Studies Of Resistance And Pathogenicity On ADH1 Gene Candida Albicans

It has been wildly accepted that Candida albicans had the characteristics that the pathogenicity was complex and the resistance is serious.And it had become the main reason that the strains were easily to infect and difficult to control.Therefore,the study of its pathogenicity and resistance had been received widespread attention.In recent years,researchers have found that some new genes may be related with drug resistance and pathogenic process,and the study of these was important for the prevention and treatment.ObjectiveIn recent years,there have been sporadic reports speculated ADH1 gene may be associated with drug resistance and pathogenic in Candida albicans,our earlier research also found that Adh1 p may participate in the azole resistance,but there is no direct evidence to support these conclusions so far.In order to understand the effects of ADH1 gene on the growth kinetics,antifungal drug susceptibility,strains pathogenicity and cellular energy metabolism of Candida albicans isolates,we successfully built the ADH1 gene deletion strain and make a series of in-depth and systemic theoretical study.These works may provide theoretical supports for screening new antifungal drug targets,preventing fungal infection,and developing new antifungal drugs for the clinical.Methods1.Construction of gene mutant strains: We constructed the ADH1 gene mutant strains by SAT1-Flipper gene deletion strategy.This method takes the standard isolate SC5314 as the parent strain,plasmid pSFS2 as the carrier,Nourseothricin(NAT)as the screening markers.And we successfully identified the srains by nested-PCR,double enzyme digestion and sequencing method respectively.2.Growth curves: we cultured the wild type,ADH1 gene deletion and reconstitution mutant strains by the same concentration,measured the OD600 of each strain at different time points,obtained the growth curves of various strains and the doubling times in the exponential growthphase.3.Antifungal drug sensitivity experiment: to study the effects of ADH1 gene on antifungal drugs sensitivity,we studied several antifungal drug sensitivity of various strains by the micro-broth dilution assay CLSI M27-A3,M27-S4 and spot-assay experiments.4.Hyphae formation experiment: to study the effects of ADH1 gene on the ability of hyphae formation,we observed the hyphae formation of strains by culturing on solid and liquid hyphae induction medium(Spider,Lee’s,YPD +10%FBS and SLAD mediums).5.Biofilm formation experiment: to study the effects of ADH1 gene on the ability of biofilm formation,XTT reduction method is used to determine the formation of biofilm in RPMI-1640 liquid medium.6.The in vitro adhesion assay: strains were cultured in spider media,added to 24-well flat bottomed plates and incubated for 24 hours at 37℃.7.Cell surface hydrophobicity(CSH)assay:strains were cultured in YPD media for 48 h and CSH of C.albicans was measured by water–hydrocarbon two-phase assay.8.Real-Time RT PCR experiment: by Real-time RT-PCR method,we studied the mRNA expression changes of pathogenicity related genes ALS1,ALS3,HWP1 and CSH1 between different strains.9.In vivo infection model experiment: to study the influence of ADH1 gene on strain pathogenicity in vivo,we experimented with mice,C.elegans and G.mellonella as infection model respectively.10.Mitochondrial energy metabolism related experiment: to study whether ADH1 genes affect mitochondrial energy metabolism in cells preliminarily,we tested the mitochondrial membrane potential in the cell level,ATP content and endogenous reactive oxygen species(ROS)level in different strains.Result1.First of all,we successfully constructed ADH1 gene deletion and reconstitution mutant strains by SAT1-Flipper gene deletion strategy.This would be help to the subsequent phenotype analysis and obtaining the information about the gene function.2.The results of the growth kinetics experimental shown ADH1 gene deletion can significantly reduce the growth rate of Candida albicans,and the concentration of the plateau phase in ADH1 gene deletion mutant was significantly lower than the other strains.3.We examined the antifungal drug sensitivity of ADH1 gene mutant strains and found that no significant change of the azoles(FLC,ITR,KCZ and VRC),propylene amine(Caspofungin),polyene(AmB)and Congo red was observed.4.The hyphae formation experiment and biofilm formation experiment results confirmed that the ADH1 gene can significantly inhibit the formation ability of hyphae formation and biofilm.5.The in vitro adhesion assay cell surface hydrophobicity(CSH)assay shown that ADH1 gene can significantly inhibit the in vitro adhesion and cell surface hydrophobicity(CSH).6.The results of Real-Time RT PCR shown that mRNA expression of the virulent related genes(ALS1,ALS3,HWP1 and CSH1)in ADH1 gene knockout strain were significantly lower.7.The results of the in vivo infection experimental confirmed that ADH1 gene deletion can significantly reduce the pathogenicity in mice,C.elegans and G.mellonella model.8.The mitochondrial energy metabolism experimental confirmed ADH1 gene obvious effect mitochondrial energy metabolism preliminary.ADH1 gene deletion would significantly reduced the level of mitochondrial membrane potential,ATP content but increased endogenous reactive oxygen species(ROS)level significantly.ConclusionsAlthough ADH1 gene has little effect on antifungal susceptibility,it involved in the pathogenesis process of Candida albicans.Deletion of the gene can significantly reduce the virulence,and has the obvious effect of energy metabolism of mitochondria in Candida albicans.The study would provide new ideas and clues for the further study.