration of compound required to decrease cell viability by 50% ( CC50 ) , was determined for triplicate measurements by nonlinear least-squares analysis using GraphPad Prism 4 . 03 . The atomic coordinates and structure factors have been deposited in the Protein Data Bank , www . pdb . org , under accession numbers 4E5E , 4E5F , 4E5G , 4E5H , 4E5I , 4E5J , and 4E5L . | Seasonal and pandemic influenza have enormous impacts on global public health . The rapid emergence of influenza virus strains that are resistant to current antiviral therapies highlights the urgent need to develop new therapeutic options . A promising target for drug discovery is the influenza virus PA protein , whose endonuclease enzymatic activity is essential for the “cap-snatching” step of viral mRNA transcription that allows transcripts to be processed by the host ribosome . Here , we describe a structure-based analysis of the mechanism of inhibition of the influenza virus PA endonuclease by small molecules . Our X-ray crystallographic studies have resolved the modes of binding of known and predicted inhibitors , and revealed that they directly block the PA endonuclease active site . We also report a number of molecular interactions that contribute to binding affinity and specificity . Our structural results are supported by biochemical analyses of the inhibition of enzymatic activity and computational docking experiments . Overall , our data reveal exciting strategies for the design and optimization of novel influenza virus inhibitors that target the PA protein . | [
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] | 2012 | Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease |
We previously utilized a Sleeping Beauty ( SB ) transposon mutagenesis screen to discover novel drivers of HCC . This approach identified recurrent mutations within the Dlk1-Dio3 imprinted domain , indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis . For the current study , we performed transcriptome and small RNA sequencing to profile gene expression in SB–induced HCCs in an attempt to clarify the genetic element ( s ) contributing to tumorigenesis . We identified strong induction of Retrotransposon-like 1 ( Rtl1 ) expression as the only consistent alteration detected in all SB–induced tumors with Dlk1-Dio3 integrations , suggesting that Rtl1 activation serves as a driver of HCC . While previous studies have identified correlations between disrupted expression of multiple Dlk1-Dio3 domain members and HCC , we show here that direct modulation of a single domain member , Rtl1 , can promote hepatocarcinogenesis in vivo . Overexpression of Rtl1 in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant ( 86% ) tumor formation . Additionally , we detected overexpression of RTL1 in 30% of analyzed human HCC samples , indicating the potential relevance of this locus as a therapeutic target for patients . The Rtl1 locus is evolutionarily derived from the domestication of a retrotransposon . In addition to identifying Rtl1 as a novel driver of HCC , our study represents one of the first direct in vivo demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis .
Hepatocellular carcinoma ( HCC ) is the third leading cause of cancer-related deaths worldwide [1] . In contrast to the downward trends in incidence observed for most cancer types , that of HCC continues to rise , particularly in the United States [2] . This is due in part to increases in obesity and hepatitis C viral infection , both of which have been implicated in HCC pathogenesis . Treatment options for patients are limited , particularly for those with advanced disease , and the five-year survival rate remains low at ∼10% . A major goal of HCC research is to develop therapies targeted at the molecular mechanisms underlying tumor development and progression . This type of approach is expected to be much more efficacious , increasing survival rates for HCC patients . Consistent with this idea , treatment with sorafenib , a multi-kinase inhibitor , has shown survival benefits for late-stage patients [3] – a rare achievement in HCC treatment . Nevertheless , sorafenib treatment is only able to extend median survival by three months , underlying the need for improved targeted therapies . Unfortunately , the molecular drivers of HCC remain poorly characterized , precluding the development of such therapeutics . Large-scale sequencing efforts currently being undertaken by The Cancer Genome Atlas ( TCGA ) project will likely characterize the recurrent genetic alterations present in human liver tumors and may identify novel therapeutic targets . However , it is becoming increasingly clear that human tumors are incredibly complex , and identifying molecular drivers of carcinogenesis among the larger number of background events has proven difficult . Comparative analysis of the information gained from human tumor profiling with data from animal models provides an improved ability to distinguish driver events contributing to human disease . The Sleeping Beauty ( SB ) transposon mutagenesis system has proven useful for identifying drivers of tumorigenesis in a wide variety of tissue types [4] . We have previously used SB mutagenesis to generate mice that developed HCC [5] . Subsequent genetic analysis of SB-induced liver tumors identified the Dlk1-Dio3 imprinted domain as a common target of transposon-induced mutations . This highly complex domain contains genes encoding protein-coding transcripts , long non-coding RNAs ( lncRNAs ) , microRNAs ( miRNAs ) , and small nucleolar RNAs ( snoRNAs ) . Expression of domain members is regulated in an allele-specific manner and depends on epigenetic modifications established in the germline [6] . Regulation of this expression pattern is maintained , at least in part , by multiple differentially methylated regions ( DMRs ) throughout the domain that are methylated on the paternally inherited allele . Maintenance of imprinting is critical for normal function , as evidenced by the fact that uniparental disomy ( UPD ) for either parental allele leads to severe and widespread developmental defects in both mouse models [7] and human patients [8] . A link between the Dlk1-Dio3 domain and HCC has previously been identified . Interestingly , it has been reported that adeno-associated viral ( AAV ) vector integration within the same region of the domain as the SB transposon integrations in our model is associated with HCC development in mice [9] , [10] . AAV integrations were found to alter expression of several domain members , preventing elucidation of a clear molecular mechanism of tumorigenesis . Other studies have also identified correlation between disrupted expression from the Dlk1-Dio3 domain and HCC [11]–[15] , often with several domain members showing aberrant expression . The majority of these studies are correlative in nature , and no attempt is made to validate tumorigenic function of domain members through direct modulation of gene expression . Here we describe a series of experiments that initially utilized deep-sequencing analyses to obtain detailed gene expression profiles of the SB-induced HCCs . This approach revealed that transposon integration within the Dlk1-Dio3 domain has variable effects on expression of several elements throughout the imprinted domain , but uniformly drives dramatic overexpression of Retrotransposon-like 1 ( Rtl1 ) . Validation experiments demonstrate that hepatic overexpression of Rtl1 promotes tumorigenesis in vivo . Additionally , we find that RTL1 is aberrantly expressed in ∼30% of human HCC samples , suggesting that it may be a relevant therapeutic target . Rtl1 is a poorly characterized gene that encodes a predicted transmembrane protein with aspartic protease activity . Interestingly , this locus is derived from domestication of a sushi-ichi-related retrotransposon [16] and is unique to placental mammals [17] . This study identifies Rtl1 as a novel oncogene involved in hepatocarcinogenesis and suggests that its expression may be used as a prognostic indicator and/or targeted therapeutically to improve outcome for patients with HCC . It also represents one of the first direct in vivo demonstrations of a role for a co-opted genetic element in driving carcinogenesis .
We previously reported the identification of a 33 kilobase region of the imprinted Dlk1-Dio3 domain as a common target of transposon insertion in an SB-induced model of HCC [5] ( Figure 1A ) . Given the domain's complexity and previous studies demonstrating altered expression of multiple domain members in response to insertion of exogenous DNA [9] , [10] , [18] , we used both transcriptome and miRNA sequencing approaches to obtain expression profiles of eight SB-induced HCCs with Dlk1-Dio3 integrations and six normal livers for comparison ( Figure 1B–1C , Figures S1 and S2 , Tables S1 and S2 ) . Expression of Dlk1-Dio3 domain miRNAs was low to undetectable in normal liver . Similar results were detected for three of eight tumors , while the remaining five tumors displayed activated expression of several imprinted miRNAs . Thus , transposon insertion in the Dlk1-Dio3 domain does not consistently alter miRNA expression . Interestingly , tumor samples with elevated expression of imprinted miRNAs also showed enhanced expression of Meg3 and Rian , suggesting a possible transposon-mediated loss of imprinting effect . Dramatic activation of expression from the locus encoding Rtl1 and Rtl1 antisense ( Rtl1as ) was observed in all eight SB-induced HCCs , while no significant expression was detected in normal liver . Notably , elevated expression from this locus is the only event that was consistently observed in all SB-induced HCCs with Dlk1-Dio3 integrations ( Figure 1B–1C ) . Because transcription can occur on either strand at this locus [19] , strand-specific RT-PCR was performed to determine whether the observed increase resulted from expression of Rtl1 , Rtl1as , or a combination of both transcripts . As shown in Figure 2A , reads from the locus encoding Rtl1 and Rtl1as detected in HCCs were derived primarily from transcription of the protein-coding sense strand ( i . e . Rtl1 ) . The lack of detectable Rtl1 in normal liver suggests that transposon integration results in activation of a normally transcriptionally silent allele . As we previously reported , SB transposon integration sites in HCC samples clustered near the 5′ end of Rian within the Dlk1-Dio3 domain [5] . Our initial characterization of transposon integrations was performed using ligation-mediated ( LM ) -PCR followed by pyrosequencing . It has been shown that this approach yields suboptimal sequencing depth for confident identification of clonal insertion sites [20] . To ensure adequate sequence coverage , the SB-induced HCCs were re-sequenced for the current study using the Illumina platform . Surprisingly , while integrations near the 5′ end of Rian were still found to be the most common event , a transposon orientation bias was revealed that had not previously been evident . For many of the tumors , multiple transposon integrations were identified in this region , and for each of the tumors at least one of these integrations was in the same orientation as Rtl1 ( Figure 1A ) . To validate the significance of transposon integrations upstream of Rtl1 in SB-induced HCCs , insertion sites from a larger set of tumors , as well as some normal livers ( Rogers et al . , in press ) , were sequenced using the Illumina platform . A quantitative analysis of all transposon integrations in the Dlk1-Dio3 domain for these samples is provided in Figure S3 . Consistent with recent studies demonstrating minimal insertion bias for SB transposon integration [21] , [22] , background insertion sites identified in normal liver and subclonal insertions in HCC samples did not show any evidence for preferential integration within the Dlk1-Dio3 domain . In contrast , clonal sites identified in tumors were highly enriched upstream of Rtl1 , suggesting positive selection for insertions in this region during the process of tumorigenesis . This analysis further confirmed that transposon integrations in the same transcriptional orientation as Rtl1 are preferentially detected specifically in HCCs . Based on these results , we hypothesized that the high levels of Rtl1 observed in tumors were driven directly by transposons integrated upstream . Amplification of transposon/Rtl1 fusion products from cDNA confirmed transposon-driven Rtl1 overexpression for each of the tumors harboring integrations in this region ( Figure 2B ) . Two different sizes of fusion products were detected , representing direct splicing of the T2/Onc3 transposon into Rtl1 ( smaller product ) or inclusion of a cryptic upstream exon ( larger product ) . Importantly , both fusion products encode the full Rtl1 open reading frame and are thus predicted to drive overexpression of functional Rtl1 protein . Two additional Sleeping Beauty screens have been reported in which liver tumors were generated and characterized [23] , [24] . Neither of these studies identified the Dlk1-Dio3 domain as a common site of integration . Both screens utilized T2/Onc mice as the source of mutagenic transposons . This transposon is similar in structure to that of the T2/Onc3 strain used in our study , but a distinct promoter is included within the transposon . T2/Onc transposons contain the murine stem cell virus ( MSCV ) 5′ long-terminal repeat ( LTR ) promoter , while T2/Onc3 transposons contain the cytomegalovirus ( CMV ) enhancer/chicken β-actin ( CAG ) promoter . Differences in promoter activities likely affect the profile of mutations that are selected for in tumors resulting from SB mutagenesis . We suspect that the MSCV promoter may be too weak to overcome the influence of imprinting within the Dlk1-Dio3 domain to drive sufficient hepatic Rtl1 expression to provide cells with a selective advantage and promote tumorigenesis . The CAG promoter , which has a much higher activity in epithelial cells like hepatocytes , may be better able to drive Rtl1 overexpression when integrated upstream , resulting in frequent selection of cells with such mutations in tumors . Consistent with this idea , insertional mutations upstream of Rtl1 have been linked to liver tumor development in two independent studies that utilized viral vectors containing promoters with high activity in hepatocytes [9] , [15] . Our RNA profiling analyses and fusion transcript detection led us to conclude that the primary tumor-driving event under positive selection in SB-induced HCCs is activation of Rtl1 . While we cannot exclude the possibility that other domain members play a role independently and/or cooperatively with Rtl1 , in our model it seems to be the dominant driver of hepatocarcinogenesis . It should be noted that other models of HCC have been described in which altered expression of maternal Dlk1-Dio3 domain members is observed in the absence of Rtl1 activation [25] , suggesting that distinct roles may exist for both paternal and maternal components of the domain in different subtypes of HCC . To study the effects of Rtl1 overexpression on hepatocyte growth and morphology in vitro , we stably overexpressed it in the murine hepatocyte cell line TIB-73 . Importantly , this cell line is non-tumorigenic and lacks endogenous expression of Rtl1 . Based on the predicted protein structure of Rtl1 , which contains an extracellular protease domain , we hypothesized that its effects may be mediated via cleavage of a substrate within the extracellular matrix ( ECM ) . To test this hypothesis , TIB-73 cells expressing either Rtl1 or an empty vector were embedded in a matrix of Matrigel , plated in 24-well plates , and cultured in serum-free medium . Two weeks after plating , cells expressing Rtl1 had grown to form dozens of cyst-like colonies composed of several cells ( Figure 3B , 3D ) . In contrast , cells lacking Rtl1 expression formed less than one colony per well on average , and colonies that did form were much denser and smaller ( Figure 3A , 3C ) . These results demonstrate that Rtl1 expression promotes growth of hepatocytes in the presence of ECM in the context of physiologically relevant levels of growth factors , and they are consistent with our hypothesis that Rtl1 acts by cleaving an ECM component . ECM is an important aspect of the tumor microenvironment , particularly in the liver . The process of liver fibrosis , which involves ECM remodeling and expansion , is strongly linked to HCC , with nearly 90% of cases developing in this context [26] . One mechanism by which fibrosis may contribute to the development of HCC is through sequestration of growth factors in the newly remodeled ECM [27] . According to this model , subsequent release of growth factors through protease-mediated cleavage of ECM components promotes proliferation of adjacent hepatocytes . Our results suggest that Rtl1 may contribute to hepatocarcinogenesis via this mechanism . We next sought to determine if Rtl1 overexpression is sufficient to promote hepatocarcinogenesis in vivo . Mice with stable hepatic expression of Rtl1 were generated by hydrodynamic tail vein injection of transposon-based expression constructs [28] into Fah-deficient male mice expressing SB transposase [24] . Selective repopulation of the liver was achieved through inclusion of a separate Fah expression vector that allowed stably transfected cells to survive withdrawal of NTBC [29] , an event that triggers the death of Fah-null hepatocytes . Mice were euthanized nine months post-injection to assess liver tumorigenesis . Of fourteen mice injected with Rtl1 overexpression constructs , twelve ( 86% ) developed liver tumors , with an average of 2 . 9 tumors per mouse ( Table 1 , Figure 4 ) . In another experimental condition , a third construct encoding a short hairpin directed against Trp53 was additionally included . Loss of p53 function is one of the most commonly observed molecular abnormalities in human HCC , occurring in ∼30% of cases and making this a relevant context in which to validate putative oncogenes . Of twelve mice injected with all three transposon constructs , ten ( 83% ) developed liver tumors , with an average of 4 . 3 tumors per mouse . Six of the mice from this cohort were sacrificed at time points earlier than nine months . When considering only those mice that were aged for nine months to allow direct comparison between the two experimental groups , five of six ( 83% ) mice with p53 knockdown in addition to Rtl1 overexpression developed liver tumors , with an average of 6 . 7 tumors per mouse . This is significantly higher ( p = 0 . 027 ) than the number of tumors per mouse developed with Rtl1 overexpression alone . Knockdown of p53 in tumors was assessed by western blot ( Figure S4A ) . Although efficiency was somewhat variable , the majority of tumors showed significant knockdown . It has been shown that following liver repopulation , the Fah mouse model is predisposed to tumor formation in the absence of any additional transgene [30] , [31] . The tumors that develop in this context uniformly lack expression of Fah . We assessed expression of both Rtl1 and Fah by RT-PCR in fourteen tumors developed following hydrodynamic injection ( Figure S4B ) . Of these fourteen tumors , eleven were found to express both genes . This result suggests that while a small subset of our tumors are likely background events developed independently of Rtl1 expression due to the model's predisposition , the majority of tumors were induced directly by overexpression of Rtl1 . Further evidence for the tumorigenic activity of Rtl1 in vivo comes from a recently published study showing that liver tumors develop in mice following hepatic lentiviral delivery [15] . In order to determine the prevalence of RTL1 activation in human disease , RT-PCR was performed on a collection of thirty-three human HCC RNA samples , along with matched benign adjacent liver tissue ( Figure 5A , Figure S5 ) . A lack of significant expression was observed for all but one of the benign liver samples . In contrast , significant activation of RTL1 was detected in 30% ( 10/33 ) of analyzed tumors . To assess RTL1 expression in another set of human HCCs , we utilized RNASeq data available through The Cancer Genome Atlas ( TCGA ) consortium . Consistent with our initial analysis , RTL1 expression was found to be significantly activated in 30% ( 10/33 ) of analyzed tumors ( Figure 5B ) . Low-level expression was detected in two of the adjacent benign tissue samples for which sequence data was available . It should be noted that four of the tumor samples included in the TCGA dataset overlap with the initial set of 33 samples analyzed by RT-PCR . No expression of RTL1 was detected in these four samples by either analysis . A notable gender disparity is observed in human HCC , wherein men are around three times more likely to develop the disease than women [1] . We analyzed our human expression data to determine if RTL1 overexpression was associated with tumors from one gender or the other , but failed to detect evidence of any bias . Based on the combined set of human samples that we analyzed , RTL1 was found to be overexpressed in samples from 12/38 males ( 32% ) and 8/24 females ( 33% ) . Unfortunately , there is very little existing data on the expression of RTL1 in disease states , including cancer . Most expression analyses utilize commercially available microarray platforms , the vast majority of which lack probes for RTL1 . While multiple studies have identified correlative links between disrupted expression of other Dlk1-Dio3 domain members and HCC [9]–[14] , expression of RTL1 has not typically been assessed . This may be due in part to the fact that RTL1 is a single exon gene , preventing straightforward design of primers that specifically amplify from cDNA and not genomic DNA . Notably , we have utilized a method for RTL1 expression analysis that adds a unique sequence tag during reverse-transcription [32] , thus allowing specific amplification from cDNA and eliminating background amplification from genomic DNA . In the setting of spontaneous hepatocarcinogenesis in humans , RTL1 activation may occur as a result of loss of imprinting ( LOI ) within the Dlk1-Dio3 domain . Epigenetic abnormalities are known to play a large role in driving tumor development and progression , in part through induction of LOI [33] . A direct causal role for LOI in cancer was demonstrated by Holm et al . , who showed that chimeric mice created using embryonic stem cells lacking imprinting-specific DNA methylation develop multiple tumor types with nearly complete penetrance [34] . The most common tumor type observed was HCC , suggesting that LOI in the liver confers a strong predisposition to cancer . While expression from the Dlk1-Dio3 domain was not examined in the study , the results we present here suggest that hepatic activation of Rtl1 may be a driving factor in the HCCs that were developed . Interestingly , Wang et al . reported loss of methylation within the Rtl1 locus in mouse HCCs resulting from AAV integration [10] , although effects on Rtl1 expression were not determined . To assess whether or not Rtl1 overexpression is associated specifically with altered expression of other imprinted genes in our SB-induced HCCs , analysis of variance ( ANOVA ) was conducted on the whole transcriptome to identify genes with differential expression between Rtl1-overexpressing tumors and normal liver . Following Bonferroni correction , 3 of 125 imprinted genes and 474 of 20 , 707 non-imprinted genes were identified as having significantly different expression between the two sample sets . By Fisher's exact test , these proportions are not significantly different ( p = 0 . 760 ) . This analysis shows that activation of Rtl1 does not correlate specifically with altered expression of other imprinted genes in our tumors . Next we sought to determine if Rtl1-induced HCCs in mice resemble a specific subtype of human HCC . An integrative meta-analysis of human HCC gene expression profiles has identified three major expression subtypes called S1 , S2 , and S3 [35] . Transcriptome sequencing data from the mouse HCCs overexpressing Rtl1 was used to determine the extent to which these SB-induced tumors resemble human HCC . Expression levels of genes defining the S1 , S2 , and S3 subclasses of human HCC were assessed for each of the SB-induced tumors and normal liver samples . Unsupervised clustering of samples based on expression of constituent genes was performed individually for each subclass . The results show that the SB-induced tumors resemble human HCCs within the S1 subclass ( Figure 6 ) . This was further supported by Gene Set Enrichment Analysis ( GSEA ) [36] , [37] that showed a statistically significant association ( p = 0 . 039 ) between Rtl1-induced HCCs and the S1 expression class . Immunohistochemistry was performed to validate protein expression of two S1 subclass genes in SB-induced HCC ( Figure S6 ) . This subclass of human HCC is associated with poor to moderate cellular differentiation , activation of the WNT signaling pathway , and early tumor recurrence . Rtl1 is a poorly characterized gene that encodes a predicted transmembrane protein with aspartic protease activity . Knockout studies in mice have demonstrated a role in the placental feto-maternal interface [38] , but functional studies in other tissues are lacking . Experiments to determine the necessity of Rtl1's protease domain for its ability to promote tumorigenesis and to identify targets of its activity will help to clarify the oncogenic mechanism . If required , RTL1's protease activity represents a promising target for therapeutic intervention in HCC patients . Pepstatin is a naturally occurring bacterial peptide that demonstrates broad potential to inhibit aspartic proteases [39] . Additionally , more specific inhibitors have successfully been developed that target the activity of other aspartic proteases , including renin [40] and HIV-1 protease [41] . It is also possible that RTL1 expression could be a useful biomarker for HCC . Based on the human samples that we analyzed , its expression appears to be highly tumor-specific . Although low-level expression was detected in three non-tumor liver samples , all of the benign samples came from HCC patients and are therefore unlikely to be representative of truly normal liver . In this study we identify Rtl1 , a co-opted imprinted gene , as a novel driver of hepatocarcinogenesis . Mutations resulting in its overexpression were highly selected for in liver tumors developed using a forward genetic screen . While several correlative results linking the Dlk1-Dio3 domain to HCC development have been reported , our study provides direct evidence that modulation of a domain member in vitro and in vivo promotes a tumorigenic phenotype . We show here that overexpression of Rtl1 in cultured hepatocytes results in an increased growth ability in extracellular matrix . We also show that overexpression via hydrodynamic gene delivery results in highly penetrant liver tumor formation in mice . Additionally , a subset of human HCCs displays overexpression of RTL1 , suggesting it may be a relevant therapeutic target for patients .
SB-induced mouse HCCs used in this study were generated as previously described [5] . All tumors used in this study came from male mice and were collected using procedures approved and monitored by the Institutional Animal Care and Use Committees at the National Cancer Institute-Frederick and the University of Minnesota . Paired tumor and benign liver tissues were obtained from 33 patients undergoing resections for HCC at Mayo Clinic between 1987 and 2003 , snap-frozen in liquid nitrogen , and stored at −80°C . The Mayo Clinic Institutional Review Board approved the study . DNA from SB-induced tumors was prepared for sequencing of transposon integration sites as previously described [20] . Stable cell lines were generated by delivery of piggyBac transposon constructs encoding either Rtl1 or an empty vector into TIB-73 ( ATCC: BNL CL . 2 ) cultured mouse hepatocytes . 24-well plates were coated with a thin layer of Matrigel basement membrane mix ( BD Biosciences ) and allowed to set up for 30 minutes at 37°C . For each stable cell line , cells were trypsinized and washed with PBS before resuspension of 5 , 000 cells in additional Matrigel . The resuspended cells were plated on top of the thin layer of basement membrane mix and allowed to set up , followed by addition of serum-free , low-glucose DMEM ( Life Technologies ) . Images were taken two weeks after plating . Hydrodynamic tail vein injection into Fah-deficient male mice expressing SB11 transposase was performed as previously described [24] . A plasmid expressing Rtl1 from the human PGK promoter and flanked by SB transposon inverted repeat/direct repeats ( IR/DRs ) was generated by amplifying the open reading frame of Rtl1 from C57Bl/6J mouse genomic DNA and subcloning it into pT2/PGK-pA . This plasmid was co-injected with PT2/PGK-FAHIL , a plasmid containing an SB IR/DR-flanked expression cassette for Fah and firefly luciferase . Some mice were additionally injected with pT2/shp53 , a plasmid containing an SB IR/DR-flanked expression cassette for a short-hairpin RNA directed against Trp53 [29] , [45] . Total protein was collected from liver tumor samples by homogenization in RIPA lysis buffer . Samples were boiled for five minutes in a reducing buffer and SDS-PAGE was performed . Proteins were transferred to nitrocellulose membranes for blotting . Primary antibodies used were anti-p53 ( Cell Signaling Technology #2524 ) , anti-GFP ( Clontech #632380 ) , and anti-β-tubulin ( Sigma-Aldrich #T4026 ) . GSEA [36] , [37] was performed using default parameters . Analyzed gene sets were comprised of all the genes defining human HCC subclasses S1 , S2 , and S3 [35] for which mouse orthologs have been annotated . Formalin-fixed , paraffin-embedded liver samples were sectioned to a thickness of 4 µm and baked onto glass slides . Samples were de-paraffinized , rehydrated , and treated with citrate antigen unmasking solution ( Vector Laboratories ) . Endogenous peroxidase activity was blocked by treatment with a 3% solution of hydrogen peroxide for fifteen minutes . The anti-rabbit ImmPRESS reagent kit ( Vector Laboratories ) was used for immunolabeling with primary antibodies anti-Fyb ( Abgent #AJ1306a ) and anti-Ier3 ( Abgent #AP11790a ) . Both primary antibodies were diluted 1∶100 and incubated with samples for one hour at room temperature . The ImmPACT DAB kit ( Vector Laboratories ) was used for detection . Sections were counterstained with hematoxylin QS ( Vector Laboratories ) and mounted in Permount ( Fisher Scientific ) for light microscopy . | HCC is the third deadliest cancer worldwide , largely due to a lack of effective treatment options . Therapeutic approaches targeted at the molecular mechanisms underlying tumor formation and progression have shown great efficacy for treating other tumor types . Unfortunately , however , much remains to be learned about the molecular pathogenesis of HCC . There is an urgent need to identify and characterize genetic alterations that drive HCC in order to facilitate the development of more effective targeted therapeutics for patients . Here , we present data showing that recurrent mutations identified in a mouse model of HCC result in overexpression of the Rtl1 gene . We have validated Rtl1 as a driver of HCC by demonstrating that its overexpression in mouse liver causes tumor formation . We also detected overexpression of this gene in a significant proportion of human HCC samples , suggesting that it may be a relevant therapeutic target for patients with this disease . | [
"Abstract",
"Introduction",
"Results/Discussion",
"Materials",
"and",
"Methods"
] | [
"oncology",
"medicine",
"cancer",
"genetics",
"basic",
"cancer",
"research",
"genomic",
"imprinting",
"genetics",
"epigenetics",
"biology",
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"neoplasms",
"gastrointestinal",
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"hepatocellular",
"carcinoma"
] | 2013 | Identification of Rtl1, a Retrotransposon-Derived Imprinted Gene, as a Novel Driver of Hepatocarcinogenesis |
Cell shape and motility are primarily controlled by cellular mechanics . The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular processes involving membrane deformation . However , little is known about the actual function of membrane-to-cortex attachment ( MCA ) in cell protrusion formation and migration , in particular in the context of the developing embryo . Here , we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm ( mesendoderm ) germ layer progenitor cells , which migrate using a combination of different protrusion types , namely , lamellipodia , filopodia , and blebs , during zebrafish gastrulation . By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy , we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration . We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors , and thus is key in controlling directed migration during gastrulation .
During development of the vertebrate body , progenitor cells must migrate from the site at which they are specified to the site where they will eventually form the different body parts . Cell migration is the direct result of mechanical forces mediating cell shape changes and cell-substrate translocation [1] . Thus , the study of cellular mechanics is a prerequisite for understanding cell migration [2]–[4] . In recent years , most studies of cell migration have focused on its molecular control [5] . To fully understand migration , the molecules controlling cell migration must be linked to the mechanics underlying this process . The attachment of the plasma membrane to the cytoskeleton ( membrane-to-cortex attachment [MCA] ) has been proposed to be an important mechanical parameter involved in cell shape changes , such as protrusion formation [6] . MCA is thought to modulate the protrusive activity of cells by providing resistance to the flow of plasma membrane into the expanding protrusion [7] . Several molecules are involved in the regulation of MCA , including Ezrin/Radixin/Moesin ( ERM ) proteins and class 1 myosins [8] , [9] . Studies in mice , Drosophila melanogaster , Caenorhabditis elegans , and cultured cells have shown that ERM proteins are critical for cell shape control during mitotic cell rounding , cell polarization , cell migration , and cell-cell adhesion [10]–[14] . Likewise , class 1 myosins in both single-celled eukaryotes and metazoans have been implicated in various morphogenetic processes , ranging from actin polymerization and microvilli formation to cell motility [15] . In zebrafish , ERM proteins , and in particular Ezrin , are essential for tissue morphogenesis during gastrulation ( [16] and Figure S1; for methods see Text S1 ) , while the role of zebrafish class 1 myosins has not yet been studied . It remains unclear whether the functions of ERM proteins and class 1 myosins in cell and tissue morphogenesis are the direct consequence of MCA modulation , or are linked to other functions of these proteins [15] , [17] . To analyze the role of MCA in cell protrusion formation and migration in vivo , we turned to zebrafish anterior axial mesendoderm progenitor cells ( prechordal plate progenitors ) , which during the course of gastrulation migrate from the germ ring margin , where they are specified , towards the animal pole of the gastrula using a combination of different protrusion types [18] , [19] . Several signaling pathways , including PDGF/PI3K and Wnt/PCP signaling , have been suggested to control protrusion formation and migration of prechordal plate progenitors [18] , [19] . Recently , we showed that ERM proteins are phosphorylated and thus activated in prechordal plate progenitor cells , and are required for prechordal plate morphogenesis ( [16] and Figure S1; for methods see Text S1 ) , alluding to the possibility that ERM proteins modulate prechordal plate cell morphogenesis by regulating MCA . Here , we show that MCA is a critical mechanical parameter determining the proportion of different protrusion types formed by prechordal plate progenitors , and thereby controlling directed migration during zebrafish gastrulation .
To test whether MCA can be modulated in prechordal plate cells by interfering with ERM protein activity , we developed an assay for measuring MCA using atomic force microscopy ( AFM ) , and compared MCA in isolated control and ERM-deficient prechordal plate cells . Control cells were obtained from embryos expressing the Nodal-ligand Cyclops ( Cyc ) , previously shown to induce prechordal plate progenitor cell fate and activate ERM proteins [16] , [20] . ERM-deficient cells were obtained from embryos expressing Cyc in combination with either a dominant negative non-phosphorylatable version of ezrin ( DNEzrin T564A; [21] ) or a combination of morpholinos ( MOs ) targeted against ezrin and moesin-a to inactivate ERM protein function ( [16]; details about MO and controls in Materials and Methods ) . To quantify MCA , we estimated the adhesion energy density between the plasma membrane and the subjacent cytoskeleton ( W0; Figure 1A and 1B; [22] , [23] ) by measuring via single cell force spectroscopy [24] the force needed to extrude single lipid-membrane nanotubes ( or tethers ) from the cell plasma membrane . Various models of tether extrusion have shown that the force required to hold a tether at a constant height ( static tether force , F0; see Figure 1A and 1B ) depends on the membrane bending rigidity ( κ ) , the plasma membrane surface tension ( σ ) , and the energy density of MCA ( W0; [22] , [23] ) : ( 1 ) where σ+W0 is also called apparent surface tension of the membrane ( Tapp; [22] ) . By extruding tethers from control and ERM-deficient prechordal plate cells , we found that the static tether force F0 was significantly reduced in ERM-deficient cells ( Figure 1C; Table S1 ) . We then used F0 to calculate the reduction of apparent tension Tapp in ERM-deficient cells ( Tapp = 18 µN•m−1 , median ) compared to control cells ( Tapp = 46 µN•m−1 , median ) , using a previously determined value for κ , which we assumed was unchanged upon ERM depletion ( [25]; details in Materials and Methods ) . To estimate the corresponding decrease in MCA energy ( W0 ) , we then measured the plasma membrane tension σ by extruding tethers from cells treated with Latrunculin A ( LatA ) to depolymerize the actin cortex , where W0 is negligible and thus Tapp≅σ [26] . We found Tapp to be strongly reduced in LatA-treated cells ( Tapp≅2 . 5 µN•m−1≅σ ) , indicating that σ is small compared to W0 and contributes very little to Tapp ( W0≅Tapp ) . Using this value of σ , we calculated W0 and found it to be strongly reduced upon ERM inactivation in prechordal plate cells ( Figure 1D ) . Inactivating ERM proteins is expected to result in a decrease in the number of molecules cross-linking the cortex to the membrane ( cross-linkers ) . To analyze whether the density of cross-linkers is indeed reduced in ERM-deficient prechordal plate cells , we extruded tethers at varying velocities in control and ERM-deficient cells ( Figure 1E and 1F; [22] , [27] ) . The tether pulling force has to counteract the friction of the cross-linkers against lipid bilayer flowing into the tether , and increases with increasing pulling velocities ( Figure 1G ) . A recent model has related pulling force–velocity profiles to the density of cross-linkers and the lipid bilayer viscosity ( [23]; details in Materials and Methods ) . By measuring the diffusion of a palmitoyl-anchored GFP ( GAP43-GFP ) within the plasma membrane as a reporter of lipid mobility [28] , we first verified that the viscosity of the plasma membrane remains unchanged between control and ERM-deficient cells ( Figure S2A–S2F; details in Text S1 ) . Using a published value for membrane viscosity ( details in Materials and Methods ) , we then deduced the density of membrane-to-cortex cross-linking molecules from the fits of the force–velocity profiles . We found that control cells displayed about 600 cross-linking molecules per square micrometer , which corresponds to a 41-nm lateral separation between molecules on average ( Figure S2G ) . In ERM-deficient cells , the density of cross-linking molecules was strongly reduced ( Figures 1H and S2G ) , indicating that the reduction of W0 in ERM-deficient cells is caused by a decrease in the density of active cross-linking molecules . Mechanical coupling of the plasma membrane to the underlying actin cortex has been proposed to influence the formation of cellular blebs [29] . Bleb-like protrusions are a common alternative to lamellipodia during migration in various cell types ranging from primordial germ cells in zebrafish to cancer cells in culture [30] , [31] . We thus compared protrusion formation in isolated control and ERM-deficient prechordal plate cells expressing membrane-anchored RFP to mark the plasma membrane and Lifeact-GFP to label F-actin [32] . Isolated control cells on nonadhesive substrates formed only blebs , recognizable by the local detachment of the plasma membrane from the underlying actin cortex ( Figure 2A; Video S1 ) . Some of these blebs propagated around the cell circumference by asymmetric assembly of the actin cortex at the bleb neck , a behavior previously described as “circus movements” [33] . In contrast , ERM-deficient cells exhibited less coordinated circus movements and formed significantly larger blebs with a higher frequency ( Figure 2A–2C; Videos S2 and S3 ) . These findings indicate that reduced MCA in isolated ERM-deficient prechordal plate cells correlates with increased blebbing activity . To determine whether similar changes in cell blebbing occur in ERM-deficient prechordal plate cells in vivo , we analyzed prechordal plate progenitor cell protrusion formation in wild type ( wt ) and ERM-deficient embryos expressing membrane-anchored RFP and Lifeact-GFP to distinguish between protrusion types ( Videos S4 , S5 , S6 ) . Three types of cellular protrusions were found in both wt and ERM-deficient prechordal plate progenitors ( Figure 2D and 2E ) : ( i ) spherical protrusions initially devoid of actin , a characteristic of blebs [34] , ( ii ) sheet-like protrusions containing actin throughout their expansion , resembling lamellipodia , and ( iii ) long , thin , actin-containing protrusions resembling filopodia . To quantify the formation of these different cellular protrusions in prechordal plate progenitors , we determined the frequencies of their formation , their respective proportions , and the mean time spent by the cell forming each type of protrusion . We found that in ERM-deficient prechordal plate progenitors , the frequency and size of blebs , the mean time spent blebbing , and the proportion of blebs were significantly increased , at the expense of lamellipodia and filopodia ( Figures 2F–2H and S3 ) . These observations indicate that , similar to isolated cells in culture , ERM-deficient prechordal plate progenitors with reduced MCA in vivo exhibit increased blebbing and that increased blebbing is accompanied by reduced filopodium and lamellipodium formation . Both cortical contractility and MCA have been previously shown to be key mechanical properties controlling bleb formation [35] , [36] . To exclude that changes in cortical tension rather than in MCA are responsible for the increased blebbing phenotype , we compared tension between control and ERM-deficient cells by colloidal force microscopy using AFM [37] . We found no significant differences in cell cortex tension between control and ERM-deficient cells ( Figure S4 ) , indicating that increased blebbing of ERM-deficient prechordal plate progenitors is not due to altered contractility . We next asked whether increased blebbing activity in ERM-deficient prechordal plate progenitors with reduced MCA changes their migratory behavior . To analyze the migratory activity of prechordal plate progenitors , we tracked the nuclei of individual progenitors at the leading edge of the prechordal plate marked with Histone-Alexa-488 from mid to late gastrulation stages ( 8–10 h post-fertilization [hpf]; Figure 3A; Video S7 ) . While the instantaneous speed of the cells remained largely unchanged , we found a significant decrease in the directional persistence and thus net speed of prechordal plate progenitor cell migration in ERM-deficient embryos ( Figure 3B–3D ) . This suggests that increased blebbing activity in ERM-deficient prechordal plate progenitors with reduced MCA leads to reduced net movement speed and directionality . To determine whether ERM proteins function cell-autonomously in mesendoderm progenitors to modulate cell migration , we co-transplanted single mesendoderm control cells ( expressing Cyc , which activates ERM proteins; [16] ) with ERM-deficient cells ( expressing Cyc in combination with ezrin-MO to inactivate ERM proteins ) into the lateral side of MZoep mutant embryos lacking most of their endogenous mesendoderm progenitors [38] . Under these conditions , transplanted cells only rarely interact with their neighbors and mostly undergo single cell migration [39] . We then tracked the movement of the cell nuclei from mid to late gastrulation stages ( 6–10 hpf; Figure 3E; Video S8 ) . Similar to the behavior observed in the prechordal plate , transplanted ERM-deficient mesendoderm cells displayed a reduced directional persistence and slower net migration speed when compared to co-transplanted control cells , while their instantaneous speed was unchanged ( Figure 3F–3H ) . This suggests that ERM proteins cell-autonomously modulate mesendoderm progenitor cell migration . We found that in ERM-deficient cells , reduced MCA correlates with increased blebbing and that increased blebbing correlates with reduced movement directionality , which suggests that these phenotypes are functionally linked . To test whether the observed changes in cell blebbing and migration are caused by the reduction in MCA rather than by potential changes in other ERM-controlled activities , we reduced MCA independent of ERM proteins . To reduce MCA in prechordal plate progenitors , we injected a MO targeted against myosin1b-like2 ( details about MO and controls in Materials and Methods ) to interfere with the activity of Myosin1b , which has been previously associated with regulating MCA [9] . Similar to ERM-deficient cells , Myosin1b-deficient mesendoderm cells exhibited reduced MCA , increased blebbing , and reduced movement directionality and net speed both within the prechordal plate and as single cells transplanted in MZoep mutant embryos ( Figures 4A–4G and S5; Video S9 ) . This supports our suggestion that reducing MCA is sufficient to enhance mesendoderm cell blebbing and interfere with movement directionality and net speed , and that these phenotypes are functionally linked . We next sought to test whether increased cell blebbing leads to the observed reduced movement directionality or whether these phenotypes are independent consequences of reduced MCA . To do so , we analyzed prechordal plate progenitor cell movement directionality when cell blebbing is increased but MCA is not reduced . To increase cell blebbing without reducing MCA , we injected a MO targeted against myosin phosphatase , target subunit 2 ( myop-MO ) , which has previously been shown to promote the formation of bleb-like protrusions in mesendoderm cells by activating Myosin2 ( [40]; details about MO and controls in Materials and Methods ) . MyoP-deficient prechordal plate progenitor cells showed increased cortex tension , as well as increased blebbing activity , reduced formation of lamellipodia and filopodia , and increased MCA ( Figures 4H–4K and S6; Video S10 ) . As in ERM- and Myosin1b-deficient cells , enhanced blebbing activity of MyoP-deficient mesendoderm cells , both within the prechordal plate and as single cells transplanted in MZoep mutant embryos , was accompanied by a significant reduction in the directional persistence and net speed of their migration , while the instantaneous speed of the cells did not change ( Figure 4L–4N ) . This indicates that increased cell blebbing leads to reduced movement directionality and net speed in mesendoderm progenitors .
We have shown that reducing MCA in prechordal plate progenitors by interfering with the function of ERM proteins and class 1 myosins leads to increased bleb formation , at the expense of filopodia and lamellipodia , and that this increased proportion of blebs leads to less directed migration during gastrulation . These findings indicate that MCA is a key mechanical parameter controlling the protrusive and migratory activity of prechordal plate progenitor cells during gastrulation . The mechanical coupling of the plasma membrane to the underlying actin cortex has been proposed to regulate various cellular processes ranging from endocytosis to cell spreading [6] . Although MCA has been directly measured in cultured cells [6] , [22] , [23] , very little is known about its actual regulation and function in cell morphogenesis in vivo , in particular in a developmental context . To directly evaluate the function of MCA in migrating prechordal plate progenitors in vivo , we developed a highly sensitive assay system based on AFM and high resolution confocal microscopy . We showed that changes in MCA lead to alterations in prechordal plate progenitor cell protrusion formation and migration . Moreover , to establish a causative relationship between MCA strength and prechordal plate progenitor cell morphogenesis , we showed that similar reductions in MCA due to inactivation of different proteins ( ERM and Myosin1b ) lead to comparable changes in cell morphogenesis . These experiments strongly support a critical function of MCA in cell protrusion formation and directed migration . Our finding that reducing MCA in prechordal plate progenitor cells leads to an increase in the formation of blebs , as compared to lamellipodia and filopodia , suggests that MCA is an important mechanical parameter determining the proportion of different protrusion types formed by migrating cells . Decreasing MCA has previously been suggested to promote the formation of cellular blebs in cultured cells [36]; however , the mechanisms of bleb formation are still poorly understood [30] . Our finding that in both ERM- and Myosin1-deficient prechordal plate progenitors , reduced MCA leads to enhanced blebbing provides direct experimental evidence for a critical function of MCA in bleb formation during prechordal plate progenitor cell migration . MCA has also been proposed to modulate the extension of lamellipodia [7] , although the role of MCA in this process in not yet clear . The observation that in Myosin1-deficient prechordal plate progenitor cells , reduced MCA increases blebbing but leaves the mean time spent forming lamellipodia unaltered ( Figures 4 and S5 ) argues against a major function of MCA in lamellipodium formation in our system . However , as the frequency of lamellipodium formation is reduced in both ERM- and Myosin1-deficient cells ( Figures S3 and S5 ) , a role of MCA in controlling certain aspects of lamellipodium extension cannot be ruled out . The observation that not only decreasing MCA , but also increasing cortical tension , which raises intracellular pressure , enhances blebbing in prechordal plate progenitors ( Figure 4 ) suggests that the balance between MCA and intracellular pressure controls bleb formation . Interestingly , lowering MCA and/or elevating cortical tension increases not only the frequency but also the size of blebs ( Figures 2 and 4 ) . We have previously shown that cortical tension , and the resulting intracellular pressure , regulate bleb size by directly determining the force driving bleb expansion [35] . MCA , on the other hand , might control bleb size by regulating the size of the bleb base , which has been shown to correlate with bleb size [35] and is enlarged upon treatments reducing MCA ( Figure 2 ) . In addition , MCA might influence bleb size by setting the mechanical resistance to membrane flow into the expanding bleb , which in turn may control bleb expansion . Future studies addressing the contribution of cytoplasmic streaming , bleb base opening , and membrane flow to the dynamics of bleb growth will help to elucidate the mechanisms by which cortical tension and MCA together control bleb size and frequency . We found that changing the proportions of blebs versus lamellipodia and filopodia by reducing MCA leads to less directed migration of prechordal plate progenitors . This finding indicates that the correct proportion of different protrusion types is critical for directed migration in these cells . Blebs are required for the directed migration of various cell types , including zebrafish primordial germ cells and cancer cells [34] , [41] , [42] . Studies in the teleost Fundulus heteroclitus have demonstrated that germ layer progenitor cells can also undergo directional migration by blebbing locomotion , suggesting that blebs are sufficient for directional migration [43] , [44] . Interestingly , these cells change from bleb- to filopodium- and lamellipodium-driven migration during the course of gastrulation , resulting in individual progenitors often simultaneously forming different protrusion types [45] . While this suggests that both blebs and lamellipodia/filopodia function in directed progenitor cell migration , it remains unclear whether these different protrusion types are interchangeable or specifically contribute to directed migration . Our finding that the proportion of different protrusion types is critical for the directed migration of prechordal plate progenitors argues against interchangeability and points to specific functions for different protrusion types in this process . Nodal/TGFβ signals are thought to be key regulators of mesendoderm cell fate specification and morphogenesis [20] . Since Nodal signaling is required for ERM phosphorylation and hence activation in mesendoderm progenitors [16] , it is conceivable that Nodal proteins control mesendoderm protrusion formation and migration by regulating ERM-dependent MCA . Future studies analyzing the function of Nodal signaling in MCA will be needed to elucidate the specific contribution of MCA in Nodal-mediated mesendoderm progenitor morphogenesis . The regulation of MCA is also likely to be important for cell migration in processes other than zebrafish gastrulation . Notably , ERM deregulation has been implicated in tumor metastasis [46] , raising the possibility that the regulation of MCA is critical for cell protrusion formation and migration during tumor progression and metastasis .
Zebrafish maintenance was carried out as described in [47] . Embryos were grown at 31°C in E3 medium and staged as described in [48] . mRNA was synthesized as described in [49] . For tether force measurements wt TL embryos were injected with 100 pg of cyc alone ( control ) or together with a combination of 4 ng of ezrin-UTR-MO [16] plus 4 ng of moesin-a-MO ( TGGTCTCTTCCTTCACGAATGTGTC ) or 300 pg of DNEzrin to generate ERM-deficient cells , 2 ng of myop-MO [40] to generate MyoP-deficient cells , and 8 ng of myo1b-UTR-MO ( CGAGCAGTGATGTTTTCACCTCCAT ) to generate Myo1b-deficient cells . For in vitro confocal microscopy , an additional 50 pg of lifeact-GFP plus 100 pg of GPI-RFP were injected in control and ERM-deficient embryos . For in vivo confocal microscopy , wt TL embryos were injected with 50 pg of lifeact-GFP plus 100 pg of GPI-RFP alone ( control ) or together with 250 pg of DNEzrin ( ERM-deficient ) , 4 ng of ezrin-UTR-MO ( ERM-deficient ) , 3 ng of myop-MO ( MyoP-deficient ) , or 8 ng of myo1b-ATG-MO ( Myo1b-deficient ) . For tracking of prechordal plate cell nuclei , wt embryos were injected with Alexa Fluor-488 conjugated histone H1 ( H13188 , Invitrogen ) and 100 pg of GPI-RFP . For tracking of cell nuclei in the transplantation experiments , wt donor embryos were injected with 100 pg of cyc together with Alexa Fluor-488 conjugated histone H1 ( H13188 , Invitrogen ) ( control ) , 100 pg of histoneH2A-zf::mcherry plus 4 ng of ezrin-UTR-MO ( ERM-deficient ) , 100 pg of histoneH2A-zf::mcherry plus 3 ng of myop-MO [40] ( MyoP-deficient ) , or 100 pg of histoneH2A-zf::mcherry plus 8 ng of myo1b-ATG-MO ( Myo1b-deficient ) . MZoep host embryos were injected with Dextran Alexa Fluor-647 ( D22914 , Invitrogen ) . The ezrin-UTR-MO and myop-MO were used and controlled as described in [16] . As a further control for the ezrin morphant phenotype , we expressed a dominant negative non-phosphorylatable zebrafish version of ezrin [21] , resulting in a phenotype similar to that observed in ezrin morphant embryos . The myo1b-ATG-MO was designed according to Gene Tools targeting guidelines against myosin1b-like2 gene . To control the myo1b morphant phenotype , we tested a second myo1b-UTR-MO and a zebrafish dominant negative myosin1b-like2 version truncated as in [50] , which produced similar prechordal plate progenitor cell blebbing phenotypes as observed with the ATG-MO . We also rescued the myo1b-UTR-MO prechordal plate progenitor cell blebbing phenotype by co-expressing mouse full-length myosin1a mRNA [50] ( data not shown ) . For in vivo experiments , images were obtained with an Andor spinning disc system equipped with a 63×/1 . 2 objective using 488-nm and 563-nm laser lines . Frames were captured at 10-s intervals for 15 min between 8 and 10 hpf . The temperature was kept constant at 28°C . For in vitro experiments , cells from Lifeact- and GPI-RFP-expressing embryos were seeded on a BSA-coated glass slide to prevent attachment and imaged using a Leica SP5 inverted microscope equipped with a 63×/1 . 2 lens using 488-nm and 561-nm laser lines for 2 min at 2-s intervals . For bleb size measurements , the projected area of the bleb at its maximal extension was measured using ImageJ and normalized to the projected area of the whole cell . Wt TL and MZoep mutant donor and host embryos were dechorionated with Pronase ( 2 mg·ml−1 in E2 ) and transferred onto an agarose plate with E3 medium . Two to three cells were taken from control and experimental donor embryos at dome stage ( 5 hpf ) and transplanted into the emerging lateral mesendoderm of a MZoep dharma::GFP host embryo labeled with Dextran Alexa Fluor-647 at shield stage ( 6 hpf ) . Time-lapse images were obtained with an upright Leica SP5 confocal microscope equipped with a 20× water immersion lens using 488-nm Argon , DPSS 561-nm , and 633-nm HeNe laser lines . Frames were captured at 90-s intervals for 3 . 5 h ( 7–10 hpf ) . The temperature was kept constant in all videos ( 28°C ) . Cell/nuclei tracking in three dimensions ( x , y , and z ) was performed with Imaris 6 . 2 . 0 software . The instantaneous and net speeds , as well as directional persistence ( ratio of the net displacement to the distance actually traveled by the cells ) , were extracted from the tracks . Tethers were extruded as described in [24] using a JPK Instruments Nanowizard equipped with a CellHesion module . In short , Olympus Biolevers ( k = 6 mN·m−1 ) were plasma-cleaned and incubated in 2 . 5 mg·ml−1 Concanavalin A ( Sigma ) for 4 h at room temperature . Before the measurements , cantilevers were rinsed in PBS plus Ca2+ and calibrated using the thermal noise method . For the measurement , cells were seeded on a glass slide in a home-built fluid chamber filled with DMEM-F12 cell culture medium and not used longer than 1 h for data acquisition . To depolymerize actin , cells were treated with 1 µM LatA for 10 min . Approach velocity was set to 5 µm·s−1 , contact time was minimized to yield an interaction in 30% of all contacts ( between 0 . 0 and 0 . 6 s ) , and contact force was set to 100 pN . For static tether force measurements , the cantilever was retracted for 6 µm at a speed of 10 µm·s−1 , and the position was kept constant for 30 s . Resulting force–time curves were analyzed using IgorPro . For dynamic tether force measurements , each cell was probed with different speeds ranging from 1 to 50 µm·s−1 in a random order . Tethers were allowed to retract completely between successive pulls . Raw data were analyzed using a home-written IgorPro procedure adapted from the Kerssemakers algorithm . Static and dynamic tether pulling experiments were used to measure the MCA energy density W0 and the density of plasma-membrane-to-cortex cross-linking molecules ν , respectively . For static tether pulling , Equation 1 , described in the Results , was used to extract W0 from the static tether force ( F0 ) . For dynamic tether pulling experiments , the force ( f ) –velocity ( ν ) profiles were analyzed using the model described in [23] , where the pulling force depends on the surface viscosity of the plasma membrane η and on ν: ( 2 ) where F0 is the static tether force , Rc is the radius of the cell , and Rt is the radius of the tether . The model was fitted to the data using a home-written least squares minimization procedure . This yielded the static tether force F0 and the coefficient characterizing the dynamics of extrusion , a . Values for cell radius were measured with light microscopy ( Figure S2H ) , and the tether radius was calculated from static tether forces according to Rt = 2πκ/F0 [22] . The other parameters of the model ( Equations 1 and 2 ) are the plasma membrane bending rigidity κ , the membrane tension σ , and the membrane surface viscosity η . All three are properties of the plasma membrane that change only if the composition of the membrane itself changes , which is unlikely to happen upon perturbations affecting proteins lying within the cortex under the plasma membrane [51] . Supporting this assumption , FRAP experiments showed that the diffusion coefficient of lipids within the plasma membrane was not changed between ERM-deficient and control cells ( Figure S2A–S2F; for methods see Text S1 ) , suggesting that membrane surface viscosity η was unchanged . Moreover , we measured the tether force ( F ) and membrane tension ( σ ) in isolated control and ERM-deficient prechordal plate progenitor cells that were treated with LatA to disassemble their actin cortex . The tether force in LatA-treated cells is determined by σ and κ only ( see also Equation 1 ) . Both the tether force F and the membrane tension σ remained unchanged in LatA-treated ERM-deficient cells relative to control LatA-treated cells ( Table S1 and data not shown ) , suggesting that κ is also unchanged . The values of κ , σ , and η were thus kept constant for all the experimental conditions . κ and η were taken from the literature with κ = 2 . 9×10−19 N•m [22] , [25] , [36] and η = 1 . 5×10−7 Pa•m•s [26] . Plasma membrane tension σ was calculated from tether pulling experiments using cells treated with LatA ( Tapp = σ = 2 . 5 µN•m−1 ) . During tether extrusion , the model assumes that the lipids flow past the cytoskeleton-bound transmembrane molecules as they are dragged into the tether ( permeation regime ) . This is true for intermediate velocities up to several 100 µm•s−1 ( Figure S2I ) , while transmembrane molecules unbind from the cortical cytoskeleton if tethers are extruded faster or membrane viscosity becomes greater [23] . Since the tether pulling velocities in our experiments were ≤50 µm•s−1 , we were most likely within the permeation regime , allowing us to investigate the density of binding molecules . No history effect was observed when sequential tethers were extruded from one cell ( Figure S2J ) . Cortex tension measurements were carried out as described previously [37] . In short , an AFM cantilever was modified with a glass bead ( diameter D = 5 µm ) and coated with heat-inactivated FCS to prevent unspecific binding with the cell during the contact measurement . The colloidal force probe was then brought into contact with the cell with 500 pN contact force at 1 µm•s−1 . A fit to the cortical shell liquid core model [37] between 125 pN and 250 pN yielded cortex tension . To depolymerize actin , cells were treated with 1 µM LatA for 10 min . Analysis of variance ( ANOVA ) and t tests were performed after data were confirmed to have normal distribution and equal variance; otherwise , Kruskal–Wallis tests or Mann–Whitney U tests were applied . p-values were computed in R . For cell transplantation experiments , ttest2 from Matlab was used , which compared our data points with a random distribution of numbers around one with the same standard deviation as our data . | Cell migration , like any event involving shape changes , is a mechanical process controlled by complex biochemical pathways . Here , we examine cell migration in developing embryos with a combination of cell biological tools and atomic force microscopy , so as to investigate how cellular mechanical properties control migration . A fundamental step during migration is the formation of a protrusion at the leading edge of the cell . In three-dimensional environments , and particularly in vivo , cells use different protrusion types: spike-like filopodia and flattened lamellipodia , whose growth is driven by actin polymerization , and spherical blebs , which grow because of intracellular pressure pushing on the membrane . It is important to understand how the formation of different protrusion types is mechanically and molecularly controlled , and how the different protrusions specifically contribute to migration . We have addressed this using the zebrafish embryo as a model system . We show that reducing the strength of the attachment between the plasma membrane and the underlying cortical network of actin filaments , or increasing intracellular pressure , increases the proportion of cellular blebs and reduces the directionality of cell migration . Our work reveals that blebs , lamellipodia , and filopodia are not interchangeable and that the relative proportion of each type of protrusion , under the control of mechanical parameters , determines migration directionality during zebrafish gastrulation . | [
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] | 2010 | Control of Directed Cell Migration In Vivo by Membrane-to-Cortex Attachment |
The apparent paucity of molecular factors of transcriptional control in the genomes of Plasmodium parasites raises many questions about the mechanisms of life cycle regulation in these malaria parasites . Epigenetic regulation has been suggested to play a major role in the stage specific gene expression during the Plasmodium life cycle . To address some of these questions , we analyzed global transcriptional responses of Plasmodium falciparum to a potent inhibitor of histone deacetylase activities ( HDAC ) . The inhibitor apicidin induced profound transcriptional changes in multiple stages of the P . falciparum intraerythrocytic developmental cycle ( IDC ) that were characterized by rapid activation and repression of a large percentage of the genome . A major component of this response was induction of genes that are otherwise suppressed during that particular stage of the IDC or specific for the exo-erythrocytic stages . In the schizont stage , apicidin induced hyperacetylation of histone lysine residues H3K9 , H4K8 and the tetra-acetyl H4 ( H4Ac4 ) and demethylation of H3K4me3 . Interestingly , we observed overlapping patterns of chromosomal distributions between H4K8Ac and H3K4me3 and between H3K9Ac and H4Ac4 . There was a significant but partial association between the apicidin-induced gene expression and histone modifications , which included a number of stage specific transcription factors . Taken together , inhibition of HDAC activities leads to dramatic de-regulation of the IDC transcriptional cascade , which is a result of both disruption of histone modifications and up-regulation of stage specific transcription factors . These findings suggest an important role of histone modification and chromatin remodeling in transcriptional regulation of the Plasmodium life cycle . This also emphasizes the potential of P . falciparum HDACs as drug targets for malaria chemotherapy .
Gene expression in the asexual intraerythrocytic developmental cycle ( IDC ) of Plasmodium falciparum and vivax occurs in a continuous cascade with the induction of most genes occurring just once in the cycle , presumably at the time when their products are required [1] , [2] , [3] . The next obvious and intriguing step is to understand how this highly specialized mode of transcriptional regulation is controlled . Emerging evidence from other eukaryotes indicates that chromatin structure regulates gene expression through histone modifications such as acetylation , deacetylation and methylation [4] . Histone acetyltransferases ( HATs ) catalyze acetylation of lysine residues located within histones , thereby reducing chromatin compaction and making the DNA more accessible to regulatory proteins resulting in transcriptional activation . The removal of acetyl groups from the lysine residues is catalyzed by histone deacetylases ( HDACs ) resulting in chromatin condensation and transcriptional repression . Specific recruitment of HAT and HDAC containing complexes to selected promoter elements generate localized domains of modified histones that influence transcriptional activity [5] , [6] . HATs and HDACs also function globally throughout the genome resulting in a highly dynamic equilibrium of histone acetylation and deacetylation reactions , which maintains a steady-state level of histone acetylation across the entire genome [7] , [8] . The HDAC super-family is grouped into different classes according to sequence similarity to yeast prototypes . Classes I , II and IV are related to the zinc-dependent yeast Rpd3 or Hda1 deacetylases [9] . Class III HDACs , are a family of NAD-dependent sirtuins related to the yeast silencing information regulator 2 ( Sir2 ) which mediates gene silencing at telomeres , mating-type loci and ribosomal DNA [10] . Homologues of a Class I and Class III HDAC , referred to as PfHDAC1 ( PFI1260c ) and PfSir2 ( PF13_0152 ) respectively , have been characterized in P . falciparum [11] , [12] . Transcripts of the nuclear localized PfHDAC1 [11] were detected throughout the asexual IDC and in the exo-erythrocytic stages [2] . The PfSir2 co-localizes with telomeric clusters generating heterochromatin at the chromosome ends . In addition , PfSir2 binding and deacetylation controls the mutual exclusive expression of the surface antigen family encoded by the telomeric var genes [12] . The genome sequence of P . falciparum has revealed three additional HDAC homologues ( PF14_0489 , PF14_0690 and PF10_0078 ) all of which have yet to be characterized [13] . To understand the regulation of gene expression in P . falciparum we took the advantage of HDAC inhibitors altering gene transcription [14] . Recently , FR235222 , a cyclic tetra-peptide was shown to inhibit HDAC3 activity in Toxoplasma gondii , resulting in nucleosomal hyperacetylation of histone 4 [15] . The Class I and II HDAC inhibitor apicidin , also a cyclic tetra-peptide , exhibits anti-proliferative activity against several Apicomplexan parasites including P . falciparum [16] and inhibits the enzymatic activity of PfHDAC1 [17] . We showed that apicidin caused profound transcriptional changes in multiple stages of the P . falciparum IDC that were characterized by specific induction of genes that are otherwise suppressed during that particular stage . We also showed that apicidin induced rapid hyperacetylation of histone lysine residues associated with promoters and coding regions of a large number of genes indicating the disruption of both targeted and non-targeted HDAC activities . Interestingly , only a partial overlap between the genetic loci with altered histone modifications and genes induced/repressed by apicidin was found . Intriguingly , induction of stage specific transcription factors and other transcription associated or chromatin binding proteins was identified . The significant enrichment of the apicidin induced histone modifications in these gene classes suggests their role in the transcriptional de-regulation in the apicidin treated cell . These findings highlight the important role of histone deacetylases in the transcriptional regulation of the Plasmodium life cycle .
A Class I and II HDAC inhibitor , apicidin , was used to analyze the global transcriptional response of P . falciparum to inhibition of its histone deacetylase activities . For this purpose , highly synchronized P . falciparum cells were treated with 70nM apicidin at the three asexual developmental stages: ring ( 6–14 hours post invasion ( hpi ) ) , trophozoite ( 20–28 hpi ) and schizont ( 34–42 hpi ) . Treatment of P . falciparum cells with 70nM of apicidin at these three developmental stages resulted in ∼90% reduction of growth ( IC90 ) that was monitored by the number of newly formed rings after the completion of the IDC ( data not shown ) . To capture the dynamics of the transcriptional response , RNA samples were collected at 0 . 5 , 1 , 2 , 4 and 6 hours post treatment and analyzed by the P . falciparum DNA microarray that represents 5363 coding sequences [18] ( Dataset S1 ) . Overall , we observed highly dynamic transcriptional changes induced by apicidin in all the three developmental stages ( Figure 1A ) . In particular , 6 hours of apicidin treatment altered the expression of 3210 ( 59 . 8% of the genome ) , 1811 ( 33 . 8% ) and 2760 ( 51 . 5% ) genes by at least 2-fold in the ring , trophozoite and schizont stages , respectively . Intriguingly , approximately half of these genes were induced or repressed as early as 1-hour post treatment . To our knowledge , this is one of the most dramatic perturbations of the P . falciparum IDC transcriptome reported so far . This is in sharp contrast with previous studies showing non-specific and low amplitude changes in mRNA abundance induced by the anti-malarial drug chloroquine [19] and the antifolate WR99210 [20] . Interestingly , the rapid and large changes in gene expression observed by inhibition of HDAC activities also contradict that of inhibition of P . falciparum HAT activities [21] . To understand the physiological relevance of the HDAC-dependent transcriptional response we carried out functional enrichment analyses of the genes induced/repressed by apicidin in all the three developmental stages . Using three types of functional terms Gene Onthology ( GO ) , Kyoto Encyclopedia of Genes and Genomes ( KEGG ) and Malaria Parasite Metabolic Pathways ( MPMP ) , we identified a large number of functional groups that are significantly enriched in the apicidin altered gene expression ( Figure S1 ) . The vast majority of the identified gene groups represented basic metabolic and cellular functions expressed during the P . falciparum IDC . In the case of apicidin induced gene expression , a considerate overlap was found among the gene groups associated with the three different stages of the IDC . These included “Fatty acid synthesis in the apicoplast” ( P value <0 . 008 ) and “Molecular motor prototypes” ( P value <0 . 004 ) . In contrast , with apicidin induced gene repression distinct functional groups were statistically overrepresented in each stage . This can be explained by the fact that any gene repression should be observed in genes that are under normal growth conditions expressed during that particular stage of apicidin treatment . For example , gene groups associated with transcription and translation that are normally induced during the ring stage were significantly repressed ( P value <0 . 01 ) during this stage [1] . Similarly , gene groups associated with invasion that are normally induced during the schizont stage were significantly repressed ( P value <10−9 ) during this stage . To investigate whether apicidin induced a specific de-regulation of the P . falciparum IDC transcriptional cascade [1] , we focused on genes with the most pronounced changes ( >3–4 fold ) in their transcript levels ( Figure 1B ) . In all three stages , this analysis revealed that essentially most of the genes induced by apicidin are under normal growth conditions suppressed during that particular stage of treatment ( Figure 1B ) . For example , genes associated with the TCA cycle that are normally expressed during the trophozoite and early schizont stage were significantly induced ( P value = 3 . 25×10−3 ) in the ring stage . Similarly , genes belonging to the functional group “Subcellular localization of proteins involved in invasion” that are normally expressed in schizonts , were strongly induced ( P value = 1 . 04×10−3 ) in the trophozoite stage by apicidin . Using the publicly available P . falciparum exo-erythrocytic transcriptome data [2] , we also examined the top 500 genes with the highest mRNA abundance in the sporozoite and gametocyte stages . A significant number of genes that are considered specific for theses stages and are normally suppressed during the IDC were induced by apicidin ( Figure S2 ) . These included genes encoding the early gametocyte markers , Pfg27 , Pfs16 , Pfpeg-3 and Pfpeg-4 [22] , Pf47: a member of the Pfs48/45 gene family [23] and Pfg377: localized in the osmiophilic bodies of gametocytes [24] . Similarly genes associated with sporozoite invasion such as circumsporozoite protein , sporozoite surface protein 2 , SPATR and Pf52 [25] were significantly induced ( >2-fold ) by apicidin . Taken together , apicidin , a specific inhibitor of HDAC activities affected expression of approximately half of the genome during the P . falciparum IDC . Therefore , as a result , a large number of functional groups were significantly affected . The induced expression of stage specific genes indicates that the changes in mRNA abundance are not compatible with arrest of the IDC , but rather a generic de-regulation of the transcriptional cascade of the P . falciparum life cycle . Intriguingly , with 70nM of the HDAC inhibitor , this de-regulation is highly dynamic affecting a large number of genes as early as 1-hour post treatment . To investigate the mechanisms by which apicidin deregulates the P . falciparum transcriptional cascade we examined the effect of this HDAC inhibitor on histone modifications . Previous studies have showed that the HDAC inhibitors Trichostatin A ( TSA ) and the two derivatives of 2-aminosuberic acid induce overall acetylation levels of P . falciparum histone 4 [26] . In P . falciparum , previous studies have found stage-specific enrichment of acetylation at histone 3 lysine 9 at putative transcriptional initiation sites , corresponding to stage-specific expression of genes [27] . Therefore , we tested the effect of apicidin on the overall levels of three distinct acetylations: histone 3 lysine 9 ( H3K9Ac ) , H4K8Ac and histone 4 acetylated on lysine residues 5 , 8 , 12 and 16 ( H4Ac4 ) ( Figure 2 ) . In addition , we also tested for tri-methylation of histone 3 lysine 4 ( H3K4me3 ) which has been linked with gene expression [28] . The most striking observation was the dramatic increase in the levels of H4K8Ac and H4Ac4 in the apicidin treated P . falciparum cells in all three stages . This strongly indicates that the histone acetylase and deacetylase activities specific for these histone 4 modifications exist in a highly dynamic equilibrium throughout the entire IDC . The increase in histone 4 acetylation observed as early as 1-hour post treatment coincides well with the rapid transcriptional changes induced by apicidin . Conversely , apicidin induced a different effect on both H3K9Ac and H3K4me3 . In rings , a dramatic decrease in the levels of both histone 3 modifications was observed after 2 hours of apicidin treatment . In the first hour of treatment , an increase or no change in H3K9Ac and H3K4me3 respectively was found . In contrast to rings , no change in the overall levels of both modifications was detected in schizonts . In trophozoites these modifications were undetected ( Figure 2 ) . In agreement with a previous study [29] , immunodetection of core histones 3 and 4 generated an extremely weak signal in both rings and trophozoites ( data not shown ) . Therefore , Pfactin 1 ( PFL2215w ) was used as a loading control for these two stages ( Figure 2 ) . In schizonts , core histones 3 and 4 as well as actin served as suitable loading controls . Interestingly protein levels of PfHDAC1 , a target of the HDAC inhibitor , were not altered by the apicidin treatment . Taken together , we observed different types of histone modifications induced by the HDAC inhibitor apicidin . While an increase in H4K8Ac and H4Ac4 was observed in all stages , the two histone 3 modifications exhibited a more complex response to HDAC inhibition . These results indicate the existence of distinct mechanisms that control the global levels of histone modifications and are directly or indirectly associated with HDAC activities . Although the western blot analyzes indicated the effect of apicidin on nucleosomal histone modifications , in our next step we wished to investigate the distribution of these induced modifications along the P . falciparum genome . The main goal was to identify chromosomal regions that are under the regulatory control of Class I and II HDACs . Thus , we carried out chromatin immunoprecipitations in conjunction with microarray analysis ( ChIP-chip ) using antibodies against the four histone modifications H4K8Ac , H4Ac4 , H3K9Ac and H3K4me3 . Using this approach , we compared changes in the chromosomal distribution of these modifications in P . falciparum cells after 1 hour of apicidin treatment ( Figure 3 ) . For the ChIP-chip analysis , we used the P . falciparum long oligonucleotide DNA microarray , utilized for the transcriptional analyzes , which represents 5363 coding sequences and features one microarray oligonucleotide element ( MOE ) per 1198 base pairs on average for all the P . falciparum coding sequences [18] . To ensure the statistical significance of the obtained data , each ChIP-chip assay was conducted in triplicate and the chromosomal regions with apicidin induced modifications were identified using the method Significance Analysis of Microarrays ( SAM ) [30] ( see materials and methods ) . To evaluate significant overlaps between the different histone modifications we utilized several different SAM threshold cutoffs ( Δ ) non of which , however , yielded data with a false discovery rate ( FDR ) greater than 0 . 11% ( supporting information ) . First , we identified 1063 and 1627 genetic loci ( represented by the MOEs ) that showed a significant increase in H4K8Ac in apicidin treated trophozoites and schizonts respectively ( Figure 3A ) . With an overlap of 287 loci , a negative correlation ( P value = 0 . 021 ) between these two groups was found . However , when overlapping genes instead of genetic loci , a most striking positive correlation ( P value <10−55 ) with an overlap of 356 genes was found . This indicates that , although , increased H4K8Ac in apicidin treated trophozoites and schizonts occurred in similar genes , the increase occurred at different loci within the same gene in the two stages . This is consistent with the model that each developmental stage is characterized by its distinct pattern of histone modifications that undergo progressive changes along the IDC [29] , [31] . In addition , in the schizont stage we identified 1216 and 1228 genetic loci that showed a significant increase in H3K9Ac and H4Ac4 , after 1 hour of apicidin treatment , respectively ( Figure 3B ) . Overlapping these two groups of genetic loci ( 372 MOEs ) showed a positive correlation ( P value <10−55 ) between H3K9Ac and H4Ac4 . Moreover , we identified 1197 genetic loci that showed a significant decrease in H3K4me3 in apicidin treated schizonts ( Figure 3C ) . The overlap of 429 MOEs between the two groups of genetic loci associated with decrease of H3K4me3 and increase of H4K8Ac ( schizonts ) showed a positive correlation ( P value = 3 . 88×10−5 ) between these two modifications . Similarly , when overlapping genes instead of genetic loci , positive correlations ( P value <10−55 ) between H3K9Ac and H4Ac4 ( Figure 3B ) as well as H4K8Ac and H3K4me3 ( Figure 3C ) were observed . However , these results are not reflected in the western blot analyzes which showed dramatic changes in the overall levels of H4Ac4/H4K8Ac but no change in H3K9Ac/H3K4me3 in apicidin treated schizonts . This apparent discrepancy can be explained by higher amplitudes of apicidin induced H4Ac4/H4K8Ac compared to H3K9Ac/H3K4me3 . Hence , these data suggests that despite the differences in their overall response to apicidin treatment across the P . falciparum IDC there is some level of association between H4Ac4 and H3K9Ac aswell as H4K8Ac and H3K4me3 in the schizont stage . Beside these two positive correlations , the majority of genetic loci associated with increased H4K8Ac ( both in trophozoites and schizonts ) and decreased H3K4me3 originate from the first 500 base pairs of the gene coding sequences . In contrast , the genomic regions associated with H3K9Ac and H4Ac4 exhibit essentially no positional preference within the individual genes ( Figure 3 , insert graphs ) . Interestingly , genetic loci associated with H4K8Ac and/or H3K4me3 showed a tendency to be mutually exclusive to loci associated with H4Ac4 and/or H3K9Ac ( supporting information ) . In summary , these results suggest that HDAC activities in P . falciparum counteract at least two chromatin-remodeling pathways . One of these is acetylation at H4K8 and de-methylation of H3K4me3 mainly in the 5′ ( possibly promoter ) regions of genes . The other pathway is acetylation at H3K9 and tetra-acetylation of histone 4 ( at lysine residues 5 , 8 12 and 16 ) which are distributed evenly throughout the coding sequences of P . falciparum genes . These two pathways appear to target two largely non-overlapping set of P . falciparum genes . It is also important to note that all four of the apicidin induced histone modifications occurred almost exclusively in the intrachromosomal regions of the P . falciparum genome . This suggests that both Class I and II HDACs act mainly in the intrachromosomal regions and do not overlap with the activity of PfSir2 , the P . falciparum Class III HDAC , which generates heterochromatin at the chromosome ends [12] . To further explore the distribution of the apicidin induced histone modifications along the P . falciparum genes , we carried out RTQ-PCR measurements with the ChIP immunoprecipitated DNA . Here we focused on four genes that were associated with increased H4K8Ac , in trophozoites treated with apicidin ( IC90 ) for 1 hour . These included circumsporozoite protein ( csp: PFC0210c ) , erythrocyte binding antigen 175 ( eba175: MAL7P1 . 176 ) , apical membrane antigen 1 ( ama1: PF11_0344 ) and merozoite surface protein 6 ( msp6: PF10_0346 ) ( Figure 4A ) . In agreement with our ChIP-chip data ( Figure 4A insert boxes ) , significant changes in H4K8Ac were detected within the 5′ extremes of the coding regions of all four genes . In all of the genes , we also detected significant increase in H4K8Ac in the regions upstream to the coding sequence . Overall , we do not detect any consistent bias towards the promoter or the 5′ regions of the coding sequences , which suggests that apicidin induced H4K8Ac spans both of these gene sections evenly . In this study , we compared the apicidin induced H4K8Ac with H4K5Ac whose levels in P . falciparum are not significantly affected by this inhibitor ( Figure 4C and data not shown ) . In addition , we investigated the inverse relationship between H4K8Ac and H3K4me3 that was observed previously in the schizont stage ( Figure 3C ) . In agreement with the ChIP-chip data ( insert box ) , the inverse relationship between these two modifications is preserved in both the 5′ coding regions as well as the upstream non-coding regions of CSP ( Figure 4B ) . Overall , the RTQ-PCR results correlated well with the ChIP-chip data indicating that the observed apicidin induced H4K8 hyperacetylation and H3K4 demethylation occurred in the promoter regions as well as the extreme 5′ regions of P . falciparum genes . In addition to their transcriptional up-regulation , both CSP and EBA-175 proteins were detected in apicidin treated trophozoites ( Figure 4D ) . CSP is N-terminally processed during sporozoite invasion of hepatocytes , resulting in a processed product that is 8–10 kD smaller [32] . Interestingly , we detected two molecular weight forms of CSP: the lower band migrates at the expected molecular weight of the CSP processed product , which suggests the presence of the putative protease in these cells . Increased protein levels of CSP , both processed and unprocessed , were also observed in apicidin treated rings and schizonts ( data not shown ) ; this was consistent with the drug induced gene expression of CSP found in all three stages of the IDC . These data indicate the likelihood that transcripts induced by apicidin are also translated . This suggests a lack of translational “checkpoints” in stage specific protein expression and re-emphasizes the importance of transcriptional control for this process . To investigate the association between genetic loci with altered histone modifications and genes induced/repressed by apicidin we determined the correlation between the RNA expression and ChIP-chip data obtained with apicidin treated schizonts . In particular , we analyzed the overlap between gene sets induced/repressed by apicidin , in each individual time point and gene sets with altered histone modifications ( Tables 1 and 2 ) . For this analysis , we used identical SAM threshold cutoffs as described in Figure 3 . To our surprise , no positive correlation between the studied histone acetylations and apicidin induced gene expression was found . Moreover , in the earlier time-points , both H4K8Ac and H3K9Ac were negatively correlated ( P value <0 . 0424 ) with increased transcription ( Table 1 ) . Conversely , a positive correlation ( P value <0 . 0326 ) between these two histone modifications and gene repression was found ( Table 2 ) . However , these findings are consistent with previous studies that show H4K8Ac , H4K12Ac and H4K16Ac are negatively correlated with increased gene expression in yeast [33] . Since H3K4me3 has been linked with gene expression [28] it was not , surprising that demethylation of H3K4me3 was found to be positively correlated ( P value <0 . 0143 ) with gene repression ( Table 2 ) . However , despite this partial correlation , the majority of the apicidin induced/suppressed genes did not associate with the four studied histone modifications . This discrepancy can be explained by two working hypotheses that are not mutually exclusive . First , additional histone modifications that act independently or in the context of each other ( e . g . histone code ) mediate the apicidin induced transcriptional changes . Given that post-translational modifications on over 60 different histone residues have been detected in eukaryotic systems [4] , it will be intriguing to pursue further studies that analyze their role in regulation of the P . falciparum life cycle . Second , some of the observed transcriptional changes could result from secondary effects where apicidin alters the expression of stage specific transcription factors and other transcription associated or chromatin binding proteins which in turn contributes to the de-regulation of the transcriptional cascade of P . falciparum . These secondary effects can be especially responsible for the transcriptional changes in the later experimental time-points of the 6-hour apicidin time course treatment during which the number of induced/repressed genes increases progressively ( Tables 1 and 2 ) . Recent reports have suggested that the Apicomplexan AP2 ( ApiAP2 ) family of putative transcriptional regulators play a major role in stage specific gene expression during the Plasmodium IDC and possibly other developmental stages . In P . falciparum , the ApiAP2 gene family consists of 26 members each of which shows stage specific gene expression spanning the IDC [34] . In addition , two of the ApiAP2 members ( PF14_0633 and PFF0200c ) were each found to bind specific DNA motifs found in the promoter regions of a number of Plasmodium genes with highly coherent expression patterns [35] . In our study , apicidin affected transcription of a number of ApiAP2 genes with the majority of these being up-regulated in the developmental stage in which they are normally suppressed ( Figure 5A ) . To determine association between genetic loci with altered histone modifications and ApiAP2 genes induced by apicidin we overlapped data from RNA expression analysis and ChIP-chip carried out with apicidin treated schizonts . Within 30 minutes of treatment , 8 ApiAP2 genes were significantly induced ( >2-fold ) and positively correlated ( P value = 0 . 0078 ) with a change in at least one acetylation or methylation in apicidin treated schizonts . Four of these ApiAP2 proteins , PF11_0404 , PF14_0079 , PF13_0097 and PFL1075w are normally only expressed during the ring or trophozoite stages . In addition , 3/8 of these ApiAP2 proteins , PF14 _0271 , PFF1100c and PFD0200c , classified as not expressed during the asexual erythrocytic stage were also up-regulated in other stages ( Figure 5A ) . It is tempting to speculate that these proteins might be responsible for the expression of the exo-erythrocytic genes that were observed in our study ( Figure S2 ) . In addition , we identified 52 genes that carry at least one predicted domain linked with DNA binding and/or transcriptional regulation and whose expression was affected by apicidin in at least one developmental stage ( Figure 5B ) . Interestingly , 45 out of the 52 genes are also associated with at least one of the apicidin induced histone modifications in at least one locus corresponding to an MOE . We were unable to find a statistically significant correlation between their change in RNA expression and association with a specific apicidin induced histone modification . Although , this is likely due to the heterogeneous character of this compiled group of 52 proteins , some of these factors may be involved in the apicidin induced transcriptional changes . Hierarchical clustering of the composite dataset revealed 21 genes that are linked with H4K8Ac in the schizont stage while their expression is altered in both schizont and ring stages ( Figure 5B cluster D ) . Interestingly , this cluster includes PFL1005c , recently identified as the heterochromatin protein 1 ( PfHP1 ) that binds to subtelomeric chromatin and is linked to mutually exclusive expression of the major virulence var gene family [36] , [37] . The sensitivity of pfhp1 gene expression to apicidin and its association with the H8K4Ac and demethylation of H3K4me3 might signal the link between subtelomeric gene silencing and the general epigenetic regulation in P . falciparum . In addition , this cluster contains 9 proteins with transcription factor-like DNA binding domains ( zinc-finger ( zf ) -B-box , zf-CCCH , zf-DHHC , zf-C2H2 , zf-Tim10_DDP , and SNF2 ) , 4 proteins with chromatin binding domains ( High Mobility Group ( HMG ) , structural maintenance of chromosomes ( SMC_N ) , and Hop1p , Rev7p and MAD2 ( HORMA ) ) , and 4 proteins with RNA recognition motif ( RRM ) which were previously implicated in RNA stability , splicing as well as posttranscriptional regulation [38] . Similar functional representation was found in the cluster of genes that are associated with H4K8Ac and over-expressed predominantly in the trophozoite stage ( Figure 5B cluster C ) . Interestingly , one of the HDAC homologues ( PF14_0690 ) is associated with increase in H4K8Ac in apicidin treated trophozoites ( Figure 5B cluster C ) and schizonts ( Figure 5B cluster D ) . This suggests an intriguing possibility that epigenetic regulation of this gene is mediated by the activity of its own protein product . The third cluster contains 23 MOE representing 11 genes which are up-regulated by apicidin in one or more developmental stage and are predominantly associated with H4Ac4/H3K9Ac ( Figure 5B cluster B ) . Finally , three proteins with transcription factor-like DNA binding domains and three proteins linked with structural maintenance of chromosomes were found to be associated with H4Ac4 or H3K9Ac and down-regulated in schizonts but up-regulated in rings ( Figure 5B cluster A ) . In conclusion , we found positive correlation between the apicidin induced histone modifications and gene repression but not with transcriptional activation . However , enrichment of these apicidin induced histone modifications in gene classes associated with transcriptional regulation suggests their role in the observed transcriptional response to the HDAC inhibitor apicidin . Thus , these proteins are suitable candidates as transcriptional regulatory factors during the P . falciparum life cycle .
The HDAC inhibitor apicidin induced profound transcriptional changes in all the stages of the P . falciparum IDC . Apicidin altered the expression of 59 . 8% , 33 . 8% and 51 . 5% of the genome in the ring , trophozoite and schizont stages respectively ( Figure 1 ) . This transcriptional response of P . falciparum to the HDAC inhibitor differs considerably from other eukaryotic organisms . Treatment of yeast cells with TSA was found to parallel the expression profile of the yeast RPD3 deletion strain ( Class I HDAC prototype ) with only a limited number ( 200–300 ) of genes exhibiting altered mRNA levels compared to the wild type strain [39] . Similarly , in cancer cell lines , HDAC inhibitors affect only limited groups of genes that typically include no more than 2–17% of the genome [14] , [40] . The vast majority of genes regulated by HDAC inhibitors in mammalian or yeast cells are involved in regulatory functions including apoptosis , mitosis and cell cycle regulation [39] , [41] . Surprisingly in P . falciparum , we found no bias in gene groups affected by apicidin associated with basic metabolic pathways , cellular pathways or biological processes essential for the parasite's development . Another feature of the apicidin-induced transcriptional response is up-regulation of genes that should be under normal growth conditions only expressed in the following or preceding developmental stage or during the exo-erythrocytic stages ( sporozoites and gametocytes ) . These findings conclude that histone deacetylase activities , that are inhibited by apicidin , play an essential role in regulation of stage specific gene expression in Plasmodium parasites . Apicidin also induced rapid histone hyperacetylation in P . falciparum parasites . The P . falciparum HATs PfGCN5 , involved in acetylation of H3K9 and H3K14 [21] , and PfMYST , implicated in acetylation of H4K5 , K8 , K12 and K16 [42] are ideal candidates to consider for the robust histone hyperacetylation observed by the apicidin treatments . The apicidin-induced histone acetylations and transcriptional changes , observed as soon as 1 hour of treatment , shows the highly dynamic role of HDAC and HAT activities in chromatin remodeling and its profound effect on transcriptional regulation . Work by Vogelauer et al [7] showed that deletion of Class I and II HDACs resulted in HAT induced global histone acetylation , spanning large chromosomal regions containing both intergenic and coding regions . They also showed that , promoter targeted histone modifications occur in a background of global acetylation and deacetylation that exists in a dynamic equilibrium across the genome controlling basal transcription . This was further explored by Katan-Khaykovich and Struhl [8] who showed that reversal of targeted histone deacetylation or acetylation , upon dissociation of a repressor or activator , to the initial state of acetylation was carried out rapidly by the non-targeted global acetylation or deacetylation respectively . Reversal of targeted acetylation , by globally acting HDACs , was found to be 3–5 times more rapid than that of targeted deacetylation . In agreement with this model , our data strongly implies that in P . falciparum inhibition of HDAC activity leads to a dramatic increase in global acetylation of histones and subsequently a general induction of basal transcription . This process then leads to a collapse of the transcriptional cascade of the P . falciparum IDC . Interestingly , only a partial overlap between the genetic loci with altered histone modifications and genes induced/repressed by apicidin was observed . Why did some genes respond transcriptionally to HDAC inhibition while others did not ? Previous studies have shown that in spite of global hyperacetylation of core nucleosomal histones induced by HDAC inhibitors , this does not result in global changes in gene expression [40] . Specific histone modifications recruit binding of non-histone proteins such as transcription factors [43] , [44] , chromatin remodeling proteins [36] , [45] and complexes [43] . Therefore particular combinations of histone modification patterns can dictate specific biological functions such as gene transcription or silencing [46] . Presumably , treatment with HDAC inhibitors would severely alter these histone modification patterns resulting in changes in the biological readout . This emphasizes the intricate and multifaceted processes that control gene expression . In agreement with our ChIP-chip analysis , Lopez-Rubio et al [47] have also showed that H3K4me3 has a broad distribution across the P . falciparum genome and is mainly absent from the heterochromatin loci found at the chromosome ends . An interesting finding of this perturbation study has been the decrease in levels of nucleosomal H3K4me3 ( Figure 3C and 4B ) . This histone modification , in particular , is intriguing since it recruits both activating and repressing effector proteins and complexes such as the yeast SAGA complex , which contains the GCN5 HAT , and the Sin3-HDAC1 deacetylation complex [48] . How the inhibition of HDAC activities results in decreased levels of H3K4me3 is yet unclear . ChIP-chip analysis of the knockout strain for the PfSir2 showed that H3K9me3 levels were reduced and H3K9Ac levels were increased , compared to the wild type strain , in the 5′UTR regions of a subset of up-regulated var and rifin genes [47] . This proposes a link between Sir2-mediated deacetylation and tri-methylation of H3K9 [36] . Similarly , in our findings inhibition of either Class I or II HDAC activities results in reduced H3K4me3 levels on nucleosomes associated with putative promoter sites ( Figure 3C and 4B ) . It will be intriguing to identify any cross-talk between HDACs and tri-methylation of histone 3 lysine 4 . Although the mechanisms of the growth inhibition of Plasmodium cells by HDAC inhibitors need further studies , our data suggest that the general de-regulation of the global transcriptional regulation might be one of its important ( if not the most important ) component . The profound effect of the HDAC inhibitor on P . falciparum growth suggests a high potential of HDAC enzymes as molecular targets for malaria intervention strategies [17] . Given the importance of transcriptional regulation in other Plasmodium developmental processes such as hepatocyte invasion and schizogony [49] and gametocytogenesis [50] , HDAC inhibitors might provide good candidates for chemotherapeutic development for these stages .
The P . falciparum 3D7 clone was cultured and synchronized as described by Bozdech et al [51] . Apicidin Fusarium sp ( Calbiochem ) was prepared as a stock in 100% di-methyl sulfoxide ( DMSO ) . Synchronized P . falciparum cells , at 5% parasitemia and 2% hematocrit , were treated with 70nM apicidin . This concentration of 70nM apicidin represents the median IC90 value determined from the individual IC90 values calculated for each stage of the IDC . The IC90 concentration resulted in 90% reduction of growth ( IC90 ) , compared to matched DMSO ( 0 . 005% ) treated controls , that was monitored by the number of newly formed rings after the completion of the IDC ( data not shown ) . Rings , trophozoites and schizonts were grown in the presence of either DMSO or apicidin for 48 , 32 and 18 hours respectively and then examined by Giemsa staining , Cells were harvested by centrifugation at 1 , 500g for 5 min , washed in phosphate buffered saline ( PBS ) and pelleted at 1 , 500g for 5 min . Cell pellets were rapidly frozen in liquid nitrogen and stored at −80°C . Synchronized P . falciparum cells: rings ( 6–14 hpi ) , trophozoites ( 20–28 hpi ) and schizonts ( 34–42 hpi ) were treated with either DMSO ( 0 . 005% ) or 70nM apicidin as described above . Cells were harvested at 0 . 5 , 1 , 2 , 4 and 6 hours post treatment . Total RNA extraction , amino-allyl cDNA synthesis and DNA microarray was carried out as described previously [51] . cDNA , synthesized from each time point sample , was hybridized against a 3D7 reference pool which was assembled from equal mass of RNA from samples representing 8 hour interval stages throughout the IDC . A P . falciparum genome-wide microarray containing 10166 MOEs representing 5363 coding sequences was used for this study [18] . The raw microarray data included mRNA abundance ratios between each time point sample and the 3D7 reference pool . These data were subjected to linear normalization and filtered as follows: signal intensity > background intensity + 2 × standard deviation of background intensity , recorded for each MOE individually . MOE ratios were averaged for genes with more than one MOE ( Dataset S1 ) . Hierarchical clustering was carried out using log-transformed ratios in Gene Cluster and visualized using Java Treeview [51] . Synchronized ring , trophozoite and schizont stage parasites were treated with either DMSO ( 0 . 005% ) or 70nM apicidin as described above . Cells collected at 1 , 2 , 3 and 4 hours post treatment were lyzed in saponin ( 0 . 15% ) and washed three times with PBS . The isolated parasites were resuspended directly in SDS sample buffer , incubated at 100°C for 10 min and centrifuged at 16 , 000g for 10 min . The supernatant was analyzed by 15% SDS-PAGE and transferred onto nitrocellulose membranes . Antibodies directed against core histone 4 , H3K9Ac , H4K8Ac , H4K5Ac , H4Ac4 and H3K4me3 were obtained from Upstate Biotechnology . Antibodies against core histone 3 and Pfactin 1 were obtained from Abcam ( 1791 ) and Sigma ( A2066 ) respectively . Antibodies against the P . falciparum CSP ( MRA-24 ) and EBA175 ( MRA-2 ) were obtained from the Malaria Research and Reference Reagent Resource Center . The polyclonal PfHDAC1 antiserum was generated by immunizing rabbits with a peptide corresponding to amino acids 421–435 , STTHHLRRKNYDDD , of PfHDAC1 ( PFI1260c ) . The peptide had a N-terminal cysteine added for conjugation purposes ( i-DNA Biotechnology ) . Horseradish peroxidase conjugated secondary antibodies and an enhanced chemiluminescence kit were used according to manufacturer's instructions ( GE Healthcare ) . Synchronized trophozoite and schizont stage parasites were treated with either DMSO ( 0 . 005% ) or 70nM apicidin . After 1 hour of treatment , the cells were lyzed in saponin ( 0 . 15% ) and washed three times with PBS . Chromatin was crosslinked by incubating the isolated parasites with formaldehyde to a final concentration of 0 . 5% for 10 min at room temperature . Immediately after , the parasites were treated with glycine ( 0 . 125M final concentration ) and washed twice with cold PBS . For nuclei isolation , parasites were resuspended in buffer A ( 25mM Tris-HCl pH 7 . 8 , 1mM EDTA , 0 . 25% Nonidet P-40 , protease inhibitor cocktail ( Roche ) , 2mM PMSF ) , incubated on ice for 1 hour and then lyzed by 200 strokes in a pre-chilled homogenizer . The nuclei were pelleted by centrifugation at 2 , 300g for 5 min . In order to obtain DNA fragments in the range of 200 to 1000 bp , the nuclei was resuspended in 200 µl of sonication buffer ( 1% SDS , 10mM EDTA , 50mM Tris , pH 8 . 0 ) , incubated on ice for 15 min and then sonicated 6 times for 10 seconds at 25% amplitude with 1 min intervals between each pulse ( Sonics Vibra Cell ) . After centrifugation at 16 , 000g for 10 min the supernatant was diluted 10-fold in ChIP Dilution Buffer ( Upstate Biotechnology ) . An aliquot of 100 µl was kept as input DNA and the remainder was subjected to chromatin immunoprecipitation as described by Upstate Biotechnology ChIP Assay Kit ( #26225 ) . For each antibody tested a 1∶200 dilution was used . An equal volume of immunoprecipitated DNA from DMSO and apicidin treated samples was amplified using random primers [52] . From each sample equal concentrations of the amplified DNA was co-hybridized against an input pool DNA . This input pool DNA was assembled from equal mass of input DNA from apicidin and DMSO treated samples . A P . falciparum genome-wide microarray representing 5363 coding sequences was used [18] . The raw microarray data included ratios between the immunoprecipitated sample and the input pool DNA . These data were filtered as follows: signal intensity > background intensity + 2 × standard deviation of background intensity , recorded for each MOE individually . To identify MOEs with statistically significant changes in levels of acetylation and methylation the filtered data was analyzed by the method Significance Analysis of Microarrays ( SAM ) [30] . SAM assigned a score to each MOE on the basis of change in acetylation or methylation level relative to the standard deviation of repeated measurements for that MOE . Various SAM threshold cutoffs ( Δ ) were used to generate sets of MOEs showing significant changes for each histone modification ( supporting information ) . For each set the percentage of MOEs identified by chance , the false discovery rate ( FDR ) , was determined as 0 . 11% or less . RTQ-PCR were carried out , with immunoprecipated and input DNA obtained from DMSO and apicidin treated cells , using the Power SYBR Green PCR Master Mix ( Applied Biosystems ) according to manufacturer's instructions . The log2 ratios were calculated by using the ΔΔCt method ( Ct of apicidin-treated immunoprecipitated target gene - Ct of apicidin-treated input target gene ) minus ( Ct of DMSO treated immunoprecipitated target gene-Ct of DMSO treated input target gene ) , where Ct is the threshold cycle . | Plasmodium falciparum , a parasitic protozoan , causes the most lethal form of human malaria , killing more than 2 million people per year . It has a complex life cycle that involves distinct morphological stages accompanied by stage specific gene expression in both the mosquito and human hosts . The lack of a vaccine for malaria and widespread resistance highlights the urgency for new anti-malarial drugs that act on different parasite targets . We show that inhibition of histone deacetylase activities results in activation and repression of transcriptionally regulated genes in multiple stages of the P . falciparum asexual life cycle . We also show that inhibition disrupts the steady-state level of histone acetylation and methylation across the P . falciparum genome . Our data strongly implies that in P . falciparum , inhibition of histone deacetylase activity leads to a dramatic increase in global acetylation of histones and subsequently disruption of stage specific gene expression . This process then leads to a collapse of the transcriptional cascade of P . falciparum . Therefore , the essential role of histone deacetylases in Plasmodium parasites suggests their high potential as molecular targets for malaria intervention strategies . | [
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] | 2010 | Histone Deacetylases Play a Major Role in the Transcriptional Regulation of the Plasmodium falciparum Life Cycle |
Over 100 million women use progesterone therapies worldwide . Despite having immunomodulatory and repair properties , their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated . Administration of exogenous progesterone ( at concentrations that mimic the luteal phase ) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus ( IAV ) infection . Progesterone treatment altered the inflammatory environment of the lungs , but had no effects on viral load . Progesterone treatment promoted faster recovery by increasing TGF-β , IL-6 , IL-22 , numbers of regulatory Th17 cells expressing CD39 , and cellular proliferation , reducing protein leakage into the airway , improving pulmonary function , and upregulating the epidermal growth factor amphiregulin ( AREG ) in the lungs . Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection . Progesterone-treatment of AREG-deficient females could not restore protection , indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection . Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone . Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection , which has important implications for women’s health .
Hormonal contraceptives are listed as an essential medication by the World Health Organization ( WHO ) [1] because of the profound benefits these compounds can have on women’s health outcomes , including decreased rates of maternal mortality and improved perinatal outcomes and child survival , by widening the intervals between pregnancies [2] . Hormonal contraceptive formulations vary , but all contain some form of progesterone ( P4 ) either alone or in combination with estrogen . There are currently over 100 million young adult women on P4-based contraceptives worldwide [3] , with the WHO projecting that over 800 million women will be using contraceptives , including P4-based contraceptives , by 2030 [2] . Despite the staggering numbers of women taking these compounds , very few studies evaluate the impact of contraceptives on responses to infection or vaccination , especially in non-sexually transmitted diseases . Natural P4 , produced by the ovaries during reproductive cycles , or synthetic P4 analogues found in contraceptives , signal primarily through progesterone receptors present on many cells in the body , including immune cells ( e . g . , NK cells , macrophages , dendritic cells ( DCs ) , and T cells ) as well as non-immune cells , such as epithelial cells , endothelial cells , and neuronal cells [4 , 5] . Human , animal , and in vitro studies show that P4 can alter the immune environment and promote homeostasis by decreasing inflammation and inducing anti-inflammatory responses . For example , in the presence of P4 , macrophages and DCs have a lower state of activation , produce higher levels of anti-inflammatory cytokines , such as IL-10 , and produce lower amounts of proinflammatory cytokines , such as IL-1β and TNF-α , as compared with placebo treated cells [6 , 7] . When either mice or cord blood cells from humans are treated with P4 , the percentages of Foxp3+ regulatory T cells ( Tregs ) increase [8 , 9] . Although the immunomodulatory effects of P4-based therapies in the form of contraception have been studied in the context of sexually transmitted infections , including HIV and herpes simplex virus [10–12] , the impact of P4 on the outcome of viral infectious diseases outside of the reproductive tract has not been considered in either humans or animal models . Influenza A viruses ( IAVs ) primarily infect respiratory epithelial cells and induce the production of proinflammatory cytokines and chemokines that recruit immune cells , causing a local proinflammatory environment [13] . Infiltration and activation of CD4+ and CD8+ T cells , while necessary for the clearance of IAVs [13–15] , can trigger inflammation and lead to tissue damage and severe outcomes from IAV infection [16] . Protection requires a balance between inflammatory responses generated to control virus replication and eliminate virus-infected cells with responses that mediate the repair of damaged areas of the lung . Repair involves a complex interplay among many cell types , cytokines , chemokines , growth factors , and extracellular matrix proteins that remodel tissue after acute injury , such as IAV infection [17] . Amphiregulin ( AREG ) is an epidermal growth factor that has emerged as a significant mediator of tissue repair at mucosal sites , including the lungs [18 , 19] , gastrointestinal tract [20 , 21] , and reproductive tract [22 , 23] . Many immune cells produce AREG , but epithelial cells are the principle producer of AREG following inflammation or tissue injury [24] . If P4 can downregulate inflammatory immune responses and promote regulatory or tissue repair responses , then this hormone , at concentrations that reflect the luteal phase of the reproductive cycle , may improve the outcome of IAV infection . Epidemiological and experimental evidence suggest that young adult females suffer a worse outcome than males following IAV infection , which in mice is associated with infection-induced suppression of reproductive hormones and excessive inflammatory immune responses in females [25–27] . In addition to influenza , young adult females suffer a worse outcome than males from several autoimmune diseases , including multiple sclerosis [28] . Paradoxically , a growing body of literature reveals that exogenous treatment of females ( both humans and mice ) with either estrogens or P4 limits inflammation and protects against infectious and autoimmune diseases by decreasing inflammation and promoting repair [25 , 29–31] . In this series of studies , we show that treatment with sustained physiological doses of P4 protects females against IAV by reducing inflammation and improving pulmonary function , primarily through upregulation of AREG in epithelial cells . The observation that P4 regulates the cellular and molecular mediators of tissue repair at a mucosal site outside of the reproductive tract to restore tissue homeostasis after infection or injury has broad implications for women’s health .
To analyze the effects of P4 on morbidity and mortality in female mice , we depleted P4 by removing the ovaries and replaced P4 with subcutaneous pellets that delivered a continuous dose of physiological levels of P4 over the course of 21 days . Mice were subsequently mock-infected or infected with a dose of IAV ( PR8 ) that is uniformly lethal for P4-depleted mice . Circulating levels of P4 and uterine horn mass , a biomarker of circulating P4 levels [32] , were assessed over the course of infection to confirm the continuous effects of hormone replacement . Exogenous replacement of P4 significantly increased and sustained plasma P4 concentrations within the normal physiological range [33] throughout the duration of the study . Both mock- and IAV-infected females treated with exogenous P4 had higher circulating concentrations of P4 , greater uterine horn mass , and higher expression of progesterone receptors ( Prs ) in the lungs than either mock or IAV-infected females treated with placebo throughout the 21 days ( Fig 1A and 1B; P<0 . 05 ) . During the course of IAV infection , treatment of female mice with P4 mitigated the effects of infection on morbidity and mortality ( Fig 1C and 1D; P<0 . 05 ) , with the average day of death being later for females treated with P4 ( 11 . 14±1 . 0 days post-infection [dpi] ) as compared to placebo-treated females ( 9 . 5±0 . 6 dpi ) ( P<0 . 05 ) . Progesterone treatment did not alter virus titers over the course of the first week of infection as compared to placebo treatment ( Fig 1E ) , suggesting that P4 did not render females more resistant to IAV infection . To test whether P4 improved survival during IAV infection by making females more tolerant to the negative consequences of infection on host health , we analyzed the interaction between virus titers and body temperature during peak disease ( 7dpi ) [34] . Females treated with P4 suffered less hypothermia relative to their pulmonary viral load than the placebo-treated females , suggesting that P4 made females more tolerant of IAV infection ( Fig 1F; P<0 . 05 ) . To test the hypothesis that P4 may increase tolerance by reducing inflammation and damage in the lung , pulmonary tissue was evaluated for vasculitis , bronchiolitis , alveolitis , and edema . In mock-infected animals , P4 alone did not result in changes in any of the parameters examined ( Fig 1G [panels 1 and 2] ) . Seven days post-infection with IAV , treatment with P4 decreased vasculitis ( Fig 1G [panels 3 and 4] and 1H ) and edema ( Fig 1G [panels 5 and 6] and 1H ) as compared to the placebo-treated mice ( P<0 . 05 ) . Progesterone improved the outcome of lethal IAV infection by limiting lung inflammation and damage , but not virus replication . Virus-specific CD8+ T cells are necessary for clearance of IAV but can also contribute to immunopathology [35 , 36] . Although the total numbers of CD8+ T cells increased in all females following IAV infection , the total number of CD8+ T cells , the number of IAV-specific CD8+ T cells , and the production of IFN-γ and TNF-α by virus-specific CD8+ T cells in the lungs did not differ between P4- and placebo-treated females ( Table 1 ) . These data indicate that P4 did not affect the cell-mediated antiviral immune response during acute IAV infection . IAV infection is characterized by the induction of a cytokine storm and excessive immunopathology , which leads to tissue damage [37] . Damage to the lung endothelium and/or epithelium results in vascular leakage into the air spaces , and can be quantified by measuring protein concentration in bronchoalveolar lavage ( BAL ) fluid . Consistent with the histopathological findings of increased vasculitis and edema ( Fig 1H ) following lethal IAV infection , treatment of females with P4 decreased the total amount of protein contained in the BAL as compared to placebo-treated mice ( Fig 2A; P<0 . 05 ) . Among infected females , treatment with P4 also increased cellular proliferation ( as measured by Ki67 expression ) in the lungs as compared to placebo treatment during peak disease ( 7dpi ) ( Fig 2B and 2C; P<0 . 05 ) . Analysis of the expression of Ki67 in the different areas of the lungs revealed greater proliferation in several regions of the lungs , but was most pronounced in the epithelial cells lining the airways during IAV infection in P4-treated mice ( Fig 2C ) . The epidermal growth factor , AREG , promotes proliferation of epithelial cells and protects mice from excessive pathology during IAV infection [18 , 19] . Analysis of AREG expression during peak disease ( 7 dpi ) revealed increased mRNA expression , as well as AREG protein in the bronchioles , but not the alveoli , in the lungs of P4-treated mice as compared to placebo-treated mice infected with IAV ( Fig 2D–2F , P<0 . 05 ) . Progesterone treatment altered inflammation during IAV infection ( Fig 1G and 1H ) and induced a repair environment through cellular proliferation and restoration of barrier integrity ( Fig 2A–2C ) . To further characterize the effect of P4 on inflammatory responses to IAV , a panel of 13 cytokines and chemokines was analyzed in the supernatant of whole lung homogenates . As expected , following infection with IAV , pulmonary concentrations of IL-1β , TNF-α , IFN-γ , and IL-12p70 were significantly increased during the first week of infection in all females , regardless of P4 treatment ( S1 Table; P<0 . 05 ) . P4 treatment decreased pulmonary production of the alarmins IL-13 and IL-33 as compared with placebo treatment during IAV infection ( S1 Table; P<0 . 05 ) . The only two cytokines that were significantly increased in P4-treated females compared with placebo-treated females during IAV infection were IL-6 and TGF-β ( Fig 3A and 3B; P<0 . 05 ) . P4 treatment of IAV-infected mice had no effect on the other canonical regulatory protein , IL-10 , as compared to placebo treatment ( S1 Table ) . Production of TGF-β and IL-6 increases differentiation of Th17 cells . Th17 cells promote repair of the gut epithelium [38] and may be similarly involved in orchestrating repair of the pulmonary epithelium . To test this hypothesis , populations of CD4+ T cells from mock- and IAV-infected mice were enumerated during peak disease ( 7 dpi ) . There was no effect of P4 treatment on total numbers of CD4+ T cells , Th1 , Th2 , or Treg cells in the lungs at 7 dpi ( Table 1 ) . In contrast , P4 treatment increased the total number of Th17 cells in the lungs during IAV infection as compared with placebo treatment ( Fig 3C; P<0 . 05 ) . The cytokine IL-23 is necessary for maintenance of Th17 cells and the expression of Il23 mRNA in the lungs was increased in P4- compared with placebo-treated females ( Fig 3D; P<0 . 05 ) . Th17 cells exert their tissue reparative effects by increasing the production of IL-22 [39] . The expression of Il22 mRNA in the lungs was greater in P4- than placebo-treated females during IAV infection ( Fig 3E; P<0 . 05 ) . Finally , one surface marker on Th17 cells that is associated with reducing inflammation ( i . e . , regulatory or suppressive Th17 cells ) is the ectonucleotidase CD39 ( ref . [40 , 41] ) . The percentage of Th17 cells that expressed CD39 was significantly increased in P4-treated as compared to placebo-treated females during IAV infection ( Fig 3F; P<0 . 05 ) . These data indicate that P4 alters the inflammatory milieu of the lungs by promoting a repair environment in IAV-infected female mice , with increased numbers of regulatory Th17 cells , elevated expression of Il22 , and upregulated expression of Areg during lethal IAV infection . To further evaluate the role of P4 in lung repair and recovery from IAV infection , P4- and placebo-treated female mice were infected with a less pathogenic IAV strain , ma2009 , at a dose ( 0 . 4mLD50 ) that allowed for monitoring of the mice over a longer duration of time . Similar to lethal IAV infection , P4-treated females infected with sublethal IAV showed less hypothermia ( Fig 4A; P<0 . 05 ) and reduced clinical disease ( Fig 4B; P<0 . 05 ) as compared to placebo-treated females . Analysis of pulmonary virus titers confirmed that P4 did not alter virus titers or clearance of infectious virus over the course of IAV infection ( Fig 4C ) . To determine if P4 reduced cell death due to IAV infection , LDH levels in the BAL fluid were quantified . Cellular damage during IAV infection was not altered by treatment with P4 as compared with placebo ( Fig 4D ) . Lung sections were evaluated for markers of inflammation and damage during the recovery ( 14 dpi ) and post-recovery ( 25 dpi ) phases of IAV infection . At 14 dpi , but not at 25 dpi , treatment of IAV-infected female mice with P4 decreased the percentage of lesioned areas , alveolitis , edema , and cumulative inflammation as compared to placebo-treated mice ( Fig 4E–4H , P<0 . 05 ) . Treatment with P4 significantly increased Ki67 expression in pulmonary cells during the recovery phase ( 14 dpi ) of IAV infection as compared with placebo treatment ( Fig 4I; P<0 . 05 ) . Based on the observation that P4 treatment promoted lung repair in IAV-infected female mice , we evaluated the impact of P4 on overall lung physiology during ( 14 dpi ) and after ( 25 dpi ) recovery from sublethal IAV infection by assessing markers of pulmonary function . Lung diffusing capacity ( DFCO ) , lung tissue compliance ( Crs ) , and resistance ( Rrs ) returned to baseline faster in P4- than placebo-treated mice infected with IAV ( Fig 4J–4L , P<0 . 05 ) . Treatment of female mice with P4 reduces inflammation and promotes faster recovery from sublethal IAV infection . Progesterone increased pulmonary AREG expression during lethal IAV infection ( Fig 2D–2F ) and increased AREG expression is associated with an improved outcome from lethal IAV infection [18 , 19] . In our sublethal IAV model , we were able to measure pulmonary expression and production of AREG over a longer duration of time to establish the effects of P4 on the kinetics of AREG production in females . P4-treatment induced a 30–70 fold greater induction of Areg mRNA and higher concentrations of AREG protein in the lungs as compared with placebo treatment over the course of IAV infection ( Fig 5A and 5B; P<0 . 05 ) . Peak production of AREG occurred at 9 dpi ( Fig 5B ) , which corresponded with peak disease ( Fig 4A and 4B ) during sublethal IAV infection . To test the hypothesis that reduced AREG production in P4-depleted females caused a more severe outcome from IAV , we treated P4-depleted female mice with recombinant AREG ( rAREG ) during the course of IAV infection . Treatment of P4-depleted mice with rAREG resulted in AREG levels that were comparable to those of P4-treated mice at 14 dpi ( Fig 5C; P<0 . 05 ) . Treatment of P4-depleted females with rAREG significantly improved the recovery from IAV infection ( Fig 5D and 5E; P<0 . 05 ) , with reduced inflammation ( Fig 5F and 5G; P<0 . 05 ) and improved pulmonary function , including lung diffusing capacity ( DFCO ) , lung compliance ( Crs ) , and resistance ( Rrs ) , to levels similar to that of P4-treated females ( Fig 5H–5J; P<0 . 05 ) . These data suggest that the protective effects of P4 on IAV disease may be mediated by an upregulation of AREG . The contribution of AREG to P4-mediated protection from IAV infection was further determined by using mice that lacked the expression of a functional Areg gene [42] . Deletion of the Areg gene in female mice ( Areg-/- ) reversed the protective effects of P4 on the outcome of IAV infection ( Fig 6A and 6B; P<0 . 05 ) . This was accompanied by increased inflammation in P4-treated Areg-/- as compared with WT female mice ( Fig 6C and 6D; P<0 . 05 ) . Improvement of pulmonary function in the presence of P4 , as measured by lung diffusing capacity ( DFCO ) , compliance ( Crs ) , and resistance ( Rrs ) , was also reversed in IAV-infected Areg-/- mice as compared with WT mice treated with P4 ( Fig 6E–6G; P<0 . 05 ) . Taken together , these data indicate that P4 treatment of IAV-infected female mice promotes a pulmonary repair environment and restoration of lung function through the induction of AREG . Treatment with P4 induces higher expression of AREG in the lungs of sublethal IAV-infected females , particularly in the epithelial cells lining the larger airways , as compared with placebo-treatment ( Fig 7A and 7B; P<0 . 05 ) . To assess the contribution of P4 treatment to the repair of damaged respiratory epithelia , we used an in vitro model system in which primary , differentiated mouse tracheal epithelial cell ( mTECs ) cultures were mechanically injured . The mTECs express the progesterone receptor ( Pr ) , which was upregulated in the presence of P4 ( Fig 7C; P<0 . 05 ) . Repair of the epithelial cell layer was measured over time to identify the return of the transepithelial resistance ( TER ) to baseline . Following injury , cultures of mTECs treated with P4 returned to baseline TER faster than vehicle-treated cultures ( Fig 7D; P<0 . 05 ) . During injury , mTEC cultures treated with P4 produced more AREG mRNA and protein than vehicle-treated mTECs cultures ( Fig 7E and 7F; P<0 . 05 ) . These data illustrate that P4 improves pulmonary repair and function by increasing AREG production and wound repair in epithelial cells .
Hosts have evolved several mechanisms for overcoming viral infections , such as the induction of antiviral defenses that increase resistance to infection , or the activation of regulatory and repair responses that increase tolerance to the negative consequences of infection . In the present study , P4 significantly protected females during IAV infection by altering inflammation , improving pulmonary function , and promoting a pulmonary repair environment , which resulted in an earlier recovery . The protective effects of P4 were primarily mediated by the induction of AREG during both lethal and sublethal infections . Progesterone did not increase resistance to infection in females as demonstrated by the lack of an effect of P4 treatment on virus titers , clearance of infectious virus , numbers of Th1 cells , and CD8+ T cell activity in lungs . Instead , P4 reduced the detrimental consequences of IAV infection in females by increasing their tolerance to infection . Several host immunological factors , including TGF-β , Tregs , and regulatory populations of CD39+ Th17 cells , are associated with maintaining the balance between protective and pathological immune responses during IAV infection . Although P4 treatment had no effect on the numbers of Tregs in the lungs during IAV infection , concentrations of TGF-β and IL-6 , the expression of Il23 and Il22 , the number of Th17 cells , as well as the proportion of Th17 cells expressing CD39 , were increased . Regulatory Th17 cells express the ectonucleotidase CD39 and are associated with repair following inflammation and infection [40 , 41] . Th17 cells also promote epithelial cell proliferation and repair in the gut , primarily through the induction of IL-22 [38] . Consequently , treatment of females with P4 increased IL-22 , a cytokine that has been shown to mediate regeneration of lung epithelial cells following IAV infection [43] . Whether the P4-induced increase in regulatory Th17 cells and IL-22 promotes cellular proliferation and repair of the lung epithelium during IAV infection by increasing AREG production requires consideration . Because P4 directly induced AREG production in respiratory epithelial cells in vitro , P4-induced AREG production may occur independent of the reparative effects of regulatory Th17 cells in the lungs during IAV infection . Progesterone induces repair of epithelial cells in the endometrium and myelin fibers in the central nervous system [44 , 45] . This repair of myelin fibers by P4 [46] is one factor mediating how this reproductive hormone mitigates the progression of multiple sclerosis [29] . Our data show that P4 promotes proliferation of pulmonary cells , including epithelial cells , and pulmonary tissue repair . The reparative effects of P4 in the reproductive tract are caused by the induction of AREG , which promotes epithelial remodeling in mammary and uterine tissues [22 , 23] . In the respiratory tract , AREG is involved in pulmonary tissue remodeling and repair during lung injury , asthma , and infection [18 , 19 , 21 , 47 , 48] . Although Areg-gene deficient mice show few abnormalities under homeostatic conditions [42] , their ability to resolve inflammation or infection is severely impaired [20 , 21] . During IAV infection , administration of rAREG protects mice from severe IAV-mediated morbidity by decreasing hypothermia , improving pulmonary function , and decreasing protein leakage into the airways [18 , 19] . The data presented are the first report of P4 induction of AREG outside of the reproductive tract and in the context of infection . The effect of other reproductive hormones on AREG expression , including differential expression between males and females , warrants further study . AREG is produced primarily by epithelial cells [49] , but type 2 innate lymphoid cells ( ILC2 ) and Tregs have also been shown to produce AREG during IAV infection and contribute to the repair during resolution of infection [18 , 19 , 49 , 50] . Because each of these cell type express progesterone receptors [5 , 51] , each is a potential producer of AREG in response to P4 treatment . Our in vivo and in vitro data suggest that respiratory epithelial cells are a predominant source of P4-induced AREG . Following IAV infection , AREG expression was predominantly localized to the bronchiolar epithelial cells , and P4 treatment of isolated mTECs increased AREG production following mechanical damage . Furthermore , P4-treatment did not activate markers of ILC2s , including IL-13 and IL-33 production , or increase numbers of Tregs in the lungs during infection , suggesting that the induction of AREG in response to P4 may not be occurring in these immune cell populations . Recovery following IAV infection is generally defined as a return of body temperature or body mass back to homeostatic levels [52] . In this study , however , we showed that pulmonary pathology and impaired pulmonary function persisted after measures of overall health , including hypothermia and clinical disease , returned to baseline . Furthermore , the impact of IAV infection was observed long after infectious virus had been cleared from the lungs . Recovery following IAV infection extended beyond 21 dpi and should be defined not only by reduced morbidity , but also by restored pulmonary function , both of which were expedited by P4 treatment in females . Progesterone concentrations fluctuate naturally during the female life span , with moderate concentrations during the menstrual cycle , high concentrations during pregnancy , and low concentrations following menopause . Progesterone is also used exogenously by over 100 million women worldwide in P4-based hormonal contraceptives , by post-menopausal women taking hormonal replacement therapy , and by both men and women in the treatment of cancer , osteoporosis , and brain injury [3 , 53] . Prior to this study , the health consequences of P4-based therapies in acute respiratory infection had not been characterized . We have demonstrated that AREG , which is a significant factor that induces tissue repair and recovery from infectious diseases , is regulated by P4 during both lethal and sublethal IAV infection . The data presented provide critical mechanistic information about how P4 and possibly synthetic P4 analogues affect women’s health outside of the reproductive tract . Contraceptives that contain P4 are listed as an essential medication by the WHO , being a safe and effective method for improving health outcomes in women , including those living with HIV [1] . During outbreaks of infectious diseases that harm pregnant women and their fetuses ( e . g . , the current Zika outbreak ) , the WHO recommends increased use of hormonal contraceptives , which according to our data could have additional beneficial consequences on the outcome of other infectious diseases .
All experiments were performed in compliance with the standards outlined in the National Research Council’s Guide to the Care and Use of Laboratory Animals . The animal protocol ( M015H236 ) was reviewed and approved by the Johns Hopkins University Animal Care and Use Committee . All efforts were made to minimize animal suffering . Adult ( 7–8 weeks old ) female C57BL/6 mice were purchased from NCI Frederick . Areg+/- ( C57BL/6 129 Sv ) mice were kindly provided by Dr . Marco Conti ( University of California San Francisco ) and bred to obtain Areg-/- and Areg+/+ female littermates . Mice were housed 5 per microisolator cages under standard BSL-2 housing condition with food and water ad libitum . At 8–12 weeks of age , mice were anesthetized with an intramuscular injection of ketamine ( 80 mg/kg ) and xylazine ( 8 mg/kg ) cocktail and ovaries were removed bilaterally as previously described [25] . All animals were given two weeks to recover prior to infection . Recombinant amphiregulin ( 10μg; R&D ) was administered intraperitoneally every other day using saline as the vehicle . Ovariectomized ( ovx ) mice were assigned to receive subcutaneous implants of placebo ( -P4 ) or 15 mg progesterone ( +P4 ) 21-day release pellets ( Innovative Research of America ) prior to IAV inoculation . Circulating concentrations of P4 were assessed from plasma using ether extraction and radiolabelled immunoassay , with P4 antibody ( MP Biomedicals ) and tracer 3H-P4 ( American Radiolabeled ) . Uterine horns were removed at several time-points upon euthanasia of mice and wet weight was quantified as a bioassay for P4 . Mouse-adapted influenza A viruses , A/Puerto Rico/8/34 ( PR8; H1N1 ) provided by Dr . Maryna Eichelberger at the Food and Drug Administration ( FDA ) and A/California/04/09 ( ma2009; H1N1 ) generated by Dr . Andrew Pekosz from a published sequence [54] , were used in these studies . Mice were anesthetized and inoculated intranasally with 30 μl of DMEM ( mock ) or H1N1 virus ( 1 . 78 50% mouse lethal dose ( MLD50 ) for PR8 and 0 . 4 MLD50 for ma2009 ) . Clinical disease scores for IAV-infected mice were based on four parameters , with one point given for each of the following: dyspnea , piloerection , hunched posture and absence of an escape response . For virus quantification , log10 dilutions of lung homogenates ( starting at 10−1 ) were plated onto a monolayer of MDCK cells in replicates of 6 for 4–6 days . Cells were stained with naphthol blue black ( Sigma Aldrich ) and scored for cytopathic effects . The 50% tissue culture infectious dose ( TCID50 ) was calculated according to the Reed-Muench method . Snap-frozen lung tissue was homogenized in DMEM supplemented with 1% penicillin/streptomycin and 1% L-glutamine ( Invitrogen ) and centrifuged to remove cellular debris . Supernatants were harvested to measure IL-1β , TGF-β , IL-4 , IL-5 , IL-13 , IL-17 , IL-33 , and AREG by ELISA ( R&D Systems and BD Biosciences ) and CCL-2 , IL-12 ( p70 ) , TNF-α , IFN-γ , IL-6 and IL-10 with the mouse inflammation cytometric bead array ( BD Biosciences ) according to the manufacturer’s protocols . Snap-frozen lung tissue or mTECs were homogenized in TRIzol and RNA was purified by chloroform extraction . RNA concentration and purity was measured using a NanoDrop ( ThermoFisher Scientific ) . The RNA concentration in each sample was standardized to 1 μg using RNAse-free water . Reverse transcription was carried out using the iScript cDNA synthesis kit ( Biorad ) according to the manufacturer’s protocol . Pre-designed Il23 ( Mm . PT . 58 . 10594618 . g ) , Il22 ( NM_016971 . 2 ) , Areg ( Mm . PT58 . 31037760 ) , Gapdh ( Mm . PT . 39a . 1 ) and Pr ( Mm . PT . 58 . 10254276 ) PrimeTime Primers were purchased from Integrated DNA Technologies . Semi-quantitative RT-PCR was performed in 96-well optical reaction plates using the SsoFast EvaGreen Supermix ( Biorad ) on the StepOnePlus RT-PCR system ( Applied Biosystems ) . Gene expression was normalized to Gapdh and mock-infected samples or wells with no injury using the ΔΔCt method . Lungs were excised and single-cell suspensions were generated following red blood cell lysis . Total viable cells were determined using a hemocytometer and trypan blue ( Invitrogen ) exclusion and resuspended at 1x106 cells/ml in RPMI 1640 ( Cellgro ) supplemented with 10% FBS ( Fisher Scientific ) and 1% penicillin/streptomycin . For IAV-specific T cells enumeration , cells were cultured for 5h with IAV peptide antigen ( CD8:NP366-374 , or CD4: HA211-255 , NP311-325 , respectively ) ( ProImmune ) in media containing Brefeldin A ( GolgiPlug , BD ) The viability of cells was determined by fixable Live/Dead violet viability dye ( Invitrogen ) and Fc receptors were blocked using anti-CD16/32A . The T cell populations were stained with the following antibodies: PerCP-Cy5 . 5 conjugated anti-CD4 ( RM4-5 ) A , PerCP-Cy5 . 5 conjugated anti-CD8 ( 53–6 . 7 ) A , FITC conjugated anti-CD25 ( 7D4 ) A , PE conjugated DbNP366-374 tetramer ( NIH Tetramer Core Facility ) , FITC conjugated anti-CD4 ( RM4-5 ) B , APC conjugated anti-CD3 ( 17A2B , and PerCP-eFluor 710 conjugated anti-CD39 ( 24DMS1 ) B . Intracellular staining with PE conjugated anti-TNF-α ( MP6-XT22 ) A , FITC conjugated anti-IFN-γ ( XMG1 . 2 ) A , PE conjugated anti-IL-4 ( 11B11 ) A , and PE conjugated anti-IL-17 ( TC11-1810 ) A , was performed following permeabilization and fixation with Cytofix/Cytoperm and Perm/Wash bufferA . Intracellular staining with PE-conjugated Foxp3 ( MF23 ) A was performed following fixation and permeabilization with a Foxp3 staining buffer setA . Data were acquired using a FACS Calibur ( Cellquest Software ) and analyzed using FlowJo ( Tree Star , Inc . ) . Total cell counts were determined by multiplying each live cell population percentage by the total live cell counts acquired prior to staining by trypan blue exclusion counts on a hemocytometer . All reagents were purchased from BD BiosciencesA or eBioscienceB unless stated otherwise . Lungs were inflated , fixed in Z-fix ( Anatech ) , embedded in paraffin , cut into 5μm sections , and mounted on glass slides . Slides were stained with hematoxylin and eosin ( H&E ) and used to evaluate lung inflammation . Histopathological scoring was performed by a single blinded veterinary pathologist on a scale from 0–3 ( 0 , no inflammation; 1 , mild inflammation; 2 , moderate inflammation; and 3 , severe inflammation ) for the following parameters: bronchiolitis , alveolitis , vasculitis , perivasculitis , necrosis , consolidation , and edema [55 , 56] . The sum of these parameters represents the cumulative inflammation score . The percentage of lesioned areas within each tissue section was also evaluated . Histopathological slides were deparafinized with xylene and rehydrated in graded ethanol . Heat-induced antigen retrieval with citrate buffer was performed and slides were blocked with 10% normal serum prior to overnight primary antibody incubation . For Ki67 , rabbit anti-Ki67 ( 1/200; Abcam ) was used , detected with the EXPOSE rabbit specific HRP/DAB detection kit ( Abcam ) , counterstained with Hematoxylin and slides were mounted using Permount ( Fisher ) . For immunofluorescence , anti-AREG ( 1/100; R&D ) and anti-β-tubulin IV ( 1/100; BioGenex ) were used and detected with appropriate secondary antibodies ( 1/400 ) conjugated to AF-555 ( Thermo ) and AF488 ( Molecular probes ) . Slides were then treated against autofluorescence using 0 . 3% Sudan Black B ( Sigma ) in 70% ethanol and mounted using anti-fade medium containing DAPI ( ProLong Gold from Cell Signaling Techonology ) . Images were taken using a Nikon Eclipse E800 ( for H&E and Ki67 stains ) or a Zeiss AxioImager M2 ( for immunofluorescence ) and analyzed using ImageJ ( NIH ) . Mice were euthanized by cervical dislocation and the lungs were lavaged twice with 0 . 5ml of a 0 . 9% saline solution . Bronchoalveolar lavage ( BAL ) fluid was centrifuged at 500g for 10 minutes to remove cells and debris and the supernatant was collected to quantify total protein leakage into the airway using a BCA assay ( Pierce ) . Cell lysis and damage was analyzed from BAL fluid by measuring lactate dehydrogenase leakage using an LDH assay kit ( Sigma ) . Lung Diffusing Capacity ( DFCO ) quantifies the ability of the lung to exchange gas , which is its primary function . Diffusing capacity is simple and quick to measure in humans and mice , and it decreases with nearly all lung pathologies , including viral infections . At the selected time points , a cohort of mice was anesthetized via an IP injection of ketamine–xylazine ( 100 mg/kg–10 mg/kg ) , and then an 18-g stub needle was secured in the trachea . 0 . 8 mL of a gas mixture containing 0 . 3% neon , 0 . 3% CO in room air was quickly injected into the lungs , held for 9 s , then quickly withdrawn . This post breathold sample was then injected into a desktop gas chromatograph ( Inficon , Micro GC model 3000A ) to measure the concentrations of Ne and CO . The DFCO in mice is analogous to the DLCO in humans , and is calculated as 1− ( CO9/COc ) / ( Ne9/Nec ) , where subscripts c and 9 refer to the calibration gas injected and the gas from the 9 s exhaled sample . DFCO is thus a dimensionless variable which varies between 0 and 1 , and is used to detect the loss and recovery of lung function after the viral infections used in this study [57] . Lung mechanics: After the DFCO is measured , the tracheostomy cannula was then connected to a Flexivent system ( Scireq ) . Ventilation was accomplished at a rate of 150 breaths/minute and a tidal volume of 10 ml/kg with a PEEP of 3 cm H2O . A deep inspiration to 30 cmH2O was done , and 1 minute later the respiratory resistance ( Rrs ) and compliance ( Crs ) were measured [58] . Increased resistance reflects increased difficulty in dynamically moving air into the lung and decreased compliance reflects increased difficulty in expanding the lung parenchyma . For mTEC cultures , tracheas were obtained from 7–9 week old C56BL/6 female mice , digested overnight in 0 . 3% pronase , and enriched by depleting fibroblasts as previously described [59 , 60] . The mTECs were cultured at a density of 2 . 22x105 cells/ml on collagen-coated 24-well transwell plates for 7 days ( i . e . , until the cultures reached a transepithelial resistance above 1000 Ω· cm2 ) and apical medium was removed to create an air-liquid interface for 14 days to induce differentiation as described previously [60] . Cells were pre-treated for 24 h with basolateral media containing vehicle ( 100% ethanol ) or 100nM P4 ( Sigma ) , and injured by scratching the cell layer with a 10ul XL pipette tip , or left uninjured , and loose cells were removed by washing with media . Transepithelial cell resistance ( TER ) was measured prior to injury , immediately after , and every 12h for 48 h by adding 100μl of warm TEC basic media to the apical chamber . New media with vehicle or P4 was added every 24h . Every 12h , basolateral media was sampled and analyzed for AREG expression by ELISA ( R&D ) according to the manufacturer’s protocol . Cells were harvested in Trizol every 12h and analyzed by RT-PCR as described above . A power and sample size calculation was used to confirm group sizes for a power of 0 . 8 and contributes to differential sample sizes for some dependent measures . Repeat measures were analyzed with a multivariate analysis of variance ( MANOVA ) followed by planned comparisons . Discrete measures were analyzed with T-tests or two-way ANOVA followed by the Tukey method for pairwise multiple comparisons . Survival was analyzed using a Kaplan Meyer survival curve followed by a log-rank test . Mean differences were considered statistically significant if P<0 . 05 . | Worldwide , the use of hormonal contraceptives is on the rise as a primary intervention for improving women’s health outcomes through reduced maternal mortality and increased childhood survival . There are many hormone contraceptive formulations , all of which contain some form of progesterone . Although the effects of hormone contraceptives and progesterone , specifically , have been evaluated in the context of infections of the reproductive tract , the effects of progesterone at other mucosal sites , including the respiratory tract have not been systematically evaluated . We have made the novel observation that administration of progesterone to female mice depleted of progesterone confers protection against both lethal and sublethal influenza A virus infection . In particular , progesterone reduces pulmonary inflammation , improves lung function , repairs the damaged lung epithelium , and promotes faster recovery following influenza A virus infection . Progesterone causes protection against severe outcome from influenza by inducing production of the epidermal growth factor , amphiregulin , by respiratory epithelial cells . This study provides insight into a novel mechanistic role of progesterone in the lungs and illustrates that sex hormone exposure , including through the use of hormonal contraceptives , has significant health effects beyond the reproductive tract . | [
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] | 2016 | Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females |
Investigation of the Vpu protein of HIV-1 recently uncovered a novel aspect of the mammalian innate response to enveloped viruses: retention of progeny virions on the surface of infected cells by the interferon-induced , transmembrane and GPI-anchored protein BST-2 ( CD317; tetherin ) . BST-2 inhibits diverse families of enveloped viruses , but how it restricts viral release is unclear . Here , immuno-electron microscopic data indicate that BST-2 is positioned to directly retain nascent HIV virions on the plasma membrane of infected cells and is incorporated into virions . Virion-incorporation was confirmed by capture of infectivity using antibody to the ectodomain of BST-2 . Consistent with a direct tethering mechanism , we confirmed that proteolysis releases restricted virions and further show that this removed the ectodomain of BST-2 from the cell surface . Unexpectedly , enzymatic cleavage of GPI anchors did not release restricted virions , weighing against models in which individual BST-2 molecules span the virion and host cell membranes . Although the exact molecular topology of restriction remains unsolved , we suggest that the incorporation of BST-2 into viral envelopes underlies its broad restrictive activity , whereas its relative exclusion from virions and sites of viral assembly by proteins such as HIV-1 Vpu may provide viral antagonism of restriction .
The innate defense against viruses includes specific host cell proteins with intrinsic abilities to restrict viral replication . In some cases these restriction factors have been linked to classic aspects of the innate immune response , such as the antiviral state induced by type I interferons . To replicate in this hostile environment , viruses encode specific antagonists of restriction factors [1] . Several of the so-called accessory proteins of primate immunodeficiency viruses have been recognized as such antagonists . For example , the HIV-1 accessory protein Vpu was long known to enhance the release of progeny virions from infected cells , potentially by antagonizing an intrinsic cellular restriction to virion-release [2] , [3] . The study of this phenomenon led to the discovery of the antiviral activity of a protein with no previously known function , BST-2 ( also known as HM1 . 24 and CD317 ) , now referred to as a “tetherin” based on its ability to retain nascent virions on the surface of infected cells [4]–[6] . BST-2 is an interferon-induced , transmembrane and GPI-anchored protein that restricts the release of a number of enveloped viruses including all retroviruses tested as well as members of the arenavirus ( Lassa ) and filovirus ( Ebola and Marburg ) families [7]–[10] . However , how BST-2 mediates the retention of nascent virions is unclear . Viral antagonists of BST-2 include the HIV-1 Vpu , HIV-2 Env , SIV Nef , Ebola glycoprotein , and KSHV K5 proteins [4] , [5] , [11]–[14] . A common feature of the antagonism of BST-2 by viral gene products is its removal from the cell surface , the presumed site of virion-tethering activity . An unusual membrane topology , localization in cholesterol enriched membrane microdomains , and homo-dimerization may each be key to BST-2's restrictive activity . BST-2 binds the lipid bilayer twice , via both an N-terminal transmembrane domain and a C-terminal GPI anchor [8] . This topology leads to the hypothesis that BST-2 retains virions by directly spanning the lipid bilayers of the virion and host cell . Many enveloped viruses including HIV-1 and Ebola bud from cholesterol-enriched membrane domains in which BST-2 is enriched [15] , [16] . These observations lead to the hypothesis that BST-2 is incorporated into virions as part of the mechanism of restriction . BST-2 forms disulfide-linked dimers [6] . This observation leads to the hypothesis that the molecular topology of restriction includes dimerization between virion- and cell-associated BST-2 . Here , we show that BST-2 is positioned to directly retain virions on the surface of infected cells and is incorporated into virions . We confirm that virions retained on the cell surface can be released by proteolysis , but find that they are not released by cleavage of GPI-anchors with phosphatidyl inositol specific phospholipase C or by disulfide reduction with dithiothreitol . Although these findings leave the precise configuration of the BST-2 molecules that restrict release unsolved , they support a model in which BST-2 incorporates into virions to directly restrict their release from the plasma membrane . This mechanism may be broadly applicable to the inhibition of enveloped viruses by BST-2 .
To test the hypothesis that BST-2 is positioned along the plasma membrane appropriately to directly tether virions , we visualized the location of the molecule using correlative fluorescence and electron microscopy . To accomplish this , we labeled the surface of HeLa cells , which express BST-2 constitutively [5] , with a specific antibody that recognizes the BST-2 ectodomain [17] . This antibody was secondarily labeled with cadmium selenide/zinc sulfide nanocrystals ( QDots ) that are both fluorescent and electron dense; this property allowed cells labeled identically to be observed by either light or electron microscopy [18] . The surfaces of cells labeled for BST-2 were characterized by a punctate staining pattern when visualized by fluorescence microscopy ( Figure 1A and Figures S1 and S2 ) . This pattern has been noted previously using routine fluorophores [9] , [19] . In cells expressing HIV-1 , these puncta appear to contain Gag as well as BST-2 and have been hypothesized to reflect sites of virion-formation; in uninfected cells their identity is unclear . Here , in cultures transfected to express the complete HIV-1 genome including the BST-2 antagonist gene vpu , some cells were characterized by reduced or absent surface staining ( Figures 1B and S1 ) . In particular , multinucleated cells resulting from virally induced cell-cell fusion were strikingly low in surface BST-2 ( Figures 1B and S1 ) , consistent with the previously described reduction in the expression of cell-surface BST-2 induced by Vpu [5] . In contrast , no reduction in surface stain was seen when cells were transfected to express a vpu-negative HIV-1 genome; in this case , multinucleated cells resulting from virally induced cell-cell fusion expressed abundant BST-2 on their surfaces ( Figures 1C and S1 ) . Together , these data indicated that the QDot-based stain for BST-2 revealed the previously noted punctate surface pattern , and it faithfully revealed the removal of BST-2 from the cell surface by Vpu as expressed in the context of the complete viral genome . To determine the distribution of BST-2 at the ultrastructural level and in relation to nascent HIV virions , cells stained in an identical manner to those shown in Figure 1A-C were processed for transmission electron microscopy . In uninfected cells , BST-2 was found in foci along the plasma membrane ( Figures 1D and S2 ) , which likely correspond to the puncta seen using immunofluorescence . Some of these foci were associated with endocytic pits , which appeared either coated or uncoated , whereas other foci were not associated with any apparent structure . In cells expressing the complete HIV-1 genome including vpu , surface labeling was often relatively sparse , even in areas of clustered viral particles ( Figure 2A ) . Such paucity of label is consistent with the reduced surface expression visualized by fluorescence microscopy . These wild type viral particles showed both immature ( crescentic electron density along the perimeter of the budding virion ) , as well as mature morphology ( electron dense cores with occasional conical shape ) . In contrast , in cells expressing vpu-negative virus , BST-2 was readily detectable at the cell surface ( Figure 2B ) . Furthermore , label was intercalated between the plasma membrane and nascent virions as well as between nascent virions found in clusters , most of which had a mature morphology . Occasionally , striking examples of label concentrated at the neck of budding virions in the case of vpu-negative virus were observed ( Figure 2B , inset ) . These electron microscopic data indicated that BST-2 is positioned appropriately to function as a direct tethering factor . To determine whether BST-2 is incorporated into virions , we looked for profiles of budding virions and for profiles of virions distant from the cell surface . Surprisingly , wild type virions were not infrequently labeled for BST-2 ( Figure 2C and E; see Figure S3 for control stain ) . This result is consistent with functional data indicating that Vpu is not a fully effective antagonist of BST-2 [5] , and it is consistent with the virion-capture and immunoblot experiments described below . In rare cases , label for BST-2 was found directly between virions that appeared linked to each other ( Figure 2E ) . In the case of vpu-negative virus , label was particularly evident among and between virions caught at a distance from the plasma membrane ( Figure 2D and F ) . Potential association of such label with the plasma membrane was excluded by electron tomography of thick sections; reconstructed three-dimensional images confirmed the presence of labeled virions that were unambiguously discontinuous with the plasma membrane ( Figure 2G and Video S1 ) . Although substantial variability was observed in the density of label for BST-2 on and between individual virions , visual inspection of 38 images yielded 358 virions with 149 virion-associated Qdots in the case of wild type virus and 327 virions with 302 associated Qdots in the case of vpu-negative virus , indicating a 2 . 3-fold greater association of label with virions in the absence of vpu . These immuno-electron microscopic data indicated that BST-2 is incorporated into virions . The data were also consistent with a model of viral antagonism in which Vpu decreases the density of BST-2 at sites of virion assembly and within virions themselves . To validate the incorporation of BST-2 into virions , we devised a bead-based virion-capture assay using the same antibody as was used above for the morphologic studies . A key feature of this assay is the virologic readout of infectivity , allowing confirmation that BST-2 is incorporated into bona fide infectious virions ( Figure 3A-C ) . Preparations of cell-free virions produced from BST-2-positive HeLa cells were mixed with antibody to the BST-2 ectodomain , or with an isotype-matched control antibody , an antibody to CD44 , or an antibody to CD45 . The virion-antibody complexes were then captured on coated magnetic microbeads and used to infect adherent CD4-positive HeLa indicator cells in an infectious center assay of HIV-1 infectivity ( Figure 3A-C ) . CD44 is incorporated into virions and served as a positive control for the capture [20] . CD45 is excluded from virions produced from hematopoietic cells [21] , but here it serves only as a second specificity control , since CD45 is not known to be expressed on HeLa cells . Strikingly , antibody to BST-2 captured infectious virus from solution , both in the case of wild type and vpu-negative genomes . In contrast , infectious virus ( wild type ) produced from HEK293T cells , which do not express BST-2 constitutively [4] , [5] , was not captured by antibody to BST-2 ( Figure 3D ) . Capture of virions produced from HeLa cells by antibody to BST-2 was confirmed by measurement of viral capsid ( p24 ) antigen by ELISA ( Figure 3E ) . The efficiency of capture as measured by infectivity or p24 ELISA was not significantly affected by Vpu; this suggests either that Vpu does not significantly decrease the incorporation of BST-2 into virions or that both wild type and vpu-negative virions incorporate a threshold amount of BST-2 sufficient for capture . Immuno-capture of three independent sets of wild type and vpu-negative virus preparations confirmed the incorporation of BST-2 into infectious virions of HIV-1 ( Figure 3 and data not shown ) . Immunoblot analysis also supported the conclusion that BST-2 is incorporated into virions and further suggested that Vpu inhibits this ( Figure 4 , in which virions produced from HeLa cells and concentrated by centrifugation were analyzed ) . Remarkably , when normalized by the volume of the original culture supernates ( Figure 4A , left panel ) , preparations of wild type virions contained more BST-2 , as well as more p24 capsid protein , than virions produced by vpu-negative virus . This difference in BST-2 contents in the volume-normalized samples suggests that the signal was derived from virions and not merely cellular debris or exosomes; if the latter were the case , then the volume normalized samples from cultures expressing wild type virus should have contained less BST-2 , due to Vpu-mediated down-regulation . In contrast , when the preparations of virions were normalized by their contents of p24 antigen , BST-2 was essentially only detectable in the absence of Vpu ( Figure 4A and B ) . The apparent association of BST-2 with virions and a relative decrease in the content of BST-2 in virions produced in the presence of Vpu was observed in three independent preparations . These observations were robust to filtration of the virion preparations through 0 . 22 µM pore size membranes , suggesting that the detection of BST-2 was not due to the presence of aggregates of BST-2-containing cellular vesicles and virions ( data not shown ) . Interestingly , the relatively greater phenotype of vpu detected in this assay ( an apparent 40-fold increase in virion output ) as compared to that detected by measurement of p24 in unfractionated culture supernatants by ELISA ( a 5–8-fold increase; see Figures 5 and 6 ) may be due to a reduced fraction of pelletable p24 when virions are produced in the absence of Vpu ( data not shown ) . Notably , virions produced in the absence of Vpu contained , in addition to a triplet of species that migrated with apparent molecular mass in the range of 27–37 kDa ( likely representing heterogeneously glycosylated BST-2 ) , two bands of under 20 kDa in apparent mass . These species are less than the predicted size of unmodified BST-2 ( 20 kDa ) , and their identity is unknown; conceivably , they could represent proteolysis of higher mass forms . Overall , these immunoblot data , like the results of immuno-electron microscopy and immuno-capture , support the conclusion that BST-2 is incorporated into virions . Furthermore , the immunoblot results suggest that Vpu reduces the virion-incorporation of BST-2 . To support further a direct tethering model , we confirmed that proteolysis with subtilisin releases virions retained on the cell surface [Figure 5A , in which the black bars indicate the fraction of the total amount of p24 capsid antigen produced by the culture that was spontaneously released into the medium after transfection with wild type or vpu-negative ( Δvpu ) proviral plasmids; the dark gray bars indicate the fraction of the total that was further eluted from the cells with buffer ( control ) or subtilisin; and the light gray bars indicate the fraction of the cell-associated p24 that was eluted with buffer or subtilisin] [22] . The fractional elution of p24 was greater in the absence of Vpu , consistent with a greater number of virions initially retained at the cell surface . Notably , these quantitative data indicated that the total fraction of p24 releasable from the cells ( adding that released spontaneously to that released by proteolysis with subtilisin; open bars in Figure 5A ) is greater in the case of wild type than Δvpu . The “non-releasable” p24 in the case of cells expressing vpu-negative virus presumably reflects virions that have been endocytosed subsequent to restricted release and are not accessible to proteolysis . We further showed that proteolysis with subtilisin indeed acts on BST-2; it largely removed the BST-2 ectodomain from the cell surface as detected by flow cytometry ( Figure 5B ) , and it degraded the ectodomain in vitro ( Figure 5C ) . These results are consistent with direct tethering mediated by BST-2 , but they do not discriminate among several potential topological models of restriction ( Figure 5 , D-F ) . The preceding data suggest that the incorporation of BST-2 into viral envelopes and a direct tethering mechanism underlie its restrictive activity . However , the topology of the BST-2 molecules that mediate the retention of nascent virions remained unclear . One hypothesis is that virion-associated BST-2 interacts directly with cell surface BST-2 , potentially via disulfide bonds but alternatively via predicted coiled-coil regions in the ectodomain of the protein ( Figure 5D and [4] ) . Alternatively , one end of BST-2 could embed in the lipid bilayer of the cell and the other in that of the virion . Such membrane-spanning models are depicted in Figure 5E and F; notably , BST-2 dimers could span the virion and host membranes in parallel or anti-parallel orientations . Here , release of nascent virions was not obtained by incubation of virus-producing cells with phosphatidyl inositol ( PI ) -specific phospholipase C ( PLC ) , which is expected to cleave the GPI-anchor of BST-2 ( Figure 6A ) . Because BST-2 remains attached to the cell surface by its transmembrane domain after cleavage of its GPI anchor ( data not shown ) , PI-PLC activity was validated using CD55 ( decay accelerating factor ) , which is a typical GPI-anchored protein ( Figure 6D; in which PI-PLC effectively removed CD55 from the cell surface ) . These data weighed against the membrane-spanning parallel dimer model of Figure 5F . Incubation of cells with dithiothreitol ( DTT ) to reduce disulfide bonds also failed to release virions ( Figure 6B ) , weighing against a self-interaction mechanism mediated exclusively by disulfide bonds . Incubation with PI-PLC followed by DTT ( Figure 6C ) also failed to release virions , weighing against an anti-parallel , disulfide linked , membrane-spanning model ( Figure 5E ) . These data do not provide direct support for any specific topology of restriction , but they leave open the possibility that the model shown in Figure 5D is operative via a coiled-coil based interaction between the ectodomains of virion- and cell-associated BST-2 .
The interferon induced , GPI-anchored and transmembrane protein BST-2 restricts the release of enveloped virions from infected cells by an unclear mechanism . Here , the prototypic restricted virus , HIV-1 , and the prototypic viral antagonist protein , Vpu , were used to investigate this mechanism . The data provide key initial support for a model in which BST-2 is a direct tethering factor that is itself incorporated into infectious virions . Recent reports have questioned the incorporation of BST-2 into virions and the co-localization of BST-2 with virion proteins , leaving a direct tethering model of restriction unsupported [23] , [24] . An inability to detect BST-2 in virions by immunoblot may be attributable to insufficient sensitivity of the assay , whereas it is more difficult to explain the reported negative data for co-localization . Here , a combination of morphologic , virologic , and biochemical approaches provided evidence supporting direct tethering and virion-incorporation of BST-2 . Evidence that BST-2 is incorporated into virions was provided by immuno-electron microscopy , immuno-capture of infectious virions , and routine immunoblot . The immuno-electron microscopic data specifically localized BST-2 as adjacent to virions , between virions and the plasma membrane , and in rare instances between virions that were linked to each other . The electron microscopic data also suggested that the punctate distribution of BST-2 seen at the cell surface by fluorescence microscopy is only partly due to the occurrence of the protein in endocytic pits . Many of the foci seen along the plasma membrane were not associated with any apparent structure . Intriguingly , these foci could represent membrane microdomains containing BST-2 , such as cholesterol-enriched lipid rafts , although we cannot exclude that they reflect antibody-induced lateral aggregation of BST-2 within the lipid bilayer . Somewhat surprisingly , immuno-electron microscopy , immuno-capture of infectious virions , and routine immunoblot each indicated that virions produced in the presence of Vpu are not devoid of BST-2 . However , immunoblot , and to a lesser extent electron microscopy , suggested that Vpu decreases the amount of BST-2 in virions . Notably , antibody to the BST-2 ectodomain captured virions produced in the presence or absence of Vpu equally well; this may reflect a threshold amount of virion-associated BST-2 required for immuno-capture that is met by virions produced in either context . Altogether , these data indicate the presence of BST-2 in virions . The data also support a relative but not absolute exclusion of BST-2 from virions by Vpu . One of two topological models has seemed likely to explain restriction mediated directly by BST-2: a membrane spanning model in which BST-2 embeds one end in the cell membrane and the other in the virion membrane , or a self-interaction model in which virion-associated and cell-surface-associated BST-2 molecules interact via their ectodomains . Here , we found no direct support for membrane spanning models; phosphatidyl inositol-specific phospholipase C ( PI-PLC ) , either with or without disulfide reduction , failed to release virions retained on the surface of BST-2-expressing cells . These results weigh against membrane spanning models involving parallel dimers or anti-parallel dimers held together by disulfide bonds . A caveat to this interpretation is that the failure of PI-PLC to release virions could be due to modification of the GPI anchor of BST-2 such that it is resistant to this enzyme , a possibility not excluded by the enzyme activity control herein ( release of CD55 ) . As noted above , PI-PLC did not release BST-2 from the cell surface , consistent with membrane anchoring by the protein's transmembrane domain ( data not shown ) ; consequently , we could not validate the activity of PI-PLC on native BST-2 . Similarly , the failure of reduction with DTT to release virions could be due to an inability of DTT to reach the key disulfide bond ( s ) at physiological temperature , for example , if they are protected within a well-folded structure . Similar attempts to release virus-like-particles of Ebola retained on the cell surface by BST-2 with as much as 500 mM DTT were also ineffective [14] . Recent mutational analyses of cysteine residues within the BST-2 ectodomain suggest that disulfide-mediated dimerization is a correlate of the restriction of HIV-1 release , but not of arenavirus release [25] , [26] . Notwithstanding these potentially conflicting findings , exactly how disulfide mediated dimerization would contribute to restriction , if not by mediating an interaction between virion- and cell-associated BST-2 , is unclear . A direct restriction mechanism that is not disfavored by any of the data herein is an ectodomain self-interaction model such as that shown in Figure 5D , but in which the driving force of tethering is a coiled-coil based interaction . To test this hypothesis , the role of the predicted coiled-coil region in the ectodomain needs to be directly and critically evaluated: are key residues in the predicted structure required for restriction ? Does the putative interaction responsible for restriction involve two , three , four , or more α-helices ? Although each of the models of Figure 5 may be too simplistic , a self-interaction model of restriction is attractive: a single plasma membrane protein , BST-2 , is localized to sites of viral assembly , incorporates into virions , and dimerizes or forms higher order multimers or aggregates to restrict release . This direct tethering , self-interaction model of restriction relies only on the localization of BST-2 to sites of viral budding and on the incorporation of BST-2 into virions . Consequently , it can potentially be generalized to all enveloped viruses that assemble on membrane microdomains that contain BST-2 . Conversely , a model of relief of restriction by removal of BST-2 from the sites of viral assembly and from virions themselves can potentially be generalized to all viral proteins that decrease the expression of BST-2 at the plasma membrane . So far , such proteins include HIV-1 Vpu , HIV-2 Env , SIV Nef , and KSHV K5 [5] , [11]–[13] . Notably , the data herein indicate the presence of BST-2 in infectious HIV-1 virions that are spontaneously released from cells , even when the viral antagonist protein Vpu is expressed . The HeLa cells used for the studies herein express BST-2 endogenously , obviating transient expression methods that may be prone to artifactual over-expression of BST-2 in individual cells . On the other hand , whether wild-type virions produced from primary T cells or produced in vivo contain BST-2 remains to be determined . The observations herein suggest the possibility that virion-associated BST-2 serves functions in addition to tethering nascent virions . In this regard , we note the potential for virion-associated BST-2 to interact with ligands , including itself , on immune effector cells . BST-2 is constitutively expressed , at least in mice , on plasmacytoid dendritic cells ( PDCs ) , as well as on B cells and activated T cells in humans [7] , [27] . Considering the incorporation of BST-2 into virions and the potential for interaction between virion- and cell-associated BST-2 , we speculate that in addition to its direct antiviral activity as a tetherin , BST-2 might flag enveloped viruses for subsequent binding to PDCs and B-cells , which are antigen-presenting cells , and so stimulate the host adaptive immune response . Recently , BST-2 was identified as a ligand for a receptor on PDCs , ILT7 , which transduces a signal for shut-off of interferon production [28] . Based in these findings and the data herein , we also speculate that virion-associated BST-2 might provide negative feedback to the interferon response . These mechanisms would place BST-2 at the interface of innate and adaptive immunity to enveloped viruses . In conclusion , the data herein advance a direct model of restriction in which BST-2 is found both at the sites of viral assembly along the plasma membrane and within budding and nascent virions . While this paper was being finalized , independent evidence for direct restriction of virus release and virion-incorporation of BST-2 was reported [29] . Biochemical data suggested a parallel dimer configuration . Strikingly , an “artificial tetherin” that lacks primary sequence homology with BST-2 , but which contains its key membrane binding and structural features , showed antiviral activity , indicating that no cellular cofactors are likely obligatory for the tethering phenomenon [29] . Directly relevant to the data herein , a mutant BST-2 lacking a GPI-anchor site was incorporated into virions but was unable to restrict virion release . These observations would leave open the role of the GPI-anchor in restriction , if not as one of the two membrane anchors in virion-cell membrane spanning models . On the other hand , this study directly demonstrated a requirement for the coiled-coil ectodomain of BST-2 in restriction and showed that a heterologous , dimeric coiled-coil could provide restrictive activity . These data can be interpreted to support a coiled-coil based self-interaction model of restriction . Alternatively , as proposed by the authors , the coiled-coil structure could be needed for an extended conformation of the ectodomain , which might facilitate spanning of the virion- and cell-membranes [29] . While the molecular topology of the BST-2 molecules that restrict virion release thus remains to be resolved , the augmentation of BST-2 activity and the inhibition of viral antagonists such as Vpu likely represent new approaches to the prevention and treatment of infections due to enveloped viruses . The development of these approaches depends on understanding the regulation of BST-2 during the immune response as well as on deciphering the structural basis of virion tethering and of the action of viral proteins that antagonize BST-2 function .
The proviral plasmid pNL4–3 was obtained from the National Institutes of Health ( NIH ) AIDS Research & Reference Reagent Program and contributed by Malcolm Martin [30] . The pNL4-3 mutant ΔVpu ( vpuDEL-1 ) was provided by Klaus Strebel [31] . The murine monoclonal antibody to BST-2/HM1 . 24/CD317 and the BST-2 ectodomain protein were gifts from Chugai Pharmaceutical Co . , Kanagawa , Japan [17] . For flow cytometry , an IgG2a antibody isotype control , a goat , anti-mouse IgG antibody conjugated to allophycocyanin ( APC ) and a FITC-conjugated antibody to CD55/DAF were obtained from BioLegend ( San Diego , CA ) . For immunofluorescence and immuno-electron microscopy , a goat anti-mouse IgG antibody conjugated to biotin was obtained from Jackson ImmunoResearch ( West Grove , PA ) , and streptavidin-conjugated cadmium selenide/zinc sulfide nanocrystals ( quantum dots; QDot 625 ) were obtained from Invitrogen ( Carlsbad , CA ) . Subtilisin was from Sigma-Aldrich . PI-specific phospholipase-C was from Prozyme , San Leandro , CA or Sigma-Aldrich . The HeLa cells used in this study were clone P4 . R5 , which express both CD4 and CCR5 and were obtained from Ned Landau; these cells are a derivative of clone P4 and were maintained in DMEM plus 10% fetal bovine serum ( FBS ) , penicillin/streptomycin , and puromycin [32] . HEK293T cells were also obtained from Ned Landau and were maintained in EMEM plus 10% FBS and L-glutamine . Cells were transfected using Lipofectamine2000 ( Invitrogen ) according to the manufacturer's instructions . For the microscopic experiments , cells were transfected in MatTek glass bottom dishes , using 0 . 8 µg pNL4-3 or pvpuDel-1 . For production of virions , cells were plated in 10 cm tissue culture dishes and transfected with 16 µg of pNL4-3 or pvpuDel-1 . HeLa P4 . R5 cells were plated on coated MatTek glass bottom dishes and transfected as indicated above . One day after transfection , the cells were fixed using 3% formaldehyde in PBS and stained using the murine monoclonal antibody to BST-2/HM1 . 24/CD317 ( 0 . 05 µg/ml ) , followed by goat anti-mouse-biotin ( 1∶100 ) and streptavidin-QDot 625 ( 1∶100 ) . For fluorescence microscopy , cells were mounted in anti-fade media containing DAPI , and image data were obtained using an Olympus laser scanning confocal microscope . Z-series of two-channel images were colored , merged and projected using Image J . For transmission electron microscopy , parallel samples were re-fixed in 2% glutaraldehyde ( EM Sciences ) in 100 mM sodium cacodylate buffer ( pH 7 . 4 ) for 30 min , post-fixed in 1% osmium tetroxide for 30 min , stained in 2% uranyl acetate in water for 1 h , dehydrated in an ethanol gradient , and embedded in Durcupan ACM ( Fluka ) . Thin sections were stained with Sato's lead . Micrographs were obtained using a JEOL 1200 transmission electron microscope operated at 80kV . For electron tomography , sections of approximately 250 nm thickness were stained with Sato's lead and 2% uranyl acetate . Series of micrographs were collected on a FEI Titan transmission electron microscope at 300 keV while the sample was tilted from −60° to +60° in 2° increments . The micrographs were digitized and aligned using IMOD software [33] . A transform-based back projection software package was then used to create the final alignment and back projection resulting in a three-dimensional volume [34] . Virus was produced from either HeLa P4 . R5 cells or HEK 293 T cells . Cells ( 6×106 ) were plated in 10 cm tissue culture dishes and transfected as described above with 16 µg of either pNL4-3 or pvpuDel-1 . Virus-containing culture supernatants were harvested 48 hours later and clarified by centrifugation at 400×g to remove cellular debris . Clarified culture supernatants were incubated with antibodies as indicated and then complexed to protein G-coated magnetic microbeads ( Miltenyi Biotec , Bergisch Gladbach , Germany ) according to the manufacturer's instructions . Bead-bound virions were captured using Miltenyi magnetic columns , washed , and eluted using DMEM plus 10%FBS and penicillin/streptomycin/puromycin in the same volume as the input supernatant . Viral protein levels in the eluate were determined using p24 capsid ELISA ( Perkin-Elmer ) . Infectious center assays of viral infectivity were performed using HeLaP4 . R5 indicator cells as targets . Infected foci were developed with X-gal , imaged using a CCD camera , and quantified using image analysis software , as described previously [35] . For analysis of surface levels of BST-2 , cells were stained before fixation in phosphate buffered saline ( PBS ) including sodium azide and 2% FBS at 4°C using an indirect method to detect BST-2: the HM1 . 24 murine monoclonal antibody ( 0 . 1 µg/ml ) was followed by a goat anti-mouse IgG conjugated to APC . The gate for BST-2 was set using an antibody isotype control ( IgG2a ) as the primary antibody . For measurement of CD55 , cells were stained before fixation either with a FITC-conjugated isotype control or a FITC-conjugated anti-CD55 . Cells were gated by forward and side-scatter characteristics . Composite data profiles were created using FlowJo software ( Tree Star , Inc . ) . A p24 antigen capture ELISA ( Perkin-Elmer ) was used to determine the concentration of viral capsid protein in culture supernatants that were first clarified by centrifugation at 400 g as well as the concentration of capsid protein in detergent lysates ( 0 . 5% Triton-X-100 in PBS ) of the adherent cells . The percentage of p24 capsid secreted into the culture media was determined as the concentration of p24 antigen in the supernatants divided by the concentration of p24 antigen in the total culture ( supernatant plus cells ) ×100 . Additional experimental details are within the figure legends . | The cellular protein BST-2 prevents newly formed particles of HIV-1 and other enveloped viruses from escaping the infected cell by an unclear mechanism . Here , we show that BST-2 is appropriately positioned to directly retain newly formed HIV-1 virions on the cell surface and is incorporated into infectious virions . We suggest that the incorporation of BST-2 into virions is a key aspect of the protein's broadly restrictive activity against enveloped viruses . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
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"Methods"
] | [
"virology/host",
"antiviral",
"responses",
"biochemistry",
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"immunity"
] | 2010 | Direct Restriction of Virus Release and Incorporation of the Interferon-Induced Protein BST-2 into HIV-1 Particles |
Aedes aegypti is the vector of a wide range of diseases ( e . g . yellow fever , dengue , Chikungunya and Zika ) which impact on over half the world’s population . Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated . Recently it was shown that conidia of M . anisopliae caused stress induced mortality in Ae . aegypti larvae , a different mode of pathogenicity to that normally seen in terrestrial hosts . Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why . In our study , using a range of biochemical , molecular and microscopy methods , the infection process of Metarhizium brunneum ( formerly M . anisopliae ) ARSEF 4556 blastospores was investigated . It appears that the blastospores , unlike conidia , readily adhere to and penetrate mosquito larval cuticle . The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel . The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion , suggests the fungus was detecting host derived cues . Similarly , immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues . The hydrophilic blastospores produce copious mucilage , which probably facilitates adhesion to the host but do not appear to depend on production of Pr1 , a cuticle degrading subtilisin protease , for penetration since protease inhibitors did not significantly alter blastospore virulence . The fact the blastospores have multiple routes of entry ( cuticle and gut ) may explain why this form of the inoculum killed Ae . aegypti larvae in a relatively short time ( 12-24hrs ) , significantly quicker than when larvae were exposed to conidia . This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae . aegypti .
Aedes aegypti is the vector of a wide range of viral diseases ( e . g . yellow fever , dengue , Chikungunya and Zika ) [1–5] . Dengue fever annually affects 284 to 528 million people around the world [6] . The range of this pest appears to be expanding due to global warming [7] . Of major concern is the establishment of Ae . aegypti and Aedes albopictus throughout Europe with the latter now firmly established in Southern Europe [7] . The success of these two species is partly due to their ability to readily adapt to urban environments and the tolerance of the eggs to desiccation [8] . Current control is still heavily dependent upon the use of chemical pesticides , which should be discouraged because of the risks they pose to human health and the environment [9 , 10] . Moreover , mosquitoes are also rapidly developing resistance to chemical insecticides as well as to the biological larvicide Bacillus thuringiensis [11–14] . Much attention is currently being focussed on the use of entomopathogenic fungi ( EPF ) such as Beauveria bassiana and Metarhizium anisopliae for the control of mosquito adults and larvae [15–24] as they are considered to be environmentally friendly and highly versatile [25] . Both aerial conidia and blastospores are highly efficacious in killing mosquito larvae [26–28] . Blastospores differ from conidia in several ways . The former are thin-walled , pleomorphic , hydrophilic spores produced relatively inexpensively due to short fermentation times within 2–3 days in liquid media , whereas conidia are uniform shaped , hydrophobic spores produced within 12–20 days on solid substrates such as rice [28 , 29] . Although aerial conidia have a comparatively longer shelf life , blastospores are normally considered more virulent against susceptible hosts [28–38] . Exactly why blastospores are more aggressive is unclear . Blastospores generally germinate faster than conidia ( 2-8hrs versus 12–24 hrs ) and this attribute is considered to be a virulence determinant [29 , 39] . Slower germination means longer exposure of propagules to deleterious biotic ( e . g . antagonistic microbes ) and abiotic ( e . g . humidity , UV , temperature ) factors that negatively affect propagule viability [40 , 41] . Furthermore , it gives the host more time to mobilise its defences and resist infection [42 , 43] . In the aquatic environment , blastospores of B . bassiana , Tolypocladium cylindrosporum and M . anisopliae were found to be more virulent against mosquito larvae than aerial conidia [27 , 28 , 44] . According to Miranpuri and Khachatourians [28] the primary infection sites of B . bassiana blastospores were the head and the anal region , although the most preferred site for invasion was the larval gut . However , none of these studies provided an explanation as to why the blastospores were more virulent than conidia . Both conidia and blastospores adhere to the surface of terrestrial arthropod hosts and penetrate the cuticle using a combination of enzymes and mechanical force [45 , 46] . Recent studies have shown that the mode of M . anisopliae pathogenesis against Ae . aegypti larvae was radically different from that observed when attacking terrestrial hosts in that the conidia failed to adhere to the cuticle surface and that death was due to stress induced in the insect gut by the spore bound proteases on the surface of ingested conidia [47 , 48] . Furthermore , the ingested conidia did not germinate and colonise the haemocoel but remained confined to the gut lumen [47] . It was postulated that M . anisopliae , which is normally found in the soil , has not evolved to infect aquatic invertebrates , hence the atypical mode of pathogenesis . To date , there has been no detailed study of the infection processes of EPF blastospores . Some fluorescence and ultrastructural studies of EPF blastospores have been conducted but mostly of those infecting terrestrial insects [49 , 50] . The current multidisciplinary study investigates various aspects of Metarhizium brunneum blastospore pathogenesis in Ae . aegypti larvae with the goal of understanding the infection process when compared to that observed in terrestrial hosts . The current study establishes why blastospores were more virulent than conidia and are highly interesting candidates for vector control , given that mortality was observed in hours rather than days . The significance of these findings as regards the use of Metarhizium for mosquito control is discussed .
Significant differences in survival were observed among treatments ( χ2 = 163 . 7 , df = 3 , overall P<0 . 001 , Fig 1 ) . Blastospores of M . brunneum ARSEF 4556 were significantly more virulent against Ae . aegypti larvae than either the wet ( χ2 = 49 . 13 , pairwise P<0 . 001 ) or dry ( χ2 = 55 . 32 , P<0 . 001 ) conidial formulations ( Fig 1 ) . There was no significant difference in survival between the wet and dry conidia ( χ2 = 0 . 568 , P = 0 . 451 , Fig 1 ) . Blastospores caused 100% mortality 2 days post inoculation ( pi ) ( LT50 = 0 . 92 days ) , while wet and dry conidia only caused 100% mortality at 5 days pi with LT50 values of 2 . 52 and 2 . 76 days respectively ( Fig 1 ) . Three lines of evidence are presented which show that proteases are not critical virulence determinants during blastospore infection . Firstly , the level of Pr1 associated with blastospore pellets was significantly lower than for wet ( diff = 41 . 97 [95% c . i . : lower = 17 . 55 , upper = 66 . 38] , P = 0 . 003 ) or dry conidia ( 50 . 49 [26 . 08 , 74 . 90] , P = 0 . 001 ) as shown in Fig 2 . Secondly , conducting assays at increased temperatures did not accelerate larval mortality with blastospores ( χ2 = 1 . 000 , df = 3 , P = 0 . 317 ) , where the respective morality rates from blastospore infection were 97% and 100% at 20°C or 27°C 24 hrs pi ( Fig 3 ) . These values are similar to those observed at 25°C ( Fig 1 ) . In contrast , conidia ( wet/dry ) bound proteolytic activity increased with temperature and corresponding mortality rates of larvae inoculated with dry or wet conidia were significantly higher at 27°C than 20°C as shown in Fig 3 ( Dry: χ2 = 5 . 214 , df = 3 , P = 0 . 022; Wet: χ2 = 6 . 513 , df = 3 , P = 0 . 011 ) . Finally , protease inhibitors did not influence blastospore virulence ( Fig 4 ) whereas they greatly affected conidial virulence [47] . Although mortality appeared to be slightly lower in suspensions containing protease inhibitors 24 hr pi , there were no statistically significant differences in virulence between M . brunneum untreated blastospores when compared to blastospores treated with α2-macroglobulin ( χ2 2 . 778 , df = 6 , P = 0 . 096 ) or chicken egg white inhibitor ( χ2 = 1 . 100 , df = 6 , P = 0 . 294 ) as seen in Fig 4 . No differences in larval survival were observed when using α2-macroglobulin and chicken egg white inhibitors ( Fig 4: χ2 = 0 . 406 , df = 6 , P = 0 . 524 , ) . Furthermore , there were no significant differences in survival between larvae exposed to heat killed blastospores and the untreated control ( Fig 4: χ2 = 1 . 000 , df = 6 , P = 0 . 317 ) . Blastospores of M . brunneum adhered to almost any part of the mosquito larval cuticle ( Fig 5A and 5B ) . There appeared to be no blockage of the mouthparts or siphons ( Fig 5A ) . Blastospores often formed clumps but they were also present as individual propagules ( Fig 5C ) . Low temperature-scanning electron microscopy ( SEM ) showed that the blastospores were often covered with copious mucilage which was present in sheet , reticulate and strand form ( Fig 5C ) . The mucilage appeared to be water insoluble since it was present when larvae were recovered from water . Mucilage strands were extruded at the fungus-cuticle interface and were particularly abundant at blastospore apices ( Fig 5C ) . Mucilage strands were strong as they resisted destruction when samples were plunged in the preparatory pre-cooled nitrogen slush and their structure was not affected by the solvents used during sample preparation for transmission electron microscopy ( TEM ) . In thin sections , mucilage appeared as an amorphous , non-uniform , matrix of fibrils that coated the relatively thin cell wall but also extended beyond the blastospores ( Fig 6A ) . Most blastospores appeared to be turgid , cylindrical cells with a smooth surface ( Fig 5C ) . They grew over the surface of the cuticle but not extensively with little evidence of branching . Blastospores rarely produced appressoria , with penetration pegs being produced sub-apically ( Fig 6B ) . The relatively short , narrow penetration pegs quickly expanded within the cuticle or soon after breaching the cuticle ( Fig 6B ) . The peg retained a relatively thin cell wall which was covered with an amorphous matrix ( Fig 6A ) . There was no obvious clearing around the peg or gross distortion of the cuticle at the penetration site . Both SEM and light microscopy confirmed that blastospores were ingested by the larvae . Numerous blastospores were present in the gut lumen ( Figs 5D and 7 ) . Many cells adhered to the peritrophic membrane , these became swollen and ultimately gave rise to penetration hyphae which penetrated the peritrophic membrane and eventually the midgut epithelium ( Fig 6C ) . The blastospores colonizing the haemocoel after breaching the gut were similar in phenotype to those produced in liquid media ( Fig 7 ) . TEM revealed that each blastospore contained dense cytoplasm , several nuclei and mitochondria but very few vacuoles . Most of the vacuoles were relatively small , containing electron opaque material . This material appeared to be extruded through the cell wall and deposited between the fungus and host cuticle ( Fig 6B ) . The electron opaque material was associated with individual blastospores as well as blastospores in groups at the cuticle surface . Electron opaque material was also observed associated with ingested blastospores in the gut lumen ( Fig 6C ) . The distribution of this material was not extensive or uniform . The cytoplasm retained a dense appearance irrespective of whether the blastospores grew on the host surface , penetrating the cuticle or midgut epithelium or when colonizing the haemocoel . Blastospores appeared to divide by budding but the size of the daughter cells varied before breakage from the mother cell . In some instances the daughter cells appeared as short filaments ( Fig 7 ) . A striking feature of blastospores was the poor staining of the cell wall with calcofluor ( Fig 8 ) . Both incipient and fully formed septa fluoresced intensely . Intense fluorescent spots were observed at the apices of some blastospores and occasionally along the length of the cell ( Fig 8 ) . The pattern of staining was similar whether the blastospores were produced in vitro or in vivo . The plasma membrane stained weakly with filipin , fluorescence being intense at septa and cell apices ( Fig 8 ) . Fluorescent spots were also observed in a number of cells ( Fig 8 ) . Expression of genes directly and indirectly linked with pathogenesis namely Pr1 , Pr2 , Mad1 , Mad2 , Mos1 , Cag8 , and nrr1 was generally higher in blastospores in the aquatic setting ( i . e . in infected larvae or in the presence or absence of the larvae ) than in Tenebrio , the terrestrial positive control host ( Fig 9 ) . Expression of the cuticle degrading enzyme Pr1 was highest in blastospores in the presence of larvae ( i . e . blastospores that were still in suspension ) , which was statistically similar to blastospores in infected but live larvae ( diff = -0 . 198 [95% c . i . : lower = -1 . 837 , upper = 1 . 440] , Tukey’s HSD: P = 0 . 993 ) . Expression was slightly lower in infected dead insects but much lower in blastospores suspended in water in the absence of Aedes larvae ( Fig 9 ) . Expression of the protease Pr2 was highest in dead infected larvae but with similar levels of expression when comparing blastospores in the presence and absence of larvae ( -2 . 320 [-7 . 699 , 3 . 060] , P = 0 . 595 ) . In infected live larvae , Pr2 expression was the lowest among all treatments excluding Tenebrio ( Fig 9 ) . Most notably there was an elevated expression of the adhesion genes Mad1 and Mad2 . Mad 1 expression was highest in M . brunneum 4556 blastospores in the presence of larvae ( Fig 9 ) . However , this was not significantly different to infected live larvae ( -5 . 735 [-12 . 39 , 0 . 924] , P = 0 . 096 ) , dead infected larvae ( -2 . 706 [-10 . 15 , 4 . 739] , P = 0 . 723 ) , and blastospores in absence of larvae ( 4 . 833 [-1 . 826 , 11 . 49] , P = 0 . 181 , Fig 9 ) . Expression of Mad2 was equally high in infected dead larvae and blastospores in presence of live larvae ( 0 . 590 [-3 . 066 , 4 . 245] , P = 0 . 980 ) . Similar expression levels of Mad2 were observed in infected live larvae , blastospores in the absence of larvae and Tenebrio ( Fig 9 ) . Expression of Cag8 and Mos1 was highest in live infected larvae followed by dead infected larvae . Cag8 was higher in blastospores in the presence of live larvae than in their absence . Mos1 was lowest in blastospores whether in the presence or absence of live larvae but similar to blastospores infecting Tenebrio ( Fig 9 ) . Expression of the nitrogen regulator gene nrr1 was high in blastospores in all treatments except for Tenebrio ( Fig 9 ) . The immune defence response of Ae . aegypti larvae was rapid following exposure to M . brunneum AFSEF 4556 blastospores; expression of all five AMPs ( AeDA , AeDB1 , Ada-defD , AeCA2 , Ada-ccg ) was elevated immediately after exposure to the pathogen ( Fig 10 ) . However , at 12 hrs pi only the expression of AeDA , AeDB1 remained high . However by 20 . 5hrs pi , all the AMP genes were down regulated ( Fig 10 ) . The expression level of AeDA , AeDB1 at 20 . 5 pi was significantly different in comparison with the control ( AeDA: -4 . 468 [-6 . 428 , -2 . 508] , P = 0 . 002 ) , ( AeDB1: -5 . 375 [-6 . 803 , -3 . 947] , P < 0 . 001 ) , or 12 hrs pi ( AeDA: -5 . 428 [-7 . 619 , -3 . 236] , P = 0 . 001 ) , ( AeDB1: -5 . 985 [-7 . 581 , -4 . 388] , P < 0 . 001 ) , where the gene expression was down regulated , with no difference between the control and 12hrs pi ( AeDA: 0 . 960 [-1 . 232 , 3 . 151] , P = 0 . 398 ) , ( AeDB1: 0 . 610 [-0 . 987 , 2 . 206] , P = 0 . 481 ) . Expression of Ada-defD , AeCA2 and Ada-ccg was significantly lower than the controls at 12 hrs ( Ada-defD: -9 . 078 [-11 . 79 , -6 . 371] , P < 0 . 001 ) , ( AeCA2: -10 . 67 [-13 . 79 , -7 . 548] , P < 0 . 001 ) , ( Ada-ccg: -10 . 54 [-12 . 59 , -8 . 496] , P < 0 . 001 ) and 20 . 5 hrs pi ( Ada-defD: -8 . 708 [-11 . 13 , -6 . 286] , P < 0 . 001 ) , ( AeCA2: -10 . 215 [-13 . 01 , -7 . 424] , P < 0 . 001 ) , ( Ada-ccg: -10 . 43 [-12 . 26 , -8 . 600] , P < 0 . 001 ) . However , between 12hrs and 20 . 5 hrs pi there was no significant difference ( Ada-defD: 0 . 370 [-2 . 337 , 3 . 078] , P = 0 . 899 ) , ( AeCA2: 0 . 454 [-2 . 667 , 3 . 574] , P = 0 . 887 ) , ( Ada-ccg: 0 . 112 [-1 . 935 , 2 . 159] , P = 0 . 983 ) . Expression of five genes associated with stress management ( HSP70 , HSP83 , GPX , Cyp6Z6 , TPX10 ) showed an unusual disjointed pattern of expression ( Fig 10 ) . Expression of the heat shock genes HSP70 and HSP83 was similar at all three time points ( HSP70: F2 , 5 = 1 . 701 , P = 0 . 273 ) , ( HSP83: F2 , 5 = 0 . 811 , P = 0 . 495 ) . Expression of GPX , Cyp6Z6 , and TPX10 was high at 0hrs pi but was low at 12 hrs pi . All three genes appeared to be up-regulated at 20 . 5hrs pi . The level of GPX at 20 . 5 hr pi was significantly higher than at 12 hrs pi ( diff = 3 . 754 [95% c . i . : lower = 1 . 844 , upper = 5 . 663] , Tukey’s HSD: P = 0 . 003 ) , but it was significantly lower than in non-infected larvae ( -1 . 1858 [-3 . 566 , -0 . 150] , P = 0 . 037 ) . Expression of Cyp6Z6 and TPX 10 was significantly lower at 12 hrs pi than in the controls ( Cyp6Z6: -7 . 178 [-10 . 46 , -3 . 893] , P = 0 . 002 ) , ( TPX 10: -2 . 543 [-4 . 330 , -0 . 757] , P = 0 . 013 ) .
Blastospores of M . brunneum strain ARSEF 4556 were significantly more virulent than conidia in killing Ae . aegypti larvae . This study shows for the first time that blastospores have specific characteristics which explain why they are more aggressive to mosquito larvae than conidia . These attributes include the ability to readily adhere to the cuticle surface , production of copious mucilage and the ability to infect without the differentiation of appressoria or excessive production of the proteases Pr1 and Pr2 . Blastospores infected larvae through integument and gut , retaining the dynamic blastospore form both on the host surface and during invasion of the haemocoel . This study also shows that the host immune and stress management responses rapidly kick in but are inadequate as they fail to prevent infection . One of the most striking features of M . brunneum blastospores is their ability to adhere to the surface of the mosquito larval cuticle whereas conidia of the same fungus are unable to do so [47 , 48] . Blastospores are hydrophilic , they readily suspend in water and have high affinity for hydrophilic surfaces [51] . This study shows for the first time that M . brunneum blastospores produce copious , robust , water insoluble mucilage , which may not only explain why these cells tended to clump but also firmly adhere to the mosquito larval surface in an aquatic environment . Exactly why the blastospores expend resources and energy in mucilage production is unclear . Fungi produce an extracellular matrix for a variety of reasons including adhesion of conidia , hyphae , appressoria and blastospores to host surfaces , protection against harmful UV radiation and creation of environments conducive for cuticle degrading enzyme activity [52] . There appears to be a relationship between mucilage production and hydrophilicity since several EPF species that produce wettable conidia often possess a mucilaginous coat [53] . Expression of the adhesin Mad1 gene was elevated in blastospores exposed to Ae . aegypti larvae . This corroborates the findings of Barelli et al . [54] who found that Mad1 was upregulated in the presence of insect cuticle . Disruption of Mad1 in M . anisopliae delays germination , suppresses blastospore formation , and greatly reduces virulence [55] . Sustained expression of Mad1 may explain why M . brunneum retained the blastospore form during invasion and colonisation of Ae . aegypti larvae . In contrast , Mad2 expression was high in dead infected Ae . aegypti larvae and blastospores in the presence of larvae . Mad2 is associated with adhesion to plant surfaces; disruption of this gene , which is under stress control , blocks adhesion of M . anisopliae to plant surfaces but has no effect on fungal differentiation or virulence [55] . Expression of Mad2 as seen here , suggests that the gene was under starvation stress control as previously reported by Barelli and colleagues [54] . It was not possible to determine the chemical composition of the mucilage but it was clear from the SEM and TEM studies that the mucilage was highly resilient and that it contained feint electron opaque fibrils . Similar material has been reported associated with appressoria of several plant pathogenic fungi [56] . None of the fluorochromes tested stained the mucilage . Calcofluor only weakly stained the cell wall of M . brunneum blastospores suggesting that its composition was substantially different to that of the septa , bud scars and growing points which fluoresced intensely . Presumably the blastospore cell wall lacked β-glucans or that these sugars were masked by mucilage . Several researchers have reported that blastospores growing in vivo possess fewer carbohydrate epitopes than conidia or blastopores incubated in artificial culture media [57 , 58] . β-glucans are powerful elicitors of the insect immune system [59] . Absence or masking of β-glucans may be an adaptation to avoid recognition by the host’s immune defences and may explain why no significant cellular or humoral defence responses were observed in Aedes larvae . Expression of AMPs was elevated immediately after exposure to the inoculum but not later suggesting that water soluble factors were being detected by the host , eliciting an immune response . Another novel finding of this study was the failure of protease inhibitors to prevent blastospores from causing larval mortality even though the Pr1 and Pr2 cuticle degrading protease genes were expressed in blastospores . Pr1 is a virulence determinant of terrestrial arthropod hosts [60] and can cause stress induced mortality in Ae . aegypti larvae following ingestion of viable conidia [47] . It is tempting to speculate that blastospores are therefore more dependent upon mechanical forces or enzymes other than Pr1 to penetrate the larval cuticle and peritrophic membrane . The current observations and the fact that EPF are able to penetrate non-host plant cells and inert substrates , suggest that mechanical force may be more important than previously realised [61 , 62] . The highest level of expression of Cag8 , nrr1 , and Mos1 was detected in infected living larvae , presumably their concerted activity enables M . brunneum blastospores to adapt to the host and cope with disparate stresses encountered particularly at the cuticle surface and gut lumen [63] . Mos1 plays a role in fungal differentiation ( appressoria , hyphal bodies ) and ability to cope with stress ( osmotic , oxidative ) . Inactivation of this gene reduces virulence [55] . Although M . brunneum blastospores failed to readily produce appressoria , Mos1 probably plays a role in the infection process such as maintaining cell turgor and stress management since the level of expression declines in dead infected insects . Cag8 plays a role in the regulation of conidiation , virulence and hydrophobin synthesis [64] . Disruption of this gene can result in the production of irregular shaped blastospores and a decline in virulence [63] . In the current study this gene may be involved in the retention of the blastospores phenotype and contribute to its virulence , hence the elevated activity during infection in the live insects but a decline in dead infected insects . Pr1 and Pr2 are subject to nitrogen derepression by nrr1 . The nrr1 protein binds to GATA sites from the promoter region of the Pr1 gene in M . anisopliae during nitrogen regulation [65] . The highest levels of Pr1 were observed in blastospores exposed to live Aedes larvae or during host colonisation suggesting that the fungus was responding to host cues before and during infection , with the level of expression remaining constant . Interestingly , Pr1 levels were depressed in dead hosts whereas Pr2 levels were highly elevated suggesting that Pr2 may have functions other than cuticle degradation . The high level of expression of all the virulence related genes examined in blastospores incubated with live Ae . aegypti larvae suggests that the blastospores can sense the presence of the insect host even before adhering to the cuticle . Just as the fungus was able to sense the insect before initiating infection , mosquito larvae could also sense the pathogen since AMP genes were elevated 0 hrs pi with only defensin A and B remaining elevated at 12 hrs pi when many insects were highly infected and even dying . In contrast , expression of genes involved with stress management showed an unusual pattern; expression was high at 0 and 22 . 5hrs pi but declined at 12 hrs pi . The only exception was Hsp70 which peaked at 12 hr pi . Investment in stress management was a high priority and remained high even at the point of death . Presumably , this was a better investment of resources than the AMPs since most of these are known to exhibit antibacterial rather than antifungal activity [63] . This study shows that the physiological and morphological adaptations of blastospores enable them to adhere to and infect mosquito larvae via the integument as well as invade through the gut following ingestion . These multiple routes of entry result in Ae . aegypti larvae being killed within hours . Blastospores unlike conidia produce copious mucilage which ensures strong adhesion to the host surface . Furthermore , blastospores are not dependent upon differentiation of appressoria or production of Pr1 for infection . These adaptations may explain why blastospores are more virulent than conidia at least when infecting mosquito larvae . However , there are other attributes which make this form of inoculum ideal for control of Ae . aegypti larvae . They are inexpensive and rapidly produced in liquid media . Their hydrophilic nature means that surfactants are not required . Aerial conidia not only take longer to produce but need surfactants to suspend them in water . Thus , M . brunneum blastospores meet most of the commercial criteria for selecting pest control products i . e . being relatively inexpensive , safe to humans and the environment , easy to use and highly efficacious ( fast acting ) against the target pest . These criteria are particularly important since mosquito control often requires treatment of large areas and most affected countries have limited resources .
Aedes aegypti ( strain AeAe ) eggs , obtained from the London School of Hygiene and Tropical Medicine ( UK ) , were hatched in tap water . Larvae were fed rabbit food ( Burgess ) and fish food ( Tetra pro ) and kept at room temperature ( 25±2°C ) in a 16L:8D photoperiod . Metarhizium brunneum ( Formerly M . anisopliae ) isolate ARSEF 4556 , identified as highly pathogenic to mosquitoes [24 , 47] , was maintained on Sabouraud dextrose agar ( SDA ) . Conidia used in assays had over 95% viability . Conidia were harvested from 15 day old sporulating cultures and suspended in 0 . 03% ( v/v ) aqueous Tween 80 . Blastospores were produced in Adamek’s medium [66] . The medium was inoculated with 107 conidia ml-1 and incubated at 27°C in a rotary shaker at 130 rpm for 3 days . Blastospores were harvested by filtering through 2 layers of lens cleansing tissue ( Whatman No . 105 ) , washed twice with sterile distilled water then centrifuged at 5000 rpm for 10 min ( IEC-Centra-3E ) and the pellet was suspended in sterile distilled water . Conidia and blastospore concentrations were determined using an improved Neubauer haemocytometer . Assays were conducted to compare the virulence of M . brunneum ARSEF 4556 blastospores and conidia against Ae . aegypti ( L3-4 ) larvae . Cohorts of ten larvae ( n = 30 ) per incubated in 280 ml plastic beakers containing 100 ml of distilled water + fungus used at a final concentration of 107 propagules ml-1 . Conidia were applied either as a dry powder dusted over the water surface ( dry conidia ) or as a suspension in 0 . 03% aqueous Tween 80 ( wet conidia ) . Blastospores were applied as a suspension in distilled water . Larval mortality was recorded every 24 hours for 7 days . Dead larvae were transferred to Petri dishes lined with moist filter paper and incubated at 27°C to encourage fungal emergence and sporulation . Controls consisted of either distilled water or 0 . 03% aqueous Tween 80 . Each treatment was replicated 3 times and the complete experiment was repeated three times . Assays were performed at 25 ± 2°C . Since conidial proteases play a key role in mosquito larval pathogenesis [47] , several complementary studies were conducted to determine if blastospore proteases also played a role in pathogenesis . The first study consisted of virulence bioassays as outlined above being conducted at 20°C and 27°C , since proteolytic activity increases with temperature . The assay was repeated 3 times . Another study was conducted using protease inhibitors in 24 well plates ( Nunc , Roskide , Denmark , n = 72 ) with one larva per well . Treatments included larvae exposed to 1 ml of 107 blastospores under the following conditions: ( 1 ) live blastospores , ( 2 ) heat killed blastospores ( autoclaved for 15 min at 121°C ) , ( 3 ) live blastospores incubated with chicken egg white Pr1 protease inhibitor ( 0 . 1 mg/ml ) , ( 4 ) live blastospores incubated with α2-macroglobulin ( 1 μg/ml ) . The latter is an inhibitor of serine , cysteine and metallo-proteases . The inhibitors were purchased from Sigma-Aldrich . Control larvae were exposed to 1ml of distilled water or the inhibitors in distilled water . Mortality was recorded daily over 4 days and the assays repeated 3 times . For more details of the assays see Text S1 in S1 File . Larvae were inoculated with blastospores as described above and examined at 12 and 24 hrs post inoculation ( pi ) . Infected larvae were examined by light microscopy to determine if there were preferential sites for blastospores adhesion and to observe the blastospores in the gut . Larvae were also prepared for examination by transmission electron microscopy ( TEM ) . Full details on the preparation and staining of material for TEM are provided in Text S2 in S1 File . Thick sections of the resin embedded larvae were also examined using a light microscope . Full details on the preparation and staining of the material for light microscopy are provided in Text S3 in S1 File . Larvae ( n = 20 ) were also examined by cryo-scanning electron microscopy ( SEM ) using a Hitachi S4800 field emission microscope equipped with a Quorum PPT2000 cryogenic stage and preparation chamber . Full details on the cryo-SEM are provided in Text S4 in S1 File . In addition to SEM and TEM , certain cell attributes of blastospores were investigated using a range of fluorochromes . Calcofluor White , Filipin , Rhodamine 123 and DAPI ( 4’ , 6-diamidino-2-phenylindole ) were used to visualize β-glucans ( in cell walls , septa , mucilage ) , ergosterol ( in cell membranes ) , mitochondria and nucleic acids , respectively . The fluorochromes were purchased from Sigma . Stained cells were examined in a Zeiss AxioCam MR3 fluorescence microscope . Details on the preparation , staining and visualization of the cell components are provided in Text S5 in S1 File . Mosquito larval survival was investigated in three treatment groups; 1 ) conidia , 2 ) blastospores and 3 ) blastospores incubated in protease inhibitors . Each experimental treatment was replicated three times . The whole study was repeated three times . Cumulative survival was quantified using Kaplan-Meier plots . Pairwise comparison were made between; 1 ) conidia vs blastospores and 2 ) blastospores vs blastospores with inhibitor using log-rank tests . Pr1 activity and molecular data sets were analysed using one-way Analysis of Variance ( ANOVA ) with Tukey´s HSD post-hoc test to assess pairwise comparisons . Prior to analysis , gene expression data was logarithm transformed , conforming to ANOVA assumption of homogeneity of variance [68] . All statistical analyses were carried out using SPSS v22 . 0 [69] , Rstudio Version 0 . 99 . 482 [70]and GraphPad Prism v5 . 0 ( GraphPad Software , USA ) . Ae . aegypti genes; Ribosomal S7 ( AAEL009496 ) , Ribosomal protein 49/L32 ( AAEL003396 ) , Defensin A ( AAEL003841 ) , Defensin B ( AF156090 . 2 ) , Defensin D ( AAEL003857 ) , Cecropin A ( AAEL000627 ) , Cecropin G ( AAEL015515 ) , heat shock protein 70 ( AAEL016995 ) , heat shock protein 83 ( AAEL011704 ) , Thiol peroxidase ( AAEL004112 ) , Cytochrome P450 ( AAEL009123 ) , and Glutathione peroxidase ( AAEL008397 ) M . brunneum; PR1a ( MBR_01491 ) , PR2 ( MBR_06579 ) , MAD1 ( MBR_08250 ) , MAD2 ( DQ338438 . 1 ) , MOS1 ( MBR_07375 ) , Cag 8 ( MBR_00569 ) , Nrr1 ( MBR_08301 ) , 18s ( DQ288247 . 1 ) , Translation elongation factor ( MBR_08275 ) | Mosquitoes transmit a range of diseases which have a profound impact on human health . Aedes aegypti vectors dengue , one of the fastest emerging diseases and , more recently , the Zika virus , which has been linked to thousands of birth defects over the last two years in Brazil . Insect pathogenic fungi such as Metarhizium brunneum are effective in killing mosquito adults and larvae . They exhibit much plasticity , producing aerial conidia on solid substrates and blastospores in liquid media . We not only show that blastospores are more virulent than conidia but present evidence explaining why they are more aggressive . The blastospore mode of pathogenesis differs from that of conidia in several ways . Firstly , blastospores appear to be more dependent on entry using mechanical force than by secretion of cuticle degrading proteases such as Pr1 . Blastospores produce copious mucilage which ensures that many spores attach to the cuticle . They are also readily ingested and able to penetrate the gut wall rapidly and colonize the haemocoel . Multiple entry points and gross damage to the cuticle and gut results in rapid larval death . Conidia neither adhere to the cuticle nor germinate in the gut but cause Pr1 stress induced mortality , which takes a slightly longer time . Blastopores , therefore , have greater potential for the control of Ae . aegypti larvae in mosquito control programmes | [
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] | 2016 | Metarhizium brunneum Blastospore Pathogenesis in Aedes aegypti Larvae: Attack on Several Fronts Accelerates Mortality |
In recent years , there have been many computational simulations of spontaneous neural dynamics . Here , we describe a simple model of spontaneous neural dynamics that controls an agent moving in a simple virtual environment . These dynamics generate interesting brain-environment feedback interactions that rapidly destabilize neural and behavioral dynamics demonstrating the need for homeostatic mechanisms . We investigate roles for homeostatic plasticity both locally ( local inhibition adjusting to balance excitatory input ) as well as more globally ( regional “task negative” activity that compensates for “task positive” , sensory input in another region ) balancing neural activity and leading to more stable behavior ( trajectories through the environment ) . Our results suggest complementary functional roles for both local and macroscale mechanisms in maintaining neural and behavioral dynamics and a novel functional role for macroscopic “task-negative” patterns of activity ( e . g . , the default mode network ) .
In recent years , empirical and theoretical work indicates that homeostatic mechanisms play an important role in the regulation of neural activity . At the microscopic level , the balance of local excitation and inhibition ( E/I ) has important computational properties [1–2] . The balance of E/I can be maintained in such circuits using relatively simple local homeostatic rules based on inhibitory plasticity ( e . g . , [3–6] ) . At a completely different scale , evidence from functional MRI suggests the balance of activity across brain regions may be an important organizing principle in macroscopic neural dynamics; networks of task associated regions typically show increased activity matched by relative de-activation of other ‘task negative’ macroscopic networks ( e . g . , [7–9] ) . The default mode network ( the classic task negative network ) is spatially situated between different networks often activated during externally focused tasks [10]; in our previous work , we suggested that at the whole brain level ‘task negative’ network activity may act to counterbalance task activation in other brain regions , forming a ‘network balance’ analogue of the local computational motifs driven by inhibition seen at smaller scales [11–14] . Computational simulations have suggested the importance of homeostatic mechanisms in regulating neural dynamics; facilitating complex patterns of neural activity ( e . g . , [15–16] ) . However , such computational models typically simulate the brain at rest or under highly constrained task settings . In the present work , we explore the regulatory role of homeostatic mechanisms underlying simple behavior . We adapt an established simulation of basic neural dynamics—the Greenberg-Hastings model [17] , incorporating information about human structural connectivity [18] ( Fig 1A ) . At rest ( i . e . , with no environmental embodiment ) , this model has been shown to approximate empirical functional connectivity patterns observed macroscopically [19] . In the model at rest , dynamics arise from low-probability random firing which propagates through recurrent connections . The large-scale dynamics of the model can be controlled by a single local inhibition parameter at each node , which regulates the propagation of incoming excitatory activity to connected nodes in the network . To explore the interaction between brain and environment , we adapt this simple model by ‘embodying’ it into a simple virtual environment . We begin by defining an agent that can move within a 2-dimensional plane , bounded by surrounding walls ( see Fig 1C ) . Movement of the agent within the virtual environment was determined by activity within two pre-defined “motor” nodes within the computational model . Sensory input to the model was defined by direct manipulation of a group of task-positive nodes ( TP ) within the model to ‘activate’ in response to “sensory” input defined using a collection of virtual sensors embedded within the agent . Two pairs of bilateral nodes reacted to “visual” input from the environment to the model and one pair of “somatosensory” nodes activated if the agent collided with the bounding walls of the environment . This set-up leads to brain/environment interactions as follows ( Fig 2 ) : This form of closed-loop interaction introduces non-stationary dynamics; in addition , it can lead to ‘traps’ where the agent moves into an area with large amounts of sensory input or no sensory input and then cannot leave that area . For example , in an area with no sensory input , activity in the model can become pathologically low , and the agent stops movement . Equally , in an area with large amounts of sensory input activity , there can be too much activity , which can lead to pathological motion ( e . g . , running forward into a wall forever ) . These types of brain-environment interaction potentially present a challenge for computational modelling approaches which focus on the emergence of spontaneous , rich dynamics only at rest . These models often rely on careful parameterization to remain in a specific dynamic regime , and so typically are investigated in static situations ( i . e . , where the input to the model is stationary , such as Gaussian noise ) . In such models , changes to the model input typically lead to destabilization of the dynamics ( i . e . , a shift to either random , saturated , or absent patterns of activity ) . To explore the effect of homeostatic control on the stabilization of neural dynamics in this system , we considered two putative mechanisms of dynamic homeostasis: We explore the extent to which these two balancing systems may perform complementary roles in maintaining flexible dynamics in the case of closed-loop brain-environment interactions . We assess the agent’s neural dynamics and trajectory through the environment , and demonstrate that these balancing mechanisms allow the agent to escape constrained environment-brain feedback loops and more completely traverse the environment .
In the absence of any homeostatic mechanisms , we explored the extent to which the dynamics of the computational model relate to the motor output of the agent ( Fig 3 ) . We started by exploring two key control parameters of the model—the threshold for incoming activity to a node to propagate ( Threshold ) , and the strength of coupling between each region ( Coupling ) . We explored the extent to which both simulated activity and movement of the agent are constrained by these factors . In the case of low coupling , the model remains in a state of low activity ( Fig 3A ) . As coupling increases the model rapidly transitions through a phase transition to a ‘high activity’ mode . In all cases , activity is either pathologically low or high , or unstable because of the interaction with the environment . As expected , in the low activity mode ( Yellow marker ) , there is very little average activity , and consequently very little “motor” activity; as such the agent remains relatively stationary over time ( Fig 3B and 3C ) . In the high activity phase ( Blue Marker ) , activity levels in the model are consistently high , involving repetitive cycling on-off activity patterns and stereotyped trajectories ( e . g . , running in a circle ) . At the phase transition ( Green maker ) , activity levels start relatively low , consistent with rich activity dynamics as reported in many neural simulations at rest ( see also S1A/S1B Fig ) ; these rich dynamics result in a relatively rich behavioral trajectory . However , when the agent reaches the boundary wall of the environment where the model receives increased ‘sensory’ input; this leads to increased activity to the model , destabilizing the dynamics and resulting in increased movement and the agent becomes ‘stuck’ ( C ) . This suggests that a model that is tuned to show rich activity dynamics can demonstrate exploratory behavioral dynamics , but that these become destabilized by external feedback into the system in the absence of other stabilizing mechanisms . To explore how rich behavioral dynamics may emerge from our computational model as a function of local homeostatic mechanisms , we started by exploring the parameter space of key variables controlling local plasticity ( see Materials and methods ) . Using the parameters described above at the optimal regime for the non-homeostatic model ( although similar results obtain in other parts of the parameter space ) we systematically varied the target rate ( i . e . , the target for tuning local inhibition ) and the learning rate . Consistent with previous results [21] , we observed that over time the threshold weights ( i . e . , the level of local inhibition ) adapt so that time-averaged excitatory activation approximates the pre-specified target activity . In Fig 4 , we illustrate four different examples of dynamic regimes , exploring both target and learning rate . We note that where the model has a low target rate , and a high rate of learning ( Fig 4 , blue marker ) , the model is both able to attain the target value over time , but also in contrast to elsewhere in the parameter space , shows rich activity dynamics ( S2 Fig ) , with the best–fit to a power-law scaling in activity cascades ( See Materials and methods ) . The model also displays non-zero but relatively weak positive correlations between the node activity time-courses which are also consistent with rich asynchronous dynamics ( Fig 4B ) . To understand how the local homeostatic plasticity stabilizes activity and how this relates to movement dynamics , we selected parameters for the local homeostatic model in a low-target activity , high-learning rate regime ( Fig 4 , Blue Marker ) , and contrasted the non-homeostatic and local homeostatic models ( Fig 5 ) . Over time the local homeostatic model moves into a regime with generally higher levels of movement ( i . e . , left/right rotation and/or forward motion ) ( Fig 4C ) , although there is considerable variability ( i . e . , the mean level of movement and activity varies considerably over time ) . Example trajectories for both the static and local-feedback model ( over 2000 epochs ) are presented in ( Fig 5 ) . The entropy of the movement dynamics ( measured using the entropy of the movement and turn motor signals generated by the model ) was significantly higher in the local homeostatic model compared to the static model , ( t58 = 2 . 68 p<0 . 05 ) . Moreover , the fractal dimension of the movement ( i . e . , the fractal dimension of the image resulting from the trajectory ) was significantly increased in the local plasticity model compared to the static model ( t58 = 11 . 76 , p<0 . 001 ) —See Also S3 Fig . Non-stationary dynamics in both the simulated neural dynamics and the behavior of the agent reflect a feedback loop arising from how the agent interacts with the environment . The level of sensory input: This brain-environment interaction can be observed by the significant anti-correlation between distance from the wall , and activity of the static model ( t26 = -3 . 93 , p< 0 . 04 ) . We can also observe how the local-homeostatic mechanism allows the model to escape from brain-environment feedback loops , by increasing or decreasing local inhibition to better approximate the target activity rate . There was a significant decrease in this anti-correlation within the local homeostatic model compared to the static model ( t58 = -2 . 78 , p<0 . 01 ) . We see that for the local homeostatic model , there was a significant anti-correlation between the mean threshold and both the distance from the wall ( t58 = -9 . 63 , p<0 . 001 ) and mean threshold and mean activity ( t58 = -12 . 76 , p<0 . 001 ) . This shows how the coupling between activity and distance from the wall was decreased in the local homeostatic model compared to the static model , as the model retunes the local thresholds to compensate for increased activity ( driven by sensory input ) and re-establish richer movement dynamics . We observed that the local-homeostatic mechanism compensates for the brain-environment feedback , by constantly readjusting weights to compensate for the non-stationary environment . By adding a complementary balancing mechanism that aims to keep activity levels approximately constant across regions with the local homeostatic mechanism ( aiming to balance activity across time ) , a more stable solution can be arrived at . We compared the local-homeostatic , macroscopic and the combined ( local and macroscopic homeostatic ) models ( see Fig 1 ) on a range of measures assessing the model’s activity and movement dynamics ( Fig 6 ) . We noted that there was a significant difference in mean activity across the network in the combined local and macroscopic model , compared to the local-homeostatic model ( t58 = -2 . 89 p< 0 . 01 ) ; the model with macroscopic balance alone , was not significantly different in terms of mean from the non-plastic model ( t58 = -0 . 49 p = 0 . 63 ) . In addition , variability of the model measured using the mean standard deviation of activity across network nodes ( t58 = -3 . 07p< 0 . 01 ) was significantly decreased in the combined model . This suggests a small decrease in mean activity in the macroscopically balanced model compared to the local-homeostasis model , with activity significantly closer to the homeostatic target function ( t58 = -2 . 87 , p<0 . 01 ) for the combined model ( Fig 6C ) . More importantly , in the combined model , threshold weight changes are significantly less variable than for the local homeostatic model when considering both the standard deviation and the coefficient of variation of the mean threshold over time ( t58 = -6 . 94 , p<0 . 001 ) and ( t58 = -2 . 60 , p<0 . 05 ) respectively ( Fig 6B ) . These results suggest that the combined model arrived at a more stable behavioral interaction than local–homeostasis alone , requiring less local weight change in response to persistently elevated or reduced activity . In addition , the relationship between the distance from the wall and mean activity is significantly less for the macroscopically-balanced model ( t58 = 2 . 46 , p<0 . 05 ) than the local homeostatic model alone—suggesting the feedback loop between brain/environment is less influential . The more stable activity and weight change of the combined model manifests itself in a richer behavior , and more complete exploration of the environment . When observing the movement of the agent we see that ( Fig 5A ) , the path of the agent has a higher fractal dimension ( t58 = 2 . 2 , p<0 . 05 ) for the combined model and significantly higher entropy for the plotted trajectories ( Fig 6A/6B ) . The fractal dimension for the macroscopic balance alone model however , was significantly higher than the non-plastic model ( t58 = -2 . 35 , p<0 . 05 ) . These results suggest that the agent has a more complex pattern of activity , that emerges from a multi-scale balancing system and covers more of the environment because behavior is less determined by brain-environment interactions alone ( S3 Fig ) .
This model is unequivocally not intended to be a detailed model of all aspects of real embodied cognition or of actual neural and sensorimotor systems; instead , in both regards , it is highly simplified . We acknowledge that there have been many arbitrary design choices , and do not intend this to be a definite presentation of how to model brain/environment/behavior interactions . Such interactions are likely to be far more complex , possibly non-stationary , and will depend on the complexity both of the neural system , but also the complexity of the motor and sensory systems . Instead , the example we present here is a useful toy example; the simplification allows us to consider the interactions between macroscopic brain networks [23] , neural dynamics and the environment to better understand possible functional roles of homeostatic systems in the brain . The presence of a local homeostatic plasticity mechanism that tunes the threshold at each node to balance excitatory input from connected nodes ensures that the agent does not stay trapped in either state for long . As the threshold for activity ( varying depending on the local homeostatic mechanism ) at individual nodes increases ( in the high activity state ) or decreases ( in the low activity state ) , the average activity level adapts to the target level . This results in the agent escaping the ‘trap’ , with resulting activity levels closer to the target level and , consequently , more stable simulated neural and movement dynamics . The model without local homeostasis is unable to cope with the sensory/motor feedback system . Local thresholds can be chosen to allow interesting dynamics ( i . e . , variable movements/neural activity ) ; however , these must be chosen to either allow rich dynamics in the presence of sensory input ( i . e . , with higher local inhibition ) or dynamics in the absence of sensory input ( i . e . , with lower local inhibition ) . Therefore , over time the agent will tend to either: a ) remain approximately stationary in a low-sensory area with local thresholds too great to allow much exploration ( i . e . , near stationary ) or b ) initially move freely , but rapidly become trapped in a high-sensory area ( e . g . , a corner or wall ) . The model suggests that modeling spontaneous dynamics at rest ( e . g . , [24–26] ) or with a simple task such as encoding a sensory stimulus ( e . g . , [15–16] ) is different to modeling sensori-motor interactions with an environment; further , the existence of closed-loop feedback made the roles of homeostatic mechanisms more important and obvious . In our case , we observed that without the local homeostatic plasticity , the agent in the environment would become trapped in either a stationary state ( with high levels of local inhibition ) or would be in a permanent state of motion ( with too little local inhibition ) . Instead , we observe that plasticity is a constant feature of the system . Initially , there are large changes in local thresholds across time points , as the model approximately balances average incoming excitation at each node . As time progresses , however , the weight changes become smaller , but never drop completely to zero . While the model with local homeostasis can compensate for this sensory-motor interaction , the addition of an explicit macroscopic balancing system across space , alongside the local homeostatic learning rule ( that balances activity across time ) , further facilitates stable simulated neural dynamics and behavioral trajectories through the environment . This occurs because the macroscopic system balances alterations in external input to the model so that the number of activated units ( sensory nodes or task negative nodes ) remains constant irrespective of interactions with the environment . The simple system we implemented , modeled on patterns of task negative deactivation from the fMRI/PET literature ( e . g . , [11] ) counteracted the destabilizing effects from the changing amount of sensory input that the model receives in different locations in the environment . Without the macroscopic system , the overall level of activity within the model is more dependent on the level of sensory input ( i . e . , “touching” and “seeing” the wall ) . This makes the task of the local homeostatic plasticity mechanism harder , since exogenous input to the system varies considerably . Instead , the task negative system simply balances the level of exogenous activity to a constant amount , such that task negative input decreases as sensory input increases and vice versa . This means that the environment/brain feedback loop does not change the overall level of incoming activity to the model , therefore facilitating the local homeostatic plasticity to find a more stable solution , i . e . , one that requires the smallest weight changes to approximate the target activation rate . Further , what we observe are different balancing systems operating at different spatial and temporal scales and with different specific mechanisms . This is consistent with the proposed description of normalization found in many neural systems [27] . Indeed , in our previous work , we described the computational complexity of behavior that emerges naturally out of systems that account for spatial and temporal interactions across a range of scales [14] . Such an architecture provides a canonical computation across scales and implementations , and results in improved neural coding efficiency and sensitivity . From a traditional cognitive neuroscience perspective , this way of thinking about task negative systems may sit somewhat uncomfortably . What we have been describing as task negative may provide a partial functional explanation for the default mode network . The default mode network is a well-characterized , frequently observed and relatively poorly understood macroscopic brain network located in areas of the brain not associated with sensorimotor activity; the default mode network has been observed across ontogeny [28] , phylogeny [29] , and found across different cognitive and sensorimotor tasks [30] and implicated in many disorders [31] . According to our findings , the default mode network can be thought of as acting as a counterweight , or as an endogenous generator of neural activity that allows the neural system to remain relatively stable in an inherently unstable world . One analogy could be to the vascular system of warm-blooded animals , which attempts to maintain a constant body temperature , irrespective of the temperature outside , to maintain a stable environment for chemical reactions to take place , ultimately allowing more flexible behavior . We note that the proposed balancing functional role for task negative brain networks does not preclude more traditional cognitive roles ascribed to them , such as mentation . We hypothesize that task negative systems could have initially evolved to perform some basic neural function , such as balancing incoming sensory activity , and eventually been adaptively repurposed over evolution to perform more specific cognitive functions that occur when external input is not present , as such , task negative systems reflect a macroscopic-scale spatial ‘mirror’ of local temporal homoeostatic normalization rules , demonstrating a multi-scale architecture in the brain for normalization processes [14] . Whilst our model only considers a relatively simple link between dynamics and functional behavior , our observations are consistent with our previous work exploring stability of neural dynamics in the brain and more complex sustained tasks [15] . From our model that suggests that complexity and flexibility of behavior is associated with efficient task-positive/negative interactions , we predict that disruption to task-negative regions of the brain such as the default mode network would be associated with disruption to sensory or motor processing; however , these disruptions could involve both increases and decreases in neural activity following sensory input; may take time before they manifest themselves and may be associated with less flexible behavior . This prediction is consistent with previous neuroimaging and behavioral work in Traumatic Brain Injury ( TBI ) [32–35] . Exploring the relationship between task-positive and task-negative balance in more complex tasks and in pathologies known to affect local E/I balance ( e . g . , the E/I disruption model of Schizophrenia [36] ) using this computational framework compared to empirical studies is the subject of ongoing work . Following this explanation of the task-negative balancing systems in general and the default mode network more specifically , we see that task-negative systems may not strictly be “necessary” for accomplishing any task . As such , lesioning task negative regions is unlikely to disturb any associated function entirely , and as such task negative systems may appear to be epiphenomenal . However , just as a sailing boat does not require a keel to move ( the keel counterbalances the forces on the sail , facilitating stability and allowing a wider range of movement and greater speed ) , the brain may have a greater range of neural state and potentially be more controllable , when it is properly counterbalanced . It might only be over longer time periods when initially adapting to a novel environment or across development that damage to task negative systems becomes particularly disabling , failing to facilitate other adaptive systems as efficiently . In the current results , we observed only a small ( but significant ) enhancement in behavioral and neural dynamics for the combined macroscopic and local-homeostatic models over the local-homeostasis only model , and no difference between the macroscopic homeostasis model and the static difference . This smaller effect suggests that the local-homeostasis is more important for promoting rich spontaneous dynamics; however , it does not mean that the macroscopic homeostasis is irrelevant . In the combined mode , macroscopic balancing was added to a local-homeostasis model that was tuned to perform optimally , unlike the macroscopic homeostasis model; this showed that the macroscopic mechanism could augment the local homeostatic model . Future work will investigate potential mechanisms that may underlie and tune the macroscopic spatial homeostasis; these have the potential to greatly increase both the independent and synergistic roles of the macroscopic homeostasis mechanism and make testable predictions about the optimal spatial organization and connectivity of task positive and negative networks . The location of the sensory input systems , motor output systems and task-negative nodes were chosen relatively arbitrarily . This is because the coarse resolution of the parcellation means that assigning sensory or motor labels to nodes is inherently very approximate . As such , we do not wish to draw parallels with specific brain regions or networks ( e . g . , from the functional imaging literature ) , however , understanding the true relationship between task positive and task negative nodes is the focus of our continued research using computational modelling approaches like that described here . Finally , to achieve a relatively stable solution with rich spontaneous dynamics and interactions with the environment , the system may have to encode ( in the local inhibitory weights ) information about the world , and the agent’s movement in it . Given the relative simplicity of the environment in the current simulation , the presence of local thresholds is adequate to facilitate a relatively stable solution . However , as the environment ( and sensory input systems ) becomes more complex , it will be necessary to use more sophisticated models with more flexibility . If the repertoire of brain states is to be more fully explored in the face of this increasing complexity , then it will be necessary to capture more information about the environment/sensory systems . This leaves open questions about the roles of other types of learning ( e . g . , longer-distance excitatory and reinforcement learning ) and their roles in supporting the system staying in a rich dynamical regime , in a complex environment , with complex sensorimotor systems and with more cognitive control mechanisms .
Simulated activity patterns were generated from a computational model constrained by empirical measures of white-matter structural connectivity between 66 cortical regions of the human brain , defined by diffusion tensor imaging ( DTI ) [18] . This structural network has been used in a range of previous computational models to demonstrate emergent properties of resting state functional connectivity [15 , 20–21 , 25 , 33] . A full methodology , describing the generation of the connectivity matrix 〈C〉 is available in [18] . In brief: measures of length and strength of stream-line based connectivity were estimated using Deterministic tractography of DSI datasets ( TR = 4 . 2s , TE = 89s , 129 gradient directions max b-value 9000s/mm2 ) of the brain in 5 healthy control subjects . A high-dimensional ROI based connectivity approach was projected though the 66 regions of the Desikan-Killianey atlas ( FreeSurfer http://surfer . nmr . mgh . harvard . edu/ ) , such that Ci . j is the number of streamlines connecting nodes i and j . | In recent years , there has been growing interest in using computational models based on the human structural connectome to better understand the brain . These simulations typically investigate spontaneous neural dynamics , in the absence of tasks , sensory input or motor output . Here , we take a different approach , embodying a computational model of spontaneous neural dynamics to control a simulated agent , with sensory input from and motor output to a simulated environment . Embodying the model radically changes how the model operates and changes how we understand the computational mechanisms . We observe interesting brain-environment feedback interactions and observe how different homeostatic systems are needed to compensate for this feedback . We observe this both in the simulated neural dynamics and the behavior of the embodied agent . These findings suggest novel functional roles for homeostatic systems in maintaining neural dynamics and behavior and for the poorly understood default mode network pattern of activity reported in functional neuroimaging in humans and animals . | [
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] | 2017 | From homeostasis to behavior: Balanced activity in an exploration of embodied dynamic environmental-neural interaction |
The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrPSc , a misfolded conformer of the normal prion protein , PrPC . However , protein-only PrPSc preparations lack significant levels of prion infectivity , leading to the alternative hypothesis that cofactor molecules are required to form infectious prions . Here , we show that prions with parental strain properties and full specific infectivity can be restored from protein-only PrPSc in vitro . The restoration reaction is rapid , potent , and requires bank vole PrPC substrate , post-translational modifications , and cofactor molecules . To our knowledge , this represents the first report in which the essential properties of an infectious mammalian prion have been restored from pure PrP without adaptation . These findings provide evidence for a unified hypothesis of prion infectivity in which the global structure of protein-only PrPSc accurately stores latent infectious and strain information , but cofactor molecules control a reversible switch that unmasks biological infectivity .
Prion diseases are a class of infectious , invariably fatal neurodegenerative diseases that affect humans and other mammals . Examples of prion diseases include Creutzfeldt-Jakob disease ( CJD ) in human patients , chronic wasting disease ( CWD ) in cervids including deer and elk , bovine spongiform encephalopathy ( BSE ) in cattle , and scrapie in sheep and goats [1] . A key pathogenic event in prion diseases is the conversion of the host-encoded prion protein from its normal , cellular conformation—termed PrPC—into a self-replicating , misfolded conformation—termed PrPSc—which is typically protease-resistant . The protein-only hypothesis posits that infectious mammalian prions are composed solely of PrPSc [1 , 2] . Pure self-replicating protein conformers have been directly shown to mediate efficient and faithful inheritance of biological traits and strain properties in fungi[3–5] . However , no similar experimental evidence has been obtained to support the protein-only hypothesis for mammalian prions[6] . Amyloid fibrils containing only wild-type recombinant ( rec ) PrP can induce prion disease in transgenic mice [7] , and induce prion formation by passage in asymptomatic wild-type mice [8] and hamsters [9] . Additionally , infectious amyloids have been generated using a disease-linked PrP truncation mutant [10] . Also , seeded propagation of recPrP without cofactors can produce prions with low levels of specific infectivity [11 , 12] . However , in each of these cases , very large quantities of pure PrP were required to induce disease , often with long incubation times and incomplete attack rates in normal hosts . In other cases , it has been shown that high concentrations of pure PrP amyloid fibrils can eventually induce the formation of prions with unusual strain characteristics after a slow in vivo adaptation process in asymptomatic animals[8 , 9] . To our knowledge , wild-type prions with significant levels of specific infectivity and faithful maintenance of parental strain properties have never been produced directly from PrP alone , raising the possibility that factors other than pure PrP may be necessary for efficient , high-fidelity replication of fully infectious prions [6] . Building upon the discovery of the membrane phospholipid phosphatidylethanolamine as an endogenous cofactor for mouse prion formation [13] , our laboratory used the serial protein misfolding cyclic amplification ( sPMCA ) technique developed by Soto and colleagues [14 , 15] to generate two self-replicating recombinant ( rec ) mouse ( Mo ) PrPSc conformers derived from the same original infectious template . The only difference between the two conformers is that one sample was produced with a substrate cocktail containing recPrP plus purified phospholipids ( Mo cofactor recPrPSc ) , while the other was produced with recPrP alone ( Mo protein-only recPrPSc ) [16] . These two autocatalytic conformers share a similar global structure but display strikingly different levels of specific infectivity in mice [16 , 17] . Based on end-point titration bioassays , the difference in specific infectivity between Mo cofactor recPrPSc and Mo protein-only recPrPSc in wild-type mice is >105 fold , with Mo protein-only recPrPSc causing no disease at all . The inability of Mo protein-only recPrPSc to infect WT mice can be explained by its inability to seed native Mo PrPC substrate in brain homogenate ( BH ) sPMCA , whereas Mo cofactor recPrPSc effectively converts native MoPrPC into PrPSc under the same conditions [16] . However , it is unknown whether a different host might be more receptive than mice to infection by Mo protein-only recPrPSc . Over the past decade , the European bank vole has emerged as an exciting model organism for prion disease research . Most animal species have transmission barriers that render them resistant to the majority of prion strains from other species . For example , humans appear to be susceptible to CJD and BSE , but not to CWD or scrapie [18–20] , while dogs appear to be resistant to nearly all naturally occurring prion strains [21] . In contrast , the bank vole ( Myodes glareolus ) appears to be uniquely susceptible to nearly all prion strains from other animal species , except BSE [22–26] . This enhanced susceptibility can be directly attributed to the bank vole ( BV ) PrPC sequence , because transgenic mice expressing BV PrPC rather than Mo PrPC are also near-universal hosts [25 , 27] . We initially sought to determine whether bank voles might be more susceptible than mice to infection by protein-only recPrPSc . This line of investigation led us to a series of unexpected results , which show that PrPSc alone can encode and propagate infectious information in a latent state , but that cofactor molecules are required to unmask biological infectivity .
The sPMCA reactions and PrPSc conformers used in this paper are illustrated in S1 Fig . We first used BV BH sPMCA to assess the potential susceptibility of bank voles to protein-only recPrPSc [14] . As expected , self-propagating PrPSc molecules were successfully produced in both BV and Mo brain homogenates seeded by RML prions ( Fig 1A , positive control ) , but not in unseeded reactions ( Fig 1A , no seed ) , confirming that both homogenates are fundamentally competent substrates for sPMCA reactions . And , as previously reported , Mo brain homogenate could be seeded by Mo cofactor recPrPSc , but not by Mo protein-only recPrPSc [28] ( Fig 1A , top row ) . Remarkably , we found that BV BH could be successfully seeded by Mo protein-only recPrPSc ( Fig 1A , bottom row; note that newly-formed native BV PrPSc product migrates at ~27–30 kDa whereas Mo protein-only recPrPSc seed migrates at ~16 kDa ) . Moreover , a substantial amount of native PrPSc could be detected immediately during the first-round sPMCA ( Fig 1A , bottom row; protein-only recPrPSc , sPMCA round 1 , indicating a rapid rate of PrPSc formation ) . Notably , the native BV PrPSc sPMCA product formed by protein-only recPrPSc seeding was identical in MW ( ~27–30 kDa ) and glycoform profile ( predominantly diglycosylated ) as the sPMCA product seeded by cofactor recPrPSc . To investigate the seed-specificity of this effect , we tested the ability of the same concentration of Mo recPrP amyloid ( a different conformer of pure recPrP[29 , 30] ) to seed BV BH , and found that it was unable to induce PrPSc in either BV or Mo BH ( Fig 1A , recPrP amyloid ) . We also tested the ability of BV recPrPSc conformers ( M109 cofactor recPrPSc , M109 protein-only recPrPSc , and I109 protein-only recPrPSc ) to seed BV BH sPMCA reactions . As expected , we found that BV M109 cofactor recPrPSc could effectively propagate in both Mo and BV BH substrates ( Fig 1B , left-hand blocs ) . Additionally , both M109 protein-only recPrPSc and I109 protein-only recPrPSc could seed sPMCA reactions containing BV BH , but not Mo BH ( Fig 1B , middle and right-hand blocs , compare bottom vs . top ) . Taken together , these results show that BV BH has a unique capacity for propagating protein-only recPrPSc seeds with various primary amino acid sequences . To determine the seeding potency of protein-only recPrPSc seeds in BV BH , we tested serial 10-fold dilutions of recPrPSc conformers in sPMCA experiments . The results show that BV BH could be seeded by all three protein-only recPrPSc seeds at high dilutions: ( 1 ) Mo protein-only recPrPSc at 10−4 ( 600 pg/mL PrPSc seed concentration ) ( Fig 2A , bottom panel ) or 10−5 ( 60 pg/mL PrPSc seed concentration ) ( S3 Fig , bottom row ) ; ( 2 ) M109 protein-only recPrPSc at 10−4 ( Fig 2B , top left panel ) ; and ( 3 ) I109 protein-only recPrPSc at 10−5 ( Fig 2B , middle left panel ) . In contrast , Mo BH could not be converted by any of the protein-only recPrPSc seeds , even at the highest concentration tested ( 0 . 6 μg/mL ) ( Fig 2A , top panel; Fig 2B , right column , top two panels ) . As expected , we found that M109 cofactor recPrPSc could seed both Mo BH and BV BH three-round sPMCA reactions at a dilution factor of 10−5 ( Fig 2B , bottom panel ) . Each sPMCA experiment also contained an unseeded control reaction to control for potential contamination . It has been previously reported that a different protein-only preparation , recPrP amyloid , is able to seed sPMCA reactions at high concentrations [8] . We determined that the minimum concentration of BV recPrP amyloid needed to seed BV BH sPMCA reactions is between 50 μg/mL ( Fig 2C ) , which is ~1 million times less potent than protein-only recPrPSc . Moreover , even at a high seeding concentration , the kinetics of PrPSc formation was slow in recPrP amyloid-seeded reactions , with a sPMCA product becoming first detectable in round 3 ( Fig 2C , right panel , last lane ) . Overall , these results show that BV BH is a uniquely susceptible substrate for the propagation of protein-only recPrPSc seeds , even at high dilutions , in BH sPMCA reactions , and that protein-only recPrPSc is a highly potent seed , especially compared to protein-only recPrP amyloid . To confirm the in vivo susceptibility of bank voles to protein-only PrPSc conformers as suggested by the sPMCA results , we performed end-point titration bioassays in M109 genotype bank voles . To our surprise , the bioassay results were completely negative despite the ability of protein-only recPrPSc conformers to potently and rapidly seed BV BH in sPMCA reactions . All bank voles inoculated with a 10−1 dilution ( 30 μL of 0 . 6 μg/mL PrPSc ) of M109 protein-only recPrPSc remained disease- and symptom-free after 570 days ( Table 1 ) . Furthermore , voles inoculated with a blind serial passage of brain homogenate prepared from an asymptomatic M109 protein-only recPrPSc-inoculated animal were also asymptomatic after 280 days ( Table 1 ) . I109 protein-only recPrPSc and Mo recPrP amyloid also failed to produce disease in bank voles at the 10−1 dilution ( Table 1 ) . The brains of M109 protein-only recPrPSc-inoculated bank voles contained minimal levels of vacuolation and PrP deposition , evident upon histopathological examination ( Fig 3 , fourth row from the top ) , but lacked protease-resistant PrP , detected by western blot ( Fig 4A , top row , left panel , samples 2–4 from the right; Fig 4B , middle row , left and middle panels ) . One out of three bank vole brains inoculated with M109 protein-only recPrPSc showed a very weak positive signal in RT-QuIC ( maximum ThT fluorescence: 8% ) ( S4 Fig ) , but the degree of fibrillization activity did not increase after blind serial passage ( S4 Fig ) . Additionally , the brains of blind serial-passaged M109 protein-only recPrPSc-inoculated animals lacked protease-resistant PrP ( Fig 4A , right panel , samples 2–4 from the left; Fig 4B , middle row , right-hand panel ) . We also inoculated C57BL/6J mice with a 10−1 dilution of M109 protein-only recPrPSc . All mice remained disease-free for the duration of their lifespans ( Table 2 ) , and their brains were histologically normal ( S5 Fig , bottom row ) . In contrast , M109 cofactor recPrPSc caused clinical scrapie in voles at all dilutions from 10−1 ( 100% attack rate , mean incubation period of 154 ± 6 days ) to 10−4 ( 100% attack rate , mean incubation period of 401 ± 46 days ) ( Table 1 ) . Upon passage of M109 cofactor recPrPSc , the mean incubation period at a 10−1 dilution dropped to 84 ± 6 days ( Table 1 ) . Clinical symptoms of disease for both primary and second passage included a disappearance of burrowing behavior , an extremely hunched posture , circling , and progressive ataxia . The course of disease lasted approximately two weeks for primary passage , but dropped to several days for second passage . The clinical diagnosis was confirmed by histopathology showing abundant vacuolation and PrP deposition ( Fig 3 , M109 cofactor recPrPSc: second row from the top , M109 cofactor recPrPSc passage: third row from the top ) , western blot showing protease-resistant PrP ( Fig 4A , left panel , right two samples; Fig 4B , top row , right two samples ) , and RT-QuIC showing fibrillization activity in brain homogenates from terminal animals ( S6A Fig ) . Importantly , bank voles inoculated with the Input PrPSc Seed Control sample were clinically asymptomatic ( Table 1 ) , histologically normal ( Fig 3 , top row ) , and lacked protease-resistant PrP in their brains ( Fig 4A , left panel , sample 4 from the left ) . We also inoculated C57BL/6J mice with a 10−1 dilution of M109 cofactor recPrPSc and observed a 100% attack rate ( 436 ± 8 days ) ( Table 2 ) , which was confirmed by pathology ( S5 Fig , middle row ) . Together , these results show that cofactor recPrPSc is potently infectious in bank voles and mice , while protein-only recPrPSc ( both M109 and I109 ) is surprisingly non-infectious in both species , even after blind serial passage . We were surprised that M109 protein-only recPrPSc failed to cause scrapie or induce significant levels of PrPSc accumulation in bank voles , despite its ability to convert BV PrPC to PrPSc in BH sPMCA quickly and potently . To explore this unexpected result further , we decided to assess the infectivity of the third-round product of BV BH sPMCA reactions seeded by protein-only recPrPSc , which we term [protein-only→BH PrPSc] for simplicity ( S1 Fig ) . Therefore , we performed an end-point titration bioassay of [protein-only→BH PrPSc] in bank voles . Remarkably , the results showed that [protein-only→BH PrPSc] is potently infectious in bank voles , causing disease at dilutions from 10−1 to 10−5 ( Table 3 ) . At a 10−1 dilution , there was a 100% attack rate and a mean incubation period of 113 ± 4 days , calculated as an average of three independent experimental inocula prepared from three separate sPMCA reactions ( Table 3 ) . Symptomatically , the disease was indistinguishable from that caused by M109 cofactor recPrPSc , but progressed more quickly ( 4–5-day clinical course ) . Clinically , we observed a disappearance of burrowing behavior , circling , followed by severe and progressive ataxia , and an extremely hunched posture . PK digestion followed by western blot revealed the accumulation of PrPSc in the brains of affected animals that was PK resistant at 64 μg/mL , the highest concentration tested ( Fig 4B , bottom row , middle panel ) . Pathology revealed the presence of vacuolation and florid PrP deposition in the brains of affected animals ( Fig 3 , bottom row ) . In contrast , animals inoculated with unseeded BH sPMCA control samples from three separate sPMCA experiments , termed [Control→BH PMCA] , remained asymptomatic for at least 320–720 days ) ( Table 3 ) , and an asymptomatic 180-day-old [Control→BH PMCA] vole lacked PK-resistant PrP in its brain ( Fig 4B , bottom row , left panel ) . This control confirms the lack of cross-contamination in sPMCA reactions used to generate [protein-only→BH PrPSc] . Given the similarity in clinical symptoms caused by M109 cofactor recPrPSc and [protein-only→BH PrPSc] , we sought to compare the strain properties of these two samples , which share a common provenance ( S1 Fig ) . We performed strain typing by examining regional vacuolation in bank voles inoculated with each strain . The two inocula produced a remarkably similar vacuolation pattern ( Fig 5 ) . Moreover , PrPSc in the brains of voles infected with either M109 cofactor recPrPSc or [protein-only→BH PrPSc] displayed similar glycoform ratios and electrophoretic mobility patterns on western blot ( Fig 4 , top row , compare lanes 11 and 12 vs . last two lanes ) , as well as similar degrees of protease resistance ( Fig 4B , middle column , top vs . bottom row ) . We also used RT-QuIC to compare the seed potency and fibrillization kinetics induced by brain homogenates prepared from animals inoculated with either M109 cofactor recPrPSc or [protein-only→BH PrPSc] [31] . Both samples showed fibrillization activity at dilutions from 10−2 to 10−8 ( S6 Fig ) . In addition , the time until a positive signal was reached was similar between the two samples: M109 cofactor recPrPSc BH-seeded brains showed a positive fluorescence signal at a 10−2 dilution after 117 minutes , while [protein-only→BH PrPSc] showed a positive fluorescence signal at a 10−2 dilution after 80 minutes . Thus , the prions induced by M109 cofactor recPrPSc and [protein-only→BH PrPSc] cannot be easily discriminated by RT-QuIC . Altogether , the results of these clinical , pathological , and biochemical analyses suggest that M109 cofactor recPrPSc and [protein-only→BH PrPSc] are very similar or identical strains . It is important to consider the possibility that the restored infectivity of [protein-only→BH PrPSc] could be due to the contamination from cofactor recPrPSc seeds; however , this explanation is unlikely for several reasons: ( 1 ) all [protein-only→BH PrPSc] and [Control→BH PMCA] inocula were prepared in dedicated , decontaminated sonicators in the absence of any other seeds , including cofactor recPrPSc; ( 2 ) special precautions were taken to prevent cross contamination ( see Methods ) [32]; ( 3 ) sentinel [Control→BH PMCA] samples would have detected contaminating cofactor recPrPSc seeds , as three-round BV BH or Mo BH sPMCA reactions detected 10−5 dilutions of M109 cofactor recPrPSc ( Fig 2 ) ; ( 4 ) native PrPSc accumulated rapidly in the first round of sPMCA ( Fig 1A , bottom row , protein-only recPrPSc , sPMCA round one ) , whereas PrPSc levels due to contamination would be expected to be negligible in the first round and only become detectable in later rounds; ( 5 ) identical positive experimental and negative unseeded control biochemical results were obtained in >15 independent experiments; and ( 6 ) identical positive experimental and negative control bioassay results were obtained in three independent experiments ( Table 3 ) . Propagating M109 protein-only recPrPSc in BV BH sPMCA ( to produce [protein-only→BH PrPSc] ) led to quantitative recovery of prion infectivity with strain properties indistinguishable from those of M109 cofactor recPrPSc . We sought to determine which biochemical factors were critical for this process . Previous studies have shown cofactor molecules to be essential for the formation of infectious prions in vitro [13 , 33] . To test whether cofactor molecules are required for M109 protein-only recPrPSc to convert native BV PrPC , we performed reconstituted sPMCA reactions using immunopurified native BV PrPC substrate ( S7 Fig ) . Positive control reactions supplemented with BH from PrP0/0 mice were able to propagate consistently for three rounds of sPMCA when seeded with either M109 protein-only recPrPSc or Mo protein-only recPrPSc ( Fig 6 , top and bottom rows , left-most panels ) . However , both M109 protein-only recPrPSc and Mo protein-only recPrPSc failed to propagate when no source of cofactor was added to the reconstituted sPMCA reaction , indicating that cofactors are essential for protein-only recPrPSc seeds to convert native BV PrPC ( Fig 6 , top and bottom rows , right-most lanes ) . Additionally , supplementing reconstituted sPMCA reactions with previously identified , specific cofactor molecules , i . e . , either poly ( A ) RNA molecules or a brain-derived lipid cofactor preparation , facilitated the propagation of both M109 protein-only recPrPSc and Mo protein-only recPrPSc ( Fig 6 , top and bottom rows , +RNA , +lipid cofactor columns ) . Taken together , these results show that cofactors are required for protein-only recPrPSc seeds to convert native BV PrPC , and that either RNA or purified phospholipid can function as the cofactor in this process . Finally , since native PrPC molecules in BV BH possess a C-terminal glycophosphatidylinositol ( GPI ) anchor and two N-linked glycans , we also sought to determine whether these post-translational modifications ( PTMs ) might be necessary for BV PrPC to restore prion infectivity in [protein-only→BH PrPSc] . To do this , we combined bacterially-expressed BV recPrP lacking post-translational modifications together with either RNA or purified phospholipid cofactor molecules as substrate cocktails for sPMCA reactions seeded with M109 protein-only recPrPSc . The results show continued propagation of recPrPSc for three rounds with either cofactor ( S8 Fig ) ; however , the MW protease-resistant cores of the sPMCA products appear to be ~16 kDa , which is the same MW as the protease-resistant core of M109 protein-only recPrPSc seed ( S8 Fig , compare lanes 2–4 vs . lanes 5–10 ) , and smaller than the core of M109 cofactor recPrPSc ( ~17 kDa ) ( S8 Fig , last three lanes ) . These biochemical results suggested that , even in the presence of cofactor molecules , BV recPrP substrate appears to continue propagating the protein-only recPrPSc confirmation rather than restore the infectious cofactor recPrPSc conformation . To test this directly , we inoculated the sPMCA products of both recPrP-RNA and recPrP-lipid reactions into bank voles . The results show that neither product is infectious ( Table 4 ) , confirming that PrPC PTMs do help facilitate the restoration of prion infectivity from protein-only PrPSc .
Our ability to restore biological infectivity from protein-only recPrPSc was critically dependent upon the remarkable susceptibility of BV PrPC to propagate protein-only recPrPSc seeds in vitro . Notably , BV BH is >100 , 000-fold more sensitive than Mo BH as substrate for propagating protein-only recPrPSc seeds in vitro , despite the fact that the amino acid sequences of BV PrPC and Mo PrPC are >96% homologous . Strikingly , BV BH could be potently seeded by Mo protein-only recPrPSc despite: ( 1 ) the inability of Mo protein-only recPrPSc to seed Mo brain homogenate; and ( 2 ) the amino acid differences between seed ( which is Mo sequence ) and substrate ( which is BV sequence ) . In particular , because native BV PrPC , but not native Mo PrPC , is susceptible to Mo protein-only recPrPSc , we can be certain that sequence similarity between seed and substrate is not responsible for the remarkable susceptibility of native BV PrPC to protein-only recPrPSc seeds , in general . This result violates the usual pattern observed for “species barriers” to prion propagation based on primary sequence , in which a perfect sequence match between substrate and seed would be expected to facilitate rather than hinder propagation [34 , 35] . Therefore , we can infer that the susceptibility of native BV PrPC substrate to protein-only recPrPSc seeds is likely due to the primary sequence of BV PrP allowing its structure to be intrinsically more accommodating than PrP sequences to a variety of templates , including protein-only recPrPSc . This interpretation is consistent with the previous observation that recombinant BV PrP substrate is able to propagate PrPSc seeds in RT-QuIC that were previously undetectable in sPMCA or RT-QuIC using PrP substrates from other species [31] . In general , the amino acid sequence of bank vole PrPC appears to greatly facilitate non-adaptive prion amplification ( NAPA ) during interspecies transmission[36] . Our data also indicate that N-linked glycans and/or the GPI anchor of BV PrPC are required for the recovery of infectivity from protein-only recPrPSc , since we were unable to restore infectivity using BV recPrP substrate lacking PTMs . Although PTMs are not absolutely required for the formation of prions with high levels of specific infectivity [28 , 37 , 38] , numerous studies have shown that these post-translational modifications can influence PrP folding pathways—sometimes in a strain-dependent manner [39–57] . In our experience , we have never been able to convert purified native PrPC substrate into a protein-resistant conformation in the absence of cofactor molecules . Therefore , we hypothesize that PTMs help prevent native BV PrPC from propagating the protein-only recPrPSc conformation , which allows it to restore the infectious [protein-only→BH PrPSc] structure instead . On the other hand , recPrP is capable of adopting the protein-only recPrPSc conformation , and likely prefers to continue propagating this state , even in the presence of cofactor molecules . In other words , continued conversion into protein-only recPrPSc may serve as a kinetic trap that sequesters recPrP substrate , effectively preventing it from converting into cofactor recPrPSc . The role played by cofactor molecules in facilitating the formation of infectious prions has been disputed . Using biochemical purification and reconstitution assays , we previously identified single-stranded RNA and PE as essential cofactor molecules for the formation of hamster and mouse prions [13 , 33 , 58] . Those studies also showed that cofactor molecules are required to produce wild-type prions with significant levels of specific infectivity , and that they restrict the strain properties of synthetic prions [13 , 33] . However , using different experimental approaches , others have argued that infectious prions can be formed in the absence of cofactor molecules [10–12] . For instance , while a different I109 protein-only recPrPSc conformer was reported to be infectious to I109 genotype bank voles [11] , its infectivity was characterized by incomplete attack rates ( 7/9 animals ) , and the need for an extremely concentrated inoculum ( i . e . , 5–10 μg/mL for I109 protein-only recPrPSc , which is 106-fold greater than the minimum concentration needed for cofactor recPrPSc ) to achieve infection [11] . These observations , combined with the lack of infectivity of I109 protein-only recPrPSc in our experiments , indicates that the specific infectivity of I109 protein-only recPrPSc is very low , and may require I109 hosts to be detected . I109 BV PrPC appears be inherently more than prone to misfolding than M109 BV PrPC , as transgenic mice overexpressing I109 BV PrPC , but not M109 BV PrPC , have been shown to develop spontaneous prion disease [59] . A different study reported that amyloid fibrils composed of Mo recPrP 23–144 could cause scrapie in mice [10]; however , an extremely concentrated inoculum ( i . e . , 100 μg/mL for recPrP 23–144 fibrils , as opposed to a minimal concentration of 60 pg/mL needed for cofactor recPrPSc ) was required to induce disease . Although end-point titration experiments were not performed , the large inoculation dose , long incubation period , and large variation in incubation times all suggest that pure recPrP 23–144 fibrils possess very low specific infectivity . Moreover , PrP 23–144 is a truncation mutant linked to Gerstmann-Staüssler Scheinker ( GSS ) syndrome , a hereditary form of prion disease , and therefore the folding requirements for this mutant may not be shared by wild type PrPC . We previously found that other disease-linked PrP mutants can misfold into self-propagating conformers in the absence of cofactor molecules , but that cofactor molecules were ultimately required for those misfolded mutant conformers to seed conversion of wild type PrPC to PrPSc [60] . Our finding that cofactor molecules are required for protein-only recPrPSc seeds to convert immunopurified native BV PrPC into PrPSc indicates that cofactor molecules work together with BV PrPC to restore prion infectivity , and therefore reinforces the concept that cofactor molecules are indeed essential components of infectious wild type prions [16] . It should be noted that although cofactor molecules are required to produce [33] , maintain [16 , 33] , and restore wild-type prions with significant levels of specific infectivity , other studies indicate they are not necessarily sufficient [61–63] , demonstrating that specific experimental conditions , i . e . , the concentrations and chemical nature of the substrates , physical parameters , etc . , must also be optimized to ensure efficient and accurate PrPSc propagation in vitro [64] . We describe , for the first time , the rapid restoration of fully infectious prions from a protein-only PrP molecule without adaptation ( defined here as a slow and inefficient PrPSc propagation process that ultimately results in a prion strain shift ) . This has allowed us to dissect the biochemical requirements for the restoration process , as discussed above . Most importantly , it also provides biological evidence that protein-only recPrPSc must be structurally similar to the infectious conformation of cofactor recPrPSc and [protein-only→BH PrPSc] prions . Other investigators have previously produced infectious prions through adaptation by blind serial passage of pure recPrP amyloid in mice [8] and hamsters [9] . It is important to distinguish that these results , although interesting in their own right , must differ fundamentally from those reported here for protein-only recPrPSc for the following reasons: ( 1 ) recPrP amyloid is formed de novo , whereas protein-only recPrPSc is produced by seeded propagation from infectious cofactor recPrPSc; ( 2 ) infectious prions induced by recPrP amyloid are formed slowly in vivo , whereas [protein-only→BH PrPSc] prions are formed immediately in vitro; ( 3 ) protein-only recPrPSc is ~1 million times more potent than recPrP amyloid at seeding formation of BV PrPSc in vitro ( compare Fig 2B to 2C ) ; and ( 4 ) infectious prions produced by adaptation from recPrP amyloid exhibit novel strain characteristics , whereas the strain characteristics of [protein-only→BH PrPSc] prions are indistinguishable from those of the original cofactor recPrPSc seed . The strain similarity between [protein-only→BH PrPSc] and cofactor recPrPSc is particularly striking because [protein-only→BH PrPSc] is composed of native PrPSc molecules , whereas cofactor recPrPSc is a recombinant protein lacking PTMs . It should also be noted that the strain similarity between [protein-only→BH PrPSc] and cofactor recPrPSc cannot be explained by having the same structural constraints imposed by a single purified cofactor [16] because [protein-only→BH PrPSc] was formed using a crude brain homogenate rather than a purified cofactor preparation . We can , therefore , conclude that strain information was successfully maintained and transmitted by the protein-only recPrPSc structure . Based on the observations listed above , we infer that recPrP amyloid induces the formation of infectious prions in vivo relatively inefficiently and slowly through an adaptation process , most likely by a deformed templating mechanism , as proposed by Baskakov and colleagues [65] . In contrast , the observations suggest that protein-only recPrPSc likely templates the formation of BV PrPSc molecules through a relatively high-fidelity , high-efficiency mechanism that requires cofactor molecules similar to the mechanism used during the replication of natural prion strains in the absence of transmission barriers or adaptation . It is unlikely that this mechanism involves selection of a rare , pre-existing protein-only recPrPSc conformer by cofactor molecules because of the rapidity , potency , and species specificity of the BV BH seeding reactions . Since [protein-only→BH PrPSc] prions with high specific infectivity can be rapidly formed without adaptation from protein-only recPrPSc seed , we infer that the global structure of protein-only recPrPSc is likely to resemble those of infectious cofactor recPrPSc and [protein-only→BH PrPSc] prions , with only small local differences that hinder biological infection . Indeed , deuterium exchange mass spectrometry ( DXMS ) experiments comparing the structures of cofactor vs . protein-only recPrPSc conformers confirm that the overall structures are similar with subtle differences [17] . An independent comparison between a different pair of infectious versus non-infectious recPrPSc conformers by Li et al . by DXMS yielded similar results[66] . Our observation that fully infectious prions with strain properties similar to cofactor recPrPSc can be rapidly restored from protein-only recPrPSc suggests a unified model of prion infectivity that reconciles the protein-only hypothesis with the ability of cofactor molecules to increase the specific infectivity of purified native and recombinant prions by many orders of magnitude ( Table 1 and previous work [16 , 33] ) . This model ( illustrated in Fig 7 ) proposes that protein-only recPrPSc molecules are able to maintain and propagate the overall global structure of infectious cofactor recPrPSc , with which it shares a similar provenance ( Fig 7 , reaction I ) . However , the lack of cofactor molecules causes a subtle conformational change of a local domain that is essential for replication in vivo ( Fig 7 , note small anomaly in blue icon ) . Consistent with this model , DXMS experiments suggest that two domains encompassing residues 91–115 and 144–163 differ in solvent accessibility between cofactor recPrPSc and protein-only recPrPSc [17] . Replacing cofactors by sPMCA propagation in BV BH ( Fig 7 , reaction II ) repairs the local conformational change [protein-only→BH PrPSc] prions , and thereby restores full prion infectivity . In the end , [protein-only→BH PrPSc] ( Fig 7 , product of sequential reactions I + II ) and cofactor recPrPSc prions ( product of reaction II ) have similar strain properties because recPrP alone is able to maintain and transmit forward the overall structure of cofactor recPrPSc prions . Ultimately , high-resolution structural determination of cofactor and protein-only recPrPSc molecules will be required to confirm this model . In addition , more work is required to determine whether cofactor molecules are also required to propagate infectious prions from other mammalian species , such as cows , deer , and humans . Our results raise an interesting conundrum: Since protein-only recPrPSc is a very potent seed for BV BH in sPMCA reactions , and native BV PrPC and cofactor molecules are both present in vivo , why isn’t protein-only recPrPSc directly infectious for bank voles ? One possibility is that protein-only recPrPSc might be degraded in vivo more rapidly than infectious conformers . However , as discussed above , protein-only recPrPSc would be expected to be structurally similar to cofactor recPrPSc , and both of these recombinant conformers lack PTMs such as sialylation , which can influence protein clearance [67 , 68] . Therefore , it difficult to envision how cellular prion clearance mechanisms , such as autophagy [69–72] or uptake by resident innate immune cells [73–75] , could specifically distinguish between these two conformers . A more likely explanation is that PMCA experimental conditions allow BV PrPC to be more structurally accommodating in vitro than in vivo . Two specific factors in PMCA experiments that could help make BV PrPC more structurally flexible ( and therefore more likely to interact with a structurally imperfect prion template , such as protein-only recPrPSc ) are the presence of detergent and cycles of intermittent sonication . Non-ionic detergents , such as Triton X-100 , disrupt the plasma membrane to which PrPC is normally attached through its GPI anchor—this disruption could allow PrPC to become more conformationally flexible than when it is anchored to an intact plasma membrane . Likewise , the intense bursts of mechanical energy during sPMCA could cause either PrPC molecules to rapidly sample conformational landscapes that it might not otherwise experience . Other investigators have also observed that sPMCA has the ability propagate PrPSc conformers that do not infect the corresponding animal hosts or tissues in vivo [76–80] . Ultimately , end-point titration bioassay in wild-type animals is the only bona fide method to measure specific infectivity [81] , and our bioassay data show that protein-only recPrPSc molecules are non-infectious , whereas [protein-only→BH PrPSc] prions appear to have restored the full specific infectivity and strain properties of cofactor recPrPSc prions . In conclusion , we report that prions with high specific infectivity can be rapidly restored from non-infectious protein-only recPrPSc molecules in vitro without adaptation . This provides the first experimental evidence that the conformation of protein-only PrPSc encodes all the information necessary for infectivity and strain properties , but paradoxically PrPSc alone is not sufficient for biological infectivity . The unique involvement of cofactor molecules in mammalian prion replication may help explain why , among various self-replicating proteins associated with neurodegenerative diseases in humans , prions are the only ones that are clinically infectious[82] .
The general sPMCA experimental method was adapted from Castilla et al . [64] . S1 Fig diagrams the sPMCA reactions and PrPSc conformers used in this paper . All PMCA reactions were sonicated in microplate horns at 37°C using a Misonix S-4000 power supply ( Qsonica , Newtown , CT ) set to power 70 for three rounds . One round of PMCA is equal to 24 hrs . The first round of PMCA was seeded with a volume of PrPSc equal to 10% of the total reaction volume . To propagate the reaction between PMCA rounds , 10% of the reaction volume was transferred into a new , unseeded , substrate mixture . Due to the sensitivity of sPMCA [32] , measures were undertaken to prevent sample contamination . Sample conical tubes were sealed with Parafilm ( Bemis Company , Oshkosh , WI ) and the sonicator horn was soaked in 100% bleach between experiments to prevent cross-contamination . Sample conical tubes were spun at 500 x g for 5 sec to remove liquid off the conical tube lids before propagation and were propagated individually using aerosol resistant pipette tips . The experimenter wore two pairs of gloves and changed the outer layer of gloves when handling a new sample . With each experiment , a sentinel conical tube ( a conical tube containing the entire sPMCA reaction mixture but lacking seed ) was also placed in the sonicator horn to detect contamination . Cofactor recPrPSc and protein-only recPrPSc were generated by sPMCA based on a previously established protocol [16] . Expression and purification of Mo recPrP 23–230 was performed as previously described [60] . Full-length BV PrP M109 1–255 on pcDNA3 . 1 and full-length BV PrP I109 1–255 on pcDNA3 . 1 were used to clone M109 BV recPrP 23–231 and I109 BV recPrP 23–231 onto pET-22b for expression . Site-directed mutagenesis using the Gene Tailor Site Directed Mutagenesis Kit ( Invitrogen , Carlsbad , CA ) was performed on full-length BV PrP M109 1–255 on pcDNA3 . 1 and full-length BV PrP I109 1–255 on pcDNA3 . 1 using the forward primer ( GCGCGCCATATGAAGAAGCGGCCAAAG ) containing the NdeI cut site and the reverse primer ( CGCGCGCTCGAGTCAGGAACTTCTCCC ) containing the XhoI cut site . Restriction digest of the PCR products and pET-22b plasmid followed by ligation created the final expression plasmids: BV M109 recPrP 23–231 on pET-22b and BV I109 recPrP 23–231 on pET-22b . These expression plasmids were used to express BV M109 recPrP 23–231 and BV I109 recPrP 23–231 proteins , which were produced and purified as previously described [60] . sPMCA reactions were performed using a previously established protocol with minor modifications [16] . Two-hundred microliter reactions containing 6 μg/mL Mo recPrP 23–230 or BV recPrP 23–231 in conversion buffer ( 20 mM Tris pH 7 . 5 , 135 mM NaCl , 5 mM EDTA pH 7 . 5 , 0 . 15% ( v/v ) Triton X-100 ) were supplemented with either purified brain-derived phospholipid cofactor [13] for cofactor recPrPSc propagation , or water for protein-only recPrPSc propagation . Four BV recPrP samples were created using either BV M109 or BV I109 recPrP , either alone or supplemented with purified brain-derived phospholipid cofactor ( S1 and S2 Figs ) . Each reaction was seeded with Mo cofactor recPrPSc and propagated for 18 rounds of sPMCA to eliminate the initial input Mo cofactor recPrPSc seed via serial dilution . Both reactions containing protein alone formed conformers containing protease-resistant cores of ~16 kDa ( S2A Fig , -cofactor ) , reminiscent of the MW of the core of Mo protein-only recPrPSc [16] ( S2B Fig , bottom panel ) . The conformers formed from reactions containing protein alone were termed I109 protein-only recPrPSc and M109 protein-only recPrPSc . The reaction containing M109 recPrP and brain-derived lipid cofactor formed a stably propagating conformer with a protease-resistant core of ~17 kDa ( S2A Fig , +cofactor ) , slightly lower than the MW of the core of Mo cofactor recPrPSc [16] ( S2B Fig ) , and was termed M109 cofactor recPrPSc . However , the MW of recPrPSc produced in reactions containing I109 recPrP plus brain-derived lipid cofactor substrate consistently shifted to ~16 kDa after 2–3 rounds of sPMCA ( S2C Fig ) . Since this conformer migrated at the same MW as the protein-only recPrPSc conformers , we decided not to include it in further experiments . All sPMCA reactions were sonicated with 15-sec pulses every 30 min . Bank vole brains were harvested from animals with M109 genotype perfused with PBS plus 5 mM EDTA . A 10% ( w/v ) perfused BH substrate was prepared in PBS , 1% ( v/v ) Triton X-100 , 5 mM EDTA , and cOmplete Mini Protease Inhibitors ( Roche , Basel , Switzerland ) . For sPMCA titrations , 10-fold serial dilutions of cofactor recPrPSc or protein-only recPrPSc seeds were created in conversion buffer [20 mM Tris pH 7 . 5 , 135 mM NaCl , 5 mM EDTA pH 7 . 5 , 0 . 15% ( v/v ) Triton X-100] . Reactions were sonicated with 20-sec pulses every 30 min . PrPC was immunopurified from BV ( genotype M109 ) brains based on a previously established protocol [33] . Using an electric potter homogenizer , 12 g of BV brains were homogenized in 80 mL Buffer A ( 20 mM MOPS pH 7 . 0 , 150 mM NaCl ) with cOmplete Protease Inhibitor Cocktail tablets ( Roche ) . The resulting homogenate was centrifuged at 3200 x g for 25 min at 4°C . The supernatant was discarded , and the pellets were resuspended to a volume of 40 mL by Dounce homogenizing in Buffer A , 1% ( w/v ) sodium deoxycholate , 1% ( v/v ) Triton X-100 . The homogenate was incubated on ice for 30 min to solubilize PrPC , then centrifuged at 100 , 000 x g for 40 min at 4°C . The solubilized supernatant was placed into a 50-mL conical tube with 1 mL of Protein A agarose ( Pierce , Rockford , IL ) and end-over-end rotated for 30 min at 4°C as a pre-clear step . Next , the supernatant/Protein A mixture was poured through an Econo-Pac ( Bio-Rad , Hercules , CA ) column and the flow-thru was collected as the pre-cleared load . The pre-cleared load was passed over a column packed with 2 mL of Protein A Agarose resin ( Pierce ) cross-linked to 6D11 mAb that was pre-equilibrated with Buffer A , 1% ( w/v ) sodium deoxycholate , 1% ( v/v ) Triton X-100 at a flow rate of 0 . 75 mL/min . The column was washed with 36 mL of Wash Buffer 1 [20 mM Tris pH 8 . 0 , 1% ( v/v ) Triton X-100 , 500 mM NaCl , 5 mM EDTA] , followed by 24 mL of Wash Buffer 2 [Buffer A , 0 . 5% ( v/v ) Triton X-100] at a flow rate of 1 . 0 mL/min . A 50-mL conical tube containing 900 μL of Neutralization Buffer [1M Tris pH 9 . 0 , 5% ( v/v ) Triton X-100 , 1 . 4 M NaCl] was placed beneath the column . The column was manually eluted using a syringe filled with Elution Buffer ( 0 . 1 M glycine pH 2 . 5 , 100 mM NaCl ) until a volume of 15 mL was reached . The eluate was brought to 50 mL with SP Equilibration/Wash Buffer [20 mM MES pH 6 . 4 , 0 . 15 M NaCl , 0 . 5% ( v/v ) Triton X-100] and applied slowly to a 1 . 5-mL SP Sepharose ( Sigma Aldrich , St . Louis , MO ) ion-exchange column that was pre-equilibrated with 10 column volumes of SP Equilibration/Wash Buffer . The column was washed with 15 mL of SP Equilibration/Wash Buffer and eluted with 5 mL of SP Elution Buffer [20 mM MOPS pH 7 . 5 , 0 . 50 M NaCl , 1% ( v/v ) Triton X-100] containing cOmplete EDTA-free Protease Inhibitor Cocktail tablets ( Roche ) . The eluate was dialyzed in 3500 MWCO Slide-a-Lyzer ( Pierce ) cassettes overnight against 4 L of Exchange Buffer [20 mM MOPS pH 7 . 5 , 150 mM NaCl , 0 . 5% ( v/v ) Triton X-100] . Reconstituted sPMCA experiments were adapted from Piro et al . [83] . Briefly , 150-μL reactions containing 20 μg/mL immunopurified BV M109 PrPC in conversion buffer ( 20 mM MOPS pH 7 . 0 , 0 . 075% Triton X-100 , 50 mM imidazole pH 7 . 0 , 5 mM EDTA pH 7 . 5 , 0 . 1 M NaCl ) were supplemented with either 45 μL of 10% ( w/v in PBS ) PrnP0/0 BH , purified brain-derived phospholipid cofactor [13] , PBS and 1% ( v/v ) Triton X-100 , or 60 μg/mL polyadenylic acid potassium salt ( Sigma Aldrich ) . Reactions were sonicated with 20-sec pulses every 30 min . Formation of PrPSc was monitored by digestion of sPMCA samples with Proteinase K ( PK ) ( Roche ) and western blotting . Samples were digested with 64 μg/mL PK at 37°C with shaking at 750 r . p . m . Samples from sPMCA reactions using recPrP as the substrate were treated for 30 min , while samples using BH or immunopurified PrPC as the substrate were treated for 60 min . Digestion reactions were quenched by adding SDS-PAGE loading buffer and heating to 95°C for 15 min . SDS-PAGE and western blotting were performed as described previously [83] using mAb 27/33 ( epitope = 136–158 mouse numbering ) . Then , 20 μL of a sPMCA reaction was subjected to PK digestion . The minus ( - ) PK lane shown in each western blot figure is used to determine the conversion efficiency of a sPMCA reaction . The amount of PrPC in the original substrate relative to the amount that was converted to PrPSc during one round of PMCA . For reactions using recPrP as the substrate , the minus PK lane contains the same volume ( 20 μL ) of a sPMCA reaction as a PK-digested sample . For reactions using BH or immunopurified PrPC as the substrate , the minus PK lane contains one-tenth ( 2 μL ) of the volume used in the PK-digested samples . Amyloid fibers were generated as previously described [84] . Briefly , a 3 . 0-mg/mL stock of recPrP was made by adding 6 . 0 M GdnHCl to the lyophilized protein . A 1 . 5-mL conical tube containing a 600-μL reaction volume ( 2 M GdnHCl , 50 mM MES buffer , pH 6 . 0 , 10 mM thiourea , and 250 μg of recPrP ) was incubated at 37°C with continuous shaking at 1700 r . p . m . for 24 h . Fibers were centrifuged at 100 , 000 x g and then washed with 10 mM NaAc pH 5 . 0 twice and stored at 4°C . Intracerebral inoculation and diagnosis of prion disease were performed as described [83] with the following modifications: PMCA mixtures and products were diluted 1:10 into PBS plus 1% ( w/v ) bovine serum albumin before inoculation . Brain homogenate samples ( 10% w/v in PBS ) were spun for 30 sec at 200 x g to remove nuclear debris , and the supernatant was collected and used as the inoculum . The inoculum volume used was 30 μL . Bank voles with the M109 genotype were bred from a colony originally established at the Istituto Superiore di Sanità ( Rome , Italy ) , and inoculated between 4–6 weeks of age . Neuropathology was performed as previously described [13] , using primary mAb 27/33 at a 1:1000 dilution and a Biocare Mouse on Mouse Horseradish Peroxidase Polymer ( Biocare Medical , Pacheco , CA ) for the immunohistochemical detection of PrP . The Guide for the Care and Use of Laboratory Animals of the National Research Council was strictly followed for all animal experiments . All experiments involving voles and mice in this study were conducted in accordance with protocol supa . su . 1 as reviewed and approved by Dartmouth College’s Institutional Animal Care and Use Committee , operating under the regulations/guidelines of the NIH Office of Laboratory Animal Welfare ( assurance number A3259-01 ) and the United States Department of Agriculture . Formation of PrPSc was monitored by digestion of BHs [10% ( w/v ) in PBS] with PK followed by western blotting . Samples were digested in a reaction containing 64 μg/mL PK ( unless otherwise specified ) , 2% ( v/v ) Tween-20 ( Fisher Scientific , Hampton , NH ) , 2% ( v/v ) NP-40 ( Fisher Scientific , Hampton , NH ) , and 2% ( w/v ) n-Octyl-β-D-Glucopyranoside ( Anatrace , Maumee , OH ) at 37°C with shaking at 750 r . p . m . for 1 hr . Digestions were quenched by adding SDS-PAGE loading buffer and heating to 95°C for 15 min . SDS-PAGE and western blotting were performed as described previously [83] using mAb 27/33 . Twenty microliters of a brain homogenate were subjected to PK digestion . The minus PK lane is used to determine the fraction of PrP that has been converted to PrPSc in the brain . The minus PK lane contains the same volume ( 20 μL ) of BH as a PK-digested sample . RT-QuIC reactions were carried out as described previously [31] , with the following modifications . Lyophilized BV M109 recPrP was resuspended in 10 mM sodium phosphate ( pH 5 . 8 ) to a concentration of 0 . 5 mg/mL . The resuspended protein was filtered through a 0 . 22-μm syringe-driven filter , and the concentration was adjusted using 10 mM sodium phosphate ( pH 5 . 8 ) to a concentration of 0 . 3 mg/mL . The resuspended protein was then diluted in a reaction buffer ( 10 mM sodium phosphate buffer pH 7 . 4 , 300 mM NaCl , 10 μM ThT , 1 mM EDTA , and 0 . 001% SDS ) to a final concentration of 0 . 1 mg/mL . Ninety-eight microliters of this reaction mixture was added to each well of a black-walled 96-well plate with a clear bottom with 2 μL of seed . Ten-fold serial dilutions of seeds were created in PBS and 0 . 025% ( v/v ) SDS . The plate was sealed and incubated at 42°C with 90-sec intervals of orbital shaking at 920 r . p . m . followed by 90 sec of rest in a FilterMax F5 Multi-Mode Microplate Reader ( Molecular Devices , San Jose , CA ) . ThT fluorescence measurements ( 430 +/- 35-nm excitation and 485 +/- 20-nm emission ) were taken every three min . Experimental samples were run in technical triplicate . Data analysis was performed as described previously [85] , except the mean baseline relative fluorescence units were calculated over a one-hr period . | Prions are unusual infectious agents that cause invariably fatal brain diseases . Unlike conventional infectious agents such as bacteria or viruses , prions do not possess nucleic acids such as DNA or RNA , and therefore it is not clear how they are able to replicate and cause infection . A leading model is that prions are composed exclusively of a specific protein molecule with an abnormal shape , which has the ability to coerce other protein molecules to change into the same abnormal shape in a self-reinforcing process . Although this model is attractive , no one has ever been able to make potently infectious prions from only pure protein . Here , we show for the first time that pure protein can faithfully store and transmit specific infectious information ( strain properties ) in a latent state even though it is non-infectious . | [
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] | 2019 | Full restoration of specific infectivity and strain properties from pure mammalian prion protein |
The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival . Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival , but functional verification was lacking . In this study , we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l ( 2 ) mbn cells . Cell viability , cell morphology , cell proliferation , and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes , including sorting nexin-like gene SH3PX1 and Sox box protein Sox14 , and 18 new pro-survival genes . Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation . We found that the pro-survival function of five genes ( Ras85D , Cp1 , CG13784 , CG32016 , and CG33087 ) , was dependent on ecdysone signaling . The TUNEL assay revealed an additional two genes ( Kap-α3 and Smr ) with an ecdysone-dependent cell survival function that was associated with reduced cell death . In vitro , Sox14 RNAi reduced the percentage of TUNEL-positive l ( 2 ) mbn cells ( p<0 . 05 ) following ecdysone treatment , and Sox14 overexpression was sufficient to induce apoptosis . In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling , including defects in larval midgut and salivary gland destruction . These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival .
Steroid hormones are small hydrophobic signaling molecules which bind to their receptors to control gene expression and initiate the regulation of growth , development , homeostasis and programmed cell death ( PCD ) [1] . Components of the steroid-regulated PCD transcriptional regulatory cascades in insects and mammals have been well characterized . For example , in vertebrates , steroid hormone glucocorticoids regulate the removal of excess thymocytes during T-cell maturation [2] , [3] . In insects , the transcriptional cascade induced by the steroid hormone 20-hydroxyecdysone ( ecdysone ) has been implicated in the activation of PCD in larval intersegmental muscle [4]–[6] , newly eclosed adult central nervous system [7] , [8] , larval salivary glands [9] , and larval midgut [10] . Deregulation of the hormonal control of PCD in humans has been associated with various pathological conditions , including cancer and the degenerative disorder Alzheimer's Disease [1] , [11] , [12] . Given the functional conservation of many genes in humans and Drosophila , experiments to identify the genes required for hormonal control of Drosophila PCD will provide not only a better molecular understanding of the process itself , but may also be valuable in the context of human disease treatment and diagnostics . During metamorphosis of Drosophila , two stage-specific sequential pulses of ecdysone activate first the transformation of larvae into pupae , and then the transformation of pupae into adult flies . The ecdysone pulses regulate the destruction of obsolete larval tissues , and the differentiation and morphogenesis of adult tissues which arise from small clusters of progenitor cells , [7] , [8] , [13]–[16] . The first ecdysone pulse occurs at the late third instar larval stage and triggers puparium formation . In addition , the larval midgut undergoes histolysis and the future adult midgut tissue envelopes it by 2 hrs after puparium formation ( APF ) [9] , [10] . A second ecdysone pulse occurs 10 hrs APF and triggers the death of larval salivary glands [9] . Previous studies indicated that larval midguts and salivary glands employ similar , yet distinct , genetic mechanisms during steroid induced programmed cell death [10] . Several studies have identified some of the components involved in the transcriptional cascade upstream of PCD of salivary glands in Drosophila . The ecdysone receptor is a heterodimer of the nuclear receptors ecdysone receptor ( EcR ) and ultraspiracle ( USP ) [17] . The heterodimer complex binds to the steroid hormone ecdysone and induces the transcription of the early genes E93 ( DNA binding protein ) , BR-C ( zinc finger transcription factor ) , E74 ( ETS-domain transcription factor ) , and E75 ( orphan nuclear receptor ) [13] , [18]–[22] . The EcR∶USP complex and E93 , BR-C and E74 proteins in turn activate transcription of several pro-death genes including reaper ( rpr ) , head involution defective ( hid ) and grim which function similarly to mammalian SMAC/DIABLO , the APAF-1 homologue ark , the initiator caspase dronc , and the CD36 receptor homologue croquemort ( crq ) [14] , [21] , [23]–[30] . The transcription factor E75B is sufficient to repress transcription of the inhibitor of apoptosis protein 2 gene , diap2 [21] , while EcR and the CREB binding protein ( CBP ) transcriptional cofactor are required for diap1 downregulation [31] . Functional studies have confirmed that at least Ecr , E93 , BR-C , hid , ark , dronc , CBP ( CREB binding protein ) , and AP-1 ( heterodimer of c-Jun and c-Fos ) are required for Drosophila salivary gland cell death [9] , [22] , [27] , [31]–[37] . Similarly , functional studies have identified a role for Ecr , BR-C , E93 , rpr and hid in larval midgut cell death [10] , [26] , [38] , [39] . While the upstream ecdysone signaling cascade and some cell death genes thus have a demonstrated function in these death processes , the results of genome-scale expression studies [23] , [24] , [40] suggest that there are many more potential effectors of ecdysone-regulated cell death and cell survival . The Drosophila cell line l ( 2 ) mbn [41] , a tumorous haemocyte cell line , is well suited for studying steroid hormone induced programmed cell death for several reasons . First , treatment of l ( 2 ) mbn ( also known as mbn2 ) cells with ecdysone was shown to induce cell death with morphological features of apoptosis ( DNA fragmentation , apoptotic bodies ) and autophagy [42] . Second , ecdysone treatment induced the expression of the transcription factor BR-C and the caspases Dronc and Drice [43] , [44] in l ( 2 ) mbn cells . And , third , following ecdysone treatment , the knock-down of E93 , BR-C and caspases by RNA interference ( RNAi ) reduced l ( 2 ) mbn cell death [43] , [44] , while the knockdown of E74B , E75A , and E75B by RNAi enhanced cell death [44] . These features indicate that ecdysone mediated cell death in l ( 2 ) mbn cells is akin , at least in part , to dying larval stage Drosophila salivary glands and midgut . Treatment of cultured Drosophila cells with double stranded ribonucleic acid ( dsRNA ) targeting specific genes depletes their corresponding transcripts and has been used as an efficient tool for genome wide loss-of- function phenotypic analyses [45]–[54] . Recent microarray , SAGE and proteomics studies [23] , [24] , [40] have identified hundreds of transcripts and proteins that are differentially regulated in Drosophila larval salivary glands immediately prior to ecdysone-induced cell death , but their functions in this process remain untested . Here we analyze the function of 460 of these gene products using RNAi in ecdysone treated l ( 2 ) mbn cells , and report the identification of many novel players in the ecdysone signaling network governing cell death and cell survival .
To validate our experimental system , we conducted cell viability , cell death , transcription and RNAi assays in ecdysone-treated l ( 2 ) mbn cells using known ecdysone signaling and apoptosis genes . First , to verify previous findings [42]–[44] of ecdysone treatment effects on Drosophila l ( 2 ) mbn cells , we employed multiple assays over a time course of ecdysone treatment . To assess cell viability , we used the trypan blue exclusion [55] and 4-[3- ( 4-iodophenyl ) -2- ( 4-nitrophenyl ) -2H-5-tetrazolio]-1 , 3-benzene disulfonate ( WST-1 ) based cell viability ( Roche Diagnostics ) assays . Both assays indicated that the majority of cells are non-viable by 72 hours following treatment with 10 uM ecdysone ( Figure 1A and 1D ) . To specifically measure cell death , nuclei were stained with DAPI and the percent TUNEL positive cells were determined 72 hours following ecdysone treatment . Our results showed that the control and ecdysone treated cells had 11% and 54% TUNEL positive cells , respectively , indicating that the reduced cell viability is due , at least in part , to increased cell death . In addition , we used electron microscopy ( EM ) to examine morphological features of l ( 2 ) mbn cells following ecdysone treatment . Consistent with previous reports [42] , we observed features representative of apoptosis , autophagy and phagocytosis in the ecdysone treated cells ( data not shown ) . To determine the expression profile of representative ecdysone regulated transcription factors and apoptosis genes in l ( 2 ) mbn cells , we employed quantitative reverse transcription PCR ( QRT-PCR ) and measured transcript levels following 24 , 48 and 72 hrs ecdysone treatment . Since we observed features of autophagy after ecdysone treatment , we also quantitated the expression levels of several autophagy genes to determine if their expression was ecdysone regulated in our experimental system . Our QRT-PCR results ( Figure 1B ) indicate that the early transcription factors Br-C and E75 had the relatively highest expression levels at 24 hrs ( 10 and 13 fold increase in expression , respectively , compared to untreated control cells ) and then decreased after 48–72 hours ( at 48 hours , 2 and 7 . 9 fold increase in expression , respectively , compared to control cells ) . As demonstrated in Figure 1B , E93 , reaper , dronc and hid demonstrated elevated expression levels by 24 hrs ( 58 , 5 . 8 , 3 . 4 , and 2 . 3 fold increase respectively ) which remained elevated or continued to increase at 48 and 72 hours ( at 48 hours , 126 , 36 , 4 . 4 and 4 . 7 fold increase in expression , respectively ) . These observations suggest that the transcriptional cascade for the representative ecdysone signaling and apoptosis genes is similar between ecdysone-treated l ( 2 ) mbn cells and dying Drosophila larval salivary glands . Although we detected expression of autophagy genes in l ( 2 ) mbn cells , we observed no significant differential expression compared to untreated cells ( ie . below the arbitrarily chosen 2 fold cut-off level ) ( Figure 1B ) up to 72 hrs following ecdysone treatment , indicating that the autophagy genes tested are not transcriptionally regulated in this system at these timepoints ( Figure 1B ) . To test the sensitivity of our RNAi strategy , we treated l ( 2 ) mbn cells with dsRNA corresponding to representative ecdysone signaling ( EcR , BR-C and E75 ) and apoptosis ( dronc , rpr , hid and diap-1 ) related genes . First , to determine the knock-down efficiency of RNAi for the genes described above , we measured their expression levels at 72 hrs by QRT-PCR in ecdysone-treated cells with or without dsRNA . For all the genes tested , the transcript knock-down ranged between 62–90% ( for examples , see Figure 1C ) . Next , the WST-1 assay was used to measure cell viability following RNAi and ecdysone treatment . We found that treatment of l ( 2 ) mbn cells with ecdysone and dsRNAs corresponding to EcR , BR-C , dronc , reaper and hid resulted in increased cell viability ( p≤0 . 05 ) compared to cells treated with a negative control , a human dsRNA NM_138278 [56] ( Figure 1D; Table 1 ) . Treatment of l ( 2 ) mbn cells with dsRNA corresponding to either E75B ( Table 1 ) or diap-1 ( Figure 1D; Table 1 ) decreased cell viability significantly ( p≤0 . 001 ) as assayed by WST-1 . We confirmed that the change in viability of the l ( 2 ) mbn cells treated with ecdysone and RNAi was due to alterations in cell death by employing the TUNEL and DAPI assay for selected genes ( Table 2 ) . To identify additional genes that function in ecdysone-mediated cell death or cell survival , we conducted an RNAi screen ( Figure 2 ) . Based on genome-wide transcript and protein expression studies conducted previously in Drosophila larval salivary glands [23] , [24] , [40] , there are a large number of genes and proteins that could affect ecdysone-mediated PCD but have not been tested functionally . Here , we conducted a systematic study of 460 of these genes which included all of the annotated genes from our previous study [24] that showed a significant ( p≤0 . 05 and 5 fold difference ) increase or decrease in expression levels in salivary glands immediately prior to PCD . The WST-1 assay was used as a primary screen to assess effects of the 460 dsRNAs on cell viability ( Table S1 ) . Using this assay , we identified five genes reported already to have a pro-survival role based on a previous RNAi screen [47] , [49] , [57]; ( Table 1 and Table S1 ) . In addition , we identified and validated another 20 genes with corresponding dsRNAs that significantly increased or decreased cell viability ( Table 1 ) . All of these 20 genes were validated with completely non-overlapping dsRNAs . In total , our final gene set for further analyses consisted of 18 genes with corresponding dsRNAs that resulted in reduced viability ( hereafter referred to as pro-survival genes ) and 7 genes with corresponding dsRNAs that resulted in increased viability ( hereafter referred to as candidate pro-death genes ) . To determine which genes are regulated by the ecdysone signaling pathway , we investigated whether the decreased cell viability phenotype caused by RNAi knock-down of the 18 pro-survival gene products was ecdysone dependent . We treated the cells with dsRNA and assessed cell viability with and without ecdysone treatment . This analysis resulted in the identification of five ecdysone dependent pro-survival genes ( CG33087 , CG13784 , CG32016 , Ras85D , Cp1; Table 1 ) . dsRNAs corresponding to these five genes reduced cell viability only in the presence of ecdysone and did not affect viability of l ( 2 ) mbn cells in the absence of ecdysone . Of the five genes identified , three ( CG33087 , CG13784 , CG32016 ) were uncharacterized previously . Of these three genes , two ( CG13784 , CG32016 ) do not have any recognizable protein domain or predicted gene function ( FlyBase ) [58] . We confirmed the ecdysone dependent pro-survival effect of two ( CG32016 , Cp1 ) of the 5 identified genes in another Drosophila cell line , S2; the other three ecdysone dependent genes identified in l ( 2 ) mbn cells did not significantly affect S2 cell viability in the presence of ecdysone ( Table S2 ) . dsRNA corresponding to 13 other genes ( Table 1 ) reduced viability of l ( 2 ) mbn cells following ecdysone treatment . However , a decreased viability phenotype , as assessed by WST-1 , was also observed for these 13 dsRNAs in l ( 2 ) mbn cells in the absence of ecdysone . Nine of the 13 dsRNAs showed similar viability effects in S2 cells in the presence or absence of ecdysone ( Table S2 ) . We initially categorized the 13 genes as ecdysone independent pro-survival genes . Among this group of genes , dsRNA corresponding to Kap-α3 resulted in different phenotypes , as assessed initially by cell morphology ( Figure 3 ) , in the absence and presence of ecdysone . In the absence of ecdysone , Kap-α3 dsRNA did not result in detectable apoptotic bodies ( up to 72 hrs ) , but in the presence of ecdysone and as early as 48 hrs following treatment , the same dsRNA resulted in a dramatic increase in apoptotic bodies compared to controls ( Figure 3; Table 2 ) . This result indicates that while the overall survival effect of this gene product may be ecdysone independent , its mechanism of action differs depending on the presence or absence of ecdysone . To determine whether the decreased viability of cells treated with ecdysone and dsRNA corresponding to the pro-survival genes is due at least in part to increased cell death , we performed the TUNEL/DAPI assay for representative genes from this category . We treated cells with dsRNA of two ecdysone dependent ( CG32016 and Ras85D ) and six ecdysone independent ( Kap-α3 , Pros26 . 4 , Smr , Sin3A , S6K , and Tor ) genes in the presence and absence of ecdysone and quantified the percent TUNEL positive cells . RNAi of six genes ( CG32016 , Ras85D , Kap-α3 , Pros26 . 4 , Smr , and S6K ) increased significantly the percentage of TUNEL positive cells ( p≤0 . 05 ) in the presence of ecdysone ( Groups A and B , Table 2 , Figure 4 ) , indicating a potential death inhibitory pro-survival role . RNAi of Sin3A and Tor did not significantly ( p>0 . 05 ) increase the percentage of TUNEL positive cells in the presence of ecdysone , indicating that their pro-survival effects in this context are likely not due to an inhibition of cell death ( Group C , Table 2 ) . However , RNAi of these same two genes did result in an increase in percent TUNEL positive cells in the absence of ecdysone compared to the controls ( Table 2 ) . In contrast , our TUNEL/DAPI assay indicated that knock-down of Kap-α3 and Smr by RNAi increased TUNEL positive cells only in the presence of ecdysone ( Group A , Table 2 ) . This result is in agreement with the previously observed increase in apoptotic bodies found only in the presence of ecdysone ( Figure 3 ) . The reduced viability caused by RNAi of Kap-α3 , and Smr in the absence of ecdysone appears not to be death-related and may instead be due to inhibition of cell proliferation . To test this possibility , we conducted a BrdU incorporation assay which indicated reduced proliferation in Kap-α3-RNAi but not in Smr-RNAi treated cells compared to control-RNAi treated cells ( p<0 . 05; Figure S2 ) . RNAi of Pros26 . 4 and S6K resulted in an increase in TUNEL positive cells ( p≤0 . 05 ) both in the absence and presence of ecdysone , distinguishing them as ecdysone independent and potential negative regulators of cell death . The TUNEL/DAPI assay also confirmed the ecdysone dependent and potential death inhibitory survival role of CG32016 and Ras85D ( Group A , Table 2 ) . Consistent with WST-1 findings , the BrdU incorporation assay for these two dsRNAs indicated no significant change in proliferation ( p = 0 . 5 and 0 . 4 , respectively; Figure S2 ) in the absence of ecdysone . In summary , based on WST-1 , TUNEL and BrdU assays , we conclude that CG32016 , Ras85D , Kap-α3 , and Smr , are ecdysone dependent pro-survival genes that result in decreased cell death , and Pros26 . 4 , S6K , Tor , and Sin3A are ecdysone independent pro-survival genes that result in decreased cell death . The observed effects on cell death following RNAi of these genes may be directly or indirectly related to gene function . Our RNAi study identified seven candidate pro-death genes , comprised of two 40S ribosomal genes ( RpS5 and RpS6 ) , three 60S ribosomal genes ( RpL13A , RpL37 and RpLP1 ) , one transcription factor Sox box protein ( Sox14 ) and one sorting nexin-like gene ( SH3PX1 ) . To determine whether their potential pro-death effects are ecdysone dependent , we performed RNAi assays with and without ecdysone . Consistent with observations by others [49] , dsRNAs corresponding to the ribosomal genes had the opposite effect in the absence of ecdysone , resulting in a significant reduction in cell viability ( Table 1 , column 3 , bold and italicized ) when compared to control cells . In agreement with the cell viability assay , the BrdU assay showed reduced proliferation in the ribosomal gene-RNAi treated cells in the absence of ecdysone ( p<0 . 05; Figure S2 ) . To confirm the putative pro-death role of the ribosomal genes observed in the presence of ecdysone in l ( 2 ) mbn cells , we employed the TUNEL/DAPI assay as described above . Knock-down of all ribosomal genes tested , with the exception of RpS6 , resulted in a decrease in the percent TUNEL positive cells ( Table 2 ) following ecdysone treatment , indicating that RpS5 , RpL13A , RpL37 and RpLP1 have a pro-death related function in l ( 2 ) mbn ecdysone-mediated death . The TUNEL/DAPI assay also indicated that the transcription factor Sox14 , and the sorting nexin-like gene SH3PX1 act as pro-death genes ( Table 2 ) . Therefore , our RNAi study which employed both cell viability ( WST-1 ) and cell death TUNEL/DAPI assays identified six new genes ( RpS5 , RpL13A , RpL37 and RpLP1 , SH3PX1 , Sox14 ) required for ecdysone-mediated cell death in l ( 2 ) mbn cells . To determine whether Sox14 expression is induced by ecdysone , we treated both l ( 2 ) mbn and S2 cells with ecdysone and determined the expression levels of Sox 14 by QRT-PCR . As shown in Figure 5A , ecdysone treatment resulted in a 5 fold and 4 fold increase in expression of Sox14 in l ( 2 ) mbn and S2 cells , respectively . To determine whether Sox14 is sufficient to decrease cell viability , we overexpressed C and N-terminal FLAG tagged Sox14 protein in l ( 2 ) mbn cells and measured cell viability using the WST-1 assay . Overexpression of Sox14 reduced cell viability , detectable 48 hrs following transfection ( Figure 5B ) . By approximately 96 hrs after transfection , apoptotic bodies were evident in Sox14 overexpressing cell cultures but not in control cells transfected with empty vector ( data not shown ) . These results indicate that Sox14 expression is sufficient to induce apoptosis in l ( 2 ) mbn cells . To examine the function of Sox14 in vivo , we used a Tubulin-GAL4 driver ( Tub-GAL4 ) to ubiquitously express Sox14 dsRNA ( Tub-GAL4/Sox14-RNAi; referred to as Tub-Sox14-RNAi ) . QRT-PCR analysis using RNA from Tub-Sox14-RNAi wandering larvae and 0 hrs APF pupae showed a reduction in Sox14 transcripts of 89+/−2% and 91+/−2% , respectively , compared to wild-type control animals ( Figure 5C ) . The Tub-Sox14-RNAi animals demonstrated lethality at 3rd instar larval , pupal or pharate adult stages . During pupation , the Tub-Sox14-RNAi animals displayed three distinct lethal phases: 14% died during early prepupal development ( i . e . prior to head eversion ) , 74% died during early to mid pupal development ( showed head eversion and/or leg elongation ) and 12% died during the pharate adult stage ( n = 104 pupae ) . In Tub-Sox14-RNAi larvae , defects in the trachea were observed ( Figure 6A ) . The branching of the tracheal system appeared normal but the dorsal tracheal trunks showed severely distorted taenidial folds , collapse of the tracheal cuticle and blackening of the cuticle ( Figure 6B ) . Tub-Sox14-RNAi animals did not eclose , but we dissected out pharate animals and found obvious alterations in the notum ( malformed; split ) and bristles ( missing and mis-oriented ) ( Figure 6C ) . The Sox14-related cellular alterations giving rise to these defects remain to be determined . To initiate investigations of Sox14 in programmed cell death , we first examined the larval midgut ( Figure 6D and E ) ; this tissue was examined since most ( 86% ) Tub-Sox14-RNAi pupae persist past the normal stage of larval midgut cell death . By 4 hrs APF , the proventriculus is significantly reduced in size and the gastric caeca are no longer detectable in wild-type animals . Head eversion occurs at approximately 10–12 hrs APF , at which time point the larval midgut is entirely destroyed , compressed and surrounded by the adult midgut [9] . As expected , in control animals ( Tub-GAL4/+; designated wild-type or wt ) we observed midgut condensation by 4 hrs APF and the gastric caecae were not detectable after 7 hrs APF ( n = 10 ) ( Figure 6E ) . The wild type larval midgut appeared degraded and the remnants were found within the adult midgut by 12 hrs APF ( n = 5 ) . Similar to BR-C mutants [10] , the Tub-Sox14-RNAi pupae showed some condensation of midguts , but a remaining proventriculus and remnants of gastric caecae were still observed even after 7–12 hrs APF ( n = 12 ) ( Figure 6E ) . A remaining proventriculus and gastric caecae remnants were observed even in animals that had clearly undergone head eversion ( ie . 10–12 hrs APF ) . These observations indicate that reduced Sox14 expression results in partially defective larval midgut cell death and thus Sox14 is normally required for complete destruction of the larval midgut . To examine the role of Sox14 in salivary gland cell death , we first examined salivary glands from head-everted Tub-Sox14-RNAi pupae ( n = 23; equivalent to >30 hrs APF at 25°C based on incubation time ) . At this timepoint , all 25 animals still had intact salivary glands . However , since Tub-Sox14-RNAi animals arrest at various developmental ages following head eversion , we used retinal pattern formation [59] as an independent morphological marker to aid in the developmental staging . Retinae were dissected and stained with phalloidin to visualize ommatidial patterning [60] . All 23 animals had fully everted eye discs consistent with development to at least 12 hrs APF ( at 25°C ) , and 8 animals had retinas with ommatidial patterning indicative of development to at least 22 hrs APF at 25°C [59] . Of these 8 animals , ommatidal patterning indicated that 5 developed to at least 30 hrs APF at 25°C ( e . g . Figure 7A ) . In rare instances ( n = 6 out of more than 100 pharate adults dissected ) , we were able to dissect intact salivary glands from Tub-Sox14-RNAi pharate adults with darkened wings and red eyes indicative of development to approximately 100 hrs APF ( 25°C ) [59] . To further analyze Sox14 function in salivary gland cell death , we employed a salivary gland GAL4 driver ( D59-GAL4 ) to express Sox14 dsRNA . A single copy of the driver did not result in a phenotype , but two copies of the driver ( D59-GAL4/D59-GAL4; Sox14-RNAi/TM6B ) resulted in a delay in salivary gland cell death compared to control animals ( D59-GAL4/D59-GAL4; MKRS/TM6B ) ( Figure 7B–D ) . In the control animals , TUNEL positive nuclei were prevalent in salivary glands equivalent to 16–17 hrs APF at 25°C ( ie . 30–32 hrs APF at 18°C ) but were not observed in salivary glands from D59-Sox14-RNAi or Tub-Sox14-RNAi animals at a comparable or later stage , respectively ( Figure 7A–C ) . Together , these results indicate that reduced levels of Sox14 expression result in either a delay or inhibition of salivary gland cell death , and thus Sox14 functions as a positive regulator of salivary gland cell death . To help place Sox14 within the context of the known signaling pathways required for ecdysone-mediated cell death , we examined gene expression in Tub-Sox14-RNAi animals . Transcript levels of two apoptosis effectors , rpr and dronc , and two ecdysone regulated transcription factors , E93 and BR-C , were examined in Tub-Sox14-RNAi wandering larvae and 0 hr APF pupae and compared to controls . While BR-C showed no changes in expression levels between control and Tub-Sox14-RNAi animals at both stages examined , rpr , dronc and E93 transcripts were reduced in Tub-Sox14-RNAi 0 hr APF pupae compared to controls ( Figure 7E ) . These results support a pro-death role for Sox14 , and indicate that Sox14 acts upstream of rpr , dronc and E93 and either downstream or in parallel to BR-C . Future studies are required to determine whether Sox14 directly regulates the transcription of any of these genes , and whether the hierarchical position of Sox14 is conserved in various developmental stages and tissues .
We performed an RNAi screen as a means of gaining new molecular insights into ecdysone induced cell death and cell survival signaling pathways . We enriched for the identification of ecdysone-dependent genes by targeting genes that were differentially expressed in Drosophila larval salivary glands immediately prior to ecdysone-induced cell death 23 , 24 . In total , we verified functionally the pro-death effects of six genes and the pro-survival effects of 18 genes , and further characterized their functions on the basis of ecdysone dependency and cell death effects . More detailed examination of one gene , Sox14 , showed that it was induced by ecydsone and its expression was sufficient to induce apoptosis in vitro . Studies in vivo revealed a role for Sox14 in larval midgut and salivary gland cell death . Potential off-target effects can be a significant issue in any RNAi screen especially when long dsRNAs are used [61] , [62] . Although Drosophila does not have interferon responses as observed in mammals , short dsRNAs ( ≥19 nt ) produced by Dicer processing that are perfect matches to non-target specific transcripts are the likely source of off-target effects [61]–[64] . To help eliminate potential false positives due to off-target effects or experimental noise , we designed a second dsRNA , free of predicted off-target effects and completely non-overlapping with the first dsRNA [65] ( in all but one case – see Materials and Methods ) . For a gene to be considered further , both of its dsRNAs had to produce an effect in the same direction with a p-value of ≤0 . 05 . While we may have eliminated some false negatives due to insufficient RNAi knockdown by this screening strategy , these stringent criteria enabled us to produce a highly reliable final list of candidate genes for further study . Many , but not all , of the dsRNAs corresponding to these candidate genes had similar effects on viability in l ( 2mbn ) and S2 cell lines ( Table S2 ) . The observed differences may be attributable to the different genotypes of these cell lines . Since both l ( 2 ) mbn and S2 cell lines are polyclonal , we also cannot rule out the possibility of an inhomogenous response to the dsRNAs tested . This could affect the overall detectable response to RNAi treatment and thus is another possible reason why results could differ in these or alternate cell lines . Recently , a role for Drosophila autophagy genes atg1 , atg2 , atg3 , atg6 , atg7 , atg8a , and atg12 in salivary gland degradation has been demonstrated [66] . Our study did not find a death related role for autophagy genes in l ( 2 ) mbn cells in the presence of ecdysone . It is possible that these genes do not have an essential death or survival related role under the conditions we tested . Since our screen was optimized to detect effects of genes that are dependent on ecdysone-regulated transcription , we cannot rule out the possibility that additional genes impacting ecdysone-mediated PCD may be detected under different experimental conditions . However , in the absence of ecdysone , knock down of several Atg genes ( atg2 , atg3 , atg5 , atg6 , atg7 , atg8a , atg8b ) resulted in decreased cell viability ( Figure S1 ) indicating a potential pro-survival role for these genes . Our screen was validated by identification of known genes and biochemical complexes with previously established cell survival or cell death phenotypes . For example , Ras85D promotes cell survival in Drosophila by down-regulating hid expression and activity [67] , [68] in vivo . Consistent with these findings , we discovered that decreased Ras85D transcripts resulted in reduced cell survival in an ecdysone dependent manner , while knockdown of hid resulted in a phenotype of increased cell survival . These results suggest that Ras pathway mediated inhibition of Hid activity may exist in the ecdysone signaling pathway . We also identified Smr , a co-repressor , and dSin3A , a transcriptional regulator , that associate with each other to mediate the transcriptional silencing of the EcR∶USP complex . Addition of ecdysone completely dissociates Smr from the EcR∶USP heterodimer complex and activates EcR∶USP mediated transcription . Elimination of repression by Smr/Sin3A on EcR∶USP activity resulted in lethality in vivo [69] . Based on these observations , we predicted that reduced expression of either Smr or Sin3A or both by RNAi in our system would release , as with ecdysone , the repression caused by these gene products on the EcR∶USP complex , resulting in increased EcR∶USP activation and subsequent increased cell death . As we expected , our cell viability/TUNEL assays in l ( 2 ) mbn cells indicated clearly that knock-down of Smr transcripts resulted in increased cell death in an ecdysone dependent manner ( Table 2 ) . The identification of such known ecdysone signaling complexes demonstrates that our assay is a viable method for functional verification and initial characterization of genes involved in ecdysone-mediated death/survival pathways The predicted or known function of several pro-survival genes identified in our screen ( Pros26 . 4 , Rpn2 , Tbp-1 and Cp1 ) was associated with protein degradation processes . Under stress conditions , down regulation of gene products associated with protein degradation processes could impair energy production and , therefore , reduce the survival of the cell/organism . The 26S proteasome complex , a major site of protein degradation , is made up of two multi-subunit sub complexes , namely the 20S Proteasome and PA700 ( 19S complex ) . The identified pro-survival genes , Pros26 . 4 , Rpn2 , and Tbp-1 all belong to the PA700 subunit of the 26S proteasome complex . Proteasome function is required for cell proliferation [70] and silencing the expression of gene products belonging to the PA700 complex by RNAi reduced cell proliferation and induced apoptosis in S2 cells [49] , [71] . Consistent with these previous findings , our results indicated that Pros26 . 4 , Rpn2 , and Tbp-1 knockdown led to reduced viability of l ( 2 ) mbn and S2 cells both in the presence and absence of ecdysone . In our RNAi screen , the pro-survival genes that were associated previously with protein degradation ( as above ) or protein transport ( Kap-α3 ) were significantly up-regulated prior to larval salivary gland histolysis [24] . During PCD , anabolic processes are reduced and , therefore , a replenishable source of carbohydrates is unavailable for energy production . Thus , it is possible that ecdysone may activate protein degradation processes in salivary glands to produce energy to complete the death process . Our RNAi screen identified five previously uncharacterized genes ( CG13784 , CG15239 , CG32016 , CG33087 , and CG7466 ) as pro-survival genes . Among these , CG13784 , CG32016 and CG33087 were ecdysone dependent for their pro-survival role . Further studies are required to determine whether these three genes affect survival in response to other agents that induce cell death; our preliminary data ( not shown ) indicates that they do not have any effects on staurosporine induced cell death . The products of CG33087 ( calcium ion binding; ATPase activity; low-density lipoprotein receptor activity ) and CG7466 ( receptor binding; cell-cell adhesion ) have predicted functions based on protein domains but CG13784 , CG15239 , and CG32016 have no illuminating sequence characteristics . We further characterized CG32016 in l ( 2 ) mbn cells by the TUNEL assay in both the presence and absence of ecdysone . Knock-down of CG32016 resulted in increased TUNEL positive cells only in the presence of ecdysone , indicating a potential cell death-related , ecdysone-dependent pro-survival role . We are the first to associate a function with these previously uncharacterized gene products ( CG13784 , CG15239 , CG32016 , CG33087 , and CG7466 ) ; additional studies will be required to elucidate their specific positions and functions in response to ecdysone . Of the 25 genes that were identified in our screen , seven genes ( Table 1 ) were identified as potential pro-death genes . Of these seven genes , five were ribosomal genes . In Drosophila , 38 small ( 40S ) and 49 large ( 60S ) ribosomal proteins have been identified [72]; the small ribosomal subunits belong to the eukaryotic pre-initiation complex and the large ribosomal subunits are usually involved in translation . We tested in our RNAi screen the five ribosomal genes that were differentially expressed in the Drosophila larval salivary glands immediately prior to PCD [24] . RNAi of both small ribosomal genes ( RpS5 , RpS6 ) and large ribosomal genes ( RpL13A , RpL37 and RpLP1 ) resulted in increased cell viability of ecdysone treated l ( 2 ) mbn cells , indicating that these genes may have a pro-death role in the presence of ecdysone . Further , with staurosporine treatment ( data not shown ) , RNAi of these ribosomal genes resulted in reduced cell viability , indicating that ecdysone is indeed required for the increased viability effect of these dsRNAs l ( 2 ) mbn cells . Ecdysone treatment induces transcription of pro-death genes such as BR-C , dronc , rpr and hid , and ribosomal gene products are required for their translation . Thus , knocking down ribosomal gene products by RNAi may affect efficient translation of pro-death genes leading to the observed phenotype of increased viability . However , in S2 cells , knock-down of these ribosomal genes in the presence of ecdysone did not increase cell viability but rather significantly decreased viability ( Table S2 ) ; further studies are required to understand these cell line dependent effects . In the absence of ecdysone , RNAi of these same ribosomal genes resulted in reduced viability in both l ( 2 ) mbn and S2 cells , supporting a pro-survival role under these conditions . This pro-survival effect is similar to that reported in S2 and Kc cells by others [47] , [49] . A pro-survival function of ribosomal proteins in the absence of ecdysone is in agreement with the key role they play in protein-synthesis and , therefore , in cell growth and cell proliferation . Our screen identified two additional gene products required for ecdysone-mediated cell death: i ) dSH3PX1 , involved in intracellular protein transport and resembling a sorting nexin with an NH2-terminal SH3 domain and a central phox homology ( PX ) domain [73] , [74] , and ii ) Sox box protein 14 ( Sox14 ) , a High mobility group ( HMG ) box-containing transcription factor related to the mammalian sex determining factor , SRY [75] . dSH3PX1 acts as a binding partner for the non-receptor Cdc-42 associated kinase ( ACK ) in Drosophila [76] . A similar interaction between ACK2 and SH3PX1 ( also called SNX9 ) occurs also in mammals where further studies showed that phosphorylation of SH3PX1 by ACK2 regulates the degradation of EGF receptor [77] . Thus , it is possible that the knockdown of dSH3PX1 by RNAi in l ( 2 ) mbn cells results in decreased cell death through enhanced EGF receptor-mediated cell survival signaling . Alternatively , the role of dSH3PX1 in cell death may be related to its associations with proteins involved in receptor trafficking and/or cytoskeletal rearrangements [78] . Our in vitro and in vivo analyses also identified for the first time a pro-death role for the transcription factor Sox14 . Previously [24] we determined that of 19 genes tested , just two genes , Sox14 and ark , were independent of E93 regulation in dying larval salivary glands . This previous finding indicates that Sox14 may act in parallel to E93 or may be acting upstream of E93 in the ecdysone induced cell death pathway . Our gene expression analyses reported here ( Figure 7E ) position Sox14 upstream of E93 , and also upstream of rpr and dronc that are known to be regulated by E93 [27] . A recent microarray study conducted during Drosophila pupariation further supports this view as Sox14 was identified as an ecdysone primary-response regulatory gene [79] . Based on comparison of the HMG box region , Drosophila Sox14 is most similar to mouse Sox4 and human Sox4 , 11 and 22 [75] , [80] , [81] . Sox proteins regulate multiple downstream targets and are involved in numerous developmental processes . In particular , human Sox 4 has been implicated in both the positive [82] , [83] and negative [84] regulation of apoptosis . Our in vivo studies using a Sox14-RNAi construct support a pro-death role for Sox14 during Drosophila ecdysone-triggered larval midgut ( Figure 6 ) and salivary gland cell death ( Figure 7 ) . During metamorphosis , the larval midgut disintegrates and a new adult gut is formed . These two events overlap and the adult gut encompasses disintegrating larval gut tissue [9] . In Tub-Sox14-RNAi animals , adult midgut cells are visible at 4 hrs APF similar to wild type gut , but complete condensation of the larval midgut and complete disintegration of the proventriculus and gastric caecae were inhibited at least up to 12 hrs APF . This observation is similar to what was observed in BR-C mutants , but different from E93 mutants which showed defects in larval midgut compaction but not destruction of the proventriculus and gastric caecae [10] . Thus , the midgut cell death defective phenotype of Sox14 is again in agreement with our prediction that Sox14 is acting upstream of E93 and downstream or parallel to BR-C . Our results using both the Tub-Sox14-RNAi ( tubulin GAL4 driver ) and D59-Sox14-RNAi ( salivary gland GAL4 driver ) animals support a role for Sox14 as a positive regulator of salivary gland cell death . Cell death was delayed in D59-Sox-14 RNAi animals and was either delayed or inhibited as late as the pharate adult stage in Tub-Sox14-RNAi animals . It is possible that the less severe phenotype in the D59-Sox14-RNAi animals is due to less efficient RNAi-mediated knockdown of Sox14 , a notion that is supported by our observed dose-dependent effects of the D59-GAL4 driver . Given the predicted function of Sox14 as a transcription factor , it is particularly likely that even reduced amounts could still lead to some wild-type function . It is also possible that Sox14 functions in a partially redundant manner in both the midgut and salivary gland so that even a complete loss of function may lead to only a partial loss or delay in cell death . Null mutants of Sox14 would be valuable for future testing of these possibilities . In addition to defects in midgut and salivary gland cell death , we observed tracheal defects in Tub-Sox14-RNAi animals , similar to defects observed in mutants of DHR3 which encodes an ecdysone responsive orphan nuclear receptor [85] . Preliminary examination of Tub-Sox14-RNAi pharate adults indicated additional roles for Sox14 in notum and bristle development . Future studies are required to determine the function of Sox14 in these and other tissues . Given its predicted role as a transcriptional regulator and its position in the ecdysone signaling cascade , it is likely that Sox14 will function in various cellular processes . In summary , we developed an RNAi-based screening system to identify genes that are required for ecdysone-mediated cell death and survival pathways . Our screen identified known and novel components of the ecdysone signaling network that act as pro-death or pro-survival genes . In particular , we have shown that in some cases the function of a gene is dependent on ecdysone , or its mechanism of action is variable depending on the presence or absence of ecdysone . In vivo studies of Sox14 support a role in ecdysone-mediated cell death . Further characterization of the novel genes identified is necessary to elucidate their specific roles and positions in the ecdysone signaling network .
For the initial screen , individual PCR products up to 735 bp in length and containing coding sequences for the transcripts to be knocked-down ( Table S1 ) were generated by RT-PCR using 500 ng of total RNA and Superscript one-step RT-PCR kit with platinum taq ( Invitrogen ) . Each primer used in the RT-PCR contained a 5′ T7 RNA polymerase binding site ( TAATACGACTCACTATAGG ) followed by sequences specific for the targeted genes ( see Table S1 ) . RT-PCR products were isopropanol-precipitated and the entire product from each reaction was used as template for in vitro transcription reactions . In vitro transcription reactions were carried out using either Megascript T7 transcription kit ( Ambion ) or T7 RiboMax Express RNAi systems ( Promega ) according to the manufacturer's instruction . dsRNAs synthesized were incubated at 65°C for 30 min followed by slow cooling to room temperature . dsRNAs were ethanol precipitated and resuspended in 50 µl nuclease free water . A 5 µl aliquot of 1/100 dilution was analysed by 1% agarose gel electrophoresis to determine the quality of dsRNA . The dsRNAs were quantitated using a picogreen assay ( Invitrogen ) and concentrations adjusted to 100 ng/µl with nuclease free water . For genes of interest identified in our initial screen ( for complete list see Table S1 ) , we designed a second non-overlapping dsRNA to confirm the observed phenotype . The German Cancer Research Center ( DKFZ ) ERNAi search tool ( Off-Target Search Tool: http://www . dkfz . de/signaling2/e-rnai/ ) [86] was used to search the RNAi probes for potential off-target effects using a 19 bp fragment length cut-off . Our final criteria for confirmation of RNAi effects was that for each gene , at least one of its dsRNAs had no predicted off-target effects ( i . e . 100% specificity ) and a second dsRNA had at least 98% specificity . Of the 20 genes that we confirmed by this method , 19 were represented by two dsRNAs that were completely non-overlapping . One additional gene confirmed by this method , RpL13A ( CG1475 ) , was represented by two dsRNAs that overlapped by 21 bp . Analysis of this 21 bp by the Off-Target Search Tool indicated 0 potential secondary targets . l ( 2 ) mbn cells [42] and S2 cells ( Invitrogen ) were grown in Schneider's ( Invitrogen ) medium supplemented with 10% FBS , 50 units/ml penicillin and 50 µg/ml streptomycin ( Gibco-BRL ) ( hereafter referred to as Schneider's medium+10%FBS ) in 25-cm2 suspension flasks ( Sarstedt ) at 25°C . All experiments were carried out 3 days after passage and the cells were discarded after 25 passages . 20-Hydroxyecdysone ( ecdysone ) was obtained from Sigma-Aldrich and resuspended in 95% ethanol at a concentration of 10 mM . Three days after passage , cells were adjusted to 1×106 cells/ml in ESF921 serum free media ( Expression systems ) and 3×105 cells ( 333 µl ) were seeded into each well of a 24 well plate . After one hour incubation , 667 µl of Schneider's medium+10%FBS and ecdysone ( 10 µM final ) ( Sigma ) was added to yield 1 ml culture in each well . Treated cells were incubated at 25°C for 24 , 48 , or 72 hours and 1 ml cultures were transferred to RNAse free eppendorf tubes ( Ambion ) and cells were pelleted at 1000 rpm for 10 min . Cell pellets were lysed in 1 ml Trizol ( Invitrogen ) and total RNA was extracted according to manufacturer's instructions . Isolated RNA was treated with RNAse free DNAse and 50 ng of total RNA was used in 15 µl QRT-PCR reactions . QRT-PCR reactions were carried out using the one-step SYBR green RT-PCR Reagent kit ( Applied Biosystems ) on an Applied Biosystems 7900 Sequence Detection System . Expression levels were calculated using the Comparative Cycle Threshold ( CT ) Method ( 2−[delta][delta]Ct method; User Bulletin #2 , ABI Prism 7700 Sequence Detection System , Applied Biosystems , 2001 ) with Drosophila rp49 , a ribosomal housekeeping gene , as the reference for normalization . To determine the fold change in expression levels of known ecdysone signaling genes , cell death related genes and Sox14 following ecdysone treatment of l ( 2 ) mbn cells , the CT values were normalized to rp49 in the same sample ( hereafter referred to as normalized CT values ) for each gene , and were compared to the normalized CT values for the same gene from untreated control cells . Similarly , for the RNAi experiments , normalized CT values for each gene from ecdysone plus dsRNA-treated cells were compared to the normalized CT values from cells treated with ecdysone plus control human dsRNA , and the knock-down efficiency was calculated . Knock-down efficiency = 100-[ ( Fold expression of targeted gene in dsRNA+ecdysone treated cells/Fold expression of targeted gene in ecdysone treated cells ) ×100] . For RNAi in l ( 2 ) mbn cells , a 33 µl volume of ESF921 media containing 3×104 cells was seeded into each well of a 96 well plate for RNAi screens . Into each well , 500 ng–1000 ng of dsRNA in a 5 µl volume was added , and incubated for one hour at room temperature . The untreated control cells received 5 µl of nuclease free water . After one hour incubation , the cells received Schneider's medium+10% FBS containing ecdysone ( 10 µM final ) to yield a final 100 µl volume . Cells were incubated for 72 hours at 25°C and 10 µl of WST-1 reagent ( Roche Scientific ) was added . A450–A650 readings were taken after overnight incubation using a 96 well spectrophotometer VersaMax ( Molecular Devices ) . A450–A650 readings of experimental samples were always compared to A450–A650 readings from cells treated with human dsRNA to control for any non-specific RNAi effects . RNAi experiments in S2 cells were essentially performed as for l ( 2 ) mbn cells with the following modifications: 1000–1500 ng of dsRNA in a 5–7 . 5 µl volume was added . Cells received 1 µM ecdysone overnight and then the ecdysone concentrations were increased to 10 µM final . Cells were incubated for a total of 48 hours ( following addition of dsRNA ) at 25°C and then 10 µl of WST-1 reagent was added . Assay readings were taken after overnight incubation . All samples were analyzed at least in triplicate . To quantitate changes in cell shape , indicative of cell differentiation , following ecdysone treatment and RNAi , five images from two biological replicates were captured using an Axiovert 200 fluorescent microscope ( Carl Zeiss ) . The observed number of spindle-shaped and rounded cells were counted manually . Percent spindle-shaped ( differentiated ) cells was calculated as the Number of spindle-shaped cells/ ( Number of spindle-shaped cells+ number of rounded cells ) *100 . RNAi experiments were carried out as described above and cell proliferation was determined using the Cell Proliferation BrdU ELISA kit ( Roche Scienitific ) . Cells received BrdU 64 hrs after dsRNA treatment and the cells were incubated for another 24 hrs . Cells were then processed as per manufacturer's instructions and A370–A492 readings were taken using a 96 well spectrophotometer VersaMax ( Molecular Devices ) . A370–A492 readings from experimental RNAi treatments were compared to the reading from cells treated with human dsRNA . For the TUNEL assay in vitro , RNAi experiments were carried out as described above except the cells were seeded into each well of 16-well CC2 coated chamber slides ( Nunc ) . After 72 hours of respective treatment , cells received 100 µl of hypotonic solution ( 75 mM KCl ) for 3–5 min . at 25°C . Cells were then fixed with 3∶1 methanol∶acetic acid solution and air dried . Cells were washed with 1XPhosphate buffer saline ( Sigma ) and processed with TUNEL using the DeadEnd fluorometric tunel system ( Promega ) . Cells were mounted with Slowfade antifade reagent with DAPI ( Invitrogen ) and viewed using a Zeiss Axioplan 2 microscope . Images were captured using a cooled mono 12 bit camera ( Qimaging ) and Northern Eclipse image analysis software ( Empix Imaging Inc . ) and the number of TUNEL positive cells ( green ) and number of DAPI positive cells ( blue nuclear stain ) were visually counted . All samples were analysed with at least two biological replicates , and three images from each replicate were taken using a 20× objective for counting the TUNEL and DAPI positive cells . Percent TUNEL positive cells were calculated as ( TUNEL positive cells/Total number of cells ) ×100 . For the TUNEL assay in tissue , dissected salivary glands were first fixed with 4% paraformaldehyde and then permeabilized with 1% TritonX-100 . The TUNEL procedure was performed using the DeadEnd fluorometric tunel system ( Promega ) and salivary glands were mounted in Slowfade antifade with DAPI ( Invitrogen ) and viewed as described above . Plasmids were constructed using the GATEWAY system ( Invitrogen ) as follows: A full length cDNA was first generated as an RT-PCR product from total Drosophila RNA using Superscript III and platinum Pfx polymerase ( Invitrogen ) with the gene-specific primers GAAGACGCGTCAAAGCATTTATTCTCGCGTTT and TATGGTACCTAGTGCACAC TCACACTCG ( underlined portions represent sequence added to create restriction enzyme sites for subsequent cloning ) . PCR products containing the Sox14 ORF flanked by AttB1and AttB2 sequences were amplified from the full length cDNA using platinum Pfx polymerase ( Invitrogen ) . The primers GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATAGCTAAGCCCAACCAGGC and GGGGACCACTTTGTACAAGAAAGCTGGGTC TCACATTTTATAATAACTTGCAAACTCG were used to amplify a product suitable for an N-terminal fusion construct and the primers GGGGACAAGTTTGTACAAAAAAGCA GGCTTCACCATGATAGCTAAGCCCAACCAG and GGGGACCACTTTGTACAAGAAA GCTGGGTCCATTTTATAATAACTTGCAAACTCGTATT were used to generate a product for the C-terminal fusion construct . ( Sox14-specific sequences are underlined . ) The PCR products were cloned into the entry clone , pDONR221 , ( Invitrogen ) which contains AttP sites . These entry clones were then used to shuttle the protein-coding region of the genes into the GATEWAY expression vectors pAFW or pAWF , containing either N-or C-terminal FLAG respectively ( Drosophila Genomics Resource Center ) , as required . Sox14-FLAG expression constructs were used for overexpression studies in l ( 2 ) mbn cells . l ( 2 ) mbn cells were grown as described above . For transfection experiments , 1 µg of plasmid DNA and 10 µl of Cellfectin ( Invitrogen ) were combined in 200 µl of serum-free Grace medium ( Invitrogen ) for 30 min . Immediately prior to transfection , 3×106 cells in 800 µl of Grace medium were prepared and incubated with the transfection medium ( a total of 1 ml culture ) overnight in a 24 well suspension culture plate ( Sarstedt ) . Cells were equally spilt into two wells ( 500 µl each ) , and each well received 1 ml of Schneider+10%FBS medium . Cells were incubated for up to 96 hrs . At 48 and 72 hrs after transfection , 100 µl of cells were plated into a 96 well plate , 10 µl of WST-1 reagent was added , and absorbance readings were taken after overnight incubation . WST-1 readings of cells transfected with Sox14 expression constructs were compared to the cells transfected with empty vector ( negative control ) . Transfected cells were monitored for the presence of apoptotic bodies up to 96 hrs after transfection . Samples were analyzed in triplicate , with two biological replicates of each construct . A UAS-Sox14-RNAi Drosophila line was obtained from the Vienna Drosophila RNAi Center . Heterozygous animals containing the Sox14-RNAi construct , balanced over TM6B , Tb1 were crossed to a stock carrying the Tubulin-GAL4 driver ( w* ; Tub-GAL4/TM6B , Tb1 , derived from TubP-GAL4LL7 ( Bloomington stock centre ) ) to drive the expression of Sox14-RNAi in vivo . The phenotype of the resulting F1 non-tubby progeny ( Sox14-RNAi/Tub-GAL4; designated Tub-Sox14-RNAi ) was compared to control animals ( Tub-GAL4/+ ) designated as wild-type ( wt ) . The knock-down efficiency of Sox14 was determined by comparing transcript levels in Sox14-RNAi/Tub-GAL4 animals to the wt +/Tub-GAL4 animals using QRT-PCR as described above . For salivary gland-specific Sox14-RNAi studies , animals containing UAS-Sox14-RNAi were crossed to a strain containing the D59 salivary gland driver [87] and a strain containing two copies of salivary gland driver and one copy of UAS-Sox14-RNAi was established ( D59-GAL4/D59-GAL4; Sox14-RNAi/TM6B; designated D59-Sox14-RNAi ) . Control animals were D59-GAL4/D59-GAL4; MKRS/TM6B . Retinae were dissected from Tub-Sox14-RNAi animals following salivary gland dissection . Retinae were fixed with 4% paraformaldehye for 20 minutes and permeabilized with 1% TritonX-100 . Phalloidin-Rhodamine ( Invitrogen ) was used to stain and reveal outline of cells . Retinae were mounted in Slowfade antifade with DAPI and viewed on a Zeiss Axioplan 2 microscope . Ommatidial organization , cell number and apical profile were used to assess developmental age [59] . Probability p-values were calculated with Student's t-test using two-tailed distribution and two-sample equal variance . Table S1 provides a complete list of genes targeted by RNAi along with their primer sequences , amplicon lengths , cell viability results and p values . Table S2 includes a comparison of WST-1 assay results in l ( 2 ) mbn and S2 cells for the identified candidate pro-death and pro-survival genes . Figure S1 shows the cell viability effects of Atg gene RNAi in the absence of ecdysone and Figure S2 shows results of the BrdU cell proliferation assay . | Hormones regulate complex signaling pathways required for the differentiation , growth , survival , and death of cells in diverse organisms . The insect steroid hormone 20-hydroxy ecdysone ( ecdysone ) triggers cell death of obsolete larval tissues , such as the midgut and salivary glands , during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival . Previous studies identified many genes and proteins associated with fruit fly salivary gland cell death , but functional verification was lacking . Here , we have analyzed 460 of those genes using RNAi , a genetic approach to inhibit gene function , to assess their possible cell death or cell survival related function . To our knowledge , this is the first large-scale functional screen for genes involved in steroid hormone regulated cell death and cell survival . We identified several novel ecdysone regulated components with a cell death/survival role , including genes with no previously known function . In vivo analyses of animals harboring an RNAi construct targeting the transcription factor Sox14 , one of the genes identified , confirmed its role as a positive regulator of ecdysone-mediated cell death . Our results provide a foundation for further studies of the molecular mechanisms by which steroid hormones control the death and survival of cells . | [
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] | 2009 | Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi |
Metabolism is central to cell physiology , and metabolic disturbances play a role in numerous disease states . Despite its importance , the ability to study metabolism at a global scale using genomic technologies is limited . In principle , complete genome sequences describe the range of metabolic reactions that are possible for an organism , but cannot quantitatively describe the behaviour of these reactions . We present a novel method for modeling metabolic states using whole cell measurements of gene expression . Our method , which we call E-Flux ( as a combination of flux and expression ) , extends the technique of Flux Balance Analysis by modeling maximum flux constraints as a function of measured gene expression . In contrast to previous methods for metabolically interpreting gene expression data , E-Flux utilizes a model of the underlying metabolic network to directly predict changes in metabolic flux capacity . We applied E-Flux to Mycobacterium tuberculosis , the bacterium that causes tuberculosis ( TB ) . Key components of mycobacterial cell walls are mycolic acids which are targets for several first-line TB drugs . We used E-Flux to predict the impact of 75 different drugs , drug combinations , and nutrient conditions on mycolic acid biosynthesis capacity in M . tuberculosis , using a public compendium of over 400 expression arrays . We tested our method using a model of mycolic acid biosynthesis as well as on a genome-scale model of M . tuberculosis metabolism . Our method correctly predicts seven of the eight known fatty acid inhibitors in this compendium and makes accurate predictions regarding the specificity of these compounds for fatty acid biosynthesis . Our method also predicts a number of additional potential modulators of TB mycolic acid biosynthesis . E-Flux thus provides a promising new approach for algorithmically predicting metabolic state from gene expression data .
Metabolism is central to cell physiology and metabolic disturbances play a role in numerous disease states . Despite its importance , the ability to study metabolism at a global scale using genomic technologies is limited . In principle , complete genome sequences describe the range of metabolic reactions that are possible for an organism , but cannot quantitatively describe the behaviour of these reactions . Gene expression data provide global insight into the regulation of metabolic reactions , but methods for inferring the behaviour of metabolic networks , and particularly metabolic flux , from these data are limited . There is thus a need to develop computational approaches that utilize available genomic data to make inferences about metabolism at the level of large scale metabolic networks . One approach to computationally studying metabolism is to develop detailed models based on coupled differential equations describing the dynamics of enzyme action . Such models , however , require measuring numerous kinetic parameters that can be prohibitively difficult for large systems and for organisms – such as infectious disease agents – that are difficult to work with experimentally . Flux balance analysis ( FBA ) is an alternative approach to modeling metabolism without developing detailed simulation models that include enzyme kinetics [1]–[4] . It exploits the fact that the stoichiometries of metabolic reactions are not organism-dependent but are fixed by chemistry and mass balance . Moreover , the availability of complete genome sequences is enabling the reconstruction of metabolic networks whose constituent reactions have known stoichiometries . Flux balance analysis also exploits the fact that enzyme dynamics occur quickly compared , for example , to regulatory changes in gene expression: when the relevant laboratory time period ( often hours ) is much longer than the chemical reaction times ( typically minutes ) , transient dynamics last for only a small portion of time period considered , after which the metabolic network functions at steady state . FBA is a method for utilizing universal reaction stoichiometries to predict a network's capability to produce a metabolic objective under steady-state conditions . Briefly , FBA represents a metabolic network by capturing the stoichiometries of constituent reactions in a stoichiometric matrix , S , and describing a flux configuration as a set of rates at which the reactions in a network are moving ( ie the set of reaction fluxes ) . FBA requires that constraints for some reactions be known , reflecting their maximum or minimum rates . These constraints can either be measured ( e . g . uptake reactions ) or calculated from physical parameters ( e . g . oxygen diffusion ) or thermodynamic constraints . In many cases , the constraints can be related to the degree of enzymatic activity for the given reaction . The matrix S and the set of reaction constraints define the set of all possible flux configurations at steady state . A flux configuration can be visualized as a vector in flux space , and all flux configurations that are feasible at steady state lie within a cone in this space , called the flux cone . The core approach of FBA is to choose a metabolic objective which is a linear function of fluxes , and then use linear programming to optimize this objective subject to the constraints . The algorithm results in one or more flux configurations that are optimal for the chosen metabolic goal , and the optimal production capacity of that objective . FBA provides a method for exploring capabilities and states of a metabolic system at steady state , and genome scale metabolic models can be reconstructed based on annotated genome sequences coupled with literature curation [1] , [2] . FBA has been used to successfully predict the metabolic phenotype of gene knockouts [1]–[3] , and the use of metabolic modeling in this case has the advantage of predicting nutrient-dependent phenotypes . FBA has also been used to predict the time courses of growth , substrate uptake , and metabolite production by both Escherichia coli and Mycobacterium tuberculosis using a pseudo-steady-state dynamic modeling approach [4]–[6] . FBA has recently been used as part of an integrated analysis scheme for drug identification; there is a recent publication ( targetTB ) by Raman et al . that reports this approach [7] . While powerful , FBA is limited in that it does not take into account the gene regulatory state , as described for example by gene expression data . In effect , the basic approach predicts metabolic capabilities assuming all reactions have the same maximum capacity . Indeed , many of the errors in the prediction of gene knockout phenotype were traced back to the lack of gene regulation in standard FBA models [1] , [2] . Incorporating a Boolean model of gene regulation with FBA allows the prediction of more biologically realistic dynamic behaviour , including for example a diauxic shift in response to changing carbon source availability [8] . However , this approach reduces gene expression to Boolean variables , using either a constant value or 0 for the upper flux bound , rather than making use of direct measurements of gene regulation through whole cell expression data . We have developed a method , which we call “E-Flux” , to predict metabolic capacity based on expression data . E-Flux extends FBA by incorporating gene expression data into the metabolic flux constraints . We applied E-Flux to M . tuberculosis ( M . tb ) , the pathogen that causes tuberculosis ( TB ) . An estimated one third of the world's population has been exposed to this disease , which is estimated to kill 1 . 6–1 . 8 million annually worldwide . Multiple drug resistant ( MDR ) and extensively drug resistant ( XDR ) strains of tuberculosis are emerging worldwide , so the development of new drugs is of the essence . Bacterial metabolism plays an important role in TB pathology , both in terms of metabolic alterations associated with intracellular growth [9]–[12] as well as through the production of metabolic products associated with virulence – including mycolic acids [13]–[15] . Given M . tb's slow growth rate , the hazards of experimenting directly with this infectious organism , and limitations in measuring all metabolites simultaneously , there is considerable motivation to augment experimental approaches with computational methods for predicting M . tb metabolism . We used E-Flux to predict the impact of drugs and environmental conditions on mycolic acid biosynthesis capacity in M . tb , based on a compendium of expression measurements from these conditions . Our method successfully identifies seven of the eight known inhibitors of mycolic acid or fatty acid production that were present in the compendium . E-Flux also correctly predicts whether conditions are directly inhibiting mycolic acid production , or inhibiting production indirectly through other mechanisms . Our method thus provides a promising approach to modeling metabolic state from whole cell measurements of gene regulation .
The key innovation underlying the E-Flux approach is that we use expression data to model the maximum possible flux through metabolic reactions . When the expression for a particular enzyme-coding gene is low ( relative to some reference ) , we place a tight constraint on the maximum flux through the corresponding reaction ( s ) . When expression is high we place a looser constraint on the flux through the reaction ( s ) . We then use FBA with the applied constraints and an appropriate objective function to determine a corresponding metabolic state or optimal metabolic capacity . Conceptually , our method can be understood as setting the width of “pipes” around particular reactions as a function of expression state . Figure 1 illustrates this for a simple metabolic model with 4 metabolites and 4 internal reactions catalyzed by enzymes corresponding to 4 genes , together with an uptake reaction and a reaction converting one metabolite into biomass . Two different sets of simulated gene expression data are shown in the two panels . In the top panel , G1 is poorly expressed . Our method models this conceptually as a thin pipe around reaction 1 , limiting the maximum flux through this reaction . Conversely , in the bottom panel , G1 is highly expressed corresponding to more possible flux ( a wider pipe ) . Under conditions in which substrate is not limited , we would predict more flux through reactions 3 and 4 , and less through reactions 1 and 2 in the top panel . Conversely , we might predict more flux through reactions 1 and 2 , and less through 3 and 4 , in the bottom panel . Geometrically , setting maximum flux constraints according to gene expression reshapes the flux cone . Different gene expression states result in different flux cone geometries , which can lead to different solutions for the same metabolic objective . Reshaping the flux cone and thus generating different flux configurations is similar to the approach used to predict phenotypes from gene knockouts using FBA [1]–[3] and for coupling Boolean regulatory models with metabolic models [8] . However , such approaches have used constraints that are the same for all reactions except those that are turned off . By contrast , E-Flux shapes the cone not by turning individual genes on or off , but by giving many or all genes in the model a range of possible flux limits . More importantly , we are reshaping the flux cone on the basis of empirical measurements of gene expression . Our method does not assume that enzyme concentrations , enzyme activities , or realized reaction fluxes are determined by mRNA expression values . Indeed , the true flux for a reaction depends on the enzyme kinetics and concentration , as well as the concentration of metabolites . The effective enzyme concentration in turn depends on gene expression , transcription and translation , post-translational modification and degradation . It would be prohibitive to determine these values for many reactions in an organism . However , the biological rationale behind our method is that expression data provide measurements on the level of mRNA for each gene . If there were limited accumulation of enzyme over the time course considered , and given a particular level of translational efficiency , the level of mRNA can be used as an approximate upper bound on the maximum available protein and hence as an upper bound on reaction rates to some level of approximation . This allows us to extend flux balance analysis from an algorithm that assumes that all reactions have the same constraint , as has been done previously , to an approach making use of condition-dependent , empirical data . E-Flux allows us to link such data directly to changes in metabolic capability . We discuss rationale behind our method further in the Discussion . Mathematically , our approach modifies FBA as follows . FBA involves solving the following optimization problem: ( 1 ) where v is a flux vector representing a particular flux configuration , S is the stoichiometric matrix , c is a vector of coefficients that defines a linear objective function cTv , and aj and bj are the minimum and maximum fluxes through reaction j . We assume that we have a set of expression measurements for some or all of the genes associated with the reactions in S . The core E-Flux method chooses the maximum flux , bj , for the jth reaction according to a function of the expression of gene j and associated genes: ( 2 ) If the reaction catalyzed by the corresponding enzyme is reversible then aj = −bj , otherwise aj = 0 . For the results presented here , “associated genes” refers both to genes that are components of the same enzyme complex , and genes associated with separate isozymes of the reaction . In the latter case , we choose f to be a monotonically increasing function of the expression of the corresponding genes . In general bj can also depend on genes that modulate the activity of the enzyme for reaction j and f can thus take on a more general form . In the Discussion section , we examine the question of which genes to associate with a particular maximum flux constraint and the functional form of f . We tested E-Flux on two metabolic models that include the biosynthesis of mycolic acids in M . tb . The first model consisted of just those reactions underlying mycolic acid production . Mycolic acids are cell wall components characteristic of mycobacteria and essential for the survival of the bacterium [13] . Because the mycolic acid biosynthetic pathway does not exist in non-actinmycetales species , including humans , it is the target of several of the most common antibiotics used to treat TB including isoniazid , thiolactoymycin and ethionamide . Moreover , a metabolic sub-model for this pathway has been previously published [7] . This model , which included 28 proteins , 219 reactions and 197 metabolites , contained four sub-pathways representing the production of malonyl CoA , the fatty acid synthase ( FAS ) I and II pathways , and the condensations of the resulting FAS products into alpha- , methoxy- and keto- mycolic acids . We augmented this model with two additional genes subsequently identified with mycolic acid biosynthesis [16] . We analyzed the microarray data from the Boshoff TB gene expression compendium [17] . This compendium consists of data from several studies , totalling 437 microarray experiments which measured the transcriptional response of M . tuberculosis to 75 different substances and conditions , including known anti-tubercular drugs , growth conditions and unknown compounds . Specifically , this set also included eight known inhibitors of mycolic acid production . Our goal was to use E-Flux to predict the impact of each of these compounds or conditions on mycolic acid biosynthetic production in M . tuberculosis . To explore the method's relevance to data from diverse groups and for a variety of experimental conditions , we also analyzed a set of expression data of Karakousis et al . [18] . These authors analyzed global gene expression profiles to study the action of isoniazid , a mycolic acid inhibitor and front-line antitubercular agent , on several models of M . tb's dormancy phase . Two genome-scale metabolic models are available for M . tuberculosis , namely those of Beste et al . [6] and Jamshidi and Palsson [19] . To validate that our method scales to genome-wide metabolic model , we applied E-Flux to the comprehensive model of M . tuberculosis metabolism of Beste et al . [6] . This was chosen because the model contains more genes and the predictions for gene essentiality were better than those of Jamshidi and Palsson , whose focus was more on growth rates . Since our analysis is comparative in nature we felt that the qualitative advantage of a model with more correct gene essentiality was relevant . Beste et al . 's model [6] was modified by merging this genome scale model with the mycolic acid submodel of Raman et al . [7] . Specifically , we removed mycolic acid reactions from the genome-scale model and replaced them with the mycolic acid reactions in Raman et al . 's model , and normalized the bounds on exchange reactions ( see Methods and Supplementary Material for more details ) . The net result was to replace Beste et al . 's representation of mycolic acids with that of Raman et al . , as the latter is more detailed and as this allows direct comparison of the results of E-Flux in the two models . As with the model of mycolic acid production , we applied E-Flux to the genome scale model to predict the impact of each of the compounds or conditions in the Boshoff TB gene expression compendium on mycolic acid biosynthetic production [17] . Our computational approach is shown in Figure 2 . We first pre-processed the expression data using a previously published analysis of variance ( ANOVA ) method [20] . This method utilizes replicates within and between conditions to estimate sources of noise including variations between binding affinities at different spots on each chip , variations from chip to chip , various binding affinities from gene to gene , dye effects , and biological variation within replicates . We also performed the method using data pre-processed with a median-adjustment to the control channel median of each chip . Our predictions were not substantially altered by the choice of pre-processing . Following pre-processing , we separated the drug or condition ( cy5 ) and control channels ( cy3 ) . For each experiment we first applied expression data from the control channel to set constraints on maximum fluxes of reactions in the model . We then used FBA to find a flux configuration that maximized overall mycolic acid biosynthesis ( bottom light blue bar in Figure 2 ) . Similarly , we predicted maximum mycolic acid production for the corresponding drug condition by applying expression from this channel ( bottom red bar ) . We compared both predictions to assess the relative impact of the drug or condition on mycolic acid biosynthetic capacity ( right blue bar ) . In the case of Figure 2 , we would predict that the drug inhibits mycolic acid production . To perform FBA for the mycolic acid biosynthetic model , we utilized an objective function representing total mycolic acid production . This model does not suggest that M . tb is in fact trying to maximize production of mycolic acids , but this objective function allows us predict the maximum amount of mycolic acid the model could produce under the given constraints . For the genome-scale model , we used the same objective function . We were also able to use the biomass objective as given in [6] ( Methods ) . Differences in predicted mycolic acid flux arising from comparing the drug and control channel could arise due to fluctuations in gene expression measurements independent of drug effects . To determine whether a particular difference could be explained by such fluctuations , we resampled data on the control channels with noise fluctuations derived from the ANOVA analysis to understand how much variation in the predictions would result from comparing two different control channels . The 95% confidence interval for predictions from resampled control channel data is represented as the dotted lines in Figure 3 and is the same for all experiments . To generate error bars for each prediction we resampled both control and drug channels with noise drawn from this distribution; the resulting error bars are shown in Figure 3 . We also wished to determine if predicted differences were specific to mycolic acid biosynthesis or reflected a more general change in metabolism . For example , a predicted inhibition of mycolic acid production might be due to an overall suppression of gene expression or metabolism . To this end , we randomly relabelled genes within each data set and found predictions using E-Flux . Repeating this permutation and computation multiple times , we calculated a null distribution associated with non-specific effects on mycolic acid production for each condition . The 95% ranges for these distributions are shown as grey bars in Figure 3 . We applied the approach shown in Figure 2 to all 437 experiments in the Boshoff data set . The results for the mycolic acid biosynthetic model are summarized in Tables 1 and 2 and details for a subset of predictions are shown in Figure 3 . The most noteworthy aspect of these results is that of the eight known inhibitors of mycolic acid tested in the Boshoff data set , E-Flux correctly predicts seven . More generally E-Flux identifies as modulators all of the drugs used against tuberculosis that are known to affect the mycolic acid pathway . The application of E-Flux to the the M . tb genome-scale model produced an identical set of predicted inhibitors and enhancers with the same specificity and predicted strength as those in Table 1 and Table 2 although the quantitative predictions differed slightly ( Supplementary Material ) . This was true regardless of whether we used the mycolic acid objective function from [7] or the biomass objective function of [6] . Our method is thus applicable to the both targeted metabolic models as well as genome-scale metabolic reconstructions . The strongest predicted inhibitors included isoniazid ( INH ) and ethionamide . Isoniazid , a first-line drug for TB , is a prodrug that is activated by the bacterial catalase-peroxidase enzyme KatG [21] . Activation leads to the development of INH-NAD and INH-NADP adducts that inhibit InhA and FabG1 respectively [22] , [23] . Both InhA and FabG1 are components FAS-II cycle of mycolic acid biosynthesis [22] , [24] . Ethionamide is a structural analog of INH and is thought to also target InhA [25] . Both isoniazid and ethionamide are predicted as strong selective inhibitors of mycolic acid biosynthesis . E-Flux also predicts thiolactomycin and ethambutol as strong selective inhibitors . Thiolactomycin is a natural product produced by both Nocardia and Streptomyces and is a potent and highly selective inhibitor of the type II dissociated fatty acid synthase of plants and bacteria [26] . Ethambutol inhibits the arabinosyl transferases in the synthesis of arabinogalactan , and prevents the attachment of mycolic acids to the 5′-hydroxyl groups of D-arabinose residues of arabinogalactan thus obstructing the formation of the mycobacterial cell well [27] . Interestingly , E-Flux only predicted inhibition for the highest concentration of ethambutol . If correct , this mechanism may lead to reduced quantities of mycolate polymer , with subsequent build-up of free mycolate , and it seems possible that mycolic acid biosynthesis would be down-regulated in response . In addition , E-Flux predicts a number of weaker inhibitors , including two drugs known to impact mycolic acid . Cerulenin is a fungal mycotoxin that is known to inhibit both FAS-I and FAS-II cycles in mycolic acid synthesis in M . tb [28] . Pyrazinamide is a pro-drug of pyrazinoic acid , and inhibits the FAS1 pathway of mycolic acid synthesis in M . tb [29] . PA-824 is a newer nitroimidazopyran drug currently in clinical trials [30] . PA-824 inhibits both lipid and protein synthesis by as yet unknown mechanisms . E-Flux predicts that PA-824 inhibits mycolic acid synthesis at the higher concentration replicates but not at lower ones . PA-824 was not predicted as a specific inhibitor of mycolic acid biosynthesis , although this may be due to the additional effect of PA-824 on protein synthesis genes or to the relative weakness of the predicted mycolic acid inhibition . E-Flux also predicts several novel compounds not previously associated with inhibition of mycolic acid biosynthesis . These include predictions of weak effects for the protein synthesis inhibitor streptomycin and the ionophore valinomycin . These compounds are predicted as non-specific inhibitors consistent with an overall impact on metabolism . PA-21 was also predicted to be a weak and marginally specific inhibitor , although the mechanism of this compound has not been reported . Of the novel predicted inhibitors , only ZnSO4 was predicted to have a strong effect with marginal specificity . However , only a single replicate for ZnSO4 is present in the Boshoff data set , and preliminary experimental data suggest that this prediction is likely a false positive . Interestingly , E-Flux also predicted a small number of compounds that may enhance fatty acid biosynthesis . Menadione and chlorpromazine are predicted to be weak non-specific enhancers , although these results are convolved with GSNO and one instance of GSNO in isolation was predicted as a strong and specific enhancer . However , it is noteworthy that menadione has been reported to increase fatty acid production in human fat cells , in addition to a number of other metabolic effects [31] . Chlorpromazine is a phenothiazine , a class of compounds recently proposed as possible drugs targeting multi-drug resistant tuberculosis . GSNO is a nitric oxide donor toxic to mycobacteria [32] whose mechanism of antimycobacterial action of GSNO is unknown [33] , [34] . Extracellular glutathione is converted to a dipeptide , which is transported into the bacterial cells by the multicomponent ABC transporter dipeptide permease [32] . Curiously , triclosan was also predicted as an enhancer . Triclosan is known to inhibit the enoyl-ACP reductase of FASII [35] . Although we predict no significant effect at low concentrations , E-Flux predicts a significant upregulation of mycolic acid production at the highest concentration . It has been observed that triclosan acts through more than one mechanism [35] and may lead to upregulation of fatty acid metabolism The data of Karakousis et al . [18] on the transcriptional response of dormant M . tb to isoniazid provide the opportunity to examine the predictions of E-Flux for dormant tuberculosis . Though it is a strong inhibitor of mycolic acid biosynthesis , isoniazid has little activity against M . tb under oxygen deprivation or nutrient starvation [36] . Consistent with this , Karakousis et al . [18] found that the transcriptional signature associated with isoniazid's activity in non-dormant tuberculosis was abolished under conditions of dormancy . The results of E-Flux applied to these data are shown in Figure 4 . E-Flux correctly shows a strong and significant inhibition of mycolic acid biosynthesis after 6 hours , but shows no effect of isoniazid for any of the four dormancy models in the dataset . This not only confirms the result for isoniazid from the Boshoff compendium [17] but provides an indication that E-Flux may be a useful tool in analyzing expression profiles for dormant M . tb under a range of treatment conditions , when such data become available . To rule out that our predictions reflect similarities in gene expression independent of metabolic modeling , we clustered the expression of the 29 genes used in the mycolic acid biosynthetic model across all 437 experiments in the Boshoff data set . As can be seen in Figure 5 , known inhibitors do not form a single cluster . This is consistent with the results of clustering all M . tb genes as reported by Boshoff et al . [17] . Similarly , inhibitors and enhancers predicted by E-Flux also do not form a single cluster . Furthermore , predicted inhibitors do not obviously fall into clusters with previously known inhibitors , suggesting that using a metabolic model allows the discovery of distinct routes to inhibition or enhancement of an objective , beyond similarity of gene expression with known inhibitors . More fundamentally , in contrast to supervised classification methods , E-Flux does not require data from compounds with a known effect to calibrate the method ( i . e . an initial training set is not required ) . In particular , no mycolic acid enhancers are currently known , and thus a method designed to classify new enhancers by comparing expression profiles to known compounds would not be applicable . We consider the differences between E-Flux and expression classification in more detail in the Discussion section .
The key principle underlying the E-Flux method is that mRNA levels for enzymes approximate an upper bound on the potential flux through the corresponding metabolic reactions , i . e . for a particular level of translation and degradation , the amount of mRNA sets an upper bound on the amount of available enzyme . The amount of available enzyme is in turn proportional to maximum flux ( e . g . Vmax ) through a particular reaction . We acknowledge that mRNA expression is not sufficient to determine fluxes or , in many cases , true upper bounds on fluxes , but nonetheless argue that including mRNA expression data into flux balance models provides a new and useful way to connect expression data with models cellular metabolism , and is an improvement upon effectively assuming that all reactions that are present have the same maximum flux . The degree of correlation between mRNA and protein levels is an area of ongoing research [37] . There are conflicting reports regarding the correlation between mRNA and protein levels [38] , but some whole genome studies have reported modest correlations . A study of 289 proteins in Saccharomyces cerevisiae , for example , reported a correlation of 0 . 61 after correcting for methodological issues [39] . Correcting for methodological noise and potential non-linearity in the mRNA-protein relationship , however , results in higher levels of mRNA-protein concordance [37] , [40] , [41] . In prokaryotes , ribosomes bind to nascent mRNAs so that translation can be synchronous with transcription; proteins levels thus depend more directly on mRNA abundance [42] . Consistent with this , a comparison of Staphylococcus aureus biofilm and planktonic cells showed qualitative agreement between transcriptomic and proteomic expression differences [43] , and an analysis of mRNA and protein levels for 400 genes from Desulfovibrio vulgaris reported correlations between 0 . 45–0 . 53 [40] , [41] . In addition , genes from different functional categories display different levels of correlation . For example , in both S . cerevisiae and D . vulgaris genes associated with central intermediary and energy metabolism display higher levels of correlation than other groups [41] , and a study of central metabolism genes in both wildtype E . coli DF11and a pgi mutant found a correlation of . 81 between the log ratio of transcripts and the log ratio of enzyme activities [44] . Studies of transcriptional regulation of metabolism in E . coli [45] also suggest tight transcription-translation coupling . In unbranched segments of amino acid biosynthetic pathways , genes for enzymes catalyzing upstream reactions are transcribed earlier and with a higher promoter activity than those for downstream reactions . This pattern is optimal for rapidly producing end-products while minimizing enzyme production when enzyme levels are a direct function of mRNA levels . Although the total amount of available enzyme sets an upper limit on the maximum flux through a particular reaction , many regulatory processes may modulate the effective levels of enzyme activity . Metabolite feedback regulation , allosteric interactions , and various covalent modifications may alter the activity of enzymes already synthesized . These modulations , however , cannot lead to more activity than is possible if all available enzyme were maximally active . For example , in the extreme case where an enzyme is completely deactivated by covalent modification ( e . g . inactivation through phosphorylation ) , the true flux through a reaction is zero whereas mRNA levels might suggest a higher bound . With respect to E-Flux , this means that the upper bound on enzyme activity set by mRNA levels is an upper bound , but not always a tight one . In such a case , the accuracy of predictions made by E-Flux may depend on the difference between the approximate and true bounds . It would be possible to generalize the E-Flux framework to take modulation of enzyme activity into account . In our application to mycolic acid production , we model maximum flux for a reaction as a function of all genes that are components of the corresponding enzymes or enzyme complexes . However , we could incorporate the expression of all genes whose products modulate the activity of a given enzyme into this function . For example , if an enzyme is inactivated through phosphorylation by a kinase , we may choose to model the corresponding maximum flux as a function of the expression of the genes for both the enzyme and kinase . This approach is conceptually similar to the coupling of metabolic models with Boolean regulatory models taken by Covert et al . [46] , although E-Flux differs in that empirically measured gene expression levels are used . Such an approach , however , would require more knowledge of regulatory interactions between proteins than we used in the analysis presented here . A number of previous methods have been developed to gain insight into metabolism from expression data . The most common method is to identify genes or sets of genes from particular pathways that are differentially expressed under different conditions [47] , [48] . Often this involves visualizing expression data on metabolic maps [49] . Although useful , this approach is limited by the need for the subjective interpretation of differentially expressed gene sets , typically by an expert on the metabolic pathways of interest . Other methods have been based on classifying gene expression by similarity to expression patterns corresponding to known metabolic or cellular states [50]–[52] . In the case of mycolic acid biosynthesis , however , different inhibitors do not cluster together based on expression similarity , either when considering all M . tb genes [17] or only the 29 genes directly involved with mycolic acid synthesis ( Figure 5 ) . This is consistent with the range of mechanisms by which mycolic acid biosynthesis can be suppressed . For example , isoniazid , ethionamide , and thiolactomycin inhibit the FAS-II fatty acid biosynthetic cycle , whereas cerulenin inhibits both FAS-I and FAS-II , and ethambutol blocks the incorporation of mycolic acids into the cell wall . It is possible that a gene expression-based classifier could be developed that would correctly identify inhibitors across this range of mechanisms . E-Flux , however , implicitly integrates across these different mechanisms by interpreting expression data through the lens of a metabolic network model . More fundamentally , our method does not require a set of training data whose effect on the pathway is known in advance . Traditional classification methods require exemplars from the categories to be classified [53] . These exemplars are used to select a decision boundary in some space that places objects of one category on a different side of the boundary from objects in other categories . Although the Boshoff data set contained conditions corresponding to known mycolic acid inhibitors , this information was not used to parameterize our method . Instead , E-Flux uses a model of the underlying chemical and biological network to simulate the effects of different regulatory states . The method can thus be used to classify new expression data sets even in the absence of previous data from the same class . Moreover , we are able to predict previously unreported effects . For example , our method predicts that a small number of compounds may act to increase overall mycolic acid production although no known mycolic acid enhancers are included in the set . Furthermore , while our goal here was to predict the metabolic impact of a known external condition , in a related manuscript [54] , we reverse this logic to predict an unknown environment , in particular to identify the most likely nutrient being metabolized , using expression data coupled to metabolic models . Since the initial development of E-Flux , two other methods for combining expression data with flux balance analysis have been described . The method of Becker and Palsson [55] utilizes a variant of the method of Covert and Palsson [46] to turn genes on or off . In contrast to this approach where genes were turned off based on a Boolean model of gene regulation , the method of Becker and Palsson [55] turns off genes whose expression is below a given threshold level . If this constrained model is incapable of achieving a given objective , genes are turned back on until the objective is reachable . The method of Shlomi et al . [56] uses a novel nested optimization method to determine an FBA solution while also maximizing the correspondence between gene expression levels and metabolic fluxes . These methods differ with respect to the degree that expression data is used to modulate constrains on an FBA model . Becker and Palsson [55] apply a hard constraint using gene expression such that genes are either on or off . On the other hand , genes not turned off are not modulated by expression data . Shlomi et al . [56] , in contrast , use expression data to influence fluxes indirectly , rather than through flux constraints . E-Flux falls in the middle of these two approaches . It is more aggressive than the method of Shlomi et al . [56] in that fluxes are directly constrained by expression . It is less aggressive than the method of Palsson et al . [55] in that genes are not turned off , although more comprehensive in that all flux constraints are modulated by the expression of the corresponding genes . Which approach is more accurate likely depends on the application . E-Flux provides a general approach for modeling metabolism from expression data . This approach has a number of potential applications , beyond the application to tuberculosis presented here . E-Flux can also be used to investigate and model other disease states where expression data are available and for which metabolic alterations are associated . For example , many cancer cells are known to grow glycolytically in the presence of oxygen and to develop a lipogenic phenotype [57]–[59] . With the availability of numerous expression data sets for various cancer cells , E-Flux may provide an opportunity to study this phenomenon computationally . E-Flux could in principle also be used as a tool for drug discovery . For example , if a drug were sought that decreased production of a particular metabolite , then genome-scale expression profiles of a large number of small molecules could be analyzed with E-Flux and subsequent study could focus on those for which E-Flux predicted the desired inhibition . This would be a valuable approach in settings where screening directly for the effect of interest is expensive relative to microarray analysis . In addition , since E-Flux can predict unanticipated effects , the approach could be used to predict possible undesirable effects including the production of toxic metabolites . Furthermore , if a set of objective functions were developed , each corresponding to a different subsystem or pathway in the metabolic network , then E-Flux could be used separately for each objective to help identify a molecule's mechanism of action . Finally , E-Flux provides a new tool for efforts to engineer metabolic systems . Flux analyses have been previously used to guide the design of metabolic networks . E-Flux enhances this approach by enabling the prediction of metabolic characteristics for specific empirically determined gene expression states .
We used two metabolic models: a model of the mycolic acid biosynthesis subsystem [7] , and a genome-scale model for M . tuberculosis [6] . The small subsystem model comprises four sub-pathways: fatty acid synthase ( fas ) I and II , the production of malonyl-CoA which is an input for each of these , and the condensation of the products of fas I and II into alpha , methoxy and keto mycolic acids . The model has 197 distinct metabolites , linked together in 219 internal reactions . There are an additional 28 external reactions corresponding to uptake of the primary input , AccB , the free exchange of water , carbon dioxide and other substances not explicitly produced and consumed in the model , and the production of the mycolate outputs . The model is available in SBML format at DOI: 10 . 1371/journal . pcbi . 0010046 . sd001 and is presented in the supplementary material of [7] . Two genes in the model remained unknown and were labeled ‘UNK1’ and ‘UNK2’ at the time Raman et al . published the mycolic acid metabolic model . One of these was the gene or complex responsible for the dehydration of ( 3R ) -hydroxyacyl-ACP in the fas II elongation cycle in M . tuberculosis . Subsequent to the publication of the orginal model by Raman et al . , Sacco et al . [16] identified two heterodimers , Rv0635-Rv0636 ( HadAB ) and Rv0636-Rv0637 ( HadBC ) which perform this role . They observed substrate specificity for these dimers , with hadAB preferentially catalyzing this reaction for shorter carbon chains and hadBC doing so for longer carbon chains . We included catalysis of reactions 68 and 74 ( length up to C-18 ) by hadAB , and reactions 80 , 86 , 92 , … , 188 ( longer lengths ) by hadBC . Our results were not substantially altered by including the hadABC genes . The genome-scale model we used was closely based on that published by Beste et al . [6] . We merged Raman et al . 's mycolic acid submodel [7] into the genome-scale model so that we could use both models with E-Flux to test for inhibition or enhancement of mycolic acid production capacity . The merging was done as follows: we identified all external metabolites of Raman et al . 's model and found the equivalent metabolite in Beste et al . 's model . We then removed exchange reactions for these metabolites so that net production and consumption of these was no longer allowed . We removed mycolic acid reactions from the genome-scale model and replaced them with the mycolic acid reactions in Raman et al . 's model , and normalized the bounds on exchange reactions so these were uniform ( +/−1 ) . The net result was to replace McFadden et al . 's representation of mycolic acids with that of Raman et al . , as the latter is more detailed and as this allows direct comparison of the results of E-Flux in the two models . The model is available as Dataset S1 . The expression data published by Boshoff et al . [17] are listed under GEO accession number GSE1642 . Boshoff et al . used clustering of the expression profiles to predict the mechanisms of action of previously unknown agents . Data are available for two channels: Cy3 ( control ) and Cy5 ( condition ) on a total of 437 spotted chips , each with mRNA expression data for M . tuberculosis strain H37Rv . The published data are in log format; these were exponentiated to obtain raw values . The data of Karakousis et al . [18] are published at http://www . ncbi . nlm . nih . gov/geo/ under accession number GSE9776 and are also two-channel data of H37Rv M . tb; the dataset contains 17 arrays for 6 unique conditions , comparing M . tb's response to isoniazid in dormancy models . We processed the expression data using MAANOVA 2 . 0 [20] , a Matlab package for analyzing data from two-dye cDNA microarray experiments . MAANOVA 2 . 0 fits an analysis of variance ( ANOVA ) model to the data to account for non-biological variation in the measurements . Briefly , let yijkg denote the log-transformed measurement from the ith channel , jth chip , kth variety ( experimental condition ) , and gth gene . Then we fit the modelwhere μij is the average measurement for channel j of chip i , Gg represents the effect of gene g , ( AG ) jg represents effects specific to chip j and gene g , ( DG ) ig represents effects specific to channel i and gene g , represents effects specific to variety k and gene g ( i . e . the biological variation ) , and εijkg is error . Thus we fit for , and subtract out , systematic , non-biological effects such as overall brightness and spot effects . We fit the model such as to minimize the residual sum of squares ( RSS ) given byThe estimate of given by this procedure is used as the ANOVA-processed data . We compared the results of our method using ANOVA-processed data with those using the published values without statistical filtering . In this approach , the published data ( log format ) were exponentiated to obtain raw values . To remove noise resulting from variation in overall brightness from chip to chip while preserving median differences between condition and control channels , we adjusted the medians of each chip according to the median of that chip's Cy3 channel . We set the median of all control channels to the maximum of the control channel medians across the dataset ( rather than a middle value ) to avoid obtaining negative flux constraint inputs . In other words , we computed the median of each chip's Cy3 channel ( denoted Mj for the j'th chip ) , and found the maximum of these , Mmax . We then added ( Mmax-Mj ) to both channels of the j'th chip , for each j . The resulting values had the same median for the Cy3 channels , and different medians for the Cy5 channels , while the difference between Cy3 and Cy5 channel medians on each chip was the same as in the raw data . We also performed E-Flux using log-transformed values; predictions using log-transformed and raw values were correlated with R = 0 . 99 after the control-channel median adjustment described here . Comparing predictions based on ANOVA-processed and raw expression data ( with median adjustment ) , our results for the top mycolic acid inhibitors ( isoniazid , thiolactomycin , ethionamide ) are preserved , as are the inhibitory predictions for cerulenin , PA-824 and valinomycin . Ethambutal was also a predicted inhibitor from the unprocessed data , but not so strongly , along with streptomycin . The enhancing effect of triclosan was more strongly predicted from the raw data than the ANOVA-processed data . ZnSO4 was inhibitory but not as strongly , and GSNO was not as strongly enhancing . Overall , results from the two data sets were positively correlated with R = 0 . 62 at a significance level p = 2 . 11e-48 . For the data on isoniazid and dormancy from Karakousis et al . [18] , the ANOVA model had fewer data points in total ( 17 microarrays ) so the corresponding error bars ( see Figure 4 ) are wider than for the Boshoff data reflecting a greater residual sum of squares . The E-Flux method consists of creating constraint vectors a and b from expression data for control and condition channels , using these as constraints for flux balance analysis with a given objective , and comparing the maximum production capacity of that objective between the control and condition . We constructed constraint vectors a and b for the control and condition channels for each condition in the compendium of expression data as follows . For each reaction that is catalyzed by only one gene , we set the upper bound bj to the expression value for the gene whose product catalyzes reaction j , taking the value from the relevant data . For example , reaction 2 in the mycolic acid sub-model is catalyzed by gene Rv3279c corresponding to birA [7] , so if the control channel expression value for Rv3279c is 15 , b2 is initially set to 15 . For each reaction catalyzed by a complex requiring two genes we set bj to the minimum of the expression of the two genes , and for reactions which can be catalyzed by either gene we set bj to the sum of their expression values . We then normalized the bj values to 1 by dividing each component of bj by M = maxj ( bj ) . For each exchange reaction , we set aj = −1 and bj = 1; in other words , these reactions were not constrained by gene expression . Changing the constraint ( for example , from +/−1 to +/−2 ) on exchange reactions to another value does not change the results presented here , as the relative production capacity from control to condition is a log ratio and is not dependent on the overall scale . Following Raman et al . , all internal reactions in the model were modeled to be irreversible , so that aj = 0 for these ( j = 1 to 219 ) . For the reactions catalyzed by the remaining unknown gene in Raman's model we set bj = 1 and aj = 0 . All of these steps were performed for the control channel expression data and then separately for the condition channel . This yields 4 vectors: acontrol and bcontrol taken from the cy3 channel of the chip , and acondition and bcondition taken from the condition channel . Linear optimization was then performed with each set of constraints and the same objective function , namely a weighted production of mycolic acids . The objective function for the mycolic acid subsystem model waswhere ei represents the vector ( 0 , 0 , … , 1 , … , 0 ) with a 1 in the i'th component and 0 in all other components . Since the linear programming tool linprog in matlab minimizes cTv , the coefficients of c were chosen to be negative so that the optimization maximizes ( −cTv ) , namely the weighted flux creating mycolic acids . The reactions included in c produce alpha , cis- and trans- methoxy mycolate and cis- and trans- keto mycolate respectively . This is the same as objective function c1 in Raman et al . Our results are insensitive to the particular balance between the α- , keto- and methoxy- mycolates in the objective function . In the genome-scale model our objective function was the same but the reactions are numbered differently . We also performed E-Flux with two alternative objective functions: biomass as given in Beste et al . , and the mycolic acid objective function with the same weights as given in [7] . This procedure , with either objective function , yields two results for the maximal production capacity: Pcontrol = max ( cTv ) using constraints taken from the control channel , and Pcondition = max ( cTv ) using constraints taken from the condition channel . The relative production , namely the results shown in Figure 3 and Supplementary Figures S1 , S2 , S3 , S4 and S5 , is given by log ( Pcondition/Pcontrol ) . In addition to setting bj equal to the expression value for the gene catalyzing the j'th reaction , we explored using sigmoidal , exponential and polynomial increasing functions to create the constraint b , so that rather than bj = expression ( j ) , we used bj = f ( expression ( j ) ) where f is sigmoidal , exponential or polynomial . The results presented in Table 1 are robust to saturation at high expression levels and suppression at low levels , as long as these retain sufficient variation in the data . To determine whether predictions were significant we resampled the control channel of each chip , adding noise sampled from the noise distribution given by the Anova model as described above . We compared the mycolic acid production capacity from one such resampled control dataset to another , and repeated the procedure 800 times . We then found the “null” interval in which 95% of the values lie . The interval is denoted by the dotted lines in Figure 3 . Predictions lying within this interval were not considered significant . To generate the error bars shown in Figure 3 , we used a similar approach . Here , we added noise ( again distributed in accordance with the anova model ) to both the control and condition channel of the chip , and applied E-Flux . After repeating this procedure 800 times , we found the intervals in which 95% of the values lay; these form the error bars shown . They represent the uncertainty in our predictions based on the Anova estimate of how much random noise there is in the data . To make a distinction between predictions that are specific to the mycolic acid pathway and those that may result from the conditions' effects on a large number of genes in M . tuberculosis , we used two approaches . For the first , we computed predictions using randomly chosen genes from each chip in place of the genes in the metabolic model . To do this , we chose a random permutation of all genes on each chip , and these randomly chosen genes ( rather than the genes actually listed in the metabolic model ) to form the constraint vectors a and b . This was done for both control and condition channels of each chip , using the same gene permutation for the two channels . We then applied E-Flux to recompute the predicted inhibition or enhancement using constraints from expression of the randomly selected genes . After repeating this procedure , we found the range in which 95% of the resulting predictions lie , for each experiment ( grey bars in Figure 3 and Supplementary Figures ) . Where the range is large , there is typically substantial overlap between the gene relabelled predictions and the predictions using the correct genes for the metabolic model . This results from the condition having affected many genes in the organism . In this case we do not consider the prediction to be specific to the mycolic acid pathway because there may be inhibition or enhancement of a number of pathways in the organism , leading to a likely inhibition when in the model when random genes are used . Alternatively , where there is considerable difference between the gene-relabelled predictions and the noise-resampled predictions from the correct genes , as is the case with isoniazid ( see Figure 3 ) , the predictions of E-Flux are considered specific to mycolic acids . Our quantitative use of “specific” required that 95% of the noise-resampled predictions ( i . e . those which give the error bars shown in Figure 3 ) lie outside the 95% range of the gene-relabelled predictions ( grey bars in Figure 3 ) . By this criterion , when a prediction is deemed “specific” it is unlikely that that prediction would be obtained with randomly relabelled genes . | The ability of cells to survive and grow depends on their ability to metabolize nutrients and create products vital for cell function . This is done through a complex network of reactions controlled by many genes . Changes in cellular metabolism play a role in a wide variety of diseases . However , despite the availability of genome sequences and of genome-scale expression data , which give information about which genes are present and how active they are , our ability to use these data to understand changes in cellular metabolism has been limited . We present a new approach to this problem , linking gene expression data with models of cellular metabolism . We apply the method to predict the effects of drugs and agents on Mycobacterium tuberculosis ( M . tb ) . Virulence , growth in human hosts , and drug resistance are all related to changes in M . tb's metabolism . We predict the effects of a variety of conditions on the production of mycolic acids , essential cell wall components . Our method successfully identifies seven of the eight known mycolic acid inhibitors in a compendium of 235 conditions , and identifies the top anti-TB drugs in this dataset . We anticipate that the method will have a range of applications in metabolic engineering , the characterization of disease states , and drug discovery . | [
"Abstract",
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"computational",
"biology/metabolic",
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] | 2009 | Interpreting Expression Data with Metabolic Flux Models: Predicting Mycobacterium tuberculosis Mycolic Acid Production |
Replication of viroids , small non-protein-coding plant pathogenic RNAs , entails reiterative transcription of their incoming single-stranded circular genomes , to which the ( + ) polarity is arbitrarily assigned , cleavage of the oligomeric strands of one or both polarities to unit-length , and ligation to circular RNAs . While cleavage in chloroplastic viroids ( family Avsunviroidae ) is mediated by hammerhead ribozymes , where and how cleavage of oligomeric ( + ) RNAs of nuclear viroids ( family Pospiviroidae ) occurs in vivo remains controversial . Previous in vitro data indicated that a hairpin capped by a GAAA tetraloop is the RNA motif directing cleavage and a loop E motif ligation . Here we have re-examined this question in vivo , taking advantage of earlier findings showing that dimeric viroid ( + ) RNAs of the family Pospiviroidae transgenically expressed in Arabidopsis thaliana are processed correctly . Using this methodology , we have mapped the processing site of three members of this family at equivalent positions of the hairpin I/double-stranded structure that the upper strand and flanking nucleotides of the central conserved region ( CCR ) can form . More specifically , from the effects of 16 mutations on Citrus exocortis viroid expressed transgenically in A . thaliana , we conclude that the substrate for in vivo cleavage is the conserved double-stranded structure , with hairpin I potentially facilitating the adoption of this structure , whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands . These results have deep implications on the underlying mechanism of both processing reactions , which are most likely catalyzed by enzymes different from those generally assumed: cleavage by a member of the RNase III family , and ligation by an RNA ligase distinct from the only one characterized so far in plants , thus predicting the existence of at least a second plant RNA ligase .
Viroids , plant pathogens with a minimal non-protein-coding circular RNA genome of 246–401 nt [1] , are classified into two families . The members of the first , Pospiviroidae , replicate in the nucleus through an asymmetric rolling-circle mechanism , have a central conserved region ( CCR ) , and cannot form hammerhead ribozymes . The members of the second , Avsunviroidae , replicate in the chloroplast through a symmetric rolling-circle mechanism , lack a CCR , and can form hammerhead ribozymes [2–4] . In Potato spindle tuber viroid ( PSTVd ) [5 , 6] , the type species of the genus Pospiviroid ( family Pospiviroidae ) , the incoming monomeric circular RNA , to which ( + ) polarity is arbitrarily assigned , is reiteratively transcribed by the nuclear RNA polymerase II into oligomeric ( − ) RNAs that in turn serve as template for synthesis of oligomeric ( + ) RNAs . These latter transcripts are then cleaved and ligated to the mature viroid circular RNA [7 , 8] . In Avocado sunblotch viroid ( ASBVd ) [9] , the type species of the family Avsunviroidae , the oligomeric ( − ) RNAs generated by a chloroplastic RNA polymerase are cleaved and ligated before serving as template for a second rolling-circle leading to the mature viroid circular RNA . In this family , the oligomeric RNA intermediates of both polarities self-cleave through hammerhead ribozymes [10 , 11] . In contrast , cleavage and ligation of oligomeric ( + ) RNAs in the family Pospiviroidae is catalyzed by host enzymes [12–14] , which recognize particular RNA motifs . Early infectivity bioassays with viroid RNAs containing repeated sequences of the upper CCR strand [15–18] implicated these sequences in processing of the oligomeric ( + ) strands of the family Pospiviroidae through the adoption of either hairpin I , a metastable motif that can be formed by the upper CCR strand and flanking nucleotides during thermal denaturation [19] , or through a thermodynamically stable double-stranded structure that can be alternatively assumed by the same sequences of a dimeric ( or oligomeric ) RNA [15 , 18] ( Figure S1 ) . More recently , in vitro and thermodynamic analyses of the products obtained by incubating a potato nuclear extract with a full-length PSTVd RNA containing a 17-nt repeat of the upper CCR strand have led to the proposal that cleavage of ( + ) strands is driven by a multibranched structure with a hairpin—different from hairpin I—capped by a GAAA tetraloop conserved in members of the genus Pospiviroid , which subsequently switches to an extended conformation with a loop E that promotes ligation [20] ( Figure S1 ) . Similar results have been obtained with a reduced version of this construction containing the GAAA tetraloop and loop E [21] . Loop E , a UV-sensitive motif of RNA tertiary structure that is conserved in PSTVd and members of its genus [3] and exists in vitro [22] and in vivo [23 , 24] , has been also involved in host specificity [25] , pathogenesis [26] , and transcription [27] . The structural model based on isostericity matrix and mutagenic analyses derived recently for PSTVd loop E [27] can be extended to CEVd . However , the proposed cleavage-ligation mechanism [20] may not apply to other members of the family Pospiviroidae that cannot form the GAAA tetraloop and loop E [3] . Moreover , alternative processing sites in the lower CCR strand , or outside this region , have been observed for several members of the family Pospiviroidae [28–32] , with a proposal even suggesting that cleavage could be autocatalytic , albeit mediated by non-hammerhead ribozymes [33] . Here we have re-examined this question in vivo using a system based in transgenic Arabidopsis thaliana expressing dimeric ( + ) transcripts of Citrus exocortis viroid ( CEVd ) , Hop stunt viroid ( HSVd ) , and Apple scar skin viroid ( ASSVd ) [34] of the genera Pospiviroid , Hostuviroid , and Apscaviroid , respectively , within the family Pospiviroidae [3] . In addition to mapping what we believe is their major processing site in vivo , data obtained with 16 CEVd mutants support a previous model involving the hairpin I/double-stranded structure formed by the upper CCR strand and flanking nucleotides in cleavage [15] , and loop E and flanking nucleotides of both strands in ligation . A corollary of our results is that the RNase and RNA ligase that catalize both processing reactions are most likely different from those generally assumed so far .
We first re-examined where the processing site occurs in vivo . This question could be tackled by mapping the 5′ termini of the monomeric linear ( ml ) viroid ( + ) strands isolated from infected propagation hosts ( e . g . , gynura for CEVd ) . However , the contribution of nicked byproducts of the monomeric circular ( mc ) viroid ( + ) RNA—the most abundant replication product—resulting from in vivo turnover or artifactual degradation during purification has precluded an unambiguous analysis so far . As an alternative , we evaluated the A . thaliana–based transgenic system established recently [34] . More specifically , an A . thaliana transgenic line expressing a CEVd ( + ) dimeric transcript ( dt ) [34] was chosen . Northern blot hybridization of RNAs separated by denaturing PAGE showed that plants of this line accumulate , besides the dt viroid ( + ) RNAs , the mc and ml CEVd ( + ) forms; however , in contrast to the situation in CEVd-infected gynura , the ml RNA was more abundant than its circular counterpart ( Figure 1A and 1C , compare lanes 2 and 4 ) . This indicates that A . thaliana can process correctly the dt CEVd ( + ) RNAs , with cleavage being more efficient than circularization , thus reducing the contribution of nicked mc ( + ) RNAs to the population of ml ( + ) RNAs present in vivo . RT-PCR amplification , cloning , and sequencing of the mc CEVd ( + ) forms extracted from the transgenic A . thaliana line confirmed that they were full-length [34] , and infectious when mechanically inoculated to tomato ( unpublished data ) . Furthermore , northern blot hybridization of another transgenic A . thaliana line expressing a dt CEVd ( − ) RNA showed low accumulation levels of the mc ( + ) RNA ( Figure 1C , lane 5 ) , indicating that despite not being a typical host , A . thaliana has the enzymatic machinery for replicating CEVd , in line with previous results for HSVd [34] . RNAs from the transgenic A . thaliana line expressing the dt CEVd ( + ) species were separated by denaturing PAGE , and the positions in the gel of the mc and ml ( + ) CEVd RNA were inferred using a purified marker stained with ethidium bromide . RNAs migrating in the region of ml CEVd ( + ) RNA were eluted and examined by northern blot hybridization with a CEVd-specific probe that excluded the presence of other viroid RNA species ( unpublished data ) . Preliminary length estimation of the CEVd-cDNAs from extensions on this RNA with 5′-end labeled primers PI , PIV , and PV , run in denaturing gels in parallel to RNA markers of known size , mapped the processing site around position 100 ( Figure S2 ) . Further analysis of the CEVd-cDNAs from extensions on the same RNA with the proximal 5′-end labeled primers PI and PII ( Figure 2C ) , also run in denaturing gels but this time in parallel to sequencing ladders , revealed with both primers single bands corresponding to stops at position G97 ( Figure 2A and 2B , lanes 5 ) . These results identified the processing site between G96 and G97 , which occupy the third and fourth positions of the tetraloop capping hairpin I ( Figure 3 ) , and two central positions of the double-stranded structure that the upper CCR strand and flanking nucleotides can form in di- or oligomeric viroid RNAs ( see below ) . The secondary structure model here presented for hairpin I ( Figure 3 ) , with a capping tetraloop [18 , 35] , differs from the original with a capping loop of 14 nt inferred from thermal denaturation studies with PSTVd [19] . As controls , ml and mc CEVd ( + ) RNAs obtained in parallel from CEVd-infected gynura were also analyzed . Prominent bands resulting from stops at G97 were also observed for the ml CEVd ( + ) RNA ( Figure 2A and 2B , lanes 6 ) , although accompanied by others ( particularly in the extension with PII ) . Some of the extra bands were also observed for the mc CEVd ( + ) RNA , suggesting that they arise from elements of secondary structure , but others were not , thus supporting the contribution of nicked circular forms to the population of linear forms ( Figure 2A and 2B , lanes 7 ) . Altogether , these results identified in CEVd ( + ) strands a major processing site in vivo located in the upper CCR strand . To explore how general this finding was , processing was also studied in two additional members of the family Pospiviroidae , each with a characteristic hairpin I/double-stranded structure: HSVd and ASSVd [3 , 35] . RNA preparations containing the ml HSVd and ASSVd ( + ) RNAs as the only viroid species were isolated from two transgenic A . thaliana lines expressing the corresponding dt viroid ( + ) RNAs . However , in contrast to the situation observed in the CEVd-expressing line , these transgenic lines accumulated similar or more mc viroid ( + ) forms than their ml counterparts [34] . Parallel RNA preparations from HSVd-infected cucumber and ASSVd-infected apple , as well as the purified mc viroid ( + ) RNAs , were also analyzed . Extension with primer PIII identified the HSVd processing site at G82-G83 and extension with primer PIV identified the ASSVd processing site at G90-A91 ( Figure 4 ) . These two sites map , like in CEVd , between the third and fourth nucleotide of the tetraloop capping hairpin I ( Figure 3 ) , and at two central positions of the double-stranded structure formed by the upper CCR strand and flanking nucleotides in di- or oligomeric viroid RNAs ( see below ) . It is pertinent in this context to note that the processing site here identified for ASSVd does not coincide with the corresponding site of Citrus viroid-III ( also of the genus Apscaviroid ) predicted from thermodynamic analysis and comparisons with PSTVd [36] . An alternative hairpin capped by a GAAA tetraloop , the RNA motif proposed to direct cleavage in a potato nuclear extract primed with a ml PSTVd ( + ) RNA containing a 17-nt repetition of the upper CCR strand [20] , can neither be formed by HSVd nor by ASSVd . However , the PSTVd processing site inferred with this in vitro system [20] was in a position equivalent to that mapped here for CEVd with the A . thaliana–based in vivo system . Collectively , these results strongly suggest a role for the hairpin I/double-stranded structure in directing cleavage in vivo of the oligomeric ( + ) RNAs in the family Pospiviroidae . If this double-stranded structure is indeed the substrate for the cleavage reaction , the enzyme involved would be most likely an RNase III . Intriguingly , the cleavage sites in each strand of the proposed substrate are separated by two 3′-protruding nucleotides , as also occurs in reactions catalyzed by enzymes of this class ( see below ) . To gain further support for the RNA motif ( s ) directing processing in vivo , we determined how 16 different mutations ( Table 1 ) affected cleavage and ligation of dt CEVd ( + ) RNAs expressed transgenically in A . thaliana . We selected the mutations according to their potential discriminatory effects on: i ) the GAAA tetraloop [20] , ii ) the hairpin I/double-stranded structure formed by the upper CCR strand , and iii ) the loop E motif formed by a subset of nucleotides of the upper and lower CCR strands ( Figure 5A ) . It should be noticed that single mutations affect one position in the GAAA-capped hairpin and in hairpin I , but two positions in the double-stranded structure; similarly , the double mutations affect two positions of the hairpin structures , and four positions of the double-stranded structure . For an easier understanding we have clustered the mutations in three groups: those located in central positions of the upper CCR strand , in peripheral positions of the upper CCR strand , and in the lower CCR strand ( the effects of the two latter groups will be presented in the two following sections ) . RNA preparations from the 16 transgenic lines , and from the line expressing wild-type ( wt ) CEVd , were analyzed by northern blot hybridization after single denaturing PAGE ( in which the dt RNAs and their ml and mc processing products are separated ) ( Figure 5B ) , or double PAGE ( in which better resolution of the ml and mc forms is achieved ) ( Figure 5C ) . Mutations in the viroid processing products were confirmed by cloning and sequencing the viroid circular RNA from different A . thaliana transgenic lines . Mutant #1 ( C95→U ) has no effect on the stem stability of hairpin I and only debilitates the stem of the GAAA-capped hairpin ( a pair G:C is converted into G:U ) ( Figure 5A ) . However , in the double-stranded structure , this mutation affects a base pair phylogenetically conserved in the family Pospiviroidae ( Figure 3 ) located in positions very close to the cleavage sites of both strands ( Figure 5A ) . Therefore , if cleavage is directed by the double-stranded structure , changes in these positions are expected to have a negative influence; this was the case , with cleavage being reduced to 38% with respect to wt ( Figure 5B ) . Results with mutant #2 ( G96→A ) also support this view because the substitution has no effect on the stem stability of hairpin I and strengthens the stem stability of the GAAA-capped hairpin ( a pair G:U is converted into A:U ) ( Figure 5A ) . But in the double-stranded structure this mutation affects a base pair also conserved in the family Pospiviroidae and adjacent in both strands to the cleavage sites , which are no longer embedded in an uninterrupted GC-rich helix ( Figure 5A ) . Cleavage was reduced to less than 20% with respect to wt , consistent with a key role of the double-stranded structure in this reaction ( Figure 5B ) . The corresponding double mutant #3 ( C95→U and G96→A ) does not essentially alter the stem stability of both hairpin I and the GAAA-capped hairpin but , in contrast to the single mutant #2 , restores the stability of the double-stranded structure ( two contiguous G:C pairs are substituted by A:U pairs ) ( Figure 5A ) . However , cleavage was not restored ( Figure 5B ) , indicating a requirement either for a particular sequence of the two nucleotides preceding the cleavage sites , or for a high thermodynamic stability of the secondary structure in the surrounding region ( in which G:C pairs are prevalent ) . Mutants #4 ( G97→A ) , #5 ( G97→U ) , and #6 ( G97→C ) , have no influence on the stem stability of hairpin I or weaken the stem stability of the GAAA-capped hairpin ( a C:G pair is disrupted ) . In the double-stranded structure mutations at this position affect nucleotides in both strands adjacent to both cleavage sites , which as in mutant #2 are no longer embedded in a double-stranded region ( Figure 5A ) . Although reduction of cleavage ( less than 25% with respect to wt , Figure 5B ) supports also the involvement of the double-stranded structure in this reaction , these data can be alternatively interpreted as resulting from a destabilization of the GAAA-capped hairpin . However , cleavage was totally recovered in the double mutant #7 ( G97→C and C94→G ) , in which the stability of the double-stranded structure was restored ( these are indeed the nucleotides existing in the corresponding positions of CbVd-1 , see Figure 3 ) , whereas the GAAA-capped hairpin was further destabilized , thus providing additional credence to the role of the double-stranded structure in directing cleavage ( Figure 5A and 5B ) . The seven mutants of this group , despite not affecting nucleotides of loop E ( Figure 5A ) , had a marked negative effect on ligation ( Figure 5C ) . These results indicate that the sequence and/or secondary structure requirements for this reaction are more demanding than those regarding cleavage , and that they include nucleotides apart from those of loop E . The adjacent bulged-U helix ( Figure 5A ) , the stability of which is affected by most of these mutants , appears particularly relevant in this respect . These mutations , besides covering alternative positions of the upper CCR strand , were anticipated as very informative because most impinge on the GAAA tetraloop capping the hairpin that according to Baumstark et al . [25] directs cleavage , and also because most of these nucleotides form part of the loop E that presumably mediates ligation [20 , 27] ( Figure 6A ) . The double mutant #8 ( C92→G and G99→C , the rationale for the second substitution is given below ) , and the single mutants #9 ( A100→U ) , #10 ( A100→C ) , #11 ( A101→C ) , #12 ( A102→U ) , and #13 ( A102→C ) , had in general a mild effect on cleavage . Excepting mutant #9 , in which cleavage was reduced to 34% with respect to wt , cleavage of the others was at least 68% , with mutants #8 , and #11 to #13 , reaching 88%–92% ( Figure 6B ) . Given that the GAAA tetraloop belongs to the family of GNRA tetraloops ( in which N is any base and R a purine ) [37] , these results do not support a role in cleavage of the GAAA-capped hairpin , because changes disrupting interactions critical for the tetraloop stability ( mutants #8 , and #11 to #13 ) had essentially no influence on cleavage . In contrast , the six mutants induced a pronounced negative effect on ligation , thus sustaining a critical function of loop E in this reaction ( Figure 6C ) . Indeed , from the structural model derived for loop E of PSTVd [27] , and considering that nucleotides critical for this motif are conserved or substituted by others preserving it in CEVd , mutants #9 , #10 , #12 , and #13 all introduce non-isosteric pairs disrupting the loop E structure . However , mutant #11 is predicted to maintain the loop E structure , suggesting that its negative effect on ligation could result from sequence rather than from structural restrictions . Consistent with this view , the nucleotide corresponding to position A101 in CEVd is phylogenetically conserved in the family Pospiviroidae ( Figure 3 ) . On the other hand , mutants #11 to #13 affect minimally the stem stability of hairpin I ( particularly of its upper portion because they map outside the 3-bp stem adjacent to the tetraloop ) and the double-stranded structure ( in which they are outside the GC-rich central region of 10 bp containing the cleavage sites ) and , therefore , their effects are consistent with a function of this latter structural motif in cleavage ( Figure 6A ) . The negative effects of mutants #9 and #10 on cleavage are also compatible with this view , because they alter the stability of both the 3-bp stem adjacent to the hairpin I tetraloop and the 10-bp central region of the double-stranded structure , although it is difficult to interpret why cleavage was significantly more reduced in mutant #9 than in #10 ( Figure 6A and 6B ) . Going back to the double mutant #8 , its high cleavage ( Figure 6B ) can be explained because , despite affecting nucleotides C92 and G99 that form a pair phylogenetically conserved in the hairpin I/double-stranded structure of the family Pospiviroidae , this base pair is just inverted ( Figure 6A ) . In mutant #14 ( C92→U ) , in which the pair between C92 and G99 was substituted by a U:G pair , cleavage still was relatively significant ( 63% ) . The differential effect in ligation of these two mutants is intriguing: ligation was essentially abolished in mutant #8 , whereas in mutant #14 was close to 10% with respect to wt ( the highest value for any of the mutants of the present study ) ( Figure 6C ) . It is worth noting that the double mutant #8 affects the nucleotide of the upper CCR strand that upon UV irradiation becomes cross-linked to U266 of the lower CCR strand ( data not shown in [22] ) and also disrupts a G:C pair of the flanking bulged-U helix , in contrast to the single mutant #14 in which this base pair is substituted by a G:U pair ( Figure 6A ) . These results again underline that ligation is influenced by nucleotides aside from those conserved in loop E . If only nucleotides of the upper CCR strand direct cleavage , its extension should not be influenced by mutations in the lower CCR strand , which in contrast should reduce ligation particularly if they impinge on nucleotides of the loop E motif that presumably directs this reaction . To test this hypothesis , we constructed two mutants in which U266 , the nucleotide of the lower CCR strand that upon UV irradiation becomes cross-linked to G99 of the upper CCR strand ( data not shown in [22] ) ( Figure 7A ) , was changed: mutants #15 ( U266→C ) and #16 ( U266→A ) . Extending to CEVd the structural model derived for loop E of PSTVd [27] , U266 and A100 in loop E of CEVd should interact via trans Watson-Crick/Hoogsteen edges and belong to the isosteric subgroup I1 . In mutants #15 and #16 , C266 and A100 , and A266 and A100 , are predicted to interact similarly; however , they belong to subgroups I2 and I4 , respectively , which are non-isosteric with respect to the original I1 and may thus disrupt the loop E structure to some extent [27] . Northern blot hybridization of RNAs from the corresponding transgenic A . thaliana lines showed that cleavage remained essentially unaffected ( 90%–95% relative to wt ) , whereas ligation was essentially abolished ( Figure 7B and 7C ) . These results support further the notion that cleavage is determined exclusively by RNA motifs formed by nucleotides of the upper CCR strand and flanking nucleotides , and also show that ligation is determined by nucleotides of loop E and by others of both CCR strands . In particular , the bulged-U helix may play a key role in aligning the termini to be ligated .
Processing of oligomeric ( + ) RNAs in the family Pospiviroidae entails cleavage to the ml ( + ) RNA , and ligation of the resulting species to the mc ( + ) RNA . Hence , the most direct way to identify the processing site is mapping where the ml ( + ) RNA intermediate is opened . Previous studies have pointed to the upper strand of the CCR , which , due to its strict conservation within each genera of the family , has been long assumed to play an essential role . Data supporting this view include infectivity bioassays with different PSTVd DNA and RNA constructs [17] , with longer-than-unit HSVd clones [16] , and with CEVd constructs containing sequence repetitions and point mutations in the upper CCR strand [18] . The latter study concluded that processing occurs at one of three consecutive Gs of the upper CCR strand , and advanced hairpin I or an alternative double-stranded structure as the putative RNA motifs directing cleavage ( Figure 5A ) . A critical reassessment of all these data led to a model involving the double-stranded structure in cleavage , although the model did not predict the mechanism of cleavage-ligation or specify the exact processing site [15] . The infectivity-based approach , however , has an important limitation: bioassays do not provide a linear dose-response , being at best semi-quantitative and making it difficult to draw accurate estimations . Reflecting this limitation , other data point to alternative processing sites in the PSTVd lower CCR strand [28] . Furthermore , transcripts with only a 4-nt repetition of the PSTVd upper CCR strand [38] or with the exact unit-length CEVd [39] are still infectious , and another work suggested that the basic requirement for infectivity of a range of unit-length CEVd in vitro transcripts starting at different domains of the molecule is their ability to form a short double-stranded region of viroid and vector sequences at the junction of the two termini [31] . Therefore , at least in some cases , infectivity is independent of duplicated viroid sequences , possibly because the exact full-length sequence is restored by strand switching of a jumping polymerase during transcription [31] . On the other hand , primer-extension on the ml viroid ( + ) RNAs isolated from infected propagation hosts also has significant constraints ( see Results ) , with this approach having mapped several processing sites in different PSTVd domains [29 , 30 , 32] . Finally , conclusions from in vitro assays in which a potato nuclear extract was primed with an ml PSTVd ( + ) RNA with a 17-nt repeat of the upper CCR strand should be interpreted with caution , because the processing complex formed in vitro may not mimic the corresponding complex in vivo . Moreover , prior to incubation with the nuclear extract , the PSTVd RNA was heated to promote the adoption of a specific secondary structure that may not parallel that existing in vivo [20] . The A . thaliana–based system reported recently [34] circumvents most of these limitations . It is an in vivo system in which the available data indicate that processing is correct: transgenically expressed dt ( + ) RNAs of typical members of the family Pospiviroidae are cleaved to the ml forms—implying recognition of two identical sites—and then ligated to the infectious mc RNAs , whereas the complementary dt ( − ) RNAs are not ( [34] , this work ) , thus reproducing the situation observed in typical hosts . However , in contrast to typical hosts in which the turnover of the longer-than-unit ( + ) replicative intermediates is difficult to follow because of their low accumulation and diverse size , the A . thaliana–based system with the viroid-expression cassette integrated in the plant genome provides a constant supply of a size-specific replicative-like intermediate that permits the easy quantification thereof and of its processing products . Moreover , despite typical members of the family Pospiviroidae being able to complete their replication cycle when expressed transgenically as dt ( + ) RNAs in A . thaliana , the replication level in this non-host plant is rather low ( see Figure 1 and [34] ) , and the ml and mc ( + ) RNAs can be assumed to come essentially from processing of the transgenically expressed dt ( + ) RNA . Therefore , the effects of specific mutations in the primary transcript on cleavage and ligation can be evaluated—regardless of whether the resulting products are infectious or not—and it is even possible to identify mutations affecting only ligation . Our results with the A . thaliana–based in vivo system mapped the cleavage site of CEVd ( + ) strands at the upper CCR strand , in a position equivalent to that inferred for PSTVd with an in vitro system [20] . However , we consider that the RNA motif directing cleavage in vivo is not the GAAA-capped hairpin proposed previously [20] , but the hairpin I/double-stranded structure . The first argument supporting this view is that whereas the cleavage sites of HSVd and ASSVd ( + ) strands also map at equivalent positions in a similar hairpin I/double-stranded structure , these viroids cannot form the GAAA-capped hairpin . In contrast , examination of the hairpin I/double-stranded structure reveals some appealing features . Hairpin I is composed by a tetraloop , a 3-bp stem , an internal symmetric loop of 1–3 nt in each strand that presumably interact by non-Watson-Crick base pairs [40] , and a 9–10-bp stem that can be interrupted by a 1-nt symmetric or asymmetric internal loop [18 , 35] ( Figure 3 ) . Remarkably , these structural features are conserved in the type species of the five genera composing the family Pospiviroidae and additionally: i ) the capping tetraloop is palindromic itself , and ii ) the two central positions of the tetraloop and the central base pair of the 3-bp stem are phylogenetically conserved ( Figure 3 ) [35] . As a consequence , a long double-stranded structure with a GC-rich central region of 10 bp containing the cleavage sites can be alternatively assumed by the same sequences in a di- or oligomeric RNA ( Figure 5A ) . The second argument supporting the hairpin I/double-stranded structure as the RNA motif directing cleavage derives from the effects on this reaction of mutants affecting differentially this motif versus the GAAA-capped hairpin . Chief among them are mutants #8 , and #11 to #13 that , despite disrupting interactions crucial for the stability of the GAAA tetraloop , did not basically modify cleavage . Furthermore , because the ml PSTVd ( + ) RNA with a 17-nt repeat of the upper CCR strand that was used to prime the potato nuclear extract [20] can also form a fragment of the proposed double-stranded structure containing the cleavage sites , the correct cleavage observed in vitro can be alternatively interpreted as being directed by this structure . Our interpretation of direct effects of the introduced mutations in viroid RNA processing is based on the weak viroid RNA-RNA amplification in A . thaliana and , therefore , side effects of this amplification in cleavage and ligation cannot be totally discarded . In summary , we believe that the substrate for cleavage in vivo of all members of the family Pospiviroidae is the double-stranded structure proposed by Diener [15] , with hairpin I playing a role in promoting the adoption of this structure ( see below ) . Although its existence in vivo remains to be fully demonstrated , we have noticed that the cleavage sites in the double-stranded structure leave two 3′-protruding nucleotides in each strand ( Figure 8 ) , the characteristic signature of RNase III enzymes [41 , 42] . The participation of an enzyme of this class , of which there are at least seven in A . thaliana [43] , is consistent with the nuclear location of some of them , which additionally have preference for substrates with a strong secondary structure resembling that of viroids . Moreover , one or more RNase III isozymes should be involved in the genesis of the viroid-derived small RNAs with properties similar to the small interfering RNAs that accumulate in viroid-infected tissues [44–48] . Going one step further , if an RNase III indeed catalyzes cleavage of the oligomeric ( + ) RNAs of the family Pospiviroidae , the resulting products should have 5′-phosphomonoester and free 3′-hydroxyl termini . Characterization of the ml ( + ) RNAs from A . thaliana transgenically expressing dt CEVd ( + ) RNAs shows that this is actually the case ( M . E . Gas , D . Molina-Serrano , C . Hernández , R . Flores , and J . Daròs , unpublished data ) . The adoption in vivo of the double-stranded structure with a GC-rich central region containing the cleavage sites could be promoted by hairpin I because prior work with PSTVd has mapped a dimerization domain at this hairpin [40] . This situation resembles that observed previously in retroviruses in which dimerization , a critical step of their infectious cycle , is mediated by a hairpin with a palindromic loop that can dimerize co- or post-transcriptionally via a kissing loop interaction between two viral RNAs [49] . During transcription of oligomeric ( + ) RNAs of the family Pospiviroidae , a kissing loop interaction between the palindromic tetraloops of two consecutive hairpin I motifs might similarly start intramolecular dimerization , with their stems then forming a longer interstrand duplex ( Figure 8 ) . Part of the negative effects on cleavage of mutants #1 to #6 ( Figure 5 ) could result from weakening dimerization . Regarding ligation , our results support that the substrate for this reaction in the genus Pospiviroid is the extended conformation containing loop E [20 , 22] ( Figure 8 ) . Therefore , whereas cleavage is solely dependent on the upper CCR strand and flanking nucleotides , ligation is dependent on nucleotides of both CCR strands that encompass those of loop E and others adjacent . This entails a switch between two conformations , one for cleavage and another for ligation , which might be facilitated by the RNA helicase activity associated with some RNase III enzymes [43] . Because within the family Pospiviroidae loop E is only formed in the genera Pospiviroid and Cocadviroid , other genera of this family must have alternative motifs playing a similar role in ligation . Potential candidates are the extended conformation of the CCR with a bulged-U helix conserved in all members of the genera Pospiviroid , Hostuviroid and Cocadviroid , and similar structures in the other genera of this family . Last but not least , the 5′-phosphomonoester and free 3′-hydroxyl termini resulting from cleavage mediated by an RNase III predict the existence of an RNA ligase able to join these ends , which is therefore different from the class represented by the wheat-germ RNA ligase that catalyzes joining between 5′-hydroxyl and 2′ , 3′-cyclic phosphodiester termini [50] . This latter RNA ligase class has been long regarded as the enzyme involved in circularization of PSTVd ( + ) strands and , by extension , of other members of its family [29 , 51] . Our results advise for a reassessment of this long-held paradigm . The A . thaliana–based system is a promising tool for dealing with this and other related questions because it combines the advantages described previously with a broad collection of mutants .
Viroid sequence variants were CEVd ( M34917 ) having a deleted G between positions 70 and 74 , HSVd ( Y09352 ) , and ASSVd ( AF421195 ) . Plasmids pBmCEVdB , pBmCEVdS , and pBmCEVdP contained monomeric CEVd-cDNAs cloned at the BamHI , SacI , and PstI sites , respectively , pBmHSVdE a monomeric HSVd-cDNA cloned at the EcoRI site , and pBmASSVdS a monomeric ASSVd-cDNA cloned at the SalI site . Plasmids containing head-to-tail dimeric cDNA inserts of CEVd , HSVd , and ASSVd have been described previously [34] . Binary vectors for plant transformation were constructed by replacing the β-glucuronidase-cDNA of pCAMBIA-2301 ( AF234316 ) by dimeric head-to-tail CEVd-cDNAs ( starting at the PstI site ) corresponding to the wt ( pCKdCEVd-wt ) and 16 mutants ( pCKdCEVd-1 to pCKdCEVd-16 ) ( Table 1 ) . Plasmid pBmCEVdP was amplified with a series of pairs of 5′-phosphorylated adjacent primers that were complementary and homologous to different regions of the wt CEVd sequence , except in some 5′-proximal positions in which changes were introduced to obtain the desired mutants ( Table 1 ) . Pwo DNA polymerase was used in the buffer recommended by the supplier ( Roche Applied Science ) . After initial heating at 94 °C for 2 min , the amplification profile ( 30 cycles ) was 30 s at 94 °C , 30 s at 58–68 °C ( depending on the predicted melting temperatures ) , and 3 . 5 min at 72 °C , with a final extension of 10 min at 72 °C . PCR products corresponding to the full-length plasmid were eluted after agarose gel electrophoresis , ligated , and used for transformation . Incorporation of the expected mutations was confirmed by sequencing . The mutated CEVd-cDNA inserts were PCR-amplified , eluted after non-denaturing PAGE , and ligated to obtain dimeric cDNAs that were cloned in pBluescript II KS ( + ) . Plasmids with dimeric head-to-tail inserts were selected by restriction analysis and subcloned in the binary vector pCAMBIA-2301 . Agrobacterium tumefaciens ( strain C58C1 ) was transformed with plasmids ( pCKdCEVd-wt and pCKdCEVd-1 to pCKdCEVd-16 ) following standard protocols . Transformation of A . thaliana ( ecotype Col-0 ) was performed by the floral dip method using midlog-grown cultures of A . tumefaciens [52] , and transgenic plants were selected by germinating the seeds from dipped A . thaliana in plates with 100 μg/ml kanamicine , 300 μg/ml cefotaxime , and 10 μg/ml benomyl . Total nucleic acids from leaves of CEVd-infected gynura ( Gynura aurantiaca DC ) , HSVd-infected cucumber ( Cucumis sativus L . ) , and transgenic A . thaliana , as well as from fruits of ASSVd-infected apple ( Malus pumilla Mill . ) , were extracted with buffer-saturated phenol and enriched in viroid RNAs by chromatography on non-ionic cellulose ( CF11 , Whatman ) [34] . RNAs from CEVd-infected gynura and ASSVd-infected apple were further fractionated with 2 M LiCl . RNA aliquots were separated by either single denaturing PAGE in 5% gels with 8 M urea in 1X TBE ( 89 mM Tris , 89 mM boric acid , 2 . 5 mM EDTA [pH 8 . 3] ) , or double PAGE , first in a non-denaturing 5% gel in TAE ( 40 mM Tris , 20 mM sodium acetate , 1 mM EDTA [pH 7 . 2] ) , with the gel segment containing the monomeric viroid RNAs being cut and applied on top of a second 5% gel with 8 M urea in 0 . 25X TBE . RNAs were electroblotted to nylon membranes ( Hybond-N , Amersham Biosciences ) , UV-fixed with a cross-linker ( Hoefer ) , and hybridized ( at 70 °C in the presence of 50% formamide ) with strand-specific 32P-labeled riboprobes obtained by transcription with T3 or T7 RNA polymerases of plasmid pBdCEVdP properly linearized . After washing the membranes , the signals of the dt RNAs and their resulting ml and mc forms were quantified with a bioimage analyzer ( Fujifilm FLA-5100 ) . Cleavage and ligation were estimated for each A . thaliana CEVd mutant from the fractions ( mc+ml ) / ( dt+mc+ml ) and mc/ ( mc+ml ) , respectively , and the results normalized with respect to those of the A . thaliana CEVd-wt ( taken as 100% ) . Two independent plants were analyzed for each transgenic line , with differences in cleavage and ligation being less than 10% in all instances . Primer extensions were carried out for 45 min at 55 °C , 10 min at 60 °C , and 5 min at 65 °C , in 20 μl containing 50 mM Tris-HCl [pH 8 . 3] , 75 mM KCl , 3 mM MgCl2 , 5 mM dithiothreitol , 0 . 5 mM each of the dNTPs , 40 U of RNase inhibitor ( Promega ) , and 200 U of SuperScript III reverse transcriptase ( Invitrogen ) . The ml and mc viroid RNAs serving as template were obtained by double PAGE and elution . Primers PI ( 5′-TTCTCCGCTGGACGCCAGTGATCCGC-3′ ) , PII ( 5′-GCTTCAGCGACGATCGGATGTGGAGCC-3′ ) , PIII ( 5′- GAGCAGGGGTGCCACCGGTCGC-3′ ) , and PIV ( 5′-GACTAGCGGCGCGAAGAGTAGGTGG-3′ ) , were 5′-labeled with T4 polynucleotide kinase ( Roche Applied Science ) and [γ-32P]ATP ( Amersham Biosciences ) . Before reverse transcription , each primer was annealed in water to the purified viroid RNA ( 10:1 molar ratio ) by heating at 95 °C for 5 min and snap-cooling on ice . Reactions were stopped at 70 °C for 15 min , and the products analyzed by PAGE on 6% sequencing gels . The exact size of the extension products was determined by running in parallel sequence ladders obtained with the corresponding primer and a recombinant plasmid containing the monomeric viroid-cDNA insert ( Thermo Sequenase cycle sequencing kit , USB ) . | Interactions of viroids with their host plants are unique because these subviral pathogenic RNAs lack protein-coding capacity . Therefore , hosts must provide all enzymes and auxiliary factors that viroids need for their infectious cycle . Replication of viroids entails reiterative transcription of their single-stranded circular genomes , cleavage of oligomeric strands to unit-length , and ligation to circular RNAs . While cleavage in chloroplastic viroids ( family Avsunviroidae ) is autocatalytic and mediated by hammerhead ribozymes , where and how cleavage of oligomeric ( + ) RNAs of nuclear viroids ( family Pospiviroidae ) occurs in vivo is controversial . We have re-examined this question in vivo , taking advantage that dimeric viroid RNAs expressed transgenically in Arabidopsis thaliana are processed correctly . Together with mapping the in vivo processing site of three members of the family Pospiviroidae , our results with 16 mutants of one of these viroids support that cleavage is directed by an RNA motif conserved in all members of the family , and ligation by an extended conformation containing a motif termed loop E . Both processing reactions are most likely catalyzed by enzymes different from those generally assumed: cleavage by an RNase III–like enzyme , and ligation by an RNA ligase distinct from the only one characterized so far in plants . | [
"Abstract",
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] | [
"virology",
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] | 2007 | Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations |
Amebiasis is caused by the protozoan parasite Entamoeba histolytica ( Eh ) , a potentially fatal disease occurring mainly in developing countries . How Eh interacts with innate host factors in the gut is poorly understood . Eh resides and feed in/on the outer colonic mucus layer and thus share an ecological niche with indigenous microbiota . As gut microbiota regulates innate immune responses , in this study we characterized the cooperative roles that microbiota and the mucus layer play in Eh-induced pro-inflammatory responses in the colon . To study this , we used antibiotics treated and non-treated specific pathogen free Muc2-/- and Muc2+/+ littermates and germ-free mice inoculated with Eh in colonic loops as a short infection model . In antibiotic treated Muc2-/- and Muc2+/+ littermates , Eh elicited robust mucus and water secretions , enhanced pro-inflammatory cytokines and chemokine expression with elevated MPO activity and higher pathology scores as compared to the modest response observed in non-antibiotic treated littermates . Host responses were microbiota specific as mucus secretion and pro-inflammatory responses were attenuated following homologous fecal microbial transplants in antibiotic-treated Muc2+/+ quantified by secretion of 3H-glucosamine newly synthesized mucin , Muc2 mucin immunostaining and immunohistochemistry . Eh-elicited pro-inflammatory responses and suppressed goblet cell transcription factor Math1 as revealed by in vivo imaging of Eh-colonic loops in Math1GFP mice , and in vitro using Eh-stimulated LS174T human colonic goblet cells . Eh in colonic loops increased bacterial translocation of bioluminescent E . coli and indigenous bacteria quantified by FISH and quantitative PCR . In germ-free animals , Eh-induced mucus/water secretory responses , but acute pro-inflammatory responses and MPO activity were severely impaired , allowing the parasite to bind to and disrupt mucosal epithelial cells . These findings have identified key roles for intestinal microbiota and mucus in regulating innate host defenses against Eh , and implicate dysbiosis as a risk factor for amebiasis that leads to exacerbated immune responses to cause life-threatening disease .
Entamoeba histolytica ( Eh ) is a human protozoa parasite that causes the disease amebiasis and that , in 2013 , was responsible for approximately 11 , 300 deaths worldwide [1] . Interestingly , of those individuals infected with the parasite , only 10% develop symptoms of the disease while the rest remain asymptomatic . To date , the variation in disease outcome has not been fully explained , but several studies have suggested that this difference could be due to host´s health conditions as well as immune fitness . More recently , with molecular tools for studying the microbiome , the idea that specific bacteria within the microbiota could modulate Eh pathogenesis and predispose to intestinal amebiasis [2] and amebic liver abscess ( ALA ) [3] has been proposed . MUC2 is the major secretory mucin in the gastrointestinal tract and forms a barrier between the epithelial cells monolayer and the luminal content that not only consist of nutrients , but is also loaded with potential pathogenic microorganisms . When Eh is ingested through contaminated food or water , it colonizes the colonic outer mucus layer , which is rich in a diverse community of bacteria . The human intestine hosts approximately 100 trillion microorganisms [4] , mainly bacteria , that form the microbiota that regulates host homeostasis by promoting digestive health as well as stimulating a balanced immune system [4 , 5] . It is known that Eh stimulates goblet cell mucus secretion [6] as an innate host defense mechanism to counter Eh adherence to intestinal epithelial surfaces [7] . In disease pathogenesis , Eh cysteine proteases cleave MUC2 in the non-glycosylated C-domain , weakening its structure and facilitating Eh contact with epithelial cells [8] . Germ-free and gnotobiotic experiments with guinea pigs have established that the presence of gut microbiota is required for Eh pathogenicity [9] . In recent studies , the link between microbiota and Eh infection in humans has been suggested with varying results depending on location and subjects of the study . A study in India have shown a decrease in mostly butyrate-producing bacteria ( Clostridium coccoides , C . leptum , Eubacterium , Lactobacillus , Bacteroides ) and an increase in Bifidobacterium in stool samples from Eh positive patients [10] . More recently , a study in Cameroon found higher α-diversity and a decrease in β-diversity in individuals positive for Eh infection with an increase in members of the Clostridiales Ruminococcaceae family and a decrease in Prevotella copri [11] . The decrease in P . copri was confirmed in a longitudinal study done in children living in a slum in Bangladesh and the presence of this bacteria was correlated with higher incidence of diarrhea when compared with asymptomatic Eh-positive children [12] . Analysis of bacterial diversity in ALA patients was not able to significantly relate its incidence with any specific bacteria , however , co-infection with bacteria was present in most of the ALA samples , with a notable higher abundance of Klebsiella [3] . Various studies [2 , 13–15] have demonstrated that microbiota can significantly alter the outcomes of different protozoan infections , however mechanisms underlying these relationship remain poorly understood . In this study , we explored the distinct contributions of microbiota and the Muc2 mucus barrier in Eh-induced innate and pro-inflammatory responses critical in disease pathogenesis . Our findings show that indigenous commensal microbiota that colonizes the outer Muc2 mucus layer plays an important role in fortifying innate host defense against Eh and that dysbiosis ( antibiotic and/or alterations in the mucus layer ) renders the colonic epithelium susceptible to Eh-induced pro-inflammatory responses and tissue injury .
To quantify the distinct roles of indigenous microbiota and/or the presence of an intact Muc2 mucus layer in Eh-induced innate host responses in closed colonic loops [16] , Muc2+/+ and Muc2-/- littermates were pre-treated or not with a broad spectrum antibiotic ( Abx ) cocktail and compared with germ-free mice ( GF ) . Eh inoculated in colonic loops in Muc2+/+ presented with watery and mucoid secretions under intense pressure and bloating ( gas bubble formation ) . Abx-treated animals showed similar responses but also presented with bloody secretions ( Fig 1A ) as compared to PBS-inoculated controls . Muc2-/- littermates in the absence of a mucus layer showed similar ballooning effects with abundant watery secretions ( Fig 1B ) . Similarly , colonic loops in GF mice presented with ballooning mucoid secretions under intense pressure ( Fig 1C ) . Overall , no significant differences were observed in gross pathology scores between the Muc2 genotypes and GF mice inoculated with Eh . Similarly , Abx treatments did not modify gross pathology scores ( Fig 1D ) . To determine whether Abx treatment altered Eh-induced host inflammatory responses in Muc2 genotypes , colonic tissues and luminal contents were analyzed for pro-inflammatory cytokines and chemokines . Interestingly , following Abx treatment , basal IFN-γ and TNF-α pro-inflammatory cytokine expression decreased in colonic tissues in both Muc2+/+ and Muc2-/- littermates as compared to untreated controls ( Fig 2A–2C ) . However , as predicted , Eh-inoculated colonic loops showed increased pro-inflammatory cytokines expression regardless of the presence or absence of a mucus layer . Surprisingly however , Abx-treated mice inoculated with Eh showed significant increase in the pro-inflammatory cytokines IFN-γ and TNF-α mRNA expression as compared with non Abx-treated Eh inoculated littermates ( Fig 2A–2C ) . A similar increase in IFN-γ and TNF-α protein levels were noted in the luminal contents of Abx-treated Muc2+/+ and Muc2-/- littermates inoculated with Eh compared as compared to none Abx-treated controls ( Fig 2D–2F ) . Likewise , chemokine levels ( MCP-1 , KC and MIP-2 ) in the luminal contents of Abx-treated animals inoculated with Eh were significantly increased compared with non Abx-treated controls ( Fig 2G–2I ) . Several other cytokines and chemokines were assessed by multiplex , however their differences were not significant . These results clearly show that a dysbiotic state induced by Abx-treatment predisposes the host for robust pro-inflammatory responses toward Eh regardless of the presence or absence of a functional mucus barrier . As an intact Muc2 mucus layer and hyper secretion of mucus are critical determinants of innate host defense against Eh [17] , we determine if Abx treatment in Muc2+/+ littermates affected mucin biosynthesis and secretion . This was monitored in colonic tissues stained with periodic acid Schiff ( PAS ) reagent to visualize the mucus layer and filled goblet cells , transcription factors for goblet cell lineage and immunostaining for Muc2 mucin within goblet cells . Muc2+/+ controls and Abx-treated animals showed normal colonic architecture with numerous goblet cells ( arrow ) filled with PAS+ material ( Fig 3A-top panel ) . However , when inoculated with Eh , Muc2+/+ showed prompt robust mucus secretory response ( Fig 3A-bottom left ) with coalescence of mucin granules and mucus streaming out in the lumen . In Abx-treated animals , Eh elicited enhanced mucus secretion ( Fig 3A-bottom right ) forming a thick mucus plug over the mucosal surface ( Fig 3A-bottom right inset ) . Mucus secretagogue effects evoked by Eh correlated well with the number of filled goblet cells per crypt . In particular , there was a significant decrease in the number of filled goblet cells in control and Abx-treated mice inoculated with Eh as compared with controls that received PBS ( Fig 3B ) . While Abx treatment alone did not affect basal Muc2 gene expression , in response to Eh , both controls and Abx treated animals showed significant upregulation of Muc2 mRNA expression ( Fig 3C ) . Interestingly , Abx treatment had no effect on basal transcription for the secretory cell lineage Math1 , however in response to Eh , Math1 gene expression was significantly decreased but had no effect in Abx-treated animals ( Fig 3D ) . As Math1 affects all secretory lineage we analyzed Spdef expression [SAM pointed domain containing ETS transcription Factor ( SPDEF ) ] , the transcription factor that is critical for terminal goblet cell differentiation [18] and observed the same trend ( Fig 3E ) . Based on the decreased number of filled goblet cells in Eh-inoculated animals with a corresponding increase in Muc2 gene expression , immunostaining of colonic loops was done to visualize Muc2 mature mucin granules within goblet cells . Even though a similar pattern to the PAS staining was observed with the Muc2 antibody , immunostaining revealed mucin granule-granule coalescence and mucus streaming from goblet cells in the deep crypts in response to Eh ( Fig 3F arrows ) . There was a paucity of filled goblet cells with mucin in Abx + Eh inoculated animals ( Fig 3F arrows ) demonstrating intense mucus secretion with cavitation and/or mucus depleted goblet cells ( Fig 3G ) . To quantify mucin and none mucin glycoprotein secretions , mucus in Muc2+/+ littermates were metabolically label with 3H-glucosamine that incorporates into galactose , N-acetylgalactosamine and N-acetylglucosamine glycans into newly synthesized mucin . The 3H-labeled glycoproteins secreted in response to Eh were then fractionated into high molecular weight Vo mucin and non-mucin components by Sepharose 4B column chromatography ( Fig 4A ) [6] . Abx treatment had no effect on constitutive mucin or non-mucin glycoproteins secreted in the colon as compared with untreated control ( Fig 4A , 4B and 4C orange panels ) . Consistent with previous studies [16] , Eh significantly stimulated not only V0 mucin but also non-mucin glycoprotein secretions ( Fig 4A , 4B and 4C yellow panels ) . Surprisingly , Abx-treated mice inoculated with Eh ( Abx + Eh ) showed enhanced secretions of both mucin and non-mucin glycoproteins ( Fig 4C , 4D and 4E purple panels ) as compared to animals that were not treated with Abx but inoculated with Eh ( Eh infected group ) . To exclude the possibility that the enhanced mucus secretory effect was due to the Abx , animals received fecal microbial transplantation ( FMT ) with their own microbiota following Abx treatment and then inoculated with Eh . Remarkably , FMT normalized both 3H-mucin and non-mucin glycoproteins secretions equivalent of animals inoculated with Eh that did not receive Abx ( Fig 4C , 4E and 4F gray ) . Taken together , these results clearly indicate that dysbiotic microbiota provokes enhanced mucin and non-mucin secretions in response to Eh . Based on the differential Math1 gene expression ( Fig 3D ) , we next investigated the fate of the secretory goblet cells in the colon following Abx treatment and in response to Eh . To interrogate this , we used Math1GFP mice containing the green fluorescent protein ( GFP ) reporter for Math1-expressing goblet cells . In the colon , Math1 is expressed in epithelial cells to differentiate into Muc2-producing goblet cells lineage . We have recently used Math1GFP mice to quantify goblet cells by flow cytometry and by imaging to follow the fate of goblet cells in DSS-induced colitis [19] . Basally , Math1GFP activity was higher in the proximal than the distal colon in control animal . However , following Abx treatment , while fluorescence activity in the proximal colon remained unchanged there was a significantly decrease in Math1 activity in the distal colon ( Fig 5A and 5B ) . Surprisingly , control mice inoculated with Eh showed a significant decrease in Math1GFP activity in the proximal colon ( area in direct contact with Eh ) with a corresponding increase in activity in the distal colon ( away from Eh interaction ) . In Abx treated mice , while Math1GFP activity in the proximal colon remained the same as control animals , Math1 activity in both the proximal and distal colon was completely silenced in response to Eh ( Fig 5A and 5B ) . These results suggest that Eh supresses Math1 activity in the proximal colon where Eh are contained within the loops and can alter Math1 activity distally . To determine if the down regulation in Math1 activity was a direct effect from Eh or initiated by Eh-induced inflammation , LS174T human colonic goblet were inoculated with Eh and MATH1 mRNA expression quantified . Within 30 min , Eh significantly decreased MATH1 mRNA expression that remained low up to 1h ( Fig 5C ) . Cells stimulated with glutaraldehyde-fixed equivalent numbers of Eh had no effect on MATH1 mRNA expression , suggesting a requirement for live parasites ( Fig 5C ) . In acute intestinal amebiasis , IL-1β is one of the most important pro-inflammatory cytokines elicited by cysteine protease 5 ( EhCP5 ) RGD motif ligating host cells integrins [20] and this cytokine had no effect on MATH1 transcription ( Fig 5C ) . These data suggest that Eh can also directly inhibit MATH1 transcription in the absence of bacteria . An important finding was that Eh infection in the proximal colon suppressed Math1 expression with a corresponding increased in Math activity in the distal colon ( Fig 5A ) . We hypothesized that the dysregulation of Math1 expression could be due to bacterial translocation . To establish if the Math1 activity was associated with increased bacterial translocation , animals were infected with bioluminescent non-pathogenic E . coli XEN-14 and inoculated with Eh in the proximal colon . Eh infection elicited significantly increased bioluminescent signals from the proximal/distal colon ( arrows shows the site of Eh inoculation ) up to the ileum and upper small intestine ( Fig 6A ) . These results suggest that in response to Eh , higher numbers of bacteria came in close contact and/or translocated in the intestinal mucosa that could potentially alter Math1 activity . Thus , to determine if bacterial LPS could regulate Math activity , Math1GFP animals were inoculated with a sublethal dose of LPS ( 5 mg/Kg BW , intraperitoneally ) and observed significantly higher levels of Math1 throughout the full-length of the gastrointestinal tract as compared to controls ( Fig 6B ) . This suggests Math1 expression in intestinal goblet cells could be stimulated via inflammation associated with sensing microbial components , ( e . g . LPS ) which triggers robust mucus secretion to reduce bacterial translocation in the gut . To address this , we first determined if Eh inoculated into colonic loops were altering gut permeability that could potentiate bacterial translocation into tissues by assessed intestinal permeability with FITC dextran . As predicted , Eh inoculated in colonic loops significant increased intestinal permeability ( Fig 6C ) associated with high levels of MPO activity in the ileum ( Fig 6D ) . We have previously demonstrated alterations in tight junction protein expression and loss of epithelial barrier function in the proximal colon inoculated with Eh [16 , 21] . As Xen-14 bacteria ( Fig 6A ) showed increased bioluminescent in the ileum and proximal colon following Eh-inoculation , fluorescent in situ hybridization ( FISH ) was done to visualize bacteria translocation . In Eh-inoculated colonic loops , most bacteria ( red ) in the ileum shifted significantly near the villi and deep in the crypts ( Fig 6E-d , Fig 6F arrow ) as compared to PBS inoculated controls ( Fig 6E-a ) . In marked contrast , Eh inoculation in the proximal colon , disrupted bacteria biofilms into patches that aggregated with mucus strands and/or translocate deep into the crypts and tissues ( Fig 6E-e arrows ) compared to control loops receiving PBS ( Fig 6E-b ) . In the distal colon of mice inoculated with Eh in the proximal colon there was increased mucus secretion with bacteria close and/or on the surface epithelium ( Fig 6E-f , Fig 6F ) compared to PBS controls ( Fig 6E-c ) . We also detected 20-fold higher bacterial counts in the mesenteric lymph nodes ( MLNs ) using 16S universal primers as compared to controls receiving PBS ( Fig 6G ) . Taken together , these data suggest that Eh-induced inflammation results in loss of epithelial barrier functions that facilitated commensal bacteria translocation that altered Math1 expressions in the ileum and proximal colon . The role microbiota plays in shaping the development of innate host defenses against Eh is not known . Based on the results above , microbial dysbiosis induced with Abx in Muc2+/+ and Muc2-/- specific pathogen-free ( SPF ) littermates markedly enhanced pro-inflammatory cytokine and mucin secretory responses towards Eh . To interrogate the distinct role of microbiota in the development of innate host defenses we quantified Eh-induced host responses in colonic loops of germ free ( GF ) mice . As expected , in response to Eh , SPF mice elicited a prompt increase in the expression of the pro-inflammatory cytokines TNF-α and IL-1β mRNA whereas no response was observed in GF mice ( Fig 7A ) . This is interesting as enhanced watery secretions were observed in Eh-inoculated colonic loops of GF mice ( Fig 1D ) . Surprisingly , basal Math1 and Muc2 mRNA expressions were very low in GF mice and Eh infection did not cause a further decrease as compared to Eh inoculated SPF animals ( Fig 7B and 7C ) . A similar decrease in myeloperoxidase ( MPO ) activity , a marker for neutrophils influx into the colon , was also noted in GF mice . This is in contrast to Eh in SPF mice that induced 4- and 3-fold increase in MPO activity in the proximal and distal colon , respectively ( Fig 7D ) . A dependency for microbiota in Eh-induced inflammation was shown by treating SPF mice with Abx that reduced MPO activity to those seen in GF mice in both the proximal and distal colon ( Fig 7D ) . As Muc2 and pro-inflammatory cytokine gene expression was associated with increased mucin secretion ( Fig 7A and 7C ) , we quantified the number of filled goblet cells ( GC ) in GF mice . In SPF , there was a significant reduction in filled GC in response to Eh as GC are actively releasing mucus in response to Eh ( see below ) whereas in GF mice we did not observe a decrease in filled GC . In fact , GF mice had less numbers of filled GC in the colon ( Fig 7E ) . Colonic tissues were fixed in Carnoy’s to preserve the mucus layers and stained with periodic acid-Schiff reagent to visualize mucus , goblet cells and Eh . In response to Eh , there was hyper secretion of mucus in SPF mice with cavitated ( empty , shown by the arrow ) GC ( Fig 8A ) , thick adherent dense inner mucus ( IM ) and a loose outer mucus layer ( OM ) with Eh ( Fig 8A ) . In GF mice the adherent mucus layer in the proximal colon was patchy with low numbers of GC . Nonetheless , Eh-induced intense mucus secretions from GC in the shallow crypts ( Fig 8B , arrows ) . Most striking however , unlike SPF were we rarely observe Eh in contact with the epithelium , in GF mice Eh were occasionally found bound to surface and adjacent epithelial cells ( Fig 8C; arrows and inset ) and at places , showed signs of epithelium erosion in direct contact with Eh ( Fig 8D arrows ) . Taken together , these results underscore a critical role for microbiota in the development of an effective mucus barrier and host pro-inflammatory cytokine responses in innate host defense against Eh .
A major deficiency in our knowledge gap is the relationship between Eh and colonic microbiota in parasite colonization , disease pathogenesis and innate host defenses . As microbiota colonizes and utilizes MUC2 mucin substrates as a food source to maintain homeostasis , it stands to reason a delicate balance must exist to sustain asymptomatic Eh infections . Eh colonizes in/on the MUC2 mucin outer layer and here it interacts freely with colonic microbiota without adverse effects on the host . At present , we do not know the distinct contribution of the microbiota and/or the MUC2 barrier in fortifying innate host defenses against Eh . This was the impetus for this study where we interrogated the distinct roles of both colonic microbiota and the mucus barrier in early responses towards Eh using colonic loops as a short-term infection model . The major findings of our study revealed that microbial dysbiosis played a critical role in Eh-induced water and mucus secretion and pro-inflammatory cytokine responses that was restored following fecal microbial transplants . Moreover , studies in germ free mice revealed that microbiota was critical for shaping the intestinal landscape for the development of goblet cells and formation of an effective mucus barrier and in educating the host pro-inflammatory cytokine responses to limit Eh binding and erosion of the surface epithelium . We have previously shown that Muc2-/- are highly susceptibility to Eh-induced secretory and pro-inflammatory responses compared to commercially bought WT animals on the same genetic background [16] . In this study , we used Muc2+/+ and Muc2-/- littermates to normalized the microbiota and surprisingly showed no differences in gross pathology scores among the genotypes . This highlights that the use of littermates are essential for microbiota studies as it greatly reduces the variability caused when using animal models in research [22] . Here , the absence of a mucus barrier did not leave Muc2-/- mice with a noticeable disadvantage to Eh as compared to mucus sufficient littermates , thus demonstrating that the protective role of the mucus barrier is intimately related to the host microbiota . It is well known that an Abx regime provokes alteration in microbial abundance [23] . This particular dysbiotic state is characterized with an increase in facultative anaerobic bacteria within the Enterobacteriaceae family , and has proven to be an indication of a non-homeostatic state in both animal models as well as in important human gastrointestinal diseases [24] . This switch to a more oxygenated luminal environment , could explain the exacerbated reaction towards Eh , characterized by an increase in pro-inflammatory cytokines IFN-γ and TNF-α , as well as chemokines MCP-1 , KC and MIP-2 . Eh , despite being a microaerophilic organism , possesses an arsenal of virulence factors to live in the colon [25] , but also has various mechanisms that not only protects it , but also allows Eh to invade into highly oxygenated environments such as in the case of extra-intestinal amebiasis [26] . In addition to mucin staining , Periodic-acid Shiff ( PAS ) reagent allowed us to visualize Eh and we consistently fail to observe Eh in close contact with the epithelium . This is interesting as the host is able to sense Eh secreted components and/or the altered environment to mount water and mucus secretions as well as pro-inflammatory cytokine and chemokine responses . Abx treatment alone did not affect basal mucus production or the numbers of filled goblet cells , Muc2 gene expression or total 3H-glycoprotein secretion . Curiously , previous studies have shown that macrolides Abx have an inhibitory effect on mucus production in airways [27] and is used therapeutically in the treatment for chronic obstructive pulmonary disease ( COPD ) , reducing airway goblet cells production of MUC5AC mucin and alleviating COPD symptoms [28] . A similar effect has been described with Muc2 mucin in the gastrointestinal tract , utilising different Abx treatment regimes that reduced the number of goblet cells and Muc2 gene expression in mice [29] , as well as mucus layer thickness [30] . At present , no other studies have reported Abx-induced mucus increase . In our study , we did not observe a reduction but rather , a slight increase in mucus production in Abx-treated mice and could be explained by the Abx regime used . The mucus layer , secreted by goblet cells , is a key player in maintaining intestinal homeostasis , mainly acting as a barrier and limiting contact between the epithelial cells and any potential hazard contained in the lumen [31 , 32] . Math1 is a transcription factor that differentiates intestinal stem cells into a secretory lineage , which includes Paneth cells , enteroendocrine cells and goblet cells [33] . Paneth cells are absent in the colon , likewise , enteroendocrine cells are more abundant in the small intestine , but some of them , like L , D and Enterochromaffin cells ( EC ) , can still be found in colon and rectum [34] , although , they form only about 1% of the cells in the colon . In our study , we conclude that Math1 activity visualized using Math1GFP had a greater effect on goblet cells than on any other secretory colonic cell lineage . There is a paucity of information on how intestinal pathogens affect the transcription factor Math1 . Studies with the nematode parasite Trichinella spiralis showed an increase in Math1 mRNA expression in the small intestine , as well as induction of goblet cell metaplasia when the parasite was present [35] , suggesting that Math1 has a protective role in the intestine . DSS-induced colitis in rats had no effect on Math1 activity [36] . Unfortunately , the exact mechanism by which this transcription factor exerts its protective activity is not yet fully described . Although the effect of the Abx cocktail we used was generic , this regime reduced Math1 activity basally in the distal colon . This regional effect could be due to the greater reduction in bacterial load and dysbiosis affecting the distal colon . An interesting finding was that Eh decreased Math1 activity in the proximal colonic loops with a corresponding increase in Math1 activity in the ileum and to a lesser extent in the distal colon . Bacterial translocation in response to Eh in colonic loops played a major role in the expression of Math1GFP activity in the ileum and proximal colon . While bacterial translocation have not been studied experimentally with Eh infection , translocation of bacteria from the genera Bacteroides , Peptostreptococcus [37] and Streptococcus [3] have been identified in samples from ALA patients . Since the liver is a sterile organ , the presence of commensal bacteria in ALA positive samples indicates that Eh infection led to bacterial translocation [3] . In this study , inoculation of Eh in colonic loops after 3h increased bacterial translocation in the proximal colon and caused shifts in bacterial populations in the ileum close to the mucosal surface and deep in crypts , this was associated with high MPO activity . Even though we did not detect bacterial translocation in the ileum , there was a significant increase in MPO activity that was not observed in Abx treated or GF animals inoculated with Eh . Based on the results of LPS administration it appears that systemic LPS administration accelerated bacterial translocation primarily in the ileum and proximal colon and to a lesser extent , in the distal colon . Commensal bacterial translocation has been reported in Giardia duodenalis infection [38] . Studies done in germ free ( GF ) guinea pigs showed that the presence of microbiota is necessary for Eh to express its pathogenicity [9] . Similarly , infection of GF mice with the parasite G . duodenalis failed to induce the characteristic pathology [39] . Likewise , GF mice infected with the protozoa Leishmania amazonensis presented with an innocuous infection and absent immune response towards the parasite [40] . The exact mechanism that explains this phenomenon is not clearly understood , but clearly suggests that microbiota plays a fundamental role in establishing the pathogenicity of these protozoa . Our results are in concordance with previous observations , as Eh inoculated in colonic loops of GF mice failed to induce the characteristic pro-inflammatory response in spite of modest water and mucus secretions in the colon . This phenomenon could be due to an undeveloped immune response in GF animals that rendered them with a limited ability to produce cytokines in response to a colonic pathogen . A requirement for cytokines was shown with Schistosoma mansoni infection in GF mice , where it is known that TNF-α was required for optimal proliferation of the parasite in the host [41] . The only parasite that showed a worst outcome in GF mice is infection with the protozoa Trypanosoma cruzi [42] . The exact mechanisms to explain this altered susceptibility are not known . Our finding on reduced number of goblet cells correlates with previous reports where a characterization of the colonic epithelium in GF mice showed decreased numbers of goblet cells [43] . Reduction in goblet cells appear to be systemic as it was also observed in paranasal sinuses [44] and in the conjunctiva [45] in these mice . GF mice have a thinner and penetrable intestinal mucus layer [46] and Muc2 monomers with shorter O-glycans [47] compared to conventional SPF mice . Based on this , we hypothesise that the reduction in thickness and changes in the biochemical structure of Muc2 rendered the mucus barrier more susceptible to Eh cleavage . It is possible that Eh glycosidases and proteases could degrade GF mucus with higher efficiency as they have shorter glycans and absence of commensal microbiota . As Eh utilizes microbiota and cleaved Muc2 substrates as its primary source of food , it is tempting to speculate that Eh in the GF colon needed to find alternatives nutrient sources , forcing the parasite to move closer to the epithelium . This could explain why we consistently see Eh in direct contact with the epithelium with epithelial erosions in the GF colon , a condition we never see in SPF mice . Taken together , these studies clearly show a requirement for colonic microbiota in forming the first line of innate host defense against Eh , independent of the Muc2 mucus layer . Disruption of microbiota with Abx , sensitized animals for exacerbated pro-inflammatory responses and high output water and mucus secretion toward Eh that was normalized with fecal microbial transplants . Eh infection in the proximal colon increased bacterial translocation and pro-inflammatory cytokine responses that influenced Math1 transcriptional activity of the goblet cell lineage . In the absence of microbiota , Eh failed to induce pro-inflammatory responses that together with a dysfunctional mucus layer allowed Eh to contact and disrupt the epithelium . This study advances critical roles for both microbiota and the mucus layer in forming layered innate host defenses against Eh invasion .
8 to 10 weeks old Muc2+/+ and Muc2-/- littermate mice on a C57BL/6 background were used in the experiments . Math1GFP mice ( also known as Atoh1tm4 . 1Hzo ) [48] were purchased from Jackson laboratory and bred in-house . Germ-free ( GF ) mice on a C57BL/6 background were purchased from the International Microbiome Centre at the University of Calgary . All animals were housed under specific pathogen-free conditions ( SPF ) in filter top cages and fed autoclaved food and water ad libitum . Throughout the study , animals were closely monitored to ensure healthy conditions; in addition , all experiments adhered to the University of Calgary Animal Care Committee standards . E . histolytica ( HM1:IMSS ) trophozoites were cultured in TYI-S-33 medium containing 100 U/ml penicillin/streptomycin at 37°C under axenic conditions . After 72h , logarithmic-growth-phase Eh cultures were harvested by chilling on ice for 9 min , pelleted at 200 × g , and washed twice with PBS . Trophozoites were subjected to routine passage through liver of gerbils to maintain high virulence . Muc2+/+ and Muc2-/- littermates were treated with Abx to decrease bacterial load as described previously [49] . Briefly , mice were gavaged every 12h with an Abx cocktail as follow: for the first 3 days mice were gavaged with amphotericin-B ( 1 mg/kg ) to suppress fungal growth . From day 4 , ampicillin ( 1 mg/mL ) was added to the drinking water , in addition , mice received orally vancomycin ( 50 mg/kg ) , neomycin ( 100 mg/kg ) , metronidazole ( 100 mg/kg ) and amphotericin-B ( 1 mg/kg ) for another 14 days . This combination ensures the safe and controlled delivery of Abx to each mouse while having a broad-spectrum effect . Due to its anti-amebic effect , metronidazole was removed from the cocktail during the last 7 days of administration . Fecal microbiota transplantation ( FMT ) was achieved by collecting 0 . 1g of mice feces ( about 4 fecal pellets ) , homogenized in 1 mL of sterile phosphate buffered saline ( PBS ) and centrifuged for 30 seconds at 1000 x g . Each mouse was gavaged with 200μl of the obtained supernatant every 48 h for a total of three times . To quantify mucin secretion in vivo , mice were fasted overnight and injected intraperitoneally with 20 μCi of 3H-glucosamine ( PerkinElmer , Waltham , MA ) in PBS for 3h to metabolically label newly synthesized mucin into galactose , N-acetyl-glucosamine and N-acetyl-D-galactosamine in the mucin monomer as described previously [16 , 50] . Colonic loops were used as a model for short-term infection studies ( 3h after infection ) , as described previously [51] . Briefly , Muc2+/+ and Muc2−/− mice were anesthetized with isoflurane inhalant anesthesia ( Pharmaceutical Partners of Canada , Richmond Hill , ON ) . A laparotomy was performed , and the colon was exteriorized and ligated with 3–0 black silk sutures ( Ethicon , Somerville , NJ; Peterborough , ON , Canada ) at the proximal end and ~2 cm below . Care was taken to keep the mesenteries , blood vessels , and nerves intact . Virulent log-phase Eh trophozoites ( 1 × 106 ) in 100 μL PBS ( pH 7 . 3 ) were inoculated into the loop . To quantify secretion of high molecular weight ( Vo ) mucin and non-mucin components , secreted 3H-labeled glycoproteins were fractionated using a Sepharose 4B columns as described previously [16 , 50] . Gross pathology of colonic loops was assessed on a scale of 1 to 4 , as follows: 1 , normal colon ( uniform thickness , no colon dilation or distension , no blood in luminal contents ) ; 2 , minimal damage ( visible mucosal thickening and colonic distension , visible mucosal exudates , and expanded loop occupying <50% of the abdominal cavity ) ; 3 , extensive damage ( thickening of the colonic mucosa , visible dilation of surface blood vessels , colon distension with visible luminal contents , mucosal exudates , and expanded loop occupying 50% of the abdominal cavity ) ; 4 , inflamed colon ( extensive colon thickening , colon surface with extensive inflamed dilated blood vessels with or without haemorrhage , extensive colon distension with or without visible brown or bloody luminal contents , mucosal exudates under extreme pressure leading to ballooning of the colon , and expanded loop occupying >50% of the abdominal cavity ) . At the endpoint of the experiments , animals were anesthetised and sacrificed by cervical dislocation and the colon was excised . For histology , colonic tissues were fixed in Carnoy’s solution , and embedded in paraffin blocks . 7μm tissue sections were rehydrated through an ethanol gradient to water and stained with Periodic acid Schiff’s reagent ( PAS , Sigma Aldrich Co . ) to visualize neutral mucins . Total RNA was isolated from snap-frozen tissue using the Trizol reagent method ( Invitrogen; Life Technologies , Burlington , ON ) as per manufacturer’s specifications , and the yield and purity determined by the ratio of absorbance at 260/280nm ( NanoDrop , Thermo Scientific ) . Only samples with a ratio of ~1 . 8 for DNA and ~2 . 0 for RNA were considered . cDNA was prepared using a qScript cDNA synthesis kit ( Quanta Biosciences ) . Real-time qPCR was performed using a Rotor Gene 3000 real-time PCR system ( Corbett Research ) . Each reaction mixture contained 100 ng of cDNA , SYBR Green PCR Master Mix ( Qiagen ) and 1μM of primers . A complete list of the primer sequences and conditions used are listed in Table 1 . Results were analyzed using the 2-ΔΔCT methods and expressed as fold changes . Luminal pro-inflammatory cytokines was analyzed using a mouse 31-plex cytokine–chemokine panel ( Eve Technologies , Calgary , AB , Canada ) . For visualizing Muc2 , 7μm sections of Carnoy´s fixed tissue were incubated with H-300 antibody [1μg/ml] at 4°C overnight and secondary anti-rabbit antibody coupled with Alexa 594 and DAPI ( Life Technologies ) was used for nuclear counterstain . Tissue sections were visualized using an Olympus FV1000 scanning confocal inverted microscope . To detect Math1 associated GFP expression , colons of differently treated Math1GFP mice were surgically removed and imaged ex vivo using an In-Vivo Xtreme 4MP-imaging platform ( Bruker , Billerica , MA , USA ) . Colons were positioned horizontally from the proximal to the distal side and imaged with the luminal side facing the camera . The imaging protocol contained two steps: reflectance imaging ( 2 sec exposure time ) and fluorescent imaging with excitation at 470 nm and emission at 535 nm ( 5 sec exposure time ) . Binning was kept constant at 2 x 2 . Images from the In-Vivo Xtreme were acquired and analyzed using Bruker molecular imaging software MI SE ( version 7 . 1 . 3 . 20550 ) . Math1 associated GFP expression in the colon under different treatment conditions was quantified by measuring the mean fluorescence ( after background subtraction ) in a constant region of interest ( ROI ) . ROI were either defined as whole colon area ( proximal to distal part ) or split into proximal , median and distal part for quantification . To determine if translocated bacteria caused Math1 expression , a sub lethal dose of LPS ( 5mg/kg BW ) was injected intra peritoneal into Math1GFP animals and the whole gut was surgically removed 24h post treatment . Small intestines were positioned vertically from duodenum to ileum and imaged using an In-Vivo Xtreme 4MP-imaging platform ( Bruker , Billerica , MA , USA ) as explained above . To avoid autoflorescence derived from diet , mice were fed a non-fluorescent diet ( Rodent Diet , AIN-93M , BioServ ) for a week before the start of the experiments . Human adenocarcinoma colonic goblet cells ( LS174T ) were cultured in Dulbecco's Modified Eagle Medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) , 20 mM HEPES , and 100 U/ml penicillin/streptomycin . Cells were passaged with 0 . 25% trypsin-EDTA ( Thermo ) once 90% confluence was reached . For experiments , LS174T cells were seeded in 24-well plates in triplicate at a density of 2 . 5 × 104 and cultured until a confluent monolayer was formed . To determine if Eh directly modulated MATH1 expression in the absence of microbiota , LS174T cells were exposed to 2 . 5x105 trophozoites/ml at 37°C for 30 and 60 min . Glutaraldehyde-fixed ( 2 . 5% for 15 min and washed twice in PBS ) Eh was used to determine a requirement for live parasites . LS174T cells were pre-treated with human IL-1β ( Peprotech , Cedarlane , Burlington , ON , Canada ) at a concentration of 20 ng/mL for 16h to determine if pro-inflammatory cytokines could modulate MATH-1 expression . To determine if Eh increased intestinal permeability , animals were gavaged with 15mg of fluorescein isothiocyanate ( FITC ) -dextran ( 3-5kDa , Sigma Aldrich ) , dissolved in 100μL of water and colonic loops were performed 2h after . Following Eh inoculation in colonic loops for 3h , animals were anaesthetized by isoflurane ( Pharmaceutical Partners of Canada , Richmond Hill , ON ) and blood was collected by cardiac puncture . Animals were sacrificed by cervical dislocation . Whole blood was allowed to clot in the dark for 3h at room temperature ( RT ) and centrifuged at 10 , 000 x g for 10 min . Serum was transferred to a clean Eppendorf tube and diluted with an equal volume of PBS . An aliquot of 100μL of each sample was loaded onto a black bottom 96-well plate in duplicate , and fluorescence was determined with a plate reader ( absorption 485nm , emission 535nm ) . For visualizing bacterial translocation animals were gavaged with 200μL of overnight grown culture of Escherichia coli XEN14 every 24h for 3 days . Colonic loops were then inoculated with Eh on E . coli XEN14 infected animals and after 3h , whole gut was surgically removed and imaged ex vivo using an in vivo Xtreme 4MP-imaging platform , as described previously . Translocated bacterial population was quantified using quantitative PCR method as described previously [52] . For visualization of bacterial translocation in tissues , fluorescence in situ hybridization ( FISH ) was performed as described previously [53] . Briefly , 7 μm sliced Carnoy´s fixed tissue was incubated with the total bacteria probe EUB338 5'-GCT GCC TCC CGT AGG AGT-3' [50ng/μL] coupled with Quasar 670 dye at 46°C overnight . FITC coupled-Ulex europaeus agglutinin ( UEA ) was used at [1:1000] to visualize the fucosylated residues in mucins and DAPI [1:1000] ( Life Technologies ) for nuclear counterstain . Tissue sections were visualized using an Olympus FV1000 scanning confocal inverted microscope . MPO activity in mouse colon samples ( 50 mg of fresh-frozen tissues ) was assessed as a marker for neutrophil influx as previously described [52] . Briefly , tissue was homogenized in 0 . 5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer ( pH 6 . 0 ) . Homogenized tissue was freeze-thawed three times , sonicated , and centrifuged ( 10 , 000 g for 10 min at 4°C ) for collection of clear supernatant . The reaction was initiated by addition of 1 mg/ml dianisidine dihydrochloride ( Sigma , St . Louis , MO ) and 1% H2O2 , and change in optical density was measured at 450 nm . The Health Sciences Animal Care Committee from the University of Calgary , have examined the animal care and treatment protocol ( AC14-0219 ) and approved the experimental procedures proposed and certifies with the applicant that the care and treatment of animals used was in accordance with the principles outlined in the most recent policies on the “Guide to the Care and Use of Experimental Animals” by The Canadian Council on Animal Care . Data was analyzed using Graphpad Prism 6 ( Graph-Pad Software , San Diego , CA ) for all statistical analysis . Treatment groups were compared using analysis of variance ( ANOVA ) when more than two groups were compared . Student’s t-test was used when only two groups were compared . Statistical significance was assumed at P < 0 . 05 , n = total number of mice per group from two independent experiments . Error bars in all the graphs represent mean ± standard error of the mean ( SEM ) . | Entamoeba histolytica ( Eh ) is a human protozoan parasite and the causative agent of amebiasis . Amebic colitis causes cellular destruction of the colonic mucosal layers allowing parasites to disseminate to the liver through blood causing amoebic liver abscess , or to other soft organs like the brain and lungs . Eh in the colon shares an environment with resident intestinal microbiota that lives , feeds and multiplies on the mucus layer . Both microbiota and mucus play critical protective roles in innate host defense however , to date , little is known about the interactions between ameba-microbiota and the mucus layers . Here we show that microbial dysbiosis worsen the outcome of Eh infection , favoring bacterial translocation , increasing pro-inflammatory responses independently of the mucus layer , as well as regulating mucus release from goblet cells . The absence of microbiota in germ-free mice altered the host from mounting a proper innate immune response towards Eh allowing parasites to bind to and disrupt mucosal epithelial cells . These data highlight critical roles for indigenous microbiota in imprinting and educating mucosal innate host defenses critical for host defense against E . histolytica invasion . | [
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Crosstalk between neurons and glia may constitute a significant part of information processing in the brain . We present a novel method of statistically identifying interactions in a neuron–glia network . We attempted to identify neuron–glia interactions from neuronal and glial activities via maximum-a-posteriori ( MAP ) -based parameter estimation by developing a generalized linear model ( GLM ) of a neuron–glia network . The interactions in our interest included functional connectivity and response functions . We evaluated the cross-validated likelihood of GLMs that resulted from the addition or removal of connections to confirm the existence of specific neuron-to-glia or glia-to-neuron connections . We only accepted addition or removal when the modification improved the cross-validated likelihood . We applied the method to a high-throughput , multicellular in vitro Ca2+ imaging dataset obtained from the CA3 region of a rat hippocampus , and then evaluated the reliability of connectivity estimates using a statistical test based on a surrogate method . Our findings based on the estimated connectivity were in good agreement with currently available physiological knowledge , suggesting our method can elucidate undiscovered functions of neuron–glia systems .
Information processing in the brain is primarily performed by neurons [1] , [2] . Some studies , however , have revealed the existence of crosstalk between neurons and astrocytes [3]–[6] , [6]–[14] that neighbor the neurons and envelop the neuronal synapses [15] . The observations in these studies suggest the involvement of glia in the brain's information processing [16] . Stimulation applied to the main type of glial cells ( i . e . , astrocytes ) may induce the exocytosis of gliotransmitters , which in turn modulates post-synaptic currents [17] and increases post-synaptic excitability [18] , [19] . Stimulation applied to neurons , on the other hand , elevates the Ca2+ activity of astrocytes [8] . This effect occurs both in culture and in acute brain slices , and is most likely mediated by astrocyte receptors for neuro-active molecules , neurotransmitters and neuromodulators [8] . In vitro astrocytes are known to exhibit relatively slow non-electrical activities ( 100 ms1 min ) [15] . In contrast , neurons exhibit rapid depolarization , or ‘spikes’ ( 1 ms ) . Furthermore , in vivo animal experiments have suggested that glia affect neural networks in the sensory cortex [20] , [21] and in the motor cortex [22] . These in vivo results imply that glia may play an important role in the information processing associated with sensory and motor functions . These findings clarify the necessity to shift our focus from pure neuronal networks to neuron–glia networks [23]–[26] . Unless otherwise noted , we will denote astrocytes as glia after this . To clarify the roles of neuron–glia interactions in brain information processing , we need to examine neuronal and glial activities in a network in an unmanipulated state . For example , some experiments have artificially generated epileptiform bursting activities of neurons and glial cells , and then examined the contributions of glial activity via further pharmacological manipulation [6] , [7] , [27] . Such approaches are very appropriate for clinical applications . However , one needs to assess the concise contribution of glial activities in networks in a resting state to elucidate their functions in information processing . In this case , the sheer complexity of the networks makes it extremely difficult to estimate neuron–glia interactions . The dissociation of glial effects from other neuronal effects is a challenging problem , especially when indirect interactions via other neurons in the network are taken into consideration . Also , such indirect interactions may themselves be important for identifying neuron–glia interactions . Generalized linear models ( GLMs ) have been developed for pure neuronal networks ( without glia ) to analyze their interactions in terms of both response functions and functional connectivity [28]–[33] . One can identify the characteristics of multivariate time series by estimating the model parameters in the GLM-based approaches . In the framework of the GLMs , the probability of spike events in a network at any given time depends on the history of the activity time series . The response functions and functional connectivity are estimated from the observed time-series of multi-neuronal spiking activities . The estimated response functions measure the extent to which the other neuronal spikes causally affect the spiking activities of target neurons . The estimated functional connectivity , on the other hand , represents the pathways over which the neuronal activities propagate . Although the functional connectivity does not necessarily correspond to a specific synaptic or non-synaptic connection ( e . g . , gap-junction ) [34] , [35] , existing studies have shown that synaptic connections are closely linked to the connections that can be functionally estimated based on Ca2+ imaging [36] and multi-electrode physiological measurements in vivo [37] , [38] . Friston et al . argued that functional connectivities , particularly the ones that depend on the context of environments and behaviors , represent information flow propagating through anatomical connectivity [39] in their research on fMRI datasets . One may then use the response functions and functional connectivity to address how each component contributes to information processing in the brain , either in a controlled environment or in the resting state . This type of data-driven approach is important in analyzing experimental data with high throughput , and in our particular case of identifying unknown neuron–glia interactions , even with a lack of a priori biological knowledge . Neuronal spiking activity is binary , while glial activity may be regarded as being graded time series [18] . Since we cannot directly apply the existing GLM-based techniques to such heterogeneous neuron-glia networks , we propose a new GLM-based statistical method in this paper to identify the interactions between neurons and glial cells . We applied this statistical method to the time-lapse imaging data of the rat hippocampal CA3 region based on high-resolution ( 18494 pixels ) and high-speed ( 100 Hz ) Ca2+ imaging [40] . We determined the response functions and functional connectivity of the neuron–glia network from spontaneous activities of neurons and glial cells , which were then quantified by measuring the Ca2+ signal averaged over each cell . The reliability of the determined connectivity was evaluated with a statistical test based on a surrogate method . Our analysis revealed several characteristics of interactions between neurons and glia , including the positive effect of glial activities on the activities of neighboring neurons . These results obtained solely by using the proposed method were compatible with existing knowledge on neuron–glia interactions , reinforcing the previous neurobiological observations and providing new insights into the functions of neuro–glia systems .
We developed a statistical method to identify the functional connectivity and response functions of neuron–glia networks in situ , which may reflect the dynamics of ionic receptors on neurons and glial cells . We applied it to a Ca2+ imaging dataset of an in vitro brain slice ( see ‘ In vitro Ca2+ imaging’ section in Methods ) , by using the Ca2+ signal ( concentration ) as an indicator of neuronal as well as glial activities . We conducted high-resolution ( 18494 pixels ) and high-speed Ca2+ imaging ( 100 Hz ) from a CA3 region ( 184 94 ) of a rat's hippocampal slice to prepare the dataset by using Nipkow-type spinning-disk microscopy [40] . We observed spontaneous Ca2+ activities of neurons and glial cells within the 10 min of a fluorescence image series . An image preprocess applied to the image series extracted binary activities of 48 neurons and graded activities of six glial cells ( Figs . 1E and 1H ) . The spike frequency of the 48 neurons was 0 . 03–1 Hz . The activity dataset thus consisted of the observation time series of 48 neurons and six glial cells . We tried to identify the neuron–glia system based on this observation time series by estimating the parameters of our neuron–glia network model ( Fig . 2A . See ‘Generative model and MAP estimation’ section of Methods ) . We developed a generalized linear model ( GLM ) of a neuron–glia network as a variation of previous GLMs used for neuronal networks [41] . We could efficiently and uniquely obtain maximum a posteriori ( MAP ) estimates of the parameters by assuming that the present activities of neurons and glial cells were independent conditional on their past . Using the MAP estimates , we could avoid ‘overfitting’ , where the model estimates were disturbed by noise involved in the relatively short observation time series . We evaluated the quality-of-fit of the estimated model to the observation time series by using -fold cross-validation ( see ‘Functional connectivity analysis’ section of Methods ) . The observation time-series dataset in the -fold cross-validation was partitioned into subseries . A single subseries was used as the dataset to evaluate the estimated model , while the remaining subseries were used as the training dataset to estimate the model parameters . Our measure of the quality-of-fit was the cross-validated likelihood , i . e . , the model's predictability of the activities of neurons and glial cells in the test dataset averaged over folds ( for more details , see ‘Functional connectivity analysis’ in Methods section ) . Since the cross-validated likelihood depended on the network structure of the model , i . e . , the connectivity pattern within the neuron–glia system , it could be used to identify the connectivity between neurons and glial cells . For a specific connection from a glial cell to a neuron ( a glia-to-neuron connection ) , we accepted the connection if a network structure with the new connection indicated a better cross-validated likelihood than the network structure that did not include the connection . In contrast , for a specific connection from a neuron to a glial cell ( a neuron-to-glia connection ) , we preferred a network structure without the connection if the reduced network structure indicated a better cross-validated likelihood than the one with the connection ( see ‘Functional connectivity analysis’ section in Methods ) . We identified the best network structure , i . e . , the connectivity and response functions of the neuron–glia system , by repeating this set of procedures ( the addition/removal of connections including MAP-based parameter estimation inside ) . The reason for our different treatment of glia-to-neuron and neuron-to-glia connections will be discussed later . We conducted surrogate analysis to verify the reliability of the extracted functional connections as follows . First , we created a set of artificial time series for neurons and glial cells by applying “cyclic” rotations in which the cross correlations were destroyed but the autocorrelations were preserved . We then applied our algorithm to this artificial data set , and compared the number of identified connections against the number of connections we had identified from the original data . This obtained a statistical evaluation of the bulk number of connections that could be identified with our method . Recent studies have shown that glial activities affect neuronal activities on various time scales , ranging from several tens of milliseconds to several hours [6] , [12] , [14] . We focused on interactions that lasted for a relatively short duration with a delay ranging between 100 and 500 ms in this study . This is because our method could not deal with interactions with longer delays in our time-lapse image dataset of 10 min ( see ‘Limitations of proposed method’ in the Discussion section ) . A detailed description of the overall method is found in Methods and Text S1 . The codes for our generative model and statistical analyses have been uploaded to GitHub ( https://github . com/nakae-k/glia-neuron ) . We estimated the response functions , , and , which corresponded to the connections between neurons , the connections from glial cells to neurons , the connections from neurons to glial cells , and the connections between glial cells ( see Fig . 2A and ‘Generative model and MAP estimation’ in Methods ) . Here , denotes the index of the “sender” cells , denotes that of the “receiver” cells , and denotes the delay time . Fig . 3A shows the identified connectivity matrix of the neuron–glia network . Here , we assumed that the functional connections between neurons and glia were directional because the neuron-to-glia and the glia-to-neuron connections are believed to depend on different biophysical processes [23] . There are small numbers of connections with substantially larger values than the other connections at the top left of the matrix , i . e . , inter-neuronal connections . This observation is consistent with existing physiological studies , which report that the strength of inter-neuronal connections in the hippocampus obeys a log-normal distribution [42] We can also see some strong glia-to-neuron connections at the top right . We took temporal averages of and , and determined connections corresponding to positive values as excitatory . We similarly determined connections corresponding to negative values as inhibitory . Approximately half of the inter-neuronal connections were found to be excitatory ( Fig . 3B ) . This may suggest some sort of a balance in inter-neuronal and inter-glial connections . Positive values for the temporal averages of and were found for 63% of the former and for 11% of the latter , suggesting that there were major excitatory effects from glial cells to neurons but minor inhibitory effects from neurons to glial cells . We determined the existence of a connection ( ) from the j-th glial cell to the i-th neuron using a newly designed t-statistic , , which determined whether the increase in the cross-validated likelihood resulting from the addition of the new connection was significant or not ( see ‘Functional connectivity analysis’ section in Methods ) . We found that 24% of the glia-to-neuron pairs increased the cross-validated likelihood , and the remaining 76% decreased the cross-validated likelihood ( Fig . S3 ) . We also found that only 17 out of 288 possible glia-to-neuron connections could significantly increase the cross-validated likelihood ( ) by performing the statistical test based on . This suggested sparsity in glia-to-neuron connections ( Fig . 4A ) . When we compared the activities of a neuron–glia pair that was identified as connected ( e . g . , neuron 6 and glial cell 2 ) with another pair that was identified as not connected ( e . g . , neuron 6 and glial cell 1 ) , the correlation between the neuronal firing rate and glial activity was higher for the connected pair ( ) than that for the non-connected pair ( ) ( Fig . S4 ) . We also identified 89 neuron-to-glia connections out of 288 neuron-to-glia pairs with a similar t-statistic , ( ) , where denotes the neuron-to-glia connection ( Fig . 5A ) ( see ‘Functional connectivity analysis’ section in Methods ) . The average response function of the identified neuron-to-glia connections suggested small and inhibitory effects of neuronal activities on glial activities . The t-test ( ) determined the temporal average of the response functions to be significantly negative . These results seemed to be inconsistent with those in experimental studies [8] , [27] , which have demonstrated excitatory neuron-to-glia connections . This inconsistency can be attributed to effects from other brain areas that were not considered in our study ( e . g . , the dentate gyrus ) , or to different experimental conditions . We need to emphasize that we observed spontaneous activities in our experiment while the preceding experiments mostly measured activities evoked by stimulation [19] , [43] ( also see Discussion ) . We examined the reliability of connectivity from each of the six glial cells to neurons , measured in terms of the bulk number of identified connections by using the surrogate method ( see ‘Surrogate method’ in Methods ) . We prepared 1000 surrogate glial activities for each glial cell . This analysis suggested that glial cells 2 and 5 had significantly large numbers of connections to neurons ( ) . We similarly examined the reliability of connectivity from neurons to each of the six glial cells , measured in terms of the bulk number of identified connections . This analysis indicated that no glial cells received a significantly large numbers of connections from neurons ( ) . The identified 17 glia-to-neuron connections out of 288 glia-to-neuron pairs are depicted in Fig . 4A . These connections had an interesting topological character , i . e . , the range of functional connectivity from glia to neurons was local ( , see Fig . S6 ) . We performed the following statistical test to statistically confirm this observation . We let be the set of identified connections from the k-th glial cell to the 48 neurons and let be the size of . The values of nk’s were = 2 , = 5 , = 3 , , and . We then randomly selected neurons from the total of 48 neurons for each glial cell , and measured the distance between the k-th glial cell and all the selected neurons . We then computed the median distance of such random glia-to-neuron connections over the six glial cells . We repeated this sampling 1000 times to obtain an empirical distribution of the median distance of randomly prepared glia-to-neuron connections . When the median distance of the glia-connected neurons from their respective glial cells was compared against this empirical distribution , it was found to be significantly lower ( ) . We found from visual inspections that each neuron had some tendency to be under the functional projection of a unique glial cell . This tendency was particularly strong for neurons under the functional projection of glial cells 1 , 2 , 3 , and 4 ( Fig . 4B ) . These findings are consistent with the anatomy of astrocytes , where they are known to occupy nonoverlapping local territories whose diameter is about 30 . The findings are also in agreement with the hypothesis of functional islands of neurons modulated by individual astrocytes [44] , [45] . Fig . 6 ( left ) suggests that the excitatory glia-to-neuron connections have a mean peak latency of around 500 ms . The t-test ( ) determined the temporal average of the response functions to be significantly positive . The 89 neuron-to-glia connections identified from 288 neuron-to-glia pairs , on the other hand , were found to be non-local ( Fig . 5B ) . When we actually applied a statistical test similar to that above to the identified neuron-to-glia connections , the p-value was 0 . 385 ( also see Fig . S6 ) . The average response function of the identified neuron-to-glia connections suggests small and inhibitory effects of neuronal activities on glial activities . The t-test ( ) determined the temporal average of the response functions to be significantly negative .
Our results suggested the existence of functional connectivity from glial cells to neighboring neurons within a 20 50 perimeter . The identified functional connectivity also exhibited a distinctive local tiling pattern with few overlaps ( Fig . 4 ) . Further , these connections had positive response functions on the time scale of 500 ms . These results are in good agreement with experimental findings [6] , [44] , [46] . For example , the activation of hippocampal CA1 astrocytes has induced an inward current to neurons for a duration of 500 ms ( e . g . , [6] ) , which is mediated by glutamate released from astrocytes [19]; this phenomenon synchronizes the activities of CA1 neurons in the same range of 100 [47] . Anatomical studies have also found that astrocytes in the hippocampus occupy non-overlapping domains [44] , [46] . The identified response functions correspond to the inward current to neurons , and the identified local connectivity corresponds to the mostly non-overlapping domain of astrocytes . This would also suggest that glial activities could affect neuronal information processing in spontaneously active situations , in concert with inter-neuronal and inter-glial interactions , like those in our in vitro experiment . The estimated glia-to-neuron response functions had a time scale of several hundred milliseconds with a peak latency of 500 ms . This relatively long duration might include the time for the activations of neuronal AMPAR and NMDAR in response to gliotransmitter release . Because the deactivation kinetics of AMAPR is known to be very rapid ( ∼5 ms ) , one may think that AMPAR activation should not appear in the response functions derived from the sampling interval of 10 ms . However , response functions not only depend on receptor kinetics , but also on the entire processes of AMPAR-mediated transmission ( i . e . , from glial vesicle release to neuronal Ca2+ signals ) . These entire processes are known to require at least several hundred milliseconds [48] . Thus , the effects of AMPA- and NMDAR-mediated transmission were most likely reflected in our response functions . Our analysis indicated the possible presence of many neuron-to-glia connections . We also found that , even if these connections really existed , the intensities of these connections were weak and they were spatially unlocalized . Indeed , neuron-to-glia interactions has been discovered in previous studies [8] , [9] , [49] . Although this has been observed in the bursting state of neuronal activities , such neuron-to-glia interactions may have been too small to observe in our spontaneously active situation . Thus , the identified weak neuron-to-glia connections were insignificant with a short observation time of 10 min . In contrast , if there were in fact no neuron-to-glia connections , those misidentified neuron-to-glia connections may have been due to spurious correlations between neuronal and glial activities . Such correlated activities may have been mediated by dentate gyrus ( DG ) neurons . DG neurons are known to relay signals to both CA3 astrocytes and CA3 neurons [50] , [51] . Thus , CA3 astrocytes and CA3 neurons could have simultaneously responded to DG neurons , which might have resulted in correlated activities for the misidentified functional connections . In either case , the significantly longer and simultaneous observation of both CA3 and DG regions is necessary to address the origin of the identified weak and spatially unlocalized neuron-to-glia connections . Ca2+ signal has been recognized to be one of the most powerful indicators of glial activities . For example , the transmission of gliotransmitter , glutamate , is known to depend on the glial Ca2+ concentration [52] . When a glial cell uptakes glutamate spilled out from synaptic clefts , the intracellular Ca2+ concentration of the glial cell is known to increase [8] , [49] , [53] . Although Ca2+ imaging is no doubt a powerful experimental methodology , our statistical method has potential applications to other types of imaging experiments . For example , we may apply our statistical technique to the dataset from intracellular pH imaging . Intracellular pH is known to reflect gliotransmitter release , which is a type of glial activity [54] , [55] . When our method is applied to electrophysiological or imaging experiments from different hippocampal areas such as CA1 , CA3 , and the entorhinal cortex , it should be modified by , for example , changing the tuning parameters in the estimation ( see ‘Tuning parameters’ section in Methods ) . Indeed , we should consider the possibility that the neuron–glia interactions are characterized by different biophysics in different brain regions [19] , [56] and hence are represented by different tuning parameter values in our method . Fig . 3B shows that about half the inter-neuronal and inter-glial interactions were positive and half were negative ( i . e . , the excitatory and inhibitory effects were balanced ) . The balanced excitatory and inhibitory effects in inter-neuronal interactions are known to lead to high levels of variability in neuronal spiking and this high variability can enable neuronal networks to embed rich information into their activity patterns [57] , [58] . Our results suggest that this balance was not only achieved in inter-neuronal interactions but also in inter-glial interactions . Balanced inputs from the glial cells might similarly provide high levels of variability to glial activities and promote efficient information processing . Our method of identifying the functional connectivity between neurons and glial cells is an extension of existing methods based on Granger causality . Granger et al . [59] presented a model-based statistical approach to explore the causality between two variables by examining whether the prediction of a time series of one variable could be improved by incorporating information on the past values of the other [60] . Kim et al . [41] applied Granger causality to functional connectivity analysis of spike sequences; they performed a statistical test based on the log-likelihood of the autoregressive model of spike sequences . Our method presented in the current study can be seen as an extension of Kim et al . 's method that utilized the cross-validated likelihood for model selection . By use of the cross-validated likelihood , we could allow the actual underlying process to be different from the process hypothesized by GLM , while the original Granger causality-based method assumed that they were exactly the same . Schleiber et al . presented another kind of model-free approach [36] to identify the causality between multiple variables . They utilized transfer entropy , which was used to measure improvements in the prediction of one time series by knowing the past values of another . No distribution of variables needs to be assumed because of the model-free computation of entropy in this approach . One possible drawback in the method of transfer entropy is that it can be difficult to incorporate effects in multiple variables and non-stationarity in the underlying stochastic process due to the lack of direct modeling . In contrast , we can apply our method to non-stationary activities of neurons and glial cells by introducing a time-varying spontaneous firing rate to the likelihood model ( Eqs . ( 1 ) and ( 2 ) ) . Our GLM is novel particularly in that it combines a Bernoulli point process model to represent binary neuronal spikes [28] , [61] and a vector autoregressive model [62] to represent graded glial activities . The vector autoregressive model has been widely accepted in the field of statistical time-series analysis [63] . Although both these models are known , there have never been any studies in neuroscience that have employed a hybrid stochastic model that could simultaneously deal with both discrete and continuous time-series like those in neuron-glia systems . The most important advantage of functional connectivity-based approaches is their high throughput . The functional connectivity-based approach enabled us to extract essential structures of the neuron–glia system even from a relatively small amount of data that consisted of 10-min time series of Ca2+ imaging in comparison with their pure anatomical connectivity-based counterparts , like those by electron microscopes [64] . The reasonable performance of our method in artificial networks ( 85% accuracy from activity time series of 1280 s; Fig . S8; see ‘Validation using artificial data’ section of Text S1 ) suggests that our identified functional connectivities are biologically and statistically plausible . The functional connections estimated with our method are expected to approach true ones in the network ( Fig . S8 ) as the amount of data increases . If there are many unobservable neurons or glial cells , on the other hand , the meaning of functional connectivity may become ambiguous . However , the advantages of functional connectivity-based approaches will increasingly grow in various neuroscientific scenarios with rapid advances in in vitro and in vivo imaging techniques and increased access to more widespread and longer measurements . A possible future direction is to explore the fusion of functional connectivity-based methods and anatomical methods . Moreover , the response functions estimated with our method have a meaning on their own; they represent the entirety of synaptic connections that not only include ionic factors but also metabotropic factors . Our functional connectivity analysis was based on an assumption that the Ca2+ activities of cells were independent conditional on their history ( see ‘Generative model and MAP estimation’ in Methods ) . This assumption was equivalent to ignoring neuron–glia interactions whose durations were shorter than the sampling interval ( 10 ms ) in this study . Nevertheless , interactions with such a short time scale can play important roles in neuron–glia networks . An existing study that has proposed the max entropy model , for example , has discussed this possibility [65] , [66] . For the following two reasons , however , we believe that our assumption will not negatively affect the reliability of our identification of the interactions with relatively long time scales ( between 100 and 500 ms ) , which is the main target of our functional connectivity analysis . First , we found that the intensity of our response functions were likely to shrink to as the delay time approached ms ( Fig . 6 ( left ) ) . This , in particular , means that high frequency responses did not take place around ms . This ruled out the possibility for major interactions on shorter time scales because such interactions most likely triggered high frequency fluctuations in the response functions . Second , our functional connectivity analysis was based on the difference in cross-validated likelihoods . It would have been unlikely that our abandonment of short term interactions would have severely deformed our computation of cross-validated likelihoods . Even if it had introduced some bias into their evaluations , the bias could be “cancelled out” as we took their differences into account . As such , our method was quite robust against bias that might have resulted from ignoring interactions on smaller time scales . It should be noted that the probability of multiple spikes in 10-ms bins was quite small because the spike frequency ( below 1 Hz ) in our observation time series was low . We conducted 10-fold cross-validation ( ) in the time-series analysis . Since we uniformly segmented the whole time series to subseries with a length of 60 s in the cross-validation procedure , interactions with time scales longer than 60 s were simply ignored . Since the optimal network structure was searched by iterative applications of local searches and hence did not necessarily assess the whole set of identified connections , the bulk number of identified connections was statistically evaluated by means of the surrogate method in which null hypothesis assumed there were in fact no connections in the network ( see ‘Surrogate method’section in Methods ) [67] . According to the surrogate method , we artificially created time series for neurons and glial cells separately by applying cyclical rotation to the original neuronal time series and phase randomization in the frequency domain to the original glial time series found in the observation dataset . The temporal relationships with other elements in the network were destroyed in the surrogate time series , while preserving important statistical features of its own like those in the distribution and autocorrelation . We then compared the number of connections identified by our method from the actual data against that with the surrogate time series , which led to a statistical evaluation of the bulk number of identified connections . Our functional connectivity analysis was based on iterative applications of local searches for the network structure with the largest cross-validated likelihood . Since multiple hypothesis testing underlies this algorithm , some connections might have been detected by chance even if there had in fact been no connections between neurons and glia . To examine the false positive detection , we used the surrogate method to determine whether the number of identified connections was larger than that found by chance ( see ‘Surrogate method’ section in Methods ) [67] . We found that the number of identified glia-to-neuron connections was significantly large through surrogate analysis , while that of the neuron-to-glia connections was not . Further , the small and inhibitory neuron-to-glia interactions were inconsistent with the excitatory interactions reported by preceding experimental studies [8] , [49] . This inconsistency may be reconciled if we consider the dependence of neuron-glia interactions on the frequency of neuronal firing . Such a frequency-dependent regulation has been discussed within the context of glia-to-neuron connections [19] , [43] , and a similar regulation might also be realized in neuron-to-glia connections . Note that clear excitatory neuron-to-glia interactions were found through experiments that induced high frequency bursting activities in neurons [8] , [49] . On the other hand , the frequency of neuronal activities in our imaging experiment was low ( 0 . 03 Hz–1 Hz ) . Thus , the excitatory neuron-to-glia interactions might have been too weak to have been detected in this low-frequency situation . It is also possible that the Ca2+ active region within the astrocyte's cell body and the sites of neuron-to-glia interactions were so far apart in our imaging experiment , which mostly measured the cell body , that it could not provide us with sufficient information to identify the actual neuron-to-glia connections . Although existing studies have shown that neural spikes cause an increase in glial Ca2+ activity [3] , our functional connectivity analysis did not take this known fact into account . The results may change when we assume that all the neuron-to-glia interactions are excitatory . This assumption is equivalent to forcing the response functions , , from neurons to glial cells to be positive ( see ‘Positivity constraints to response functions from neuron to glia’ section in Methods ) . We identified nine neuron-to-glia connections out of 288 pairs with the positivity constraints; we found functional connections from neurons to glial cells 2 , 4 , and 5 , but no connections to other glial cells ( Fig . S9 ) . When we validated the set of identified connections with the surrogate method , the p-value of the number of connections was too large to accept any neuron-to-glia connections . This suggests that , even under the new constraint , neurons do not directly affect glial cells when neurons and glial cells are spontaneously behaving . We compared the cross-validated likelihood between our original model ( without the positivity constraints ) and the modified model with the positivity constraints on the basis of the distribution of . We only considered the set of 's corresponding to the pair of cells for which our method detected a functional connection . The standard error of the mean ( SEM ) of these was for the original model , and for the modified model . These results indicate that the original model was better than the modified model support our speculation that the model with the positivity constraints did not necessarily capture the nature of the spontaneous in vitro activities of neurons and glial cells in the hippocampal CA3 circuit .
We prepared the hippocampal slice cultures from postnanal , day 7 Wistar/ST rats ( SLC ) . We applied refrigeration anesthesia to the rat pups prior to extracting their brains . We sliced the brains into 300 thick slices in aerated , ice cold Gay's balanced salt solution supplemented with 25 mM of glucose . Entorhino-hippocampal stumps including the CA3 region were excised and cultivated on Omnipore membrane filters ( JHWP02500 , Millipore ) placed on plastic O-ring disks . The cultures were fed with 1 ml of 50% minimal essential medium , 25% Hanks' balanced salt solution , 25% horse serum , and antibiotics in a humidified incubator at in 5% CO2 . They were used for the experiments on days 7 to 14 in vitro , and the medium was changed every 3 . 5 days . We washed the slices three times on the day of the experiment with oxygenated artificial cerebrospinal fluid ( aCSF ) consisting of ( mM ) 127 NaCl , 26 NaHCO3 , 3 . 3 KCl , 1 . 24 KH2PO4 , 1 . 2 MgSO4 , 1 . 2 CaCl2 , and 10 glucose and bubbled them with 95% O2 and 5% CO2 . The slices were transferred to a 35-mm dish filled with 2 ml of dye solution and incubated for 40 min in a humidified incubator at in 5% CO2 with 0 . 0005% Oregon Green 488 BAPTA-1AM ( Invitrogen ) , 0 . 01% Pluronic F-127 ( Invitrogen ) , and 0 . 005% Cremophor EL ( Sigma-Aldrich ) . The slices were then recovered in aCSF for >30 min , mounted in a recording chamber at , and perfused with aCSF at a rate of 1 . 5–2 . 0 ml/min for >15 min . The hippocampal CA3 pyramidal cell layer was imaged at 100 Hz using a Nipkow-disk confocal microscope ( CSU-X1 , Yokogawa Electric ) equipped with a cooled CCD camera ( iXonEM+DV897 , Andor Technology ) , and an upright microscope with a water-immersion objective lens ( 16 , 0 . 8 numerical aperture , Nikon ) [40] . The area we observed is depicted in Fig . 1A . Fluorophores were excited at 488 nm with a laser diode and visualized with a 507-nm long-pass emission filter . We did not see any photodamage during the period of observation; however , we did observe weak photo-bleaching ( Figs . 1D and G . Also see [68] , [69] ) . We removed the effect of photo-bleaching by preprocessing the data as described below . We performed the Ca2+ imaging ( Fig . 1B ) for 10 min ( 600 s ) according to the experimental procedure above . Our imaging yielded a time-lapse image dataset that consisted of 60 , 000 image frames . The visual field of single image frames was 184 94 ( 18494 pixels ) . We extracted regions of interest ( ROIs ) in the first step of image preprocessing , as follows . We applied a spatial smoothing filter ( 2D Gaussian filter with = 1 ) to each image in the time lapse . We calculated the average and standard deviation ( SD ) of fluorescence signals over the observation period for each pixel along this filtered image series . We then specified the neighborhood ( a ball with a radius of 3 ) of each local maximum of the average fluorescence intensity as an ROI . We identified a total of 170 ROIs ( Fig . 1C ) . We computed the average signal intensity over the pixels in each of the 170 ROIs , and arranged the average signal intensity along the 60 , 000 frames that constituted the signal time series of the ROIs ( Fig . 1D ) . We then decomposed the signal time series into a baseline series and activity series on all the ROIs by iteratively applying the following procedure until the baseline series converged . Beginning with the initial baseline series set as flat at the average , we detected all the timepoints inside one SD of the baseline series as inliners , and replaced the baseline series with the new one connecting the inliners . We re-calculated the SD based on the new baseline series in the next application of this procedure . We then dissociated another baseline series . This baseline detection was in essence a detrending procedure; it removed the trends due to possible photo-bleaching . We defined spiking events as peaks of time series with substantially larger intensities than the baseline ( with a fixed difference ) for each ROI . We detected 48 ROIs out of the 170 ROIs , which indicated sufficient numbers of spiking events ( 0 . 03–1 Hz , Fig . 1E ) . We confirmed these 48 ROIs corresponded to neuronal soma by visually inspecting them . The peaks of the neuronal Ca2+ spikes were found to have similar intensities , and we observed no buildup activities ( Fig . 1D ) . We therefore deemed it safe to interpret each Ca2+ spike with a width of 10 ms to be a single spike . As such , the activity over each of the 48 ROIs was recorded as a binary time series . We selected six ROIs , other than the 48 neuronal ROIs , as regions representing glial cells , based on their morphologies ( by visual inspection ) and fluorescence levels . We particularly selected small cells with high fluorescence levels because such cells were likely to be astrocytes [34] . The radius of each ROI was re-set individually to a smaller value than that of the neurons because we only found six glial ROIs . We used the signal average over each glial ROI as the measure of glial activity ( Fig . 1G ) and arranging it over 60 , 000 frames constituted the activity time series . We applied individual linear detrending to each glial time series to remove slow trends possibly induced by photo-bleaching . We then applied a temporal Gaussian filter ( ) to remove high frequency noise and shot noise . The glial time series thus obtained is depicted in Fig . 1H . We assumed that the activity of astrocytes had a linear relationship in the analysis that followed with the signal intensity measured by Ca2+ imaging . Generative modeling was adopted to statistically describe the Ca2+ signals of neurons and glial cells . We introduced a prior distribution to avoid overfitting due to the finite/small size of collected data in the experiments . The model parameters were estimated with the MAP method . Let index the image sampling time over the observation , ; in our particular case , . We have activity series of neurons and glial cells after preprocessing , where and correspond to the numbers of neuronal and glial ROIs . As glial activity is continuous , is a series of discrete values sampled from a continuous function of time . can be seen as a unit point process; when the i-th neuron emits a spike at time , or otherwise . Our sampling interval was 10 ms within which every neuron was well assumed to have produced at most one spike in our imaging experiment ( see ‘Pre-processing’ section ) . We normalized the activity time series of the j-th glial cell individually , so that its average was zero and variance was one . This normalization was performed because glial cells exhibited different initial fluorescence levels due to variations in light absorption . For simplicity , let denote the activities of all the elements , , where T is a transpose . The vector , , will be called the observation time series after this . We assumed that would obey a stationary and conditionally independent Markov chain of order , which included an autoregressive process of order as a special case . When we use the term Markov , our models of interest may include those in which the dependence of the current state on past states is non-linear . Below , we provide the likelihood of , based on our generative model , where is the parameter vector . Let be its prior distribution . Bayes' theorem tells us that the posterior distribution of the parameter vector is given by . Given an observation time series , , the parameter-vector estimate , , is the that maximizes the posterior distribution ( i . e . , the MAP estimation ) . Our generative model is based on a Markov chain model where the neuronal and glial activities at present are assumed to be mutually independent but dependent on their past activities . More precisely , , where is the history of activities of all the components with a maximum time lag , . We allowed all neurons to have their own parameters and all glial cells to have their own parameters . That is , . Moreover , the maximum time lag , , could be differently set for individual types of interactions ( see below ) . A spike production by the i-th neuron with a fixed time interval was assumed to obey a Bernoulli process with logistic regression [32] , [70] ( 1a ) ( 1b ) where denotes the maximum time lag ( history window sizes ) from neurons and denotes the maximum time lag from glial cells ( Fig . 2A ) . The generative model above is an instance of GLMs , in which the parameter vector of neuron is given by , where represents the spontaneous firing rate of neuron , and and denote the response functions from neuron to neuron and from glial cell to neuron , which are defined over the history window sizes and , respectively . The activity of glial cell is given by a vector autoregressive ( VAR ) model disturbed by white Gaussian observation noise , which is another instance of GLMs . More precisely , ( 2a ) ( 2b ) where denotes the maximum time lags ( history window sizes ) from neurons and denotes the maximum time lags from glial cells . The parameter vector of glial cell in this VAR model is given by , where is the bias of glial cell and is its variance . Also , and denote the response functions from neuron to glial cell and from glial cell to glial cell , which are defined over the history window sizes and , respectively . We have used the notations , and , in this paper to represent the sets of response functions between neurons , from glial cells to neurons , from neurons to glial cells , and between glial cells , respectively . The whole GLM for the neuron-glia system above is a state-space model with internal deterministic processes based on a combination of logistic regression and VAR models . The model reduces to a couple of independent GLMs if there are no interactions between the neuronal and glia networks , i . e . , . Here , we explain our prior setting of the model parameters in our GLM . We introduced a prior distribution to the parameters representing the response functions , and , to make the response functions sparse , which is preferred in avoiding overfitting to relatively small datasets , in addition to smoothing with respect to the lag time . Such a prior distribution is given by ( 3 ) where tuning constant controls the L2-sparseness of the response functions and controls their smoothness . We granted independent , noninformative priors and to parameters and ( Eqs . ( 1 ) and ( 2 ) ) . In summary , we put , , and . These parameters and their prior distribution are summarized in Table S3 . The prior based on L2-sparseness would be preferable for increasing the cross-validated likelihood of the model [71] by effectively reducing the sensitivity of the model to noise inevitably involved in a relatively small dataset . The smoothness prior would reduce the effective space in which the response functions exist and hence would be beneficial to improve the cross-validated likelihood . Although the time scales of neuron-glia interactions may span a wide range , fluctuating from several tens of milliseconds to several hours [6] , [12] , [14] , our current study focused on specific types of interactions that lasted for several hundreds of milliseconds . Our prior setting that preferred smooth response functions was also considered to work in removing neuron-glia interactions with shorter time scales . By applying Bayes' theorem to the likelihood and the prior distribution above , we have the following log posterior ( 4 ) We obtained the parameter vector , , that maximized the log posterior above; the expression above suggests that this MAP estimation can be individually performed for each and for each . This individuality also suggests the ability to apply parallel computation to the estimation of parameters . Fortunately , our set of MAP estimates is unique because our generative model is an instance of GLM [72] and a strictly convex prior distribution also makes the posterior distribution convex . This allows us to use efficient optimization algorithms . When maximizing the first term in Eq . ( 4 ) with respect to , we used a limited-memory Broyden-Fletcher-Goldfarb-Shanno ( BFGS ) method [73] , which is a variation of a quasi-Newton method , to conserve the memory necessary for optimization . The second term in Eq . ( 4 ) is a convex quadratic function . We can therefore use a simple linear algebra to estimate . Our functional connectivity analysis between neurons and glial cells was based on a comparison of the cross-validated likelihood , i . e . , the model's reproducibility for the activities in a validation dataset , between two different network structures . If there were two different network structures , one with a certain neuron-glia connection and another without the connection , and the latter demonstrated a larger cross-validated likelihood than the former , then , the connection was not considered to be included in our neuron-glia system . According to the -fold cross-validation with being 10 , we partitioned the time series into 10 subseries; we used nine of these subseries to train the model ( “training dataset” ) , and calculated the model-likelihood of the one remaining subseries ( “test dataset” ) as the cross-validated likelihood of the model . The neuron-wise , test-dataset-wise cross-validated likelihood of the activity of the i-th neuron , evaluated on the k-th test dataset for a network structure , , was given by , where indexes the re-arranged sampling time ( sample number ) in the k-th test dataset , and the parameter vector was determined by using the training dataset other than the k-th test dataset under network structure . By taking the average of the neuron-wise , test-dataset-wise cross-validated likelihood over the 10 test datasets , we have the neuron-wise cross-validated likelihood of the i-th neuron , . Then , taking the average over all the neurons , we have the cross-validated likelihood of network structure as . Similarly , we defined as the glia-wise , test-dataset-wise cross-validated likelihood of the activity of the i-th glial cell evaluated on the k-th test dataset for network structure . We also defined the i-th glia-wise cross-validated likelihood , , and likewise the cross-validated likelihood of network structure as . When evaluating the connections from the j-th glial cell to neurons , we compared the cross-validated likelihood between two different network structures , and , to which different constraints were introduced . The constraint given to was , i . e . , there were no connections from any glial cell to any neuron . The constraint given to was for all , i . e . , there were no connections from glial cells to neurons other than from the j-th glial cell . We evaluated the neuron-wise cross-validated likelihood , , , for each of the two network structures after we had estimated their individual model parameters . Observe that yields where the expectation is with respect to the GLM ( Eq . ( 1 ) ) with the true parameter vector plugged in . This observation suggests that we can use the difference in the cross-validated likelihood , , to evaluate the effect from a specific functional connectivity from glial cell to neuron , which is represented by the response function , . As it is difficult to obtain the analytical form of the distribution for the stochastic variable , , there is no theoretical way to perform a statistical test based on it . To construct a statistical test in a practical manner , therefore , we assumed that the difference in the neuron-wise , test-dataset-wise cross-validated likelihood , , would obey a normal distribution with a zero mean and variance , and designed a t-statistic: ( 5 ) where is the unbiased variance of the difference in the cross-validated likelihood , , calculated in the cross-validation process . By simply assuming the normality of the stochastic variable , , we can make to follow a t-distribution . We can then rely on the standard t-test , when evaluating each connection from glial cell to neuron . Indeed , this t-statistic assumption is not very accurate because the cross-validation samples are not independent of one another and the stochastic variable does not obey a normal distribution . However , the advantages of the t-statistic assumption on outweigh the disadvantages; we can evaluate the stochastic uncertainty of up to the second order moment by using this token . We took the opposite approach when evaluating connections from neurons to a particular single glial cell , . We compared two different network structures in a similar way to that above with a fixed glial cell of interest , i . e . , the i-th glial cell: the network with no neuronal connections to the glial cell ( i . e . , ) , and the network consisting of all possible connections . We defined the mean of differences in the glia-wise , test-dataset-wise cross-validated likelihood of the i-th glial cell by . A t-statistic of the difference in the glia-wise cross-validated likelihood was similarly defined as ( 6 ) We treated the connections from glia to neurons differently from those from neurons to glia in this study . The main principle of our search for the optimal network structure was to begin the search from a network structure with the highest cross-validated likelihood possible ( see ‘Methods Overview’ section in Results . Some details are also given in Text S1 ) . While the network structure with no neuron-to-glia connections exhibited a higher glia-wise cross-validated likelihood than the network structure with full neuron-to-glia connections ( full network ) , the neuron-wise cross-validated likelihood of the full network was lower than that of the structure with no glia-to-neuron connections ( Fig . S1 ) . Also , we resorted to an incremental search algorithm by considering the intractability of a full search over the whole space of all possible network structures . The search algorithm we adopted converges to an optimal network structure if we begin the search from a heuristically chosen structure with a high cross-validated likelihood . The cross-validated likelihood of the network structure monotonically increases and necessarily converges in this search algorithm because we only adopt a new structure when the cross-validated likelihood increases whereas the number of possible network structures is huge but still finite . We explored a statistical test based on the surrogate method to statistically examine the number of detected connections under the null hypothesis of no causal connectivity . We need to construct surrogate neuronal or glial activities that might have been observed under the null hypothesis , only from the observation time series . In order to evaluate the number of detected connections from the i-th glial cell to neurons , we generated the surrogate glial activity ( Ca2+ signals ) of the i-th glial cell ( called the original glial cell below ) 1000 times based on the Iterated Amplitude Adjusted Fourier Transform ( IAAFT ) method ( for details , see Text S1 ) [74] . This surrogate glial cell was assumed to have no connections to any neurons in the neuron-glia system , but all other parts of the system remained untouched . Surrogate glial activity in the IAAFT method was generated based on the randomization of phases in the activity time series of the original glial cell . Application of IAAFT to glial activity destroyed the mutual correlation between the original glial cell and all the other network components while preserving the amplitude distribution and the autocorrelation of the activity of the original glial cell ( see Fig . S2 ) . We obtained 1000 surrogate datasets by replacing the activity time series of the original glial cell with each of the 1000 surrogate glial activities . We then applied functional connectivity analysis to each of the 1000 surrogate datasets and computed the number of detected connections from the surrogate glial cell to neurons . The empirical distribution constructed from the 1000 surrogate datasets could serve as a null distribution built on the hypothesis that there were no functional connectivities from the original glial cell to neurons . We compared the number of actually detected connections based on the original glial cell's activity against the empirical distribution to compute the p-value of the original glial cell's activity . In the construction of each surrogate neuronal activity , on the other hand , we applied a circular shift to the original neuronal spike time series . This type of implementation is preferable [75] because it can perturb the temporal relationship between neurons , whose activities are surrogated , and other components of the network while preserving its own statistics , such as the distribution of inter-spike intervals , autocorrelation , and self-dependence of the original neuronal activity . We determined the tuning parameters ( tuning constants ) , to optimize the cross-validated likelihood by applying heuristic constraints to reduce the space to search for their optimal combination . The parameters to be tuned were maximum time lags ( history window sizes ) under the heuristic constraints , and , shrinkage parameters of the response functions under the heuristic constraints , and , and smoothness parameters of the response functions under the heuristic constraints , and . More concretely , we searched discretized candidates , and for the best values for both and , , and for both and , and , and for both and , to maximize the cross-validated likelihoods , and . Consequently , we found the optimal values for the tuning parameters were , , and . Here , we applied the heuristic constraints to mainly reduce the search space of the tuning parameters . Such application of constraints is equivalent to having assumed that similar mechanisms govern all receptors on neuronal and glial cells . However , some studies have indicated the possibility that glial receptors might respond differently to neurons and glia [15] . Therefore , we recomputed and independently ( with no constraints ) to validate our heuristic constraint , while clumping all the other tuning parameters , and we found that the recomputed parameter values were equal to that with the constraint . When we carried out the same validation for the constraint , , the optimal values without the constraint also yielded the same value as that with the constraint . Further , the overall characteristics of the response functions were found to be fairly robust against the large diversion in the smoothing parameter from its optimal value ( Fig . S7 ) . We attempted to introduce a specific constraint , for any , to our GLM , i . e . , the connection from neuron to glial cell is required to be strictly positive . The parameter optimization ( the MAP estimation ) of the log posterior with our likelihood and prior distribution is equivalent to the minimization of a specific quadratic cost function . The parameter estimation under the additional constraint , for any , can then be performed by quadratic programming [76] , so as to minimize the cost function under the constraint . Based on the thus computed , we can compute the neuron-wise cross-validated likelihood , as well as t-statistic for any network structure . We explained how the introduction of the positivity constraint above affected the results of functional connectivity analysis at the end of the Discussion section . | Many neuroscientists believe that neurons mainly perform information processing in the brain . Glial cells have traditionally been regarded as passive cells , whose roles have been limited to mechanical support and energy transfer to neurons . However , some studies have recently demonstrated the existence of interactions between neurons and glial cells and implied the involvement of crosstalk between neuronal and glial systems in information processing . Nevertheless , the details on neuron–glia communication largely remain unknown . One way of addressing this issue is to use a powerful statistical methodology to identify the network structure based on high-throughput time-lapse imaging from neuron–glia networks . We developed a new statistical method for functional connectivity analysis that was suitable for examining neuron–glia interactions . We applied the method to multicellular Ca2+ imaging data , where neurons and glial cells carried out spontaneous activities in a rat hippocampal CA3 culture . We found in a data-driven manner that each glial cell facilitated the activities of neighboring neurons with a peak latency of 500 ms . Our study is the first of its kind to present a statistical framework to investigate the functional connectivity between neurons and glial cells . Our statistical method is thus capable of identifying neuron–glia interactions by utilizing the high-throughput imaging technique . | [
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] | 2014 | A Statistical Method of Identifying Interactions in Neuron–Glia Systems Based on Functional Multicell Ca2+ Imaging |
Ribosomopathies are a family of inherited disorders caused by mutations in genes necessary for ribosomal function . Shwachman-Diamond Bodian Syndrome ( SDS ) is an autosomal recessive disease caused , in most patients , by mutations of the SBDS gene . SBDS is a protein required for the maturation of 60S ribosomes . SDS patients present exocrine pancreatic insufficiency , neutropenia , chronic infections , and skeletal abnormalities . Later in life , patients are prone to myelodisplastic syndrome and acute myeloid leukemia ( AML ) . It is unknown why patients develop AML and which cellular alterations are directly due to the loss of the SBDS protein . Here we derived mouse embryonic fibroblast lines from an SbdsR126T/R126T mouse model . After their immortalization , we reconstituted them by adding wild type Sbds . We then performed a comprehensive analysis of cellular functions including colony formation , translational and transcriptional RNA-seq , stress and drug sensitivity . We show that: 1 . Mutant Sbds causes a reduction in cellular clonogenic capability and oncogene-induced transformation . 2 . Mutant Sbds causes a marked increase in immature 60S subunits , limited impact on mRNA specific initiation of translation , but reduced global protein synthesis capability . 3 . Chronic loss of SBDS activity leads to a rewiring of gene expression with reduced ribosomal capability , but increased lysosomal and catabolic activity . 4 . Consistently with the gene signature , we found that SBDS loss causes a reduction in ATP and lactate levels , and increased susceptibility to DNA damage . Combining our data , we conclude that a cell-specific fragile phenotype occurs when SBDS protein drops below a threshold level , and propose a new interpretation of the disease .
Ribosomopathies are inherited diseases due to the haploinsufficiency of ribosomal proteins or ribosome processing factors [1 , 2] . Patients affected by ribosomopathies present multiorgan phenotypes [2] . Some relatively common features are bone marrow and skeletal deficits , and cancer predisposition [3 , 4] . At the cellular level , ribosomal haploinsufficiency may cause the induction of tumor suppressor p53 [2 , 5] . Consequently , in some models the depletion of p53 reduces the deleterious effects of ribosomal haploinsufficiency [6–9] leading to the hypothesis that abnormal p53 is the pathogenic culprit . However , this is not true for all cases [10 , 11] . Shwachman-Diamond Syndrome ( SDS ) is a recessive ribosomopathy affecting 1 in 76 , 000 births . SDS is a multisystem disorder presenting in the first year of life and characterized by the hallmark of exocrine pancreatic dysfunction . Another common symptom is the susceptibility to chronic infections accompanied by neutropenia [12 , 13] . With variable penetrance , patients affected by SDS may have low stature , skeletal defects and cognitive impairment [14 , 15] . Finally , high risk of acute myeloid leukemia ( AML ) is associated with older patients [16] . Loss-of-function mutations in the SBDS gene have been identified as the cause of the disease [17] . Several studies addressed the function of SBDS protein in mammals and of its yeast homolog . A concise survey will be presented . SBDS has a role in the maturation of 60S ribosomal subunits . Deletion of the yeast homolog sdo1 is quasi-lethal , leading to pre-60S nuclear export defects . Importantly , point mutations of tif6 , a gene necessary for 60S biogenesis [18] , revert the quasi-lethal phenotype [19] . These and other studies , have led to a general model in which SBDS is necessary for the maturation of 60S subunits , in order to remove eIF6 ( mammalian Tif6 ) from mature 60S subunits [20–22] . Since eIF6 controls 60S availability [23] and full translational activation [24 , 25] , manipulation of the binding of eIF6 to the 60S may be critical for restoring SBDS-mutant cells . A recent report suggested that SBDS contributes to the efficient translation of C/EBPα and C/EBP-β mRNAs , uORF-containing mRNAs that are , among others , indispensable regulators of granulocytic differentiation [26] . A general analysis of translated mRNAs depending from SBDS is still lacking . We do not know in detail whether other steps of 60S maturation beside eIF6 release are affected . Several studies have attempted to pinpoint other functions of the SBDS protein , in the obvious effort to explain the multiorgan phenotype of patients . At the cellular level , several phenotypes associated to SBDS loss have been described . These phenotypes have been largely observed either in primary cells from patients , or upon shRNA experiments in cell lines . Increased apoptosis driven by FAS was seen in HeLa upon SBDS shRNA [27] , and increased ROS production [28] . Increased apoptosis was also seen in SBDS-depleted HEK293 cells upon DNA and chemically induced endoplasmic reticulum stress [29] , and in hemopoietic cells [30] . SBDS association with mitotic spindle has been proposed [31] and the lack of SBDS has been associated with genetic instability [32] . In general , reduced clonogenic potential is observed in hematopoietic precursors upon SBDS depletion [9 , 33] . Reduced respiratory capability has been observed in mammalian and yeast cells lacking SBDS or its homolog , Sdo1p [34 , 35] . Overall , it is unclear whether the cellular phenotypes ascribed to the loss of SBDS activity are direct or indirect , general or cell-specific , and have a relationship with protein synthesis . Recently , a mouse strain in which one of the most frequent missense mutation found in SDS patients is modeled , R126T , has been produced [36] . This mouse model reproduces the clinical symptoms of SDS patients . We exploited the availability of this model to address the cell autonomous effects due to hypomorphic SBDS alleles . We have derived from these mice embryonic fibroblast cell lines , we have reconstituted control MEFs with wild type SBDS and then performed a full characterization of their properties . We addressed in SBDS mutant cells their predisposition to oncogenic transformation , changes in eIF6 binding , transcriptional and translational changes , metabolic parameters , and sensitivity to drugs and stresses . We unveiled several features due to the loss of SBDS . We provide a pathogenic model of SBDS deficiency that focuses on a diminished anabolic and energetic status of Sbds mutant cells due to reduced protein synthesis , which may be useful to design rational therapeutic ameliorative strategies .
Swhachman-Diamond ( SDS ) patients have an higher incidence of blood tumors , mainly acute myeloid leukemia ( AML ) [37] . This observation raises the question whether SBDS mutant cells are intrinsically susceptible to oncogene-mediated transformation . SBDS point mutation R126T corresponds to a common mutation found in SDS patients ( c . 377G>C ) and it has been modeled in mice [36] . We immortalized R126T mouse embryonic fibroblasts ( MEFs ) , together with their matched wild type controls , and we evaluated their capability to form colonies . Two strategies can be employed for immortalization of MEFs , a ) sequential subcloning [38] or b ) immortalization and transformation with oncogenes and tumor suppressor inhibitors [39] . By sequential subcloning , we were unable to derive SbdsR126T/R126T MEFs due to early senescence ( Fig 1A ) , whereas we normally derived wt cells . This result is in line with a recent work describing early senescence in the pancreas of SBDS mutants [8] . In contrast , immortalization of SbdsR126T/R126T MEFs by infection with a vector carrying the dominant negative p53 and Ras G12V was successful ( Fig 1A; S1A and S1B Fig ) . The growth of immortalized SbdsR126T/R126T MEFs was virtually identical to the one of wt cells ( S1C Fig ) . Transformation of immortalized cells is induced by long-term growth at confluency . Next , we analyzed the capability of immortalized SbdsR126T/R126T MEF cells to form transformed colonies respect to wild type cells , upon long term culture at 100% confluency . We observed that SbdsR126T/R126T MEFs formed less foci respect to the wild type cells ( Fig 1B and 1C ) . We then plated wt MEFs and SbdsR126T/R126T MEFs in soft agar , another indicator of transformation efficiency , to test their capability to grow in anchorage independent condition . We found that surviving clones of SbdsR126T/R126T MEFs grew as well as wt cells ( Fig 1D ) , but their overall number was lower than the one of wt MEFs ( Fig 1E ) . In order to evaluate the capability of these transformed colonies to induce tumor in vivo , we injected 500 . 000 transformed cells in nude mice and monitored tumor mass growth . Surprisingly and strikingly ( Fig 1F ) , formally transformed SbdsR126T/R126T cells were inefficient ( n = 1 ) or unable ( n = 6 ) to grow in vivo respect to the wild type cells . We demonstrate that SBDS deficiency induces in a cell-autonomous fashion a growth and clonogenic deficit that can be unveiled when cells are challenged by environmental conditions . We established a further model for studying SBDS function by generating , from immortalized SbdsR126T/R126T MEFs , new clones retransduced with either wild type Sbds ( SbdsRESCUE ) or mock control ( SbdsMOCK ) vectors ( S1D Fig ) . By comparing parental SbdsR126T/R126T clones to their wild type counterparts , and the SbdsMOCK with the SbdsRESCUE clones , we can discriminate direct events due to SBDS lack from indirect effects . We describe the most important observations and discuss later a model that explains the pathogenic effect of SBDS deficiency . Since SBDS deficiency leads to a ribosomal defect [19] , we performed a complete analysis of translation . Polysomal profiles can be used to analyze defects in initiation as well as in ribosome maturation . We observed , in line with previous reports [20] , a strong unbalance in 60S and 80S peaks in SbdsR126T/R126T MEFs respect to the wild type cells ( Fig 2A ) . SbdsRESCUE cells showed a complete rescue of the profile ( Fig 2B ) , confirming a direct action of SBDS on ribosome maturation , and validating our model for discriminating direct versus clonal or indirect effects . We analyzed rRNA precursors with a pulse-chase assay by monitoring the incorporation of 5 , 6 3H-uridine in the nascent ribosomal RNA ( S2A Fig ) . We did not observe differences in rRNA maturation associated with the SBDS mutation , a result consistent with the idea that only the late maturation of 60S is affected by SBDS deficiency . The nucleolus is the nuclear compartment where both ribosome biogenesis and early maturation occur . A defect in ribosomal export can be assessed by measuring the number and the size of nucleoli . We did not observe differences in the number of nucleoli between SbdsR126T/R126T and wild type cells ( Fig 2C ) or in SbdsMOCK and SbdsRESCUE cells ( Fig 2D ) . In addition , we did not observe differences in co-localization of SBDS and nucleolar marker nucleophosmin ( NPM ) between SbdsR126T/R126T and wild type cells ( Fig 2E and 2F ) . eIF6 nucleolar localization was relatively similar in SbdsR126T/R126T and wild type cells ( S2B Fig ) . eIF6 binds 60S subunits , blocking 80S formation and increasing free 60S peak [24] . It has been proposed that SBDS deficiency blocks eIF6 release [20] . This model is consistent with the accumulation of free 60S that we observed ( Fig 2A ) . We recently developed a Ribosomes Interaction Assay ( Fig 2G ) , able to quantify eIF6 binding sites on the 60S [40] . We immobilized equal ribosomes from SbdsR126T/R126T and wild type cells , and measured eIF6 binding sites . We found a 25% reduction in eIF6 binding sites on the ribosomes of SbdsR126T/R126T , compared to wild type cells ( Fig 2H ) . Thus , we conclude that SBDS deficiency leads to a late maturation deficit of 60S consistent with the generation of a reduced pool of functional 60S subunits . It is worth to note that 60S peak increases at least 2-fold ( Fig 2A and 2B ) , whereas eIF6 binding sites drop only 25% ( Fig 2H ) . We asked the consequences of the maturation deficit on translation . We developed a fully reconstituted in vitro model , in which translation competent extracts are prepared from equal amount of cells , normalized to the number of ribosomes and transduced with defined amounts of exogenous mRNA . This experiment allows to measure the maximal translational capability per cell/per ribosome . Ribosomal extracts from SbdsR126T/R126T MEF showed around 70% reduction in the translational capability of a cap-dependent reporter ( Fig 3A ) . Cells rescued with SBDS in vivo ( SbdsRESCUE ) recovered their translational capability ( Fig 3B ) . Importantly , adding wild type SBDS in vitro did not rescue the translational capability of extracts prepared from SbdsR126T/R126T MEFs ( S2C Fig ) . This result indicates an overall translational impairment . Next , we adapted the canonical SUnSET protocol to citofluorimetry analysis . We observed both in SbdsR126T/R126T MEFs respect to wild type ( Fig 3C ) and in SbdsMOCK MEFs compared to SbdsRESCUE MEFs ( Fig 3D ) about 10% reduction in the number of cells incorporating medium to high levels of puromycin . Taken together , our results demonstrate that R126T mutation leads to a strong reduction of the pool of 60S subunits competent for translation . An obvious question is whether the impaired maturation of 60S ribosomes , associated with a reduced translational capability in SbdsR126T/R126T cells , results in a qualitative difference of translation . We decided to proceed with an RNASeq study on total RNA extracted from sucrose gradient collected fractions . We studied 1 ) RNAs associated to polysomes , 2 ) RNAs associated to the 80S and 3 ) steady-state mRNA levels ( total RNA ) ( Fig 4 ) . The isolated fractions are shown in Fig 4A and 4B . The combination of these parameters allowed us to define the overall translational and transcriptional status associated with SbdsR126T/R126T mutation , assuming that mRNAs differentially localized to either polysomes or 80S are controlled at the translational level . We have decided to use this strategy over ribosome profiling because a ) altered 60S subunits may lead to abnormal RNA protection , b ) the big change in the 80S monosomal peak found in SbdsR126T/R126T cells is difficult to be normalized , and c ) to efficiently reach deepness of more than 2x107 reads . S1 File contains read counts of polysomes and total RNAs . S2 File contains read counts of 80S . By comparing the polysomes of SbdsRESCUE and SbdsMOCK , we identified 844 modulated genes ( Fig 4C; S1 File ) , most of them enriched in presence of mutant SBDS . However , when we estimated the translational efficiency , i . e . bona fide polysomal enrichment , by normalizing each mRNA level on the polysome to the amount present on the total , we found only 74 mRNAs with a significant modulation . Of these 74 , 31 had less than 10 normalized read counts average expression , suggesting that fluctuations of poorly expressed mRNAs may contribute to the observed effects . The analysis of translation efficiency of SbdsRESCUE and SbdsMOCK confirmed that they do not differ in qualitative translational regulation ( Fig 4D ) . The analysis on RNAs enriched on 80S subunits unveiled 250 genes modulated in 80S SbdsMOCK compared to 80S from SbdsRESCUE cells ( Fig 4E; S2 File ) . However , identical to what we observed in the polysomal fraction , also in this case the mRNAs modulation on 80S followed the steady state levels ( S3A and S3B Fig ) as well the polysomal . Taken together , the data suggest that SBDS loss induces a solid transcriptional rewiring due to a general impairment of translation rather than specific translational regulation . By analyzing polysomal versus steady state mRNAs and 80S modulated in Sbds mutated cells we made two major observations: a ) a 4-fold enrichment of snoRNAs ACA8 and ACA31 in the ribosomes of SbdsMOCK cells compared to SbdsRESCUE . ACA8 and ACA31 drive the pseudouridylation of 28S rRNA U3832 and U3713 on the 60S . Other snoRNAs were not changed ( Fig 4F; S1 and S2 Files ) . Intriguingly , there was no overlap between the genes regulated at the polysomal level by Sbds mutation ( S1 File ) , to the ones we previously found regulated by eIF6 deficiency [41] . In conclusion , our data ( Figs 2G , 2H , 3 and 4 ) suggest that the lack of mature 60S leads to a general reduction of translational capability associated with transcriptional rewiring . The limited specific translational changes seen at the polysomal level ( Fig 4 ) , and the strong reduction in the maximal translational capability ( Fig 3 ) were mirrored by a complex rewiring of gene expression and metabolism of SBDS deficient cells ( Fig 5 ) . Hereafter , we describe the transcriptional and metabolic changes directly due to SBDS deficiency , i . e . fully rescued by SBDS readministration in the SbdsR126T/R126T background . To simplify , 527 genes were at least 2-fold altered at the transcriptional level in SbdsMOCK cells , all of them presenting concomitant changes in the polysomal pool . Functional analysis by classical Gene Ontology ( GO ) for the Molecular Function Domain was performed on these 527 genes . Twentyseven ontology terms grouped in ten emerging categories were found as significantly enriched . Gene Set Association Analysis ( GSAA ) on both polysomal and total fractions confirmed the findings of the classical ( GO ) gene ontology ( S3 File ) . Globally , we found that SBDS mutant cells had a decrease in genes encoding for the ribosomal and respiratory chains , and an increase in the lysosomal capability . We will specifically describe some of them . We found upregulated ( both at the polysomes and at the steady-state level ) mRNAs with peptidase activity including lysosomal cathepsins such as Ctsb , Ctsd , Ctsk and Ctsl ( Fig 5A ) , and lysosomal genes with vacuolar ATPase activity , including Atp6ap1 , Atp6ap2 and Atpv0b ( Fig 5A , S1 File ) . Validation was confirmed by quantitative PCR ( Fig 5B ) . M6pr , Lamp1 and Atp6ap1 upregulation suggests increased lysosomal activity associated with Sbds mutation , whereas increased CtsB suggests higher degradation activity . Consistently , we found in SbdsMOCK cells an increase in cell acidification by the Lysotracker assay ( Fig 5C ) and an increase in Lamp1 positive cells by immunofluorescence ( Fig 5D and 5E ) and by western blot ( S5A Fig ) . Other coordinated gene expression changes observed in SbdsMOCK cells were a puzzling upregulation of membrane transporters for aminoacids and other intermediates ( S4A Fig ) , a downregulation in most ribosomal components ( S4B Fig ) , and a drop of expression of several genes important for mitochondrial function ( S1 and S2 Files ) . These findings , in line with a reduction in the global protein synthesis capability observed in Fig 2G and 2H and in Fig 3 , further predicted that SbdsR126T/R126T cells might have a decrease in the level of high energy molecules like ATP . Therefore , we measured intracellular ATP levels and found a decrease in ATP levels both in SbdsR126T/R126T and SbdsMOCK cells respect to their controls ( Fig 5F ) . The reduction of ATP levels was also confirmed in HEK cells carrying an shRNA for SBDS ( S5B and S5C Fig ) . In addition , we did not observe any significant difference 1 . AMPK and 2 . phospoAMPK levels , whereas a mild difference was appreciate in 3 . PhosphoAMPK substrates by western blots ( S5D and S5E Fig ) . We then measured the levels of secreted lactate and pyruvate ( S6A and S6B Fig ) , as index of the glycolytic flux , and we found a reduction in the Lactate/Pyruvate ratio both in SbdsR126T/R126T and SbdsMOCK respect to their controls ( Fig 5G ) . To further understand the metabolism of these cells we measured the levels of a . glycolytic activity , b . respiration and c . ROS production . We found that both SbdsR126T/R126T and SbdsMOCK cells show a decrease in glycolysis ( S6C Fig ) and a reduction in respiration ( S6D Fig ) , while ROS levels remained unchanged ( S6E Fig ) . In conclusion , we demonstrate that the reduction of SBDS activity causes a reduction of a global translational capability and a cellular adaptation with less energy production and more compensatory catabolic capability . We focused our attention on information deriving from LINCS/CMap project to evaluate if the gene expression profile found altered in the total fraction of SbdsR126T/R126T MEFs cells was similar to transcriptional changes induced by drugs , observed in other cell types and to eIF6 deficiency [41] . Analysis performed by Query web tool on the complete list of modulated genes , and on a subset of the most changed ones identifies three common PKC activators: ingenol , phorbol-12-myristate-13-acetate and prostratin ( S3 File ) . Overall the analysis on SBDS deficient cells suggests that they have an impaired translational capability that leads to a compensatory transcriptional and metabolic rewiring that favors a catabolic processes and a state of low energy versus high synthetic capability . We reasoned that the transcriptional signature generated by SBDS deficiency could lead to a differential sensitivity to drugs or stressors which may be exploited at a therapeutic level . In principle , inhibitors that preferentially repress the growth of SbdsR126T/R126T immortalized MEFs may be of use in treating SDS patients who are affected by Acute Myeloid Leukemia ( AML ) . On the contrary , selective stimulators of growth could be potentially interesting for early-phase of the disease , for instance in the context of neutropenia or pancreatic insufficiency . We decided to proceed with two different screenings , the first based on 100 compounds selected from commercial oral drugs ( S4 File ) . We evaluated the response signature to drugs associated to R126T mutation by measuring cell viability after 48h of treatment . We found that overall SbdsR126T/R126T MEFs were similar to SbdsMOCK , and wt to SbdsRESCUE . This said , the signature profile that we found was surprisingly similar between wt and SBDS deficient cells ( Fig 6A ) , with only one exception . In particular , we found increased sensitivity of SbdsR126T/R126T and SbdsMOCK cells to chlorambucil ( Fig 6B ) , a DNA alkylating agent . Microtubule-targeting drugs mebendazole and colchicine were more toxic to SbdsR126T/R126T cells ( S7 Fig ) , instead niclosamide had a stimulatory effect on SbdsR126T/R126T cells ( S7 Fig ) , but the effects were not rescued in the SbdsRESCUE , suggesting indirect effects . The second screening with a Prestwick Library including 1280 compounds aimed at finding growth stimulators identified clindamycin ( S7B Fig ) , but the effect was not rescued in SbdsRESCUE cells . In conclusion , 1 . Sbds mutation cause limited effects unless cells are challenged , 2 . chlorambucil data predict that SbdsR126T/R126T cells might be more sensitive to stress causing DNA damage . The increased toxicity of the alkylating agent chlorambucil in SbdsR126T7R126T cells hinted a possible increased susceptibility to cell death associated to other forms of DNA damage . We first measured the basal level of apoptosis in wild type and in SbdsR126T/R126T cells by measuring the AnnexinV/7AAD positive population , and we did not find any differences between the two genotypes ( Fig 7A ) . Treatment of cells with UV pulses caused an increase in cell death ( Fig 7A ) , that was significantly more marked in SbdsR126T/R126T and in SbdsMOCK respect to wild type or SbdsRESCUE cells ( Fig 7B ) . We analyzed DNA damage by examining cells with the COMET assay ( Fig 7C and 7D ) , where the tail moment revealed a slight but significant increase in DNA damage in SbdsMOCK respect to wild type or SbdsRESCUE cells . This effect , even if small , is consistent with increased sensitivity of SbdsMOCK to two external stresses acting on DNA like chlorambucil and UV rays . These data , together with a decrease in energy levels and the uncapability to grow in vivo , show that Sbds mutated cells have an intrinsic fragility , that makes them weaker respect to their wild type counterpart .
Our study suggests a model ( Fig 8A ) that may explain the pathogenic culprit of SDS and will be first described . The table in Fig 8B outlines the phenotypes that we have observed . A simple model for SBDS-driven pathological symptoms is based on a threshold concept . Total null SBDS mutations are never observed in humans indicating that a zero threshold of SBDS is incompatible with life . Mutant SBDS has a residual activity . This activity is sufficient for , i . e . , normal proliferation , but becomes limiting for efficient colony formation or escaping senescence . If senescence is bypassed by oncogene-driven immortalization and transformation , then , SBDS remains still limiting for survival in nude mice . We can therefore imagine that each individual cell , in each moment , may or may not meet the condition of insufficient SBDS activity , with subsequent effects . Indeed , by the SUnSET protocol we see that only some SBDS mutant cells do not reach high translation levels . If the threshold is not reached , our study supports a model in which SBDS deficiency leads primarily to a delayed maturation of 60S ribosomal subunits . We confirm that the number of eIF6 binding sites on 60S ribosomes is reduced , consistent with reduced generation of free 60S and with a proposed model of SBDS-mediated eIF6 release in the late maturation of 60S , in cooperation with EFL1 [26] . In detail , we quantify a 25% decrease of eIF6 binding sites on 60S , similar to [42] . We also report that the increase of free 60S is 2-fold , the amount of accumulated ACA8/ACA31 snoRNAs on immature 60S is 4-fold , and ribosomal extracts from SBDS mutants have a 70% reduction in translational capability . Apparently , the ribosomal deficit due to SBDS mutation is more pervasive than expected and suggests the generation of a bulk of immature 60S that are poorly active in translation , even if eIF6 is released . This said , eIF6 release may be critical in the rescue of some mature ribosomes and the alleviation of pathological consequences . A variety of proxyes due to reduced SBDS activity are evident . Indeed , adaptation is evident in the absence of overt signs of SBDS insufficiency . Even if SBDS mutant cells proliferate normally in rich media , they show a signature with reduced energy level , increased lysosomal capability , decreased ribosomal production and less oxygen consumption . The drug screening identified a subtle signature of fragility which may contribute to the pathology . Previously , it was reported excess protease secretion in SDS-derived iPSC [43] , reduced respiration in yeasts and mammalian cell models [34] , and less ATP [35] that together with our extended gene signature define a status of reduced anabolic capability . This may be a keyfactor for rescuing the phenotype . Stimulators of ATP production , for instance , may have an impact on the disease . Our initial drug screening suggests that efforts in this direction may be valuable: we screened drugs for survival activity and found that SBDS mutants are remarkably similar to wt cells . However , a screening for a closer proxy of SBDS deficiency such as ATP levels , may lead to successful compounds . We think that increased AML in SDS patients is a defect due to impaired host-cancer interplay , rather than due to genetic instability . If anything , SBDS-deficient cells are very resistant to oncogenic transformation and unable to grow in unfavourable conditions like the ones generated in tumors ( see xenografts ) . In the bone marrow of SDS models , the neutrophil lineage can be impaired by the absence of sufficient endogenous SBDS activity [9] . SDS patients have neutropenia [44] . It is known that severe congenital neutropenia is associated with the progression to acute myeloid leukemia [45] . Therefore , we speculate that neutropenia in the bone marrow niche [30 , 42] may lead to expansion of tumor cells that cannot be eliminated by proper immunosurveillance . Similar suggestions came from mouse work [46] . In summary , for therapy , we need to address which cell is directly affected by SBDS deficiency . A divergence between our studies and previously published works relates to ROS [28] production . In our hands we did not find evidence for increased ROS production . It is possible that impaired ROS balance is observed as an indirect effect driven by mitochondrial alterations in specific cells , but is not a direct proxy of SBDS deficiency . Finally , we would like to comment on translational control driven by SBDS and eIF6 . SBDS insufficiency generates a unique phenotype . Similarly to SBDS mutants , eIF6 depletion protects from oncogene-induced transformation [25 , 47] , whereas its amplification is oncogenic [48 , 49] . eIF6 depletion does not induce senescence [25] , but induces a different transcriptional rewiring [41] , a divergent bone marrow phenotype affecting platelets [50] , and a generally improved metabolic status [41] . SBDS depletion reduces the translation of mRNAs undergoing reinitiation , as CEBP/β derived LIP peptide [26] . In our study the decrease of CEBP/β mRNA on polysomes driven by SBDS deficiency is around 10% ( S1 Table ) . eIF6 depletion reduces both reinitiation and LIP expression as well as the translation of G/C rich mRNAs [41] . To summarize , the specific effects of eIF6 loss and SBDS loss are different , but overall they suggest that both eIF6 and SBDS have a stimulatory role in protein synthesis . In yeast and Dictyostelium eIF6 mutants revert the SBDS loss [19 , 20 , 22] . We speculate that these mutants are gain-of-function genes and that eIF6 agonists may be beneficial to the disease . In conclusion , our results support the idea that cells with reduced SBDS activity are able to grow and cycle as well as wild type cells in proper conditions , but if they drop below a SBDS threshold level they show a phenotype . However , the adaptation signature suggests logical roads for improving their fitness . In the long run a screening for compounds which may accelerate the release of eIF6 from 60S subunits can be helpful to discover specific drugs for treating SDS ( assuming that a modest increase in active 60S can strongly ameliorate the phenotype ) . Alternatively , screening on proxyes such as ATP levels may lead to faster therapeutic options .
Primary wild type and SbdsR126T/R126T MEFs ( E12 . 5 ) were grown in DMEM ( Lonza ) , supplemented with 10% Fetal Bovine Serum ( FBS ) and 1% penicillin , streptomycin , L-glutamine , and maintained at 37°C and 9% CO2 . Mycoplasma testing was performed before experiments . These cells were infected at early passages through retroviral vectors carrying DNp53 + oncogenic H-rasV12 [25] . After immortalization , cells were maintained at 37°C and 5% CO2 . Immortalized SbdsR126T/R126T MEFs were infected with lentiviral vectors carrying the wild type Sbds to generate the SbdsRESCUE line or with the corresponding empty vector to obtain the SbdsMOCK line . The lentiviral vectors ( pHAGE-CMV-dsREDexpressIRESzsGreen backbone vectors ) used to reconstitute SBDS wild type protein were kindly provided by A . Shimamura ( Fred Hutchinson Cancer Research Center , Seattle ) . HEK-293T and HeLa cells from ATCC were cultured in DMEM ( Euroclone ) supplemented with 10% FBS and penicillin/streptomycin/glutamine solution ( GIBCO ) at 37°C and 5% CO2 . Mycoplasma testing was performed before experiments . 293T cells were transfected at 60–70% confluence with pFCY , SBDS shRNA and pFCY scramble lentiviral vector ( a generous gift from the lab of DC . Link , Washington University School of Medicine ) to produce lentiviral particles . HeLa cells were infected with lentivirus at a confluence of 50% . Experiments were performed one week after infection . Silencing of protein was measured by western blot analysis . All animal experiments were carried out under the guidelines of the Canadian Council on Animal Care , with approval of procedures by The Animal Care Committee of the Toronto Centre for Phenogenomics , Toronto , AUP #0093 . For the transformation analysis , primary fibroblasts were infected at early passage with DNp53 + H-rasV12 retroviral vectors and left to grow at overconfluency . Foci were counted 3 weeks after infection and cells were recovered for in vivo experiments . Eight-weeks old CD1 athymic nude mice were used for detecting tumor growth after a subcutaneous injection of in vitro transformed MEFs ( 500 , 000 cells/mouse ) . Tumor growth was monitored and animals were euthanized when the tumor reached the size of 400 mm3 . All experiments involving mice were performed in accordance with Italian National Regulations . Experimental protocols were reviewed by local Institutional Animal Care and Use Committees . The soft agar formation assay and the focus formation assay were performed as described previously [51] . In vitro assays were performed at least three times , each in triplicate . Growing cells were lysed in 50 mM Tris-HCl , pH 7 . 5 , 100 mM NaCl , 30 mM MgCl2 , 0 . 1% Nonidet P-40 , 100 μg/ml cycloheximide and 40 U/mL RNasin . After centrifugation at 12 , 000 r . p . m . for 10 min at 4°C , cytoplasmic extracts with equal amounts of RNA ( 10 OD260 ) were loaded on a 15–50% ( or 10–30% ) sucrose gradient dissolved in 50 mM NH4Cl , 50 mM Tris-Acetate , 12 mM MgCl2 , 1 mM DTT and centrifuged at 4°C in a SW41Ti Beckman rotor for 3 h 30 min at 39 , 000 r . p . m . Absorbance at 254 nm was recorded by BioLogic LP software ( BioRad ) and fractions ( 1 . 5 mL each ) were collected for subsequent proteins or RNA extraction . Each experiment has been performed at least six times , for each condition . Total RNA was extracted from sucrose gradient aliquots . For the 15–50% gradient , we pulled fractions corresponding to polysomes in one fraction , and we pulled 100 μL from each fraction from the whole gradient in one fraction ( total ) . For the 10–30% gradient , we pulled fractions corresponding only to the 80S peak in one fraction , named 80S , and we used as total RNA aliquots from the pre-gradient loading extract ( pre-load ) . Afterward , we added to samples proteinase K ( to a final concentration 100μg/mL ) and SDS ( to a final concentration of 1% ) and we incubated them for 1 h at 37°C . Total RNA was then extracted by phenol/chloroform/isoamyilic acid method ( https://tools . thermofisher . com/content/sfs/manuals/trizol_reagent . pdf ) . Libraries for Illumina sequencing were constructed from 100 ng of total RNA with the Illumina TruSeq RNA Sample Preparation Kit . The generated libraries were loaded on to the cBot ( Illumina ) for clustering on a HiSeq Flow Cell v3 . The flow cell was then sequenced using a HiScanSQ ( Illumina ) . A paired-end ( 2×101 ) run was performed using the SBS Kit v3 ( Illumina ) . Experiments were performed in biological triplicates . For quantitative PCR , 150 ng of RNA was retrotranscribed according to SuperScriptTM III First-Strand Synthesis SuperMix manufacturer protocol ( 18080400 , Life Technologies ) . For RNASeq validation , Taqman probes specific for Atp6ap1 ( Mm01187488_g1 ) , Lamp1 ( Mm00495262_m1 ) , Ctsb ( Mm01310506_m1 ) and M6pr ( Mm04208409_gH ) were used . Target mRNA quantification was performed by using ΔCt-method with 18S rRNA as an internal standard , performed on a StepOne Puls System ( Applied Biosystems ) . Results are represented as means + s . d . of three independent experiments . Gene set association analysis for polysomal and total fractions was performed by GSAA software ( version 1 . 2 ) [57] . Raw reads for about ~ 22000 genes identified by Entrez Gene ID were analyzed by GSAASeqSP , using gene set C5 ( mouse version retrieved from http://bioinf . wehi . edu . au/software/MSigDB ) and specifying as permutation type ‘gene set’ and as gene set size filtering min 15 and max 800 . Analysis on CMap/LINCS gene signatures was performed using the ‘Query’ web tool ( http://apps . lincscloud . org/query ) , that , taking as input a list of genes , computes the connectivity between this set and the gene expression signatures of the LINCS database . Human orthologs of mouse genes were retrieved relying on Biomart web tool ( http://www . ensembl . org/biomart ) ; only genes with a one-to-one orthologs relationship were maintained for downstream computations . Query tool was first applied on the list of 726 mouse genes significantly modulated in the total fraction; chemical compounds showing a mean rank value of at least 90 on 6 cell lines were selected as interesting . As second strategy , the same procedure was applied on the genes showing a robust modulation ( 143 genes with a fold-change higher than 3 as absolute value ) and then selecting chemical compounds with a mean rank value of at least 90 on 4 cell lines . To find perturbations that are consistently retrieved , the overlap between the two analyses was considered as final result . SDS-PAGEs were performed on protein extracts obtained with RIPA buffer ( 10 mM Tris-HCl , pH 7 . 4 , 1% sodium deoxycholate , 1% TritonX-100 , 0 . 1% SDS , 150 mM NaCl and 1 mM EDTA , pH 8 . 0 ) . Protein concentration was determined with BCA analysis ( Thermo Fisher Scientific ) . Equal amounts of proteins were loaded on each lane and separated on a 10% SDS-PAGE , then transferred on a PVDF membrane . The membranes were blocked in 10% Bovine Serum Albumin ( BSA ) in Phosphate Buffer Saline ( Na2HPO4 10 mM , KH2PO4 1 . 8 mM , NaCl 137 mM , KCl 2 . 7 mM , pH 7 . 4 ) ( PBS ) with Tween ( 0 , 01% ) for 30 minutes at 37°C . The following primary antibodies were used: β-actin ( CST 4967L 1:4000 ) , SBDS ( Santa Cruz S15 SC49257 1:500 ) H-RAS ( Santa Cruz SC520 , 1:1000 ) , Lamp1 ( Santa Cruz SC20011 , 1:200 ) , AMPK ( CST 5831 1:1000 ) , phospho-AMPK ( CST 2535 1:1000 ) , phospho-AMPK Substrates ( CST 5759 , 1:1000 ) . The following secondary antibodies were used: donkey anti-goat IgG HRP ( Santa Cruz SC2020 , 1:2000 ) , donkey anti-rabbit IgG HRP ( Amersham NA934 1:5000 ) and donkey anti-mouse IgG HRP ( Santa Cruz , SC2005 1:5000 ) . Each experiment was performed at least three times , each time in triplicate . Cells were seeded the day before the staining . The next day , cells were rinsed three times with PBS then fixed with ice cold methanol 100% for 10 minutes at -20°C . After three washes with PBS , cells were blocked in Normal Goat Serum 5% , for 1 hour at room temperature . Primary antibodies were incubated overnight at 4°C , and after three washes with PBS , secondary antibodies were incubated 3h at room temperature in the dark . After three washes with PBS , cells were incubated with DAPI ( Molecular Probes NucBlue Live ReadyProbes Reagent R37605 ) as manufacturer protocol , then washed and mounted on slides with Mowiol 20% . All the antibodies were diluted in blocking solution . The following primary antibodies were used: NPM [25] , SBDS ( Santa Cruz S15 SC49257 1:25 ) , eIF6 [58] ( 1:100 ) , Lamp1 ( Santa Cruz sc-20011 1:100 ) . The following secondary antibodies were used: donkey anti-goat , donkey anti-mouse , donkey anti-rabbit ( Alexa Fluor secondary antibodies , Molecular Probes 1:500 ) . The cells were examined by confocal microscopy ( Leica SP5 ) and analyzed with Volocity 6 . 3 software ( Perkin Elmer ) . Immunofluorescence experiments were performed at least three times , in triplicate . Comet assay was performed following the manufacturer protocol ( Trevigen , 4250-050-K ) . The stained nuclei were then examined by confocal microscopy ( Leica SP-5 ) , and analyzed ( n≥30 for each condition ) with Comet Assay IV software ( Perceptive Instruments ) . The experiment was performed three times in triplicate . Ribosome biogenesis was analyzed by pulse-chase experiments by adding 5 , 6-3H-Uridine to the medium ( final concentration 3uCi/mL , NET367001MC PerkinElmer ) . 5 , 6-3H -Uridine was removed after 0 , 10 , 20 and 40 minutes of incubation and RNA was extracted with Trizol Reagent and hybridized on nitrocellulose membrane . This experiment was reproduced twice . For in vitro translation assays , we used cell extracts prepared as described [59] with some optimizations . 80% confluent cells were trypsinized and lysed for 45 minutes at 4°C in 10 mM HEPES pH 7 . 6 , 10 mM potassium acetate , 0 . 5 mM magnesium acetate , 5 mM DTT and protease inhibitor ( Promega ) . Lysates were homogenized by syringing through a 27G , 3/4-inch needle . Lysates were clarified by centrifugation at 18 . 000 g for 1 minute and protein concentration was determined by BCA quantification assay . To make translation fully dependent on exogenously added mRNA , lysates were treated with 15 U/ml Micrococcal nuclease ( MN Boehringer ) and 0 . 75 mM CaCl2 and incubated at 25°C for 7 minutes . EGTA was added to terminate the reaction . For the translation assay , 6 μl of cell extract were mixed to 1 . 2 μl of Master Mix ( 125 mM HEPES , 10 mM ATP , 2 mM GTP , 200 mM creatine phosphate , 0 . 2 mM aminoacid mix without methionine ( Promega ) , 0 . 25 mM spermidine , 20 mM of L-methionine , 50 mM potassium acetate , 2 . 5 mM magnesium acetate , 20 U of RNAsin ( from Promega ) and 0 . 5 μg of purified reporter-encoding mRNA ) . The mRNA was obtained by using the Megascript T7 kit ( Ambion ) to perform the in vitro transcription reaction supplemented with 2 mM cap analog [M7G ( 5' ) PPP ( 5' ) G] ( Ambion ) . The mix for the in vitro translation reaction was then incubated for 90 minutes at 30°C . Dual-Glo Luciferase Assay kit ( Promega ) was used to read Firefly and Renilla luciferase output with GloMax Luminometer ( Promega ) . In vitro translation experiments were performed at least three times , in triplicate . For protein synthesis measurements , we adapted SUnSET protocol [60] . 70% confluent cells were treated with 5μg/ml puromycin for 10 minutes , then trypsinized and centrifugated 5 minutes at 500 g . Then , cell were fixed and permeabilized using reagents from the Foxp3/Transcription Factor Staining Buffer Set ( Affymetrix , 5523–00 ) , incubated with anti-puromycin 12D10 ( 1:2000 , Millipore , MABE343 ) and finally with secondary antibody Alexa Fluor 488 Goat anti-Mouse ( Invitrogen ) and DAPI , according to manufacturer protocol . Cells were then analyzed with FACSCanto II ( BD Bioscience ) and analyzed with FlowJo8 . 8 . 7 software . SUnSET experiments were performed at least three times , in triplicate . 1280 approved drugs were analyzed for their impact on SbdsR126T/R126T cells growth . Cells were seeded using a Multidrop Combi ( Thermo Scientific ) in 384 well plates ( Perkin Elmer Cell Carrier , collagen coated ) at a density of 4000 cells/well . After 24 hours the drugs were supplemented using a Hamilton Starlet liquid handler at a final concentration of 10 μM and cells were treated for 30 hours . The reference compound for toxicity was 5 μM Trichostatin A . Cytotoxicity was than evaluated by measuring in vivo nuclear shrinkage and loss of cells using Hoechst33342 , and cell membrane disruption using the cell-impermeant nuclear dye BOBO-3 . Briefly , following incubation with the drugs diluted in 50 μl growth medium , 25 μl of a dye cocktail containing 3 μM Hoechst33342 and 2 . 25 μM BOBO-3 were added and incubation was continued for further 45 min at 37°C , 5% CO2 . Images were then immediately acquired and analyzed using the Operetta microscope ( Perkin Elmer ) . For the validation of hits , 6-point dose-responses for each compound in duplicate were used ( ranging from 30uM to 0 . 12uM ) . The number of dead cells/total number of cells was the readout of the assay , where dead cells correspond to nuclei stained with BOBO-3 and the total number of cells correspond to the nuclei stained with Hoechst33342 . All analysis were performed using the FACSCanto II Flow cytometer ( BD ) and analyzed with FlowJo software ( BD ) . All experiments were performed when cells reached the confluence of 70% . For the apoptosis analysis , cells were UV irradiated with a cross-linker ( 9999 μJ/cm2 , three times ) and cell death was detected by using the 7AAD-Annexin V kit ( 640926 , BioLegend ) . For ROS measurement , cells were incubated with 50 μM H2DCF-DA ( 2’ , 7’ , -dichlorodihydrofluorescein diacetate , AbCam Ab113851 ) for 30 min at 37°C and 5% CO2 and immediately analyzed by flow citometry . All experiments were performed at least three times , in triplicate . iRIA assay was performed as described in [40] . Briefly , 96-well plates were coated with a cellular extract diluted in 50 μL of PBS , 0 , 01% Tween-20 , O/N at 4°C in humid chamber . Coating solution was removed and aspecific sites were blocked with 10% BSA , dissolved in PBS , 0 , 01% Tween-20 for 30 minutes at 37°C . Plates were washed with 100 μl /well with PBS-Tween . 0 , 5 μg of recombinant biotynilated eIF6 was resuspended in a reaction mix: 2 , 5 ;mM MgCl2 , 2% DMSO and PBS-0 . 01% Tween , to reach 50 ;μl of final volume/well , added to the well and incubated with coated ribosomes for 1 hour at room temperature . To remove unbound proteins , each well was washed 3 times with PBS , 0 , 01% Tween-20 . HRP-conjugated streptavidine was diluted 1:7000 in PBS , 0 , 01% Tween-20 and incubated in the well , 30 minutes at room temperature , in a final volume of 50 μl . Excess of streptavidine was removed through three washes with PBS-Tween . OPD ( o-phenylenediamine dihydrochloride ) was used according to the manufacturer’s protocol ( Sigma-Aldrich ) as a soluble substrate for the detection of streptavidine peroxidase activity . The signal was detected after the incubation , plates were read at 450 nm on a multiwell plate reader ( Microplate Bio-Rad model 680 ) . This experiment was performed at least three times , in triplicate . Cells were seeded the day before the assay . The next day , cells were incubated with Lysotracker DeepRed ( Thermo Fisher Scientific L12492 ) according to the manufacturer protocol . The cells were examined with Nikon Ti-Eclipse microscope and analyzed with Volocity 6 . 3 software ( Perkin Elmer ) . For subsequent analysis , cells were then fixed with paraformaldehyde 3% for 10 minutes at room temperature and nuclei were stained with DAPI ( Molecular Probes NucBlue Live ReadyProbes Reagent R37605 ) as manufacturer protocol , then washed and mounted on slides with Mowiol 20% . The cells were examined by confocal microscopy ( Leica SP5 ) and analyzed with Volocity 6 . 3 software ( Perkin Elmer ) . Lysotracker experiments were performed at least three times , in triplicate . The RNA seq data are available at www . ebi . ac . uk/arrayexpress with accession number ID: E-MTAB-5089 . | Shwachman Diamond syndrome ( SDS ) is an inherited disease . SDS presents , as hallmarks , exocrine pancreatic insufficiency , increased rate of infections , and higher incidence of leukemia . Most cases are due to mutations in the SBDS gene . SBDS encodes for a ribosome maturation factor . In this study , we immortalized mouse fibroblasts carrying one of the most common mutation of SDS patients and performed a thorough analysis of their properties . We show that the loss of SBDS activity causes a rewiring of gene expression and cellular metabolism . Overall we find a reduction of protein synthesis capability , a lower energy status , and increased lysosomal capability . SBDS mutant cells have an increased susceptibility to various forms of stress , but are strikingly resistant to oncogene-induced transformation . We propose a model that explains the complex phenotype of SDS patients and suggests roads for a rationale treatment . | [
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] | 2017 | SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention |
Hosts repeatedly bitten by sand flies develop antibodies against sand fly saliva and screening of these immunoglobulins can be employed to estimate the risk of Leishmania transmission , to indicate the feeding preferences of sand flies , or to evaluate the effectiveness of vector control campaigns . Previously , antibodies to sand fly saliva were detected using whole salivary gland homogenate ( SGH ) or recombinant proteins , both of which also have their disadvantages . This is the first study on sand flies where short peptides designed based on salivary antigens were successfully utilized for antibody screening . Specific IgG was studied in hosts naturally exposed to Phlebotomus orientalis , the main vector of Leishmania donovani in East Africa . Four peptides were designed by the commercial program EpiQuest-B , based on the sequences of the two most promising salivary antigens , yellow-related protein and ParSP25-like protein . Short amino acid peptides were synthesised and modified for ELISA experiments . Specific anti-P . orientalis IgG was detected in sera of dogs , goats , and sheep from Ethiopia . The peptide OR24 P2 was shown to be suitable for antibody screening; it correlated positively with SGH and its specificity and sensitivity were comparable or even better than that of previously published recombinant proteins . OR24 P2 , the peptide based on salivary antigen of P . orientalis , was shown to be a valuable tool for antibody screening of domestic animals naturally exposed to P . orientalis . We suggest the application of this promising methodology using species-specific short peptides to other sand fly-host combinations .
The specific IgG antibody response against salivary proteins is induced in repeatedly exposed hosts after being bitten by the female sand fly ( reviewed by Ribeiro and Francischetti [1] and Lestinova et al . [2] ) . In sand flies this antibody response is species-specific [3 , 4] and correlates with the biting intensity [5–8] . IgG values decrease after the hosts are protected against sand flies [9] , therefore the detection of antibodies can be used for testing the efficacy of vector control campaigns [10 , 11] . Antibody detection with the whole salivary gland homogenate ( SGH ) as antigen is impractical in large epidemiological studies due to the possibility of crossreactivity with other insects [9] , variability of saliva composition during sand fly aging [12 , 13] , and the workload required to obtain sufficient quantity of the antigen . In the past decade , sand fly SGH was replaced by several antigenic recombinant proteins , expressed in bacterial or mammalian cells , and with various degrees of success ( reviewed by Lestinova et al . [2] ) . In humans , successful detection of anti-sand fly IgG with recombinant proteins was described by Teixeira et al . [14] and by Souza et al . [15] for Lutzomyia longipalpis and by Marzouki et al . [16 , 17] and Mondragon-Shem et al . [18] for Phlebotomus papatasi . In domestic animals , using recombinant antigens , antibodies against sand fly saliva were detected in sera of dogs bitten by L . longipalpis or P . perniciosus [14 , 19 , 20] and in sera of dogs , sheep , and goats exposed to P . orientalis [21] . In wild animals these studies were performed with rabbits and hares bitten by P . perniciosus [22] and with foxes exposed to L . longipalpis [14] . However , production of recombinant proteins requires cell expression and a complicated purification procedure . Therefore , we focused on linear B-cell epitopes ( synthetic peptides , representing short amino acid sections of the antigenic proteins ) , which can be produced in large amounts with high purity . This approach was previously applied to mosquitoes as well as to tsetse flies . In Anopheles gambiae the peptide designed based on the salivary protein gSG6 was validated in many field studies [23–26] and promising results were also achieved with peptide based on the salivary protein of Aedes aegypti and human serum samples [27] . In tsetse flies , peptides originating from saliva of Glossina palpalis gambiensis and G . morsitans specifically bound anti-tsetse fly antibodies in human and cattle sera , respectively [28–30] . In this study we applied , for the first time , this novel approach to sand flies and used short peptides to detect specific IgG response in domestic animals ( dogs , goats , and sheep ) naturally exposed to P . orientalis . Our main aim was to compare the peptides with previously described recombinant proteins [21] and to assess whether this methodology is also applicable to large scale surveillance .
BALB/c mice were maintained and handled in the animal facility of Charles University in accordance with institutional guidelines and the Czech legislation ( Act No . 246/ 1992 coll . on Protection of Animals against Cruelty in present statutes at large ) , which complies with all relevant European Union and international guidelines for experimental animals . The experiments were approved by the Committee on the Ethics of Animal Experiments of the Charles University ( Permit Number: MSMT-10270/2015-6 ) and were performed under the Certificate of Competency ( Registration Number: CZ 02457 ) in accordance with the Examination Order approved by Central Commission for Animal Welfare of the Czech Republic . Sera of domestic animals were collected within the study by Rohousova et al . [31] . Their collection was approved by the Ethiopian National Research Ethics Review Committee ( NRERC ) under approval no . 3 . 10/3398/04 . Sera of domestic animals naturally exposed to P . orientalis in Ethiopia were obtained during the previous study by Rohousova et al . [31] and include 40 sheep , 94 goats , and 30 dogs . Sera from 10 sheep , 15 goats , and 10 dogs from non-exposed animals originating from the Czech Republic served as negative controls . More details of all the samples are provided by Rohousova et al . [31] . Twenty laboratory Balb/c mice were divided into four groups of five animals . Three groups were exposed at least ten-times to about 150 insectary-bred sand fly females ( at two-week intervals ) of either P . orientalis , P . papatasi , or Sergentomyia schwetzi; the fourth group was used as the non-exposed control . The Phlebotomus orientalis colony originating from Ethiopia ( for more details see Seblova et al . [32] ) was reared under standard conditions as described by Volf and Volfova [33] . Salivary glands were dissected from 4–6 day old female sand flies in 20mM Tris buffer with 150mM NaCl and stored at -20°C . Before use , salivary glands were disrupted by freeze-thawing three times in liquid nitrogen . Peptides were designed from amino acid sequences based on the two most suitable recombinant proteins of P . orientalis ( rPorSP24 and rPorSP65 ) as previously described [21] . Two peptides from each protein were selected in the software EpiQuest-B ( Aptum Biologics Ltd . , www . epiquest . co . uk ) . In EpiQuest-B , immunodominant parts of protein sequences were distinguished and their antigenicity indices were calculated based on three algorithms–the peptide immunogenicity , the probability of antibody-accessibility ( exposure on the protein surface ) , and the uniqueness of protein sequence . The probability of peptide-antibody binding increases with the antigenicity index . These four generated sequences ( Table 1 ) were sent to a commercial laboratory ( Genosphere Biotechnologies , France ) , where they were synthesised and conjugated with two molecules of polyethylene glycol , which acts as a spacer on ELISA plates and facilitates improved accessibility of antibodies . After the spacer , one molecule of biotin was added , which enabled avidin-biotin peptide binding to ELISA plates coated with diluted avidin . Peptides were diluted in sterile PBS at a concentration 1 mg/ml and stored in -80°C . ELISA Clear Flat-Bottom Plates ( 3855: Thermo Fisher Scientific , USA ) were coated with avidin ( A9275: Sigma-Aldrich , UK ) at a concentration 5 0μg/well , diluted in 20mM carbonate-bicarbonate buffer ( pH 9 . 5 ) and incubated overnight at 4°C . Plates were washed three times with PBS-Tw ( 0 . 05% Tween 20 ) , blocked with 6% blocking medium diluted in PBS ( see S1 Table ) for 2 hours at 37°C and then washed twice . Peptides diluted in 2% blocking medium in PBS-Tw were added to the wells at a concentration 5 μg/well and the plates incubated for one hour at 37°C . After washing three times , sera diluted in 2% blocking medium in PBS-Tw were incubated on the plates for one hour at 37°C . Plates were washed five times and secondary antibodies diluted in PBS-Tw were added and incubated for one hour at 37°C . Finally , plates were washed six times , the reaction developed with phosphate-citrate buffer ( pH 5 . 5 ) in the dark for six minutes at room temperature and stopped with 10% sulfuric acid . The optical density was measured at 492 nm using the Infinite M200 microplate reader ( Tecan , Switzerland ) . At each step , 100 μl of each solution per well was used and all serum samples were tested in duplicate . When the salivary gland homogenate ( SGH ) was used as antigen , ELISA plates were coated with 0 . 2 gland/well [21] . The step with peptide incubation was replaced by incubation with 2% blocking medium in PBS-Tw and the rest of the protocol remained the same . Blocking media , sera and conjugate dilutions for individual host species are indicated in S1 Table . The non-parametric Spearman test was used to assess correlations between total anti-SGH and anti-peptide IgG levels using GraphPad Prism version 6 ( GraphPad Software , Inc . , San Diego , CA , USA ) . For evaluation of the possible crossreactivity with other sand fly species the non- parametric Wilcoxon Rank-Sum test in GraphPad Prism version 6 was used . Statistical significance was considered when the p-value was < 0 . 05 . Cut-off values were calculated from the mean optical density of control sera plus 3 standard deviations . The optical density values of anti-SGH antibodies were used as the gold standard to validate peptides in ELISA tests using positive ( PPV ) and negative predictive values ( NPV ) , sensitivity , and specificity .
For designing the peptide sequences , two of the most antigenic proteins previously tested in recombinant form were used: rPorSP24 ( yellow-related protein ) and rPorSP65 ( ParSP25-like protein ) . The antigenicity was calculated for both protein sequences in EpiQuest-B and two peptides with the higher antigenicity indices ( Fig 1 ) were chosen from each protein: OR24 P1 , OR24 P2 , OR65 P1 and OR65 P2 . First , the synthetic peptides were tested by ELISA for possible crossreactivity with antibodies against salivary antigens of sympatric sand fly species ( P . papatasi and Sergentomyia schwetzi ) using sera of experimentally bitten Balb/c mice . Five mice were exposed to single sand fly species–either P . orientalis , P . papatasi , or S . schwetzi , and five mice served as non-exposed controls . Significant differences in OD values were detected with sera of mice bitten by P . orientalis compared to all the other three groups , as shown in Fig 2 . No differences were observed in non-exposed controls and mice exposed to P . papatasi or S . schwetzi ( Fig 2 ) . The four aforementioned peptides were used as antigens in ELISA experiments to detect the specific anti-P . orientalis SGH antibodies from three animal species–dogs , goats , and sheep . Their antigenicities were compared with the whole SGH , and the statistical values calculated were cut-off , positivity , correlation coefficient , PPV , NPV , specificity , and sensitivity ( Table 2 ) . OR24 P1 showed the closest cut-off value to SGH with canine sera , as well as high correlation coefficient ( > 0 . 75 ) , PPV , specificity ( > 0 . 65 ) and very high NPV and sensitivity ( > 0 . 9 ) . With the goat sera , the correlation ( 0 . 6 ) was lower as were other statistical values ( all between 0 . 6–0 . 7 ) . With sheep sera , all statistical values were very high ( > 0 . 9 ) except for the low sensitivity ( 0 . 25 ) . Peptide OR24 P2 had a high correlation coefficient with the SGH for dog and goat sera . It reached comparable PPV , NPV , specificity , and sensitivity as OR24 P1 for dogs and the highest PPV , NPV , specificity and sensitivity for goats ( all above 0 . 75 ) . Correlation and PPV ( < 0 . 45 ) were low for sheep sera . OR65 P1 showed similar statistical values as the previous two peptides for dogs but the specificity was slightly lower ( 0 . 5 ) . With goat sera , there was a very high cut-off value and a low correlation coefficient ( 0 . 55 ) . The second highest correlation ( 0 . 7 ) was achieved with this peptide and with sheep sera but the PPV and sensitivity ( < 0 . 35 ) were low . Despite high statistical values for OR65 P2 and canine sera ( 0 . 8 ) , high cut-off and the lowest correlation coefficient ( 0 . 65 ) were observed with this host species . In contrast , OR65 P2 was the second best antigen for goats , with the lowest cut-off value , high correlation , PPV , NPV , specificity , and sensitivity ( all above 0 . 65 ) . Although high PPV , NPV , and specificity ( > 0 . 9 ) were detected with sheep sera , this peptide achieved low correlation and NPV ( < 0 . 6 ) . The correlation analysis for the most promising peptide ( OR24 P2 ) with dogs and goats is shown in Fig 3 .
Previously , anti-sand fly IgG was detected by using SGH or recombinant proteins prepared based on the most antigenic proteins ( reviewed by Lestinova et al . [2] ) . In this study , we focused on the novel approach of detecting IgG with short amino acid chain synthetic peptides . These peptides can be synthesised in large amounts with very high purity and without the need for cell expression of recombinants . We designed four peptides , two from each of the most promising P . orientalis salivary proteins: yellow-related protein ( PorMSP24 , ACCN: AGT96461 ) and ParSP25-like protein ( PorMSP65 , ACCN: AGT96466 ) , and tested them with sera of domestic animals naturally exposed to Phlebotomus orientalis . Phlebotomus orientalis is the main East African vector of Leishmania donovani–the causative agent of visceral leishmaniasis [35] . Previous studies revealed that salivary antigens of P . orientalis belong to several protein families , specifically yellow-related proteins , odorant-binding proteins , apyrases , antigen 5-related proteins and ParSP25-like proteins [34] . Recombinant yellow-related proteins and ParSP25-like proteins were used to replace P . orientalis SGH for antibody screening of domestic animals in Ethiopia [21] . Application of recombinant yellow-related proteins of Lutzomyia longipalpis was also described for canine , fox , and human sera [14 , 15] , and of P . perniciosus for hare , rabbit , and canine sera [19 , 20 , 22] . However , so far , the recombinant ParSP25-like protein has not been used for other sand fly species except P . orientalis [21] . Peptides based on salivary antigens have not previously been used for studies on sand flies but they have been applied to detection of specific antibodies in hosts bitten by mosquitoes or tsetse flies . For mosquitoes , the peptides were first used for Anopheles gambiae , to study specific IgG responses among humans living in different foci of Plasmodium falciparum transmission [23 , 25 , 36] , to correlate IgG levels with the risk of malaria transmission [26] , and to monitor the effect of vector control campaigns [24] . Ndille et al . [27] used salivary peptide of Aedes aegypti to describe a positive correlation between specific IgG responses in humans with rainfall and mosquito seasonality . In tsetse flies , differences in anti-salivary peptide IgG titers were observed between two human populations with diverse abundance of Glossina palpalis gambiensis [28] , and before and after vector control [29] . Somda et al . [30] suggested that the peptide based on salivary protein of G . morsitans was not suitable for IgG screening of domestic animals in areas with high tsetse fly abundance , because it was only recognized by sera of cattle with low exposure . Our study is the first , for sand flies , in which antigenicity and IgG detection are compared for peptides , whole SGH and recombinant proteins . The peptides designed for P . orientalis are species-specific; no crossreactivity was observed with sera of mice exposed to the sympatric sand fly species P . papatasi and Sergentomyia schwetzi or with sera of non-exposed controls; there was similar high species specificity for the SGH and recombinant proteins of P . orientalis [21] . Previous work on dogs with recombinant proteins showed that the best protein-SGH performing recombinant ( ParSP25-like protein ) had very low specificity . This implied high probability of false positivity among non-exposed animals . Higher specificity was achieved with recombinant yellow-related protein [21] . Comparable correlation with recombinant yellow-related protein was detected with three peptides–OR24 P1 , OR24 P2 , and OR65 P1 . The first two of these peptides also showed much higher specificity ( 0 . 7 ) than both of the recombinants . With goats , low correlation was observed with both recombinant proteins [21] . In contrast , peptide OR24 P2 based on the sequence of yellow-related protein reached high correlation with SGH ( 0 . 8 ) as well as values > 0 . 75 for PPV , NPV , specificity , and sensitivity . Results with sheep sera were difficult to interpret due to the very low positivity with SGH ( 10% ) . Similar positivity was found with all four peptides: even a relatively small change in the number of false positives or false negatives would significantly change calculation of PPV , NPV , specificity , or sensitivity . Although the correlation with two peptides ( OR24 P1 and OR65 P1 ) was above 0 . 7 , we therefore do not recommend these peptides for screening sheep sera . However , promising results for sheep sera have previously been achieved with both recombinant proteins [21] . In summary , we tested four short amino acid sequence peptides , designed based on two most antigenic P . orientalis salivary proteins , for detection of antibodies to sand fly saliva , in three species of domestic animals from Ethiopia . One of the peptides , OR24 P2 , showed promising results with sera of dogs and goats . We therefore suggest that this peptide may replace SGH or recombinant proteins in surveillance for anti-P . orientalis IgG . As it was shown , synthetic peptides might work only for some host species . For future detection of human antibodies to sand fly saliva , we recommend comparison of the efficacy of recombinant proteins and synthetic peptides . | Previously , two types of antigens were used for detection of antibodies to sand flies: 1 ) salivary gland homogenate ( SGH ) , which requires maintaining a sand fly colony and laborious work to obtain a sufficient amount of antigen or 2 ) recombinant proteins with the need to use cell expression and a complicated purification procedure . In contrast , synthetic peptides have never been studied for sand flies despite it being easier to produce them in sufficient quantities and purity . In this study , we screened specific antibodies to sand flies in domestic animals using synthetic peptides based on the two most antigenic salivary proteins of Phlebotomus orientalis . This sand fly is the main East African vector of Leishmania donovani , causative agent of visceral leishmaniasis , and we detected specific anti-P . orientalis IgG in naturally exposed dogs , goats , and sheep from Ethiopia . We showed that , in dogs and goats , the peptide named OR24 P2 is more suitable for antibody detection then the recombinant proteins . Therefore , we recommend this peptide to replace SGH in larger epidemiological studies for evaluation of the effectiveness of vector control programmes or to estimate the risk of Leishmania transmission . | [
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] | 2019 | Synthetic peptides as a novel approach for detecting antibodies against sand fly saliva |
DNA damage observed during plant immune responses is reported to be an intrinsic component of plant immunity . However , other immune responses may suppress DNA damage to maintain host genome integrity . Here , we show that immunity-related DNA damage can be abrogated by preventing cell death triggered by Nucleotide-binding , Leucine-rich-repeat immune Receptors ( NLRs ) . SNI1 ( suppressor of npr1-1 , inducible 1 ) , a subunit of the structural maintenance of chromosome ( SMC ) 5/6 complex , was reported to be a negative regulator of systemic acquired resistance ( SAR ) and to be necessary for controlling DNA damage . We find that cell death and DNA damage in sni1 loss-of-function mutants are prevented by mutations in the NLR signaling component EDS1 . Similar to sni1 , elevated DNA damage is seen in other autoimmune mutants with cell death lesions , including camta3 , pub13 and vad1 , but not in dnd1 , an autoimmune mutant with no visible cell death . We find that as in sni1 , DNA damage in camta3 is EDS1-dependent , but that it is also NLR-dependent . Using the NLR RPM1 as a model , we also show that extensive DNA damage is observed when an NLR is directly triggered by effectors . We also find that the expression of DNA damage repair ( DDR ) genes in mutants with cell death lesions is down regulated , suggesting that degraded DNA that accumulates during cell death is a result of cellular dismantling and is not sensed as damaged DNA that calls for repair . Our observations also indicate that SNI1 is not directly involved in SAR or DNA damage accumulation .
Plants rely on a dual layered innate immune system to defend against pathogens . In a first layer , pattern recognition receptors ( PRRs ) detect pathogen associated molecular patterns ( PAMPs ) , leading to PAMP triggered immunity ( PTI ) [1] . To avoid PTI and establish infection , pathogens deliver effectors to modify host proteins . In a second immune layer , cytoplasmic Nucleotide-binding , Leucine-rich-repeat Receptors ( NLRs ) may detect these modifications and activate immunity . Some plant NLRs thus ‘guard’ host ‘guardees’ and activate effector triggered immunity ( ETI ) [2 , 3] . NLR activation and ETI are dependent on signaling components including ENHANCED DISEASE SUSCEPTIBILITY 1 ( EDS1 ) and NON RACE-SPECIFIC DISEASE RESISTANCE ( NDR1 ) [4] . ETI can be accompanied by a type of programmed cell death at infection sites called the hypersensitive response ( HR ) [5] . HR cell death shares features with apoptosis , including changes in membrane integrity , cysteine protease mediated cleavage of key proteins , and DNA degradation [6–8] . Following local cell death , systemic acquired resistance ( SAR ) can be induced to boost immunity in uninfected parts of the plant . HR and SAR are regulated by the phytohormone salicylic acid ( SA ) , whose accumulation promotes defense responses . Consequently , interference with SA accumulation impairs defense responses [9] . Numerous Arabidopsis autoimmune mutants exhibit accumulation of SA and inappropriate activation of immunity in the absence of infection [2 , 10] . These mutants can be broadly divided into those with de-repressed , SA-dependent defense responses and those that also exhibit accelerated cell death [2] . Importantly , autoimmunity in many such mutants can be suppressed by mutations in EDS1 , NDR1 , or specific NLRs [11–13] . In line with this , Lolle et al . [14] recently reported a screen to systematically abrogate autoimmunity in selected mutants by disruption of NLR function [14] . DNA damage repair ( DDR ) is essential to maintain genome stability , and compelling evidence links DNA damage responses with innate immune programs in mammals [15] and plants [16] . Foreign and damaged host DNA , including DNA breaks generated by viral integration , can trigger innate immune responses [16–18] . Thus , both alien and damaged host DNA function as danger signals that can alert the immune system . Interestingly , DNA damage accompanying infection was reported to be an intrinsic component of plant immunity . For example , SA pretreatment that reportedly caused DNA damage was concluded to boost immune responses [19] . This conclusion was supported by the finding that loss-of-function mutants of SNI1 , a subunit of the DDR complex SMC5/6 earlier indicated as negative regulator of SAR [20] , accumulated significant levels of DNA damage under normal growth conditions [19] . This increased DNA damage was proposed to be caused by decreased DDR activity in the absence of SNI1 . In contrast , Song and Bent [21] demonstrated that DNA damage was induced by pathogen infection and that the plant immune system tried to diminish this damage to preserve genome integrity . While these and other studies indicate that DNA damage may trigger immune responses , it seems unclear whether DNA damage is actively induced in infected host cells or is a consequence of infection . Here we report that activation of NLR signalling and ETI are sufficient to trigger DNA damage accumulation observed during plant immune responses . We demonstrate this using autoimmune mutants that display accumulation of DNA damage in the absence of pathogen infection . We show that such DNA damage is abrogated by shutting down NLR mediated signaling , and thus immunity . We also provide evidence that DNA damage accumulation observed in sni1 mutants is not due to faulty DDR but is dependent on NLR signaling components .
To investigate if accumulation of DNA damage is a common feature among mutants with constitutive immune activation , we performed the alkaline version of the single cell gel electrophoresis ( Comet assay ) to estimate the amount of DNA damage in autoimmune mutants including pub13 , vad1 and dnd1 . The alkaline version of the comet assay is capable of detecting DNA double-strand breaks , single-strand breaks , alkali-labile sites , DNA-DNA/DNA-protein cross-linking , and incomplete excision repair sites [22] . PUB13 ( Plant U-Box 13 ) encodes an E3 ubiquitin ligase implicated in ubiquitination and degradation of the PRR FLS2 [23] , VAD1 ( Vascular Associated Death 1 ) encodes a membrane-bound protein [24] , and DND1 ( Defense No Death 1 ) encodes a cyclic nucleotide gated channel [25] While pub13 , vad1 and dnd1 all over accumulate SA , only pub13 and vad1 also exhibit accelerated cell death . We found that vad1 and pub13 had more DNA damage ( P<0 . 05 ) than wild type ( Fig 1A and 1B ) . Interestingly , the level of DNA damage observed in dnd1 was not significantly different from the level in wild type ( Fig 1B ) . However , it should be mentioned that dnd1 was reported to display macroscopic cell death when grown under certain conditions , and it is thus possible that in other conditions it would also display elevated DNA damage . We also performed an immunoblot against the phosphorylated version of Histone 2AX ( γ-H2AX ) , a common marker for DNA double strand breaks , which corroborated our comet assay data , i . e . while vad1 strongly accumulated γ-H2AX , this was not detected in Col-0 or dnd1 ( Fig 1C and 1D ) . These results point to a connection between macroscopic cell death and DNA damage , and provide indirect evidence that increased SA levels may not be the major reason for DNA damage accumulation in autoimmune mutants . Many autoimmune mutant phenotypes can be partly or fully rescued by loss-of-function of key immune signaling proteins such as EDS1 or NDR1 [2] . We speculated that DNA damage accumulation in autoimmune mutants might also be dependent on such signaling components . To address this , we compared the levels of DNA damage in another autoimmune mutant , camta3 , caused by loss-of-function of the CAMTA3 calmodulin-binding transcription factor [26] to camta3 eds1-2 double mutants . This showed that introducing eds1-2 into the camta3-1 background completely rescues the DNA damage accumulation observed in the camta3-1 single mutant ( Fig 2A and 2B ) . We recently reported that transgenic expression of dominant negative ( DN ) forms of Arabidopsis NLRs specifically disrupt the function of the corresponding wild type alleles [14] . That study showed that a DN mutant of an NLR named Dominant suppressor of camta3 2 ( DSC2S ) fully suppressed autoimmunity in camta3 [14] . Consequently , we also did the comet assay with camta3-1 expressing DN-DSC2 and observed that DNA damage accumulation was reduced to control levels ( Fig 2A and 2B ) . Immunoblotting of γ-H2AX showed that camta 3 accumulation of this DSB marker is mediated by the NLR DSC2 ( Fig 2C and 2D ) . These results indicate that DNA damage accumulation in camta3 is dependent on an intact NLR signaling pathway and the induction of immunity triggered by DSC2 . DNA damage accumulation thus seems to be a common feature of autoimmune mutants with accelerated cell death including pub13 , vad1 and camta3 . Our data also suggest that constitutive accumulation of SA is insufficient to cause DNA damage since dnd1 mutants have no signs of increased DNA damage . This conclusion is based on the observation that all the mutants tested accumulate SA but only camta3 , vad1 and pub13 have macroscopic cell death lesions [24–28] and DNA damage . In contrast to a previous report [17] , Song and Bent [21] , could not detect significantly increased DNA damage in WT plants treated with SA , and we verified this with SA and its analogs BTH and INA ( Fig 3A–3D ) . NLRs are thought to guard host proteins against tampering by microbial effectors , and many NLRs require EDS1 for signaling . Because the camta3-1 phenotype is dependent on EDS1 and DSC2 , we tested if detection of a single effector would be sufficient to induce accumulation of DNA damage . Song and Bent [21] showed that P . fluorescens , a bacterium known to induce systemic resistance in plants , does not cause DNA damage accumulation when infiltrated into Arabidopsis . We therefore infected rpm1-3 , a loss-of-function mutant of the RPM1 NLR which detects AvrRPM1 , and wild type Col-0 with P . fluorescens expressing the effector AvrRPM1 . As expected , while Col-0 triggers ETI and accumulates DNA damage upon recognition of avrRPM1 , the rpm1-3 mutant does not ( Fig 4A and 4B ) . We corroborated this by estimating DNA damage in plants expressing AvrRPM1 under the control of a DEX inducible promoter . While DEX treatment did not induce DNA damage accumulation in wild type Col-0 , plants expressing DEX-induced AvrRPM1 had higher levels of DNA damage compared to their untreated counterparts ( Fig 4C and 4D ) . This experiment demonstrates that DNA damage can be induced by triggering an NLR pathway based on the recognition of a single effector . Thus , in this case , DNA damage is first found after the induction of immunity . We then wanted to determine if DNA damage observed was part of an early response to effector recognition . To this end we performed a time course in DEX-induced AvrRPM1 expressing plants and verified that γ-H2AX accumulated upon DEX induction and was more than doubled after 8h ( Fig 4E and 4F ) . Since sni1 is an autoimmune mutant that exhibits accelerated cell death [19 , 20] , we tested if sni1 could be rescued by shutting down immunity . To this end , we crossed sni1 to eds1-2 and verified that the doubly homozygous plants had their growth partially restored when compared to sni1 ( Fig 5A ) . Furthermore , cell death ( by trypan blue staining ) and PR1 transcript accumulation of transcripts of marker PATHOGENESIS-RELATED 1 ( PR1 ) were completely abrogated in sni1 eds1-2 plants ( Fig 5B and 5C ) . These results , together with comet assay data from sni1 and sni1 eds1-2 ( Fig 6A and 6B ) , confirmed that DNA damage accumulation in sni1 is due to autoimmunity and not to defective DNA damage repair [19] . SNI1 was proposed to be a negative regulator of RAD51 , a key DDR gene involved in double strand break repair , because RAD51 accumulates in sni1 mutants [29] . Since sni1 phenotypes are suppressed by mutation of EDS1 , we also tested if the involvement of SNI in RAD51 regulation could be linked to sni1 autoimmunity . Using the same antibody as Wang et al . [29] , we observed that accumulation of RAD51 in sni1 mutants was diminished in the sni1 eds1 double mutant ( Fig 7A and 7B ) . This result again points to an immunity related origin for sni1 phenotypes . In mammals , activation of apoptosis leads to Caspase 3 mediated cleavage of RAD51 to inactivate the DNA damage repair machinery [30 , 31] . We therefore tested if AtRAD51 was cleaved during effector triggered immunity , and if such cleavage could be affected by Caspase 3 inhibitors . To this end , we infiltrated Col-0 plants with P . syringae AvrRPM1 in the presence or absence of the Caspase 3 inhibitor Z-DEVD-FMK , which was recently shown to inhibit protease activity in Arabidopsis [7] . Infection with P . syringae led to rapid accumulation of RAD51 ( Fig 7C and 7D ) 2 hours post infection ( hpi ) for all conditions tested . With the establishment of ETI ( 4 hpi ) only co-infiltration with Z-DEVD-FMK stabilized RAD51 . This observation that RAD51 is degraded upon induction of ETI is in keeping with the shutdown of DDR responses during apoptosis [30 , 31] and the accumulation of γ-H2AX seen in Fig 4E . Since it is reasonable to assume that cells shut down DDR when undergoing programmed cell death such as that during the HR in plants , we also analyzed the relative transcript accumulation of a subset of DDR genes in sni1 and other autoimmune cell death mutants . While DDR genes were previously shown to be upregulated in sni1 [19] , we found that several DDR genes were downregulated in sni1 ( Fig 7E ) . Such genes were also downregulated in other autoimmune mutants with accelerated cell death ( Fig 7E and 7F ) , but not in dnd1 which does not exhibit cell death ( Fig 7F ) . In addition , the apparent reduction in the levels of DDR gene transcripts in sni1 and camta3 were dependent on EDS1 ( Fig 7E ) . These results again indicate that the suppression of DDR in sni1 is caused by NLR signaling .
A model has been proposed in which pathogen infection induces SA accumulation which leads to increased DNA damage that acts as an intrinsic component of plant immune responses [19] . This model is based on observations that SA treatment induced DNA damage , and that DNA damage accumulated in uninfected loss-of-function mutants of SNI1 encoding a subunit of the SMC5/6 complex required for controlling DNA damage . In contrast , we ( Fig 3 ) find that SA or its analogues BTH and INA do not cause an increase in DNA damage . Similarly , Song and Bent [21] found that SA treatment prior to pathogen infection reduced the accumulation of damaged DNA . We note that application of 1mM SA can be phytotoxic [32] and could consequentially cause DNA damage accumulation under certain growth conditions . We also demonstrate here that other immune-related cell death mutants accumulate DNA damage . Such damage is therefore not an exclusive feature of the sni1 mutant . Notably , the dnd1 mutant , which over-accumulates SA but exhibits ‘Defense No Death’ , does not accumulate damaged DNA . This indicates that processes involved in immune-related cell death , rather than constitutive defense responses , cause DNA damage . Immune-related cell death encompasses DNase mediated oligonucleosomal DNA fragmentation which is normally seen as DNA laddering [33] . The comet assay , which is able to detect DNA strand breaks , would thus also ‘score’ oligonucleosomal DNA fragmentation as damaged DNA . This may explain the accumulation of putatively damaged DNA in autoimmune mutants . Our analyses of infections with P . syringae avrRPM1 , and of plants expressing DEX inducible avrRPM1 , further confirm that NLR triggered cell death is sufficient to induce DNA damage accumulation , even in the absence of a pathogen ( Fig 3A and 3B ) . Since avrRPM1 is not recognized in the rpm1-3 mutant , rpm1-3 fails to trigger ETI and consequently does not accumulate significant amounts of damaged DNA as measured by the Comet assay . Thus , it is the host immune system that in this case causes DNA damage . We note that we do not rule out the possibility that pathogens , or their activities , may cause DNA damage , as it is well described in other systems that diverse pathogens affect host genome integrity [34] . Importantly , mutations in the NLR signaling component EDS1 completely suppress DNA damage accumulation , as measured by the comet assay , in both the sni1 ( Fig 6 ) and camta3 ( Fig 2 ) autoimmune mutants . Likewise , expression of a dominant negative mutant form of the NLR DSC2 is sufficient to prevent DNA damage accumulation in the single camta3-1 mutant . Thus , the DNA damage seen in these autoimmune mutants is indirect . That such damage occurs in the four unrelated autoimmune mutants described here supports a model in which DNA damage is a consequence of cell death . It could be argued than in an alternative model for sni1 , defective DNA damage repair causes DNA damage accumulation which in turn induces upregulation of immune responses , e . g . activation of NLRs due to damaged DNA . In this model , however , the double sni1 eds1 mutant should retain the DNA damage accumulation seen in the single sni1 mutant while losing all of the enhanced immune responses . Because DNA damage accumulation is restored to basal levels in the double mutant we maintain that DNA damage in these mutants is a consequence of autoimmunity . SNI1 was originally identified in a screen for suppressors of NPR1 , a known positive regulator of SAR . Because sni1 mutants restore PR1 gene expression and pathogen resistance in npr1 backgrounds , SNI1 was proposed to be a negative regulator of SAR . However , neither macroscopic nor microscopic cell death was originally reported in sni1 , even after INA treatment [20] . Surprisingly , sni1 was later reported to exhibit cell death in the absence of pathogens [19] . We also find that sni1 displays cell death ( Fig 4B ) and , more importantly , that increased PR1 expression , stunted growth , and HR PCD in sni1 are dependent on the NLR signaling component EDS1 ( Fig 4A–4C ) . Autoimmunity in sni1 may therefore be better explained by a guard model in which SNI1 and/or other components related to the SMC5/6 complex are guarded by an NLR ( s ) . While it is still possible that SNI1 plays a role in immune responses , these effects are overshadowed by EDS1-dependency . For example , partial suppression of sni1 growth defects by eds1 could be due to an intermediate phenotype between eds1 mutants ( which can be larger than wild type plants ) and sni1 , and therefore not directly linked to autoimmunity . A potential caveat to a SNI1 guard model is that mutations in the upstream DDR components RAD17 and ATR rescue the sni1 phenotype [19] . An explanation could be that the NLR ( s ) which may recognize sni1 loss-of-function needs to be associated with other components of the SMC complex to become activated and trigger immune responses . If so , such components or the complex may be so severely altered or absent in sni1 rad17 or sni1 atr double mutants as to abrogate the function of the NLR guard . Tangential support of a model in which the whole SMC5/6 complex is guarded comes from the finding that the mutant of MMS21 , another member of the SMC5/6 complex , also displays stunted growth , spontaneous cell death and accumulation of damaged DNA [35] . Future work could characterize double mms21 eds1 and mms21 atm/atr double mutants to check if the mms21 phenotype is suppressed , as with sni1 . It is also possible that , like RAD51 and BRCA2 , SNI1 could be positively involved in immunity by maintaining genome integrity during infection . This would make sni1 and other components of the DDR machinery potential targets for pathogen effectors and thus likely candidates for guarding by NLRs . Considering the involvement of SNI1 in RAD51 regulation , our observation that transcripts of DDR genes are downregulated in sni1 ( Fig 7E and 7F ) again fits with a model in which autoimmunity , and not a regulatory function on SNI1 , affects the levels of DDR transcripts and RAD51 protein . In contrast , Yan et al . and Xu et al . [19 , 35] observed increased DDR gene transcripts in mms21 and sni1 . An explanation for these differences is that they used 2 week-old plants , while we used plants at a more advanced developmental stage ( 6 week-old ) to allow the onset of runaway cell death in some of the mutants tested . At early developmental stages , a constitutive defense phenotype would lead to an increase in DDR which would later be switched off as plant tissues start to succumb to HR PCD . In addition , Wang et al . [29] showed that RAD51 and BRCA2 are actively recruited and bind to the promotors of defense related genes during SAR . This could explain the initial upregulation of these genes in young sni1 and mms21 plants . The recruitment of the DDR machinery to defense genes during SAR may be necessary to protect actively transcribed regions of the genome , or it may be a strategy to prevent pathogens from tampering with defense responses by interfering with genome integrity . It is well established in humans that pathogens can affect host genome integrity [34] , so it is probable that DDR genes which maintain genome integrity would be upregulated during initial stages of defense . However , once the balance between life and death has shifted towards the latter , the DDR machinery is shutdown to allow for cellular dismantling . In conclusion , we demonstrate that activation of NLR-mediated immunity leads to DNA damage accumulation as an effect of the execution of HR PCD . We provide evidence of sni1 autoimmunity and propose that this autoimmunity underlies some previous misconceptions about the function of SNI1 as a negative regulator of SAR , its involvement in RAD51 regulation , and the accumulation of DNA damage in sni1 loss-of-function mutants .
Sterilized seeds were placed on soil supplemented with vermiculite , perlite , and fertilizer . Plants were grown in chambers at 21°C under 8 hours of light and 16 hours of darkness . The mutants camta 3–1 ( SALK_00152 ) , vad1 ( SALK_00782 ) , pub13 ( SALK_093164 ) and sni1 ( SAIL_298_H07 ) were obtained from the European Arabidopsis Stock Center ( NASC ) and genotyped ( primers listed in Table 1 ) . camta 3–1 x DSC2-DN ( At5g18370 ) mutants were obtained as described in [14] . Comet assays were performed as described by [22] . In brief , tissue was finely cut with a new scalpel in 300 μl of Tris Buffer ( 0 , 4 M pH 7 , 5 ) in the dark on ice . The nuclear suspension obtained was mixed 1:1 with 1% low melting point ( LMP ) agarose , added to a pre-coated slide ( 1% agarose ) and incubated at 4°C for 10 mins . Afterwards , nuclear unwinding was done in alkaline solution ( 200 mM NaOH , 1 mM EDTA pH >13 ) for 20 minutes , and the slides were then electrophoresed in alkaline solution ( 300 mM NaOH , 1 mM EDTA , pH >13 ) at 0 . 7 V/cm for 20 mins . The slides were then neutralized , washed with Tris Buffer followed by water , then stained with SYBR Green I ( Invitrogen , California , USA ) . Comet images were captured with a Zeiss Axioplan microscope with a CoolSnap camera CF ( Photometrics , Arizona US ) or with a Zeiss LSM 700 upright confocal microscope . DNA damage quantification was performed with Open Comet plug-in for ImageJ . Col-0 and rpm1-3 leaves were syringe infiltrated with Pst DC3000 ( AvrRpm1 ) strain ( 1 × 108 CFU mL−1 ) or with P . fluorescens ( 1x107 CFU mL−1 ) in 10mM MgCl2 . For SA analog assays , Col-0 plants were sprayed with 100 μM BTH ( Syngenta , UN 3077 ) , 100 μM INA ( Sigma ) , 1mM SA ( Sigma ) or water and analyzed 4 or 24h after exposure . For the protease inhibitor assay , 10 μM Z-DEVD-FMK ( Santa Cruz was infiltrated with or without Pst DC3000 ( AvrRpm1 ) . Leaves were excised from 2 week-old plants from the genotypes given and stained with Lactophenol-Trypan blue , followed by distaining in chloral hydrate as described previously [36] . Total proteins were extracted as described in [29] . In brief , tissue was flash frozen and protein extracted in 50 mM Tris-HCl ( pH 7 , 5 ) , 150 mM NaCl , 5 mM EDTA 0 , 1% Triton x-100; 0 , 2% Nonidet P-40 , 1 mM PMSF , 1 cOmplete ULTRA Tablet , Mini , EDTA-free , ( Roche ) and 3 x SDS buffer was added . The extract was centrifuged at 16 . 000 ×g for 10 min at 4°C . The supernatant was transferred to a new tube and centrifuged twice more before SDS/PAGE analysis on a 12% gels . The samples were transferred onto nitrocellulose membrane , blocked with 5% BSA in TBS-T and sequentially probed with rabbit polyclonal Anti-Rad51 ( Abcam ab63801 ) and anti-rabbit horseradish-conjugated antibody ( Promega , W4028 ) . Histone extraction was performed as previously described [37] . In brief , 3 grams of tissue were ground in nuclear isolation buffer ( 15 mM PIPES , pH 6 . 8; 5 mM MgCl2; 0 . 25 M sucrose; 15 mM NaCl , 1 mM CaCl2; 0 . 8% triton X-100; 1 mM PMSF; 0 . 7 μg/ml pepstatin A; 30 mM NaF; 60 mM KCl , 1 tablet of cOmplete ULTRA Tablets , Mini , EDTA-free , and 1 tablet of PhosSTOP from Sigma ) . The liquid was passed through Miracloth and then spun at 10 . 000 x g for 20 mins at 4°C . The pellet was resuspended and incubated in 0 . 4 M H2SO4 at 4°C for at least 1h . Samples were then spun at 15 . 000 x g for 5 mins at 4°C . Histones were precipitated with acetone at -20°C overnight . Samples were then spun at 16 . 000 x g for 5 mins at 4°C . After centrifugation , the pellet was resuspended in 4M urea . Protein samples were subjected to SDS-PAGE , blotted and immunodetected with rabbit anti-human γ-H2AX antibody at 1∶1000 dilution ( Sigma-Aldrich , St . Louis , MO ) . Band intensity quantification was done with ImageJ , normalizing specific bands to input control . Total RNA was extracted using TRI reagent ( Sigma ) and performed according to the manufacturer’s recommendations . 1μg of total RNA was used for DNase treatment with TURBO DNA-free Kit ( Ambion Life technologies ) . cDNA synthesis was then performed using RevertAid First Strand cDNA Synthesis Kit ( Thermo Fisher ) according to the manufacturer’s recommendations . qPCR was done using the Luminaris SYBR ROX qPCR Master Mix ( ThermoFisher ) and expression level was normalized to UBQ10 ( primers listed in Table 1 ) . | DNA is constantly subjected to damaging agents that can cause mutations , disease and cell death . Not surprisingly , cells have evolved mechanisms to repair damaged DNA and preserve its integrity . Recent reports suggested that the innate immune system in plants might actively damage host DNA as a mechanism to boost resistance to pathogens . This process was shown to be mediated by the phytohormone Salicylic Acid and the DNA damage repair protein and immunity regulator SNI1 . In contrast , we report that the DNA damage observed in sni1 is independent of its role in DNA damage repair and is instead linked to autoimmunity and shared with other , unrelated autoimmune mutants . We demonstrate , genetically , that cellular dismantling triggered by NLR-mediated immunity is a more plausible explanation for the DNA damage observed in those mutants . Our results provide clarification and new insight into the role of DNA damage and immunity . | [
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] | 2018 | DNA damage as a consequence of NLR activation |
Visual scene category representations emerge very rapidly , yet the computational transformations that enable such invariant categorizations remain elusive . Deep convolutional neural networks ( CNNs ) perform visual categorization at near human-level accuracy using a feedforward architecture , providing neuroscientists with the opportunity to assess one successful series of representational transformations that enable categorization in silico . The goal of the current study is to assess the extent to which sequential scene category representations built by a CNN map onto those built in the human brain as assessed by high-density , time-resolved event-related potentials ( ERPs ) . We found correspondence both over time and across the scalp: earlier ( 0–200 ms ) ERP activity was best explained by early CNN layers at all electrodes . Although later activity at most electrode sites corresponded to earlier CNN layers , activity in right occipito-temporal electrodes was best explained by the later , fully-connected layers of the CNN around 225 ms post-stimulus , along with similar patterns in frontal electrodes . Taken together , these results suggest that the emergence of scene category representations develop through a dynamic interplay between early activity over occipital electrodes as well as later activity over temporal and frontal electrodes .
Categorization , the act of grouping like with like , is a hallmark of human intelligence . Scene categorization in particular is incredibly rapid [1 , 2] and may even be automatic [3] . Despite the importance of this problem , little is known about the temporal dynamics of neural activity that give rise to categorization . A common conceptual framework in the visual neuroscience community considers representations in each visual area as a geometric space , with individual images represented as points within this space [4 , 5] . According to this account , early visual processing can be described as a tangled geometric surface in which individual categories cannot be easily separated , and that over the course of processing , the ventral visual stream disentangles these manifolds , allowing for categories to be distinguished [6 , 7] . Although this view has provided a useful descriptive framework , we still know little about how this disentangling occurs because it has been difficult to examine the representations at each stage of processing . In a parallel development , work in computer vision has resulted in the creation of deep convolutional neural networks ( CNNs ) whose categorization abilities rival those of human observers [8] . Although CNNs do not explicitly seek to model the human visual system , their architectures are inspired by the structure of the human visual system [9]: like the human visual system , they are arranged hierarchically in discrete representational layers , and they apply both linear and nonlinear operations across the whole of visual space . As CNNs are explicitly trained for the purpose of visual categorization , and as they achieve near human-level performance at this task , they provide neuroscientists with an unprecedented opportunity to interrogate the types of intermediate level representations that are built en route to categorization . Indeed , there is a growing literature detailing the similarities between aspects of CNNs and activity in biological brains [10–17] . Of particular interest to the current study is the correspondence between the stages of processing within a CNN and the temporal order of processing in the human brain , as assessed with M/EEG . Studies have demonstrated that the brain activity elicited by individual images can be predicted by the CNN [12 , 18] and that upper layers of the CNN can predict semantically-relevant spatial properties such as overall scene volume [19] . However , these studies pool across all sensors at a given time point , discarding potentially informative scalp patterns . While this capitalizes on the fine temporal scale of M/EEG , a complete understanding of the neural dynamics of scene understanding requires the characterization of information flow across the cortex . Additionally , key questions remain open , including understanding the development of scene category membership and how intermediate stages of visual representation allow for these complex abstractions to take place . Therefore , the goal of the current study is to assess the extent to which sequential representations in each layer of a pre-trained deep convolutional neural network predict the sequential representations built by the human brain using high-density time-resolved event-related potentials ( ERPs ) . Previewing our results , we show a spatiotemporal correspondence between sequential CNN layers and the order of processing in the human visual system . Specifically , earlier layers of the CNN correspond better to early time points in human visual processing and to electrodes over occipital and left temporal scalp regions . Later layers of the CNN correspond to later information and best match electrodes in the frontal half of the scalp , as well as over the right occipitotemporal cortex . The correspondence between computer models and human vision provides neuroscientists with the unique opportunity to probe intermediate-level representations in silico , allowing for a more complete understanding of the neural computations generating visual categorization .
In order to understand the potential contributions of each CNN layer to category-specific neural responses , we assessed the extent to which each CNN layer contained decodable category information . All layers contained above-chance information for decoding the 30 scene categories ( see Fig 2 ) . The classification accuracy ranged from 40 . 8% in the first layer ( Conv1 ) to 90 . 3% correct in the first fully-connected layer ( FC6 ) . Somewhat unexpectedly , this is higher accuracy than we observed in the top layer ( FC8: 84 . 6% ) . It is also noteworthy that this level of classification is higher than the 69–70% top-1 classification accuracy reported in [20] . This is likely due to the fact that some of the images in our dataset were from the training set for this CNN . In sum , we can expect to see category-specific information in each of the eight layers of the CNN , with maximum category information coming from the fully connected layers , peaking in the sixth layer . To get an overall picture of human/CNN correspondence , we examined the extent to which the set of all CNN layers have explanatory power for visual ERPs . Our data-driven clustering method identified five groups of electrodes . We averaged ERPs within each cluster and then examined the variance explained by all layers of the CNN within that cluster’s representational dissimilarity matrices ( RDMs ) over time . For each cluster , we examined the onset of significant explained variance , the maximum explained variance , and the latency of maximum explained variance . We observed no statistically significant differences between the clusters for onset ( F ( 4 , 60 ) <1 ) , maximum explained variance ( F ( 4 , 60 ) = 1 . 11 , p = 0 . 36 ) , nor latency at maximum ( F ( 4 , 60 ) <1 ) . Thus , we show the average of all 256 electrodes in Fig 3 . We found that the CNN could predict neuroelectric activity starting at 54 ms after scene presentation ( 55 ms for internal replication set ) , and it achieved maximal predictive power 93 ms after stimulus onset ( 75 ms for internal replication set ) . To assess how much of the explainable ERP variance was captured by the CNN , we estimated the noise ceiling using a method suggested by [21] . We found that the maximum explainable variance to be 48–54% ( internal replication set: 45%-48% ) . Therefore , the average maximum r2 of 0 . 10 across all electrodes does not account for all variability in this dataset . As CNNs are exclusively feedforward models , the remaining variability to be explained may reflect the role of feedback in human scene categorization [22 , 23] . In order to gain additional insight into the relative successes and failures of the CNN as a model for visual scene categorization , we examined the regression residuals between 50 and 250 ms after stimulus onset . We averaged the residuals over participant , resulting in a 30x30 matrix . We then averaged over superordinate category ( indoor , urban outdoor , and natural outdoor ) in order to visualize broad patterns of CNN/neuroelectric difference . As seen in Fig 4 , we observed negative residuals among and between the two outdoor superordinate categories . This pattern indicates that the CNN predicted more similarity between these categories than what was observed neurally . In other words , that the human category representations of outdoor categories are more distinct compared to the predictions made by the CNN . By contrast , the residuals between indoor and urban categories were positive , suggesting that the CNN had a finer-grained representation of these category differences compared to the human brain . The residuals for the replication set demonstrated the same broad pattern ( see Supporting Material ) . In order to examine the correspondence between the processing stages of the CNN and the representational stages in human visual processing , we next examined the variance explained by each individual CNN layer . Averaged across all 256 electrodes , we observed a correspondence between the onset of explained variability and CNN layer , with earlier layers explaining ERP variability earlier than later layers ( r = 0 . 38 , p<0 . 001 for the main set and r = 0 . 40 , p<0 . 001 for the replication set , see Fig 5 ) . Additionally , we observed a negative relationship between CNN layer and explained variability , with earlier layers explaining more ERP variability than later layers . This effect was pronounced in the first 100 ms post-stimulus ( r = -0 . 51 , p<0 . 0001 for the main set and r = -0 . 49 , p<0 . 0001 for the replication set ) , and was also observed between 101 and 200 ms ( r = -0 . 26 , p<0 . 005 for the main set and r = -0 . 23 , p<0 . 05 for the replication set ) . A one-way ANOVA found a significant difference in the onset of significant explained variability as a function of CNN layer ( F ( 7 , 96 ) = 5 . 46 , p<0 . 0001 ) for the main dataset and F ( 7 , 112 ) = . 4 . 81 , p<0 . 001 for the replication set ) Additionally , a one-way ANOVA revealed significant differences in the maximum explained variability across CNN layers: F ( 7 , 96 ) = 15 . 7 , p<0 . 0001 for the main dataset and F ( 7 , 112 ) = 10 . 1 , p<0 . 0001 for the replication set ) . A chief advantage of conducting the encoding analyses at each electrode , rather than collapsing across all sensors as has been done in previous work [12 , 18] , is that we are able to examine the spatiotemporal patterns of explained variability rather than just temporal . Using data-driven electrode clustering ( see Methods ) , we identified five groups of spatially contiguous electrodes with differing voltage patterns . The clustering took microvolt patterns in the topographic maps at each time point as input and tested for spatially continuous groups of electrodes that had similar microvolt patterns . We tested these against a chance level defined by a baseline topographic map . In each group , we examined the maximum explained variability for each layer of the CNN . As shown in Fig 6 , we observed a striking dissociation . Central occipital electrodes were better explained by the earlier layers of the CNN at all time points ( Spearman’s rank-order correlation between layer and maximum explained variance: ( rho = -0 . 64 , t ( 12 ) = -13 . 5 , p<0 . 0001 for main set; rho = -0 . 68 , t ( 14 ) = -13 . 3 , p<0 . 0001 for replication set ) . A similar trend was seen in left occipitotemporal electrodes for time points before 200 ms in the main dataset , with a trend in the replication dataset ( 80–110 ms: rho = -0 . 53 , t ( 12 ) = -6 . 31 , p<0 . 0001 for main set; rho = -0 . 78 , t ( 14 ) = -10 . 3 , p<0 . 0001 for replication set; 120–200 ms: rho = -0 . 54 , t ( 12 ) = -6 . 4 , p<0 . 0001 for main dataset , rho = -0 . 38 , t ( 14 ) = 1 . 2 , p = 0 . 13 ) . By contrast , the right occipitotemporal cluster was best explained by early layers in early time bins ( 80–110 ms: rho = -0 . 57 , t ( 12 ) = -8 . 37 , p<0 . 0001 for the main dataset and rho = -0 . 88 , t ( 14 ) = 10 . 5 , p<0 . 0001 for the replication dataset ) , then by mid-to-late-level layers between 120–200 ms ( peak in layer 4 for main set , layer 5 for replication set; rho = 0 . 44 , t ( 12 ) = 1 . 29 , p = 0 . 11 for main; rho = -0 . 08 , t ( 14 ) <1 for replication ) and between 200–250 ms post stimulus onset ( maximum in layer 6 for main set , maximum in layer 3 for replication set; rho = 0 . 17 , t ( 12 ) <1 ) . As evident in Fig 6 , the later time bins did not have a significant rank-order correlation because of non-monotonic patterns between CNN layer and explained variability . Post-hoc t-tests ( Benjamini-Hochberg corrected for multiple comparisons ) revealed that for 120–200 ms , the numerical peak in layer 4 was statistically significant from both earlier layer 1 ( t ( 12 ) = 4 . 78 , p<0 . 001 ) and later layer 7 ( t ( 12 ) = 3 . 66 , p<0 . 001 ) and layer 8 ( t ( 12 ) = 4 . 08 , p<0 . 001 ) . For 200–300 ms , the numerical peak in layer 6 was significantly different from layer 1 ( t ( 12 ) = 5 . 17 , p<0 . 0001 ) and layer 8 ( t ( 12 ) = 3 . 3 , p<0 . 005 ) . Statistics for the replication set can be found in the Supporting Materials . Similarly , while the frontal cluster best reflected lower layers of the CNN early 120–200 ms time bin ( rho = -0 . 62 , t ( 12 ) = -4 . 98 , p<0 . 0005; see Supporting Materials for replication set ) , it best reflected information from the later layers , particularly the sixth CNN layer ( FC6 ) in the 200–300 ms time bin ( rho: 0 . 48 , t ( 12 ) <1 . ) Post-hoc t-tests ( corrected ) revealed significantly more variance explained by layer 3 compared with layer 1 ( t ( 12 ) = 2 . 29 , p<0 . 05 ) , and more in layer 6 compared with layer 7 ( t ( 12 ) = 1 . 92 , p<0 . 05 ) or layer 8 ( t ( 12 ) = 1 . 87 , p<0 . 05 ) . Similarly , the replication dataset also had a local maximum of explained variance at the sixth CNN layer ( see Supporting Materials for more information ) . Interestingly , the sixth layer had the maximum decodable category information ( see Fig 2 ) , suggesting that these signals reflect category-specific information . When comparing the latencies of maximum explained variability in these two clusters , we observed that the right occipitotemporal cluster peaked about 10 ms earlier than the frontal cluster ( 227 versus 236 ms ) . However , this difference was not statistically reliable ( t ( 12 ) <1 ) .
Human subjects research was approved by the Colgate University Internal Review Board ( IRB ) . Fourteen observers ( 6 female , 13 right-handed ) participated in the study . One of the participant’s data contained fewer than half valid trials following artifact rejection and was removed from all analyses . An additional 15 participants ( 9 female , 12 right-handed ) were recruited as part of an internal replication study ( specific results from those participants are reported as Supporting Material ) . The age of all participants ranged from 18 to 22 ( mean age = 19 . 1 for main experiment , and 19 . 4 for replication study ) . All participants had normal ( or corrected to normal ) vision as determined by standard ETCRS acuity charts . All participants gave Institutional Review Board-approved written informed consent before participating , and were compensated for their time . The stimuli consisted of 2250 color images of real-world photographs from 30 different scene categories ( 75 exemplars in each category ) taken from the SUN database [24] . Categories were chosen to include 10 indoor categories , 10 urban categories , and 10 natural landscape categories , and were selected from the larger set of 908 categories from the SUN database on the basis of making maximally different RDMs when examining three different types of visual information ( layer 7 features from a CNN , a bag-of-words object model , and a model of a scene’s functions , see [25] for more details on category selection ) . When possible , images were taken from the SUN database . In cases where this database did not have 75 images , we sampled from the internet ( copyright-free images ) . Six of the 30 categories ( bamboo forest , bar , butte , skyscraper , stadium , and volcano ) were represented in the Places-205 database that comprised the training set for the CNN . Although the SUN and Places databases were designed to be complementary with few overlapping images [20] , it is possible that some images in our set were included in the training set for the Places CNN . Care was taken to omit images with salient faces in them . All images had a resolution of 512 x 512 pixels ( which subtended 20 . 8° of visual angle ) and were processed to possess the same root-mean-square ( RMS ) contrast ( luminance and color ) as well as mean luminance . All images were fit with a circular linear edge-ramped window to obscure the square frame of the images , thereby uniformly distributing contrast changes around the circular edge of the stimulus [26 , 27] . All stimuli were presented on a 23 . 6” VIEWPixx/EEG scanning LED-backlight LCD monitor with 1ms black-to-white pixel response time . Maximum luminance output of the display was 100 cd/m2 , with a frame rate of 120 Hz and resolution of 1920 x 1080 pixels . Single pixels subtended 0 . 0406° of visual angle ( i . e . 2 . 43 arc min . ) as viewed from 32 cm . Head position was maintained with an Applied Science Laboratories ( ASL ) chin rest . Participants engaged in a 3 alternative forced-choice ( 3AFC ) categorization task with each of the 2250 images . As it was not feasible for participants to view all images in one sitting , all images were randomly split into two sets , keeping roughly equal numbers of images within each category . Each image set was presented within a different ~50-minute recording session , run on separate days . The image set was counterbalanced across participants , and image order within each set was randomized . Participants viewed the windowed scenes against a mean luminance background under darkened conditions ( i . e . the only light source in the testing chamber was the monitor ) . All trials began with a 500 ms fixation followed by a variable duration ( 500–750 ms ) blank mean luminance screen to enable any fixation-driven activity to dissipate . Next , a scene image was presented for 750 ms followed by a variable 100–250 ms blank mean luminance screen , followed by a response screen consisting of the image’s category name and the names of two distractor categories presented laterally in random order ( distractor category labels were randomly sampled from the set of 29 and therefore varied on a trial-by-trial basis ) . Observers selected their choice by using a mouse to click on the correct category name . Performance feedback was not given . Continuous EEGs were recorded in a Faraday chamber using EGI’s Geodesic EEG acquisition system ( GES 400 ) with Geodesic Hydrocel sensor nets consisting of a dense array of 256 channels ( electrolytic sponges ) . The on-line reference was at the vertex ( Cz ) , and the impedances were maintained below 50 kΩ ( EGI amplifiers are high-impedance amplifiers–this value is optimized for this system ) . All EEG signals were amplified and sampled at 1000 Hz . The digitized EEG waveforms were band-pass filtered offline from 0 . 1 Hz to 45 Hz to remove the DC offset and eliminate 60 Hz line noise . All continuous EEGs were divided into 850 ms epochs ( 100 ms before stimulus onset and the 750 ms of stimulus-driven data ) . Trials that contained eye movements or eye blinks during data epochs were excluded from analysis . Further , all epochs were subjected to algorithmic artifact rejection of voltage exceeding ± 100 μV . These trial rejection routines resulted in no more than 10% of the trials being rejected on a participant-by-participant basis . Each epoch was then re-referenced offline to the net average , and baseline corrected to the last 100 ms of the luminance blank interval that preceded the image . Grand average event-related potentials ( ERPs ) were assembled by averaging all re-referenced and baseline corrected epochs across participants . Topographic plots were generated for all experimental conditions using EEGLAB [28] version 13 . 4 . 4b in MATLAB ( ver . R2016a , The MathWorks , MA ) . For all analyses , we improved the signal-to-noise ratio of the single trial data by using a bootstrapping approach to build sub-averages across trials for each trial ( e . g . , [19] ) . Specifically , for each trial within a given scene category , we randomly selected 20% of the trials within that category and averaged those to yield a sub-averaged ERP for that trial . This was repeated until all valid trials within each category were built . This process was repeated separately for each participant . This approach is desirable as we are primarily interested in category-level neuroelectric signals that are time-locked to the stimulus . In order to assess the representations available in a deep convolutional neural network at each processing stage , we extracted the activations in each of eight layers in a pre-trained network . Specifically , we used a CNN based on the AlexNet architecture [29] that was pre-trained on the Places database [20] and implemented in Caffe [30] . The first five layers of this neural network are convolutional , and the last three are fully connected . The convolutional layers have three operations: convolution , pooling , and a rectified linear ( ReLu ) nonlinearity . For these layers , we extracted features after the convolution step . This CNN was chosen because it is optimized for 205-category scene classification , and because the eight-layer architecture , loosely inspired by biological principles , is most frequently used when comparing CNNs and brain activity [12 , 15 , 16] . For each layer , we averaged across images within a category , creating 30-category by N-feature matrices . In order to assess the amount of category-related information available in each layer of the CNN , we performed a decoding procedure on the feature vectors from each CNN layer using a linear multi-class support vector machine ( SVM ) implemented as LIBSVM in Matlab [31] . Decoding accuracies for each layer were calculated using 5-fold cross validation . For all analyses , statistical testing was done via permutation testing . Specifically , we fully exchanged row and column labels for the RDMs ( 1000 permutation samples per participant ) to create an empirical chance distribution . To correct for multiple comparisons , we used cluster extent with a threshold of p<0 . 05 , Bonferroni-corrected for multiple comparisons ( similar to [12] ) . A forward pass of activations through the CNN was collected for each image and each layer , and reshaped into a feature vector . Feature vectors were averaged across category to create eight 30-category by N-feature matrices . From these feature matrices , we created 30-category by 30-category correlation matrices . Representational dissimilarity matrices ( RDMs ) were created by transforming the correlation matrices into distance matrices using the metric of 1-Spearman rho [12 , 14] . Each RDM is a symmetrical matrix with an undefined diagonal . Therefore , in all subsequent analyses , we will use only the lower triangle of each RDM to represent patterns of category similarity . To create neural RDMs , for each participant and for each electrode , we extracted ERP signals within a 40 ms sliding window beginning 100 ms before image presentation , and extending to the entire 750 ms image duration . For each 40 ms window , we created a 30 x 30 correlation matrix from the voltage values at that electrode , averaged over category . A 30 x 30 RDM using the same 1-minus-correlation distance metric described above . The window size was truncated at the end of each trial as to not extend beyond image presentation . Thus , RDMs were created from each of 256 electrodes for each of 850 ms time points . The upper and lower bounds of the noise ceiling for the data were computed as recommended in [21] . It is worth noting that while previous MEG RDM results have performed time-resolved analyses on a time point by time point manner taking the value at each sensor at each time point as a feature ( e . g . [12] ) , our approach allows for the understanding of feature correspondence at each electrode , also enabling spatiotemporal analysis . We computed a noise ceiling for the results following the method detailed in [14 , 21] . Briefly , the noise ceiling contains both lower- and upper-bound estimates of the group-average correlation with an RDM that would be predicted by an unknown true model . The upper bound consisted of the average correlation of each single participant to the group mean . As this includes all participants , it is overfit and therefore an overestimation of the true model’s fit . By contrast , the lower bound used a leave-one-participant out approach , computing each single-participant RDM’s correlation with the average RDM of the other participants . This avoids overfitting , but underestimates the true model’s average correlation due to the limited data . In order to examine spatial relations in encoding patterns across electrodes , we adopted a data-driven approach based on random field theory . We modified an algorithm initially created by Chauvin and colleagues used to assess statistical significance of pixel clusters in classification images [32] . This allowed us to identify spatially contiguous electrode clusters based on voltage differences , while remaining agnostic to any encoding differences with the CNN . Specifically , we submitted participant-averaged and z-transformed voltage difference topographic maps to the algorithm time point by time point . The spatial clustering algorithm then grouped electrodes that contained normalized voltage differences that significantly deviated from baseline noise ( p < . 001 ) . As this spatial grouping procedure is based on assessing the statistically significant peaks and troughs of the voltage patterns at a given point of time , its advantage is that we did not need to set the number of clusters in advance . We selected clusters that persisted for more than 20 ms , resulting in five clusters: an early central occipital cluster ( 100–125 ms ) , an early central cluster ( 80–105 ms ) , two bilateral occipitotemporal clusters ( 70–500 ms ) , and a large frontal cluster ( 135–500 ms ) . We assessed the relative encoding strength of each of the eight CNN layers within each of these five clusters in all analyses .
In this work , we demonstrated that there is a substantial resemblance between the sequential scene category representations from each layer of a pre-trained deep convolutional neural network ( CNN ) , and those in human ERP activity while observers are engaged in categorization . Early layers of the CNN best predicted early ERP activity , while later layers best predicted later activity . Furthermore , the total variability captured by the CNN was slightly less than half of the variability given by the noise ceiling of the data . Furthermore , we observed a spatial correspondence between CNN layers and ERP variability . While electrodes over central- and left- occipitotemporal cortex were robustly predicted early by early CNN layers , electrodes over right occipitotemporal cortex had sequential representations that resembled the sequential representations of the CNN . Specifically , activity in these electrodes was best predicted by the second and third CNN layers before 100 ms post-stimulus , by the fourth layer between 120–200 ms , and by the sixth layer after 200 ms . A similar striking dissociation was observed over the frontal electrode cluster: early CNN layers best predicted early activity , but the more conceptual , fully-connected layers captured activity at the frontal electrodes about 100 ms later . Taken together , these results suggest a deeper homology between the human visual system and the deep neural network than the few biological principles that inspired the architecture [29] . This homology provides the unique intellectual opportunity to probe the mid- and late- stages of visual representation that have thus far been difficult to ascertain . Because the CNN is an exclusively feedforward model , comparing the representations in the CNN and the human brain allows us to speculate on the extent to which human category representations are processed in a feedforward manner ( such as [33] ) . We observed that earlier layers of the CNN explained more ERP variance compared with later layers of the CNN . This may be evidence that earlier visual processing is predominantly feedforward , while later visual processing requires feedback , consistent with other relatively early accounts of top-down feedback [22 , 34–36] . However , we also observed that the neural variability explained by the CNN only reached about half of the maximum possible explained variability given by the noise ceiling . This may suggest that feedback or recurrent processing plays a significant role in categorization [22 , 34 , 37] . Of course , it is possible that a different feedforward model , or a different weighting of features within this model may explain more variability in human ERP patterns . However , recent literature suggests that performance differences between different deep CNN architectures is smaller than the difference between CNNs and human observers , or between CNNs and previous models [14 , 15 , 25 , 38 , 39] . Future work will examine both recurrent and feedforward architectures to disentangle these possibilities . In examining the residuals of the model fits , we found that the CNN over-estimated the similarity of natural landscape images while simultaneously under-estimating the similarity between different types of manufactured environments ( indoor versus urban ) . This suggests a coarser-grained representation for natural landscape images in the CNN compared to human observers . This pattern may reflect the fact that only 29% of images and 28% of scene categories in Places-205 are natural landscape environments [20] . Having more training data for the CNN may have resulted in the ability to form finer-grained representations for manufactured environments . Our results are largely in agreement with previous MEG studies that examined the correspondence between CNNs and time-resolved neural signals [12 , 18] . These studies examined whole-brain signals in a time point by time point manner , losing any spatial response pattern . By contrast , by using a sliding window on each electrode , we were able to retain the patterns across the scalp while still examining time-resolved data . Given the known representational differences between MEG and EEG [40] , this work provides unique but complementary information about visual category representations . Specifically , [40] found that compared to EEG , MEG signals had decodable information earlier , and more driven by early visual cortex . This suggests that the time points reported here might constitute an upper bound on information availability . A second difference between this work and previous is that we examined category-specific information instead of image-level information . As the act of recognition is generally an act of categorization [41] , and because the CNN we used was pre-trained specifically to make category-level distinctions [42 , 43] , we argue that this is the most natural comparison for human and artificial neural networks . Accordingly , we assessed the amount of category-level information available in each layer of the CNN . While it is unsurprising that significant category information exists in all eight layers , or that the amount of information increases across layers , we were surprised to observe that category information peaked in the sixth layer . Given that the utility of layer depth is still controversial within the computer vision community [44 , 45] , this result may be of interest to this community as well . Furthermore , that both right occipitotemporal and frontal electrodes also had explained variability that peaked in the sixth CNN layer corroborates the view that a shallower artificial neural network might outperform deeper ones on scene classification tasks . Although CNNs differ significantly from biological networks , they are of interest to neuroscientists because they allow us to see a solution to a difficult information-processing problem in a step-by-step manner . The extent to which hard problems such as visual recognition have unique solutions is an open question . Thus , the growing number of similarities between biological and neural networks may indicate that artificial neural networks have honed in on the same solution found by evolution and biology . | We categorize visual scenes rapidly and effortlessly , but still have little insight into the neural processing stages that enable this feat . In a parallel development , deep convolutional neural networks ( CNNs ) have been developed that perform visual categorization with human-like accuracy . We hypothesized that the stages of processing in a CNN may parallel the stages of processing in the human brain . We found that this is indeed the case , with early brain signals best explained by early stages of the CNN and later brain signals explained by later CNN layers . We also found that category-specific information seems to first emerge in sensory cortex and is then rapidly fed up to frontal areas . The similarities between biological brains and artificial neural networks provide neuroscientists with the opportunity to better understand the process of categorization by studying the artificial systems . | [
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] | 2018 | Shared spatiotemporal category representations in biological and artificial deep neural networks |
Urogenital schistosomiasis remains highly endemic in Africa . Current control is based on drug administration , targeted either to school-age children or to high-risk communities at-large . Urine dipsticks for detection of microhematuria offer an inexpensive means for estimating infection prevalence . However , their diagnostic performance has not been systematically evaluated after community treatment , or in areas with continuing low prevalence . The objective of the present study was to perform meta-analysis of dipstick accuracy for S . haematobium infection in endemic regions , with special attention to performance where infection intensity or prevalence was low . This review was registered at inception with PROSPERO ( CRD42012002165 ) . Included studies were identified by computerized search of online databases and hand search of bibliographies and existing study archives . Eligible studies included published or unpublished population surveys irrespective of date , location , or language that compared dipstick diagnosis of S . haematobium infection to standard egg-count parasitology . For 95 included surveys , variation in dipstick sensitivity and specificity were evaluated according to study size , age- and sex-specific participation , region , local prevalence , treatment status , and other factors potentially affecting test performance . Independent of prevalence , accuracy was greater in surveys of school-age children ( vs . adults ) , whereas performance was less good in North Africa , as compared to other regions . By hierarchical ROC analysis , overall dipstick sensitivity and specificity for detection of egg-positive urine were estimated at 81% and 89% , respectively . Sensitivity was lower among treated populations ( 72% ) and in population subgroups having lower intensity infection ( 65% ) . When the insensitivity of egg count testing was considered ( and diagnosis inferred instead from combined hematuria and egg-count findings ) , overall dipstick sensitivity/specificity were 82%/97% , with significantly better sensitivity ( 92% ) in high prevalence settings . This analysis suggests that dipsticks will continue to serve as very useful adjuncts for monitoring community prevalence following implementation of population-based control of urogenital schistosomiasis .
Urogenital schistosomiasis caused by Schistosoma haematobium infection is highly endemic in many developing areas of Africa and the Middle East that lack adequate sanitation and safe water supply [1]–[5] . The urinary dipstick for detection of hematuria has long been recommended as a relatively inexpensive and potentially accurate proxy for detection of S . haematobium infection [6]–[12] , but as with any diagnostic test , performance characteristics can vary with the underlying population prevalence of the targeted disease [13] , [14] . Multi-center trials by the Red Urine Study Group and others have documented the utility of using gross hematuria or a questionnaire-derived history of visible hematuria to identify schools or communities with a high prevalence of S . haematobium infection [15]–[19] . Current WHO guidelines for preventive chemotherapy recognize hematuria prevalence , in addition to egg count-based criteria , as an effective means for identifying communities with high , moderate , or low risk for schistosomiasis [20] . However , reports from programs performing repeated intervention for schistosomiasis indicate that visible ( gross ) hematuria dramatically declines following therapy , and so it can no longer be used as an indicator of local prevalence of urogenital schistosomiasis as campaigns proceed [21] . The question remains: does microscopic hematuria , detectable by dipstick , continue to be a good marker for S . haematobium infection after treatment ? Whereas heme dipstick performance , per se , has been extensively examined in S . haematobium-endemic communities when tested before therapy [22] , dipstick performance after mass treatment , or in communities with marginal transmission , has remained uncertain [23] , [24] . In the present meta-analysis , we sought to assess systematically the diagnostic performance of urinary ‘chemical reagent dipsticks’ ( those that detect urine heme ) for the diagnosis of S . haematobium in both high- and low-prevalence areas , using available published and unpublished evidence . In particular we sought to estimate the utility of dipstick diagnosis for estimation of community prevalence in ongoing campaigns for schistosomiasis control . Our systematic review focused on results of studies that specifically compared dipstick hematuria to urine egg positivity at the individual subject level in population-based surveys for S . haematobium infection . The studies included were single or repeated cross-sectional surveys of either school-age or community-based groups in any endemic area , and our summary estimates of dipstick diagnostic performance were based on meta-analysis of data from 95 surveys identified in our systematic review .
The protocol for this project was developed prospectively by the authors then registered and published in the International Prospective Register of Systemic Reviews ( PROSPERO ) online database , http://www . crd . york . ac . uk/prospero/index . asp , number CRD42012002165 , on 03 January 2012 . The data used in this project were aggregated , anonymized data from previously published studies; as such , this study does not constitute human subjects research according to U . S . Department of Health and Human Services guidelines ( http://www . hhs . gov/ohrp/policy/checklists ) . In assessing the diagnostic performance of chemical reagent strips ( urine dipsticks ) for the detection of hematuria as a proxy diagnosis for S . haematobium infection , we aimed to include any available published or unpublished school-age or community-based population surveys , irrespective of date , location , or language of report . Studies had to include paired data for comparison of both dipstick hematuria and egg output , at the per-subject level , in order to provide study-specific estimates for true positive/true negative/false positive/false negative categories . Hospital-based or case-series , in which representativeness of the sample to the general population was unknown , were excluded . We aimed to include any studies in English , Spanish , French , Arabic , Chinese , or Portuguese in which dipstick performance was quantitatively measured for the period of 1 Jan 1966 to 31 July 2012 . Both observational studies and prospective therapeutic trials were eligible for inclusion . We identified published studies using PubMed , Google Scholar , Web of Science , African Journals Online , and private archives . Where published bibliographies of the recovered studies were found to contain promising citations ( including grey literature ) not included in online searches , these papers were obtained , whenever possible , and screened for inclusion in the meta-analysis . Contact with authors who had not presented full test performance data in their papers yielded individual level data sufficient to include two additional survey studies . We started with the wildcard keywords schistosom# and bilharz# ( e . g . , ‘Schistosoma’ , ‘schistosomiasis’ , ‘schistosome’ , ‘bilharzia’ , or ‘bilharziasis’ ) , combined with ‘dipstick’ , ‘hematuria’ , ‘chemical reagent strip ( s ) ’ , ‘reagent strips/diagnostic use’ , ‘bandelettes/urinaires’ and the terms ‘comparative study’ , ‘evaluation study’ , ‘diagnosis’ or ‘mass screening’ . As relevant articles were identified , we broadened our search by accessing additional titles through the online databases' automated ‘related articles’ links . Full titles and abstracts were recovered for initial review for inclusion . Two reviewers independently screened each study recovered in our search lists for inclusion in the systematic review . Those studies found suitable for inclusion were then obtained from online or library sources for full-text review . Where a single report contained data on multiple individual community surveys , each survey was also separately abstracted for inclusion in some of the sub-group comparison analysis . Both observational and prospective studies were considered eligible for inclusion in the overall analysis . We excluded studies where comparator parasitological diagnosis was not reported , or when the data on individual level hematuria and egg count status was not sufficiently detailed to confirm the reported sensitivity and specificity of dipstick testing . Cases of duplicate publication or extended analysis of previously published studies were also excluded . Included papers were abstracted and their relevant features entered into a purpose-built database created in Microsoft Access 2010 software ( Redmond , WA ) . In addition to full citation information and year of publication , information was collected on the country and region where the study was performed , the target population studied ( children , school-age children , adults , community , etc . ) , their prior treatment status , the sex and age distribution of included subjects , study size , local infection prevalence in general , dipstick manufacturer , definition of hematuria ( dipstick cutoff value , i . e . , ≥trace , 1+ , 2+ , or 3+ , as scored by study technicians , based on the manufacturer's instructions using a color chart supplied with the dipsticks ) , method of parasite egg detection , number of urine samples tested , and the study's definition of ‘light infection’ . Whenever possible , raw data for true positives , false positives , true negatives , and false negatives were extracted from the study's text or tables , or back calculated from the included summary data . These values were entered into separate 2 by 2 tables and the dipstick diagnostic performance was reconfirmed for each reported survey . Data entries were fully verified by a second reviewer before final data analysis was carried out . As practiced in a previous systematic quantitative review of S . haematobium diagnostics [22] , we allowed each included research report to contribute multiple observations to the analysis , i . e . , data reported from individual communities or schools were included as independent observations . Once study eligibility was determined , test results from each study and sub-study were entered into RevMan 5 software ( available from the Cochrane collection at http://ims . cochrane . org/revman ) for calculation of study prevalence , sensitivity , specificity , positive predictive value and negative predictive values , along with their confidence intervals . Dipstick performance was assessed initially according to study size , infection prevalence , and region , by visual comparison of forest plots . Summary Receiver Operating Curves ( SROC ) , were also graphed using the RevMan5 analysis module . Heterogeneity among studies was determined using Higgins's and Thompson's I2 statistic . In exploratory data analysis , multivariable meta-regression examined the impact of additional factors ( study era , dipstick brand , age grouping ( i . e . , school age vs . community ) , male∶female ratio , and world region ) on diagnostic odds ratios associated with dipstick testing . For this analysis , a Moses-Shapiro-Littenberg method study size-weighted model of test performance in the ROC plane was implemented in Meta-DiSc Software v . 1 . 4 ( provided by Hospital Ramon y Cajal , Madrid at its website , http://www . hrc . es/investigacion/metadisc_en . htm ) [25] . Final pooled summary estimates for sensitivity and specificity were calculated by using hierarchical summary ROC ( HSROC ) regression following a Bayesian Monte Carlo Markov Chain approach as described by Dendukuri , et al , [26] and implemented using their programs ( available at http://www . nandinidendukuri . com ) in SAS v . 9 . 3 statistical software . This approach was selected because of its ability to adjust for heterogeneity by assessing within- and between-study variability in test performance , while also including the effects of imperfect sensitivity and specificity of the various different reference tests ( e . g . , egg counts by filtration , centrifugation , or sedimentation ) included in the meta-analysis . Sub-group analysis examined the impact of i ) population prevalence of S . haematobium egg positivity in urine , ii ) class of intensity of infection ( ‘light’ vs . ‘heavy’ , as determined in each study using measured egg counts/10 mL urine ( see Results section , below , for details ) , and iii ) pre- vs . post-treatment status , on test accuracy in terms of sensitivity and specificity .
Our search strategy recovered a total of 537 listings , of which 409 were identified by online database searches , and an additional 128 were found through searches of bibliography listings and private archives ( see Figure 1 ) . Of these , 307 were excluded after initial review as not relevant to the area of dipstick diagnostic performance , or as duplicate entries . 230 were selected for full review , and 71 reports , containing data on 95 separate surveys , were included in the meta-analysis ( see supporting information files , Table S1-Included Studies and Table S2-Excluded Studies , for study listings and their citations ) . Details of inclusion and exclusion criteria for the analysis are provided in the Methods section , above . All studies were identified through peer-reviewed publications . Eighty-eight were in English , and seven were in French . Because criteria for performance and reporting of clinical trials have continued to evolve over the last two decades , we included study age as a covariate that might potentially influence results . Sixty-four ( 67% ) of the 95 included studies were reported after 1989 , whereas 31/95 were from the pre-1990s era ( 1979–1989 ) . Five studies focused on adults alone , one focused on infants alone , 27 were reported as community-based surveys , and 62 were performed with school-aged children alone ( Figure 1 ) . Included studies ranged from 5%–100% female participation ( where reported , the median female participation was 50% , IQR = 47–56% ) , and only 3/95 ( 3% ) of studies excluded women over 11 years of age . Geographic variation in parasite strains has also been suggested as a source of variation in infection-associated morbidity—among the included studies , 47 were from East Africa , 29 from West Africa , 8 from southern Africa , 8 from North Africa , but only 3 from central African countries . Eighty-two of the reported studies were performed before any mass anti-schistosomal treatment had been given to the study population , whereas 13 of the included studies were performed on previously treated populations , usually at a 1 year post-treatment interval follow-up . There were 8 paired surveys performed on the same populations before and after treatment . Egg output in subject urine was detected by filtration in 83/95 studies , by centrifugation in 8 studies , and by sedimentation in 3 studies . One study did not report on their egg detection technique . Twenty-seven studies reported on dipstick performance among subjects with ‘light’ intensity infection . Light intensity infection was most commonly defined as <50 or 51 eggs per 10 mL aliquot of urine ( N = 29 ) , but varied in definition from <11 eggs to <500 eggs per 10 mL . [N . B . in published studies , the general definition of ‘light infection’ shifted from ≤100 eggs/10 mL urine in the 1970s , to ≤50 eggs/10 mL in the 1980s . For this paper , any studies using a definition of ‘light’ as ≤100 eggs/10 mL ( or lower ) were included for the light infection subgroup analysis] Prevalence of urine egg positivity among the tested study populations ranged from 0 . 76% to 88% ( median 34% , IQR = 15–55% , see Figure 2 ) . Studies in 29/95 of included reports had prevalence of egg positivity ( <20% ) and these were analyzed as a separate ‘low prevalence’ subgroup in some of the analysis below . Urine egg testing was based on a single daily urine in 79/95 of the included studies , multiple ( 2–6 ) daily urines were tested in 10/95 studies , whereas 6 studies did not provide specific information about the number of urines tested . Of the studies with multi-day testing , most provided results for the first day's egg count and heme dipstick testing . To harmonize their inclusion in the meta-analysis , only these first day results were included in the analytic database used in this paper's results . For performance analysis of multi-day testing , readers are referred to Savioli at al . [27] , and Tiemersma et al . , [28] The surveys selected for inclusion in the meta-analysis and meta-regression were felt to be representative , i . e . , those used in screening of schools , whole communities , school-age children , or adult women , within target communities . Partial or differential verification bias did not apply to these studies . However , in nearly all studies , data were not available on non-participation , on delays in testing , or on the number of uninterpretable results , nor were there descriptions of efforts to mask the results of the two tests being compared during the performance of the studies .
Our meta-analysis indicates that commercial dipsticks , designed for rapid detection of heme in the urine , can provide an effective proxy for detection of S . haematobium infection in disease-endemic areas . The study populations included in our meta-analysis were felt to be representative of S . haematobium endemic communities likely to be targeted in regional and national control programs . Overall , dipstick performance showed 81% sensitivity and 89% specificity for detection of egg positive urines , and an estimated 82% sensitivity and 97% specificity for detection of active S . haematobium infection , as estimated via the combination of dipstick and egg count results . Whereas dipsticks were less sensitive in detecting egg-positive urines among post-treatment studies and among subject sub-groups with lower intensity infections ( Table 1 , Figures 3 and 4 ) , evaluation of paired pre- and post-treatment studies did not show a consistent post-treatment effect on dipstick diagnostic performance ( Figure 5 ) . When the limitations of egg-count diagnosis of S . haematobium infection were taken into account , diagnostic performance was not significantly different between pretreatment and post-treatment study populations ( Table 2 ) . Significant differences in dipstick diagnostic performance were noted based on the age range of included survey subjects ( school-age vs . community-wide ) . In addition , studies from North Africa were found to have significantly lower dipstick performance , independent of age , local prevalence , treatment status , and other measured co-factors , suggesting a possible differential in risk for Schistosoma-associated morbidity between North African and sub-Saharan populations [32] , [33] . Previous reports have commented on differences in dipstick performance between Egypt/Ghana vs . Zambia [34]and between Liberia and Tanzania [35] in parallel survey studies . There have been continuing concerns about ‘false positives’ for heme dipsticks , resulting in an apparently low specificity in some populations [36]–[40] . In their 2004 mathematical synthesis of dipstick performance , van der Werf and de Vlas [22] estimated an average 18% ‘false positive’ rate for microhematuria for the diagnosis of S . haematobium ( i . e . , heme positive but egg negative ) , based on an assumed low likelihood of infection in this class of patients . They presumed that this 18% had to be due to non-schistosome causes , because “…at low prevalence of infection…only few or no cases have an infection intensity high enough to cause morbidity . ” [22] This assumption , i . e . , that low-intensity infection is non-morbid , has been critically challenged over the last decade [41]; with the use of complementary , more sensitive diagnostics , it now appears that many heme-positive egg-negative subjects in endemic areas are , in fact , S . haematobium-infected [27] , [42]–[45] . In a ‘low prevalence , low-intensity’ community in Nigeria ( 9 . 5% egg positive , but 52% heme positive ) infection prevalence determined by Schistosoma-specific PCR was , in fact , 93–98% [46] . As an unmeasured cofactor , differential circadian variation in egg output and hematuria [47] , [48] might account for some of the variation among studies observed in our analysis . In non-endemic areas such as North America , a cumulative 3% of ambulatory school children have hematuria between 6 and 12 years of age [49] and 3–13% among otherwise healthy adults [50] , [51] , suggesting that in S . haematobium-endemic areas , the balance of 5–15% of the population with heme-positive/egg-negative urines actually do suffer from active schistosomiasis that is not detected by egg counting . In summary , the specificity of dipstick heme diagnosis is likely to be more specific in S . haematobium transmission zones than previously assumed [22] . Contamination of urine by menses , or by inflammation from bacterial cystitis , sexually-transmitted infection , or genital mutilation , has been suggested as a source of false positive dipstick diagnosis among girls and women [36] , [37] , [52] . Studies of adult women , particularly focused on menstrual status , indicate that there is a risk of false positive dipstick results based on this factor . In S . haematobium-endemic communities , prevalence of heme-positive urine among women who are urine egg negative is 33–53% during menses , and 10–17% when not having menses [52] . However , the current meta-analysis did not find evidence that the proportion of participating females in a survey had an effect on overall dipstick performance at the population level . It is possible that women having menses self-exclude from screening surveys for cultural , religious , or personal reasons [36] . In practice , the effect of menses has been minimal in surveys of 4–20 year olds South Africa [53] , in community-wide surveys in Zanzibar [27] , [54] , [55] , and among adult age groups ( >15 years old ) screened in communities in Cote d'Ivoire [56] . Where studied , subject age has been suggested to have an important effect on dipstick diagnostic performance . In several studies , for each infection category , children consistently had a greater prevalence of hematuria than adults with the same infection intensity [57]–[61] . The hypothesis is that adults experience less inflammation or release less blood in response to parasite egg deposition around the urinary tract . Koukounari et al . [62] specifically observed that dipstick sensitivity for detection of S . haematobium infection was not as good among adults >50 years old in village surveys in Ghana . By contrast , Eltoum , et al . [63] in Sudan , and Poggensee , et al . [52] , in Tanzania found disproportionately higher rates of hematuria among older adult groups despite their lower prevalence of egg-positive urines . Although there was a high degree of heterogeneity in dipstick test performance across all studies , this sort of variation is common in comparisons among diagnostic trials [64] . The HSROC approach used to estimate the summary sensitivity and specificity values reported in Tables 1 and 2 allowed for the influence of both inter- and intra-study variability , and is believed to provide a robust estimation of overall dipstick performance [26] , [64] . Bayesian HSROC also allowed us to examine the likely sensitivity and specificity values for dipsticks given the imperfections of urine egg detection methods used as the comparison diagnostic test [26] . Considering the lack of a true gold standard for diagnosis of S . haematobium infection [27] , the analysis in Table 2 allows us to infer that dipsticks retain their validity as a diagnostic tool , even when eggs in the urine are scarce or become so after a round of therapy . In this latent construct analysis , for low prevalence areas the estimated sensitivity for detection of active S . haematobium infection was higher for dipsticks ( 79% ) than for egg detection ( 51% ) while specificity was the virtually the same ( 98–99% ) . In post-treatment populations , sensitivity for dipsticks was 79% , compared with 58% for egg-detection . This suggests that reagent dipsticks may be the preferred community-level diagnostic screening tool as programs reduce parasite prevalence and move towards elimination . This meta-analysis had several limitations . Risk of bias in the included studies was for the most part , unknown . Many of the included studies did not aim to evaluate dipstick performance as their primary outcome , but rather reported on detected hematuria as a marker of S . haematobium-associated morbidity . No study reported in much detail on inclusion/exclusion criteria , subject adherence , or on masking of observers to the results of comparator diagnostic tests . As such , these studies did not meet the usual criteria for ‘high-quality’ diagnostic test clinical research [64] . However , we recognize the limitations of such studies in this underfunded area of neglected tropical diseases research–given the need for a working summary of the ‘state of the art’ for S . haematobium screening , we elected to include all available data from comparable field studies meeting our inclusion criteria . Across all studies , we recognize the possibility that certain countries were under-represented , as evidenced by lack of data inputs from lusophone Africa and an absence of data from the Arabian Peninsula or other Middle Eastern countries , a circumstance that could have biased our assessment . While there was broad variation in the performance of heme dipstick diagnosis of S . haematobium infection , our meta-analysis could identify certain important themes: Overall , on an individual patient basis , it appears from our meta-analysis that , after treatment has been given , dipsticks become less efficient in detecting a person who has eggs in his or her urine . However , given the insensitivity of egg counting for low intensity infections , dipsticks may ultimately prove more sensitive for detection of low-level persistent S . haematobium infection than testing for egg output on any given single day . In the studies included in our review , dipstick performance after treatment was seen to change in several different ways , depending on location . This finding points to the need for formal detailed diagnostic trials against a truly sensitive and specific ‘gold standard’ of active S . haematobium infection , particularly after one or more rounds of treatment have been given . This applies particularly to pre-school children and adults , for whom egg output may be relatively limited . Ongoing studies combining anti-parasite serologies and high-sensitivity detection of circulating parasite antigens should help to clarify the actual diagnostic performance of dipsticks and egg-filtration in detecting early and low-intensity S . haematobium infections . If and when population-based treatment is restricted to only ‘infected’ persons [62] , neither the urine egg count nor the dipstick heme test could be considered sufficiently sensitive to accurately identify a full fraction of ‘true infections’ to effect full prevention of morbidity [24] . On a population basis , dipsticks remain very valid tools for use in public health campaigns for control of urogenital schistosomiasis . As current preventive chemotherapy campaigns move forward , the prevalence and infection intensity profiles of S . haematobium-endemic communities undoubtedly will change . Overall , based on our synthesis of the available data , we expect that the urine dipstick for detection of microscopic hematuria will retain its usefulness as a tool for estimation of S . haematobium prevalence in treated and untreated communities [21] , [29] , [67]–[69] . The strong link between infection , urinary tract ulceration , and resultant hematuria means that the inexpensive and rapid detection of urine hematuria can continue to be a useful estimator of local prevalence . As such , dipsticks will serve as useful adjuncts for monitoring the impact of schistosomiasis control programs on prevalence , and can guide us in determining the need for further program interventions . | Schistosomiasis is a chronic human disease caused by infection with multicellular trematode parasites of Schistosoma species . In particular , Schistosoma haematobium colonizes the veins in the pelvis , in and around the urinary tract , and causes inflammation that leads to ulceration and bleeding into the urine . One low-tech , inexpensive means of quickly identifying blood in the urine ( hematuria ) is to use a chemical reagent strip ( dipstick ) to test a patient's urine . While many studies have confirmed the usefulness of dipstick-detected hematuria as a proxy for infection in diagnosing infected people before treatment is given , their diagnostic performance after treatment has been uncertain . The current study systematically reviewed 95 available reports of dipstick performance in S . haematobium-endemic areas of Africa and determined , based on a meta-analysis of the primary data , that dipsticks appear to retain their diagnostic accuracy in low prevalence areas and after one or more rounds of treatment . This suggests that dipsticks will continue to be very useful tools in tracking and targeting regional requirements for treatment and prevention of S . haematobium infection as current school- and community-based programs go forward . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [] | 2013 | Meta-analysis of Urine Heme Dipstick Diagnosis of Schistosoma haematobium Infection, Including Low-Prevalence and Previously-Treated Populations |
Loss-of-function mutations in the Caenorhabditis elegans gene sup-18 suppress the defects in muscle contraction conferred by a gain-of-function mutation in SUP-10 , a presumptive regulatory subunit of the SUP-9 two-pore domain K+ channel associated with muscle membranes . We cloned sup-18 and found that it encodes the C . elegans ortholog of mammalian iodotyrosine deiodinase ( IYD ) , an NADH oxidase/flavin reductase that functions in iodine recycling and is important for the biosynthesis of thyroid hormones that regulate metabolism . The FMN-binding site of mammalian IYD is conserved in SUP-18 , which appears to require catalytic activity to function . Genetic analyses suggest that SUP-10 can function with SUP-18 to activate SUP-9 through a pathway that is independent of the presumptive SUP-9 regulatory subunit UNC-93 . We identified a novel evolutionarily conserved serine-cysteine-rich region in the C-terminal cytoplasmic domain of SUP-9 required for its specific activation by SUP-10 and SUP-18 but not by UNC-93 . Since two-pore domain K+ channels regulate the resting membrane potentials of numerous cell types , we suggest that the SUP-18 IYD regulates the activity of the SUP-9 channel using NADH as a coenzyme and thus couples the metabolic state of muscle cells to muscle membrane excitability .
Hypothyroidism , one of the most common endocrine disorders , can cause many different symptoms and can lead to defects in brain development and maturation and retarded postnatal development [1] . For thyroid hormone biosynthesis , iodide is recycled by iodotyrosine deiodinase through the deiodination of monoiodotyrosine and diiodotyrosine , two byproducts in the generation of thyroid hormones [2]–[6] . In humans , this deiodination is catalyzed by human iodotyrosine dehalogenase ( DEHAL1 ) /iodotyrosine deiodinase ( IYD ) , an NADH oxidase/flavin reductase [7]–[10] . Mutations in IYD cause congenital hypothyroidism [11]–[13] . How the activity of IYD is regulated in vivo and whether IYD has other functions remain to be elucidated . Four transmembrane/two-pore domain K+ channels play a key role in establishing the resting membrane potentials of many cell types and in modulating their responses to neurotransmitters and second messengers [14]–[16] . To date , 15 human two-pore domain K+ channels have been identified [14] , [16] , [17] . The activities of two-pore domain K+ channels can be regulated by multiple chemical and physical factors , including temperature [18] , membrane stretch [19] , [20] , arachidonic acid [21] , pH [22] , [23] , volatile anesthetics [24] , [25] and neurotransmitters [26] , [27] . The gene sup-9 of the nematode Caenorhabditis elegans encodes a two-pore domain K+ channel [28] . sup-9 ( n1550 ) gain-of-function ( gf ) mutants are egg-laying defective and display a flaccid paralysis and a rubberband uncoordinated ( Unc ) behavior: when prodded on the head , a sup-9 ( n1550gf ) worm contracts and relaxes along its entire body without moving backwards , while a wild-type worm contracts its anterior end and moves away [29] . Loss-of-function ( lf ) mutations in sup-9 or two other genes , sup-10 and unc-93 , completely suppress these sup-9 ( n1550gf ) defects [29]–[31] . In addition , gf mutations in sup-10 and unc-93 themselves induce a rubberband Unc paralysis , which in turn are suppressed by lf mutations in sup-9 , sup-10 and unc-93 [30]–[32] . lf mutants of unc-93 , sup-9 and sup-10 do not have obviously abnormal phenotypes [29]–[31] , [33] . The SUP-9 two-pore domain K+ channel is most closely related to human TASK-3 [28] , [34] , [35] . unc-93 encodes a conserved multi-pass transmembrane protein [33] . An UNC-93 homolog , UNC93b1 , is involved in innate immune responses in mammals [36] , [37] . sup-10 encodes a novel type-I transmembrane protein [35] . Genetic analyses and the molecular identities of these genes suggest that in vivo SUP-10 and UNC-93 form a protein complex with the SUP-9 two-pore domain K+ channel and modulate its activity as regulatory subunits [28] , [33] . Mutations in the gene sup-18 suppress the muscle defects caused by gf mutations in these three genes , strongly suppressing the locomotory defects of sup-10 ( n983gf ) mutants , partially suppressing the locomotory defects of the strong unc-93 ( e1500gf ) mutants , the weak unc-93 ( n200gf ) mutants and the strong sup-9 ( n1550gf ) /+ heterozygous mutants , and suppressing only the lethality of sup-9 ( n1550gf ) mutants [29] , [30] ( also see Table 1 below ) . In this study we report that sup-18 encodes the C . elegans ortholog of mammalian iodotyrosine deiodinase/dehalogenase ( IYD ) [7] , [8] , [10] . Our findings suggest that SUP-18 is a functional regulator of the SUP-9/SUP-10/UNC-93 two-pore domain K+ channel complex in vivo and that IYD might function with two-pore domain K+ channel complexes in mammals .
sup-10 ( n983gf ) mutants have a reduced locomotory rate ( Table 1 ) . A loss-of-function mutation in sup-18 , n1030 , restores wild-type locomotion to sup-10 ( n983gf ) mutants ( Table 1 ) [30] . unc-93 ( n200gf ) causes a less severe rubberband Unc phenotype than sup-10 ( n983gf ) , yet the unc-93 ( n200gf ) phenotype is still only partially suppressed by sup-18 ( n1030 ) ( Table 1 ) . unc-93 ( e1500gf ) mutants , which have a more severe rubberband Unc phenotype than sup-10 ( n983gf ) mutants , similarly are only weakly suppressed by sup-18 ( n1030 ) . These results suggest that the differential suppression of the rubberband Unc mutants by sup-18 ( n1030 ) is caused by gene-specific effects rather than by differential severity of paralysis in these mutants . We further tested this notion using weakly paralyzed double mutants carrying the unc-93 ( e1500gf ) mutation and a partial lf allele of sup-10 . Introduction of the sup-18 ( n1030 ) mutation into partially suppressed unc-93 ( e1500gf ) ; sup-10 ( n4025 ) or unc-93 ( e1500gf ) ; sup-10 ( n4026 ) mutants only weakly improved their locomotory rates from approximately 14 to 19 body-bends/minute ( Table 1 ) . These results confirm that sup-18 ( n1030 ) only weakly suppresses gf mutations in unc-93 . The suppression of sup-10 ( n983gf ) depends on the dosage of the sup-18 allele [29] . We found that sup-18 ( n1030 ) /+; sup-10 ( n983gf ) males exhibit an intermediate phenotype ( 15 . 2 bends/min ) between those of the more severely paralyzed sup-10 ( n983gf ) males ( 4 . 7 bends/min ) and the strongly suppressed sup-18 ( n1030 ) ; sup-10 ( n983gf ) males ( 31 . 7 bends/min ) ( Table 2 ) . This dose-dependent effect was observed for all lf alleles of sup-18 tested ( Table 2 ) . By contrast , the suppression of sup-10 ( n983gf ) by sup-9 ( n1913 ) , a channel null allele , was recessive . Because the weak suppression of the locomotory defect of unc-93 ( e1500gf ) mutants by sup-18 ( lf ) mutations ( Table 1 ) [30] makes a dosage analysis of sup-18 ( lf ) suppression of unc-93 ( e1500gf ) difficult , we examined weakly paralyzed unc-93 ( e1500gf ) ; sup-10 ( n4025 ) males , which are more visibly suppressed by sup-18 ( n1030 ) ( Table 2 ) . We found that the locomotory rate of unc-93 ( e1500gf ) ; sup-10 ( n4025 ) males heterozygous for sup-18 ( n1030 ) was similar to that of males wild-type for sup-18 ( 10 . 0 vs . 9 . 8 , respectively ) ( Table 2 ) . Similarly , sup-9 ( n1550gf ) /+; sup-18 ( n1030 ) /+ males had only slightly improved locomotion compared to sup-9 ( n1550gf ) /+ males ( 5 . 0 vs . 3 . 8 , respectively ) ( Table 2 ) . We conclude that the dose-dependent suppression of rubberband Unc mutants by sup-18 alleles is also gene-specific: the sup-10 ( n983gf ) phenotype is much more sensitive to sup-18 levels than is that of the other rubberband mutants . sup-18 had previously been mapped to the interval between daf-4 and unc-32 on LGIII [30] . Using three-point mapping we further localized sup-18 to the interval between ncl-1 and unc-36 ( see Materials and Methods ) ( Figure 1A ) . Transgene rescue experiments with cosmids spanning the ncl-1-to-unc-36 interval and with smaller cosmid subclones identified a 4 . 5 kb minimal rescuing fragment from cosmid C02C2: as a transgene , this fragment restored the rubberband Unc phenotype to sup-18 ( n1010 ) ; sup-10 ( n983gf ) mutants ( Figure 1A ) . This rescuing fragment contained a single predicted gene , C02C2 . 5 [www . wormbase . org] . We screened a mixed-stage cDNA library [38] using the smallest cosmid subclone with sup-18 rescuing activity and obtained a single partial cDNA of this predicted gene . We defined the structure of this gene from RT-PCR and RACE experiments ( see Materials and Methods ) ( Figure 1B ) . sup-18 encodes a predicted protein of 325 amino acids . This protein is the only C . elegans ortholog of mammalian iodotyrosine deiodinase ( IYD ) , which belongs to the NADH oxidase/flavin reductase superfamily ( Fig . 1C ) [7]–[10] . IYD catalyzes the recycling of iodide by deiodinating 3′-monoiodotyrosine and 3′ , 5′-diiodotyrosine , the main byproducts in the process of thyroid hormone biogenesis [2]–[5] , [7] , [8] . The identity between SUP-18 and human IYD protein variant 2 ( also named DEHAL1 ) [8] is 31% overall and 45% over the NADH oxidase/flavin reductase domain ( Figure 1C ) . Like IYDs of Drosophila , mouse and human , SUP-18 has a hydrophobic region that precedes the NADH oxidase/flavin reductase domain and might serve as a transmembrane domain . We identified molecular lesions in the sup-18 coding sequence of all 18 mutant strains analyzed ( Table 3 , Fig . 1C ) . The sup-18 ( n1033 ) mutation leads to the substitution of an isoleucine for the initiator methionine , which should cause any translational products to be nonfunctional . ( The next three ATG sequences in the sup-18 cDNA are out of frame . ) The sup-18 ( n1030 ) and sup-18 ( n1548 ) mutations cause premature stop codons that likely generate truncated protein products . Four mutations ( n1038 , n527 , n463 , n1539 ) cause a frameshift . Another four mutations ( n1036 , n1035 , n1015 , n1558 ) affect splice donor or acceptor sites . The remaining seven missense mutations ( n1010 , n1554 , n1471 , n1556 , n1014 , n1022 , n528 ) disrupt residues within the NADH oxidase/flavin reductase domain . To examine the expression pattern of sup-18 , we introduced the coding sequence of gfp between codons 88 and 89 of a genomic clone of sup-18 , generating a sup-18 translation fusion transgene ( see Materials and Methods ) . Similar to transgenic animals expressing a Psup-10::gfp translational fusion transgene , Psup-18::gfp transgenic animals displayed GFP fluorescence in body-wall ( Fig . 2A , D ) , defecation ( Fig . 2B , E ) and vulval muscles ( Fig . 2C , F ) . In body-wall muscle cells ( Fig . 2A , D ) , the SUP-10::GFP and SUP-18::GFP fusion proteins both localized to cell-surface regions aligned with dense bodies , the functional analogs to vertebrate Z-lines that connect the myofibril lattice to the cell membrane [39] . In addition to muscles , three neurons in the head of Psup-18::gfp transgenic animals also displayed GFP staining ( I . de la Cruz and H . R . Horvitz , unpublished observations ) . We previously reported expression of a Psup-9::gfp reporter in the four SIA interneurons [28] . We stained the Psup-18::gfp transgenic animals with an anti-CEH-17 antibody , which labels the four SIA neurons and the ALA neuron [40] , and found that the neurons expressing the SUP-18::GFP fusion protein were not the SIAs ( I . de la Cruz and H . R . Horvitz , unpublished observations ) . We generated a rabbit anti-SUP-18 antibody ( see Materials and Methods ) . In immunostained animals , this antibody could detect overexpressed SUP-18 but failed to detect endogenous SUP-18 , probably because of the low level of SUP-18 expression . We next generated transgenic animals co-expressing a Psup-10::gfp fusion transgene and sup-18 under control of a myo-3 promoter [41] and examined the subcellular expression of SUP-18 using the antibody and of SUP-10::GFP using GFP fluorescence . We found that SUP-10 and SUP-18 colocalize in subcellular structures , including the dense bodies in the body-wall muscles ( Fig . 2G , H , I ) . Since GFP fusions to SUP-9 and UNC-93 localize similarly [28] , this result suggests that SUP-18 colocalizes with a SUP-9/UNC-93/SUP-10 complex . Mammalian IYD is a transmembrane protein [7] , [8] . The presence of a possible transmembrane domain in the predicted SUP-18 protein sequence ( Fig . 1C ) suggests that SUP-18 is also a transmembrane protein . To distinguish whether the NADH oxidase/flavin reductase domain of SUP-18 resides intracellularly or extracellularly , we generated transgenic animals expressing different SUP-18::β-galactosidase fusion proteins and assayed β-galactosidase activity in vivo in fixed animals ( Fig . 3A ) . When β-galactosidase is localized intracellularly it is enzymatically active , whereas extracellular localization results in loss of β-galactosidase activity [42] , [43] . The use of β-galactosidase activity to elucidate the membrane topology of C . elegans proteins in vivo has been reported previously for the presenilin SEL-12 protein [44] and for the MEC-4 sodium channel subunit [45] . Fixed transgenic animals expressing β-galactosidase fused to either the C-terminal region of SUP-18 or immediately C-terminal to the putative transmembrane domain showed robust β-galactosidase activity ( Fig . 3A ) . Introduction of a synthetic transmembrane domain [45] between SUP-18 and β-galactosidase in these chimeras eliminated β-galactosidase enzymatic activity , presumably because the membrane orientation of β-galactosidase had been reversed ( Fig . 3A ) . These results strongly suggest that SUP-18 is a transmembrane protein and that the NADH oxidase/flavin reductase domain of SUP-18 resides intracellularly . But they do not distinguish between a type-I transmembrane protein ( single-pass transmembrane protein with the N-terminal domain located extracellularly ) and a cytoplasmic protein that simply localizes at the cell surface , e . g . , by interacting with another membrane protein or by linking to a GPI anchor [46] . To test if the putative transmembrane domain of SUP-18 can indeed behave as a transmembrane domain , we inserted a signal sequence at the N-terminus of SUP-18 ( see Materials and Methods ) . While a fusion containing the presumptive extracellular domain of SUP-18 but lacking the putative transmembrane domain resided intracellularly as expected , the introduction of a signal sequence led to its secretion and loss of β-galactosidase enzymatic activity ( Fig . 3A ) . By contrast , when either the SUP-18 putative transmembrane domain or the synthetic transmembrane domain [45] was added to this SUP-18::β-galactosidase fusion , the enzymatic activity was restored . These results indicate that the putative transmembrane domain of SUP-18 can indeed function as a transmembrane domain and suggest that SUP-18 is likely a type-I integral membrane protein , like IYD . To establish an assay for in vivo SUP-18 activity , we expressed the sup-18 coding sequence under the control of the myo-3 promoter [41] in sup-18 ( n1033 ) ; sup-10 ( n983 ) mutant animals . While sup-10 ( n983gf ) mutant animals are defective in locomotion , double mutants carrying the sup-18 ( n1033 ) null mutation had improved locomotory rates ( Fig . 3B ) . Expression of Pmyo-3 gfp in sup-18 ( n1033 ) ; sup-10 ( n983gf ) animals had little effect on their locomotory rate , whereas expression of a Pmyo-3 sup-18 ( + ) transgene restored sup-10 ( n983gf ) paralysis ( Fig . 3B ) . By contrast , expression of two Pmyo-3 sup-18 mutant constructs containing either the n1554 missense mutation or the n1010 mutation ( which affects a conserved amino acid in the NADH oxidase/flavin reductase domain; Fig . 1C and Table 3 ) did not restore the rubberband Unc phenotype to sup-18 ( n1033 ) ; sup-10 ( n983gf ) mutants ( Fig . 3B ) . We found that the mouse IYD gene could not substitute for sup-18 in vivo in restoring the rubberband Unc phenotype of sup-18 ( n1033 ) ; sup-10 ( n983gf ) animals ( Figure 3B ) . We tagged mouse IYD with GFP at its C-terminus and found that C . elegans expressing the fusion protein displayed GFP fluorescence in body-wall muscle structures similar to that observed for the SUP-18::GFP fusion ( I . de la Cruz and H . R . Horvitz , unpublished observations ) . These results suggest that mouse IYD had been expressed properly and that mouse IYD might be inactive or otherwise incapable of substituting for SUP-18 in C . elegans . Interestingly , transgenic expression of the SUP-18 intracellular domain alone ( amino acids 66–325 ) was sufficient to restore rubberband Unc paralysis to sup-18 ( n1033 ) ; sup-10 ( n983gf ) animals , although the rescue was less robust than that conferred by full-length SUP-18 ( Fig . 3B ) . This finding suggests that the extracellular and transmembrane domains of SUP-18 are not essential for its in vivo function and is consistent with the conclusion that the NADH oxidase/flavin reductase domain is intracellular . The overexpression of sup-18 ( + ) from a Pmyo-3 sup-18 ( + ) transgene in sup-18 ( n1033 ) : sup-10 ( n983gf ) mutants not only restored the rubberband Unc phenotype but also apparently enhanced that phenotype beyond that of sup-10 ( n983gf ) single mutants ( Fig . 3B ) . This finding indicates a dose-dependent effect of sup-18 ( + ) and is consistent with our gene-dosage observation that sup-18 ( lf ) /+ can partially improve the locomotory rate of sup-10 ( n983gf ) mutants ( Table 2 ) . Overexpression of sup-18 ( + ) with the coinjection marker lin-15 ( + ) in lin-15 mutant animals did not cause obvious differences in locomotion compared to animals injected with lin-15 ( + ) alone ( Table 4 ) , indicating that overexpression of sup-18 ( + ) itself did not slow locomotion . We introduced the extrachromosomal arrays containing the transgenes from two independently-derived strains carrying sup-18 ( + ) and the lin-15 ( + ) coinjection marker into sup-10 ( n983gf ) lin-15 double mutants by mating , so that each resulting strain would contain the same transgenes as the parental strain and therefore would overexpress sup-18 ( + ) at equivalent levels . sup-18 ( + ) overexpression caused a severe paralysis of sup-10 ( n983gf ) lin-15 animals relative to control transgenic animals expressing lin-15 alone ( 0 . 1 and 0 . 0 vs . 5 . 7 and 5 . 4 , bends/minute , respectively ) ( Table 4 ) . sup-10 ( n983gf ) mutants overexpressing sup-18 ( + ) were smaller in size ( Fig . 4A–D ) and resembled severely paralyzed mutants carrying a sup-9 ( n1550gf ) mutation ( compare Figs . 4B and 4D ) . We next tested if overexpression of sup-9 ( + ) , unc-93 ( + ) or sup-10 ( n983gf ) itself could enhance the sup-10 ( n983gf ) defect as did overexpression of sup-18 ( + ) . Overexpression of these other genes under the control of the myo-3 promoter did not affect the locomotory rate of transgenic sup-10 ( n983gf ) mutant animals compared to animals transgenic for lin-15 alone ( Table 4 ) . These results suggest that the activity of SUP-18 might be enhanced by increased expression , while increased expression of SUP-9 , UNC-93 and SUP-10 does not increase the biological effects of these proteins . We tested if overexpression of sup-18 ( + ) could enhance the defects of unc-93 ( e1500gf ) mutants and found no obvious difference in appearance compared to control animals overexpressing lin-15 alone ( Fig . 4E , F ) . Because the locomotory rate of unc-93 ( e1500gf ) mutants transgenic for either sup-18 ( + ) or lin-15 ( + ) transgenes was zero ( Table 4 ) and an enhancement of locomotory defects could not be scored , we turned to a different aspect of the phenotype of rubberband mutants , a reduced brood size [29] . Consistent with the enhancement of locomotory defects , overexpression of sup-18 ( + ) reduced the brood size of sup-10 ( n983gf ) mutants by three-fold , from an average of 74 and 75 progeny for the two transgenic lines , to 17 and 27 , respectively ( Table 4 ) . These low brood sizes are comparable to those of severely paralyzed sup-9 ( n1550gf ) ; sup-18 ( n1030 ) mutants ( Table 4 ) . By contrast , the brood sizes of unc-93 ( e1500gf ) mutants did not change in response to sup-18 ( + ) overexpression ( 35 and 43 vs . 37 and 40 , respectively ) . Thus , the effects of sup-18 ( + ) overexpression on the locomotion and brood size of rubberband Unc mutants are gene-specific: the sup-10 ( n983gf ) phenotype is more sensitive to increased sup-18 levels than is that of unc-93 ( e1500gf ) mutants . Like sup-18 mutations , the sup-9 allele n1435 strongly suppresses the locomotory defects of sup-10 ( n983gf ) but not those of unc-93 ( e1500gf ) mutants ( Table 5 ) [29] . By contrast , null mutations in sup-9 , such as sup-9 ( n1913 ) , completely suppress the defects caused by gf mutations in both sup-10 and unc-93 ( Table 5 ) [30] , [31] . To determine if other sup-9 alleles exhibit similar gene-specific effects , we assayed 13 previously isolated sup-9 missense mutations [34] , [35] , [36] , [39] . Four sup-9 mutations that had been isolated as sup-10 ( n983gf ) suppressors and nine that had been isolated as unc-93 ( e1500gf ) suppressors all strongly suppressed unc-93 ( e1500gf ) and sup-10 ( n983gf ) defects equally well ( Table 5 ) , confirming that sup-9 ( n1435 ) represents a rare class of sup-9 mutations . The similarity of sup-18 ( lf ) mutations and sup-9 ( n1435 ) in preferentially suppressing sup-10 ( n983gf ) defects compared to those of unc-93 ( e1500gf ) mutants suggests that sup-18 ( lf ) mutations and the sup-9 ( n1435 ) mutation might act via the same mechanism . If so , n1435 might have no suppressive activity in the absence of sup-18 . Indeed , the locomotory rate of the sup-9 ( n1435 ) ; unc-93 ( e1500gf ) sup-18 ( n1030 ) triple mutant was similar to that of either the sup-9 ( n1435 ) ; unc-93 ( e1500gf ) or the unc-93 ( e1500gf ) sup-18 ( n1030 ) double mutant ( Fig . 5A ) . This effect appears to be specific for sup-9 ( n1435 ) , as a different weak sup-9 allele , n264 , was enhanced by sup-18 ( n1030 ) ( Fig . 5A ) . We also assayed the brood size of unc-93 ( e1500gf ) mutants in the presence of either or both sup-18 ( n1030 ) and sup-9 ( n1435 ) . For example , although the low brood size of unc-93 ( e1500gf ) mutants was restored to wild-type levels by the null mutation sup-9 ( n1913 ) ( Fig . 5B ) , sup-9 ( n1435 ) and sup-18 ( n1030 ) single mutations or sup-9 ( n1435 ) ; sup-18 ( n1030 ) double mutations only partially rescued the brood size of unc-93 ( e1500gf ) mutants and the double mutations acted similarly to the sup-18 ( n1030 ) single mutation ( Fig . 5B ) . As was the case for locomotion , for brood size sup-18 ( n1030 ) enhanced the effect of the weak loss-of-function allele , sup-9 ( n264 ) on unc-93 ( e1500gf ) mutants ( Fig . 5B ) . The lack of an additive effect of sup-18 ( n1030 ) and sup-9 ( n1435 ) in suppressing the locomotion and brood size defects of unc-93 ( e1500gf ) mutants suggests that sup-9 ( n1435 ) and sup-18 ( n1030 ) mutations likely act through the same pathway . To further examine this hypothesis , we tested for an additive effect between sup-18 ( n1030 ) /+ and sup-9 ( n1435 ) /+ in their suppression of the locomotory defects of sup-10 ( n983gf ) mutants . ( A test for an additive effect of sup-18 ( n1030 ) and sup-9 ( n1435 ) homozygous mutations would not be informative , as both mutations fully suppress the locomotory defect of sup-10 ( n983gf ) mutants . ) We found that sup-10 ( n983gf ) males heterozygous for either sup-9 ( n1435 ) /+ or sup-18 ( n1030 ) /+ are partially suppressed for the locomotory defects ( Fig . 5C ) . The sup-9 ( n1435 ) /+; sup-18 ( n1030 ) /+; sup-10 ( n983gf ) male triple mutant moved only slightly better than sup-9 ( n1435 ) /+; sup-10 ( n983gf ) mutants ( 23 . 7±0 . 6 vs . 21 . 6±0 . 9 , mean ± SEM , respectively ) ( Fig . 5C ) , suggesting a very weak additive effect of sup-18 ( n1030 ) /+ and sup-9 ( n1435 ) /+ . [This small effect might be caused by the presence in these animals of wild-type SUP-9 dimers at a fourth the wild-type level; this SUP-9 would respond to sup-18 ( n1030 ) /+ effects . ] To verify the specificity of the interaction between sup-18 ( n1030 ) and sup-9 ( n1435 ) , we tested sup-9 ( n264 ) . sup-9 ( n264 ) /+ is as strong as sup-9 ( n1435 ) /+ in suppressing the locomotory defects of sup-10 ( n983gf ) mutants . However , unlike sup-9 ( n1435 ) /+; sup-18 ( n1030 ) /+; sup-10 ( n983gf ) mutants , sup-9 ( n264 ) /+; sup-18 ( n1030 ) /+; sup-10 ( n983gf ) mutants moved better than sup-9 ( n264 ) /+; sup-10 ( n983gf ) mutants ( 28 . 5±0 . 5 vs . 21 . 3±0 . 6 bends/minute , mean ± SEM , respectively ) ( Fig . 5C ) . This result is consistent with the finding that sup-18 ( n1030 ) and sup-9 ( n1435 ) lack an obviously additive effect in suppressing the locomotion and egg-laying defects of unc-93 ( e1500gf ) mutants ( Fig . 5A and B ) and supports our conclusion that sup-9 ( n1435 ) and sup-18 ( lf ) alleles act in the same pathway in affecting rubberband Unc mutants . We determined the sup-9 coding sequences in sup-9 ( n1435 ) mutants and identified a C-to-T transition within codon 292 , leading to a serine-to-phenylalanine substitution within the predicted intracellular C-terminal domain of SUP-9 ( Fig . 6A ) . Although SUP-9 is 41%–47% identical in amino acid sequence over its entire region to several TASK-family two-pore domain K+ channels [28] , the C-terminal cytoplasmic domain of SUP-9 is poorly conserved among these channels ( Fig . 6A ) . However , the serine affected by the n1435 mutation is located in a small conserved stretch of amino acids with the sequence SxxSCxCY ( Fig . 6A ) . We named this region the SC ( Serine-Cysteine-rich ) -box . The residues in the SC-box do not correspond to any reported motifs , including phosphorylation sites , as defined by the protein motif database PROSITE [47] . Variations of the SC-box are found in the human TASK-1 and TASK-3 channels and in two Drosophila two-pore domain K+ channels ( Fig . 6A ) . We have not found an SC Box in other human two-pore domain K+ channels ( I . de la Cruz and H . R . Horvitz , unpublished observations ) or in TWK-4 ( C40C9 . 1 ) , a C . elegans two-pore domain K+ channel that is 41% identical to and the most closely related C . elegans channel to SUP-9 ( Fig . 6A ) . To determine if other residues in the SC-box of SUP-9 might function like the S292F substitution , we performed an in vivo mutagenesis study of the SC-box . We mutated residues S289 , C293 , C295 and Y296 to alanine individually and compared their effects in suppressing the egg-laying defects of the sup-10 ( n983gf ) and unc-93 ( e1500gf ) sup-18 ( n1030 ) double mutants . When assayed over a 3 hr period , both mutant strains laid fewer than three eggs , and a sup-9 ( n1913 ) null mutation drastically increased egg laying by both strains ( Fig . 6B , C ) . As a control , overexpression of a sup-9 ( + ) cDNA driven by the myo-3 promoter ( Pmyo-3 sup-9 ( + ) ) in either sup-10 ( n983gf ) or unc-93 ( e1500gf ) sup-18 ( n1030 ) mutants did not increase egg-laying in each of three independent transgenic lines . By contrast , overexpression of a sup-9 cDNA containing the n1435 mutation ( Pmyo-3 sup-9 ( n1435 ) ) dominantly suppressed the egg-laying defects of sup-10 ( n983gf ) mutants ( Fig . 6B ) but not those of unc-93 ( e1500gf ) sup-18 ( n1030 ) animals ( Fig . 6C ) . These results establish an in vivo assay for identifying mutations in sup-9 that preferentially suppress sup-10 ( n983gf ) over unc-93 ( e1500gf ) mutations . A Pmyo-3 sup-9 ( S289A ) and a Pmyo-3 sup-9 ( Y296A ) transgene suppressed the defects of sup-10 ( n983gf ) mutants but not of unc-93 ( e1500gf ) sup-18 ( n1030 ) mutants , suggesting that the S289A and Y296A mutations act similarly to n1435 to mediate the gene-specific effects of sup-18 ( lf ) mutations . By contrast , the cysteine-to-alanine mutations at residues 293 and 295 of SUP-9 suppressed both sup-10 ( n983gf ) and unc-93 ( e1500gf ) sup-18 ( n1030 ) mutants ( Fig . 6B , C ) . We suggest that these mutations when overexpressed have a dominant-negative effect on the wild-type sup-9 allele . To further understand how its C-terminal domain affects SUP-9 activity , we deleted in the sup-9 cDNA the region encoding the SUP-9 C-terminal cytoplasmic domain . We also replaced this region with the corresponding region of twk-4 , which encodes a two-pore domain K+ channel without an SC-box , or of TASK-3 , a mammalian homolog that contains an SC-box ( Fig . 6 ) . Deletion of the SUP-9 C-terminal domain caused suppression of both the sup-10 ( n983gf ) and unc-93 ( e1500gf ) sup-18 ( n1030 ) mutant phenotypes , suggesting that the truncated form of SUP-9 acts as a dominant-negative protein . Interestingly , both the sup-9::twk-4 and sup-9::TASK-3 fusion transgenes suppressed the sup-10 ( n983gf ) egg-laying defect ( Fig . 6B ) but failed to suppress that of the unc-93 ( e1500gf ) sup-18 ( n1030 ) mutants ( Fig . 6C ) , suggesting that these fusion transgenes act similarly to sup-9 ( n1435 ) and affect rubberband Unc mutants in a gene-specific manner . To identify more sup-9 mutations that act similarly to sup-9 ( n1435 ) , we performed a genetic screen for mutations that semidominantly suppressed the sup-10 ( n983gf ) rubberband phenotype ( see Materials and Methods ) . We isolated eight mutations of sup-9 that define seven novel alleles ( n3975 ( n4265 ) , n3976 , n3977 , n3935 , n4259 , n4262 and n4269 ) ( Fig . 7A ) and three additional mutations ( n3942 , n4253 , n4254 ) that contained the same C-to-T transition and therefore caused the same S292F substitution as sup-9 ( n1435 ) . As heterozygotes , five of the seven novel alleles ( n3977 , n3935 , n4259 , n4262 , n4269 ) were stronger suppressors of sup-10 ( n983gf ) mutants like sup-9 ( n1435 ) /+ ( ∼23 bends/minute ) , while the other two ( n3975 , n3976 ) were weaker ( Fig . 7B ) . These mutations affect six different regions of SUP-9 ( Fig . 7A ) , including the first ( n3975 ) and second ( n3977 ) transmembrane domains , the first pore domain ( n3976 ) , the beginning of the C-terminal cytoplasmic domain ( n3935 ) , the SC-box ( n4259 and n4262 ) , and a region C-terminal to the SC-box ( n4269 ) To determine if these novel sup-9 mutations conferred resistance to sup-18 activation or if they were simply dominant-negative lf mutations , we tested their responsiveness to changes in sup-18 levels in a similar manner to that used for testing sup-9 ( n1435 ) ( Table 2 and Fig . 5 ) . By comparing the locomotion of sup-9 ( mut ) /+; sup-18 ( n1030 ) /+; sup-10 ( n983gf ) mutants to that of sup-18 ( n1030 ) /+; sup-10 ( n983gf ) mutants , we found that sup-9 ( n3935 ) /+ , sup-9 ( n4259 ) /+ , sup-9 ( n4262 ) /+ and sup-9 ( n4269 ) /+ caused a weak effect similar to that by sup-9 ( n1435 ) /+ , while n3975/+ , n3976/+ and n3977/+ caused a significant improvement in locomotory rate in response to a change in sup-18 levels ( Fig . 7B ) . This result suggests that the channels generated by the three mutations n3975 , n3976 and n3977 have impaired ability to generate K+ currents but retain regulation by SUP-18 . In addition to its sup-18 insensitivity , sup-9 ( n1435 ) was also a weak suppressor of the unc-93 ( e1500gf ) locomotory defect , while the null mutation sup-9 ( n1913 ) completely suppressed the unc-93 ( gf ) defect ( Tables 1 and 5 ) . Similarly , sup-9 ( n4259 ) , sup-9 ( n4262 ) and sup-9 ( n4269 ) only weakly suppressed the locomotory defects of unc-93 ( e1500gf ) animals ( Fig . 7C ) , suggesting that these mutations belong to the class of sup-9 alleles defined by sup-9 ( n1435 ) . However , sup-9 ( n3935 ) completely suppressed the locomotory defects of unc-93 ( e1500gf ) animals ( Fig . 7C ) , indicating that sup-9 ( n3935 ) was not only insensitive to sup-18 but also resistant to the activating effects of unc-93 ( e1500gf ) . Thus , mutations affecting different residues of SUP-9 confer differential channel sensitivity to its regulatory subunits .
Two-pore domain K+ channels are widely expressed and play important roles in regulating resting membrane potentials of cells [15] , [17] . However , very little is known about protein factors with which these channels interact . We previously identified UNC-93 and SUP-10 as presumptive regulatory subunits of the SUP-9 two-pore domain K+ channel . We now suggest that SUP-18 also regulates the SUP-9/UNC-93/SUP-10 channel complex . sup-18 encodes the C . elegans ortholog of mammalian iodotyrosine deiodinase ( IYD ) , which belongs to the NADH oxidase/flavin reductase superfamily [7] , [8] . By oxidizing NADH using flavin mononucleotide ( FMN ) as a cofactor , IYD catalyzes the recycling of iodide from monoiodotyrosine and diiodotyrosine , two major byproducts in the synthesis of thyroid hormones [7] , [8] . Lack of IYD function can lead to congenital hypothyroidism [12] , [13] . In C . elegans , no SUP-18 function besides regulating the SUP-9 channel has been identified . The enzymatic activity of SUP-18 remains to be defined . Little is known about the metabolism and function of iodide in nematodes . The C . elegans genome contains two genes , ZK822 . 5 and F52H2 . 4 , that encode homologs of the mammalian sodium/iodide symporter , which enriches iodide in the thyroid cells by active membrane transport [48] . The presence of both SUP-18 IYD and sodium/iodide symporter-like proteins suggests that iodide functions biologically in C . elegans . Although iodide appears not to be an essential trace element in the culture medium of C . elegans [49] , it is possible that residual iodide in components of that medium can provide sufficient nutritional support for survival . C . elegans lacks homologs of mammalian iodothyronine deiodinase ( I . de la Cruz , L . Ma and H . R . Horvitz , unpublished observations ) , enzymes that remove the iodine moieties from the precursor thyroxine ( T4 ) and generate the more potent thyroid hormone 3 , 5 , 3′-triiodothyronine [50] , which suggests that thyroid hormones might not be synthesized in C . elegans . IYDs across metazoan species share a similar enzymatic activity in reductive deiodination of diiodotyrosine [51] , and it seems likely that SUP-18 acts similarly in C . elegans . Like mammalian IYDs , SUP-18 contains a presumptive N-terminal transmembrane domain that is required for full activity . Interestingly , the SUP-18 intracellular region lacking the transmembrane domain could still partially activate the SUP-9 channel , suggesting that membrane association is not absolutely required for SUP-9 activation by SUP-18 . Membrane association is important for mammalian IYD enzymatic activities [5] , [52] , [53] . The presence of a transmembrane domain suggests that SUP-18 IYD might interact with other transmembrane proteins . The genetic interactions we observe between sup-18 and the genes that encode the SUP-9/UNC-93/SUP-10 two-pore domain K+ channel complex support this hypothesis . Based on expression studies , we conclude that SUP-18 and SUP-10 localize to similar subcellular structures within muscle cells , further supporting the idea that SUP-18 and the channel complex interact physically . We found that transgenic expression of the SUP-18 intracellular domain could enhance the expression of the rubberband phenotype , suggesting that plasma membrane localization is not essential for SUP-18 function . Nonetheless , the expression of the full-length SUP-18 was more potent than the expression of the SUP-18 intracellular domain in rescuing the rubberband Unc phenotypes of sup-18 ( lf ) ; sup-10 ( n983gf ) mutants , suggesting that the presence of a transmembrane domain in SUP-18 IYD could enhance the activity of SUP-18 by targeting SUP-18 to the plasma membrane . The crystal structure of mouse IYD reveals that eight residues contact the FMN cofactor: R96 , R97 , S98 , R100 , P123 , S124 , T235 and R275 [54] . Except T235 , which is replaced by a serine in SUP-18 , these residues are completely conserved ( Figure 1C , yellow boxes ) . Furthermore , the sup-18 ( n1010 ) missense mutation leads to an S137N substitution at the position equivalent to the mouse S98 residue , likely disrupting the binding of FMN . This high degree of conservation at the cofactor binding site suggests that SUP-18 likely retains the ability to bind FMN and likely has a catalytic activity . Three IYD missense mutations that cause hypothyroidism ( R101W , I116T , and A220T ) affect residues that are conserved in SUP-18 [12] , [55] ( Fig . 1C , red boxes ) . A fourth human mutation replaces F105 and I106 with a leucine [8] . The phenylalanine at position 105 is conserved in SUP-18 ( Fig . 1C ) . The conservation of residues associated with IYD function supports the hypothesis that SUP-18 regulates the SUP-9 two-pore domain K+ channel complex via an enzymatic activity . The SUP-18 substrate remains to be elucidated . That SUP-18 might function as a NADH oxidase/flavin reductase raises the intriguing possibility that SUP-18 might couple the metabolic state of muscle cells with membrane excitability . Mammalian Kvβ voltage-gated K+ channel regulatory subunits [56] , which belong to the aldo-keto reductase superfamily [57] , [58] , have similarly been proposed to couple metabolic state with cell excitability based on indirect evidence . Kvβ2 has an NADP+ cofactor bound in its active site and a catalytic triad spaced appropriately to engage in enzymatic activity [58] . Although suggestive of an enzymatic activity , no substrate has been reported for Kvβ subunits . While Kvβ2 knockout mice have seizures and reduced lifespans , mice carrying a catalytic null mutation in Kvβ2 have a wild-type phenotype , suggesting that if an enzymatic activity for Kvβ2 exists , it is functionally dispensable in vivo [59] . By contrast , the predicted catalytic mutation sup-18 ( n1010 ) behaves like a null mutation in its inability to activate the SUP-9 channel , even though the SUP-18 ( n1010 ) protein is synthesized and localized normally to the cell surface of muscle cells ( I . de la Cruz and H . R . Horvitz , unpublished observations ) . Five other sup-18 mutations affecting highly conserved residues in the NADH oxidase/flavin reductase domain also behave like null mutations , consistent with the hypothesis that SUP-18 enzymatic activity is essential for its function . sup-18 ( lf ) mutations strongly suppress sup-10 ( n983gf ) mutants and weakly suppress unc-93 ( e1500gf ) mutants . Certain specific mutations of sup-9 , including n1435 , n4259 , n4262 , and n4269 , act similarly to sup-18 ( lf ) and are strong suppressors of sup-10 ( n983gf ) mutants and weak suppressors of unc-93 ( e1500gf ) mutants . Together these sup-9 mutations and sup-18 ( lf ) mutations represent a novel class of mutations that exhibit gene-specific suppression of the rubberband Unc mutants and are distinct from another class of gene-specific suppressors we identified previously , mutations in three splicing factor genes that strongly suppress unc-93 ( e1500gf ) and sup-10 ( n983gf ) but do not obviously suppress unc-93 ( n200gf ) or sup-9 ( n1550gf ) [60]–[62] . The difference between sup-18 ( lf ) and sup-9 ( n1435 , n4259 , n4262 , n4269 ) mutations and the splicing factor mutations in their patterns of suppressing the rubberband Unc mutants suggests that these two classes of suppressors function by distinct mechanisms . SUP-9 is closely related to the subfamily of two-pore domain K+ channels that include human TASK-1 and TASK-3 [28] . TASK-1 is activated by multiple factors , including extracellular pH [22] , [23] , [63] , inhalational anesthetics such as halothane [24] and oxygen [64] . TASK-1 is directly inhibited by sub-micromolar levels of the cannabinoid neurotransmitter anandamide [65] and by neuromodulators such as thyrotropin releasing hormone ( TRH ) [27] . A histidine residue in the first P-domain of TASK-1 modulates its sensitivity to pH [66] , while a six amino acid stretch following its fourth transmembrane domain is required for both halothane activation and TRH suppression [24] , [67] . Deletion of the TASK intracellular C-terminal domain , which contains the SC-box , does not change its basal activity or activation by halothane [24] , [67] , suggesting that the TASK-1 C-terminal domain and probably the SC-box represent an activation region that is required by some types of channel activator ( e . g . , human IYD ) but not by others ( e . g . , halothane and pH ) . It remains to be determined whether IYD is involved in the inhibition of TASK-1 channel activity by TRH . From our genetic analysis of the sup-9 ( n1435 ) mutation and site-directed mutagenesis of sup-9 , we have defined the SC-box , a domain of SUP-9 required for SUP-10 ( n983gf ) -specific activation . The importance of the SC-box in mediating this activation is supported by the results of a genetic screen in which we isolated additional sup-9 mutations ( Fig . 7 ) that act like sup-9 ( n1435 ) and cause distinct amino acid changes in ( n4259 ( S292A ) , n4262 ( S294A ) ) or near ( n4269 ( L303P ) ) the SC-box . Although conserved in the human TASK-1 and TASK-3 channels ( Fig . 6A ) , no function has yet been assigned to the SC-box . Our analyses suggest that the SC-box and the C-terminal domain of SUP-9 likely mediate the functional interaction between SUP-9 and SUP-10/SUP-18 but are dispensable for interaction with UNC-93 . We found that replacing the C-terminal domain of SUP-9 with the corresponding region of TWK-4 ( which lacks an SC-box ) or of TASK-3 ( with an SC-box ) makes the fusion channels behave like SUP-9 ( n1435 ) , consistent with the model that the SC-box is required for SUP-9 activation by SUP-10 ( n983gf ) and SUP-18 ( based on the TWK-4 data ) and suggests that SC-box-dependent activation requires one or more nearby residues in the C-terminal domain ( based on the TASK-3 data ) . The unc-93 ( e1500gf ) mutation results in a glycine-to-arginine substitution at amino acid 388 in one of the putative transmembrane domains [33] , suggesting that the UNC-93 ( gf ) protein activates SUP-9 through an interaction involving transmembrane domains , without a need for the SC-box or the rest of the cytoplasmic domain . We describe three important properties of the unusual sup-9 ( n1435 ) mutation . First , SUP-9 ( n1435 ) channels cannot be activated by SUP-10 ( n983gf ) . Second , SUP-9 ( n1435 ) channels are insensitive to SUP-18 activity . Third , SUP-9 ( n1435 ) channels can be activated by UNC-93 ( e1500gf ) . The existence of a channel mutation that is insensitive to both SUP-18 and SUP-10 ( n983gf ) suggests that these two inputs act through a common pathway . A mutant channel that can be activated by UNC-93 ( e1500gf ) but not by SUP-10 ( n983gf ) suggests that there is an independent pathway for SUP-9 activation by UNC-93 . We propose a model to explain the functional interactions between SUP-18 and SUP-9/UNC-93/SUP-10 ( Fig . 8 ) . In this model , SUP-10 and UNC-93 have an essential role in and are both required for activating SUP-9 channel , since the n1550 gf mutation in sup-9 is completely suppressed by sup-10 ( lf ) and unc-93 ( lf ) mutations [38] . SUP-18 activates SUP-9 only weakly and relies on SUP-10 for this activation ( Fig . 8 ) . SUP-10 ( n983gf ) enhances the activity of SUP-18 and results in over-activation of SUP-9 by SUP-18 . Our model is consistent with the genetic and molecular evidence described in this and previous studies [28]–[31] , [33] and should provide a framework for understanding the interactions of SUP-18 and the SUP-9/UNC-93/SUP-10 channel complex . Our results do not distinguish whether SUP-18 regulates the SUP-9/UNC-93/SUP-10 complex via a direct physical interaction or indirectly through an unknown factor or factors . In short , we identified SUP-18 IYD as a functional regulator of the SUP-9/UNC-93/SUP-10 two-pore domain K+ channel complex . We also defined an evolutionarily conserved serine-cysteine-rich domain , the SC-box , in the C-terminal region of SUP-9 and showed that this region is required for activation of the channel by SUP-18 . Since IYD is likely to be an NADH oxidase/flavin reductase that uses the ubiquitous energy carrier molecule NADH as a coenzyme , our study suggests that IYD might couple cellular metabolic state to two-pore domain K+ channel activities . Future molecular analyses should reveal the mechanism underlying the interaction between the SUP-9 two-pore domain K+ channels and SUP-18 IYD .
C . elegans strains were cultured as described [49] , except that E . coli strain HB101 was used instead of OP50 as a food source . Strains were grown at 20°C unless otherwise noted . The following mutations were used in this study: LGII sup-9 ( n213 , n233 , n264 [31] , n1016 , n1025 [30] , n1435 , n1550gf [29] , lr35 , lr38 , lr45 , lr100 , lr129 , lr142 , n1472 , n1557 , n1913 [28] , n3935 , n3942 , n3975 , n3976 , n3977 , n4253 , n4254 , n4259 , n4262 , n4265 , n4269 ( this study ) ) . LGIII unc-93 ( e1500gf , n200gf ) [31] , sma-3 ( e491 ) [49] , mec-14 ( u55 ) [68] , ncl-1 ( e1865 ) [69] , unc-36 ( e251 ) [49] . sup-18 ( n463 , n527 , n528 , n1010 , n1014 , n1015 , n1022 , n1030 , n1033 , n1035 , n1036 [30] , n1038 , n1471 , n1539 , n1548 , n1554 , n1556 , n1558 ( this study ) ) . LGX sup-10 ( n183 [31] , n1008 , n983gf [30] , n4025 , n4026 ( this study ) ) , lin-15 ( n765ts ) [70] . Since lf mutations in sup-10 completely suppress the paralysis of unc-93 ( e1500gf ) mutants [31] , we reasoned that partial lf mutations of sup-10 would partially suppress the unc-93 ( e1500gf ) locomotory phenotype . To isolate such partial lf sup-10 mutations , we performed an EMS F2 genetic screen for partial suppressors of the locomotory defects of unc-93 ( e1500gf ) mutants . From 17 , 500 haploid genomes screened , we isolated over 30 strong suppressors and seven weak suppressors . We assigned two of the seven weak suppressors , n4025 and n4026 , to the sup-10 locus by complementation tests and three others to the unc-93 locus . All seven were saved for future analyses . 34 Sma non-Unc and 23 Unc non-Sma progeny were isolated from a sma-3 mec-14 ncl-1 unc-36/sup-18 parent . Scoring of the ncl-1 and sup-18 phenotypes identified the 57 recombination events to be distributed in the three relevant intervals as follows: sma-3 ( 30/57 ) ncl-1 ( 3/57 ) sup-18 ( 24/57 ) unc-36 . A pool of cosmids C33C3 , C08C3 , C27D11 , C02C2 , C39F10 and C44C9 at 1 ng/µL each and a rol-6 marker [71] at 80 ng/µL were injected into sup-18 ( n1010 ) ; sup-10 ( n983gf ) animals . Two Rol transgenic lines were obtained , one of which generated rubberband Unc animals . The four middle cosmids were injected separately , and C02C2 yielded 5/5 rescued lines , while transgenes containing cosmids C08C3 ( 0/7 ) , C27D11 ( 0/5 ) or C39F10 ( 0/9 ) showed no rescue . RT-PCR was performed on cDNA from the wild-type N2 strain using the primers 5′-TTGAAAACCCCTGTTAAATAC-3′ and 5′-CGAGTTTCTAATAAAAATAAACC-3′ . PCR products were cloned into pBSKII ( Stratagene ) , and their sequences determined . 5′ and 3′ RACE were performed using the corresponding kits ( Gibco ) . Genomic subclones of cosmid C02C2 were generated in pBSKII ( Stratagene ) . The subclones , in the order shown in Figure 1 , spanned the following sequences ( Genebank Acc#L23649 ) : EcoRV ( 9 , 790 ) - EcoRV ( 21 , 098 ) ; PstI ( 23 , 699 ) - PstI ( 32 , 833 ) ; PstI ( 23 , 699 ) - SacI ( 28 , 185 ) ; BstBI ( 24 , 448 ) -SacI ( 28 , 185 ) ; and HindIII ( 24 , 671 ) - HindIII ( 27 , 169 ) . All PCR amplifications used in plasmid constructions were performed using Pfu polymerase , and the sequences of their products were determined . The Pmyo-3 sup-18 vectors for ectopic expression of wild-type or mutant sup-18 alleles were generated by PCR amplification of the respective coding regions from sup-18 cDNAs using primers that introduced NheI and SacI sites at the 5′ and 3′ ends , respectively , and cloned into vector pPD95 . 86 ( from A . Fire ) . Pmyo-3 sup-18 ( intra ) was similarly constructed , except that the 5′ primer began at codon 66 of sup-18 . The gfp-tagged version of this vector was created by PCR amplification of the gfp coding sequence from vector pPD95 . 77 ( from A . Fire ) and subcloned into Pmyo-3 sup-18 ( intra ) just prior to the start codon of the sup-18 sequence . Pmyo-3 mIYD ( mouse IYD ) was generated by PCR amplification of the coding region of the mouse cDNA ( Gene Bank AK002363 ) with 5′ and 3′ primers containing NheI and EcoRV sites , respectively , and subcloning the PCR products into pPD95 . 86 at the NheI and SacI ( blunted ) sites . Pmyo-3 mIYD::gfp was generated by a similar strategy using a 5′ primer containing an NheI site and a 3′ primer that did not include the stop codon of mIYD but instead contained a BamHI site . The myo-3 promoter from pPD95 . 86 was subcloned into pPD95 . 77 , such that upon subcloning of the mIYD PCR fragment into the NheI and BamHI sites of the vector the myo-3 promoter drove expression of the mIYD gene fused in-frame at its 3′ end to gfp . The sup-18::gfp genomic fusion was constructed by introducing SphI sites at the ends of a gfp cassette by PCR amplification of plasmid pPD95 . 77 ( from A . Fire ) and subsequent subcloning into the single SphI site contained within a 9 . 1 kb PstI genomic sup-18 rescuing fragment . The resulting fusion contained 6 . 5 kb of promoter sequence , the entire sup-18 coding region with gfp inserted between the transmembrane and NADH oxidase/flavin reductase domains and 1 . 1 kb of 3′ UTR and downstream sequence . The sup-10::gfp fusion used in colocalization studies was constructed by subcloning a 7 . 3 kb MfeI genomic fragment from cosmid C27G6 containing sup-10 into the EcoRI site of pBSKII . A 6 . 4 kb Pst I fragment was subcloned from this vector into pPD95 . 77 , which contained 3 . 5 kb of promoter sequence and the sup-10 coding region . Using PCR , we introduced a SalI site immediately preceding the stop codon of sup-10 to create an in-frame fusion with the gfp coding sequence . sup-18::β-galactosidase fusions were created by PCR amplification of 1869 bp of 5′ sup-18 promoter sequence and subcloning the product into the SphI and PstI sites of pPD34 . 110 ( from A . Fire ) to generate Psup-18 TM-β-Gal , which contains a synthetic transmembrane sequence [45] followed by the β-galactosidase coding sequence [72] . sup-18 genomic coding sequence spanning codons 1–42 , 1–70 and 1–301 were PCR-amplified from the minimal rescuing fragment with 5′ and 3′ primers that contained PstI and BamHI sites , respectively , and subcloned into these sites in Psup-18 TM-β-Gal . The synthetic transmembrane domain was deleted from these plasmids by excising the KpnI fragment containing this domain . A signal sequence [73] was inserted into these vectors using standard PCR techniques . The GST::sup-18 ( N ) and MBP::sup-18 ( N ) fusion genes used to generate and purify anti-SUP-18 antibodies were generated by PCR amplification of codons 1–258 of the sup-18 cDNA and subcloning the products into pGEX-2T ( Pharmacia ) and pMal-2c ( NEB ) vectors . The full-length twk-4 cDNA was cloned by RT-PCR with primers 5′-CTCTGCTAGCAATGCATCAAATTGACGGAAAATCTGC-3′ and 5′-AGAGGATCCATATAGTTCAAGATCCACCAGATG-3′ from wild-type mixed-stage RNA . The sequence of the twk-4 cDNA obtained was in agreement with its predicted sequence ( GenBank Acc#AF083646 ) . The C-terminal cytoplasmic domain of sup-9 from the Pmyo-3 sup-9 vector ( codons 257–329 of sup-9 ) was replaced by twk-4 codons ( 265–365 ) using standard PCR ligation techniques to generate Pmyo-3 sup-9::twk-4 . Site-directed mutagenesis of the SC-box in the Pmyo-3 sup-9 vector was likewise performed . Young adults were individually picked to plates with HB101 bacteria , and body-bends were counted for one minute using a dissecting microscope as described [74] . A GST::SUP-18 ( N ) fusion protein was expressed in E . coli and the insoluble protein was purified by SDS-PAGE and used to immunize rabbits . Antisera were purified by binding to the MBP::SUP-18 protein immobilized on nitrocellulose strips and elution with 100 mM glycine-HCl ( pH 2 . 5 ) . This antibody could detect SUP-18 overexpressed in the body-wall muscles ( Fig . 2H ) but failed to detect endogenous SUP-18 . For immunofluorescence experiments , worms at mixed stages were fixed in 1% paraformaldehyde for 2 hrs at 4°C and permeabilized as described [75] . For colocalization studies , transgenic worms were stained with primary antibodies at 1∶200 dilution and a secondary goat-anti-rabbit antibody conjugated with Texas Red ( Jackson Labs ) . Worms were viewed using confocal microscopy . Germline transformation experiments were performed using standard methods [71] . Transgenic strains carrying the lin-15 ( n765ts ) mutation contained the coinjection marker pL15EK ( lin-15 ( + ) ) at 50 ng/µL [70] , and transgenic animals were identified by their non-Muv phenotype at 22 . 5°C . The dominant rol-6 plasmid [71] was used at 100 ng/µl during cosmid rescue experiments , and transgenic animals were identified by their Rol phenotype . The dominant myo-3::gfp fusion vector pPD93 . 97 ( from A . Fire ) was used where indicated at 80 ng/µl , and transgenic animals were identified by GFP fluorescence . Experimental DNA was injected at 30–50 ng/µl . One plausible genetic strategy for isolating sup-9 alleles similar to sup-9 ( n1435 ) would be to perform an F2 screen for suppressors of the sup-10 ( n983gf ) locomotory defect and then test these suppressors for their effects on the locomotory defect of unc-93 ( e1500gf ) mutants . Most sup-9 alleles isolated from such a screen would be typical lf alleles rather than rare alleles that would result in a SUP-9 protein specifically impaired in activation by SUP-10 ( gf ) and SUP-18 ( + ) . We therefore opted for an alternative strategy based on the semidominance of the sup-9 ( n1435 ) mutation . While sup-9 null mutations , such as n1913 , recessively suppress the locomotory defects of sup-10 ( n983gf ) mutants , sup-9 ( n1435 ) caused a strong semidominant suppression ( Fig . 5C ) . As two-pore domain K+ channels are homodimers [66] , [76] , this semidominance likely reflects the formation of nonfunctional heterodimers composed of n1435 and wild-type SUP-9 proteins . The strength of this semidominance ( ∼23 vs . ∼5 bends/minute for sup-9 ( n1435 ) /+; sup-10 ( n983gf ) vs . sup-10 ( n983gf ) mutants , respectively ) formed the basis of an F1 screen for suppressors of the sup-10 ( n983gf ) locomotory defect . sup-10 ( n983gf ) L4 hermaphrodites were mutagenized with EMS , and approximately 550 , 000 F1 progeny ( 1 . 1×106 genomes ) were screened for improved locomotion on agar plates . From 89 candidate suppressors , 35 mutants retested in the next generation , representing at least 31 independent isolates . To quantify the semidominant character of these mutants ( sup ( new ) ) , wild-type males were crossed with homozygous mutant hermaphrodites to generate sup ( new ) /+; sup-10 ( n983gf ) /0 males , and their locomotory rate was scored . Because sup-10 is on the X chromosome , this strategy generates males hemizygous for sup-10 ( n983gf ) while heterozygous for autosomal mutations , providing a convenient assay of semidominance . Four mutations completely suppressed the rubberband Unc phenotype of males , with locomotory rates very similar to that of wild-type animals ( ∼33 bends/minute ) . We reasoned that these four mutants were likely lf alleles of sup-10 , as such animals would be hemizygous for sup-10 . We confirmed this assignment by determining the sequences of the sup-10 locus and found mutations in all four strains ( I . de la Cruz and H . R . Horvitz , unpublished observations ) . For the remaining strong mutants , we performed complementation tests with sup-9 , sup-18 and unc-93 strains and identified 11 semidominant alleles of sup-9 ( see Results ) . | Iodotyrosine deiodinase ( IYD ) controls the recycling of iodide in the biogenesis of thyroid hormones that regulate metabolism . Defects in IYD result in congenital hypothyroidism , a multisystem disorder that can lead to growth failure and severe mental retardation . We identified the gene sup-18 of the nematode Caenorhabditis elegans as a regulator of the SUP-9/UNC-93/SUP-10 two-pore domain potassium channel complex and showed that SUP-18 is an ortholog of IYD , a member of the NADH oxidase/flavin reductase family . SUP-18 IYD is required for the activation of the channel complex by a gain-of-function mutation of the SUP-10 protein . SUP-9 channel activation by SUP-18 requires a conserved serine-cysteine-rich region in the C-terminus of SUP-9 and is independent of the function of the conserved multi-transmembrane protein UNC-93 . We propose that SUP-18 uses NADH as a coenzyme to activate the SUP-9 channel in response to the activity of SUP-10 and the metabolic state of muscle cells . | [
"Abstract",
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] | 2014 | The Caenorhabditis elegans Iodotyrosine Deiodinase Ortholog SUP-18 Functions through a Conserved Channel SC-Box to Regulate the Muscle Two-Pore Domain Potassium Channel SUP-9 |
LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase ( AMPK ) and other AMPK-related kinases , including the salt-inducible kinases ( SIKs ) . However , the roles and regulation of LKB1 in lipid metabolism are poorly understood . Here we show that Drosophila LKB1 mutants display decreased lipid storage and increased gene expression of brummer , the Drosophila homolog of adipose triglyceride lipase ( ATGL ) . These phenotypes are consistent with those of SIK3 mutants and are rescued by expression of constitutively active SIK3 in the fat body , suggesting that SIK3 is a key downstream kinase of LKB1 . Using genetic and biochemical analyses , we identify HDAC4 , a class IIa histone deacetylase , as a lipolytic target of the LKB1-SIK3 pathway . Interestingly , we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions . In short-term fasting , the adipokinetic hormone ( AKH ) pathway , related to the mammalian glucagon pathway , inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation , and consequently induces HDAC4 nuclear localization and brummer gene expression . However , under prolonged fasting conditions , AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses . We also identify that the Drosophila insulin-like peptides ( DILPs ) pathway , related to mammalian insulin pathway , regulates SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity . Overall , these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in Drosophila .
Perturbation of energy homeostasis either directly or indirectly causes human health problems such as obesity and type II diabetes [1] . Lipid stores are the major energy reserves in animals and are dynamically regulated by alternating between the lipogenesis and lipolysis cycles in response to food availability . Dissecting the regulatory mechanisms of lipid homeostasis is therefore essential to our understanding of how energy metabolism is maintained . Drosophila has emerged as a useful genetic model organism for studying lipid homeostasis and energy metabolism [2] . Drosophila lipid reserves are mainly stored as triacylglycerol ( TAG ) in the fat body , the insect equivalent of mammalian adipose tissue . In addition , lipolytic factors are evolutionarily conserved between insects and mammals . Brummer ( Bmm ) is the Drosophila homolog of ATGL , a key regulator of lipolysis . bmm mutant flies are obese and display partial defects in lipid mobilization [3] . Furthermore , hormonal regulation of lipid metabolism is also highly conserved in Drosophila . Under starvation conditions , the primary role of AKH , the functional analogue of glucagon and β-adrenergic signaling in mammals [4 , 5] , is to stimulate lipid mobilization by activating AKH receptor ( AKHR ) [6] and consequently inducing cAMP/PKA signaling in the fat body [7] . A report demonstrated that AKH acts in parallel with Bmm to regulate lipolysis and that AKHR mutation leads to obesity phenotypes and defects in fat mobilization [7] . However , bmm expression is hyperstimulated in starved AKHR mutants [7] , implying the existence of an unknown regulatory mechanism between Bmm and AKHR in Drosophila . LKB1 ( liver kinase B1 , also known as STK11 ) is a serine/threonine kinase that was first identified as a tumor suppressor gene associated with Peutz-Jeghers syndrome [8 , 9] . LKB1 phosphorylates and activates AMP-activated protein kinase ( AMPK ) in response to cellular energy status , thus controlling cell metabolism , cell structures , apoptosis , etc . [10–13] . Moreover , LKB1 is the master upstream protein kinase for 12 AMPK-related kinases , including salt-inducible kinases ( SIKs ) [14] , suggesting that it plays diverse roles . Although the metabolic functions of AMPK have been highly studied , the in vivo functions of LKB1 and AMPK-related kinases in metabolism , including lipid homeostasis , are still largely unknown [15] . Recent reports showed that LKB1 is required for the growth and differentiation of white adipose tissue [16] and that SIK3 maintains lipid storage size in adipose tissues [17] . In addition , we and others determined that Drosophila SIK family kinases regulate lipid levels and starvation responses [18 , 19] . However , to further understand the roles and mechanisms of LKB1 signaling in lipid metabolism , proper genetic animal models are urgently required . Here we demonstrate the role of LKB1 and its downstream SIK3 in the regulation of lipid homeostasis using Drosophila as an in vivo model system . We demonstrated that LKB1-activated SIK3 regulates the nucleocytoplasmic localization of HDAC4 to control lipolytic gene expression . We also identified that DILPs modulate SIK3 activity via Akt-dependent phosphorylation and the AKH pathway regulates LKB1 activity in phosphorylating SIK3 to control its lipolytic responses upon short-term fasting . Furthermore , we identified that AKH-independent signaling modulates the LKB1-SIK3-HDAC4 pathway upon prolonged fasting . Altogether , these studies showed that the LKB1-SIK3 signaling pathway plays a crucial regulatory role in maintaining lipid homeostasis in Drosophila .
LKB1 functions in a complex with two scaffolding proteins , STE20-related adaptor ( STRAD ) and mouse protein 25 ( MO25 ) [20 , 21] . As the first step toward elucidation of the role of LKB1 in lipid metabolism , we demonstrated the gene expression of each component of the LKB1 complex in the fat body ( Fig 1A ) , suggesting that Drosophila LKB1 forms the heterotrimeric complex when activated in tissues . Additionally , we characterized an LKB1-null mutant line , LKB1X5 [22] , and found that these flies showed markedly decreased lipid storage compared to wild-type flies , despite having similar food intake and retaining expression of the lipogenic genes ( SREBP , FAS , and ACC ) ( Figs 1B , 1C and S1A ) . However , expression of bmm and lipolysis activity were elevated in LKB1X5 mutants ( Figs 1C and S1B , respectively ) . Moreover , transgenic expression of wild-type LKB1 with two different fat body drivers ( FB-Gal4 and cg-Gal4 ) rescued the decreased lipid levels and increased bmm expression phenotypes of LKB1X5 mutants , whereas expression of the kinase-dead form of LKB1 ( LKB1 K201I ) did not ( Figs 1E , 1F , S2A and S2B ) . Additionally , overexpression of LKB1 induced significant increases in the lipid levels and decreases in bmm expression in a dose-dependent manner ( S3A and S3B Fig ) . The implication behind these observations is that LKB1 plays a critical role in lipid storage in Drosophila by regulating the lipolysis pathway in a kinase activity-dependent manner . To identify the lipolytic target of LKB1 among AMPK-related kinases in Drosophila , we determined mRNA levels of SIKs and AMPK , which are heavily involved in various metabolic pathways . As shown in Fig 1D , SIK3 and AMPKalpha were more highly expressed in the fat body . Furthermore , transgenic expression of constitutively active SIK3 ( SIK3 T196E ) in the fat body rescued the lipid accumulation and bmm expression defects of LKB1-null mutants , whereas expression of constitutively active AMPK ( AMPK T184D ) or inactive SIK3 with a mutation in the LKB1 phosphorylation site ( SIK3 T196A ) failed to rescue the lipid levels of the null mutants ( Figs 1E , 1F , S4A and S4B ) . These results clearly suggest a specific role for SIK3 in the LKB1-mediated regulation of lipid storage in the fat body of Drosophila . Supporting this conclusion , overexpression of LKB1 highly augmented the phosphorylation of conserved Thr196 in SIK3 ( Fig 1G ) , but this phosphorylation was completely lost in LKB1X5 mutants ( Fig 1H ) . Drosophila SIK3 , one of the AMPK-related kinases , shares considerable sequence homology with the kinase domain of mammalian SIK3 ( Fig 2A ) . To assess the in vivo role of SIK3 , SIK3 loss-of-function mutants were generated by mobilizing the EP-element from SIK3G7844 ( Fig 2B ) . From 600 EP excision alleles , we generated SIK3Δ5–31 mutant , which lacks 2 , 476 bp ( 2R14578001~14580477 ) that encodes for the translation start site and the ATP-binding site of SIK3 ( Fig 2B and 2C ) . Confirming that SIK3Δ5–31 is a null mutant , SIK3 mRNA was not detected in the mutant ( Fig 2D ) . However , the internal gene ( CG15071 ) in the coding region of SIK3 was not affected ( Fig 2D ) . SIK3Δ5–31 mutant flies died before the mid-pupal stage and showed a decreased survival rate ( Fig 2E and 2F ) . The SIK3 null mutant also exhibited a lipodystrophic phenotype ( Fig 2G ) , and FB-Gal4-driven EGFP expression further confirmed the lean fat body phenotype of SIK3Δ5–31 mutants compared to control flies ( Fig 2H ) . Consistently , SIK3Δ5–31 mutant had decreased lipid stores despite having a similar food intake in the larval stage ( Figs 2I and S1A , respectively ) . Surprisingly , the fat body-specific expression of exogenous wild-type SIK3 rescued the lethality of SIK3Δ5–31 mutant , while the expression of a kinase-dead SIK3 ( SIK3 K70M ) failed to rescue the mutant ( Fig 2E and 2F ) . These results demonstrated that the phosphotransferase activity of SIK3 in the fat body is crucial for its function . To further investigate the role of SIK3 in lipid metabolism , we analyzed bmm gene expression in SIK3Δ5–31 mutant . Expectedly , the mutant showed markedly increased expression of bmm and increased lipase activity ( Figs 2J and S1B , respectively ) , a phenotype similar to the LKB1 null mutant . Transgenic expression of either wild-type ( SIK3 WT ) or constitutively active SIK3 ( SIK3 T196E ) in the fat body of SIK3Δ5–31 mutant resulted in full recovery of lipid levels and bmm expression compared to wild-type controls ( Figs 2K , 2L , S2C and S2D ) . In contrast , expression of either inactive SIK3 harboring a mutation in the LKB1 phosphorylation site ( SIK3 T196A ) or a kinase-dead mutant ( SIK3 K70M ) failed to rescue the SIK3Δ5–31 mutant phenotypes ( Fig 2K and2L , respectively ) . Therefore , the kinase activity of SIK3 controlled by LKB1 is critical for the lipid storage in Drosophila fat body . LKB1 and AMPK-related kinases play a major role in the inhibition of hepatic gluconeogenesis in response to high glucose levels via phosphorylation of the class IIa HDACs and the CREB co-activator CRTC [23–25] . To test whether HDAC4 or CRTC is involved in the LKB1 and SIK3 pathway , we analyzed the genetic interactions of LKB1 and SIK3 with HDAC4 and CRTC in Drosophila . We found that ablation of CRTC exacerbated the lethality of LKB1 and SIK3 null mutants ( S5A and S5B Fig ) . However , strikingly , the loss of HDAC4 rescued the lethality of SIK3 null mutants , but did not affect the lethality of LKB1 null mutants ( S6A–S6D Fig ) , suggesting that HDAC4 participates in LKB1-SIK3 signaling of Drosophila . To evaluate whether HDAC4 is crucial for the regulation of lipid storage by LKB1 and SIK3 , we expressed HDAC4 RNAi in the fat body of LKB1 and SIK3 mutants . Surprisingly , knockdown of HDAC4 in the fat body fully rescued the TAG levels and bmm gene expression of LKB1 and SIK3 null mutants ( Fig 3A and 3B , respectively ) , indicating that HDAC4 is indeed a critical downstream target of LKB1 and SIK3 in lipid metabolism of Drosophila . SIKs can regulate target gene expression by directly phosphorylating the class IIa HDACs and consequently inhibiting their translocation to the nucleus [26 , 27] . Expression of wild-type SIK3 ( SIK3 WT ) or constitutively active SIK3 ( SIK3 T196E ) augmented the phosphorylation of HDAC4 but not of the phosphorylation-defective HDAC4 ( HDAC4 3A ) , demonstrating that SIK3 induces HDAC4 phosphorylation in Drosophila ( Fig 3C ) . HDAC4 localized to both the cytoplasm and nuclei of larval fat body cells under feeding conditions , but localized mostly to the nucleus under fasting conditions ( Fig 3D ) . However , HDAC4 accumulated in the nuclei of the fat body cells of LKB1 and SIK3 null mutants even under feeding conditions ( Fig 3D ) . In addition , HDAC4 3A was retained in the nuclei of the fat body cells under both feeding and fasting conditions ( Fig 3D ) . These results indicated that Drosophila SIK3 , under the control of LKB1 , phosphorylates HDAC4 in the fat body and regulates its nucleocytoplasmic localization in different dietary conditions . The class IIa HDACs deacetylate and activate FOXO transcription factors [19 , 24] , and the activated FOXO then induces ATGL/Bmm expression [19 , 28] . Overexpression of wild-type HDAC4 increased the mRNA levels of bmm ( Fig 3E ) , indicating that HDAC4 regulates bmm gene expression in the fat body . Furthermore , overexpression of constitutively active SIK3 completely blocked the increased bmm expression induced by HDAC4 overexpression ( Fig 3E ) , and bmm knockdown in the fat body blocked the decreases in TAG levels induced by LKB1 or SIK3 null mutation ( Fig 3F and 3G ) . Altogether , these results suggested that the LKB1-SIK3 signaling pathway controls HDAC4-dependent Bmm activity in Drosophila fat body . Under fasting conditions , AKH activates the mobilization of fat body triglyceride by triggering AKHR and consequent activation of cAMP signaling in the fat body [7] . Consistently , we showed that AKHR mutation highly increased TAG levels ( Fig 4A ) and decreased bmm gene expression ( Fig 4B ) . To determine the functional interaction between AKHR signaling and the LKB1-SIK3 signaling pathway , we crossed LKB1 or SIK3 null mutant flies with AKHR mutant flies . Interestingly , deletion of LKB1 or SIK3 reversed both the lipid accumulation and the reduced bmm expression phenotypes of AKHR mutant flies ( Fig 4A and 4B , respectively ) , suggesting that the LKB1-SIK3 pathway likely acts downstream of AKHR . Furthermore , SIK3 Thr196 phosphorylation was reduced in both fasting and AKH overexpression conditions compared to that in feeding conditions ( Fig 4C ) , supporting that the AKH pathway inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation . On the basis of the observation that fasting induces the nuclear translocation of HDAC4 , we also examined subcellular localization of HDAC4 in AKHR mutant flies . Intriguingly , HDAC4 in AKHR mutants localized to both the cytoplasm and the nucleus of larval fat body cells in 4 hr fasting condition compared to control ( Figs 3D and 4D ) , suggesting that AKHR-dependent regulation is critical for HDAC4 localization . In addition , overexpression of the phosphorylation-defective HDAC4 by SIK3 ( HDAC4 3A ) suppressed the TAG levels and enhanced bmm expression in AKHR mutants ( Fig 4E and 4F , respectively ) . Collectively , these results suggested that the LKB1-SIK3-HDAC4 pathway acts downstream of the AKH pathway to control lipolysis activity in Drosophila . We showed that AKHR-dependent regulation of LKB1-SIK3 activity is critical for HDAC4 nuclear localization in ~4 hr fasting condition ( Fig 4D ) . However , Gronke et al . showed that bmm gene expression is stimulated in AKHR mutant flies in 6 hr fasting condition [7] . Notably , in contrast to 4 hr fasting condition ( Fig 4D ) , HDAC4 accumulated in the nuclei of the fat body cells in AKHR mutant flies after prolonged fasting ( ~10 hr ) ( Fig 5A ) , indicating that there should be AKHR-independent HDAC4 regulation during prolonged fasting . Furthermore , knockdown of HDAC4 in the fat body blocked the increased bmm gene expression in AKHR mutant flies after 10 hr fasting ( Fig 5B ) , indicating that AKHR-independent signaling promotes HDAC4 nuclear localization to induce bmm gene expression under prolonged fasting conditions . Interestingly , expression of constitutively active SIK3 blocked the prolonged fasting-induced nuclear localization of HDAC4 ( Fig 5C ) , suggesting that LKB1-SIK3 activity is critical for bmm expression under prolonged fasting . Taken together , these results demonstrated that the LKB1-SIK3-HDAC4 pathway acts as the primary lipolytic signaling upon both short-term and prolonged fasting while AKH plays a major role only in short-term fasting . Thus , it is of particular interest to investigate novel signaling mechanisms regulating the LKB1-SIK3-HDAC4 pathway under prolonged fasting conditions . Our study provides evidence that LKB1 is necessary for maintaining Drosophila lipid storage via the regulation of lipolysis through the activation of SIK3 . Consistent with our results in Drosophila , adipose tissue-specific LKB1 knockout mice showed decreased serum triglycerides [16] , and the basal lipogenesis activity of adipocytes was significantly lower in LKB1 hypomorphic mice [29] . Recently , SIK3 null mice were also found to display a malnourished phenotype with lipodystrophy and were resistant to high-fat diets [17] . Thus , the LKB1-SIK3 pathway is indeed an evolutionally conserved regulatory mechanism for lipid homeostasis . LKB1 is ubiquitously expressed and constitutively active in mammalian cells [15] , which raises the question of how dietary conditions change the activity of LKB1 and SIK3 to control lipid homeostasis . Our findings suggested that fasting and the AKH pathway inhibit LKB1 activity to regulate SIK3 Thr196 phosphorylation ( Figs 4C and 6C ) . It is possible that fasting- and AKH-induced inhibition of LKB1 activity can be achieved by altered subcellular localization , protein conformation , stability , and/or protein-protein interactions of LKB1 and its associated proteins . Interestingly , in HEK-293 cells , fasting triggers autophosphorylation of human LKB1 at Thr336 [30] that corresponds to Thr460 in Drosophila LKB1 [31] . This phosphorylation promotes the protein-protein interaction between LKB1 and 14-3-3 proteins [30] and inhibits the ability of LKB1 for suppressing cell growth [31] . In addition , the AKH pathway activates cAMP/PKA signaling in Drosophila [7] . Mammalian PKA inhibits SIK activity by phosphorylating a conserved serine residue [32 , 33] that corresponds to Ser563 in Drosophila SIK3 [19] , suggesting that the AKH pathway also controls SIK3 activity via PKA-dependent phosphorylation ( Fig 6C ) . On the other hand , the Drosophila insulin-like peptides ( DILPs ) did not increase SIK3 Thr196 phosphorylation ( Fig 6A ) , but induced Akt-mediated SIK3 phosphorylation ( Fig 6B ) , suggesting that DILPs directly regulate SIK3 activity independently of affecting LKB1 activity [19 , 34] ( Fig 6C ) . Interestingly , these Drosophila signaling circuits are highly similar to mammalian insulin and glucagon pathways in controlling lipid metabolism and storage , raising questions regarding whether the LKB1-SIK3-HDAC4 signaling pathway is also conserved in mammalian systems as a converging point between feeding and fasting signals to control lipid homeostasis . Is SIK3 also involved in the modulation of other LKB1 functions , such as the regulation of cell polarity and mitosis ? SIK3 null mutants showed normal epithelial polarity and mitosis ( S7A and S7B Fig ) . Additionally , transgenic expression of constitutively active SIK3 ( SIK3 T196E ) failed to suppress the cell polarity and mitosis defects of LKB1 mutants ( S8A and S8B Fig ) , suggesting that SIK3 does not participate in the regulation of cell polarity and mitosis by LKB1 . In addition , both fat body-specific expression of LKB1 and ablation of HDAC4 failed to rescue the lethality of LKB1 null mutants ( S6C Fig ) , indicating that LKB1 has SIK3/HDAC4-independent roles and additional targets in other tissues and developmental processes . In summary , we have demonstrated that the LKB1-SIK3 pathway is important for maintaining lipid homeostasis in Drosophila . As alterations in lipolysis are closely associated with human obesity [35] , future studies will be required to unravel the relationship between LKB1-SIK3-HDAC4 signaling and obesity-related metabolic diseases .
The following fly stocks were used in this study: LKB1X5 , UAS-LKB1WT , and UAS-LKB1KI [22] , HDAC4KG09091 ( Bloomington #15159 ) , UAS-HDAC4 RNAi ( VDRC #20522 ) , UAS-bmm RNAi ( Bloomington #25926 ) , UAS-InRCA ( Bloomington #15159 ) , cg-Gal4 ( Bloomington #7011 ) , hs-Gal4 ( Bloomington #1799 ) , UAS-2xEGFP ( Bloomington #6874 ) , UAS-HA-AMPKTD [12] , CRTC25-3 [36] , UAS-FLAG-HDAC4WT and UAS-FLAG-HDAC43A [19] , AKHR1 [7] , and FB-Gal4 [37] . SIK3Δ5–31 was generated by imprecise excision of SIK3G7844 line ( KAIST Drosophila Library Facility , Daejeon , Korea ) . To generate UAS-SIK3 flies , SIK3 EST cDNA ( Berkeley Drosophila Genome Project accession no . LD07105 ) was cloned into the Myc-tagged pUAST vector and microinjected into w1118 embryos . All flies were grown on food containing approximately 35 g cornmeal , 70 g dextrose , 5 g agar , 50 g dry active yeast ( Ottogi , Inc . , Korea ) , 4 . 6 ml propionic acid , and 7 . 3 ml Tegosept ( 100 g/l in ethanol ) per liter at 25°C . All flies were backcrossed for a minimum of 6 generations into w1118 background . The QuickChange kit ( Stratagene ) was used for site-directed mutagenesis . For generation of a kinase-dead mutant SIK3 ( Lys70Met , SIK3K70M ) , 5’-CAAGACAAAGGTGGCCATCATGATCATAGACAAAACATGTC-3’ and 5’-GACATGTTTTGTCTATGATCATGATGGCCACCTTTGTCTTG-3’ primers were used . For generation of a SIK3 mutant non-phosphorylatable by LKB1 ( Thr196Ala , SIK3 T196A ) , 5’-GGGTGCCACCTTAAAAGCTTGGTGTGGATCAC-3’ and 5’-GTGATCCACACCAAGCTTTTAAGGTGGCACCC-3’ primers were used . For generation of a SIK3 mutant mimicking LKB1-dependent phosphorylation ( Thr196Glu , SIK3 T196E ) , 5’-GAGGGTGCCACCTTAAAAGAATGGTGTGGATCACCGCCC-3’ and 5’-GGGCGGTGATCCACACCATTCTTTTAAGGTGGCACCCTC-3’ primers were used . Larvae were collected , and RNA was extracted using the RNeasy Mini Kit ( QIAGEN ) . Total RNA ( 1 μg ) was reverse-transcribed by M-MLV Reverse Transcriptase ( Promega ) to generate cDNA for quantitative real-time RT-PCR ( Bio-Rad CFX96 Real-Time PCR detection system , SYBR Green ) using a 500 nM primer concentration and 2 ng of cDNA template . The primers were used in S1 Table . The relative values were calculated using the ΔΔCt method via normalization to rp49 mRNA levels . Results were expressed in arbitrary units , with each control value as 1 unit . Third instar larvae were dissected in Drosophila Ringer’s solution and fixed with 4% formaldehyde in phosphate buffered saline ( PBS ) for 10 min at room temperature . After being washed with 0 . 1% Triton X-100 in PBS ( PBST ) , the samples were blocked for 1 hr incubation at room temperature with 5% bovine serum albumin ( BSA ) in PBST . The samples were further incubated at 4°C for 16 hr with the indicated antibodies: anti-FLAG-M2 ( Sigma , F1804 ) , anti-Myc ( Cell Signaling Technology , #2272 ) , anti-aPKC ( Santa Cruz , sc-216 ) , and anti-PH3 ( Millipore , 06–570 ) . Following three washes with PBST , the samples were incubated with appropriate secondary antibodies ( and with Hoechst 33258 used for staining DNA , if required ) for 3 hr at room temperature . The samples were washed with PBST and mounted with 80% glycerol in PBS , then observed by a confocal microscope LSM710 ( Zeiss ) . Feeding assay was performed according to previously described with minor modifications [38] . Blue food dye ( Erioglaucine Disodium Salt , Sigma , #861146 ) was added at 1% ( w/v ) to fly food . Larvae were switched from normal food to blue-color food for 2 hr . After feeding , larvae were frozen immediately . Samples were homogenized in PBS buffer and centrifuged for 25 min at 13 , 200 rpm . The absorbance of the supernatant was measured at 625 nm using Infinite M200 spectrophotometer ( Tecan ) . Larvae were lysed in a lysis buffer ( 20 mM Tris-HCl ( pH 7 . 5 ) , 1 mM EDTA , 5 mM EGTA , 150 mM NaCl , 20 mM NaF , 1% Triton X-100 , 1 μg/ml leupeptin , and 1mM PMSF ) for 30–60 min on ice . After centrifugation for 15 min at 13 , 200 rpm , supernatants were reserved for SDS-PAGE analysis , and proteins were then transferred to nitrocellulose membranes ( GE Healthcare , #BA85 ) . Membranes were incubated in a blocking solution ( Tris-buffered saline ( TBS ) containing 0 . 1% Tween-20 , 5% BSA ) for 1hr . The primary antibodies used were anti-LKB1 [22] , anti-phospho-Thr196 SIK3 [39] , anti-phospho-Ser239 HDAC4 ( Cell Signaling Technology , #3443 ) , anti-phospho-Akt substrate ( Cell Signaling Technology , #9614 ) , anti-FLAG-M2 ( Sigma , #F1804 ) , anti-Myc ( Cell Signaling Technology , #2272 ) , anti-AKH ( a gift from Dr . Veenstra ) , and anti-β-tubulin antibody ( Developmental Studies Hybridoma Bank , E7 ) . Protein detection was done using the LAS-4000 imaging system ( Fujifilm ) , and densitometric analysis was performed using Multi Gauge 3 . 0 software . TAG measurement was performed according to previously described methods using Free Glycerol Reagent ( Sigma , #F6428 ) and Triglyceride Reagent ( Sigma , #T2449 ) [40] . A standard curve was generated with a glycerol standard solution ( Sigma , #G7793 ) . Samples were assayed at 540 nm using Infinite M200 spectrophotometer ( Tecan ) . In each homogenate , amounts of TAG ( in mg ) were normalized to those of protein ( in mg ) using Bradford protein assay ( Bio-Rad ) . Lipase activity was determined according to the manufacturer’s instructions with QuantiChrom kit ( BioAssay Systems , DLPS-100 ) . The analysis for survival rate was performed as previously described [12] . The eggs from flies with the appropriate genotypes were laid on 60 mm dishes containing standard apple juice-agar with yeast paste for 4 hr . The hatched larvae were collected using selection markers and transferred to plates containing normal food media . The green balancer chromosome ( CyO , Actin-GFP ) was used to select the homozygous SIK3Δ5–31 mutant . The number of larvae was scored for viability at each developmental stage , and dead larvae were removed . At least 100 larvae were studied per genotype . All quantitative data are analyzed using Student’s t tests or ANOVA with a post Tukey’s multiple comparison test , and P < 0 . 05 was considered statistically significant . Each experiment was repeated at least three times , data are presented as the average ± standard error of the mean ( SEM ) . The P values given in the survival data are the result of a log rank test using GraphPad Prism 5 software . | Liver kinase B1 ( LKB1 ) , a serine/threonine kinase , controls 14 different AMP-activated protein kinase ( AMPK ) family kinases , including salt-inducible kinase 3 ( SIK3 ) , suggesting that it plays a variety of roles . Using the fruit fly as an in vivo model system , we reveal that LKB1 kinase activity is critical for lipid storage and controls the lipolysis pathway in the fat body , which is equivalent to mammalian adipose and liver tissue . We find that the lipolytic defects of LKB1 mutants are rescued by the expression of constitutively active SIK3 in the fat body . We show that LKB1 and SIK3 regulate lipid storage by altering the gene expression of brummer , the Drosophila homolog of human adipose triglyceride lipase ( ATGL ) , a critical lipolytic gene . We also identify that LKB1-SIK3 signaling controls the nuclear and cytosolic localization of the class IIa deacetylase HDAC4 via SIK3-dependent phosphorylation in feeding and fasting conditions , respectively . Collectively , these data suggest that the LKB1-SIK3-HDAC4 pathway plays a critical role in maintaining fly lipid homeostasis in response to dietary conditions . | [
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] | [] | 2015 | Feeding and Fasting Signals Converge on the LKB1-SIK3 Pathway to Regulate Lipid Metabolism in Drosophila |
Candida glabrata is one of the most common causes of candidemia , a life-threatening , systemic fungal infection , and is surpassed in frequency only by Candida albicans . Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body . Here we addressed the flexibility and adaptability of C . glabrata during interaction with macrophages with a serial passage approach . Continuous co-incubation of C . glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures – a phenotype which was stable over several generations without any selective pressure . Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture . This altered growth form permitted faster escape from macrophages and increased damage of macrophages . In addition , the evolved strain ( Evo ) showed transiently increased virulence in a systemic mouse infection model , which correlated with increased organ-specific fungal burden and inflammatory response ( TNFα and IL-6 ) in the brain . Similarly , the Evo mutant significantly increased TNFα production in the brain on day 2 , which is mirrored in macrophages confronted with the Evo mutant , but not with the parental wild type . Whole genome sequencing of the Evo strain , genetic analyses , targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration . A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested . These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C . glabrata to distinct host niches and even lead to hypervirulent strains .
Candida glabrata , like C . albicans , is both a fungal commensal and an opportunistic pathogen of humans . The fungus normally co-exists with its host without causing damage , but it can also elicit life-threatening diseases under predisposing conditions , such as prolonged hospitalization , use of central venous catheters and immunosuppression [1] . In fact , Candida species have become the third most frequent cause of nosocomial bloodstream infections , and pose a severe risk to intensive care patients and other susceptible individuals [1] . Among different Candida species , C . glabrata has risen to become the second most common cause of bloodstream infections , surpassed only by C . albicans . During infection , this fungus is able to colonize virtually all organs , reflecting a strong capacity to adapt to the many different niches inside the human host . Moreover , the rise in relative incidence is attributable in part to the ability of C . glabrata to tolerate or resist many antifungals commonly used in the clinical setting [2]–[4] . This reduced susceptibility is often due to a high intrinsic resistance of most C . glabrata strains to many antifungals [5] , which , in many cases , can be further increased by genetic and genomic mutations [6]–[8] . In vitro experiments have shown that susceptible C . glabrata strains can become resistant after less than four days of continuous culture with low doses of fluconazole [2] . Therefore , it is not surprising that a rapid acquisition of increased resistance has also been observed in vivo , when patients were treated with azoles for longer periods [3] , [4] , [9] , [10] . The main driving force for this phenomenon is microevolution . For diverse pathogens , such small-scale evolution has been shown to occur during the course of infections: it has been observed for viruses [11] , bacteria [12] , and also fungi . While macroevolution leads to new species or subspecies , microevolution generates new variants of a given species [13] . This can happen by small- to large-scale genome alterations like point mutations , loss-of-heterozygosity , translocations , and many other mechanisms . All of these may cause changes in the expression pattern or in the amino acid sequence of proteins . This kind of microevolutionary adaptation enables pathogens to ‘fine-tune’ to their current environment . Similarly , microorganisms are also known to delete large DNA segments , so-called ‘anti-virulence genes’ , which are incompatible with a pathogenic lifestyle [14] . While the adaptation of C . glabrata to antifungals is well investigated , microevolution during long-term or persistent infections has not been addressed so far . In previous experiments , we observed that C . glabrata microcolonies were regularly found in close proximity to mononuclear cells within murine tissues [15] and speculated that C . glabrata may face constant exposure to these phagocytes during infection . In fact , we and others have shown that C . glabrata can survive and replicate inside macrophages for days in vitro [16]–[19] . During commensal growth , fungi are repeatedly exposed , at least transiently , to host-induced stresses ( e . g . oxidative and nutritional stress ) . Therefore they should have the potential – as commensals or as pathogens – to adapt to these stresses and the ever-present effector cells of the immune system . In previous studies by the Casadevall group , it was postulated and shown that exposure to predatory amoeba can pre-adapt the fungus Cryptococcus neoformans to resist phagocytic immune cells [20] , [21] . Only recently , the genetic basis for a phenotypic change induced by exposure of C . neoformans to amoeba has been elucidated: there , distinct genetic alterations led to a transition from yeast- to pseudohyphal growth , accompanied by a loss in murine virulence [22] . Similarly , experimental evolution approaches in Legionella pneumophila and Escherichia coli led to an adaptation and specialization to hosting macrophages or a better escape from phagocytosis and killing , respectively [23] , [24] . Thus , our study aimed at simulating aspects of in vivo interactions , by constantly challenging C . glabrata with macrophages . We then investigated how the fungus adapts to stresses caused by this long lasting exposure to immune effector cells . While this constitutes a significantly more complex selection pressure than a single antifungal compound , we still expected C . glabrata to adapt , possibly revealing novel potential pathogenicity attributes necessary to counteract this part of the innate immune system . In our approach , we continuously exposed C . glabrata to macrophages for six months with a daily transfer of yeast cells to fresh macrophages . This microevolution experiment led to the generation of a C . glabrata variant which had evolved to a genetically stable , pseudohyphae-like growth phenotype , with a significantly increased virulence in different infection models . Here , we describe the generation of this strain , the characteristics of the evolved phenotype , and the genetic basis of the phenotypic change that allowed the adaptation to phagocytes .
In previous works , we and others have addressed the ability of C . glabrata to not only resist the conditions inside the macrophage phagosome , but to even persist and thrive inside [16]–[19] . Yet , these investigations have mainly focused on the short-term interaction between host and fungus , lasting for hours up to days . To investigate the long-term ability of C . glabrata to cope with the stresses of exposure to the phagosome , we devised a continuous co-culture model: we co-incubated the C . glabrata wild type strain ATCC 2001 ( WT ) with the murine macrophage-like cell line RAW 264 . 7 ( referred to as macrophages in the following sections ) , and transferred all phagocytosed and attached C . glabrata cells to fresh macrophages every day ( see Materials & Methods ) . The daily transfer , before substantial amounts of phagocytosed yeasts were released from the macrophages [16] , and the use of DMEM , which does not support long-term growth of non-phagocytosed C . glabrata ( not shown ) , ensured that the vast majority of the fungal population was continuously growing inside the RAW 264 . 7 cells . We continued the experiment for over six months . After nearly one month of daily transfers of 106–107 yeasts each , C . glabrata cells with an unusual morphology appeared , and then were enriched over the following weeks . ( Fig . 1A , left ) . This evolved strain of C . glabrata ( here called ‘Evo’ strain ) showed a striking alteration in growth form: it changed from the typical single spherical yeasts ( Fig . 1A , middle ) to chains of tightly connected yeasts , reminiscent of pseudohyphal growth ( Fig . 1A , right ) . Colonies on agar plates appeared strongly wrinkled instead of smooth ( Fig . 1B , left ) . We verified the strain to be C . glabrata by genetic tests and the color reaction on CHROMagar ( Fig . 1B , right ) . To exclude a temporary , reversible phenotype , like the ‘irregular wrinkle’ phenotype of the switching system of C . glabrata [25] , the Evo strain was grown for 15 passages on YPD complex medium in the absence of phagocytic cells ( not shown ) . Even under these non-selecting conditions , the wrinkled colony growth form remained . This shows that the phenotype is both genetically stable and expressed independently of the culture conditions . The ultrastructure of the evolved strain was analyzed by transmission electron microscopy ( Fig . 1C ) . Compared to the parental strain , major differences in the cell wall structure were observed: most strikingly an abnormal septum formation , with a thick layer forming between mother and daughter cell at the bud neck . The primary septum was not visible in the micrographs of the Evo strain . Additionally , the cell wall of the Evo strain appeared thicker than the wall of the parental strain , and displayed a thicker and more prominent outer layer , most likely corresponding to extracellular mannoproteins . Overall , the micrographs showed strong signs of a cytokinesis defect in the evolved strain . We were interested to know whether the appearance and expansion of the Evo strain in our original co-culture experiment was due to a high selective advantage of this strain in macrophage co-culture . Therefore , we inoculated fresh RAW 246 . 7 cells with the wild type and the evolved strain at a ratio of 100∶1 , and performed daily transfers as in the original co-culture , but with an additional sonification step to ensure similar uptake ratios of WT and Evo strain ( see below ) . After an average of 7 passages ( in three independent experiments ) , the ratio completely inverted to approximately 1∶100 wild type to evolved strain ( Fig . 2 ) . This corresponds to a mean competitive fitness ratio of 4 . 75 d−1 for the wrinkled strain under macrophage exposure . The Evo strain had been exposed to the phagosome with its prevailing stressors , such as reactive oxygen species , for a long time . We reasoned that the relative selective advantage may be based on an increased resistance to these stressors . Therefore , we tested the stress resistance of wild type and evolved yeasts in vitro , by growing them on agar plates containing either high concentrations of sodium chloride to induce osmotic stress , H2O2 or menadione to induce oxidative stress , the cell wall stressor calcofluor white or congo red , or the β- ( 1 , 3 ) -glucan synthase inhibitor caspofungin . Differences between the parental and the evolved strain were not apparent when comparing stress versus non-stress conditions ( Fig . S1 ) , excluding an altered sensitivity of the Evo strain towards all stress conditions tested here . The observed alterations in the cell wall structure of the Evo strain may also have affected recognition and/or uptake by macrophages . Therefore , we quantified the uptake rates of parental and evolved cells . The larger cell masses of the Evo strain could not be completely phagocytosed by RAW 264 . 7 macrophages . Only after sonication , single cells and smaller aggregates were internalized well . Therefore , we quantified the uptake rate as the number of macrophages that had phagocytosed sonicated parental or evolved yeast cells . We did not detect any differences between the Evo strain and the parental strain ( Fig . 3A ) . Additionally , we investigated whether the remaining aggregates in the Evo strain inoculum were taken up by macrophages less avidly than the single wild type yeast cells ( Fig . S2A–C ) . After 15 minutes , the distribution of different aggregate sizes inside the macrophages was nearly identical to the inoculum ( Fig . S2A ) , with a slight decrease in larger ( >3 yeasts ) clumps for the Evo strain . The sonication hence allowed uptake of the Evo similar to the WT strain . Still , after 15 minutes of co-incubation , there were more macrophages infected with two or more yeast cells in the Evo strain than in the WT strain . However , after six hours , these differences were severely reduced and the number of yeast cells per macrophage showed a highly similar distribution between wild type and Evo strain ( Fig . S2A&B ) . Any large differences in macrophage damage after this time point should therefore not be due to differences in the inoculum distribution . We then tested whether the evolved yeast cells were better at escaping from the macrophage phagosome after uptake . This was done by incubating sonicated evolved or parental yeast cells with macrophages , followed by measuring the release of lactate dehydrogenase ( LDH ) as a marker for host cell damage due to escaping fungi . Similarly , we co-incubated sonicated evolved or parental yeast cells with TR146 epithelial cells for 24 h and 48 h to quantify epithelial damage . Within the first 24 h , the Evo strain specifically damaged macrophages , but not epithelial cells , to a significantly higher degree than the parental strain ( Fig . 3B ) . After 48 h , the damage levels equalized , and both strains reached the same levels of damage in both types of host cells . To exclude that these differences are because of a better survival of the Evo strain due to its small aggregates in the inoculum , we compared it to a strain ( cbk1Δ , [26] ) with a similar phenotype and similarly sized aggregates ( Fig . S3A ) . This strain did not elicit the same damage as the Evo strain ( Fig . S3B ) . Another yeast-chain forming mutant ( dse2Δ , [26] ) which separated into single cells by sonication ( Fig . S3A ) similarly failed to induce Evo levels of damage to macrophages ( Fig . S3B ) . Additionally , we controlled for the possibility that even small clumps are protecting their individual yeast cells better from macrophage killing . We estimated the survival rates of single cells and cells within clumps by video microscopy and found no benefit for yeast cell survival in small clumps ( Fig . S3C ) . Hence , any remaining initial differences in aggregate distribution between the Evo and the WT strain should have no impact on yeast survival and LDH release by host cells . Altogether , this indicated that the host cell-type specific adaptation during the microevolution experiment also correlated with a host cell-type specific damage of macrophages by C . glabrata . We were next interested to know whether the Evo strain can not only damage , but also better escape macrophages due to its pseudohyphal growth form , similar to C . albicans cells producing hyphae or pseudohyphae . By using time lapse microscopy we monitored interactions of evolved yeast cells with macrophages ( Video S1 ) . Internalized Evo yeasts survive intracellularly and start to replicate within the first 24 h . Clumps of escaped yeast cells are immediately attacked by macrophages , leading to aggregates of yeasts and host cells . Finally , within 48 h , most macrophages burst and released yeast cells which continued to grow in the pseudohyphal morphology , finally overgrowing the macrophage cell layer . In contrast , the majority of yeasts of the parental strain , which was phagocytosed with an equal efficiency , were cleared by macrophages within 48 hours . Here , fewer yeasts survived and started to replicate intracellularly . But after approximately four days , the number of extracellular yeasts increased and macrophages finally died , likely due to cellular sensecence or the overgrowing fungal mass ( Video S2 ) . To gain further insights into host-pathogen interactions , we examined the evolved and the parental strain in a chicken embryo model [27] . Chicken embryos on developmental day 10 were chosen for infection , as their developing immune system is comparable to a naturally immunocompromised state . In this stage , differences in the virulence of C . glabrata variants should be best detectable . Consistent with previous results [27] , infection with the parental strain resulted in only minor killing , as after 7 days of infection more than 80–90% ( depending on experimental run ) of all embryos were still alive ( Fig . S4 ) . In contrast , the Evo strain consistently showed a moderately increased propensity to kill chicken embryos , as at the end of the observation period about 20–40% of all embryos were killed . This indicates a generally higher virulence of the Evo strain in an in ovo infection . We continued our investigation of the virulence by using our established murine model of C . glabrata infections [15] . In this intravenous infection model , C . glabrata does not cause mortality , and therefore mouse weight , fungal burden ( colony forming unit , cfu ) in different organs , and histopathological alterations are used as parameters for determining fungal virulence . The inocula as well as all samples retrieved from organs were sonicated as described above to separate cell aggregates . As expected , mice infected with the C . glabrata wild type strain remained clinically healthy throughout the experiment . In contrast , animals infected with the Evo strain displayed weight loss and unspecific symptoms of illness ( ruffled fur and moderate lethargy ) for 48 h after infection , but then recovered ( Fig . 4A ) . Coinciding with differences in clinical presentation of infected mice , the distribution of fungal burdens differed significantly between the wild type and the evolved strain at the early time point ( Fig . 4B ) . In the brain , the burden on day 2 post infection ( p . i . ) was more than 100× higher for the Evo strain than for the wild type ( median 1 . 1×107 cfu/ml vs . 4 . 9×104 cfu/ml , respectively; p<0 . 005 ) . This difference disappeared at later time points ( days 7 and 21 p . i . ) , and both strains persisted at comparable levels in the organ . Significant differences were also observed in the spleen , where the evolved strain had a lower cfu count than the wild type at day 2 and 7 p . i . ( day 2 , p<0 . 01 and day 7 , p<0 . 005 ) . Similarly , for the evolved strain there was an approximately two-log reduction in fungal burden in the liver at day 7 and 21 ( day 2 , p<0 . 01 ) , but not at day 2 . Finally , in the kidney , although no significant differences were observed , the evolved strain also showed a tendency towards lower fungal burden at day 7 and 21 [15] . Finally , we tested a different clone from the evolution experiments , bearing the same mutation in CHS2 , but also some additional SNPs distinguishing it from both Evo and WT strain . In mice , this strain elicited similar weight loss after infection , and importantly exhibited nearly identical patterns of fungal burden in the organs ( Fig . S5 ) . To explain the differences in murine virulence , we measured cytokine and myeloperoxidase levels as markers for inflammation in the different organs . In all organs with similar fungal burden of wild type and evolved strain , we found the tissue concentrations of these markers to be indistinguishable . Yet strikingly , the evolved strain induced significantly increased levels of the proinflammatory cytokines TNFα and IL-6 specifically in the brain at day two , mirroring the high fungal burden in this organ ( Fig . 5A ) . Similarly , IL-1β and the inflammation marker MPO were found at increased levels , albeit not statistically significant . This increased inflammation also coincided with the most severe clinical symptoms . At later time points , the brain cytokine levels of mice infected with either strain became similar again , paralleling the fungal burden of the evolved and parental strain in this organ . We concluded that the transiently increased virulence of the evolved strain was likely caused at least in part by a strongly increased inflammatory response in the brain . Colonies of the re-isolated evolved strain showed the typical wrinkled colony phenotype , while the wild type grew as smooth colonies ( not shown ) . This indicated that the growth form of the strain was retained in vivo . Histological analyses revealed that , in comparison to the wild type , infection with the evolved strain led to larger and more numerous microcolonies , in agreement with the higher number of cfu found in the organ ( Fig . 4C ) . In each histological section of the brain two days p . i . , we found a mean of 35 . 7 microcolonies after infection with the Evo strain , but only 0 . 31 microcolonies in WT-infected animals ( Table S1 ) . Smaller aggregates ( 1–5 cells ) were found in a similar ratio of 4 to 0 . 15 . These ratios are in very good agreement with our cfu data . The total area in each brain section which these colonies occupied likewise differed strikingly by a factor of 176fold , with a mean 72 , 245 µm2 for the Evo strain and only 410 µm2 for the WT strain . Additionally , histology suggested that the evolved strain may have a higher invasion potential into brain tissue ( typical picture shown in Fig . 4C ) . We then investigated the genetic basis for the evolved phenotype . First , chromosomes were separated by pulsed field gel electrophoresis ( PFGE ) ( Fig . S6A ) . No large-scale chromosomal aberrations were detectable with PFGE in the evolved strain , excluding loss or gain of whole chromosomes or large rearrangements to be the cause for the morphological changes . PCR fingerprinting analysis using M13 and ( GACA ) 4 primers also revealed no major differences between the two strains ( Fig . S6B ) . Complete genome sequences were then obtained by Solexa/Illumina technology from the parental strain and the Evo strain . The 36 bp single-end reads were aligned to the ATCC2001 reference genome [28] , with 98 . 5% of the reference genome covered for the two strains and an average sequencing depth of 69 . 6-fold and 73 . 1-fold for the parental strain and Evo strain , respectively ( Table S2 ) . Sequencing depth was plotted over the chromosomes to detect duplication or deletion events . Sequencing depth for both strains was homogeneous across all chromosomes , except for chromosome K that showed a duplication of ca . 130 kb on its left arm , consistent with previous karyotyping [chromosome K* in 7] , and chromosomes C and L that showed over-covered regions corresponding to a tandem array of genes encoding adhesin-like proteins and rDNA , respectively ( Fig . 6 ) . Additionally , several under-covered regions were detected; these were mainly located at subtelomeres and telomeres , known to harbor gene families and tandem repeats [29] , [30] . Importantly , no obvious difference could be observed between the wild type and the evolved strain with respect to regions showing increased or decreased sequencing depth ( Fig . 6 ) . Similarly , when the sequencing depth over individual ORFs was analyzed , no obvious difference was observed between the two strains ( Fig . S7 ) . Taken together , these results suggest that no major regional amplifications or deletions had occurred during the microevolution process . This is in agreement with results obtained from karyotyping via PFGE and microsatellite analysis ( Fig . S6 ) . Resequencing of our parental C . glabrata ATCC2001 isolate revealed 56 single-nucleotide polymorphisms ( SNPs; Table S3 ) compared to the published reference sequence [28] . 54 of these SNPs were also observed when comparing the genome of the Evo strain with the reference genome . Sanger sequencing of selected genes confirmed the corresponding SNPs inferred from our sequencing of C . glabrata ATCC2001 and Evo strains ( Table S3 ) . In particular , ORFs that had been annotated as pseudo-genes appeared as complete coding sequences in our resequencing . For instance , the pseudogene CAGL0L09955g , highly similar to S . cerevisiae stress regulator gene WHI2 , shows a mid-protein stop codon in the reference genome , while the SNP we identified in this gene restores the full length open-reading frame . Taken together , these results suggest that the 56 SNPs observed in our C . glabrata ATCC2001 isolate may be due to mutations , sequencing errors in the reference genome , or a combination of both . In addition to the 54 SNPs present in the parental and evolved genomes ( see above ) , we identified nine SNPs unique to the evolved strain ( Table 1 ) . Four of these SNPs were located in intergenic regions , while the remaining five were located in coding regions , among which 3 were non-synonymous , leading to a change in the amino acid sequence of the protein ( Table 1 ) . These were located in the genes CHS2 , NSP1 and PYC2 . We hypothesized that one or more of these changes in protein sequence between parental and evolved strains was likely responsible for the phenotypic alterations in the Evo strain . The Saccharomyces cerevisiae orthologues of NSP1 and PYC2 encode an essential nucleoporin and a pyruvate carboxylase , respectively . Interestingly , in S . cerevisiae , CHS2 encodes a chitin synthase . Moreover , S . cerevisiae cells lacking CHS2 grew in clumps and exhibited thick septa , which lacked an intact primary septum [31] . Given these notable similarities between S . cerevisiae chs2Δ and our Evo strain , we reasoned that the Asn→Lys exchange in the Chs2 chitin synthase ( Fig . 7A ) may be responsible for the altered growth phenotype of the Evo strain . Indeed , the corresponding ( Asn556 ) residue in ScChs2 lies within the chitin synthase catalytic domain and is essential for full enzymatic activity in S . cerevisiae [32] . We therefore introduced the evolved Asn→Lys mutation into a C . glabrata wild type strain , replacing the original copy of CHS2 ( strain CHSEvo , Fig . 7B and 7C ) . The resultant CHSEvo strain exhibited the same pseudohyphae-like growth morphology as the original ( macrophage-evolved ) Evo strain ( Fig . 7D ) and a similar growth rate in YPD medium ( not shown ) . In contrast , ( re ) -introducing a wild type copy of CHS2 to the same strain ( CHSWT ) did not lead to visible changes in morphology ( Fig . 7D ) or generation time . We were interested in possible cell wall alterations due to the mutation in the chitin synthase gene CHS2 . Therefore , we performed FACS analyses to determine the accessible surface mannan , β-glucan and chitin levels ( Fig . S8 ) . Indeed , we observed a significant reduction in mannan and β-glucan signals by the Evo strain , but also by the CHSEvo and an Δace2 mutant , which we used as a control strain with a known cell-separation defect [33] . In addition , a reduction in accessible chitin was observed for Evo , CHSEvo and Δace2 , but not the CHSWT strain . Hence , the mutation in the CHS2 gene likely induced cell surface alterations in the Evo strain which are superficially comparable to the Δace2 strain . We next performed a counter-selection experiment to reverse the original microevolution . We incubated the Evo strain in liquid medium , a condition where the cell clumps readily sink to the bottom of the flask . By continuously subculturing samples from the upper phase of the culture flask , we were able to select for a strain that again grew as single-celled yeasts and formed smooth colonies . Sequencing revealed that the CHS2 gene in this strain had reverted to its original sequence ( Fig . 7A , Rev ) , while other SNPs detected in the Evo strain remained . Together , these experiments confirm that the single nucleotide exchange in CHS2 , selected for by growth within macrophages , is sufficient to induce pseudohyphae-like growth in C . glabrata . To determine whether the introduction of this mutation into the wild type increases its damage capacity to the level of the Evo strain , we measured macrophage damage by the LDH assay . As before , the Evo strain elicited approximately six-fold more damage than the wild type after 24 hours ( Fig . 8 ) . Strikingly , wild type C . glabrata harboring the asparagine to lysine substitution ( CHSEvo ) caused virtually identical macrophage damage as the evolved strain ( Fig . 8 ) . In contrast , the control replacement of the CHS2 wild type allele ( CHSWT ) did not lead to a significant increase in the damage potential after 24 hours . A similar picture emerged when we tested the CHS2 mutant strains in the embryonated chicken egg model ( Fig . S4 ) . Here , the CHSWT strain caused the same low final mortality rate as the wild type strain . In contrast , the original Evo and the CHSEvo strain , both carrying the mutated CHS2 allele , showed the same increased virulence in ovo . Moreover , we investigated possible explanations for the increased cytokine response in the murine model . When we tested for cytokine induction in mouse macrophages by the different strains we found identically increased levels of TNFα release after infection with both , the evolved and the CHSEvo strain ( Fig . 5B ) . In contrast , the CHSWT strain only induced low TNFα levels which were nearly identical to the parental control strains . Interestingly , this effect was specific for TNFα , as IL-6 , IL-1β and GM-CSF secretion by macrophages never significantly exceeded medium control levels for any strain . In summary , we conclude that the increased cytokine induction and increased virulence in ovo by the evolved strain depends on the point mutation in CHS2 , which induced the pseudohyphae-like growth .
For pathogenic microorganisms , the host niches they infect and the innate and adaptive immune response raised against them can provide the selective pressure for driving evolution and adaptation . In addition , in the case of humans and domesticated animals , medical interventions such as drug therapy can select for resistant isolates . Indeed , previous studies have elegantly shown the potential of pathogens to adapt to clinical drug regimens [4] , [34] , [35] . Here , we used an in vitro microevolution approach to evaluate the potential of C . glabrata to adapt to an important player in the host immune system: macrophages . By continuously exposing yeast cells to macrophages , we aimed at recreating a scenario that the fungus potentially encounters within tissues during persistent infection . C . glabrata can be found in close proximity to , and possibly within , these phagocytes in vivo during murine infections [15] . Although C . glabrata can reside and replicate inside the macrophage phagosome [36] , this hostile environment likely represents a substantial selective pressure . The short-term survival strategies of C . glabrata inside macrophages have been addressed in previous investigations [16] , [18] , [19] . However , longer-term interactions likely pose different challenges to the fungus . We predicted that C . glabrata , which has the ability to persist in the organs of fully immunocompetent mice for several weeks [15] , [37] , [38] , has the potential to overcome these challenges in the long term by ( micro ) evolutionary adaptation . During the months-long exposure in our experiment , C . glabrata evolved to a stable phenotype with a pseudohyphae-like growth morphology accompanied by abnormal septum formation and an overall thicker cell wall . Morphologically , this phenotype seemed akin to the reversible irregular wrinkle phenotype of C . glabrata's core switching system [25] , [39] . Our in vitro analyses and the genome sequence , however , showed that this phenotype is not dependent on this system , and instead represents a genetically stable , micro-evolved strain of C . glabrata . Genome sequencing revealed only nine SNPs between the parental and the evolved strain , with only three of them resulting in altered protein sequence . In addition , no large-scale genomic changes , such as translocations , duplications , or deletions were observed . Previous works have addressed the genome plasticity and dynamics of C . glabrata . It has been shown , for example , that C . glabrata can form new chromosomes by extensive chromosomal rearrangements [6] , [8] , [40] , probably due to the loss of genes involved in telomere end protection [8] . Such events have been linked to the emergence of antifungal resistance and adaptation to the human host [6] , [8] . Furthermore , the genomic plasticity of C . glabrata encompasses copy number variations in tandem gene repeats [41] , but also chromosomal rearrangements and recombination events triggered by mini- and megasatellites . Affected genes often encode putative or known cell wall proteins , such as cell wall anchored aspartyl proteases , suggesting a role for these gene tandems in adaptation to the environment and in cell–cell interaction [17] . Furthermore , changes in copy numbers of minisatellites in potential virulence genes were shown to alter cell adhesion and pathogenicity in C . glabrata [42] . We observed such rearrangements , including a large duplication of ca . 130 kb on the left arm of chromosome K – chromosome K* in [7] – and an increase in copy number of a tandem array of adhesin genes on chromosome C . However , these rearrangements were present in both the original wild type and Evo strains , compared to the reference genome , indicating that they were not responsible for the phenotype of the Evo strain . In fact , the Evo strain exhibited a relatively low number of nucleotide exchanges compared to its parental wild type , and maintained chromosomal stability over several months of continuous culture . This may be due to the lower division rate within the phagosome in comparison to classical in vitro cultures . Alternatively , the parental strain may have already been relatively well adapted for growth within macrophages [16] , rendering most genomic rearrangements detrimental . This would be in agreement with recent data on the commonly used C . albicans wild type strain SC5314 , which was shown to be well adapted to growth in the kidney , its main target organ in mice [43] . We were able to successfully pin down the genetic basis for the filament-like phenotype of the evolved C . glabrata strain: a single nucleotide exchange in the CHS2 gene . This mutation likely rendered Chs2 non-functional , or significantly reduced in function , as the phenotype of the cell wall architecture of the C . glabrata Evo strain was similar to a S . cerevisiae mutant lacking CHS2 [31] . A targeted single nucleotide exchange in the wild-type background resulted in a mutant which produced pseudohyphae-like structures , damaged macrophages and induced TNFα secretion to a similar extent as the experimentally evolved strain . Despite the stability of the evolved phenotype under standard growth conditions , we were able to readily restore the parental phenotype ( and wild type CHS2 allele ) by counter-selecting for yeast-like growth which led to a reversal of the single nucleotide exchange in the original Evo strain . Interestingly , when investigating gain of function mutations in the regulator of ABC transporters of C . glabrata , Pdr1 , Ferrari et al . found clinical strains in which these mutations mediated both antifungal resistance and enhanced virulence in mice [35] . Like in our in vitro microevolution experiment , these were based on single nucleotide exchanges . While – to our knowledge – this is the first described microevolution experiment using fungi and macrophages , a few comparable experiments have been performed with alternative host cells . In earlier work , it has been shown that C . neoformans , passaged through phagocytic amoeba , developed pseudohyphae-like structures which were genetically unstable [44] . Furthermore , certain hypermutator strains of C . neoformans show phenotypic switching from yeast to pseudohyphae and back [22] , [45] . Recently , targeted sequencing of selected genes was employed to associate these phenomena with spot mutations of single RAM ( Regulation of Ace2 and Morphogenesis ) pathway genes [22] . These mutants formed pseudohyphae , and were not taken up by macrophages as efficiently as the wild type . It is interesting to note that adaptation of C . neoformans in non-mammalian host cells ( amoeba ) attenuated virulence in mice [22] , whilst adaptation of the normally commensal C . glabrata in mammalian cells ( macrophages ) increased its virulence . Interestingly , Ace2 also plays a role in cell separation in C . glabrata . The C . glabrata Δace2 mutant grows as clumps of cells and is hypervirulent in immunosuppressed mice [33] . However , in contrast to our Evo strain , Δace2 virulence and organ burden was not affected in immunocompetent animals , albeit after infection with a lower dose [46] . Most likely , Ace2 regulates chitinase gene expression in C . glabrata [33] , which is necessary for complete mother-daughter cell separation . Indeed , the ACE2 orthologue in S . cerevisiae is necessary for the correct expression of chitinases [47] , [48] . Our data indicates that the cell wall alterations of both the Evo and the Δace2 mutant are similar , with reduced β-glucan , mannan and possibly chitin accessibility on the surface . It seems possible that the increased pro-inflammatory cytokine release elicited by both mutants ( measured in serum in [33] and directly in differents organ in this work ) has a similar basis in these cell wall alterations . Alternatively , both the Δace2 and Evo mutations lead to similar cell wall alterations , but the increase in cytokines is due to increased host damage by the filament-like growth of the two mutants . Interestingly , however , in immunocompromised , but importantly not immunocompetent animals , the Δace2 mutant had an increased tissue burden in lung and liver [33] , [46] , whereas the Evo strain was found ( in immunocompetent mice ) specifically in the brain in unusually high numbers . While experimental conditions such as the infection dose differed , this indicates important differences in the host-pathogen interaction between the two strains . The C . glabrata phenotype that was generated in our microevolution experiment appears to share certain properties with the filamentous ( i . e . pseudohyphae and true hyphae ) morphologies of C . albicans . Filamentous growth , especially true hypha formation , is considered to be a key virulence attribute of C . albicans and is essential for escape from macrophages [36] , [49] , [50] . Analogously , we observed rapid escape from macrophages and increased phagocyte damage by the C . glabrata Evo strain and the reconstructed ( CHSEvo ) mutant . It would appear likely that this is in part due to mechanical stresses exerted by the growing fungus , similar to the mechanism which has been proposed for C . albicans-macrophage piercing [50] . Alternatively , it seemed feasible that the remaining clumps of 2–3 cells after sonication allowed for a better survival of the Evo strain in the macrophages . After uptake , these small aggregates may have protected the yeast cells in the phagosome and allowed a faster outgrowth . Our data , however , show that this is not the case , and furthermore that the number of yeast cells per macrophage equalizes between WT and Evo strain within hours , before macrophage damage commences . In addition , an unrelated mutant with similar sized clumps in the inoculum showed no significant increase in macrophage damage . The reason for the host cell damage hence most probably lies in the pseudohyphae-like growth form after uptake , although the differences in inoculum aggregates may still play a more subtle role . Whether the increased virulence of the Evo strain in vivo is directly linked to the altered morphology is unclear . In our embryonated egg model , however , the presence of an altered allele of the CHS2 gene was sufficient to elicit an increased mortality rate similar to the Evo strain . This seems to link the single nucleotide exchange and its accompanying phenotypic alterations to the increased virulence . It is possible that , after the initial infection and escape from immune cells , the growth as large aggregates of pseudohyphae-like structures allowed the Evo strain to avoid uptake by phagocytes due to sheer size . In the mouse model , we detected strong increased levels of the pro-inflammatory cytokines TNFα and IL-6 in the brain during early infection , which coincided with a massive increase in C . glabrata cell number in this organ . Histological analysis furthermore indicated that the Evo strain grew in more abundant and larger microcolonies in the brain , which possibly have contributed to the increased fungal burden and clinical symptoms . The precise mechanism of the altered tissue tropism is unclear , and it may be attributed to the increased invasive growth itself or to the alterations in the cell wall we observed in the electron micrographs and via FACS analyses . Indeed , the mechanism ( s ) by which C . glabrata ( and other pathogenic yeast ) cells access the brain and other organs are so far poorly understood . It has been proposed that C . neoformans may hijack immune cells , using them as a “Trojan Horse” to invade the central nervous system [51] . However , this is unlikely to account for the increased brain tropism of the Evo strain given its propensity to escape more rapidly from macrophages . Interestingly , we detected an increased production of TNFα by macrophages infected with the evolved strain . The CHSEvo strain had the same effect on TNFα production , showing that the single nucleotide exchange in the CHS2 gene is sufficient for increased macrophage activation . TNFα , produced by macrophages or microglial cells , is a potent inducer of chemokine production by cells of the central nervous system . It seem therefore likely that one or several of the phenotypic alterations elicited by the CHS2 mutation are at least indirectly responsible for the increased pro-inflammatory response in the brain . A similar effect was observed for the Δace2 mutant , where murine serum levels of pro-inflammatory cytokines were highly increased after infection coinciding with the changes in morphology [33] and altered cell wall composition ( our data ) . The localized effect of the Evo strain in the brain may therefore have a similar basis . Like for the Δace2 mutant , it is unlikely that cellular aggregations in the inoculum account for hypervirulence , as our C . glabrata cells were sonicated and thus separated before injection . Moreover , physical blockage of capillaries following intravenous administration of the fungus would have led to an immediate effect , which was not observed in our experiment . Hence , the early increase in murine virulence by the Evo strain , with the associated clinical symptoms , such as weight loss and other clinical scores , was likely caused by a strong inflammatory response in the brain . In this respect , the evolved strain also resembled the neurotropic fungus C . neoformans , which likewise induces local TNFα and IL-6 responses in the brain , correlating with its presence in the CNS and the progression of meningoencephalitis [52] . However , while in C . neoformans this culminates in murine mortality , we observed a reduction of fungal burden by the evolved strain . The decline to wild-type levels from day 7 on is possibly due to this increased immune response , which the less-well adapted C . glabrata likely cannot withstand as well as C . neoformans . Finally , considering the ability of C . glabrata to persist within mouse organs for weeks [15] , it is tempting to speculate that similar microevolutionary adaptations may also occur in the clinical setting . The evolved strain has a clear selective advantage in an in vitro macrophage cell line , and possesses a higher virulence potential in mice . It seems reasonable to assume that similar adaptations may also occur during an in vivo infection . If such a phenotype arose during long-term colonization or infection in a patient , it may be adaptive due to the presence of host immune cells , similar to the selection of azole resistant isolates under drug regimens [3] , [4] , [9] , [10] . In future work , it would therefore be of interest to carefully analyze the pheno- and genotypes of clinical isolates from candidiasis patients for alterations similar to those observed in our study . In fact , even though the vast majority of C . glabrata clinical isolates grow as yeast cells , pseudohyphal growth has been observed under certain conditions in vitro [53] and clinical isolates with comparable phenotypes exist [54] . Notably , a C . glabrata clinical isolate with pseudohyphae-like morphology caused increased macrophage damage in our hands ( Fig . S9 ) , similar to the Evo strain . Sequencing of the CHS2 locus , however , revealed no non-synonymous mutations . Hence , the genetic basis is different , but the effect of the pseudohyphal growth form on the interaction with macrophage was similar . It is tempting to speculate that these phenotypic traits may have been selected for in this clinical isolate . A systematic analyses of clinical C . glabrata isolates with aberrant morphologies and their interaction with phagocytes could shed more light on this possibility . In summary , the pathogenic fungus C . glabrata has the ability to adapt to distinct microniches such as macrophages by evolutionary processes . Under conditions of stress , mutations which may hinder growth in standard culture conditions can become beneficial . These stresses can include antifungal treatments or challenges by the host's immune system . Our data suggest that C . glabrata , and likely other pathogenic microbes , has the potential to develop and express ‘hidden’ or ‘silent’ pathogenicity factors in response to environmental challenges . These adaptations may require relatively few genetic steps to manifest: in this case , a single nucleotide exchange in CHS2 . Whether this short evolutionary route is the rule or the exception cannot be resolved by the experiments presented here . Future investigations with C . glabrata and other fungi may shed more light on how frequent such adaptations are and whether they are realized in vivo by infection-related selection processes .
All animal experiments were in compliance with the German animal protection law and were approved by the responsible Federal State authority ( Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz ) and ethics committee ( beratende Komission nach § 15 Abs . 1 Tierschutzgesetz; permit no . 03-006/09 ) . The C . glabrata wild type strain ATCC2001 ( WT ) , the evolved strain ( Evo ) and the deletion mutants cbk1Δ and dse2Δ [26] were routinely grown overnight in YPD ( 1% yeast extract , 2% peptone , 2% dextrose ) at 37°C and 180 rpm in a shaking incubator . When indicated , clumps of fungal cells were separated by sonication: cells were sonicated for 30 s in PBS at 40% amplitude setting with a Sonopuls HD2070 ( Bandelin , Germany ) . Cell viability was checked routinely by methylene blue staining after sonication and the clumps appeared separated into single or pairs of yeasts upon microscopic inspection . The murine RAW 264 . 7 macrophage-like cell line used in this study was routinely cultured in Dulbecco's Modified Eagle's Medium ( DMEM ) with 4 mM L-glutamine and 4 . 5 g/l glucose ( PAA Laboratories , Austria ) and supplemented with 10% heat-treated fetal bovine serum ( PAA ) at 37°C and 5% CO2 . Cells were passaged every three days by scraping and diluting 1∶5 in fresh media up to 15 passages . For some experiments , RAW 264 . 7 cells were inoculated in 24 or 96 well plates at an initial concentration of approximately 1×105 cells/well or 5×104 cells/well , respectively , and then incubated overnight at 37°C and 5% CO2 to near confluency ( 60%–80% ) . TR-146 cells were routinely grown and maintained ( passages 4 to 20 ) in DMEM medium with 4 mM L-glutamine , 4 . 5 g/l glucose and 10% FBS at 37°C in 5% CO2 . For infection experiments with C . glabrata , TR-146 cells were detached by trypsin ( PAA ) treatment , 2×104 cells/well seeded in 96 well plates and incubated overnight at 37°C and 5% CO2 to near confluency ( 60%–80% ) . Macrophages were grown in cell culture flasks ( 75 cm2 , PAA ) to near confluency ( 80% ) and infected with 5×107 yeast cells . The following day , the supernatant containing non-phagocyte-associated yeast cells was removed , and the adhering macrophages were scraped off and lysed in 2 ml lysis buffer ( 50 mM Tris , 5 mM EDTA , 150 mM NaCl , 0 . 5% Nonidet-P40 ) . The cell debris and phagocytosed yeast cells were pelleted by centrifugation . The pellet was washed two times in fresh DMEM , resuspended in 1 ml DMEM , and centrifuged for 1 min at 50 rcf to pellet the remaining debris and retain the yeasts in the supernatant . Yeasts were counted , and 5×107 yeast cells were again transferred to fresh macrophages . This procedure was repeated daily . The parental strain and the evolved strain were grown overnight in YPD at 37°C in a shaking incubator ( 180 rpm ) . Yeast cells were washed in PBS and fixed with Karnovsky fixative ( 3% paraformaldehyde , 3 . 6% glutaraldehyde , pH 7 . 2 ) . After centrifugation , the sediment was embedded in 3 . 5% agarose at 37°C , solidified at room temperature , and fixed again in Karnovsky fixative . After post-fixation of samples ( 1% OsO4 containing 1 . 5% K-ferrocyanide in aqua bidest , 2 h ) , they were rinsed with distilled water , block-stained with uranyl acetate ( 2% in distilled water ) , dehydrated in alcohol ( stepwise 50–100% ethanol ) , immersed in propylenoxide , and embedded in glycide-ether ( polymerized 48 h at 60°C , Serva , Germany ) . Ultra-thin sections were examined with a LIBRA 120 transmission electron microscope ( Carl Zeiss SMT AG , Germany ) at 120 kV . The competition experiment was carried out using a similar protocol to the continuous co-culture in the microevolution experiment , with the following modifications: Initial inoculation was performed with a mixture of sonicated WT/Evo-cells ( ratio: 100: 1 ) and an additional sonication step was included before the 50 rcf centrifugation step . The competitive advantage was determined by daily measurements of the relative amount of WT and Evo cells by colony morphology after plating on YPD . To calculate the competitive fitness ratio , the best fit for x was determined for the change in relative amounts ( Evo and WT ) according to the formula Evot = ( x Evot-1 ) / ( x Evot-1+[1-x] WTt-1 ) , and vice versa for the WT . The advantage was expressed as the ratio of the x for Evo and WT , and expresses the ratio of daily increase between the strains . Yeast cells were grown overnight in YPD , washed with PBS , sonicated , resuspended in carbonate buffer ( 0 . 1 M Na2CO3 , 0 . 15 M NaCl , pH 9 . 0 ) , and labeled with 100 µg/ml fluorescein isothiocyanate ( FITC , Sigma-Aldrich ) for 30 min at 37°C . After washing with PBS , yeast cells were counted using a hemocytometer . RAW 264 . 7 macrophages , seeded on cover slips in 24 well plates with serum-free DMEM , were infected at a multiplicity of infection ( MOI ) of 2∶1 and co-incubated for 15 min , 45 min , or 90 min . After washing three times with PBS , samples were fixed with 4% paraformaldehyde , followed by washing and counterstaining with 25 µg/ml Alexa Fluor 647-conjugated concanavalin A ( ConA , Life Technologies , UK ) to differentially visualize extracellular ( non-phagocytosed ) yeast cells . Coverslips were mounted in ProLong Gold Antifade Reagent with DAPI ( Life Technologies ) . At least 200 macrophages were counted and scored as containing or not containing internalized yeast cells . This experiment was performed three times independently . The release of lactate dehydrogenase ( LDH ) into the culture supernatant was monitored as a measure of host cell damage . RAW 264 . 7 and TR146 cells were seeded in 96 well plates and infected with sonicated WT or Evo yeast cells at an MOI of 2∶1 . For control samples , host cells or yeast cells were incubated with medium only . After 24 h and 48 h of co-incubation , culture supernatants were collected , and the amount of LDH was determined using a Cytotoxicity Detection Kit ( Roche Applied Science , Germany ) according to the manufacturer's instructions . LDH activity was determined spectrophotometrically at 492 nm , and LDH concentration was calculated using a standard curve obtained from dilutions of an LDH control . All experiments were performed in triplicate for each condition and performed three times independently . RAW 264 . 7 cells were seeded in 35 mm diameter petri dishes ( Ibidi , Germany ) at a density of 1 . 5×106 cells per dish in DMEM with 10% FBS , and infected with sonicated yeast cells at a MOI of 2∶1 . After 60 min of phagocytosis , cells were washed with pre-warmed DMEM with 10% FBS to remove excess yeast cells . The setup was placed under an Axio Observer . Z1 microscope ( Zeiss , Germany ) with a plexiglas box and heating to keep incubation conditions constant at 37°C and 5% CO2 . Phase-contrast pictures were taken every 5 min at a magnification of 630 . For differentiation between killed and surviving yeast cells , the fate of about 150 yeasts per strain were followed in these videos . Degradation of dead yeasts in the phagosome was determined visually by comparison to a previous positive control video of heat killed yeasts in macrophages . Replication or ( more rarely ) the absence of any sign of degradation of non-replicating yeasts served as indicators for live yeast cells . The sensitivity of the parental strain ( Wt ) and the evolved C . glabrata strain ( Evo ) to various stress conditions was tested by spotting serial dilutions of pre-sonicated Wt ( 1×105 to 1×101 cells/spot ) and Evo ( 2×105 to 2×101 cells/spot , to reach comparable cfu for better comparison ) yeasts on agar plates containing different stressors , followed by incubation for two days at 30°C . Plates containing H2O2 ( 10 mM , Roth , Germany ) , NaCl ( 1 M , Roth ) , or caspofungin ( Cancidas , 150 ng/ml diluted in 125 mM NaOH , MSD , NJ , USA ) were prepared with SD agar ( 1× Difco yeast nitrogen base [YNB , BD Diagnostic Systems , MD , USA] , 2% glucose , 0 . 5% ammonium sulfate , 2% agar ) ; plates containing calcofluor white ( 800 µg/ml , Sigma Aldrich , MO , USA ) or Congo red ( 500 µg/ml , Sigma Aldrich ) were prepared with buffered SD agar ( 1× YNB , 2% glucose , 0 . 5% ammonium sulfate , 100 mM potassium phosphate pH 6 . 0 , 2% agar ) . Plates containing menadione ( 200 µM , Sigma Aldrich ) were prepared with YPD agar . Yeasts were grown overnight in YPD , washed with PBS , sonicated , and adjusted to 1×108 yeast cells/ml ( inoculum ) . Embryonated eggs were infected as previously described [55] . Briefly , a 100 µl inoculum ( 107 yeasts ) was applied via an artificial air chamber to the chorioallantoic membrane ( CAM ) using a sterile 1-ml syringe . Twenty eggs were infected for each group on developmental day 10 . The holes were sealed with paraffin and survival was monitored for up to seven days post infection ( p . i . ) by candling . Six week old outbred , female , specific-pathogen free CD-1 mice ( 18–22 g , Charles River , Germany ) were used in the experiments . Animals were kept in groups of five in individually ventilated cages , and cared for in accordance with the principles described in the European . Mice were infected with 5×107 cfu ( sonicated to separate yeasts ) in 200 µl PBS via the lateral tail vein on day 0 . On days 2 , 7 , and 14 p . i . five mice per group were sacrificed . Animals were monitored at least twice daily and humanely sacrificed if moribund ( defined by severe lethargy and/or hypothermia ) . Fungal burden and blood marker enzyme levels were analyzed as described previously [15] . For colony forming unit determination , organ homogenates were again sonicated before plating appropriate dilutions on YPD medium . For histology , parts of organs were fixed with buffered formalin and paraffin-embedded sections were stained with Periodic acid-Schiff ( PAS ) according to standard protocols . For brain histopathology , longitudinal sections close to the midline of the brain were used for semi-quantitative analysis: For each animal , PAS stained slides with two to four sections were scanned at 40× magnification with a Hamamatsu NanoZoomer 2 . 0 slide scanner . The resulting images of the sections ( n≥13 ) were evaluated in a blinded fashion for the number of single aggregates ( 1–5 yeasts ) and microcolonies ( >5 yeasts ) , and the colony size using the measuring tools of the Hamamatsu NDP . view2 software . Tissue homogenates of infected mice were diluted 1∶1 to 1∶7 in tissue lysis buffer ( 200 mM NaCl , 5 mM EDTA , 10 mM Tris , 10% glycerol , 1 mM phenylmethylsulfonyl fluoride [PMSF] , 1 µg/ml leupeptin , and 28 µg/ml aprotinin [pH 7 . 4] ) and centrifuged twice ( 1500 g , 15 min , 4°C ) , and the supernatants were stored at −80°C until measurement . Myeloperoxidase ( MPO ) and cytokine levels were determined by commercially available murine enzyme-linked immunosorbent assay ( ELISA ) kits ( for MPO , the Mouse MPO ELISA kit [Hycult Biotechnology , the Netherlands]; for IL-1β , IL-6 , tumor necrosis factor alpha [TNFα] , and granulocyte-macrophage colony-stimulating factor [GM-CSF] , ELISA Ready SET Go ! [eBioscience , United Kingdom] ) according to the manufacturer's recommendations . RAW264 . 6 macrophages were infected in 24 well plates . LPS ( Sigma-Aldrich ) was used as a control and applied at concentration of 1 µg/ml . After 24 h , samples of surrounding medium were centrifuged ( 10 min , 1000 g ) and stored at −80°C until measurement . The amount of TNFα , IL-1β , IL-6 and GM-CSF was determined by ELISA according to the manufacture's protocol ( ELISA Ready SET Go ! [eBioscience , United Kingdom] ) . All experiments were performed in triplicate and normalized to LPS samples . Wild type and evolved strains were preheated to 50°C at a concentration of 7×108 cells/ml in 250 µl double distilled H2O and 20 µl zymolase solution ( 100 mg/ml zymolase 20T [Medac , Germany] in 10 mM Tris , 50 mM EDTA , pH 7 . 2 ) was added . The suspension was mixed at 50°C with prewarmed 2-fold concentrated agarose in 2×TE and allowed to solidify at room temperature . Agarose blocks were then incubated for 3 h at 37°C with zymolase ( 5 mg/ml zymolase in 10 mM Tris , pH 7 . 2 , 20 mM NaCl , 50 mM EDTA ) under gentle agitation , washed twice with 5 ml washing buffer ( 20 mM Tris , pH 8 . 0 , 50 mM EDTA ) for 30 min , and further treated with 5 ml proteinase K solution ( 1 mg/ml proteinase K in 100 mM EDTA , pH 8 . 0 , 0 . 2% sodium deoxycholat , 1% sodium lauryl sarcosine ) over night at room temperature . After four washing steps with washing buffer , the blocks were stored in TE buffer ( 10 mM Tris , 50 mM EDTA , pH 7 . 2 ) at 4°C . A 1% agarose gel was cast around pre-cut agarose samples and electrophoresis was performed in a CHEF DR II chamber ( BioRad ) with the settings: pulse A∶B ratio 1∶1; initial pulse time 60 , final pulse time 120 sec 120 s for 22 h at 14°C and 200 V The band pattern was visualized by ethidium bromide staining . Total genomic DNA ( gDNA ) was recovered from fungal cells by a standard nucleic acid extraction protocol adapted from [56] , using phenol∶chloroform and glass beads in a Precellys 24 homogeniser ( PeqLab , Germany ) . The fingerprinting PCR reaction was performed as described previously [57] . Primers used were the M13 primer ( GAGGGTGGCGGTTCT ) and a primer consisting of the repeat sequence ( GACA ) 4 . Products were separated by 1 . 2% agarose gel electrophoresis for 6 h at 3 V/cm and detected by ethidium bromide staining . Telomere length determination was performed as previously described [58] with minor modifications . Briefly , 7 . 5 µg genomic DNA from the parental and the evolved strain was digested with either ApaLI , MseI , RsaI , Sau3AI , or XbaI , run on a 0 . 8% agarose gel , and transferred to positively charged nylon membrane ( Roche ) . The blot was hybridized with the digoxigenin-labeled 32-mer probe TCTGGGTGCTGTGGGGTCTGGGTGCTGTGG-DIG , that binds to the telomere sequence of C . glabrata . Detection with an anti-digoxigenin antibody followed standard procedures [59] . Libraries were prepared using the TruSeq DNA Sample Prep kit ( Illumina , Eindhoven , The Netherlands ) according to the manufacturer's recommendations . Genomic DNA of C . glabrata strains ATCC2001 and the Evo strain were sheared by sonication to an average fragment length of 500 base pairs . Illumina adapters were blunt-end ligated , and libraries were amplified by PCR . Each sample was sequenced on an Illumina Genome Analyzer platform ( Illumina GAII ) . 36 bp single-end reads were obtained for strain ATCC2001 while 60 bp single-end reads were obtained for the Evo strain . In order to avoid bias between the two types of reads , only the first 36 bp of each 60 bp read were used . Sequencing reads were aligned to the C . glabrata strain CBS138/ATCC2001 reference genome [28; downloaded at http://www . genolevures . org on March 28 , 2011] using shore 0 . 5 . 0 [60] . Sequencing depth scores were computed for each 1 kb region across the genomes and for ORFs using sequencing depth data for each nucleotide located within the 1 kb region or the ORF . Sequencing depth scores were normalized based on the overall sequencing depth obtained for each genome . Single nucleotide polymorphisms ( SNPs ) between the genomes and/or the reference genome were identified using shore 0 . 5 . 0 [60] . To be assigned as a SNP , positions had to be covered at least 20 times with a minimum quality of 25 and the polymorphism had to be present in at least 90% of the calls . Data were evaluated using GraphPad Prism ( version 6 . 00 , GraphPad Software , La Jolla , CA , USA ) and are reported as the mean ± SD . Yeast uptake assays , cell wall property and damage assays were analyzed using two-tailed , unpaired Student's t-test corrected for multiple comparisons where necessary . In the murine infection model , cfu counts in different organs were compared between strains at different time points using Holm-Sidak corrected t-tests . Resulting values are indicated in the figures as: * , p<0 . 05 . ; ** , p<0 . 01; *** , p<0 . 005 ) . | Evolution is not limited to making new species emerge and others perish over the millennia . It is also a central force in shorter-term interactions between microbes and hosts . A good example can be found in fungi , which are an underestimated cause of human diseases . Some fungi exist as commensals , and have adapted well to life on human epithelia . But as facultative pathogens , they face a different , hostile environment . We tested the ability of C . glabrata , a pathogen closely related to baker's yeast , to adapt to macrophages . We found that by adaptation , it changed its growth type completely . This allowed the fungus to escape the phagocytes , and increased its virulence in a mouse model . Sequencing the complete genome revealed surprisingly few mutations . Further analyses allowed us to detect the single mutation responsible for the phenotype , and to recreate it in the parental strain . Our work shows that fungi can adapt to immune cells , and that this adaptation can lead to an increased virulence . Since commensals are continuously exposed to host cells , we suggest that this ability could lead to unexpected phenotype changes , including an increase in virulence potential . | [
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] | 2014 | One Small Step for a Yeast - Microevolution within Macrophages Renders Candida glabrata Hypervirulent Due to a Single Point Mutation |
The rod-shaped fission yeast Schizosaccharomyces pombe , which undergoes cycles of monopolar-to-bipolar tip growth , is an attractive organism for studying cell-cycle regulation of polarity establishment . While previous research has described factors mediating this process from interphase cell tips , we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle . Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site , but not at interphase cell tips , resulted in many cells failing to grow at new ends created by cell division . This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells . Moreover , additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects , supporting that robust completion of cell division contributes to new end-growth competency . To test this model , we genetically manipulated S . pombe cells to undergo new end take-off immediately after cell division . Intriguingly , such cells elongated constitutively at new ends unless cytokinesis was perturbed . Thus , cell division imposes constraints that partially override positive controls on growth . We posit that such constraints facilitate invasive fungal growth , as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae . Collectively , these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites , and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state .
Many cells polarize in response to intrinsic and extrinsic signals . As cell polarization is generally multifaceted , cells must integrate both negative and positive cues for successful cellular morphogenesis . In various organisms , the cell cycle provides a platform on which these cues are organized ( for reviews , see [1] , [2] ) , thereby ensuring distinct polarization events occur at the appropriate location , time , and context . The fission yeast Schizosaccharomyces pombe represents a genetically tractable organism for studying cell cycle regulation of growth polarity ( for reviews , see [3] , [4] ) . Wild-type S . pombe extend solely at their two cell tips , lengthening their rod-shaped bodies while retaining fairly constant widths . After cell division , S . pombe grow only at old ends , so-called because they served as ends of the dividing mother cell . Then , at a point in G2 known as new end take off ( NETO ) , new ends , which arise from cell division , also initiate growth [5] . NETO is not required for cell viability , and myriad mutants defective in this process have been identified [3] , [4] . Yet , beyond requirements for S-phase completion and a minimal interphase cell size [5] , additional cell cycle controls on NETO have not been identified . As in other cell polarization events , cytoskeletal rearrangements accompany growth transitions in S . pombe . Prior to NETO , microtubule plus end-associated proteins Tea1 and Tea4 ride growing microtubule ends to both cell tip cortices [6]–[9] , where they anchor based on their association with membrane proteins [10] , [11] . Upon NETO , Tea4 recruits formin For3 , which had before only been tethered to old ends , into a complex with itself and Tea1 at new ends [8] . As over-expression of a Tea1-For3 fusion can drive NETO prematurely [8] , this association likely brings For3 into the proximity of formin activators at new ends , stimulating For3 catalysis of F-actin cables that will deliver growth cargo to this tip . Not surprisingly , loss of Tea1 , Tea4 , and/or For3 impairs fission yeast polarization and elongation [8] , [9] , [12] , [13] . Actin patches , which guide endocytic vesicle internalization and constitute a second F-actin structure , also re-polarize to both cell tips upon NETO [14] . Disruption of proteins comprising these structures similarly jeopardizes growth polarity establishment [15]–[17] . Thus , alteration in protein composition at cell tips is coupled tightly to cytoskeletal rearrangements . In addition to promoting cell tip growth , several tip-localized cell polarity factors , including Tea1 and Tea4 , direct the cell division plane away from cell ends and towards the cell middle for cytokinesis [18] , the process by which daughter cells undergo physical separation following nuclear division . However , whether the process of cytokinesis reciprocally modulates cell polarity is unclear . Some observations hint that the cell division machinery may play a role in directing cell polarity . As was previously noted , new ends formed by cell division initiate growth well after old ends . In mutants in which cells remain physically connected at division sites for multiple cell divisions , internal cells can grow , though this occurs sub-apically adjacent to septa [19] , [20] . Moreover , many polarity factors localize to the cell division site [4] , [21]–[23]; nonetheless , only cell tip-localized populations of these polarity proteins have been demonstrated to contribute to growth polarity in S . pombe . As in most eukaryotes , cytokinesis occurs in S . pombe through the assembly and constriction of an actomyosin-based cytokinetic ring ( CR ) [24] . In addition to actin and myosin , several accessory proteins regulate the dynamics and organization of this structure . For one , Cdc15 , which contains an N-terminal F-BAR domain and a C-terminal SH3 domain characteristic of the pombe Cdc15 homology protein family [25] , has been posited to link CR proteins to the cortical membrane at the division site [26] . Cdc15-binding proteins at the CR include formin , myosin , and the C2 domain protein Fic1 [27] , [28] . Fic1 localizes to both interphase cell tips and the cell division site [28] , though its specific functions at these sites have not been described . Fic1's budding yeast ortholog , Inn1 , contributes to cytokinesis by linking the CR to the ingressing membrane and by participating in septum formation [29] , [30] . Septa form in both budding and fission yeasts as cell wall is deposited behind the constricting CR [31] . A conserved signaling network , known as the septation initiation network ( SIN ) in S . pombe , triggers septum deposition during cytokinesis [32] . Together with the CR , septa provide mechanical force for membrane closure at the cell division site [33] . Subsequent septum degradation allows for abscission [34] , [35] . Clearly , various remodeling events must occur at the cell division site for cytokinesis to complete efficiently . Whether such remodeling events also influence daughter cell behavior has never been examined . While wild-type S . pombe classically grow in a single-celled form , multiple fission yeasts , including S . pombe , possess the ability to assume an invasive , hyphal-like state [20] , [36] . The ability of pathogenic fungi to undergo such a morphogenetic switch contributes significantly to fungal infections [37] . Though non-pathogenic , S . pombe , similar to the budding yeast Saccharomyces cerevisiae [38] , can transition into invasive growth as a foraging response to low nutrients [36] . Invasive S . pombe form structures that technically qualify as pseudohyphae , for , unlike as in hyphal growth , cytokinetic constriction occurs [39] , [40] . Pseudohyphae likely maintain their hyphal-like structure due to cellular adherence and preferential growth at old ends [39] , [40] . Intriguingly , it has been postulated that single-celled fission yeast evolved from multicellular , filamentous fungi , with transcriptional networks that ensure efficient cell separation playing predominant roles in the evolution of a single-celled state [41] . Though S . pombe pseudohyphae do not commonly exhibit aborted cytokineses or multicellularity , it is an attractive hypothesis that inefficient , but not entirely defective , cytokinesis might somehow mark new ends to impair their growth and promote the dimorphic switch in S . pombe . In this manuscript , we define a novel cell cycle control on S . pombe growth polarity , namely that the process of cytokinesis imposes limitations on new end growth competency . Here , we focus on Fic1 , which we show to be involved in the re-establishment of polarized cell growth at new ends following cell division . Specifically , we demonstrate that Fic1 polarity function requires its localization to the CR but not to interphase cell tips , and that its protein-protein interactions at the CR , including that with Cdc15 , promote bipolar cell growth in the ensuing cell cycle . We present evidence that loss of Fic1 impairs disassembly of the cell division apparatus , with parts of this machinery persisting at new ends following CR constriction . Additional mutants defective in late cytokinesis also exhibit impaired new end growth . Importantly , premature activation of NETO signaling does not fully rescue bipolar growth in cells with late cytokinesis defects , suggesting that cytokinesis-based constraints on S . pombe growth polarity play a central role in defining new end growth competency . We propose that such constraints can provide a mechanistic understanding of how S . pombe and possibly other fungi transition into invasive hyphal-like growth .
Recently , our laboratory identified Fic1 , which was implicated in cytokinesis based on its protein and genetic interactions and its localization to the CR [28] . In addition to defects in cytokinesis , deletion of S . pombe fic1+ , which is a non-essential gene , resulted in an abnormally high percentage of cells that grew only from one end ( i . e . , monopolar cells ) ( Figure 1A–1C ) . Tip growth was judged using calcofluor staining , as birth scars formed at previous division sites do not stain well with calcofluor and growth can be assessed using the position of these scars relative to cell tips ( Figure S1A ) [5] . The growth defects observed upon fic1+ disruption suggested that Fic1 not only participates in cytokinesis but also in the establishment of bipolar cell growth . Although the upstream NETO factors Tea1 and Tea4 localized normally to both cell tips in fic1Δ cells ( Figure S1B–S1C ) , other cell tip proteins implicated in growth polarity regulation exhibited unusual localization patterns in this mutant . Unlike wild-type cells with mostly bipolar actin patch distribution ( Figure 1D–1E ) , a variety of mutants defective in bipolar cell growth exhibit monopolar actin patches [8] , [21]–[23] . As in such mutants , the actin patch marker Crn1-GFP [42] accumulated preferentially at one cell end in a high percentage of fic1Δ cells ( Figure 1D–1E ) . Signaling through Rho GTPases controls actin patch organization in S . pombe [13] , [43] , and the Rho1 activator Rgf1 [21] , which was GFP-tagged and imaged with the spindle pole body marker Sid4-RFP [44] , likewise predominated on one end of many fic1Δ cells ( Figure 1F–1G ) . Not surprisingly , in both wild-type and fic1Δ cells , Rgf1-GFP and Crn1-RFP concentrated at the same ends ( Figure S1D ) , which were confirmed by calcofluor staining to be the growing ends of fic1Δ cells ( Figure S1E ) . Consistent with Fic1 affecting both actin and Rho networks , deletion of fic1+ was synthetically sick with deletion of genes encoding factors involved in F-actin nucleation ( WASp Wsp1 ) and Rho GTPase regulation ( RhoGEF Rgf1 and RhoGAP Rga1 ) ( Figure S1F ) . Thus , we conclude that the absence of Fic1 upsets patterning of some but not all polarity factors . To discern whether new and/or old ends were defective in resuming growth following cell division in fic1Δ cells , we performed time-lapse DIC imaging to trace birth scars in live cells . As expected , nearly all wild-type cells underwent NETO prior to subsequent septation ( Figure 2A and 2C ) . However , following roughly two-thirds of fic1Δ cell divisions , either one or both daughter cells failed to initiate new end growth prior to the next septation ( Figure 2B–2C ) . The most predominant growth pattern in fic1Δ cells was that in which one daughter cell underwent NETO while the other did not ( Figure 2B–2C ) , with nearly 70% of those daughter cells that did not exhibit NETO being the younger daughter cell . Unlike tea1Δ and tea4Δ cells , in which one daughter cell commonly fails at its new end and the other daughter cell fails at its old end ( Figure 2D ) [8] , [22] , [23] , fic1Δ cells were specifically defective in the re-establishment of growth at new ends following cell division ( Figure 2B–2C ) . Intriguingly , tea1Δ fic1Δ double mutants grew mainly in a tea1Δ pattern , though nearly one-fifth of cell divisions produced a T-shaped daughter cell ( Figure 2D–2E ) . Consistent with this , roughly 10% of tea1Δ fic1Δ cells were T-shaped at 25°C , while T-shaped tea1Δ cells were almost never observed at this temperature ( Figure 2F ) . T-shapes always arose in cells that the tea1Δ growth pattern dictated should grow at their new ends ( Figure 2D–2E ) but that actually grew at neither ( Figure 2E and 2G ) , suggesting these cells polarize at sites other than their tips because growth is inhibited at both ends . These data confirmed that the polarity defect caused by loss of Fic1 stochastically impacts new end growth in a variety of genetic backgrounds . Importantly , fic1Δ new ends that failed to extend in one cell cycle initiated growth as an old end in the next cell cycle , suggesting the defect in growth polarity caused by loss of Fic1 was not permanent . Consistent with a delay but not a block in growth , new ends that initiated growth prior to the next septation did so much later on average in fic1Δ cells than in wild-type cells ( 120 min versus 75 min ) ( Figure 2H ) . To test whether fic1Δ's polarity defect was independent of S phase completion , we arrested fic1Δ cells in late G2 using cdc25-22 , a temperature-sensitive allele of the phosphatase that activates cyclin-dependent kinase at the G2-M transition . As was previously observed [5] , otherwise wild-type cells blocked in G2 almost always underwent NETO ( Figure 2I–2J ) . However , roughly half of fic1Δ cells remained monopolar ( Figure 2I–2J ) , indicating that the fic1Δ polarity defect occurs irrespective of S phase completion . To test whether fic1Δ cells were too small to initiate NETO , we measured cell lengths at division . Though slightly shorter on average than wild-type cells ( 13 . 3 µm versus 15 . 3 µm ) , all fic1Δ cells were longer at division than the minimum length required for NETO ( ∼9 µm ) ( Figure 2K ) [5] . Therefore , it is unlikely that the fic1Δ growth polarity defect is caused by reduced cell length . These data underscore that loss of Fic1 disrupts the establishment and timing of NETO independently of previously described cell cycle controls . Though many cell polarity factors localize to the cell division site in addition to interphase cell tips , only the actions of these proteins at interphase cell tips have been demonstrated to be relevant to polarity regulation . As was observed previously [28] , cytoplasmic Fic1-GFP localizes to cell tips during interphase and later to the CR during cell division ( Figure 3A ) . We also detected another pool of Fic1-GFP lining the division site as the CR constricted ( Figure 3A ) . Given this localization pattern and the specific new end growth defect of fic1Δ cells , we asked whether Fic1 affected the timing of NETO via its functions at the cell division site . Like S . cerevisiae Inn1 [30] , Fic1 is comprised of an N-terminal C2 domain and a C-terminal stretch of PxxP motifs ( Figure 3B ) . As was found for Inn1 [29] , [30] , the C terminus of Fic1 ( “Fic1C” , amino acids 127–272 ) , expressed from its endogenous locus and GFP-tagged , was sufficient for CR localization , as judged by co-localization with the CR protein Cdc15-mCherry ( Figure 3C ) . In contrast , a GFP-tagged N-terminal C2 domain fragment ( “Fic1N” , amino acids 1–126 ) was never observed at the CR ( Figure 3C ) though it was produced in vivo ( Figure S2 ) . Importantly , medial-localizing Fic1C , unlike Fic1N , supported proper growth polarity establishment ( Figure 3D–3F ) , and , in contrast to full-length Fic1-GFP , Fic1C-GFP was not detected at tips of interphase cells ( Figure 3G ) . We thus conclude that Fic1 , unlike other characterized growth polarity factors , does not exert its polarity function at cell tips during interphase , but instead does so at the cell division site during cytokinesis . Because Fic1's C terminus was necessary and sufficient for proper growth polarity , we examined whether protein-protein interactions at the CR mediated by Fic1's C-terminal PxxP motifs , which bind SH3 domains , govern Fic1's polarity function . Fic1 was originally identified based on its interaction with Cdc15's SH3 domain [28] . As would be expected if association of Cdc15 with Fic1's C terminus is important in establishing the timing of NETO , calcofluor-stained cdc15ΔSH3 cells , which are viable but lack Fic1-Cdc15 interaction [28] , exhibited growth polarity defects ( Figure 4A–4C and Figure S3A–S3B ) . To address the consequence of specifically disrupting Fic1-Cdc15 interaction , we determined which of Fic1's C-terminal PxxP motifs interact ( s ) with Cdc15's SH3 domain . Previous yeast-two hybrid data indicated Fic1 amino acids 190–269 mediate direct association with Cdc15's SH3 domain [28] . This region contains four of the eleven PxxP motifs within Fic1's C terminus ( Figure S3C ) . To identify which are relevant for Cdc15 interaction , yeast two-hybrid assays using single and combinations of proline to alanine mutations were performed ( Figure S3D ) . Mutation of PxxPs 10 and 11 in combination , or P257 of PxxP 11 alone , abolished the two-hybrid interaction ( Figure S3D ) , and the P257A mutation eliminated co-immunoprecipitation of Fic1-FLAG3 with Cdc15 in vivo ( Figure 4D ) . Supporting the idea that the Fic1-Cdc15 interaction is most relevant during cell division , Fic1-GFP did not accumulate preferentially in Cdc15-mCherry puncta at interphase cell tips ( Figure S3E ) and co-immunoprecipitation of Fic1-FLAG3 with Cdc15 was considerably stronger in mitosis than in interphase ( Figure 4D ) . This is similar to other Cdc15 protein-protein interactions , which become enriched upon Cdc15 dephosphorylation at mitosis [26] . fic1-P257A cells exhibited monopolar growth defects similar to fic1Δ and cdc15ΔSH3 cells ( Figure 4A–4C ) , confirming that binding of Fic1's C terminus to Cdc15 is critical for Fic1's polarity function . Even so , Fic1-P257A-GFP still localized to the CR ( Figure S3F ) , indicating that medial localization of Fic1 during cytokinesis is necessary but not sufficient for re-establishment of proper growth polarity following cell division . To corroborate that PxxP-mediated protein-protein interactions at the cytokinetic ring play a predominant role in Fic1's polarity function , we tested whether other interactors participate in S . pombe polarity regulation . The SH3 protein Imp2 has previously been shown to function redundantly with Cdc15 and bind Fic1 during cytokinesis [28] . Consistent with additional Fic1 interactions guiding growth polarity , loss of Imp2 also severely compromised bipolar cell growth ( Figure 4A–4C and Figure S3A–S3B ) . In S . cerevisiae , the Fic1 ortholog Inn1 binds to another SH3 protein , Cyk3 [29] , [45] , and complexing of these two proteins with the Cdc15 homolog Hof1 has been suggested to direct septum formation and cell separation [29] . We found that S . pombe Cyk3 co-immunoprecipitated with Fic1 in mitosis ( Figure 4E ) , and we also detected direct interaction between S . pombe Cyk3's SH3 domain and Fic1 via yeast two-hybrid ( Figure S3G ) . Accordingly , these interactions appear to be conserved . As was also described in a recent study [46] , we found that Cyk3-GFP localized to the CR and division site during cytokinesis , and it was retained at new ends immediately following cell division ( Figure 4F ) . Consistent with these proteins performing a common function , loss of Cyk3 resulted in growth polarity defects similar to those seen upon loss of Fic1 or its interaction with Cdc15 or Imp2 ( Figure 4A–4C ) . Thus , Fic1 collaborates with associated proteins at the CR to execute its growth polarity function , and we postulate that its C terminus acts as an adaptor molecule for SH3 proteins to ensure integration of distinct processes during cytokinesis . Of note , Fic1-P257A-GFP still localized to the CR in imp2Δ cyk3Δ cells ( Figure S3H ) , indicating other CR proteins besides Cdc15 , Imp2 , and Cyk3 bind Fic1 and likely participate in polarity-relevant events at the division site . To discern how loss of Fic1 scaffold function during cytokinesis impacts subsequent new end growth , we next defined what aspects of cytokinesis are perturbed in fic1Δ cells . Previous data demonstrated that fic1Δ was synthetically lethal with sid2-250 [28] , a temperature-sensitive allele of the SIN kinase Sid2 . Consistent with Fic1 and associated factors working in parallel to the SIN , we found that fic1Δ and cyk3Δ suppressed the hyperactive SIN allele cdc16-116 ( Figure S4A ) , and that fic1Δ and cyk3Δ were synthetically sick or lethal with a variety of SIN alleles conferring loss of SIN function ( Figure S4A–S4B ) . These genetic data implied that Fic1 most likely functions during late stages of cytokinesis . In line with this idea , the percentage of fic1Δ cells that had undergone ingression but were still joined at their division sites was more than four times that of wild-type cells ( Figure 5A–5B ) . When cells were arrested in G2 using the cdc25-22 allele , this difference increased , with the percentage of joined cells roughly 15 times greater in the absence of Fic1 ( Figure 5A–5B ) . Similar to S . cerevisiae inn1Δ cells [29] and S . pombe cdc15ΔSH3 cells [28] , many G2-arrested fic1Δ daughter cells that were still joined at division sites exhibited membranous bridges ( Figure S4C ) . These findings verified that the completion of cell division is perturbed in the absence of Fic1 . Consistent with early cytokinesis events proceeding appropriately without Fic1 , time-lapse imaging of myosin regulatory light chain Rlc1-GFP [47] , [48] along with spindle pole body marker Sid4-GFP revealed that the CR formed and constricted normally in fic1Δ cells ( Figure 5C–5D ) . However , at the termination of CR constriction , parts of the CR persisted at the division plane ( Figure 5E–5G and Figure S4D ) . During cytokinesis , the septum closes behind the constricting CR , and septum closure can be visualized using the β-glucan synthase GFP-Cps1 [49] , [50] . As cytokinesis progresses , two GFP-Cps1 dots marking the leading edge of the septum can be seen getting progressively closer in the division plane , and these dots eventually join into one just as the CR completes constriction ( Figure 5F ) . We found that Rlc1-mCherry3 remained at the division site following septum closure on average longer in fic1Δ cells compared to wild-type cells ( 22 min versus 8 min ) ( Figure 5E–5F ) . Consistent with these remnants representing the CR as a whole and not just Rlc1 , phalloidin staining revealed atypical actin-rich masses , in addition to normal actin patches , flanking septa in fic1Δ cells ( Figure 5G ) . By expressing LifeAct-GFP , we verified that these abnormal actin masses co-localized to a high degree with Rlc1-mCherry3 in a fic1Δ genetic background ( Figure S4D ) . Thus , we conclude that the CR does not disassemble properly at the conclusion of cell division in fic1Δ cells . In addition to CR-associated factors , glucanase Eng1-GFP [35] persisted at ingressed division sites significantly longer in fic1Δ cells compared to wild-type cells ( on average , 51 min versus 21 min ) ( Figure S4E–S4F ) . Because glucanases execute septum degradation [34] , [35] , these data suggest that cell wall turnover is inefficient at fic1Δ septa . We thus conclude that loss of Fic1 jeopardized the completion of cell division , stalling remodeling of new ends in the next cell cycle . Because faulty cytokinesis led to persistence of parts of the cell division machinery at fic1Δ division planes , we speculated that these remnants might deter subsequent polarized growth at new ends . If this were the case , one would expect other mutants with late cytokinesis defects to also show erroneous new end growth . Previous data had indicated that Fic1-associated Imp2 contributes to CR disassembly , with imp2Δ cells exhibiting abnormal actin structures flanking previous division sites [51] . Though we had shown that imp2Δ cells are defective in bipolar cell growth ( Figure 4A–4C ) , we wanted to confirm that their growth defect was specific to new ends . Using time-lapse DIC imaging , we found that roughly 75% of imp2Δ cell divisions produced at least one daughter cell that failed at new end growth ( Figure 6A ) . Interestingly , both imp2Δ daughter cells failed at new end growth in the majority of cases ( Figure 6A–6B ) . Therefore , proper disassembly of CR components correlates with new end competency for polarized growth . In addition to showing CR disassembly defects , fic1Δ cells also exhibited delays in septum remodeling at the division site . We therefore tested if disruption of septum degradation could likewise impact polarized growth . Loss of Eng1 or its cooperating glucanase , Agn1 [34] , resulted in high percentages of monopolar growth ( Figure 6C–6D and Figure S5A ) . Moreover , the growth defect of eng1Δ daughter cells was specific to new ends ( Figure 6A–6B ) , and , similar to fic1Δ cells , eng1Δ daughter cells that initiated NETO prior to the next septation did so on average later than wild-type cells ( 129 min versus 75 min ) ( Figure S5B ) . Anillin-like Mid2 and the septin ring , of which Spn1 and Spn4 form the core [52] , target these glucanases into a ring structure around septa [53] . Loss of any of these proteins likewise impaired bipolar cell growth ( Figure 6C–6D and Figure S5A ) . In addition , though the majority of spn1Δ daughter cells failed at new end growth ( Figure 6A–6B ) , those that initiated NETO prior to the next septation took longer on average to do so than wild-type cells ( 95 min versus 75 min ) ( Figure S5B ) . We therefore conclude that defective completion of cell wall remodeling at the division site , in addition to improper disassembly of CR components , compromises NETO efficiency . The SIN coordinates many aspects of CR and septum regulation during late cytokinesis . Not only does SIN signaling oversee maintenance of a mature , homogenous CR [54] , it mediates Cps1 targeting and accumulation at the division site [49] , [50] . Loss of SIN signaling during cytokinesis can thus lead to CR fragmentation [54] and abortive septation [49] . Given these phenotypes and the synthetic genetic interactions between fic1Δ and SIN mutants ( Figure S4A–S4B ) , we examined the relevance of the SIN to new end growth control . Temperature-sensitive alleles of genes encoding the SIN kinases Cdc7 and Sid2 caused mild but statistically significant growth polarity defects at semi-restrictive temperature ( Figure 6C–6D and Figure S5A ) . A temperature-sensitive allele of the gene encoding Cps1 , which functions downstream of the SIN , caused dramatic defects in establishing bipolar cell growth even at permissive temperature ( Figure 6C–6D and Figure S5A ) . Additionally , a high proportion of cps1-191 cells failed specifically at new end growth ( Figure 6A–6B ) , and those that were able to trigger NETO prior to subsequent septation did so on average later than wild-type cells ( 107 min versus 75 min ) ( Figure S5B ) . Not surprisingly , we were able to detect incomplete ingression of cps1-191 cells shifted to the restrictive temperature during cytokinesis ( Figure S5C ) , again suggesting that these mutants experience remodeling errors at the division site . Currently , the mechanism of membrane remodeling and scission at the S . pombe division site is unclear . In a variety of other organisms , endosomal sorting complex required for transport ( ESCRT ) -III factors contribute to this process [55] . ESCRT-III components have not been implicated in S . pombe cytokinesis regulation , though ESCRT-III-associated AMSH ( S . pombe Sst2 ) localizes to the division site [56] . We found that deletions of genes encoding ESCRT-III components Vps2 and Vps24 or ESCRT-III-associated Sst2 were synthetically sick with a variety of loss-of-function cytokinesis alleles , including imp2Δ and cps1-191 ( Figure S5D ) . Interestingly , loss of Vps2 , Vps24 , or Sst2 resulted in monopolar percentages significantly greater than observed for wild-type cells ( Figure 6C–6D and Figure S5A ) , and nearly half of vps24Δ cell divisions resulted in one or both daughter cells that failed at new end growth prior to the next septation ( Figure 6A–6B ) . Though these phenotypes were less penetrant than in other mutants , we speculate that ESCRT-III function guides membrane remodeling at the conclusion of S . pombe cell division to impact new end polarized growth . Of note , deletion of rlc1+ or paxillin pxl1+ , which function primarily in early actomyosin function at the CR [47] , [48] , [57] , [58] , did not alter growth polarity percentages as significantly as other mutations or deletions ( Figure 6C–6D and Figure S5A ) . Indeed , less than half of non-septated rlc1Δ and pxl1Δ cells were monopolar ( Figure 6C ) , and the monopolar septated percentages of these genotypes were more similar to wild-type percentages than were those of the other mutants examined ( Figure 6D ) . We therefore conclude that early steps in cytokinesis do not impact subsequent polarized cell growth as much as the terminal steps in cell division . If faithful remodeling of the division site is important for growth competency of new ends , then one would expect that prematurely triggering NETO signaling just after cell division should not fully rescue the growth polarity defects of late cytokinesis mutants . To test this , we constructed a mutant that would undergo constitutive NETO . As over-expression of a fusion protein linking cell tip-associated Tea1 with formin For3 induces NETO in G1 [8] , we integrated a Tea1-For3 fusion ( Figure 7A ) into the endogenous tea1+ locus and deleted the single copy of the for3+ gene . We confirmed that the Tea1-For3 fusion protein was produced in vivo ( Figure 7B ) and verified that this fusion was sufficient to induce NETO in a cdc10-V50 G1 arrest ( Figure S6A–S6B ) . As previously reported [7] , double deletion of tea1+ and for3+ resulted in general cell rounding ( Figure 7C ) . However , expression of the Tea1-For3 fusion protein in the absence of Tea1 and For3 individually caused cells to regain their rod-shaped appearance ( Figure 7C ) . Intriguingly , a high percentage of tea1-for3 cells were either septated or exhibited cytokinesis defects ( Figure 7C–7D ) , and tea1-for3 cells were significantly longer at division than wild-type cells ( on average , 18 . 3 µm versus 15 . 3 µm ) ( Figure 7C and 7E ) . Thus , though the endogenous Tea1-For3 fusion protein functioned in prematurely triggering NETO , it also affected cell division . To analyze tea1-for3 cells in real-time , we performed time-lapse DIC imaging . As expected , most tea1-for3 cells underwent NETO before the next cell division ( Figure 8A ) , with nearly 75% of new ends initiating growth within 50 minutes of septum splitting ( Figure 8B ) . Nonetheless , some tea1-for3 outliers took much longer to extend at tips created by cell division ( Figure 8B ) . After grouping the times needed for tip growth to occur at previous division sites relative to the amount of time needed for the mother cell to complete cytokinesis , we found that newly-formed tips that took longer to initiate growth had been formed by more inefficient cytokinesis ( Figure 8C–8D ) . As distal tip growth continued in cells undergoing division ( Figure 8D ) and appeared unimpeded by additional factors , these findings suggested that faulty cytokinesis imposes constraints at previous division sites that counteract positive polarizing cues . We corroborated this model by expressing the Tea1-For3 fusion in fic1Δ cells . Although tea1-for3 cells were mostly bipolar , tea1-for3 fic1Δ cells showed a high percentage of monopolar growth ( Figure 8E–8G ) . These findings confirmed that efficient completion of cytokinesis is critical for new end growth , even when signaling networks responsible for NETO are prematurely activated . S . pombe undergoing a dimorphic switch from single-celled to invasive form grow primarily in a monopolar fashion at old ends [39] , [40] . Moreover , it has been postulated that cytokinesis errors might contribute to a hyphal-like transition in S . pombe [41] . We therefore considered that cytokinesis-based constraints on S . pombe growth polarity might facilitate invasive growth transitions . Using techniques similar to those described previously [40] , [59] , we tested whether various cytokinesis mutants displaying defective bipolar growth could form pseudohyphae into 2% agar . Cells lacking Fic1 or its interacting partners Cyk3 or Imp2 were significantly more invasive than wild-type cells ( Figure 9A–9B ) . Like other invasive S . pombe mutants [39] , [40] , these mutants formed pseudohyphae composed of single cells oriented in filament-like projections ( Figure 9C and Figure S7A ) . In addition to these strains , we found other cytokinesis mutants exhibiting high degrees of monopolar growth ( spn1Δ , cdc7-24 , and vps24Δ ) to also be highly invasive and to form pseudohyphal projections into 2% agar ( Figure 9A–9B and Figure S7A ) . Of note , the vps24Δ strain showed drastically more invasive growth than the others , though the reasons for this are currently unclear . rlc1Δ and pxl1Δ , which possess cytokinesis defects that do not considerably impact polarized cell growth ( Figure 6C–6D and Figure S5A ) , invaded less efficiently on 2% agar than cytokinesis mutants exhibiting NETO defects ( Figure 9A–9B ) . This supports the notion that defective cytokinesis promotes the dimorphic switch most robustly when it results in faulty NETO . As has previously been observed , tea1Δ also invaded well on 2% agar ( Figure S7B–S7D ) . Thus , though cytokinesis-based constraints on growth polarity support enhanced S . pombe invasiveness , other polarity defects , which are not entirely specific to new ends , can do so as well . Consistent with bipolar growth defects accompanying pseudohyphal growth , tea1-for3 cells , which experience constitutive NETO induction , almost never extended pseudohyphae into 2% agar ( Figure 9D–9E ) . Because cytokinesis-based constraints on growth polarity partially override tip-based NETO signaling , we reasoned that tea1-for3 cells should become more invasive upon loss of Fic1 . Indeed , on 2% agar tea1-for3 fic1Δ cells formed pseudohyphae ( Figure S7E ) , which were more numerous than those observed for wild-type and tea1-for3 strains ( Figure 9D–9E ) . Thus , perturbations in cytokinesis cause growth polarity errors that facilitate pseudohyphal growth even upon constant NETO signaling . Lastly , we asked whether loss of polarity-relevant cytokinesis factors could partially rescue invasiveness of an asp1Δ strain , which is unable to undergo the dimorphic switch due to an inability to sense external cues [40] . Previously , it was demonstrated that asp1Δ cells form a biofilm-like colony on 0 . 3% agar ( Figure 9F ) [40] . Growth on 0 . 3% agar is more sensitive for assaying invasiveness of strains that invade less efficiently , as wild-type colonies form protrusions on 0 . 3% agar but extend relatively few pseudohyphal projections into 2% agar ( Figure 9A–9B and 9F ) [40] . We therefore assessed the effect of cytokinesis defects on asp1Δ invasiveness by testing whether asp1Δ strains that also lacked relevant cytokinesis factors still formed biofilms on 0 . 3% agar . Intriguingly , double deletion strains of asp1Δ with fic1Δ , spn1Δ , or vps24Δ did not form biofilms on 0 . 3% agar but instead made projections into and on the surface of the agar ( Figure 9F ) . In contrast , double deletion strains of asp1D with either rlc1Δ or pxl1Δ still formed biofilms on 0 . 3% agar ( Figure 9F ) . We thus conclude that cytokinesis-based constraints on polarized cell growth in S . pombe can foster invasiveness even in the absence of typical nutritional signals .
The majority of S . pombe monopolar mutants previously analyzed fail at old end growth [4] . However , the cytokinesis mutants studied here were predominantly new end growth defective . As in other organisms [60] , numerous S . pombe proteins known to affect growth polarity localize to the division site; this has fostered speculation that signaling at both cell tips and the division site might impact growth zones [23] . However , whether or not the cytokinesis functions of these proteins can specifically impact cell polarity has received little attention , especially in S . pombe . Our data provide evidence directly linking division site organization to S . pombe growth polarity . Because many factors involved in completing cell division likely also impact subsequent polarized growth , we believe our data could explain the involvement of diverse proteins in this process . In other organisms , cytokinesis proteins appoint local regions of the cell cortex for growth following cell division [61]–[63] . For example , several budding yeast proteins , which remain at the cell cortex following cell division , have been reported to convey a cortical “tag” that marks the position of the next bud site [61] , [62] . Similarly , during Drosophila melanogaster neurogenesis , cytokinetic furrow components mark the site from which the first dendrite will sprout [63] . In these cases , cytokinesis factors confer a positive polarizing cue adjacent to previous division sites , which contrasts with our findings in S . pombe where the cell division machinery impedes polarization and growth at new ends created by cell division . The fact that S . pombe grow at old but not new ends after cell division is somewhat counterintuitive [4] , especially because the cell growth machinery concentrates at the division site . Upon the completion of cytokinesis , the S . pombe growth machinery mysteriously shuttles to old ends rather than remaining at new ends . Why does the growth machinery relocate from the division site to old ends ? One explanation is that new end cortices must be re-structured to become competent for tip growth . Indeed , specific lipid and cell wall variants contribute to S . pombe cytokinesis [64] , [65] , and local rearrangements of these may be required for growth activation . Moreover , the persistence of CR factors at new ends might create physical barriers to cytoskeletal elements , such as actin cables , required for tip growth . An inhibitory role for cell division in polarization is supported by studies of mutants that undergo multiple rounds of cytokinesis without physically separating , because internal cells in these structures do not grow into septa but branch adjacently . In a single-celled context , we speculate that when late cytokinetic events are perturbed , inherent delays in cortical re-structuring are exacerbated , causing growth polarity defects at new ends . In cases in which one daughter cell fails at NETO while the other is successful , we suspect these arise due to unequal partitioning of cytokinesis remnants and/or differences in cell cycle stages or life histories of daughter cells . Consistent with furrow remodeling affecting polarization in other organisms , initial cellular protrusions of dividing mammalian cells orient away from the midbody linking daughter cells during abscission [66] . Only after the completion of cell division does polarization also occur near the latent division site [66] . Moreover , forced entry of HeLa cells with monopolar spindles into cytokinesis results in anucleate daughter cells that , similar to their nucleated counterparts , exhibit membrane protrusions only distal to cleavage furrows [67] . Thus , similar to our model in S . pombe , some factor at the division site cortex , and not a cell's cytosolic constituents , requires remodeling for post-cytokinetic polarization . Recent evidence indicates that mechanosensory pathways can direct cell polarization away from points of tension [68] . As modeling predicts that cortical tension peaks at the division plane during cytokinesis [69] , it will likewise be important to assess the relevance of mechanical cues to cytokinesis-based polarization events . Previous work has implied that association of microtubule-associated protein Tea1 with formin For3 at new ends is sufficient for NETO [8] . In our study , we expressed an endogenous Tea1-For3 fusion that could induce premature NETO . However , when cytokinesis was perturbed in tea1-for3 mutants , NETO was delayed . We posit that local cortical abnormalities in cell wall , membrane , or associated factors can partially override typical growth cues in S . pombe , as has been observed in some plants [70] . Upon defective cytokinesis , such abnormalities at the division site may physically inhibit cell growth at new ends . These defects can lead to the formation of T-shaped cells when old end growth is also blocked , as in tea1Δ fic1Δ mutants . Our data underscore robust completion of cytokinesis as a major determinant of S . pombe NETO . Is it beneficial for a cell to halt new end growth until well after cytokinesis completion ? As mentioned previously , human cells undergoing division initially move away from each other , creating a pulling force that could contribute to abscission [66] . Highly adherent mammalian cells can actually complete cytokinesis , with some defects , in the absence of cortical myosin from the cleavage furrow [71] . Constriction-independent cytokinesis was first observed in the amoeba Dictyostelium discoideum [72] , which accomplishes this task by likewise polarizing and growing distally to the division site [73] . One could imagine that in cases where S . pombe cell separation is delayed , tip growth at old ends might contribute similar forces to aid in abscission . Premature new end growth signaling might unbalance these forces , leading to exacerbated cytokinesis delays as in some tea1-for3 cells . Premature new end growth might also interfere with remodeling during cytokinesis and thereby result in cell division defects . These findings highlight interdependence between the cell polarization and division machineries in S . pombe . Our data indicate that Fic1's C terminus , and not its C2 domain , represents its major cytokinetic functional domain , contrasting with data reported for S . cerevisiae Inn1 [29] , [30] . Why is Fic1's C2 domain dispensable for Fic1's cytokinesis , and thus polarity , functions ? Sequence alignment indicates that there is in fact very low sequence identity between Fic1 and Inn1 C2 domains [30] . If Fic1's C2 domain is unable to perform functions or mediate interactions that Inn1's C2 domain can , it seems reasonable that Fic1-interacting proteins may be able to compensate . Consistent with this idea , over-expression of S . cerevisiae Cyk3 suppresses cytokinesis defects of inn1Δ mutants , suggesting Cyk3 function overlaps with Inn1 [29] . These data support that Fic1's C terminus is an efficient signaling platform , which scaffolds SH3 domain proteins through its PxxP motifs to ensure coherent integration of late cytokinesis signals . What is the specific function of the Fic1 scaffold during cytokinesis ? Our data indicate that loss of Fic1 leads to faulty CR disassembly and prolonged persistence of factors at the division site . CR disassembly defects were also observed in inn1Δ S . cerevisiae mutants , leading to speculation that Inn1 might stabilize the constricting CR by physically linking it to the ingressing membrane [30] . Subsequent findings countered that Inn1's C2 domain cannot bind phospholipids , and it was postulated instead that Inn1 cooperates with Cyk3 to coordinate cell wall deposition [29] . As in fic1Δ cells , septation and CR disassembly defects commonly accompany one another [51] , [74] . Because these processes are inextricably linked , it is currently difficult to tease apart which defect precedes the other in fic1Δ cells . Moreover , completion of cytokinesis also requires lipid rearrangements in both animal cells and S . pombe [64] , [75] , and membrane bridges were observed in fic1Δ cells . We thus envision that Fic1's C terminus links signaling pathways that guide completion of multiple tasks during late cytokinesis and thereby affect new end remodeling . Of note , we believe that defects in early cytokinesis do not significantly alter bipolar growth establishment , as later defects more directly impinge on division site remodeling and have less time to be remedied before the next cell division . Our data furthermore support the notion that CR constriction and disassembly occur independently [74] , as CR constriction but not disassembly proceeded appropriately in fic1Δ cells . As fungal hyphae consist of long chains of cells , the transition into hyphal growth requires strict inhibition of cytokinesis . In some yeasts , Cdc14 phosphatase activates the Ace2 transcriptional program [76] , which triggers expression of cell separation enzymes [77] . Upon the hyphal transition in Candida albicans , this signaling cascade is disrupted [78] , and other transcription factors suppress expression of Ace2 targets [79] . Therefore , cytokinetic inhibition in hyphae is believed to operate largely on a transcriptional level , and reactivation of the Ace2 transcriptional program is thought to be responsible for the evolution of single-celled yeast growth [41] . In this study , we showed that fission yeast cells that undergo defective , yet not wholly abortive , cytokinesis exhibit enhanced invasive capacity . We believe cytokinesis-based constraints on growth polarity assist the transition into pseudohyphal growth because they force S . pombe to orient outwards and grow predominantly from old ends , a pattern commonly observed in S . pombe pseudohyphal growth [39] , [40] . However , as demonstrated by tea1Δ cells , other changes in polarity can also enhance S . pombe invasiveness . Though not specifically defective at new end growth , tea1Δ cells grow predominantly in the direction of the mother cell , and these alterations in polarity might likewise favor growth orientations that are more conducive than bipolar growth to the invasive process . We believe our data suggest that manipulation of cytokinesis proteins , and not necessarily signaling cascades that feed into downstream transcriptional pathways , can directly modulate the dimorphic switch . We thus speculate that the cytokinetic machinery might represent a direct target of the pseudohyphal developmental program . Intriguingly , loss of cytokinesis proteins that affect NETO rescued invasiveness of an asp1Δ mutant , which lacks the ability to detect nutritional cues [40] deemed important for the S . pombe dimorphic switch [36] . Because various environmental cues also regulate hyphal morphogenesis in pathogenic fungi [80] , it will be important to assess the relative significance of cytokinesis-based controls on polarized growth for invasiveness in these species .
The S . pombe strains used in this study ( Table S1 ) were grown in either yeast extract ( YE ) or Edinburgh minimal media with relevant supplements . fic1+ , fic1N , fic1C , fic1-P257A , crn1+ , tea4+ , rlc1+ , tea1+ , for3+ , and tea1-for3 were tagged endogenously at the 3′ end with GFP:kanR , FLAG3:kanR , mCherry3:kanR , RFP:hygR , V5:kanR , or V5:hygR cassettes as previously described [81] . A lithium acetate method [82] was used in S . pombe tagging transformations , and integration of tags was verified using whole-cell PCR and/or fluorescence microscopy . Introduction of tagged loci into other strains was accomplished using standard S . pombe mating , sporulation , and tetrad dissection techniques . For blocking of cdc25-22 strains in G2 , cells were grown at 25°C and then shifted to 36°C for 3 h . For blocking of nda3-KM311 strains in prometaphase , cells were grown at 32°C and then shifted to 18°C for 6 . 5 h . For blocking of cdc10-V50 strains in G1 , cells were grown at 25°C and then shifted to 36°C for 4 h . For blocking of cps1-191 cells in a cytokinesis arrest , cells were grown at 25°C and then shifted to 36°C for 3 h . Mutants and truncations of fic1 were expressed from the endogenous fic1+ locus . To make these strains , a pIRT2 vector was originally constructed in which fic1+ gDNA with 5′ and 3′ flanks was inserted between BamHI and PstI sites of pIRT2 . Mutations were then introduced via site-directed mutagenesis . The fic1 ( aa1-126 ) construct was made by inserting a stop codon after residue 126 . The fic1 ( aa127-272 ) construct was created by inserting XhoI sites before both the start codon and residue 127 , digesting with XhoI to release the internal fragment , re-ligating the plasmid , and adding a start codon after the remaining XhoI site . fic1Δ was then covered by these pIRT2-fic1 constructs , and stable integrants resistant to 5-FOA were isolated and confirmed by whole-cell PCR and western blotting . To make the tea1-for3 fusion , a pIRT2 vector was originally constructed in which tea1+ gDNA with 5′ and 3′ flanks was inserted between SacI and SphI sites of pIRT2 . Site-directed mutagenesis was performed to replace the tea1+ stop codon with a SmaI/SalI/PstI multiple cloning site . for3+ gDNA was amplified with a small N-terminal linker sequence and inserted between SmaI and PstI in this multiple cloning site ( linker residues are Pro-Gly-Ade-Gly-Ade-Gly-Ade accounting for restriction site and added residues ) . tea1Δ was then covered by this pIRT2-tea1-for3 construct , and stable integrants resistant to 5-FOA were isolated and confirmed by whole-cell PCR and western blotting . An integrant was subsequently mated with for3Δ , such that we could isolate tea1-for3 strains in which tea1+ and for3+ were lacking . Expression of acyl-GFP [83] was controlled by the thiamine-repressible nmt1 promoter of pREP3 [84] , [85] . Expression of LifeAct-GFP [86] was controlled by the thiamine-repressible nmt81 promoter of pREP81 [87] . Expression from these nmt promoters was kept off by addition of 5 µg/mL thiamine to the medium , and expression was induced by washing and culturing in medium lacking thiamine for at least 24 h . Spot assays to analyze genetic interactions were performed as previously described [88] , except that all were done on YE agar . Synthetic interactions were judged based on differences in growth between double mutants and relevant single mutants . Yeast two-hybrid analysis was performed as previously described [89] , except that the bait and prey plasmids were either empty or encoded Cdc15 SH3 ( aa843–927 ) [28] , Cyk3 SH3 ( aa1–59 ) , wild-type [28] or mutant Fic1 ( aa190–269 ) fragments , or full-length Fic1 . Cells were lysed by bead disruption in NP40 lysis buffer in either native or denaturing conditions as previously described [90] , except with the addition of 0 . 5 mM diisopropyl fluorophosphate ( Sigma-Aldrich ) . Proteins were immunoprecipitated by anti-FLAG ( Sigma-Aldrich ) , anti-Cdc15 [28] , or anti-V5 ( Invitrogen ) antibodies . Immunoblot analysis of cell lysates and immunoprecipitates was performed using anti-FLAG , anti-Cdc15 , anti-V5 , anti-GFP ( Roche ) , or anti-Cdc2 ( Sigma-Aldrich ) antibodies as previously described [88] . Live-cell bright field images as well as all still images of cells expressing proteins endogenously-tagged with GFP , RFP , or mCherry were acquired on a spinning disc confocal microscope ( Ultraview LCI; PerkinElmer ) equipped with a 100X NA 1 . 40 PlanApo oil immersion objective , a 488-nm argon ion laser ( GFP ) , and a 594-nm helium neon laser ( RFP , mCherry ) . Images were taken via a charge-coupled device camera ( Orca-ER; Hamamatsu Phototonics ) and processed using Metamorph 7 . 1 software ( MDS Analytical Technologies; Molecular Devices ) . Bright field images were used in determining cell lengths at division . Time-lapse GFP images of cyk3-GFP sid4-GFP cells secured on agar pads , which were sealed by Valap ( a Vaseline , lanolin , and paraffin mixture ) , were acquired every 3 min using this system . Z-sections were acquired for all fluorescence images and combined into maximum projections . Cells were grown to log phase at 25°C before such imaging . Images of yeast cells and pseudohyphae on YE agar plates were acquired by focusing a camera ( PowerShot SD750; Canon ) through a microscope ( Universal; Carl Zeiss ) equipped with a 20X NA 0 . 32 objective . All other microscopy was performed using a personal DeltaVision microscope system ( Applied Precision ) . This system includes an Olympus IX71 microscope , 60X NA 1 . 42 PlanApo and 100X NA 1 . 40 UPlanSApo objectives , fixed- and live-cell filter wheels , a Photometrics CoolSnap HQ2 camera , and softWoRx imaging software . The microscopy performed using this system was as follows: To assay pseudohyphal invasion into 2% agar , 5 µl containing a total of 105 cells were spotted on 2% YE agar and incubated at 29°C for 20 days . Colonies were subsequently placed under a steady stream of water and surface growth was wiped off using a paper towel . These methods were established in previous studies [40] , [59] . To assay whether specific mutants rescued invasiveness of an asp1Δ strain on 0 . 3% agar [40] , 1 µl containing 106 cells was spotted on 0 . 3% YE agar as well as onto 2% agar as a control . Plates were incubated at 29°C for 12 days , at which point colony growth and/or biofilm formation were visualized . | Many processes , including cell growth , are often regulated differently in distinct cellular regions . In the rod-shaped fission yeast Schizosaccharomyces pombe , new cell ends created by cell division initiate growth long after old cell ends inherited from mother cells . Though distributions of cell tip factors contribute to this growth pattern , we have found that the process of cytokinesis , which executes physical separation of daughter cells at the end of the cell cycle , also plays an important role in defining new end-growth competency . Defects in completing cytokinesis and remodeling the division site curb new end growth even when protein complexes that drive tip elongation constitutively associate with new cell ends . Moreover , when parts of the cytokinetic machinery persist at the division plane following constriction , S . pombe cells become highly invasive . We believe that these findings provide insight into growth transitions in pathogenic fungi , as well as into the evolution of the single-celled state from multicellular hyphal forms . Additionally , we speculate that cytokinesis-based constraints on growth polarity might be conserved in mammalian cells , which have been reported to likewise polarize only distally to the cleavage furrow at the conclusion of cell division . | [
"Abstract",
"Introduction",
"Results",
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"Materials",
"and",
"Methods"
] | [] | 2012 | Cytokinesis-Based Constraints on Polarized Cell Growth in Fission Yeast |
TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates . All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5α but they have distinct lentiviral specificities , conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques . Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages , resulting in species-specific alleles that target different viruses . Structural , thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain , R69H , occurred early in macaque TRIMCyp evolution , expanding restriction specificity to the lentiviral lineages found in African green monkeys , sooty mangabeys and chimpanzees . Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity . We reveal how specificity is altered by a scaffold mutation , E143K , that modifies surface electrostatics and propagates conformational changes into the active site . Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations .
Mammals have evolved antiviral proteins called restriction factors , which contribute to their protection from pathogenic viral infections . Expression of restriction factors is invariably enhanced by the innate immune cytokines of the type one interferon family suggesting that restriction factors are an integral part of the innate immune system [1] . Pathogenic viral infections are thought to be a significant source of selective pressure on restriction factor evolution . Evidence for positive selection is found in the sequences of the intracellular antiviral restriction factors APOBEC3G , TRIM5α and tetherin and the positively selected amino acids have been shown to influence antiviral specificity [2]-[5] . Positions under positive selection tend to be in patches on the protein that directly contact the pathogen . Mutation of these residues alters which viruses are restricted . Variability and evidence for positive selection in regions of contact between host and pathogen illustrate the evolutionary conflict during which both constantly evolve under pressure from the other , with each alternately gaining the advantage . This ongoing arms race is described by the Red Queen hypothesis [6] , [7] which has also been elegantly demonstrated by the study of bacteria/phage coevolution [8] . The restriction factor TRIM5α contains an N terminal tripartite motif comprising RING , Bbox2 and coiled coil domains and a C terminal PRYSPRY or B30 . 2 sequence that constitutes the virus-binding domain [9]–[13] . TRIM5α exhibits potent species-specific antiviral activity against retroviruses . This activity is mediated , in part , by recruiting proteasomes to incoming retroviral capsids leading to their premature uncoating and destruction [13]–[16] . This is observed as a potent and early block to viral DNA synthesis by reverse transcription . TRIM5α dimers are thought to recruit to the retrovirus via interactions between their B30 . 2 domain and retroviral capsid molecules [17] . Patches of amino acids with evidence for positive selection are found in the B30 . 2 domain in exposed loops on the very end of the molecule [3] , [18] . The differences between species variants of TRIM5α in this region dictate antiviral specificity by determining which capsids can be recruited . Similarly , sequence differences between the viral capsids from various retroviruses , particularly in the exposed loop region referred to as the cyclophilin binding loop , influence sensitivity of the virus to restriction by TRIM5α , presumably by influencing the ability of TRIM5α to bind [10]–[12] , [19] . TRIM5-CypA chimeric proteins , referred to as TRIMCyps , have evolved through modification of the TRIM5 genetic locus on two independent occasions during primate evolution . [20]–[26] . Retrotransposition events , indicated by typical target site duplications , have inserted complete CypA cDNA sequences into the TRIM5 loci in different positions in New World owl monkeys and Old World macaques . These modifications have caused replacement of the virus binding B30 . 2 domain with a cyclophilin A ( CypA ) cDNA ( Fig . 1A ) CypA is a ubiquitous and highly conserved protein , whose sequence is identical in humans and macaques [27] . However , the CypA sequences in owl monkey and macaque TRIMCyps are not the same . Furthermore , like the parental CypA domain , owl monkey TRIMCyp targets HIV-1 M and O-group viruses but not HIV-2 , whereas rhesus macaque TRIMCyp targets HIV-2 and HIV-1 O group but not HIV-1 M group virus . HIV-1 and HIV-2 are derived from zoonoses of different lineages of primate lentivirus , HIV-1 from chimpanzees and HIV-2 from sooty mangabeys [28] , [29] . HIV-1 O and M group viruses are also distinct viruses , derived from different zoonotic transfers from chimpanzees and with radically different frequencies in the human population [29] . Recently , we showed that the change in lentiviral specificity in rhesus macaque TRIMCyp is mediated by two mutations at positions 369 and 372 ( equivalent to positions 66 and 69 of the Cyp domain ) that alter the conformation of the active site [30] . From hereon we use the CypA numbering for example TRIMCyp residue 369 is referred to as Cyp 66 . Importantly , previous work showed that TRIMCyp encoding a Cyp domain changed at a single amino acid from the natural cyclophilin A sequence ( Cyp R69H ) restricts a broader range of viruses , including HIV-1 M and O groups and HIV-2 [30] . Since a broad anti-viral specificity would confer a selective advantage , we hypothesised that a TRIMCyp encoding this single change may be present as a natural allele . Investigation of naturally occurring TRIMCyp alleles in Asian Macaques reveals that an ancestral TRIMCyp bearing this broad-specificity mutation ( Cyp R69H ) has been specialised towards different lentiviruses in different species , giving rise to distinct specificities in present day macaques .
A TRIMCyp encoding the mutation R69H but not D66N in its Cyp domain has previously been identified in Macaca fascicularis [24] . To characterise Macaca fascicularis TRIMCyp ( Mafa TRIMCyp ) we RT PCR cloned a TRIMCyp cDNA from RNA purified from peripheral blood lymphocytes from an Indonesian Cynomolgus macaque . The protein sequence of the Mafa TRIMCyp cDNA that we amplified differed from that described by Brennan and colleagues at 4 positions I77T , E209K , D247E , and H357R ( CypH54R ) . We refer to Brennan's allele as Mafa TRIMCyp1 and the allele we cloned as Mafa TRIMCyp2 . The protein sequences of the two Mafa TRIMCyps are shown aligned to the rhesus macaque ( Macaca mulatta ) ( Mamu ) TRIMCyp and CypA protein sequences ( Fig . 1 ) . In order to examine the antiviral specificity of Mafa TRIMCyp2 we cloned it into a murine leukaemia virus ( MLV ) based expression vector and expressed it in feline CRFK cells as described [22] . These cells naturally encode a truncated TRIM5 and are thus highly permissive to VSV-G pseudotyped , GFP encoding , lentiviral vectors and exhibit no detectable post-entry restriction activity to retroviral vectors [31] , [32] . We infected the Mafa TRIMCyp2 expressing CRFK cells with lentiviral vectors derived from HIV-1 , HIV-2 , Simian Immunodeficiency Virus from Tantalus monkeys ( SIVtan ) and Feline Immunodeficiency Virus ( FIV ) as described [33] , [34] . CRFK expressing empty vector were infected as a control and infected cells were enumerated 48 hours later by flow cytometry ( Fig . 2A ) . Mafa TRIMCyp2 restricted HIV-1 and SIVtan but not HIV-2 infectivity . Mafa TRIMCyp2 also restricted FIV infectivity . Furthermore , HIV-1 , SIVtan and FIV infectivity were rescued by adding the CypA inhibitor cyclosporine ( Cs ) at 5 µM , as has been described [22] , [23] . We were surprised that Mafa TRIMCyp2 restricted HIV-1 and not HIV-2 because a previous study has shown that Mamu TRIMCyp protein encoding identical residues at Cyp domain positions 66 ( aspartate ) and 69 ( histidine ) can restrict both viruses [30] . Comparison of the Mafa and Mamu sequences ( Fig . 1 ) indicates that the Mafa TRIMCyp proteins differ from the Mamu TRIMCyp sequence in the CypA domain at position Cyp 143 , encoding a lysine rather than a glutamate . We therefore mutated the Mafa TRIMCyp2 to resemble the Mamu sequence at this position ( Cyp K143E ) and expressed it in CRFK cells as before . Cells expressing the MafaTRIMCyp2 Cyp K143E were able to restrict both HIV-1 and HIV-2 by around 2 orders of magnitude ( Fig . 2B ) . This TRIMCyp mutant was also much more potent against SIVtan infectivity . Treatment with 5 µM Cs partially rescued HIV-1 and SIVtan infectivity and completely rescued HIV-2 infectivity ( Fig . 2B ) . We hypothesised that the Cyp E143K ( E446K ) mutation specifically prevents binding of the HIV-2 capsid , whilst HIV-1 binding is unaffected . To test this , we measured interaction between the HIV-1 and HIV-2 N-terminal capsid domains and the CypA domains from the Mafa TRIMCyp2 allele and K446E ( Cyp K143E ) mutant by isothermal titration calorimetry ( ITC ) . Whilst the K143E mutant bound both HIV-1 and HIV-2 , Mafa TRIMCyp2 was only able to bind HIV-1 ( Table 1 ) . These data demonstrate that the Cyp E143K change in Mafa TRIMCyp2 switches specificity from being able to bind and restrict HIV-1 and HIV-2 to being able to bind and restrict only HIV-1 . Cyp E143K also influences antiviral specificity against the African green monkey lineage virus SIVtan , restricting it much more weakly than its ancestral TRIMCyp protein . This change therefore influences antiviral activity against members of three lineages of extant primate lentiviruses . In a previous study comparison of the crystal structure of the N terminal capsid domain of HIV-2 with HIV-1 indicated that the HIV-2 CypA binding loop is shorter by a single alanine residue at the position equivalent to HIV-1 CA88 [30] . This underlies a significant conformational difference between the CypA binding loops of HIV-1 and HIV-2 capsids and dictates binding to TRIMCyp variants , which in turn correlates with restriction sensitivity . We therefore tested whether the alanine at CA88 also dictated sensitivity to Mafa TRIMCyp . To do this we used a previously described HIV-1 mutant in which the alanine at CA88 had been removed ( HIV-1 CA-A88 ) , and an HIV-2 mutant in which the missing alanine has been inserted ( HIV-2 CA+A88 ) . GFP encoding HIV-1 and HIV-2 mutant vectors were prepared and used to infect CRFK cells expressing Mafa TRIMCyp2 and cells bearing empty vector as a control ( Fig . 2C ) . In fact , whilst HIV-1 was sensitive to Mafa TRIMCyp2 the mutant with a shorter CypA binding loop was unrestricted . Furthermore , Mafa TRIMCyp2 restricted the HIV-2 mutant with an extra alanine , whereas the wild type virus remained unrestricted . The HIV-1 mutant was as infectious as the wild type virus whereas the HIV-2 mutant with an extra alanine was around an order of magnitude less infectious than wild type on feline cells bearing empty vector . However , the switch in sensitivity to TRIMCyp on adding or removing the extra alanine residue in these viruses is clear . Furthermore , corresponding binding experiments by ITC support the restriction data; the loss or gain of restriction sensitivity upon addition or removal of A88 directly correlates with loss or gain of binding ( Table 1 ) . Sensitivity to Mafa TRIMCyp is therefore dictated by the sequence of the CypA binding loop with the alanine at CA88 being an important determinant . In order to understand the structural basis for these restriction specificities we solved the crystal structure of the Mafa TRIMCyp2 Cyp domain in complex with the HIV-1 capsid N terminal domain ( NTD ) ( Table S1 ) . As stated above , there are two amino acid differences between the Cyp domain of Mafa TRIMCyp and the genomic CypA sequence , R69H and E143K . Superposition of the Mafa:HIV-1 complex with the previously solved wild-type CypA:HIV-1 structure ( 1AK4 ) [35] reveals that the two Cyp mutations ( R69H and E143K ) do not provoke large structural rearrangements ( Fig . 3A ) . This is in contrast to the two mutations in Cyp from Mamu TRIMCyp ( D66N and R69H ) that have previously been shown to create a large ( >16 Å ) rearrangement of the active site loop around positions 66–69 ( loop66–69 ) and switch specificity of binding and restriction from HIV-1 M group to HIV-2 [30] . Mafa TRIMCyp thus maintains binding to HIV-1 despite the R69H mutation because it conserves the closed loop66–69 conformation observed in wild type CypA . The closed-loop conformation allows interactions with capsid position CA88 , enabling several hydrogen bond interactions to be made , including between the CA88 peptide nitrogen and the peptide oxygen from G72 in Cyp and a water-mediated interaction between the peptide oxygen of CA88 and Cyp residue H54 . The loss of these interactions resulting from removal of A88 from HIV-1 explains why this mutant virus neither binds nor is restricted by Mafa TRIMCyp2 ( Fig . 3A ) . The E143K mutation is located in a helix outside of the active site at the opposite end of the Cyp domain to R69H . How does such a distant mutation impact on viral specificity ? The Mafa complexed structure shows that the change from a negatively charged glutamate to a positively charged lysine at 143 causes a knock on effect through the molecule which impacts on the stereochemistry of the active site . Mutation to lysine at position 143 causes changes to the backbone of a neighbouring active-site loop around P58 by attracting the peptide oxygen and flipping it from its orientation in the wild-type CypA structure ( Fig . 3A ) . This propagates changes to other Cyp active-site residues , including R148 that is coordinated by the P58 peptide oxygen in the wild-type structure ( Fig . 3A ) . The altered position of P58 releases the side chain of R148 , allowing it to interact with capsid residues downstream of residue A88 . The E143K mutation also modifies the surface electrostatics ( Fig . 3B ) leading to a significant increase in the positive charge of the active site . Surface electrostatics are also shown for rhesus TRIMCyp and CypA for comparison . E143K therefore impacts on viral specificity through propagated conformational changes and long-range electrostatic effects . HIV-1 binding is unaffected by the charge alterations introduced by R69H and E143K ( Fig . 3B ) , whereas binding to HIV-2 is conferred by a reduction in positive charge ( R69H ) and abrogated by an increase in positive charge ( E143K ) . HIV-2 can be made insensitive to these charge changes by introducing A88 into the capsid , resulting in susceptibility to Mafa TRIMCyp2 . This suggests that the two viruses normally utilise different binding mechanisms but that insertion of A88 confers an HIV-1 like binding mode . To test this we solved the structure of HIV-2 with an alanine inserted at position 88 ( Table S1 ) . The introduction of A88 increases the structural dynamics of the HIV-2 CypA-binding loop and allows it to adopt alternative conformations ( Fig . 4A ) . Importantly , this allows the main-chain nitrogen of the introduced A88 to adopt an orientation that in HIV-1 permits hydrogen bond interaction with cyclophilin residues G72 and H54 ( Fig . 4B ) . These hydrogen bond interactions are not affected by the E143K mutation , explaining why HIV-2 with an inserted A88 is no longer affected by the charge change . It is unlikely that these interactions are normally possible in HIV-2 , since it has a deletion at this position and the preceding proline lacks a hydrogen on its amide group . Position CA88 is therefore a critical determinant of sensitivity even to mutations that are over 20 Å away as in the E143K Mafa TRIMCyp2 allele . In contrast to Mafa TRIMCyp2's ability to restrict HIV-1 the Mafa TRIMCyp1 protein identified by Brennan and colleagues was shown to not restrict HIV-1 [24] . Mafa TRIMCyp 1 and 2 differ at four positions including Cyp position 54 . We therefore tested whether this difference is responsible for Mafa TRIMCyp1's lack of anti-HIV-1 activity . We mutated Cyp H54 to arginine in Mafa TRIMCyp2 and expressed it in CRFK cells . Infection of the modified cells with HIV-1 , HIV-2 and FIV derived GFP encoding vectors indicated that Cyp H54R ablates Mafa TRIMCyp2's ability to restrict both HIV-1 and FIV , explaining the different restriction activities ( Fig . 5 ) . HIV-2 is also unrestricted by Mafa TRIMCyp2 Cyp H54R . Consideration of the Cyp-CA complex structure ( Fig . 3A ) explains this observation . The peptide oxygen of CA88 makes a water-mediated interaction with cyclophilin residue H54 ( H357 in TRIMCyp ) . The importance of this interaction is indicated by the fact that the water molecule is conserved in both the wild-type ( 1AK4 ) [35] and E143K structures ( Fig . 3A ) . In order to consider the differences in Asian Macaque TRIMCyp sequence in the context of Asian macaque evolution , we mapped the changes onto the recently described macaque phylogeny [36] ( Fig . 6 ) . We assume that the CypA sequence retrotransposed into the macaque TRIM5 locus was wild type . TRIMCyp with a wild type CypA sequence has been shown to restrict HIV-1 but HIV-2 only weakly [23] , [30] . Intriguingly , the phylogeny suggests that the R69H mutation arose early in a macaque common ancestor , conferring broad antiviral specificity including against both HIV-1 and HIV-2 . Following speciation events giving rise to present day macaque species , subsequent TRIMCyp mutations were either selected or underwent fixation . D66N mutation may have arisen independently in two different macaque species , Macaca mulatta and Macaca nemestrina . This is suggested by the fact that the D66N change is not found in Macaca fascicularis TRIMCyps ( Fig . 1 ) . D66N switches antiviral activity from restriction of both HIV-1 and HIV-2 to restriction of HIV-2 only ( Fig . 2 ) . In contrast , the Macaca fascicularis TRIMCyp sequence has acquired an alternate change , E143K , which switches antiviral specificity from HIV-1 and HIV-2 to HIV-1 alone , the opposite direction to nemestrina/mulatta ( Figs . 2–3 ) . The allelic diversity in present day macaque species represents functional TRIMCyps of defined lineage-specificity , strongly suggesting selection pressure from different lentiviruses .
Here we have considered the restriction of HIV-1 , HIV-2 and SIVtan as well as the feline lentivirus FIV by TRIMCyp proteins from Cynomolgus macaques ( Macaca fascicularis ) . Analysis of TRIMCyp antiviral specificity has revealed that a surprising diversity of antiviral activity can be achieved using a cyclophilin A-like domain to target lentiviral capsids . The antiviral activity of TRIMCyps can be very specific , distinguishing between HIV-1 and HIV-2 , which are members of different lentiviral lineages , as well as between HIV-1 M and O group viruses , which are derived from independent zoonoses of SIVcpz from chimpanzees to humans [23] , [30] . TRIMCyps can thus distinguish between closely related lentiviruses for example the HIV-1s , whilst also being able to restrict distantly related lentiviruses , for example FIV . By studying the details of the structural changes that underlie the switching of antiviral specificity we can gain a better appreciation of the flexibility of CypA-lentiviral interactions . The Cyp E143K change found in the TRIMCyp from Cynomolgus macaques ( Macaca fascicularis ) is surprisingly distant from the region of contact between Cyp and the lentiviral capsids . Nonetheless , by propagating structural changes across the molecule this change can switch a Cyp capable of binding and restricting HIV-1 and HIV-2 to one that only restricts HIV-1 . The selection of a mutation that affects specificity through propagated changes is reminiscent of the antibody immune response . Affinity maturation has been shown to utilise distant indirect mutations to alter the specificity and affinity of more generalized primary antibodies [37]–[39] . Remarkably , the potency of single amino acid changes in Cyp is mirrored by the effect of single amino acid changes in the lentiviral capsids . Altering HIV-1 or HIV-2 by a single amino acid can switch either virus cleanly between exquisite sensitivity and complete insensitivity to restriction by Mafa TRIMCyp2 ( Fig . 2C ) . These observations reinforce the notion that there is a fine balance between restriction and replication . We imagine that the host , which evolves relatively slowly , can compete successfully with rapidly evolving lentiviruses because it relies on a large number of innate and adaptive pathways to combat viral infection . The host can also make complex adaptive changes , illustrated for example , by the exchange of the B30 . 2 viral binding domain for CypA placed in the TRIM5 locus by retrotransposition . There are three possible explanations for the present day allelic distribution of TRIMCyp in macaque species . First , modern macaque species may have originated from a common ancestor that was multi-allelic . Incomplete lineage sorting during speciation may then have given rise to the limited allelic diversity in each species . A second possibility is that following speciation , each species may have been placed under selection by a different virus , causing fixation of different alleles with different viral specificities . Finally , the macaque common ancestor may have had limited allelic diversity , for instance the broad specificity R69H allele , and different viruses subsequently fixed new mutations in emerging macaque species . In this last scenario , the D66N mutation that switches specificity to HIV-2 , would represent convergent evolution in M . nemestrina and M . mulatta . It is likely that a combination of the above processes - selection and random fixation - underlie present day species diversity . Irrespective of the evolutionary sequence of events , the data presented here illustrate that specific changes made in the Cyp domain of TRIMCyp during Asian monkey evolution make important , and surprisingly potent , alterations to its antiviral specificity [23] , [30] . Given that the D66N , R69H and E143K TRIMCyp mutations all affect viral specificity it is tempting to speculate that the Mafa TRIMCyp1 allele with its H54R mutation in its Cyp domain , though not active against the lentiviruses tested , restricts viruses that we have yet to determine . Cyp position H54 is an active site residue that forms a water-mediated interaction with HIV-1 ( Fig . 3A ) . A larger arginine side-chain could be expected to form a similar interaction but without an intermediate water . This makes H54R a good candidate for fixation through selective pressure from a viral infection in the distant past , as is likely the case for the other TRIMCyp mutations . The conclusion that lentiviruses have exerted selection pressure in Asian macaques presupposes that Asian macaques have lost the selective virus , or viruses , permanently as there are currently no extant lentiviruses described in these animals . However , we cannot be certain that lentiviruses do not exist in Asian macaque populations as they have not been surveyed systematically and rare viruses may yet be found . The fossil record suggests that macaques migrated across Europe around 5 . 5 mya [36] , whilst lentiviruses are known to have been present in European lagomorphs for at least 12 million years [40] , [41] , It is therefore possible that lentiviruses may have undergone interspecies transmission into macaques at around this time . In contrast , the Asian macaque-tropic SIVmac appears to be a cross-species transmission from sooty mangabeys that occurred in captivity and was probably the inadvertent result of forced passage experiments during investigation of kuru transmission [42] , [43] . It is difficult to find evidence for the loss of a lentivirus from a species but it is clear that the numbers of HIV-1 O group infected individuals declined sharply between 1988 and 1998 in Cameroon , although the frequency now appears stable [44] . The number of HIV-2 infected individuals has also fallen suggesting that in time these viruses could disappear altogether from the human population [45] . The rarity of endogenous lentiviral sequences has suggested that lentiviruses are relatively young compared to related retroviruses that are commonly found as endogenous sequences but it may be that they are simply less efficient at entering the germ lines of their hosts [40] , [41] , [46] . Thus , our observations suggest that whilst lentiviruses may not be present in current populations of Asian macaques they may have been important pathogens in the past . Of course , other , as yet unidentified , cyclophilin binding pathogens could have contributed to the selective pressures driving TRIMCyp evolution although the switches in primate lentiviral specificity that we have described lead us to favour lentiviruses as an important selective force . In conclusion , the structural changes mediated by Cyp residue E143K illustrate the remarkable adaptability of CypA as a viral binding domain , providing the necessary flexibility to respond to a rapidly changing pathogen . Seemingly innocuous scaffold mutations such as Cyp E143K can propagate structural changes within CypA , resulting in sufficient rearrangement of the active site to potently alter viral restriction specificity . The ease with which the conformation and hence specificity of CypA can be changed with even a single mutation explains why it has been selected as a targeting domain for a primate restriction factor . The characterisation of the different TRIMCyp homologues and their alleles present in the Macaca species – nemestrina , mulatta and fascicularis – show that each TRIMCyp mutation has a direct structural , thermodynamic and functional effect on the restriction of viruses from three lentiviral lineages . This strongly suggests that these mutations are not the effect of random drift , but instead that primate TRIMCyps have undergone positively selected changes that have impacted specifically on antiviral activity .
Feline CRFK cells were a gift from Yasuhiro Ikeda and were maintained in DMEM ( Invitrogen ) with 10% FCS ( Biosera ) . VSV-G pseudotyped lentiviral vectors encoding GFP derived from HIV-1 [47] , [48] , HIV-2 [49] and FIV [50] were prepared as described [51] . SIVtan with GFP in place of Nef was a gift from Paul Bieniasz and was VSV-G pseudotyped as described [33] . Viral mutants HIV-1 CA –A88 and HIV-2 CA +88A have been described [30] . Infections were performed on 25000 cells/well in 24 well plates or 200 , 000 cells/well in 6 well plates . Serial dilutions of virus were used to infect cells and the percentage of green cells expressing GFP was measured 48 hours later by flow cytometry as described [52] . Data are presented as mean titres derived from two doses and errors are standard errors of the mean . Data are representative of two independent experiments . Cyclosporine ( Sandoz ) was diluted in DMSO and added to cells at the time of infection at 5 µM . Viral doses were measured by reverse transcriptase enzyme linked immunosorbant assay ( Roche ) . RNA was purified from peripheral blood mononuclear cells ( PBMC ) from an Indonesian Cynomolgus macaque ( RNAeasy , Qiagen ) and reverse transcribed to cDNA ( Superscript , Invitrogen ) . Mafa TRIMCyp was then cloned by PCR using pfu turbo ( Stratagene ) into the murine leukaemia virus based vector EXN [53] , a gift from Paul Bieniasz . Three independent clones were sequenced and the Mafa TRIMCyp2 sequence has Genbank accession number FJ609415 . MLV vector was prepared and TRIMCyp expressed as described [22] , [34] . Mutagenesis was performed as described [54] . EXN encodes an N terminal HA tag allowing detection of protein expression by western blot , detecting the HA tag . CAN domains of HIV-1 M-type ( NL4 . 3 ) and HIV-2 ( ROD ) were expressed and purified as previously described [30] . The CypA domain from MafaTRIMCyp was expressed as an N-terminal His-tagged protein in BL21 ( DE3 ) cells and protein purified by capture on Ni-NTA resin ( Qiagen ) followed by gel filtration . All mutant proteins were expressed and purified as per the wild-type proteins . ITC experiments were conducted on a MicroCal ITC200 . Proteins were dialysed overnight against phosphate buffer ( 50 mM KPO4 ( pH 7 . 4 ) , 100 mM NaCl , 1 mM DTT ) and experiments carried out at 15°C , with cyclophilin ( typical concentration 1 . 4 mM ) in the syringe and capsid ( typical concentration 120 µM ) in the cell . Binding isotherms were fitted using the standard one-state model within the MicroCal instrument software , as previously described [30] . Crystals of Mafa TRIMCyp2 CypA domain in complex with HIV-1 CAN were grown at 17 °C in sitting drops . Protein solution ( 0 . 33 mM each of Mafa Cyp and HIV-1 CAN in 20 mM HEPES pH 7 , 50 mM NaCl , 1 mM DTT ) was mixed with reservoir solution ( 30% PEG 6000 , 1 M LiCl , 0 . 1 M HEPES pH 7 , 0 . 5% ethyl acetate ) in a 1:1 mix , producing 0 . 2 mm 5 0 . 12 mm 5 0 . 02 mm crystals within 48 h . Crystals of HIV-2 CAN +A88 were grown at 17 °C in sitting drops . Protein solution ( 0 . 6 mM in 20 mM HEPES pH 7 , 50 mM NaCl , 1 mM DTT ) was mixed with reservoir solution ( 2 . 8 M sodium acetate trihydrate pH 7 , 3% 1 , 4-dioxane , 1% PEG 3350 ) in a 1:1 mix , producing 0 . 08 mm 5 0 . 04 mm 5 0 . 04 mm crystals within 48 h . Data was collected on an in-house FR-E SuperBright high-brilliance rotating anode linked to an automated crystal mounting system ( ACTOR; Rigaku ) . Data were processed and refined using programmes from the CCP4 package [55] . Molecular replacement ( Phaser ) was performed using structure 1AK4 [35] for the HIV1:Cyno complex and the free HIV-2 structure [30] for the HIV-2 +A88 structure . Figures were prepared using Pymol and the adaptive Poisson-Boltzmann solver ( APBS ) for electrostatics . Protein Data Bank: Coordinates for HIV-2 +A88 capsid mutant and Mafa TRIMCyp Cyp domain in complex the HIV-1 CA have been deposited with accession codes 2X82 and 2X83 , respectively . Mafa TRIMCyp2 sequence has Genbank accession number FJ609415 . | Retroviruses have constantly been infecting mammals throughout their evolution , causing them to evolve defensive mechanisms to protect themselves . One of these mechanisms utilises intracellular antiviral molecules referred to as restriction factors . Restriction factor sequences have changed through primate evolution , suggesting an ongoing battle between retroviruses and their hosts as described by the Red Queen hypothesis . TRIM5 is an important restriction factor able to protect some monkeys , but not humans , from HIV infection . Certain monkeys have modified their TRIM5 genes by swapping the virus binding B30 . 2 domain with a cyclophilin A domain inserted into the TRIM5 locus by retrotransposition . This leads to expression of a TRIMCyp protein with antiviral activity against viruses , such as HIV-1 , that recruit cyclophilins . It appears that cyclophilin makes a particularly flexible virus-binding domain able to restrict divergent lentiviruses from primates as well as cats . Here we characterise the molecular details of Cyclophilin-Capsid interactions focusing on TRIMCyp proteins from Macaca Fascicularis . Using a structure/function approach we can show the molecular details of how adaptive changes in the TRIMCyp sequence switch specificity between members of different primate lentiviral lineages . Mapping these changes onto the macaque phylogeny reveals a history of TRIMCyp evolution that directs restriction to a variety of diverse lentiviruses . | [
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] | 2010 | Conformational Adaptation of Asian Macaque TRIMCyp Directs Lineage Specific Antiviral Activity |
SNAPc is one of a few basal transcription factors used by both RNA polymerase ( pol ) II and pol III . To define the set of active SNAPc-dependent promoters in human cells , we have localized genome-wide four SNAPc subunits , GTF2B ( TFIIB ) , BRF2 , pol II , and pol III . Among some seventy loci occupied by SNAPc and other factors , including pol II snRNA genes , pol III genes with type 3 promoters , and a few un-annotated loci , most are primarily occupied by either pol II and GTF2B , or pol III and BRF2 . A notable exception is the RPPH1 gene , which is occupied by significant amounts of both polymerases . We show that the large majority of SNAPc-dependent promoters recruit POU2F1 and/or ZNF143 on their enhancer region , and a subset also recruits GABP , a factor newly implicated in SNAPc-dependent transcription . These activators associate with pol II and III promoters in G1 slightly before the polymerase , and ZNF143 is required for efficient transcription initiation complex assembly . The results characterize a set of genes with unique properties and establish that polymerase specificity is not absolute in vivo .
The human pol II snRNA genes and type 3 pol III genes have the particularity of containing highly similar promoters , composed of a distal sequence element ( DSE ) that enhances transcription and a proximal sequence element ( PSE ) required for basal transcription . In pol II snRNA promoters , the PSE is the sole essential core promoter element whereas in type 3 pol III promoters , there is in addition a TATA box , which determines RNA pol III specificity [1] , [2] . The PSE recruits the five-subunit complex SNAPc , one of the few basal factors involved in both pol II and pol III transcription . Basal transcription from pol II snRNA promoters requires , in addition , TBP , TFIIA , GTF2B ( TFIIB ) , TFIIF , and TFIIE , and from pol III type 3 promoters TBP , BDP1 , and a specialized GTF2B-related factor known as BRF2 [3] , [4] , [5] . The DSE is often composed of an octamer and a ZNF143 motif ( Z-motif ) that recruit the factors POU2F1 ( Oct-1 ) and ZNF143 ( hStaf ) , respectively [1] , [2] . POU2F1 activates transcription in part by binding cooperatively with SNAPc and thus stabilizing the transcription initiation complex on the DNA ( see [6] , and references therein ) . In addition to requiring some different basal transcription factors for transcription initiation , pol II and pol III transcription at SNAPc-recruiting promoters differ in the way transcription terminates . In pol III genes , there are runs of T residues at various distances downstream of the RNA-coding sequence , which direct transcription termination ( [7] and references therein ) . In pol II snRNA genes , a “3′ box” starting generally 5–20 base pairs downstream of the RNA coding sequence directs processing of the RNA , with transcription termination reported to occur either just downstream of the 3′ box [8] , or over a region of several hundreds of base pairs [9] . Although model snRNA promoters have been extensively studied , it is unclear how broadly SNAPc is used , and to what extent the highly similar pol II and pol III PSE-containing promoters are selective in their recruitment of the polymerase . It is also unclear how generally the use of the basal factor SNAPc is coupled to that of the activators POU2F1 and ZNF143 , and by which mechanisms ZNF143 activates transcription . To address these questions , we performed genome-wide immunoprecipitations followed by deep sequencing ( ChIP-seq ) to localize four of the five SNAPc subunits , GTF2B , BRF2 , and a subunit of each pol II and pol III . These studies define a set of SNAPc-dependent transcription units and show that although most loci are primarily bound by one or the other polymerase , the RPPH1 ( RNase P RNA ) gene is occupied by both enzymes . Pol II is detectable up to 1 . 2 kb downstream of the end of the RNA-coding regions of pol II snRNA genes , thus defining a broad region of transcription termination . Localization of POU2F1 and ZNF143 shows widespread usage of these activators by PSE-containing promoters , and we find that several of these promoters also bind the activator GABP [10] , which has not been implicated in snRNA gene transcription before . Activators are recruited before the polymerase in G1 , and this process is less efficient when ZNF143 levels are decreased by RNAi .
We performed ChIP-seq with antibodies against SNAPC4 ( SNAPC190 ) , the largest SNAPc subunit , SNAPC1 ( SNAP43 ) , and SNAPC5 ( SNAP19 ) in IMR90Tert cells . To localize SNAPC2 ( SNAP45 ) , we used an IMR90Tert cell line expressing both biotin ligase and SNAPC2 tagged with the biotin acceptor domain for chromatin affinity purification ( ChAP ) -seq ( see [11] ) . We also used antibodies against GTF2B , which should mark pol II snRNA promoters , BRF2 , which should mark type 3 pol III promoters , and POLR2B ( RPB2 ) , the second largest subunit of pol II . We used POLR3D ( RPC4 ) ChIP-seq data [11] to localize pol III . Most of the human pol II snRNA and type 3 pol III genes are repeated and/or have given rise to large amounts of related sequences within the genome . We therefore aligned tags as described before [11] , excluding tags aligning with one or more mismatches but including tags with several perfect matches in the genome ( see Methods ) . We selected regions containing at least two SNAPc subunits and either BRF2 and pol III , or GTF2B and pol II , as described in Methods . We obtained loci encompassing all known type 3 pol III genes as well as most annotated pol II snRNA genes . In addition , we obtained a few novel loci occupied by SNAPc and pol II . Table S1 shows these loci as well as the annotated snRNA genes that did not display any tags , namely four RNU1 and one RNU2 snRNA genes ( in red in the first column ) . It also shows , in grey , RNU2 genes that are still in the “chr17_random” file of the human assembly and were thus not in the reference genome used for tag alignment . In some cases , we noticed adjacent POLR2B peaks separated by only one or a few nucleotides , which often corresponded to annotated SNP positions . Inclusion of tags aligned with ELAND , which allows for some mismatches , often resulted in the fusion of adjacent peaks , as for the SNORD13 gene shown in Figure S1A ( compare upper and lower panels ) . Such loci are likely to be occupied by POLR2B –indeed their promoter regions are occupied by significant amounts of GTF2B and SNAPc subunits– and they are labeled in yellow in the first column of Table S1 . In a few cases , however , this did not result in fusions of adjacent peaks , as shown in Figure S1B for a RNU1 gene ( U1-12 ) . Such peaks probably result from attribution of tags with multiple genomic matches to an incorrect genomic location and are thus likely to be artifacts . Consistent with this possibility , U1-11 , U1-12 , U1-like-8 , U3-2 , U3-2b , U3-4 , and U3-3 , all labeled in orange in Table S1 , had POLR2B , GTF2B , and SNAPc subunits scores with either 0% or , in the cases of U3-4 , less than 15% , unique tags . We consider these loci unlikely to be occupied by pol II in vivo . In contrast , the POLR2B peak on the RNU2 snRNA gene on chromosome ( chr ) 11 , even though interrupted about 500 base pairs downstream of the snRNA coding region , is constituted mostly of unique tags , as are the GTF2B and SNAPc subunit peaks . This gene is likely , therefore , to be indeed occupied by pol II and other factors , and is labeled in striped yellow in the first column ( Table S1 ) . We calculated occupancy scores for all loci by adding tags covering peak regions , as described in Methods ( see legend to Table S1 for exact regions ) . We first examined the POLR2B , POLR3D , GTF2B , and BRF2 scores . For most genes there was a clear dominance of either POLR2B and GTF2B or POLR3D and BRF2 ( Figure 1A ) . Further , there was a good correlation between POLR2B and GTF2B ( 0 . 89 ) or POLR3D and BRF2 ( 0 . 80 ) scores , but not between POLR2B and BRF2 ( 0 . 075 ) , or POLR3D and GTF2B ( 0 . 22 ) ( Figure S2 ) . This is consistent with GTF2B and BRF2 being specifically dedicated to recruitment of pol II and pol III , respectively , and indicates that most SNAPc-occupied genes are transcribed primarily by a single polymerase . Strikingly , among SNAPc-occupied promoters , only thirteen loci were occupied primarily by BRF2 and pol III ( listed on top of Table S1 ) , corresponding to the known type 3 genes previously shown to be occupied by pol III in IMR90hTert and other cell lines [11] , [12] , [13] , [14] . We identified a larger number of SNAPc-bound loci occupied primarily by GTF2B and pol II . They included genes coding for the U1 , U2 , U4 and U5 snRNAs , all involved in splicing of pre-mRNAs; U11 , U12 , and U4atac snRNAs , which have similar functions as U1 , U2 , and U4 but participate in the removal of a smaller class of introns referred to as AT-AC introns; U7 snRNA , involved in the maturation of histone pre-mRNAs; U3 , U8 , and U13 small nucleolar RNAs ( snoRNAs ) , involved in the maturation of pre-ribosomal RNA , as well as snRNA-derived sequences . The relationship of these loci with previously described snRNAs and snoRNA genes is described in the Results section of Text S1 . We also uncovered a few non-annotated loci harboring SNAPc subunits , as well as GTF2B and POLR2B , peaks constituted by at least 20% of unique tags and , therefore , likely to correspond to new actively transcribed regions . These are labeled Unknown-1 to 7 ( rows 76–82 in Table S1 ) . As described below , these sequences harbor a PSE as well as some other sequence elements typical of pol II snRNA promoters , and contain similarities to the 3′ box . Although most genes were occupied mostly by either BRF2 and POLR3D , or GTF2B , and POLR2B , there were a few exceptions . The most notable was the RPPH1 gene , which is considered a type 3 pol III gene [15] but was in fact occupied not only by BRF2 and POLR3D but also by significant amounts of POLR2B and GTF2B , comparable to those found on the RNU4 snRNA genes ( Figure 1A and 1B ) . This suggested that this gene could be transcribed in vivo by either of two RNA polymerases , pol II or pol III . To explore this possibility further , we treated cells with a concentration of α-amanitin known to inhibit pol II but not pol III transcription [16] . As expected , this treatment reduced the POLR2B signal of the pol II RNU2 gene but not the POLR3D signal on the pol III hsa-mi-886 gene ( Figure 1C , upper panels ) . To determine the effects of α-amanitin for the RPPH1 gene and the U6-2 gene , which also displayed some POLR2B signal in addition to the expected POLR3D signal ( see Figure 1A ) , we set the POLR2B and POLR3D signals obtained in the absence of α-amanitin at 1 . In each case , addition of α-amanitin to the medium reduced the POLR2B but not the POLR3D signal ( Figure 1C , lower panels ) . Thus , the RPPH1 gene can be transcribed either by pol II or pol III in vivo . One of the criteria used to select the genes in Table S1 was the presence of at least two of the four SNAPc subunits examined . We obtained a good correlation between scores for the four SNAPc subunits tested ( Figure S3 ) , consistent with SNAPc binding as a single complex to snRNA promoters [17] . Figure 2A shows the peaks obtained for the SNAPc subunits , BRF2 , GTF2B , POLR3D , and POLR2B on the pol III TRNAU1 gene and the pol II RNU4ATAC gene , and Figure 2B shows two non-annotated genomic loci occupied by POLR2B , GTF2B , and SNAPc subunits . Whereas the polymerase subunits were detected over the entire RNA coding sequence of the corresponding genes ( and further downstream in the case of POLR2B ) , the other factors were located within the 5′ flanking region , with GTF2B and BRF2 close to , or overlapping , the TSS . Although peaks were sometimes constituted of too few tags to allow an unambiguous determination of the peak summit location ( see for example the SNAPC4 peak in Figure 2A ) , we could nevertheless detect clear trends . The GTF2B or BRF2 peaks were generally the closest to the TSS , the SNAPC4 , SNAPC1 , and SNAPC5 peaks were within the PSE sequence , and the SNAPC2 peak was upstream of the PSE ( Figure 2C ) . Figure S4 shows an alignment of the PSEs and TATA boxes of the 14 pol III type 3 promoters ( including the RPPH1 gene ) , and Figure S5 an alignment of the PSEs of all pol II loci listed in Table S1 . The non-annotated loci occupied by POLR2B and factors contain clear PSEs . Moreover , as noted previously [1] , [2] , the PSE is located further upstream of the TSS in pol III than in pol II snRNA genes . The corresponding LOGOs revealed similar but not identical consensus sequences for the PSEs of pol II and pol III genes ( Figure 2D ) ; for example , adenines were favored in positions 11 and 12 of pol III , but not pol II , PSEs . Thus , although the TATA box is the dominant element specifying RNA polymerase specificity –indeed the U2 and U6 PSEs can be interchanged with no effect on RNA polymerase recruitment specificity [16]– the exact PSE sequence may also contribute to specific recruitment , for example in the context of a weak TATA box . The U1 and U2 snRNA genes are followed by a processing signal known as the 3′ box [18] , [19] , which is also found downstream of several other pol II snRNA genes [1] . We could identify 3′ boxes in most of the pol II genes in Table S1 . An alignment of these motifs allowed us to generate a matrix with GLAM2 [20] , which we then used to search for 3′ boxes in all pol II with GLAM2SCAN [20] . As shown in Figure S6 , we could identify putative 3′ boxes downstream of all annotated pol II genes in Table S1 ( except for the non-expressed RNU1 ( U1-9 ) and RNU1 ( U1-13 ) genes ) , as well as for the non-annotated genes . For the RPPH1 gene , the best match to a 3′ box was located within the RNA coding sequence , from −73 to −61 relative to the end of the RNA coding sequence ( Figure S6 ) . The resulting 3′ box LOGO derived from all sequences aligned in Figure S6 is shown in Figure 3A . Pol II transcription termination has been reported to occur either shortly after , or several hundred base pairs downstream of , the 3′ box [8] , [9] . Our POLR2B ChIP-seq data reveal the extent of pol II occupancy downstream of the RNA coding region . Whereas on average , the POLR3D ChIP-seq signal dropped quite abruptly downstream of the RNA coding region of pol III genes ( see [7] ) , POLR2B could be detected as far as about 1200 base pairs past the RNA coding region of pol II snRNA genes ( Figure 3B ) . Moreover , examination of the POLR2B peak downstream of individual pol II genes revealed a gradual decrease of tag counts over regions of 500 or more base pairs ( see for example Figure 2A and 2B , and Figure 4A below ) . Thus , transcription termination occurs well downstream of the 3′ box and over a broad region . snRNA promoters are characterized by an enhancer element ( DSE ) typically containing an octamer motif and a ZNF143 binding site ( Z-motif ) , which in some specific genes has been shown to recruit , respectively , the POU domain protein POU2F1 and the zinc finger protein ZNF143 ( see [1] , [2] and references therein ) . To determine how general the binding of POU2F1 and ZNF143 is among SNAPc-binding promoters , we localized POU2F1 by ChIP-seq in HeLa cells and we analyzed ChIP-seq data obtained by others in HeLa cells ( JM , VP , and Winship Herr , personal communication ) for ZNF143 and , as ZNF143 was found to bind often together with GABP ( JM , VP , and Winship Herr , personal communication ) , for the α subunit of GABP ( GABPA ) . The scores for all genes are listed in Table S1 and , in a summarized form , in Table S2 . The pol III genes in Table S1 , which were all occupied by basal factors ( see above ) , were each occupied by at least one activator . Among pol II genes , those not occupied by basal factors ( labeled in red in the first column of Tables S1 and S2 ) did not display peaks for any of the activators , and those with interrupted POLR2B peaks ( orange in the first column ) had peaks composed solely of tags with multiple matches in the genome , consistent with the possibility raised above that these genes are , in fact , not occupied by factors . Of the genes clearly occupied by basal factors , all displayed peaks for at least one activator with three exceptions , U1-like-11 , unknown-2 , and unknown-3; these last three loci had basal factor peaks with relatively low scores and thus may bind some of these activators at levels too low to be detectable in our analysis . Most genes had a POU2F1 peak ( 93% ) , a large majority had a ZNF143peak ( 81% ) , and about half had a GABPA peak ( 45% ) . Interestingly , some genes had specific combinations of activators; for example the RNU5 and U5-like genes as well as most pol III genes had peaks for both POU2F1 and ZNF143 but not for GABPA . In contrast RNU6ATAC , SNORD13 , and RNU3 genes had POU2F1 and GABPA peaks but no ZNF143 peak . Only few genes had only one activator ( RMRP , RNY4 , RNU2-2 , U3b2-like , RNU7 , and Unknown-5 ) suggesting that most snRNA genes require some combination of the three activators tested for efficient transcription . Indeed , altogether 23 genes had peaks for all three factors and 23 had peaks for both ZNF143 and POU2F1 but not GABPA . Thus , the very large majority ( 79% ) of SNAPc-binding genes bound both POU2F1 and ZNF143 . The scores for the various activators were surprisingly correlated ( see Figure S7 ) , perhaps indicating that these factors bind to snRNA promoters interdependently . Figure 4A shows two examples ( RNU4ATAC and U1-like-5 ) with the three factors present , and two examples ( Unknown-6 and tRNAU1 ) with only POU2F1 and ZNF143 . In all cases , the factors bound upstream of the PSE with GABP , when present , generally binding the furthest upstream . We analyzed 5′ flanking sequences for motifs and identified POU2F1 ( octamer , see [21] ) , ZNF143 [22] , [23] , and GABP [24] , [25] , [26] binding sites ( Figure 4B , Figure S8A and S8B ) . This analysis revealed a high concordance between occupancy as determined by ChIP-seq and presence of the corresponding motif , with only a few cases ( GABP and ZNF143 for U1-like-10 , and GABP for U5E-like , U4-1 , and unknown-7 genes ) where no convincing motif could be identified . We then aligned all occupied motifs ( see Figures S9 , S10 , and S11 ) to generate the LOGOs shown in Figure 4C , which thus reflect the ZNF143 , POU2F1 , and GABP binding sites in SNAPc-recruiting genes . Transcription of RNU6 and probably RNU1 and RNU2 is known to be low during mitosis and to increase as cells cycle through the G1 phase [27] , [28] , [29] , [30] , [31] , hence we measured the levels of U1 , U2 , and U6 snRNA during mitosis and at several times after entry into G1 . Since snRNA transcripts are very stable , making it difficult to measure transcription variability , we generated HeLa cell lines containing RNU1 or RNU6 reporter construct expressing unstable transcripts whose levels therefore better reflect ongoing transcription . For U2 snRNA , we measured its precursor , which has a short half-life [16] . Cells were blocked in prometaphase with Nocodazole and released with fresh medium . RNA levels were low during mitosis and , in the case of the U1 reporter RNA and pre-U2 RNA , increased to a maximum 6–7 h after release , around the middle of the G1 phase ( as determined by FACS analysis , see Methods ) . For the U6 reporter RNA , RNA levels reached a maximum 3 h after release , at the beginning of the G1 phase ( Figure 5A ) . POLR2B occupancy was apparent 4 h after the mitosis release and peaked after 6 h , as measured by ChIP-qPCR analysis of both RNU1 and RNU2 loci ( Figure 5B ) . This was specific , as no significant amounts of POL2RB were detected on the control region . In comparison , increased POLR3D occupancy of RNU6 ( but not the control region ) was apparent 3 h after release and peaked after 6 h , consistent with the accumulation of U6 RNA earlier in G1 than U1 and U2 RNA . We then examined promoter occupancy by transcription activators ( Figure 5B ) . ZNF143 occupancy increased over time on both the RNU1 and RNU6 promoters , becoming clearly detectable at 3 h and reaching a maximum at 6 h for RNU1 and 4 h for RNU6 . In contrast , ZNF143 was undetectable on the RNU2 promoters . POU2F became detectable at 3 h on the RNU1 , RNU2 , and RNU6 promoters and then remained at a more or less constant level . GABP was detected only on the RNU1 promoters and was recruited early , starting 2 h after the release and reaching a maximum at 5 h . Thus , activators were recruited on the promoters expected from the ChIP-seq data above , with kinetics slightly faster than the polymerase . Among activators , GABP was recruited the earliest , followed by concomitant recruitment of ZNF143 and POU2F1 . Some basal transcription factors such as TBP are thought to remain bound to chromatin , and hence probably promoters , during mitosis [32] , [33] . To explore whether this is the case for SNAPc , GTF2B , and BRF2 , we monitored occupancy by these factors at mitosis ( 1 h after release ) and in mid-G1 ( 7 h after release ) . On the pol II RNU1 snRNA promoter , we observed enrichment of GTF2B and SNAPc subunits , as well as the pol II subunit POLR2B , the activators ZNF143 , POU2F1 , and GABP , and H3 acetylated on lysine 18 ( H3K18Ac ) at mid-G1 compared to mitosis ( Figure 5C , upper panel ) . This was specific as the pol III subunit POLR3D was not enriched . On the pol III RNU6 promoter , we observed enrichment of POLR3D , BRF2 , SNAPc subunits , ZNF143 , POU2F1 and H3K18Ac , but not POLR2B nor GABP , as expected ( Figure 5C , lower panel ) . This suggests that at snRNA promoters , both basal transcription factors and activators are removed from promoter DNA during mitosis and are recruited de novo upon transcription activation in G1 . To explore the role of ZNF143 in transcription factor recruitment , we targeted endogenous ZNF143 by siRNA and synchronized the cells as above . Total protein levels measured both at mitosis and in mid-G1 were reduced by more than 70% ( Figure 6A ) , and in mid-G1 , ZNF143 bound to the U1 promoter was decreased by 50% ( Figure 5B ) . Under these conditions , binding of the activators POU2F1 and GABP , the basal transcription factors GTF2B and SNAPC1 , and POL2RB were reduced by 40 to 70% . In contrast , the H3K18Ac levels were not reduced ( Figure 6B ) . Thus , ZNF143 contributes to efficient recruitment of other activators , basal transcription factors , and the RNA polymerase , but not to H3K18 acetylation , at the pol II U1 promoter .
Using stringent criteria of co-occupancy by two SNAPc subunits and either GTF2B and pol II , or BRF2 and pol III , we identified a surprisingly small number of SNAPc-occupied promoters comprising the 14 known type 3 pol III promoters , some 40 pol II snRNA genes , and 7 novel pol II-occupied loci . It seems , therefore , that in cultured cells , SNAPc is a very specialized factor participating in the assembly of transcription initiation complexes at fewer than 100 promoters . We have not explored , however , the possibility that some of the SNAPc subunits participate in transcription of other genes or in other functions as part of complexes other than SNAPc . Indeed , in a previous localization of SNAPc subunits on genomic sites also binding TBP , a correlation analysis on non-CpG islands split the SNAPc subunits into two subgroups , one containing SNAPC1 and SNAPC5 and the other SNAPC2 , SNAPC3 , and SNAPC4 [34] , consistent with the possibility that other SNAP -subunit-containing complexes exist . A peculiarity of SNAPc is its involvement in transcription from both pol II and pol III promoters , promoters that differ from each other mainly by the presence or absence of a TATA box . We found that most SNAPc-occupied promoters were predominantly occupied by either pol II or pol III with two exceptions , the U6-2 and most notably the RPPH1 genes , which were occupied not only by BRF2 and pol III , as expected , but also by levels of GTF2B and pol II comparable , in the second case , to those found on some pol II snRNA genes . We showed that pol II occupancy of the RPPH1 gene was obliterated by levels of α-amanitin shown before to inhibit pol II transcription in cultured cells [16] . Previous experiments comparing the 3′ ends of pol II and pol III transcripts derived from wild-type and mutated versions of the human RNU2 and RNU6 promoters have shown that pol II-synthesized transcripts end downstream of a signal referred to as the “3′ box” whereas pol III-synthesized transcripts are not processed at such boxes and instead end at runs of T residues [16] . The best similarity to a 3′ box lies within the RPPH1 RNA coding region . However , we detect only one type of transcript , terminated at the run of T residues downstream of the RPPH1 gene , in endogenous RNA from proliferating IMR90Tert cells ( data not shown ) , suggesting that the transcript synthesized by pol II is highly unstable , at least under the conditions tested . It is conceivable that the ratio of RPPH1 genes transcribed by pol II and pol III , as well as the ratio of stable pol II and pol III RNA products , change in different cell types or under different conditions . The observation that a gene can be transcribed by two different polymerase in vivo thus raises the possibility of an added layer of complexity in the regulation of gene expression . It is not clear why the U6-2 and RPPH1 promoters are capable of recruiting significant levels of pol II . The RPPH1 promoter has a short TATA box , but the U6-7 and U6-8 promoters have the same TATA box and are not promiscuous . An intriguing possibility is that the presence of a 3′ box at a correct distance downstream of the TSS , together with a weak TATA box , allow pol II recruitment . The locations of the occupancy peaks for the four SNAPc subunits we tested are remarkably consistent with what is known about the architecture and DNA binding of SNAPc . SNAPC4 , the largest SNAPc subunit and the backbone of the complex , binds directly to the PSE through Myb repeats located in the N-terminal half of the protein [35] . SNAPC1 and SNAPC5 associate directly with SNAPC4 , N-terminal of the Myb repeats ( aa 84–133 , see [36] ) . Consistent with this architecture , we find that SNAPC4 , SNAPC1 , and SNAPC5 generally peak very close to each other within the PSE . In contrast , SNAPC2 , which associates with the C-terminal part of SNAPC4 ( aa 1281–1393 , see [36] ) , peaks upstream of the PSE . This suggests that the N-terminus of SNAPC4 is oriented facing the transcription start site whereas the C-terminal part is oriented towards the upstream promoter region . This is consistent with the orientation of D . melanogaster SNAPC4 [37] on the U1 and U6 D . melanogaster snRNA promoters as determined by elegant studies combining site-specific protein-DNA crosslinking with site-specific chemical protein cleavage ( [38] , see also [39] and references therein ) . The 3′ end of pol II snRNAs is generated by processing at a sequence called the 3′ box [2] , [40] . The 3′ box is efficiently used only by transcription complexes derived from snRNA promoters , suggesting that the polymerase II recruited on these promoters is somehow different from that recruited on mRNA promoters . Indeed , the C-terminal domain of pol II associated with snRNA genes carries a unique serine 7 phosphorylation mark , which recruits RPAP2 , a serine 5 phosphatase , as well as the integrator complex , both of which are required for processing ( [41] and references therein; [42] , [43] ) . Moreover , pol II transcription of snRNA genes requires a specialized elongation complex known as the Little Elongation Complex ( LEC ) [44] . It has been unclear , however , how far downstream of the 3′ box processing signal transcription continues , with one report indicating a very sharp drop in transcription within 60 base pairs past the U1 3′ box [8] and another reporting continued transcription for several hundreds of base pairs downstream of the U2 3′ box [9] . Our ChIP-seq data indicate that pol II can be found associated with the template more than 1 Kb downstream of the 3′ box , for both the RNU1 and RNU2 genes as well as all other pol II snRNA genes . This suggests that transcription termination downstream of snRNA gene 3′ boxes does not occur at a precise location but rather over a broad 1 . 2 Kb region , and is triggered by passage of the polymerase through the processing signal , reminiscent of transcription termination downstream of the poly A signal , in this case in a region of several Kbs [45] . Activation of several SNAPc-dependent promoters has been shown to depend on a DSE and on the binding of POU2F1 and ZNF143 ( see [1] , [2] and references therein , [23] ) . Our ChIP-seq analyses show that POU2F1 and ZNF143 are associated with the large majority of SNAPc-dependent promoters and identify GABP as a new factor binding to a subset of these promoters . During transcription activation in G1 , we observed binding of ZNF143 and POU2F1 preceding binding of RNA pol II and pol III , consistent with the possibility that binding of these activators prepares the promoters for polymerase recruitment . Indeed , lowering the amount of ZNF143 by siRNA strongly affected recruitment of POU2F1 , GABPA , basal factors , and the polymerase itself on the U1 promoter . Thus , ZNF143 could either recruit and stabilize POU2F1 by direct protein-protein contact , or affect chromatin structure to allow recruitment of POU2F1 , or both . In support of the first hypothesis , ZFP143 , the mouse homolog of ZNF143 , recruits another POU-domain protein , Oct4 ( the mouse homolog of POU5F1 ) by direct association [46] . On the other hand , ZNF143 and POU2F1 do not bind cooperatively to the human U6-1 promoter [47] , but then U6-1 is weakly POLR3D-occupied compared to other human RNU6 genes [11] . In support of the second possibility , we have shown before that ZNF143 can bind to an snRNA promoter , in this case the pol III U6 snRNA promoter , preassembled into chromatin [48] , suggesting that it is an early player in the establishment of a transcription initiation complex . However , promoter H3K18 acetylation , which is low just after mitosis and increases during G1 , was unaffected . This suggests that SNAPc-dependent promoters are targeted very early in G1 by as yet unidentified factors that lead to histone modifications , in particular H3K18 acetylation . It will be interesting to determine how this modification combines with the H3K4me3 mark observed on pol III promoters , including type 3 pol III promoters [12] , [13] , [14] , [49] .
ChIPs were performed as described [11] . The antibodies used ( rabbit polyclonal antibodies except where indicated ) were as follows: POLR3D , CS682 , directed against the C-terminal 14 aa [50]; POLR2B , H-201 from Santa Cruz Biotechnology; BRF2 , 940 . 505 #74; GTF2B , CS369 #10 , 11; SNAPC4 , CS696 #4 , 5; SNAPC5 , CS539 #7 , 8; SNAPC1 , CS47 #7 , 8; GABP , sc-22810 X from Santa Cruz Biotechnology; POU2F1 , mix of YL8 and YL15 [51] , [52] or mix of two polyclonal antibodies ( A310-610A from Bethyl Laboratories ) ; ZNF143 , antibody 19164 raised against ZNF143 aa 623–638 , [48] . The ChAPs have been described [11] . The sequence tags obtained after ultra-high throughput sequencing were mapped onto the UCSC genome version Hg18 , corresponding to NCBI 36 . 2 , as before [11] except that we included tags mapping to up to 500 rather than 1000 different locations in the genome . Table S3 shows the total number of tags sequenced for each ChIP and the percentages of tags mapped onto the genome . In all cases , 75 . 5% or more of the total tags mapped onto the genome had unique genomic matches . Peaks were detected with sissrs ( www . rajajothi . com/sissrs/ ) [53] with a false discovery rate set at 0 . 001% , as previously described [11] . We identified 77312 POLR2B , 4838 GTF2B , 1366 POLR3D , and 2526 BRF2 peaks . We then selected the POLR2B peaks within 100 base pairs of a GTF2B peak ( 3878 peaks ) , and the POLR3D peaks within 100 base pairs of a BRF2 peak ( 125 peaks ) . The ChIPs with the anti-SNAPc subunit antibodies gave relatively weak signals . We therefore divided the genome into 200 nucleotide bins , counted tags obtained for each of the four SNAPc subunits analyzed , and retained only bins displaying an enrichment for at least two of the SNAPc subunits . Bins were considered positive only if the tag number in bin reached at least the minimum tag count determined by sissrs for enriched regions with a 0 . 001 false discovery rate as the one used in sissrs set at the default parameters . We then considered genomic regions containing POLR2B and GTF2B , or POLR3D and BRF2 , sissrs peaks as well as a bin positive for two SNAPc subunits within 100 nucleotides of the polymerase sissrs peak . We obtained 157 and 58 loci for the POLR2B and POLR3D lists , respectively , which were all visually inspected . We eliminated peaks in regions of high background , with shapes never found in known snRNA genes ( for example peaks with rectangular shapes resulting from artefactual accumulation of tags ) , or with identical shape and location in all samples . The most convincingly occupied loci are listed in Table S1 , which also shows all annotated pol II snRNA genes , whether or not they were found occupied by POLR2B , GTF2B , and SNAPc subunits . Scores were calculated as described in [49] and contained a component consisting of the sum of tags with unique matches in the genome and another representing tags with multiple matches in the genome: such tags were attributed a weight corresponding to the number of times they were sequenced divided by the number of matches in the genome , with a maximum weight set at 1 . In Table S1 , the score percentage contributed by unique tags is indicated in separate columns . Scores and peak shapes are more reliable for scores consisting mostly of unique tags , as in these cases there is no ambiguity as to where in the genome tags should be aligned . For the SNAPc subunits , we confirmed the results of the first analysis by performing a second analysis in which we counted tags in 200 nucleotide bins as before , then fitted a normal distribution to the data , and used the normal distribution's standard deviation and mean to attribute a P-value for each SNAPc subunit to each genomic bin . We then adjusted it with Benjamini & Hochberg ( BH ) correction and kept the bins with an adjusted P-value under 0 . 005 that were located within a 100 nucleotides of either a RPB2 and TF2B positive region , or a RPC4 and BRF2 positive region ( as defined by sissrs ) . We then applied a second filter to keep only the bins containing at least two ( of the four mapped ) SNAPc subunits . This gave us a total of 275 bins , which contained all the genes listed in Table S1 except for 10 loci . Of these 10 loci , 5 of them are flagged Table S1 as being not occupied ( U1-7 , U1-9 , U1-10 , U1-13 , U2-1 ) . The remaining five ( U1-like-1 , U1-like-11 , RNU5 ( U5F ) , UNKNOWN-2 , and RNU6-7 ( U6-7 ) ) have low scores . The additional regions with positive bins ( 93 regions ) corresponded to regions of high background and were eliminated after visual inspection . To measure RPPH1-dependent transcription in vivo , 1 . 2×106 HeLa cells were transiently transfected ( 48 hours ) with pU6/Hae/RA . 2 [16] or derivatives containing the wild-type RPPH1 promoter , or the RPPH1 promoter harboring a mutation in the TATA box ( TTATAA changed to TCGAGA ) , as well as the RPPH1 3′ flanking region . To specifically inhibit POLR2B transcription , the cells were treated with 50 µg/ml of α-amanitin ( Santa Cruz Biotechnology , sc-202440 ) for two or six hours before harvesting . Clonal cell lines expressing U1 or U6-promoter-directed unstable RNA were established by transfection of HeLa cells with plasmid derivatives of pU6/RA . 2+U6end-Dsred [48] ( see Methods section of Text S1 for details ) . Individual clones were expanded and tested for expression of the U1 or U6 construct . HeLa cell lines were synchronized as described [54] . Briefly , cells were first incubated for 24 h with 2 mM of Thymidine , then 3 h with normal medium , then 14 h with 0 . 1 mg/ml of Nocodazole . Cells were then harvested ( M phase ) or transferred to normal medium and harvested at different time points . The cell cycle stage of each sample was determined by flow cytometry analysis with the UV precise T kit ( Partec , Germany ) , which involves isolation of nuclei followed by DAPI staining . RNA was extracted from HeLa cells with TRIzol reagent ( Invitrogen ) according to the manufacturer's protocol and analyzed by RNase T1 protection as before ( see Methods section of Text S1 for details ) . To reduce levels of endogenous ZNF143 , a siRNA duplex was generated ( Microsynth ) to target the ATAAGCTGTGGTACCATCTTCCAGCTG region of the ZNF143 gene . HeLa cells were seeded at 2×106 cells per 10 cm plate the day before transfection . Thirty µl of INTERFERin transfection reagent ( Polyplus ) was added to 1 ml of DMEM serum-free medium containing 60 nM of siRNA duplex , incubated for 15 minutes , and added to the 10 cm plate containing 10 ml of medium . As negative control , we used a siRNA directed against the firefly luciferase [55] ( Dharmacon ) . Two other siRNA treatments were performed 12 and 24 h after the first transfection . Thirty hours after the 1st transfection , the cells were synchronized as described above . The data can be accessed at NCBI Gene expression Omnibus ( http://www . ncbi . nlm . nih . gov/geo ) under accession number GSE38303 . | SNAPc-dependent promoters are unique among cellular promoters in being very similar to each other , even though some of them recruit RNA polymerase II and others RNA polymerase III . We have examined all SNAPc-bound promoters present in the human genome . We find a surprisingly small number of them , some 70 promoters . Among these , the large majority is bound by either RNA polymerase II or RNA polymerase III , as expected , but one gene hitherto considered an RNA polymerase III gene is also occupied by significant levels of RNA polymerase II . Both RNA polymerase II and RNA polymerase III SNAPc-dependent promoters use a largely overlapping set of a few transcription activators , including GABP , a novel factor implicated in snRNA gene transcription . | [
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] | 2012 | Genomic Study of RNA Polymerase II and III SNAPc-Bound Promoters Reveals a Gene Transcribed by Both Enzymes and a Broad Use of Common Activators |
Ionotropic glutamate receptors ( iGluRs ) are a highly conserved family of ligand-gated ion channels present in animals , plants , and bacteria , which are best characterized for their roles in synaptic communication in vertebrate nervous systems . A variant subfamily of iGluRs , the Ionotropic Receptors ( IRs ) , was recently identified as a new class of olfactory receptors in the fruit fly , Drosophila melanogaster , hinting at a broader function of this ion channel family in detection of environmental , as well as intercellular , chemical signals . Here , we investigate the origin and evolution of IRs by comprehensive evolutionary genomics and in situ expression analysis . In marked contrast to the insect-specific Odorant Receptor family , we show that IRs are expressed in olfactory organs across Protostomia—a major branch of the animal kingdom that encompasses arthropods , nematodes , and molluscs—indicating that they represent an ancestral protostome chemosensory receptor family . Two subfamilies of IRs are distinguished: conserved “antennal IRs , ” which likely define the first olfactory receptor family of insects , and species-specific “divergent IRs , ” which are expressed in peripheral and internal gustatory neurons , implicating this family in taste and food assessment . Comparative analysis of drosophilid IRs reveals the selective forces that have shaped the repertoires in flies with distinct chemosensory preferences . Examination of IR gene structure and genomic distribution suggests both non-allelic homologous recombination and retroposition contributed to the expansion of this multigene family . Together , these findings lay a foundation for functional analysis of these receptors in both neurobiological and evolutionary studies . Furthermore , this work identifies novel targets for manipulating chemosensory-driven behaviours of agricultural pests and disease vectors .
Ionotropic glutamate receptors ( iGluRs ) are a conserved family of ligand-gated ion channels present in both eukaryotes and prokaryotes . By regulating cation flow across the plasma membrane in response to binding of extracellular glutamate and related ligands , iGluRs represent an important signalling mechanism by which cells modify their internal physiology in response to external chemical signals . iGluRs have originated by combination of protein domains originally encoded by distinct genes ( Figure 1A ) [1]–[2] . An extracellular amino-terminal domain ( ATD ) is involved in assembly of iGluR subunits into heteromeric complexes [3] . This precedes the ligand-binding domain ( LBD ) , whose two half-domains ( S1 and S2 ) form a “Venus flytrap” structure that closes around glutamate and related agonists [4] . Separating S1 and S2 in the primary structure is the ion channel pore , formed by two transmembrane segments and a re-entrant pore loop [5] . S2 is followed by a third transmembrane domain of unknown function and a cytosolic carboxy-terminal tail . Animal iGluRs have been best characterised for their essential roles in synaptic transmission as receptors for the excitatory neurotransmitter glutamate [1] , [6] . Three pharmacologically and molecularly distinct subfamilies exist , named after their main agonist: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( AMPA ) , kainate and N-methyl-D-aspartate ( NMDA ) . AMPA receptors mediate the vast majority of fast excitatory synaptic transmission in the vertebrate brain , while Kainate receptors have a subtler modulatory role in this process . NMDA receptors require two agonists for activation , glutamate and glycine , and function in synaptic and neuronal plasticity . Representatives of these iGluR subfamilies have been identified across vertebrates [7] , as well as invertebrates , such as the fruit fly Drosophila melanogaster , the nematode worm Caenorhabditis elegans and the sea slug Aplysia californica [8]–[10] . While most iGluRs have exquisitely tuned synaptic functions , identification of iGluR-related genes in prokaryotic and plant genomes provided initial indication of more diverse roles for this class of ion channel . A bacterial glutamate receptor , GluR0 , was first characterised in the cyanobacterium , Synechocystis PCC6803 [11] . GluR0 conducts ions in response to binding of glutamate and other amino acids in vitro , suggesting a potential function in extracellular amino acid sensing in vivo . The flowering plant Arabidopsis thaliana has 20 iGluR-related genes , named GLRs [12]–[13] . Genetic analysis of one receptor , GLR3 . 3 , has implicated it in mediating external amino acid-stimulated calcium increases in roots [14] . We recently described a family of iGluR-related proteins in D . melanogaster , named the Ionotropic Receptors ( IRs ) [15] . Several lines of evidence demonstrated that the IRs define a new family of olfactory receptors . First , the IR LBDs are highly divergent and lack one or more residues that directly contact the glutamate ligand in iGluRs . Second , several IRs are expressed in sensory neurons in the principal D . melanogaster olfactory organ , the antenna , that do not express members of the other D . melanogaster chemosensory receptor families , the Odorant Receptors ( ORs ) and Gustatory Receptors ( GRs ) [16] . Third , IR proteins localise to the ciliated endings of these sensory neurons and not to synapses [15] . Finally , mis-expression of an IR in an ectopic neuron is sufficient to confer novel odour-evoked neuronal responses , providing direct genetic evidence for a role in odour sensing [15] . The identification of the IRs as a novel family of olfactory receptors in D . melanogaster provides a potential link between the well-characterised signalling activity of iGluRs in glutamate neurotransmitter-evoked neuronal depolarisation and a potentially more ancient function of this family in environmental chemosensation . In this work , we have combined comparative genomics , molecular evolutionary analysis and expression studies to examine the evolution of the IRs . Four principal issues are addressed: first , when did olfactory IRs first appear ? Are they a recent acquisition as environmental chemosensors in D . melanogaster , or do they have earlier origins in insect or deeper animal lineages ? Second , what is the most recent common ancestor of IR genes ? Do they derive from AMPA , Kainate or NMDA receptors , or do they represent a distinct subfamily that evolved from the ancestral animal iGluR ? Third , what mechanisms underlie the expansion and diversification of this multigene family ? Finally , do IRs function only as olfactory receptors or are they also involved in other sensory modalities ? Through answers to these questions , we sought insights into IR evolution in the context of the origins of iGluRs , the appearance and evolution of other chemosensory receptor repertoires and the changing selective pressures during animal diversification and exploitation of new ecological niches .
iGluRs and IRs are characterised by the presence of a conserved ligand-gated ion channel domain ( the combined Pfam domains PF10613 and PF00060 [17] ) ( Figure 1A ) . All iGluRs additionally contain an ATD ( Pfam domain PF01094 ) , which is discernible , but more divergent , in only two D . melanogaster IRs , IR8a and IR25a . Most IRs have only relatively short N-terminal regions preceding the LBD S1 domain ( Figure 1A ) . To identify novel iGluR/IR-related genes , we therefore constructed a Hidden Markov Model ( HMM ) from an alignment of the conserved iGluR/IR C-terminal region , which is specific to this protein family . In combination with exhaustive BLAST searches , we used this HMM to screen raw genomic sequences and available annotated protein databases of 32 diverse eukaryotic species and 971 prokaryotic genomes ( see Materials and Methods and Table S2 in Supporting Information ) . These screens identified all previously described eukaryotic iGluRs and all D . melanogaster IRs , as well as 23 prokaryotic iGluRs . Novel sequences were manually reannotated and classified by sequence similarity , phylogenetic analysis and domain structure as either non-NMDA ( i . e . AMPA and Kainate ) or NMDA subfamily iGluRs , or IRs ( Figure 1B , Table S3 , and Datasets S1 and S2 ) . Like D . melanogaster IRs , newly annotated IRs have divergent LBDs that lack some or all known glutamate-interacting residues , supporting their distinct classification from iGluRs . iGluRs are widespread in eukaryotes , present in all analysed Metazoa ( except the sponge , Amphimedon queenslandica [18] ) and Plantae , but absent in unicellular eukaryotes ( Figure 1B , Table S3 , and Datasets S1 and S2 ) . Analysis of iGluR subfamilies on the eukaryotic phylogeny suggests that NMDA receptors may have appeared after non-NMDA receptors , as we identified them in Eumetazoa but not in the placozoan Trichoplax adhaerens . Further support for this conclusion will require additional genome sequences . One member of the Eumetazoa , the sea urchin Strongylocentrotus purpuratus , may have secondarily lost NMDA receptors . Different species contain distinct numbers of each iGluR subfamily: vertebrates , for example , have more NMDA receptor subunits than invertebrates . Notably , IRs were identified throughout Protostomia , encompassing both Ecdysozoa ( e . g . nematodes and arthropods ) and Lophotrochozoa ( e . g . molluscs and annelids ) ( Figure 1B , Table S3 , and Datasets S1 and S2 ) . There is substantial variation in the size of the IR repertoire , from three in C . elegans to eighty-five in the crustacean Daphnia pulex . Amongst insects , Diptera ( i . e . flies and mosquitoes ) generally had a larger number of IRs than other species . We did not identify IRs in Deuterostomia , Cnidaria or Placozoa . To explore the evolutionary origin of the IRs , we examined phylogenetic relationships of the identified protostome IRs . Reciprocal best-hit analysis using D . melanogaster sequences as queries revealed that a subset of this species' IRs was conserved in several distant lineages , allowing us to define putative orthologous groups . These include one group containing representatives of all protostome species ( IR25a ) , one represented by all arthropods ( IR93a ) , nine by most or all insects , and three by dipteran insects ( Figure 2A and 2B ) . For most orthologous groups , a single gene for each species was identified . In a few cases , for example the IR75 group , certain species were represented by several closely related in-paralogues , some of which appeared to be pseudogenes ( Figure 2A and 2B , Table S3 , and Datasets S1 and S2 ) . Consistent with its conservation in Protostomia , IR25a is the IR with the most similar primary sequence to iGluRs , suggesting that it is the IR gene most similar to the ancestral IR . Analysis of the phylogenetic relationship of IR25a and eukaryotic iGluRs locates it clearly together with the animal iGluR family , in the non-NMDA receptor clade ( Figure 2C ) . To substantiate this conclusion , we asked whether the IR25a gene structure resembles more closely that of NMDA or non-NMDA receptors . Intron positions and numbers are extremely variable across IR25a orthologues , with multiple cases of intron loss , gain and putative intron sliding events by a few nucleotides ( Figure 2D ) . Nevertheless , we identified eight intron positions that are conserved between at least subsets of IR25a orthologues and D . melanogaster non-NMDA receptor genes , some of which may represent intron positions present in a common ancestral gene . By contrast , only a single intron that was conserved in position ( but not in phase ) was identified between DmelIR25a ( but not other IR25a orthologues ) and DmelNMDAR1 ( Figure 2D ) . A phylogram of intron positions in IR25a , non-NMDA and NMDA sequences reveals greater similarity of IR25a intron positions to those of non-NMDA receptors than NMDA receptors ( Figure 2D ) . Together , these observations support a model in which IR25a evolved from a bilaterian non-NMDA receptor gene . The conserved D . melanogaster IRs encompass the entire subset of its IR repertoire that is expressed in the antenna [15] . Moreover , evidence for antennal expression of the three additional genes , DmelIR41a , DmelIR60a and DmelIR68a , has been obtained by reverse transcription ( RT ) -PCR analysis , although we have not yet been able to corroborate this by RNA in situ hybridisation ( data not shown ) . These combined phylogenetic and expression properties led us to designate this subfamily of receptors the “antennal IRs” . We examined whether antennal expression of this subfamily of IRs is conserved outside D . melanogaster by performing a series of RT-PCR experiments on the honey bee , Apis mellifera , for all six putative antennal IR orthologues: IR8a , IR25a , IR68a , IR75u , IR76b and IR93a ( see Materials and Methods for the nomenclature of newly-identified IRs ) . As in D . melanogaster , we could reproducibly amplify all of these bee genes from antennal RNA preparations but not in control brain RNA , except for AmelIR68a and AmelIR75u , which are also detected in the brain ( Figure 2E ) . Thus , antennal expression of this subgroup of IRs is conserved across the 350 million years separating dipteran and hymenopteran insect orders [19] , and therefore potentially in all insects . To investigate whether IRs are likely to have an olfactory function beyond insects , we examined expression of the IR repertoire from a representative of a distantly related protostome lineage , Aplysia molluscs , whose last common ancestor with D . melanogaster probably existed 550–850 million years ago [20] . We first used RT-PCR to analyse the expression of the ten Aplysia IR genes in a variety of sensory , nervous and reproductive tissues ( Figure 3A ) . Notably , the Aplysia IR25a orthologue is predominantly expressed in the olfactory organs , the rhinophore and oral tentacle [21] . Two other Aplysia-specific IR genes , IR214 and IR217 , are expressed in the rhinophore and oral tentacle , respectively , and not detected in other tissues , except for the large hermaphroditic duct ( IR214 ) and skin ( IR217 ) . Five additional IRs are also expressed in the oral tentacle , but displayed broader tissue expression in skin and the central nervous system; both of these tissues are likely to contain other types of chemosensory cells [22]–[23] . Expression of two IR genes , IR209 and IR213 , was not detected in this analysis ( data not shown ) . To further characterise Aplysia IR25a , we analysed its spatial expression in the mature A . dactylomela rhinophore by RNA in situ hybridisation . An antisense probe for AdacIR25a labels a small number of cells in rhinophore cryosections . Their size and morphology is typical of neurons , although we lack an unambiguous neuronal marker to confirm this identification ( Figure 3B–3D ) . These cells are found either singly or in small clusters adjacent or close to the sensory epithelial surface in the rhinophore groove , in a similar position to cells expressing other types of chemosensory receptors [21] . A control sense riboprobe showed no specific staining ( Figure 3E ) . Together , these results are consistent with at least some of these molluscan IRs having a chemosensory function . The expression of putative IR25a orthologues has previously been reported in two other Protostomia . An IR25a-related gene from the American lobster , Homarus americanus , named OET-07 , is specifically expressed in mature olfactory sensory neurons [24]–[25] . In C . elegans , a promoter reporter of the IR25a orthologue , GLR-7 , revealed expression in a number of pharyngeal neurons [9] , which might have a role in food sensing [26] . While both crustacean and nematode genes were classified in these studies as iGluRs , there is no evidence that they act as canonical glutamate receptors , and we suggest that they fulfil instead a chemosensory function . The antennal IR subfamily accounts for only a small fraction of the IR repertoire in most analysed insects and only 1–2 genes in other Protostomia . The remaining majority of IR sequences are - amongst the genomes currently available - largely species-specific , with low amino acid sequence identity ( as little as 8 . 5% ) with other IR genes in either the same or different species . We refer to this group of genes here as the “divergent IRs” . Dipteran insects have particularly large expansions of divergent IRs ( Figure 1B ) . Phylogenetic analysis revealed no obvious orthologous relationships of these genes either between D . melanogaster and mosquitoes or amongst the three mosquito species ( Aedes aegypti , Culex quinquefasciatus and Anopheles gambiae ) ( Figure 4 ) . Instead , this subfamily of IRs displays a number of species-specific clades , perhaps reflective of the distinct ecological niches of these insects . By contrast to antennal IRs , divergent IR expression has not been detected in D . melanogaster olfactory organs [15] , leading us to test whether these genes are expressed in other types of chemosensory tissue . As endogenous transcripts of non-olfactory chemosensory genes , such as GRs , are difficult to detect [27]–[28] , we employed a sensitive transgenic approach to investigate divergent IR expression . We transformed flies with constructs containing putative promoter regions for these genes upstream of the yeast transcription factor GAL4 and used these “driver” transgenes to induce expression of a GAL4-responsive UAS-mCD8:GFP fluorescent reporter [29] . We sampled divergent IRs from several distinct clades , including IR7a , IR11a , IR52b , IR56a and IR100a ( Figure 4 ) . All IR promoter-GAL4 constructs were inserted in the same genomic location using the phiC31 integrase system [30] , eliminating transgene-specific position effects on expression resulting from their site of integration . Expression of three of these divergent IR reporters was observed in highly selective populations of neurons in distinct gustatory organs ( Figure 5A ) . In the adult , IR7a is expressed in at least eleven neurons in the labellum , a sense organ involved in peripheral taste detection ( Figure 5B ) [31] . Two reporters labelled neurons in internal sense organs in the pharynx: IR11a is expressed in one neuron in the ventral cibarial sense organ and IR100a is expressed in two neurons in the dorsal cibarial sense organ ( Figure 5C and 5D ) . These internal pharyngeal neurons are thought to play a role in assessment of ingested food prior to entry into the main digestive system [16] . Expression was not detected in any other neurons or other cell types in the adult head ( data not shown ) , although we cannot exclude expression in other regions of the body . IR52b and IR56a reporters were not detected in these experiments . We also examined expression of these reporters at an earlier stage in the D . melanogaster life cycle , third instar larvae , which display robust gustatory responses [16] . The same three IR reporters were exclusively detected in unique bilaterally-symmetric larval gustatory organs: IR7a was expressed in two neurons in the terminal organ at the periphery , IR11a in a single neuron in the ventral pharyngeal sense organ and IR100a in two neurons in the posterior pharyngeal sense organ ( Figure 5E–5H ) . Notably , all of these neurons in both adult and larval tissues ( except for a single IR7a-expressing cell in the terminal organ ) co-express IR25a , as revealed by a specific antibody against this receptor ( Figure 5 ) [15] . IR25a is also expressed in several other cells in each of the gustatory organs , which may express other divergent IRs not examined here . Together these results support a role for divergent IRs as taste receptors in distinct taste organs and stages of the D . melanogaster life cycle . To obtain more detailed insights into the processes underlying the expansion and diversification of IR repertoires , we investigated their evolution over a shorter timescale by comparative analysis of D . melanogaster with 11 additional sequenced drosophilid species [32]–[33] . The last common ancestor of these drosophilids is estimated to have existed 40 million years ago [34] , by contrast to the ∼250 million years since the last common ancestor of D . melanogaster and the mosquito A . gambiae [35] . Certain species may have diverged much more recently , such as D . simulans and D . sechellia , whose last common ancestor may have existed only 250 , 000 years ago [36] . We used D . melanogaster sequences as queries in exhaustive BLAST searches of the drosophilid genomes . Retrieved sequences were manually reannotated to unify gene structure predictions across species and , in some cases , genes were partially resequenced to close sequence gaps or verify them as pseudogenes ( see Materials and Methods , Table S3 , and Datasets S1 and S2 ) . Although predicted full-length gene sequences could be annotated for most genes , 28 sequences remain incomplete - but assumed in further analysis to be functional - because of a lack of sequence data or difficulty in precise annotation of exons in divergent regions of these genes . Of the 926 drosophilid sequences identified ( including those of D . melanogaster ) , 49 genes were classified as pseudogenes because they consisted of only short gene fragments or contained frameshift mutations and/or premature stop codons . We clustered all genes into orthologous groups by examining their sequence similarity , phylogenetic relationships and , in the case of IR47a , IR47b , IR47c , IR56e and IR60f , their micro-syntenic relationships ( Table S1 and Figure 6 ) . For drosophilid species that are most distant from D . melanogaster , definition of precise orthologous relationships was not always possible , particularly for groups of closely related IR genes ( e . g . IR52a–f , IR60b–f ) ( Table S1 ) . Orthologous groups were named after their D . melanogaster representatives or a logical variant in groups where no D . melanogaster gene was identified ( see Materials and Methods ) . This analysis identified 14 iGluR and 58–69 IR genes in each of the twelve drosophilid species ( Figure 6A and Table S1 ) . iGluRs are highly conserved , with a mean amino acid sequence identity of 89±1% s . e . m . , and a single representative for each species in every orthologous group . Antennal IRs are also well conserved ( mean sequence identity = 76±2% ) and amongst these genes we identified only a single pseudogenisation event , in D . sechellia IR75a , and a single gene duplication event , of D . mojavensis IR75d . By contrast , divergent IRs , though also largely classifiable into monophyletic groups , display a more dynamic pattern of evolution ( mean sequence identity = 61±2% ) , with multiple cases of gene loss , pseudogenisation or duplication ( Figure 6 and Table S1 ) . We reconciled the gene phylogeny with the drosophilid species phylogeny to estimate the number of IR gene gain and loss events . While this analysis is necessarily constrained by our ability to accurately define gene orthology , we estimated across the entire phylogeny there to be sixteen gene gain events ( gene birth rate , B = 0 . 0006/gene/million years ) and 76 gene loss events ( gene death rate , D = 0 . 0030/gene/million years ) ( Figure 7A , see Materials and Methods ) . Most ( 46/76 ) gene losses are pseudogenisation events , which indicates that many of these events must have occurred relatively recently , as drosophilid species appear to eliminate pseudogenes rapidly from their genomes [37]–[38] . Notably , 13 gene loss events – 12 of which reflect the presence of just one or a small number of premature stop codons or frameshift mutations – occur on the branch leading to the specialist D . sechellia . Consequently , the gene loss rate on this branch is remarkably high compared with its generalist sister species D . simulans ( Figure 7A and 7B ) . We studied the selective forces acting on drosophilid iGluRs and IRs by calculating the ratio of nonsynonymous to synonymous nucleotide substitution rates ( dN/dS , ω1 ) in these genes from all 12 species . All tested iGluR , antennal IR and divergent IR genes are evolving under strong purifying selection ( ω1<<1 ) ( Figure 7C , left and Table S4 ) , suggesting that they all encode functional receptors . iGluRs have the lowest estimated dN/dS ratio ( median ω1 = 0 . 060 ) , consistent with a conserved role in synaptic communication . Antennal IRs have an intermediate dN/dS ratio ( median ω1 = 0 . 107 ) and divergent IRs the highest ( median ω1 = 0 . 149 ) , suggesting that divergent IRs have evolved under weaker purifying selection and/or contain more sites that have been shaped by positive selection . Amongst the IRs , IR25a has the lowest dN/dS ratio ( ω1 = 0 . 028 ) , consistent with its high sequence conservation in and beyond drosophilids ( Figure 2 ) . To compare these properties with those of other insect chemosensory receptor families ( ORs and GRs ) [39] , we also calculated dN/dS ratios for IR genes from only the five sequenced species of the melanogaster subgroup ( D . melanogaster , D . sechellia , D . simulans , D . erecta and D . yakuba ) . For this subset of sequences , the relative differences between median dN/dS ratios ( ω2 ) for the iGluR and IR gene subfamilies observed with all twelve species was reproduced ( Figure 7C , right ) . The GR gene family has previously been noted to evolve under weaker purifying selection than ORs [39] . Notably , we found that the median dN/dS ratios for antennal IRs ( ω2 = 0 . 120 ) is statistically indistinguishable from that of ORs ( ω2 = 0 . 137 ) ( p>0 . 4 , Wilcoxon rank-sum test ) , and that the median dN/dS ratio of divergent IRs ( ω2 = 0 . 176 ) is statistically indistinguishable from that of GRs ( ω2 = 0 . 217 ) ( p>0 . 5 , Wilcoxon rank-sum test ) . Thus , the selective forces acting on the IR receptor gene subfamilies parallel those on the ORs and GRs and appear to correlate with their putative distinct chemosensory functions in olfaction and gustation ( Figure 7C , right ) . The reason for this difference is unknown , but might reflect reduced evolutionary constraints on co-expressed and partially redundant taste receptor genes or selection for higher diversity in taste receptor sequences to recognise more variable non-volatile chemosensory ligands in the environment . Most residues of IR proteins can be expected to have evolved under purifying selection to maintain conserved structural and signalling properties , which may mask detection of positive selection ( ω>1 ) at a small number of sites that contribute to their functional diversity . To obtain evidence for site-specific selection we applied site class models M7 and M8 in PAML to analyse 49 sets of orthologous IR genes of the six species of the melanogaster group . This test did not identify any sites significantly under positive selection after Bonferroni correction ( Table S4 ) , a result consistent with orthologous IR genes having the same function across drosophilids . Site-specific positive selection may be more easily detectable in relatively recent IR gene duplicates potentially undergoing functional divergence . We therefore analysed the sole duplication of an antennal IR , IR75d . 1 and IR75d . 2 in D . mojavensis . Assuming an estimated divergence time of 35 My between D . virilis and D . mojavensis [40] , and based on analysis of dS of IR75d genes in these species ( see Materials and Methods ) , we estimated this duplication to have occurred relatively recently , approximately 2 . 6–5 . 1 My ago . Using a branch-site test we identified two sites ( p<0 . 05 ) that have evolved under positive selective pressure , where DmojIR75d . 1 and DmojIR75d . 2 appear to contain the ancestral and derived residues , respectively: DmojIR75d . 2-S670 maps to the third transmembrane domain and DmojIR75d . 2-Q365 maps to the putative ligand binding domain . Functional characterisation of these variant receptors will be required to determine their significance . From potentially one ancestral IR , what genetic processes underlay the generation of large repertoires of IR genes ? We initially sought evidence for these mechanisms through analysis of the D . melanogaster IR family . Several monophyletic groups of IR genes exist in clusters in the genome suggesting an important role of gene duplication by non-allelic homologous recombination . For example , eight divergent IRs of the IR94 orthologous groups are located in three close , but separate , tandem arrays on chromosome arm 3R ( Figure 8A ) . Other genes in the same clade are also found scattered on other chromosome arms ( X , 2R , 3L ) ( Figure 6 and Figure 8A ) , indicating that interchromosomal translocation has also occurred frequently , most likely both during and after formation of the tandem arrays . Similar patterns are observed in the orthologous/paralogous sequences of these IRs in other drosophilid species ( Figure 8A ) , as well as for other IR clades ( data not shown ) . These features are also observed in IR repertoires in other insects , although incomplete genome assembly prevented a more precise analysis . For example , in Aedes aegypti the 23 IR7 clade members are found in arrays of 1 , 1 , 2 , 5 , 7 and 7 genes on 6 different supercontigs ( data not shown ) . We also noticed an unusual pattern in D . melanogaster IR gene structures , in which antennal IRs ( as well as iGluRs ) contain many ( 4–15 ) introns , while the vast majority of divergent IRs are single exon genes ( Figure 8B ) . Drastic intron loss in multigene families is a hallmark of retroposition , where reverse-transcription of spliced mRNAs from parental , intron-containing genes and reinsertion of the resulting cDNA at a new genomic location may give rise to a functional , intronless retrogene [41] . The few introns that are present in these IRs in D . melanogaster have a highly biased distribution towards the 5′ end of the gene ( 19/25 introns in the first 50% of IR gene sequences ) ( Figure 8C ) , which is characteristic of recombination of partially reverse-transcribed cDNAs ( a process which initiates at the 3′ end ) with parental genes [42] . Sequence divergence of IRs prevented us from identifying parental gene-retrogene relationships . Nevertheless , these observations together suggest that divergent IRs arose by at least one , and possibly several , retroposition events of ancestral antennal IRs . Once “born” , single exon IRs could presumably readily further duplicate by non-allelic homologous recombination .
Our comprehensive survey and phylogenetic analysis of iGluR/IR-like genes permits development of a model for their evolution ( Figure 9 ) . The shared , unusual “S1-ion channel-S2” domain organisation of prokaryotic GluR0 and eukaryotic iGluRs is suggestive of a common ancestor of this family by fusion of genes encoding the separate domains that were present in very early life forms ( Figure 9 ) [11] . However , we have found prokaryotic glutamate receptors in only a very small number of bacterial species . Thus , if an iGluR evolved in the common ancestor of prokaryotes and eukaryotes , it must have subsequently been lost in a large number of prokaryotic lineages . It is possible , therefore , that iGluRs only originated in eukaryotes and were acquired by certain prokaryotic species by horizontal gene transfer [43] . If the latter hypothesis is true , the presence of closely related iGluRs in both plants and animals implies their early evolution within eukaryotes , potentially in the last common eukaryotic ancestor [44] . However , the absence of iGluRs in sponges and all examined unicellular eukaryotes raises the alternative possibility that animal and plant receptors evolved independently , or were acquired by horizontal transmission , perhaps from prokaryotic sources . Whatever the precise origin of iGluRs in animals , their subsequent divergence into AMPA , Kainate and NMDA subfamilies also occurred early , although variation in the size of these subfamilies suggests continuous adaptation of the synaptic communication mechanisms they serve to nervous systems of vastly different complexities . Several outstanding issues regarding IR evolution can now be addressed . First , we have shown that the IRs were very likely to have been present in the last common ancestor of Protostomia , an estimated 550–850 million years ago [20] . IR25a represents the probable oldest member of this repertoire and conservation of chemosensory organ expression of IR25a orthologues in molluscs , nematodes , crustaceans and insects strongly suggests that this receptor may have fulfilled a chemosensing function in the protostome ancestor . Second , the apparent absence of IRs in Deuterostomia suggests the parsimonious model that IRs evolved from an animal iGluR ancestor rather than representing a family of chemosensing receptors that was present in a common ancestor of Animalia and lost in non-protostomes . Our phylogenetic and gene structure analysis suggests that IR25a may have derived from a non-NMDA receptor gene . The transition from an iGluR to an IR may not have involved drastic functional modifications: both receptor types localise to specialised distal membrane domains of neuronal dendrites ( post-synaptic membranes and cilia , respectively ) and , in response to binding of extracellular ligands , depolarise these domains by permitting transmembrane ion conduction which in turn induces action potentials [45] . Thus , it is conceivable that IRs arose simply by a change in expression of an iGluR from an interneuron ( where it detected amino acid signals from a pre-synaptic partner ) to a sensory neuron ( where it could now detect chemical signals from the external environment ) . Third , our analyses of IR repertoires across both divergent and relatively closely related species provide insights into the mechanistic basis for the expansion and functional diversification of the IR repertoire . Gene duplication by non-allelic homologous recombination is a widespread mechanism for growth of most multigene families in chemosensory systems [46] , and this is also true for the IRs . Our implication of retroposition as a second mechanism in the evolution of IR repertoires offers two advantages for functional diversification . First , by arising from random re-insertion of reverse transcribed copies of parental genes , retrogenes normally lack endogenous promoter sequences , and can therefore potentially acquire novel expression patterns from genomic sequences flanking their insertion site that are distinct from their parental ancestor [41] . Indeed , in D . melanogaster , retrogene or retrogene-derived IRs - the divergent IRs - are apparently no longer expressed in antennal neurons like their ancestors , but instead in gustatory ( and perhaps other ) tissues . Second , release from the evolutionary constraints of the preservation of splicing signals near exon boundaries may have contributed to the more rapid divergence of the protein sequences of these intronless IRs [47] . Analysis of IR repertoires across the well-defined drosophilid phylogeny provides clear evidence for a birth-and-death model of evolution , in which , following gene duplication , individual family members progressively diverge in sequence and , in some cases , are lost by pseudogenisation and/or deletion [48]–[49] . Differential rates of these processes will ultimately shape the precise IR repertoire of an individual species ( discussed below ) . Our molecular evolutionary analysis has distinguished two subfamilies in the IR repertoire: conserved , antennal IRs and the species-specific , divergent IRs . Their distinct evolutionary properties may correspond to fundamental functional differences , as we provide here the first evidence , to our knowledge , for expression of divergent IR subfamily members in subsets of neurons in both peripheral and internal gustatory organs at both adult and larval stages of D . melanogaster . The selective and non-overlapping expression patterns observed in the small sample of IR genes examined indicate that a large fraction of the divergent IR repertoire may be expressed in gustatory neurons . It is also possible that some of these IRs may be expressed in non-chemosensory tissues . Although subsets of GR genes have been implicated in the detection of sweet or bitter compounds in peripheral taste bristles in D . melanogaster [31] , a comprehensive understanding of the physiological breadth and molecular logic of taste detection is lacking . Our results introduce further complexity into the molecular mechanisms of taste detection and demand comprehensive and comparative expression and functional analysis of divergent IRs and GRs in this sensory system . Although many gustatory-expressed divergent IRs in D . melanogaster are recently derived in drosophilids , the ancestral chemosensory function of IRs is likely to be not in the detection of airborne volatiles but rather water-soluble , non-volatile compounds , as the last common ancestor of Protostomia was probably aquatic . Indeed , the strikingly similar expression of IR genes in internal pharyngeal neurons in D . melanogaster and C . elegans suggests a conserved role for these receptors in sensing chemical signals from ingested food . In this light , the derivation of IRs from receptors detecting amino acid-related neurotransmitters invites the attractive hypothesis that ligands for these gustatory IRs ( as well as species-specific IRs in other protostomes ) are also amino acids . Almost nothing is known about sensory responses to this class of chemical signals in D . melanogaster , despite their vital importance for normal insect physiology and metabolism [50] , but amino acids are chemosensory stimulants in other insects , lobsters and molluscs [51]–[53] . Our evolutionary and expression studies have highlighted IR25a as an atypical member of the repertoire , displaying deep conservation and broad expression in many olfactory and gustatory neurons . While we cannot exclude the possibility that IR25a recognises a specific chemical ligand , co-expression of this receptor with other cell-type specific IRs favours a model in which this acts as a co-receptor , analogous both to the heteromeric assembly of iGluR subunits into functional complexes [1] , as well as to the pairing of ligand-specific ORs with the common OR83b co-receptor [54]–[55] . An insect- and antennal-specific homologue of IR25a , IR8a , may play a similar role specifically for olfactory IRs . In addition to IR25a and IR8a , many other D . melanogaster antennal IRs are highly conserved in insects , both in sequence and expression pattern . These properties contrast starkly with the insect OR repertoires , which probably evolved only in terrestrial insects [56] , and which contain only one member displaying orthology across multiple orders , the atypical OR83b co-receptor [57] . ORs are an expanded lineage of the ancestral GR repertoire whose evolutionary origins are unknown [56] . Homologues of GR genes exist in D . pulex and C . elegans [56] , [58] , but in the latter species these receptors may not be involved in chemosensation [59]–[60] . These observations suggest that , in insects , the IRs represent the first olfactory receptor family , whose members were fixed functionally early in their evolution to detect olfactory stimuli that are important for all species of this animal class . Consistent with this , the antenna of the mayfly Rhithrogena semicolorata – an insect belonging to the Paleoptera and not the Neoptera that encompasses all species described here – bears coeloconic sensilla ( potentially housing IR-expressing neurons ) but not trichoid or basiconic sensilla ( which house OR-expressing neurons in all other insects examined ) [61] . Available data on ligands for IR sensory neurons - and the role of specific IRs within these neurons - are limited , but include stimuli such as carboxylic acids , water and ammonia , which are known to be physiologically and behaviourally important in many insect species [62] . ORs , by contrast , may be primarily dedicated to detection of species-specific odour cues . In this light , the IRs are attractive molecular targets for novel , broad-spectrum chemical regulators of insect odour-driven behaviours , with applications in the control of disease vectors , such as mosquitoes , and agricultural pests . Given the general conservation of the antennal IRs , what is the significance of the more recently evolved , species-specific variation in this family of chemosensory receptors ? It is particularly informative to consider this question in the evolutionarily closely related drosophilid species . These display prominent differences in their global geographical distribution and chemosensory-driven behaviours [63]–[64] , and include both generalists , which feed and breed on a wide range of substrates , and specialists , which have highly restricted ecological niches . The chemical ecology is best-understood for D . sechellia , a species endemic to the Seychelles that utilises the acid-rich fruit of Morinda citrifolia as its sole food source and oviposition site , a remarkable specialisation as this fruit is repulsive and toxic for other drosophilids [64]–[65] . Genetic hybrids between D . sechellia and D . simulans indicate that host specialisiation is due to loss-of-function mutations , rather than gain of new chemosensory perception abilities [65] . The accelerated rate of IR gene loss in D . sechellia compared to its sibling D . simulans ( and other drosophilids ) bears the hallmark of genetic adaptation of this chemosensory repertoire to the restricted host fruit . Notably , one of the D . sechellia pseudogenes is IR75a , an antennal IR expressed in a neuron responsive to several acids [62] . Thus , DsecIR75a represents an interesting gene whose mutation may be directly linked to host specialisation of this species . Future study of this receptor , and other species-specific IRs , may offer novel models to link genetic changes with phenotypic adaptation during animal evolution . Finally , our results may shed light into the outstanding question of the evolutionary origin of animal olfactory systems . Common neuroanatomical features have long been appreciated in animal olfactory circuitry , notably glomeruli , which represent sites of synaptic connection of OSNs of identical molecular and physiological specificity with second order neurons [66] . Whether these represent homologous or analogous structures across phyla is unclear . Revelations of fundamental distinctions in the structure , function and regulation of mammalian and insect ORs support a theory of convergent evolution of the neuronal circuits in which these receptors act [67]–[68] . Our demonstration that most , if not all , insect olfactory systems comprise two molecularly distinct receptor families , the ORs and IRs , indicates that the evolution of receptor repertoires can be uncoupled from a presumed common origin of the OR and IR neuronal circuits within the insect ancestor . Thus , during a significantly greater timescale across animal phyla , profound molecular differences between olfactory receptor genes do not necessarily imply distinct evolutionary origins of the neuronal circuitry in which they are expressed . Our discovery of IRs in mollusc olfactory organs reveals this to be an interesting potential “hybrid” organism in olfactory system evolution . The A . californica rhinophore and oral tentacle also express a large family of GPCR-family candidate chemosensory receptors , belonging to the same Rhodopsin superfamily as vertebrate ORs [21] . The co-existence of both insect-like and vertebrate-like olfactory receptors in this species provides evidence for the occurrence of an evolutionary transition between these distinct olfactory receptor families . Thus , while extant animal olfactory systems display an enormous diversity in their receptor repertoires , there may remain - perhaps unexpectedly - a sufficient genetic trace within receptor gene families themselves to open the possibility of a common evolutionary origin of this sensory system .
IR genes were named according to a unified nomenclature system based upon a foundation of the cytologically derived D . melanogaster IR gene names [15] . Receptor names are preceded by a four-letter species abbreviation consisting of an uppercase initial letter of the genus name and three lower case initial letters of the species name ( e . g . Anopheles gambiae = Agam; Daphnia pulex = Dpul ) . Orthologues of D . melanogaster sequences are given the same name ( e . g . CquiIR25a , AcalIR25a ) . If multiple copies of an orthologue of a D . melanogaster gene exist for a species ( based on sequence , not function ) , they are given the same name followed by a point and a number ( e . g . ApisIR75d . 1 , ApisIR75d . 2 ) . If several in-paralogues exist both in D . melanogaster and other species , these are all given the same number ( indicating their grouping within a common clade ) , but different final letterings . For novel , species-specific IRs , we defined new names numbering from 101 upwards to avoid confusion with D . melanogaster gene names , which number up to IR100a . For species-specific IRs that form monophyletic clades and had high ( >60% ) amino acid identity , we gave these the same name with an additional number suffix after a point ( e . g . AaegIR75e . 1 , AaegIR75e . 2 ) . We did not rename genes with previously published names ( e . g . C . elegans GLR-7 and GLR-8 [9] ) . For vertebrate iGluRs , we used the NC-IUPHAR nomenclature [81]: each species name is followed by “Glu” , a letter representing the subtype of the receptor ( K for Kainate , A for AMPA and N for NMDA ) , and a number , reflecting predicted orthology with mammalian iGluRs . We did not name ( or rename ) invertebrate iGluRs in this study , except for newly predicted gene sequences ( Table S3 ) , where logical variants of NC-IUPHAR nomenclature were assigned . Genomic DNA was extracted from the sequenced drosophilid genome strains ( obtained from the Drosophila Species Stock Center , University of California-San Diego ) using a standard DNA extraction protocol . PCR primers were designed to amplify ∼500 bp regions covering putative nonsense or missense mutations or spanning gaps in the genome sequence ( oligonucleotide sequences are listed in Table S5 ) . PCR amplifications were performed using Taq DNA Polymerase ( PEQLAB Biotechnologie GmbH ) in a MasterCycler Gradient Thermocycler ( Eppendorf ) with the following programme: 95°C for 3 min , 35 cycles of ( 95°C for 30 sec , 55°C for 1 min , 72°C for 1 min ) and 72°C for 10 min , with minor modifications of annealing temperature and elongation times for different primer pairs and amplicon sizes . Products were gel purified ( Machery-Nagel ) and sequenced with BigDye Terminator v3 . 1 according to the manufacturers' protocols . Insects: total RNA was extracted from hand-dissected tissues of wildtype A . mellifera and D . melanogaster ( w1118 strain ) using the RNeasy Mini Kit ( Qiagen ) , and reverse-transcribed using oligo-dT primers and the SuperScript III First-Strand Synthesis System ( Invitrogen ) . Genomic DNA was extracted using standard procedures . Primers were designed to amplify short regions overlapping an intron , if possible at the 3′ end of the coding sequence ( Table S5 ) . PCR product amplification and purification were performed as described above and sequenced to verify their identity . Multiple independent cDNA preparations were analysed for each primer pair . Primers were designed to amplify putative promoter regions from Oregon-R D . melanogaster genomic DNA with flanking restriction sites , extending from immediately upstream of the predicted start codon to the following 5′ extents: IR7a ( 2318 bp ) , IR11a ( 2099 bp ) , IR52b ( 446 bp ) , IR56a ( 2400 bp ) and IR100a ( 512 bp ) ( Table S5 ) . Gel purified PCR products were T:A cloned into pGEM-T Easy ( Promega ) , end-sequenced , and sub-cloned into a pGAL4-attB vector , comprising the GAL4 ORF-hsp70-3′UTR in the pattB vector [30] . These constructs were integrated into the attP2 landing site [88] , by standard transformation procedures ( Genetic Services , Inc . ) . IR-GAL4 transgenic flies were double-balanced and crossed with flies bearing a UAS-mCD8:GFP transgene [89] to visualise driver expression . | Ionotropic glutamate receptors ( iGluRs ) are a family of cell surface proteins best known for their role in allowing neurons to communicate with each other in the brain . We recently discovered a variant class of iGluRs in the fruit fly ( Drosophila melanogaster ) , named Ionotropic Receptors ( IRs ) , which function as olfactory receptors in its “nose , ” prompting us to ask whether iGluR/IRs might have a more general function in detection of environmental chemicals . Here , we have identified families of IRs in olfactory and taste sensory organs throughout protostomes , one of the principal branches of animal life that includes snails , worms , crustaceans , and insects . Our findings suggest that this receptor family has an evolutionary ancient function in detecting odors and tastants in the external world . By comparing the repertoires of these chemosensory IRs among both closely- and distantly-related species , we have observed dynamic patterns of expansion and divergence of these receptor families in organisms occupying very different ecological niches . Notably , many of the receptors we have identified are in insects that are of significant harm to human health , such as the malaria mosquito . These proteins represent attractive targets for novel types of insect repellents to control the host-seeking behaviors of such pest species . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"evolutionary",
"biology",
"genetics",
"and",
"genomics/comparative",
"genomics",
"neuroscience/sensory",
"systems"
] | 2010 | Ancient Protostome Origin of Chemosensory Ionotropic Glutamate Receptors and the Evolution of Insect Taste and Olfaction |
The male-specific region of the human Y chromosome ( MSY ) includes eight large inverted repeats ( palindromes ) in which arm-to-arm similarity exceeds 99 . 9% , due to gene conversion activity . Here , we studied one of these palindromes , P6 , in order to illuminate the dynamics of the gene conversion process . We genotyped ten paralogous sequence variants ( PSVs ) within the arms of P6 in 378 Y chromosomes whose evolutionary relationships within the SNP-defined Y phylogeny are known . This allowed the identification of 146 historical gene conversion events involving individual PSVs , occurring at a rate of 2 . 9–8 . 4×10−4 events per generation . A consideration of the nature of nucleotide change and the ancestral state of each PSV showed that the conversion process was significantly biased towards the fixation of G or C nucleotides ( GC-biased ) , and also towards the ancestral state . Determination of haplotypes by long-PCR allowed likely co-conversion of PSVs to be identified , and suggested that conversion tract lengths are large , with a mean of 2068 bp , and a maximum in excess of 9 kb . Despite the frequent formation of recombination intermediates implied by the rapid observed gene conversion activity , resolution via crossover is rare: only three inversions within P6 were detected in the sample . An analysis of chimpanzee and gorilla P6 orthologs showed that the ancestral state bias has existed in all three species , and comparison of human and chimpanzee sequences with the gorilla outgroup confirmed that GC bias of the conversion process has apparently been active in both the human and chimpanzee lineages .
The male-specific region of the human Y chromosome ( MSY ) is constitutively haploid , yet contains a high proportion ( ∼35% ) of pseudo-diploid duplicated regions , eight of which are arranged as large inverted repeats ( ‘palindromes’ , known as P1 - P8; Figure 1a ) , with arms in most cases separated by non-duplicated spacers [1] . The arms of each palindrome are >99 . 9% similar in sequence due to the homogenising effect of gene conversion . Human-chimpanzee sequence divergence within palindrome arms is significantly lower than that within spacers , and compared to the MSY ( non-palindrome , non-spacer ) average [2] . This suggests that gene conversion since speciation may have been directional , tending to return new mutations that arise within arms to their ancestral states . Most palindrome arms are enriched in testis-specific genes , important in spermatogenesis , and the suggestion has been made that directional gene conversion between pseudo-diploid copies may protect these genes against evolutionary decay [2] . It is becoming increasingly recognised that such palindromic structures are far from being a peculiarity of great ape Y chromosomes , but have more general biological significance as a feature of independently arising constitutively haploid sex chromosomes in other mammals [3]–[5] , birds [6] , [7] and insects [8] , as well as of the mammalian X chromosome [9] , [10] , which is haploid in males . Yet despite this general importance , and despite some theoretical analyses of palindrome evolution [11] , [12] , little is known about the dynamics of conversion within these remarkable structures . Large stretches of sequence identity between palindrome arms represent compelling evidence for rapid gene conversion , and yet , paradoxically , provide a barrier to understanding the dynamics of the conversion process . Conversion rate , tract length , and directionality cannot be examined when there are no sequence differences ( paralogous sequence variants; PSVs ) between arms that might allow specific conversion events to be recognised . However , when a PSV does exist ( e . g . the ‘pseudoheterozygous’ state G/A ) , then the observation in other chromosomes of the two other possible genotypes , the ‘pseudohomozygous’ G/G and A/A , indicates that gene conversion must have occurred within the history of the examined sequences ( Figure 2a ) , assuming that recurrent mutation can be neglected . Such an observation tells us nothing about how many independent conversion events underlie the three genotypes . But the availability of a detailed and robust Y phylogeny , defined by stable single nucleotide polymorphisms ( SNPs ) outside the palindromic regions , allows the evolutionary relationships of palindrome sequences to be known , and genotyping within this phylogenetic context can then provide an estimate of the minimum number of conversion events ( Figure 2b–d ) . Genotyping PSVs within a phylogenetic context provides evidence for past gene conversion events , but the resulting genotypes ( pursuing the analogy of diploidy ) are ‘unphased’ - we do not know which allele of a PSV lies on which palindrome arm . Because of the high degree of sequence identity and the scarcity of PSVs within palindromes , phasing is technically challenging , but nonetheless important if we are to gain an understanding of the lengths of conversion tracts , suggested by sets of co-converted adjacent PSVs ( Figure 2e ) . If phased PSV data for palindromes were available , it would also be possible to address an additional important aspect of the dynamics of these structures: the ratio of non-reciprocal exchanges ( conversions ) to reciprocal exchanges ( inversions; Figure 2f ) . Here , we analyse paralogous sequence variants ( PSVs ) within the arms of human palindrome P6 , taking the approaches outlined above . We demonstrate through a phylogenetic analysis of conversion events five cardinal features of the palindrome conversion process during human evolution: ( i ) the conversion process has been rapid throughout the evolution of modern human Y-chromosomal lineages; ( ii ) it shows significant bias to the fixation of GC base pairs; ( iii ) it is biased towards the retention of ancestral states of PSVs; ( iv ) conversion tracts can encompass several kilobases; and ( v ) despite the high frequency of recombination events within palindrome arms , these resolve overwhelmingly via non-reciprocal exchange ( conversions ) rather than reciprocal exchange ( inversions ) . We then extend our findings to deeper evolutionary time by determining the sequence of gorilla P6 , showing that ancestral state bias has existed in the gorilla lineage as well as in humans and chimpanzees , and allowing us to ascertain the direction of evolutionary changes in the human and chimpanzee lineages , revealing a possible long-term GC conversion bias .
To study gene conversion dynamics we first sought a segment of a palindrome carrying a suitable number and density of PSVs . Arm-to-arm alignment of the reference sequence ( belonging to haplogroup R1b1b2* [13] ) for palindrome P6 ( Figure 1b ) demonstrated a 99 . 97% sequence similarity between its 110-kb arms , but revealed a total of 49 discrete sequence differences , which we supplemented with two additional single-nucleotide PSVs identified from the sequencing of a flow-sorted Y chromosome from a different source , belonging to haplogroup O3a [14] . Twenty-nine of these represent simple single-nucleotide PSVs ( SN-PSVs ) that are unlikely to undergo mutational reversion or recurrence . Furthermore , 16 SN-PSVs lie within 20 kb of the outer arm boundaries , potentially allowing arm-specific PCR anchored in flanking single-copy DNA to determine in which arm a particular variant lies ( ‘phasing’ ) . Two additional factors favour P6: chimpanzee and gorilla orthologs exist that allow the ancestral state of its PSVs to be determined; and P6 lacks protein-coding genes , meaning that direct effects of natural selection are less likely than for other palindromes . We sought to design reliable typing assays for all SN-PSVs , and this was successfully accomplished ( see Materials & Methods ) for ten , indicated in Figure 1b . In order to identify gene conversion events between the arms of P6 , we required a set of Y chromosomes for which detailed phylogenetic relationships were well established . We exploited the availability of the CEPH-Human Genome Diversity Project ( HGDP ) panel of DNA samples [15] , which has good global coverage and for which data were available for 184 Y-chromosomal binary markers ( [16]–[18]; www . cephb . fr/en/hgdp/ ) , supplementing this by typing an additional 23 binary markers , to define a total of 63 haplogroups . The tree thus defined , the markers , the haplogroup nomenclature and the sources of data are shown in Figure S1 . A simplified version of the tree is shown in Figure 3 . The ten PSVs were analysed in a subset of 378 of the 684 HGDP male samples , chosen to cover the haplogroup diversity of the sample set . Each PSV genotype was recorded as pseudoheterozygous ( e . g . G/A ) or pseudohomozygous ( e . g . G/G or A/A ) , and , by comparison to the orthologous sequences in chimpanzee [19] and gorilla ( [20] , and our own gorilla sequence – see below ) each PSV allele coded as ancestral ( 0 ) or derived ( 1 ) . Figure 3 illustrates the patterns of variation observed in the sample , and full details are given in Table S1 . Some PSVs ( e . g . PSV6 ) are variable across all haplogroups , suggesting that the variant arose at the root of the Y phylogeny . Others show variability only in specific haplogroups ( hg ) , suggesting ( assuming maximum parsimony , and no recurrent mutation ) that they arose in their founders ( e . g . PSV2 in hgF , PSV5 in hgP , and PSV9 in hgO3a ) . PSV10 was monomorphic in all 378 cases tested , suggesting that it represents a recently arising variant . For any haplogroup , we can deduce whether the founder was pseudoheterozygous ( 0/1 ) ; when this is so , the finding of pseudohomozygous states ( 0/0 or 1/1 ) among chromosomes within the haplogroup indicates that conversion must have occurred ( Figure 2d ) . Treating each PSV as an independent site of gene conversion , it is thus possible to both count the total number of conversion events , and to ask what proportion of these are conversions to the ancestral state ( i . e . 0/1 to 0/0 ) , or the derived state ( i . e . 0/1 to 1/1 ) . This analysis identified a total of 146 converted SN-PSVs , of which 86 represent conversion to the ancestral , and 60 to the derived state ( Tables S1 and S2 ) . This difference is statistically significant ( p = 0 . 0314; Chi-square test ) , which is consistent with published observations based on human-chimpanzee comparisons [2] . We can also ask if there is a tendency towards the fixation of GC base-pairs rather than AT base-pairs: this is so-called GC-biased gene conversion , and results from a bias in the repair of AC and GT mismatches that form in heteroduplex recombination intermediates [21] . Of the 146 converted SN-PSVs , some are uninformative because they involve transversions ( from CG to GC , or AT to TA ) , but among the 79 informative cases 59 involve the fixation of GC , and 20 of AT ( p = 1 . 1×10−5; Chi-square test ) . From these observations , gene conversion among the studied PSVs appears to be strongly GC-biased , and slightly but significantly biased towards the retention of ancestral state . Having counted the number of observed gene conversion events in our dataset , we can estimate an average rate of gene conversion by dividing by the number of generations encompassed in the phylogeny that relates the studied Y chromosomes ( Materials & Methods ) . For a 25-year generation time , this yields a rate of 2 . 9–8 . 4×10−4 events per generation . The above analysis provides evidence of a highly active gene conversion process within P6: but does the frequent formation of recombination intermediates that this implies also give rise to frequent inversions of the palindrome arms ? As explained in the Introduction ( Figure 2f ) , identification of inversion events requires the palindrome arms to be ‘phased’ at pseudoheterozygous sites . In order to do this , an arm-specific long-range PCR approach was developed , using one universal primer binding within the arm , and another binding to a distal-arm-specific region outside the outer palindrome boundary . This generated a product of ∼18 . 9 kb incorporating seven of the studied PSVs ( PSV1–7 ) that could then be typed in an arm-specific manner , thus determining their phase . Arm-specific haplotypes from 83 selected DNA samples representing all of the haplogroups were compared to the Y-chromosome reference sequence , whose phase is known from BAC clone sequencing [1] . All but five samples were found to have identical phase to the reference sequence ( Figure 4 ) at informative ( pseudoheterozygous ) sites; this corresponds to just three independent inversion events , in haplogroups A3b2* , B2a , and D2 . Where phase information is available for several chromosomes within a haplogroup , these are always concordant – in other words , inversions are rare . This strong preponderance of conversion over inversion allows us to infer the phase of unphased chromosomes within haplogroups . Among the 83 phased chromosomes , the three inversion events compare with 56 gene conversion events ( assuming each converted PSV represents a single event ) . In the same set of chromosomes , and under the same assumptions , the per-generation rate of inversion is 1 . 36–1 . 72×10−5 , compared to 2 . 54–3 . 21×10−4 for conversion . The latter rate differs from that given in the section above due to the smaller number of chromosomes phased and analysed here . All of the analysis above assumes that PSVs are independently converted , but from simple inspection of the behaviour of the adjacent PSVs 1 and 2 , separated by only 81 bp , it is evident that co-conversions must be occurring: for example , of the 14 instances where conversion affecting PSV1 and PSV2 is informative , 11 involve apparent co-conversion of both markers ( e . g . in hgM1*; Figure 3 ) . We therefore wished to examine co-conversion more systematically , and the phasing information allows us to do this ( as shown in Figure 2e ) . The true number of co-conversion events is impossible to estimate , because the apparent co-conversion of adjacent variants could actually reflect the sum of two independent events . However , we can estimate the minimum number of co-conversions that explain the observed data: first , we identify adjacent pairs of pseudohomozygous PSVs within a haplogroup whose founder is deduced to be pseudoheterozygous for the same PSVs; and second , to exclude independent opposite conversions as an explanation ( Figure 2e ) , we count only those PSV pairs that match a single arm-specific haplotype of the reference sequence . We then assume that these reflect a single conversion tract . On this basis , 49 of the 107 ( 45 . 8% ) individual conversion events among ‘phased’ PSVs ( 1–7 ) can be explained by a minimum of 18 co-conversion tracts . We cannot arrive at a useful estimate of maximum co-conversion tract length , because most tracts are not flanked by informative genotyped markers that would indicate their outer limits . However , we can estimate minimum lengths by considering the distance between the outer converted markers within each tract . The mean value of these minimum estimates is 2068 bp: this is much longer than most recorded gene conversion events , which are typically a few hundred bp in length , and rarely exceed 1 kb [22] . Some apparent co-conversion tracts are very long indeed . For example , within hgQ1a* we observe PSVs 1–8 in the pseudoheterozygous state , but also a case where the first seven of these variants are pseudohomozygous . This case seems unlikely to have arisen as a result of a series of consecutive small-scale conversion events , because the allelic state of the variants matches a single arm-specific haplotype in the same haplogroup . An alternative trivial explanation is that one arm in this chromosome has been lost by deletion , and that the PSVs are being observed in a pseudohemizygous , rather than pseudohomozygous state . To eliminate this possibility we confirmed that two arms were present , and were of the expected length , using two methods: a paralog-ratio test ( PRT ) to measure the copy-number of the palindrome arm with respect to a reference sequence on the X chromosome; and a long-PCR assay specific for each arm in turn . The most parsimonious explanation for the observed genotype in this chromosome is therefore a massive conversion event that spans at least 9023 bp ( the distance between PSVs 1 and 7 on the proximal arm ) . The analysis carried out above , to detect biases in gene conversion towards retention of the ancestral state and fixation of GC base-pairs , treated each converted nucleotide as an independent replicate in a statistical test . However , since we have inferred that co-conversions occur , some variants are not independent; we therefore repeated both tests after removing the putative co-conversion events . In both cases , the statistical significance of the bias is retained ( Table S2 ) . In order to study the deeper history of gene conversion activity and its impact on palindrome evolution , we required an outgroup to human and chimpanzee P6 . A high-quality MSY sequence is available for rhesus macaque that contains three palindromes , but a P6 ortholog is not among them [3] . A gorilla Y-chromosome reference sequence is not yet available , but this species is known to carry both P6 arm-spacer boundaries with almost identical sequence to human and chimpanzee [2] . We constructed a partial sequence of gorilla P6 by merging Illumina paired-end sequencing data from two whole-genome-sequenced male gorillas [20] and from an independent male analysed in a sequence capture experiment . A total of 88 , 031 bp of merged gorilla P6 arm and 31 , 206 bp of gorilla spacer were assembled using the human Y-chromosome sequence as a reference . These data represent 80% of the human proximal arm and 67 . 5% of human spacer . The presence of both P6 arms in gorilla is confirmed by the fact that the mean coverage of proximal arm for all three gorillas is approximately twice that of the spacer ( Protocol S1 ) . Pairwise alignments between human , chimpanzee and gorilla show that nucleotide divergence in all three comparisons is highly significantly reduced in the arms of P6 compared to spacer ( Table 1 ) . This is consistent with previous results [2] showing a similar pattern when comparing segments of Y-chromosome palindromes between human and chimpanzee . Our findings therefore confirm that the processes influencing palindrome evolution are active in both human and chimpanzee lineages , and also probably active in gorilla . Availability of an outgroup sequence also allows possible long-term GC-bias to be examined in human and chimpanzee lineages . We used a phylogenetic approach to study nucleotide replacements in palindrome arms and spacer . Since the universally low ( ∼0 . 02% ) arm-to-arm divergence suggests that conversion is highly active within each species , all replacements found in arms can be assumed to be due to mutation followed by gene conversion; in spacers the divergence is expected to arise solely from mutational processes . From the alignment of human , chimp and gorilla P6 sequences ( Dataset S1 ) , we determined the types of all fixed differences , noting G or C ( S ) nucleotides that changed to A or T ( W ) nucleotides , and vice versa . We also determined the evolutionary direction of each of these fixed differences: if a nucleotide was identical between chimpanzee and gorilla but divergent in human , a replacement on the human lineage was assumed; if human and gorilla were identical , a replacement in chimpanzee was assumed . Table 2 summarises the numbers and types of nucleotide replacements in both the human and chimpanzee arms and spacers . In the arm , the proportion of W to S changes slightly exceeds that in the spacer , but the proportion of S to W changes is significantly lower than that in the spacer . Furthermore , in human the proportion of W to S and S to W changes in the arm are approximately equal , while in the spacer S to W changes significantly predominate ( as has been observed previously for substitutions not associated with gene conversion [23] ) . These observations indicate a relative bias towards W to S changes in arms . In chimpanzee P6 , the proportion of W to S changes in the arm is significantly higher than that of S to W . In order to eliminate the potential influence of hypermutable CpG dinucleotides , all sites in CpG , TpG , or CpA sequences were removed from the raw sequence alignment , and the comparisons repeated . In both human and chimpanzee P6 , the differences between arm and spacer remain . These striking differences in substitution patterns in arms and spacer seems likely to reflect the preferential fixation of GC base-pairs in arms due to the action of GC-biased gene conversion .
We observe a conversion rate of 2 . 9–8 . 4×10−4 events per generation among the 10 surveyed PSVs . This equates to a per-PSV rate of 2 . 9–8 . 4×10−5 events per generation , though this represents a minimum estimate , since not all of the PSVs are informative in all studied chromosomes . Based on a measure of inter-arm divergence and an estimate of the base-substitution rate , Rozen et al . [2] estimated a conversion rate per generation per nucleotide in Y palindromes of 2 . 2×10−4 . Although gene conversion tracts several kilobases in length occur frequently in yeast [33] , in mammals tracts are short , typically ranging from a few tens of base pairs [34] to 1 kb [22] . In palindrome P6 , we infer minimum gene conversion tract lengths up to 9023 bp with mean minimum length of 2068 bp . These lengths do not represent direct measurements , and it remains possible that the inferred patterns of long conversion tracts could be created by multiple independent events . However , the longest inferred tract , including 7 PSVs , would require several independent events all in the same direction ( from proximal to distal arm ) , so the most parsimonious explanation is a single event . It is possible that long conversion tracts are a typical characteristic of palindromes , but this remains to be tested by future studies . Recombination is initiated by double-strand breaks ( DSBs ) , and their repair can result in either reciprocal crossover , or non-reciprocal conversion . In considering the effects of these different pathways in P6 , we need to differentiate between inter- and intramolecular events since , while conversion between or within chromatids will have the same molecular outcomes , this is not the case for crossover . Inter-chromatid crossover is expected to lead to an isodicentric chromosome and an acentric fragment , both of which are selected against . For example , 7/8 human palindromes are involved in crossover events between sister chromatids resulting in large-scale rearrangements in patients with disorders ranging from spermatogenic failure to sex reversal and Turner syndrome [35] . By contrast , intra-chromatid crossover will lead to simple inversion of palindrome arms , which seems unlikely to have strong effects on fitness . As an example , crossover between IR3 inverted repeats on Yp , resulting in apparently asymptomatic inversion , has occurred at least twelve times in the history of the Y phylogeny [36] . This different consequences of the two pathways means that while observed conversions reflect both inter- and intramolecular events , observed inversions are the result of intra-chromatid events only , and this complicates the interpretation of conversion: crossover ratios . Phylogenetic detection of intra-chromatid crossovers leading to palindrome inversions is possible if the phase of the PSVs is known . Phasing of seven of the studied PSVs , located within the first ∼19 kb from the outer palindrome boundaries provides evidence of only three independent inversions among the studied chromosomes ( Figure 4 ) . The deduced rate of inversion , 1 . 36–1 . 72×10−5 per generation , compares to a published rate of 2 . 3×10−4 for the IR3 inverted repeats [36] . Notably , we have ascertained only those inversions with breakpoints occurring in the outer ∼16% of the arms of P6 , whereas the published study was able to ascertain all intra-chromatid inversions by determining the orientation of markers between the IR3 repeats . Our finding of 56 conversion events in the same chromosome set indicates that observed recombination events in P6 are strongly biased towards conversions rather than crossovers . Among the studied chromosomes , intra-chromatid inversions are comparatively well ascertained , because a crossover in the interval between any pair of informative PSVs will be detected reliably . Conversion , however , is under-ascertained because it is only observed when it transfers a particular informative PSV . The scarcity of PSVs means that the observed conversion: intra-chromatid crossover bias is actually an underestimate of the true value . Additional uncertainty is introduced by our inability to accurately identify co-conversion . A bias towards non-crossovers is commonly observed in recombination analysis . According to cytological studies the repair of only 10% of DSBs in mammals results in crossovers , while the remainder are assumed to be repaired as non-crossovers [37] . Most mammalian data on conversion: crossover ratios come from studies of meiotic recombination hotspots in humans and mice . The ratio varies significantly between different human hotspots ( from 2 . 7∶1 at hotspot DNA3 to <1∶12 at the β-globin hotspot ) ; there are also considerable differences among individuals , driven in part by variation in trans-acting factors [38]–[41] . In comparing MSY gene conversion with conversion affecting other chromosomes , its singular status as a constitutively haploid chromosome must be remembered . As discussed above , both inter- and intra-chromatid conversion can occur , but neither of these processes is linked with the highly regulated ‘normal’ processes of synapsis and meiotic crossing over . Many questions therefore remain about the timing and mechanism of MSY conversion processes . In a number of organisms recombination has been associated with GC-bias arising from biased repair of mismatches in heteroduplex DNA [42] . Consistent with this , we found evidence of highly statistically significant GC-bias among the P6 gene conversion events within the Y phylogeny . We also asked whether GC-bias in gene conversion had a deeper evolutionary history by comparing the patterns of nucleotide replacements among human , chimpanzee and gorilla P6 sequences . Spacers show a statistically significantly greater proportion of replacements of S nucleotides by W nucleotides than arms do ( Table 2 ) . This is true for both human- and chimpanzee-specific nucleotide replacements . It is possible that these differences could be due to regional variation in GC-content , repeat content , mutation rates or some other factors , but the observed replacement patterns in palindrome arms are nonetheless consistent with the action of GC-biased gene conversion . We might expect the long-term action of such bias to lead to elevated GC-content in arms compared to spacers . For P6 , this is the case ( Table S6 ) : 38 . 8% ( arms ) is significantly greater ( p = 2 . 7×10−11; Chi-square test ) than 37 . 0% ( spacer ) . We can make the same comparisons for other palindromes , setting aside P1 and P2 , which have very large arms and very small spacers . P3 also shows a significant elevation of GC-content in its arms ( p = 1 . 0×10−56 ) , while P4 , P5 , P7 and P8 show no significant differences; the pattern is therefore complex , but notably none of these palindromes shows significantly higher GC-content in spacer compared to arms . The observed differences could in principle reflect the enrichment of protein-coding genes in palindrome arms compared to spacers; however , the observed pattern persists when the genes are removed ( Table S6 ) . Our comparisons of human , chimpanzee and gorilla P6 sequences concur with previous observations [2] in revealing significantly lower inter-specific divergence among arms than among spacers , in all three possible comparisons ( Table 1 ) . This suggests either that the rate of initial mutation in arms is lower than that in spacers , or that gene conversion is acting to preferentially return new mutations arising in one arm to the ancestral state , via conversion from the unmutated arm . Our observation that individual gene conversion events among human Y chromosomes are significantly biased towards retention of the ancestral states of PSVs tends to support the second explanation . Natural selection acting directly on the PSV sites seems an unlikely explanation for the bias: examination of ENCODE [43] data ( as represented in the UCSC Genome Browser; April 2013 ) shows P6 to be devoid of functionally significant elements , apart from a 107-bp snRNA gene in the arms ∼20 kb proximal to the inner arm boundary . There is no evidence for functional elements overlapping the variants tested . An alternative explanation is that the ancestral state bias emerges from the GC-bias . Notably , of the six PSVs that are informative about GC-bias acting at individual sites , five have a G or C nucleotide as their ancestral state . Whether GC-bias provides a more general explanation for the conservation of palindrome sequences will require more data on a larger number of palindrome sequence variants . Y-chromosomal palindromes are not alone in showing apparent ancestral-state bias in conversion: comparison of human and chimpanzee orthologs of an X-chromosomal palindrome [44] also display significantly reduced interspecific divergence in arms compared to spacers . This bias in conversion may therefore be a general property of palindromic repeats . Its consequence is that palindromes are ‘hard wired’ for conservation; although this will be largely beneficial because most mutations are deleterious , it may also ultimately limit adaptability of genes in palindromes by limiting the opportunity for fixing beneficial mutations . Our understanding of the molecular evolution of the Y chromosome would be greatly improved by the availability of additional accurate sequences both from non-human primates and humans . In principle , next-generation sequencing technologies offer the opportunity to generate such sequences , but in practice the complex repetitive structure of the Y chromosome means that sequence assembly is impossible with current methods . Successful generation of useful Y-chromosome sequences from humans and other species [1] , [3] , [15] , [19] has required shot-gun sequencing of assembled tiling arrays of BAC clones , an expensive and laborious process . An additional problem is that genome sequencing projects in non-human primates focus on females , in order to provide good coverage of the X chromosome . The structures of palindromes , the phase of variants within them , and gene conversion tract lengths will be illuminated by the advance of third-generation sequencing methods that have very long read lengths , and also high-throughput haplotyping of single sperm molecules , a method that has already proved successful in identifying the longest known allelic gene conversion tract of 22 kb [45] .
We analysed a total of 378 male samples chosen from the CEPH-HGDP Cell Line Panel ( Table S1 ) [15] . Choice was motivated by existing information on haplogroup , and practicality: we wanted to ensure representation of several members of each known haplogroup in order to detect gene conversion events ( Figure 2 ) , but to avoid analysing all 684 males in the panel due to the laborious nature of PSV typing and phasing . Y-chromosome binary polymorphism data for HGDP samples were compiled as follows: 145 SNPs from CEPH 2011 ( www . cephb . fr/en/hgdp/ - data supplement 10 ) , 37 from Shi et al . 2010 [16] and Peter de Knijff ( unpublished observations ) , 10 from Li et al . 2008 [18] and three from Sengupta et al . 2006 [17] . In addition , 23 SNPs ( M112 , M119 , M150 , M182 , M217 , M223 , M231 , M267 , M285 , M287 , M3 , M32 , M35 , M38 , M6 , M75 , M78 , M8 , P15 , P2 , P37 , P45 and P312 ) were typed as part of a GoldenGate custom genotyping assay ( see section below ) . Eleven samples representing haplogroup K* ( xL , M , N , O , P ) [16] were typed for M254 and P204 using published PCR primers [13] and Sanger sequencing . The whole dataset is described in Figure S1 and Table S1 . For the phylogeny , the total of 200 mutational events gave rise to 122 possible Y-chromosome haplogroups , of which we observed 63 among the 378 samples analysed . Haplogroup nomenclature is as described [13] , with shorthand names for some haplogroups , as described in Table S1 . There were two inconsistencies between data sources: ( i ) The phylogenetic relationships of markers P7 and M169 within hgB2 were consistent with the data of [46] rather than the original description [13]; ( ii ) Four samples ( HGDP numbers 541 , 542 , 553 and 662 ) are listed in the data of [16] as belonging to hgK ( xL , M1 , NO , P ) , with the hg-M1-defining marker M106 ancestral; however , these same samples are listed under CEPH 2011 ( www . cephb . fr/en/hgdp/ - data supplement 10 ) as derived for both M106 and the phylogenetically equivalent marker M189 . Given that two markers are in agreement in this dataset , we regard them here as hgM1 chromosomes . This study uses human DNA samples from the CEPH-HGDP panel , a widely available anonymised set of lymphoblastoid cell-lines ( LCLs ) . The original paper describing this panel [15] states that the blood specimens that served as sources of the LCLs were freely donated under conditions of informed consent and confidentiality by reviewing consent forms , institutional review board approvals , or detailed reports from those who organised collections . The ten typed PSVs were labelled PSV1 to PSV10 based on their proximal-to-distal order on the proximal palindrome arm in the reference sequence ( Figure 1b , Table S4 ) . As a convenient medium-throughput system for typing SN-PSVs , we chose the Illumina GoldenGate Genotyping Assay ( Illumina , San Diego , CA ) . This system does not allow assay design or reliable calling for some variants in particular sequence contexts , and was eventually used for the successful typing of seven analysed PSVs ( PSV2 , 3 , 5 , 7–10 ) . Experiments were carried out at the Genomics Core Facility of the University of Leicester . Genotypes were called with the Illumina GenomeStudio software version 3 . 1 . 0 . 0 ( Illumina ) . Results were validated by Sanger sequencing of 5% of samples ( n = 19 ) for each PSV ( 133 sequencing reads in total ) . PSV1 and PSV4 were typed by PCR-RFLP analysis using the restriction enzyme TstI ( Fermentas ) for the former and Hpy166II ( NEB ) for the latter . PSV6 was typed by allele-specific PCR . In order to phase the palindrome arms an arm-specific long-range PCR approach was developed , using one universal primer binding within the arm and an arm-specific primer binding outside the outer palindrome boundary , generating a distal-arm-specific fragment of 18 , 893 bp incorporating seven of the studied PSVs . This fragment was then used as a template in nested PCR followed by re-typing of the seven PSVs . Five of the PSVs were typed by PCR-RFLP analysis using the following restriction enzymes ( all NEB except PSV1 ) : PSV1 - TstI , PSV2 - AcuI , PSV3 - HinfI , PSV4 - Hpy166II and PSV7 – MnlI . Sanger sequencing and allele-specific PCR were used for PSV5 and PSV6 , respectively . All primer sequences are listed in Table S5 . Arm-specific haplotypes were compared to the known phase of the human Y-chromosome reference sequence . In total 83 samples were examined and all but three found to have identical phase to the reference sequence ( Table S1 ) . In order to ascertain the presence of both palindrome arms in samples with long apparently pseudohomozygous stretches , a paralog ratio test ( PRT ) [47] was developed . PRT primers were designed to amplify fluorescently labelled 390-bp test fragments from both arms of P6 ( Figure 1b ) , plus a single 387-bp reference region from chromosome X ( Table S5 ) . Products were resolved and quantified using an ABI3130xl Genetic Analyzer and GeneMapper software v4 . 0 ( Applied Biosystems , Carlsbad , CA ) . A normal male is expected to have two palindrome arms and one X chromosome , resulting in a test-to-reference ratio of 2∶1 . In total 50 samples were tested , each at least twice , including pseudoheterozygous controls known to contain both palindrome arms ( Table S1 ) . All samples showed the expected ∼2∶1 ratio except one ( HGDP00445 ) , which showed a ratio of ∼1∶1 . Semi-quantitative analysis using the amelogenin sex test [48] , which simultaneously amplifies different-sized X- and Y-specific fragments , showed an X∶Y ratio of 2∶1 , consistent with this cell-line having a 47 , XXY karyotype . The presence of both palindrome arms was also checked by an additional PCR-based approach . Firstly , PCR primers were designed to specifically extend over and amplify both the inner and outer boundaries of the palindrome . Secondly , long-range PCR primers were used to amplify ∼10-kb fragments arm-specifically from the outer boundary of both arms followed by gel electrophoresis to check for changes in product length . The presence of all four palindrome boundaries and expected lengths of arm-specific PCR products was confirmed for all samples tested . Mean gene conversion rate ( assuming each converted SN-PSV represented an independent event ) was estimated by dividing the number of conversion events ( n ) , by the number of generations ( g ) encompassed in the phylogeny relating the 378 tested Y chromosomes . Estimation of g was based on a study [36] in which ∼80 kb of DNA were resequenced in 47 Y chromosomes covering most of the major branches of the Y phylogeny to ascertain unbiased nucleotide divergence , revealing a total of 95 base substitutions . Assuming a TMRCA of 118 , 000 years ( supported by more recent large-scale resequencing [49] ) , a generation time of 25 years , and a human-chimpanzee divergence time of 6 . 5 million years , the 47 chromosomes encompassed 52 , 000 generations [36] . The 378 Y chromosomes we studied also included most haplogroups in the phylogeny , but also multiple examples in individual haplogroups . We estimated the number of additional generations contributed by these: for the lower bound we assumed that all chromosomes sharing major haplogroups contributed no additional base substitutions in excess of the haplogroup-specific branch lengths; for the upper bound we assumed that each additional chromosome in a given haplogroup contributed an additional number of base substitutions equivalent to its descending from the root of the clade independently . This led to a range of total base substitutions of 323–935 , corresponding to ∼175 , 000–505 , 500 generations ( Table S3 ) . A partial consensus sequence of gorilla P6 was constructed from Illumina paired-end sequencing reads from: ( i ) whole genomes of two male gorillas giving an overall ∼6× Y-chromosome coverage [20]; ( ii ) a sequence-capture library ( SureSelect , Agilent ) , using a repeat-masked probe-design based on the human reference sequence , of a male gorilla giving a mean coverage of targetable portions of P6 of 232× ( Protocol S1 ) . Reads from all samples were mapped against the spacer and proximal arm of P6 in the human reference ( GRCh37 ) and a consensus sequence for a given nucleotide called where it was covered by at least 5 concordant reads and minimum base quality score 20 . Chimpanzee MSY sequence used in interspecific comparisons was taken from [19] . Sequence alignments were performed using the web-based ClustalW2 ( http://www . ebi . ac . uk/Tools/msa/clustalw2 ) and Stretcher implemented in the EMBOSS package ( http://emboss . sourceforge . net/ ) . | The sex-determining role of the human Y chromosome makes it male-specific , and always present in only a single copy . This solo lifestyle has endowed it with some bizarre features , among which are eight large DNA units constituting about a quarter of the chromosome's length , and containing many genes important for sperm production . These units are called palindromes , since , taking into account the polarity of the DNA strands , the sequence is the same read from either end of the unit . We investigated the details of a process ( gene conversion ) that transfers sequence variants in one half of a palindrome into the other , thereby maintaining >99 . 9% similarity between the halves . We analysed patterns of sequence variants within one palindrome in a set of Y chromosomes whose evolutionary relationships are known . This allowed us to identify past gene conversion events , and to demonstrate a bias towards events that eliminate new variants , and retain old ones . Gene conversion has therefore acted during human evolution to retard sequence change in these regions . Analysis of the chimpanzee and gorilla versions of the palindrome shows that the dynamic processes we see in human Y chromosomes have a deep evolutionary history . | [
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] | 2013 | Recombination Dynamics of a Human Y-Chromosomal Palindrome: Rapid GC-Biased Gene Conversion, Multi-kilobase Conversion Tracts, and Rare Inversions |
A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine . We previously identified a participant in the CAPRISA cohort , CAP248 , who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection . Here , we report the isolation by B cell culture of monoclonal antibody CAP248-2B , which targets a novel membrane proximal epitope including elements of gp120 and gp41 . Despite low maximum inhibition plateaus , often below 50% inhibitory concentrations , the breadth of CAP248-2B significantly correlated with donor plasma . Site-directed mutagenesis , X-ray crystallography , and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus . While this epitope is distinct , it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151 , VRC34 , 35O22 , 3BC315 , and 10E8 . CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region . Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane . Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope , and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors , furin co-transfection , or pre-incubation with soluble CD4 . Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites . Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B , but enhanced neutralization sensitivity to 35O22 , 4E10 , and 10E8 by 10-100-fold . Altogether , this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41 , with potential utility in HIV vaccine design .
The HIV-1 envelope glycoprotein trimer ( Env ) is the only known target for neutralizing antibodies and is thus a focus for vaccine design efforts . However , the development of an effective HIV-1 vaccine has been thwarted by the complex nature of Env , and the inability to produce soluble Env immunogens able to elicit broadly neutralizing antibodies ( bNAbs ) [1] . Env is expressed as a single gp160 protomer that is extensively glycosylated and trimerized in the endoplasmic reticulum [2 , 3] . These gp160 oligomers are cleaved into gp120 ( receptor binding subunit ) and gp41 ( transmembrane subunit ) , resulting in a trimer of heterodimers that is subjected to extensive glycan processing in the Golgi apparatus [3 , 4] . The cleaving of gp160 occurs primarily through furin activity at position R511 , but a fraction of Env is also cleaved at position R504 [4–6] . During this process gp120 is often shed from non-covalently associated gp120-gp41 trimers , and the entry-competent form of Env may comprise only a small portion of the total Env content on the viral membrane [3 , 4 , 7] . The remainder often exists as gp41 “stumps” , and incorrectly processed or prematurely triggered monomers and oligomers . The abundance of these aberrant forms of Env on the viral surface , and the consequent exposure of immunodominant regions not normally present on entry competent trimers , misdirects the humoral immune response toward non-protective epitopes [8] . In addition Env is highly sequence variable , particularly within the V1-V5 variable loop regions , and heterogeneously glycosylated ( even at conserved NxS/T sequons ) because the densely packed glycans afford each other varied protection from glycan processing enzymes . This combination of factors favours the formation of strain-specific neutralizing antibodies [9–13] . Nevertheless most people develop some level of cross-neutralizing antibodies [14] , and after several years of infection these can mature into HIV-1 bNAbs that are able to target more conserved regions of Env , and thus neutralize diverse viral strains [15–17] . In some instances these bNAbs can neutralize 50%—99% of globally circulating strains , and have been shown to prevent infection in animal models [18–24] . HIV-1 bNAbs often display unusual characteristics such as high levels of somatic hypermutation , unusually long complementarity determining regions ( CDRs ) , and insertions/deletions in both the CDRs and antibody framework regions [25–27] . These features enable HIV-1 bNAbs to access epitopes that are often recessed or contain conserved glycan or membrane components . Interestingly , bNAbs that include glycan ( s ) in their epitope frequently display incomplete neutralization , usually because they have evolved to recognise a specific glycoform not present on every trimer [28–32] . More recently , incomplete neutralization has also been described for HIV-1 bNAbs with epitopes that do not appear to depend on glycan , suggesting that additional factors may contribute to neutralization potency [33 , 34] . To understand how these unusual bNAb specificities might be elicited by an HIV-1 vaccine , there has been a concerted effort to isolate bNAbs from HIV-1 infected individuals and to define their targets and developmental pathways [29 , 35–48] . The identification of bNAb-mediated immune selection pressures on Env , coupled with mutagenesis and structural biology techniques have been instrumental in our understanding of the epitopes susceptible to broad neutralization [49] . These include the CD4 binding site ( CD4bs ) , a cluster of epitopes surrounding the N332 glycan , the membrane proximal external region of gp41 ( MPER ) , and a number of quaternary structure specific epitopes in either the V2 trimer apex or the gp120-gp41 interface . The more recently discovered gp120-gp41 interface bNAbs 8ANC195 , PGT151 , VRC34 , 35O22 , and 3BC315 target distinct , usually glycan dependent epitopes , that overlap in gp41 [29 , 44 , 47 , 48 , 50] . For example , 8ANC195 requires glycans at positions N234 and N276 , PGT151 at positions N611 or N637 , and VRC34 and 35O22 at position N88 . The identification of additional bNAbs that have epitopes within the gp120-gp41 interface could further enhance our understanding of this new site of vulnerability . In a previous study we described CAP248 , an HIV-1 subtype C infected participant in the CAPRISA 002 cohort who developed bNAbs to a trimer-specific epitope that could not be defined at the time [15] . Here , we have isolated a monoclonal antibody ( mAb ) that was representative of the broadly neutralizing plasma response , but lacked potency due to low neutralization plateaus that could not be accounted for by glycan heterogeneity . Through the interrogation of autologous selection pressure in Env , together with X-ray crystallography and electron microscopy , we mapped the CAP248-2B target to a glycan independent epitope in gp120 and gp41 that overlaps with previously identified gp120-gp41 interface bNAbs , but is distinct in its recognition of the gp160 cleavage motifs in the gp120 C terminus . Escape from this antibody conferred a viral phenotype that was exceptionally sensitive to neutralization by MPER directed antibodies , suggesting a role for the C terminus of gp120 in the exposure of important bNAb epitopes in gp41 .
CAP248 first developed cross-neutralizing antibodies after one year of infection . By three years , the neutralization breadth of CAP248 plasma increased incrementally , from 7% to 60% ( Fig 1A ) when tested against a 45 pseudovirus panel [15] . CAP248 plasma neutralized 78% of 27 subtype C , and 50% of 6 subtype A pseudoviruses , but only 25% of 12 subtype B pseudoviruses at titres >1:100 ( Fig 1B ) . As mapping of polyclonal plasma may be complicated by the presence of multiple overlapping specificities [51 , 52] , we used a stored peripheral blood mononuclear cell ( PBMC ) sample from 3 . 5 years post-infection to isolate a monoclonal antibody , called CAP248-2B . B cells were enriched by negative selection with immunomagnetic beads , and B cell culture supernatants were screened after 14 days for neutralization of CAP45 , a tier-2 pseudovirus sensitive to CAP248 plasma at ID50 titres of ~1:4 , 000 . CAP248-2B was predicted to be derived from the IGHV4-31 and IGHJ3 heavy and IGLV2-14 and IGLJ1 lambda chain germline genes , and displayed modest levels of affinity maturation , with 13 . 5% and 9 . 7% nucleotide mutation in the heavy and light chains respectively ( S1 Fig ) . CAP248-2B potently neutralized CAP45 with an IC50 of 0 . 04 μg/mL ( IC80 of 0 . 19 μg/mL ) , but against the same panel on which CAP248 plasma displayed 60% neutralization breadth at three years post-infection , CAP248-2B neutralized only 22% of viruses with IC50 titres ( Fig 1C ) . Examination of the neutralization curves indicated that the poor breadth of CAP248-2B was due to incomplete neutralization , e . g . the maximum inhibition of CAP45 , CNE52 , CAP228 , and ZM249 plateaued at 95% , 69% , 54% , and 32% respectively ( Fig 1B and 1D ) . When IC20 values were examined , CAP248-2B neutralized a much larger fraction of the panel , showing 58% breadth , equivalent to the plasma breadth ( Fig 1B and 1C ) . In contrast , weakly neutralizing antibodies 447-52D ( that targets V3 ) , 17b ( co-receptor binding site ) , and HK20 ( gp41 ) , had sporadic , equivalent neutralization at both IC20 and IC50 ( S2 Fig ) . No neutralization at IC20 was observed using Palivizumab ( a negative control antibody ) indicating that CAP248-2B IC20 titres were not the result of background activity in the assay ( Fig 1B—right column ) . This phenomenon of neutralization at IC20 due to low neutralization plateaus has been observed for other HIV-1 antibodies , such as the PGT151-158 bNAb lineage [29] . For CAP248 , there was significant concordance ( p<0 . 00001 ) between pseudoviruses neutralized by the plasma at 3 years ( measured as ID50 ) and those neutralized by the monoclonal antibody at IC20 . This suggested that CAP248-2B was representative of the dominant bNAb specificity in CAP248 plasma , though more potent variants of this mAb lineage likely exist and remain to be isolated . Previously , we have shown that CAP248 plasma bNAbs could not be adsorbed using recombinant monomeric gp140 or MPER peptides , and were not sensitive to V2 mutations at positions N160 and L165 [15] . This suggested that CAP248 bNAbs targeted a quaternary epitope distinct from the V1V2 epitope . To identify potential escape mutations in response to CAP248 bNAbs , autologous gp160 sequences from nine weeks ( study enrolment ) , as well as 1 , 2 , 3 , and 3 . 5 years post-infection were examined for accumulating mutations ( indicative of selection pressure ) in normally conserved regions of the envelope . Several autologous mutations were identified in , or proximal to , known bNAb epitopes within V2 ( E164I/V , L165I/F ) , C1 ( G87R/E ) / C2 ( D230N , N234T/S , T236K ) , V3 ( D321E/G , N325D/K , E328K/D ) , and the MPER ( N674G , K677N/Q , K683R ) ( S3A Fig ) . However when these changes were made in the heterologous virus CAP45 , none affected CAP248-2B neutralization ( S3B Fig ) . We also assessed the effect of mutations known to confer resistance to bNAbs targeting these four regions , but these mutations also failed to abrogate CAP248-2B neutralization ( S3C Fig ) . There were slight effects following the T303A and F672L/W673L mutations , but these are also known to affect overall Env conformation [30 , 53] . A cluster of unusual mutations were identified in the C terminus of gp120 at positions 500 , 502 , 505 , 507 , 508 , and 509 within the gp160 furin cleavage motifs that separate gp120 from gp41 ( Fig 2A ) . This region has not previously been implicated in viral escape from neutralizing antibodies , but analysis of 2 , 558 sequences from the Los Alamos National Laboratory HIV-1 database showed that while sequence variation at position 500 is common , mutations at the other five sites are rare , particularly amongst clade C viruses ( Fig 2B ) . The simultaneous presence of these mutations in CAP248 viral sequences therefore suggested strong immune pressure on this region . When the six most common mutations at 3 . 5 years post-infection ( E500K , R502Q , V505M , E507G , R508K , and E509G ) were introduced individually into the heterologous virus CAP45 , only the V505M mutation substantially reduced CAP248-2B neutralization ( Fig 2C—blue line ) . However , the simultaneous introduction of all six mutations into CAP45 , hereafter referred to as CAP45 ( CS-Mut ) , conferred complete resistance to both CAP248-2B neutralization ( Fig 2C—red line ) , as well as CAP248 broadly neutralizing plasma ( S3D Fig ) , confirming their collective role in escape from CAP248 bNAbs . The gp120 C terminus is proximal to gp41 , suggesting that the CAP248-2B epitope might be in the gp120-gp41 interface . To determine whether autologous gp41 mutations might contribute towards escape from CAP248-2B , we examined CAP248 gp41 ectodomain sequences over time . Of the thirteen changes identified ( I515M , L519F , K588Q , N607T , Q619L , D621E , D624G , D632E , S636H , G640D , K644Q , N651I , and D659E ) , none affected CAP248-2B neutralization appreciably when introduced into CAP45 , however these mutations may play a role in resistance to other members of the CAP248-2B lineage present in CAP248 plasma ( Fig 2D ) . Altogether , these data suggest that major escape mutations from the CAP248-2B lineage accumulated in the gp160 cleavage motifs , with the role of additional mutations accumulating in proximal regions of gp41 still to be defined . Antibodies such as PGT151 and VRC34 that recognize the gp41 N terminus bind only to fully cleaved Env [29 , 50] , while other gp120-gp41 interface bNAbs that do not recognize the peptide termini ( 35O22 and 3BC315 ) bind equally well to both cleaved and uncleaved Env [44 , 48] . To assess the cleavage dependence of CAP248-2B , we compared the binding of CAP248-2B to cell surface expressed CAP45 envelope trimers , and an R508S/R511S mutant ( SEKS mutant ) [54] that is incompletely cleaved ( Fig 2E ) . Both CAP248-2B and PGT151 bound less efficiently to the SEKS mutant Env , while 3BC315 and CAP256-VRC26 . 09 ( a V2-directed bNAb ) bound to both . CAP248-2B is escaped by mutations in the gp120 C terminus , and the R508S/R511S changes could directly cause resistance to CAP248-2B by altering contact residues in the gp120 C terminus . Therefore , to further confirm the role of cleavage in forming the CAP248-2B epitope , protein A coupled antibodies were used to capture soluble SOSIP trimers ( described below ) that were not co-transfected with furin ( which is usually used to enhance cleavage efficacy ) . A single preparation of SOSIP trimer ( containing both cleaved and uncleaved trimer ) was divided into two , and passed over either CAP248-2B or CAP256-VRC29 . 09 antibody columns . SDS-PAGE showed that CAP248-2B was only able to capture completely cleaved SOSIP trimers , while CAP256-VRC26 . 09 could capture both cleaved and uncleaved Env from the same preparation ( Fig 2F ) . These data confirm proper cleavage of Env is required for the formation of the CAP248-2B epitope . CAP248-2B possessed an average length CDR-H3 of 15 amino acids , but had an unusually long CDR-L3 of 19 amino acids ( Fig 3A ) . The typical length of a CDR-L3 is 8–12 amino acids , and there are no antibodies with CDR-L3s of greater than 15 amino acids in the Abysis database ( http://www . bioinf . org . uk/abysis/index . html ) . In addition to the CDRs , there was also substantial maturation away from germline in the framework region three ( FR3 ) of the heavy chain ( Fig 3A—blue ) . We determined two crystal structures of the unliganded CAP248-2B antigen binding fragment ( Fab ) to resolutions of 2 . 0 Å and 3 . 1 Å respectively ( S1 Table and Fig 3B ) . The CDR-H3 and CDR-L3 loops of the 2 . 0 Å resolution structure were influenced slightly by crystal packing ( S4A Fig ) , and a comparison of the CDR-H3 between the two structures revealed a potentially dynamic loop that flipped between two distinct conformations bent at residues Gly100D and Gly100E to flip the Asp100B and Asp100C anionic pair ~180° between CDR-L3 proximal and distal orientations ( Fig 3B—top inset ) . In the 3 . 1 Å resolution structure ( not influenced by crystal packing ) , the 19 amino acid CDR-L3 formed a β-hairpin which was stabilized along its length by seven hydrogen bonds , and protruded ~15 Å at a right angle relative to the other CDR’s . The tip of the CDR-L3 ended in hydrophobic residues Phe95C and Phe95D that were angled by Pro95F and immediately flanked by residues Ser95B and Gly95E which may confer a degree of plasticity to this region ( Fig 3B—bottom inset ) . In both structures , the angle at which the CDR-L3 extended from the Fab was stabilized at its base by germline conserved hydrogen bonding interactions with CDR-L1 , as well as a salt bridge formed between CDR-L3 residue Arg95I , and Asp50 in the CDR-H2 . Overall , the CAP248-2B antigen binding site displayed substantial structural plasticity , an attribute that likely contributes to its mode of neutralization . To identify the binding site of CAP248-2B , we obtained structural information about the Fab bound to a soluble pre-fusion Env trimer by electron microscopy ( EM ) ( Fig 4 and S4 Fig ) . CAP248-2B was unable to bind monomeric gp120 , or gp145 proteins which contain the entire Env ectodomain ( S4B Fig ) . Furthermore CAP248 plasma was unable to neutralize BG505 , and consistent with these data CAP248-2B failed to bind recombinant BG505 SOSIP trimer in ELISA ( Fig 4A—top graph ) . In the pre-fusion SOSIP structure , the gp120 C terminus is immediately proximal to gp41 , and accordingly the CAP248-2B epitope could be engineered into the BG505 SOSIP trimer by designing a BG505 ( gp120 ) -CAP45 ( gp41 ) chimera . BG505 and CAP45 differed in gp41 by only 6 . 7% ( 23 amino acids ) , with most of these mutations not predicted to interact with gp120 in the pre-fusion structure . This chimeric SOSIP trimer was efficiently cleaved , and bound well to trimer-specific bNAbs CAP256-VRC26 . 09 and CAP248-2B , but not to the non-neutralizing antibody F105 , suggesting that it retained a native-like pre-fusion conformation ( Fig 4A—bottom graph ) . Single particle negative stain EM reconstructions at ~20 Å showed a maximum stoichiometry of three CAP248-2B Fabs bound to the BG505 ( gp120 ) -CAP45 ( gp41 ) SOSIP trimer ( Fig 4B and S4C–S4F Fig ) . Docking of a SOSIP trimer crystal structure into the EM 3D reconstruction revealed a binding site for the CAP248-2B Fab that was extremely close to the viral membrane , similar to 35O22 and 3BC315 [44 , 48] , but approaching from an angle that was proximal to the gp120 C terminus ( Fig 4B and 4C ) . The approximate Fab footprint bridged the gp41-gp41 interface , but did not encompass the gp120-gp41 interface bound by previously described bNAbs . Rather CAP248-2B binds to a second more membrane proximal interface between gp41 and the gp120 N- and C- termini ( Fig 4C ) . Since this is also the site of viral escape mutations , these data support the hypothesis that CAP248-2B binds to the C terminus of gp120 , as well as to parts of gp41 . The CAP248-2B Fab structure could be docked into the EM reconstructions in two possible orientations . These placed the hydrophobic CDR-L3 tip in close proximity to either the viral membrane ( Fig 5A—right inset ) , or the fusion peptide ( FP ) of gp41 ( Fig 5B—left inset ) , with approximately 1 , 100 Å2–900 Å2 of surface area buried by the paratope in the Env SOSIP trimer respectively . The viral membrane bound model suggested a role for the heavy chain CDR-H1 in specific interactions with gp41 , where two residues selected through somatic hypermutation , Glu32 and Asp33 , are situated in close proximity to position N656 ( Fig 5A—left inset ) . In the FP bound model , these residues do not interact with the SOSIP trimer which is truncated at Asp664 of gp41 ( Fig 5B—right inset ) . When the CDR-H1 Glu32/Asp33 residue pair were reverted to germline ( Gly32/Gly33 ) , the mutant antibody failed to bind to SOSIP trimers or neutralize the CAP45 virus ( Fig 5C and 5D –pink curves ) , providing evidence for the model where the CDR-L3 interacts with the viral membrane . In this lipid binding model , Phe95C and Phe95D would insert into the core of the viral membrane , while Lys94 and Lys95 would be positioned to interact with the polar membrane lipid heads ( Fig 5A ) . To provide further evidence for this binding orientation , we replaced the phenylalanine residues at the CAP248-2B CDR-L3 tip with either tryptophan or alanine ( Fig 5C and 5D ) . Bulky , hydrophobic tryptophan side chains could interfere with specific protein-protein FP interactions , potentially impacting negatively on binding and neutralization . These same mutations would be expected to enhance interactions with the viral lipids by increasing the hydrophobicity of the CDR-L3 , improving neutralization but not binding to soluble SOSIP trimers where the viral membrane is absent . The Trp95C/Trp95D CDR-L3 mutant was substantially more potent against viruses CAP45 , CNE52 , and CAP228 in neutralization assays ( Fig 5C ) . Similarly , mutating only one of the CDR-L3 Phe residues to generate Trp95C/Phe95D , or Phe95C/Trp95D mutants , also resulted in enhanced neutralization , with Trp95C showing the greater effect . In contrast the CDR-L3 Ala95C/Phe95D , Phe95C/Ala95D , and Ala95C/Ala95D mutants all showed decreased neutralization potency against the same three viruses , confirming the importance of a hydrophobic CDR-L3 tip in effective neutralization of HIV-1 . Despite these effects on neutralization , binding of CAP248-2B to soluble SOSIP trimers ( in the absence of viral membrane ) was not affected by CDR-L3 hydrophobicity ( Fig 5D ) , supporting the docking model where the CDR-L3 enhances CAP248-2B neutralization by burying in the viral membrane . Other neutralizing antibodies that interact with the viral membrane ( such as MPER antibodies 4E10 and 2F5 ) are often autoreactive [55] , however CAP248-2B bound only very weakly to cardiolipin ( Fig 5E ) and HEp-2 epithelial cells ( Fig 5F ) compared to the 4E10 positive control , indicating no significant autoreactivity . Altogether , these data support a docking orientation where the CAP248-2B heavy chain makes contact with gp41 , while the CDR-L3 makes productive hydrophobic interactions primarily with the viral membrane . A number of bNAbs target distinct epitopes in gp41 , including the gp120-gp41 or gp41-gp41 interfaces , and the MPER . When the CAP248-2B EM reconstructions were superimposed with similar EM reconstructions of these antibodies , CAP248-2B bound in close proximity to the epitopes of almost all gp41 directed bNAbs ( Fig 6A ) . These data suggested overlap with 35O22 and 3BC315 near the gp41-gp41 interface , with PGT151 and VRC34 near the fusion peptide , and with 10E8 near the MPER . We therefore assessed the ability of gp41 targeted antibodies to compete with CAP248-2B binding to cell surface expressed CAP45 envelope trimers by flow cytometry ( Fig 6B ) . The gp120-gp41 interface antibodies VRC34 , PGT151 , and 35O22 ( but not 8ANC195 ) competed for CAP248-2B binding , but 3BC315 only showed slight competition at the highest concentrations tested . In the reverse competition assay , CAP248-2B competed more substantially with 3BC315 , consistent with the overlap observed by EM ( S5 Fig ) . MPER bNAbs 4E10 and 10E8 also competed for CAP248-2B binding to Env , while CAP248-2B binding did not affect 4E10 in the reverse assay ( Fig 6B and S5 Fig ) . Plotting the epitopes for these gp41 targeted antibodies , together with the CAP248-2B footprint , onto a model of the HIV-1 Env trimer with MPER ( shown in the 10E8 bound form ) shows how the distinct CAP248-2B epitope extends the membrane proximal target defined by previously identified bNAbs . Altogether , these data highlight the importance of the pre-fusion solvent exposed region of gp41 as a target for overlapping bNAb epitopes ( Fig 6C ) . With the exception of 3BC315 , bNAbs identified to date targeting the gp120-gp41-gp41 interfaces depend on various highly conserved glycosylation sites for neutralization . Docking of the CAP248-2B Fab into the EM 3D reconstruction showed that CAP248-2B does not use long CDRs to penetrate the glycan shield , but instead glycans proximal to the epitope ( particularly N88 and N611 ) need to shift to facilitate CAP248-2B binding ( Fig 7A ) . In this model , the 35O22 bound orientation of the N88 glycan was incompatible with CAP248-2B binding , suggesting that similarly to 3BC315 [48] , the N88 glycan must first be relocated ( Fig 7B ) . Given the close proximity of the CAP248-2B epitope to several N-linked glycans in Env , particularly N88 , N611 , N625 , and N637 , we assessed whether CAP248-2B binds one/more glycans as part of its epitope . Glycan binding arrays showed that CAP248-2B only bound significantly to one of 230 glycans tested ( with fluorescence intensity of greater than 500 a . u . ) , a biantennary mono-sialylated complex N-glycan ( Fig 7C ) . This differs from the tri- and tetra- antennary complex glycans required by the PGT151 lineage [29] . No binding was detected to high mannose glycan . Both N88 and N611 exist predominantly as biantennary complex type glycans on BG505 SOSIP trimers [56] , making these glycans candidates for CAP248-2B binding . To assess whether glycans in gp120 or gp41 that were proximal to the CAP248 epitope were required for CAP248-2B neutralization , knock-out mutants were generated in CAP45 and tested for altered sensitivity to CAP248-2B , 8ANC195 , 35O22 , PGT151 , and 3BC315 ( Fig 7D and S6A Fig ) . As expected , 8ANC195 neutralization was abrogated by T236K and N276A mutations but enhanced by an N230A mutation , 35O22 neutralization was reduced by N88A , N230A , T236K , and N625A mutations , and PGT151 was negatively affected by N611D and N637A mutations ( S6A Fig ) [29 , 44 , 57] . However unlike these three bNAbs , but similar to 3BC315 , CAP248-2B neutralization titres were not negatively affected by any of these glycan deleting mutations ( Fig 7D ) . The N276D and N611D changes resulted in slightly lower CAP248-2B neutralization plateaus , but did not affect CAP248-2B IC50 . To test whether CAP248-2B neutralization was dependent on the N611 glycan , an S613A mutation ( that also removes the N611 glycan ) was introduced into CAP45 . While similarly resistant to PGT151 ( when compared to N611D ) , the S613A mutation had no effect on CAP248-2B neutralization , suggesting that unlike PGT151 , CAP248-2B does not interact with the N611 glycan but rather with the amino acid side chain at position 611 ( Fig 7D and S6A Fig—dashed red line ) . In contrast to N611 , the N88A , N230A , and N625A glycan mutants did not affect neutralization plateaus , but were more potently neutralized by CAP248-2B at IC50 than the wild-type CAP45 , supporting the observation that these glycans obscure the CAP248-2B epitope . The N88A glycan mutant was also more sensitive to 3BC315 neutralization , consistent with published data showing that this glycan partially occludes the 3BC315 epitope [48] . These data suggest that CAP248-2B was not critically dependent on any single glycan in the gp120-gp41 interface for neutralization , despite the ability to bind a complex glycan . PGT151 is only partially affected by the individual N611D/S613A or N637A mutations , and simultaneous mutation at both N611 and N637 glycan sites is required to completely abrogate neutralization [29] . To test whether the removal of an additional glycan , in conjunction with the N611D mutation , was similarly required to knock out CAP248-2B activity , each of the glycan mutants was also made in the N611D mutant backbone ( Fig 7E and S6B Fig ) . None of these double glycan mutant pseudoviruses showed increased resistance to CAP248-2B , though all showed partially elevated plateaus relative to the N611D mutant for CAP248-2B , with a T90A mutation ( that removes the N88 glycan ) having the greatest effect . Altogether these data suggest that CAP248-2B can bind to a complex glycan , perhaps at position N88 or N611 , but this binding is not required for neutralization . While CAP248-2B did not appear to be critically dependent on glycans surrounding the gp120-gp41 interface , incomplete inhibition by HIV-1 bNAbs is often attributed to heterogeneous glycosylation within or proximal to bNAb epitopes [33] . To test whether CAP248-2B had a preference for particular N-linked glycoforms of Env , we evaluated CAP248-2B neutralization of pseudoviruses grown in the presence of N-glycosylation pathway inhibitors kifunensine or swainsonine , or grown in a GnTI deficient cell line ( Fig 7F ) . Four sensitive strains representing different neutralization maxima ( CAP45 , CNE52 , CAP228 , and ZM249 from Fig 1D ) were used . Except for the slightly enhanced sensitivity of swainsonine-grown CNE52 to CAP248-2B , there was no substantial change in the CAP248-2B neutralization plateaus when enriching for high mannose ( kifunensine ) , medium-high mannose ( GnTI ( -/- ) ) or pre-complex only ( swainsonine ) glycans ( Fig 7F ) . Therefore , the unusually low neutralization plateaus of CAP248-2B could not be completely explained by overall envelope N-linked glycan heterogeneity . Other factors influencing intra-strain heterogeneity could potentially include O-linked glycosylation of Env , inefficient gp160 cleavage , or an exclusive preference for a CD4 activated form of envelope . While the potential role of O-linked glycosylation in envelope heterogeneity has not been extensively characterized , recombinant gp120 expressed as a monomer can be O-glycosylated in the C terminus at position T499 , and at least some forms of gp160 might be O-glycosylated at position T606 in gp41 [58–62] . Both of these sites were proximal to the CAP248-2B epitope , but manipulating Env O-linked glycosylation pathways by growing pseudoviruses in the presence of benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside ( a modulator of mucin-like O-linked glycosylation pathways , preventing N-acetyl glucosamine addition ) did not affect CAP248-2B maximum inhibition plateaus ( Fig 7F—orange dashed curves ) . Similarly , increasing gp160 cleavage efficacy by co-transfecting pEnv and pFurin during pseudovirus production ( Fig 7F—pink dashed curves ) , increasing the sampling of a CD4-bound conformation during the neutralization assays by pre-incubating pseudovirions with soluble CD4 at a previously determined IC40 value for 30 minutes ( Fig 7F—green dashed curves ) , or increasing the incubation time between virus and antibody from 1 hour to 24 hours to increase the sampling of less frequent Env conformations ( Fig 7F—red curves ) , did not substantially affect CAP248-2B neutralization plateaus . These data suggest that gp160 cleavage , sampling of non-native pre-fusion conformations , or O-linked glycan processing also do not contribute to the low neutralization plateaus of CAP248-2B . Most of the CAP248-2B affinity maturation occurred in the heavy chain CDRs and FR3 , with very limited maturation occurring in the CDR-L1 ( Figs 3A and 8A ) . In accordance with these data , docking CAP248-2B onto an Env model that includes the MPER suggests that most of the protein-protein interactions are made by the heavy chain CDRs as well as the heavy chain FR3 ( Fig 8B ) . There were some predicted peptide interactions for the CDR-L1 and L2 near the gp41-gp41 interface , and the CAP248-2B CDR-L3 was of the correct length to traverse the gap between the α9 helix of gp41 and the viral membrane ( Fig 8B and 8C ) . To characterize the CAP248 epitope in finer detail , we made mutants in the gp120 C terminus ( the location of escape mutations ) and proximal regions of gp41 ( the fusion peptide , C-C loop region , HR-2 / α9 helix , and MPER ) and compared neutralization of CAP248-2B to gp41-directed bNAbs ( Fig 8C and 8D ) . None of the mutations universally abrogated neutralization of all the bNAbs tested , suggesting that trimer conformation was not compromised , however the A501C mutation negatively affected the neutralization of PGT151 , CAP248-2B , and 35O22 . Mutations in the fusion peptide at positions 513 and 514 negatively affected CAP248-2B neutralization , similar to bNAbs PGT151 and VRC34 . However unlike these latter antibodies , mutations in the membrane proximal face of the α9 helix of gp41 at position Q652 substantially affected CAP248-2B neutralization , while N656D completely abrogated its activity . These sites are close enough to interact with CDR-H1 residues Glu32/Asp33 that were shown above to be critical to the CAP248-2B interaction with SOSIP trimer ( Figs 5C , 5D and 8D ) . Mutations between positions 640–660 in the membrane distal face of the α9-helix of gp41 affected PGT151 neutralization , consistent with its epitope . Lastly , mutations in the MPER at position 666 abrogated CAP248-2B neutralization , similar to MPER antibody 2F5 , and mutations at 672/673 also substantially affected neutralization , similar to 10E8/4E10 . Several of the mutations in the fusion peptide , MPER , and gp120 C terminus appeared to affect CAP248-2B maximum inhibition percentages , but did not affect CAP248-2B IC50 values more than three-fold . Mutations in the gp120 C terminus affected both CAP248-2B and PGT151 neutralization , and despite observations that PGT151 does not bind the gp120 C terminus directly ( its access is occluded by gp41 ) the CAP45 ( CS-Mut ) virus , that is completely resistant to CAP248-2B , was also resistant at IC50 to PGT151 ( neutralization plateaus just under 50% ) . Conversely , 35O22 and 10E8 neutralization of CAP45 ( CS-Mut ) was considerably more potent than the wild-type CAP45 virus . All of the mutations affecting CAP248-2B overlapped with the predicted antibody binding footprint , confirming their importance in the CAP248-2B epitope ( Fig 8C—black spheres ) . Altogether , these data confirm the overlap between the CAP248-2B epitope and the epitopes for PGT151 , VRC34 , and 10E8 , but also highlight the distinct nature of the CAP248 plasma bNAb epitope which includes the membrane proximal half of the α9 helix in gp41 , and the gp120 C terminus . The enhancement of 35O22 neutralization after the introduction of cleavage site mutations into CAP45 suggested a role for the gp120 C terminus in Env conformation . To investigate this , the neutralization of CAP45 ( CS-Mut ) was compared to wildtype CAP45 for bNAbs with diverse epitopes on HIV-1 Env ( Fig 9A ) . CAP45 ( CS-Mut ) neutralization by V2 and N332 antibodies remained unchanged relative to wildtype . The neutralization of CD4bs antibodies VRC01 and b12 was marginally enhanced , but neutralization of MPER bNAbs 4E10 and 10E8 was enhanced by 26 and 38 fold respectively . CAP45 is resistant to 2F5 and Z13e1 , so these bNAbs were not tested . To determine whether this effect was specific to CAP45 , the six CAP248 autologous escape mutations ( CS-Mut ) were simultaneously introduced into four additional heterologous pseudoviruses . Similar to CAP45 , all four of these mutant pseudoviruses were between 10 and 100 fold more sensitive to 35O22 , 4E10 , and 10E8 neutralization ( Fig 9B ) . In some instances introduction of the CS-mutations conferred sensitivity to 35O22 or 4E10 , where the wild-type virus was completely resistant at the concentrations tested . These data show that mutations in the C terminus of gp120 induce localised effects in trimer conformation that specifically enhance sensitivity to MPER bNAbs .
The identification of HIV-1 bNAbs with overlapping epitopes in the gp120-gp41 interface has greatly expanded our knowledge of the regions of the envelope trimer susceptible to neutralization [29 , 44 , 47 , 48 , 63–65] . Here , we isolated a monoclonal antibody called CAP248-2B that targeted a membrane proximal epitope in the gp120 C terminus-gp41 and gp41-gp41 interfaces . This epitope overlapped with , but was distinct from the epitopes for bNAbs PGT151 , VRC34 , 35O22 , 3BC315 , and 10E8 . Together with 8ANC195 , these epitopes surround the base of the HIV-1 envelope trimer , forming a continuum of neutralization vulnerability ( Fig 6C ) [49] . Unlike many other bNAbs , CAP248-2B neutralization plateaus could not be completely explained by glycan heterogeneity . Furthermore , rare mutations in the gp120 C terminus that mediated escape from CAP248-2B increased the sensitivity of HIV-1 viruses to MPER antibodies . Thus the identification of new antibodies targeting this supersite of vulnerability may provide important insights for vaccine design . CAP248-2B was isolated from a CAPRISA donor ( CAP248 ) who showed plasma neutralization breadth of nearly 60% against a multi-subtype panel and 80% against subtype C viruses . While CAP248-2B did not recapitulate the donor’s plasma breadth at IC50 , the neutralization profile at IC20 strongly suggested that this lineage was responsible for CAP248 broad neutralization . The CAP248-2B epitope was structurally proximal to glycans at N88 , N230 , N611 , and N625 , and was able to bind to a biantennary complex glycan , but CAP248-2B neutralization was not dependent on any single glycan , or any double glycan knock-outs that included N611D . It is possible that other members of this antibody lineage in CAP248 plasma have matured to become glycan dependent , however the ability to bind glycans that are not critical for neutralization has also been described for other bNAbs [66 , 67] . These data would support recent evidence for the common occurrence of neutralization plateaus across all bNAb classes , including those that do not require glycan for effective neutralization [33 , 34] . Potential mechanisms for incomplete neutralization were explored , such as the rate of furin cleavage or random sampling of CD4-induced transition intermediates , but did not affect the low plateaus for CAP248-2B . It is possible that CAP248-2B neutralization plateaus were the result of varied accessibility of the gp120 C terminus , unfavourable binding kinetics ( eg: varied off-rates between various strains ) , or a preference for different glycoforms at two or more N-linked glycan sites . This could explain why shifting the overall glycosylation profile of a viral strain in one direction ( e . g . by growing a pseudovirus in the presence of a glycosylation pathway inhibitor ) had no substantial effect on the neutralization plateau . Alternatively , our data suggest an additional contributor to envelope heterogeneity , which could be an important confounder for vaccine immunogen design . Structural analysis indicated that the CAP248-2B paratope was conformationally variable . Divergent CDR-H3 conformations that were only seen because of crystal packing may represent a level of plasticity that could have evolved to better accommodate Env sequence variation . Many bNAb epitopes are composed of structurally dynamic components , such as lipid membranes or large glycan moieties . For instance , the N88 glycan is oriented towards the viral membrane when bound by 35O22 , but shifts into an orientation that would clash with 35O22 binding to allow for 3BC315 to access its epitope [48 , 65] . As a result , bNAb affinity maturation often rigidifies the paratope by hydrogen bonding and/or disulphide bonds within the CDRs . This optimization of the “lock-and-key” fit between antigen and antibody can result in increased potency by reductions in binding entropy . Conversely the additional flexibility observed for CAP248-2B may contribute to its low potency . The unusually long CDR-L3 of CAP248-2B was specifically angled towards the viral membrane but its tip retains a level of plasticity that may assist in interacting with dynamic viral lipids . In this way , the CAP248-2B light chain CDR-L3 performs a similar function to the heavy chain CDR-H3 of MPER targeting bNAbs 2F5 , Z13e1 , 4E10 , and 10E8 which all extend hydrophobic residues at the CDR-H3 loop tip to anchor the antibody in the viral membrane . While 2F5 and 4E10 are significantly autoreactive , we saw no evidence of this for CAP248-2B , similar to 10E8 and 35O22 . In addition to sharing a common mechanism of lipid recognition , CAP248-2B , 35O22 , and MPER bNAbs all approach the HIV-1 trimer very close to the viral membrane . These antibodies may require the trimer to alter its orientation or position relative to the viral membrane for them to access their epitopes [44] . As a consequence of this epitope occlusion , MPER bNAbs may not bind as well to pre-fusion native trimers [68] . From our EM docking analyses , it appears that CAP248-2B recognizes the pre-fusion “closed” state of the HIV-1 envelope trimer . It remains to be determined what structural rearrangements ( if any ) in the trimer are required for CAP248-2B to properly access its epitope . In accordance with its low angle of binding , escape from CAP248-2B occurred in both the gp160 cleavage motifs . Of the six identified mutations , only V505M individually affected CAP248-2B neutralization . In autologous viruses , mutations at positions 500 , 502 , 507 , 508 , and 509 all occurred by two years post-infection , while variants at position 505 were only detectable after three years of infection . Based on these kinetics , it is likely that mutations at positions 500 , 502 , 507 , 508 , and 509 accumulated first in response to earlier members of the CAP248 bNAb lineage , with the eventual selection of extremely rare mutations at position 505 by later members of the antibody lineage . It is also possible that the other mutations affect local Env conformation , conferring escape through an indirect mechanism . Similarly , we identified a cluster of autologous gp41 mutations overlapping the CAP248-2B epitope that may have played a role in escaping earlier lineage members . Future experiments to isolate these early members of the lineage will help to understand how virus-antibody co-evolution led to the development of CAP248-2B . In addition , the isolation of more potent variants of CAP248-2B should help to explain the mechanisms of incomplete neutralization for this new interface targeting antibody . In addition to mediating escape from CAP248-2B , mutations in the gp120 C terminus conferred partial resistance to PGT151 . Based on EM 3D reconstructions PGT151 does not bind the gp120 C terminus [63] , suggesting that these mutations have the ability to affect envelope conformation in a way that confers resistance to PGT151 . Conversely , the gp120 C-terminal mutations also had the unexpected effect of dramatically enhancing the neutralization of 35O22 ( targeting a membrane proximal epitope ) and bNAbs targeting the MPER , suggesting that these conformational effects may assist in raising gp41 relative to the membrane [44 , 69–72] , perhaps by increasing the frequency at which membrane associated Env trimers sample a CD4-induced conformation , without first having to engage the CD4 receptor . Importantly , this effect did not make viruses globally sensitive to HIV-1 antibodies , but was specific for bNAbs with membrane proximal epitopes such as 4E10 , 10E8 , and 35O22 . Incorporating these mutations into membrane bound HIV-1 trimer immunogens may therefore improve the antigenicity of bNAb epitopes in gp41 . Overall these data expand the recently identified gp120-gp41 interface supersite to include the gp120 C terminus , highlighting the importance of this region as a vaccine target . This region of Env was also the target for neutralizing antibodies elicited in rabbits by SOSIP trimer immunogens [73] . Further characterization of the CAP248 bNAb epitope could therefore inform pathways through which these sorts of antibodies might achieve neutralization breadth . Future experiments should also aim to determine whether membrane-bound CS-Mut trimers successfully engage MPER bNAb precursors , thus overcoming an important barrier to the induction of MPER bNAbs . Furthermore , defining additional glycan independent mechanisms of envelope heterogeneity will have implications for the use of bNAbs in both passive and active immunization strategies .
The CAPRISA Acute Infection study in adult women received ethical approval from the Universities of KwaZulu-Natal ( E013/04 ) , Cape Town ( 025/2004 ) , and the Witwatersrand ( MM040202 ) . CAP248 provided written informed consent for study participation . The CAPRISA Acute Infection cohort is comprised of women at high risk of HIV-1 infection in Kwa-Zulu Natal , South Africa [15] . Blood samples collected at regular intervals from seroconversion through to the initiation of antiretroviral therapy were cryopreserved as individually processed PBMC , serum and plasma samples . Cryopreserved CAP248 PBMC were thawed , washed and suspended in medium containing 10% foetal bovine serum ( FBS ) and antibiotics . B cells were enriched by negative selection with immunomagnetic beads ( Miltenyi ) , and were cultured at 25 cells per well in Iscove’s Modified Dulbecco’s Medium ( IMDM ) containing 10% FBS , 2 μg/mL CpG2006 , 100 units/mL rIL-2 , rIL-21 ( 50 ng/mL ) with 3T3msCD40L as feeder cells ( a gift of Mark Connors ) [45] , plated at 2500 cells/well in multiple 96 well plates . rIL-2 was obtained from the NIH AIDS Reagent Program as provided by Maurice Gately ( Hoffmann-La Roche ) . Fresh medium containing growth factors was added after 7 days of culture and after each antibody screening procedure . B cell culture fluids were screened from day 14 for neutralizing activity against CAP45 pseudovirus in an adaptation of the single-cycle TZM-bl neutralization assay as previously described [74] . B cells from wells testing positive for antibody were stored in RNAlater ( Ambion ) . VH , Vκ , or Vλ genes were amplified in separate reactions from RNA using a one-step RT-PCR ( Invitrogen SuperScript III kit with Platinum Taq High Fidelity polymerase ) with previously described primer mixes [75] . For expression vector assembly , forward primers included a 25 nucleotide non-annealing 5’ tag sequence , which was homologous to the immunoglobulin leader sequence at the 3’ end of the CMV promoter fragment . Reverse primers were designed to overlap the 5’ end of the immunoglobulin constant region for each vector . Linear expression constructs were assembled by overlapping PCR between two DNA fragments containing the CMV promoter and immunoglobulin leader sequence or the constant region sequences for IgG1 , kappa or lambda genes followed by a C-terminal BGH poly A sequence . These were co-transfected into 293T cells , and supernatant fluids were tested for neutralization activity . This step allowed rapid detection of pairs of VH and VL chain genes that functioned together to produce antibody . To produce monoclonal antibodies , the In-Fusion cloning system ( Clontech ) was used to insert re-amplified pairs of VH and VL gene fragments into pLM2 expression plasmids similar to previously described [76] , but modified to contain the immunoglobulin leader sequence in the linear vectors . Expression plasmids were linearized by restriction enzymes acting on sites within the multiple cloning site of plasmid ( EcoRI for IgG1 and lambda , BsiWI for kappa ) . Linearized vectors were then PCR amplified with primer pairs designed to create terminal sequences that were homologous to 5’ and 3’ terminal sequences of the variable region insert fragments , allowing insertion of the VH and VL fragments into linearized plasmids by the activity of the In-Fusion enzyme as described [77] . The resulting plasmids were transformed in JM109 cells . A previously described strategy was used to identify the correct pair of VH and VL clones responsible for antibody production [78] . Subsequent sequencing of multiple clones showed that only one heavy and one light chain were capable of directing mAb synthesis . CD4+/CCR5+ TZM-bl HeLa cells were obtained from the NIH AIDS Research and Reference Reagent Program , Division of AIDS , NIAID , NIH ( developed by Dr . John C . Kappes , and Dr . Xiaoyun Wu [79 , 80] ) . 293T cells were obtained from Dr George Shaw ( University of Alabama , Birmingham , AL ) . Adherent cell lines were cultured at 37°C , 5% CO2 , in DMEM containing 10% heat-inactivated fetal bovine serum ( Gibco BRL Life Technologies ) and supplemented with 50 ug/ml gentamicin ( Sigma ) . Cells were routinely disrupted at confluency with 0 . 25% trypsin in 1 mM EDTA ( Sigma ) every 48–72 hours . 293F suspension cells were cultured in 293Freestlye media ( Gibco BRL Life Technologies ) at 37°C , 10% CO2 , 125RPM and diluted twice a week to between 0 . 2 and 0 . 5 million cells/mL . The single genome amplification of HIV Env has been previously described [79] . Briefly , CAP248 viral RNA was isolated using the Viral RNA Extraction Kit ( QIAGEN ) , to serve as a template for Superscript III Reverse Transcriptase ( Invitrogen ) in cDNA generation . Residual RNA was degraded with RNaseH ( Invitrogen ) and CAP248 envelope genes were amplified by a nested PCR approach using Platinum Taq ( Invitrogen ) . PCR products were cleaned up ( QIAGEN ) and sequenced with the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit ( Applied Biosystems ) on the ABI 3100 automated genetic analyser , assembled using Sequencher v . 4 . 5 ( Genecodes ) , and compiled into working alignments in Bioedit v . 7 . 0 . 5 . 3 . Plasmids expressing the HIV Env of interest were co-transfected with pSG3DEnv backbone expressing plasmids ( obtained from the NIH AIDS Research and Reference Reagent Program , Division of AIDS , NIAID , NIH ) into 293T cells using PEI-MAX 40 , 000 ( Polysciences ) . Cultures were incubated for 48 hours at 37°C , then filtered through 0 . 45 μm and frozen in DMEM , 20% FBS to yield Env-pseudotyped viruses capable of a single round of infection only . Mutant envelope genes were generated with the QuikChange Lightning Kit ( Stratagene ) , confirmed by DNA sequencing , and transfected as above . For the glycan heterogeneity experiments , pseudoviruses were grown as above in the presence of 25M glycosylation inhibitor , or in 293S GnTI ( -/- ) cells . Neutralization assays were performed in TZM-bl cells as previously described [12 , 80] . Neutralization is measured as a reduction in relative light units after a single round of pseudovirus infection in the presence of the monoclonal antibody or plasma sample of interest . Samples were serially diluted 1:3 and the ID50/IC50 calculated as the dilution at which the infection was reduced by 50% . For CAP248-2B antibody expression , plasmids separately encoding heavy and light chain genes were co-transfected into 293F cells with PEI-MAX 40 , 000 ( Polysciences ) . To make CAP248-2B Fab , the HRV-3C protein cleavage site ( GLEVLFQGP ) was introduced into the heavy chain gene between Fab and Fc fragments by PCR . Expressed full length mAb was digested with HRV-3C enzyme ( Merck Millipore ) at 25°C for four hours , and then the separated Fab fragments were purified by sequential negative selection over protein A , and positive selection by gel filtration using a superdex 200 ( GE Healthcare ) . Cells were cultured for seven days in 293Freestyle media at 37°C , 10% CO2 , then harvested supernatants were 0 . 22 μm filtered and purified using protein A . Trimers were expressed previously described [65] , and purified from 0 . 22 μm filtered supernatants with an Ni-NTA column ( 30mM Imidazole wash and 400mM Imidazole elution buffers at pH7 ) , and then by CAP248-2B mAb bound to protein A . The Fab-trimer complexes were eluted by digestion with HRV-3C ( which also removed the His6 tag from gp41 ) , and further purified by gel filtration using a superdex 200 column ( GE Healthcare ) . HisTagged trimers were coated at 2 μg/mL in PBS onto nickel coated 96 well plates ( Thermo ) for one hour at 25°C . Plates were washed and then probed with serial dilutions of HIV-1 monoclonal antibody for one hour at 25°C . This process was repeated using an anti-Fc/HRP conjugate to detect trimer-bound antibodies . Antigen-antibody complexes were detected by incubating with 100 μL of enzyme substrate for five minutes and then the reaction was stopped with 25 μL of 1 M HCl . Absorbance was read at 450 nm . Concentrated aliquots were stored at 4°C . 576 crystallization conditions were screened in 96 well plates ( Corning ) using the Cartesian Honeybee crystallization robot by sitting drop vapour diffusion in 400 nL drops at 25°C containing 50% mother liquor . Crystal hits were hand-optimised in 15 well hanging drop diffusion plates at 25°C in 1 μL drops containing 50% mother liquor . All crystallographic diffraction data was collected at the Advanced Photon Source ( Argonne National Laboratory ) SER-CAT ID-22 beamline , at a wavelength of 1 . 00 Å , 100K , and processed with HKL2000 . Model building and refinement was handled with COOT v0 . 8 and PHENIX v1 . 9–1692 software packages respectively , using 5% of the data as an R-free cross validation test set , and hydrogens were refined to minimise clashes . The unliganded CAP248-2B Fab was first crystallized in 10 . 25% PEG4000 , 87 . 5 mM ammonium sulphate , and flash frozen in 25% PEG400 as a cryoprotectant . This crystal diffracted to a resolution of 2 Å ( PDB-ID: 5MP6 ) and phasing by molecular replacement was done using PDB-IDs: 4QHK and 3B2U as search models . We could not reliably build the constant domain for one of the two Fabs in the asymmetric unit , which appeared to have considerable mobility within the crystal lattice , resulting in poor RSRZ scores for regions of these chains . This first structure served as the search model for the second crystal structure obtained in 7 . 5% PEG4000 , 12 . 5% isopropanol , 0 . 1 M sodium citrate ( pH5 . 6 ) , and flash frozen in 30% ethylene glycol as a cryoprotectant , which diffracted with I/αI >2 to a resolution of 3 . 1 Å , with data up to 2 . 8 Å ( PDB-ID: 5F89 ) . All structural images were generated in PyMOL Molecular Graphics System , Version 1 . 3r1edu , Schrodinger LLC . , or UCSF Chimera [81] . BG505-CAP45 SOSIP trimers were incubated with a 6 molar excess of CAP248-2B Fab overnight at room temperature and the complexes were diluted to ~0 . 03 mg/mL in Tris-buffered saline prior to application onto a carbon-coated 400 Cu mesh grid ( Electron Microscopy Sciences ) that had been glow discharged at 20 mA for 30 seconds . The grids were stained with 2% ( w/v ) NanoW ( Nanoprobes ) for 7 s , blotted , and stained for an additional 15 s . Samples were imaged on an FEI T12 electron microscope operating at 120 keV , with an electron dose of ~25 electrons/Å2 and a magnification of 52 , 000x that resulted in a pixel size of 2 . 05 Å at the specimen plane . Images were acquired with Leginon [82] , using a Tietz TemCam F416 camera and a nominal defocus range of 1000–1500 nm . Stage tilts between -50° and 0° using 10° increments were performed to increase the amount of unique views to aid with 3D reconstruction . Automated particle picking , stack creation , and initial 2D classification were performed in the Appion software suite [83] . Classes representing noisy alignments , neighboring particles , unbound Fab , or ligand-free trimers were discarded and representative class averages with unique views of the SOSIP-CAP248-2B complex were used to generate an initial common-lines model using EMAN2 [84] , followed by refinement against all 28 , 215 particles in Sparx [85] , with C3-symmetry imposed . The resolution of the final reconstruction is ~20 Å based on a Fourier shell correlation of 0 . 5 . Two-dimensional back projections of the final 3D models were generated using EMAN [84] . Antibody binding to cardiolipin , or reactivity with Hep-2 epithelial cells ( ZEUS Scientific ) was assessed as previously described [35] per the manufacturer’s protocol . Antibodies were scored as positive or negative at 50 μg/mL when compared to a no antibody control . The monoclonal antibodies 4E10 and 35O22 were included as positive and negative controls respectively . CAP45 . 2 . 00 . G3J ( Genbank: EF203960 ) env plasmid was codon optimised ( GenScript ) and truncated at the cytoplasmic tail to increase surface Env content [86] . Following restriction digest cloning this plasmid was transiently transfected using TrueFect-MAX ( United Biosystems ) into HEK/293T cells . Two days after transfection , cells were labelled with Live/Dead Fixable Aqua Dead Cell Stain ( Life Technologies ) followed by biotinylated CAP248-2B and serially diluted unlabelled competitor antibodies ( CAP248-2B , 3BC315 , 35O22 , PGT151 . 8ANC195 , 4E10 , and control antibody Palivizumab ) . After incubation and three washes with 5% FBS in PBS , cells were stained with Streptavidin-PE ( Life Technologies ) at a 1:300 dilution . Reverse competition assays were also performed with biotinylated 3BC315 , 35O22 , PGT151 , 8ANC195 and 4E10 and serially diluted CAP248-2B or Palivizumab . Cells were analysed on a BD FACS Aria II ( Becton Dickinson ) and binding was measured as the median fluorescence intensity ( MFI ) for each sample minus the MFI of the cells stained with the detection antibody only . BG505-CAP45 chimeric SOSIP trimers ( expressed without additional pFurin to reduce cleavage efficacy , and therefore containing both cleaved and uncleaved trimers ) were captured from 293F supernatants by monoclonal antibodies CAP256-VRC26 . 09 and CAP248-2B covalently bound to protein A . Eluted proteins were assessed on SDS-PAGE with and without dithiothreitol to determine the ratio between cleaved and uncleaved gp160 . Pure amine-functional glycans were printed onto NHS-activated glass slides , blocked with ethanolamine , and probed with CAP248-2B monoclonal antibody as described previously [29 , 87] . Reactivity was calculated by the mean intensity minus the mean background , and preformed with different secondary antibodies to limit signal: noise ratio , where binding with a fluorescence intensity of greater than 500 a . u . was considered positive . No binding was detected against high-mannose glycans . | Our understanding of which regions of the HIV-1 envelope are targets for broadly neutralizing antibodies ( likely required for an HIV-1 vaccine ) has expanded greatly in recent years , offering insights for vaccine design . For instance , much of solvent-exposed gp41 is now known to be targeted by antibodies at the gp120-gp41 interface . In this study , we isolated the neutralizing monoclonal antibody CAP248-2B , and used structural biology to characterize its epitope which spanned both the gp120-gp41 and gp41-gp41 interfaces in a manner distinct from other antibodies . CAP248-2B extends the interface target to include the gp120 C terminus , effectively encircling the base of native pre-fusion trimers . While CAP248-2B recapitulated the donor’s plasma breadth , it had poor potency against some isolates as a consequence of low neutralization plateaus . Unlike many broadly neutralizing antibodies , these plateaus did not appear to be a consequence of glycan heterogeneity , suggesting additional mechanisms that contribute towards incomplete neutralization . Finally , we characterized viral escape pathways from CAP248-2B , and identified a cluster of unusual mutations in the gp160 cleavage sites that made HIV-1 viruses more sensitive to antibodies targeting highly conserved membrane-proximal epitopes . These mutations might improve the immunogenicity of gp41 , and thereby inform HIV-1 immunogen design . | [
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] | 2017 | Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape |
Gene regulatory information guides development and shapes the course of evolution . To test conservation of gene regulation within the phylum Nematoda , we compared the functions of putative cis-regulatory sequences of four sets of orthologs ( unc-47 , unc-25 , mec-3 and elt-2 ) from distantly-related nematode species . These species , Caenorhabditis elegans , its congeneric C . briggsae , and three parasitic species Meloidogyne hapla , Brugia malayi , and Trichinella spiralis , represent four of the five major clades in the phylum Nematoda . Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes , all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns . We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C . elegans orthologs than would be expected by chance , but they do harbor motifs that are important for proper expression of the C . elegans genes . These motifs are too short to be distinguished from the background level of sequence similarity , and while identical in sequence they are not conserved in orientation or position . Functional tests reveal that some of these motifs contribute to proper expression . Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements .
Similar expression patterns of orthologous genes imply similarity of developmental programs in different species . Numerous such examples have been uncovered , including hox [1] , dlx [2] , and dpp/BMP [3] genes , as well as genetic programs regulating photoreceptor [4] and muscle [5] development in distantly related bilaterian animals . Largely based on these and similar findings , a current view of evolution of development emerged that emphasizes the conservation of the genetic “toolkit” within animals and the relative importance of regulatory changes in driving morphological change [6] . The mechanisms responsible for expression pattern conservation are less clear , however . One possibility is that ancestral gene regulatory programs are strictly retained . An alternative is that expression similarity is mediated by divergent regulatory processes [7 , 8] , a phenomenon known as “developmental system drift” [9] . Regulatory rewiring of the latter type is known to occur even when individual components of the diverged networks are highly conserved developmental regulators [10–12] . One way to probe the evolution of regulatory linkages is with enhancer swap experiments , in which cis-regulatory DNA from one species is used to drive expression of a reporter gene in another species ( reviewed in [13] ) . The resulting pattern of gene expression can be compared to the pattern driven by the endogenous regulatory element , with the similarities and differences giving evidence of conservation and divergence in the gene regulatory network . We wanted to assess the conservation of gene regulatory programs among distantly-related members of the phylum Nematoda , a group of morphologically similar worms with mostly small , vermiform bodies . This body plan is largely conserved , with numbers of certain neuronal subtypes nearly identical in even deeply diverged taxa [14 , 15] , and the intestine arising from a clonal cell lineage [16] in most ( but not all , see [17] ) nematodes studied . However , instances of developmental divergence have been documented in this clade [18–23] . We therefore performed enhancer swap experiments with regulatory elements of genes expressed in two subsets of neurons and in the developing intestine . By examining the function of cis regulatory sequences from four different nematode species in transgenic C . elegans , we sought to determine the extent of cis-regulatory conservation within this phylum .
The phylum Nematoda is comprised of animals with simple vermiform body plans and diverse life-history strategies . To look for evidence of gene regulatory conservation across this phylum , we carried out a series of enhancer-swap experiments between several distantly-related nematodes and a C . elegans host . Regulatory regions from orthologous C . elegans genes driving the mCherry reporter were co-expressed as controls with the exogenous cis elements driving expression of the GFP gene . This approach allows us to isolate and compare cis-regulatory functions of the two orthologous regulatory elements in a common trans-regulatory background . Any observed differences can then be attributed to the divergence of the cis-regulatory DNA . We sought broad coverage of the phylum , which is hypothesized to have diversified in the Silurian [24] . Representatives from two basally branching nematode groups have sequenced genomes [25] . These are the Chromodorea ( comprised of Clades III-V ) and the Dorylaimia ( Clade I ) . No Enoplia ( Clade II ) genomes have been sequenced to date . For this study we used C . elegans [26] as the transgenic host species , and its congeneric C . briggsae [27] to test divergence of regulatory elements among close relatives ( both are from Clade V ) . The next most closely related nematode species is Meloidogyne hapla ( Clade IV , [28 , 29] ) , followed by Brugia malayi ( Clade III , [30 , 31] ) . Finally , as a representative of Clade I , we used Trichinella spiralis [32] . Divergence of Clade I was one of the earliest events in nematode evolution . The relationships among these five species are shown in Fig 1 . We leveraged both this phylogeny and the amenability of C . elegans to genetic manipulation to create a series of comparisons of expression of cis-regulatory elements from progressively more distantly-related species in transgenic C . elegans . C . elegans have been used as transgenic hosts of regulatory DNA from a number of different species ( reviewed , along with similar studies using Drosophila melanogaster , in [13] ) , however , to our knowledge , this study is the first explicit test of the relationship between evolutionary relatedness and conservation of cis-regulatory function among a set of genes . While our selection of species gave us unprecedented ability to test the phylogenetic limits of regulatory conservation , it also rendered reciprocal transgenesis infeasible due to the complex modes of reproduction of the parasitic species . We selected genes that have considerable conservation of their coding sequences and are single-copy orthologs among the species . Two genes are expressed in GABAergic neurons in Caenorhabditis nematodes , unc-47 [33 , 34] and unc-25 [35] . We have previously investigated the evolution of their regulation within this clade [36–39] . The third gene , mec-3 , is expressed in another neuronal cell type , the touch-receptor neurons , in C . elegans [40 , 41] . The regulatory region of the C . briggsae ortholog of mec-3 has previously been shown to drive gene expression in C . elegans [42] . Finally , we chose the gene elt-2 , which is expressed in the endoderm [43 , 44] , and shows evidence of regulatory conservation outside the genus Caenorhabditis [45] . These cis-regulatory elements are expressed in different cell types , and drive expression of terminal differentiation genes ( unc-47 and unc-25 ) as well as transcription factors ( mec-3 and elt-2 ) . Where possible ( see Materials and Methods ) , the putative regulatory regions we investigated ranged from the start of recognizable protein-coding sequence conservation with C . elegans on the 3’ end to the next upstream coding element on the 5’ end . This choice of putative regulatory sequences in no way depended on non-coding conservation between species . All but one of the 12 regulatory sequences from distantly related species that we tested in C . elegans directed expression in at least a subset of the expected cells , so some degree of functional conservation is preserved even at these great phylogenetic distances . Since the putative regulatory regions from the distant relatives were selected without regard for non-coding conservation , we next examined them for sequence similarity with the C . elegans orthologs . We did not know , a priori , what types of sequence similarity to expect , and did not find any extended sequence conservation . For this reason , we conducted three types of sequence comparison to ascertain the extent of sequence similarity between C . elegans and each of the distantly-related nematodes . First , we created dotplots , which depict the positions of nucleotide strings of a certain length that are shared by the C . elegans unc-47 sequence and a sequence from another nematode ( 10 bp examples shown in Fig 6A–6D ) . Only the C . briggsae cis element displayed substantial evidence of sequence conservation , represented by collinear blocks of sequence with conserved spacing upstream of the translation start site ( upper right diagonal , Fig 6A ) . Not only do the distantly-related nematodes lack any such collinear blocks of sequence ( evidence of conservation ) , they lack much in with way of sequence similarity as well , with only a few scattered motifs found in both the C . elegans unc-47 upstream region and those upstream regions from M . hapla , B . malayi , and T . spiralis ( Fig 6B–6D ) . We looked more closely at the few 10 bp motifs in each of the divergent sequences that are shared with the C . elegans cis element ( Fig 6B–6D ) . Since the functional units of cis-regulatory elements are thought to be short binding sites , we next hypothesized that the divergent cis elements might be enriched for such short , shared motifs . We tested this in two ways . First , we broke the sequences down into their component k-mers , and asked what percentage of the total sequence length was made up of k-mers shared with the C . elegans sequence . For example , by definition , 100% of the M . hapla unc-47 cis element is made up of 1-mers ( A , T , G , or C ) that are also found in the C . elegans unc-47 cis element . Approximately 40% of the examined cis elements of C . briggsae and the other 3 nematodes are made up of 8-mers that are also found in the C . elegans sequence ( Fig 6E ) , suggesting that window sizes shorter than 9 nucleotides are not likely to be informative for this comparison . For 9-mers , slight differences in the proportion of shared sequence can be detected among species; at window sizes of 10–12 nucleotides , the difference between C . briggsae and the distantly-related nematodes becomes apparent ( Fig 6E ) . Note that the B . malayi unc-47 cis element , while it functions remarkably better than the T . spiralis ortholog ( Fig 2 ) , is not substantially more similar in sequence to the C . elegans regulatory element . None of the three distantly-related nematodes had any identical sequence blocks longer than 12 nucleotides , and blocks longer than 10 nucleotides were primarily low-complexity polynucleotide sequences ( S6 Fig ) , while C . briggsae had identical sequences of up to 23 nucleotides in length ( Fig 6E ) . Alignments showing all of the identical sequence matches in the unc-47 upstream regions that are 9 nucleotides or longer can be found in S6 Fig . These identical blocks are not enriched proximal to the start of the coding sequence . Similar levels of conservation were found for unc-25 , mec-3 , and elt-2 as well ( S7–S9 Figs ) . The next method that we used to test whether the orthologous cis elements were enriched for short motifs shared with the C . elegans unc-47 upstream region compared the number of shared motifs detected with the number that might be expected by chance . Here , “chance” refers to a random reordering of the C . elegans sequence that preserves nucleotide , dinucleotide , or trinucleotide frequencies . For each of the four genes , we reshuffled the C . elegans sequence 1000 times . The cis elements from C . briggsae , M . hapla , B . malayi , and T . spiralis were compared to each of the 1000 reshuffled C . elegans sequences , and we calculated the numbers of nucleotide blocks ( length 8–12 ) that were identical between each reshuffled C . elegans cis element and each of the orthologs . This provided empirically derived distributions of sequence identity that could be expected solely as a result of basic nucleotide composition properties . The results for tests of 10 nucleotide blocks are shown in Fig 6F–6I . For the 1000 comparisons between the reshuffled C . elegans sequences and the other nematode’s upstream unc-47 sequence , the number of identical motifs was plotted ( Fig 6F–6I ) . The number of motifs form distributions centered between about 10–20 motifs per reshuffled sequence , depending on the length of the ortholog . Comparing the actual number of conserved blocks of various lengths between C . elegans cis elements and their orthologs revealed that only C . briggsae had more sequence identity than our “chance” rearrangements , with 63 identical 10-mers ( Fig 6F ) . The other three distantly-related nematodes’ sequences had no more similarity than expected by chance , with numbers of shared 10-mers that fell close to the means of the distributions ( Fig 6G–6I ) . The same was true for the upstream noncoding sequences of unc-25 , mec-3 , and elt-2 ( S10 Fig ) . Comparisons of noncoding sequence identity did not reveal any substantially conserved regions likely to be responsible for the functional conservation of orthologous cis elements . And yet , 11 of the 12 cis-regulatory elements from deeply diverged nematodes drove gene expression in C . elegans that recapitulated at least some of the expected endogenous expression pattern . We therefore searched the orthologous sequences for motifs known to be functionally important in the C . elegans sequences . Expression of unc-47 is regulated by direct binding of UNC-30 [47] to TAATCC sites . Mutations to this motif abolish expression in the D-type neurons [47] . Perhaps functional conservation of the unc-47 cis elements from distantly-related nematodes is due to the presence of this and other short sequences below the level of detection in our naive sequence comparison . Searching for the TAATCC site revealed a perfect match , including one flanking base pair on either side in C . briggsae , with similar spacing from the translational start site ( Fig 7 ) . The noncoding sequence upstream of M . hapla unc-47 has three instances of this motif , all on the reverse strand , with additional identical nucleotides flanking the core site ( Fig 7 ) . The upstream sequences from B . malayi and T . spiralis lack perfect matches to this consensus , but do have 5/6 bp core matches with some additional flanking identity ( Fig 7 ) . Either these close matches are divergent cis-regulatory sites , hinting at evolved differences in TF-TFBS recognition , or else there is more to the control of expression in D-type neurons than we have recognized in C . elegans thus far . The C . elegans UNC-30 binding site controls expression in D-type neurons , but not in DVB in the tail or AVL , RIS , or the RMEs in the head [47] . One site that contributes to expression in DVB , RIS , and AVL is the AHR-1-like motif [37] . This motif has the sequence CACGC and is conserved in sequence and position between C . elegans , C . briggsae , C . brenneri , and C . remanei [37] . A match to this motif is found on the reverse strand of the M . hapla unc-47 cis element ( Fig 7 ) . A palindromic sequence CACGCGTG , that is , two overlapping AHR-1-like motifs on opposite strands , along with an additional single instance of this motif , are present upstream of the T . spiralis unc-47 gene ( Fig 7 ) . Similar motif-matching analyses were carried out for the other three sets of orthologous cis-regulatory elements . Matches to motifs known to be necessary for function in C . elegans were identified in almost all tested orthologs from distantly-related nematodes ( S1 Text; S11–S13 Figs ) . However , the occurrence of even multiple instances of motifs corresponding to transcription factor binding sites should not be construed as evidence of conservation . First , these motifs are not found any more frequently than in randomly reshuffled C . elegans sequences . We explicitly estimated the probability of finding these motifs in the randomly reshuffled C . elegans sequences . The probability of finding TAATCC ( the UNC-30 binding site ) in sequences preserving the single-nucleotide composition of the C . elegans cis element was 0 . 558 , conserving dinucleotides it was 0 . 378 , and trinucleotides it was 0 . 566 . The probabilities of finding CACGC ( the AHR-1-like motif ) in these same sequences were 0 . 632 , 0 . 606 , 0 . 674 , respectively . Second , these motifs were routinely found in the cis elements of the other genes we examined ( S3 Table ) . Third , these motifs are often found on the opposite strand , suggesting that , while individual motifs are born and die , this sequence turnover maintains at least one instance of the motif in each of the orthologous regulatory elements . Therefore , identical motifs are not , strictly speaking , conserved . It is suggestive that the unc-47 regulatory sequence from a distantly-related nematode that retains the best function in D-type neurons , that of M . hapla , has the best match to the UNC-30 binding site . Similarly , the regulatory sequence with the best function in DVB—T . spiralis unc-47—has the best matches to the AHR-1-like motif . We therefore tested the contribution of these motifs to functional conservation . We introduced mutations into an UNC-30 binding motif in the M . hapla unc-47 cis element . This motif was selected because it shares the longest similarity in the flanking sequences with the UNC-30 binding site of the C . elegans unc-47 cis element ( Fig 8A ) . The mutant M . hapla unc-47 sequence directed less consistent expression in the D-type neurons than its wild-type counterpart ( Fig 8B , 8C and S14A ) , suggesting that this UNC-30 motif contributes to control of gene expression in the D-type neurons . Next , we introduced mutations into the palindromic double AHR-1-like motif of the T . spiralis unc-47 element , eliminating the consensus sequence on both strands ( Fig 8D ) . This resulted in a substantial decrease in the fraction of animals expressing the transgene in RIS and DVB ( Figs 8E–8H and S14B ) . This suggests that the palindromic AHR-1-like motif upstream of the T . spiralis unc-47 gene is partially responsible for expression in DVB and RIS , just as the AHR-l-like motif is in C . elegans [37] . Neither mutation eliminated expression in the affected cells entirely , implying that these sites contribute to but are not strictly essential for expression . This could be due to the redundancy of these binding sites in both cis elements ( Fig 7 ) . As another possible explanation , consider the case of the B . malayi unc-47 element that lacks good matches for either the UNC-30 or the AHR-1-like motifs , and yet is reasonably well expressed in both the D-type neurons and in DVB . It is possible that some orthologous cis elements retain functional conservation via sequences that can be recognized by C . elegans transcription factors , but that we currently cannot recognize as functional .
We investigated cis-regulatory function in an explicitly evolutionary framework . The extent of divergence between the species involved in this study ranged from that of congenerics ( C . elegans and C . briggsae ) to the deepest in the phylum Nematoda ( C . elegans and T . spiralis ) . This allowed us to test how regulatory information breaks down over time . Transgenic experiments were conducted in the “common garden” of C . elegans to control for the effects of trans-regulatory divergence and to focus comparisons on the cis elements ( see Potential Caveats in Materials and Methods ) . We selected the putative regulatory regions without regard for non-coding sequence similarity , which then permitted us to comment on the relationship between functional and sequence conservation . We used a standard methodology to look at the cis elements of four genes , allowing us to make four generalizations . First , despite the vast spans of evolutionary time that we sampled—the most distantly-related species diverged perhaps as long as 400 million years ago [24]—the majority of the cis-regulatory elements exhibited appreciably conserved gene regulatory function in C . elegans ( Table 1 ) . Two reasons compelled us to focus explicitly on the conserved , rather than divergent , aspects of expression . First , at such great phylogenetic distances , any conservation might be less expected than divergence . Second , for technical reasons , we can only know the endogenous function of the C . elegans regulatory elements , not the patterns driven by the divergent cis elements in their native species ( see Potential Caveats in Materials and Methods ) . Our findings are consistent with previous reports of functional conservation of cis-regulatory elements between distantly-related members of the same phylum , most extensively tested in arthropods and chordates [48 , 49] . Although there have been a number of reports of functional conservation of cis elements between different phyla [50–56] , this is not true for cis elements of all genes tested [39 , 57] . It is possible that the evolutionary dynamics of regulatory elements may be sufficiently idiosyncratic to preclude general conclusions about the “outer limits” of cis-regulatory conservation . Second , in most cases cis-regulatory elements from more distant relatives have retained less function than elements from closer relatives . However , there are notable exceptions and , importantly , the pattern of functional divergence that we observed reflects modular organization of cis-regulatory elements—separable elements control different aspects of expression [58–60] . Due to modularity of cis elements , evolution can “tinker” with some functions while avoiding pleiotropic effects on others [61] . In C . elegans , expression of unc-47 is controlled by different mechanisms in D-type neurons and DVB , RIS , and AVL [37 , 47] . Accordingly , we see that whereas the T . spiralis unc-47 element is not expressed in D-type neurons , it functions relatively well in DVB and RIS ( Figs 2 and S1 ) . In contrast , the M . hapla unc-47 element is expressed well in the D-type neurons , but not in DVB ( Fig 2 ) . Similarly , the elt-2 elements from M . hapla and B . malayi are expressed reasonably well during embryogenesis , but not in later stages ( Fig 5 ) . We consider this good evidence for separate regulation of pattern , timing , and levels of expression , as well as substantiating evidence that the weak expression of some of these regulatory elements is due to genuine divergence of regulatory information rather than experimental artifacts of weak transgene expression . We conclude that modular organization of cis elements manifests in different rates of divergence for different aspects of expression patterns [62] and may be quite common [13] . Mechanisms controlling spatial , temporal , and levels of expression may be particularly prone to different rates of divergence ( e . g . [45 , 63] ) . Third , despite their substantially conserved functions , the regulatory elements of all species but C . briggsae have not retained more sequence similarity than would be expected by chance . This finding is consistent with previous reports that suggested that conservation of cis-regulatory function does not , strictly speaking , require extended sequence conservation [64–70] . Since different types of regulatory elements evolve under different constraints [36] , relying on sequence conservation to find cis-regulatory elements might bias discovery to only particular types of elements with highly constrained sequences [71] . Additionally , because sequences of different elements evolve at different rates [38] , it is not a priori clear how distant the species to be compared should be to discover cis elements of different types . Even when some short stretches of identical nucleotides are discovered between distantly-related orthologous cis elements , this should not be taken as evidence of conservation . This is because many short matches will always be found by chance , particularly in regions with biased nucleotide composition . For instance , the co-occurrence of the UNC-30 and AHR-1-like motifs upstream of unc-47 orthologs ( Fig 7 ) is more plausibly explained by a birth-and-death process rather than strict conservation , considering that these motifs are found on opposite strands of DNA in different species . Fourth , despite the lack of extended sequence conservation , for all four genes we could readily identify motifs corresponding to transcription factor binding sites previously identified as functionally important for regulation of C . elegans orthologs . The motifs that we tested contributed to gene regulation of the orthologous cis elements , implying that gene regulatory output can be conserved , even among distantly-related organisms , as long as key gene regulatory connections—“kernels” [10 , 72] or “input-output devices” [73]—are maintained . This further reinforces the view that when developmental programs evolve , the regulatory “toolkit” controlling major patterning and cell-type specification programs remains relatively static [6] . Of course , the mere presence of these short motifs is not likely to be sufficient to explain regulatory output . For instance , we can find chance matches to GATA motifs important for elt-2 expression in many of the other sequences we tested , which do not drive expression in the intestinal precursor cells . Similarly , we can find matches to the AHR-1-like motif ( that regulates unc-47 expression ) in the elt-2 cis elements of C . elegans , C . briggsae , and T . spiralis , none of which drive expression in DVB , RIS , or AVL . In this study we aimed to understand how the patterns of divergence of gene regulatory mechanisms between closely related species scale up over long evolutionary times . Models have predicted [74] that regulatory control can be shifted from one site to another within a cis-regulatory sequence; if these sites arise somewhat stochastically , longer wait times increase the likelihood of new sites originating and being optimized . These new sites could diminish the strength of purifying selection acting on ancestral motifs [75] . On shorter evolutionary time scales , new motifs do not have the time to arise , so function relies on conservation of existing sites [76] . As the same process plays out over different timespans , cis-regulatory conservation remains common among close relatives , but is mostly absent among more distantly-related species . Naturally , the rates of divergence and motif turnover are different for different genes . An important factor determining the rate of evolution could be the organization of a cis element , whether it is flexible [77 , 78] or constrained [79] , a billboard or an enhanceosome [80 , 81] . Modeling suggests that some enhancer sequences are inherently more prone to higher rates of turnover than others [74] . Better understanding of the structure of cis-regulatory elements may provide clues to their evolution [70 , 82 , 83] . Practically , our results advocate the use of C . elegans as a convenient and reliable experimental system for testing the functions of putative regulatory elements from nematode species , many of them parasites of major economic and medical significance , that are not amenable to transgenic studies [45] . Furthermore , the fact that C . elegans has been a genetic model system for decades means that the wealth of information about gene regulation in this species could be leveraged into hypothesis-driven investigation of non-model organisms . As discussed above , functionally conserved sequences can retain no more sequence conservation than would be expected by chance . Indeed , motifs that mediate functional conservation , namely transcription factor binding sites , are short enough that they would be likely to be found by chance in sequences of the lengths of these cis elements . By the measures of sequence conservation we applied , including alignment-free methods , M . hapla does not have appreciably greater sequence similarity to C . elegans than does T . spiralis . Nevertheless , M . hapla cis elements of all four tested genes drive more consistent and correct expression in transgenic C . elegans than elements from T . spiralis do . This means that some sequence properties were retained to a greater extent by the more closely related species . Identification of these properties would lead to a better understanding of function and evolution of gene regulatory elements .
Orthologous genes from C . briggsae , M . hapla , B . malayi , and T . spiralis were identified as best tblastn/blastx matches with the C . elegans protein sequence . For C . briggsae , B . malayi , and T . spiralis , the genome browser on Wormbase was used . For M . hapla , the genome browser at www . hapla . org was used . Forward primers were designed proximal to the next upstream gene , or failing that the 5’-most part of the contig on which the orthologous coding sequence was found . Reverse primers were selected to make in-frame translational fusions with GFP in the 5’-most part of the gene with protein coding sequence similarity with C . elegans . The only cases in which this was not possible were B . malayi and T . spiralis elt-2 , in which protein-coding conservation started deep in the protein-coding sequence , and the fusions were generated in the first exon . A previous study of elt-2 from a parasitic nematode , the less divergent Haemonchus contortus [84] , found that despite protein sequence divergence from C . elegans , the H . contortus protein retained function when expressed transgenically in C . elegans by a C . elegans heat shock promoter , so this increases our confidence that these can be elt-2 orthologs despite coding sequence divergence . In all cases , the start codon of the ortholog was included in the fusion . To generate reporter transgenes , upstream non-coding sequences were PCR amplified from genomic DNA and cloned upstream of GFP into the Fire vector pPD95 . 75 , or upstream of mCherry ( for C . elegans genes ) , which was inserted in place of GFP in a modified vector pPD95 . 75 [85] . elt-2 transgenes carried a nuclear localization signal upstream of GFP or mCherry . Prior to injection , all transgenes were sequenced to ensure accuracy . We injected a mixture ( 5 ng/μL ( for C . briggsae; 10 ng/μL for the other species ) promoter::GFP plasmid , 5 or 10 ng/μL promoter::mCherry plasmid , 5 ng/μL pha-1 rescue transgene , 100 ng/μL salmon sperm DNA ) into temperature-sensitive C . elegans pha-1 ( e2123 ) strain [86] . Transformants were selected at 25°C . Multiple strains were examined for each transgenic construct . Statistical analyses of consistency of expression patterns between strains and individuals are presented in S2 Table , since extrachromosomal transgenes are known to have more variable expression than integrated transgenes . Our previous reports [36 , 37] thoroughly addressed the similarity of expression driven by transgenes of different types—extrachromosomal , multicopy integrated , and single-copy integrated . We found that while the strength of the signal increases with multiple copies , and variability increases with extrachromosomal transgenes , the patterns generated by these different methods are consistent . The structures of extrachromosomal transgene arrays are generally not known . Although there is a possibility of cross-talk between promoters from different species if they land close enough when the DNA is concatenated , we mitigate against this by including an excess of salmon sperm DNA and vector sequence to create distance between the promoters and reduce the repetitiveness of the arrays . We measure expression in multiple independent strains . We also tested several of the highly divergent promoters alone , without a coexpressed C . elegans-DNA-driven reporter ( S15 Fig ) . Without the coexpressed mCherry marker , cells were more difficult to identify , so counts were not attempted for these strains , but expression was observed in the same subsets of cells that it was observed in coexpressing lines . The coexpressing strains also allowed us to control for the mosaicism inherent in extrachromosomal transgenes . Since the transgenes are concatenated , mCherry and GFP are inherited together by cells , and if array loss or silencing causes the loss of expression of one marker , the other will also disappear . This is why , for most of our quantification , we describe expression as the ratio of mCherry ( control ) positive cells that also express GFP ( see Figs 2–5 and 7 ) . We tested the functions of motifs corresponding to consensus sequences of binding sites of UNC-30 [47] ( TAATCC ) and AHR-1-like [37] ( CACGC ) . Motifs were identified using the ConsensusSequence feature on the GeneGrokker web server ( https://genegrokker . biology . uiowa . edu ) . Of the several UNC-30 motifs in the M . hapla unc-47 element , we selected for mutagenesis the longest extended match to the C . elegans sequence: aTAATCCcc ( reverse complement , since the motif is found on the ( - ) strand ) . This motif was mutagenized to aTAGGCGac ( changes highlighted ) . Of the several matches to the AHR-1-like motif in the T . spiralis unc-47 element , the motif selected for mutagenesis was a palindromic sequence ( CACGCGTG ) , which matches two overlapping instances of the AHR-1-like motif ( one on each strand ) . This sequence was mutagenized to CACAAGTG , changing the CACGC sequence on the ( + ) strand to CACAA and on the ( - ) strand to CACTT . All mutations were introduced by PCR with overlapping , opposite-facing primers carrying the mutant sequence . Primers were used to amplify plasmid DNA carrying the wild-type sequence . Following PCR , the reaction was digested with the methylation sensitive restriction enzyme DpnI to selectively digest the wild-type plasmid template . A second PCR reaction was performed , amplifying the mutagenized cis element and some flanking vector sequence . This PCR product was purified and digested for directional cloning back into the expression vector . Mutations were verified by sequencing before microinjection . Mixed-stage populations of C . elegans carrying transgenes were grown with abundant food . Worms of appropriate stages were selected . These were immobilized on agar slides with 10 mM sodium azide in M9 buffer . The slides were examined on a Leica DM5000B compound microscope under 400-fold magnification , except in S4 and S15 Figs , which include micrographs taken at 1000-fold magnification ( as labeled ) . Exposure times varied as necessary for each transgene . Each photograph showing worms in figures is composed of several images of the same individual capturing anterior , middle , and posterior sections , as well as shallow and deep focus . False-colored composite images were generated with QCapturePro . Brightness , contrast , and scaling of images were adjusted where necessary in final display items . The stronger background visible in the GFP images relative to their mCherry counterparts may have several explanations . First , GFP has higher background relative to mCherry , and the autofluorescence of the gut is detectable with GFP filters . Second , longer exposure times were necessary to capture expression of the more weakly expressing exogenous cis-regulatory elements . Finally , GFP fluorescence in the gut is a known site of off-target expression [38] . Worms were also injected with a subset of the GFP transgenes carrying the other nematode’s cis elements alone ( without a C . elegans mCherry control ) , and results were consistent ( S1 and S2 Tables , S15 Fig ) . Young adult individuals were examined for gene expression , except for elt-2 , in which case pretzel stage embryos and L1 larvae were counted . Worms without any visible fluorescence were assumed to have lost the transgene and were ignored . Presence of mCherry was a precondition for the worm to be counted , but without regard for the strength or completeness of the mCherry expression pattern . Motifs matching between C . elegans and each orthologous cis element ( identified by the Mirror tool on the GeneGrokker web server https://genegrokker . biology . uiowa . edu ) were mapped back to the orthologous sequence , and the total amount of the sequence covered by blocks of conservation of different sizes is plotted in Fig 6A . Empirical p-values for the sequence similarity of the C . elegans elements to their orthologs were calculated by generating 1000 reshuffled replicates of the C . elegans sequence . Replicates were generated using single , di- , and tri-nucleotide sampling from the C . elegans sequence . Each replicate was compared to each ortholog and scored for similarity in windows of different sizes . The distributions of these similarity scores were plotted ( Fig 6F–6I ) . The actual number of observed motif matches between the C . elegans sequence and its relevant orthologs were indicated on those distributions . The reported p-value is equal to the number of shuffled replicates that had more motif matches than the actual number , divided by 1000 . Only C . briggsae had more similar motifs than would be expected by chance . We used multicopy extrachromosomal transgenes , which could have made the detected levels of expression higher and less consistent than what would have been produced by single-copy transgenes . In previous work [36 , 37] we did determine that , at least in the case of unc-47 from C . elegans and C . briggsae , the nature of the transgene ( multi- vs . single-copy , extrachromosomal vs . integrated ) did not change the pattern , but rather the amount and consistency of expression . If the same principle holds for the genes examined here , the conserved patterns we detected represent the cell types where the foreign cis elements are truly active in C . elegans , but the expression levels could be overestimated . The fact that in most instances only subsets of the overall pattern were conserved suggests that artificially higher expression levels were not solely responsible for the conserved expression patterns we detected . Any apparent divergence—i . e . incongruence between the pattern driven by the C . elegans cis element and its orthologs—could be due to cis-regulatory changes ( in the function of the donor element ) , trans-regulatory changes ( in the function of transcription factor ( s ) in C . elegans ) , or due to the experimental combination of the two . In addition , endogenous expression patterns may have diverged between C . elegans and other species . For technical reasons , it is difficult to determine endogenous patterns of gene expression in divergent parasitic nematodes used in this study . It is even more difficult to generate transgenic animals in these species . These technical limitations make it essentially impossible to assess divergence in endogenous expression patterns or to disentangle their causes ( that is , cis vs . trans changes ) . For these reasons , we focused on enumerating similarities , rather than differences in expression . Our tests actually underestimate the extent of regulatory conservation , because a failure of a cis element from a distant nematode when tested in C . elegans may reflect a genuine divergence in cis-regulation in that species that was compensated in trans , therefore maintaining the same overall expression pattern . | To explore the phylogenetic limits of conservation of cis-regulatory elements , we used transgenesis to test the functions of enhancers of four genes from several species spanning the phylum Nematoda . While we found a striking degree of functional conservation among the examined cis elements , their DNA sequences lacked apparent conservation with the C . elegans orthologs . In fact , sequence similarity between C . elegans and the distantly related nematodes was no greater than would be expected by chance . Short motifs , similar to known regulatory sequences in C . elegans , can be detected in most of the cis elements . When tested , some of these sites appear to mediate regulatory function . However , they seem to have originated through motif turnover , rather than to have been preserved from a common ancestor . Our results suggest that gene regulatory networks are broadly conserved in the phylum Nematoda , but this conservation persists despite substantial reorganization of regulatory elements and could not be detected using naïve comparisons of sequence similarity . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
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"Methods"
] | [] | 2015 | Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence |
Leptospirosis is a zoonotic bacterial disease that affects more than one million people worldwide each year . Human infection is acquired through direct or indirect contact with the urine of an infected animal . A wide range of animals including rodents and livestock may shed Leptospira bacteria and act as a source of infection for people . In the Kilimanjaro Region of northern Tanzania , leptospirosis is an important cause of acute febrile illness , yet relatively little is known about animal hosts of Leptospira infection in this area . The roles of rodents and ruminant livestock in the epidemiology of leptospirosis were evaluated through two linked studies . A cross-sectional study of peri-domestic rodents performed in two districts with a high reported incidence of human leptospirosis found no evidence of Leptospira infection among rodent species trapped in and around randomly selected households . In contrast , pathogenic Leptospira infection was detected in 7 . 08% cattle ( n = 452 [5 . 1–9 . 8%] ) , 1 . 20% goats ( n = 167 [0 . 3–4 . 3%] ) and 1 . 12% sheep ( n = 89 [0 . 1–60 . 0%] ) sampled in local slaughterhouses . Four Leptospira genotypes were detected in livestock . Two distinct clades of L . borgpetersenii were identified in cattle as well as a clade of novel secY sequences that showed only 95% identity to known Leptospira sequences . Identical L . kirschneri sequences were obtained from qPCR-positive kidney samples from cattle , sheep and goats . These results indicate that ruminant livestock are important hosts of Leptospira in northern Tanzania . Infected livestock may act as a source of Leptospira infection for people . Additional work is needed to understand the role of livestock in the maintenance and transmission of Leptospira infection in this region and to examine linkages between human and livestock infections .
Leptospirosis is a zoonotic disease caused by infection with a pathogenic serovar of Leptospira bacteria . Worldwide , leptospirosis is estimated to affect more than one million people and result in the loss of 2 . 9 million Disability Adjusted Life Years ( DALYs ) each year [1] . The greatest burden of leptospirosis occurs in tropical and sub-tropical areas , where people live in close contact with animal hosts and warm humid conditions facilitate environmental survival of the bacteria [1 , 2] . The clinical presentation of leptospirosis ranges from a mild febrile illness to severe disease with secondary manifestations including renal failure , multiple organ dysfunction , and severe pulmonary haemorrhagic syndrome ( SPHS ) [3] . The reported median case fatality ratio is around 2% for uncomplicated leptospirosis and 12–40% in patients with more severe disease manifestations such as jaundice and renal failure [4] . Under-reporting of leptospirosis is thought to be common , particularly as human leptospirosis can be difficult to distinguish clinically from other tropical causes of fever such as malaria or dengue fever [5 , 6] . Human infection with Leptospira occurs following direct or indirect contact with the urine of an infected mammalian host [5] . To date , more than 250 pathogenic Leptospira serovars belonging to 10 different Leptospira species have been described , which infect a wide variety of animal hosts [7 , 8] . Rodents are common hosts of pathogenic Leptospira and are often considered as the most important source of human infection [3 , 5] . However , many other animals including companion animals , production livestock species such as cattle and pigs , or wildlife can also carry the infection [9] . In settings where multiple hosts and serovars are present , determining the epidemiology of leptospirosis and identifying sources of human infection can be complex and challenging . Acute leptospirosis is an important cause of human febrile disease in Tanzania . Hospital-based surveillance conducted in the Kilimanjaro Region of northern Tanzania demonstrated acute leptospirosis in 2–9% of febrile admissions [10 , 11] . Estimates of the population-level incidence of leptospirosis in the Kilimanjaro Region vary over time with 11–18 cases per 100 , 000 per year in 2012–14 [11] and 75–102 cases per 100 , 000 per year in 2007–08 [12] . A large number of different Leptospira serogroups have been implicated in human disease although the most common predominant serogroups vary by year and by study [11] . Little is known about sources of infection for people in northern Tanzania . Leptospira bacteria have been isolated from cattle , pigs and a variety of small mammal species elsewhere in Tanzania [13] . However , the roles of these animal hosts as a source of infection for people in the Kilimanjaro Region remains unclear . This study was performed to identify hosts of pathogenic Leptospira bacteria in northern Tanzania . To assess the role of rodents in the epidemiology of Leptospira infection , a cross-sectional survey of peri-domestic rodents was conducted in two districts with a high reported incidence of human leptospirosis . Sampling of cattle , sheep and goats was also performed in local slaughterhouses . The prevalence of Leptospira infection was determined by qPCR testing of kidney samples . Molecular typing of Leptospira bacteria was performed to characterise circulating Leptospira species and genotypes in animal hosts . Here , we discuss the results of these studies and their implications for our understanding of human and animal Leptospira infection in northern Tanzania .
Ethical approval for the study was granted by the Tanzania Commission for Science and Technology ( COSTECH 2012-471-ER-2005-141 ) ; Kilimanjaro Christian Medical Centre ( KCMC ) Ethics Committee ( 537 ) ; National Institute of Medical Research ( NIMR ) , Tanzania ( NIMR/HQ/R . 8a/Vol . IX/1499 ) ; Tanzania Wildlife Research Institute ( TAWIRI ) ; University of Glasgow College of Medicine , Veterinary Medicine and Life Sciences Ethics Committee ( 200120020 ) , and University of Glasgow Faculty of Veterinary Medicine Ethics and Welfare Committee ( 01a/13 & 02a/13 ) . Written consent for study participation was obtained for each participating household . Rodent sampling was performed in accordance with UK and international guidelines for humane euthanasia [14 , 15] . The study was conducted in the Kilimanjaro Region in northern Tanzania . The climate in this region follows a pattern of long rains from March to May and short rains from October to December with the coolest months coinciding with the long dry season from June to September . The region has a population of 1 . 64 million people , and an estimated population density of 124 people per km2 ( national average: 51 per km2 ) [16] . The region is divided into seven districts . Two districts , Moshi Municipal and Moshi Rural ( Fig 1 ) , were chosen as the site of the study due to the high reported incidence of human leptospirosis [12] and on-going febrile disease surveillance at local hospitals ( Fig 1 ) . Moshi Municipal District is the administrative centre of the Kilimanjaro Region . In the 2012 Tanzania National Census , the district was classified as urban and had a population of approximately 184 , 000 people [16] . Moshi Rural District has a population of approximately 467 , 000 people and is dominated by small-scale agriculture and smallholder livestock farming [16] . The environment ranges from lush high-altitude mountainous areas where coffee , bananas , and avocados dominate cash crop production , to drier low-altitude pasture land and plains where maize and beans are cultivated . Subsistence livestock farming is common . In the most recent livestock census ( 2008 ) , the populations of ruminant livestock reported were 139 , 000 cattle and 353 , 000 small ruminants ( sheep and goats combined ) for Moshi Rural District and 2 , 100 cattle and 7 , 300 small ruminants for Moshi Municipal District ( population size given to nearest 100 ) [17] . A cross-sectional survey was performed to determine the prevalence of Leptospira infection in peri-domestic rodents within the catchment area of two hospitals ( Kilimanjaro Christian Medical Centre ( KCMC ) or Mawenzi Regional Referral Hospital ( MRRH ) ) that previously identified a high prevalence of acute leptospirosis in patients with febrile illness [10 , 11] . The geographical sampling frame was composed of villages within Moshi Municipal and Moshi Rural Districts from which people had sought health care and been enrolled in fever surveillance studies at KCMC and MRRH in the preceding years ( 2012–2014 ) . One village was selected by convenience as a pilot village ( 2013 ) and eleven study villages were selected at random ( Fig 2 ) . Consent for study participation was obtained from the Village Chairperson of each study village , who also provided a list of sub-villages within their villages . A single sub-village was selected at random as the sampling location within each study village . The population size of selected sub-villages ranged from 916 to 4320 people ( Moshi Municipal: 1039–4320 people; Moshi Rural: 916–3926 people ) [18] . Using a reported average household size of 4 , this equates to approximately 229 to 1080 households per sub-village ( Moshi Municipal: 260–1080 households; Moshi Rural: 229–935 households ) [16 , 18] . Study maps ( Figs 1 & 2 ) were made using Quantum Geographic Information System ( QGIS ) open access software [19] . Shapefiles for Tanzania country boundaries , regions and districts from the most recent census were obtained from Tanzania National Bureau of Statistics [16 , 20] . A single representative location for each study village was defined by recording the GPS co-ordinates for the administrative centre of each sampled sub-village . Rodent trapping was performed in three sampling periods: 1 ) May-June 2013 ( wet season ) ; 2 ) May-June 2014 ( wet season ) ; and 3 ) August-September 2014 ( dry season ) . The target sample size was 50 rodents per sub-village to give sufficient power ( α = 0 . 95 , β = 0 . 8 ) to detect a minimum Leptospira infection prevalence of 10% [21–24] . Based on a predicted average trap success of 12 . 5% [22 , 25] , 100 traps were set for a target of four nights to give a trapping effort of 400 trap nights per sub-village with the exception of the pilot village ( A ) , where only 50 traps were used . Following initial trapping ( villages A & B ) , the number of nights was increased to an average of eight ( trapping effort of 800 trap nights ) per sub-village due to lower than expected trapping success . Sampling transects were established in each sub-village using a method based on the World Health Organization ( WHO ) Expanded Program for Immunization ( EPI ) random walk method for cluster sampling [26 , 27] . The administrative centre of each sub-village was used as the starting point for sampling transects . The direction of each transect was determined at random within the sub-village ( defined by spinning a pen in the field ) and ran from the centre of the sub-village to its peripheral boundary . Households were recruited along each transect ensuring a minimum distance of 50 metres between each household until 20 households had been recruited . Five rodent traps were set in each participating household . In 2013 , four large Sherman traps ( HB Sherman Traps , Tallahassee , USA . Dimensions: 7 . 6 x 8 . 9 x 22 . 9 cm ) and one small Sherman trap ( dimensions: 5 . 1 x 6 . 4 x 22 . 9 cm ) were set in each household . In 2014 , the trapping approach was adjusted and one large Sherman trap per household was replaced with a Tomahawk trap ( Tomahawk Live Trap , Hazelhurst , USA . Model 602; dimensions 12 . 7 x 12 . 7 x 40 . 6 cm ) . Traps were placed in kitchens , food storage areas , and animal housing areas within each household and in sheltered outdoor areas within each compound ( e . g . adjacent to animal houses , fence lines and in log piles ) . A stiff mixture of peanut butter and oats and chopped carrots was used to bait Sherman traps . Dried fish was used to bait Tomahawk traps . Traps were checked and reset each morning . Traps containing rodents were removed and replaced . Trapped rodents were euthanised by terminal halothane anaesthesia and cervical dislocation . The species of each trapped rodent was determined by observation of phenotypic characteristics and measurement of morphometric features [28 , 29] . Rodent sex and age class ( mature or immature ) was determined based on external sexual characteristics [29] . A full necropsy and tissue sampling was performed . For detection of Leptospira infection , one kidney from each rodent was collected and preserved in 70–96% ethanol at room temperature prior to testing by real-time PCR ( qPCR ) . For a subset of rodents , kidney tissue was also collected for Leptospira culture . Culture was attempted opportunistically during the randomised cross-sectional survey in Villages C , D , E & M based on availability of culture media . In addition , to maximise the chance of Leptospira culture success , the village with the highest trap success in the cross-sectional survey ( Village F ) was re-visited in September 2014 for repeat rodent trapping and sampling for culture . In this village , trapping was repeated in the 20 previously recruited households using the same strategy ( 100 traps x 8 nights ) . Rodent sampling was performed as described above , and kidney tissue was collected for qPCR and culture . Ruminant livestock ( cattle , goats and sheep ) was sampled in slaughterhouses within the Moshi Municipal District . Five slaughterhouses were selected for livestock sampling in liaison with the District Veterinary Officer based on high slaughter throughput ( ranging from 14 and 210 cattle per week ) , accessibility of location and cooperation from livestock field officers responsible for meat hygiene inspection at each of the slaughterhouses . GPS co-ordinates were recorded at each participating slaughterhouse ( Fig 2 ) . The target sample size for cattle ( n = 323 ) was selected to give the study sufficient power to estimate the prevalence of infection with a precision of 5% based on seroprevalence estimates of 30% [30] . Goat and sheep sampling was performed opportunistically at the same slaughterhouses . Livestock sampling was performed between May 2013 and September 2014 . A maximum of ten animals per species were sampled per slaughterhouse per day . The source ( region , district and market of origin ) , approximate age ( adult vs . juvenile ) , gender , and breed ( indigenous , exotic or cross-breed ) were recorded for each animal . Kidney samples were collected during evisceration into a clean , labelled , single-use Ziplok bag . Following surface sterilisation with a flamed blade , samples of kidney tissue ( approximately 3 x 1 x 1 cm ) spanning the cortico-medullary junction were taken using a sterile blade and placed directly into 70–96% ethanol prior to testing by qPCR . Samples of kidney tissue were also collected for Leptospira culture from an opportunistically selected subset of cattle and goats . The prevalence of renal Leptospira infection in livestock and rodents was determined by qPCR testing . DNA was extracted from 25 milligrams ( mg ) of kidney tissue preserved in ethanol using the QIAamp DNA Mini Kit spin-column protocol for DNA purification from tissues ( Qiagen , Maryland , USA ) . The DNA concentration was quantified using a NanoDrop spectrophotometer ( ThermoScientific , Waltham , MA ) and stored at -20°C prior to qPCR testing . DNA extracts were tested for pathogenic Leptospira spp . using a lipL32 TaqMan qPCR assay run on the ABI 7500 Real-Time PCR system ( Applied Biosystems , Foster City , CA ) as previously described [31 , 32] . Amplification of a 245 bp product was performed using the primer set: lipL32-45F ( 5’-AAG CAT TAC CGC TTG TGG TG-3’ ) and lipL32-286R ( 5’-GAA CTC CCA TTT CAG CGA TT-3’ ) , and a 19-bp 5’FAM-labelled probe with a 3’BHQ quencher dye ( FAM-5’-AA AGC CAG GAC AAG CGC CG-‘3-BHQ1 ) . Low concentration ROX ( 50nmol/L ) was added to the final reaction mix as a passive reference to improve the diagnostic sensitivity and specificity of the assay [33] . DNA extracts were diluted 1:10 in PCR grade water to reduce the effects of PCR inhibitors . Amplifications were performed using 5μl of diluted template DNA ( approximately 50 to 150ng ) per 25μl qPCR reaction . Samples were tested in duplicate . Two replicates of a Leptospira positive control , L . interrogans serovar Copenhageni Strain Wijnberg were also run per reaction plate . Control DNA was sourced from the WHO/FAO/OIE Collaborating Leptospirosis Reference Laboratory in Amsterdam and tested at a concentration of 1 pg of DNA ( approximately equal to 102 genomic equivalents ) per 25μl qPCR reaction . In addition , two replicates of a non-template extraction control , and two replicates of PCR-grade water were included on each test plate . Reaction profiles were analysed using Applied Biosystems 7500 System Sequence Detection ( SDS ) Software Version 1 . 2 . 4 ( Applied Biosystems , Carlsbad , CA 2001–2004 ) . A qPCR plate run was considered valid when all negative controls were negative and at least one replicate of the Leptospira positive controls amplified with cycle threshold ( Ct ) value < 40 . Samples were considered positive when at least one test well amplified the lipL32 target with a Ct value < 40 . For qPCR-positive samples , the infecting Leptospira species was determined through amplification and sequencing of a conserved 470-bp segment of the secY gene previously shown to have phylogenetic discriminatory power for pathogenic Leptospira species [34 , 35] . PCR assays optimized for use in eastern Africa were run at the University of Aberdeen following published protocols [36] . Amplifications were performed using 5μl undiluted template DNA in a 25μl PCR reaction using the primer set: secYFd ( 5’-ATG CCG ATC ATY TTY GCT TC-3’ ) and secYR3 ( 5’-TTC ATG AAG CCT TCA TAA TTT CTC A-3’ ) . All PCR assays included one non-template control ( PCR grade water ) per five test samples and a positive control of DNA extracted from a pure isolate of L . interrogans or L . borgpetersenii . PCR products were visualised by gel electrophoresis on a 1 . 5% agarose gel and purified using the QIAquick PCR Purification Kit following manufacturer’s instructions ( Qiagen , Maryland , USA ) . Purified products were quantified using a Nanodrop ND1000 spectrophotometer ( ThermoScientific , Massachusetts , USA ) and sequenced by Eurofins Genomics GmbH ( Ebersburg , Germany ) . Leptospira culture was performed from kidney tissue samples collected from a total of 98 rodents , 100 cattle , and 49 goats . Following kidney collection , the renal capsule was sterilised using a hot flamed blade and approximately 25 mg of kidney tissue was dissected across the cortico-medullary junction . Tissue was immediately homogenised in 1ml of Ellinghausen-McCullough-Johnson-Harris ( EMJH ) culture media supplemented with 0 . 4mg/ml of fluorouracil ( 5’FU ) ( EMJH-5FU media ) supplied by the WHO/FAO/OIE Collaborating Leptospirosis Reference Laboratory in Amsterdam . A ten-fold dilution series ( 1:10 , 1:100 , 1:1000 ) was prepared in three tubes with 5 ml of EMJH-5FU . Inoculated aliquots of culture media were shipped to the WHO/FAO/OIE Collaborating Leptospirosis Reference Laboratory in Amsterdam for Leptospira isolation . Cultures were incubated at 30°C and checked for Leptospira growth by dark-field microscopy every four weeks for three months and then again after six months of incubation . Positive cultures were confirmed by secY qPCR [37] and sub-cultured in EMJH media prior to typing . Leptospira isolated by culture were typed using serological and genetic methods at the WHO/FAO/OIE Collaborating Leptospirosis Reference Laboratory in Amsterdam . Serological typing of pathogenic Leptospira isolates was performed by microscopic agglutination test in two stages . First , a panel of polyclonal rabbit antisera raised against 24 Leptospira serogroups was used to determine the serogroup of isolates [38] . Subsequently , a panel of 18 serovar-specific mouse monoclonal antibodies was used to determine the isolate serovar [39 , 40] . Sequence type was determined using a multi-locus sequence typing ( MLST ) scheme targeting seven Leptospira housekeeping genes ( glmU , pntA , sucA , tpiA , pfkB , mreA and caiB ) following published protocols [41] . PCR amplicons were sequenced by Macrogen Europe ( Amsterdam , Netherlands ) . Trimmed sequences were aligned against reference sequences for the MLST scheme ( obtained from PubMLST; Leptospira Scheme #1: http://pubmlst . org/leptospira/ ) to generate a unique allelic profile for each isolate [42 , 43] . Finally , each allelic profile was compared to an online database of 223 profiles to determine the sequence type ( ST ) and Leptospira serovar [41] . Phylogenetic analysis was performed using MEGA7 . 0 software [44] . Leptospira secY sequences from qPCR positive samples and Leptospira isolates obtained in this study were trimmed and then aligned using the ClustalW algorithm in MEGA with secY sequences from 128 Leptospira reference serovars obtained through GenBank [34 , 45] . The model test function in MEGA was used to select the most appropriate nucleotide substution model for the aligned sequences . Phylogenetic analysis was performed using a maximum likelihood method with 500 bootstrap repeats to generate the final phylogenetic tree . Adjusted trap success was used as a measure of relative rodent abundance in each sub-village [46] . Adjusted trap success was calculating by dividing the total number ( n ) of rodents caught per sub-village by the corrected number of trap nights ( Total number of trap nights ( number of traps x number of nights ) minus lost trap nights ( sum of number of closed , damaged or lost traps / 2 ) and expressed as a percentage ) . Statistical analysis was performed in R [47] . Two-sample T-tests were used to compare the adjusted trap success and proportion of households with rodents between the two study districts . Binomial confidence intervals for point prevalence estimates ( Wilson method ) were calculated using the Hmisc package [48] . Fisher’s exact tests were performed to compare the prevalence of infection between animal species , and between sex and age groups within-species .
Overall , five villages in Moshi Municipal District and seven villages in Moshi Rural District were selected for inclusion in this study . A summary of selected village details is given in Table 1 . During the randomised cross-sectional survey , 351 rodents were trapped across the 12 selected villages . Rodents were trapped in 60 . 0% of all participating households . The adjusted trap success by village ranged from 1 . 94 to 10 . 4% ( median = 4 . 42% ) . Overall , no significant differences were observed in the adjusted trap successes ( two sample t-test: p = 0 . 690 ) or average proportion of households with trapped rodents ( two-sample t-test: p = 0 . 124 ) between the two study districts . In addition , a further 33 rodents ( R . rattus: n = 21 , 63 . 6% and M . musculus: n = 12 , 36 . 4% ) were trapped from 80 . 0% of households during repeat sampling in village F ( adjusted trap success of 4 . 42% ) . In total , 384 rodents were trapped in this study and were tested for Leptospira infection . Of these , 221 ( 57 . 6% ) were female and 225 ( 58 . 6% ) were classified as sexually mature based on external sexual characteristics . The most common species trapped was the black rat ( Rattus rattus ) ( n = 320 , 85 . 1% ) . Other species included house mice ( Mus musculus: n = 44 , 11 . 5% ) ; multimammate mice ( Mastomys natalensis: n = 8 , 2 . 08% ) ; spiny mice ( Acomys spp . : n = 6 , 1 . 56% ) ; African pygmy mice ( Mus minutoides: n = 3 , 0 . 781% ) ; and striped bush squirrels ( Paraxerus flavovittis: n = 3 , 0 . 781% ) . Kidney samples were collected from 452 cattle , 167 goats , and 89 sheep . Cattle were sampled at all five slaughterhouses included in this study ( median per site = 70; range = 6–273 ) . Opportunistic sampling of sheep was performed at three slaughterhouses ( median = 40; range = 2–47 ) and goats at two slaughterhouses ( range = 12–141 , slaughterhouse information not recorded for 14 animals ) . Based on visual assessment of physical characteristics , 439 ( 97 . 1% ) cattle , 165 ( 98 . 8% ) goats and 88 ( 98 . 9% ) sheep were classified as indigenous breeds . The majority of animals were male ( cattle: n = 370 , 81 . 9%; goats: n = 117 , 70 . 1%; and sheep: n = 47 , 53 . 8% of sheep ) and 93 . 2% of animals were adult ( cattle: n = 405 , 89 . 6%; goats: n = 135 , 80 . 8%; and sheep: n = 77 , 86 . 5% ) . Almost all ruminant livestock sampled in this study originated from areas outside the core study districts of Moshi Municipal and Moshi Rural ( S1 Table ) . Of 452 cattle sampled , 381 ( 84 . 3% ) originated from the Manyara Region ( Fig 1 ) , mainly from the districts of Mbulu ( n = 296 ) and Babati ( n = 65 ) . Of five cattle that originated from the Kilimanjaro Region , only one originated from either of the Moshi districts ( Moshi Rural District , n = 1 ) . All small ruminants sampled in this study originated from either the Arusha or Manyara Regions ( S1 Table ) . Renal infection with pathogenic Leptospira spp . was detected by lipL32 qPCR in 32 ( 7 . 1% ) cattle , 2 ( 1 . 2% ) goats , and 1 ( 1 . 1% ) sheep ( Table 2 ) . Leptospira infection was not detected in any of 384 rodent kidney samples tested by lipL32 qPCR ( Table 2 ) . Statistically significant differences in the prevalence of infection were detected in pairwise comparisons between cattle and small ruminants , and cattle and rodents ( Fisher’s Exact Test , p < 0 . 05 ) . The odds ratio ( OR ) of cattle Leptospira infection was 6 . 26 ( 95% confidence interval ( CI ) : 1 . 57–54 . 5 ) when compared to goat infection; and 6 . 75 ( 95% CI: 1 . 10–278 ) when compared to sheep infection . Compared to rodents , cattle were also significantly more likely to be infected with Leptospira ( 95% CI: 7 . 41 –Inf ) . No significant differences in infection prevalence were observed in pairwise comparisons between goats , sheep or rodents ( Fisher’s Exact Test , p > 0 . 05 ) . For ruminant livestock species , no significant differences were observed in infection prevalence by qPCR between male and female , or adult or juvenile animals ( Fisher’s exact tests; p > 0 . 05 ) . Leptospira was successfully isolated from four cattle kidneys from the subset of cattle tested by Leptospira culture ( n = 100 ) . All four Leptospira isolates derived from cattle kidneys were typed as L . borgpetersenii serovar Hardjo ( Hardjo-bovis ) , serogroup Sejroe ( ST 152 ) [43] . No Leptospira growth was detected from the subset of rodents ( n = 98 ) or goat samples ( n = 49 ) that were tested for Leptospira infection by culture . Identification of infecting Leptospira species by amplification and sequencing of the secY gene was successful for 19 ( 54 . 3% ) of 35 qPCR-positive kidney samples ( Table 3 ) . L . borgpetersenii was the most common infecting Leptospira species and was identified in 13 ( 72 . 2% ) of 17 cattle samples with secY sequence available for analysis . Phylogenetic analysis revealed two distinct clades of L . borgpetersenii sequence ( Fig 3 ) . Sequences from eight cattle samples showed 100% sequence identity with L . borgpetersenii serovar Hardjo isolates obtained in this study ( Fig 3: Isolate C0097 and C0101 ) . Sequences from five cattle samples formed a separate clade within the L . borgpetersenii species , which was distinct from all reference sequences . Leptospira kirschneri , was identified in qPCR-positive samples from one cattle , one goat , and one sheep . Sequences from small ruminants ( Fig 3: C0417 and C0481 ) and one bovine ( Fig 3: C0059 ) showed 100% identity to each other as well as to several reference serovars including three serovars isolated human leptospirosis cases in the Democratic Republic of Congo ( DRC: Kambale ( EU358030 ) , Ndambari ( EU358001 ) and Ndahambukuje ( EU358002 ) ) . Infecting Leptospira species could not be determined by secY sequence analysis for a clade of three cattle samples ( Fig 3: C0221 , C0223 and C0236 ) . In the final phylogenetic tree , the clade containing these sequences appeared most closely related to L . kirschneri but showed only 95% similarity with the closest available reference sequences . GenBank searches also failed to identify any more similar Leptospira species or serovars .
In this investigation of animal hosts of pathogenic Leptospira in northern Tanzania , Leptospira infection was detected in ruminant livestock but not in rodents sampled in two districts with a high reported incidence of human leptospirosis [10 , 11] . No evidence of infection was detected in any of 384 peri-domestic rodents trapped in a cross-sectional survey conducted across a two-year period at 12 randomly selected sites . In contrast , slaughterhouse sampling of ruminant livestock detected Leptospira infection in cattle ( 7 . 06% ) , goats ( 1 . 20% ) and sheep ( 1 . 11% ) . Two infecting Leptospira species were detected in ruminant livestock , including L . borgpetersenii in cattle and L . kirschneri in cattle , goats and sheep . A novel Leptospira genotype was also detected in cattle that showed relatively little sequence similarity ( 95% ) to known Leptospira species . The absence of Leptospira infection in the rodents is a notable finding of this study . Worldwide , rodents are frequent carriers of pathogenic Leptospira bacteria [3 , 6] and are often described as the most common source of human infection [3] . However , the lack of detectable infection in our study , which was conducted in two districts where the incidence of human leptospirosis is known to be high [10 , 11] , indicates that peri-domestic rodents are not a major source of Leptospira infection for people in this area . Although these results were unexpected , we consider that they are robust . Diagnostic protocols used to test rodent samples were consistent with those used in other species ( e . g . cattle ) that yielded positive results . Rodent sampling was performed at 12 randomly selected villages over a two-year period and the total sample size achieved by our study ( n = 384 ) had sufficient statistical power to demonstrate freedom from infection at the 95% confidence level , even allowing for a low prevalence of infection ( e . g . 1 . 0% ) [21 , 49] . The reasons for a lack of detectable Leptospira infection in the rodents sampled in our study are unclear . Rattus rattus , the most common species sampled in our study , is globally widespread invasive rodent species that has been demonstrated as a carrier host of Leptospira infection in other settings [23 , 50 , 51] . Infection has been reported in these species in other African countries [52] , including in a study conducted by the authors ( KJA , JEBH , AA , RAH ) in neighbouring Kenya , where Leptospira was detected in R . rattus ( 9 . 1%; n = 33 ) in the Kibera slums [22] . However , to date , no published studies of R . rattus in Tanzania ( e . g . [13 , 24 , 53] ) have demonstrated Leptospira infection by culture or PCR . Therefore , despite their prominent role in other settings , there is very little evidence to suggest that this species are important hosts of Leptospira in northern Tanzania . To date , Leptospira infection has only been reported in indigenous rodent species such as the African pouched rats ( Cricetomys spp . ) and multimammate mice ( Mastomys natalensis ) [13 , 54] that typically live outside of domestic environments . Although both rodent species are reported to live in the Kilimanjaro Region [28] , Cricetomys was not trapped in our study and M . natalensis was trapped in very low numbers ( n = 8 ) that may have been insufficient to detect low levels of infection in this host population . Another notable absence in the study was the lack of Norway rats ( Rattus norvegicus ) , which is considered the definitive maintenance host of several Leptospira serovars including L . interrogans serovars Copenhagenii and Icterohaemorrhagiae worldwide [9 , 55] . The apparent absence of key maintenance hosts of rodent-associated Leptospira serovars such as Cricetomys or R . norvegicus at our study sites is one possible explanation for the lack of infection in the rodents trapped and tested in this study . In contrast , cattle Leptospira infection appears to be widespread across Tanzania . In this study , bovine Leptospira infection was detected in cattle originating from Manyara , Arusha , Dodoma , Singida and Tanga Regions ( S1 Table ) . Infection has also been reported in cattle sampled in the Morogoro Region [56] . A degree of caution should be exercised in extrapolating estimates of cattle Leptospira prevalence from slaughterhouse studies to the source population . Selection biases for animals sent for slaughter and the potential for increased probability of infection associated with mixing of animals in markets and during transport may increase the prevalence of some infections in slaughterhouse populations [57 , 58] . Further sampling of resident livestock in the study districts is necessary to understand the local prevalence and epidemiology of infection in these populations . Demonstration of renal Leptospira carriage in small ruminant hosts in this study is a novel finding for sub-Saharan Africa . Leptospira infection is well-documented in small ruminants in other parts of the world ( e . g . goats in Brazil [59] and sheep in New Zealand [60] ) but there have been few studies of small ruminants as hosts of Leptospira infection in the African continent . Goats and sheep are important production livestock in Tanzania [61] . Small ruminant ownership is common and people live in close contact with their livestock in our study area [62] . Detection of renal infection in goats and sheep demonstrates that small ruminants in this setting also carry and shed pathogenic Leptospira in this setting and corroborates serological findings from elsewhere in Tanzania [63] . Small ruminants therefore also have the potential to act as sources of infection for people . Multiple species and genotypes of pathogenic Leptospira were detected in infected ruminant livestock sampled in this study . Leptospira borgpetersenii was the predominant species infecting cattle . L . borgpetersenii serovar Hardjo was isolated from four cattle , supporting previous serological evidence for the presence of this serovar in Tanzania [63–66] . L . borgpetersenii sequence was also detected in 13 ( 76 . 5% ) of 17 qPCR cattle with successful secY amplification . Sequences derived from eight qPCR-positive cattle samples were identical to those from L . borgpetersenii serovar Hardjo isolates . A second L . borgpetersenii genotype was detected in 5 ( 29 . 4% ) cattle samples , which showed only 98% identity to the most similar reference serovars . GenBank BLAST searches identified Leptospira qPCR-positive samples with identical secY sequences in cattle from Brazil ( KP862647 . 1 ) [67] . The presence of this L . borgpetersenii type in multiple international cattle populations suggests that this Leptospira type could be globally widespread in cattle . Leptospira kirschneri was the second Leptospira species identified in ruminant livestock species . L . kirschneri sequences derived from cattle , goats and sheep in this study showed 100% identity to each other and to seven other reference serovars ( serovars Bim , Bogvere , Kambale , Mozdok , Ndambari , Ndahambukujue , Tsaratsovo ) . Two serovars , L . kirschneri serovar Grippotyphosa and L . kirschneri serovar Sokoine , have previously been isolated from Tanzanian cattle and showed a high degree of similarity to L . kirschneri genotypes detected in this study ( > 99% ) [13 , 56] . Notably , a clade of novel secY sequences was also detected in cattle qPCR-positive samples that could not be attributed to any Leptospira species by phylogenetic analysis . Sequences derived from three cattle infections were identical to each other but distinct from any reference sequences used in the phylogenetic analysis for this study . BLAST searches conducted in GenBank also failed to identify any similar sequences from other studies . Two possible explanations exist to describe the relationship of this clade of novel sequences to the rest of the Leptospira genus . First , these sequences could represent a divergent clade of L . kirschneri , which is the most similar known Leptospira species . However , sequence variation of 5% in the secY gene is the reported threshold of the difference observed between Leptospira species [34] . Therefore , an alternative explanation is that this clade represents a new and previously undescribed Leptospira species . Further work is needed to determine the species and fully characterise this novel Leptospira genotype . The secY single-locus genotyping approach is this study provides a robust initial assessment of the diversity of Leptospira species circulating in Tanzanian livestock . The high degree of similarity between some of the livestock sequences identified in this study and sequences from human infections elsewhere in sub-Saharan Africa ( e . g . DRC and Kenya , see Fig 3 ) suggests that livestock may be an important source of Leptospira infection for people across the eastern and central African region . To date , there are no secY sequences derived from human Leptospira infection in northern Tanzania , limiting our ability to use genomic data to compare infecting Leptospira species between human and livestock populations . Serological data from human cases in Tanzania does exists [10 , 11] but the poor correlation between genotype and serogroup for Leptospira bacteria limits our ability to robustly link these data to attribute sources of Leptospira infection [7 , 68] . However , epidemiological studies have identified milking cattle , feeding and cleaning cattle and handling cattle waste as significant risk factors for human Leptospira infection in Moshi and neighbouring regions [69 , 70] . These findings suggest that cattle are indeed an important source of Leptospira infection for people in northern Tanzania and provide a strong rationale for further investigation linked human and cattle populations to better understand the relationship between human and bovine infection . Overall , our study makes a substantial contribution to the growing body of evidence that livestock play an important role in the epidemiology of human leptospirosis in sub-Saharan Africa . Although the contribution of other species cannot be ruled out , contact with livestock has been demonstrated as an important risk factor for human Leptospira infection in northern Tanzania [70] . Occupational exposure to infected livestock is known to be an important risk factor for human leptospirosis in other settings [71] and currently more than 75% of the Tanzanian population is estimated to be employed in the agriculture sector [61] . Given the importance of leptospirosis as a cause of human febrile illness in Tanzania [72] , quantifying the contribution of livestock-associated leptospirosis to human health and understanding the factors that support the maintenance and transmission of pathogenic Leptospira in livestock populations are important priorities for future leptospirosis and public health research . | Leptospirosis is a globally important disease that is transmitted from animals to people and affects more than 1 million people worldwide each year . Leptospirosis is an important cause of febrile illness in northern Tanzania but little is known about the animal hosts of Leptospira infection for people in this area . This study aimed to evaluate the role of rodents and ruminant livestock ( cattle , sheep and goats ) in the epidemiology of Leptospira infection in northern Tanzania . The results of our study showed that ruminant livestock but not rodents are commonly infected with pathogenic Leptospira infection . Genetic typing identified four distinct types of Leptospira in livestock , including three types that were only identified in cattle , and one type that was identified in cattle , goats and sheep sampled in our study . These results indicate that livestock are a potential source of infection for people in Tanzania . This finding is important as a large proportion of the human population are employed in farming activities or keep ruminant livestock at home . Further work is needed to understand which Leptospira types are transmitted in our setting and to understand how livestock infection contributes to human disease . | [
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] | 2018 | Assessment of animal hosts of pathogenic Leptospira in northern Tanzania |
Opisthorchiasis is a parasitic infection caused by the liver flukes of the Opisthorchiidae family . Both experimental and epidemiological data strongly support a role of these parasites in the etiology of the hepatobiliary pathologies and an increased risk of intrahepatic cholangiocarcinoma . Understanding a functional link between the infection and hepatobiliary pathologies requires a detailed description a host-parasite interaction on different levels of biological regulation including the metabolic response on the infection . The last one , however , remains practically undocumented . Here we are describing a host response on Opisthorchiidae infection using a metabolomics approach and present the first exploratory metabolomics study of an experimental model of O . felineus infection . We conducted a Nuclear Magnetic Resonance ( NMR ) based longitudinal metabolomics study involving a cohort of 30 animals with two degrees of infection and a control group . An exploratory analysis shows that the most noticeable trend ( 30% of total variance ) in the data was related to the gender differences . Therefore further analysis was done of each gender group separately applying a multivariate extension of the ANOVA—ASCA ( ANOVA simultaneous component analysis ) . We show that in the males the infection specific time trends are present in the main component ( 43 . 5% variance ) , while in the females it is presented only in the second component and covers 24% of the variance . We have selected and annotated 24 metabolites associated with the observed effects and provided a physiological interpretation of the findings . The first exploratory metabolomics study an experimental model of O . felineus infection is presented . Our data show that at early stage of infection a response of an organism unfolds in a gender specific manner . Also main physiological mechanisms affected appear rather nonspecific ( a status of the metabolic stress ) the data provides a set of the hypothesis for a search of the more specific metabolic markers of the Opisthorchiidae infection .
Opisthorchiasis is a parasitic infection caused by the liver flukes of the Opisthorchiidae family ( Trematoda; Platyhelminthes ) . The family includes the three most important species for human health: C . sinensis , O . viverrini , O . felineus; together they are responsible for more than 45 million infections worldwide; 600–750 million people are currently at risk [1 , 2] . O . viverrini and C . sinensis are endemic to the Far East regions and South East Asia remaining an important public health problem [3] . O . felineus infection is highly prevalent in Eastern Europe ( Ukraine and the European part of Russia ) , Central Asia ( northern Kazakhstan ) and North Asia ( Siberia ) [2] . Both experimental and epidemiological data strongly support a role of liver fluke infections in the etiology of the hepatobiliary pathologies such as chronic forms of cholecystitis , cholangitis , pancreatitis , and cholelithiasis [2–5] . However , the most threatening effect of the Opisthorchiidae infection is an increased risk of intrahepatic cholangiocarcinoma [6] . Unlike other pathogens , e . g . viruses and microbes , parasitic helminthes do not proliferate within their mammalian host . Accordingly , the intensity of infection is one of the leading causes of the morbidity . For instance , O . viverrini associated symptoms occurred more frequently in those with high intensities of infection [7] and positive association between hepatobiliary pathology and O . viverrini infection intensity is well documented [4] . Moreover , the biliary tract abnormalities more often detected by ultrasonography in the patients with heavy O . viverrini infections than light or moderate ones [8] . However , despite clear phenomenological and epidemiological evidence linking Opisthorchiidae infection and hepatobiliary pathology , our understanding of the mechanistic and descriptive biochemistry of the association is still rather poor . Only the humoral response to O . viverrini is reasonably well documented; for instance , a study on the infected hamsters showed that a titer of the parasite-specific IgG in the acute stage is directly correlated to the intensity of infection , as determined by both worm burden and eggs per gram ( EPG ) counts . A more discriminative approach where the pathogen specific antibodies were divided according to their specificity to egg , excretory–secretory and somatic antigens , shows an interesting kinetic of humoral response: while in the acute phase of infection the antibody response was higher in the animals infected with higher dose of metacercariae , in chronic phase higher responses , particularly to somatic and egg antigens , were found in the lightly infected hamsters [9] . A recent report of Khoontawad et al [10] represents the most detailed proteomics work on O . viverrini infection published so far . The authors explore the differential protein expression in the host tissue with a chronic inflammation versus the tissue samples from the subjects with O . viverrini induced cancer . With regard to the metabolomics , a post-genomic discipline aiming at studying the metabolites—the end points and the intermediate products of the metabolism , the data is simply not available . The metabolome of body fluids is the closest approximation of the physiological phenotype of an organism and , as such , it represents an important , but still undervalued , source of clinical/physiological information [11] . The dynamic character of the metabolome , its ability to change in response to the external stimuli makes it an optimal “readout” for exploratory studies describing the systemic responses of an organism . Whereas in schistosomiasis a number of exploratory metabolomics studies have been performed in animal models and patient material[12–16] , there are so far no such studies in Opischthorchiasis . Here we present the first exploratory metabolomics study of an experimental model of O . felineus infection . Using an established hamster infection model we conducted a nuclear magnetic resonance ( NMR ) based metabolomics study involving a cohort of 30 animals with two degrees of infection ( severe and mild ) and a control group . Urine samples were collected every two weeks for a period over several months . Using a combination of unsupervised and supervised multivariate statistical analysis we were able to discriminate the time-resolved urinary metabolic patterns of the infection .
All hamsters used in the study were handled according the recommendations of the national guidelines for animal caring: 12 . 08 . 1977 N 755 "On measures to further improve the organizational forms of work using experimental animals" . The study was approved by the local ethical committee of Siberian State Medical University with a license number 3296 issued on 29 . 04 . 2013 . Hamsters Mesocricetus auratus were purchased from the animal facility of the Institute of Bioorganic Chemistry Academicians M . M . Shemyakin and U . A . Ovchinnikov . Metacercariae of O . felineus were obtained from naturally infected fishes sold in the local supermarkets as a consumption product . The muscular tissue and the subcutaneous tissue were digested by pepsin-HCl and viable metacercariae were collected and identified by microscopy . Hamsters were divided in 3 groups: high intensity of infection ( 50 metacercariae /hamster ) , low intensity of infection ( 15 metacercariae/hamster ) and uninfected ( vehicle—PBS ) groups . Each group consisted of 10 animals- five males and five females . Hamsters were housed in separate cages , maintained on a 12:12 light-dark cycle ( 0600–1800:1800–0600 hs ) , and provided food and water ad libitum for the duration of the experiment . Hamsters were approximately five weeks old at the time of infection . Animals were sacrificed after 46 weeks of infection Livers were collected at the time of the sacrifice to count the adult worms . Urine and feces were collected every two weeks , specifically at the weeks 0 , 2 , 4 , 6 , 8 , 10 , 12 , 14 , 16 , 18 , 20 , 22 , 24 , 26 , 28 , 30 and 32 after infection . For urine and feces collection hamster were placed individually into sterile empty glass crater and observed until urine and feces pellets were generated . Urine and fecal samples were collected into individually labelled Eppendorf tubes , and transferred to a freezer ( -80°C ) for long term storage . The mean number of eggs per gram of feces was calculated following the modified Kato method ( Katz et al . 1972 ) . The fecal sample from each animal in the examined time point were mixed by Mini-Beadbeater-16 ( Bio-Spec ) for 5 minutes . A sample 25 mg of the feces was weighed with an electronic scale ( Sartorius type 1702 , sensitivity 0 . 1 mg ) immediately after homogenization . Two slides were prepared for each 25 mg of feces . The extrapolation of the egg counts per 25 mg sample to the eggs per gram values was done by a simple multiplication ( x 40 ) . All chemicals used for the buffer solution were purchased from Sigma-Aldrich except for the 2H2O which was purchased from Cortecnet and the 3- ( trimethylsilyl ) propionic-2 , 2 , 3 , 3-d4 acid sodium salt ( TSP ) from Cambridge Isotope Laboratories Inc . 96-well plates and NMR tubes were purchased from Bruker Biospin Ltd . ( Germany ) . Aliquots of 0 . 5 ml urine per sample were thawed overnight at 4°C . Cellular components and other insoluble material were then spun down by centrifugation for 10 min at 3184 g and 4°C and the supernatants were transferred into 96-well plates . 270 μL of urine from each sample were mixed with 30 μL buffer solution in 2H2O ( pH = 7 . 4 ) containing 1 . 5 M K2HPO4 , 2 mM NaN3 and 4 mM of TSP-2 , 2 , 3 , 3-d4 as an internal standard and chemical shift reference ( 0 . 4 mM final concentration in each sample ) . Finally , 165 μL of each urine-buffer mixture were transferred to 3 mm SampleJet NMR tubes and placed in refrigerated racks ( 6°C ) of a SampleJet system until the NMR measurements . Both mixing of urine with buffer and transfer of the mixture to NMR tubes were performed by two 215 Gilson liquid handler robots and controlled by the SampleTrack software ( Bruker Biospin Ltd . ) . NMR data were recorded using a Bruker 14 . 1T AVANCE II spectrometer for 1H 600 MHz , equipped with a triple resonance inverse cryoprobe ( TCI ) . Each sample was allowed to sit in the probe for 5 min to adopt a stable temperature at 27°C before starting the calibration routines and data acquisition . The probe was then automatically tuned and matched , followed by shimming and proton pulse calibration . One-dimensional ( 1D ) 1H NMR spectra were recorded using the first increment of a NOESY pulse sequence[17] ( noesygppr1d in Topspin 3 . 0 library ) . Water signal suppression was achieved with pre-saturation , a continuous wave irradiation of 50 Hz soft pulse during the relaxation delay of 4 s and the mixing time of 10 ms . The spectral width was set to 20 ppm ( 12335 Hz ) and 16 scans of 65536 points were collected . The recorded free induction decays ( FIDs ) were Fourier transformed and a line broadening of 1 . 0 Hz was applied . The spectra were automatically phased and baseline corrected and referenced to the internal standard chemical shift ( TSP; δ 0 . 0 ppm ) . An evaluation of spectra quality was performed after processing . Peaks line-width was evaluated with the TSP singlet and Alanine’s methyl protons doublet . In addition , the efficiency of water suppression and the quality of the baseline were also checked . Spectra which failed to fulfil the quality criteria were discarded from further analysis . Two dimensional J-resolved spectra ( 2D Jres ) were also collected for each sample using the same water suppression scheme as described above during the relaxation delay of 2 s . The spectral width was set to 16 . 66 ppm ( 12288 Hz ) for the direct dimension and 78 Hz for the indirect one and 2 scans were acquired over 40 increments . The FIDs were automatically processed with Fourier transformation and spectra were referenced to the TSP signal at 0 . 0 ppm in the F2 dimension and at 0 . 0 Hz in the F1 dimension . For assignment purpose , 2D NMR spectra were also acquired for a sample is made as mix all urine samples . The set of 2D experiments included 1H-1H correlation spectroscopy ( COSY ) , 1H-1H total correlation spectroscopy ( TOCSY ) , 1H-13C heteronuclear single quantum correlation ( HSQC ) and 1H-13C heteronuclear multiple bond correlation spectroscopy ( HMBC ) using the standard parameters implemented in Topspin 3 . 0 library ( Bruker Biospin Ltd . ) . Pre-processing of NMR data ( S2 File ) to be suitable for statistical analysis was performed with in house routines written in Matlab 2014a ( The Mathworks , Inc . , USA ) and Python 2 . 7 ( Python Software Foundation , www . python . org ) . All 1D NMR 1H spectra were re-evaluated for incorrect baselines and corrected using a polynomial fit of degree 5 . The spectral region from 0 . 5 to 9 . 5 ppm was binned using an in-house algorithm for adaptive intelligent binning[18] . Initial bin width was set to 0 . 02 ppm and final variable bins sizes were calculated based on the peaks edges in the spectra by using a lowest standard deviation criterion . The spectral region including the residual water and the urea peaks ( δ 4 . 5–6 . 2 ppm ) was excluded from the data . The final data consisted of 392 bins of variable size × 490 observations ( samples ) , which were normalized by the Probabilistic Quotients Normalization method ( PQN ) [19] to correct for dilution differences from sample to sample . Finally , the normalized data was scaled to unit variance for the statistical analysis . The data analysis was performed with R statistical environment ( ( http://www . r-project . org/ , R versions 3 . 3 . 2 ) . For exploratory analysis “Rcpm” , “pcaMethods” and “caret” packages were used . ASCA modeling was performed using “lmdme” package [20] . The visualizations were made using “ggplot2” , “cowplot” and “gridExtra” packages . Identification of metabolites was performed by exhausting search of the total 1D and 2D Jres data using the proprietary Bbiorefcode ( Bruker Biospin Ltd . ) and ChenomX NMR suite 8 . 1 ( Chenonx Inc . ) databases . The IDs of the annotated resonances were further verified by the collected 2D NMR data .
The study design involves 30 animals divided in three groups: a control uninfected group and the two experimental groups infected with fifteen and fifty metacercaria Fig 1 . Each experimental group consisted of an equal number of male and female animals . The dataset described in the current manuscript includes the samples from the baseline up to the thirty-two weeks collected every two weeks . The median of adult worm count at the end of the study was 35 for severe and 6 for mild infection intensity group respectively ( p-value = 0 . 003 ) ( S1A Fig ) . Eggs of O . felineus were detected in all fecal samples starting from 4 weeks post infection . S1B Fig shows the time course of the egg production over the entire period of the experiment . It shows a coherent increase in the egg production for both experimental groups over a period from 4 till 10 weeks . We consider changes in the egg production related to the different stage of the infection , namely an acute ( till 10 weeks ) and chronic one ( from 10 weeks on ) . From week 12 till week 30 of the experiment , egg output is stable and the group with high infection has constantly higher egg output . The output is significantly higher at the weeks 12 ( p-value = 0 . 002 ) , 14 ( p-value = 0 . 004 ) , 16 ( p-value = 0 . 030 ) , 20 ( p-value = 0 . 001 ) , 26 ( p-value = 0 . 001 ) and 28 ( p-value = 0 . 009 ) . The first step of an exploratory study is to identify the main sources of the variance in the data and influence of the confounding factors . The principal component analysis ( PCA ) is a commonly accepted approach . Fig 2 shows the score plots of the first two principle components of a PCA model built on the entire data set . The model required 5 components to explain 50% of the variance with 34% explained by the first two components . Every block of the figure represents the same model colored according to different factors . Surprisingly , the intensity of the infection ( Fig 2A ) is not contributing to the main sources of the variance in the data . Gender influences the profile profoundly , the influence is clearly represented in the first two principal components ( Fig 2C ) . Another recognizable source of the variance is the time of sample collection ( Fig 2B and 2D ) . The S2 Fig shows the PCA models built separately for male ( A , B ) and female ( C , D ) animals . The models have similar characteristics to the “global” one , both require 5 components to explain 50% of the variance; the first two components explain 37% and 32% of the variance in the male and female models , respectively . Again the intensity of infection is not explaining the main variance within the data ( S2A and S2C Fig ) , but the time trend is clearly visible ( S2B and S2D Fig ) . Moreover , the directions of the time trends in the models built on gender specific subsets appear to be different . The data shown in Fig 2 indicates clearly that a straightforward analysis of the infection dependent changes will be hampered by the influence of the gender and time trend . Thus , to reduce the complexity of the analysis we decided to concentrate on the period from 0 to 10 weeks of the experiment: a time frame when egg output was steadily increasing ( S1B Fig ) . The analysis was performed separately on the male and female subsets . Fig 3 shows the PCA models built on the data from week 0 up to the week 10 of the experiment . The “male model” ( Fig 3A , 3B and 3C ) required four components to explain 50% of the variance with 42% explained by the first two . The model build of the data including only females ( Fig 3D , 3E and 3F ) required 6 components to explain 50% of the variance with the first two explain 34% . Both models appear quite similar , however applying the geometric trajectory analysis [21] ( Fig 3C and 3F ) some underlying differences can be revealed . The geometric time trajectories for the infected animals ( groups E1 and E2 ) are clearly pronounced in the male model ( Fig 3C ) . The same time in the female subset ( Fig 3F ) only a trajectory for highly infected animals ( E1 ) has a distinct form , a trajectory for the lower infection group appears as random as the control one . To dissect the metabolic features related to the infection one needs a data analysis approach which enables simultaneous analysis of the experimental design and time . The choice is rather limited . One of the “stress tested” methods is ANOVA simultaneous component analysis or ASCA [22 , 23] . In a nutshell , ASCA is a multivariate extension of the ANOVA and the strongest asset of the method is a possibility of modelling of the experimental designs with several factors . With regards to our study those factors are the time , intensity of the infection and gender . To avoid a complex interpretation of the multiple cross-factor interactions we applied ASCA separately to each gender subset . Fig 4 shows the results of the analysis for the first two components . The trends are looking similar , but not identical . In the male subset the infection specific time trends are present in the main component ( 43 . 5% variance ) . The time trends for non-infected and infected with 50 metacercaria animals have almost opposite behavior in the first component showing somewhat random pattern in the second one . In the female subset , the infection related trends are not presented in the first component , but well presented in the second one . The last one , however , covers only 24% of the variance , almost the half of what was seen in males . Fig 4 shows the time/infection specific trends and the systemic differences between the males and females in the reaction on the infection challenge . Yet , it gives no information on the nature of the biochemical entities involved in the observed phenomena . Thus , using a leverage as a measure of the variable importance we extracted a subset of the variables influencing the models shown at Fig 4 . Table 1 summarizes the selected variables . Most of the variables can be annotated , but few complex regions ( 1 . 63–1 . 69 , 1 . 71–1 . 74 and 2 . 46 ppm ) simply cannot be assigned to a single metabolite . S1 File shows the 24 week time trends of the every individual feature included in the table . The traces are plotted as the median values per experimental group , the original values are included in the plots in a form of transparency graphics . A visual inspection of the graphs shows that only few compounds demonstrate a consistent trend over the entire period . For most of them , the changes are evident only in the early weeks; some compounds ( e . g . 2−aminoadipic acid and nicotinuric acid ) shows a distinct trend for the group of the animals infected with 50 metacercaria .
Here we present for the first time an exploratory analysis of the metabolic response to O . felineus infection in an animal model . Following an established routine of a descriptive study we started the analysis by exploring the main sources of the variance in the data . Using unsupervised projections methods namely PCA we have shown that neither infection status nor the time are representing the major sources of the variance in the data . In fact , the most noticeable trend in the data was related to the gender differences . At first glance , this observation may appear contradictory to the earlier publications on the animal models infected with other trematodes S . japonicum[13] and S . mansoni [12] as well as to our own report on a human study[15] where clear differences between infected and non-infected subjects were reported . Yet , to reveal the infection related differences authors of the above mentioned studies had to use the supervised modeling techniques and an unbiased assessment of the main sources of the variance in the data as a rule showed the influence of the physiological confounding factors such as for example age [15 , 24] . A gender related bias in human studies was concealed by age factor due to the broad age range of the participants . In the animal studies the single gender models were used: the female animals for mice [12 , 14 , 25] and the males for hamsters [13 , 26] . Since the animals included in our study were age matched , gender is now the strongest physiological cofounder [27]; consequently the gender bias observed in the data should be expected . Of course one should keep in mind the all the above metioned examples are taken from the publications on the metabolic effects of Schistosomiasis , no information on Opisthorchiasis has been published yet . A gender related trend explains approximately 30% of variance in the data; therefore further analysis was done of each sub-group separately . Both unsupervised multivariate modelling and ASCA-based analysis showed that a time related response to the infection unfolds differently in the males and females . Moreover , the Fig 4 which provides an overview of the main patterns associated with time-infection interaction clearly shows that in the male model the distinct trend is present in the first principle component covering twice as much variance as the female model where the similar trend is visible only in the second component . Naturally , a discussion of the biological relevance of the observed effects is only possible if we know the identity of the metabolites influencing the models . A subset of the most influential entities is presented in the Table 1 . In overall it appears as a more or less standard set of the metabolites which are regularly reported in the NMR-based metabolomics studies of urine . It also has a strong overlap with the metabolites reported in the publications on Schistosomiasis mentioned above . In the context of an acute Opisthorchiasis infection , a state of the energetic stress , one particular trend , namely the bile acids becomes interesting . S1 File shows that in the group of heavy infected animals urinary excretion of the bile acids is increasing staring from the beginning of the experiment reaching the peak at four weeks and going down after that point . A current physiological paradigm links an increased urinary excretion of the bile acids with the possible obstructions of the main duodenal path [28] . We can only speculate about the exact physiological mechanisms which leads to the increase of the bile acids secretion but the timing of the observed effect overlaps perfectly with a period when the parasite “settles down” in the host’s bile ducts . Of course our method catches only a gross effect and cannot provide the exact annotation of the bile acids . From the literature we know that the bulk of the urinary bile acids are excreted as the sulphonated species [29] but to our best knowledge there is no published report on urinary bile acids profiles in Opisthorchiasis . A similar transient pattern to the bile acids was also observed for the spectral areas where resonate lysine and a product of its catabolism 5-aminopentanoic acid are located . Despite an intriguing similarity , a “simple” interpretation where lysine would be considered as a breakdown product of the conjugated bile acids is difficult to accept; the lysine conjugates are rather unusual and were reported only as the minor products of lithocholic acid in the liver [30] . Another group of the metabolites which are related to the physiological control of the lipid metabolism consist of nicotinic acid and its metabolites nicotinuric acid and nicotinamide-N-oxide . The last two compounds show a transient decrease with a peak at four weeks . The effect is more pronounced in the male group . It is logical to assume that the host energy metabolism works in a stress mode during invasion of the parasite the bulk of nicotinic acid pool will be utilized for NAD+ production , in turn this can lead to a reduced production and urinary clearance of nicotinuric acid and nicotinamide-N-oxide . Another metabolites which nicely fits into a pattern of the stress mode of the energy metabolism is guanidinoacetate—a precursor of creatine [31] . Of course , an interpretation of the data from a point of view of the metabolic stress has a weakness . None of the described changes can be considered as specific for Opisthorchiasis; all the changes reflect a general reaction of an organism to acute infection . Yet , an exploratory study , especially the one that enters a relatively uncharted territory should provide the leads and hypothesis for further investigation . To this end , we have fulfilled a purpose of the study . A logical follow up of our study appears to be an in depth analysis of the urinary and fecal profiles of the bile acids using more sensitive techniques namely mass spectrometry . Already the last decades of twentieth century the analytical methods enabled detection and quantitation of several of the bile acids [32] , the state of the art technology offers a reliable analysis of more than hundred species [33] . Besides , the existing reports on alteration in the urinary bile acids profiles during the pathological conditions of liver [34] supports our hypothesis . Considering the other mentioned compounds and trends they fit a context of the energetic stress and metabolic redistribution of the bulk metabolites in the circulation , it remains to be seen however whether our findings can be translated to the human infection . | Opisthorchiasis is a parasitic infection caused by the liver flukes of the Opisthorchiidae family ( Trematoda; Platyhelminthes ) . The liver fluke infections trigger development of the hepatobiliary pathologies such as chronic forms of cholecystitis , cholangitis , pancreatitis , and cholelithiasis . However , the most threatening effect of the Opisthorchiidae infection is an increased risk of intrahepatic cholangiocarcinoma . With this work we are getting an insight into a host response on Opisthorchiidae infection using a metabolomics approach . Metabolomics is a post-genomic discipline studying the metabolome . The dynamic character of the metabolome , its ability to change in response to the external stimuli makes it an optimal “readout” for exploratory studies aiming for the description of the systemic responses of an organism . Using this approach we demonstrate that that early response to the O . felineus infection unfolds in a gender-dependent manner . Moreover , with this first exploratory analysis of the metabolic response to O . felineus infection in an animal model we present a subset of the metabolites changing during the early phase of the infection and offer a possible physiological interpretation . | [
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] | 2017 | Exploratory metabolomics study of the experimental opisthorchiasis in a laboratory animal model (golden hamster, Mesocricetus auratus) |
ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes , thereby orchestrating skeletal muscle excitability . Malfunction of ClC-1 is associated with myotonia congenita , a disease impairing muscle relaxation . Here , we present the cryo-electron microscopy ( cryo-EM ) structure of human ClC-1 , uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters . The chloride conducting pathway exhibits distinct features , including a central glutamate residue ( “fast gate” ) known to confer voltage-dependence ( a mechanistic feature not present in ClC-K ) , linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule . These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels . Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase ( CBS ) domains and the intracellular vestibule ( “slow gating” ) . The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating , opening avenues for rational design of therapies against ClC-1–related diseases .
CLC proteins comprise a large family of chloride ( Cl− ) -transporting integral membrane proteins with diverse physiological functions [1–3] . The first identified human member , ClC-1 , is essential for maintaining the permeability of Cl− across the plasma membrane of skeletal muscle fibers , gCl , accounting for approximately 80% of the resting membrane conductance and assuring precise neuronal control of muscle contraction [3] . Mutations of the ClC-1 gene cause myotonia congenita , a disease that allows a single nerve action potential to trigger a series of muscle action potentials ( myotonic runs ) , leading to prolonged muscle contraction [4–7] . Despite distinct roles as passively conducting Cl− channels and stoichiometrically coupled secondary active Cl−/H+ antiporters [2 , 3] , members of the CLC family share a common homodimeric core architecture , with each subunit harboring an independent ion translocation pathway [8 , 9] . The molecular mechanisms of ion transport in CLC antiporters have been extensively studied functionally and structurally [8 , 10–15] . Yet it is poorly understood how the antiporters and channels establish their separate functions . In addition , the complex gating processes that regulate CLC channel activity remain elusive , with only a single available structure of a channel member , namely , that of bovine ClC-K [9] . Each CLC monomer has a gate that operates independently from the other ( also known as “protopore” or “fast gate” ) , structurally attributed to a specific glutamate , “GluGATE” [10] . A slower gate controls both conducting pathways simultaneously ( “common” or “slow gate” ) [16] , but the principles and determinants of this regulation are enigmatic . Furthermore , activity of ClC-1 is modulated by cellular cues such as phosphorylation [17] , pH , and nucleotides [18 , 19] in an unknown manner . Such regulation is , however , physiologically essential because intense muscle exercise leads to acidosis , resulting in an increased nucleotide sensitivity of ClC-1 and consequent reduction of gCl , thereby assisting in preventing muscle fatigue [20 , 21] . The recent ClC-K structure provided the first insights into the differences between CLC channels and transporters; in particular , it revealed a pore widening on the intracellular side . Yet there are surprisingly few known structural differences between the CLC channels and transporters . However , ClC-K channels exhibit only limited gating as GluGATE is missing [2 , 3] , and their activity has not been reported to depend on nucleotide binding [22] . Therefore key questions concerning CLC channel function and regulation remain unanswered . Furthermore , a deeper understanding on structure–phenotype relationships of myotonia-causing mutations in ClC-1 is required to shed further light on how the muscle disease is manifested at a molecular level .
Here , we have determined structures of full-length human ClC-1 using single-particle cryo-electron microscopy ( cryo-EM ) , exploiting a purified protein sample that displays Cl−-dependent single-channel–derived ion conductance ( S1 Fig and S1 Data ) . For structural characterization , sample in the presence of 100 mM Cl− at pH 7 . 5 and in the absence of nucleotides or antibodies was initially employed ( Fig 1 ) . Three-dimensional ( 3D ) classification of particles resulted in several different groups , of which one yielded a 3 . 6 Å overall resolution density map for the transmembrane domain , allowing confident model building ( S2–S4 Figs ) . The final model represents the membrane-spanning portion ( note that the N terminus and intracellular αA helix are lacking ) as well as parts of two C terminal’s so-called cystathionine-β-synthase ( CBS ) domains present per monomer ( for which some cryo-EM density is left unmodeled ) and includes several features that were not observed in the ClC-K structure ( S5 Fig ) . The homodimeric architecture of ClC-1 is reminiscent of that of bovine ClC-K and available structures of CLC proteins from lower organisms ( Fig 2A ) . The monomers consist of membrane-spanning helices and half-helices ( αB to αR ) with connecting loops ( e . g . , αB–C , between αB and αC ) as well as the CBS domains ( Fig 1 ) . Each protomer holds a separate chloride conducting pathway across the membrane , established by a vestibule on either side of the membrane , and an interconnecting narrow and short pore . In CLC transporters , the Cl− conducting pore ( Fig 2B ) is marked by distinct Cl− binding sites ( denoted sext , scen , and sint , respectively , but no Cl− ions are resolved in the current structure ) , and the constricting Glu232 ( of αF , also known as GluGATE; ClC-1 numbering throughout ) and Tyr578 ( of αR , TyrC ) [9] . Furthermore , Ser189 ( of αC-D , SerC ) is located in the vicinity of the pore ( Fig 2B , 2C and 2E and S5A Fig ) . In ClC-1 , voltage-dependent gating is established by GluGATE , which is perhaps being displaced by competing Cl− ions and/or protonation . In contrast , in voltage-independent ClC-K channels , GluGATE is replaced by a valine , and , indeed , substitutions of GluGATE with uncharged residues render ClC-1 similarly voltage independent [24] . Unfortunately , the GluGATE side chain is not visible in our cryo-EM density maps ( S4 Fig ) , but carboxylate groups of interacting acidic residues are known to be frequently undetectable using cryo-EM due to radiation damage . A similar orientation of the side chain as observed in ClC-K would be in agreement with Cl− passage through a maintained scen , as a concomitant adaptation of αR significantly shifts the position of TyrC and thus maintains the GluGATE-TyrC distance ( Fig 2B–2F and S5G Fig ) . However , we cannot exclude that the side-chain of GluGATE is buried deep into the hydrophobic pocket established by Phe279 , Phe288 , and Phe484 ( S5H Fig ) . The pore aperture of the extracellular vestibule is constricted by a hydrophobic barrier with Met485 ( Met427 in ClC-K ) , but in contrast to ClC-K , the gate opening is also controlled by Lys231 ( of αE–F ) and Arg421 ( of αL ) ( Fig 2B–2F ) that may orchestrate Cl− permeation to or from the extracellular environment [25–27] . This difference can be attributed to αE–F , with its GluGATE and Lys231 adopting a more CLC-transporter–like configuration because this loop is considerably shorter than in ClC-K , alongside a side-chain reorientation of Arg421 ( Fig 2C–2F ) . We also observe a structural adjustment on the intracellular side of the pore , with αC–D being displaced as compared to the corresponding loop in ClC-K . This rearrangement opens the vestibule even deeper toward GluGATE ( Fig 2C–2F ) , providing intracellular access beyond the sint site present in CLC antiporters and suggesting that no tight Cl− binding occurs on the intracellular side , in agreement with electrophysiological data [28] . The wider intracellular vestibule of the CLC channels , as compared to the transporters , has been proposed to allow for the higher Cl− conductance in channels , lowering the kinetic barrier between scen and the cytosol [9] . We note that the vestibule width of ClC-1 is similar to that of ClC-K at SerC , with the side chain of this residue being positioned away from the Cl− permeation pathway in both channels , establishing the SerC location as another of the distinguishing features between CLC channels and transporters . It remains obscure whether the channel has been captured in the open configuration , a priori induced by the experimental conditions ( 0 mV , 100 mM Cl− ) . Molecular dynamics simulations of the ClC-1 structure suggest that Cl− from the intracellular side spontaneously interacts with GluGATE upon protonation of its side chain but that free energy is required to complete the passage across the membrane ( S6 Fig ) . We anticipate that GluGATE and the Lys231–Arg421 constricting interactions attenuate chloride flux , in agreement with the smaller conductance of ClC-1 versus ClC-K [2 , 3] , and we cannot exclude that Cl− shuttling occurs directly between protonated GluGATE and Lys231 across the Met485 barrier ( GluGATE overlays sext in some CLC transporters [8 , 14 , 29] ) ; chloride interaction with the latter may be unfavorable , however . The molecular mechanisms that govern slow gating in CLC proteins remain elusive . It is known that CBS nucleotide binding and low pH inhibit ClC-1 activity by favoring closure of the common gate [19 , 29] . Assessment of the 3 major cryo-EM maps obtained in our structural classification ( see also S2 Fig and Methods ) reveals different arrangements of the CBS domains , suggesting intrinsic domain flexibility at pH 7 . 5 ( Fig 3A and 3B and S7 and S8 Figs ) . To test this , we determined the structure of ClC-1 also at lower pH ( 6 . 2 ) in the presence of 0 . 3 mM of the nucleotide nicotinamide adenine dinucleotide ( NAD ) to unravel the regulation mechanism ( S2 , S3 and S8 Figs ) . In these conditions , the CBS domains appear significantly more rigid ( in comparison to pH 7 . 5; Fig 3A and 3B and S7 and S8 Figs ) . This observation is also supported by ClC-1 size-exclusion chromatography profiles ( S9 Fig ) , with samples at low pH being shifted toward lower molecular weight ( more compact ) . Therefore , the CBS arrangements seem to correlate with slow gating , being rigid at low pH in the presence of nucleotides and more flexible at higher pH in the absence of nucleotides , bringing to mind a mechanism that has been proposed based on electrophysiological data [29] . The complete effects of such putative rearrangements are , however , not demonstrated experimentally by our structures , because they remain closed also at the higher pH ( determined from particles in detergent environment ) . How then can the Cl− conductance of 2 separate pores be affected by structural shifts of the CBS domains ? Examination of the interface between the CBS and the transmembrane domain suggests that CBS2 interacts with αD–E , a loop previously shown to affect slow gating ( Fig 3C and 3D ) [25 , 31] . Nucleotides may also interact directly with the transmembrane domain when bound in the cleft between CBS1 and CBS2 ( the latter observed in structures of isolated CBS domains [13]; Fig 3E ) . It is conceivable that these structural arrangements and the direct physical connection between CBS and αR—all structural elements leading to the GluGATE constrictions site—allow structural adjustment of the transport pathway and thus chloride conductance regulation ( Fig 3C ) . Such structural effects will be propagated between the monomers via the CBS domains , in agreement with concurrent modulation of the 2 conducting pathways in the dimer [16] . We note that the CBS portions that interact with the transmembrane and the CBS domain of the adjacent monomer are structurally ( and at interaction sites also sequencewise; S10 Fig ) conserved ( Fig 3E ) , and therefore this may represent a unifying mechanism of slow gating for CLC proteins . ClC-1 defects cause recessive ( Becker type ) or dominant ( Thomsen type ) myotonia congenita , typically associated with complete disruption of channel function or with a dominant negative effect in heterodimeric wild-type ( WT ) -mutant complexes [7] , respectively . Our structure now allows mapping of such ( or other experimental ) ClC-1 substitutions for evaluation of structure–function–disease and -phenotype relationships ( Fig 4 ) . Several dominant and recessive mutations induce an alteration of the overall gating from depolarization to hyperpolarization activated , yielding a similar intracellular Cl−-sensitive gating as described for ClC-2 [32] . Therefore , the different gating profiles of ClC-1 and ClC-2 likely do not necessitate major structural differences . These residues are generally surface exposed and localized to the extracellular half , including the vestibule and the pore-constricting residues Lys231 and Arg421 ( Fig 4B ) [26 , 27 , 32–35] . In contrast , many dominant mutations exert a “shift” of the common gate to open probability to positive voltages , leading to significant reduction of gCl at the physiological membrane potential [36] . Such mutations cluster primarily at the dimer interface and in the intracellular vestibule and pore region ( Fig 4C , and 4D and S5D Fig ) . One is located in CBS2 , close to the membrane domain , in agreement with the above-mentioned mechanism of slow-gating regulation exerted via CBS2 . Residues that affect binding of one of the most commonly used ClC-1 inhibitors , the lipophilic 9-anthracene-carboxylic acid ( 9-AC ) , are all buried into a CAVER [37]-computed membrane-embedded cavity on the intracellular side that stretches to GluGATE , in agreement with the intracellular mechanism of action proposed for this compound ( Fig 4E and 4F and S11 Fig ) [24] . Because this pocket is lined by multiple hydrophobic and a few negatively charged residues , it is unlikely to allow chloride conductance ( proton access is possible ) but rather 9-AC–induced interference of flux across GluGATE and may thus represent a suitable site for future drug-discovery efforts .
In summary , we report the molecular structure of Cl−-conducting human ClC-1 , sharing an overall fold similar to other CLC proteins , with a narrow connecting pore and positively charged vestibules attracting Cl- ions similar to CFTR [38] . The structure exhibits several unique features , including shifts in the central GluGATE-TyrC pair , a more closed extracellular vestibule , and a wider penetration profile from the intracellular side , the latter representing a distinct feature of CLC channels separating them from transporters . We propose a model for adenine nucleotide and pH regulation of the common gate via CBS2 and the intracellular loops congruent with previous functional data . Overall , these findings significantly increase our understanding of Cl− conductance in physiology and open new opportunities for biomedicine . For example , the positively charged constriction of the extracellular vestibule and the putative 9-AC pocket may serve as favorable target sites for stimulators or inhibitors from outside or inside the cell , respectively . During the course of the preparation of this manuscript , the structure of human ClC-1 was reported by another group [39] . The ClC-1 structures display only limited differences despite that different overproduction hosts were exploited . The authors detected a similar putative 9-AC binding pocket ( the alternative pathway ) and conformational flexibility in the CBS region ( determined at pH 7 . 4 ) , in agreement with our findings . We anticipate that the pH-dependent conformational changes reported here—in conjunction with mutational efforts using , e . g . , single-channel recordings , as for the first time demonstrated in this work , will allow for more refined studies to further resolve the mechanism of slow-gating in CLC proteins .
Yeast codon-optimized cDNA encoding human ClC-1 ( UniProt accession P35523 ) was purchased from Genscript ( Genscript , USA ) . cDNA was inserted into pEMBLyex4 [40] along with yeast-enhanced GFP by homologous recombination to encode ClC-1 , followed by a Tobacco Etch Virus ( TEV ) cleavage site , GFP , and a His10 tag . The correct nucleotide sequence of the expression construct was verified by DNA sequencing ( Eurofins MWG Operon , Germany ) . Human ClC-1 was produced in the PAP1500 strain [41] grown in computer controlled 15-L bioreactors as previously reported but without addition of any chloride salts ( such as NaCl ) [42] . Yeast cells were harvested approximately 90 hours after induction of ClC-1 expression . For crude membrane preparations , approximately 25 g of yeast cells were resuspended in 25 mL lysis buffer ( 25 mM imidazole [pH 7 . 5] , 1 mM EGTA , 1 mM EDTA , 10% glycerol , 5 mM β-mercaptoethanol ) supplemented with protease inhibitors ( 1 μg/mL leupeptin , pepstatin , and chymostatin , and 1 mM PMFS ) . Cells were disrupted by addition of glass beads ( 0 . 4–0 . 8 mm ) and vortexed in 50-mL Falcon tubes 8 times for 1 minute . The supernatant was collected , and glass beads were washed several times in ice-cold lysis buffer . The cell lysate was centrifuged at 1 , 000g for 10 minutes to remove cell debris . Crude membranes were pelleted from the supernatant by ultracentrifugation at 160 , 000g for 90 minutes; resuspended in a buffer containing 50 mM Tris ( pH 7 . 5 ) , 300 mM NaCl , 10% glycerol , 1 mM PMSF , and EDTA-free protease inhibitors ( Sigma ) ; and homogenized in a Potter-Elvehjem homogenizer . Subsequently , membranes were solubilized by adding dodecyl-β-maltoside ( DDM ) and cholesteryl semi succinate ( CHS; from Anatrace ) at final concentrations of 1% and 0 . 33% , respectively , and incubated at 4°C for 3 hours under gentle stirring . Nonsolubilized material was removed by ultracentrifugation at 30 , 000 rpm for 30 minutes in a Beckman Ti 60 rotor . Ni-beads from 5 mL of slurry ( Thermofisher ) were incubated with the supernatant for 2 hours under gentle stirring . To prevent unspecific binding , 30 mM imidazole was added . Resin was transferred to a 5-mL Econo column ( Bio-Rad ) and washed with 10 column volumes of high-salt buffer ( 50 mM Tris [pH 7 . 5] , 800 mM NaCl , 5% glycerol , 0 . 4 mg/mL DDM , and 0 . 04 mg/mL CHS ) followed by 10 column volumes of low-salt buffer ( 50 mM Tris [pH 7 . 5] , 300 mM NaCl , 5% glycerol , 0 . 4 mg/mL DDM , and 0 . 04 mg/mL CHS ) . ClC-1 protein was liberated from the beads by overnight incubating in 10 mL low-salt buffer containing 0 . 2 mg of TEV protease . Ni-beads were washed twice with 5 mL of low-salt buffer , and all collections were pooled and concentrated to approximately 1 mL using a 100 , 000 kDa cutoff concentrator device ( Sartorius ) . Amphipol PMAL-C8 ( Anatrace ) was added to the purified protein at a mass ratio of 1:5 and incubated overnight . To remove DDM , protein was dialyzed overnight against final buffer ( 20 mM Tris [pH 7 . 5] , 100 mM NaCl , 0 . 2 mM TCEP ) supplemented with 100 mg of SM-2 Bio-Beads ( Bio-Rad ) . The protein-amphipol complex was applied to a Superdex-200 column equilibrated with final buffer . Peak fractions were collected and concentrated to approximately 0 . 5 mg/mL . For the low pH samples , the purification procedure was identical except for using 20 mM BisTris ( pH 6 . 2 ) ( instead of Tris [pH 7 . 5] ) in the final buffer ( final protein concentration only reached approximately 0 . 3 mg/mL due to precipitation ) . Single-channel ion current was recorded using 2 separate methods , as follows: Cryo-EM grids were prepared with the Vitrobot Mark IV ( FEI ) operated at 100% humidity at 4°C . Immediately prior to sample vitrification , Quantifoil 1 . 2/1 . 3-μm holy carbon grids were glow-discharged with Easyglow ( TedPella ) , and fluorinated fos-choline-8 ( Anatrace ) was added to the protein sample to a final concentration of 3 mM , which was an essential step for producing good quality thin ice . For each grid , an aliquot of 3 . 5 μL was applied and incubated for 20 seconds inside the Vitrobot . Blotting time was set to 2 . 5 seconds with 2 seconds of drain time . The low pH sample was treated identically , except for incubation with 0 . 3 mM NAD before freezing ( and that no fluorinated fos-choline-8 was added to obtain one of the pH 6 . 2 data sets ) . Cryo-EM data sets were collected on a Titan Krios electron microscope ( FEI ) operating at 300 keV with a Gatan K2 Summit direct electron detector attached to a Gatan imaging filter ( GIF ) . Movies were recorded under super-resolution counting mode at a pixel size of 0 . 535 Å and a dose rate of 0 . 876 e/pixel/frame for a total of 60 frames . The total electron dose was 45 electrons per Å2 per movie for 9 seconds . Cryo-EM movies were first gain-corrected and 2× binned to a final pixel size of 1 . 07 Å . Dose-weighted and nondose-weighted summed micrographs were generated with MotionCorr2 [43] using all frames except the first one . Defocus values were calculated with the nondose-weighted micrographs using Gctf [44] . Next , image processing was conducted using dose-weighted micrographs with the predetermined defocus . Template-free particle picking was done using Kai Zhang’s Gautomatch software ( https://www . mrc-lmb . cam . ac . uk/kzhang/Gautomatch ) . All following processing steps were done in Relion 2 . 0 [45] using a box size of 288 pixels . For the pH 7 . 5 data set , a total of 594 , 609 auto-picked particles from 4 , 475 micrographs with a defocus range of −1 . 0 to −3 . 0 μm were subjected to several rounds of reference-free 2D classification to remove defective particles . The selected 477 , 729 particles were sorted using 3D classification . Selected classes were refined using masks , either with the complete protein excluding the amphipol belt or with the membrane domain only . Multiple cryo-EM density maps were calculated demonstrating structural heterogeneity of the protein . 3D classification of particles into 5 classes provided the best class consisting of 176 , 871 particles ( representing more than 37% of all particles ) . A soft mask covering the entire protein without amphipol belt yielded a map with an overall resolution of 4 . 00 Å , and a tighter mask only containing the membrane domain resulted in map with resolution of 3 . 63 Å . To further investigate the structure heterogeneity in the cytoplasmic domain , the 2D selected particles were first refined , and then the refined per-particle parameters were applied for 3D classification , only performing local angular searches within ±10 degrees . This local 3D classification resulted in 9 classes , and the 2 major classes differed primarily in the cytoplasmic domain . Refinement of these 2 classes , each representing approximately 15% of all selected particles , yielded overall map resolutions of 4 . 34 Å and 4 . 28 Å , respectively . For the pH 6 . 2 data set collected with fluorinated fos-choline-8 , 552 , 914 particles were autoselected from 4 , 119 motion-corrected micrographs , and 300 , 572 particles were selected after 2D classification for further processing; 3D classification into 5 classes generated the best class , which eventually was refined to a final resolution of 4 . 47 Å . Combination of the data collected at pH 6 . 2 with and without fluorinated fos-choline-8 , and a similar local angular search strategy as for the pH 7 . 5 data set , generated a final map of 4 . 2 Å of the best class ( based on approximately 30% of the total particles ) . C2 symmetry was applied for all classification procedures , and all maps were sharpened with a B-factor of −100 Å2 . Local resolution was calculated using the postprocessed map , and the map was filtered according to the local resolution and used for model building . The initial model was generated using the SWISS-MODEL online server and the ClC-K structure [9] ( PDB-ID 5TQQ ) as a template . The model was first fitted into the cryo-EM density map and later manually built in COOT [46] . The 3 . 6 Å membrane domain density map was sufficient for building the entire membrane domain ( residues 115 to 589 ) with only 1 loop missing ( residues 254–261 ) . The built model was refined using phenix . real_space_refine of the Phenix software package [47] . C2 symmetry was imposed during the refinement by using strong non-crystallographic symmetry ( NCS ) restraints . Secondary structure restraints and Ramachandran restraints were also imposed during refinement . The resolution and connectivity of the cytoplasmic domain was insufficient for de novo model building . Instead , a homology model based on the available structure of the CBS domains of ClC-0 ( PDB-ID 2D4Z [30] ) was generated and docked into different maps . The refinement of the cytoplasmic domain was conducted by local grid minimization , model morphing , and simulated annealing implemented in the phenix . real_space_refine software [47] . To prevent overfitting , the map resolution was restricted to 5 Å , the local resolution of the cytoplasmic domains as determined by Relion postprocessing . After model building , the models were trimmed to only include the minimal CBS architecture , consisting of 2 helices and a β-sheet . The quality of the models were validated assessed using Molprobity [48] ( see S1 Table for statistics ) . All figures except for Fig 3A and 3B were generated using the model based on the 4 . 0 Å ( Map 1 ) . The ClC-1 dimer with Glu232 either protonated or deprotonated was inserted into a palmitoyloleoylphosphocholine ( POPC ) membrane , and CHARMM36 force field parameters [49 , 50] were generated using CHARMM-GUI [51] . The simulations were performed using the GROMACS 2016 . 4 simulation software [52] . Each system was energy minimized and equilibrated in a stepwise manner using 25-ps NVT simulations with decreasing restraints on the protein and lipid heavy atoms . In these simulations , a 1-fs time step was used and the temperature was maintained at 310 K with a Berendsen temperature-coupling scheme [53] . The following set of NPT simulations further released heavy-atom restraints for 0 . 1 ns , 10 ns , and 10 ns , respectively . Here , a 2-fs time step was used and the pressure was kept constant at 1 bar using a Berendsen pressure barostat [53] . In a 100 ns production simulation , all atoms were unrestrained , and the temperature and pressure coupling schemes were Nose-Hoover [54 , 55] and Parrinello-Rahman [56 , 57] , respectively . The GROMACS pull code with a force constant of 1 , 000 kJ mol−1 nm−2 was applied for 300 ps to the Cl− ion in closest vicinity of Glu232 in 1 monomer . The pull rate was 0 . 1 Å per ps , and the pull force was directed along the vertical axis of the membrane . The potential of mean force ( PMF ) was calculated using umbrella sampling from 1 Å windows along the ion path . The figures were generated using VMD software[58] . | Chloride transporting CLC proteins are expressed in a wide range of organisms , and the family encompasses several members with numerous roles in human health and disease by allowing movement of chloride ions across the membranes that encapsulate cells and cellular organelles . Structurally , CLCs form dimers possessing a separate ion translocation pathway in each monomer , and they can operate as either channels or transporters that exchange chloride for protons . The CLC channel ClC-1 is critical to skeletal muscle excitability and has been proposed as a target to alleviate neuromuscular disorders . Here , we have analyzed the structure of human ClC-1 and revealed the high similarity of its ion conducting pathway to those observed in other CLC members , including prokaryotic and algal transporters . Our data suggest how ClC-1 is regulated by environmental cues to allow opening and closure , thereby permitting attenuation of muscle function . Our results help with understanding the principal determinants that govern CLC proteins and may guide downstream translational applications to combat muscle pathologies . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [] | 2019 | Structure of the human ClC-1 chloride channel |
Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation . Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females , which have two X-chromosomes . Dosage compensation is mediated by the Male-Specific Lethal ( MSL ) histone acetyltransferase complex . Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females . However , because dosage compensation must interface with the general transcriptional machinery , it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level . Such regulators would not have been recovered from previous male-specific lethal screening strategies . Therefore , we have performed a cell culture-based , genome-wide RNAi screen to search for factors required for MSL targeting or function . Here we focus on the discovery of proteins that function to promote MSL complex recruitment to “chromatin entry sites , ” which are proposed to be the initial sites of MSL targeting . We find that components of the NSL ( Non-specific lethal ) complex , and a previously unstudied zinc-finger protein , facilitate MSL targeting and display a striking enrichment at MSL entry sites . Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila .
X-chromosome dosage compensation in male Drosophila provides a model for understanding how a large number of diversely regulated genes along the length of a single chromosome can be targeted for coordinate regulation . In Drosophila , the transcript levels of genes along the length of the single male X-chromosome are upregulated approximately two-fold , a process mediated by the MSL ( Male-specific lethal ) histone acetyltransferase complex [1] . A model , initially based on genetic observations [2] , posits that the MSL complex binds the X-chromosome in a two-step process . First , high levels of the MSL complex accumulate at approximately 150–300 “chromatin entry sites” ( CES ) containing GA-rich MRE ( MSL Recognition Element ) sequences that are ∼two-fold enriched on the male X [3][4] . Second , a sequence-independent spreading occurs in which the MSL complex associates with the bodies of active X-linked genes via general features associated with transcription , including H3K36me3 [5] . MSL complex increases transcript levels of its target genes by increasing the density of RNA Polymerase II ( RNAP II ) over transcription units , likely via MSL-dependent H4K16 acetylation [6] . While significant progress has been made towards understanding how MSL complex is targeted and functions , much remains to be understood . For example , since MREs are less than two-fold enriched on the X-chromosome [3] and the core MSL complex does not appear to have sequence-specific DNA binding activity [7] , it is likely that additional factors are involved in the key process of CES recognition . Seminal genetic screens identified five core components of the MSL complex by isolating genes that were specifically required for male viability [8][9] . However , these approaches would not recover a potentially key class of regulators that might be required for viability in both sexes , and act in dosage compensation by providing an interface with the core transcriptional machinery . In fact , based on experiments from budding yeast [10][11] the essential SET2 H3K36 methyltransferase was identified as a potential regulator of MSL complex association with active genes [5][12] . SET2 contributes to targeting of the MSL complex to the bodies of active genes [5][12] , but it is likely that other factors also participate . Furthermore , there are no candidates for direct binding to the MRE sequence , which is proposed to be a key first step in MSL targeting . Therefore , we designed a cell-based reporter system that specifically monitors MSL function at chromatin entry sites , and conducted a genome-wide RNAi screen to identify genes required to regulate this reporter . We identified the NSL [13][14] and PAF [15][16] general transcriptional regulators , as well as a previously unstudied zinc finger protein , CG1832 , that emerges as a strong candidate to function as the previously unknown link between MSL complex and MRE sequences within chromatin entry sites .
In order to identify genes that regulate MSL complex recruitment or function , we performed a genome-wide RNAi screen using an MSL-dependent cell-based reporter system for which we previously reported functionality [3] ( Figure 1A ) . This reporter system includes the following three elements: 1 ) promoter of roX2 gene ( 370 bp ) ; 2 ) Firefly luciferase reporter gene; 3 ) MSL complex binding site for roX2 ( DNase I Hypersensitivity Site: 280 bp ) . The system is based on a similar one that functions as a male-specific MSL-dependent reporter in vivo [17] . A genome-wide RNAi library generated by the Drosophila RNAi Screening Center ( DRSC ) that contains approximately 21 , 000 dsRNAs was screened in duplicate ( www . flyrnai . org ) . To eliminate off-target effects , secondary screens were conducted using a set of validation RNAi constructs in addition to rescreening the original constructs . In addition to direct regulators of MSL complex recruitment or function , several additional types of genes could be identified by our RNAi screen . Therefore , we used the following steps to identify potential direct activators of MSL complex targeting: 1 ) To reduce the number of non-specific regulators of luciferase gene transcription and protein stability that were identified , we calculated the ratio of activity from our MSL-dependent Firefly luciferase reporter to a heterologous Renilla luciferase reporter . 2 ) We used multiple MSL-dependent reporters to further narrow the list of genes to those that function independently of the roX2 promoter and specific CES used in the genome-wide screen; 3 ) We then used known functional information about the genes to narrow the list to those that have potential direct activating roles; 4 ) We determined whether new proteins affect MSL complex recruitment at endogenous sites without altering transcription of MSL complex components; 5 ) We performed genome-wide localization analysis to determine whether new proteins are enriched at CES loci . As briefly described above , we first normalized the MSL-dependent Firefly luciferase reporter activity to an MSL-independent reporter including the Renilla luciferase gene that can be independently assayed using a different substrate . This dual Firefly/Renilla approach also controls for technical variation in transfection efficiencies among wells and differences in cell number caused by altered viability after RNAi treatments . We used a Renilla construct that was regulated by an Actin promoter as previously described [18] . Therefore , by calculating the ratio of Firefly/Renilla luciferase activity , RNAi conditions that specifically alter the MSL-dependent reporter but not the Actin-Renilla construct could be identified . To conduct the RNAi screen , sixty-two screening plates containing approximately 21 , 000 dsRNAs arrayed in individual wells of a 384-well plate were assayed in duplicate . Each plate contained two positive control ( msl2 RNAi ) and two negative control ( GFP RNAi ) constructs , that were previously validated . After confirming that at least a 5-fold dynamic range was observed in the expected activity of positive and negative controls on each plate , the Firefly/Renilla ratios of the two plates were averaged and screening hits were identified relative to the median Firefly/Renilla ratio for each plate ( See Methods ) . We also conducted the same computational analysis for the Renilla values and removed hits in which the Renilla activity was dramatically altered by the RNAi treatment ( See Methods ) . Our screening approach identified 322 RNAi constructs that altered MSL-dependent reporter activity ( Table S1 ) . Importantly , we found all known components of the MSL complex in an unbiased way , validating our screen ( Figure 1B ) . We identified 254 RNAi constructs that reduced MSL-dependent reporter activity and 68 RNAi constructs that increased reporter activity . Gene Ontology analysis revealed several functional categories that were enriched including the following: protein synthesis , cell cycle control , and transcriptional regulation ( p<0 . 05 ) ( Table 1 ) . Similar analysis of publically available data sets from other RNAi screens performed using the same library indicated that the transcriptional regulation Gene Ontology category was not enriched in any other publically available screens . In contrast , the protein synthesis category and cell cycle control categories were commonly enriched in the majority of screens conducted with this RNAi library . Next , candidate genes were chosen for further analysis and validation with additional RNAi constructs . Three classes of genes were removed from the original list of candidates: 1 ) Ribosomal protein genes that are identified by many RNAi screens ( 41 genes ) ; 2 ) Genes with a large number of predicted RNAi off targets using 17 bp as an overlap cutoff [19] ( 19 genes ) 3 ) RNAi amplicons that did not target a specific gene and/or target multiple predicted genes ( 21 amplicons ) . We rescreened the 241 remaining candidates using both original and validation RNAi constructs with the original screening system and two additional MSL-dependent reporters that we previously validated [3] . These additional MSL-dependent reporters had a different promoter or CES compared with the original RNAi screening construct and were previously described ( Figure 1A ) . In this way , we identified 72 genes that were validated with an additional RNAi construct and regulated all three MSL-dependent reporters ( Table S2 ) . We do not wish to over-interpret any negative results; however , we did not identify the genes encoding HP1 , SU ( VAR ) 3–7 , or ISWI . These each have a mutant phenotype that differentially affects the polytene X chromosome [20][21] , but may not affect MSL function . Likewise , we did not identify JIL-1 , SCF , NUP153 , or Megator , all previously implicated in MSL interaction [22][23][13] . To narrow our list of candidate genes , we classified genes into categories based on their activity in our assay and previously determined functional information about the gene products as follows ( Table S2 ) : 1 ) Class 1 ( 18 genes ) : General transcriptional regulators that activate our reporter; 2 ) Class 2 ( 6 genes ) : Proteins that have potential sequence-specific DNA binding activity and activate our reporter; 3 ) Class 3 ( 7 genes ) : General transcriptional regulators that repress our reporter; 4 ) Class 4 ( 3 genes ) : Proteins that have potential sequence-specific DNA binding activity and repress our reporter; 5 ) Class 5 ( 7 genes ) : Proteins that regulate RNA metabolism ( e . g . splicing , non-sense mediated decay ) ; 6 ) Class 6 ( 26 genes ) : Proteins that are previously unstudied and/or have functions unrelated to transcription or RNA metabolism . The remaining genes are known MSL complex components . Members of each of these classes could directly modulate MSL recruitment or activity or indirectly effect MSL complex by altering complex levels . Due to the large number of genes identified , we focused on potential direct regulators of MSL complex recruitment or function that had known roles in transcriptional regulation or potential DNA binding domains ( Classes 1–4 ) . This analysis identified 34 potential transcriptional regulators . We validated the activity of these candidate genes using our MSL-dependent reporter assay in a more accurate 96-well format ( Figure 2 ) . Overall , we identified 24 activators ( Classes 1 and 2 ) and 9 repressors ( Classes 3 and 4 ) with known or predicted roles in transcriptional regulation . In addition to identifying all core components of MSL complex as activators of our reporter system ( Figure 1B ) , we identified a known repressor of MSL function , NURF301 , which we previously determined represses roX gene transcription in vivo [24] ( Table S2 ) . Also , NURF301 is known to associate with the roX1 CES [24] . Therefore , our RNAi screening strategy identified all MSL complex components and a known direct repressor of MSL-dependent transcription , validating our ability to identify activators and repressors with direct functions . The transcription of MSL complex components could be modulated and thus indirectly affect its function . Therefore , we selected candidates and examined whether they function directly to recruit MSL complex to CES loci using several approaches: 1 ) Determine whether RNAi against candidate genes affects recruitment of MSL complex to endogenous CES; 2 ) Define whether RNAi knockdown of candidates alters transcription of MSL complex components; 3 ) Examine the recruitment of candidate proteins to CES loci . We hypothesized that direct regulators of MSL complex recruitment will be recruited to CES loci and alter MSL complex recruitment but not transcription of MSL complex components . Using these approaches , we examined the function of three factors: 1 ) the NSL complex; 2 ) the PAF complex; and 3 ) CG1832 . We selected NSL complex for further study because six of its components ( NSL1 , NSL3 , CG1135 , z4 , Chro , and MBD-R2 ) were identified in our screen and NSL and MSL complexes share the MOF catalytic component . NSL1 is one of the structural components of NSL complex that directly contacts MOF using a similar protein-protein interaction surface as MSL1 [25] . Although this biochemical relationship would predict that NSL complex would compete with MSL complex for the MOF subunit , NSL complex was identified as an activator of our MSL-dependent reporter . Therefore , we further characterized the relationship between the two complexes to determine whether NSL complex functions positively during MSL complex recruitment . We also examined the PAF transcription complex that associates with RNAP II across gene bodies to promote transcription elongation [26][16] . Again , we identified multiple subunits of this complex in our screen , making the PAF complex a strong candidate . The PAF complex has been implicated in facilitating H2B ubquitylation catalyzed by the S . cerevisiae SAGA complex component Ubp8 [27][28] . Intriguingly , recent work has identified a new activity for the MSL2 protein as an E3 ligase that targets H2BK34 for ubiquitylation ( H2BK31 in Drosophila ) [29] . Therefore , it is possible that the PAF complex facilitates the H2B ubiquitin ligase activity of MSL2 . Also , MSL complex associates with gene bodies of active genes like the PAF complex [30][31] . The previously-unstudied CG1832 zinc finger protein was chosen for further study because it was identified three independent times using different double-stranded RNA constructs and exhibits a very strong reporter activation phenotype . Furthermore , unlike other potential DNA binding proteins , CG1832 RNAi does not decrease cell viability significantly and was not identified by other RNAi screens . Similarly , loss of MSL complex components does not significantly alter cell viability although it causes male-specific lethality in flies . The CG1832 protein has a glutamine rich N-terminus and a seven zinc finger domain at its C-terminus . Because each finger is likely to recognize three bases [32] , CG1832 is a candidate for recognition of a 21-mer sequence such as the MRE element . CG1832 is highly conserved among insect species and orthologs are present in other species such as mouse ( znf80 ) and human ( znf429 ) . Furthermore , CG1832 is maternally loaded into the early embryo and ubiquitiously expressed ( FlyAtlas ) and therefore it could potentially target MSL complex early in development . In addition , CG1832 is likely to be an essential gene because P-element insertions at its 5′ end are lethal in both sexes ( data not shown ) . Therefore , CG1832 would not have been identified by the seminal male-specific lethal screens in vivo . To determine whether candidate genes are needed for MSL complex recruitment to endogenous CES loci , we used chromatin immunoprecipitation ( ChIP ) of the MSL2 core component in male SL2 cells . We found that both NSL1 and CG1832 RNAi knockdowns significantly reduce MSL2 recruitment to several CES loci in vivo ( Figure 3A ) . Next , we conducted ChIP on the H4K16ac histone modification to examine the roles of NSL1 and CG1832 in depositing this key modulator of dosage compensation . In parallel with the effect on MSL2 recruitment , both NSL1 and CG1832 RNAi caused reduced H4K16ac at several CES loci . In contrast , PAF RNAi treatment did not alter MSL complex recruitment and therefore it is likely that PAF functions to alter MSL complex function at a step subsequent to recruitment ( Figure 3A ) . To examine whether NSL1 and CG1832 RNAi treatments disrupt MSL complex recruitment due to regulation of MSL complex component mRNA levels , we assayed these by qRT-PCR ( Figure S1 ) . roX1 is not expressed at significant levels in SL2 cells [33] . We noted effects on roX2 RNA levels as expected because roX2 is activated by MSL complex , but little effect on the mRNA levels of other MSL complex components . Also , NSL1 and CG1832 RNAi treatments do not affect mRNA splicing of MLE as reported for the Znf72D zinc finger protein ( data not shown ) [34] . MLE splicing was quantified using splice-site specific primers that were previously validated [34] . Because improper targeting of MSL complex reduces complex stability [35] , precise measurements of MSL protein levels do not address the point at which NSL1 or CG1832 acts to promote MSL complex activity . Instead , we tested the hypothesis that these proteins promote MSL recruitment by assessing their own localization to CES loci . To define NSL complex and CG1832 localization on chromatin , we analyzed the genome-wide occupancy of both factors . Genome-wide localization data were available for the Chromator/Chriz and MBD-R2 components of the NSL complex ( www . modENCODE . org ) . Therefore , we analyzed the enrichment of these proteins at CES loci compared with other sites that contain MRE sequences . Results indicate that there is a broad enrichment of NSL complex components within a 10 kb region surrounding CES compared to additional genomic loci on the X and autosomes that contain MREs ( Figure 4A ) , consistent with a local or regional function . To assess the genome-wide occupancy of CG1832 , we generated a polyclonal antibody that targets the N-terminus of the protein ( see Methods ) . We validated that the CG1832 antibody recognizes a protein of the predicted size and its RNAi knockdown strongly reduces its protein and mRNA levels and association with chromatin ( Figures S1 , S2 , S3 ) . CG1832 is expressed in both males and females and binds to polytene chromosomes in both sexes , consistent with it having a general , essential function in addition to its potential role in facilitation of MSL targeting ( Figures S2 , S4 ) . We performed ChIP-seq analysis on the CG1832 protein in male SL2 cells and mapped 2 , 695 CG1832 sites on the X and 10 , 009 on autosomes using inclusive statistical criteria for defining binding sites ( see Methods ) . Overall , MREs on the X chromosome are approximately 1 . 5-fold more likely to be occupied by CG1832 than MREs on autosomes ( 49 . 7% on X: 1832/3683; 32 . 5% on autosomes: 3787/11619 ) . Although CG1832 occupancy is not X-specific , it is highly enriched at CES loci . In fact , 98 . 5% ( 135/137 ) of the genetically defined CES that contain MRE sequences [3] have a CG1832 binding site . Also , 92 . 5% ( 446/484 ) of all MSL complex binding sites that contain an MRE also have a CG1832 binding site . This expanded set would include likely CES not characterized previously . Most compelling , CG1832 occupancy at MREs is strongly enriched at CES compared with locations on the X and autosomes that contain MRE sequences but fail to recruit MSL complex , as visualized by heat maps for enrichment at various classes of MREs ( Figure 4B ) . Comparison of ChIP-seq and ChIP-chip profiles of CG1832 , MBD-R2 , and Chromator show similar localization to 5′ ends of active genes , with CG1832 more focused at CES ( Figure 4C ) . The precise pattern of enrichment over MREs makes CG1832 a strong candidate to directly recognize these sequences . These results demonstrate that our genetic screen has identified at least two new regulators of MSL complex recruitment that are implicated in functioning directly at chromatin entry sites .
Using a novel cell-based RNAi screening approach , we have identified new candidate regulators of MSL complex function . The screen took advantage of the ∼five fold regulation of a roX2-luciferase reporter construct by MSL complex , rather than relying on a direct assay of dosage compensation ( <two fold ) or on more laborious assays for MSL occupancy . Many of the regulators that we have identified are likely to have additional functions in both male and female flies and therefore could not have been recovered from classical male-specific lethal genetic screens . In addition to direct regulators of MSL complex recruitment , it is possible that transcriptional regulators identified from our screening approach could function indirectly to regulate transcription of MSL complex components themselves . Therefore , we used chromatin immunoprecipitation and existing modEncode data to demonstrate that two factors , the NSL complex and CG1832 , are likely to have local roles in MSL complex recruitment to CES . Moreover , CG1832 is a strong candidate to recruit MSL complex directly to CES due to its strong enrichment at MRE sequences . Interestingly , we identified a previously unstudied function of the highly-conserved NSL complex at CES loci . NSL and MSL complex share the MOF histone acetyltransferase as their catalytic component [13][36] and there are conserved MOF protein-protein interaction interfaces between MSL1 and NSL1 [25] . Therefore , it is possible that MSL complex and NSL complex would compete for the MOF subunit , suggesting that NSL complex would antagonize MSL complex . However , we instead found that NSL complex facilitates MSL complex recruitment to CES ( Figure 3 ) . Therefore , it is possible that MOF may be recruited to MSL entry sites via NSL complex components such as MBD-R2 or RCD-5 that are known to be involved in MOF recruitment to 5′ ends of active genes [37][36] . Subsequently , MOF could be transferred to newly assembling MSL complexes at chromatin entry sites as previously suggested [25] . Alternatively , since functional CES are enriched in the vicinity of active genes [38] , this could indicate a role for NSL-dependent active gene expression in CES function . Many additional activators were identified from our RNAi screening approach including all components of the PAF transcription complex that associates with RNAP II across gene bodies to promote transcription elongation and polyadenylation [26][16] . However , RNAi treatment targeting the PAF complex did not alter MSL complex recruitment to entry sites or MSL target genes ( Figure 3A ) . There are several possible mechanisms by which PAF complex could regulate our MSL-dependent reporter gene . The PAF complex has been implicated in facilitating H2B ubquitylation catalyzed by the S . cerevisiae SAGA complex component Ubp8 [27][28] . H2B was also identified as an activator in our screen potentially implicating H2B ubiquitylation in facilitating MSL-dependent activity of the reporter ( Table S1 ) . Moreover , recent work has identified a new activity for the MSL2 protein as an E3 ligase that targets H2B for ubiquitylation [29] . Therefore , it is possible that the PAF complex facilitates the H2B ubiquitin ligase activity of MSL2 . Alternatively , PAF has been implicated in regulating H3K36me3 [39] suggesting that it could also mediate MSL complex activity by modulating deposition of this known regulator of MSL complex recruitment to active genes . In addition to identifying activators of MSL-dependent reporter activity , we identified several repressors including NURF301 , a known repressor of MSL complex function at the roX CES loci [24] . In this way , we validated that additional candidates have potential to directly function in repressing MSL complex recruitment or activity . Other repressors of known function include the Sfmbt protein that recruits the Polycomb repressor complex and the large TRRAP protein that mediates interactions between gene specific regulators and large transcription complexes [40] . It is possible that these proteins and other potential repressors directly antagonize MSL complex function or alter the chromatin environment around CES to reduce transcriptional activation . Our most exciting new candidate is CG1832 , a previously unstudied zinc-finger protein that is strongly and precisely enriched at MRE sequences within CES and facilitates MSL complex recruitment to these sites . This protein has seven zinc-fingers at its C-terminus that could potentially bind DNA and a glutamine-rich N-terminus that may interact with MSL complex . Therefore , future analyses will focus on the exciting possibility that CG1832 functions as a previously unknown link between MSL complex and MRE sequences . In summary , our novel screening approach identified two regulators that facilitate MSL complex recruitment at CES: NSL complex and CG1832 . Furthermore , our genetic screen provides an extensive dataset of additional candidate genes that may facilitate MSL targeting and function . We expect that future genetic , genomic , and biochemical approaches will define new mechanisms by which many of these candidate genes modulate coordinate gene regulation .
A genome-wide RNAi screen was performed in duplicate using the version 2 RNAi libraries generated by the Drosophila RNAi Screening Center ( www . flyrnai . org ) [41] . Transient transfection of MSL-dependent and independent luciferase reporter plasmids was conducted as described previously [3] . For screening , all volumes of plasmids and luciferase reagents were reduced by 4-fold to accommodate a 384-well format from the original 96-well format . As controls , GFP dsRNA was added to two wells and MSL2 dsRNA was added to two additional wells on every screening plate . GFP and MSL2 dsRNA was prepared as described previously [42] . All RNAi treatments were conducted for 5 days . First , the ratio of the Firefly luciferase to the Renilla luciferase activity was computed for each well in each of the 124 screening plates . Second , the Firefly/Renilla activity ratios for the GFP dsRNA wells and the MSL2 dsRNA wells were compared to assure that at least a 5-fold dynamic range was present for each plate . All plates fulfilled these criteria . Third , we calculated the median Firefly/Renilla ratio for each plate and its standard deviation . Our criterion for identifying a well as a positive screening hit was that its Firefly luciferase/Renilla luciferase ratio is more that two standard deviations above or below the plate median for both replicate plates . Most 384-well plates had 2–5 screening hits . To eliminate screening hits that cause non-specific cell lethality or low transfection efficiency , we did not consider wells with Renilla luciferase values that were more than three-fold below the plate median . Secondary screens using validation dsRNAs and additional plasmids described in [3] were performed using the same protocol and analysis approach . All dsRNA amplicons are identified in Table S1 by their DRSC number . mRNA analysis and qRT-PCR was performed after the following RNAi treatments: 1 ) DRSC03718 to target CG1832; 2 ) DRSC15625 to target NSL1 ( CG4699 ) ; and 3 ) DRSC27502 to target PAF1 ( atms ) . All RNAi treatments were conducted for 5 days in SL2 cells as previously described [42] . An additional CG1832 RNAi construct ( DRSC 29935 ) was also tested with similar results ( data not shown ) . Western blotting was conducted as previously described using standard protocols [42] . We generated the rabbit polyclonal CG1832 antibody against part of the glutamine-rich domain of the protein ( amino acids: #22-121 ) that was determined to be unique in the Drosophila genome . For Western blotting , the CG1832 antibody was used at a 1∶1000 dilution , the tubulin antibody ( Sigma ) was used at a 1∶10 , 000 dilution , and the histone H3 antibody ( Abcam ) was used at a 1∶1000 dilution . Chromatin immunoprecipitation from SL2 cells was conducted as described previously [42] using antibodies targeting the MSL2 protein , H4K16ac ( Millipore ) , H3 ( Abcam ) , and CG1832 ( SDI ) proteins . RNAi treatments were performed using the following DRSC RNAi constructs ( www . flyrnai . org ) : 1 ) DRSC03718 to target CG1832; 2 ) DRSC15625 to target NSL1 ( CG4699 ) ; and 3 ) DRSC27502 to target PAF1 ( atms ) . To assure reproducibility , large scale RNAi experiments were performed in which 225 ug of each dsRNA was added to a T225 flask containing 45 mls of SL2 cells . An additional CG1832 RNAi construct ( DRSC 29935 ) was also tested with similar results ( data not shown ) . Three independent chromatin preparations were performed for each experiment and qPCR was performed on input and IP samples using primers that are specific for MSL target genes and CES loci . Primer sequences were previously described [42] . Standard deviations among the three replicates were calculated after normalization to a sample that was treated with the GFP dsRNA construct as a control within each chromatin preparation . mRNA extraction followed by qRT-PCR was performed in parallel with each ChIP experiment to assure that the RNAi treatments were effective . Duplicate ChIP-seq libraries from independent chromatin preparations were generated using a protocol adapted from the Illumina ChIP-seq sample preparation guide as follows ( www . illumina . com ) . To obtain sufficient starting material , three IPs were pooled and concentrated using Qiagen MinElute columns such that 15 ng of starting material were obtained prior to library preparation . Libraries from matching input samples were also prepared for each chromatin preparation . Each enzyme reaction was followed by purification using AMPure XP Beads ( Agilent ) . The fragmented DNA was 3′ end repaired and 5′ phosphorylated using End-it Kit ( Epicentre ) . A 3′ adenosine was added using Klenow Fragment ( 3′→5′ exo ) ( NEB ) . The annealed adapters were ligated in a 1∶50 dilution of the 10 mM stock using T4 DNA Ligase Quick Ligase Kit ( Enzymatics ) . The libraries were PCR amplified using Phusion high fidelity polymerase ( NEB ) for 15 cycles . The libraries were then size selected on a 2% agarose gel using the E-Gel system ( Invitrogen ) selecting a range from 200 bp to 500 bp followed by concentration on a MinElute Column ( Qiagen ) . Sequencing was performed on the Illumina GaIIx platform . Reads were aligned to the dm3 assembly of the D . melanogaster genome using the Bowtie aligner [43] . A summary of the sequencing statistics is provided below: Replicate #1 input: 29495696 raw reads , 17591247 aligned reads ( 59 . 6% ) Replicate #1 CG1832 IP: 27820142 raw reads , 19210177 aligned reads ( 69 . 1% ) Replicate #2 input: 22498098 raw reads , 13510882 aligned reads ( 60 . 1% ) Replicate #2 CG1832 IP: 28231400 raw reads , 15194724 aligned reads ( 53 . 8% ) Pearson R value for CG1832 ChIP-seq replicates , 1 kb bins: 0 . 95 Reads aligned with more than one mismatch , and reads that did not align uniquely were discarded . Peaks were called in each ChIP-seq sample using the SPP software package with an FDR threshold of 0 . 05 [44] . For comparisons with MSL complex ChIP-seq data , MSL complex ChIP-seq data [3] were realigned with Bowtie and peaks were called with SPP in the same manner as the CG1832 ChIP-seq samples , for more direct comparison . The locations of CES loci and MRE sequences were previously reported [3] . Data will be available at NCBI GEO Short Read Archive ( SRA ) upon publication . The average enrichment profiles of proteins around the MREs ( +/−5 kb ) are shown in Figure 4 . The five columns in the heatmap are the following: ‘MRE in CES’ consists of experimentally obtained 140 MREs ( located within 137 CES ) described in [3]; ‘Best MRE on X’ and ‘Best MRE on 2L’ consist of 150 MREs that have the best consensus motif match on the X or 2L , respectively; and ‘Random MRE on X’ and ‘Random MRE on 2L’ consist of 150 MREs randomly chosen from the X or 2L , respectively . More details can be found in Alekseyenko et al , 2012 ( submitted ) . ChIP-chip data using Genomic DNA Tiling Arrays v2 . 0 ( Affymetrix ) are publicly accessible online through the modENCODE project ( www . modencode . org ) . Data analysis was performed in R statistical programming environment ( http://www . r-project . org ) . For the visualization of the heatmap ( e . g . , Figure 4 ) , the +/−5 kb region surrounding each MRE was separated into non-overlapping bins of 200 bp . The smoothed probe value within each bin is averaged to obtain the enrichment value for that bin . Heatmaps were generated using the same classes of MRE containing loci as described above for the CG1832 ChIP-seq data . | Gene regulation is essential to all living things . For example , levels of gene expression in individual cells must be fine-tuned during development and in response to changing environmental conditions . Genes are regulated by DNA binding proteins and by factors that influence DNA packaging into chromatin . The MSL complex in Drosophila melanogaster is a chromatin-modifying complex that specifically regulates a large number of genes . The MSL complex targets active genes on the single male X chromosome to upregulate their output to match both female X chromosomes . How the MSL complex specifically targets the X chromosome and upregulates active genes is only partially understood . In order to increase our understanding of gene regulation at a mechanistic level , we performed a genome-wide genetic screen in male cells to identify factors that facilitate MSL targeting and function . Our results identify two chromatin-associated protein complexes and a new candidate DNA binding protein as key factors in MSL–based regulation . We also provide an extensive list of additional candidate genes to be examined in future studies . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [
"biology"
] | 2012 | Identification of Chromatin-Associated Regulators of MSL Complex Targeting in Drosophila Dosage Compensation |
Covalent intermolecular cross-linking of collagen is essential for tissue stability . Recent studies have demonstrated that cyclophilin B ( CypB ) , an endoplasmic reticulum ( ER ) -resident peptidyl-prolyl cis-trans isomerase , modulates lysine ( Lys ) hydroxylation of type I collagen impacting cross-linking chemistry . However , the extent of modulation , the molecular mechanism and the functional outcome in tissues are not well understood . Here , we report that , in CypB null ( KO ) mouse skin , two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type ( WT ) or heterozygous ( Het ) mice . Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice . Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO . Lys hydroxylation at other sites was also lower in KO but to a lesser extent . A key glycosylation site , α1 ( I ) Lys-87 , was underglycosylated while other sites were mostly overglycosylated in KO . Despite these findings , lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het . However , the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased , respectively , in KO . The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT . These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes . Together , our study underscores the critical role of CypB in Lys modifications of collagen , cross-linking and mechanical properties of skin .
Collagens comprise a large family of structurally related extracellular matrix proteins in vertebrates [1 , 2] . Among the family members , fibrillar type I collagen is the most predominant type , providing connective tissues with form and tensile strength . Type I collagen is a heterotrimetric molecule composed of two α1 chains and one α2 chain forming a long uninterrupted triple helix with short nonhelical domains ( telopeptide ) at both N- and C-termini . One of the functionally important characteristics of fibrillar collagens is its set of unique post-translational modifications such as hydroxylation of proline ( Pro ) and lysine ( Lys ) residues , O-glycosylation of hydroxylysine ( Hyl ) [3] in the helical domain , oxidative deamination of Lys and Hyl in the telopeptides , and subsequent covalent intra- and intermolecular cross-linking [2] . These intra- and extra-cellular modifications require intricate coordination of a large number of biochemical events involving specific enzymes and their chaperone molecules [2] . Imbalance of the modification events results in various connective tissue diseases [4–6] . Lysyl hydroxylase 1–3 ( LH1-3 ) catalyze the hydroxylation of specific Lys residues in the procollagen α chains [7] . It is generally accepted that LH1 primarily hydroxylates Lys residues in the helical domains , while LH2 hydroxylates in the telopeptidyl domains [7–11] . LH3 is a multifunctional enzyme possessing LH , hydroxylysyl galactosyltransferase [12] , and galactosylhydroxylysyl glucosyltransferase ( GGT ) activities , but mainly functions as GGT in type I collagen [13–16] . These modifications are critical in determining the type and maturation of covalent intermolecular cross-links and , thus ultimately , collagen stability . In soft connective tissues such as skin and cornea , the major cross-links are derived from non-hydroxylated Lys residues in the C- and N-telopeptides . These residues are converted to aldehyde by the action of lysyl oxidase ( Lox ) and the Lysald generated then reacts with the juxtaposed helical Hyl on a neighboring molecule to form an aldimine intermolecular cross-link , dehydro-hydroxylysinonorleucine ( HLNL ) . Another major cross-link in these tissues is derived from an aldol condensation product ( ACP ) formed between two residues of telopeptidyl Lysald within the same molecule; the ACP then intermolecularly condenses with the helical histidine ( His ) and Hyl residues to produce a tetravalent cross-link , dehydro-histidinohydroxymerodesmosine ( HHMD ) [17] . Notably , both cross-links are derived from the non-hydroxylated telopeptidyl Lysald and helical Hyl . Recent studies on recessive osteogenesis imperfecta ( OI ) have provided significant insight into the mechanism by which LH activities are regulated by several endoplasmic reticulum ( ER ) chaperones [18] . Cyclophilin B ( CypB ) , encoded by the PPIB gene , is an ER-resident peptidyl-prolyl cis-trans isomerase ( PPIase ) that functions as a component of the collagen prolyl 3-hydroxylation complex [19 , 20] . In tissues of CypB-null ( hereafter referred to as CypB KO ) mice , a model of recessive type IX OI [21] , prolyl 3-hydroxylation at α1 ( I ) -986 , the major site for this modification , is severely suppressed [21–24] . Furthermore , a series of recent studies on the CypB KO mouse tissues have provided evidence that CypB also modulates Lys hydroxylation in a tissue- and molecular site-specific manner affecting cross-linking chemistry , fibrillogenesis and tissue formation [21 , 23 , 24] . However , the extent of alterations in Lys modifications , the molecular mechanism of CypB-controlled LH functions and the functional outcome in different tissues are still not well understood . In the present study , we performed in-depth analysis of Ppib KO mouse skin collagen , and report the profound effects of CypB deficiency on Lys post-translational modifications of type I collagen , cross-linking , Lys modifying enzymes and their ER chaperones , and tissue mechanical property .
The effect of CypB deficiency on skin collagen matrix was first examined by light microscopy with H&E and picrosirius red staining ( S1 Fig ) . Under polarized light , KO skin showed sporadically distributed , thick but less dense collagen fibers than WT skin , indicating poorly organized collagen matrices . Since the structure of skin showed no difference between WT and Het , images only from WT and KO were shown . Gelatinized skin samples were subjected to proteomic analysis using LC-quadrupole time-of-flight ( QTOF ) -MS/MS after trypsin digestion . Type I and III collagens were identified as major protein components in all WT , Het , and KO mice , while no other types of collagen were identified ( S1A Table ) . The type I/III ratio was further estimated by LC-QTOF-MS analyzing tryptic marker peptides using stable isotope-labeled collagen ( SI-collagen ) as an internal standard [25] . Type III collagen content in the skin did not differ significantly among three genotypes ( 14 . 2 ± 2 . 6% for WT , 15 . 0 ± 1 . 0% for Het , and 13 . 9 ± 1 . 0% for KO ) ( S1B Table ) . The majority of skin collagen was type I collagen ( >85% ) , which is similar to tail tendon [24] and dentin [23] . The distributions of Lys hydroxylation/glycosylation at specific sites within the triple helical region of type I collagen were semiquantitatively estimated by LC-QTOF-MS using tryptic digests of skin samples as described in previous studies [21 , 23 , 24] . Although the values of WT and Het were essentially identical to each other at all analyzed sites , Lys modifications were affected in a site-specific manner in the absence of CypB ( Table 1 ) . The most notable difference in Lys hydroxylation between KO and WT/Het was observed at all four helical cross-linking sites: At α1 Lys-87 , only ~19 . 2% was hydroxylated in KO whereas it was almost completely ( ~99 . 8% ) hydroxylated in WT/Het . At α2 Lys-87 , hydroxylation was 29 . 3% in KO while 95–97% in WT/Het . At α2 Lys-933 , it was 30 . 4% in KO and 100% in WT/Het . For α1 Lys-930 , using collagenase/pepsin digest as previously reported [23] , we analyzed Lys modifications in the peptide containing α1 Lys-918/930 ( GDKGETGEQGDRGIKGHR ) . In WT/Het , these sites were almost fully hydroxylated ( Lys + Lys = 0% , Lys + Hyl = 4 . 1% , and Hyl + Hyl = 95 . 9% ) , however , at least 66 . 4% of those in KO was nonhydroxylated ( Lys + Lys ) , indicating that α1 Lys-930 in KO was significantly underhydroxylated compared to WT/Het ( Table 1 ) . Though to a lesser extent , significant underhydroxylation was also observed at other sites in the helical domain: α1 Lys-99 ( ~17% for WT/Het vs 9 . 3% for KO ) , α1 Lys-603 ( 86 . 1% for WT vs 84 . 3% for KO ) , and α2 Lys-174 ( 61–63% for WT/Het vs 8 . 2% for KO ) . In contrast , Lys hydroxylation at α1 Lys-564 was significantly increased ( 51 . 2% in KO vs 23–24% in WT/Het ) , and that at α1 Lys-174 and α1/2 Lys-219 was unchanged in KO type I collagen . These results show that the largest relative changes in Lys hydroxylation occur at specific cross-linking sites , α1/2 Lys-87 , α1 Lys-930 , and α2 Lys-933 . We next analyzed Lys hydroxylation in the telopeptides of type I collagen . We previously analyzed telopeptidyl Lys hydroxylation at N-telopeptide ( Lys-9N ) and C-telopeptide ( Lys-16C ) of the α1 ( I ) chain after sequential digestion by Grimontia collagenase and pepsin [23] . In the present study , we further identified Lys-5N-containing peptide ( pQYSDKGVSSGPGPM; pQ indicates pyroglutamic acid ) from N-telopeptide of α2 ( I ) chain ( S2 Fig ) . Furthermore , Lysald-containing peptides were identified for the three telopeptidyl cross-linking sites ( S2–S5 Figs ) . These aldehydes are likely derived from dissociation of labile cross-linking bonds during the sample preparation for MS analysis , e . g . heat denaturation , since hydroxynorleucine , the reduced product of Lysald , was not detected by cross-link analysis performed on separate aliquots of the same samples . Neither Hyl nor Hylald-containing peptides were detected in KO , WT and Het skin samples . In Table 1 , Lys represents Lys and Lysald . Furthermore , eight glycosylation sites , α1 Lys-87 , α1 Lys-99 , α1 Lys-174 , α1 Lys-564 , α1 Lys-603 , α2 Lys-174 , α2 Lys-219 , and α2 Lys-933 were identified ( Table 2 ) . The effect of CypB KO on glycosylation patterns was found to be site-specific . When calculated as percentages of glucosylgalactosyl ( GG ) - , galactosyl ( G ) - , and free-Hyl in total Hyl , the relative abundance of GG-Hyl at α1 Lys-87 , the major glycosylation site , was significantly lower in CypB KO skin collagen compared to those of WT/Het . Free-Hyl at this site , in contrast , was significantly higher in KO ( Table 2 ) , which is different from those in CypB KO bone , tendon and dentin [21 , 23 , 24] . In contrast , at almost all other sites , i . e . α1 Lys-99 , α1 Lys-174 , α1 Lys-564 , α2 Lys-174 , and α2 Lys-219 , the relative abundance of GG-Hyl was significantly higher in KO than those of WT/Het , and free-Hyl was significantly lower in KO compared to WT and Het ( Table 2 ) . Thus , except for α1 Lys-87 , Hyl glycosylation was higher in CypB KO type I collagen than WT/Het . The CypB protein levels in dermal tissues were assessed by Western blot analysis ( Fig 1 ) . An immunoreactive band was observed at the expected molecular mass of CypB at ~19 kDa in the tissues of the WT while that in KO skin was absent ( Fig 1A ) . Since Lys hydroxylation and Hyl glycosylation of type I collagen in KO were significantly affected , we then examined the protein levels of the responsible enzymes , i . e . LH1-3 and glycosyltransferase 25 domain containing 1 ( GLT25D1 ) , by Western blot analyses , and found that all of these modifying enzymes were significantly upregulated in KO skin ( Fig 1B–1E ) . In order to pursue the alternative control mechanisms of Lys modifications by CypB , we examined the recently proposed LH-associated chaperone components . They included: a positive modulator for LH2 , FK506-binding protein 65 ( Fkbp65 ) [26] , a negative modulator for LH2 , heat shock protein 47 ( Hsp47 ) , a stabilizer of these molecular complexes , Immunoglobulin heavy-chain-binding protein ( Bip ) [27] , as well as positive LH1 modulators CypB , Synaptonemal Complex 65 ( Sc65 ) and prolyl 3-hydroxylase 3 ( P3h3 ) [28] . The results demonstrated that Fkbp65 , Sc65 , and P3h3 were severely suppressed in KO to less than 8% ( Fkbp65 ) , 11% ( Sc65 ) , and 11% ( P3h3 ) of those in WT ( Fig 1F , 1G and 1H ) , respectively . The level of Bip was slightly diminished in KO ( Fig 1I ) , whereas Hsp47 was significantly upregulated in KO ( p<0 . 05 , Fig 1J ) . Thus , while all LHs were upregulated , all of their positive modulators were suppressed , a negative modulator for LH2 , Hsp47 was upregulated . To examine the levels of Lox family members , i . e . Lox and Loxl1-4 , we performed Western blot analysis with respective antibodies . The results demonstrated that the immunoreactivities for mature Lox , Loxl1 and Loxl4 were comparable between WT and KO ( S6 Fig ) . Immunoreactivities for Loxl2 and 3 were not detected in WT and KO under the conditions used . The gene expression level of Lox also showed no difference between WT and KO fibroblasts . We next performed immunohistochemical analyses for LH1-3 , GLT25D1 , Fkbp65 , Sc65 , P3h3 , Hsp47 , Bip , Lox and its isoforms ( Loxl1and Loxl4 ) of WT and KO skin tissues . More intense immunoreactivities in and around fibroblasts were evident for all LH1-3 , GLT25D1 and Hsp47 in KO skin when compared to those in WT ( Fig 2 ) . In contrast , immunoreactivity of Fkbp65 , Sc65 , and P3h3 in KO was markedly diminished in comparison with WT . Slightly decreased immunoreactivity for Bip was seen in KO . Immunoreactivities for Lox , Loxl1 and 4 were comparable between WT and KO ( S7 Fig ) . These results are consistent with Western blot analyses described above ( Fig 1 and S6 Fig ) . Cross-link analysis detected radioactive peaks in the KO acid hydrolysate that were not detected in WT/Het . To identify these reducible compounds , potential cross-links generated by the unusual Lys hydroxylation pattern in CypB KO type I collagen , the compounds were enriched by a molecular sieve column chromatography and subjected to cross-link and mass spectrometric analyses . Fig 3 shows the typical chromatographic patterns of collagen cross-links obtained from the acid hydrolysates of reduced WT , Het , and KO skin samples . In all three groups , two Lysald-derived reducible cross-links , HLNL and HHMD , were identified . WT and Het showed essentially identical cross-linking pattern exhibiting these two cross-links with no statistical difference . In KO skin , in contrast , both of these cross-links were markedly diminished ( i . e . HLNL , 0 . 53±0 . 10 in WT , 0 . 47±0 . 07 in Het , and 0 . 08±0 . 01 mol/mol of collagen in KO , p<0 . 001; for HHMD , 0 . 72±0 . 15 in WT , 0 . 75±0 . 16 in Het , and 0 . 08±0 . 03 mol/mol of collagen in KO , p<0 . 001 , respectively ) ( Table 3 ) . However , two additional radioactive peaks were detected ( unidentified peaks 1 and 2 in Fig 3 ) . The peak 1 that eluted after HHMD represented the highest peak and the structure was identified as deoxy ( d ) -HHMD ( see below ) . The peak 2 was also identified as LNL ( see below ) . The amounts of these compounds were 0 . 28±0 . 07 and 0 . 09±0 . 02 mol/mol of collagen , respectively . Using the acid hydrolysates of reduced WT and KO skin samples , cross-linking amino acids were enriched by molecular sieve column chromatography [29] . The eluent was collected and an aliquot of each fraction collected was measured for radioactivity . There were two major radioactive peaks from both WT ( R1 and R2 , Fig 4A ) and KO ( R3 and R4 , Fig 4B ) samples . The fractions encompassing these peaks were pooled , lyophilized and subjected to cross-link analysis by HPLC equipped with a strong cation exchange column [30] . R1 was eluted at the position corresponding to HHMD ( Fig 4C ) and R2 to HLNL ( Fig 4D ) , respectively , as single peaks . However , the pattern of KO was significantly different . R3 showed two radioactive peaks , including a major peak eluting after HHMD ( unidentified peak 1 ) and a minor peak corresponding to HHMD ( Fig 4E ) , indicating that both compounds possess similar molecular weights but the unidentified compound is more basic than HHMD . R4 also contained a slightly larger radioactive peak eluting at the position after HLNL but before HHMD ( unidentified peak 2 ) , and a smaller peak corresponding to HLNL ( Fig 4F ) . This indicates that the compound showing a larger radioactive peak possesses a similar molecular weight as HLNL , i . e . eluted at the same position on a molecular sieve column , but is more basic compared to HLNL . We suspected that these unidentified compounds were collagen cross-links similar to HHMD and HLNL , respectively , but associated with very low Lys hydroxylation at the helical cross-linking sites in KO collagen ( Table 1 ) . The chemical structures of these crosslinks were then determined by mass spectrometric analyses . To determine the structures of these compounds detected in the KO samples , we reduced the KO skin samples with non-radioactive NaBH4 , hydrolyzed , subjected to the same column chromatography in the same manner as above and collected the fractions corresponding to those of R3 and 4 . These fractions were analyzed by high resolution QTOF-MS . In the R3 fraction , MS/MS spectra were obtained for theoretical m/z of d-HHMD ( C24H43N7O8 + H = m/z 558 . 32 ) and HHMD ( C24H43N7O9 + H = m/z 574 . 32 ) ( Fig 5A and 5C ) . Similar MS/MS fragmentation patterns were obtained for the two precursor ions , and some fragment peaks ( m/z 156 . 08 and m/z 245 . 16 ) were determined to be commonly derived from d-HHMD and HHMD . In addition , fragment peaks at m/z 159 . 12 and m/z 403 . 27 characteristic for the precursor ion at m/z 558 . 32 were assigned to be Lys-containing fragments of d-HHMD ( Fig 5A ) , while other fragment peaks at m/z 175 . 11 and m/z 419 . 26 observed only for the precursor ion at m/z 574 . 32 were assigned to be Hyl-containing fragments of HHMD ( Fig 5C ) . Similarly , in the R4 fraction , the structure of LNL ( C12H25N3O4 + H = m/z 276 . 19 ) and HLNL ( C12H25N3O5 + H = m/z 292 . 19 ) were confirmed by MS/MS fragmentation of corresponding precursor ions as shown in Fig 5B and 5D . Fragmented peaks due to loss of NH3 , H2O , and/or HCOOH were observed for LNL , HLNL , Lys ( fragmented from LNL and HLNL ) , and Hyl ( fragmented from HLNL ) . These results demonstrated the presence of d-HHMD/HHMD in the R3 fraction and LNL/HLNL in the R4 fraction . We conclude that the d-HHMD and LNL cross-links found in KO skin were formed by the same mechanisms as HHMD and HLNL , respectively , but involved the helical Lys residues instead of Hyl . Table 3 shows the levels of glycosylated and non-glycosylated forms of HLNL . The amount of HHMD cross-link did not change with base hydrolysis , indicating that the Hyl residue involved in this cross-link is not glycosylated , consistent with the cross-linking pattern of mouse tail tendon collagen reported previously [24] , and presumably due to HHMD cross-links being derived from Hyl residues located at α1 ( I ) -930 or α2 ( I ) -933 , both of which are essentially non-glycosylated ( Table 1 ) . In CypB KO skin , there were significant decreases in the level of non-glycosylated- , G- , and GG-HLNL ( p<0 . 001 ) compared with those in WT and Het skins . S8 Fig shows the typical chromatographic pattern of the base hydrolysates obtained from reduced WT , Het and KO . In WT and Het , the relative amounts ( HLNL + G-HLNL + GG-HLNL = 100% ) of glycosylated ( G- and GG- ) and non-glycosylated HLNL were identical , and the non-glycosylated form was abundant ( ~49% ) with relatively low amounts of GG- ( ~34% ) and G-HLNL ( ~22% ) ( S8 Fig ) . In KO skin , the relative amount of GG-HLNL was higher ( ~47% ) and non-glycosylated HLNL lower ( ~37% ) when compared to those of WT/Het ( S8 Fig ) . Collagen was serially extracted from dissected skin using acetic acid and pepsin to estimate extractability of collagen in CypB KO skin ( S2 Table ) . Extracted collagen was quantified by LC-MS analysis of 4-hydroxyproline ( Hyp ) after acid hydrolysis with SI-collagen as an internal standard [25] . Since we noticed that a substantial amount of 4-Hyp was present in the commercial pepsin used here as peptide or gelatin form ( S9 Fig ) , we performed salt precipitation of the pepsin-soluble fraction to remove the pepsin-derived 4-Hyp . Although 0 . 5 M acetic acid extracted only trace amounts of collagen ( 14 . 2% in WT , 14 . 3% in Het , and 14 . 9% in KO , p>0 . 05 , respectively ) , 70 . 8% of collagen was extracted following digestion with pepsin from KO skin ( p>0 . 05 ) , similarly to WT/Het ( 78 . 4% in WT and 78 . 7% in Het , respectively ) . Thus , extractability of collagen in KO skin was not different from WT/Het . To evaluate the changes in the nanomechanical properties of skin , AFM-based nanoindentation was performed on the four regions ( S1 Fig ) of the cryo-sectioned , unfixed skin samples . The results were compared between WT and KO skin . AFM-nanoindentation detected markedly lower modulus ( Eind ) in the KO when compared to that in WT . In all four regions tested , we found that KO skin was significantly softer than that of WT ( i . e . epidermis , 12 . 04±1 . 16 KPa in WT and 4 . 70±0 . 36 KPa in KO , p<0 . 0001; for upper reticular dermis , 9 . 60±0 . 99 KPa in WT and 4 . 73±0 . 47 KPa in KO , p<0 . 0001; for middle reticular dermis , 6 . 89±0 . 46 KPa in WT and 4 . 11±0 . 30 KPa in KO , p<0 . 0001; for lower reticular dermis , 6 . 46±0 . 51 KPa in WT and 5 . 50±0 . 58 KPa in KO , p<0 . 05 , respectively ) ( Table 4 ) .
CypB KO mice have been generated as a model of recessive OI [21] . The function of CypB as a component of the P3H complex is well-known and , in all tissues reported thus far , CypB deficiency causes a marked decrease in prolyl 3-hydroxylation at the primary modification site , α1 ( I ) Pro-986 [21 , 23 , 24] . In addition , these recent studies have also revealed a novel role of CypB in Lys hydroxylation that impacts collagen cross-linking chemistry . In the present study , we further examined the effect of CypB KO on skin collagen by conducting extensive analyses and found: marked decreases in Lys hydroxylation of type I collagen at all of the helical cross-linking sites resulting in the formation of unusual cross-links , varying but less pronounced effects on Lys hydroxylation at other helical sites , altered site-specific changes in glycosylation , significant alterations in the levels of Lys modifying enzymes and their chaperone complex components , and impaired mechanical properties . The lack of CypB did not alter the major collagen types but has a major effect on post-translational modifications within collagen molecules . In the past , we performed analyses of Lys modifications ( hydroxylation and Hyl glycosylation ) in CypB KO type I collagen and collagen cross-links in bone [21] , tendon [24] and dentin [23] . The data from these studies demonstrated that , in these tissues , CypB deficiency caused decreased levels of Lys hydroxylation at α1/2 ( I ) -87 by 20–40% , but hydroxylation at other sites was either unchanged in bone and dentin , or slightly lower ( tendon ) when compared to WT/Het . In the present study , by employing trypsin and collagenase/pepsin digestion in combination with high resolution MS , we analyzed Lys modifications including all of the cross-linking sites , i . e . both in the telopeptidyl and helical sites , and non-cross-linking helical sites . In the CypB KO skin , a marked decrease in Lys hydroxylation at all of the helical cross-linking sites was observed ( Table 1 ) and the extent of decrease was far more pronounced ( Table 1 ) than any other CypB KO tissues reported [21 , 23 , 24] . We also found that , in skin type I collagen , Lys hydroxylation in other helical sites was also significantly affected to varying extents . It is generally accepted that LH1 is primarily responsible for helical Lys hydroxylation and LH2 for telopeptidyl Lys hydroxylation [2] . The involvement of LH3 in the helical Lys hydroxylation is also possible but less clear at this point . Thus , suppressed levels of helical Lys hydroxylation and absence of telopeptidyl Lys hydroxylation seen in CypB KO skin type I collagen indicate the impaired LH activities . To determine whether or not defective Lys hydroxylation seen in CypB KO collagen is due to the diminished LH proteins , we examined their levels in KO skin by Western blot and immunohistochemical analyses . To our surprise , we found that all of these LH isoforms are significantly higher in the KO skin than those of WT/Het ( Fig 1 ) . These data clearly indicate that upregulation of LH proteins alone is not sufficient to rescue the deficiency in Lys hydroxylation seen in CypB KO skin collagen . Heard et al . recently proposed that ER components Sc65 ( Synaptoenamel Complex 65 or P3H4 ) , P3h3 ( prolyl 3-hydroxylase 3 ) , LH1 and possibly CypB form a complex to regulate the LH1 activity at the helical cross-linking sites , i . e . α1/2-87 and α1–930 , in skin and bone [28] . Hudson et al . further proposed that all of these four ER components are essential for normal triple helix Lys hydroxylation [31] . Our results demonstrated that , in the absence of CypB , though LH1 protein level was higher , its chaperone complex components , Sc65 and P3h3 , were both markedly diminished . This likely caused the impaired LH1 activity leading to a marked decrease in helical Lys hydroxylation . It is possible that CypB may contribute to the stability of the CypB/Sc65/P3h3 complex that is critical to regulate LH1 activity , i . e . helical Lys hydroxylation . Since the level of LH1 protein was still high in KO skin , LH1 protein is stable regardless of the complex . However , a question still remains: why are the helical cross-linking sites much more affected than other helical sites ? Significantly varying effects on helical Lys hydroxylation in CypB KO type I collagen ( e . g . α1–564 Lys hydroxylation in KO type I collagen was even higher than that of WT/Het , while other sites were lower or unchanged ) , though their biological consequences are not clear , suggest that Lys hydroxylation at the helical cross-linking sites could be regulated by a mechanism distinct from other helical sites . Possibly , the unique sequence around the cross-linking sites , e . g . the presence of KGH at or near cross-linking sites , may provide preferential interaction sites for the CypB-involved Sc65/P3h3 ER complex to facilitate LH1 activity , while other sites may not necessarily require the presence of such a complex . It is still unclear why Lys hydroxylation at the helical cross-linking sites in CypB KO type I collagen in skin is far more affected than other tissues . Mineralized tissue type I collagen in general seems less affected in CypB KO mice [21 , 23] . Similarly , skin collagen in Sc65 and P3h3 null mice appeared more affected than mineralized tissues [28 , 31] . In mineralized tissue cells , this modification could be more protected by an unknown mechanism . O-linked glycosylation of Hyl is another post-translational modification that impacts collagen cross-linking [2 , 31] . Note that there are tissue-specific differences in the Hyl glycosylation at the helical cross-linking site , α1–87 , the major glycosylation site in type I collagen [23] . Our present and previous studies showed that , in WT mice , almost all Lys at this site are hydroxylated but the Hyl residue is glycosylated in varying degrees in different tissues , i . e . >98% in skin ( Table 2 ) , ~92% in bone [21] , ~80% in dentin [23] , and ~25% in tendon [24] . The type of glycosylation also varies: the ratio of GG-Hyl to G-Hyl at this site in skin is ~27:1 ( Table 2 ) while it is ~3:1 in bone [21] , ~2 . 5:1 in dentin [23] and ~4:1 in tendon [24] . These data demonstrate that , in normal skin collagen , Hyl at this site is highly glycosylated in the form of GG-Hyl , which is distinct from other major collagenous tissues ( also see Hudson et al [31] ) . This indicates that , in mouse skin type I collagen , Lys at α1 ( I ) -87 is almost quantitatively and sequentially modified by three enzymes , i . e . first hydroxylated by LH1 , then galactosylated by GLT25D1 [13 , 32] and finally glucosylated by LH3 [14 , 15] . Since LH1 activity at this site is positively regulated by a complex including CypB , Sc65 and P3h3 ( see above ) , and CypB also interacts with LH3 [24] , it is possible that all of these three enzymes are a part of this complex , and sequentially catalyze the respective modifications . It remains unclear as to why this site is different from the rest of the glycosylation sites where relative glycosylation , especially GG- form , is higher in KO than those of WT/Het ( Table 2 ) . A possible explanation is that , while a CypB-involved specific ER complex ( see above ) binds and modifies Lys at the helical cross-linking sites , the rest of the sites may not involve such a complex but rather depends on the accessibility of the substrate , e . g . folding rate of procollagen α chains , as CypB deficiency causes a significant delay in this process [21] . Another intriguing question is related to Lys hydroxylation in the telopeptides: why are the telopeptidyl Lys residues not hydroxylated in both WT and KO skin collagen ? We previously reported that CypB may differentially regulate Lys hydroxylation between helical and telopeptidyl domains , i . e . positively for the former and negatively for the latter [24] . We now know that LH2 catalyzes the telopeptidyl Lys hydroxylation [9–11 , 33 , 34] and its activity is regulated by Fkbp65 [26 , 35] together with other ER chaperones , i . e . Hsp47 and BiP [27] . Our present study has shown that the lack of CypB led to increased LH2 levels ( Fig 1 ) , but hydroxylation of telopeptidyl Lys resides was still incomplete . The increased LH2 supports our previous findings on CypB KO tendon in which Hylald-derived cross-links were formed while they were absent from WT [24] . However , in KO skin , despite an increase of LH2 , telopeptidyl Lys is not hydroxylated ( Table 1 ) and none of the Hylald-derived cross-links is formed ( Table 3 ) . This now can be partially explained by marked suppression of Fkbp65 , a slight decrease in Bip and a significant increase of Hsp47 [27] . Duran et al has recently proposed that Fkbp65 is a positive modulator , Hsp47 a negative regulator and Bip a stabilizer of the LH2 complex [27] . Our present data indicate the involvement of CypB in the LH2 chaperone complex as a negative regulator for LH2 , likely via its interaction with LH2 and Fkbp65 [24] . Possibly , in normal/WT skin , a low LH2 level and the presence of its negative regulators , Hsp47/CypB , may be sufficient to prevent Lys hydroxylation in telopeptides . In the case of KO skin , though LH2 level is increased , severely suppressed Fkbp65 and increased Hsp47 may prevent the LH2 activity for telopeptidyl Lys hydroxylation . A mechanism by which CypB differentially regulates Fkbp65 and Hsp47 is unclear . It is possible that CypB stabilizes Fkbp65 through direct interaction [24] , independent of Hsp47 , and the absence of CypB may cause rapid degradation of Fkbp65 , though this is not consistent with the report from Ishikawa et al . [36] . Together , it is likely that the absence of CypB causes imbalance of LH1 and 2 chaperone complexes resulting in marked reduction of Lys hydroxylation at all the helical cross-linking sites and absence of Lys hydroxylation in all the telopeptides . Further studies are warranted to elucidate the mechanism by which levels and stability of these LH chaperone complexes are regulated , leading to well-known tissue-specific cross-linking chemistry . It has been well established that the major collagen cross-links in normal skin are all derived from the telopeptidyl Lysald residues [2] , which is consistent with the MS data on WT/Het/KO showing there is no detectable Hyl in telopeptides ( Table 1 ) . In the N-telopeptides , as an intramolecular cross-link , ACP ( aldol condensation product; Lysald × Lysald ) is formed between two residues of Lysald and then cross-linked to His by Michael addition and Hyl by aldimine addition to produce a tetravalent reducible cross-link , dehydro-histidinohydroxymerodesmosine ( HHMD; Lysald × Lysald × His × Hyl ) [17] . This cross-link was originally isolated and identified by Tanzer and co-workers [17] . Though Robins et al soon claimed that this cross-link is an artifact of the NaBH4 reduction procedure [37] , Bernstein and Mechanic later provided evidence demonstrating it is indeed a natural cross-link present in vivo [38] . The molecular loci of this cross-link have not been conclusively determined at this point . The aldol condensation ( ACP ) can be derived from the N-telopeptides [17] or C-telopeptides [38] . The former would then involve the Hyl at α1 ( I ) -930 or α2 ( I ) -933 , and the latter at α1/2 ( I ) -87 . However , considering the fact that HHMD is not glycosylated , it is likely that the cross-linking Hyl is from α1 ( I ) -930 or α2 ( I ) -933 ( i . e . these residues are essentially non-glycosylated , see Table 1 ) rather than α1–87 , which is almost entirely glycosylated . Another interesting item to note is that collagen extractability of KO skin was essentially identical to WT/Het skin ( S2 Table ) despite the fact that the total amounts of cross-links in KO collagen are lower than those of WT/Het ( less than 50% ) ( Table 3 ) . Recently , Kalamajski et al [39] and Hudson et al [31] reported that ACP under abnormal conditions could be formed intermolecularly and render collagen more stable . If the ACP in CypB KO collagen is formed intermolecularly between the N-telopeptide-derived Lysald residues from the two parallel molecules in register [31] , even lower number of cross-links may be sufficient to maintain the overall insolubility . The total number of aldehydes involved in cross-linking was significantly lower in KO , which is different from other tissues like bone [21] , tendon [24] and dentin [23] , suggesting that Lox activity was diminished by the loss of CypB specifically in skin . However , this is not due to the lower levels of Lox and its isoforms in KO skin ( S6 and S7 Figs ) . Previously we reported that , when GG-Hyl was diminished by lowered level of LH3 , the total number of cross-links also decreased . This was not due to the diminished gene expression of Lox or its isoforms , suggesting that Lox binding to collagen in the extracellular space could be impaired due to the lower GG-Hyl form [15] . In the KO tissues we previously reported , i . e . bone , tendon and dentin , the GG-Hyl levels at α1 ( I ) -87 were even higher or similar compared to those of WT ( see Supplemental data in Terajima et al [23] ) and the total cross-links in KO collagens were higher or similar to those of WT . Thus , lower levels of cross-links seen in CypB KO skin collagen are possibly due in part to the low level of GG-Hyl . AFM-nanoindentation results highlighted the critical roles of CypB in the local micromechanical properties of skin extracellular matrix . Here , mechanical properties are an integrated response of the matrix composition and structural integrity . Specifically , given the dominance of fibrillar collagen in skin tissue , the modulus is a direct manifestation of type I collagen fibrillar organization . Here , AFM-nanoindentation was performed normal to the fiber axis with a microspherical indenter tip ( R ≈ 5 μm ) , resulting in tip-sample contact area ~ 10 μm2 . At this length scale , the indentation modulus represents the local resistance of collagen fibrils to uncrimping and sliding [40] . The lower Eind of the CypB KO skin tissue ( Table 4 ) is consistent with the reduction in cross-links ( Table 3 ) , since reduced covalent cross-linking results in collagen fibrils with higher compliance and less stability [41] . Further , the fact that modulus reduction was observed in all four domains suggested that the CypB-mediated regulation of collagen cross-linking is a ubiquitous phenomenon across all the anatomical regions of the skin . Taken together , our results clearly highlighted an essential role of CypB in the proper biomechanical function of skin tissue . Unfortunately , the deficits in biomechanical function of murine PPIB KO skin cannot be compared to data in human type IX OI . This is an especially rare and severe form of OI . Of the 10 reported cases , 7 survived the perinatal period . However , skin histology was not reported for any of the surviving children [19 , 20 , 42–45] , nor were they noted to have dermatological problems similar to the skin findings reported in horses with a substitution ( p . G6R ) at the N-terminal end of CyPB ( HERDA , hereditary equine regional dermal asthenia ) which is marked by sloughing and ulcerations [18 , 46] . Functional studies of the contribution of abnormal Lys hydroxylation and crosslinking of collagen to skin mechanics in humans are certainly warranted . In conclusion , our study demonstrates that , in skin tissue , deficiency of CypB profoundly affects collagen Lys modifications at all cross-linking sites generating unusual cross-links , altered levels of Lys modifying enzymes and dysregulation of ER complexes regulating LH1 or LH2 activity , skin tissue formation and mechanical property . These results underscore the critical role of this ER protein in skin-specific collagen post-translational modifications and tissue integrity .
Animal care and experiments were performed in accordance with a protocol approved by the NICHD , National Institutes of Health , animal care and use committee . CypB KO mice have recently been [21] generated as a mouse model of recessive OI . In this model , Ppib transcripts and CypB protein were not detected in primary cells and tissues . Dorsal skin was harvested from 2 month old WT , Het and CypB KO mouse and the histological sections were prepared ( 3 samples/group ) . The specimens were immersed for 3 days with 10% formalin and then immersed in 70% ethyl alcohol , dehydrated through ascending gradations of ethanol , embedded in paraffin , and sectioned into 5 μm thick slices . After hydration , the slices were stained with hematoxylin and eosin ( H&E ) and observed under light microscopy ( Olympus BX40; Olympus , Tokyo , Japan ) . In addition , to evaluate the organization and maturation of skin collagen matrices , the sections were also stained with 0 . 1% solution of Sirius Red in saturated aqueous picric acid ( Electron Microscopy Sciences , Hatfield , PA , USA ) for 60 min , washed with 0 . 01 N HCl , dehydrated and mounted . The Sirius Red stained sections were observed under a polarized light microscopy ( BX40 microscope , Olympus Co . , Center Valley , PA , USA ) and photographed as previously reported [47] . Dorsal skin was harvested from 2 month old WT , Het , and CypB KO mouse . All operations were carried out on ice or at 4°C . The dissected skin was pulverized with a pestle and mortar to a fine powder under liquid nitrogen . Pulverized samples were washed several times with cold phosphate-buffered saline ( PBS ) , and cold distilled water by repeated centrifugation at 4 , 000×g for 30 min , and lyophilized . Lyophilized skin from WT , Het and KO mice was heated at 60°C for 15 min in 50 mM sodium phosphate buffer ( pH 7 . 2 ) and the supernatants ( gelatin ) were collected by centrifugation . The gelatin samples were digested with sequencing grade trypsin ( Promega , Madison , WI , USA; 1:100 enzyme/substrate ratio ) in 100 mM Tris-HCl/1 mM CaCl2 ( pH 7 . 6 ) at 37°C for 16 h . The tryptic digest was analyzed by LC-MS on an ultra-high resolution QTOF mass spectrometer ( maXis II , Bruker Daltonics , Bremen , Germany ) coupled to a Shimadzu Prominence UFLC-XR system ( Shimadzu , Kyoto , Japan ) . Peptide separation was performed using an Ascentis Express C18 HPLC column ( 5 μm particle size , L × I . D . 150 mm × 2 . 1 mm; Supelco , Bellefonte , PA , USA ) at a flow rate of 500 μl/min with a binary gradient as follows: 100% solvent A ( 0 . 1% formic acid ) for 2 . 5 min , linear gradient of 0–50% solvent B ( 100% acetonitrile ) for 12 . 5 min , 90% solvent B for 2 . 5 min , and 100% solvent A for 2 . 5 min . The acquired MS/MS spectra were searched against the UniProtKB/Swiss-Prot database ( release 2014_08 ) using ProteinPilot software 4 . 0 ( AB Sciex , Foster City , CA , USA ) . Type I and III collagens were further quantified using SI-collagen as an internal standard [25] . In brief , SI-collagen was first mixed into the gelatin samples , and trypsin digestion was performed as described above . Generated marker peptides of type I and III collagens ( two peptides for each α chain ) were monitored by LC-QTOF-MS using a BIOshell A160 Peptide C18 HPLC column ( 5 μm particle size , L × I . D . 150 mm × 2 . 1 mm; Supelco ) . Concentrations of type I and type III collagens were estimated by the peak area ratio of extracted ion chromatograms ( EICs ) of the marker peptides relative to that of the corresponding stable isotopically heavy peptides derived from SI-collagen ( mass precision range = ±0 . 02 ) . Dried skin samples ( ~2 . 0 mg each ) were suspended in buffer containing 0 . 15 M N-trismethyl-2-aminoethanesulfonic acid , and 0 . 05 M Tris-HCl , pH 7 . 4 , and reduced with standardized NaB3H4 . The specific activity of the NaB3H4 was determined by the method described previously [48 , 49] . The reduced samples were washed with cold distilled water several times by repeated centrifugation at 4 , 000×g and lyophilized . The tryptic digests of gelatinized skin samples prepared above were subjected to LC-QTOF-MS to analyze the Lys post-translational modifications at the specific molecular sites within the triple helical domain of type I collagen . In addition , to analyze Lys hydroxylation at the telopeptide domains of type I collagen , the lyophilized skin samples were sequentially digested with bacterial collagenase and pepsin as previously reported [23] . In brief , the samples were digested with 0 . 01 mg/ml of collagenase from Grimontia hollisae ( Wako Chemicals , Osaka , Japan ) [50] in 100 mM Tris-HCl/5 mM CaCl2 ( pH 7 . 5 ) at 37°C for 16 h after heating at 60°C for 30 min . After addition of acetic acid ( final 0 . 5 M ) , the collagenase-digests were further digested with 0 . 01 mg/ml of pepsin ( Sigma-Aldrich , St . Louis , MO , USA ) at 37°C for 16 h . The peptide solutions digested with trypsin or collagenase/pepsin were subjected to LC-QTOF-MS analysis using the Ascentis Express C18 HPLC column under the same conditions as described above . Site occupancy of Lys hydroxylation/glycosylation ( Lys , Hyl , G-Hyl , and GG-Hyl ) were calculated using the peak area ratio of EICs ( mass precision range = ±0 . 02 ) of peptides containing the respective molecular species as previously reported [21 , 23 , 24] . Reduced collagen was hydrolyzed with 6 N HCl and subjected to cross-link analysis as described previously [30 , 51] . Upon reduction , dehydrohydroxylysinonorleucine ( dehydro-HLNL ) / its ketoamine , dehydrolysinonorleucine ( dehydro-LNL ) /its ketoamine , and dehydrohistidinohydroxymerodesmosine ( dehydro-HHMD ) are reduced to stable secondary amines , HLNL , LNL , and HHMD . The reducible cross-links were analyzed as their reduced forms ( i . e . HLNL , LNL , and HHMD ) . Hereafter , the terms HLNL , LNL , and HHMD will be used for both the unreduced and reduced forms . The levels of cross-links were quantified as mole/mole of collagen . The glycosylated immature reducible cross-links were analyzed employing base hydrolysis of the reduced samples [48] . By applying the hydrolysates to the HPLC system , the glycosylated ( GG- and G- ) and non-glycosylated cross-links were separated . These forms of cross-links were quantified as mole/mole of collagen as previously reported [15] . Molecular sieve chromatography of the acid hydrolysates of reduced skin samples were performed on a standardized column ( 1 . 0 × 50 cm ) filled with Bio-Gel P-2 ( Extra Fine , Bio-Rad , Hercules , CA , USA ) equilibrated with 0 . 1 N acetic acid at room temperature [29] . Aliquots of the acid hydrolysate ( ~4 mg ) of NaB3H4 reduced skin collagen were applied to the column and separated at a flow rate of 0 . 3 ml/min . Fractions of 0 . 6 ml were collected and an aliquot of each fraction was measured for radioactivity . The fractions encompassing two main radioactive peaks from WT ( R1 , 2 ) and KO ( R3 , 4 ) were pooled , lyophilized , and subjected to cross-link analysis as described above . Furthermore , to determine the structures of cross-links , acid hydrolysates of ( nonradioactive ) NaBH4-reduced WT and KO skin collagen were applied to the same column , the fractions eluted at the positions corresponding to R1-R4 were collected , lyophilized and analyzed by mass spectrometry ( see below ) . The structure of cross-links present in the radioactive fractions purified by molecular sieve chromatography were confirmed by the high resolution QTOF mass spectrometer . The R3 and R4 fractions from CypB KO skin were dissolved in 0 . 1% formic acid/50% acetonitrile , and MS/MS spectra of d-HHMD/HHMD ( R3 fraction ) and LNL/HLNL ( R4 fraction ) were obtained by direct infusion analysis . Extractability of collagen from lyophilized skin samples were evaluated by sequential extraction using acetic acid and pepsin [39] . Collagen was first extracted using 0 . 5 M acetic acid at 4°C for 24 h . After centrifugation at 20 , 000 xg for 30 min at 4°C , the supernatants were collected as acetic acid-soluble fraction . The pellets were further treated with 5 mg/ml pepsin in 0 . 5 M acetic acid at 4°C for 24 h and were then centrifuged to collect the supernatants as pepsin-soluble fraction and the pellets as residual fraction . Collagen in the pepsin-soluble fraction was purified by salt precipitation ( 2 M NaCl ) to remove pepsin-derived gelatin or peptides that contains 4-Hyp . The three fractions were subjected to acid hydrolysis ( 6 N HCl , 110°C for 20 h in the gas phase under N2 ) after addition of SI-collagen as an internal standard [25] . The acid hydrolysates were subjected to multiple reaction monitoring analysis of 4-Hyp on a hybrid triple quadrupole/linear ion trap 3200 QTRAP mass spectrometer ( AB Sciex ) coupled to an Agilent 1200 Series HPLC system ( Agilent Technologies , Palo Alto , CA , USA ) using a ZIC-HILIC column ( 3 . 5 μm particle size , L × I . D . 150 mm × 2 . 1 mm; Merck Millipore , Billerica , MA , USA ) as previously described [52] . Concentration of collagen was estimated by the peak area ratio of 4-Hyp to stable isotopically heavy 4-Hyp derived from SI-collagen . Tissues were lysed with radio-immunoprecipitation assay ( RIPA ) lysis buffer ( 50mM Tris-HCl , 150 mM NaCl , 0 . 5% sodium deoxycholate , 0 . 1% SDS , and 1% NP-40 ) containing a cocktail of protease inhibitors including EDTA ( cOmplete Mini Protease Inhibitor Cocktail , Roche City , IN , USA ) . Lysates were centrifuged at 12 , 000 xg and the supernatant was collected . The total protein concentration was measured by the Pierce BCA Protein Assay Kit ( Pierce Biotechnology , Rockford , IL , USA ) according to the manufacturer’s protocol . The cell lysate was mixed with 2x Laemmli Sample Buffer containing 2-mercaptoethanol ( BIO-RAD ) and 10 μg of total protein was applied to a 4–20% Mini-PROTEAN TGX Stain-Free Protein Gel ( BIO-RAD ) . The separated proteins were transferred to a polyvinylidene fluoride ( PVDF ) membrane ( Immobilon-P , Millipore Corp . , Bedford , MA , USA ) and probed with primary antibodies followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG ( Cell Signaling Technology ) . The immunoreactivities were detected with SuperSignal West Pico Chemiluminescent Substrate ( Thermo Fisher Scientific ) . Protein loading of cell lysate was confirmed by Western blotting with anti-GAPDH rabbit monoclonal antibody ( Clone 14C10 , Cell Signaling Technology ) . The immunoreactivities of these protein bands were scanned using an Odyssey Infrared Imaging System ( LI-COR ) . Quantitation of proteins was performed using the Image Studio software version 4 . 0 ( LI-COR ) with normalization to GAPDH levels . The primary antibodies used in this study were as follows; rabbit polychlonal PLOD1 antibody ( 1:200 , cat# 12475-1-AP , Proteintech ) , rabbit polychlonal PLOD2 antibody ( 1:100 , cat# 21214-1-AP , Proteintech ) , rabbit polychlonal PLOD3 antibody ( 1:200 , cat# 11027-1-AP , Proteintech ) , rabbit polychlonal GLT25D1 antibody ( 1:200 , cat# 16768-1-AP , Proteintech ) , rabbit polychlonal Fkbp65 antibody ( 1:200 , cat# 12172-1-AP , Proteintech ) , rabbit polychlonal CypB antibody ( 1:10 , 000 , cat# PA1-027A , Thermo Fisher Scientific ) , rabbit polyclonal Sc65 antibody ( 1:100 , cat# 15288-1-AP , Proteintech ) , rabbit polyclonal P3h3 antibody ( 1:100 , cat# 16023-1-AP , Proteintech ) , rabbit polyclonal Hsp47 antibody ( 1:100 , cat# 10875-1-AP , Proteintech ) , rabbit polyclonal Bip antibody ( 1:100 , cat# 11587-1-AP , Proteintech ) , rabbit polyclonal Lox antibody ( 1:100 , cat# NBP2-24877 , Novus Biologicals ) , rabbit polyclonal Loxl1 antibody ( 1:100 , cat# ab81488 , Abcam ) , rabbit polyclonal Loxl2 antibody ( 1:100 , cat# ab96233 , Abcam ) , rabbit polyclonal Loxl3 antibody ( 1:100 , cat# ab232884 , Abcam ) , and rabbit polyclonal Loxl4 antibody ( 1:100 , cat# ab88186 , Abcam ) . Total RNA was extracted from primary fibroblast cultures established from PPIB wildtype and knockout 3-days old pups ( n = 3/genotype ) using TriReagent ( Molecular Research Center Inc , Cincinnati , OH , USA ) , according to the manufacture’s protocol . Total RNA was treated with DNA-free Kit ( Life Technologies , Carfsbad , CA , USA ) and cDNA library was prepared by High Capacity cDNA Achieve Kit ( Life Technologies ) . Taqman assay probes , Lox , Mm00495386_m1 and Gapdh , Mm99999915_g1 ( Life Technologies ) were used for gene expression analysis . Real-time PCR was performed by using Taqman Fast Universal PCR Master Mix and the reactions were carried by QuanStudio 6 Flex ( Applied Biosystems , Foster City , CA , USA ) : 95°C 20 sec , then 40 cycles of 95°C 1 sec , 60°C 20 sec . Relative expression of Lox was normalized to Gapdh . To determine the distribution of Lys modifying enzymes and a molecular chaperone Fkbp65 at the histological level , immunohistochemical analysis was performed using the avidin-biotin complex method . The serial sections were deparaffinized , treated with 10 mM citric acid buffer ( pH 6 . 0 ) for antigen retrieval [53] and incubated with 0 . 3% H2O2 in methanol . The sections were then incubated overnight with the primary antibodies , washed several times with PBS , and incubated with biotinylated anti-rabbit IgG ( 1:400 , cat# PK-6101 , Vector Laboratories ) for 30 min . The sections incubated without primary antibodies were served as negative controls . After several washes with PBS , the sections were further incubatetd with avidin-biotin-HRP mixture for 30 min , and the immunoreactivity was visualized by 3 , 3’ diamino benzidine tetrahydrochloride ( DAB; Vector Laboratories ) . The sections were observed under light microscopy and photographed . The primary antibodies used in this study were the same as those used for Western blot analysis; rabbit polychlonal PLOD1 antibody ( 1:100 ) , rabbit polychlonal PLOD2 antibody ( 1:100 ) , rabbit polychlonal PLOD3 antibody ( 1:100 ) , rabbit polychlonal GLT25D1 antibody ( 1:200 ) , and rabbit polychlonal Fkbp65 antibody ( 1:200 ) , rabbit polyclonal Sc65 antibody ( 1:100 ) , rabbit polyclonal P3h3 antibody ( 1:100 ) , rabbit polyclonal Hsp47 antibody ( 1:100 ) , rabbit polyclonal Bip antibody ( 1:100 ) , rabbit polyclonal Lox antibody ( 1:100 ) , rabbit polyclonal Loxl1 antibody ( 1:100 ) , and rabbit polyclonal Loxl4 antibody ( 1:100 ) . Dorsal skin was harvested from 2 month old WT and CypB KO mouse ( 3 samples/group ) and cut into about 0 . 5 cm wide by 1 cm long pieces . The long axis of sample coincided with the cranio-caudal axis of the mouse . Samples were embedded in optimum cutting temperature medium ( OCT ) to produce 40 μm-thick , unfixed cross-sections using Kawamoto’s tape-assisted cryo-sectioning [54] . To quantify the modulus of each anatomical region on the cross-section , AFM-based nanoindentation was performed with a microspherical tip ( R ≈ 5 μm , k ≈ 1 N/m , μMasch ) and a Dimension Icon AFM ( Bruker ) in PBS with protease inhibitors ( Sigma-Aldrich ) , following our established procedures [55] . The effective indentation modulus ( Eind ) was calculated by fitting the loading portion of force-indentation depth curve with the finite thickness-corrected Hertz model ( Dimitriadis Ref 65 ) by assuming the Poisson’s ratio ν ≈ 0 . 45 [56] . | Deficiency of cyclophilin B ( CypB ) , an endoplasmic reticulum-resident peptidyl-prolyl cis-trans isomerase , causes recessive osteogenesis imperfecta type IX , resulting in defective connective tissues . Recent studies using CypB null mice revealed that CypB modulates lysine hydroxylation of type I collagen impacting collagen cross-linking . However , the extent of modulation , the molecular mechanism and the effect on tissue properties are not well understood . In the present study , we show that CypB deficiency in mouse skin results in the formation of unusual collagen cross-links , aberrant tissue formation , altered levels of lysine modifying enzymes and their chaperones , and impaired mechanical property . These findings highlight an essential role of CypB in collagen post-translational modifications which are critical in maintaining the structure and function of connective tissues . | [
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] | 2019 | Cyclophilin B control of lysine post-translational modifications of skin type I collagen |
The formation of vesicles is essential for many biological processes , in particular for the trafficking of membrane proteins within cells . The Endosomal Sorting Complex Required for Transport ( ESCRT ) directs membrane budding away from the cytosol . Unlike other vesicle formation pathways , the ESCRT-mediated budding occurs without a protein coat . Here , we propose a minimal model of ESCRT-induced vesicle budding . Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds . The model , cast in the framework of membrane elasticity theory , reproduces the experimentally observed vesicle morphologies with physically meaningful parameters . In this parameter range , the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck , consistent with experiment . The minimum energy configurations agree with those seen in the fluorescence images , with respect to both bud shapes and ESCRT protein localization . On the basis of our model , we identify distinct mechanistic pathways for the ESCRT-mediated budding process . The bud size is determined by membrane material parameters , explaining the narrow yet different bud size distributions in vitro and in vivo . Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding .
Lipid membranes enclose the cytosol of biological cells and compartmentalize their interior . The structure and contents of cellular membranes are actively controlled to sustain the vital functions of the cell . Transport vesicles are used to traffic membrane-bound proteins between cellular compartments . The best-characterized pathways of vesicle formation include those facilitated by BAR domain proteins [1] and by the coat protein clathrin with its adaptors [2] . Coat proteins impose their curved shape onto the membrane , thereby promoting vesicle curvature in an intuitively straightforward and computationally well-characterized process [3]–[5] . In the degradative transport of membrane proteins from endosomes to lysosomes ( Fig . 1 ) , small patches of the endosomal membrane bud into the interior ( lumen ) of the endosome and detach , forming intralumenal vesicles ( ILVs ) [6] , [7] . This pathway is catalyzed by the cytosolic Endosomal Sorting Complex Required for Transport ( ESCRT ) [8]–[10] . The ESCRT proteins are not internalized in ILVs , but rather recycled continuously in the cytosol [11] , [12] . To avoid the consumption of ESCRT proteins within the ILVs , these vesicles contain no protein coat to template their shape . Rather , these vesicles are initially formed as buds whose necks are stabilized by an assembly of ESCRT-I and -II [13] . As the assembly matures with the incorporation of ESCRT-III , the bud neck is cleaved from the cytosolic side , leaving ESCRTs in the cytosol and the detached spherical ILVs in the lumen of the endosome . Because this process involves no protein coat , the shape and energy of the mature buds must be governed primarily by membrane mechanical properties . Until recently , the membrane remodeling functions of ESCRT were attributed only to ESCRT-III proteins , which assemble on liposomes in vitro [14] , induce the ILV formation in giant unilamellar vesicles ( GUVs ) [12] and cause the plasma membrane to bud and tubulate when overexpressed in cells [15] . Based on these experimental observations , membrane deformations induced by ESCRT-III have been modeled [12] and studied theoretically [16] . An unresolved problem of these models was their inability to explain how the ESCRT-III proteins can be recycled from self-induced buds or tubes . Recent in vitro experiments showed , however , that ESCRT-I and -II together are responsible for vesicle budding [13] . In these experiments , vesicle budding could be induced at physiological concentrations ( 15 nM ) of ESCRT-I and -II . Fluorescence microscopy showed ESCRT-I and -II to be colocalized in the neck region of the buds ( Fig . 1C ) , where they were shown to recruit ESCRT-III . The latter proteins then induced membrane scission [12] . Importantly , the ESCRT proteins were not found in the bud lumen . In this way , the ESCRT machinery that facilitates membrane budding and scission is not consumed in the process of ILV formation ( Fig . 1D ) . Moreover , with ESCRT binding restricted to the neck region , the principal part of vesicle buds in vitro is thus bare lipid membrane . Fluorescence microscopy [13] also showed that membrane-bound ESCRT proteins form microdomains on vesicle membranes ( Fig . 1B and 1C ) . The lipid composition of these domains likely differs from that in the ESCRT-free membrane portions because ESCRTs bind specifically to certain charged lipids ( PI3P ) and could employ raft-favoring lipids and cholesterol to facilitate membrane budding [17] , [18] . These two experimental observations of ( i ) the formation of ESCRT microdomains on the membrane and ( ii ) the formation of coatless buds ( with ESCRT proteins localized in the bud neck ) jointly form the basis for a phenomenological model of ESCRT-induced budding . Observation ( i ) provides us with the starting point for the budding process and motivates a mechanism that can provide the large energy necessary for membrane deformation . Observation ( ii ) determines the end point of the budding pathway . In our model , we assume that the ESCRT-I-II supercomplexes have an enhanced affinity for binding to saddle-shaped membrane regions , and that a line tension acts on the outer boundary of an ESCRT-sequestered membrane domain . We cast our model in the framework of membrane elasticity theory . A bending elastic model of lipid bilayers was previously used to study the energetics of a possible mechanism of ESCRT-III induced fission of nascent vesicles [19] . Here we employ a similar approach to study the ESCRT-I-II induced formation of vesicle buds . The analytical solutions of our model allow us to map out different membrane morphologies over a range of possible physical parameters . We identify a regime of membrane bending parameters and line tensions for which the minimum energy configurations are coatless membrane buds with ESCRTs localized in the bud neck . These minimum energy configurations closely resemble the fluorescence images observed in experiment [13] . Within our model , we also identify energetically and kinetically feasible budding pathways , and propose a three-stage mechanism of ESCRT-driven budding: ( i ) membrane-bound ESCRT-I-II complexes form clusters , or domains , and induce a line tension on the domain boundaries through local segregation of lipids; ( ii ) as the domain boundary energy exceeds a threshold level , the membrane patch sequestered by the ESCRT assemblies buckles and forms a bud; ( iii ) the ESCRT-I-II complexes scaffold the bud neck and thus stabilize a neck diameter optimized for ESCRT-III protein binding and bud scission . To validate the model , we compare its predictions for bud shapes , sizes , and formation kinetics to experiment [13] , [20] . We also relate our model to recent experiments probing ESCRT-induced lipid segregation in membranes [21] .
In the framework of membrane elasticity theory , the energy of membrane deformations consists of the mean curvature term ( 1 ) and the Gaussian curvature term ( 2 ) where and are the mean and Gaussian bending rigidity moduli , respectively; and are the principal local curvatures; is the spontaneous curvature; and the integrals are performed over the membrane surface [22] . For uniform membranes with fixed topology , the energy term does not depend on membrane shape . Consistent with in vitro experiments [13] , we will consider symmetric lipid bilayers with no spontaneous curvature , . Eq . ( 1 ) implies that the energetic cost of forming a vesicle bud [23]–[27] is approximately , which exceeds for typical membrane bending rigidities . This large energetic cost effectively eliminates random thermal fluctuations as a main factor . Instead , the energetic cost of the nascent vesicle bud has to be offset by the molecular machinery that drives the budding process . In ESCRT-induced budding , the source of this offset energy is provided by the binding of the ESCRT proteins to the membrane . But unlike BAR domains or clathrin proteins , the ESCRT machinery does not seem to use the scaffold mechanism as the primary mode of action . Indeed , the scaffold mechanism cannot easily account for the significantly different bud radii in vitro ( m [13] ) and in vivo ( [20] ) , and appears to be contradicted by the experimental observation that the ESCRT proteins colocalize in the neck of buds . How does the ESCRT machinery deform a flat membrane patch into a bud [28] ? There are two relevant observations that help answer this question . Firstly , there is increasing evidence that the insertion of amphipathic , cationic peptide segments into the lipid bilayer can induce negative Gaussian curvature [29]–[31] , which is a characteristic of the bud necks where the ESCRT proteins localize . The Vps37 subunit of ESCRT-I has such a cationic helix at its N-terminus [32] , as does the Vps22 subunit of ESCRT-II [33] . It is therefore possible that the ESCRT-I-II proteins generate negative Gaussian curvature by inserting amphipathic and cationic segments into the membrane . In the lowest order of approximation , the resulting curvature-dependent binding energy , ( 3 ) is proportional to the membrane Gaussian curvature integrated over an ESCRT-rich membrane patch , where is a coupling constant . Remarkably , we also arrive at an energy term that is formally equivalent to Eq . ( 3 ) on an entirely different route . If we assume that by binding to the membrane , the ESCRT-I-II proteins locally perturb its Gaussian bending modulus according to Eq . ( 2 ) , then the parameter can be understood as the Gaussian bending modulus contrast . The energy term in Eq . ( 3 ) thus accounts for both the preferential binding to saddle-shaped features of the membrane and for changes in the Gaussian bending modulus in response to binding , with different interpretations of the constant . The second relevant observation is that late endosomes contain raft-like domains that are rich in cholesterol and sphingomyelin [34]–[36] . The in vitro experiments [13] were performed on vesicles containing cholesterol , raft-favoring lipids and PI3P , which is consistent with the possibility that ESCRTs could use lipid rafts or domains to promote membrane budding [17] , [18] . The ESCRT proteins could bind to pre-existing rafts or induce lipid domain formation . In fact , proteins with multiple lipid binding sites can induce lipid segregation in membranes close to the demixing point [37]–[40] . Lipid segregation has been suggested as a factor driving scission in endocytosis [41] . The enhancement of lipid segregation upon protein binding is primarily an entropic effect . Lipid segregation has to overcome an entropic penalty to move away from a perfectly mixed state . This penalty is reduced by concentrating certain lipids through preferential binding because the entropic cost of rearranging the resulting clusters of protein-bound lipids is smaller than that of rearranging individual lipids . We consider a single lipid-segregated domain . A line tension acts on the boundary of this domain , where the corresponding energy is proportional to the length of the boundary line ( 4 ) To quantify the preferential binding of ESCRTs to lipid rafts or domains , we introduce an additional energy term in the spirit of mean field theory ( 5 ) Here , is an effective ESCRT binding free energy per unit area , and denotes the intersection of the lipid-segregated domain and the ESCRT-rich domain on the membrane , where we assume that the latter is a subset of the former , . In the following , we assume the shapes of and to be circular and ring-shaped , respectively . As we show below , a minimal model of ESCRT proteins inducing or enhancing lipid segregation can explain central experimental results , in particular the narrow yet different bud-size distributions in vivo and in vitro . Moreover , the line tension on the boundaries of the lipid-segregated membrane domains [42]–[44] can also provide the large energetic driving force for vesicle budding [45]–[48] . Our model is defined by the energy functional ( 6 ) which combines the energy terms in Eqs . ( 1 ) , ( 3 ) , ( 4 ) , and ( 5 ) . The four corresponding parameters , , , and depend on membrane lipid composition , on external conditions such as temperature , and on ESCRT molecular properties , which are not entirely understood yet . Therefore , the model parameters can take different values depending on the molecular details of the ESCRT-membrane system . We thus use the spherical cap approximation [45] , [49] to derive analytical solutions that allow us to map out the entire parameter space of the model . We then minimize the energy functional Eq . ( 6 ) with respect to the membrane shape and the ESCRT cluster location , and investigate the membrane states , or morphologies , that are stable in different parameter regimes . Interestingly , we find a range of physically meaningful parameters in which the minimum energy states closely resemble the fluorescence images in Ref . [13] . We also investigate possible budding pathways that lead without activation barriers from a flat membrane state to the coatless membrane bud with ESCRTs localized in the neck .
To make our model analytically tractable , we applied the spherical cap approximation , which is known to capture the energetics of unassisted membrane budding [45] , [49] . In the spherical cap approximation , the energy functional Eq . ( 6 ) becomes a simple function whose minima can be found analytically ( see Methods ) . The phase diagram that results from the energy minimization for displays three regimes which are shown in Fig . 2 . For small line tensions ( left side of Fig . 2 ) , the membrane resists bending and remains flat . The lipid-segregated domain and the ESCRT-rich domain colocalize entirely . For large line tensions and large binding energies ( top right corner of Fig . 2 ) , the membrane buckles and forms an ESCRT-covered bud . Again and colocalize . For large and small ( bottom right corner of Fig . 2 ) , the minimum energy configurations are coatless buds with ESCRTs localized in the bud neck . To test the spherical cap approximation and to calculate bud shapes , we minimized the energy functional Eq . ( 6 ) numerically with respect to the membrane shape and the location of the ESCRT cluster ( see Methods ) . To distinguish different morphologies of the minimum energy configurations , we introduced the area fraction , where and denote the areas of the lipid segregated domain and the ESCRT-rich domain , respectively . We grouped all the minimum energy configurations found numerically into three distinct categories: ( i ) flat membrane domains with zero bending energy and area fraction , ( ii ) ESCRT-coated buds with bending energy and area fraction , and ( iii ) coatless buds with , small area fraction , and the ESCRT cluster located primarily in the bud neck region . The resulting morphology diagram agrees very well with the diagram based on the spherical cap approximation ( Fig . 2 ) . The spherical cap model predicts discontinuous transitions between the different morphological phases , with a kink in the minimum energy at the transition lines . In the complete model , however , the kink is rounded at the transition line due to the finite-size of the bud neck ( Fig . 3A ) . To additionally quantify the bud morphologies , we determined the tangent angle ( see Methods ) along the longitude at the boundary of the ESCRT-coated domain . The angle as well as the area fraction exhibit an abrupt change at the transition line , as shown in Fig . 3 , which further justifies the distinction between ESCRT-coated buds and uncoated buds . It is instructive to have a closer look at the membrane buds shown in the bottom right corner of Fig . 2 . The principal part of these buds is coatless membrane , and the ESCRT cluster is localized only in the bud neck . These coatless buds assume the shape of a sphere . The geometry of the bud neck follows that of a catenoid , a minimal surface with no mean curvature , , and thus zero bending energy [24]–[27] . As a result , the bending energy of the buds approaches the bending energy of a spherical vesicle , . We note that our calculated bud shapes , as shown in the top right corner of Fig . 2 , resemble the tomographic images of early endosomes [20] and the fluorescence images of GUV buds observed in vitro [13] . Within the framework of the spherical cap approximation , we also analyzed possible budding pathways . We focused in particular on activation-less pathways along which the buds grow without having to cross significant energy barriers ( see Methods ) . This analysis revealed a three-stage mechanism of ESCRT-mediated membrane budding . In stage ( i ) the ESCRT-rich domain and the lipid-segregated domain colocalize entirely on the flat membrane if . As the domain radius increases beyond , the energy of the flat domain becomes larger than the energy of an ESCRT-covered bud . As the domain radius increases to , the barrier between these membrane states vanishes entirely and the system enters into stage ( ii ) in which the flat membrane domain buckles and forms an ESCRT-covered bud . The resulting bud has a radius ( 7 ) which , interestingly , is determined by membrane material parameters . In stage ( iii ) the ESCRTs coalesce into the bud neck with no energy barriers if . The third stage is driven by the energy of the ESCRT preferential binding to membrane regions with negative Gaussian curvature . Importantly , in stage ( iii ) , the bud is cleared of ESCRTs for possible recycling . If , the mechanism of ESCRT-mediated membrane budding is different . The flat membrane with ESCRTs bound is then unstable and the system evolves spontaneously to a transient state , in which ESCRT-free buds with radius are formed . The mini-buds , induced by a strong binding preference to saddle-shaped membrane structures , are next squeezed bigger by line tension if the radius of the ESCRT-coated membrane patch is smaller than . The latter condition puts an upper bound on the ESCRT-free bud radius . Interestingly , on this budding pathway the ESCRTs never coat the bud . We note , however , that this pathway is somewhat problematic since the linear approximation in Eq . ( 3 ) may not be applicable for large parameters . In addition , effects of finite membrane thickness cannot be ignored in this regime .
The detailed physical mechanisms used by the ESCRT machinery to control membrane budding are currently unknown . This lack of knowledge has motivated us to consider a simple , phenomenological model for ESCRT-induced membrane budding . Our model is based on well-established physical principles to ensure robustness . Following earlier studies of membrane tubulation [16] and fission [19] driven by ESCRT-III proteins , we have cast our model in the framework of membrane elasticity theory . The key assumption in our model is that ESCRTs induce or enhance the formation of raft-like domains in lipid membranes . Indeed , recent total internal reflection fluorescence ( TIRF ) images show that the ESCRT-II proteins induce lipid phase separation in model membranes with endosome-like composition [21] . These direct experimental observations thus support a central element of our theoretical model . However , to test and further quantitate our model will require additional studies of ESCRT-membrane interactions . In particular , line tension measurements would provide critical input in building a fully quantitative description of the budding process . In addition , studies of the different components of the ESCRT system , including modifications such as Vps20 myristoylation , should provide further guidance at which stage and to what extent lipid phase separation is induced . The TIRF experiments [21] indicate interesting variations , with the yeast ESCRT-II protein complex forming somewhat smaller and more numerous clusters than the human ESCRT-II protein complex under the same conditions . Ultimately , it will be important to determine if membrane curvature is induced in a way consistent with our theoretical analysis . Such measurements would provide critical tests to what extent our model captures ESCRT-induced budding , and would help in its quantitation and refinement . The energetic analysis of our model suggests a three-stage mechanism of vesicle budding induced by the ESCRT proteins . In the first stage , the ESCRT-I-II binding induces lipid phase segregation , consistent with the TIRF experiments [21] . We speculate that in this stage , ESCRT-I-II complexes loosely assemble on the membrane to form clusters or domains . The ESCRT-membrane interactions enhance lipid segregation in the membrane and induce a line tension on the domain boundaries . Once the lipid-segregated membrane domain exceeds a critical size , as determined by membrane material parameters according to our model , the process enters the second stage . Beyond a size threshold set by the ratio , the energy barrier to membrane shape deformation disappears , and the membrane patch sequestered by ESCRTs buckles and forms a bud . In the third stage , according to our energy function , the ESCRT-I-II complexes concentrate in the neck of the newly formed bud . This final step is driven the ESCRT binding energy to the membrane portions with negative Gaussian curvature . While our model lacks molecular detail of this curvature-dependent binding , recent experiments [29]–[31] suggest possible mechanisms . In particular , it could be caused by the insertion of amphipathic and cationic segments of the membrane-bound ESCRT proteins into the membrane , or by the formation of an assembly whose shape is complementary to that of the bud neck [50] . The formation of neck-coating assemblies by the ESCRT-I-II complexes not only would help recruit ESCRT-III proteins for scission , but could also clear the ESCRT-I-II proteins from the bud lumen and set them up for recycling . The third stage of the budding process described above is dynamically feasible within the overall time scale of the process . The diffusion coefficient of membrane proteins is of order [51] . Provided a gradient to the bud neck region , the process of clearing in vitro buds with radius [13] would be completed within . The clearing process would be two orders of magnitude faster for the in vivo buds with radius [20] . One immediate consequence of the three-stage mechanism is that the complete buds have a radius that is determined by membrane material parameters , namely membrane rigidity and line tension . Therefore , the bud radius attains a particular value for a vesicle at given conditions , which explains the narrow distribution of bud sizes observed both in vitro [20] and in vivo [13] . We note , however , that both the line tension and the bending rigidity may be affected by details of the ESCRT-membrane interactions , which could explain small ILV size variations in response to mutations [52] . Despite the 40-times smaller radius , the in vivo buds have the same bending energies and shapes as the buds observed in vitro . This conclusion is a direct consequence of the scale invariance of the membrane bending energy Eq . ( 1 ) . However , the line tensions necessary to induce vesicle budding , , are different . For in vivo buds with radius and bending rigidity [53] , we estimate ; for in vitro buds with , we get assuming the same membrane rigidity . Line tensions in this range have been measured in GUVs [42] , [47] . We note that the bending rigidity of early endosomes [53] is an order of magnitude lower than the bending rigidity of lipid membranes in the liquid-ordered phase [47] , [54] . This difference in mechanical properties may be caused by variations in the composition and by the presence and activity of proteins in the membrane of endosomes . The membrane-sculpting protein Sar1 of the COPII complex , for example , has been shown to lower the membrane rigidity below [55] . The rigidity of fluid membranes has also been shown to decrease gradually with increasing concentration of the HIV-1 fusion peptide in the bilayer [56] . The three-stage budding process discussed above occurs if the Gaussian bending modulus contrast is limited to a window . This relation puts an upper bound on the binding energy . For typical bending rigidities and line tensions , the required energy is of order per or smaller . If the affinity of ESCRTs to regions with negative Gaussian curvature is high , , our model predicts a different budding pathway . In this case , flat ESCRT-coated membrane domains are unstable , and small ESCRT-free buds form spontaneously . The mini-buds induced by a strong binding preference to saddle-like membrane shapes can next be squeezed bigger by line tension . The latter transition occurs if the radius of the ESCRT-coated domain does not exceed . The resulting ESCRT-free buds have a radius . For and as above , we get the bud radius smaller than approximately , which is consistent with the size of late endosome buds , [20] . In contrast to the three-stage mechanism , in the pathway starting with mini-buds the ESCRTs never enter the bud lumen . It is thus tempting to picture this alternative budding route as follows: ESCRT-0 and -I bind to the membrane with and the membrane remains flat . When ESCRT-II proteins bind to ESCRT-I and to the membrane , becomes larger than and the ESCRT-free membrane buds are formed spontaneously . The condition for forming a finite-size , ESCRT-free bud is that the radius of the ESCRT patch at the ESCRT-II arrival can not exceed . We notice , however , that this pathway might be an artifact of our model since the linear approximation in Eq . ( 3 ) could be inapplicable for large . Nevertheless , we cannot entirely exclude this alternative pathway . Overall , we favor the three-stage mechanism as a possible route for ESCRT-induced budding . It concisely explains the uniform bud sizes seen in experiment , and makes quantitative and testable predictions for their dependence on physical parameters . In particular , according to Eq . ( 7 ) the vesicle bud radius should increase linearly as a function of the ratio . Since the line tension depends sensitively on temperature [42] , it should be possible to test this theoretical prediction in future experiments on GUVs . A key element of the model is the Gaussian bending energy . Because of experimental difficulties , our understanding of Gaussian curvature effects is rather limited . However , factors affecting the Gaussian bending modulus [57] , [58] or enhancing the binding to membranes with negative Gaussian curvature [29]–[31] have been established . As detailed structural models of the full-length ESCRT protein supercomplexes emerge [50] , [59] , it will be important to identify the interactions responsible for the preferential association of the ESCRT proteins to saddle-shaped bud-neck membrane regions . It may also be possible to study binding of ESCRT proteins or protein fragments to model systems with negatively curved membranes [29]–[31] . In this way , one could gain a microscopic quantification of the parameter , putting it on similar footing as the other membrane-bending terms .
It is convenient to use dimensionless variables in numerical calculations . Here we introduce ( 13 ) and with . With these dimensionless variables , the boundaries of the ESCRT-occupied membrane patch are described by parameters and , where . The energy functional ( 14 ) depends then on the bud shape , which is described by ( 15 ) on the domain location as given by and ; and on three dimensionless parameters ( 16 ) ( 17 ) and ( 18 ) In deriving the equations above we used a relation between the contour length and the membrane area , namely ( 19 ) The membrane energy ( 14 ) is minimized numerically with respect to the bud shape and the domain boundaries and using a simulated annealing method . To this end , the function is assumed to be smooth at and approximated by a Fourier series ( 20 ) that fulfills the boundary conditions and . Here , is the number of Fourier amplitudes . In simulated annealing Monte Carlo moves , the amplitudes and the boundary positions and are varied . We performed the numerical calculations with up to Fourier modes as in [62] , [63] . For any set of parameters , , and , numerical calculations were repeated twice with different initial configurations to ensure convergence to the global minimum . To make our model analytically tractable , we approximate the nascent membrane bud by a spherical cap of radius with a budding angle . A convenient budding coordinate is . We also define the area ratio where and denote the areas of the lipid-segregated domain and the ESCRT-rich domain , respectively . The domain of the two dimensionless parameters and is the unit square . In the following , we write the energy of the bud in terms of and , considering two cases: Case I: The whole lipid-segregated domain forms a spherical cap and thus the membrane surface parametrization is given by for with . Then and the energy functional Eq . ( 6 ) becomes a two-variable function ( 21 ) Case II: Only the ESCRT-free membrane patch forms a bud and therefore the membrane surface parametrization is given by for and for with . Then and ( 22 ) In both cases I and II , the determinant of the Hessian matrix is always negative within the unit square , , which implies that the local minima of can be found only at the square boundaries . A simple analysis of Eqs . ( 21 ) and ( 22 ) shows that the local minima of the energy function are located only at the corners of the unit square in the − plane . First , . Second , , and . Third , . We compare these energies to find the global minimum of the energy function and , in this way , construct the phase diagram in Fig . 2 . Point corresponds to the flat membrane state , which is obviously the starting point of the budding process . Point on energy surface corresponds to an ESCRT-coated bud , whereas point on energy surface corresponds to coatless buds with radius . Point corresponds to a coatless membrane bud with finite radius and ESCRTs localized in the neck . Fluorescence microscopy shows that this state is the end point of the ESCRT-induced budding . The transition from the initial , flat membrane state at point to the end point at occurs spontaneously with no energy barriers if either ( i ) and , or ( ii ) and . In case ( i ) , the transition occurs on energy surface . In case ( ii ) , the system evolves on energy surface . In both cases ( i ) and ( ii ) the transitions occur from point to along the line and next from point to along the line . We checked that for some parameter combinations , a saddle point can emerge within the unit square , separating local minima at and . However , if the saddle point is in the unit square , it is higher than . We also checked that for positive parameters and , the saddle point cannot merge with point . So the barrier-free path should indeed go from point to to . In the case , the system stays on energy surface . It is then instructive to analyze the energy landscape as the circular domain grows . As the domain radius increases beyond , the minimum at becomes higher than the energy at . As the domain radius increases to , the barrier between and disappears entirely . However , if , the point is not a local minimum , with an energy already higher than the minimum at . So if budding is induced at the radius , then the ESCRTs either stay on the bud , if , or they coalesce into the neck immediately after bud formation if . Another budding scenario is possible if and . The system evolves then on energy surface from point to with no energy barriers , which means that flat , ESCRT-coated membrane domains are unstable and small , ESCRT-free buds with radius form spontaneously . The mini-buds induced by a strong binding preference to membrane portions with negative Gaussian curvature are next squeezed bigger by line tension if . The system evolves then from point to . The resulting ESCRT-free buds have a radius . In contrast to the three-stage mechanism , in this budding pathway the ESCRTs never enter the bud lumen . | Lipid membranes enclose the cytosol of biological cells and compartmentalize their interior . Vesicles are used to transport membrane proteins between cellular compartments . The ESCRT protein machinery induces the creation of such vesicles away from the cytosol . The resulting vesicles are uncoated by protein . Upon vesicle scission and release into the endosome , the ESCRT proteins are recycled into the cytosol . We develop a membrane-elasticity model that captures this budding process . The model reproduces the vesicle morphologies observed in fluorescence microscopy images , and identifies the energetic driving force of vesiculation . We also characterize possible mechanisms of ESCRT-induced membrane budding . The size of the resulting vesicles is determined by membrane material parameters , explaining the narrow yet different bud size distributions in vitro and in vivo . Our membrane elasticity model thus provides insight into the energetics and mechanisms of uncoated vesicle formation . | [
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] | 2012 | Membrane-Elasticity Model of Coatless Vesicle Budding Induced by ESCRT Complexes |
Pathogen-associated molecular patterns ( PAMPs ) trigger host immune response by activating pattern recognition receptors like toll-like receptors ( TLRs ) . However , the mechanism whereby several pathogens , including viruses , activate TLRs via a non-PAMP mechanism is unclear . Endogenous “inflammatory mediators” called damage-associated molecular patterns ( DAMPs ) have been implicated in regulating immune response and inflammation . However , the role of DAMPs in inflammation/immunity during virus infection has not been studied . We have identified a DAMP molecule , S100A9 ( also known as Calgranulin B or MRP-14 ) , as an endogenous non-PAMP activator of TLR signaling during influenza A virus ( IAV ) infection . S100A9 was released from undamaged IAV-infected cells and extracellular S100A9 acted as a critical host-derived molecular pattern to regulate inflammatory response outcome and disease during infection by exaggerating pro-inflammatory response , cell-death and virus pathogenesis . Genetic studies showed that the DDX21-TRIF signaling pathway is required for S100A9 gene expression/production during infection . Furthermore , the inflammatory activity of extracellular S100A9 was mediated by activation of the TLR4-MyD88 pathway . Our studies have thus , underscored the role of a DAMP molecule ( i . e . extracellular S100A9 ) in regulating virus-associated inflammation and uncovered a previously unknown function of the DDX21-TRIF-S100A9-TLR4-MyD88 signaling network in regulating inflammation during infection .
Pathogen-associated molecular patterns ( PAMPs ) are molecular signatures of pathogens which facilitate induction of the host immune response [1] , [2] . PAMPs activate cellular pattern-recognition-receptors ( PRRs ) such as toll-like receptors ( TLRs ) to induce immunity [1] , [2] . Wide arrays of pathogens activate PRRs in the absence of PRR-specific PAMPs . It is thought that during infection cellular factors can activate PRRs and thus indirectly fulfill the function of PAMPs . The mechanism regulating the activity and function of non-PAMP dependent immune response during virus infection is still an enigma . Damage-associated molecular patterns ( DAMPs ) , which are molecules produced from damaged or dead cells induce an inflammatory response in paracrine fashion via TLR activation [3] . However , whether DAMPs can function as a host-derived molecular pattern during virus infection is not known . In this study , we determined that during influenza A ( IAV ) virus infection , S100A9 protein ( also known as Calgranulin B or MRP-14 ) , which is classified as a DAMP , is released from undamaged infected cells to activate the TLR4/MyD88 pathway for induction of innate and inflammatory responses against IAV . Thus , we have identified extracellular S100A9 as a critical host-derived molecular pattern during IAV infection . This protein has an essential role in enhancing the inflammatory response , which culminates in exacerbated IAV pathogenesis and lung disease . Influenza A virus ( IAV ) is a negative-sense , single-stranded RNA virus that causes severe respiratory tract infection [4]–[6] . Infection among high-risk people such as elderly and immuno-compromised individuals manifests in massive airway inflammation , which leads to the development of pneumonia [4]–[6] . Furthermore , there is a constant threat from naturally evolving IAV strains in avian and animal reservoirs that can lead to an epidemic or pandemic . Death of more than 200 , 000 individuals due to swine IAV ( 2009 H1N1 IAV ) associated infection [7] is an example of the catastrophic nature of IAV infection . Innate immunity , comprised of antiviral activity ( via type-I interferons , IFN-α/β ) and a controlled inflammatory response , is critical host defense machinery for virus clearance and the resolution of virus-induced disease [8]–[16] . PRRs recognize PAMPs to induce innate immunity in response to pathogen invasion . During IAV infection , both membrane-bound ( e . g . , TLRs ) and cytosolic ( e . g . , RIG-like receptors such as RIG-I and Nod-like receptors such as NLRP3 and Nod2 ) PRRs are required to launch an effective innate response [13] , [17]–[31] . Activation of PRRs could serve as a double-edged sword: While operating as host defense factors , activated PRRs can also contribute to the progression of virus-induced disease . For example , although TLR4 is activated during IAV infection , studies with TLR4 KO mice have shown that TLR4 contributes to exacerbated lung disease and mortality in IAV-infected animals [22] , [23] . Since pneumonia is an inflammatory disease [6] , [32] , it is imperative to characterize the molecular mechanisms and cellular factors responsible for uncontrolled inflammation mediated by TLR4 during IAV infection [23] . Although activated TLR4 is a key contributor to exacerbation of disease , the mechanism by which TLR4 is activated in IAV-infected cells is unknown , especially since IAV does not have TLR4-specific PAMP ligand lipopolysaccaride ( LPS ) . Therefore , it is crucial to identify and characterize “non-PAMP” host-derived molecular pattern , which can activate PRRs during virus infection . We expect that this line of investigation will illuminate the role of host-factors in contributing , either positively or negatively , to IAV-associated disease and pathogenesis . These studies will be a stepping stone for development of therapeutics to combat IAV-associated lung disease . We are interested in understanding the role of secreted soluble factors ( e . g . defensins , interferon-alpha induced soluble factor ) in viral innate immunity [15] , [21] , [33] , [34] . During our studies to further understand how TLR response modulates expression/production of soluble secreted factors during infection , we found that cells lacking TLR adaptor TRIF failed to release S100A9 following IAV infection . We specifically focused on S100 proteins , since expression of both defensins and S100 proteins are concurrently enhanced during various cellular stimuli [35]–[37] and S100 proteins ( S100A9 and S100A8 ) has implicated in activation of TLR4 pathway during LPS stimulation [38] . In the current study , we have identified extracellular S100A9 protein as a host-derived molecular pattern regulating the pro-inflammatory response , cell death , and pathogenesis during IAV infection . We also show that DDX21/TRIF and TLR4/MyD88 pathways are respectively required for S100A9 gene expression and activity . In addition , we have uncovered DDX21-TRIF-S100A9-TLR4-MyD88 signaling network as a critical regulator of inflammation . This network may also contribute to inflammation and disease during both infection-associated and noninfectious inflammatory diseases and disorders .
Macrophages are essential immune cells that modulate host defense , inflammation , and disease pathogenesis during IAV infection . Macrophages are also the major cytokine- and chemokine-producing cells during IAV infection and thus contribute to lung tissue damage [39]–[42] . To investigate whether IAV infection stimulates S100A9 secretion , we infected macrophages with IAV for 4–16 h . After infection , medium supernatant was collected to assess S100A9 protein levels by ELISA . We found that following IAV infection both human ( U937 cells ) ( Figure 1A ) and mouse [J774A . 1 macrophage cell-line , primary alveolar macrophages and primary bone marrow-derived macrophages ( BMDMs ) ] macrophages ( Figure 1B–D ) secreted high levels of S100A9 . The physiological significance is evident from the ability of primary macrophages ( i . e . alveolar macrophages and BMDMs ) ( Figure 1C and D ) to secrete S100A9 upon IAV infection . Interestingly , S100A9 secretion was detected as early as 4–8 h postinfection . Release of S100A9 is not due to cell cytotoxicity or damage , since an LDH release cytotoxicity assay showed minimal cytotoxicity in macrophages at 8 and 16 h postinfection ( Figure S1A ) . Similarly , no cell death ( apoptosis or necrosis ) was observed during the 8–16 h postinfection period ( not shown ) . These results demonstrated that following IAV infection , S100A9 is released to the extracellular environment from undamaged macrophages . There have been no studies to date on the signaling mechanism that regulates gene expression of S100 family of proteins . We examined the signaling mechanism involved in S100A9 expression . We infected BMDMs derived from wild-type ( WT ) , TLR2 knockout ( KO ) , TLR4 KO and TRIF KO mice with IAV . At 24 h postinfection , we evaluated S100A9 levels in the medium . TLR2 and TLR4 were not involved , since comparable S100A9 secretion was noted in WT and TLR KO BMDMs ( Figure 2A ) . A similar result was obtained with TRAM KO and TIRAP KO cells ( not shown ) . In contrast , significant reduction in S100A9 secretion was observed in IAV-infected TRIF KO BMDMs ( Figure 2A ) . RT-PCR analysis showed that loss of S100A9 secretion was caused by the absence of S100A9 mRNA in infected TRIF KO cells ( Figure 2B ) . Apart from TLR4 , which uses TRIF for MyD88-independent signaling , TLR3 also recruits TRIF for TLR3-mediated signal transduction . However , TLR3 is not involved in this process , as shown by the fact that S100A9 secretion was not reduced in TLR3 KO BMDMs ( Figure 2C ) . These results demonstrated that S100A9 gene induction occurs via the TLR-independent TRIF-dependent pathway . Recently , DEAD box proteins ( also known as DDX protein ) having RNA helicase activity has been implicated in innate immunity [43] . DDX proteins ( e . g . DDX21 ) can function as cytosolic PRR in mouse dendritic cells ( mDCs ) to induce type-I interferon during infection [43] . Interestingly , DDX signaling was TRIF-dependent and DDX21 interacted with TRIF during signaling [43] . Therefore , we examined whether DDX21 has a role in S100A9 expression during IAV infection of macrophages . Since KO animals lacking DDX proteins do not exist , we used siRNA technology to silence DDX21 expression in macrophages . Mouse alveolar macrophages ( MH-S cell line ) were transfected with DDX21-specific siRNA or control scrambled siRNA , after which these cells were infected with IAV . DDX21 expression was monitored by RT-PCR . We observed induction of DDX21 expression following IAV infection ( Figure 2D ) . The silencing efficiency was evident from the loss of DDX21 expression in IAV-infected cells transfected with DDX21-specific siRNA ( Figure 2D ) . We used the silenced cells to deduce the role of DDX21 in S100A9 gene expression following IAV infection . DDX21 is critical for S100A9 gene expression , since drastic loss of S100A9 mRNA was observed in IAV-infected DDX21 silenced cells ( Figure S1B ) . Accordingly , reduced S100A9 mRNA expression in DDX21 silenced cells led to diminished S100A9 secretion following IAV infection of these cells ( Figure 2E ) . The DDX/TRIF dependent S100A9 expression was independent of virus replication , since IAV hemagglutinin ( HA ) mRNA levels were similar in DDX21 silenced and TRIF KO cells ( Figure S2A and S2B ) . Moreover , S100A9 expression ( not shown ) and production ( Figure S2C and S2D ) was not significantly altered in IAV infected MyD88 KO and MAVS KO cells , which implicated MyD88 aααnd MAVS independent expression/production of S100A9 during IAV infection . In addition , we failed to observe significant difference in S100A9 expression/production from IAV infected WT vs . TLR7 KO cells ( Figure 2F ) . It is interesting to note that DDX21 expression was undetected at 48 h postinfection ( Figure 2D ) , which co-relates with loss of S100A9 production during that time frame ( not shown ) . This suggests that to maintain homeostasis and to avoid hyper-inflammation cells may negatively regulate DDX21 expression to reduce S100A9 production . These results demonstrated that the DDX21/TRIF pathway is required for S100A9 gene induction and the resulting S100A9 secretion following IAV infection . In the preceding studies , the high levels of S100A9 secretion during infection suggested that secreted extracellular S100A9 may have some role during IAV infection . Therefore , we focused on the role and function of extracellular S100A9 during IAV infection . Earlier studies have shown that the S100A9/S100A8 complex is required for optimal LPS-induced TLR4-dependent TNF-α ( TNF ) production in bone marrow cells ( comprised of undifferentiated monocytes and DCs ) [38] . However , few studies have shown the pro-inflammatory activity of S100A9 in the absence of S100A8 and LPS . Moreover , it is not known whether S100A9 can launch a pro-inflammatory response in macrophages . Since our studies are focused on the innate responses of IAV-infected macrophages , we investigated whether extracellular addition of purified S100A9 protein ( to mimic secreted S100A9 ) promotes the release of pro-inflammatory cytokines IL-6 and TNF-α ( TNF ) . These pro-inflammatory cytokines are produced early during IAV infection , a period that corresponds with S100A9 secretion kinetics . Mouse ( J774A . 1 ) and human ( U937 cells ) macrophages were incubated with purified mouse or human S100A9 proteins , respectively for 6–12 h ( Figure 3 ) . After treatment , medium supernatant was collected to analyze TNF and IL-6 levels by ELISA . S100A9 alone stimulates a pro-inflammatory response in macrophages , as is evident from high levels of TNF ( Figure 3A and C ) and IL-6 ( Figure 3B and D ) production by macrophages treated with purified S100A9 protein . Both human ( Figure 3A and B ) and mouse ( Figure 3C and D ) macrophages produced pro-inflammatory cytokines upon incubation with human and mouse S100A9 protein . Interestingly , the response was rapid , since substantial TNF and IL-6 production occurred within 6 h of treatment with S100A9 protein . RT-PCR analysis showed that production of TNF and IL-6 by S100A9 was due to activation of their corresponding genes ( Figure S3 ) . Since the pro-inflammatory activity of purified S100A9 protein could be inhibited by anti-S100A9 blocking ( neutralizing ) antibody ( not shown ) , the observed response was due to S100A9 protein . Moreover , the effect observed with purified S100A9 protein was not due to LPS contamination ( Figure S4A and S4B ) . These studies demonstrated that S100A9 functions as an extracellular host factor to launch a pro-inflammatory response in macrophages . We next examined the role of secreted S100A9 in eliciting a pro-inflammatory response during IAV infection . We used blocking antibody against S100A9 to neutralize the activity of extracellular ( secreted ) S100A9 . Previously , it was shown that this blocking antibody specifically inhibited the activity of the secreted extracellular form of S100A9 both in vitro and in vivo [44]–[50] . J774A . 1 cells were infected with IAV in the presence of either control antibody ( control IgG ) or S100A9-specific blocking antibody . At various postinfection time points , IL-6 and TNF levels were examined by ELISA . Extracellular S100A9 plays a key role in inducing the pro-inflammatory response during IAV infection , since significant reduction in IL-6 ( Figure 4A ) and TNF ( Figure S4C ) levels were observed in infected cells treated with S100A9 blocking antibody . RT-PCR showed that loss of IL-6 and TNF production was due to diminished gene expression ( not shown ) . Similar results were obtained following treatment of IAV-infected primary macrophages ( BMDM ) with S100A9 blocking antibody ( Figure S4D and S4E ) . Diminished IL-6 ( Figure S4D and S4E ) and TNF ( not shown ) production ( Figure S4D ) and expression ( Figure S4E ) was observed in infected BMDM treated with S100A9 blocking antibody . The loss of pro-inflammatory response was not due to reduced IAV replication , since IAV HA expression was similar in control antibody and S100A9 blocking antibody treated J774A . 1 cells ( Figure S5A ) and BMDMs ( Figure S5B ) . Thus , extracellular S100A9 modulates pro-inflammatory response independent of IAV replication . Our finding that S100A9 contributes to the pro-inflammatory response during IAV infection was validated by using BMDMs derived from S100A9 KO mice . WT and S100A9 KO BMDMs were infected with IAV , after which TNF and IL-6 levels in the medium supernatant were measured by ELISA . As compared to WT cells , there were significant reductions in IL-6 ( Figure 4B ) and TNF ( Figure 4C ) production from infected S100A9 KO cells . Once again , this was a consequence of the loss of pro-inflammatory gene expression in IAV-infected S100A9 KO BMDMs ( Figure S5C ) . The critical function of secreted ( extracellular-form ) S100A9 during this response was apparent from the observation that addition of purified mouse S100A9 protein to S100A9 KO BMDMs restored the pro-inflammatory response in IAV-infected S100A9 KO BMDMs ( Figure 4D ) . This result also suggested that intracellular S100A9 does not play a role in inducing a pro-inflammatory response . Treatment of WT or S100A9 BMDMs with S100A9 protein did not alter IAV replication status in the corresponding cells ( not shown ) . We also observed production of S100A9 following treatment of BMDMs with synthetic dsRNA ( poly-IC ) ( Figure S6A ) . The pro-inflammatory activity of S100A9 was specific for IAV and dsRNA ( which serves as a replicative intermediate during IAV infection and induces DDX21/TRIF pathway ) since dsRNA ( poly-IC ) dependent TNF and IL-6 release was significantly diminished in S100A9 KO cells ( Figure S6B and S6C ) , and treatment of KO cells with purified S100A9 protein restored the pro-inflammatory response in poly-IC treated S100A9 KO cells ( Figure S6D and S6E ) . In contrast , TNF and IL-6 release from S100A9 KO BMDMs was not affected following imiquimod ( which activates TLR7 dependent pro-inflammatory response ) treatment ( Figure S6F and S6G ) . In addition , treatment of WT and S100A9 KO BMDMs with TNF ( to induce NF-κB dependent inflammatory response via TNF receptor ) revealed similar levels of IL-6 production from both WT and KO cells ( Fig . S6H ) . During these studies we observed that IAV replication ( as deduced from IAV HA mRNA expression ) was significantly reduced in S100A9 KO BMDMs compared to WT cells ( Figure S7A ) . This result suggested that although extracellular S100A9 plays a critical role in modulating pro-inflammatory response ( Figure 4D ) , intracellular S100A9 may be involved in negatively regulating antiviral factor expression/production or it is required for efficient virus infection/replication . This is not surprising in light of previous reports illustrating differential function of extracellular vs . intracellular S100 proteins . It is important to mention that we observed S100A9 production from IAV-infected BMDMs at 4 h postinfection ( Figure 1C ) and that TNF and IL-6 are produced from IAV-infected BMDMs at 8–12 h postinfection ( not shown ) ; these cytokines are undetectable at 4 h postinfection ( not shown ) . Thus , S100A9 secretion and production of early pro-inflammatory mediators ( e . g . TNF , IL-6 ) are temporally regulated during IAV-infection . Therefore , extracellular S100A9 is a key regulator of the pro-inflammatory response during IAV infection . Macrophages undergo apoptosis during IAV infection [41] , [42] . Several studies have demonstrated that S100A9 has a pro-apoptotic function in epithelial cells , muscle cells , and neutrophils [51]–[55] , but no apoptosis-inducing activity of S100A9 ( or any other S100 proteins ) in macrophages has been reported . Since IAV infection resulted in high levels of S100A9 secretion , we examined the ability of extracellular S100A9 to induce apoptosis in macrophages and the role of secreted S100A9 in apoptotic induction during IAV infection . We treated J774A . 1 and MH-S macrophages with purified S100A9 protein for 48 and 72 h , then examined the apoptotic status of cells by monitoring annexin V and PI staining . The apoptosis rate was calculated based on the number of annexin V positive/PI negative cells ( denoting early apoptosis ) +number of annexin V positive/PI-positive cells ( denoting late apoptosis ) per total number of cells . We noted apoptosis in S100A9 protein-treated mouse macrophages ( Figure 5A and B ) . The result obtained with annexin V and PI staining was confirmed by performing TUNEL analysis ( Figure S7B and S7C ) . Similar results were obtained following treatment of human U937 macrophages ( not shown ) . Since IAV infection triggers S100A9 secretion , we next examined whether extracellular S100A9 has a role in apoptosis of IAV-infected macrophages . J774A . 1 macrophages were infected with IAV for 48 h in the presence of either control antibody ( control IgG ) or S100A9 blocking antibody . Significantly diminished apoptosis ( reduction of 27% ) occurred in macrophages treated with S100A9 antibody ( Figure 5C ) . These results were further confirmed by TUNEL analysis ( Figure S7D ) . Thus , extracellular S100A9 has a critical function in regulating apoptosis of IAV-infected macrophages . Previous studies have found that optimal TLR4 activation by LPS in bone-marrow cells required the activity of extracellular S100A9/S100A8 complex [38] . However , it is not known whether S100A9 alone activates TLR4 , especially in macrophages . In addition , there has been no report of DAMP proteins like S100A9 activating PRR signaling during virus infection . Therefore , we investigated the role of the TLR4/MyD88 pathway in the macrophage pro-inflammatory response by S100A9 alone ( in the absence of S100A8 ) , and the function of the S100A9/TLR4/MyD88 pathway in regulating the pro-inflammatory response in IAV-infected macrophages . We incubated WT and TLR4 KO BMDMs with purified S100A9 protein , and then measured IL-6 ( Figure 6A ) and TNF ( Figure 6B ) levels by ELISA . Drastic loss of IL-6 and TNF production was detected in S100A9 protein-treated TLR4 KO BMDMs ( Figure 6A and B ) , indicating that TLR4 is absolutely required for the S100A9-mediated response . Drastic reductions in IL-6 ( not shown ) and TNF ( Figure S8A ) transcripts occurred in S100A9 protein treated TLR4 KO cells . Since MyD88 is one of the critical adaptors for activated TLR4 , we next investigated the role of MyD88 by using MyD88 KO BMDMs . Incubation of WT and MyD88 KO BMDMs with purified S100A9 protein significantly reduced production of IL-6 ( Figure 6C ) and TNF ( not shown ) from MyD88 KO cells . The loss of cytokine protein production was due to reduced TNF ( Figure S8B ) and IL-6 ( not shown ) gene expression in MyD88 KO BMDMs , thus , demonstrating that the TLR4/MyD88 pathway is required for the S100A9-mediated pro-inflammatory response . We next studied the role of TLR4/MyD88 in stimulating the pro-inflammatory response following IAV infection . After WT , MyD88 KO , and TLR4 KO BMDMs were infected with IAV , IL-6 levels were assessed by ELISA . Our study revealed that TLR4/MyD88 is an essential regulator of pro-inflammatory response during IAV infection , since significant reduction in IL-6 ( Figure 6D ) and TNF ( Figure 6E ) production was noted in IAV infected TLR4 KO ( Figure 6D and E ) and MyD88 KO ( Figure 6D ) BMDMs . RT-PCR analysis demonstrated diminished expression of IL-6 mRNA in TLR4 KO ( Figure S8C ) and MyD88 KO ( not shown ) BMDMs . Similarly , we noted significant reduction in TNF production from IAV-infected TLR4 KO ( Figure 6E ) and MyD88 KO ( not shown ) BMDMs . The observed effect was independent of virus replication since compared to WT cells , no change in HA mRNA expression was noted in TLR4 KO ( Figure S8D ) and MyD88 KO ( not shown ) cells . Thus , TLR4/MyD88 activation is a key step for inducing the S100A9-mediated pro-inflammatory response . Also , the S100A9/TLR4/MyD88 pathway is a crucial regulator of the pro-inflammatory response during IAV infection . Our study showed that extracellular S100A9 uses TLR4/MyD88 signaling for the pro-inflammatory response during IAV infection . TLR4 activation has been associated with apoptosis induction via various mechanisms , including activation of the pro-apoptotic function of NF-κB , modulation of tumor-suppresser expression or function etc [56]–[62] . To assess the role of TLR4 in S100A9-mediated apoptosis , we treated WT and TLR4 KO BMDMs with purified S100A9 protein for 72 h . Treatment of WT BMDMs with S100A9 protein induced apoptosis ( Figure 7A ) , which was consistent with our previous findings . However , significant loss of apoptosis was observed in S100A9 protein-treated TLR4 KO BMDMs ( Figure 7A ) . We next examined the role of TLR4 and MyD88 in apoptosis induction during IAV infection . We infected WT and TLR4 KO BMDMs with IAV and evaluated apoptosis 48 h later . Apoptosis analysis by annexin V staining ( Figure 7B ) and TUNEL ( Figure 7C ) revealed that while IAV infection resulted in apoptosis of WT macrophages , a significant reduction in apoptosis was detected in TLR4 KO cells . Diminished apoptosis was also observed in infected MyD88 KO BMDMs ( Figure 7D ) , indicating that MyD88 is also required during this event . Thus , the S100A9/TLR4/MyD88 pathway constitutes one of the mechanisms that modulate apoptosis of IAV-infected cells . To establish the in vivo role of S100A9 in regulating innate response during IAV infection of the airway , we next evaluated S100A9 expression and its secretion in the IAV-infected mouse respiratory tract . Mice were intratracheally inoculated with IAV and , at 1–6 days postinfection , lungs were harvested . S100A9 mRNA expression in the lungs were analyzed by RT-PCR . S100A9 transcripts were observed in infected lungs but not in lungs from uninfected animals ( Figure 8A ) , indicating that IAV infection led to robust induction of S100A9 gene expression . We also detected high levels of S100A9 protein in the lungs of IAV-infected mice ( Figure 8B ) . Immunohistochemical analysis of lung sections confirmed the presence of S100A9 protein in IAV-infected animals ( Figure 8C ) , while S100A9 was nearly undetectable in mock-infected lungs . Analysis of bronchoalveolar lavage fluid ( BALF ) by ELISA confirmed the presence of S100A9 protein in the airway of IAV-infected animals ( Figure 8D ) . Thus , IAV infection of the respiratory tract results in induction of S100A9 gene expression and secretion of S100A9 protein in the airway . Macrophages play a vital role in the innate response to IAV infection by producing pro-inflammatory mediators that determine the inflammation status in the lung [39]–[42] . Moreover , debris from dead cells , originating from apoptosis of immune cells , contributes to airway inflammation [40]–[42] , [63]–[68] . Since extracellular S100A9 acted as a positive regulator of pro-inflammatory response and induced apoptosis , we hypothesized that extracellular S100A9 exacerbates IAV-associated lung disease . To test this , we used anti-S100A9 blocking antibody , which neutralizes extracellular ( secreted ) S100A9 protein . We used anti-S100A9 antibody instead of doing our in vivo studies with S100A9 KO mice because S100 proteins have both intracellular functions ( such as cytoskeletal rearrangement , cell metabolism , intracellular calcium response etc ) and extracellular functions [69] , [70] . Since we have elucidated a role of extracellular ( secreted form ) S100A9 , results from KO mice might not distinguish whether the observed effects are due to activity of extracellular S100A9 or intracellular S100A9 function . Most importantly our studies demonstrated that intracellular S100A9 could be involved in negatively regulating antiviral response or it is required for IAV infection/replication , since reduced virus replication was noted in S100A9 KO BMDM compared to WT cells ( Figure S7A ) . In that scenario , S100A9 KO mice may not serve as an appropriate model to study IAV-induced pro-inflammatory ( and apoptotic ) response in vivo , since viral burden in the lung is directly proportional to the degree of pro-inflammatory ( and apoptotic ) response ( i . e . if there is less viral burden then concomitantly reduced pro-inflammatory response and apoptosis will occur ) . However , neutralizing the activity of extracellular S100A9 with S100A9 blocking antibody did not alter IAV replication in vitro ( Figure S5A and S5B ) and in vivo ( please see below ) . We therefore used anti-S100A9 blocking antibody to specifically inhibit the activity of extracellular S100A9 in mice . We have previously shown that anti-S100A9 blocking antibody has extracellular S100A9 blocking activity [44]–[50] . Specifically , intraperitoneal ( i . p ) injection of S100A9 blocking antibody inhibited the activity of mouse S100A9 during S . pneumoniae infection [49] . Thus , this antibody [44] , [45] , [48]–[50] is useful to assess the functional role of extracellular ( secreted form ) S100A9 . Furthermore , similar levels of S100A9 protein were detected in the BALF of control IgG-treated and S100A9-antibody treated mice ( Figure S9A ) . Thus , i . p . -injected anti-S100A9 antibody did not significantly affect S100A9 protein production in the airway-lumen following IAV infection . As in previous reports , we detected anti-S100A9 antibody ( administered i . p . ) in lung homogenate ( Figure S9B ) . Thus , anti-S100A9 antibody could effectively block lung-localized S100A9 during IAV infection [49] , [71] , [72] . The clinical significance of utilizing neutralizing antibody is obvious from possible passive immunization with S100A9 antibody as a new therapeutic strategy to control lung inflammation and associated lung disease during IAV infection . Initially , we investigated the role of secreted S100A9 in regulating IAV susceptibility . For these studies , mice were i . p . injected with either control IgG or anti-S100A9 blocking antibody . One day later , mice were infected with IAV via intra-tracheal ( I . T ) inoculation . Survival of IAV-infected mice was monitored until 8 days postinfection . Blocking S100A9 activity significantly reduced the mortality of IAV-infected mice ( Figure 9A ) , demonstrating that extracellular S100A9 is a key regulator of IAV susceptibility . Extracellular S100A9 also contributes to morbidity since mice treated with S100A9 blocking antibody exhibited reduced weight loss upon IAV infection ( Figure S9C ) . Thus , extracellular S100A9 contributes to both IAV-induced mortality and morbidity . In addition , inflammation was reduced following inhibition of extracellular S100A9 activity ( Figure 9B and S10A ) . These results demonstrated that extracellular S100A9 contributes to the severity of IAV-associated lung inflammation and serves as a critical host factor for heightened IAV susceptibility and IAV-induced death . The clinical significance of our result is borne out by the possibility of passive immunization with anti-S100A9 antibody to reduce the severity of respiratory disease associated with IAV infection . We have identified extracellular ( secreted ) S100A9 as a critical regulator of the pro-inflammatory response following IAV infection of macrophages . To examine the physiological role of secreted S100A9 in lung inflammation , we tested whether intratracheal ( I . T . ) administration of purified S100A9 protein would trigger a pro-inflammatory response in the lungs . Indeed , this led to production of TNF ( Figure 9C ) and IL-6 ( Figure S10B ) in the respiratory tract due to S100A9-mediated upregulation of TNF and IL-6 gene expression in the lung ( not shown ) . The ability of S100A9 protein to trigger pro-inflammatory mediators in the lung is further reflected by observing airway inflammation in S100A9 protein administered ( via I . T ) mice ( Figure S10C ) . Based on this observation , we next examined the role of extracellular S100A9 in airway pro-inflammatory response following IAV infection . Mice were given i . p . injections of control IgG antibody or anti-S100A9 blocking antibody . At 1 d post-antibody treatment , mice were infected with IAV via I . T route . Levels of IL-6 and TNF in the lung were measured by ELISA . Extracellular S100A9 contributes to production of pro-inflammatory mediators during infection as evident from reduced TNF ( Figure 9D ) and IL-6 ( Figure S11A ) levels in the lung of S100A9 antibody treated mice . Reduced pro-inflammatory cytokine production was caused by loss of TNF ( Figure S11B ) and IL-6 ( data not shown ) mRNAs in the lungs of IAV-infected mice treated with S100A9 blocking antibody . Diminished pro-inflammatory response is not due to reduced IAV infection , since both control antibody and S100A9 antibody treated mice exhibited similar IAV infection status ( i . e . viral burden ) ( Figure S12A ) . Interestingly , S100A9 antibody could also be utilized as therapeutics to control IAV-associated disease , since administration of S100A9 blocking antibody after IAV infection significantly reduced pro-inflammatory response and lung inflammation ( Figure S12B and S12C ) . In order to provide evidence for direct neutralization of S100A9 activity in the airway following i . p . administration of S100A9 antibody , we administered S100A9 antibody ( via i . p . ) to mice and after one day ( to exactly mimic IAV infection studies ) mice were inoculated with S100A9 protein via I . T route . Significant inhibition in pro-inflammatory activity was noted in the presence of S100A9 antibody ( Figure S13A ) , which shows that i . p . administered blocking antibody can neutralize S100A9 protein in the airway . The role of extracellular S100A9 was further validated by conducting ex vivo experiment with BALF-associated cells derived from IAV infected mice administered ( via i . p ) with either control antibody or S100A9 blocking antibody . Significant reduction in IL-6 and TNF production from BALF cells was observed in S100A9 blocking antibody treated mice ( Figure 9E ) . This result once again validates blocking of S100A9 activity in the alveolar space localized ( i . e . present in the BALF ) cells . These studies illustrate the importance of secreted S100A9 in regulating pro-inflammatory cytokine gene expression and production during IAV infection of the airway . Our studies with macrophages have illuminated a vital role of extracellular S100A9 in inducing apoptosis of IAV-infected cells . We have extended those observations in mice to establish the in vivo physiological relevance of extracellular S100A9 as a regulator of apoptosis . Further , it is known that apoptosis significantly contributes to IAV infection severity and associated lung disease [40] , [63]–[68] . Therefore , reduced apoptosis in IAV-infected S100A9-blocked mice may contribute to reduced susceptibility and diminished airway disease ( as shown in Figure 9A and 9B and S9C ) . To examine this possibility , mice treated with control IgG and S100A9 blocking antibody were inoculated with IAV via the I . T route . On the third day post-infection , we performed in situ TUNEL assay with lung sections to determine the apoptotic status of the IAV-infected respiratory tract . We found significantly less apoptosis in the lungs of mice given S100A9 blocking antibody than in the lungs of control mice ( Figure 10A and B ) . These results demonstrated that secreted S100A9 is a pivotal regulator of lung apoptosis following IAV infection .
The role of DAMPs as a host-derived molecular pattern during virus infection is not known . In the current study we have demonstrated that extracellular S100A9 protein functions as a host-derived molecular pattern during infection . Although S100A9 is classified as a DAMP , using clinically important influenza A virus ( IAV ) infection model , we show release of S100A9 from “undamaged” cells during IAV infection; which triggered PRR ( i . e . , TLR ) signaling . Surprisingly , we observed that extracellular ( secreted ) S100A9 regulates two key mechanisms that contribute to inflammation during IAV infection . These are pro-inflammatory cytokine production during early infection and induction of apoptosis . We also found that S100A9-mediated activation of the TLR4/MyD88 pathway resulted in increased inflammation , which culminated in exacerbated IAV pathogenesis . Thus , our study shows a role of “non-PAMP” PRR-activating DAMPs in modulating immunity and inflammation during virus infection . We also have identified DDX21-TRIF-S100A9-TLR4-MyD88 as a novel signaling “network” that regulates inflammation . It is possible that a similar pathway is used to promote disease during infection with other viruses including highly pathogenic RNA viruses like SARS , Ebola virus , Marburg virus . Based on our results , we propose a model ( Figure 10C ) whereby the S100A9 gene is activated by the DDX21-TRIF pathway and the resulting S100A9 protein is secreted during IAV infection . Extracellular S100A9 activates the TLR4/MyD88 pathway via an autocrine or paracrine mechanism . As a consequence , S100A9/TLR4 activity exacerbates lung disease by promoting a pro-inflammatory response and inducing cell-death . Our studies are the first to highlight the role of S100A9 protein and DDX21-TRIF-S100A9-TLR4-MyD88 signaling network in modulating inflammation during virus infection . The clinical significance of our study is borne out by detection of S100A9 protein in mucosal secretions from IAV-infected individuals [73] . The clinical significance of utilizing neutralizing antibody is obvious from possible passive immunization with S100A9 antibody ( as shown in Figure 9A and B and S9C ) as a new therapeutic strategy to control lung inflammation and associated lung disease during IAV infection . Mortality among IAV-infected individuals is associated with pneumonia , a disease characterized by massive lung inflammation leading to tissue damage and endothelial barrier disruption , resulting in fluid leakage in the airway and the development of edema . Highly pathogenic IAV strains have greater propensity to launch a hyper-inflammatory response in the respiratory tract upon infection , culminating in the development of pneumonia . Among the cellular factors that regulate IAV-induced lung disease , TLR4 is a major contributor to susceptibility and exacerbated pathophysiology associated with IAV infection [22] , [23] . TLR4 is activated during IAV infection and reduced mortality and diminished lung disease ( and inflammation ) was observed in IAV infected TLR4 KO mice [22] , [23] . The mechanism of TLR4 activation by IAV is unknown , especially since IAV doses not posses TLR4 ligand LPS . In that regard , our current study has elucidated a role of extracellular S100A9 in activation of TLR4/MyD88 pathway during IAV infection . Previous studies reported that S100A9-S100A8 complex optimizes LPS-mediated TLR4 activation [38] . Bone marrow cells , including undifferentiated monocytes and DCs , and mice were treated/infected with LPS and LPS- expressing bacteria ( E . coli 018:K1 ) to demonstrate augmentation of LPS activity by extracellular S100A9-S100A8 complex [38] . However , we show for the first time that S100A9 alone can directly activate TLR4 ( in the absence of LPS ) and contribute to the regulation of inflammation during infection with IAV , a non-LPS-expressing pathogen . Thus , extracellular S100A9 can directly modulate immune response via TLR4 activation . We also demonstrated that S100A9 alone ( independent of S100A8 ) is a critical regulator of pro-inflammatory response in vitro and in vivo . Although few studies have been done on the mechanism of S100 gene induction during biological responses , we have shown that the S100A9 gene is induced by the DDX21-TRIF pathway . These studies also demonstrated the critical function of the DDX family PRRs in regulating expression of a host factor ( S100A9 ) that is not a cytokine ( i . e . IFNs ) . We have determined that the DDX21-TRIF pathway is required for S100A9 gene expression . A recent study has shown that the DDX1-DDX21-DHX36/TRIF pathway triggers a type-I interferon response in myeloid dendritic cells ( mDCs ) during virus infection and treatment of cells with dsRNA ( poly-IC ) [43] . In these studies , direct interaction of viral dsRNA with DDX proteins was not shown . Thus , during virus infection viral dsRNA can directly or indirectly activate DDX proteins for signaling . In that context , S100A9 related S100A8 gene expression was induced by dsRNA via MAPK pathway [74] . In accord with the previous studies [43] , [74] , we noted that IAV replication , which will generate viral dsRNA , is required for S100A9 production , since UV inactivated IAV failed to secrete S100A9 from macrophages ( Figure S13B ) . Surprisingly , our study demonstrated that apart from the reported DDX/TRIF-dependent IFN production in mDCs [43] , the DDX/TRIF pathway is also important in inducing a pro-inflammatory response during IAV infection of macrophages , which is mediated by DDX21-TRIF dependent activation of S100A9 gene expression and resulting autocrine/paracrine action ( via TLR4/MyD88 pathway ) of secreted S100A9 protein . Thus , our studies have illustrated a role of two PRRs in modulating inflammation during IAV infection – a cytosolic PRR ( i . e . DDX21 ) regulating S100A9 gene expression and membrane-localized PRR ( i . e . TLR4 ) transducing the biological activity ( i . e . pro-inflammatory response and cell death ) of S100A9 . This shows how concerted activity of two PRRs is used to control inflammation , since the inflammatory response has to be “regulated” at several levels due to the detrimental effect of uncontrolled inflammation on promoting cell and tissue damage . We have also identified extracellular S100A9 as one of the host factors that regulate apoptosis during IAV infection . Apoptosis is a key contributor to pathogenesis and the pathology associated with IAV infection [40] , [63]–[68] , [75] . Cell death intensifies inflammation in the respiratory tract , culminating in exacerbated lung disease . Previous studies have shown the ability of S100 proteins to induce cell death by various mechanisms [51]–[55] . Similar mechanisms may contribute to S100A9-mediated cell-death following IAV infection . Two arms of viral innate immunity consist of antiviral and inflammatory responses . Our studies have indicated that extracellular and intracellular S100A9 may function differently in terms of innate immune response during IAV infection , whereby extracellular S100A9 modulates pro-inflammatory response ( independent of viral replication ) and intracellular S100A9 is involved in orchestrating antiviral response to reduce viral burden . The differential activity of extracellular vs . intracellular S100A9 protein has been noted previously [69] , [70] . In that regard , two different pools ( i . e . extracellular and intracellular ) of S100A9 exist in IAV infected macrophages ( Table S1 ) . During infection , while 10%–25% of S100A9 protein is released , the rest is localized inside the cell ( Table S1 ) . In the current study we demonstrated that extracellular S100A9 ( secreted form ) triggers pro-inflammatory response and apoptosis . In contrast , we speculate that intracellular S100A9 may be involved in negatively regulating antiviral response or it is required for efficient IAV infection/replication . This conclusion was based on the observation that - a ) treatment of macrophages ( and mice ) with S100A9 blocking antibody diminished inflammatory response , while virus replication/infection was unchanged ( Figure S5A , S5B and S12A ) , b ) virus replication in S100A9 KO macrophages is reduced compared to WT cells ( Figure S7A ) , and c ) addition of S100A9 protein ( to mimic extracellular S100A9 protein ) to IAV infected S100A9 deficient ( KO ) cells led to a pro-inflammatory response even in the absence of intracellular S100A9 ( i . e . in S100A9 KO BMDMs ) ( Fig . 4D ) . Thus , extracellular S100A9 regulates inflammatory response independent of virus replication , while intracellular S100A9 may negatively regulate expression/production of antiviral factor ( s ) or it functions as a host factor required for efficient IAV infection/replication . In the future we will further dissect the exact mechanism ( s ) by which intracellular S100A9 modulates IAV infection/replication . S100A9 confers protective immunity during Klebsiella pneumoniae infection , since enhanced bacterial dissemination , lung damage , and susceptibility was observed in mice deficient in S100A9 expression [76] . In these studies , no distinction was made in terms of unique and differential function of extracellular vs . intracellular S100A9 . However , our studies have shown that extracellular S100A9 is one of the factors that dictate detrimental host ( inflammatory and apoptotic response ) response during IAV infection , and this response is independent t of virus replication . In contrast , we show that intracellular S100A9 ( which constitutes majority of S100A9 protein in IAV infected cells ) is required for efficient IAV infection/replication . Therefore , our studies have illustrated a distinct role of extracellular vs . intracellular S100A9 during IAV infection . Thus , in accord with the previous study with Klebsiella we speculate that IAV infection of S100A9 KO mice will result in diminished IAV replication and as a consequence , these mice will exhibit reduced inflammation , susceptibility and pathogenesis . We will conduct these studies in the future to elucidate the exact role ( and underlying mechanism ) of intracellular S100A9 in controlling IAV infection/replication . Apart from macrophages , epithelial cells ( primary mouse lung epithelial cells ) also produced S100A9 upon IAV infection ( Figure S14 ) . Interestingly , lower levels of S100A9 was released from primary lung epithelial cells compared to primary alveolar macrophages [please compare Figure S14 ( lung epithelial cells ) vs . Figure 1D ( alveolar macrophages ) ] . In the future we plan to perform in-depth study to investigate the role of macrophages and lung epithelial cells ( and their cross-talk ) during S100A9 mediated inflammatory response following IAV infection . In summary , we have identified extracellular S100A9 as a host-derived molecular pattern that regulates inflammation during virus infection . In addition , we have uncovered DDX21-TRIF-S100A9-TLR4-MyD88 as a novel signaling “network” that regulates inflammation . Future studies dealing with identification and characterization of host-derived molecular patterns ( e . g . DAMPs ) during virus invasion may lead to the development of measures to combat infection-associated inflammatory diseases .
Animal studies were performed according to housing and care of laboratory animals guidelines established by National Institutes for Health . All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee ( IACUC ) of University of Texas Health Science Center at San Antonio . The Animal Welfare Assurance # is A3345-01 . Influenza A [A/PR/8/34 ( H1N1 ) ] virus was grown in the allantoic cavities of 10-day-old embryonated eggs [21] , [75] . Virus was purified by centrifuging two times on discontinuous sucrose gradients [21] , [75] , [77] . J774A . 1 cells were maintained in DMEM supplemented with 10% fetal bovine serum ( FBS ) , penicillin , streptomycin , and glutamine . U937 cells were maintained in RPMI 1640 medium supplemented with 10% FBS , 100 IU/mL penicillin , 100 µg/mL streptomycin , 1 mM sodium pyruvate , and 100 nM HEPES . MH-S cells were maintained in RPMI 1640 medium supplemented with 10% FBS , 100 IU/mL penicillin , and 100 µg/mL streptomycin . Bone-marrow-derived macrophages ( BMDMs ) were obtained from femurs and tibias of wild-type ( WT ) and knock-out mice and were cultured for 6–8 days as described earlier [21] , [75] . Cells were plated on 12-well plates containing RPMI , 10% FBS , 100 IU/mL penicillin , 100 µg/mL streptomycin , and 20 ng/ml GM-CSF . Alveolar macrophages were obtained from the broncho-alveolar lavage fluid ( BALF ) of wild-type C57BL/6 mice . The IAV titer was monitored by plaque assay analysis with MDCK cells . S100A9 KO mice were generated at University of Laval , Quebec , Canada . Other KO mice ( TLR4 , TLR2 , TRAM , TRIF , TIRAP ) were originally provided by Dr . Doug Golenbock ( University of Massachusetts Medical School , Worcester , MA ) under a Materials Transfer Agreement with Dr . Shizuo Akira ( Osaka University , Osaka , Japan ) . TLR3 KO , TLR7 KO and MyD88 KO mice were obtained from Jackson Laboratory , Bar Harbor , ME . Murine S100A9 neutralizing antibody purified IgG from the serum of S100A9 immunized rabbits was generated as described previously [44]–[45] . This antibody has been successfully used to block the activity of extracellular mouse S100A9 [44]–[50] . Human S100A9 antibody was acquired from AbCam , Cambridge , MA ( goat anti-human S100A9 antibody ) and R&D Systems ( mouse anti-human antibody ) . Recombinant human and mouse S100A9 proteins were generated as previously described [44]–[50] . Briefly , full length human S100A9 cDNA was cloned into pET28 expression vector ( Novagen , Madison , WI ) . S100A9 protein expression was induced with 1 mM isopropyl β-D-thiogalactoside ( IPTG ) in E . coli HMS174 ( Boehringer Mannheim , Mannheim , Germany ) for 16 h at 16°C . After IPTG treatment , the bacteria were centrifuged at 5000×g for 10 min and the pellet was re-suspended in PBS [ ( containing NaCl ( 0 . 5 M ) and imidazole ( 1 mM ) ] and lysed by sonication . Upon centrifuging the lysate at 55 , 000×g for 30 min at 4°C , the supernatant was collected . Recombinant His-Tag S100A9 was purified by using a nickel column . S100A9 bound to the column was incubated with 10 U of biotinylated thrombin ( Novagen ) ( for 20 h at room temperature ) to free S100A9 from its His-Tag . Recombinant S100A9 was then eluted with PBS . The digestion and elution processes were repeated one more time to cleave the remaining undigested recombinant proteins , and streptavidin-agarose ( Novagen ) was added to remove contaminating thrombin . Finally , the protein preparation was passed through a polymyxin B-agarose column ( Pierce , Rockford , IL ) to remove endotoxins . Recombinant proteins were prepared in Hank's buffered salt solution ( HBSS ) buffer . The absence of endotoxin contamination in antibody and protein preparations was confirmed using the limulus amebocyte assay ( Cambrex ) . Total RNA was extracted using Tri Reagent ( Invitrogen ) . cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit ( Applied Biosystems ) . PCR was done using 0 . 25 units of Taq polymerase , 10 pmol of each oligonucleotide primer , 1 mM MgCl2 , and 100 µM deoxynucleotide triphosphates in a final reaction volume of 25 µl . Following amplification , the PCR products were analyzed on 1 . 5% agarose gel . Equal loading in each well was confirmed by analyzing expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) . The primers used to detect the indicated genes by RT-PCR were: GAPDH forward , 5′-GTCAGTGGTGGACCTGACCT , GAPDH reverse , 5′-AGGGGTCTACATGGCAACTG , Mouse GAPDH forward , 5′-GCCAAGGTCATCCATGACAACTTTGG , Mouse GAPDH reverse , 5′-GCCTGCTTCACCACCTTCTTGATGTC Mouse S100A9 forward , 5′-GTCCTGGTTTGTGTCCAGGT , Mouse S100A9 reverse , 5′-TCATCGACACCTTCCATCAA Mouse DDX21 forward , 5′-GATCCCCCTAAATCCAGGAA , Mouse DDX21reverse , 5′-TTCGGAAGGCTCCTCTGTTA Mouse TNF-α forward , 5′-CCTGTAGCCCACGTCGTAGC , Mouse TNF-α reverse , 5′-TTGACCTCAGCGCTGAGTTG Mouse IL-6 forward , 5′-TTGCCTTCTTGGGACTGATGCT , Mouse IL-6 reverse , 5′-GTATCTCTCTGAAGGACTCTGG IAV HA forward , 5′- CCCAAGGAAAGTTCATGG , IAV HA reverse , 5′-GAACACCCCATAGTACAAGG U937 cells , alvelolar macrophages , BMDM , MH-S , and J774A . 1 were infected with purified IAV [1 multiplicity of infection ( MOI ) −2 MOI as indicated] in serum-free , antibiotic-free OPTI-MEM medium ( Gibco ) . Virus adsorption was done for 1 . 5 h at 37°C , after which cells were washed twice with PBS . Infection was continued in the presence of serum containing DMEM or RPMI medium for the specified time points . In some experiments , cells were infected in the presence of 2 ng–10 ng/ml control IgG ( purified rabbit IgG , Innovative Research , Novi , MI ) or 2 ng–10 ng/ml anti-S100A9 blocking antibody . Following virus adsorption , antibodies were added to the cells and the infection was carried out in the presence of the antibodies . In addition , in some experiments infection was done in the presence of purified S100A9 protein or HBSS buffer ( vehicle control ) . Purified S100A9 protein ( 5 µg/ml ) was added to S100A9 KO BMDMs following virus adsorption . Purified protein was present during infection . Control siRNA and mouse DDX21 siRNA were purchased from Santa Cruz Biotechnology . MH-S cells were transfected with 40 pmol of siRNAs using Lipofectamine 2000 ( Invitrogen ) . At 48 h posttransfection , the cells were infected with IAV . Medium supernatant and mouse lung homogenate were analyzed for TNF and IL-6 levels by using a TNF and IL-6 specific ELISA kit ( eBioscience , San Diego , CA ) . For S100A9 ELISA , Costar High-Binding 96-well plates ( Corning , NY ) were coated overnight at 4°C with 800 ng/well of purified rabbit IgG against mouse S100A9 or 100 ng/well of goat polyclonal human S100A9 antibody ( Abcam ) diluted in 0 . 1 M carbonate buffer , pH 9 . 6 . The wells were blocked with PBST+1% BSA for 1 h at room temperature . The samples were added and incubated overnight at 4°C . The plates were washed three times with PBST and incubated with either goat anti-mouse IgG ( 300 ng/well ) ( R&D ) ( for mouse S100A9 ) or mouse anti-human IgG ( 50 ng/well ) ( R&D ) ( for human S100A9 ) in PBST+0 . 1% BSA for 2 h at room temperature . The plates were then washed three times in PBST . To detect mouse S100A9 , rabbit anti-goat HRP ( Bio-Rad ) was added to the plates . To detect human S100A9 , goat anti-mouse HRP ( Bio-Rad ) was added . After 1 h incubation at room temperature , the plates were washed three times with PBST . TMB-S substrate ( 100 µl/well ) ( Sigma-Aldrich ) was added to the plates according to the manufacturer's instructions . The ODs were detected at 450 nm , using a Modulas micro-plate reader . To detect i . p . -injected S100A9 antibody in the lung homogenate , Costar High-Binding 96-well plates were coated overnight at 4°C with mouse S100A9 protein diluted in 0 . 1 M carbonate buffer , pH 9 . 6 . The wells were blocked with PBST+1% BSA for 1 h at room temperature . The lung homogenate was added and incubated overnight at 4°C . The plates were washed three times with PBST and goat anti-rabbit HRP ( Bio-Rad ) was added . After 1 h of incubation at room temperature , the plates were washed three times with PBST . TMB-S substrate ( 100 µl/well ) ( Sigma-Aldrich ) was added to the plates according to the manufacturer's instructions . ODs were detected at 450 nm by using a Modulas micro-plate reader . For survival experiments , 6–8-week old pathogen-free WT C57BL/6 mice ( Jackson Laboratory ) were injected i . p . with 2 mg/mouse of either control IgG or anti-S100A9 antibody . One day later , mice were anesthetized and inoculated via the intratracheal or I . T route with IAV ( 1×105 pfu/mouse ) in 100 µl of PBS ( Invitrogen ) . Control mice were sham-inoculated with 100 µl of PBS . Survival was monitored until 8 days postinfection . For pathogenesis assay , mice were inoculated with IAV ( 2×104 pfu/mouse via the I . T route ) at 1 day after antibody treatment . At 3 days after infection , lungs and BALF were collected . Lung tissue sections were used for H&E analysis and in-situ TUNEL analysis . Lung homogenate was used for ELISA analysis ( for TNF and IL-6 ) . RT-PCR analysis for TNF and IL-6 expression was done with RNA isolated from mouse lungs . BALF was used for Western blotting with S100A9 antibody and S100A9 ELISA analysis . In some experiments , purified mouse S100A9 protein ( 15 µg/mouse ) diluted in PBS or HBSS buffer diluted in PBS ( vehicle control ) was administered to mice via the I . T route . At 8 h posttreatment , TNF and IL-6 expression and production in the lung was monitored by RT-PCR and ELISA . Lung sections from mock- or IAV-infected mice were stained with goat anti-mouse S100A9 antibody ( 1∶100 dilution ) ( R&D ) for 2 h at room temperature . After washing five times with PBS , lung sections were incubated with anti-goat Texas Red ( 1∶50 dilution ) ( Vector Labs ) for 1 h at room temperature . After washing three times with PBS , sections were mounted with DAPI containing mounting solution ( Invitrogen ) . Sections were visualized by fluorescence microscopy . To study apoptosis in the respiratory tract , TUNEL assays were done . Formalin-fixed lungs from IAV-infected mice were used . The TUNEL assay was done using an ApopTag Peroxidase In Situ Apoptosis Detection Kit ( Milipore , MA ) . Digital images of TUNEL-stained lung sections were examined by light microscopy . Digital images were used to count the number of TUNEL-positive cells , using Image J software from NIH ( http://rsbweb . nih . gov/ij/ ) as described previously by us [75] . For each analysis , an area of 5 . 39×102 µm×4 . 09×102 µm of TUNEL-stained lung section was scanned by Image J software . Gross apoptotic area was expressed as pixels per micron . This value was used to calculate the percentage of the apoptotic area in each analysis . Three IAV- infected mice treated with control IgG and three IAV-infected mice treated with S100A9 antibody were used . Data were collected from 9 areas per mouse from each experimental group . The values obtained from the 27 lung section areas of each experimental group were used for statistical analysis . Hematoxylin and eosin ( H&E ) staining was performed on paraffin-embedded mouse lung sections . Briefly , slices of lung were sequentially rehydrated in 100% and 95% ethanol followed by xylene deparaffinization . After rinsing with distilled water , sections were stained with hematoxylin for 8 min and counterstained in eosin for 1 min followed by serial dehydration with 95% and 100% ethanol . Sections were then mounted on coverslips . IAV-infected and S100A9 protein-treated cells were examined for apoptosis by annexin V labeling , using an annexin V/propidium iodide ( PI ) apoptosis detection kit ( BioVision , CA ) [75] , [78] , [79] . For TUNEL assay cells were grown in cover slips ( 12 mm diameter ) ( Ted-Pella , CA ) . TUNEL assay with macrophages was performed by using DeadEnd Colorimetric TUNEL System ( Promega , WI ) . Digital images of TUNEL-stained macrophages were examined by light microscopy . Digital images were used to count the number of TUNEL-positive cells using Image J software ( please see above ) . At least eight different fields were counted for each cover slip and two cover slips ( duplicate ) were examined for each experiment . Furthermore , each experiment was repeated independently three times . | The lung disease severity following influenza A virus ( IAV ) infection is dependent on the extent of inflammation in the respiratory tract . Severe inflammation in the lung manifests in development of pneumonia . Therefore , it is very critical to identify cellular factors and dissect the molecular/cellular mechanism controlling inflammation in the respiratory tract during IAV infection . Knowledge derived from these studies will be instrumental in development of therapeutics to combat the lung disease associated with IAV infection . Towards that end , in the current study we have identified a cellular factor S100A9 which is responsible for enhanced inflammation during IAV infection . In addition , we have characterized a signal transduction pathway involving various cellular receptors and signaling adaptors that are involved in mediating S100A9-dependent inflammatory response . Thus , our studies have illuminated a cellular/molecular mechanism that can be intervened by therapeutics to reduce and control IAV-associated lung inflammatory disease like pneumonia . | [
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] | [] | 2014 | DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway |
Airway infection by the Gram-positive pathogen Streptococcus pneumoniae ( Sp ) leads to recruitment of neutrophils but limited bacterial killing by these cells . Co-colonization by Sp and a Gram-negative species , Haemophilus influenzae ( Hi ) , provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model . Products from Hi , but not Sp , also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells . Here we identify the stimulus from Hi as its peptidoglycan . Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 ( Nod1 ) , which is involved in recognition of γ-D-glutamyl-meso-diaminopimelic acid ( meso-DAP ) contained in cell walls of Hi but not Sp . Neutrophils from mice treated with Hi or compounds containing meso-DAP , including synthetic peptidoglycan fragments , showed increased Sp killing in a Nod1-dependent manner . Moreover , Nod1−/− mice showed reduced Hi-induced clearance of Sp during co-colonization . These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo .
Successful pathogens have mechanisms both to avoid triggering inflammatory responses and/or to evade the inflammatory response they induce in their host . In the case of the Gram-positive Streptococcus pneumoniae ( Sp ) , a major pathogen of the human respiratory tract , infection involving normally sterile parts of the airway is characterized by acute inflammation with a marked and brisk recruitment of neutrophils [1] . This neutrophil influx , however , is often insufficient to clear the infection until type-specific antibody promotes opsonophagocytic killing . Before such antibody is generated , pneumococci are relatively resistant to neutrophil-mediated killing even when opsonized by complement [2] . The inability of phagocytes to eliminate pneumococci in this period may account for the rapid and often overwhelming progression of pneumococcal pneumonia , a disease responsible for more than a million deaths a year [3] . In fact , in experimental acute pneumonia , neutrophils enhance the likelihood of death without impacting bacterial clearance [4] . Likewise , in a murine model of carriage , intranasal inoculation of Sp induces recruitment of neutrophils into the nasal spaces , yet systemic depletion of neutrophils has little effect on the density of colonizing bacteria [5 , 6] . In contrast , when co-colonized with the Gram-negative respiratory tract bacteria Haemophilus influenzae ( Hi ) , the neutrophil influx is sufficient to rapidly clear Sp from the mucosal surface [6] . Clearance during co-colonization is not seen if either neutrophils or complement are systemically depleted , indicating that killing occurs through neutrophil-mediated phagocytosis of Sp opsonized by complement . These in vivo observations demonstrate that one microbe can co-opt the innate immune response of the host to prevail over a competitor that resides within a similar niche . Enhanced killing of Sp can be modeled ex vivo using neutrophils derived from peritoneal exudates cells ( PECs ) treated in vivo with Hi or its products . Thus , components of Hi are sufficient to stimulate neutrophil activity that overcomes the resistance of complement-opsonized Sp to phagocytic killing . The focus of this study is to define the mechanism leading to effective neutrophil-mediated killing of Sp that occurs in the absence of specific antibody . We observed that peptidoglycan fragments from Hi are sufficient to promote neutrophil-mediated phagocytosis of opsonized Sp . Pathways for the recognition of and response to peptidoglycan fragments leading to NF-κB-dependent transcriptional activation and pro-inflammatory responses have been partially characterized [7] . Peptidoglycan fragments containing the minimal structure γ-D-glutamyl-meso-diaminopimelic acid ( meso-DAP ) found in Gram-negative bacteria , including Hi , act through a cytoplasmic signaling molecule , nucleotide-binding oligomerization domain-1 ( Nod1 ) [8–10] . In the peptidoglycan of most Gram-positive bacteria , including Sp , meso-DAP is replaced by lysine , a structural difference of a single carboxyl group that is sufficient to prevent effective signaling involving Nod1 [11] . In addition , another peptidoglycan fragment , muramyl dipeptide ( MDP ) , common among most Gram-negative and Gram-positive bacteria , is the minimal structure needed for responses involving a separate cytoplasmic immune signaling molecule , Nod2 [12] . Our findings provide a demonstration of the contribution of Nod1-mediated signaling to the anti-bacterial activity of neutrophils and their ability to clear mucosal infection .
The increased ability of ex vivo PECs to kill Sp when elicited following intraperitoneal ( i . p . ) administration of heat-killed Hi ( HKHi ) allowed us to examine the mechanism whereby one species stimulates the killing of another . When HKHi-stimulated PECs were divided by density gradient centrifugation into mononuclear cell– and neutrophil-containing fractions , only the neutrophil-enriched fraction demonstrated killing of Sp ( unpublished data ) . This result correlated with the absence of killing by HKHi-stimulated PECs when elicited from mice depleted of neutrophils by prior treatment with RB6-8C5 , an antibody to murine Ly6 . G [6 , 13] . Addition of HKHi correlated with increased neutrophil activation as confirmed by increased expression of the marker Mac-1 ( complement receptor 3 [CR3] , CD11b/CD18 ) in cells co-expressing Ly6 . G [6] . Moreover , increased killing of Sp following administration of HKHi was observed with neutrophil-enriched PECs derived from parental but not congenic Mac-1−/− mice ( Figure 1 ) . This finding pointed to the requirement of complement-mediated opsonization for neutrophil recognition . When heat-inactivated serum or serum from C3−/− mice was used as a complement source , no killing by HKHi-stimulated neutrophil enriched PECs was seen , confirming the requirement of active complement . Although C3 may be activated by either classical or alternative pathways , killing in the presence of serum from scid mice lacking antibody made it less likely that complement was being activated by the classical pathway [6] . The requirement for the alternative pathway was confirmed by showing a lack of Sp killing when serum from factor B–deficient mice was used as a complement source ( Figure 1 ) . Thus , results using PECs indicated that products of Hi stimulate neutrophil-mediated phagocytic killing of Sp opsonized primarily by activation of the alternative pathway of complement . Products of Hi have previously been shown to signal pro-inflammatory responses through toll-like receptor ( TLR ) 2 and TLR4 , through recognition of its lipopolysaccharide ( LPS ) and lipoproteins , respectively [14 , 15] . In addition , platelet-activating factor receptor ( rPAF ) -mediated signaling has been described for those Hi phase variants expressing the cell surface ligand phosphorylcholine [16] . Opsonophagocytic killing was assessed in neutrophil-enriched PECs derived from TLR2−/− mice . These showed increased killing in response to HKHi and were as active as cells derived from the TLR2-expressing mouse strain ( Figure 2 ) . Opsonophagocytic killing was also compared in neutrophil-enriched PECs derived from CH3/OuJ and C3H/HeJ mice , which express functional and non-functional TLR4 , respectively . TLR4 did not contribute to Sp killing in response to HKHi stimulation . Moreover , HKHi derived from isogenic mutants expressing or not expressing phosphorylcholine stimulated similar levels of Sp killing by neutrophil-enriched PECs ( unpublished data ) [17] . Together , these results showed that the enhancement of opsonophagocytic killing occurs independently of non-redundant signaling involving known cell surface pattern recognition receptors for Hi , including TLR2 , TLR4 , and rPAF . This unexpected finding led us to characterize the signal from Hi that enhances the opsonophagocytic killing of Sp . Neither lysis of HKHi by sonication , nor prior treatment with proteinase K , diminished stimulation of killing by neutrophil-enriched PECs , which indicates the involvement of a non-proteinaceous bacterial product . However , there was no stimulation of neutrophil-enriched PECs by purified LPS ( in doses up to 50 μg/animal ) extracted from Hi or Escherichia coli ( Figure 3 ) . These findings were also consistent with a signaling pathway other than recognition of Hi components by TLR2 , TLR4 , or rPAF . In contrast , purified Hi peptidoglycan at a dose as low as 1 μg/animal was sufficient to stimulate increased killing of Sp by neutrophil-enriched PECs ( activity equivalent to 107 HKHi ) . Purified peptidoglycan from Sp ( or Staphylococcus aureus ) was less active even when administered at a 10-fold higher dose ( Figure 3 and unpublished data ) . The greater potency of Hi peptidoglycan correlated with the stimulation of killing by HKHi but not HKSp [6] . This observation indicated that structural differences between cell wall fragments of these species may be an important determinant of their peptidoglycan-mediated signaling . To confirm this hypothesis , FK-156 , a synthetic muropeptide containing meso-DAP , was tested and showed a level of stimulatory activity equivalent to purified Hi peptidoglycan when administered at an equivalent concentration . Experiments with FK-156 also demonstrated that Hi peptidoglycan could provide a sufficient stimulus to neutrophil-enriched PECs that accounts for their enhanced killing of Sp and makes it unlikely that a contaminant in the peptidoglycan preparation could explain our findings . In contrast , MDP at the equivalent concentration was a relatively poor stimulus . The potency of Hi peptidoglycan , as well as that of FK-156 , suggested that stimulation of opsonophagocytic killing involved recognition of Hi components by Nod1 . In order to examine this possibility , neutrophil-enriched PECs from Nod1−/− mice were analyzed for their response to HKHi and FK-156 . As predicted , administration of FK-156 ( 10 μg/animal ) stimulated Sp killing by cells in parental , but not in Nod1−/− mice ( Figure 4A ) . Neutrophil-enriched PECs from Nod1−/− mice also showed a diminished response to HKHi , demonstrating that Nod1 accounts for a significant proportion of the signaling generated by innate recognition of this organism . To further confirm this observation , a meso-DAP-containing peptide , murNAcTRIDAP , was synthesized using the Mur enzymes of Gram-negative bacteria [18] . As predicted , its ability to stimulate Sp killing by neutrophil-enriched PECs was equivalent to that of FK-156 and dependent on Nod1 ( Figure 4A ) . In contrast , a synthetic form of the corresponding lysine-containing tripeptide found in Sp peptidoglycan , murNAcTRILYS , lacked stimulatory activity at the same concentration . In Figure 4B , the effect of these peptides on killing is compared in neutrophil-enriched PECs elicited with peptide in buffer alone without the addition of casein to show that administration of murNAcTRIDAP is sufficient and murNAcTRILYS is insufficient to enhance killing of Sp . Our findings using neutrophil-enriched PECs stimulated in vivo and tested in killing assays ex vivo suggested that co-colonization with Hi in competition experiments with Sp should promote clearance of Sp in a Nod1-dependent manner . Indeed , a significant decrease in the density of Sp colonization was observed in Nod1+/+ but not in Nod1−/− mice co-infected with Hi ( Figure 5 ) . The reduced interspecies competition in the absence of Nod1 demonstrated an important role for peptidoglycan recognition in the innate response to Gram-negative bacteria on the mucosal surface . Next , we explored the mechanism for increased opsonophagocytic killing stimulated through Nod1 . Levels of the proinflammatory chemokine MIP-2 , which functions as a murine neutrophil attractant and activator , were previously shown to correlate with neutrophil influx into the nasal spaces [6] . MIP-2 levels increased in response to co-colonization , but were not significantly different between co-colonized Nod1−/− and parental mice ( Figure 6A ) . Analysis of tissue sections from co-colonized mice , both Nod1−/− and parental , showed an intimate association of both Sp ( and Hi ) with neutrophils in the lateral nasal spaces ( Figure 6B ) . These results suggested that the recruitment of neutrophils and their migration to mucosal sites with bacteria were not affected by the expression of Nod1 in this model . Additional evidence that Nod1 did not impact neutrophil migration came from comparisons by flow cytometry of PECs elicited by HKHi or FK-156 ( Figure 6C ) . No difference between Nod1−/− and parental mice was seen in the proportion of total cells expressing Ly6 . G ( neutrophils ) . For both Nod1−/− and parental mice , Ly6 . G positive cells also expressed the markers CD18/CD11b ( activated neutrophils ) . We next considered whether Nod1 signaling affected uptake or killing of bacteria . Gentamicin sulfate was added at the end of killing assays to determine the proportion of Sp surviving within neutrophils as a measure of phagocytic activity and killing . Comparison of neutrophil-enriched PECs elicited by HKHi from Nod1+/+ or Nod1−/− mice showed no difference in the proportion of viable intracellular bacteria ( Figure 6D ) . Preincubation of neutrophil-enriched PECs with cytochalasin D , to inhibit actin rearrangements and block phagocytosis , resulted in minimal survival after gentamicin treatment . These findings confirmed the role of phagocytosis in neutrophil-mediated killing and suggested that Nod1 signaling did not affect the uptake of Sp . Killing of Sp by neutrophil-enriched PECs elicited by HKHi from Nod1+/+ or Nod1−/− mice was also not affected by pretreatment with dibenziodolium chloride ( DPI ) , a blocker of the oxidative burst . Together , these observations suggest that Nod1 signaling acts on events following phagocytosis on a non-oxidative pathway for killing Sp .
Although numerous studies have defined Nod1-mediated effects of bacteria or their cell wall products in vitro , our understanding of its contribution to innate immune responses to bacterial infection in vivo remains limited ( reviewed in [7 , 19] ) . We demonstrate here that the Nod1 signaling pathway can respond to meso-DAP-containing compounds to increase clearance of Sp from the mucosal surface of the airway . Thus , Nod1 was shown to be important in dictating the outcome of competition between two pathogens that occupy a similar niche in their host [6] . Enhanced killing of Sp required products from another organism , since cell wall fragments from Sp , like most Gram-positive species , do not signal through Nod1 . Our findings are relevant to polymicrobial infection and situations in which products from multiple types of organisms are present . This information adds to our previous report , which describes how combinations of microbes and microbial products synergize to enhance inflammatory responses [20] . Mucosal surfaces , in particular , are generally colonized simultaneously with multiple species . The paradigm of one species promoting an innate immune response that affects a competitor may be underappreciated , because most models of infection typically examine responses to individual microbial species . While our model was useful in revealing a role for Nod1 in vivo , it also demonstrates that bacteria that succeed in such environments must have mechanisms to evade its clearance-promoting effects . The specificity for bacterial cell wall components that act through Nod1 suggests a mechanism whereby many Gram-positive pathogens that lack meso-DAP may avoid signaling events that lead to neutrophil-mediated killing . Likewise , the density of colonizing Hi during co-infection was not affected by Nod1 signaling , in contrast to clearance of Sp during co-colonization . This suggests that Hi may be resistant to the response induced by its meso-DAP-containing peptidoglycan , and also to the enhancement of opsonophagocytic killing by neutrophils seen against Sp . In addition to the synthesis of stem peptides without meso-DAP , there may be multiple mechanisms to evade peptidoglycan recognition and stimulation of immune signaling through Nod1 [21] . For example , it has recently been reported that modification of the α-carboxylic acid group of iso-glutamic acid , the residue proximal to meso-DAP , to an amide diminishes signaling through Nod1 and may be a mechanism for immune invasion by some pathogens [22] . Both Sp and Hi are considered extracellular pathogens , which are unable to effectively access intracellular pathways [23] . In the case of epithelial cells , pore-forming toxins or delivery via the type IV pilus have been shown to be necessary for peptidoglycan to gain access to the cytoplasm [24 , 25] . Moreover , Nod1-deficient mice were shown to be more susceptible to infection by Helicobacter pylori expressing the cag pathogenity island type IV secretion apparatus than were wild-type mice [25] . Our observation in this report that peptidoglycan fragments alone are sufficient to induce Nod1-dependent effects shows that access to these cytoplasmic pathways may not be similarly limited for professional phagocytes . In killing assays , however , bacteria or peptidoglycan fragments were delivered in vivo and their activity tested ex vivo . Thus , we cannot confirm whether the effect of injected compounds or bacterial products on neutrophil function was direct . Attempts to treat neutrophils in vitro with immunostimulatory fragments that are active when provided in vivo were not sufficient to elicit a direct effect in killing assays . It is unlikely that this is due to a lack of Nod1 expression in these cells , because in contrast to other members of the Nod protein family , Nod1 expression is ubiquitous [7] . It remains possible that Nod1-mediated signaling requires other cell types , such as epithelial cells , and that its effects on neutrophil function are indirect . A further consideration is that neutrophils have cell wall–degrading enzymes , such as lysozyme , that may generate more biologically active peptidoglycan fragments . This could account for the effects of purified peptidoglycan in our study , which contrasts with prior reports where only synthetic products are active . Thus , both the processing of peptidoglycan and the ability of cell wall fragments to access the cytoplasm may be important factors for signaling events involving neutrophils . In this regard , it has been suggested that Sp and other Gram-positive pathogens synthesize modified peptidoglycan that is resistant to lysozyme [21 , 26 , 27] . Thus , a number of adaptations may contribute to minimizing Nod-mediated signaling by Gram-positive bacteria despite their greater quantity of peptidoglycan per cell . Our study demonstrates that the resistance of Sp to killing by neutrophils ( Figure 4B ) can be overcome by a specific immune signaling pathway . Findings in this study with microbial products and synthetic meso-DAP-containing peptidoglycan fragments add to a prior report that systemic administration of FK-156 enhanced host resistance to various microbial infections [28] . Bacterial killing in our system required opsonization , which for Sp strain Sp1121 occurred through activation of the alternative pathway of complement , followed by phagocytosis by activated , Mac-1 ( CR3 , CD11b/CD18 ) -expressing neutrophils . One of the ligands of Mac-1 , or CR3 , is iC3b [29] . It remains unclear how Nod1-mediated signaling enhances Mac-1-dependent opsonophagoytic killing of complement-opsonized Sp . It has been suggested that Nod1 transduces signals that can stimulate chemokine production and neutrophil recruitment [30] . We did not observe , however , a Nod1-related effect on the increase in MIP-2 levels or influx of neutrophils into either the peritoneal cavity or the nasal spaces in response to bacteria . Likewise , no effect of Nod1 on the uptake of bacteria or generation of an oxidative burst was detected . Rather , killing of Sp in our model resulted from stimulation of non-oxidative activity of neutrophils . Reduced killing in the presence of inhibitors of actin polymerization and rearrangement , and the requirement for complement , argue against Nod1-mediated enhancement of previously described mechanisms for extracellular killing of Sp by neutrophils [31] . We are currently characterizing this oxidative burst–independent anti-pneumococcal effect of neutrophils and the contribution of Nod1 to stimulation of this biological activity . Findings in this study also show a limited role of other signaling pathways in clearance of Sp from the mucosal surface of the murine airway . Sp has previously been shown to activate cellular NF-κB-dependent immune responses through Nod2 . However , the effect of fragments acting through Nod2 , including MDP , purified Hi or Sp peptidoglycan , and live or killed Hi or Sp , was minor in comparison to those acting through Nod1 [6 , 32] . Moreover , the Hi-induced increase in Sp killing by neutrophil-enriched PECs was not influenced by the pathogen-associated molecular pattern receptors , TLR2 or TLR4 , in a non-redundant manner . Thus , our study provides an example where the predominant signaling response of the innate immune system to a bacterial challenge appears to be through Nod1 .
Hi and Sp strains were grown as previously described [33] . Strains used in vivo were selected because of their ability to colonize efficiently the murine nasal mucosa and included Hi636 ( a type b capsule-expressing , spontaneously streptomycin-resistant mutant of Hi strain Eagan ) , and Sp1121 ( a type 23F capsule-expressing Sp isolate from the human nasopharynx [34] ) . Genetically modified Hi mutants of strain Eagan that constitutively express or lack phosphorylcholine on its LPS were previously described [17] . Six-week-old mice used in the study were housed in accordance with Institutional Animal Care and Use Committee protocols . Mouse strains included C57Bl/6J and congenic Nod1−/− ( Millennium Pharmaceuticals , http://www . mlnm . com/ ) , B6 . 129S4-Itgamtm1Myd/J ( Jackson Laboratories , http://www . jax . org/ ) , and TLR2−/− ( provided by H . Shen , University of Pennsylvania ) . Mac-1 ( CD11b/CD18 ) -deficient mice ( Jackson Laboratories ) have a targeted mutation in the gene for integrin alpha M or CR3 [35] . Neutrophils from these animals are deficient in phagocytosing complement-opsonized particles and in several Fc-mediated functions . The genotype of Nod1−/− ( CARD4-deficient ) mice was confirmed by PCR using primers CARD4-F2 ( 5′-CTTAGGCATGACTCCCTCCTGTCG-3′ ) , CARD4-R1 ( 5′-GATCTTCAGCAGTTTAATGTGGGAGTGAC-3′ ) , and CARD4-RB ( 5′-CCATTCAAGCTGCGCAACTGTTG-3′ ) . Sequences and PCR protocols were supplied by Charles River Laboratories Genetic Testing Services ( http://www . criver . com/ ) , where the colony was derived . TLR2−/− mice have a targeted disruption of the gene encoding the C-terminus of the extracellular domain of TLR2 and display an increased susceptibility to bacterial infections [36] . Serum was also obtained from factor B–deficient and C3-deficient mice ( provided by J . Lambris , University of Pennsylvania ) [37 , 38] . TLR4-sufficient and -deficient mice were obtained from Jackson Laboratories . C3H/HeJ ( TLR4-deficient ) mice have a spontaneous mutation that occurred in wild-type C3H/HeOuJ ( TLR4-sufficient ) mice at an LPS response locus ( mutation in TLR4 gene ) , making C3H/HeJ mice resistant to endotoxin [39] . Mice were used in a previously described model of nasal colonization with Sp and Hi [34] . Briefly , groups of at least ten mice per condition were inoculated intranasally with 10 μl containing 1 × 107 CFU of PBS-washed , mid-log phase Hi , Sp , or both applied separately to each naris . Then , 24 h post-inoculation , the animal was sacrificed , the trachea cannulated , and 200 μl of PBS instilled . Lavage fluid was collected from the nares for determination of viable counts of bacteria in serial dilutions plated on selective medium containing antibiotics to inhibit the growth of contaminants ( 100 μg/ml streptomycin to select for Hi636 , and 20 μg/ml neomycin to select for Sp1121 ) . Neutrophil-enriched PECs were isolated as previously described [40] . Briefly , phagocytes were obtained by lavage of the peritoneal cavity ( 8 ml/animal with PBS containing 20 mM EDTA ) of mice treated 24 h and again 2 h prior to cell harvest by i . p . administration of 10% casein in PBS ( 1 ml/dose ) . Administration of casein provided for a higher and more consistent yield of cells . Cells collected from the peritoneal cavity lavage ( PECs ) were enriched for neutrophils or monocytic cells using separation by a Ficoll density gradient centrifugation according to the manufacturer's protocol ( MP Biomedicals , http://www . mpbio . com/ ) . Neutrophil or monocytic cell-enriched fractions were collected and washed with 5 ml of Hank's buffer without Ca++ or Mg++ ( GIBCO , http://www . invitrogen . com/ ) plus 0 . 1% gelatin . An aliquot of these cells was characterized using FACS for staining of granulocytes with anti-mouse Gr-1 mAb to Ly6 . G ( BD Biosciences , http://www . bdbiosciences . com/ ) and showed >90% positively stained cells following enrichment . Additional characterization involved staining for CD11b/CD18 ( BD Biosciences ) . Where indicated , heat-inactivated Hi ( Hi636 ) , bacterial components , FK-156 ( an analog of meso-DAP provided by Astellas Pharmaceuticals , http://www . us . astellas . com/ ) , or synthetic peptidoglycan fragments were co-administered intraperitoneally with or without the casein solution as indicated . PBS-washed , mid-log phase bacteria ( 107 cells/animal ) were heat-inactivated by treatment at 65 °C for 30 min and shown to be non-viable . Neutrophil-enriched PECs were counted by trypan blue staining and adjusted to a density of 7 × 106 cells/ml . Killing during a 45-min incubation at 37 °C with rotation was assessed by combining 102 PBS-washed , mid-log phase bacteria ( in 10 μl ) with complement source ( in 20 μl ) , 105 mouse phagocytes ( in 40 μl ) , and Hank's buffer with Ca++ and Mg++ ( GIBCO ) plus 0 . 1% gelatin ( 130 μl ) . Earlier time points and fewer effector to target cells were shown in pilot experiments to result in less killing . The complement source consisted of fresh mouse serum from C57Bl/6 mice unless indicated otherwise . After stopping the reaction by incubation at 4 °C , viable counts were determined in serial dilutions . Percent killing was determined relative to the same experimental condition without i . p . administration of bacterial products or FK-156 ( casein alone ) . For groups without co-administered casein , the percent killing was calculated by comparison to controls with inactivated complement ( 56 °C for 30 min ) where there was no loss of bacterial viability . Additional controls consisting of heat-inactivated Hi636 administered without casein gave similar levels of killing , confirming that killing was stimulated by bacterial products rather than by casein . Where indicated , neutrophils were preincubated with 10 μM DPI , an NADPH-ubiquinone oxidoreductase inhibitor , for 15 min at 37 °C . The respiratory burst of activated neutrophils and its inhibition by DPI was assessed by cytochrome C oxidation with activation by treatment with 25 nM phorbol 12-myristate 13-acetate ( PMA; Sigma , http://www . sigmaaldrich . com/ ) as a control . To inhibit phagocytosis , neutrophils were pretreated with cytochalasin D ( 20 μM , Sigma ) for 15 min at 37 °C . Intracellular pneumococci were quantified using viable counts following the addition of gentamicin sulfate ( final concentration 300 μg/ml ) . After a 20-min incubation at 37 °C , the antibiotic was removed by serial washing prior to plating for viable counts . Hi LPS was purified by hot-phenol extraction from strain Eagan as previously described [41] . E . coli LPS and Staphylcoccus aureus peptidoglycan were purchased from Sigma . Preparation of peptidoglycan from Hi was modified from a previously described protocol [42] . Briefly , strain Hi636 was grown overnight in sBHI , pelleted at 6 , 000g at 4 °C , and washed with Tris-buffered saline ( TBS ) . The pellet was resuspended in 5 ml of cold dH2O , and cells were lysed in boiling SDS ( 5% ) for 30 min . Lysates were collected at 150 , 000g , resuspended in dH2O , and washed once with TBS . Glycogen and nucleic acids were removed by treatment with α-amylase ( Fluka 10070 , from Bacillus subtilis ) and DNAse/RNAse A ( Sigma ) for 2 h at 37 °C , followed by overnight incubation at 37 °C with agitation and 100 μg/ml of trypsin ( Worthington Biochemical , http://www . worthington-biochem . com/ ) in the presence of 10 mM CaCl2 . To stop the reaction , 10 mM EGTA was added and the peptidoglycan preparation was boiled in 5% SDS for 30 min . After extensive washing , Hi peptidoglycan was lyophilized and resuspended at 5 mg/ml in endotoxin-free water . For preparation of peptidoglycan from Sp , bacteria were grown in tryptic soy medium and treated as above , except cells were also treated in 0 . 5% Na-layrilsarcosin prior to boiling in SDS ( 5% ) [43] . Sp peptidoglycan was additionally treated with hydrofluoric acid ( 49% for 48 h at 4 °C with agitation ) to remove teichoic acid as described [44] . The pellet was washed extensively with dH2O , twice with acetone , and lyophilized . N-acetlymuramyl-L-alanyl-γ-D-glutamyl-meso-2 , 6-diaminopimelic acid ( murNAcTRIDAP ) , N-acetlymuramyl-L-alanyl-γ-D-glutamic acid ( murNAcDI ) , and N-acetlymuramyl-L-alanyl-γ-D-glutamyl-L-lysine ( murNAcTRILYS ) were prepared as described previously [18 , 45 , 46] . Briefly , recombinant Pseudomonas aeruginosa ( Pa ) MurA , MurB , MurC , and MurD were used to synthesise uridine 5′diphosphoryl-N-acetlymuramyl-L-alanyl-γ-D-glutamic acid ( UDP-murNAcDI ) ; additionally , Pa MurE was used to synthesise uridine 5′diphosphoryl-N-acetlymuramyl-L-alanyl-γ-D-glutamyl-meso-2 , 6-diaminopimelic acid ( UDP-murNAcTRIDAP ) , and Sp MurE was used to synthesise uridine 5′diphosphoryl-N-acetlymuramyl-L-alanyl-γ-D-glutamyl-L-lysine ( UDP-murNAcTRILYS ) . Electrospray ionization mass spectrometry ( negative ion ) was used to confirm the molecular weight of synthesized compounds . Purity was assessed by analytical anion exchange chromatography using a GE Healthcare Mono Q HR5/5 column ( http://www . gelifesciences . com/ ) and by continuous spectrophotometric enzyme assay with MurE for UDP-murNAcDI , and MurF for UDP-murNAcTRIDAP and UDP-murNAcTRILYS . N-acetlymuramyl-peptides were produced by mild acid hydrolysis ( 0 . 1 M HCl , 100 °C , 1 h ) of the corresponding uridine 5′diphosphoryl-N-acetlymuramyl-peptides . Complete hydrolysis was confirmed by continuous spectrophotometric enzyme assay with MurE for murNAcDI ( MDP ) , and MurF for murNAcTRIDAP and murNAcTRILYS . Peptides were analysed by electrospray ionisation mass spectrometry ( positive ion ) . The concentration of other hydrolysis products ( UDP , UMP , and Pi ) was established by continuous spectrophotometric enzyme assay . Corresponding concentrations of UDP , UMP , and Pi were added to the casein-only control . At 24 h post-inoculation , the animal was sacrificed and decapitated , and the head was fixed for 48 h in 4% paraformaldehyde in PBS . The head was then decalcified by serial incubations in 0 . 12 M EDTA ( pH 7 . 0 ) at 4 °C over 1 mo before freezing in Tissue-Tek O . C . T . embedding medium ( Miles , Elkhart , Indiana , United States ) in a Tissue-Tek Cryomold . Then , 5-μm-thick sections were cut , air dried , and stored at −80 °C . Frozen-imbedded tissue sections were stained with hematoxylin and eosin ( H&E ) following a 10-min fixation step in 10% neutral buffered formalin ( NBF ) . Sections were then dehydrated in alcohol , cleared in xylene , and mounted in cytoseal ( Richard-Allan Scientific , http://www . rallansci . com/ ) . Immunofluorescent staining on frozen tissue was performed and visualized as previously described [47] . Neutrophil-like cells were stained using rat anti-mouse Ly6G mAb ( BD Biosciences ) followed by anti-rat Ig secondary antibody [6] . To detect Sp1121 , sections were incubated with antisera to Sp type 23F ( Statens Serum Institut , http://www . ssi . dk/ ) followed by anti-rabbit Ig secondary antibody . To detect Hi636 , sections were incubated with antisera to Hi type b ( DIFCO Laboratories , http://www . bd . com/ds/ ) followed by anti-rabbit Ig secondary antibody . Upper respiratory tract lavage fluid was assayed for the concentration of macrophage inhibitory protein ( MIP-2 ) by ELISA in duplicate according to the manufacturer's instructions ( Pharmingen , http://www . bdbiosciences . com/ ) . Statistical comparisons of colonization among groups were made by the Kruskal–Wallis test with Dunn's post-test ( GraphPad Prism 4; GraphPad Software , http://www . graphpad . com/ ) . In vitro killing assays were compared by ANOVA with Tukey post-tests as appropriate .
The GenBank ( http://www . ncbi . nlm . nih . gov/Genbank/index . html ) accession number for murine Nod1 is NM_172729 . | Pathogens are generally studied in the laboratory one species at a time . Most exist , however , in complex environments where they must adapt not only to their host but also to other members of the microbial flora . Using a mouse model of co-colonization , we have shown that one bacterial species ( Haemophilus influenzae ) can take advantage of the innate immune response of its host to outcompete and eliminate another species ( Streptococcus pneumoniae ) that resides in the same microenvironment of the upper respiratory tract . The molecular mechanism for this effect involves recognition of a cell wall fragment found on H . influenzae , but not on S . pneumoniae . The response to this immunostimulatory fragment requires Nod1 , a host molecule that transmits inflammatory signals in response to specific peptides of the bacterial cell wall . This Nod1-mediated inflammatory stimulation triggers an increase in the ability of a type of white blood cell ( neutrophil ) to engulf and then kill S . pneumoniae , effectively removing it from its niche on the mucosal surface of the host airway . Our study , therefore , provides a demonstration of the importance of Nod1 in neutrophil-mediated clearance of bacterial infection . In addition , we have described a mechanism for interspecies competition between microbes that occurs through selective stimulation of host innate immune responses . | [
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] | 2007 | Nod1 Signaling Overcomes Resistance of S. pneumoniae to Opsonophagocytic Killing |
The yeast Dekkera bruxellensis is a major contaminant of industrial fermentations , such as those used for the production of biofuel and wine , where it outlasts and , under some conditions , outcompetes the major industrial yeast Saccharomyces cerevisiae . In order to investigate the level of inter-strain variation that is present within this economically important species , the genomes of four diverse D . bruxellensis isolates were compared . While each of the four strains was shown to contain a core diploid genome , which is clearly sufficient for survival , two of the four isolates have a third haploid complement of chromosomes . The sequences of these additional haploid genomes were both highly divergent from those comprising the diploid core and divergent between the two triploid strains . Similar to examples in the Saccharomyces spp . clade , where some allotriploids have arisen on the basis of enhanced ability to survive a range of environmental conditions , it is likely these strains are products of two independent hybridisation events that may have involved multiple species or distinct sub-species of Dekkera . Interestingly these triploid strains represent the vast majority ( 92% ) of isolates from across the Australian wine industry , suggesting that the additional set of chromosomes may confer a selective advantage in winery environments that has resulted in these hybrid strains all-but replacing their diploid counterparts in Australian winery settings . In addition to the apparent inter-specific hybridisation events , chromosomal aberrations such as strain-specific insertions and deletions and loss-of-heterozygosity by gene conversion were also commonplace . While these events are likely to have affected many phenotypes across these strains , we have been able to link a specific deletion to the inability to utilise nitrate by some strains of D . bruxellensis , a phenotype that may have direct impacts in the ability for these strains to compete with S . cerevisiae .
Dekkera ( Brettanomyces ) bruxellensis has been described in the population ecology of various fermented beverages , such as wine , beer and cider [1]–[3] , and is of increasing relevance to the biofuel industry [4] . Recent genomic sequencing of this species is beginning to reveal the mechanisms by which it is able to survive the harsh environment of alcoholic fermentation , primarily through gene-family expansions in membrane transporters and oxidoreductase enzyme classes that are predicted to facilitate nutrient scavenging and maintain redox homeostasis respectively [5] . However , our understanding of how other industrially important traits have evolved in D . bruxellensis lags well behind what is known for S . cerevisiae [6] . In general , D . bruxellensis utilises a make-accumulate-consume strategy similar to that found in S . cerevisiae [7] , however traits , including carbon and nitrogen source utilisation [8] , vary considerably between D . bruxellensis strains . For example , it was recently shown that nitrate utilisation enables D . bruxellensis to out-compete S . cerevisiae in continuous industrial fermentations [9] , and key genes involved in nitrate assimilation were found in a cluster in the partial genome sequence of D . bruxellensis strain CBS2499 [10] . Nonetheless , nitrate utilisation is not a defining feature of this species . Nearly one third of D . bruxellensis isolates from a range of sources do not grow on nitrate as a sole nitrogen source [8]; presumably nitrate assimilation is less important for D . bruxellensis in some fermentation ecosystems . In another recent study , variation in sulphite tolerance in D . bruxellensis was linked to amplified fragment length polymorphism ( AFLP ) and 26S rDNA genetic markers [11] , inferring a genetic basis for previously reported regional variation and groupings of this yeast across Australian wineries [1] . To date , de-novo assemblies exist for genomes of two D . bruxellensis wine isolates , AWRI1499 [5] and CBS2499 [12] , from Australia and France , respectively . Unlike AWRI1499 , CBS2499 has the same 26S rDNA sequence as the D . bruxellensis type strain CBS 74 ( unpublished data ) , a lambic-beer isolate . This data , in combination with AFLP genotyping [1] , infers the two sequenced strains are likely to be highly divergent . AWRI1499 , a representative isolate of a sulphite-tolerant genotype group [11] , is an allotriploid comprising a moderately heterozygous diploid , and divergent haploid complements [5] . Thus far it remains unclear to what degree intra-specific differences observed using methods such as AFLP may simply reflect presence or absence of all or part of the divergent haploid genome found in AWRI1499 . CBS2499 was assembled as a pseudo-haploid [12] , preventing such comparison with other de-novo assemblies . To improve our understanding of genome diversity amongst D . bruxellensis wine isolates and gain insights into the evolution of industrially relevant traits in this important microorganism , we have performed mapping-assemblies of CBS2499 and two newly sequenced Australian D . bruxellensis wine strains against the reference genome sequence of AWRI1499 . Comparative genomics of the four strains reveals that presence of a divergent haploid genome is not a feature restricted to AWRI1499 , but has arisen through at least two independent ‘hybridisation’ events . In addition to large-scale ploidy variation , gene conversion and allelic expansion appear to be key molecular mechanisms driving strain divergence . Some phenotypes , such as nitrate/nitrite utilisation , on the other hand , are determined by genomic insertions and deletions ( InDels ) .
In order to compare the genomic complements of D . bruxellensis strains , a re-sequencing strategy was used to align genome data from short-read sequencing ( 2×100 bp ) for three strains against the published draft genome assembly of D . bruxellensis strain AWRI1499 [5] . Two of the assemblies were for strains sequenced specifically for this work; AWRI1608 and AWRI1613 . For the third , CBS2499 , comparable data ( 2×100 bp format genome data ) used as part of the D . bruxellensis CBS2499 draft genome assembly [12] was obtained from the NCBI short read archive . The two newly sequenced strains were chosen because they have divergent AFLP genotypes and , with AWRI1499 represent 98% of D . bruxellensis isolates associated with Australian wineries [1] . In all , the divergence between all four strains , as determined from AFLP analysis , is considerable and therefore should provide insights into the genomic landscape of wine isolates of this important yeast . Given the unusual nature of the D . bruxellensis AWRI1499 genome ( triploid hybrid comprised of a closely related diploid set of alleles and a third distantly related genomic complement ) , it was of interest to determine whether this genomic organisation is a defining characteristic of this species . Sequence alignments were therefore interrogated globally to determine genomic ploidy and the levels of both inter- and intra-allelic genetic diversity ( Fig . 1 , Datasets S1 and S2 ) . Ploidy levels across the genomes were estimated by taking advantage of allele proportions . In a triploid genome , it is expected that the maximum average frequency of a particular allele at a heterozygous site will be approximately 0 . 66 ( due to a base difference in a single allele ) , while this number will be closer to 0 . 5 for heterozygous sites in a diploid . The observed average major allele frequency was therefore calculated across the entire genome of each isolate using a sliding window approach ( Fig . 1A ) . As a triploid control , RNA-seq data for AWRI1499 was mapped to the AWRI1499 genome and this showed a maximum average allele proportion consistent with its triploid state ( 0 . 68±0 . 04 , data not shown ) . AWRI1608 also displayed an average allele proportion ( 0 . 69±0 . 03 ) , consistent with this isolate being triploid . However , both CBS2499 ( 0 . 58±0 . 05 ) and AWRI1613 ( 0 . 57±0 . 03 ) displayed maximum allele frequencies consistent with these isolates being diploid . For all strains , while the average maximum allele frequency approximated to either 0 . 66 or 0 . 5 there were many localised regions that differed from these values , including significant portions of the genomes that displayed loss of heterozygosity ( LOH; 17 . 9% of AWRI1613 , 16 . 3% of CBS2499 and 3% of AWRI1608 ) ( Fig . 1A , Fig . 2 ) . As these differences in local allele proportions may be due to copy number variation ( CNV ) , such as heterozygous deletions or genomic duplications , CNV was also determined globally for each of the genomes ( Fig . 1B ) . While copy number was relatively stable , there were many instances of localised copy number variation in each strain , and of opposing copy number changes in the same genomic region between strains ( Fig . 2A ) . Copy number amplification was especially prominent in CBS2499 with several genomic regions displaying effective copy numbers of 4 n or greater ( Fig . 1B , Fig . 2B ) . Regions of increased copy number in CBS2499 appear to coincide with the ends of genomic scaffolds in both the AWRI1499 and CBS2499 [12] assemblies ( Fig . S1 ) , a feature not described in the CBS2499 de-novo assembly . This may be indicative of sub-telomeric amplification of sequences , which is common to other yeasts including S . cerevisiae and Cryptococcus neoformans [13] , [14] . Examination of the functional annotation for the genes in these expanded regions revealed a statistically significant enrichment for those encoding proteins involved in carbohydrate metabolic processes ( p = 6 . 5×10-7 ) and may therefore indicate adaptation to utilisation of specific carbon sources by this strain . It was also apparent that there was co-localisation of copy number variation and alterations in allele frequencies ( including the majority of LOH events ) . This is consistent with gene conversion , rather than heterozygous deletion , being responsible for the majority ( 95% ) of genomic regions displaying LOH across the strains . For example , Fig . 2C describes a 100 kb genomic locus in which loss of heterozygosity has occurred in AWRI1613 and CBS2499 without altering the normal diploid genomic complement of these strains , while in AWRI1608 , the otherwise triploid state is predicted to have been amplified to a tetraploid complement . Interestingly , this amplification in AWRI1608 is complicated by the fact that the allelic ratios change from 2∶2 to 3∶1 within the amplified region . This change in allelic ratios is indicative of either gene conversion of one allele following amplification of the region , or two amplification events that each amplified adjacent parts of the region carrying different homologs . It has been shown previously that the allotriploid genome of AWRI1499 consists of two highly related sets of chromosomes in addition to a third , more distantly related , set [5] . As the genomic analyses of AWRI1608 , AWRI1613 and CBS2499 predicted only one of these strains to also be a triploid , it was of interest to determine the relationship between each of the haplotypes across all of the strains in order to ascertain whether either of the diploid strains contained the “divergent” haplotype of AWRI1499 . Seven loci that displayed three clearly defined haplotypes in AWRI1499 [5] were selected with individual haplotypes derived for each locus in each strain by taking advantage of co-occurring SNPs within individual reads . This resulted in a total of ten possible haploid sequences ( 3+3+2+2 ) for each locus for which maximum-likelihood phylogenies [15] , [16] were constructed ( Fig . 3 , Dataset S3 ) . Consistent with whole genome alignments ( Fig . S2 ) , AWRI1613 and CBS2499 alleles were identical for five of seven loci , and exhibited only minor differences for the remaining two ( AWRI1499_1134 and AWRI1499_1822 ) . Furthermore , in the majority of cases , the phylogeny was resolved into a relationship whereby two of the alleles from AWRI1499 and AWRI1608 and both alleles from AWRI1613 and CBS2499 formed a highly related clade , while the third alleles from AWRI1499 and AWRI1608 were both divergent from this conserved clade and also distinct from one another . For the remaining two loci , it appears likely that gene conversion has resulted in either one or both of these divergent alleles being replaced by an allele that is consistent with the conserved clade . Maximum likelihood phylogenies for individual haplotypes derived at five additional genomic loci ( Fig . S3 , Dataset S3 ) , previously sequenced for a collection of international D . bruxellensis strains [17] , revealed similar topologies . Together with a 26S rDNA phylogeny ( Fig . S4 ) , these data provide evidence that predominant Australian D . bruxellensis strain AWRI1499 [1] is similar to South African wine-related D . bruxellensis strains CBS4481 ( Y900 ) and CBS5206 ( Y908 ) . Of note , the DbHAD phylogeny ( Fig . S3E ) suggested that the horizontal transfer of an adenyl deaminase from an unknown Proteobacterial species [10] occurred prior to divergence of the AWRI1499 and AWRI1608 ‘third haplotype’ donors . A protein-based phylogeny ( Fig . S5 ) suggests that DbHAD1 may have descended vertically from the common progenitor of D . bruxellensis and Ogataea parapolymorpha . While the initial analysis of the AWRI1499 genome failed to identify a potential ‘donor’ species for the divergent alleles [5] , we sought to determine if there was sequence data now available that would shed new light on this . Protein-based maximum-likelihood phylogenies were therefore produced for each of the haplotype groups from D . bruxellensis for three of the open reading frames ( ORFs ) presented in Fig . 3 , in addition to homologs identified in the Genbank non-redundant protein database ( Fig . S6 ) . This analysis clearly shows all of the D . bruxellensis alleles to be far more closely related to each other than to any other available protein sequences . A small number of gene sequences available for D . anomala , the closest known relative of D . bruxellensis according to 26S rDNA based phylogenies [18] , were then used as nucleotide queries against the AWRI1499 blast database . Two accessions , annotated as ATP2 and PGK1 , were strong positive matches to AWRI1499 open reading frames , with 92% and 93% nucleotide identity . Nucleotide-based maximum-likelihood phylogenies for these ORFs , with haplotypes extracted from AWRI1499 , 1608 and 1613 , were performed ( Fig . 4 ) . The D . anomala sequences were not closely related to any of the D . bruxellensis haplotypes . As such , the potential source of the divergent alleles in the triploid strains remains to be determined . While there was significant variation in SNP diversity across strains , strain-specific genomic deletions were found to be far less common , with an average of only 0 . 15% of the genome lost , across the three strains relative to AWRI1499 . The majority ( 97% ) of these deletions were found in AWRI1613 . Of the genomic loci that did display strain-specific deletions , one region that was lost specifically in AWRI1613 was of particular interest as it involved the D . bruxellensis nitrate assimilation cluster ( Fig . 5 ) . While nitrate utilisation is common throughout Ascomycota , genes associated with the nitrate assimilation cluster are generally confined to Pezizomycotina; Dekkera , Ogataea , Wickerhamomyces and Blastobotrys are the only genera within the Saccharomycotina where this cluster has been identified . In these species the nitrate cluster appears to have been retained from the last common ancestor with the Pezizomycotina ( Fig . S7 ) . However , despite this ancient evolutionary conservation , it is apparent that both the nitrate and nitrite reductase genes have been lost in AWRI1613 , along with an adjacent β-galactosidase gene . Furthermore , while this cluster is present in AWRI1608 it is predicted to have undergone LOH via gene conversion , resulting in three identical alleles . In contrast , the nitrate cluster of CBS2499 is predicted to have undergone a duplication event resulting in four copies of this genomic region being present in a 1∶1 ratio of two alleles ( Fig 2A , Fig 5A ) . Consistent with presence or absence of ORFs encoding these key assimilatory enzymes , AWRI1499 and CBS2499 displayed robust growth on nitrate as a sole nitrogen source whereas AWRI1613 was unable to grow on this medium ( Fig . 5B ) . Interestingly , despite the presence of the nitrate assimilation locus , AWRI1608 was also unable to utilise nitrate . In order to determine if this loss of nitrate utilisation was due to frameshift or nonsense mutations in the homozygous coding sequences of the AWRI1608 cluster , the ORFs of the nitrate and nitrite reductases were compared to haplotyped sequences from CBS2499 ( Dataset S4 ) . In each case , there were no obvious truncated coding regions or non-synonymous mutations present in the AWRI1608 ORFs , compared to either of the CBS2499 haplotypes that would be expected to cause the drastic changes to enzyme function that could account for the loss of nitrate utilisation in AWRI1608 . To further investigate the cause of the non-nitrate utilisation phenotype of AWRI1608 , a high-affinity nitrate transporter gene that lies adjacent to the two reductases and the genomic region surrounding the two putative nitrate assimilation transcription factors of D . bruxellensis [10] was also investigated in these strains . The gene encoding the nitrate transporter was shown to be homozygous in both strains that could not grow on nitrate ( AWRI1608 and AWRI1613 ) but was heterozygous in AWRI1499 and CBS2499 ( data not shown ) . However , even in the two homozygous strains the ORF is predicted to encode a full-length , functional , nitrate transporter . Similarly , the genomic region encompassing both putative regulatory proteins was predicted to be present in all four strains; as for the nitrate and nitrite reductase genes in AWRI1608 , both ORFs displayed LOH in AWRI1608 , AWRI1613 and CBS2499 ( data not shown ) . In contrast , this region was shown to be heterozygous in AWRI1499 .
The advent of next generation sequencing has enabled a significant increase in knowledge regarding the genomic makeup of important , but often genetically intractable , industrial yeasts . This manuscript describes the first analysis of inter-strain variation in the genomic landscape of D . bruxellensis . The most prominent finding of these genome comparisons was the common occurrence of triploid hybrids in the strains examined . AWRI1613 and CBS2499 were determined to be diploid in this study , with each strain possessing a pair of closely related chromosomes with moderate levels of heterozygosity . However , AWRI1499 and AWRI1608 , the most common strains found in Australian wineries , were shown to be triploid . While triploid , both AWRI1499 and AWRI1608 contain pairs of chromosomes that are closely related to those found in the diploid strains suggesting that a diploid complement comprises the basis of the D . bruxellensis genome . The third , complete , set of more distantly related chromosomes present in AWRI1499 and AWRI1608 are therefore likely to have been introduced via hybridisation with a distantly related strain of D . bruxellensis or possibly another closely related but as yet undescribed species ( Fig . 6 ) . Furthermore , the divergent third sets of chromosomes present in each triploid are , in-turn , distantly related to each other , indicating that the two triploid strains likely arose from independent hybridisation events . The results of this genomic study therefore suggest that the D . bruxellensis genomic landscape is similar to counterparts in the Saccharomyces sensu stricto clade , where inter-specific hybrids between S . cerevisiae , S . kudriavzevii , S . uvarum and S . eubayanus can be found in natural environments and in industrial fermentations [19]–[22] . Furthermore , Saccharomyces spp . interspecific hybrids are often allotriploid , with a ‘diploid’ complement coming from S . cerevisiae and a ‘haploid’ input from a non-cerevisiae parent . These Saccharomyces sensu stricto hybrids have been isolated from cold winemaking and brewing environments , where it is suggested the hybrid has a selective advantage over its parents . In these situations , the S . cerevisiae genomic component provides the means to efficiently ferment sugar to ethanol while genomic contributions from cold tolerant Saccharomyces spp . allow the hybrid strains to ferment at temperatures that are normally too low for S . cerevisiae [6] , [19] , [23] . In fact , strains of S . pastorianus , the yeast species responsible for the vast majority of lager beer fermentations , are hybrids generated from matings between S . cerevisiae and Saccharomyces eubayanus . At least one line of these hybrids has allotriploid origins , as was observed for the two D . bruxellensis hybrids analysed in this work [19] , [20] , [24] . At this time it is not possible to determine whether the ‘additional haploid’ inputs in the karyotypes of the two triploid D . bruxellensis strains described in this paper originate from one or more non-D . bruxellensis species or distantly related D . bruxellensis strains . However , as for lager brewing , it appears that the formation of these triploid hybrid strains may have resulted in a population replacement event , with the hybrid strains representing 92% of isolates from across the Australian wine industry . Based upon a limited multi-locus analysis , some previously analysed international strains [17] bear resemblance to AWRI1499 and AWRI1608 , therefore it is possible that the current population structure in Australian wineries reflects historical gene flows and bottlenecks . It remains to be determined whether the additional sets of chromosomes in AWRI1499 and AWRI1608 confer a selective advantage in the winery environment , although increased levels of resistance to sulphite , the primary means of D . bruxellensis control in a winery setting , may be at least partially responsible [11] . As for presumptive selective pressures underpinning hybrid prevalence , the driver for loss of nitrate assimilation ability in specific strains of D . bruxellensis remains unclear . Ammonium is fully utilised by S . cerevisiae and other wine yeast species during alcoholic fermentation [25] . However D . bruxellensis appears to be the only wine yeast species that can assimilate nitrate , which is reported to be at levels of between 0 . 9 and 53 . 7 mg/l in Californian wine [26] . One might predict therefore that , in ecological settings where nitrate is available and other nitrogen sources are limited , nitrate assimilation would provide D . bruxellensis with a selective advantage . Yet up to a third of D . bruxellensis wine isolates fail to grow on nitrate [8] . Interestingly , it was recently shown that during anaerobic fermentation nitrate assimilation in D . bruxellensis favours the production of acetic acid over ethanol while partially abolishing the Custers effect [27] . This impact of nitrate assimilation may be detrimental in some environmental settings , thereby providing a selective pressure for its loss . This phenomenon of loss of nutrient utilisation is not unknown in nature . For example , there is a concerted loss of the galactose ( GAL ) catabolism cluster in Japanese isolates of Saccharomyces kudriavzevii when compared to European relatives . In this example , the Japanese strains show degeneration of the genes involved in the utilization of galactose to pseudogene equivalents , while this cluster is completely active in European strains [28] . Evidence was also presented for a selective pressure driving loss of function for all members of the GAL pathway thereby producing a GAL− phenotype , as the presence of partial function in the pathways were suggested to have fitness costs . At face value , this does not appear to be the case for loss of nitrate assimilation in AWRI1613 , which still retains the coding region for the nitrate transporter . However , it is currently not known whether the presence of this transporter may be due to pleiotropy; it may , for example , be required for secondary transport functions . It is also possible that it may be non-functional , although given that the nucleotide sequence of the nitrate transporter ORF in AWRI1613 represents the opposite haplotype group to the homozygous transporter sequence from AWRI1608 ( Fig . S8 ) , it would be expected that at least one of these strains would have a functional transporter as evidenced by the nitrate assimilation phenotype of CBS2499 , which has one copy of each haplotype . Further study of nitrate assimilation in D . bruxellensis will reveal the molecular mechanisms driving the phenotype towards , or away from , utilisation of this nitrogen source , augmenting knowledge gained through detailed studies of the preferred yeast model system for nitrate assimilation , Ogataea parapolymorpha [29] .
D . bruxellensis strains AWRI1608 and AWRI1613 were obtained from The Australian Wine Research Institute Microorganisms Culture Collection . For nitrate assimilation tests , strains were grown on solid YPD for 2 days at 30°C , then plated onto either YNB+ nitrate , YNB+ ammonium as a positive control , or YNB with no nitrogen source as a negative control . Plates were then incubated for 7 days at 30°C . Genomic DNA was prepared using a standard zymolyase and phenol-chloroform extraction from cultures grown under standard conditions . DNA sequencing was performed using 2×100 bp paired-end chemistry on the Illumina HiSeq2000 ( Ramaciotti Centre , Sydney Australia ) . AWRI1499 genome sequences were obtained from Genbank ( Accession number AHIQ0100000 ) . CBS2499 short-read sequences were obtained from the NCBI short-read archive ( Accession number SRR065689 ) . Sequence data for AWRI1608 and AWRI1613 have been deposited in the NCBI short-read archive under the Bioproject accession PRJNA213658 . 26S rDNA sequences for AWRI1499 , 1608 and 1613 have been deposited with GenBank ( accessions KF781196 , KF781197 and KF781198 , respectively ) . Short read sequences were mapped to the AWRI1499 genome using Novoalign v2 . 08 . 01 ( www . novocraft . com ) . The . sam files produced by Novoalign ( default parameters; -F ILM1 . 8 –ILQ_SKIP -i PE 100-1000 -o SAM ) were converted to sorted . bam files using samtools view v0 . 1 . 18 [30] . SNPs , and regions of LOH and gain-of-heterozygosity ( GOH ) were identified from the alignments using the pileup2snp functionality of Varscan v2 . 3 ( default parameters; -min-coverage 10 ) [31] combined with custom python scripts and presented relative to a concatenated AWRI1499 genome sequence . The position of individual AWRI1499 Genbank contigs and ORF annotations within the concatenated genome sequence are provided in Datasets S5 and S6 respectively . Sequence alignments were visualized using the Integrated Genome Browser v2 . 0 [32] . Any region displaying a maximum allele frequency of >95% was classed as being homozygous for that allele . Sequencing coverage was extracted from alignments in . bam format using mpileup from the samtools v0 . 1 . 18 package [30] with actual coverage values converted to changed ploidy levels in sliding windows using custom python scripts . Segmental smoothing of copy number alterations calculated final copy number based on rounding the average value across 21 adjacent genomic windows . To provide additional robustness against single outliers producing small false-positive intervals of altered ploidy levels , a difference of ±0 . 75 was required between the average ploidy of the current 21 window genomic segment and the predicted ploidy level of the previous genomic segment in order to trigger a change in the final predicted ploidy level . If this threshold was not met , the average value obtained for the 21 segment window was rounded to the ploidy level of the previous genomic segment . Phylogenies were constructed using PhyML v3 . 0 ( GTR model; default parameters ) and visualized using Seaview v4 . 0 [15] , [16] . | The yeast D . bruxellensis is of great importance in biofuel and fermented beverage industries , largely as a contaminant and/or spoilage organism . Its lifestyle is not unlike that of the wine/brewing/baking yeast S . cerevisiae , with independent evolutionary pathways having led to this convergence; these species are phylogenetically very distant . Unlike S . cerevisiae , D . bruxellensis is highly intractable in the laboratory; it is difficult to mate and to transform , making even the most basic genetic analysis very difficult . Thus we still have a great deal to learn about this economically important yeast . The latest gene sequencing technologies are , however , providing a means of addressing these limitations . The current manuscript describes a comparative genomics approach to providing insights into inter-strain variations that shape the genomic landscape of D . bruxellensis . Like other industrial yeasts , it has a diploid core genome , but there are also triploid isolates which possess the core diploid complement with an additional , more distantly related , full set of chromosomes . Evidence presented in this paper suggests that this form of triploidy has arisen more than once in the evolutionary history of D . bruxellensis , and it confers a selective advantage for strains of this yeast isolated from wineries . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"applied",
"microbiology",
"genome",
"sequencing",
"genome",
"complexity",
"mycology",
"industrial",
"microbiology",
"genome",
"evolution",
"comparative",
"genomics",
"biology",
"genomics",
"evolutionary",
"biology",
"genomic",
"evolution",
"microbiology",
"yeast"
] | 2014 | Insights into the Dekkera bruxellensis Genomic Landscape: Comparative Genomics Reveals Variations in Ploidy and Nutrient Utilisation Potential amongst Wine Isolates |
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