Role Of The Cofilin Protein In Growth,Mitochondrial Function,and Chitin Synthetase Regulation Of Aspergillus Fumigatus

Aspergillus fumigatus is a saprophytic mold that produces a lot of small spores,and causes pulmonary infection in immunocompromised individuals.Azoles are widely used as antifungal drugs for the treatment of aspergillosis because they inhibit fungal cytochrome P450 sterol 14?-demethylase?Cyp51?,leading to reduced ergosterol in fungal pathogens.Nonetheless,A.fumigatus has evolved several azole resistance mechanisms including mutation of cyp51A,overexpression of cyp51A and/or cyp51B,and activation of efflux pumps.Thus,it is urgent to screen promising targets for antifungal therapy.In our preliminary study,we considered A.fumigatus cofilin as a promising target of antifungal therapy.Cofilin belongs to the actin-depolymerizing factor family in eukaryotic cells,and has long been known to modulate the dynamic balance of the actin cytoskeleton,to maintain mitochondrial function,and to regulate genes expression.We found that the amino acid sequence of A.fumigatus cofilin is very different from that of Homo sapiens.Besides,the expression of cofilin hold a vital role in virulence of A.fumigatus.Moreover,overexpression of cofilin increased the expression levels of chitin synthase gene?chsA-chsG?and this event may enhance stress resistance and virulence of A.fumigatus.Nevertheless,the biological function of A.fumigatus cofilin need further investigation,and mechanistic details behind the protein'function remain unclear.The objectives of this study are to investigate the biological function of some charged residues in cofilin,and to study the role of cofilin in the regulation of expression of chitin synthase genes in A.fumigatus.We have constructed 9 A.fumigatus mutants,including D11A-E12A,D19A-R21A,K26A,K36A,K41A-K42A,E48A,D54A,E56A,and R59A-R61A?meaning strains harbor cofilin mutation D11A;E12A,D19A;R21A,K26A,K36A,K41A;K42A,E48A,D54A,E56A,or R59A;R61A,respectively?expressing cofilin in which some charged residues were mutated to alanine.We found that the growth rate of mutant strains D11A-E12A,K26A,K41A-K42A,E56A,and R59A-R61A was slower that the wild-type strain?WT?at 37?C.Besides,K26A and K41A-K42A manifested reduced growth rate at 48?C,while the growth rate of E56A and R59A-R61A increased at this temperature.Our data revealed that the actin cytoskeleton in hypha of K26A,K41A-K42A,D54A,and R59A-R61A was abnormal,implying that the charged residues in cofilin are important for the regulation of the dynamic balance of actin.Taken together,we found that some charged residues in cofilin play a critical role in the regulation of growth,thermotolerance,and the dynamic balance of actin of A.fumigatus.Here,we found that mutating some charged residues in cofilin to alanine?D19A;R21A,E48A,and K36A?affected mitochondrial function and thus increased formation of reactive oxygen species and induced apoptosis along with typical hallmarks including mitochondrial membrane potential depolarization,the cytochrome c release,upregulation of metacaspases,and DNA cleavage in A.fumigatus.Moreover,two of these mutations?D19A;R21A and K36A?increased acetyl-coenzyme A and ATP concentrations by triggering fatty acid?-oxidation.The upregulated acetyl-coenzyme A affected the ergosterol biosynthetic pathway,leading to overexpression of cyp51A and-B,while excess ATP fueled efflux pumps.Both reduced susceptibility to azoles.Besides,these two mutations enhanced virulence in a Galleria mellonella infection model.Besides,we found that mutant strains D19A-R21A and K36A manifested reduced susceptibility to H₂O₂ and menadione.Our result showed that inhibition of catalases abrogated the oxidation resistance of these two mutants,whereas inhibiting superoxide dismutases was unable to completely eliminate this phenome.In keeping with this phenomenon,we found that the expression levels of catA and cat1 increased.For sod1 and sod2,a reduction in the expression of both genes were observed.Besides,our data revealed that the expressions levels of skn7 and yap1 decreased.Taken together,our results show that key charged residues in cofilin play an essential role in the regulation of mitochondrial function involved in azole susceptibility,apoptosis,virulence,and oxidation adaption of A.fumigatus.To investigate the molecular events behind the fact that overexpression of cofilin increases chitin synthase genes expression in A.fumigatus,we knocked out sakA in both Cofilin OE?A.fumigatus strain that overexpresses cofilin?and the WT.The results showed that only the expression of chsA and chs E in Cofilin OE?sakA was still higher than that in WT?sakA;by contrast,the expression of chsB,chsC,chsD,chsF,and chsG in Cofilin OE?sakA reduced when compared with that in WT?sakA.More,the transcription factor ZipD was also deleted in Cofilin OE and the WT.Our data showed that the expression of chsD,chsE,and chsF in Cofilin OE?zipD increased when compared with that in WT?zipD.Nonetheless,the expression of chsB and chsC in Cofilin OE?zipD is lower than that in WT;besides,there is no significant difference in the expression profiles of chsA and chsG in Cofilin OE and WT.It was found that the expression of zipD,which encodes calcium/calcineurin-responsive transcription factor,in Cofilin OE is lower than that in the WT,while the expression of crzA,another transcription factor in calcium/calcineurin-responsive pathway,is comparable to that in the WT.Moreover,there is no significant different in the expression of rlmA between Cofilin OE and the WT.Taken together,we found that overexpression of cofilin had a significant influence on hog signaling pathway and calcium/calcineurin-responsive pathway,but not on cell wall signaling pathway.Taken together,we found that some charged residues in cofilin holds important biological roles in A.fumigatus.Some charged residues are associated with growth,thermotolerance,and the regulation of dynamic balance of actin;especially,other residues are linked to mitochondrial function involved in metabolism,azole susceptibility,oxidative stress adaption,apoptosis,and virulence of A.fumigatus.Besides,it is suggested that overexpression of cofilin increasing expression of chitin synthesis genes is involved in hog signaling and calcium/calcineurin-responsive pathway,which needs further investigation.Through the above study,it will provide scientific basis for considering A.fumigatus cofilin as a promising target for developing antifungal agents,and will be important for treatment of aspergillosis,and alleviation of economic burden on society.