a. fumigatus af293 ( wt ) , flba4 ( pyrg1 , flba4::pyrg ) , and tjh4.04 ( pyrg1 , gpaa , pyrg ) strains were used . glucose minimal medium with 0.1% ( w / v ) yeast extract ( mmy ) with supplements was used for general culture of fungal strains [ 11 , 12 ] . for liquid submerged culture , approximately 5  10 conidia / ml were inoculated into liquid mmy and incubated at 37 , 250 rpm . the susceptibility of wt and mutant strains to varying concentrations of h2o2 was tested by performance of plate assays . drop dilution assay was performed in a series of 10-fold dilutions derived from a starting inoculum of 1.0  10 conidia per spot . for the hyphal sensitivity test , mycelial plugs cut from the growing edge of five - day old colonies from mmy agar medium were transferred to plates containing 10 mm h2o2 and incubated at 37 for four days . five micrograms ( 5 g ) of total rna was reverse - transcribed to cdna using ecodrytm premix ( clontech , mountain view , ca , usa ) . quantitative pcr ( qrt - pcr ) assays were performed according to the manufacturer 's instructions ( qiagen , valencia , ca , usa ) using 96-well optical plates and a rotor - gene q ( qiagen ) . each run was assayed in triplicate in a total volume of 20 l containing the dna template , 2 qpcr sybr green mix ( doctor protein , seoul , korea ) , and 100 mm of each primer . pcr conditions were : 95 , 15min followed by 95 , 30 sec and 55 , 30 sec for 40 cycles . amplification of one single specific target dna was checked by melting curve analysis ( + 0.5 ramping for 10 sec , from 55 to 95 ) . the expression ratios were normalized to ef1 expression , and calculated according to the ct method . to verify the absence of genomic dna contamination , negative controls , in which reverse transcriptase was omitted , were used for each gene set . 250ml flasks containing 100ml liquid mmy were inoculated with wt and mutant strain conidia and grown at 37 for two days . after washing with phosphate buffered saline ( ph 7.4 ) , mycelia were suspended in lysis buffer ( 50 mm tris - hcl ph 7.2 , 150 mm nacl , 1% triton x-100 , 1 mm edta ) and homogenized using a mini bead - beater ( biospec products , bartlesville , ok , usa ) . the homogenate was centrifuged in a microcentrifuge for 5 min at 15,000 rpm at 4 , and the supernatant was used for further analyses . for detection of catalase activity on gels , the mycelial extracts were subjected to non - denaturing polyacrylamide gel electrophoresis , and the ferricyanide - negative stain was used to locate bands containing catalase activity . peroxidase activity on gels was detected according to the method of wayne and diaz   with a modification of using o - dianisidine instead of diaminobezindine as the substrate . a. fumigatus af293 ( wt ) , flba4 ( pyrg1 , flba4::pyrg ) , and tjh4.04 ( pyrg1 , gpaa , pyrg ) strains were used . glucose minimal medium with 0.1% ( w / v ) yeast extract ( mmy ) with supplements was used for general culture of fungal strains [ 11 , 12 ] . for liquid submerged culture , approximately 5  10 conidia / ml were inoculated into liquid mmy and incubated at 37 , 250 rpm . the susceptibility of wt and mutant strains to varying concentrations of h2o2 was tested by performance of plate assays . drop dilution assay was performed in a series of 10-fold dilutions derived from a starting inoculum of 1.0  10 conidia per spot . for the hyphal sensitivity test , mycelial plugs cut from the growing edge of five - day old colonies from mmy agar medium were transferred to plates containing 10 mm h2o2 and incubated at 37 for four days . five micrograms ( 5 g ) of total rna was reverse - transcribed to cdna using ecodrytm premix ( clontech , mountain view , ca , usa ) . quantitative pcr ( qrt - pcr ) assays were performed according to the manufacturer 's instructions ( qiagen , valencia , ca , usa ) using 96-well optical plates and a rotor - gene q ( qiagen ) . each run was assayed in triplicate in a total volume of 20 l containing the dna template , 2 qpcr sybr green mix ( doctor protein , seoul , korea ) , and 100 mm of each primer . pcr conditions were : 95 , 15min followed by 95 , 30 sec and 55 , 30 sec for 40 cycles . amplification of one single specific target dna was checked by melting curve analysis ( + 0.5 ramping for 10 sec , from 55 to 95 ) . the expression ratios were normalized to ef1 expression , and calculated according to the ct method . to verify the absence of genomic dna contamination , negative controls , in which reverse transcriptase was omitted , were used for each gene set . for protein extraction , 250ml flasks containing 100ml liquid mmy were inoculated with wt and mutant strain conidia and grown at 37 for two days . after washing with phosphate buffered saline ( ph 7.4 ) , mycelia were suspended in lysis buffer ( 50 mm tris - hcl ph 7.2 , 150 mm nacl , 1% triton x-100 , 1 mm edta ) and homogenized using a mini bead - beater ( biospec products , bartlesville , ok , usa ) . the homogenate was centrifuged in a microcentrifuge for 5 min at 15,000 rpm at 4 , and the supernatant was used for further analyses . for detection of catalase activity on gels , the mycelial extracts were subjected to non - denaturing polyacrylamide gel electrophoresis , and the ferricyanide - negative stain was used to locate bands containing catalase activity . peroxidase activity on gels was detected according to the method of wayne and diaz   with a modification of using o - dianisidine instead of diaminobezindine as the substrate . catalase is regarded as a putative virulence factor with the capability of scavenging h2o2 produced by host phagocytes . phylogenetic analyses place the catalases into four distinct clades : clade p ( peroxisomal catalases ) , clade c ( cytoplasmic catalases ) , clade a ( conidia - specific catalases ) , and clade b ( secreted catalases ) . there are three types of catalases in a. fumigatus : cata ( afu6g03890 ) , produced in conidia ; and cat 1 ( afu3g02270 ) and cat2 ( afu8g01670 ) , produced in hyphae [ 17 , 18 ] . both the cata deletion and cat1 disrupted mutants showed increased sensitivity to h2o2 [ 17 , 18 ] . the susceptibilities of the flba and gpaa mutants against h2o2 were determined in comparison with wt . drop dilution assay showed that the two mutants were more resistant than wt , where the flba mutant exhibited higher tolerance than the gpaa mutant ( fig . the radial growth of wt was approximately 62% of that of flba strain , suggesting approximately 1.6 fold enhanced growth by the absence of flba ( fig . these results led us to hypothesize that expression and activities of catalases are elevated in the flba and gpaa mutants . we performed qrt - pcr for examination of mrna levels of the three catalases . according to one report , catap is the only catalase present in the resting conidia required for conidial resistance to heat , denaturing agents , and metal ions . accordingly , cata mrna accumulation at low levels was observed in hyphae of all tested strains ( fig . , levels of cat1 mrna in flba and gpaa strains appeared to be low at 24 hr , highly increased at 48 hr , and then slightly decreased at 72 hr . the highest level of accumulation of cat1 mrna was observed in flba strain at 48 hr of growth ( fig . 2 ) . however , the expression pattern of cat2 mrna was somewhat different from that of cat1 . accumulation of the cat2 mrna began to occur at 24 hr and remained at high levels until 48 hr in both mutants and then became indistinguishable among the three strains , including wt ( fig . these results suggest that the absence of flba and the activation of gpaa result in enhanced expression of the cat1 and cat2 genes . we then performed the zymogram assay for determination of catalase activity ; according to our findings , two catalases ( cat1p and cat2p ) , while at different levels , were present in all tested strains ( fig . the cat1p activity of flba strain was higher than those of gpaa mutant and wt , and the highest activity was observed in flba strain at 72 hr ( fig . distinct from catap and cat1p , the cat2p protein is a bifunctional catalase - peroxidase , conferring resistance to heat , heavy metals , and sodium dodecyl sulfate . in all tested strains , cat2p activities were not detected at 24 hr , but began to be detectible at 48 hr , and reached the highest level at 72 hr . the patterns of peroxidase activities were similar to those of catalase activity , suggesting that cat2p has both peroxidase and catalase activities , as reported ( fig . , we found that flba strain exhibits enhanced sod activity and elevated resistance to menadione and paraquat , suggesting that flba - mediated signaling control results in down - regulation of cellular responses associated with detoxification of ros in a. fumigatus . incorporating the findings of the current study , we propose that gpaa - mediated vegetative growth signaling is involved in increased protection of the fungus from various ros by increasing the levels of sod , catalases , and glit , which are negatively controlled by flba . we also propose that the active cellular growth of a. fumigatus requires a large amount of energy generated by respiration leading to increased production of various ros ; thus , the fungus has evolved to couple the enhanced production of defensive cellular enzymes with proliferation .
vegetative growth signaling of the opportunistic human pathogenic fungus aspergillus fumigatus is mediated by gpaa ( g ) . 
 flba is a regulator of g protein signaling , which attenuates gpaa - mediated growth signaling in this fungus . 
 the flba deletion ( flba ) and the constitutively active gpaa ( gpaaq204l ) mutants exhibit enhanced proliferation , precocious autolysis , and reduced asexual sporulation . in this study 
 , we demonstrate that both mutants also show enhanced tolerance against h2o2 and their radial growth was approximately 1.6 fold higher than that of wild type ( wt ) in medium with 10 mm h2o2 . 
 we performed quantitative pcr ( qrt - pcr ) for examination of mrna levels of three catalase encoding genes ( cata , cat1 , and cat2 ) in wt and the two mutants . according to the results , 
 while levels of spore - specific cata mrna were comparable among the three strains , cat1 and cat2 mrna levels were significantly higher in the two mutants than in wt . 
 in particular , the flba mutant showed significantly enhanced and prolonged expression of cat1 and precocious expression of cat2 . 
 in accordance with this result , activity of the cat1 protein in the flba mutant was higher than that of gpaaq204l and wt strains . 
 for activity of the cat2 protein , both mutants began to show enhanced activity at 48 and 72 hr of growth compared to wt . 
 these results lead to the conclusion that gpaa activates expression and activity of cat1 and cat2 , whereas flba plays an antagonistic role in control of catalases , leading to balanced responses to neutralizing the toxicity of reactive oxygen species .